Mikhail Varlamov

Hypoxia arises in most growing solid tumors and can lead to pleotropic effects that potentially increase tumor aggressiveness and resistance to therapy through regulation of the expression of genes associated with the epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET). The main goal of the current work was to obtain and investigate the intermediate phenotype of tumor cells undergoing the hypoxia-dependent transition from fibroblast to epithelial morphology. Primary breast cancer fibroblasts BrC4f, being cancer-associated fibroblasts, were subjected to one or two rounds of “pulsed hypoxia” (PH). PH induced transformation of fibroblast-shaped cells to semi-epithelial cells. Western blot analysis, fluorescent microscopy and flow cytometry of transformed cells demonstrated the decrease in the mesenchymal markers vimentin and N-cad and an increase in the epithelial marker E-cad. These cells kept mesenchymal markers αSMA and S100A4 and high ALDH activity. Real-time PCR data of the cells after one (BrC4f_Hyp1) and two (BrC4f_Hyp2) rounds of PH showed consistent up-regulation of TWIST1 gene as an early response and ZEB1/2 and SLUG transcriptional activity as a subsequent response. Reversion of BrC4f_Hyp2 cells to normoxia conditions converted them to epithelial-like cells (BrC4e) with decreased expression of EMT genes and up-regulation of MET-related OVOL2 and c-MYC genes. Transplantation of BrC4f and BrC4f_Hyp2 cells into SCID mice showed the acceleration of tumor growth up to 61.6% for BrC4f_Hyp2 cells. To summarize, rounds of PH imitate the MET process of tumorigenesis in which cancer-associated fibroblasts pass through intermediate stages and become more aggressive epithelial-like tumor cells.

The ability of dying cancer cells to induce an anticancer immune response can increase the effectiveness of anticancer therapies, and such type of death is termed immunogenic cell death (ICD). Cells can die along the ICD pathway when exposed not only to chemo- and immunotherapeutics, but also to various types of radiation, such as ionizing radiation and cold atmospheric plasma jets (CAP). We have previously shown that CAP, lactaptin, and a recombinant vaccinia virus encoding lactaptin induce in vitro molecular changes typical of ICD in cancer cells. In the current work, we treated MX-7 rhabdomyosarcoma cells with CAP and lactaptin-based anticancer drugs and evaluated the immunological effects of the treated cells. We showed that dendritic cells (DCs) captured cells treated with various ICD inducers with different efficiency. CAP-treated cells were weakly potent in inducing the maturation of DCs according to MHC II externalization. Moreover, CAP-treated cells were worse in the stimulation of IFN-α release in vitro and were poorly captured by spleen DCs in vivo. Under the irradiation conditions used, CAP was not capable of activating a significant immunological anti-tumor effect in vivo. It is possible that modifications of the CAP irradiation regimen will enhance the activation of the immune system.

Multicellular spheroids with 3D cell–cell interactions are a useful model to simulate the growth conditions of cancer. There is evidence that in tumor spheroids, the expression of various essential molecules is changed compared to the adherent form of cell cultures. These changes include growth factor receptors and ABC transporters and result in the enhanced invasiveness of the cells and drug resistance. It is known that breast adenocarcinoma MCF7 cells can spontaneously form 3D spheroids and such spheroids are characterized by high expression of EGFR/HER2, while the natural phenotype of MCF7 cells is EGFR^low/HER2^low. Therefore, it was interesting to reveal if high epidermal growth factor receptor (EGFR) expression is sufficient for the conversion of adherent MCF7 to spheroids. In this study, an MCF7 cell line with high expression of EGFR was engineered using the retroviral transduction method. These MCF7-EGFR cells assembled in spheroids very quickly and grew predominantly as a 3D suspension culture with no special plates, scaffolds, growth supplements, or exogenous matrixes. These spheroids were characterized by a rounded shape with a well-defined external border and 100 µM median diameter. The sphere-forming ability of MCF7-EGFR cells was up to 5 times stronger than in MCF7^wt cells. Thus, high EGFR expression was the initiation factor of conversion of adherent MCF7^wt cells to spheroids. MCF7-EGFR spheroids were enriched by the cells with a cancer stem cell (CSC) phenotype CD24^−/low/CD44⁻ in comparison with parental MCF7^wt cells and MCF7-EGFR adhesive cells. We suppose that these properties of MCF7-EGFR spheroids originate from the typical features of parental MCF7 cells. We showed the decreasing of HER3 receptors in MCF7-EGFR spheroids compared to that in MCF^wt and in adherent MCF7-EGFR cells, and the same decrease was observed in the MCF7^wt spheroids growing under the growth factors stimulation. To summarize, the expression of EGFR transgene in MCF7 cells stimulates rapid spheroids formation; these spheroids are enriched by CSC-like CD24⁻/CD44⁻ cells, they partly lose HER3 receptors, and are characterized by a lower potency in drug resistance pomp activation compared to MCF7^wt. These MCF7-EGFR spheroids are a useful cancer model for the development of anticancer drugs, including EGFR-targeted therapeutics.

The application of cold atmospheric plasma (CAP) in cancer therapy could be one of the new anticancer strategies. In the current work, we used cold atmospheric plasma jet for the treatment of cultured cells and mice. We showed that CAP induced the death of MX−7 mouse rhabdomyosarcoma cells with the hallmarks of immunogenic cell death (ICD): calreticulin and heat shock protein 70 (HSP70) externalization and high-mobility group box 1 protein (HMGB1) release. The intensity of HMGB1 release after the CAP treatment correlated directly with the basal extracellular HMGB1 level. Releasing from dying cells, HMGB1 can act as a proinflammatory cytokine. Our in vivo study demonstrated that cold atmospheric plasma induces a short-term two-times increase in serum HMGB1 level only in tumor-bearing mice with no effect in healthy mice. These findings support our hypothesis that CAP-dependent HMGB1 release from dying cancer cells can change the serum HMGB1 level. At the same time, we showed a weak cytokine response to CAP irradiation in healthy mice that can characterize CAP as an immune-safety physical antitumor approach.

Natural compounds of various origins are intensively investigated for their antitumor activity. Potential benefits of antitumor therapy can be achieved when cytotoxic agents kill cancer cells and these dying cancer cells drive adoptive immunity to the tumor. This strategy was successfully demonstrated for chemotherapeutic drugs that induce immunogenic type of cell death (ICD) with release of DAMPs (danger associated molecular patterns) and exposure of “eat me” signals. In this study, we demonstrated that recombinant human milk peptide lactaptin (RL2) induces death of cancer cells with ICD hallmarks in vitro with the release of ATP and high-mobility group box 1 protein (HMGB1) and exposure of calreticulin and HSP70 on the external cell membrane. RL2-treated cancer cells were efficiently engulfed by phagocytic cells. Using the syngeneic mouse model, we demonstrated that RL2-treated MX-7 rhabdomyosarcoma cells confer long-term immune-mediated protection against challenge with live MX-7 cells. We also analyzed the combinatorial antitumor effect of vaccination with RL2-treated cells and the inhibition of indoleamine 2,3-dioxygenase (IDO) with ethyl pyruvate. Compared to solo anti-tumor immunization with RL2-treated cells, additional chemical inhibition of IDO demonstrated better long-term antitumor responses than vaccination alone.