ABSTRACT Background Immunotherapy has significantly improved outcomes for cancer patients; however, its clinical benefits vary among patients and its effectiveness across breast cancer subtypes remains uncertain. To enhance its efficacy, it is important to gain more insight into tumor-intrinsic immunomodulatory factors that could be used as therapeutic targets. We previously identified Lactate Dehydrogenase C (LDHC) to be a promising anti-cancer target due to its role in regulating cancer cell genomic integrity. In this study, we investigated the effects of tumor LDHC expression on immune responses. Methods TIMER AND TIDE deconvolution methods were used to investigate the relationship between tumor LDHC expression, immune cell infiltration and T cell dysfunction. Multiplex cytokine assays and flow cytometry analyses of breast cancer cell monocultures, and direct and indirect cancer cell-immune cell co-culture models were performed to assess the effect of LDHC knockdown on the secretion of inflammatory mediators and the expression of immune checkpoint molecules. T cell activity was determined by IFN-γ ELISPot assays and 7-AAD viability flow cytometry of cancer cells in direct co-culture. Results TIMER and TIDE analyses revealed that tumor LDHC expression is associated with T cell dysfunction in breast cancer and worse post-immunotherapy survival in melanoma. Depletion of LDHC in three breast cancer cell lines (MDA-MB-468, BT-549, HCC-1954) enhanced T cell activation and cytolytic function (4-hour direct co-culture). Analysis of cancer cell monocultures revealed an increase in secreted pro-inflammatory cytokines (IFN-γ, GM- CSF, MCP-1, CXCL1), a decrease in immunosuppressive factors (IL-6, Gal-9) and a reduction in tumor cell surface PD-L1 expression following LDHC knockdown. Using 72-hour direct co- cultures with LDHC-silenced cancer cells, we observed a decrease in tumor-promoting cytokines (IL-1β, IL-4 and IL-6) and an increase in the tumor-inhibiting cytokine CXCL1. Furthermore, LDHC knockdown reduced the number of CD8+ T cells expressing PD-1 and CTLA-4, as well as the cell surface expression of CTLA-4, TIGIT, TIM3, and VISTA. Conclusions Our findings suggest that targeting LDHC may improve anti-tumor immune responses by modulating the secretion of pro- and anti-tumorigenic cytokines and impairing immune checkpoint signaling. Further studies are needed to elucidate the molecular mechanisms by which LDHC modulates these responses in breast cancer.

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re-expression in poor prognostic solid tumours. PRAME has been extensively investigated as a target for immunotherapy, however, its role in modulating the anti-tumour immune response remains largely unknown. Here, we show that PRAME tumour expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune-unfavourable tumours. Using direct and indirect co-culture models, we found that PRAME overexpressing MDA-MB-468 breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduced expression of several immune checkpoints and their ligands, including PD-1, LAG3, PD-L1, CD86, Gal-9 and VISTA. Interestingly, silencing of PRAME induced cancer cell killing to levels similar to anti-PD-L1 atezolizumab treatment. Comprehensive analysis of soluble inflammatory mediators and cancer cell expression of immune-related genes showed that PRAME tumour expression can suppress the expression and secretion of multiple pro-inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. Together, our data indicate that targeting of PRAME offers a potential, novel dual therapeutic approach to specifically target tumour cells and regulate immune activation in the tumour microenvironment.
© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.

Cell-cell communication involves a large number of molecular signals that function as words of a complex language whose grammar remains mostly unknown. Here, we describe an integrative approach involving (1) protein-level measurement of multiple communication signals coupled to output responses in receiving cells and (2) mathematical modeling to uncover input-output relationships and interactions between signals. Using human dendritic cell (DC)-T helper (Th) cell communication as a model, we measured 36 DC-derived signals and 17 Th cytokines broadly covering Th diversity in 428 observations. We developed a data-driven, computationally validated model capturing 56 already described and 290 potentially novel mechanisms of Th cell specification. By predicting context-dependent behaviors, we demonstrate a new function for IL-12p70 as an inducer of Th17 in an IL-1 signaling context. This work provides a unique resource to decipher the complex combinatorial rules governing DC-Th cell communication and guide their manipulation for vaccine design and immunotherapies.
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