Figure 4. Chemotherapy-stimulated CAFs promote CIC growth and chemoresistance. The effect of CAFs on CICs was assessed by (A) co-culture and (B) conditioned media treatment. (A) 104 CICs were cultured with 2 × 104 CAFs or normal fibroblasts treated with vehicle (DMSO) or chemotherapy for 10 d. Cellular viability was measured by Cell Titer-Glo assay. Data are presented as mean ± SD (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) 1.5 × 105 CICs and CAFs were treated with vehicle (DMSO) or chemotherapy for 3 d. Conditioned media were collected and use to culture CICs for 10 d. Cellular viability was measured by Cell Titer-Glo assay. Data are presented as mean ± SD (n = 3); *, P < 0.1; ***, P < 0.001. Student’s t-test was used to assess the significance. The experiment was performed three times and representative data are shown. (C) The ability of the CAFs to affect the tumorigenic capacity of CICs from two different human specimens was tested in vivo. 3 × 104 CAFs treated with vehicle (DMSO) or with chemotherapy for 3 d were subcutaneously co-implanted in immunocompromised mice with 3 × 103 CICs. Tumors were monitored every day to evaluate the latency, harvested simultaneously, and volume measured to evaluate their development. **, P < 0.01; ***, P < 0.001. ANOVA, followed by Bonferroni’s post-hoc test was used to assess the significance. As indicated in the table, three to six mice were used in each group. The contribution of CAFs to post-therapy tumor growth was assessed by immunostaining of the xenografts for Ki-67 (D). Nuclei were counterstained with DAPI. Quantification was done by counting four high-power fields per condition. Data are presented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Student’s t-test was used to assess the significance. Bars, 50 µm. (E) Xenografts derived from data in C were dissociated and analyzed for the expression of CD44 by FACS. Colorectal cancer cells conditioned by the presence of the CAFs were enriched in the CD44 compartment. (F) CICs sorted from xenografts in C were plated in 96-well plates in limiting dilution (50, 10, or 1 cells per well) and analyzed for sphere formation. Data are presented as mean ± SD (n = 20); **, P < 0.01; ***, P < 0.001. The likelihood ratio test was used to assess the significance. The experiment was performed once. (G) 3 × 104 CICs and non-CICs from the same xenografts (C) were plated in 96-well plates and treated with chemotherapy. The effect of CAFs on CICs and non-CICs cells chemosensitivity was tested by evaluating cell viability (absolute number) measured by CellTiter-Glo assay. Data are mean ± SD (n = 3); **, P < 0.01; ***, P < 0.001; unlabeled, P > 0.05. Student’s t test was used to assess the significance. The experiment was performed twice and representative data are shown.
Figure 4.

Chemotherapy-stimulated CAFs promote CIC growth and chemoresistance. The effect of CAFs on CICs was assessed by (A) co-culture and (B) conditioned media treatment. (A) 104 CICs were cultured with 2 × 104 CAFs or normal fibroblasts treated with vehicle (DMSO) or chemotherapy for 10 d. Cellular viability was measured by Cell Titer-Glo assay. Data are presented as mean ± SD (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) 1.5 × 105 CICs and CAFs were treated with vehicle (DMSO) or chemotherapy for 3 d. Conditioned media were collected and use to culture CICs for 10 d. Cellular viability was measured by Cell Titer-Glo assay. Data are presented as mean ± SD (n = 3); *, P < 0.1; ***, P < 0.001. Student’s t-test was used to assess the significance. The experiment was performed three times and representative data are shown. (C) The ability of the CAFs to affect the tumorigenic capacity of CICs from two different human specimens was tested in vivo. 3 × 104 CAFs treated with vehicle (DMSO) or with chemotherapy for 3 d were subcutaneously co-implanted in immunocompromised mice with 3 × 103 CICs. Tumors were monitored every day to evaluate the latency, harvested simultaneously, and volume measured to evaluate their development. **, P < 0.01; ***, P < 0.001. ANOVA, followed by Bonferroni’s post-hoc test was used to assess the significance. As indicated in the table, three to six mice were used in each group. The contribution of CAFs to post-therapy tumor growth was assessed by immunostaining of the xenografts for Ki-67 (D). Nuclei were counterstained with DAPI. Quantification was done by counting four high-power fields per condition. Data are presented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Student’s t-test was used to assess the significance. Bars, 50 µm. (E) Xenografts derived from data in C were dissociated and analyzed for the expression of CD44 by FACS. Colorectal cancer cells conditioned by the presence of the CAFs were enriched in the CD44 compartment. (F) CICs sorted from xenografts in C were plated in 96-well plates in limiting dilution (50, 10, or 1 cells per well) and analyzed for sphere formation. Data are presented as mean ± SD (n = 20); **, P < 0.01; ***, P < 0.001. The likelihood ratio test was used to assess the significance. The experiment was performed once. (G) 3 × 104 CICs and non-CICs from the same xenografts (C) were plated in 96-well plates and treated with chemotherapy. The effect of CAFs on CICs and non-CICs cells chemosensitivity was tested by evaluating cell viability (absolute number) measured by CellTiter-Glo assay. Data are mean ± SD (n = 3); **, P < 0.01; ***, P < 0.001; unlabeled, P > 0.05. Student’s t test was used to assess the significance. The experiment was performed twice and representative data are shown.

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