Effect of Interferon α-2b on Multicellular Tumor Spheroids of MCF-7 Cell Line Enriched with Cancer Stem Cells

Abstract

Background. The main problem of cancer treatment is that the disease prediction depends on subpopulations of the primary tumor and the ability of the tumor to recover and form metastases. Cancer stem cells (CSCs) provide self-renewal of the tumor after irradiation and chemotherapy. One way to isolate these cells is to form multicellular tumor spheroids. The method of enrichment of such spheroids by CSCs can be used to test complex antitumor therapy aimed at the CSCs population in vitro. Some studies have shown a close relationship between CSCs and interferon in tumor proliferation and progression. Therefore, it is important to assess the sensitivity of the CSCs population to the action of interferon on a model of multicellular tumor spheroids.

Objective. The aim of the article is to study the sensitivity of MCF-7 cell populations to the effect of IFNα-2b on a model of multicellular tumor spheroids enriched with CSCs.

Methods. To enrich multicellular spheroids with CSCs, adherent cells of monolayer culture MCF-7 (human breast adenocarcinoma) were trypsinized and cultured on an orbital shaker under serum-free conditions with the addition of growth factors (fibroblast growth factor, epidermal growth factor, insulin, and hydrocortisone). In order to study the direct effect of IFNα-2b on cell populations, this cytokine was added to plates with cell aggregates at a concentration of 10² U/mL and 10⁴ U/mL and incubated for 48 h. Counting the number of cells was performed using the routine method with a trypan blue and hemocytometer. Morphometric analysis was performed by measuring the volumes of spheroids using the software Stemi2000 (Zeiss, Germany) and the Bjerkvig formula. Receptor expression was analyzed after two days of incubation using primary monoclonal antibodies CD24 (Sigma, USA), CD44 (Sigma, USA), CD133 (Sigma, USA), bmi-1 (Sigma, USA). For statistical data processing, one-factor analysis of the variance and t-Student testing with the Statistica 8 software package were used. The differences were considered significant at p < 0.05. For each result obtained, we determined the indices of the arithmetic mean (M) and the standard deviation of the arithmetic mean (m).

Results. The presence of CSCs expressing the receptors characteristic of mammary gland (CD24, CD44, CD133, bmi-1) in the culture of multicellular tumor spheroids, which were cultured under serum-free conditions with the addition of growth factors, was shown using immunohistochemistry method. Since cells are obtained from monoculture of tumor cells, it can be assumed that CSCs are detected by appropriate markers, but not stromal cells, for which expression of these receptors is also possible. The data on the effect of IFNα-2b on the heterogeneous population of tumor and stem-like cells were obtained. It turned out that this cytokine effects the reduction of the average volume of cell aggregates by 16% and 25% at a concentration of 10² and 10⁴ U/mL, respectively. It also suppresses cell proliferation by 13% at a concentration of IFNα-2b 10⁴ U/mL, compared with control samples. Further incubation of spheroids with IFNα-2b leads to a decrease in the average and median volumes of spheroids by 48% and 74% at a cytokine concentration of 10⁴ U/mL and 10⁴ U/mL, respectively, compared to control samples. IFNα-2b dose-dependently contributes to the disintegration of multicellular tumor spheroids enriched with CSCs and to the formation of a large number of small cell aggregates and individual cells of the MCF-7 cell line.

Conclusions. IFNα-2b can be considered as an auxiliary agent for the development and testing of complex antitumor therapy aimed at activating СSCs with simultaneous effects of chemotherapy on models of multicellular tumor spheroids of the MCF-7 line, enriched with this cells in vitro