Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment

Abstract

Many types of tumors are organized in a hierarchy of heterogeneous cell populations. The cancer stem-like cells (CSCs) hypothesis suggests that tumor development and metastasis are driven by a minority population of cells, which are responsible for tumor initiation, growth and recurrences. The inability to efficiently eliminate CSCs during chemotherapy, together with CSCs being highly tumorigenic and invasive, may result in treatment failure due to cancer relapse and metastases. CSCs are emerging as a promising target for the development of translational cancer therapies. Ideal panacea for cancer would kill all malignant cells, including CSCs and bulk tumor cells. Since both chemotherapy and CSCs-specific therapy are insufficient to cure cancer, we propose combination therapy with CSCs-targeted agents and chemotherapeutics for improved breast cancer treatment. We generated in vitro mammosphere of 2 breast cancer cell lines, and demonstrated ability of mammospheres to grow and enrich cancer cells with stem-like properties, including self-renewal, multilineage differentiation and enrichment of cells expressing breast cancer stem-like cell biomarkers CD44(+)/CD24(-/low). The formation of mammospheres was significantly inhibited by salinomycin, validating its pharmacological role against the cancer stem-like cells. In contrast, paclitaxel showed a minimal effect on the proliferation and growth of breast cancer stem-like cells. While combination therapies of salinomycin with conventional chemotherapy (paclitaxel or lipodox) showed a potential to improve tumor cell killing, different subtypes of breast cancer cells showed different patterns in response to the combination therapies. While optimization of combination therapy is warranted, the design of combination therapy should consider phenotypic attributes of breast cancer types.

Keywords: Breast cancer; cancer stem-like cell; combination therapy; mammosphere; salinomycin.

Figure 1.

The self-renewal (A-D) and the differential (E-G) potential of stem-like cells enriched in vitro as cancer mammospheres of 2 breast cancer cell lines. (A) MCF-7 and (B) MDA-MB-231 grown under undifferentiated conditions in an anchorage-independent manner to form floating 3D mammospheres of generation 1 (G1). In vitro serial passages of mammosphere of MCF-7 (C) and MDA-MB-231 (D) up to generation 4 (G4), indicating cells with stem cell-like properties and self-renewal potential. The differential potential of breast cancer stem cell-like cells from mammospheres tested on a 3 dimensional clonal cultivation system containing Matrigel under differentiated conditions. MCF-7 showing branched, ductal-acinar structure (E) and acinar structures (F); MDA-MB-231 showing mixed lineage colonies with existence of clear branched, ductal-acinar structure (G). Photographs were taken at 10x magnification.

Figure 2.

Expression of biomarker CD44 and CD24 of a MCF-7 adherent monolayer culture and different generations of mammospheres, showing that breast cancer stem-like cells with CD44⁺/CD24^−/1ow were enriched by mammosphere passage to the third generation.

Figure 3.

Examination of breast cancer stem-like marker CD44 and CD24 of MDA-MB-231, showing high CD44 and low CD24 expression in both, monolayers and mammospheres of different generations. Red: Unstained cells; Green: Cell with CD44 or CD 24 isotype Antibody stained; Blue: Cells with CD44- or CD 24 Antibody staining.

Figure 4.

Effect of drug treatment on the formation of MCF-7 mammospheres. Salinomycin treatment inhibited the formation of mammospheres, while paclitaxel treatment generated mammospheres, which were smaller compared to the untreated group.

Figure 5.

Viability of breast cancer cells after exposure to monotherapy or a combination therapy of lipodox with salinomycin. Combination therapy of salinomycin with lipodox produced synergistic effects on the viability of (A) MDA-MB-231 cells and (B) MCF-7 cells. Mean ± SD, n = 5. * P < 0.05.

Figure 6.

Viability of breast cancer cells exposed to monotherapy or with a combination therapy of paclitaxel with salinomycin. (A) MDA-MB-231; (B) MCF-7 cells. Mean ± SD, n = 5. * P < 0.05.