Mica Nanoparticle, STB-HO Eliminates the Human Breast Carcinoma Cells by Regulating the Interaction of Tumor with its Immune Microenvironment

Abstract

Mica, an aluminosilicate mineral, has been proven to possess anti-tumor and immunostimulatory effects. However, its efficacy and mechanisms in treating various types of tumor are less verified and the mechanistic link between anti-tumor and immunostimulatory effects has not been elucidated. We sought to investigate the therapeutic effect of STB-HO (mica nanoparticles) against one of the most prevalent cancers, the breast cancer. STB-HO was orally administered into MCF-7 xenograft model or directly added to culture media and tumor growth was monitored. STB-HO administration exhibited significant suppressive effects on the growth of MCF-7 cells in vivo, whereas STB-HO did not affect the proliferation and apoptosis of MCF-7 cells in vitro. To address this discrepancy between in vivo and in vitro results, we investigated the effects of STB-HO treatment on the interaction of MCF-7 cells with macrophages, dendritic cells (DCs) and natural killer (NK) cells, which constitute the cellular composition of tumor microenvironment. Importantly, STB-HO not only increased the susceptibility of MCF-7 cells to immune cells, but also stimulated the immunocytes to eliminate cancer cells. In conclusion, our study highlights the possible role of STB-HO in the suppression of MCF-7 cell growth via the regulation of interactions between tumor cells and anti-tumor immune cells.

Figure 1. Suppressive effect of STB-HO on MCF-7 growth in athymic nude mice.

(a–c) From 1 week of stabilization period after MCF-7 xenotransplantation, STB-HO was orally administrated daily in two different dose (35 mg/kg or 70 mg/kg) for 12 weeks and tumor size was measured twice a week. (a) Photographs of tumors in mice. (b) Tumor volume measurement in a time course. (c) Tumor volume at 12 weeks. (d–f) Tumors from xenograft mouse model were isolated and measured. (d) Photographs of isolated tumors. (e) Volume and (f) Mass of dissected tumors. Seven to eight mice per group were used. *P < .05, **P < .01. Results are shown as mean ± SD.

Figure 2. Regulation of crucial signaling in MCF-7 tumor formation by STB-HO.

Dissected tumors were processed for further histological analysis and specific protein detection. (a) Isolated tumors were stained with hematoxylin and eosin. (b,c) Tumors were lysed and determined for the expression of crucial proteins in tumor growth. (b) ERα and COX-2 expression in tumor samples were detected by western blotting and quantified. (c) Expressions of four EP receptors were determined and quantified. Results are shown as mean ± SD.

Figure 3. Direct effects of STB-HO treatment on MCF-7 cell proliferation and apoptosis.

(a) MCF-7 cells were treated with two different concentrations of STB-HO for 3 days and photographs were taken. (b–d) MCF-7 proliferation in response to STH-HO was determined with various methods (b) A trypan blue exclusion test was performed. (c) Concentration of total protein from lysed cell was measured. (d) BrdU incorporation assay was performed. (e) Apoptotic rate in MCF-7 cells was measured by staining Annexin V after STB-HO treatment. (f) Apoptosis-related proteins were detected by western blot analysis. Results are one representative experiment of three independent experiments. Results are shown as mean ± SD. Cas-3; caspase-3, PUMA; p53 upregulated modulator of apoptosis, BAK; Bcl-2 homologous antagonist/killer.

Figure 4. Regulation of immune evasive components in MCF-7 cells by STB-HO treatment.

(a) The expression of HLA class I on the surface of MCF-7 cells was detected by flow cytometry after STB-HO treatment. (b) The secretion of soluble factors related with immune evasion was measured using commercial kits. Production of PGE₂, IL-10, IL-6 and IL-8 from MCF-7 cells was detected from culture supernatant. *P < .05, **P < .01. Results are one representative experiment of three independent experiments. Results are shown as mean ± SD.

Figure 5. Polarization of macrophages by STB-HO treatment.

(a) A time line for the differentiation of THP-1 into macrophage-like cells. (b) Photographs of THP-1-derived macrophages after STB-HO treatment. STB-HO engulfed by macrophages is indicated by red arrow. (c) TNF-α, IL-1β and IL-12 secretion was measured in culture media of THP-1-derived macrophages. (d) Production of IL-12 from MNC-derived macrophages was detected. Treatment of LPS with IFN-γ was used to induce the polarization of macrophages. *P < .05, **P < .01, ***P < .001. Results are one representative experiment of three independent experiments. Results are shown as mean ± SD.

Figure 6. Activation of DCs by STB-HO treatment.

(a) Outline for DC induction from human MNCs. (b) The expression of CD83, DC activation marker, was detected by flow cytometry after STB-HO treatment. (c) IL-12 concentration in culture media of DCs was measured. Treatment of LPS with IFN-γ was used to induce the maturation of DCs. **P < .01, ***P < .001. Results are one representative experiment of three independent experiments. Results are shown as mean ± SD.

Figure 7. Enhancement of NK cell cytotoxicity upon STB-HO treatment.

(a) Isolated NK cells were determined for the purity through the detection of CD56 and CD3 on cell surface using flow cytometry. CD3^- CD56⁺ cells represent NK cells. (b) IFN-γ  secretion from isolated NK cells was measured. (c,d) CFDA-labeled MCF-7 cells were co-cultured with NK cells in the presence of STB-HO for 4 hours. (c) Co-cultured cells were stained with PI and PI-expressing dead cells were detected by flow cytometry. (d) Culture media used in co-culture was determined for the production of IFN-γ. IL-12 treatment was used to induce the activation of NK cells. **P < .01, ***P < .001. Results are one representative experiment of three independent experiments. Results are shown as mean ± SD.

Figure 8. Increase in the number of NK cells residing in mouse spleen by STB-HO administration.

(a–d) The alteration in the number of NK cells in mice after oral STB-HO administration was determined. The cells from spleen and lymph nodes of (a,b) athymic nude mice or (c,d) BALB/c mice were isolated and NKp46⁺ cells in isolated cells were detected by flow cytometry. Five to seven mice per group were used. *P < 0.05.