Variable Intrinsic Expression of Immunoregulatory Biomarkers in Breast Cancer Cell Lines, Mammospheres, and Co-Cultures

Abstract

Advances in immunotherapy have increased interest in knowing the role of the immune system in breast cancer (BC) pathogenesis. Therefore, immune checkpoints (IC) and other pathways related to immune regulation, such as JAK2 and FoXO1, have emerged as potential targets for BC treatment. However, their intrinsic gene expression in vitro has not been extensively studied in this neoplasia. Thus, we evaluated the mRNA expression of tumor-cell-intrinsic CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2, and FoXO1 in different BC cell lines, derived mammospheres, and co-cultures with peripheral blood mononuclear cells (PBMCs) by real-time quantitative polymerase chain reaction (qRT-PCR). Our results showed that intrinsic CTLA-4, CD274 (PD-L1), and PDCD1LG2 (PD-L2) were highly expressed in triple-negative cell lines, while CD276 was predominantly overexpressed in luminal cell lines. In contrast, JAK2 and FoXO1 were under-expressed. Moreover, high levels of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2 were found after mammosphere formation. Finally, the interaction between BC cell lines and peripheral blood mononuclear cells (PBMCs) stimulates the intrinsic expression of CTLA-4, PCDC1 (PD1), CD274 (PD-L1), and PDCD1LG2 (PD-L2). In conclusion, the intrinsic expression of immunoregulatory genes seems very dynamic, depending on BC phenotype, culture conditions, and tumor-immune cell interactions.

High CTLA-4 gene expression is an independent good prognosis factor in breast cancer patients, especially in the HER2-enriched subtype

“…However, the association was not signi cant, probably because of the limited number of analyzed cases [73]. We have recently reported an in vitro intrinsic expression of CD276 (B7-H3) in luminal cell lines [23]. However, contrary to our results, Kim et al [78] have described in a clinical series of 198 tumors a signi cant overexpression of B7-H3 in the TN/BL phenotype.…”

“…Previous studies have also shown that PDCD1 (PD1) mRNA levels are elevated in neoplastic tissue relative to healthy tissue [62,63]. However, although the intrinsic expression of this gene has not been studied in depth in BC, the few available data show that it remained stable in different BC cell lines [23] and tends to be overexpressed in HER2-enriched and TN/BL phenotypes [64]. Furthermore, contrary to our results, no correlation with ER expression has been observed [63].…”

“…Regarding CD274 (PDL1), several studies of both mRNA and protein have shown an association with HER2enriched and TN/BL phenotypes [23,[68][69][70][71][72]. However, few studies correlate PDCD1LG2 (PDL2) expression with BC molecular subtypes.…”

“…However, the association did not reach statistical signi cance. In our previous in vitro studies, the correlation between intrinsic PDCD1LG2 (PDL2) expression and BC phenotypes is unclear [23].…”

“…These proteins inhibit cell proliferation, cytokine production, and cytotoxic activity of effector T cells, leading to immune evasion [19,20]. Recent studies have also demonstrated the intrinsic expression of CTLA-4 and PD-1 in different neoplasm and cancer cell lines [21][22][23], suggesting that these ICs may be involved in other functions beyond the immune system regulation. PD-L1 and PD-L2 not only modulate the immune response but also participate in tumor-intrinsic functions such as neoplastic cell proliferation, migration, and invasion [24,25], epithelial-to-mesenchymal transition (EMT) regulation [25,26], stem cell properties acquisition [26-28], apoptosis inhibition, and chemotherapy resistance [29].…”