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. 2016 Dec 12;213(13):2967–2988. doi: 10.1084/jem.20151665

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© 2016 Chan et al.

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Figure 4. — The pleiotropic effects of MTD chemotherapy–treated CAFs mediated through the ELR⁺ chemokine−CXCR-2 paracrine signaling axis. (A) Bar graphs showing the increased endothelial tube formation induced by MTD-doxorubicin– or MTD-paclitaxel–treated BCAF-011 CAFs that could be reduced by treatment with the CXCR-2 inhibitor SB225002 (1 µM) or prevented when LDM-mimetic regimens as described in Fig. 5 (A and C) were used to treat CAFs. Data from two independent experiments (n = 3 in each group) are shown. (B, left) Representative photomicrographs showing pronounced vascularity in tumors formed by orthotopically implanted BC-011 carcinoma cells along with MTD-doxorubicin (MTD-CAF)– or vehicle (vehicle-CAF)-treated CAFs with or without concurrent intraperitoneal injections of SB225002 (0.5 mg/kg/day) in NOG mice. Also shown are immunohistochemical images of von Willebrand factor (vWF) staining of the endothelial cells in the tumors. Bar, 50 µm. (Right) quantification of microvessel density (MVD) per high-power field (HPF) in tumors. At least 10 different areas were examined in each tissue. Data from one experiment (n = 3 in each group) are shown. (C, left) The invasive capacities of U937-derived macrophages in response to vehicle-CAFs, MTD-CAFs, or LDM-CAFs in the absence or presence of 1 µM SB225002 in a Transwell invasion assay. Shown are representative immunofluorescence images of the invaded cells, with cell nuclei stained with CYTOX-green. Bars, 100 µm. (Right) The numbers of invaded macrophages. Data from two independent experiments (n = 3 in each group) are shown. (D, left) Representative immunohistochemical images of F4/80 staining in tumors formed by subcutaneous inoculation of BC-011 cells along with vehicle-, MTD-, or LDM-CAFs in the flank of nude mice. Bar, 50 µm. (Right) Percent F4/80- or Fizz-1–positive cells in the microscopic field. At least 10 different areas were examined in each tissue. Data from two independent experiment (n = 3 tumor tissues in each group) are shown. (E) BCAF-011 CAFs were treated with vehicle (vehicle-CAF), MTD, (MTD-CAF), or LDM-doxorubicin or paclitaxel (LDM-CAF) as in Fig. 5 and then co-cultivated with BC-011 carcinoma cells in the presence or absence of 1 µM SB225002 in a dual chamber culture apparatus for 5 d, after which the carcinoma cells were subjected to flow cytometric analyses. Shown are the percentages of the CD44⁺CD24^−/low carcinoma cells. Data from two independent experiments (n = 3 in each group) are shown. (F) Freshly sorted CD44⁺CD24^−/low BC-011 carcinoma cells were cultured in the presence of human recombinant CXCL-1, -2, -5, and -6 (each at 1 µg/ml) or vehicle in low-attachment culture plates for 10 d, and the diameters of the tumorspheres generated were quantified. Data from two independent experiments (n = 3 in each group) are shown. (G) The ability of CD44⁺CD24^−/low BC-011 carcinoma cells to invade through reconstituted basement membrane in response to human recombinant CXCL-1, -2, -5, and -6 (each at 1 µg/ml) in a Transwell invasion assay. Data from two independent experiments (n = 3 in each group) are shown. (H) The percentages of CD44⁺CD24^−/low cells in BC-011 carcinoma cells co-cultivated with vehicle- or MTD-CAFs along with the neutralizing antibody directed against individual ELR⁺ chemokines. Data from two independent experiments (n = 3 in each group) are shown. (I) Limiting dilution assay demonstrating the tumorsphere formation efficiency of BC-011 carcinoma cells cultured in the conditioned medium from MTD- or LDM-CAFs. The arrow indicates change of slope of the trend line, suggestive of differential tumorsphere formation ability. Data from one experiment (n = 6 in each group; mean ± SEM; Student’s t test; ***, P < 0.001 vs. MTD) are shown. (J) The invasive capacities of BC-011 cells in response to vehicle-, MTD-, or LDM-CAFs, with or without 1 µM SB225002, in a Transwell invasion assay. Shown are the numbers of the invaded cancer cells per microscopic field. Data from two independent experiments (n = 3 in each group) are shown. (A–H and J) Data are mean ± SEM; Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vehicle. †, P < 0.05 versus MTD.