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. Author manuscript; available in PMC: 2015 Apr 1.

Published in final edited form as: Mol Cancer Res. 2014 Jan 24;12(4):607–621. doi: 10.1158/1541-7786.MCR-13-0469

CAFs induce EMT to cE1 mouse prostate cancer cells in vitro. A, phase contrast images of representative spheroids formed in cE1, after co-culturing with either control media, NPFs or CAFs. Bar 100 µm. B, cE1 cells were FACS sorted into subpopulations based on high, medium and no expression of SCA-1 (S) and CD49f (C; SC^hi, SC^med and SC^none, respectively). C, Sorted cells grown in post-FACS 3-D culture displayed differential response to CAF CM. cE1 SC^hi and SC^med spheroids increased in size in the presence of CAF CM. Phase contrast images show gross spheroid morphology and incidence of EMT-like phenotypic changes in CAF CM treated cE1 SC^med cells. Sections of spheroids were analyzed by coimmunofluorescence (co-IF) using antibodies against the basal/transit-amplifying cell marker CK5 (red) and luminal cell marker CK8 (green). 4’,6-Diamidino-2-phenylindole (DAPI) was used to label cell nuclei. Phase contrast, bar 100 µm; IF, bar 50 µm. D, Grown in maintenance media, SC^hi and SC^med displayed distinguishing molecular characteristics as assessed by expression levels of pluripotency transcription factors Oct4, Nanog and Sox2, and Snail. E, Expression of EMT/pluripotency transcription factors increases after 14 days in SC^med via CAF CM treatment. SC^hi, SC^med and SC^none cells were subjected to control media (CTL), NPF CM (N) or CAF CM (C) for 14 days. SC^med + CAF CM was compared to SC^hi + CAF CM and SC^none + CAF CM (indicated above bars) as described in the text, SC^med + control (indicated above lines), and SC^med + NPF CM (data not shown). Induction of EMT in response to CAF CM in cE1 SC^med cells was confirmed by qRT-PCR using EMT transcription factor markers Snail, Slug and Twist, pluripotency transcription factors Oct4, Sox2 and Nanog, epithelial marker E-cadherin, and mesenchymal markers N-cadherin and Vimentin. F, Up-regulation of EMT and pluripotency transcription factors, as well as mesenchymal markers, and decrease in E-cadherin in SC^med cells exposed to CAF CM was observed between 7 and 14 days of treatment, compared to control. All qRT-PCR ratios are expressed as fold change normalized to β-Actin expression. In all panels, CAFs 1 and 3 were used in independent repeats. NS, not significant; *, P < 0.05; •, P < 0.01; **, P < 0.001.

CAFs induce EMT to cE1 mouse prostate cancer cells in vitro. A, phase contrast images of representative spheroids formed in cE1, after co-culturing with either control media, NPFs or CAFs. Bar 100 µm. B, cE1 cells were FACS sorted into subpopulations based on high, medium and no expression of SCA-1 (S) and CD49f (C; SC^hi, SC^med and SC^none, respectively). C, Sorted cells grown in post-FACS 3-D culture displayed differential response to CAF CM. cE1 SC^hi and SC^med spheroids increased in size in the presence of CAF CM. Phase contrast images show gross spheroid morphology and incidence of EMT-like phenotypic changes in CAF CM treated cE1 SC^med cells. Sections of spheroids were analyzed by coimmunofluorescence (co-IF) using antibodies against the basal/transit-amplifying cell marker CK5 (red) and luminal cell marker CK8 (green). 4’,6-Diamidino-2-phenylindole (DAPI) was used to label cell nuclei. Phase contrast, bar 100 µm; IF, bar 50 µm. D, Grown in maintenance media, SC^hi and SC^med displayed distinguishing molecular characteristics as assessed by expression levels of pluripotency transcription factors Oct4, Nanog and Sox2, and Snail. E, Expression of EMT/pluripotency transcription factors increases after 14 days in SC^med via CAF CM treatment. SC^hi, SC^med and SC^none cells were subjected to control media (CTL), NPF CM (N) or CAF CM (C) for 14 days. SC^med + CAF CM was compared to SC^hi + CAF CM and SC^none + CAF CM (indicated above bars) as described in the text, SC^med + control (indicated above lines), and SC^med + NPF CM (data not shown). Induction of EMT in response to CAF CM in cE1 SC^med cells was confirmed by qRT-PCR using EMT transcription factor markers Snail, Slug and Twist, pluripotency transcription factors Oct4, Sox2 and Nanog, epithelial marker E-cadherin, and mesenchymal markers N-cadherin and Vimentin. F, Up-regulation of EMT and pluripotency transcription factors, as well as mesenchymal markers, and decrease in E-cadherin in SC^med cells exposed to CAF CM was observed between 7 and 14 days of treatment, compared to control. All qRT-PCR ratios are expressed as fold change normalized to β-Actin expression. In all panels, CAFs 1 and 3 were used in independent repeats. NS, not significant; *, P < 0.05; •, P < 0.01; **, P < 0.001.

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