Figure 3.

Cxcl-12 is mainly produced by tumor-associated macrophages in colon tumors. (A) Chronic colon inflammation was induced by 2.5% DSS administration in the drinking water for 5 days, every 21 days for a total of 5 cycles. 10 mg/kg AOM was intraperitoneally administered at the start of the treatment to generate colon tumors. Macrophages were immunodepleted with anti-CSFR1 once weekly from day 68 to the end of the protocol. IgG was used as isotype control. (B) Representative pictures from distal colons portions from IgG controls (left) and macrophage-depleted (right) mice at the time of tumor evaluation. Note the smaller tumor size in macrophage-depleted mice. Scale bar 5mm. (C) All tumors in all of the mice (6 mice total per group) were blindly counted under the dissecting microscope and tumor areas quantified using ImageJ. (D) Representative pictures of macrophage marker F4/80, M2 marker Arginase-1 and blood vessels marker CD31 detected by immunohistochemistry on macrophage-depleted and corresponding isotype controls. Stained slides were scanned, and positive cells were automated counted within tumor areas using the positive pixel or nuclei positive algorithm in ImageScope. Strong positive counts were normalized by area of tumor analyzed in mm². Each data point reflects a single quantified tumor area from all tumors in all mice within the same treatment group. Scale bar 300 µm. (E) Representative pictures of angiogenic factors immunodetection on macrophage-depleted mice and corresponding isotype controls. Scanned pictures were analyzed as in (D) Scale bar 300µm *p-value < 0.05; ***p-value < 0.001; ****p-value < 0.0001; ns, not significant. Student’s t test.