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. 2016 Jan 11:6:18772.
doi: 10.1038/srep18772.

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

Cynthia Morata-Tarifa  1   2 , Gema Jiménez  1   2   3 , María A García  1   2   4 , José M Entrena  5   6 , Carmen Griñán-Lisón  1   2 , Margarita Aguilera  2   7 , Manuel Picon-Ruiz  1   2   8 , Juan A Marchal  1   2   3
Affiliations

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

Cynthia Morata-Tarifa et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. Differential trypsinization protocols.
(A) Isolation of TS1, TS2 and TR1 subpopulations, used for Aldefluor assays, Hoechst 33342, surface markers, spheres and colonies formation ability. (B) Improved protocol to isolate highly attached cells (TR2) used for Aldefluor, qRT-PCR and in vivo assays. Red balls represent cells with higher CSC-like properties and green balls cells with a non CSC-like phenotype.
Figure 2
Figure 2. Phenotypic properties of breast and colon cancer cell subpopulations isolated by differential tripsinization.
Percentage of ALDH+ (A) and SP (B) cells determined in subpopulations of MCF7, MDA-MB-231, HT-29 and T84 cancer cell lines. (C,D) Percentage of CD44+/CD24− cells in TR1, TP, TS1 and TS2 isolated from MCF7 (C) and MDA-MB-231 (D) breast cancer cells measured by flow cytometry. (E) Co-expression of CD44, CD133 and CD326 in HT-29 cells isolated by differential trypsinization and in the TP. (F) Flow cytometry for CD44 and CD326 expression, alone or together, in T84 cells. Data are graphed as mean ± SD from at least two different experiments carried-out by triplicates (**P < 0.01; *P < 0.05). See also Supplementary Fig. S1.
Figure 3
Figure 3. Tumorsphere- and colony-forming ability of breast and colon cancer cell subpopulations isolated by differential trypsinization.
(A) Representative images of mammospheres formed from different MCF7 subpopulations. (B) Number of spheres formed by different subpopulations of each cell line. (C) Secondary sphere formation assay performed in MDA-MB-231 and T84 cells selected by differential trypsinization and compared to the TP. Data shown as mean ± SD (**P < 0.01; *P < 0.05). (D) Number of colonies formed by TP and TS1 of each cell line. Data are represented as mean ± SD (**P < 0.01; *P < 0.05).
Figure 4
Figure 4. ALDH activity, mRNA and miRNA expression in enriched subpopulations of MDA-MB-231 cells isolated by their different adhesion capacity.
(A) Aldefluor assay performed in TR2, TP and TS1 MDA-MB-231 cells. See also Supplementary Fig. S2. (B) qRT-PCR analysis for the expression of CSCs and EMT-related genes in different subpopulations of MDA-MB-231 cells. Data are normalized to 1 for TP using GAPDH as internal control, and graphed as mean ± SEM (n = 3) (**P < 0,01; *P < 0,05). (C) Differential expression of miRNAs related to CSC phenotype and/or EMT in MDA-MB-231 cells isolated by differential trypsinization. Data are normalized to 1 for TP using miR-24c-3p as internal control, and graphed as mean ± SEM (n = 3) (**P < 0.01; *P < 0.05).
Figure 5
Figure 5. Proliferation assay.
Proliferation curves of TS1, TR2 and TP cells cultured for 6 days in media containing FBS, and measured using MTT every 2 days in cultures seeded with an equal number of cells at day 0. Data are represented as absorbance at 570 nm and shown as mean ± SD (**P < 0.01; *P < 0.05).
Figure 6
Figure 6. Overall survival and subcutaneous xenograft tumor formation
(A) Kaplan-Meier curve for overall survival of NSG mice injected with 5 × 103 cells (left) and 1.5 × 104 cells (right) from TS1, TR2 or TP. (B) Tumor volume (mm3) of subcutaneous xenograft tumors formed by 5 × 103 and 1.5 × 104 cells from TS1, TR2 and TP in NSG mice. Data are shown as mean ± SD (**P < 0.01; *P < 0.05).
Figure 7
Figure 7. Experimental lung metastasis assay.
(A) IVIS images of lung metastasis formed after tail vein injection of TP, TR2 and TS1 MDA-MB-231 L2T cells (up). Measure of photon flux is graphed as mean ± SEM (down). (B) Fluorescence of metastatic lungs ex vivo. (C) Representative optical images of lung metastasis for a healthy mice (control) and lungs extracted from NSG mice injected with different subpopulations of MDA-MB-231 cells obtained by differential trypsinization. (D,E) Histological (D) and immunohistochemical (E) images of lungs obtained from healthy mice (control) and mice injected with TR2, TP and TS1 cells (left to right).
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