Triple-negative breast cancer (TNBC) is characterized by a lack of receptor expression of estrogen receptor (ER) and progesterone receptor (PgR) and lack of gene expression for human epidermal growth factor receptor 2 (HER2) [1, 2]. Chemotherapy remains the only possible therapeutic option in the adjuvant or metastatic setting in the TNBC. Importantly, the prognostic effect of TNBC is independent of poor grade, nodal status, tumor size and treatment . The aggressiveness of TNBC is further indicated by the fact that: (i) the peak risk of recurrence occurs within the first 3 years after initial treatment of the disease with the majority of deaths occurring in the first 5 years  and (ii) after diagnosis of metastatic disease, a significantly shorter survival is observed in TNBC . Conversely, the risk for late recurrences (i.e. beyond 5 years of diagnosis) is decreased by 50% compared with HER2-positive disease . More than 90% of TNBC exhibit an invasive ductal histology and high histological grade, present with high mitotic index and carry central necrotic zones and pushing borders as well as a conspicuous lymphocytic infiltrate.
TNBC are typically observed in young African-American women, Hispanic women and Caucasian women who carry a mutation in the breast cancer 1 (BRCA1) early onset gene . While some TNBC respond to chemotherapy, subsets of TNBC are chemotherapy-resistant and highly metastatic carrying with it an extremely poor prognosis . The molecular mechanism, which governs the aggressive behavior of this subset of TNBC, is a matter of intense speculation, particularly since TNBC frequently express markers of epithelial mesenchymal transition (EMT). EMT is a normal developmental process in which cells of epithelial origin lose epithelial characteristics and polarity and acquire a mesenchymal phenotype associated with increased migratory behavior [7, 8].
Self-renewing cancer cells are reported to be the only cell types within a tumor with an unlimited ability to initiate tumor growth and are therefore known as tumor-initiating cells (TICs), cancer stem cells or tumor-propagating cells [9–11]. The majority of carcinoma cells express a CD44low/CD24high phenotype; however, a small subpopulation of carcinoma cells present within human breast cancers that exhibit a CD44high/CD24low antigenic phenotype are highly enriched for TICs . TNBC have been reported to have a higher percentage of CD44high/CD24low expressing cells than other breast cancer subtypes .
The mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) belongs to the family of small HSP and is the murine homologue of human twenty-seven kDa heat shock protein (Hsp27/human HspB1) which operates through ATP-independent mechanisms [14, 15]. Elevated human HspB1 levels have been found in various tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumors arising from the nervous system and urinary system. Elevated levels of human HspB1 in ER-α positive benign neoplasia have been shown to promote the progression to more malignant phenotypes , increased anchorage independent tumor growth , increased resistance to chemotherapeutic drugs (including cisplatin and doxorubicin) and increased metastatic potential in vitro [18–20]. Furthermore, elevated human HspB1 expression in tumors correlated with shorter disease-free survival and recurrence in node-negative breast cancer [21, 22], whereas its induction following chemotherapy also predicted poor prognosis and shorter disease-free survival .
Surface bound/expressed and total Hsp25 (mouse HspB1) and Hsp72 (HspA1A) on 4T1 murine breast adenocarcinoma tumors suggest that tumor development and metastatic spread favors cells that express high levels of mouse HspB1 on their plasma surface. However, tumors that express Hsp72/HspA1A on their plasma surface are sensitive to anti-tumor effector cells, which indicate increased surface expression of Hsp72/HspA1A on tumors and down regulation of human HspB1 expression could inhibit tumor growth and eliminate metastasis [24, 25]. Enhanced expression of intracellular Hsp72/HspA1A has shown to be anti-inflammatory , anti-apoptotic , induce cell cycle arrest  and protect cells from stressful stimuli . In contrast, enhanced extracellular expression of Hsp72/HspA1A either on the surface of tumors or in the extracellular milieu either enhances natural killer (NK) cell-mediated lysis  or upregulates antigen presenting cells (APC)-mediated acute phase responses [30–33], respectively. A study in which the mouse hspb1 gene has been silenced using interference ribonucleic acid (RNAi) technology suggests that mouse HspB1 has a profound effect on tumor proliferation and migration [24, 25].
In this study, we constructed TNBC and TPBC by the stable transfection of parental 4T1 cells with the rat HER2, ER and PgR genes and sorted for cells with high expression of HER2, ER and PgR. We further produced TNBC-TICs and TPBC-TICs by sorting CD24+/CD44high/aldehyde dehydrogenase 1 (ALDH1)+ expressing cells from TNBC and TPBC. Functional analysis demonstrated that TNBC-TICs exhibits a more aggressive phenotype than TNBC, TPBC, TPBC-TICs or parental 4T1 cells. Taken together, our studies suggest that these cells could be important preclinical model to characterize and effectively target cancer stem cells or TICs, and to further our understanding of the aggressive nature of TNBC found in humans.