Targeting CCL2/VEGFA via antibodies or miRNAs administration inhibits lung tumor growth.
(A) NFs-Scr, NFs-TM, CAFs-TM, or CAFs-Scr was co-cultured with LCCs for 48h and CM was collected. CM from LCCs-NFs-TM co-culture was treated with or without anti-CCL2/VEGFA; CM from LCCs-CAFs-TM co-culture was treated with or without CCL2/VEGFA. Cell migration assay was performed to assess LCCs migration efficiency in response to those CM. (B) Colony formation assay was performed to determine LCCs colony formation ability when co-cultured with NFs-Scr, NFs-TM, CAFs-TM, or CAFs-Scr with or without addition of anti-CCL2/VEGFA or CCL2/VEGFA as indicated in the Figure B. (C-F) A549 cells were subcutaneously co-injected with CAFs into immunodeficient mice. For neutralizing antibodies treatment, after 7 days of tumor implantation, the mice were treated with mouse IgG or anti-mouse CCL2 and/or anti-VEGF 164 at 2 mg/kg/dose by intraperitoneal injection (i.p.) injections twice weekly for up to 6 weeks (C). Representative CD31-stained section of tumors. The relative tumor microvessel density were presented as Mean ± SEM; (n = 8) (D). Representative HE-stained section of lungs of mice bearing the indicated tumors. The lung metastasis indices pooled within each cohort of mice in (C) are expressed (mean ± SEM; n = 8) (E). The percentage of CSF-1R+F4/80+ in tumors was determined by flow cytometry assay (F) (mean ± SEM; n = 8). *p < 0.05, **p < 0.001 vs IgG. (G-J) For miRNAs injection treatment, miRNAs were formulated with MaxSuppressor in vivo RNALancerII and formulated miRNAs were administered intravenously (i.v.) by tail vein injections every 5 days starting on day 5 after tumor cells implantation (G). Representative CD31-stained section of tumors. The relative tumor microvessel density were presented as Mean ± SEM; (n = 8) (H). Representative HE-stained section of lungs of mice bearing the indicated tumors. The lung metastasis indices pooled within each cohort of mice in (G) are expressed (mean ± SEM; n = 8) (I). The percentage of CSF-1R+F4/80+ in tumors was determined by flow cytometry assay (J) (mean ± SEM; n = 8). *p < 0.05, **p < 0.001 vs miR-Scr.
