Introduction
CD44 is a transmembrane glycoprotein that is widely expressed across various human cell types and plays crucial roles in numerous biological processes, particularly in mediating cell–cell interactions [ 1 ]; its interaction with hyaluronic acid (HA) enables tumor cells to evade apoptosis, resist chemotherapeutic effects, and sustain proliferation [ 2 ]. Several recent studies have demonstrated that elevated CD44 expression in tumor cells is associated with a diminished treatment response and a poor prognosis across multiple cancer types [ 3 – 10 ].
In multiple myeloma (MM), CD44 is frequently overexpressed and contributes to disease progression by promoting interactions between malignant plasma cells and the bone marrow (BM) microenvironment [ 11 ]. CD44 also strengthens stromal–tumor crosstalk, enhancing cytokine signaling and angiogenesis that sustain MM cell survival and proliferation [ 12 ]. High CD44 expressions are linked to extramedullary spread [ 13 ] and contribute to cell adhesion-mediated drug resistance [ 14 ]. Among the key downstream effectors of CD44 are matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, which play critical roles in tumor invasion and systemic dissemination [ 15 , 16 ]. By localizing and activating these MMPs, CD44 facilitates degradation of the extracellular matrix, enabling myeloma cells to infiltrate surrounding tissues and disrupt bone architecture. This process contributes to osteolytic lesion formation and supports dissemination of malignant plasma cells beyond the BM. Therefore, the cooperative activity of CD44 and MMPs promotes tumor cell escape from the primary site, vascular invasion, and extramedullary spread.
To overcome these mechanisms, differentiation-inducing agents such as all- trans retinoic acid (ATRA) have been investigated. ATRA downregulates CD44 expression and inhibits MMPs activity, reducing cellular invasiveness and enhancing chemosensitivity [ 14 , 17 – 19 ]. In MM, ATRA has been and continues to be extensively studied with various drugs in both preclinical [ 20 – 22 ] and clinical trials ( NCT06158412 ; NCT06536413 ). Bortezomib (Bor), a proteasome inhibitor, is a cornerstone of current MM therapy. It suppresses myeloma cell growth and migration, overcomes cell-adhesion-mediated drug resistance, and augments the cytotoxic effects of conventional chemotherapeutic agents [ 23 – 25 ]. Notably, several recent studies indicate that ATRA induces protease inhibitors sensitivity in MM [ 26 , 27 ] and combined treatment with ATRA and Bor at low dose exhibits synergistic effects in promoting cells differentiation and inhibiting tumor proliferation [ 28 , 29 ].
Beyond conventional chemotherapy, the immune system—particularly natural killer (NK) cells—plays a critical role in controlling MM. NK cells are innate lymphocytes that are capable of recognizing and eliminating tumor cells without prior sensitization, effectively suppressing tumor metastasis [ 30 – 32 ]. However, MM cells frequently evade immune surveillance by establishing an immunosuppressive microenvironment. Notably, both ATRA and Bor may help restore NK cell function by modulating cytokine profiles, attenuating inhibitory signaling, and upregulating NK cell activation ligands [ 33 – 36 ].
In this study, we evaluated the prognostic significance of CD44 overexpression in MM, its association with systemic dissemination through MMPs activation, and the therapeutic potential of combining NK-cell-based immunotherapy with ATRA and Bor. Our findings indicate that this combination strategy effectively targets CD44-high myeloma cells, attenuates extramedullary spread potential, and improves overall survival in MM (Graphic Abstract).
Materials and methods
Cell lines, cytokines, and drugs
Human MM cell lines (U266B1 [cat no. TIB-196 ™], NCI-H929 [cat no. CRL-3580 ™], MM.1S [cat no. CRL-2974 ™]) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA). Modified K562-OX40L-mb-IL-18/IL-21 cells were used as feeder cells for NK cell activation and expansion. The U266-green fluorescent protein–firefly luciferase (U266-GFP-FLuc) cell line was generated by transducing U266 cells with firefly luciferase-2A-GFP lentivirus (LVP020-PBS, San Diego, CA, USA). U266-Fluc, H929-Fluc, and MM1S-Fluc cell lines were generated by transducing cells with firefly luciferase lentivirus (LVP325-PBS, San Diego, CA, USA). CD44-high and CD44-low subsets of U266 and H929 cells were isolated using anti-CD44 antibody-coupled CytoFLEX SRT (Beckman Coulter, Brea, CA, USA). All cell lines were cultured in Roswell Park Memorial Institute (RPMI)−1640 medium (Welgene, Daegu, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Gibco). Cultures were maintained at 37°C in a humidified incubator containing 5% CO₂. Recombinant human interleukin (IL)−2 and IL-15 (PeproTech, Rocky Hill, NJ, USA) were used for NK cell culture. ATRA (cat no. R2625; Sigma Aldrich, St. Louis, MO, USA) and Bor (Chong Kun Dang, Seoul, Korea) were used as indicated. Bor was dissolved in phosphate-buffered saline (PBS), and ATRA was dissolved in dimethyl sulfoxide at a concentration of 30 mM and stored frozen as a stock solution.
MM samples
Peripheral blood (PB) and BM samples from 7 MM patients (Table S1 ). Fresh samples were collected, with approximate volumes of 10 mL for PB and 20 mL for BM. CD138 + cells are isolated from BM using CD138-specific magnetic microbeads (Miltenyi Biotec, BG, Germany). BM mononuclear cells from patients 1–4 were used to evaluate ATRA/Bor-mediated CD44 modulation, whereas CD138 + primary myeloma cells and autologous expanded NK cells from patients 5–7 were used to evaluate CD44 modulation after ATRA/Bor plus autologous NK-cell coculture.
Ex vivo NK cell activation and expansion
NK cells were expanded and activated following a previously established protocol using K562-OX40L-mbIL-18/−21 feeder cells [ 37 ]. Briefly, peripheral blood mononuclear cells isolated from healthy donors and patients were cocultured with γ-irradiated (100 Gy) K562-OX40L-mbIL-18/−21 feeder cells in NK cell culture medium (RPMI-1640 supplemented with 10% FBS, 1% penicillin–streptomycin, and 4 mM L-glutamine). During the first 7 days, the culture medium contained recombinant IL-2 (10 IU/mL). From days 8 to 14, the medium was replaced with recombinant IL-2 (100 IU/mL) and recombinant IL-15 (5 ng/mL). The cytokine-containing medium was refreshed every 48–72 h. On day 14, expanded NK cells with a purity exceeding 90% were harvested for subsequent experiments (Fig. S1 ).
Surface and intracellular staining for flow cytometry
Expression levels of CD44 and NK cell ligands on MM cells, NK cell markers, cytotoxicity, and degranulation activity, were analyzed using flow cytometry. Cells were stained with the specific antibodies listed in Table S2 . For BD FACSCalibur acquisition, FITC and CFSE signals were collected in FL1, PE and propidium iodide signals in FL2, and APC signals in FL4. Mean fluorescence intensity (MFI) ratios were calculated by dividing the MFI of the stained cell population by that of the corresponding isotype control.
Transfection of CD44 siRNA
Three distinct CD44-targeting small interfering RNAs (siRNAs; siCD44-960–1, siCD44-960–2, and siCD44-960–3) and a non-targeting control siRNA (siNC) were obtained from Bioneer Corp. (Daejeon, Korea). U266 and H929 cells were transfected with these siRNAs using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA). Cells were harvested at 3 days after transfection, and CD44 knockdown efficiency was evaluated using flow cytometry. The siCD44-2 construct demonstrated optimal knockdown efficiency in cell lines and was used for subsequent experiments (Fig. S2A ).
Western blotting
Protein extraction from cancer cells was performed using radioimmunoprecipitation assay buffer (R2002; Biosesang, Seongnam, Korea). Protein concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (10 µg) were separated on 7.5% SDS–polyacrylamide gels using a Bio-Rad electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto polyvinylidene difluoride membrane (cat no. IPVH00010; MilliporeSigma, Burlington, MA, USA). Membranes were blocked with 3% bovine serum albumin (BioShop Canada, Burlington, Ontario, Canada) in tris‑buffered saline containing 0.1% Tween‑20 (TBST) at room temperature for 1 h, followed by overnight incubation at 4 °C with primary antibodies. Primary antibodies included: CD44 (1:1,000; cat no. ab189524; Abcam, Cambridge, MA, USA), MMP9 (1:1,000; cat no. 7-11C; Santa Cruz Biotechnology, Inc.), MMP2 (Novus Biologicals, CO, USA), phosphorylated-protein kinase B (p-AKT) (1:1,000; cat no. 05–669; MilliporeSigma), AKT1 (1:1,000; cat no. 5298; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nuclear factor kappa B (NF-κB; 1:1,000; cat no. 372; Santa Cruz Biotechnology, Inc.), β-catenin (1:1,000; cat no. 393501; Santa Cruz Biotechnology), and β-actin (1:2,000; cat no. 26628–22-8; MilliporeSigma). After three washes with TBST, the membranes were incubated at room temperature for 1 h with horseradish peroxidase (HRP)–conjugated secondary antibodies: anti-mouse IgG (1:5,000; cat no. 7076S) and anti-rabbit IgG (1:10,000; cat no. 7074; Cell Signaling Technology, Danvers, MA, USA). Following three additional washes, the membranes were developed using a 1:1 mixture of HRP substrate peroxide solution and HRP substrate luminol reagent (cat no. WSKLS0500; MilliporeSigma). Protein bands were visualized using the Fusion Solo imaging system (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany) and quantified using ImageJ software (version 1.53 m; National Institutes of Health, Bethesda, USA).
Cell proliferation assay
Cell viability was evaluated using a water-soluble tetrazolium salt (WST-1) assay (EZ-Cytox cell viability assay kit; ITSBIO, Seoul, Korea). Briefly, cells were seeded in 96-well plates at 100 μL per well in RPMI medium containing 10% FBS and incubated for 24, 48, or 72 h, depending on the experimental design. Subsequently, 10 μL of the WST-1 reagent was added to each well, followed by incubation at 37°C for 30 min. Absorbance was measured at 450 nm using a VersaMax microplate reader (Molecular Devices, MD, USA) to determine cell viability.
Cell migration and invasion assays
Transwell chambers (cat no. 3422; Corning, NY, USA) were used to assess cell migration and invasion. For the invasion assay, Transwell inserts were pre-coated with 50 μL of extracellular matrix gel (1.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 2 h at 37°C to allow the gel to solidify before the experiment. The next steps in these two experiments are the same. Briefly, cells subjected to siRNA-mediated knockdown or treated with ATRA (10 μM for 72 h), Bor (5 nM for 24 h), or a combination of ATRA (10 μM for 72 h) and Bor (5 nM for 24 h) were washed twice with PBS and seeded (10 5 cells) in 200 μL of serum-free medium into the upper chamber. The lower chambers were filled with 600 μL of RPMI medium supplemented with 10% FBS. After 24 h of incubation, non-migrated cells or non-invaded cells on the upper surface of the membrane were gently removed with a cotton swab. Migrated cells on the lower surface were stained using Diff-Quik stain solution (cat no. 38721; Sysmex Corporation, Kobe, Japan) at room temperature. After drying, stained cells were imaged under a light microscope (Olympus IX51; Olympus Corporation, Tokyo, Japan), and cell counts were obtained from five randomly selected fields per chamber.
Cytotoxicity assay
Quantitative cytotoxicity and degranulation assays were performed as previously described [ 38 ]. Briefly, tumor cells (U266, H929, and MM1S) or primary cells were pretreated with ATRA (10 μM for 72 h), Bor (5 nM for 24 h), or a combination of ATRA (10 μM for 72 h) and Bor (5 nM for 24 h). Subsequently, cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies Corp., Carlsbad, CA, USA) for 10 min and cocultured with ex vivo–expanded NK cells at various effector-to-target (E:T) ratios at 37°C for 4 h in a 5% CO₂ incubator. After incubation, 1 μL of propidium iodide (Life Technologies Corp.) was added to each sample, and flow cytometric analysis was performed using a BD FACSCalibur. The percentage of dead cells among CFSE-positive cells was calculated to determine cytotoxicity.
CD107a degranulation assay
To assess NK cell degranulation capacity, target cells pretreated with ATRA, bortezomib, or their combination were cocultured with NK cells at an E:T ratio of 0.5:1 in 96-well flat-bottom plates at 37°C in a 5% CO₂ incubator. Phycoerythrin-conjugated anti-CD107a antibodies were added at the beginning of coculture. After 1 h, Monensin (BD Biosciences, San Jose, CA, USA) was added to the wells. Following a total of 4 h incubation, the cells were stained with antibodies against CD3 and CD56 for 20 min and analyzed on a BD FACSCalibur flow cytometer, collecting 10,000 events per sample. Flow cytometric data were processed and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Luciferase assays
The quantitative cytotoxicity assay based on luciferase activity was previously described [ 39 ]. Shortly, the luciferase-expressing MM cell lines (U266-Fluc, H929-Fluc, and MM1S-Fluc) were co-cultured with NK cells at a 1:1 ratio in a white 96-well plate (SPL Life Sciences Co., Gyeonggi-do, Korea) for 4 h at 37°C in a 5% CO₂ atmosphere. After incubation, 1 μL of D-luciferin (15 mg/mL; PerkinElmer, MA, USA) was added to each well, and luminescence was read in endpoint mode using Synergy H1 (BioTek, VT, USA). Percent specific lysis is equal to (1-percent viability)*100 and is calculated as follows:
Human interferon (IFN)-γ enzyme-linked immunoabsorbent assay (ELISA)
Mice were deeply anesthetized with 5% isoflurane (Ifran; Hana Pharm, Gyeonggi, Korea) to minimize pain and distress, followed by euthanasia via cervical dislocation. Blood samples were collected from the left ventricle using a 25G needle, and plasma was immediately separated and stored at ˗ 20°C until analysis. Human IFN-γ concentrations were quantified using a human IFN-γ ELISA Set (BD Biosciences).
Hematoxylin and eosin staining
After systemic dissemination analysis, small portions of lung, liver, and kidney tissue were excised and stored in 4% of neutral buffered formalin. Tissue sample was then dehydrated by graded ethanol sequentially and embedded in paraffin wax. Paraffin-embedded tissue was sectioned serially about 5 μm size and were stained with hematoxylin and eosin dyes (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Stained tissue sections were documented using camera equipped microscope (Leica DM3000, Wetzlar, Germany).
MM xenograft model
NSG mice (6 weeks old, 17–22 g) obtained from Bar Harbor, MA, USA, were maintained under specific-pathogen-free conditions at the Hwasun Biomedical Convergence Center. The mice were housed under a 12 h light/dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) at a controlled temperature of 22–26°C and relative humidity of 45–55%. A commercial rodent diet and water were sterilized before use. The Institutional Animal Care and Use Committee of Chonnam National University Medical School approved the study protocol (CNU IACUC-H2022-35). The procedures were performed in accordance with the ARRIVE guidelines. Overall survival (OS) and physiological status were monitored daily. Mice were euthanized upon reaching humane endpoints, including body weight loss exceeding 20%, significantly reduced mobility or activity, moribund condition, or signs of wasting syndrome.
On day 0, NSG mice were intravenously injected with 5 × 10⁶ U266-GFP-Fluc cells. Seven days after tumor inoculation, 48 mice were randomly divided into four groups (n = 12 per group; 4 mice per cage): untreated control, ATRA + Bor, NK, and ATRA + Bor + NK. The mice received the following treatments: vehicle control, intraperitoneal, Bor (0.5 mg/kg/day) once weekly (days 10 and 17), and intragastric ATRA (10 mg/kg/day; dissolved in corn oil, cat no. C8267; Sigma-Aldrich) three times weekly (days 8–10 and 15–17). Freshly expanded NK cells (5 × 10 6 cells/mouse) were administered via tail vein injection once weekly (days 11 and 18). Tumor growth was monitored weekly by bioluminescence imaging of both dorsal and ventral views. Ten minutes prior to imaging, the mice were intraperitoneally injected with D-luciferin (150 mg/kg; PerkinElmer, Waltham, MA, USA), and images were captured using the NightOWL imaging system (Berthold Technologies, Bad Wildbad, Germany). After 7 weeks of tumor inoculation, four mice from each group were sacrificed for systemic dissemination analysis. Disseminated disease was visualized using the FOBI fluorescence in vivo imaging system (Cellgentek, Daejeon, Korea). The remaining eight mice per group were monitored for tumor progression and OS.
Statistical analysis
Statistical analyses were performed using R software version 4.5.0 (R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org ) and GraphPad Prism (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were conducted using a two-tailed unpaired Student’s t -test for normally distributed data or the Mann–Whitney U test for nonparametric data. For multiple-group comparisons, one-way analysis of variance followed by Tukey’s post hoc test was applied. Data are presented as mean ± standard deviation (SD). P- values < 0.05 were considered statistically significant.
Results
CD44 overexpression is associated with inferior OS in MM patients
To assess the prognostic significance of CD44 expression in MM, clinical data from 837 patients in the CoMMpass database were analyzed [ 40 ]. Patients were stratified into two groups based on the median CD44 expression level. The CD44-high group (n = 419) exhibited significantly worse OS compared to the CD44-low group (n = 418) (Fig. 1 A; P < 0.0001). Further analysis by Revised International Staging System (R-ISS) stage revealed that high CD44 expression was consistently associated with inferior OS across all stages. The lowest survival probability was observed in patients with high CD44 expression and R-ISS stage III disease. In contrast, low CD44 expression correlated with improved OS in MM patients (Fig. 1 B; P < 0.0001).
CD44 drives proliferation, migration, and invasion of MM cells via NF-κB/MMP signaling
To investigate the mechanistic role of CD44 in MM, patients were divided into three groups according to CD44 expression levels. Analysis revealed that transcription factors, including NF-κB, as well as the mRNA expression of MMP9 and MMP2, were significantly higher in the CD44-high group compared to the CD44-low group (Fig. 2 A, B; P < 0.05). Analysis of the CoMMpass database revealed a positive correlation among NF-κB, AKT, MMP9, MMP2, and CD44 expression (Fig. S3 ). To assess further the regulatory effects of CD44 on MMP9 and MMP2, CD44 expression was silenced using small interfering RNA (siCD44) in U266 and H929 cells. CD44 silencing significantly suppressed cell proliferation from day 2 onward in U266 and H929 cells (Fig. 2 C; P < 0.0001). Moreover, inhibition of CD44 significantly reduced MMP9 and MMP2 expression, as well as AKT and NF-κB activity, compared to the siNC group, total β-catenin levels changed insignificantly (Fig. 2 D; P < 0.0001). Finally, the effect of CD44 knockdown on cell motility was evaluated using Transwell migration and invasion assays. In MM cell lines, CD44 silencing resulted in > 50% reduction in migrated and invaded cells compared to controls (Fig. 2 E; P < 0.0001).
ATRA and Bor exert anti-proliferative and pro-apoptotic effects in CD44-high myeloma cell lines
To evaluate the effects of ATRA and Bor on CD44-overexpressing MM cell viability, three cell lines with abundant CD44 surface expression (U266, H929, and MM1S) were selected (Fig. 3 A). Cell viability and half-maximal inhibitory concentration (IC 50 ) values were determined using the WST-1 assay following treatment with increasing concentrations of ATRA (1, 10, 20, and 40 μM for 72 h) or Bor (5, 10, 15, and 20 nM for 24 h). H929 and MM1S cells exhibited significant sensitivity to both agents, whereas U266 cells demonstrated relative resistance. At the highest concentrations (40 μM ATRA and 20 nM Bor), cell viability was reduced by 68 and 87% in H929 cells, 60 and 89% in MM1S cells, and 54 and 56% in U266 cells, respectively, compared to untreated controls. The calculated IC₅₀ values for ATRA were 19.85 μM in H929, 32.82 μM in MM1S, and 50.73 μM in U266 cells, whereas those for Bor were 10.88 nM, 11.83 nM, and 19.67 nM, respectively (Fig. 3 B). To determine whether sublethal drug concentrations modulate CD44 expression, MM cells were treated with 10 μM ATRA for 72 h and 5 nM Bor for 24 h, either alone or in combination, for all subsequent assays. Combined treatment significantly enhanced apoptosis across all MM cell lines, as evidenced by an increased Annexin V/PI-positive population, most prominently in H929 cells, which exhibited a 7.5-fold increase compared to untreated controls (Fig. 3 C). In addition, U266 and H929 cells were sorted into CD44-high and CD44-low subsets (each representing 15% of the population; Fig. S2 B) and subjected to identical treatments. The ATRA-Bor combination significantly inhibited proliferation in CD44-high myeloma cells, reducing cell growth from 158 to 53% in U266 and from 260 to 51% in H929 compared to untreated controls (Fig. 3 D).
Combination of ATRA and Bor suppresses CD44 expression, leading to inhibition of invasion and migration of MM cells through downregulation of MMP2 and MMP9
We used flow cytometry to further investigate the inhibitory effects of the combination treatment on CD44 expression. The combination significantly reduced CD44 levels, as indicated by MFI, with decreases of approximately 75% in U266 cells, 53% in H929 cells, and 46% in MM1S cells (Fig. 4 A). To elucidate the underlying mechanism, we assessed β-catenin, a key signaling molecule that regulates CD44 expression and metastasis [ 41 , 42 ]. Reduced CD44 protein levels were accompanied by a corresponding decrease in β-catenin expression. Moreover, ATRA and Bor synergistically downregulated p-AKT, total AKT, NF-κB, MMP9, and MMP2 protein levels across all cell lines, consistent with our previous findings from CD44 knockdown experiments (Fig. 4 B; Fig. S4 ). These findings indicate that CD44 depletion attenuates AKT activation in MM cells, correlating with reduced MMP9 and MMP2 expression, implicating CD44-mediated AKT/NF-κB signaling in systemic dissemination. Finally, Transwell migration assays demonstrated that single-agent and combination treatments significantly suppressed the migratory capacity of all myeloma cell lines (Fig. 4 C; P < 0.01).
ATRA and Bor enhance NK cell cytotoxicity by upregulating NK cell-activating ligands in CD44-high MM cells
To evaluate changes in NK cell-activating ligands following ATRA and Bor treatment, we analyzed the surface expression of natural killer group 2 member D (NKG2D) ligands (MICA and MICB), intercellular adhesion molecule 1 (ICAM-1), and death receptors (Fas and TRAIL-R2) on MM cells. Four experimental groups were established: untreated control, ATRA alone, Bor alone, and the combination of ATRA and Bor. The combined treatment significantly increased the expression of MICA, MICB, ICAM1, Fas, and TRAIL-R2 compared with the single-agent or untreated groups (Fig. 5 A; P < 0.05).
To assess further the susceptibility of CD44-overexpressing tumor cells to NK cell-mediated cytotoxicity, we compared the upper and lower 33% fractions of viable tumor cells expressing CD44 following ATRA + Bor treatment. Although the two groups exhibited minimal differences in baseline ligand expression, the CD44-high group exhibited significantly greater induction of MICA, MICB, ICAM1, Fas, and TRAIL-R2 after treatment compared to the CD44-low group (Fig. 5 B; P < 0.01).
Next, NK cell–mediated cytotoxicity against MM cell lines (U266, H929, and MM1S) was assessed. NK cells alone induced approximately 40% tumor lysis within 4 h, which increased to about 80% when tumor cells were pretreated with ATRA + Bor. NK-mediated cytotoxicity was significantly enhanced at all E:T ratios following pretreatment (Fig. 6 A; P < 0.05). CD107a degranulation assays further demonstrated that NK cell activation was significantly augmented by ATRA + Bor pretreatment compared to all other groups (Fig. 6 B; P < 0.01), whereas direct drug effects at similar doses on NK cells were negligible (Fig. S5 ). To examine which signaling pathway was preferred, all three luciferase-expressing MM cell lines were evaluated to determine whether blocking the NKG2D recognition receptor and/or the LFA-1 adhesion receptor could affect the cytotoxicity of NK cells to the untreated or ATRA + Bor treatment. The sharp decrease in cytotoxicity when combined NKG2D and LFA-1 blocking at U266-fluc and MM1S-fluc cells (from 50 to 20%) demonstrates that these are still the main tumor-killing receptors rather than death receptor (Fig. S6 ).
Flow cytometric analysis revealed that NK cells effectively targeted CD44-high MM cells after ATRA + Bor pretreatment. CD44 expression decreased from 96.6 to 33.0% in U266 cells and from 99.6 to 74.0% in H929 cells (E:T = 0.5:1), indicating efficient depletion of CD44-high cells (Fig. 6 C). Across all three MM cell lines, the combination of NK cells and ATRA + Bor resulted in the lowest CD44 MFI values in viable tumor cells compared to all other groups (Fig. 6 D; P < 0.05). To extend these findings to clinically relevant primary specimens, we evaluated CD44 modulation in primary samples from seven newly diagnosed MM patients. A significant decrease in expression (nearly 60%) was observed in both experiments, as assessed by the CD44 MFI relative (Fig. S7 ). Because primary CD138 + cell numbers and autologous NK-cell yields were limited, these experiments were designed to evaluate ex vivo CD44 modulation rather than to provide a powered assessment of patient-level cytotoxicity.
Combined ATRA, Bor, and NK cell therapy inhibits extramedullary spread and prolongs survival in the U266-GFP-FLuc xenograft model
To evaluate whether ATRA, Bor, and NK cells act synergistically in vivo, we assessed their antitumor efficacy in a MM xenograft model (Fig. 7 A). In this model, untreated tumor-bearing mice survived no longer than 8 weeks, whereas all treatment groups exhibited extended survival. Notably, the ATRA + Bor + NK combination produced the slowest tumor progression and markedly reduced bioluminescent tumor signals compared with ATRA + Bor or NK treatment alone at 8 weeks post-injection (Fig. 7 B, C; Fig. S8 A, B). Moreover, ATRA + Bor + NK treatment yielded the longest survival (> 16 weeks), whereas the ATRA + Bor and NK-alone groups survived 10.61 ± 1.91 weeks and 9.60 ± 1.17 weeks, respectively (Fig. 7 D; P < 0.01). No significant body weight loss and signs of damage on histopathology analysis was observed in mice treated with ATRA + Bor or ATRA + Bor + NK, indicating good tolerability (Fig. S8 C, D).
To assess systemic dissemination, GFP signals in secondary organs were analyzed using the FOBI system at 7 weeks after tumor inoculation (n = 4). The ATRA + Bor + NK combination significantly inhibited MM spread from the bone marrow to distant organs, with no brain involvement detected (0/4 mice), in contrast to NK therapy alone (3/4 mice) and untreated controls (4/4 mice). ATRA + Bor treatment alone resulted in weak GFP signals in 1 of 4 mice (Fig. 7 E).
To assess further the impact on CD44, the experiment was repeated in a progressive MM model, which delayed treatment until day 22 (Fig. S9 A). Consistent with previous model, tumor signals were significantly reduced in the ATRA + Bor + NK group compared to other treatments after 2 weeks (Fig. S9 B, C). The proportion of MM cells in femurs decreased from 46.5 to 15% following ATRA + Bor + NK treatment. CD44 expression declined from 93.5 to 73.9% with ATRA + Bor and further to 58.7% with ATRA + Bor + NK (Fig. S9 D; P < 0.05). In parallel, plasma MMP9 levels were significantly reduced ( P < 0.01), whereas changes in MMP2 were negligible (Fig. S9 E). Moreover, ATRA + Bor pretreatment enhanced NK cell-mediated IFN-γ secretion by up to 50% compared to NK cells alone (Fig. S9 F; P < 0.05).
Discussion
Recent studies have reported associations between CD44 expression and tumor recurrence or mortality in various cancers [ 43 , 44 ]. Consistent with these reports, analysis of the CoMMpass database showed that MM patients with CD44 overexpression at diagnosis had significantly inferior OS. This adverse association was observed across R-ISS stages I, II, and III, suggesting that CD44 expression provides prognostic information beyond stage alone. Prior studies have reported that CD44 overexpression correlates with osteolytic lesions, advanced disease features, and contributes to drug resistance in MM by enhancing cell adhesion, migration, and interactions with the BM microenvironment, protecting malignant cells from therapy [ 2 , 9 , 45 ]. In addition, CD44 has been associated with aggressive diseases biology, serving as a key driver in the transition from intramedullary growth to extramedullary disease (EMD) [ 9 , 46 ]. High CD44 expression facilitates the detachment of plasma cells from the BM and their dissemination into peripheral sites, a process strongly correlated with high-risk cytogenetic profiles such as 1q21 (+ 1q) and t(4;14). This synergy between CD44-mediated adhesion plasticity and underlying genomic instability promotes a chemo-resistant phenotype and poor outcomes, particularly in advanced R-ISS stages [ 14 ]. Supporting this, CD44 has been found to be enriched in EMD sites compared to matched BM samples, reinforcing its role as a functional marker for high-risk, extramedullary progression [ 46 ].
ATRA or Bor, individually, inhibited proliferation in all three MM cell lines exhibiting abundant CD44 expression, and their combination exerted synergistic pro-apoptotic effects across these cell lines. This is consistent with previous studies. In particular, Alonso et al. [ 26 ] showed that retinoic acid depletion in the BM niche promotes a proteasome inhibitor–resistant phenotype in myeloma. More recently, Wang et al. [ 27 ] demonstrated that retinoid receptor activation sensitizes myeloma cells to proteasome inhibitor via the OAS1–RNase L pathway. In our study, combined treatment also reduced surface CD44 expression on myeloma cells. Prior reports have described CD44 downregulation via ATRA- mediated inhibition of β-catenin signaling [ 47 , 48 ], and our data are consistent with this regulatory axis.
Mechanistically, ATRA and Bor co-treatment decreased total β-catenin levels and was accompanied by reduced expression of CD44 and selected downstream effectors, including AKT, NF-κB-associated signaling components and MMP9, and MMP2. While earlier research established CD44 as a Wnt/β-catenin target driving cell adhesion, our findings support that targeting this molecule also disrupts pro-survival signaling, invasive potential, and also primes CD44+ myeloma cells for NK cell-mediated lysis [ 14 ]. This represents a transition from a mechanical adhesion focus to a dynamic immune-susceptibility focus.
Beyond tumor-intrinsic effects, we found that ATRA/Bor pretreatment enhanced NK cell–mediated cytotoxicity against CD44-positive MM cell lines and primary MM cells (Fig. 6 ; Fig. S6 ). Although baseline NK susceptibility varied among samples, pretreatment reproducibly increased NK-mediated killing and was associated with lower CD44 levels in the post-treatment tumor cell population. Notably, a CD44 downregulation was also observed on surviving myeloma cells after NK-cell exposure in both NK-only and ATRA/Bor-pretreated conditions. This finding may reflect at least two distinct biological mechanisms; trogocytosis and selective clonal elimination. Through trogocytosis, NK cells may physically shave membrane patches containing CD44 during immune synapse formation, directly reducing surface density on the target cell [ 49 ]. Alternatively, the lower CD44 levels in the residual tumor population may reflect the preferential killing of CD44-high cells, which we have shown are specifically primed for NK-mediated lysis. This NK-driven modulation suggests that NK cells preferentially target the more aggressive, CD44-high myeloma subpopulations that are typically associated with worse clinical outcomes.
To evaluate therapeutic activity in vivo, we used the U266 cells, a commonly employed MM model with high CD44 expression [ 50 – 53 ]. U266 cells showed comparatively lower sensitivity to ATRA and Bor compared to other MM cell lines in vitro (Fig. 3 B), providing a stringent setting to test combination strategies. In the U266-GFP-FLuc mouse model, NK cell therapy alone suppressed tumor growth and prolonged survival, yet disseminated disease remained frequent, consistent with known limitations of NK monotherapy such as limited persistence and susceptibility to immunosuppressive tumor microenvironment [ 54 – 56 ]. Importantly, the combination of ATRA + Bor with NK cells produced the most potent anti-myeloma response, accompanied by a marked reduction in CD44 expression in the ATRA + Bor + NK group (Fig. S9 D). These findings support the concept that pharmacologic priming with ATRA + Bor can enhance the efficacy of adoptive NK cell therapy against CD44-high MM and may reduce dissemination in this intravenous mouse model.
Currently, CAR-T and CAR-NK cell therapies targeting CD44v6 are being explored in cancers, including MM [ 57 – 59 ]. However, CAR-T cell therapy in MM (especially in elderly patients) can be limited by clinically significant toxicities such as cytokine release syndrome and neurotoxicity [ 60 ]. While CAR-NK cells may offer a favorable safety profile, genetic modification of NK cells remains technically challenging [ 61 ]. Low viral transduction efficiency necessitates repeated gene transfer procedures, which can impair NK cell functionality [ 62 ]. In contrast, our approach of combining ATRA and Bor with NK cell therapy maybe relatively simple, employs existing drugs, and is economically feasible. ATRA and Bortezomib are already approved in hematologic malignancies with well-established dosing schedules in humans. Moreover, clinical studies have shown that multiple infusions of NK cells (autologous or allogeneic) are feasible in myeloma patients, and that IL-15 agonists can be used to extend NK cell persistence. Our data suggest that ATRA + Bor could function as a targeted tumor-priming regimen before NK cell infusion, rather than relying solely on high-dose chemotherapy for lymphodepletion. This may be particularly valuable in the autologous NK cell setting to minimize graft-vs-host risks, or could be combined with cytokine support (e.g., IL-15) to boost NK longevity in vivo. By specifically targeting the CD44-mediated resistance niche, this regimen provides a practical framework for integrating NK-cell adoptive transfer into existing myeloma treatment protocols, especially for patients with CD44-driven high-risk disease.
Despite these promising findings, certain limitations warrant consideration. While our results link the synergistic effects of ATRA and Bortezomib to the CD44–MMP axis, other contributing signaling pathways remain to be fully elucidated. Furthermore, although the U266 xenograft model effectively demonstrates the efficacy of this triple combination in a high-CD44, drug-resistant context, the use of a single cell line may not capture the extensive genetic and subclonal heterogeneity of MM. Future studies utilizing a broader panel of cell lines and patient-derived xenograft models are therefore necessary to confirm the generalizability of our findings. Finally, regarding clinical translation, the favorable safety profile observed in vivo must be balanced against potential overlapping toxicities in elderly or frail populations, such as bortezomib-induced neuropathy and thrombocytopenia, ATRA-related metabolic or mucocutaneous effects, and cytokine-related inflammation in NK therapy. Implementing risk-adapted dosing will be crucial to maximize the economic and therapeutic benefits of this regimen without compromising patient quality of life.
In conclusion, our findings suggest that CD44 overexpression was associated with inferior survival in MM. CD44 may contribute to MM progression and extramedullary spread, at least in part, by promoting MMP9 and MMP2 expression through activation of the AKT/NF-κB signaling pathway. In addition, the combination of NK cell therapy with ATRA and bortezomib showed enhanced activity against CD44-overexpressing MM cells and was associated with improved survival in a xenograft mouse model. These results support further investigation of CD44 as a potential therapeutic target and suggest that CD44-directed strategies, particularly in combination with NK-cell-based approaches, may help improve treatment outcomes in MM patients with CD44 overexpression.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary file1 (PDF 1043 KB)
Supplementary file2 (PDF 750 KB)
Supplementary file3 (PDF 337 KB)
Supplementary file4 (PDF 341 KB)
Supplementary file5 (PDF 745 KB)
Supplementary file6 (PDF 229 KB)
Supplementary file7 (PDF 351 KB)
Supplementary file8 (PDF 1295 KB)
Supplementary file9 (PDF 6163 KB)
Supplementary file10 (DOCX 19 KB)
Acknowledgements
The authors acknowledge the use of Microsoft Copilot, an AI-powered writing assistant, for grammar and style refinement during manuscript preparation.
Abbreviations
Antibody
Protein kinase B
All-trans retinoic acid
Bortezomib
Bone marrow
Carboxyfluorescein succinimidyl ester
Effector-to-target
Enzyme-linked immunosorbent assay
Extramedullary disease
Fetal bovine serum
Firefly luciferase
Green fluorescent protein
Hyaluronic acid
Interleukin
Mean fluorescence intensity
Multiple myeloma
Matrix metalloproteinases
Matrix metalloproteinase 2
Matrix metalloproteinase 9
Nuclear factor kappa B
Natural killer
Overall survival
Peripheral blood
Propidium iodide
Untreated
Author contributions
SHJ and JJL contributed to conceptualization, supervision, funding acquisition, review, revision, and final approval of the manuscript. VTN performed experiments, data analysis, and drafted the original manuscript. TNT, VDH, CHJ, MCV and THC contributed to investigation, methodology, review, and editing of the final version. MK, GYS, SYA, JSA, DHY, CHL, HJK and DC reviewed the manuscript. All authors have read and approved the final version of this manuscript.
Funding
This work was supported by a grant (HCRI23029) from the Chonnam National University Hwasun Hospital Institute for Biomedical Science, by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023–00207920), and by a grant from Yuhan Corporation.
Data availability
All data generated in this study are included in this manuscript and its Supplementary Information files. Additional datasets used and analyzed during our study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
The authors have no conflict of interest to declare.
Informed consent
This study used cryopreserved peripheral blood and bone marrow samples that were collected from patients after obtaining informed consent and stored at human biobank in Chonnam National University Hwasun Hospital.
Patient consent for publication
Not applicable.
Human or animal rights
All animal procedures were approved by the Institutional Animal Care and Use Committee of Chonnam National University Medical School (CNU IACUC-H2022-35).
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Sung-Hoon Jung, Email: shglory@hanmail.net.
Je-Jung Lee, Email: drjejung@chonnam.ac.kr.
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Associated Data
Supplementary Materials
Supplementary file1 (PDF 1043 KB)
Supplementary file2 (PDF 750 KB)
Supplementary file3 (PDF 337 KB)
Supplementary file4 (PDF 341 KB)
Supplementary file5 (PDF 745 KB)
Supplementary file6 (PDF 229 KB)
Supplementary file7 (PDF 351 KB)
Supplementary file8 (PDF 1295 KB)
Supplementary file9 (PDF 6163 KB)
Supplementary file10 (DOCX 19 KB)
Data Availability Statement
All data generated in this study are included in this manuscript and its Supplementary Information files. Additional datasets used and analyzed during our study are available from the corresponding author upon reasonable request.