Effects of Free and Liposomal Doxorubicin Combined with Inductive Moderate Hyperthermia on Multimodal Intratumoural Heterogeneity in Sarcoma-45.

Background/Objectives: Sarcomas exhibit marked intratumoural heterogeneity at the molecular, cellular and tissue levels, thereby limiting drug delivery and treatment response. Herein, we evaluated the effects of free doxorubicin (FDOX) and liposomal doxorubicin (LDOX) combined with inductive moderate hyperthermia (IMH) on intratumoural heterogeneity in an experimental sarcoma model using magnetic resonance imaging (MRI), histology and immunohistochemistry image analysis. Methods: Female non-inbred rats bearing sarcoma-45 were divided into six groups: control (no treatment), IMH, FDOX, LDOX, FDOX + IMH and LDOX + IMH. FDOX or LDOX was administered every other day for a total of five times, beginning from day 2 after inoculation. IMH was applied locally following drug administration using a 42 MHz radiofrequency electromagnetic field. Treatment-induced changes were assessed by T1- and T2-weighted MRI, haematoxylin-eosin-orange (H&E) staining, and Ki-67 and p53 immunohistochemistry. Moran's spatial autocorrelation index was used as a measure of tumour phenotypic heterogeneity in medical images. Results: Combination treatment with different doxorubicin formulations resulted in distinct patterns of intratumoural heterogeneity in MRI and in H&E-, Ki-67- and p53-stained images. FDOX or LDOX combined with IMH produced greater deviations from the control group in multimodal spatial organisation of sarcoma-45 than the corresponding drug treatments alone. LDOX + IMH reduced tumour growth by 34% relative to the control group and was associated with distinct histological and immunohistochemical remodelling, including connective tissue replacement and the lowest Ki-67 staining level. The lowest p53 staining levels were observed in the LDOX and LDOX + IMH groups. Conclusions: FDOX and LDOX combined with IMH induced distinct tumour remodelling patterns in sarcoma-45. Multimodal analysis of intratumoural heterogeneity in sarcomas may provide additional quantitative information for assessing treatment response.