Figure 3.

Expression of OLFML3 in inhibitory effects of anti-OLFML3 mAbs and Olfml3 gene deletion on CRC tumor growth and vascularization. (A) C57Bl6/J mice received a subcutaneous implant of MC38 tumor cells, followed by treatment with anti-OLFML3 or isotype control mAbs on days 0, 5, 8, 11, 14, and 17. (B) The volume of MC38 tumors in mice treated with rec9F8 (n = 7), rec46A9 (n = 7), or IgG2a isotype control mAb (Ctrl IgG, n = 8) was measured every 2–4 days (starting on day 3) using a caliper (left) or ex vivo after sacrifice on day 19 (right). Bars (right) represent the mean ± SEM; * p < 0.05. (C) Right, representative images (scale bar, 100 µm), and left, quantification of Pecam1-positive events after Pecam1 immunostaining of tumor sections from (B). Results represent mean values ± SEM; * p < 0.05; ** p < 0.01. (D) Gating strategy used for flow cytometry analysis of CD45⁻/CD31⁺/PDPN⁻ vascular endothelial cells and CD45⁻/CD31⁺/PDPN⁺ lymphatic endothelial cells in single-cell suspensions derived from MC38 tumors from mice treated with rec9F8 or control IgG antibodies (left). Quantification of vascular CD45⁻/CD31⁺/PDPN⁻ (middle) and lymphatic CD45⁻/CD31⁺/PDPN⁺ (right) endothelial cells. (E) Growth curve (left) and ex vivo measurement of the volume (right) of MC38 tumors growing in wild-type (WT) (n = 9) or Olfml3 knockout mice (n = 14). Bars (right) represent the mean ± SEM; * p < 0.01. (F) Quantification of Pecam1-positive events after Pecam1 immunostaining of the tumor sections (E). Results represent the mean value ± SEM; * p < 0.05; ** p < 0.01. (G) Gating strategy used for flow cytometry analysis of CD45⁻/CD31⁺/PDPN⁻ vascular endothelial cells and CD45⁻/CD31⁺/PDPN⁺ lymphatic endothelial cells in MC38 tumor-derived single-cell suspensions from WT and Olfml3 knockout mice (left). Quantification of vascular CD45⁻/CD31⁺/PDPN⁻ (middle) and lymphatic CD45⁻/CD31⁺/PDPN⁺ (right) endothelial cells. Results represent the mean value ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.01.