Bortezomib-induced gastrointestinal dysfunction, peripheral neuropathy and neuroimmune signalling are influenced by the gut microbiota in mice.

Background

Bortezomib is a first-in-class proteasome inhibitor that is integral to optimal treatment of the malignant plasma cell disorder multiple myeloma; representing a standard-of-care therapy in both transplant-eligible and -ineligible newly diagnosed patients [ 1 ]. The drug is also a front-line therapy for light-chain amyloidosis [ 2 ]. Bortezomib is a dipeptidyl boronic acid that reversibly inhibits the chymotrypsin-like activity of the 26 S proteasome, a key component of the ubiquitin-proteasome system. This mechanism is central to the success of bortezomib in the treatment of myeloma, which is characterised by high immunoglobulin production, rendering malignant cells particularly vulnerable to proteasome inhibition, subsequent endoplasmic reticulum stress and, ultimately, induction of apoptosis [ 3 ].

While considered to be a more “targeted” agent compared with conventional cytotoxic therapies, bortezomib is neurotoxic and causes clinically challenging side effects that reduce quality of life and can necessitate drug dose reductions or treatment cessation, leading to suboptimal clinical outcomes [ 4 – 7 ]. Peripheral neuropathy is the major dose-limiting toxicity of bortezomib that is typically characterised by sensory dysfunction in the extremities, but can also impact motor function. Symptoms include uncomfortable and sometimes painful sensations (pins-and-needles, burning or tingling), numbness and sensory loss in hands and feet, loss of balance and proprioception and, in some cases, muscle weakness [ 4 , 8 , 9 ]. Peripheral neuropathy, of any grade, is reported in approximately 30–50% of people treated with bortezomib, with 8–12% experiencing severe symptoms (grades 3–4), defined by intolerable pain and disabling sensory loss, marked muscle weakness and, rarely, paralysis [ 10 – 12 ]. Previous studies implicate activation of neuroimmune signalling within the peripheral and central nervous system in the pathogenesis of bortezomib-induced peripheral neuropathy [ 13 – 15 ].

While peripheral neuropathy is a well-documented clinical challenge, gastrointestinal side effects are also highly prevalent; being reported in over 50% of people undergoing bortezomib therapy, but are less understood. Gastrointestinal side effects commonly associated with bortezomib use include diarrhoea, constipation, nausea and vomiting [ 11 , 16 – 20 ]. A rarer, but more serious, complication of bortezomib therapy is paralytic ileus, characterised by abdominal pain, distention and absence of bowel movements, which can be life-threatening [ 21 – 23 ]. Recent data highlight the impact of bortezomib-associated gastrointestinal symptoms, with a significant decrease in two-year progression-free and overall survival observed in people with severe grade 3 gastrointestinal symptoms in comparison to those with milder grade 1 or 2 symptoms [ 18 ]. Both peripheral neuropathy and the gastrointestinal consequences of bortezomib therapy remain without effective intervention. Although current clinical practice guidelines recommend duloxetine for the managememt of painful peripheral neuropathy, the magnitude of benefit is small and, hence, implementation is low [ 24 , 25 ]. This, combined with the complete lack of specific interventions for gastrointestinal symptoms associated with bortezomib, highlights a major gap in care. Thus, there is a need to better understand the aetiology of these side effects to develop novel, more efficacious supportive care strategies that enhance bortezomib tolerability and adherence.

The ecosystem of micro-organisms in the gut (the gut microbiota) is rapidly emerging as a key modulator of anti-cancer drug toxicity, owing to its ability to modulate drug metabolism, host immune function and cellular repair processes [ 26 – 30 ]. The gut microbiota is increasingly recognised for its ability to influence both central and peripheral nerve function, including autonomic nerves, and, as a result, has been implicated in the development of neuropathic and visceral pain across various clinical contexts [ 31 – 35 ]. Within the oncology setting, the gut microbiota has been causally implicated in the development of peripheral neuropathy and gastrointestinal toxicity caused by some conventional chemotherapeutic agents [ 36 – 38 ]. Of note, mice lacking a gut microbiome (through antibiotic depletion or germ-free status) are resistant to oxaliplatin-induced peripheral neuropathy [ 36 ]. Moreover, the transfer of microbiota from mice sensitive to paclitaxel-induced peripheral neuropathy into peripheral neuropathy-resistant mice induces nociceptive hypersensitivity [ 37 ]. Beyond these findings, the gut microbiota’s role in mediating anti-cancer drug-induced neuropathy, including those induced by bortezomib, is unclear. Hence, we aimed to develop a mouse model in which to investigate the involvement of the gut microbiota in the development of bortezomib side effects.

Methods

Animals and bortezomib treatment

Male C57BL/6 mice (6–8 weeks old, weight: 16–26 g), were bred and housed in standard, individually ventilated cages ( N = 4–5/cage) in the South Australian Health and Medical Research Institute (SAHMRI) specific pathogen-free (SPF) Bioresources Facility (Adelaide, South Australia). Male gnotobiotic (germ-free; GF) C57BL/6 mice (Translational Research Institute, Queensland, Australia) were housed in positively pressurised, high-efficiency particulate air (HEPA)-filtered ISO-P cages (Techniplast, Italy) at the SAHMRI Preclinical, Imaging and Research Laboratories (PIRL). All side effect studies were performed in C57BL/6 mice. All mice were housed at an ambient temperature of 21 °C ± 3.0 °C with a 12:12 h light-dark cycle and had access to gamma-irradiated (SPF mice) or autoclaved (GF) commercially pelleted food and sterilised water ad libitum . Bortezomib (Selleckchem, USA) or vehicle (3.9% DMSO in PBS) alone was given intravenously via tail vein injection twice weekly (days 1, 4, 8 and 11) for two weeks, representing a clinical dosing schedule [ 11 , 12 ]. Forty eight hours after final treatment (unless otherwise stated), mice were humanely killed via isoflurane anaesthesia, cardiac puncture and cervical dislocation.

Von Frey test

To assess paw sensitivity to mechanical stimulation, the manual von Frey method was performed using filaments ranging from 0.07 to 2 g of force (Stoelting, USA). Unless otherwise stated, mice were acclimatised in the testing room for 15 min and habituated for 45 min on testing grids using modular holding cages 24 h, and then immediately, prior to mechanical sensitivity testing, with testing performed at baseline (24 h prior to first treatment) and at the study end-point (48 h after final treatment) [ 39 ]. The ‘ascending stimulus’ method was used with a 50% withdrawal response rate indicative of animals reaching mechanical withdrawal threshold [ 40 ]. Baseline mechanical sensitivity assessments in SPF mice informed randomisation into treatment groups.

Behavioural phenotyping

To evaluate non-stimulus-evoked, spontaneous behaviour, automated behavioural analyses were performed 24 h after final treatment using a behavioural spectrometer (Biobserve, Germany) [ 41 ]. Animals were acclimatised in the testing room for 15 min prior to being individually placed in the testing arena and recorded for 20 min, as previously described [ 39 , 42 ]. The time engaged in 23 different behaviours including grooming, rearing and locomotor behaviours were recorded, as well as total track length and zone duration during the testing period.

Rotarod test

The rotarod apparatus (LE8200, Harvard Apparatus) was used to assess animal motor function and coordination. Before each training and testing session, mice were acclimatised in the testing room for 15 min. Mice were trained using an acceleration mode protocol (4–40 rpm) over four consecutive days with increasing time spent on the rod each day, starting from 70 s on day 1, 145 s on day 2, 220 s on day 3 and 300 s on day 4. Rotarod testing (acceleration mode for up to 5 min) was performed 24 h prior to first treatment to randomise mice, and then 24 h after final drug treatment. The mean of 3 tests, performed at least 15 min apart, was recorded for each animal [ 43 ]. A normal (time to) fall was defined as being within the range of that observed in vehicle-treated mice.

Intestinal barrier permeability and integrity assessments

Intestinal barrier function was assessed 48 h after final drug treatment by the administration of 4 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, USA) at a 500 mg/kg dose (75 mg/mL) via oral gavage. Plasma citrulline, a biomarker of enterocyte mass and thus intestinal barrier integrity, was quantified in plasma (collected at the study end-point) by liquid chromatography mass spectrometry, using an API 5500 QpQ mass spectrometer (Sciex, USA) and Acquity UPLC system (Waters Corporation, USA).

Total gastrointestinal transit time assessment

Mice were fasted overnight for 17 h (4pm-9am) before being placed in individual cages and receiving 0.1 mL of a semi-liquid solution containing 5% Evans blue dye, 0.9% NaCl, and 0.5% methylcellulose in PBS, via oral gavage. Mice were monitored for the presence of a blue faecal pellet for up to 6 h after gavage. Total gastrointestinal transit time was calculated as the time from gavage until the time of visualisation of the first blue pellet.

Neurofilament light chain analysis

Neurofilament light chain (NfL), a biomarker of neurotoxicity [ 44 , 45 ], was quantified in serum (collected at the study end-point) by GeneWorks (Australia) using the Simoa NF-light Advantage v2 Assay following manufacturer’s instructions (Quanterix, USA).

Histopathological analysis of the gastrointestinal tract

Immediately after humane killing, the stomach and entire gastrointestinal tract, from pyloric sphincter to rectum, were dissected. Stomachs were weighed full (i.e. with contents) and empty (after contents were removed). The tissue was then laid flat and one half of the corpus section was excised prior to fixation in 10% neutral buffered formalin (NBF). Intestinal contents were removed by flushing with ice-cold PBS (pH 7.4), before weighing both the small and large intestines. Two samples each (0.5 cm long) of duodenum, jejunum, ileum and colon were collected and fixed in 10% NBF for 24 h, and then processed and embedded in paraffin wax. Sections (5 μm) were stained with haematoxylin and eosin (H&E) and subsequently imaged using the NanoZoomer S60 Digital Slide Scanner (Hamamatsu Photonics, Japan). Images were evaluated in a blinded fashion on NDP.view2 software, such that 10 well-oriented crypts and villi were measured per slide, and an average for both was calculated for each animal.

Nerve histopathology

Segments of sciatic nerve from the mid-femoral region and the superior half of the left cervical vagal nerve were excised and fixed in 2.5% glutaraldehyde (ProSciTech, Australia) for 24 h, before washing in 50 mM sodium cacodylate buffer (pH 7.4). Samples were then stained with 1% osmium tetroxide (ProSciTech) for 1 h at room temperature, dehydrated in graded concentrations of ethanol, embedded into EPON resin (ProSciTech) and incubated at 70 °C overnight to allow polymerisation. Sections (0.5 μm) were cut on an Ultracut E Ultramicrotome (Reichert-Jung, USA) and stained with toluidine blue, before being imaged on the NanoZoomer S60. Fibre and axon diameter were measured in three and five symmetrically-spaced regions of interest from sciatic and vagal nerve sections, respectively, using NDP.view or Image J software (National Institutes of Health, USA, Version 2.14.0). Mean myelin thickness ([fibre diameter] - [axon diameter]) and g ratio ([axon diameter]/[fibre diameter]) were then calculated for each sample.

Inflammatory cytokine gene expression in dorsal root ganglia and nodose ganglia

Dorsal root ganglia (DRG) at levels 3–5 (L3-L5) and nodose ganglia pairs, containing nerve cell bodies of the mouse sciatic nerve and vagus nerve, respectively [ 46 , 47 ], were resected and snap frozen in liquid nitrogen. RNA was extracted using the Invitrogen PureLink™ RNA Micro Kit following the manufacturer’s instructions (Thermo Fisher Scientific, USA). Briefly, samples were lysed using the TissueLyser II (Qiagen, Germany) and stainless steel beads (Qiagen). RNA was isolated using TRIzol (Thermo Fisher Scientific), including on-column DNase digestion, eluted in 22 µL of RNase-free water and stored at -20 °C. Total RNA yield and purity were quantified using the NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific). Next, cDNA was synthesised using Superscript IV reverse transcriptase (Invitrogen), from 650 ng (per three DRG [L3-L5]), or 100 ng (per two nodose ganglia) of RNA template. Real-time quantitative PCR was performed using QuantStudio 3 Real-time PCR system (Applied Biosystems, USA) to determine the mRNA expression of the genes encoding mouse IL-1β, IL-6, TNF-ɑ and F4/80 using RT 2 SYBR ® Green reagent (Qiagen) and 10 µM primer pair (primer sequences shown in supplementary methods section) in 15 µL reaction volume. Ct values were obtained and analysed using QuantStudio 3 (Thermo Fisher Scientific) and changes in gene expression were calculated relative to Gapdh expression, using the 2 −ΔCT method [ 48 ].

Gut microbiota 16S rRNA gene sequencing

Faecal pellets were collected longitudinally on day 0 (baseline; F0), day 4 (after one treatment; F1), day 7 (after two treatments; F2), day 11 (after three treatments; F3) and day 12 (after four treatments; F4), and stored at -80 °C. Faecal DNA was extracted using the DNeasy PowerSoil Pro Kit (Cat #47014, Qiagen), as per manufacturer’s guidelines. Briefly, samples and the lysis buffer were added to PowerBead Pro Tubes (Qiagen) and incubated at 65 °C for 10 min, before being homogenised using the TissueLyser II at maximum speed for 10 min and DNA was eluted in 50 µL of Tris (10 mM). DNA yield and purity were determined using a NanoDrop 8000 Spectrophotometer. Sequencing of the amplicon library was performed using an Illumina Miseq system (2 × 300 bp run) at the South Australian Genomics Centre (SAHMRI, Adelaide, South Australia). Bioinformatic analysis, including demultiplexing of paired end 16S rRNA gene sequences, error correcting to exact sequence variants and taxonomic assignment was performed, as previously described [ 49 ]. Alpha and beta diversity values were determined as previously described [ 49 ].

Germ-free mouse studies

Caecal content was collected from five seven-week-old healthy male SPF C57BL/6 mice, pooled and resuspended in PBS with 15% glycerol using a Stomacher ® 80 Bag (Thomas Scientific, USA) under anaerobic conditions, as previously reported [ 50 ]. Forty GF C57BL/6 mice (four to five weeks old) were divided into two groups ( N = 20/group), and one group was colonised with gut microbiota using faecal microbiota transplantation (FMT) via administration of two 100 µL caecal slurries (via oral gavage) [ 49 – 51 ]. At seven weeks of age, GF and FMT mice were treated with vehicle or high-dose bortezomib (HD-BTZ; 1 mg/kg) twice weekly for two weeks, as described earlier ( N = 10/group). The GF status of mice was routinely assessed throughout the experiment via amplification of 16S rRNA gene in faecal DNA samples [ 52 ]. Following final treatment, GF and FMT mice were transported to SAHMRI Bioresources in a sterile cage, before overnight acclimation. The next day, mice were individually removed from their cages in a biosafety cabinet and behavioural spectrometry was performed (as described above). Mice (six at a time) were then acclimatised in the testing room for 15 min and habituated for 45 min on von Frey testing grids before performing mechanical sensitivity testing (as described above). Sub-cohorts of GF and FMT mice ( N = 4/group) were designated for real-time quantitative PCR and stomach weight analyses only to minimise potential contamination from exposure to ambient conditions. Control SPF mice (vehicle and HD-BTZ-treated) were housed at SAHMRI and underwent behavioural phenotyping and von Frey testing 24 h after final treatment in line with GF and FMT mice. Mice were then humanely killed (as described above).

Statistical analysis

Unless otherwise specified, data were analysed using GraphPad Prism 10.4.1. Data were analysed for normality using the Shapiro-Wilk test. For parametric data, multiple-group analyses were performed using a one-way or two-way ANOVA with Dunnett’s or Tukey’s multiple comparisons test or uncorrected Fisher’s LSD test (as appropriate) and two-group analyses were analysed using a t-test. When normality was not confirmed, non-parametric tests were used (Mann-Whitney test to compare two groups or Kruskal-Wallis with Dunn’s multiple comparisons test for multiple-group comparisons). Real-time quantitative PCR data in GF/FMT mice were analysed using individual Mann-Whitney U tests. Tumour burden, behavioural spectrometry ‘time engaged in’ and total gastrointestinal transit time data were log-transformed prior to conducting statistical analyses. For rotarod studies, a Fisher’s exact test was performed. All microbial sequencing data were analysed using R version 3.6.3, as previously described [ 49 ]. Correlative analyses between gut microbiota read-outs and behavioural phenotypes were performed using Pearson’s correlation for parametric data and Spearman’s correlations for non-parametric data.

Results

High-dose bortezomib alters weight and spontaneous behaviour in mice indicative of reduced well-being

Side effects of bortezomib therapy adversely affect patient quality of life [ 53 , 54 ]. To initially explore the effect of bortezomib on animal well-being, C57BL/6 mice were treated with high-dose bortezomib (HD-BTZ; 1 mg/kg), which induces 5TGM1 myeloma tumour regression in the related C57BL/KalwRij mouse strain [ 55 , 56 ], equivalent to a very good partial or complete response observed clinically, or low-dose bortezomib (LD-BTZ; 0.5 mg/kg) which induces stable disease (Fig. S1A-C). C57BL/6 mice treated with HD-BTZ displayed cyclical, self-limiting weight loss and end-point weight change (-7.00 ± 1.60% relative to baseline weight) was significantly different compared with both vehicle-treated mice (7.30 ± 0.81%, P < 0.01) and LD-BTZ-treated mice (3.22% ± 2.54%, P < 0.001; Fig. 1 A, B).

Fig. 1 Evaluation of changes in weight and spontaneous behaviour in SPF mice treated with bortezomib. A Daily body weight as percentage change relative to baseline. Arrows indicate treatment days. Data represent mean ± SEM ( N = 11–13 mice/treatment group). B End-point body weight as percentage change relative to baseline. Data represent mean ± SEM ( N = 11–13 mice/treatment group) **** P < 0.0001, *** P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). C Total distance travelled by mice over 20 min and representative track map images. Data represent mean ± SEM ( N = 11–21 mice/treatment group) **** P < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test). D Total time engaged in still, locomotor, rearing, orienting and grooming behaviour over 20 min. Data represent mean ± SEM ( N = 11–21 mice/treatment group) **** P < 0.0001, *** P < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). E Time engaged in individual grooming behaviours over 20 min. Data represent mean ± SEM ( N = 11–21 mice/treatment group) **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 (two-way ANOVA with Tukey’s multiple comparisons test)

Automated behavioural phenotyping was then performed to evaluate changes in spontaneous animal behaviour related to reduced well-being. Notably, mice treated with HD-BTZ displayed profound changes in behaviour with a 40% reduction in total distance travelled (track length: 3264 ± 297.7 cm), total time engaged in locomotor activity (113.6 ± 9.8 seconds [s]) and time spent in centre zone of testing arena (15.8 ± 2.8 s) compared with vehicle-treated animals (track length: 5476 ± 218.1 cm; P < 0.0001, locomotor time: 192.2 ± 6.4 s; P < 0.001 and centre zone time: 26.6 ± 2.9 s; P < 0.0001; Fig. 1 C, D and S2A, B). In addition, HD-BTZ-treated mice spent 52% and 32% less time engaged in total rearing (59.7 ± 6.5 s) and orienting (237.3 ± 17.7 s) behaviour compared with vehicle-treated mice (rearing time: 124.6 ± 4.2 s; P < 0.0001, orienting time: 351.3 ± 6.6 s; P < 0.0001), respectively (Fig. 1 D and S2C, D). Consistent with this, HD-BTZ-treated mice spent 139% more time still (437.6 ± 32.4 s) compared with vehicle-treated counterparts (182.8 ± 10.3 s; P < 0.0001; Fig. 1 D). Strikingly, while there was no difference in total time spent grooming, HD-BTZ-treated mice demonstrated a 77% increase in time engaged in paw grooming (107.4 ± 23.4 s) compared with vehicle-treated animals (60.6 ± 13.9 s; P < 0.0001) but spent less time engaged in grooming of other body parts including legs, back and genitalia ( P < 0.0001) compared with control mice (Fig. 1 D, E). In contrast, we found no difference in spontaneous behaviour between LD-BTZ- and vehicle-treated mice (Fig. 1 B-D and S2A-D). Taken together, HD-BTZ, but not LD-BTZ, alters weight and spontaneous behaviour of mice that is indicative of reduced well-being.

High-dose bortezomib induces peripheral neuropathy, inflammation in dorsal root ganglia and neuronal damage in mice

Numbness and sensory loss in hands and feet, changes in proprioception and reduced fine motor function are clinical features of bortezomib-induced peripheral neuropathy [ 4 , 8 , 9 ]. To assess whether bortezomib induces a similar neuropathic phenotype in mice, von Frey and rotarod testing was performed. Notably, mice treated with HD-BTZ displayed a significant decrease in paw sensitivity to mechanical stimulus (paw withdrawal threshold median and range: 1 g [0.16–1.4 g]) compared with vehicle-treated mice (0.6 g [0.16–1 g], P < 0.01). In contrast, LD-BTZ-treated mice showed no significant change in mechanical sensitivity (0.6 g [0.16–2 g]) compared with vehicle-treated and HD-BTZ-treated mice (Fig. 2 A). In addition, HD-BTZ-treated mice demonstrated a marked reduction in rotarod performance. While time spent on rod was not significantly reduced in HD-BTZ-treated mice compared with vehicle-treated mice (data not shown), 43% (6/13) of HD-BTZ-treated mice fell off the rod more rapidly than the vehicle-treated mice (43% vs. 0%, P < 0.01; Fig. 2 B). In contrast, there was no significant difference in rotarod performance in mice treated with LD-BTZ compared with vehicle-treated mice, as assessed by the percentage of mice falling off the rod rapidly (14% [2/12] vs. 0%) (Fig. 2 B).

Fig. 2 Evaluation of sensory function, motor function, neuroimmune signalling and neurotoxicity in SPF mice treated with bortezomib. A Hind paw sensitivity to mechanical stimulus, as assessed by von Frey testing. Box and whiskers plot depicts median, interquartile range, minimum and maximum values and individual values (dots) ( N = 19–28 mice/treatment group) ** P < 0.01 (Kruskal-Wallis test with Dunn’s multiple comparisons test). B Motor co-ordination in mice, as assessed by rotarod performance testing. Contingency plot depicts the number of mice with normal or rapid fall ( N = 12–14 mice/treatment group) ** P < 0.01 (Fisher’s exact test). C Representative images of toluidine blue-stained sciatic nerve sections (Scale bar represents 25 μm). D - E Myelinated nerve fibre number ( D ) and size distribution ( E ) in toluidine blue-stained sciatic nerve sections. Data represent mean ± SEM ( D ) and mean ( E ) ( N = 4–5 mice/treatment group) (one-way ANOVA with Tukey’s multiple comparisons test (D)). F - I Gene expression of proinflammatory markers F4/80 ( Adgre1 ) ( F ), TNF-α ( Tnfa ) ( G ), IL-6 ( Il6 ) ( H ) and IL1-β ( Il1b ) ( I ) in DRGs. Data represent mean ± SEM ( N = 6–10 mice/treatment group) ** P < 0.01, * P < 0.05 (Kruskal-Wallis test with Dunn’s multiple comparisons test). J NfL levels in serum. Data represent mean ± SEM. ( N = 9–10 mice/treatment group) **** P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test)

Previous studies have shown that bortezomib induces histological and inflammatory changes in the sciatic nerve of mice [ 13 , 57 – 59 ]. While there was a trend towards reduced sciatic nerve myelinated fibre number with HD-BTZ treatment compared with vehicle-treated mice, this did not reach statistical significance (Fig. 2 C-E). There were also no differences in sciatic nerve myelinated fibre morphology in HD-BTZ-treated mice compared with vehicle-treated mice (Fig. S3). Dorsal root ganglia (DRG) receive and process sensory information from the periphery and internal organs to the central nervous system (CNS) to control voluntary and involuntary bodily functions [ 60 , 61 ]. Notably, HD-BTZ induced significant neuroimmune signalling in sciatic nerve dorsal root ganglia (DRG; L3-L5). Specifically, HD-BTZ-treated mice demonstrated a 4-fold increase in Adgre1 (encoding the mouse macrophage marker F4/80) expression in DRG relative to vehicle-treated mice ( P < 0.05; Fig. 2 F). Pro-inflammatory cytokine gene expression was also significantly increased in DRG from HD-BTZ-treated mice, including a 3.8-fold increase in Tnfa ( P < 0.05) and Il6 ( P < 0.01) expression, and a 4.8-fold increase in Il1b ( P < 0.01) expression, relative to vehicle-treated mice (Fig. 2 G-I). In contrast, we found no significant change in DRG expression of Adgre1 , Tnfa , Il6 and Il1b between LD-BTZ- and vehicle-treated mice. Expression of Il1b was significantly higher in HD-BTZ-treated mice than LD-BTZ-treated mice ( P < 0.05; Fig. 2 F-I). These data show that HD-BTZ, but not LD-BTZ, induces peripheral neuropathy in mice that is accompanied by neuroimmune signalling in sciatic nerve DRG. Neurofilament light chain (NfL) is a neuron-specific cytoskeletal protein and serum biomarker of axonal damage associated with neurodegenerative conditions and chemotherapy-induced peripheral neuropathy [ 44 , 45 ]. Serum NfL is also elevated in MM patients treated with bortezomib, with levels correlating with electrophysiological measures of acute axonal damage in these patients [ 62 ]. Notably, we found serum NfL levels to be dramatically elevated in mice treated with HD-BTZ (mean: 791 ± 179 pg/mL) compared with both vehicle-treated mice (76.5 ± 22.8 pg/mL; P < 0.0001) and LD-BTZ-treated mice (51.3 ± 5.6 pg/mL; P < 0.0001) while treatment with LD-BTZ had no significant effect on serum NfL levels (Fig. 2 J).

High-dose bortezomib alters gastrointestinal motility and permeability, but does not alter gastrointestinal micro-architecture or induce intestinal inflammation

To explore whether bortezomib induces gastrointestinal dysfunction in mice, we initially assessed wet weights of gastrointestinal tissues at necropsy 48 h after final drug treatment. Notably, HD-BTZ-treated mice displayed a 52% increase in full stomach weight (1.61 ± 0.148% of body weight [%BW]) compared with vehicle-treated mice (1.06 ± 0.665%BW, P < 0.005; Fig. 3 A). This increase in full stomach weight was observed as early as 6 h after a single treatment with HD-BTZ (Fig. S4A). Empty stomach weight was also elevated in both HD-BTZ- and LD-BTZ-treated mice compared with control animals ( P < 0.05; Fig. 3 A). In addition, HD-BTZ-treated mice showed an increase in small intestine (4.42 ± 0.171%BW) and caecum wet weight (1.74 ± 0.093%BW), compared with vehicle-treated mice (3.49 ± 0.120%BW, P < 0.0001 and 1.22 ± 0.057%BW, P < 0.0001, respectively; Fig. 3 B). Notably, we found no change in daily food intake in HD-BTZ-treated mice over time (Fig. S4B). Stomach (full), small intestine, caecum and colon wet weights did not differ between LD-BTZ- and vehicle-treated mice (Fig. 3 A, B).

Fig. 3 Structural and functional changes in the gastrointestinal tract of SPF mice following bortezomib treatment. A , B Wet weights of full and empty stomach ( A ), small intestine, colon and caecum ( B ) as a percentage of total body weight (%BW). Data represent mean ± SEM ( N = 10–11 mice/treatment group) **** P < 0.0001, ** P < 0.001, ** P < 0.01, * P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). C Representative images of stomach and intestinal histopathology. Scale bar represents 50 μm. D Stomach pit depth and intestinal villus height and crypt depth. Data represent mean ± SEM ( N = 11 mice/treatment group) (one-way ANOVA with Dunnett’s multiple comparisons test). E Enterocyte mass, as assessed by plasma citrulline. Data represent mean ± SEM ( N = 9–10 mice/treatment group). F Intestinal permeability, as assessed using FITC-dextran. Data represent mean ± SEM ( n = 4–5 mice/treatment group). * P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). G Representative images of claudin-1 immunofluorescence staining in the duodenum. White arrows indicate evidence of claudin-1 internalisation. Scale bar represents 50 μm. H Intestinal motility, as assessed by total gastrointestinal transit time (TGITT). Data represent mean ± SEM ( N = 5–6 mice/treatment group). * P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test)

Histopathological assessment of gastrointestinal tissues revealed no significant differences in stomach or intestinal architecture (villus height, pit depth or crypt depth) between HD-BTZ-, LD-BTZ- and vehicle-treated mice (Fig. 3 C, D). In addition, no change in enterocyte mass was observed, with plasma citrulline levels being comparable across treatment groups (Fig. 3 E). Moreover, there were no changes in expression of inflammatory markers Adgre1 , Tnfa , Il6 and Il1b in gastrointestinal tissues (Fig. S5) or detectable increases in circulating levels of inflammatory cytokines and chemokines, IFN-γ, IL-1ɑ, IL-6, IL-10, IL-17, CXCL1 and CCL2 (Fig. S6). In contrast, circulating levels FITC-dextran (4 kDa) following oral gavage of the fluorescent probe were significantly elevated in mice treated with HD-BTZ (407 ± 51.0 ng/mL) compared with vehicle-treated mice (191 ± 39.2 ng/mL; P < 0.05; Fig. 3 F) indicating increased intestinal permeability [ 63 ] which corroborated the observed internalisation of claudin-1, a tight junction protein, in the small intestine of HD-BTZ-treated mice (Fig. 3 G). In addition to permeability changes, HD-BTZ caused a decrease in total gastrointestinal transit time (TGITT) (146 ± 26.0 min), indicative of hypermotility, compared with both vehicle-treated (237 ± 22.9 min, P < 0.05) and LD-BTZ-treated (247 ± 14.6 min, P < 0.05) mice. Conversely, LD-BTZ-treated mice did not differ in TGITT in comparison to vehicle-treated mice (Fig. 3 H). Collectively, these data suggest bortezomib induces gastrointestinal dysfunction in mice independently of local and systemic inflammation or overt architectural disruption of the gastrointestinal tract.

High-dose dose bortezomib causes inflammation in nodose ganglia of the vagus nerve

While autonomic nerve dysfunction is speculated to play a role in bortezomib-induced gastrointestinal dysfunction [ 19 , 21 ], the effect of bortezomib on autonomic nerves that control gastrointestinal function is yet to be explored. To investigate this, we examined the micro-architecture of the vagus nerve, a critical communication pathway in the gut-brain axis controlling gastrointestinal function [ 64 ]. We found no differences in the number of myelinated or unmyelinated nerve fibres, myelinated fibre size or morphology between treatment groups (Fig. 4 A-D and S7). However, HD-BTZ-treated mice demonstrated a significant increase in expression of Adgre1 in nodose ganglia (housing sensory cell bodies of the vagus nerve [ 47 ], with a 4.31-fold increase relative to vehicle-treated mice ( P < 0.01; Fig. 4 E). Nodose ganglia expression of pro-inflammatory markers Tnfa , Il6 and Il1b was not significantly elevated in HD-BTZ-treated mice compared with vehicle-treated animals (Fig. 4 F-H). These findings suggest that neuroimmune signalling in the vagal nerve could, at least in part, be associated with gastrointestinal dysfunction associated with bortezomib treatment.

Fig. 4 Assessment of vagus nerve histopathology and inflammation in nodose ganglia of SPF mice following bortezomib treatment. A Representative images of toluidine blue-stained vagus nerve sections. Scale bars represent 20 μm. B , C Myelinated fibre number ( B ) and size distribution ( C ) in toluidine blue-stained vagus nerve sections. Data represent mean ± SEM ( N = 4–5/treatment group). D Unmyelinated fibre number in toluidine blue-stained vagus nerve sections. Data represent mean ± SEM ( N = 4–5/treatment group). E - H Gene expression of proinflammatory markers F4/80 ( Adgre1 ) ( E ), TNF-α ( Tnfa ) ( F ), IL-6 ( Il6 ) ( G ) and IL1-β ( Il1b ) ( H ) in nodose ganglia. Data represent mean ± SEM ( N = 10/treatment group) ** P < 0.01 (Kruskal-Wallis test with Dunn’s multiple comparisons test)

High-dose bortezomib induces a transient alteration in gut microbiota diversity and composition

Previous studies have demonstrated that some anti-cancer treatments can induce changes in gut microbiota composition, with these changes increasingly implicated in the development of drug side effects [ 65 , 66 ]. However, the effects of bortezomib on gut microbiota composition are yet to be investigated. To address this, we performed 16S rRNA gene sequencing on longitudinally-collected faecal samples from mice with and without HD-BTZ treatment. Analysis of Shannon index identified a transient decrease in alpha diversity four days after initial bortezomib administration compared with vehicle-treated mice (F1; P < 0.05) (F1) and baseline (F0; P < 0.001) and which was not evident at the study end-point (F4; Fig. 5 A). Relative abundance analyses identified modest but statistically significant shifts in the microbial composition of HD-BTZ-treated mice compared with vehicle-treated mice at F1 (Fig. 5 B), with Lactobacillus gasseri ( L.gasseri ) expansion observed at this time-point in HD-BTZ-treated mice (HD-BTZ: 27.7 ± 3.72% vs. vehicle: 13.5 ± 3.02%, P < 0.05; Fig. 5 C) aligning with the decrease in microbial diversity. Indeed, Shannon index inversely correlated with L.gasseri relative abundance at F1 ( r =-0.905; P < 0.0001). The abundance of other taxa were not significantly different between groups. PERMANOVA identified differences in beta diversity between HD-BTZ- and vehicle-treated mice at F1 and F4 (Fig. 5 D-F). Correlation analyses revealed that Shannon index inversely correlated with time engaged in orientation behaviour at F1 in HD-BTZ-treated mice ( r =-0.601; P < 0.05). In addition, paw withdrawal threshold to mechanical stimulus positively correlated with Shannon index at F1 in HD-BTZ-treated mice ( r = 0.598; P < 0.05). Conversely, paw withdrawal threshold inversely correlated with L.gasseri relative abundance ( r =-0.613; P < 0.05) at F1 in HD-BTZ-treated mice (Fig. S8).

Fig. 5 Bortezomib-induced dysbiosis in SPF mice is characterised by an increase in the abundance of Lactobacillus and a decrease in microbial diversity. A Alpha diversity in microbiota composition (Shannon index) longitudinally assessed over experimental time-course. Faecal sampling days are indicated (F0-F4) *** P < 0.001 * P < 0.05. B Relative abundance of microbiota longitudinally assessed over experimental time-course for individual mice. C Relative abundance of Lactobacillus gasseri (L. gasseri) at time-point F1. Data represent mean ± SEM ( N = 10–12/treatment group) ** P < 0.01 (unpaired t-test). D - F Principal component analyses of beta diversity in microbiota of treatment groups at F0, F1 and F4. Dots represent individual mice and ovals represent 95% confidence intervals. PERMANOVA analyses are presented for all principal component plots

The gut microbiota modulates the development of bortezomib side effects in germ-free mice

The gut microbiota has previously been implicated in the aetiology of chemotherapy side effects, with gnotobiotic (germ-free, GF) and antibiotic-treated mice reported to be less vulnerable to the toxic effects of numerous chemotherapeutic agents, including oxaliplatin, paclitaxel and irinotecan [ 36 – 38 , 67 ]. In light of our data demonstrating that bortezomib alters gut microbiota composition, we next sought to interrogate whether the presence or absence of a gut microbiota influenced bortezomib-induced side effects. To do this, we used GF mice; considered the gold standard for studying gut microbiota contributions to disease [ 68 , 69 ], and antibiotic (ABX)-treated mice. For GF mice, we compared the development of bortezomib side effects in these mice with conventional SPF mice and GF mice colonised with a healthy gut microbiota from SPF mice (“FMT” mice). 16S rRNA PCR confirmed the germ-free status of GF mice prior to the commencement of bortezomib treatment, and that this status was maintained throughout the course of the study (Fig. S9). FMT mice were successfully colonised with an SPF-derived gut microbiota (Fig. S9). Treatment with HD-BTZ led to significantly decreased end-point weight in SPF mice (end-point change [mean ± SEM]: -10.62 ± 1.36% relative to baseline weight), compared with vehicle-treated couterparts (4.31 ± 1.55%, P < 0.01; Fig. 6 A) in line with data presented in Fig. 1 B. While this was not observed in GF or FMT mice (Fig. 6 A), HD-BTZ significantly decreased end-point weight in both ABX ( P < 0.01) and non-ABX ( P < 0.01) mice compared with vehicle-treated couterparts (Fig. S10A). Notably, behavioural phenotyping demonstrated that HD-BTZ altered spontaneous behaviour in both GF and SPF mice, although the magnitude of effect was generally milder in GF mice. Specifically, bortezomib-treated GF mice showed a 39.6% reduction in total distance travelled (track length: 3909 ± 694 cm) compared with vehicle-treated GF mice (6468 ± 397 cm, P < 0.01), while SPF mice exhibited a 62.2% reduction with HD-BTZ treatment (vehicle: 6581 ± 333 cm vs. bortezomib: 2489 ± 269 cm, P < 0.0001). Notably, treatment with HD-BTZ did not alter total distance travelled in FMT mice (Fig. 6 B) but did in both ABX ( P < 0.001) and non-ABX ( P < 0.05) mice compared with vehicle-treated mice (Fig. S10B). Bortezomib also caused reductions in time engaged in overall locomotor and orienting behaviours in both GF and SPF mice compared with their vehicle-treated counterparts, with effects less pronounced in GF mice. In contrast, locomotor and orienting behaviour was not affected by HD-BTZ treatment in FMT mice (Fig. 6 C). HD-BTZ-treated ABX mice displayed significantly more time spent still and less time engaged in exploratory behaviour (i.e. locomotor, orienting and rearing) relative to vehicle treated mice ( P < 0.01), although these effects did not reach statistical significance in non-ABX mice (Fig. S10C). Notably, HD-BTZ caused a 223% increase in time engaged in paw grooming (198 ± 47.5 s) in SPF mice relative to vehicle-treated SPF mice (61.2 ± 5.84 s, P < 0.001), while there was no significant effect in either GF or FMT mice. In fact, time engaged in paw grooming was significantly decreased in both HD-BTZ-treated GF (83.4 ± 25.0 s; P < 0.05) and FMT (56.7 ± 21.0 s; P < 0.001) mice compared with HD-BTZ-treated SPF mice (Fig. 6 D). Time engaged in paw grooming was not significantly elevated with HD-BTZ treatment in ABX or non-ABX mice (Fig. S10D).

Fig. 6 Assessment of bortezomib side effect development in GF, FMT and SPF mice. A End-point (day 12) body weight change relative to baseline. Data represent mean ± SEM ( N = 10–12 mice/treatment group) *** P < 0.001 * P < 0.05 (two-way ANOVA with Tukey’s multiple comparisons test). B Total distance travelled by mice after 20 min and representative track map images. Data represent mean ± SEM ( N = 6 mice/treatment group) **** P < 0.0001, *** P < 0.001, ** P < 0.01 (two-way ANOVA with Tukey’s multiple comparisons test). C Total time engaged in still, locomotor, rearing, orienting and grooming behaviour over 20 min. Data represent mean ± SEM ( N = 6 mice/treatment group) **** P < 0.0001, *** P < 0.001, ** P < 0.01 * P <0.05 (two-way ANOVA with Tukey’s multiple comparisons test). D Time engaged in grooming of paws over 20 min. Data represent mean ± SEM ( N = 6 mice/treatment group) *** P < 0.001, ** P < 0.01, * P < 0.05 (two-way ANOVA with Tukey’s multiple comparisons test). E Hind paw sensitivity to mechanical stimulus, as assessed by von Frey testing. Box and whiskers plot depicts median, interquartile range, minimum and maximum values and individual values (dots) ( N = 6 mice/treatment group) (Mann-Whitney U tests). F Full stomach wet weight as a percentage of total body weight (%BW). Data represent mean ± SEM. N = 4 mice/treatment group. G NfL levels in serum. Data represent mean ± SEM (N = 4–8 mice/treatment group) **** P < 0.0001, ** P < 0.01 (two-way ANOVA with Tukey’s multiple comparisons test)

To assess the contribution of the gut microbiota to the specific feature of sensory loss associated with bortezomib-induced peripheral neuropathy, we performed von Frey testing in GF and FMT mice, and a parallel cohort of SPF mice ( N = 6/group). Unlike previous data from a larger cohort of SPF mice ( N = 12/group; Fig. 2 A), treatment with HD-BTZ did not significantly increased paw withdrawal threshold relative to vehicle treatment in SPF mice ( P = 0.067). Elevated paw withdrawal threshold was also not observed in GF and FMT mice treated with HD-BTZ compared with vehicle-treated controls (Fig. 6 E). With respect to gastrointestinal dysfunction, HD-BTZ-treated GF mice demonstrated a 2.25-fold increase in full stomach weights (2.83 ± 0.660%BW) compared with vehicle-treated GF mice (1.26 ± 0.220%BW), but this did not reach statistical significance ( P = 0.0634) (Fig. 6 F). Treatment with HD-BTZ did not affect full stomach weights in FMT mice (Fig. 6 F). Collectively, these data indicate that gut microbiota depletion using ABX does not profoundly influence bortezomib toxicity, yet GF mice display a milder symptom profile than SPF mice while bortezomib-induced side effects are not evident in FMT mice.

Faecal microbiota transplantation in GF mice partially restores neuronal sensitivity to bortezomib toxicity

Neuroimmune signalling is central to the aetiology of numerous neurological disorders affecting both the central and peripheral nervous system and is well documented to be influenced by the gut microbiota [ 70 , 71 ]. We have previously shown that HD-BTZ induces neuroimmune signalling in both DRG and nodose ganglia in SPF mice (Figs. 2 F-I and 4 E). In contrast, no significant changes in Adgre1 , Tnfa , Il6 and Il1b expression in the DRG or nodose ganglia were observed in GF mice treated with HD-BTZ compared with vehicle (Fig. S11A-D). However, Tnfa ( P < 0.05) and Il1b ( P < 0.05) expression were significantly elevated in the nodose ganglia of FMT mice treated with HD-BTZ compared with vehicle (Fig. S11E-H).

Given that neuroimmune signalling is considered a driver of chemotherapy-induced nerve damage, we then assessed the effect of bortezomib on serum NfL levels in GF and FMT mice. Consistent with previous experiments (Fig. 2 J), serum NfL was profoundly elevated in HD-BTZ-treated SPF mice (1506 ± 133 pg/mL), compared with vehicle-treated counterparts (48.6 ± 10.3 pg/mL, P < 0.0001). In contrast, there was no significant increase in serum NfL in GF mice (HD-BTZ: 270 ± 32.1 pg/mL vs. vehicle: 85.4 ± 7.10 pg/mL). Notably, FMT mice had a partially re-established sensitivity to bortezomib-induced nerve damage, with serum NfL levels 7.36-fold higher in HD-BTZ-treated FMT mice (697 ± 174 pg/mL) than in vehicle-treated FMT mice (94.7 ± 16.1 pg/mL, P < 0.0001), and 2.59-fold higher than in HD-BTZ-treated GF mice ( P < 0.01). However, serum NfL levels in HD-BTZ-treated FMT mice was 2.16-fold lower than in HD-BTZ-treated SPF mice (Fig. 6 G). Collectively, these data provide evidence for a causal role of the gut microbiota in mediating bortezomib-induced neuroimmune signalling and nerve damage. Moreover, these data highlight that despite FMT re-establishing host sensitivity to bortezomib-induced nerve damage, it impedes overt symptomatic presentation of bortezomib side effects.

Discussion

Despite the rapidly evolving landscape in treatment of myeloma over the past two decades, the first-in-class proteasome inhibitor bortezomib remains integral to optimal treatment of the cancer. Peripheral neuropathy and gastrointestinal dysfunction are common side effects of bortezomib that lack effective intervention and thereby impact patient quality of life, treatment adherence and overall survival. Here, for the first time, we have assessed the gastrointestinal side effects induced by bortezomib in rodents and identified that neuroimmune changes in the vagus nerve may be related to their aetiology. Moreover, our studies in GF mice suggest that the gut microbiota is required for the induction of bortezomib-induced nerve damage and can modulate neuropathic symptoms and behavioural deficits associated with the drug, providing a rationale to explore microbiota-directed interventions for their utility in myeloma care.

An important feature of our study is the use of automated behavioural phenotyping to evaluate the effect of bortezomib treatment on spontaneous animal behaviour. Our behavioural phenotyping data demonstrate that HD-BTZ-treated SPF mice were more reluctant, or less able, to explore the testing arena, indicating a significant decline in animal well-being. This is consistent with the clinical situation where bortezomib side effects significantly reduce patient physical and mental functioning, substantively diminishing their quality of life [ 53 , 54 ]. Self-limiting, cyclic weight loss experienced by mice treated with HD-BTZ is also a novel observation, likely reflecting the impact of individual bortezomib treatments on animal well-being. Another novel finding revealed by behavioural phenotyping was increased paw grooming in mice treated with HD-BTZ. This appears to align with the clinical presentation of peripheral neuropathy, which causes sensory changes and spontaneous pain in the hands and feet [ 4 , 8 , 9 ]. Whilst we cannot specifically attribute this to animal pain in paws, we speculate that this may relate, at least in part, to abnormal sensations or the sensory loss we observed in hind paws of HD-BTZ-treated SPF mice. Unlike current modelling of bortezomib-induced peripheral neuropathy that relies on stimulus-evoked sensory and motor function tests [ 13 , 57 – 59 ], this is the first time that a spontaneous behavioural trait of increased paw grooming associated with bortezomib treatment has been reported in an animal model.

In addition to using novel behavioural analyses, our study is the first to model bortezomib-induced gastrointestinal side effects in mice. A striking and robust observation was a significant increase in full stomach weights in SPF mice. Considering that daily food intake did not change in HD-BTZ-treated mice over time and full stomach weights did not significantly differ to baseline weights 48 h after treatment, this suggests acute gastric retention; a phenotype consistent with delayed gastric emptying (gastroparesis) that is a documented clinical consequence of bortezomib treatment [ 72 ]. Indeed, enlarged and heavier stomachs in the absence of elevated food intake has previously been associated with gastroparesis in rodents treated with the neurotoxic chemotherapeutics oxaliplatin and cisplatin [ 73 , 74 ]. Gastroparesis is commonly associated with nerve dysfunction, particularly to the autonomic vagus nerve, which plays a central role in controlling gastrointestinal function including gastric emptying [ 64 , 75 – 77 ]. Thus, bortezomib-induced gastric dysfunction supports previous reports of bortezomib-related gastrointestinal autonomic neuropathy in people with myeloma, as well as other symptom profiles indicative of autonomic neuropathy, such as syncope, postural orthostatic hypotension and neurogenic bladder that have been associated with bortezomib use [ 18 , 72 , 78 ]. While our histological analyses did not identify vagus nerve damage, we did observe neuroimmune signalling changes in nodose ganglia, suggesting possible dysfunction of the vagus nerve induced by bortezomib could contribute to the observed gastric dysfunction and faster gastrointestinal transit time. This is further supported by the absence of overt inflammatory or anatomical changes in the gastrointestinal tract induced by bortezomib, which contrasts gastrointestinal damage induced by mucotoxic chemotherapy drugs such as cyclophosphamide, melphalan and irinotecan [ 63 , 79 , 80 ]. While there is currently no specific evidence of neuroimmune signalling in the nodose ganglia contributing to gastrointestinal dysfunction, general dysfunction of autonomic ganglia is well documented to alter autonomic function in the gastrointestinal tract. For example, acute autonomic ganglionopathy, a disorder characterised by the production of ganglionic acetylcholine receptor autoantibodies affecting autonomic ganglia, causes symptoms such as constipation and gastroparesis [ 81 ]. Furthermore, there is evidence of ganglionic neuroimmune signalling resulting in symptoms of gastrointestinal dysmotility including gastroparesis, although this specifically relates to changes in enteric ganglia of the myenteric plexus [ 82 ]. Thus, it is plausible that bortezomib may affect enteric nerves, or even spinal sensory nerves, to alter gastrointestinal function. Moreover, while we speculate that drug-neuroimmune interactions are a predominant cause of gastrointenstinal dysfunction associated with bortezomib, we cannot exclude the possibility that increased intestinal permeability observed in drug-treated mice may also contribute to the phenotype. Studies are required to investigate this possibility and whether increased permeability may promote the transfer of signals from the gut lumen (e.g. hormones, cytokines or bacterial products) to the circulation to alter nerve function. Ultimately, however, it is likely that gastrointestinal symptoms of bortezomib are multifactorial in nature, with these events co-occurring to dictate the clinical manifestation.

Previous studies suggest that elevated serum neurofilament light chain (NfL) levels are associated with the extent of chemotherapy-induced sensory peripheral nerve damage and electrophysiological dysfunction in rodents [ 83 ]. Despite no change reported in serum NfL levels in rats treated with bortezomib [ 84 ] and the lack of overt neuropathology observed in either the sciatic and vagus nerves in our studies, serum NfL was elevated in our SPF mice treated with bortezomib; representing the first report of elevated NfL levels in rodents undergoing bortezomib treatment. What remains unclear is the source of NfL, as it is not exclusive to sensory peripheral nerves and may therefore reflect axonal injury in the autonomic nervous system and even the CNS. While the ability of bortezomib to cross the blood-brain-barrier is limited [ 85 ], peripheral nerve inflammation and damage can trigger CNS neuroimmune signalling via indirect mechanisms [ 86 ]. Thus, NfL could potentially originate from CNS neurons (e.g. in the brain) affected by peripheral inflammatory signals induced by bortezomib. Previous studies have also reported neuroimmune signalling changes at L4-L6 spinal cord dorsal horns in bortezomib-treated rodents [ 13 , 15 ], suggesting immune signalling in spinal cord dorsal horns (where central terminals of DRG neurons reside) could contribute to elevated serum NfL levels in our bortezomib-treated mice. However, the clinical utility of NfL as a biomarker of drug-induced peripheral neuropathy and association with bortezomib-induced peripheral nerve dysfunction in myeloma patients [ 45 , 62 ] supports that elevated NfL levels in our bortezomib-treated mice likely reflect, at least in part, sensory peripheral nerve damage. Whilst our inclusion of NfL as an objective measure of nerve damage represents a significant advance in the field of modelling bortezomib side effects in mice, further studies are required to identify its precise source(s) and also its specificity as a biomarker of bortezomib-induced peripheral neuropathy in mice given the neuroimmune changes we observed in the vagus nerve of drug-treated animals.

The gut microbiota is rapidly emerging as a central modulator of cancer drug toxicity. Previous studies in GF mice have causally implicated the gut microbiota in chemotherapy side effects, with FMT restoring oxaliplatin- and paclitaxel-induced peripheral neuropathy and irinotecan-induced gastrointentinal toxicity to that observed in conventional mice [ 36 – 38 ]. Our observation that FMT mice did not recapitulate bortezomib side effect profiles seen in SPF mice suggests that the gut microbiota may not be a major driver of this symptom profile. In support of this, antibiotic-treated SPF mice demonstrated, overall, a similar side effect profile to non-antibiotic-treated mice. However, by performing a battery of assessments (behavioural, performance, molecular and biomarker) in GF mice, complexity in the gut microbiota’s role becomes evident. Our studies in GF mice provide two lines of evidence that the gut microbiota may modulate the development of bortezomib side effects. Firstly, peripheral neuropathy was not as evident in GF mice compared with SPF mice. The gut microbiota is well established to shape host immune system development, with GF mice having defective innate and adaptive immune responses to physiological challenges [ 87 , 88 ]. Indeed, neuroimmune signalling changes were not observed in DRGs and nodose ganglia of bortezomib-treated GF mice, consistent with low serum NfL levels observed in these mice. As neuroimmune signalling contributes to the pathogenesis of bortezomib-induced peripheral neuropathy [ 14 , 59 ], it is likely that the absence of a pro-inflammatory response within DRGs of bortezomib-treated GF mice minimised peripheral nerve injury and, subsequently, the development of peripheral neuropathy. Secondly, bortezomib-induced nerve damage and neuroimmune signalling was partially restored in FMT mice relative to GF mice, suggesting the gut microbiota may, at least in part, modulate the neurotoxicity of bortezomib. Despite this, overt behavioural deficits caused by bortezomib were not observed in FMT mice. Whilst this may reflect an insufficient level of nerve damage in FMT mice to induce symptomatic neuropathic effects of bortezomib, it is plausible that colonisation of GF mice with a healthy gut microbiota from SPF mice induced a level of host resilience to the neurotoxic effects of the drug. This begs the question: how can bortezomib-treated FMT mice have clear biochemical evidence of nerve damage without associated functional deficits? Notably, the gut microbiota is well established to influence central and peripheral nervous system function and processing, involving immune, endocrine and metabolic pathways [ 89 , 90 ], with previous studies showing that healthy-donor FMT restores normal gut function and innate and adaptive immune function in GF mice [ 91 – 93 ]. Indeed, the restorative effects of microbial colonisation on host immune function may account for the partially restored neuroimmune signalling and neurotoxic response to bortezomib in FMT mice. The utility of FMT to protect against chemotherapy-induced peripheral neuropathy has also previously been reported. Specifically, FMT mediated protection against paclitaxel-induced peripheral neuropathy in conventional rats which was associated with maintenance of gut microbiota stability, inhibition of colonic TLR4 signalling and reduced glial cell reactivity in the colon and spinal dorsal horn [ 94 ]. As bortezomib activates TLR4 signalling and glial cells in both central and peripheral nerves [ 95 ], it is plausible that reduced TLR4 signalling or modulation of microglial cell activity could, at least in part, mediate the protective effects of FMT in the context of bortezomib side effects seen in our study. Moreover, short-chain fatty acids; the major metabolic by-product of commensal gut microbiota, have been shown to decrease microglial cell reactivity and promote anti-inflammatory processes in the CNS [ 96 ]. Thus, FMT-mediated short-chain fatty acid production in GF mice could dampen microglial cell reactivity and neuronal signalling in the CNS to limit its response to peripheral nerve injury. In addition, serotonin; a neurotransmitter that is important for mood, behaviour and overall well-being, influences CNS function through diverse mechanisms [ 97 , 98 ]. Notably, serotonin is significantly lower in GF mice than in SPF counterparts, with the introduction of a gut microbiota via FMT in GF mice restoring serotonin levels [ 99 ]. Hence, it is plausible that the restoration of this “feel good” hormone may mask underlying neuropathic events and explain the lack of behavioural deficits observed in FMT mice, despite the presence of nerve injury.

Some chemotherapy drugs, in particular high-dose conditioning agents such as melphalan, are associated with profound gut microbiota changes, including expansion of enteric pathogens [ 80 ]. In comparison, the microbial changes we observed with bortezomib in SPF mice appeared mild and transient. Nonetheless, the decrease in alpha diversity and expansion of Lactobacillus gasseri ( L. gasseri ) at F1 were significant. Moreover, these gut microbiota read-outs were inversely correlated, suggesting L.gasseri expansion is likely responsible for the decrease in alpha diversity. Notably, an inverse correlation between Lactobacillus abundance and alpha diversity, together with Lactobacillus expansion, has previously been reported in mice treated with the neurotoxic drug paclitaxel [ 100 ]. However, why microbial changes with bortezomib resolve despite ongoing treatment remains to be determined. Another important question arising from this work is whether gut dysbiosis contributes to the etiology of bortezomib side effects. Given the gut microbiota is a dynamic regulator of the intestinal barrier [ 101 ], bortezomib-induced gut microbiota changes could potentially account for the observed increase in intestinal permeability seen in drug-treated SPF mice. Disruption or changes in the composition of the gut microbiota are widely associated with gastrointestinal dysfunction and neurodegenerative conditions including Alzheimer’s and Parkinson’s disease [ 102 , 103 ]. Moreover, gut dysbiosis is increasingly implicated in chemotherapy-induced peripheral neuropathy through modulation of immune cell function and neuroimmune signalling [ 71 , 104 ]. Notably, our analyses revealed an inverse correlation between L.gasseri relative abundance and paw withdrawal threshold at F1 in bortezomib-treated mice. Interestingly, L.gasseri has been used as a probiotic, demonstrating beneficial gastrointestinal effects in people with dyspepsia and irritable bowel syndrome [ 105 – 107 ]. Thus, it is plausible that the expansion of L.gasseri at F1 with bortezomib treatment could potentially be a protective response intended to limit or overcome drug side effects in the host.

This study also has some limitations that warrant consideration. Firstly, 16S rRNA sequencing does not provide deep taxonomic or functional insights into the gut microbiota. Given the high degree of functional redundancy within microbial communities, compositional changes may not necessarily reflect altered biological activity [ 108 ]. Accordingly, future metagenomic or metabolomic analyses are likely required to determine whether bortezomib induces functionally relevant gut microbiota perturbations. Secondly, our study does not investigate potential mechanisms of FMT-mediated protection against bortezomib side effects in GF mice. Whilst we speculate this could be driven by immune, metabolic and hormonal events, further studies are required to decipher potential effects of FMT on host GF mouse biology that induce resilience to bortezomib side effects. Finally, our side effects studies are limited to male mice. While the use of male mice is consistent with a high proportion of existing bortezomib- and chemotherapy-induced peripheral neuropathy studies performed in rodents, pain responses, neuroimmune signalling and gut microbiota composition can differ between male and female mice [ 109 – 111 ]. Thus, future studies assessing the role of the gut microbiota in bortezomib side effects in female mice are warranted.

Conclusions

We have, for the first time, described the effect of bortezomib on spontaneous animal behaviour indicative of reduced well-being and generated new knowledge on how to model and measure concurrent neuropathologies associated with bortezomib in a clinically-relevant manner. Using this model, we have revealed potential mechanisms underpinning gastrointestinal dysfunction associated with bortezomib, implicating neuroimmune changes in the vagus nerve. Our data indicate that the gut microbiota is not a primary driver of bortezomib side effects. However, the absence of a symptom profile in bortezomib-treated FMT mice, despite clear evidence of nerve damage, suggests that the gut microbiota may shape host biology in GF mice in a manner that protects against the manifestation of overt side effects related to bortezomib neurotoxicity. The paradoxical dissociation between behavioural symptoms and nerve injury in FMT mice is highly relevant, both methodologically and translationally. Firstly, it underscores the importance of using a comprehensive battery of assessments, including behavioural, molecular and biomarker, to fully characterise the phenotype associated with bortezomib toxicity. Secondly, it highlights a potential opportunity to decrease symptomatic bortezomib side effects independently of underlying neuropathology. Further studies are required to unravel the underlying mechanisms of this observation and to investigate whether the protective effects of FMT are recapitulated in a non-GF mouse context or could be harnessed in the clinical setting of myeloma treatment.

Supplementary Information

Supplementary Material 1. [ 55 , 113 , 114 ].

Supplementary Material 2: Figure S1. Tumour response to bortezomib treatment in the C57BL/KalwRij/5TGM1 mouse model of myeloma. (A) Representative bioluminescence images of whole-body tumour burden at day 31. (B) Longitudinal assessment of whole-body tumour burden, as assessed by bioluminescence imaging. Data represent mean ± SEM ( N =10-14 mice/treatment group) **** P <0.0001, *** P <0.001 (two-way ANOVA with Tukey’s multiple comparisons test). (C) End-point tumour burden in hind-limb bone marrow, as assessed by flow cytometric analysis of GFP+ cells at day 32. Data represent box and whiskers plot depicting median, interquartile range and individual values (dots) ( N =5-10 mice/treatment group) **** P <0.0001, *** P <0.001 (one-way ANOVA with Tukey’s multiple comparisons test).

Supplementary Material 3: Figure S2. Effect of bortezomib on spontaneous behaviour of SPF mice. (A) Time engaged in individual categories of locomotor behaviour over 20 minutes. Data represent mean ± SEM ( N =11-21 mice/treatment group) **** P <0.0001, *** P <0.001, ** P <0.01 (two-way AVOVA with Tukey’s multiple comparisons test). (B) Time spent at periphery and centre zone over 20 minutes. Data represent mean ± SEM ( N =11-21 mice/treatment group) **** P <0.0001 (two-way AVOVA with Tukey’s multiple comparisons test). (C) Time engaged in individual categories of rearing behaviour over 20 minutes. Data represent mean ± SEM ( N =11-21 mice/treatment group) **** P <0.0001, ** P <0.01, * P <0.05 (two-way ANOVA with Tukey’s multiple comparisons test). (D) Time engaged in individual categories of orienting behaviour over 20 minutes. Data represent mean ± SEM ( N =11-21 mice/treatment group) **** P <0.0001, *** P <0.001, ** P <0.01 (two-way ANOVA with Tukey’s multiple comparisons test).

Supplementary Material 4: Figure S3. Assessment of sciatic nerve fibre morphology in SPF mice following bortezomib treatment. (A-D) Sciatic nerve fibre diameter (A), axon diameter (B), myelin thickness (C) and g ratio (D). Data represent mean ± SEM ( N =4-5/treatment group).

Supplementary Material 5: Figure S4. Time course stomach weight and food intake in bortezomib-treated SPF mice. (A) Full stomach wet weight, as a percentage of total body weight (%BW), over time. Data represent mean ± SEM. ( N =3-4 mice/time-point) *** P <0.05 (one-way ANOVA with Dunnett’s multiple comparisons test). (B) Daily (24 hour) food intake over time. Data represent mean ± SEM ( N =8-12 mice/group).

Supplementary Material 6: Figure S5. Effect of bortezomib on inflammatory marker gene expression in gastrointestinal tissue in SPF mice. (A-D) Expression of Adgre1 (A), Tnfa (B), Il6 (C) and Il1b (D) in stomach tissue. Data represent mean ± SEM (N=5/treatment group). (E-H) Expression of Adgre1 (E), Tnfa (F), Il6 (G) and Il1b (H) in duodenum tissue. Data represent mean ± SEM ( N =4-5/treatment group). (I-L) Expression of Adgre1 (I), Tnfa (J), Il6 (K) and Il1b (L) in jejunum tissue. Data represent mean ± SEM ( N =5/treatment group). (M-P) Expression of Adgre1 (M), Tnfa (N), Il6 (O) and Il1b (P) in ileum tissue. Data represent mean ± SEM ( N =5/treatment group). (Q-T) Expression of Adgre1 (Q), Tnfa (R), Il6 (S) and Il1b (T) colon tissue. Data represent mean ± SEM ( N =5/treatment group).

Supplementary Material 7: Figure S6. Effect of bortezomib on plasma concentrations of inflammatory cytokines and chemokines in SPF mice. (A-G) Plasma concentration of IFN-γ (A), IL-1α (B), IL-6 (C), IL-10 (D), IL-17 (E), CXCL1 (F), CCL2 (G). IL-1β and TNF-α concentrations were outside the detectable range, precluding further analysis. Data represent mean ± SEM ( N =13 mice/treatment group).

Supplementary Material 8: Figure S7. Assessment of vagus nerve fibre morphology in SPF mice following bortezomib treatment. (A-D) Vagus nerve fibre diameter (A), axon diameter (B), g ratio (C) and myelin thickness (D). Data represent mean ± SEM ( N =4-5/treatment group).

Supplementary Material 9: Figure S8. Correlation analyses between gut microbiota read-outs and behavioural phenotypes. Heatmap summarises correlations of alpha diversity (Shannon index) and relative abundance of Lactobacillus gasseri ( L. gasseri ), with avalaible behavioural parameters at baseline (F0) and throughout drug treatment (F1-F4). Pearson’s correlations were performed for all behavioural spectrometry parameters (track length, and time engaged in still, locomotor, rearing, orientation, grooming and paw grooming behaviour), and Spearman’s correlations were used for correlations with 50% paw withdrawal threshold (PWT). Correlation strength and co-efficient (r-value) are denoted by right scale bar (green = r>0, purple = r<0) and asterisks denote significance (* P <0.05).

Supplementary Material 10: Figure S9. Assessment of mouse germ-free and colonisation status. Copies of the 16S rRNA gene in faecal samples collected from GF and FMT mice at F0, F1, F2, F3 and F4, as assessed by 16S rRNA gene qPCR. Individual dots represent pooled faecal samples from a single cage.

Supplementary Material 11: Figure S10. Assessment of bortezomib side effects in antibiotic-treated mice. (A) End-point (day 12) body weight change relative to baseline. Data represent mean ± SEM (N=4-7 mice/treatment group) ***P<0.001 (two-way ANOVA with uncorrected Fisher’s LSD test). (B) Total distance travelled by mice after 20 minutes and representative track map images. Data represent mean ± SEM (N=4-7 mice/treatment group) ***P<0.001, *P<0.05 (two-way ANOVA with uncorrected Fisher’s LSD test). (C) Total time engaged in still, locomotor, rearing, orienting and grooming behaviour over 20 minutes. Data represent mean ± SEM (N=4-7 mice/treatment group) **P<0.01 (two-way ANOVA with uncorrected Fisher’s LSD test). (D) Time engaged in grooming of paws over 20 minutes. Data represent mean ± SEM (N=4-7 mice/treatment group).

Supplementary Material 12: Figure S11. Effect of bortezomib on inflammatory marker gene expression in dorsal root ganglia and nodose ganglia in GF and FMT mice. (A) Expression of Adgre1 , Tnfa , Il6 and Il1b in dorsal root ganglia. Data represent mean ± SEM ( N =3-4 mice/group). (B) Expression of Adgre1 , Tnfa , Il6 and Il1b in nodose ganglia. Data represent mean ± SEM ( N =3-4 mice/group) * P <0.05 (Mann-Whitney U tests).

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jacqui S. Scott and Sadia Munir are co-first authors.

Hannah R. Wardill and Krzysztof M. Mrozik are co-senior and corresponding authors.

Acknowledgements

We acknowledge the support of staff at the South Australian Health and Medical Research Institute (SAHMRI) Bioresources, flow cytometry and Preclinical, Imaging and Research Laboratories (PIRL) germ-free facilities. We thank Dylan Harnas and Vicki Wilczek (Adelaide University) and Dr Todd Norton (Flinders University) for technical assistance. We also acknowledge the South Australian Genomics Centre (SAGC) for conducting 16S rRNA gene sequencing.

Authors’ contributions

J. S. S. and S. M.: investigation, analysis, writing, and editing; M. A. L.: design, investigation, analysis and editing; J. A. D., S. S. and P. T.: investigation and analysis; J. C. and S. M. B.: design, analysis and editing; A. M. H., G. S., D. R. H., C. B. C., K. C and B. G.: methodology and editing; M. R. H.: methodology, analysis and editing; A. Y. and S. P. C.: design and editing; S. B. P. and F. J. R.: analysis and editing; A. C. W. Z.: supervision and editing; D. J. L.: conceptualisation, design, methodology, analysis and editing; K. V.; conceptualisation, design, methodology, supervision, analysis and editing; K. M. M. and H. R. W.: conceptualisation, design, supervision, methodology, investigation, analysis, writing and editing.

Funding

This project was supported by grant 1163245 awarded to K. Vandyke through the Priority-driven Collaborative Cancer Research Scheme and co-funded by Cancer Australia, Cure Cancer, and Leukaemia Foundation of Australia, and further supported by a The Hospital Research Foundation (THRF) Innovations in Pain Management grant. The work was also produced with the financial and other support of Cancer Council SA’s Beat Cancer Project on behalf of its donors and the State Government of South Australia through the Department of Health and Wellbeing (awarded to K. M. Mrozik). K. M. Mrozik was supported by a THRF Myeloma Fellowship. H. R. Wardill was supported by a THRF Research Fellowship. H. R. Wardill and S. M. Brierley were supported by a National Health and Medical Research Council of Australia (NHMRC) Investigator Leadership Grants. S. M. Brierley was supported by a NHMRC Development Grant. J. Castro was supported by a NHMRC Ideas Grant. S. B. Park was supported by a Rebecca Cooper Medical Research Foundation Fellowship.

Data availability

The datasets generated and/or analysed during the current study are not publicly available but may be made available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were performed in accordance with protocols approved by the SAHMRI Animal Ethics Committee (SAM-21-012 and SAM-22-056) and complied with the Australian code for the care and use of animals for scientific purposes [ 112 ].

Consent for publication

Not applicable.

Competing interests

S. P. Costello is a shareholder in BiomeBank and Microbiotica.

References

1. Rajkumar SV Multiple myeloma: 2024 update on diagnosis, risk-stratification, and management Am J Hematol 2024 99 1802 24 10.1002/ajh.27422 38943315 PMC11404783 Rajkumar SV. Multiple myeloma: 2024 update on diagnosis, risk-stratification, and management. Am J Hematol. 2024;99:1802–24. 38943315 10.1002/ajh.27422 PMC11404783

2. Palladini G Milani P Diagnosis and Treatment of AL Amyloidosis Drugs 2023 83 203 16 10.1007/s40265-022-01830-z 36652193 Palladini G, Milani P. Diagnosis and Treatment of AL Amyloidosis. Drugs. 2023;83:203–16. 36652193 10.1007/s40265-022-01830-z

3. Gandolfi S Laubach JP Hideshima T Chauhan D Anderson KC Richardson PG The proteasome and proteasome inhibitors in multiple myeloma Cancer Metastasis Rev 2017 36 561 84 10.1007/s10555-017-9707-8 29196868 Gandolfi S, Laubach JP, Hideshima T, Chauhan D, Anderson KC, Richardson PG. The proteasome and proteasome inhibitors in multiple myeloma. Cancer Metastasis Rev. 2017;36:561–84. 29196868 10.1007/s10555-017-9707-8

4. Yang Y Zhao B Lan H Sun J Wei G Bortezomib-induced peripheral neuropathy: Clinical features, molecular basis, and therapeutic approach Crit Rev Oncol Hematol 2024 197 104353 10.1016/j.critrevonc.2024.104353 38615869 Yang Y, Zhao B, Lan H, Sun J, Wei G. Bortezomib-induced peripheral neuropathy: Clinical features, molecular basis, and therapeutic approach. Crit Rev Oncol Hematol. 2024;197:104353. 38615869 10.1016/j.critrevonc.2024.104353

5. Cengiz Seval G Beksac M The safety of bortezomib for the treatment of multiple myeloma Expert Opin Drug Saf 2018 17 953 62 10.1080/14740338.2018.1513487 30118610 Cengiz Seval G, Beksac M. The safety of bortezomib for the treatment of multiple myeloma. Expert Opin Drug Saf. 2018;17:953–62. 30118610 10.1080/14740338.2018.1513487

6. Jagannath S Barlogie B Berenson JR Updated survival analyses after prolonged follow-up of the phase 2, multicenter CREST study of bortezomib in relapsed or refractory multiple myeloma Br J Haematol 2008 143 537 40 10.1111/j.1365-2141.2008.07359.x 18783399 Jagannath S, Barlogie B, Berenson JR, et al. Updated survival analyses after prolonged follow-up of the phase 2, multicenter CREST study of bortezomib in relapsed or refractory multiple myeloma. Br J Haematol. 2008;143:537–40. 18783399 10.1111/j.1365-2141.2008.07359.x

7. Mateos MV Richardson PG Dimopoulos MA Effect of cumulative bortezomib dose on survival in multiple myeloma patients receiving bortezomib-melphalan-prednisone in the phase III VISTA study Am J Hematol 2015 90 314 9 10.1002/ajh.23933 25557740 Mateos MV, Richardson PG, Dimopoulos MA, et al. Effect of cumulative bortezomib dose on survival in multiple myeloma patients receiving bortezomib-melphalan-prednisone in the phase III VISTA study. Am J Hematol. 2015;90:314–9. 25557740 10.1002/ajh.23933

8. Argyriou AA Iconomou G Kalofonos HP Bortezomib-induced peripheral neuropathy in multiple myeloma: a comprehensive review of the literature Blood 2008 112 1593 9 10.1182/blood-2008-04-149385 18574024 Argyriou AA, Iconomou G, Kalofonos HP. Bortezomib-induced peripheral neuropathy in multiple myeloma: a comprehensive review of the literature. Blood. 2008;112:1593–9. 18574024 10.1182/blood-2008-04-149385

9. Cata JP Weng HR Burton AW Villareal H Giralt S Dougherty PM Quantitative sensory findings in patients with bortezomib-induced pain J Pain 2007 8 296 306 10.1016/j.jpain.2006.09.014 17175202 Cata JP, Weng HR, Burton AW, Villareal H, Giralt S, Dougherty PM. Quantitative sensory findings in patients with bortezomib-induced pain. J Pain. 2007;8:296–306. 17175202 10.1016/j.jpain.2006.09.014

10. Li T Timmins HC King T Kiernan MC Goldstein D Park SB Characteristics and risk factors of bortezomib induced peripheral neuropathy: A systematic review of phase III trials Hematol Oncol 2020 38 229 43 10.1002/hon.2706 31873944 Li T, Timmins HC, King T, Kiernan MC, Goldstein D, Park SB. Characteristics and risk factors of bortezomib induced peripheral neuropathy: A systematic review of phase III trials. Hematol Oncol. 2020;38:229–43. 31873944 10.1002/hon.2706

11. Richardson PG Barlogie B Berenson J A phase 2 study of bortezomib in relapsed, refractory myeloma N Engl J Med 2003 348 2609 17 10.1056/NEJMoa030288 12826635 Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 2003;348:2609–17. 12826635 10.1056/NEJMoa030288

12. Jagannath S Barlogie B Berenson J A phase 2 study of two doses of bortezomib in relapsed or refractory myeloma Br J Haematol 2004 127 165 72 10.1111/j.1365-2141.2004.05188.x 15461622 Jagannath S, Barlogie B, Berenson J, et al. A phase 2 study of two doses of bortezomib in relapsed or refractory myeloma. Br J Haematol. 2004;127:165–72. 15461622 10.1111/j.1365-2141.2004.05188.x

13. Moschetti G Amodeo G Maftei D Targeting prokineticin system counteracts hypersensitivity, neuroinflammation, and tissue damage in a mouse model of bortezomib-induced peripheral neuropathy J Neuroinflammation 2019 16 89 10.1186/s12974-019-1461-0 30995914 PMC6471808 Moschetti G, Amodeo G, Maftei D, et al. Targeting prokineticin system counteracts hypersensitivity, neuroinflammation, and tissue damage in a mouse model of bortezomib-induced peripheral neuropathy. J Neuroinflammation. 2019;16:89. 30995914 10.1186/s12974-019-1461-0 PMC6471808

14. Meregalli C Marjanovic I Scali C High-dose intravenous immunoglobulins reduce nerve macrophage infiltration and the severity of bortezomib-induced peripheral neurotoxicity in rats J Neuroinflammation 2018 15 232 10.1186/s12974-018-1270-x 30131066 PMC6103882 Meregalli C, Marjanovic I, Scali C, et al. High-dose intravenous immunoglobulins reduce nerve macrophage infiltration and the severity of bortezomib-induced peripheral neurotoxicity in rats. J Neuroinflammation. 2018;15:232. 30131066 10.1186/s12974-018-1270-x PMC6103882

15. Chen K Fan J Luo ZF Yang Y Xin WJ Liu CC Reduction of SIRT1 epigenetically upregulates NALP1 expression and contributes to neuropathic pain induced by chemotherapeutic drug bortezomib J Neuroinflammation 2018 15 292 10.1186/s12974-018-1327-x 30342528 PMC6195754 Chen K, Fan J, Luo ZF, Yang Y, Xin WJ, Liu CC. Reduction of SIRT1 epigenetically upregulates NALP1 expression and contributes to neuropathic pain induced by chemotherapeutic drug bortezomib. J Neuroinflammation. 2018;15:292. 30342528 10.1186/s12974-018-1327-x PMC6195754

16. Richardson PG Sonneveld P Schuster MW Bortezomib or high-dose dexamethasone for relapsed multiple myeloma N Engl J Med 2005 352 2487 98 10.1056/NEJMoa043445 15958804 Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005;352:2487–98. 15958804 10.1056/NEJMoa043445

17. Durie BGM Hoering A Abidi MH Bortezomib with lenalidomide and dexamethasone versus lenalidomide and dexamethasone alone in patients with newly diagnosed myeloma without intent for immediate autologous stem-cell transplant (SWOG S0777): a randomised, open-label, phase 3 trial Lancet 2017 389 519 27 10.1016/S0140-6736(16)31594-X 28017406 PMC5546834 Durie BGM, Hoering A, Abidi MH, et al. Bortezomib with lenalidomide and dexamethasone versus lenalidomide and dexamethasone alone in patients with newly diagnosed myeloma without intent for immediate autologous stem-cell transplant (SWOG S0777): a randomised, open-label, phase 3 trial. Lancet. 2017;389:519–27. 28017406 10.1016/S0140-6736(16)31594-X PMC5546834

18. Zhao W Liu J Wang L Wang W Study on autonomic neuropathy of the digestive system caused by bortezomib in the treatment of multiple myeloma Hematology 2023 28 2210907 10.1080/16078454.2023.2210907 37166129 Zhao W, Liu J, Wang L, Wang W. Study on autonomic neuropathy of the digestive system caused by bortezomib in the treatment of multiple myeloma. Hematology. 2023;28:2210907. 37166129 10.1080/16078454.2023.2210907

19. Stansborough RL Gibson RJ Proteasome inhibitor-induced gastrointestinal toxicity Curr Opin Support Palliat Care 2017 11 133 7 10.1097/SPC.0000000000000266 28333868 Stansborough RL, Gibson RJ. Proteasome inhibitor-induced gastrointestinal toxicity. Curr Opin Support Palliat Care. 2017;11:133–7. 28333868 10.1097/SPC.0000000000000266

20. Papandreou CN Daliani DD Nix D Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer J Clin Oncol 2004 22 2108 21 10.1200/JCO.2004.02.106 15169797 Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol. 2004;22:2108–21. 15169797 10.1200/JCO.2004.02.106

21. Mele G Coppi MR Melpignano A Quarta G Paralytic ileus following subcutaneous bortezomib therapy: focus on the clinical emergency-report of two cases Clin Exp Med 2016 16 99 101 10.1007/s10238-015-0337-6 25600700 Mele G, Coppi MR, Melpignano A, Quarta G. Paralytic ileus following subcutaneous bortezomib therapy: focus on the clinical emergency-report of two cases. Clin Exp Med. 2016;16:99–101. 25600700 10.1007/s10238-015-0337-6

22. Perfetti V Palladini G Brunetti L Bortezomib-induced paralytic ileus is a potential gastrointestinal side effect of this first-in-class anticancer proteasome inhibitor Eur J Gastroenterol Hepatol 2007 19 599 601 10.1097/MEG.0b013e32811ebffe 17556909 Perfetti V, Palladini G, Brunetti L, et al. Bortezomib-induced paralytic ileus is a potential gastrointestinal side effect of this first-in-class anticancer proteasome inhibitor. Eur J Gastroenterol Hepatol. 2007;19:599–601. 17556909 10.1097/MEG.0b013e32811ebffe

23. Fukushima T Nakamura T Iwao H Efficacy and safety of bortezomib plus dexamethasone therapy for refractory or relapsed multiple myeloma: once-weekly administration of bortezomib may reduce the incidence of gastrointestinal adverse events Anticancer Res 2011 31 2297 302 21737655 Fukushima T, Nakamura T, Iwao H, et al. Efficacy and safety of bortezomib plus dexamethasone therapy for refractory or relapsed multiple myeloma: once-weekly administration of bortezomib may reduce the incidence of gastrointestinal adverse events. Anticancer Res. 2011;31:2297–302. 21737655

24. Loprinzi CL Lacchetti C Bleeker J Prevention and Management of Chemotherapy-Induced Peripheral Neuropathy in Survivors of Adult Cancers: ASCO Guideline Update J Clin Oncol 2020 38 3325 48 10.1200/JCO.20.01399 32663120 Loprinzi CL, Lacchetti C, Bleeker J, et al. Prevention and Management of Chemotherapy-Induced Peripheral Neuropathy in Survivors of Adult Cancers: ASCO Guideline Update. J Clin Oncol. 2020;38:3325–48. 32663120 10.1200/JCO.20.01399

25. Hertz DL Tanay M Tofthagen C Patient-reported strategies for prevention and treatment of chemotherapy-induced peripheral neuropathy Support Care Cancer 2025 33 142 10.1007/s00520-025-09190-6 39899120 Hertz DL, Tanay M, Tofthagen C, et al. Patient-reported strategies for prevention and treatment of chemotherapy-induced peripheral neuropathy. Support Care Cancer. 2025;33:142. 39899120 10.1007/s00520-025-09190-6

26. Weersma RK Zhernakova A Fu J Interaction between drugs and the gut microbiome Gut 2020 69 1510 9 10.1136/gutjnl-2019-320204 32409589 PMC7398478 Weersma RK, Zhernakova A, Fu J. Interaction between drugs and the gut microbiome. Gut. 2020;69:1510–9. 32409589 10.1136/gutjnl-2019-320204 PMC7398478

27. Li S Zhu S Yu J The role of gut microbiota and metabolites in cancer chemotherapy J Adv Res 2024 64 223 35 10.1016/j.jare.2023.11.027 38013112 PMC11464465 Li S, Zhu S, Yu J. The role of gut microbiota and metabolites in cancer chemotherapy. J Adv Res. 2024;64:223–35. 38013112 10.1016/j.jare.2023.11.027 PMC11464465

28. Blake SJ James J Ryan FJ The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota Cell Rep Med 2021 2 100464 10.1016/j.xcrm.2021.100464 35028606 PMC8714857 Blake SJ, James J, Ryan FJ, et al. The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota. Cell Rep Med. 2021;2:100464. 35028606 10.1016/j.xcrm.2021.100464 PMC8714857

29. Bhatt AP Pellock SJ Biernat KA Targeted inhibition of gut bacterial beta-glucuronidase activity enhances anticancer drug efficacy Proc Natl Acad Sci U S A 2020 117 7374 81 10.1073/pnas.1918095117 32170007 PMC7132129 Bhatt AP, Pellock SJ, Biernat KA, et al. Targeted inhibition of gut bacterial beta-glucuronidase activity enhances anticancer drug efficacy. Proc Natl Acad Sci U S A. 2020;117:7374–81. 32170007 10.1073/pnas.1918095117 PMC7132129

30. Blake SJ Wolf Y Boursi B Lynn DJ Role of the microbiota in response to and recovery from cancer therapy Nat Rev Immunol 2024 24 308 25 10.1038/s41577-023-00951-0 37932511 Blake SJ, Wolf Y, Boursi B, Lynn DJ. Role of the microbiota in response to and recovery from cancer therapy. Nat Rev Immunol. 2024;24:308–25. 37932511 10.1038/s41577-023-00951-0

31. Calabro S, Kankowski S, Cescon M, et al. Impact of Gut Microbiota on the Peripheral Nervous System in Physiological, Regenerative and Pathological Conditions. Int J Mol Sci. 2023;24:8061. 10.3390/ijms24098061 PMC10179357 37175764

32. Ding W You Z Chen Q Gut Microbiota Influences Neuropathic Pain Through Modulating Proinflammatory and Anti-inflammatory T Cells Anesth Analg 2021 132 1146 55 10.1213/ANE.0000000000005155 32889847 Ding W, You Z, Chen Q, et al. Gut Microbiota Influences Neuropathic Pain Through Modulating Proinflammatory and Anti-inflammatory T Cells. Anesth Analg. 2021;132:1146–55. 32889847 10.1213/ANE.0000000000005155

33. Lin B Wang Y Zhang P Yuan Y Zhang Y Chen G Gut microbiota regulates neuropathic pain: potential mechanisms and therapeutic strategy J Headache Pain 2020 21 103 10.1186/s10194-020-01170-x 32807072 PMC7433133 Lin B, Wang Y, Zhang P, Yuan Y, Zhang Y, Chen G. Gut microbiota regulates neuropathic pain: potential mechanisms and therapeutic strategy. J Headache Pain. 2020;21:103. 32807072 10.1186/s10194-020-01170-x PMC7433133

34. Pusceddu MM Gareau MG Visceral pain: gut microbiota, a new hope? J Biomed Sci 2018 25 73 10.1186/s12929-018-0476-7 30309367 PMC6182804 Pusceddu MM, Gareau MG. Visceral pain: gut microbiota, a new hope? J Biomed Sci. 2018;25:73. 30309367 10.1186/s12929-018-0476-7 PMC6182804

35. Lucarini E Di Pilato V Parisio C Visceral sensitivity modulation by faecal microbiota transplantation: the active role of gut bacteria in pain persistence Pain 2022 163 861 77 10.1097/j.pain.0000000000002438 34393197 PMC9009324 Lucarini E, Di Pilato V, Parisio C, et al. Visceral sensitivity modulation by faecal microbiota transplantation: the active role of gut bacteria in pain persistence. Pain. 2022;163:861–77. 34393197 10.1097/j.pain.0000000000002438 PMC9009324

36. Shen S Lim G You Z Gut microbiota is critical for the induction of chemotherapy-induced pain Nat Neurosci 2017 20 1213 6 10.1038/nn.4606 28714953 PMC5575957 Shen S, Lim G, You Z, et al. Gut microbiota is critical for the induction of chemotherapy-induced pain. Nat Neurosci. 2017;20:1213–6. 28714953 10.1038/nn.4606 PMC5575957

37. Ramakrishna C Corleto J Ruegger PM Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic Pain Sci Rep 2019 9 20324 10.1038/s41598-019-56832-x 31889131 PMC6937259 Ramakrishna C, Corleto J, Ruegger PM, et al. Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic Pain. Sci Rep. 2019;9:20324. 31889131 10.1038/s41598-019-56832-x PMC6937259

38. Pedroso S Vieira AT Bastos RW Evaluation of mucositis induced by irinotecan after microbial colonization in germ-free mice Microbiol (Reading) 2015 161 1950 60 10.1099/mic.0.000149 26224606 Pedroso S, Vieira AT, Bastos RW, et al. Evaluation of mucositis induced by irinotecan after microbial colonization in germ-free mice. Microbiol (Reading). 2015;161:1950–60. 10.1099/mic.0.000149 26224606

39. Castro J Maddern J Grundy L A mouse model of endometriosis that displays vaginal, colon, cutaneous, and bladder sensory comorbidities FASEB J 2021 35 e21430 10.1096/fj.202002441R 33749885 Castro J, Maddern J, Grundy L, et al. A mouse model of endometriosis that displays vaginal, colon, cutaneous, and bladder sensory comorbidities. FASEB J. 2021;35:e21430. 33749885 10.1096/fj.202002441R

40. Deuis JR Dvorakova LS Vetter I Methods Used to Evaluate Pain Behaviors in Rodents Front Mol Neurosci 2017 10 284 10.3389/fnmol.2017.00284 28932184 PMC5592204 Deuis JR, Dvorakova LS, Vetter I. Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci. 2017;10:284. 28932184 10.3389/fnmol.2017.00284 PMC5592204

41. Brodkin J Frank D Grippo R Validation and implementation of a novel high-throughput behavioral phenotyping instrument for mice J Neurosci Methods 2014 224 48 57 10.1016/j.jneumeth.2013.12.010 24384067 PMC4305388 Brodkin J, Frank D, Grippo R, et al. Validation and implementation of a novel high-throughput behavioral phenotyping instrument for mice. J Neurosci Methods. 2014;224:48–57. 24384067 10.1016/j.jneumeth.2013.12.010 PMC4305388

42. Castro J, Harrington AM, Lieu T, et al. Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity. JCI Insight. 2019;4(20):e131712. 10.1172/jci.insight.131712 PMC6824308 31536477

43. Huehnchen P Boehmerle W Endres M Assessment of paclitaxel induced sensory polyneuropathy with Catwalk automated gait analysis in mice PLoS ONE 2013 8 e76772 10.1371/journal.pone.0076772 24143194 PMC3797113 Huehnchen P, Boehmerle W, Endres M. Assessment of paclitaxel induced sensory polyneuropathy with Catwalk automated gait analysis in mice. PLoS ONE. 2013;8:e76772. 24143194 10.1371/journal.pone.0076772 PMC3797113

44. Khalil M Teunissen CE Lehmann S Neurofilaments as biomarkers in neurological disorders - towards clinical application Nat Rev Neurol 2024 20 269 87 10.1038/s41582-024-00955-x 38609644 Khalil M, Teunissen CE, Lehmann S, et al. Neurofilaments as biomarkers in neurological disorders - towards clinical application. Nat Rev Neurol. 2024;20:269–87. 38609644 10.1038/s41582-024-00955-x

45. Andersen NE Boehmerle W Huehnchen P Stage TB Neurofilament light chain as a biomarker of chemotherapy-induced peripheral neuropathy Trends Pharmacol Sci 2024 45 872 9 10.1016/j.tips.2024.08.001 39242335 Andersen NE, Boehmerle W, Huehnchen P, Stage TB. Neurofilament light chain as a biomarker of chemotherapy-induced peripheral neuropathy. Trends Pharmacol Sci. 2024;45:872–9. 39242335 10.1016/j.tips.2024.08.001

46. Rigaud M Gemes G Barabas ME Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain Pain 2008 136 188 201 10.1016/j.pain.2008.01.016 18316160 PMC2700063 Rigaud M, Gemes G, Barabas ME, et al. Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain. Pain. 2008;136:188–201. 18316160 10.1016/j.pain.2008.01.016 PMC2700063

47. Browning KN Excitability of nodose ganglion cells and their role in vago-vagal reflex control of gastrointestinal function Curr Opin Pharmacol 2003 3 613 7 10.1016/j.coph.2003.06.011 14644013 Browning KN. Excitability of nodose ganglion cells and their role in vago-vagal reflex control of gastrointestinal function. Curr Opin Pharmacol. 2003;3:613–7. 14644013 10.1016/j.coph.2003.06.011

48. Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 2001 25 402 8 10.1006/meth.2001.1262 11846609 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8. 11846609 10.1006/meth.2001.1262

49. Lynn MA Eden G Ryan FJ The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life Cell Rep 2021 36 109564 10.1016/j.celrep.2021.109564 34433065 Lynn MA, Eden G, Ryan FJ, et al. The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life. Cell Rep. 2021;36:109564. 34433065 10.1016/j.celrep.2021.109564

50. Lynn MA Ryan FJ Tee YC Lynn DJ Protocol to colonize gnotobiotic mice in early life and assess the impact on early life immune programming STAR Protoc 2022 3 101914 10.1016/j.xpro.2022.101914 36595955 PMC9763935 Lynn MA, Ryan FJ, Tee YC, Lynn DJ. Protocol to colonize gnotobiotic mice in early life and assess the impact on early life immune programming. STAR Protoc. 2022;3:101914. 36595955 10.1016/j.xpro.2022.101914 PMC9763935

51. Secombe KR Al-Qadami GH Subramaniam CB Guidelines for reporting on animal fecal transplantation (GRAFT) studies: recommendations from a systematic review of murine transplantation protocols Gut Microbes 2021 13 1979878 10.1080/19490976.2021.1979878 34586011 PMC8489962 Secombe KR, Al-Qadami GH, Subramaniam CB, et al. Guidelines for reporting on animal fecal transplantation (GRAFT) studies: recommendations from a systematic review of murine transplantation protocols. Gut Microbes. 2021;13:1979878. 34586011 10.1080/19490976.2021.1979878 PMC8489962

52. Lynn MA Ryan FJ Tee YC Benson SC Lynn DJ Protocol to assess the impact of early-life antibiotic exposure on murine longevity STAR Protoc 2022 3 101220 10.1016/j.xpro.2022.101220 35284839 PMC8904607 Lynn MA, Ryan FJ, Tee YC, Benson SC, Lynn DJ. Protocol to assess the impact of early-life antibiotic exposure on murine longevity. STAR Protoc. 2022;3:101220. 35284839 10.1016/j.xpro.2022.101220 PMC8904607

53. Selvy M Kerckhove N Pereira B Prevalence of Chemotherapy-Induced Peripheral Neuropathy in Multiple Myeloma Patients and its Impact on Quality of Life: A Single Center Cross-Sectional Study Front Pharmacol 2021 12 637593 10.3389/fphar.2021.637593 33967771 PMC8101543 Selvy M, Kerckhove N, Pereira B, et al. Prevalence of Chemotherapy-Induced Peripheral Neuropathy in Multiple Myeloma Patients and its Impact on Quality of Life: A Single Center Cross-Sectional Study. Front Pharmacol. 2021;12:637593. 33967771 10.3389/fphar.2021.637593 PMC8101543

54. Beijers AJ Vreugdenhil G Oerlemans S Chemotherapy-induced neuropathy in multiple myeloma: influence on quality of life and development of a questionnaire to compose common toxicity criteria grading for use in daily clinical practice Support Care Cancer 2016 24 2411 20 10.1007/s00520-015-3032-y 26634561 Beijers AJ, Vreugdenhil G, Oerlemans S, et al. Chemotherapy-induced neuropathy in multiple myeloma: influence on quality of life and development of a questionnaire to compose common toxicity criteria grading for use in daily clinical practice. Support Care Cancer. 2016;24:2411–20. 26634561 10.1007/s00520-015-3032-y

55. Mrozik KM Cheong CM Hewett DR LCRF-0006, a small molecule mimetic of the N-cadherin antagonist peptide ADH-1, synergistically increases multiple myeloma response to bortezomib FASEB Bioadv 2020 2 339 53 10.1096/fba.2019-00073 32617520 PMC7325588 Mrozik KM, Cheong CM, Hewett DR, et al. LCRF-0006, a small molecule mimetic of the N-cadherin antagonist peptide ADH-1, synergistically increases multiple myeloma response to bortezomib. FASEB Bioadv. 2020;2:339–53. 32617520 10.1096/fba.2019-00073 PMC7325588

56. Williams CMD Noll JE Bradey AL Myeloperoxidase creates a permissive microenvironmental niche for the progression of multiple myeloma Br J Haematol 2023 203 614 24 10.1111/bjh.19102 37699574 PMC10952523 Williams CMD, Noll JE, Bradey AL, et al. Myeloperoxidase creates a permissive microenvironmental niche for the progression of multiple myeloma. Br J Haematol. 2023;203:614–24. 37699574 10.1111/bjh.19102 PMC10952523

57. Bruna J Udina E Ale A Neurophysiological, histological and immunohistochemical characterization of bortezomib-induced neuropathy in mice Exp Neurol 2010 223 599 608 10.1016/j.expneurol.2010.02.006 20188093 Bruna J, Udina E, Ale A, et al. Neurophysiological, histological and immunohistochemical characterization of bortezomib-induced neuropathy in mice. Exp Neurol. 2010;223:599–608. 20188093 10.1016/j.expneurol.2010.02.006

58. Boehmerle W Huehnchen P Peruzzaro S Balkaya M Endres M Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice Sci Rep 2014 4 6370 10.1038/srep06370 25231679 PMC5377307 Boehmerle W, Huehnchen P, Peruzzaro S, Balkaya M, Endres M. Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice. Sci Rep. 2014;4:6370. 25231679 10.1038/srep06370 PMC5377307

59. Ale A Bruna J Morell M Treatment with anti-TNF alpha protects against the neuropathy induced by the proteasome inhibitor bortezomib in a mouse model Exp Neurol 2014 253 165 73 10.1016/j.expneurol.2013.12.020 24406455 Ale A, Bruna J, Morell M, et al. Treatment with anti-TNF alpha protects against the neuropathy induced by the proteasome inhibitor bortezomib in a mouse model. Exp Neurol. 2014;253:165–73. 24406455 10.1016/j.expneurol.2013.12.020

60. Grundy L Erickson A Brierley SM Visceral Pain Annu Rev Physiol 2019 81 261 84 10.1146/annurev-physiol-020518-114525 30379615 Grundy L, Erickson A, Brierley SM. Visceral Pain. Annu Rev Physiol. 2019;81:261–84. 30379615 10.1146/annurev-physiol-020518-114525

61. Berta T Strong JA Zhang JM Ji RR Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain: an update Expert Opin Ther Targets 2023 27 665 78 10.1080/14728222.2023.2247563 37574713 PMC10530032 Berta T, Strong JA, Zhang JM, Ji RR. Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain: an update. Expert Opin Ther Targets. 2023;27:665–78. 37574713 10.1080/14728222.2023.2247563 PMC10530032

62. Cebulla N Schirmer D Runau E Neurofilament light chain levels indicate acute axonal damage under bortezomib treatment J Neurol 2023 270 2997 3007 10.1007/s00415-023-11624-2 36802032 PMC10188420 Cebulla N, Schirmer D, Runau E, et al. Neurofilament light chain levels indicate acute axonal damage under bortezomib treatment. J Neurol. 2023;270:2997–3007. 36802032 10.1007/s00415-023-11624-2 PMC10188420

63. Wardill HR Gibson RJ Van Sebille YZ Irinotecan-Induced Gastrointestinal Dysfunction and Pain Are Mediated by Common TLR4-Dependent Mechanisms Mol Cancer Ther 2016 15 1376 86 10.1158/1535-7163.MCT-15-0990 27197307 Wardill HR, Gibson RJ, Van Sebille YZ, et al. Irinotecan-Induced Gastrointestinal Dysfunction and Pain Are Mediated by Common TLR4-Dependent Mechanisms. Mol Cancer Ther. 2016;15:1376–86. 27197307 10.1158/1535-7163.MCT-15-0990

64. Cao Y Li R Bai L Vagal sensory pathway for the gut-brain communication Semin Cell Dev Biol 2024 156 228 43 10.1016/j.semcdb.2023.07.009 37558522 Cao Y, Li R, Bai L. Vagal sensory pathway for the gut-brain communication. Semin Cell Dev Biol. 2024;156:228–43. 37558522 10.1016/j.semcdb.2023.07.009

65. Fei Z Lijuan Y Xi Y Gut microbiome associated with chemotherapy-induced diarrhea from the CapeOX regimen as adjuvant chemotherapy in resected stage III colorectal cancer Gut Pathog 2019 11 18 10.1186/s13099-019-0299-4 31168325 PMC6489188 Fei Z, Lijuan Y, Xi Y, et al. Gut microbiome associated with chemotherapy-induced diarrhea from the CapeOX regimen as adjuvant chemotherapy in resected stage III colorectal cancer. Gut Pathog. 2019;11:18. 31168325 10.1186/s13099-019-0299-4 PMC6489188

66. Mafe AN, Busselberg D. Microbiome Integrity Enhances the Efficacy and Safety of Anticancer Drug. Biomedicines. 2025;13:422. 10.3390/biomedicines13020422 PMC11852609 40002835

67. Kurita A Kado S Matsumoto T Streptomycin alleviates irinotecan-induced delayed-onset diarrhea in rats by a mechanism other than inhibition of beta-glucuronidase activity in intestinal lumen Cancer Chemother Pharmacol 2011 67 201 13 10.1007/s00280-010-1310-4 20354702 Kurita A, Kado S, Matsumoto T, et al. Streptomycin alleviates irinotecan-induced delayed-onset diarrhea in rats by a mechanism other than inhibition of beta-glucuronidase activity in intestinal lumen. Cancer Chemother Pharmacol. 2011;67:201–13. 20354702 10.1007/s00280-010-1310-4

68. Thomson CA Morgan SC Ohland C McCoy KD From germ-free to wild: modulating microbiome complexity to understand mucosal immunology Mucosal Immunol 2022 15 1085 94 10.1038/s41385-022-00562-3 36065057 Thomson CA, Morgan SC, Ohland C, McCoy KD. From germ-free to wild: modulating microbiome complexity to understand mucosal immunology. Mucosal Immunol. 2022;15:1085–94. 36065057 10.1038/s41385-022-00562-3

69. Gheorghe CE Ritz NL Martin JA Wardill HR Cryan JF Clarke G Investigating causality with fecal microbiota transplantation in rodents: applications, recommendations and pitfalls Gut Microbes 2021 13 1941711 10.1080/19490976.2021.1941711 34328058 PMC8331043 Gheorghe CE, Ritz NL, Martin JA, Wardill HR, Cryan JF, Clarke G. Investigating causality with fecal microbiota transplantation in rodents: applications, recommendations and pitfalls. Gut Microbes. 2021;13:1941711. 34328058 10.1080/19490976.2021.1941711 PMC8331043

70. Solanki R Karande A Ranganathan P Emerging role of gut microbiota dysbiosis in neuroinflammation and neurodegeneration Front Neurol 2023 14 1149618 10.3389/fneur.2023.1149618 37255721 PMC10225576 Solanki R, Karande A, Ranganathan P. Emerging role of gut microbiota dysbiosis in neuroinflammation and neurodegeneration. Front Neurol. 2023;14:1149618. 37255721 10.3389/fneur.2023.1149618 PMC10225576

71. Shatunova S Aktar R Peiris M Lee JYP Vetter I Starobova H The role of the gut microbiome in neuroinflammation and chemotherapy-induced peripheral neuropathy Eur J Pharmacol 2024 979 176818 10.1016/j.ejphar.2024.176818 39029779 Shatunova S, Aktar R, Peiris M, Lee JYP, Vetter I, Starobova H. The role of the gut microbiome in neuroinflammation and chemotherapy-induced peripheral neuropathy. Eur J Pharmacol. 2024;979:176818. 39029779 10.1016/j.ejphar.2024.176818

72. Stratogianni A Tosch M Schlemmer H Bortezomib-induced severe autonomic neuropathy Clin Auton Res 2012 22 199 202 10.1007/s10286-012-0164-8 22532274 Stratogianni A, Tosch M, Schlemmer H, et al. Bortezomib-induced severe autonomic neuropathy. Clin Auton Res. 2012;22:199–202. 22532274 10.1007/s10286-012-0164-8

73. Rahman AA, Masango P, Stavely R, Bertrand P, Page A, Nurgali K. Oxaliplatin-Induced Damage to the Gastric Innervation: Role in Nausea and Vomiting. Biomolecules. 2023;13:276. 10.3390/biom13020276 PMC9952961 36830645

74. Cabezos PA Vera G Castillo M Fernandez-Pujol R Martin MI Abalo R Radiological study of gastrointestinal motor activity after acute cisplatin in the rat. Temporal relationship with pica Auton Neurosci 2008 141 54 65 10.1016/j.autneu.2008.05.004 18579450 Cabezos PA, Vera G, Castillo M, Fernandez-Pujol R, Martin MI, Abalo R. Radiological study of gastrointestinal motor activity after acute cisplatin in the rat. Temporal relationship with pica. Auton Neurosci. 2008;141:54–65. 18579450 10.1016/j.autneu.2008.05.004

75. Koch CA Uwaifo GI Are gastrointestinal symptoms related to diabetes mellitus and glycemic control? Eur J Gastroenterol Hepatol 2008 20 822 5 10.1097/MEG.0b013e3282f5f75e 18794593 Koch CA, Uwaifo GI. Are gastrointestinal symptoms related to diabetes mellitus and glycemic control? Eur J Gastroenterol Hepatol. 2008;20:822–5. 18794593 10.1097/MEG.0b013e3282f5f75e

76. Shafi MA Pasricha PJ Post-surgical and obstructive gastroparesis Curr Gastroenterol Rep 2007 9 280 5 10.1007/s11894-007-0031-2 17883974 Shafi MA, Pasricha PJ. Post-surgical and obstructive gastroparesis. Curr Gastroenterol Rep. 2007;9:280–5. 17883974 10.1007/s11894-007-0031-2

77. Lu KH Cao J Oleson S Vagus nerve stimulation promotes gastric emptying by increasing pyloric opening measured with magnetic resonance imaging Neurogastroenterol Motil 2018 30 e13380 10.1111/nmo.13380 29797377 PMC6160317 Lu KH, Cao J, Oleson S, et al. Vagus nerve stimulation promotes gastric emptying by increasing pyloric opening measured with magnetic resonance imaging. Neurogastroenterol Motil. 2018;30:e13380. 29797377 10.1111/nmo.13380 PMC6160317

78. Shimura K Shimazaki C Taniguchi K Inaba T Horiike S Taniwaki M Bortezomib-induced neurogenic bladder in patients with multiple myeloma Ann Hematol 2009 88 383 4 10.1007/s00277-008-0614-5 18836719 Shimura K, Shimazaki C, Taniguchi K, Inaba T, Horiike S, Taniwaki M. Bortezomib-induced neurogenic bladder in patients with multiple myeloma. Ann Hematol. 2009;88:383–4. 18836719 10.1007/s00277-008-0614-5

79. Wei R Liu X Wang Y (-)-Epigallocatechin-3-gallate mitigates cyclophosphamide-induced intestinal injury by modulating the tight junctions, inflammation and dysbiosis in mice Food Funct 2021 12 11671 85 10.1039/D1FO01848E 34730149 Wei R, Liu X, Wang Y, et al. (-)-Epigallocatechin-3-gallate mitigates cyclophosphamide-induced intestinal injury by modulating the tight junctions, inflammation and dysbiosis in mice. Food Funct. 2021;12:11671–85. 34730149 10.1039/d1fo01848e

80. Wardill HR de Mooij CEM da Silva Ferreira AR Translational model of melphalan-induced gut toxicity reveals drug-host-microbe interactions that drive tissue injury and fever Cancer Chemother Pharmacol 2021 88 173 88 10.1007/s00280-021-04273-7 33877390 PMC8236460 Wardill HR, de Mooij CEM, da Silva Ferreira AR, et al. Translational model of melphalan-induced gut toxicity reveals drug-host-microbe interactions that drive tissue injury and fever. Cancer Chemother Pharmacol. 2021;88:173–88. 33877390 10.1007/s00280-021-04273-7 PMC8236460

81. Vernino S Low PA Fealey RD Stewart JD Farrugia G Lennon VA Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies N Engl J Med 2000 343 847 55 10.1056/NEJM200009213431204 10995864 Vernino S, Low PA, Fealey RD, Stewart JD, Farrugia G, Lennon VA. Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies. N Engl J Med. 2000;343:847–55. 10995864 10.1056/NEJM200009213431204

82. De Giorgio R Guerrini S Barbara G Inflammatory neuropathies of the enteric nervous system Gastroenterology 2004 126 1872 83 10.1053/j.gastro.2004.02.024 15188182 De Giorgio R, Guerrini S, Barbara G, et al. Inflammatory neuropathies of the enteric nervous system. Gastroenterology. 2004;126:1872–83. 15188182 10.1053/j.gastro.2004.02.024

83. Meregalli C Fumagalli G Alberti P Neurofilament light chain: a specific serum biomarker of axonal damage severity in rat models of Chemotherapy-Induced Peripheral Neurotoxicity Arch Toxicol 2020 94 2517 22 10.1007/s00204-020-02755-w 32333051 Meregalli C, Fumagalli G, Alberti P, et al. Neurofilament light chain: a specific serum biomarker of axonal damage severity in rat models of Chemotherapy-Induced Peripheral Neurotoxicity. Arch Toxicol. 2020;94:2517–22. 32333051 10.1007/s00204-020-02755-w

84. Balayssac D, Durif J, Lambert C, et al. Exploring Serum Biomarkers for Neuropathic Pain in Rat Models of Chemotherapy-Induced Peripheral Neuropathy: A Comparative Pilot Study with Oxaliplatin, Paclitaxel, Bortezomib, and Vincristine. Toxics. 2023;11:1004. 10.3390/toxics11121004 PMC10747971 38133405

85. Huehnchen P Springer A Kern J Bortezomib at therapeutic doses poorly passes the blood-brain barrier and does not impair cognition Brain Commun 2020 2 fcaa021 10.1093/braincomms/fcaa021 32954282 PMC7425526 Huehnchen P, Springer A, Kern J, et al. Bortezomib at therapeutic doses poorly passes the blood-brain barrier and does not impair cognition. Brain Commun. 2020;2:fcaa021. 32954282 10.1093/braincomms/fcaa021 PMC7425526

86. Sun Y Koyama Y Shimada S Inflammation From Peripheral Organs to the Brain: How Does Systemic Inflammation Cause Neuroinflammation? Front Aging Neurosci 2022 14 903455 10.3389/fnagi.2022.903455 35783147 PMC9244793 Sun Y, Koyama Y, Shimada S. Inflammation From Peripheral Organs to the Brain: How Does Systemic Inflammation Cause Neuroinflammation? Front Aging Neurosci. 2022;14:903455. 35783147 10.3389/fnagi.2022.903455 PMC9244793

87. Hapfelmeier S Lawson MA Slack E Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses Science 2010 328 1705 9 10.1126/science.1188454 20576892 PMC3923373 Hapfelmeier S, Lawson MA, Slack E, et al. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses. Science. 2010;328:1705–9. 20576892 10.1126/science.1188454 PMC3923373

88. Round JL Mazmanian SK The gut microbiota shapes intestinal immune responses during health and disease Nat Rev Immunol 2009 9 313 23 10.1038/nri2515 19343057 PMC4095778 Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol. 2009;9:313–23. 19343057 10.1038/nri2515 PMC4095778

89. Cook TM Mansuy-Aubert V Communication between the gut microbiota and peripheral nervous system in health and chronic disease Gut Microbes 2022 14 2068365 10.1080/19490976.2022.2068365 35482894 PMC9067538 Cook TM, Mansuy-Aubert V. Communication between the gut microbiota and peripheral nervous system in health and chronic disease. Gut Microbes. 2022;14:2068365. 35482894 10.1080/19490976.2022.2068365 PMC9067538

90. Lu S Zhao Q Guan Y The communication mechanism of the gut-brain axis and its effect on central nervous system diseases: A systematic review Biomed Pharmacother 2024 178 117207 10.1016/j.biopha.2024.117207 39067168 Lu S, Zhao Q, Guan Y, et al. The communication mechanism of the gut-brain axis and its effect on central nervous system diseases: A systematic review. Biomed Pharmacother. 2024;178:117207. 39067168 10.1016/j.biopha.2024.117207

91. Frith ME Kashyap PC Linden DR Theriault B Chang EB Microbiota-dependent early-life programming of gastrointestinal motility iScience 2024 27 110895 10.1016/j.isci.2024.110895 39351201 PMC11440258 Frith ME, Kashyap PC, Linden DR, Theriault B, Chang EB. Microbiota-dependent early-life programming of gastrointestinal motility. iScience. 2024;27:110895. 39351201 10.1016/j.isci.2024.110895 PMC11440258

92. Gaboriau-Routhiau V Rakotobe S Lecuyer E The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses Immunity 2009 31 677 89 10.1016/j.immuni.2009.08.020 19833089 Gaboriau-Routhiau V, Rakotobe S, Lecuyer E, et al. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Immunity. 2009;31:677–89. 19833089 10.1016/j.immuni.2009.08.020

93. El Aidy S van Baarlen P Derrien M Temporal and spatial interplay of microbiota and intestinal mucosa drive establishment of immune homeostasis in conventionalized mice Mucosal Immunol 2012 5 567 79 10.1038/mi.2012.32 22617837 El Aidy S, van Baarlen P, Derrien M, et al. Temporal and spatial interplay of microbiota and intestinal mucosa drive establishment of immune homeostasis in conventionalized mice. Mucosal Immunol. 2012;5:567–79. 22617837 10.1038/mi.2012.32

94. Shi H Chen M Zheng C Yinglin B Zhu B Fecal Microbiota Transplantation Alleviated Paclitaxel-Induced Peripheral Neuropathy by Interfering with Astrocytes and TLR4/p38MAPK Pathway in Rats J Pain Res 2023 16 2419 32 10.2147/JPR.S415642 37483406 PMC10361291 Shi H, Chen M, Zheng C, Yinglin B, Zhu B. Fecal Microbiota Transplantation Alleviated Paclitaxel-Induced Peripheral Neuropathy by Interfering with Astrocytes and TLR4/p38MAPK Pathway in Rats. J Pain Res. 2023;16:2419–32. 37483406 10.2147/JPR.S415642 PMC10361291

95. Yardim A Gur C Comakli S Investigation of the effects of berberine on bortezomib-induced sciatic nerve and spinal cord damage in rats through pathways involved in oxidative stress and neuro-inflammation Neurotoxicology 2022 89 127 39 10.1016/j.neuro.2022.01.011 35121005 Yardim A, Gur C, Comakli S, et al. Investigation of the effects of berberine on bortezomib-induced sciatic nerve and spinal cord damage in rats through pathways involved in oxidative stress and neuro-inflammation. Neurotoxicology. 2022;89:127–39. 35121005 10.1016/j.neuro.2022.01.011

96. Silva YP Bernardi A Frozza RL The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication Front Endocrinol (Lausanne) 2020 11 25 10.3389/fendo.2020.00025 32082260 PMC7005631 Silva YP, Bernardi A, Frozza RL. The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication. Front Endocrinol (Lausanne). 2020;11:25. 32082260 10.3389/fendo.2020.00025 PMC7005631

97. Tahiri J Mian M Aftan F Serotonin in depression and Alzheimer’s disease: Focus on SSRI’s beneficial effects Ageing Res Rev 2024 101 102537 10.1016/j.arr.2024.102537 39389238 PMC11531385 Tahiri J, Mian M, Aftan F, et al. Serotonin in depression and Alzheimer’s disease: Focus on SSRI’s beneficial effects. Ageing Res Rev. 2024;101:102537. 39389238 10.1016/j.arr.2024.102537 PMC11531385

98. Liu QQ Yao XX Gao SH Role of 5-HT receptors in neuropathic pain: potential therapeutic implications Pharmacol Res 2020 159 104949 10.1016/j.phrs.2020.104949 32464329 Liu QQ, Yao XX, Gao SH, et al. Role of 5-HT receptors in neuropathic pain: potential therapeutic implications. Pharmacol Res. 2020;159:104949. 32464329 10.1016/j.phrs.2020.104949

99. De Vadder F Grasset E Manneras Holm L Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks Proc Natl Acad Sci U S A 2018 115 6458 63 10.1073/pnas.1720017115 29866843 PMC6016808 De Vadder F, Grasset E, Manneras Holm L, et al. Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks. Proc Natl Acad Sci U S A. 2018;115:6458–63. 29866843 10.1073/pnas.1720017115 PMC6016808

100. Loman BR Jordan KR Haynes B Bailey MT Pyter LM Chemotherapy-induced neuroinflammation is associated with disrupted colonic and bacterial homeostasis in female mice Sci Rep 2019 9 16490 10.1038/s41598-019-52893-0 31712703 PMC6848141 Loman BR, Jordan KR, Haynes B, Bailey MT, Pyter LM. Chemotherapy-induced neuroinflammation is associated with disrupted colonic and bacterial homeostasis in female mice. Sci Rep. 2019;9:16490. 31712703 10.1038/s41598-019-52893-0 PMC6848141

101. Dmytriv TR Storey KB Lushchak VI Intestinal barrier permeability: the influence of gut microbiota, nutrition, and exercise Front Physiol 2024 15 1380713 10.3389/fphys.2024.1380713 39040079 PMC11260943 Dmytriv TR, Storey KB, Lushchak VI. Intestinal barrier permeability: the influence of gut microbiota, nutrition, and exercise. Front Physiol. 2024;15:1380713. 39040079 10.3389/fphys.2024.1380713 PMC11260943

102. Ullah H Arbab S Chang C Gut microbiota therapy in gastrointestinal diseases Front Cell Dev Biol 2025 13 1514636 10.3389/fcell.2025.1514636 40078367 PMC11897527 Ullah H, Arbab S, Chang C, et al. Gut microbiota therapy in gastrointestinal diseases. Front Cell Dev Biol. 2025;13:1514636. 40078367 10.3389/fcell.2025.1514636 PMC11897527

103. Mou Y Du Y Zhou L Gut Microbiota Interact With the Brain Through Systemic Chronic Inflammation: Implications on Neuroinflammation, Neurodegeneration, and Aging Front Immunol 2022 13 796288 10.3389/fimmu.2022.796288 35464431 PMC9021448 Mou Y, Du Y, Zhou L, et al. Gut Microbiota Interact With the Brain Through Systemic Chronic Inflammation: Implications on Neuroinflammation, Neurodegeneration, and Aging. Front Immunol. 2022;13:796288. 35464431 10.3389/fimmu.2022.796288 PMC9021448

104. Beales JT Mohanty V Seng MM Gut microbiota and metabolite disruption during breast cancer chemotherapy is associated with peripheral neuropathy sensory symptoms and pain Brain Behav Immun 2026 135 106489 10.1016/j.bbi.2026.106489 41722712 PMC13227551 Beales JT, Mohanty V, Seng MM, et al. Gut microbiota and metabolite disruption during breast cancer chemotherapy is associated with peripheral neuropathy sensory symptoms and pain. Brain Behav Immun. 2026;135:106489. 41722712 10.1016/j.bbi.2026.106489 PMC13227551

105. Ohtsu T Takagi A Uemura N The Ameliorating Effect of Lactobacillus gasseri OLL2716 on Functional Dyspepsia in Helicobacter pylori-Uninfected Individuals: A Randomized Controlled Study Digestion 2017 96 92 102 10.1159/000479000 28768250 PMC5637312 Ohtsu T, Takagi A, Uemura N, et al. The Ameliorating Effect of Lactobacillus gasseri OLL2716 on Functional Dyspepsia in Helicobacter pylori-Uninfected Individuals: A Randomized Controlled Study. Digestion. 2017;96:92–102. 28768250 10.1159/000479000 PMC5637312

106. Takagi A Yanagi H Ozawa H Effects of Lactobacillus gasseri OLL2716 on Helicobacter pylori-Associated Dyspepsia: A Multicenter Randomized Double-Blind Controlled Trial Gastroenterol Res Pract 2016 2016 7490452 10.1155/2016/7490452 27478434 PMC4958476 Takagi A, Yanagi H, Ozawa H, et al. Effects of Lactobacillus gasseri OLL2716 on Helicobacter pylori-Associated Dyspepsia: A Multicenter Randomized Double-Blind Controlled Trial. Gastroenterol Res Pract. 2016;2016:7490452. 27478434 10.1155/2016/7490452 PMC4958476

107. Nobutani K Sawada D Fujiwara S The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome J Appl Microbiol 2017 122 212 24 10.1111/jam.13329 27761980 Nobutani K, Sawada D, Fujiwara S, et al. The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome. J Appl Microbiol. 2017;122:212–24. 27761980 10.1111/jam.13329

108. Jiang Y Che L Li SC Deciphering the personalized functional redundancy hierarchy in the gut microbiome Microbiome 2025 14 17 10.1186/s40168-025-02273-w 41345980 PMC12781464 Jiang Y, Che L, Li SC. Deciphering the personalized functional redundancy hierarchy in the gut microbiome. Microbiome. 2025;14:17. 41345980 10.1186/s40168-025-02273-w PMC12781464

109. Vacca V Marinelli S Pieroni L Urbani A Luvisetto S Pavone F Higher pain perception and lack of recovery from neuropathic pain in females: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice Pain 2014 155 388 402 10.1016/j.pain.2013.10.027 24231652 Vacca V, Marinelli S, Pieroni L, Urbani A, Luvisetto S, Pavone F. Higher pain perception and lack of recovery from neuropathic pain in females: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice. Pain. 2014;155:388–402. 24231652 10.1016/j.pain.2013.10.027

110. Ho MFS Farkas O Faria AV Plemel JR Kerr BJ A recent history of immune cell sex differences in the peripheral nervous system in persistent pain states Brain Behav Immun 2025 128 766 75 10.1016/j.bbi.2025.05.004 40345628 Ho MFS, Farkas O, Faria AV, Plemel JR, Kerr BJ. A recent history of immune cell sex differences in the peripheral nervous system in persistent pain states. Brain Behav Immun. 2025;128:766–75. 40345628 10.1016/j.bbi.2025.05.004

111. Org E Mehrabian M Parks BW Sex differences and hormonal effects on gut microbiota composition in mice Gut Microbes 2016 7 313 22 10.1080/19490976.2016.1203502 27355107 PMC4988450 Org E, Mehrabian M, Parks BW, et al. Sex differences and hormonal effects on gut microbiota composition in mice. Gut Microbes. 2016;7:313–22. 27355107 10.1080/19490976.2016.1203502 PMC4988450

112. Australian code for the. care and use of animals for scientific purposes. 8th edition 2013 (updated 2021). 2021.

113. Noll JE Williams SA Tong CM Myeloma plasma cells alter the bone marrow microenvironment by stimulating the proliferation of mesenchymal stromal cells Haematologica 2014 99 163 71 10.3324/haematol.2013.090977 23935020 PMC4007935 Noll JE, Williams SA, Tong CM, et al. Myeloma plasma cells alter the bone marrow microenvironment by stimulating the proliferation of mesenchymal stromal cells. Haematologica. 2014;99:163–71. 23935020 10.3324/haematol.2013.090977 PMC4007935

114. Zeissig MN Hewett DR Panagopoulos V Expression of the chemokine receptor CCR1 promotes the dissemination of multiple myeloma plasma cells in vivo Haematologica 2021 106 3176 87 10.3324/haematol.2020.253526 33147936 PMC8634189 Zeissig MN, Hewett DR, Panagopoulos V, et al. Expression of the chemokine receptor CCR1 promotes the dissemination of multiple myeloma plasma cells in vivo. Haematologica. 2021;106:3176–87. 33147936 10.3324/haematol.2020.253526 PMC8634189