Canine Mammary Cancer Stem Cells are Radio- and Chemo- Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype

Abstract

Canine mammary carcinoma is the most common cancer among female dogs and is often fatal due to the development of distant metastases. In humans, solid tumors are made up of heterogeneous cell populations, which perform different roles in the tumor economy. A small subset of tumor cells can hold or acquire stem cell characteristics, enabling them to drive tumor growth, recurrence and metastasis. In veterinary medicine, the molecular drivers of canine mammary carcinoma are as yet undefined. Here we report that putative cancer stem cells (CSCs) can be isolated form a canine mammary carcinoma cell line, REM134. We show that these cells have an increased ability to form tumorspheres, a characteristic of stem cells, and that they express embryonic stem cell markers associated with pluripotency. Moreover, canine CSCs are relatively resistant to the cytotoxic effects of common chemotherapeutic drugs and ionizing radiation, indicating that failure of clinical therapy to eradicate canine mammary cancer may be due to the survival of CSCs. The epithelial to mesenchymal transition (EMT) has been associated with cancer invasion, metastasis, and the acquisition of stem cell characteristics. Our results show that canine CSCs predominantly express mesenchymal markers and are more invasive than parental cells, indicating that these cells have a mesenchymal phenotype. Furthermore, we show that canine mammary cancer cells can be induced to undergo EMT by TGFβ and that these cells have an increased ability to form tumorspheres. Our findings indicate that EMT induction can enrich for cells with CSC properties, and provide further insight into canine CSC biology.

Figure 1.

Isolation and characterization of putative cancer stem cells. Tumorsphere formation from the REM134 canine mammary carcinoma cell line. Single cells (A) and sphere (B). Non-quantitative RT-PCR analysis of mRNA expression of the embryonic stem cell markers Oct4 and Nanog (C).

Figure 2.

Tumorspheres exhibit increased resistance to conventional chemo- and radiation therapies. Adherent cells and tumorspheres were treated with increasing concentrations of doxorubicin and cell viability was assayed 72 hours post-treatment (★ p = 0.008; ★ ★ p = 0.038; #p < 0.001) (A). Radiation sensitivity was determined by assaying for cell viability 72 hours post-treatment (★ p = 0.003; ★ ★ p = 0.026; ★ ★ ★ p = 0.002) (B) and by determining colony forming ability (★ p = 0.01; ★ ★ p = 0.009; ★ ★ ★ p < 0.001) (C).

Figure 3.

Putative cancer stem cells exhibit mesenchymal characteristics. Tumorspheres derived from the REM134 canine mammary carcinoma cell line express mesenchymal markers (A). Representative images of invading cells, stained purple, embedded within the membrane of a boyden chamber (C) and quantified by colorimetric measurement at 560 nm (★ p < 0.008) (B).

Figure 4.

Treatment of canine mammary carcinoma cells with TGFβ can induce an epithelial to mesenchymal transition, as indicated by changes in cell morphology (A), protein expression levels (B), and increased migration ability (★ p = 0.018; #p = 0.014; ★ ★ p = 0.004; ##p = 0.002; up = 0.001) (C, D).

Figure 5.

TGFβ treated cells show an increased tumorsphere forming ability compared to untreated cells. Tumorspheres resulted less frequently from untreated cells (A), compared to cells treated with 10 ng/ml TGFβ (B). The resultant number of spherical colonies were counted (★ p < 0.001; ★ ★ p < 0.001; ★ ★ ★ p < 0.01) (C).