Background Depletion of tumor-associated macrophages (TAMs), which are regarded as M2, pro-tumor cells, is one of the strategies for cancer treatment. However, repolarization of TAMs to the M1 anti-tumor phenotype could constitute an immunotherapeutic alternative for tumors with defective major histocompatibility complex class I (MHC-I), where the anti-tumor effect of cytotoxic CD8+ T cells could be limited.
Materials and Methods In this study, we characterized TAMs from mouse tumor models of human papillomavirus 16-associated tumors, characterized by either reversibly (TC-1/A9) or irreversibly (TC-1/dB2m) downregulated MHC-I expression. Tumors were treated with DNA immunization against the papillomaviral E7 oncoprotein combined with intraperitoneal injection of the synthetic oligodeoxynucleotide ODN1826, a Toll-like receptor 9 agonist. TAMs were characterized ex vivo by flow cytometry. In vitro, F4/80+ TAMs from naïve tumors were stimulated to M1 or M2 phenotype and co-cultures with TC-1/A9 or TC-1/dB2m cells were established. The cytotoxic effect of polarized TAMs was investigated, and the role of nitric oxide (NO) and tumor necrosis factor (TNF)-α was examined. Finally, interleukin (IL)-10, IL-12 and TNF-α concentrations were determined by ELISA in the culture media from polarized TAMs.
Results We demonstrated that TAMs infiltrated both tumor types and this effect was moderately enhanced after combined immunotherapy. Increase in MHC-II molecules, broadly regarded as an M1 marker, was observed solely in TAMs from treated TC-1/A9 tumors. In contrast, TAMs from TC-1/dB2m tumors expressed high MHC-II levels, regardless of the treatment. Therefore, the new CD38+/Egr2+ classification1 was applied and showed to be a better descriptive parameter for M1/M2 TAMs, respectively, because the number of Egr2+ TAMs decreased in both tumor types after combined immunotherapy. While CD38+ TAMs were significantly increased after treatment of TC-1/A9 tumors, they did not increase substantially in TC-1/dB2m tumors. In vitro, co-cultures with tumor cells resulted in increase of NO production by M1 TAMs. However, NO and TNF-α contributed to the cytotoxic effect only in TAMs from TC-1/A9 tumor. Finally, in vitro polarized M1 TAMs were able to produce TNF-α and IL-10 but not IL-12.
Conclusions Our results showed different effects of immunostimulation on cytotoxicity of TAMs from tumors with distinct MHC-I expression. While TAMs from TC-1/A9 tumors acquired M1 phenotype and became cytotoxic, TAMs from TC-1/dB2m tumors were more resistant to repolarization. This project was supported by grants GA19–00816S provided by the Czech Science Foundation and LQ1604 provided by the Ministry of Education, Youth and Sports of the Czech Republic.
Jablonski KA, Amici SA, Webb LM, Ruiz-Rosado JdD, Popovich PG, Partida-Sanchez S, Arellano M. Novel Markers to Delineate Murine M1 and M2 Macrophages. PLoS ONE 2015; 10(12); 1–25.
Disclosure Information A. Piataková: None. I. Poláková: None. M. Šmahel: None.
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