Cabozantinib and IL-27 Combinatorial Therapy for Bone-Metastatic Prostate Cancer

Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. We hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27 (IL-27)) could be used to treat bone-metastatic prostate cancer and exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. To test this hypothesis, in vivo experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells that are able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), and/or a therapeutic gene (IL-27). Sonoporation was used to aid gene delivery. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing was used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone quality by enhancing osteoblast differentiation and inhibiting osteoclast differentiation. Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach will enable a significant reduction in chemotherapy-associated toxicity, enhance sensitivity to immunotherapy, and improve bone quality. Mouse adenocarcinoma cell line TC2Ras was cultured in Dulbecco’s modified Eagle’s medium–nutrient mixture F-12 (DMEM-F12) (Gibco, Waltham, Massachusetts) supplemented with 10% FBS (Hyclone, Cytiva, Marlborough, MA) and 1% antibiotic-antimycotic (Anti-Anti) (Gibco). Male C57/BL6 mice (8-10 weeks of age) were obtained from Envigo. Mice were handled in accordance with an approved study protocol by the Purdue Animal Care and Use Committee (PACUC). TC2ras cells were prepared for implantation by adding trypsin (0.25% Trypsin-EDTA, Gibco, Billings, MT) to the cells and washing with sterile 1x DPBS. The pellet was resuspended in 1x DPBS at an appropriate concentration. For the intratibial model, 105 TC2Ras-Luc cells were injected in each tibia in 10ul sterile 1xDPBS. For the subcutaneous model, 3x105 TC2Ras cells were injected in each flank in 50uL sterile 1xDPBS. Implantation was performed under 2% inhaled isoflurane anesthesia delivered by high-flow vaporizer (VetFlo, Kent Scientific, Torrington, CT). In the subcutaneous model, tumors were harvested one week post implantation for RNA-sequencing analysis. In the intratibial model, three mice per group were euthanized 14 days post implantation, whereas the rest of the mice (n=4) were allowed to progress to the ethical endpoint. Post-euthanasia, tumors were collected in RNAlater (Invitrogen, Waltham, MA) and preserved for further RNA isolation and analysis. In short, RNA was isolated using RNeasy kit (Qiagen) according to manufacturer's protocol. Poly(A) RNA sequencing library was prepared following Illumina's TruSeq-stranded-mRNA protocol and conducted by LC Sciences (Houston, TX, USA). Poly(A) tail-containing mRNAs were purified using oligo-dT Briefly, magnetic beads with two rounds of purification, and fragmented using divalent cation buffer at elevated temperature. Quality control analysis and quantification of the sequencing library were performed using an Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illumina’s NovaSeq 6000. For transcript assembly and estimating transcript expression levels, software is described by Figueiredo et al. (2020) (21). For differential expression analysis of mRNAs, StringTie was used by calculating fragments per kilobase million (FPKM). The differentially expressed mRNAs were selected with log2 (fold change) > 1.5 or log2 (fold change) < -1.5 and with statistical significance (p < 0.05) by edgeR.