Head-to-Head Comparison of CCN4, DNMT3A, PTPN11, and SPARC as Suppressors of Anti-tumor Immunity.

Purpose: Emergent cancer cells likely secrete factors that inhibit anti-tumor immunity. To identify such factors, we applied a functional assay with proteomics to an immunotherapy resistant syngeneic mouse melanoma model. Four secreted factors were identified that potentially mediate immunosuppression and could become targets for novel immunotherapies. We tested for consistent clinical correlates in existing human data and verified in vivo whether knocking out tumor cell production of these factors improved immune-mediated control of tumor growth. Methods: Existing human data was analyzed for clinical correlates. A CRISPR/Cas9 approach to generate knockout cell lines and a kinetic analysis leveraging a Markov Chain Monte Carlo (MCMC) approach quantified the various knockouts' effect on cells' intrinsic growth rate. Flow cytometry was used to characterize differences in immune infiltration. Results: While all four gene products were produced by malignant melanocytes, only increased CCN4 expression was associated with reduced survival in primary melanoma patients. In immunocompetent C57BL/6 mice the CCN4 knockout increased survival while the other knockouts had no effect. This survival advantage was lost when the CCN4 knockout cells were injected into immunocompromised hosts, indicating that the effect of CCN4 may be immune mediated. Parameter estimation from the MCMC analysis shows that CCN4 was the only knockout tested that decreased the net tumor growth rate in immunocompetent mice. Flow cytometry showed an increase in NK cell infiltration in CCN4 knockout tumors. Conclusions: The results suggest that CCN4 is a mediator of immunosuppression in the melanoma tumor microenvironment and a potential collateral immunotherapy target. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00787-7.

Data-driven learning how oncogenic gene expression locally alters heterocellular networks.

Developing drugs increasingly relies on mechanistic modeling and simulation. Models that capture causal relations among genetic drivers of oncogenesis, functional plasticity, and host immunity complement wet experiments. Unfortunately, formulating such mechanistic cell-level models currently relies on hand curation, which can bias how data is interpreted or the priority of drug targets. In modeling molecular-level networks, rules and algorithms are employed to limit a priori biases in formulating mechanistic models. Here we combine digital cytometry with Bayesian network inference to generate causal models of cell-level networks linking an increase in gene expression associated with oncogenesis with alterations in stromal and immune cell subsets from bulk transcriptomic datasets. We predict how increased Cell Communication Network factor 4, a secreted matricellular protein, alters the tumor microenvironment using data from patients diagnosed with breast cancer and melanoma. Predictions are then tested using two immunocompetent mouse models for melanoma, which provide consistent experimental results.

Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma.

Cell Communication Network factor 4 (CCN4/WISP1) is a matricellular protein secreted by cancer cells that promotes metastasis by inducing the epithelial-mesenchymal transition. While metastasis limits survival, limited anti-tumor immunity also associates with poor patient outcomes with recent work linking these two clinical correlates. Motivated by increased CCN4 correlating with dampened anti-tumor immunity in primary melanoma, we test for a direct causal link by knocking out CCN4 (CCN4 KO) in the B16F0 and YUMM1.7 mouse melanoma models. Tumor growth is reduced when CCN4 KO melanoma cells are implanted in immunocompetent but not in immunodeficient mice. Correspondingly, CD45+ tumor-infiltrating leukocytes are significantly increased in CCN4 KO tumors, with increased natural killer and CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC). Among mechanisms linked to local immunosuppression, CCN4 suppresses IFN-gamma release by CD8+ T cells and enhances tumor secretion of MDSC-attracting chemokines like CCL2 and CXCL1. Finally, CCN4 KO potentiates the anti-tumor effect of immune checkpoint blockade (ICB) therapy. Overall, our results suggest that CCN4 promotes tumor-induced immunosuppression and is a potential target for therapeutic combinations with ICB.