Enriquecimiento de subpoblaciones de células madre cancerígenas en base a su capacidad de adhesión y estudio de micrornas específicosdesarrollo de terapias selectivas mediante fármacos y nanopartículas

Resumen

Cancer stem cells (CSCs) are responsible of tumor progression, metastasis, therapy resistance and cancer recurrence, being their identification and isolation of special relevance. Here, we show a new methodology to isolate breast and colon cancer stem-like cells based on their adherence ability to the cell culture surface. Our results demonstrate that trypsin-sensitive (TS) 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. Further studies in MDA-MB-231 breast cancer cells revealed that TS subpopulations express 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-mesenchymal transition (EMT). In the TS subpopulation, CXCL10, BMI1 and OCT4 were upregulated, being associated their expression with a poor prognosis of cancer. Furthermore, in vivo studies in NOD scid gamma (NSG) mice demonstrated that MDA-MB-231 TS cells formed more and bigger xenograft tumors with shorter latency and had higher metastatic potential when were injected in the tail vein. In addition, we have made bioinformatic analysis in order to select the expression of several microRNAs (miRs) related with CSC phenotype that were finally validated in different subpopulations. In fact, subpopulations selected by differential adhesion capacity differ in the expression of several miRs, such as miR-34a-5p, miR-34c-5p, miR-21-5p, miR-93-5p and/or miR-100-5p, which are also involved in EMT and/or cell self-renewal. Also, colon cancer cell lines, HCT 116 and HT-29, and normal colon cell line, CCD-18Co, differ between them in their miRs expression profile. Both colon cancer cell lines overexpressed miR-142-3p, but miR-15b-5p and miR-590 were only overexpressed in HT-29. In contrast, miR-199a-5p and miR-370 were downregulated in both cancer colon cell lines with respect to CCD-18Co. These different profiles encourage future investigations to use the selected miRs with prognostic and/or therapeutic value in clinic. The presence of CSCs within tumor is in part responsible of drug resistance to traditional therapies targeting cell with high division rates. Therefore, it is necessary to develop new therapies that target these subpopulations. We studied the use of innocuous polyethylene nanoparticles (NPs) that can be use for anti-miR delivery. Anti-miR-21-5p loaded NPs diminished melanosphere formation in enriched CSCs subpopulation. Other therapeutic strategy consisted in the development of new compounds against CSCs. In this way, we showed that compounds derivated of p-Nitrobenzenosulfonamides were more active that purine and 5-FU derivatives, and they showed a high activity against melanoma CSCs. Drug distribution is critical for the design of more selective and efficient agents in cancer therapy. Fluorescence compounds analyzed in the present work were very useful to elucidate the mechanism of action of drugs inside the cell. In conclusion, we have developed a non-aggressive, easy, cheap and reproducible methodology to prospectively isolate breast and colon cancer stem-like cells. This study has shown an important point for subsequent biological and preclinical studies, as well as the analysis of specific miR expression profile or the screening of new prospective therapies such as new antiproliferative compounds or NPs linked to therapeutics anti-miRs.