Breast cancer is the most common cancer in women worldwide and metastatic dissemination is the principal factor related to death by this disease. Breast cancer stem cells (bCSC) are thought to be responsible for metastasis and chemoresistance. In this study, based on whole transcriptome analysis from putative bCSC and reverse engineering of transcription control networks, we identified two networks associated with this phenotype. One controlled by SNAI2, TWIST1, BNC2, PRRX1 and TBX5 drives a mesenchymal or CSC-like phenotype. The second network is controlled by the SCML4, ZNF831, SP140 and IKZF3 transcription factors which correspond to immune response modulators. Immune response network expression is correlated with pathological response to chemotherapy, and in the Basal subtype is related to better recurrence-free survival. In patient-derived xenografts, the expression of these networks in patient tumours is predictive of engraftment success. Our findings point out a potential molecular mechanism underlying the balance between immune surveillance and EMT activation in breast cancer. This molecular mechanism may be useful to the development of new target therapies.

The presence of tumor-initiating cells (CD44(+)/CD24(-)) in solid tumors has been reported as a possible cause of cancer metastasis and treatment failure. Nevertheless, little is know about the presence of CD44(+)/CD24(-) cells within the primary tumor and metastasis. The proportion of CD44(+)/CD24(-) cells was analyzed in 40 samples and in 10 lymph node metastases using flow cytometry phenotyping. Anti-human CD326 (EpCam; FITC), anti-human CD227 (MUC-1; FITC), anti-human CD44 (APC), and anti-human CD24 (PE), anti-ABCG2 (PE), and anti-CXCR4 (PeCy7) were used for phenotype analysis. The mean patient age was 60.5 years (range, 33-87 years); mean primary tumor size (pT) was 1.8 cm (0.5-3.5 cm). The Wilcoxon or Kruskal-Wallis test was used for univariate analyses. Logistic regression was used for multivariate analysis. The median percentage of CD44(+)/CD24(-) cells within primary invasive ductal carcinomas (IDC) was 2.7% (range, 0.2-71.2). In lymph node metastases, we observed a mean of 6.1% (range, 0.07-53.7). The percentage of CD44(+)/CD24(-) cells in IDCs was not associated with age, pT, tumor grade and HER2. We observed a significantly enrichment of CD44(+)/CD24(-) and ABCG2(+) cells in ESA(+) cell population in patients with positive lymph nodes (P = 0.02 and P = 0.04, respectively). Our data suggest that metastatic dissemination is associated with an increase in tumor-initiating cells in stage I and II breast cancer.

The existence of tumor-initiating cells (TICs) within solid tumors has been hypothesized to explain tumor heterogeneity and resistance to cancer therapy. In breast cancer, the expression of CD44 and CD24 and the activity of aldehyde dehydrogenase 1 (ALDH1) can be used to selectively isolate a cell population enriched in TICs. However, the ideal marker to identify TICs has not been established. The aim of this study was to evaluate the expression of novel potential markers for TIC in breast carcinoma. We prospectively analyzed the expression of CD44, CD24, ABCG2, and CXCR4, and the activity of ALDH1 by using flow cytometry in 48 invasive ductal carcinomas from locally advanced and metastatic breast cancer patients who were administered primary chemotherapy. A mammosphere assay was employed in 30 samples. The relationship among flow cytometric analyses, ABCG2 gene expression, and clinical and pathological responses to therapy was analyzed. The GSE32646 database was analyzed in silico to identify genes associated with tumors with low and high ABCG2 expression. We observed that the presence of ABCG2(+) cells within the primary tumor was the only marker to predict the formation of mammospheres in vitro (R (2) = 0.15, p = 0.029). Quantitative polymerase chain reaction (qPCR) revealed a positive correlation between ABCG2 expression and the presence of ABCG2(+) cells within the primary tumor. The expression of ABCG2 was predictive of the response to neoadjuvant chemotherapy in our experiments and in the GSE32646 dataset (p = 0.04 and p = 0.002, respectively). The in silico analysis demonstrated that ABCG2(Up) breast cancer samples have a slower cell cycle and a higher expression of membrane proteins but a greater potential for chromosomal instability, metastasis, immune evasion, and resistance to hypoxia. Such genetic characteristics are compatible with highly aggressive and resistant tumors. Our results support the hypothesis that the presence of ABCG2(+) cells in breast carcinomas is a marker of resistance to chemotherapy, and based on in vitro assays and the genetic profile, we show, for the first time, that ABCG2 protein can be used as an independent marker for TIC identification in breast cancer.

The therapy of colon and rectum tumors based on microRNAs remains under investigation reserving huge potential for future applications and clinical interventions in conjunction with existing therapies. We expect, based on the exposed data, to stimulate the development of new therapeutic possibilities, making the treatment of these tumors more effective.

e12007 Background: The identification of cancer stem cell (CSC) markers gives opportunity to develop new target therapies. ABCG2 protein is reported as a predictive marker for breast cancer resistance to chemotherapy and as a potential marker for CSC in solid tumors. Methods: We included prospectively 41 patients with locally advanced or metastatic (LAMBC) invasive ductal carcinomas treated with NACT. The expression of CD44/CD24 and ABCG2 was analyzed by flow cytometry after fresh tumor sample digestion. The mammosphere assay (Mammocult) was carried out in 23 samples. We analyzed the correlation between the percentage of ESA+/ABCG2+ and ESA+/CD44+/CD24- cells within the primary tumor with the number of spheres in culture and their relationship with clinical and pathological parameters. ABCG2+ and ABCG2- cells from four primary tumors and from MDA-MB231 cell line were sorted and cultured in triplicate in Mammocult medium for 7 days. The ability to form sphere was analyzed. Patients’ mean age was 52.8 ± 10.3 yo. The ER, PgR and HER2 positive expression rates were 65%, 58% and 45%, respectively. Patients were treated with docetaxel/anthracycline combination (n = 33) of FEC75 combination (n = 8). Results: Complete pathological response was observed in 10 patients (24%). ER negative patients and patients with lower percentage of ABCG2+ cells within the tumor were more likely to achieve near pCR (44% versus 11%, p = 0.04; and 50% versus 14%, p = 0.04, respectively). We did not observe a significant association between pCR and PgR and HER2 expression or the percentage of CD44+/CD24- cells within the tumor. There was a strong correlation between the percentage of ESA+/ABCG2+ cells and the number of mammospheres in culture (r = 0.63; p= 0.0007). This correlation was not significant comparing to CD44+/CD24- cells. Additionally, ABCG2+ cells from primary tumors and MB-231 cell line form a higher number of spheres in culture than ABCG2- (mean difference = 1.15±0.45 /1000 cells, p = 0.04; mean difference = 0.48±0.04 /1000cells, p = 0.008, respectively). Conclusions: ABCG2 is a potential marker for CSC in breast cancer, and the percentage of ABCG2+ cancer cells within the tumor is a predictive factor for pCR in LAMBC patients subjected to NACT.