Ex vivo Optimization of Glucose-Regulated Protein 94/Glycoprotein 96 Expressions in Mammospheres; Implication for Breast Cancer Immunotherapy

The induction of immunity against cancer stem cells (CSCs) can boost the efficiency of cancer vaccines. Heat shock proteins (HSPs) are required for the successful activation of anti-tumor immune responses. Glycoprotein 96 (gp96) is a well-known HSP that promotes the cross-presentation of tumor antigens. The aim of the present study was to optimize the temperature for induction of gp96 in grade 3 breast cancer spheres. In the experimental study, CSCs were enriched from breast tumor tissue samples and cultured in DMEM-F12 with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B27, and bovine serum albumin (BSA) for 22 days. The expression level of CD24 and CD44 as CSC markers was measured by flow cytometry in secondary mammospheres, and the expression of NANOG, SOX2, and OCT4 genes in CSCs was also analyzed using the real-time polymerase chain reaction (PCR). To find the optimal temperature regulation of gp96, the mammosphere was incubated at different temperatures for 1 hour, and gp96 expression was measured using the western blotting assay. Primary mammospheres were obtained after seven days of culture, and secondary spheres formed 22 days after passage. Flow cytometry analysis showed that cells with CD24- CD44+ phenotype were enriched in the culture period (from 2.6% on day 1 to 32.6% on day 22). Real-time PCR indicated that OCT4, NANOG, and SOX2 expression in mammospheres were increased by 3.8 ± 0.6, 17.8 ± 0.6, and 7.7 ± 0.8 fold respectively in comparison to the MCF-7 cell line. Western blot analysis showed that gp96 production was significantly upregulated when mammospheres were incubated at both 42°C and 43°C in comparison to the control group. Altogether, we found that heat-induced upregulated expression of gp96 in CSCs enriched mammospheres from breast tumor tissue might be used as a complementary procedure to generate more immunogenic antigens in immunotherapy settings.