Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells

Abstract

Cancer stem cells are responsible for tumor progression, metastasis, therapy resistance and cancer recurrence, doing their identification and isolation of special relevance. Here we show that low adherent breast and colon cancer cells subpopulations have stem-like properties. Our results demonstrate that trypsin-sensitive (TS) breast and colon cancer cells subpopulations show increased ALDH activity, higher ability to exclude Hoechst 33342, enlarged proportion of cells with a cancer stem-like cell phenotype and are enriched in sphere- and colony-forming cells in vitro. Further studies in MDA-MB-231 breast cancer cells reveal that TS subpopulation expresses higher levels of SLUG, SNAIL, VIMENTIN and N-CADHERIN while show a lack of expression of E-CADHERIN and CLAUDIN, being this profile characteristic of the epithelial-to-mesenchymal transition (EMT). The TS subpopulation shows CXCL10, BMI-1 and OCT4 upregulation, differing also in the expression of several miRNAs involved in EMT and/or cell self-renewal such as miR-34a-5p, miR-34c-5p, miR-21-5p, miR-93-5p and miR-100-5p. Furthermore, in vivo studies in immunocompromised mice demonstrate that MDA-MB-231 TS cells form more and bigger xenograft tumors with shorter latency and have higher metastatic potential. In conclusion, this work presents a new, non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells for subsequent biological and preclinical studies.

Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity

“…In addition, it has been validated in bone marrow and peripheral blood samples from patients with neuroblastoma compared to healthy patients28, and used as normalizer for pancreatic ductal tumour assays29. Although hsa-miR-24c-3p was a valid normalizer for comparing cancer stem-like subpopulations, as we previously published16, however it did not show stability in tumour cells compared to normal cells. Therefore, our results encourage the need to set standards miR normalizers considering normal or cancer cell lines, different cell sub-populations or normal or cancer tissue samples.…”

“…201616. We compared the expression of selected miRs between those two enriched CSC subpopulations, HCT-116 ALDH + human colon cancer cells and MDA-MB-231 trypsin sensitive (TS) human breast cancer cells, with their corresponding ALDH − and trypsin resistant (TR) subpopulations with not stemness properties.…”

“…The analysis of selected miRs in cancer stem-like subpopulations using the hsa-miR-24c-3p normalizer was previously published in Morata-Tarifa C et al . 201616.…”

“…ALDH activity has been extensively used for CSCs identification and isolation15. Recently, we have developed a non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells based on their differential sensibility to trypsin exposure16. Trypsin-sensitive (TS) cancer cell subpopulation shows increased CSC properties when compared with the total population (TP) and/or the trypsin-resistant (TR) subpopulation16.…”

“…Recently, we have developed a non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells based on their differential sensibility to trypsin exposure16. Trypsin-sensitive (TS) cancer cell subpopulation shows increased CSC properties when compared with the total population (TP) and/or the trypsin-resistant (TR) subpopulation16. Aberrant miR expression and the implication of these miRs on the biological complexities of CSCs, makes miR determination a powerful tool with great clinical potential, encouraging further studies with this approach.…”