Treatment of breast cancer stem cells with oncolytic herpes simplex virus

Abstract

Cancer stem cells have recently been isolated from several different solid tumors. In breast cancer, the CD44(+)CD24(-/low) population is considered to comprise stem-like cells. The identification of cancer stem cells has provided new targets for the development of therapeutics. Oncolytic herpes simplex viruses (oHSVs) are an effective strategy for killing breast cancer cells and treating breast tumors in preclinical models. Here, we examined the efficacy of the oHSV G47Δ in killing breast cancer stem cells. Human breast cancer cell line SK-BR-3 and human primary breast cancer cells were cultured in suspension under conditions conducive to the growth of stem cells. They generated mammospheres, which had cancer stem cell properties. The proportion of CD44(+)CD24(-/low) cells in these mammospheres exceeded 95%, as determined by flow cytometry. The mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. G47Δ contains the LacZ gene, and X-gal staining of infected cells in vitro and in vivo showed the replication and spread of the virus. G47Δ was found to be highly cytotoxic to the CD44(+)CD24(-/low) population in vitro, even when injected at low multiplicities of infection, and G47Δ treatment in vivo significantly inhibited tumor growth compared with mock treatment. This study demonstrates that oHSV is effective against breast cancer stem cells and could be a beneficial strategy for treating breast cancer patients.

Figure 1

Identification of human primary breast cancer cells. Flow cytometric analysis for endothelial marker CD31 (a) and hematopoietic marker CD45 (b) on human breast cancer cells. The percentage of CD31- or CD45-positive cells is indicated. Immunofluorescent staining of vimentin, fibronectin and cytokeratin in primary cancer cells (c) and foreskin-derived fibroblasts (d). Fibroblasts served as a positive control for vimentin and fibronectin. We found that foreskin-derived fibroblasts strongly expressed fibroblastic markers vimentin and fibronectin, but these cells were negative for cytokeratin. In contrast, human primary breast cancer cells strongly expressed cytokeratin, whereas these cells were negative for vimentin or fibronectin. Scale bar, 50 μm.

Figure 2

Mammosphere cells were analyzed by flow cytometry. (a) The proportion of CD44⁺CD24^−/low cells among the SK-BR-3 mammosphere cells in suspension culture was 99.6±0.07%. (b) Microscopic image of mammosphere in suspension culture. (c) The proportion of CD44⁺CD24^−/low cells among the SK-BR-3 mammosphere cells in adherent culture was 96.7±1.7%. (d) Microscopic image of mammosphere in adherent culture.

Figure 3

Stem cell features of mammosphere cells. Immunofluorescent staining of Oct4 (a) and Sox2 (b) in SK-BR-3 mammosphere cells. Oct4 and Sox2 mRNA was detected by RT-PCR (c). Lanes 1–4 show mammospheres of primary breast cancer cells from different tumors, lane 5 shows non-mammosphere cells, lane 6 show mammosphere cells of SK-BR-3, M is the molecular size ladder. β-Actin served as a control for total RNA.

Figure 4

ALDEFLUOR-positive cell population of SK-BR-3 mammosphere cells and parental cells. The percentage of ALDEFLUOR-positive SK-BR-3 mammosphere cells was 37.6±1.5%, whereas the percentage of ALDEFLUOR-positive SK-BR-3 parental cells was 2.9±0.5% (P<0.001, independent samples t-test).

Figure 5

In vitro killing of mammosphere cells and parental cells of SK-BR-3 by G47Δ. Adherent cultured mammosphere cells and parental cells of SK-BR-3 in six-well dishes were infected with G47Δ at an MOI of 0.01 or 0.1 or with PBS (mock), and the average number of cells from duplicate wells is plotted as the percent in the mock wells.

Figure 6

X-gal staining of human primary breast cancer mammospheres infected with G47Δ. Adherent human primary breast cancer mammospheres cultures in six-well dishes were infected with G47Δ at an MOI of 0.01 or 0.1 or with PBS (mock), and the cells were stained with X-gal to identify infected cells (blue).

Figure 7

Treatment of subcutaneous SK-BR-3 mammosphere-derived tumors. (a) Example of tumors from G47Δ- (lower) or mock-treated (upper) mice at 35 days after treatment. (b) The virus spread in the tumor after intratumoral inoculation. The tumor (1.2 cm³) was injected with G47Δ (1 × 10⁷ p.f.u.), removed 13 days later, sectioned and stained with X-gal to identify replicating G47Δ (blue). (c) SK-BR-3 mammosphere cells were implanted in the left flank of female nude BALB/c mice. when the maximal tumor diameter reached 0.5 cm (29 days after implantation), the mice were injected intratumorally with G47Δ (1 × 10⁷ p.f.u. per 50 μl; n=9) or PBS (mock; n=8) on days 0, 3, 7 and 10 (arrows). Tumor size was measured using an external caliper twice a week, and the tumor volume was calculated. Each data point is the average tumor volume in each group, and error bars represent standard deviation. Tumors treated with G47Δ were significantly smaller than those treated with PBS from 14 days after treatment onward (P<0.05, independent samples t-test).

Figure 8

Single-cell suspensions from mock- and oHSV-treated tumors analyzed by flow cytometry. (a) The proportion of CD44⁺CD24^−/low cells in tumors from mock-treated mice (n=3) was 3.9±0.79%. (b) The proportion of CD44⁺CD24^−/low cells in tumors from oHSV-treated mice (n=2) was 4.5±0.3% (P>0.05, independent samples t-test).