Targeting endosialin/CD248 through antibody-mediated internalization results in impaired pericyte maturation and dysfunctional tumor microvasculature

Abstract

Over-expression of endosialin/CD248 (herein referred to as CD248) has been associated with increased tumor microvasculature in various tissue origins which makes it an attractive anti-angiogenic target. In an effort to target CD248, we have generated a human CD248 knock-in mouse line and MORAb-004, the humanized version of the mouse anti-human CD248 antibody Fb5. Here, we report that MORAb-004 treatment significantly impacted syngeneic tumor growth and tumor metastasis in the human CD248 knock-in mice. In comparison with untreated tumors, MORAb-004 treated tumors displayed overall shortened and distorted blood vessels. Immunofluorescent staining of tumor sections revealed drastically more small and dysfunctional vessels in the treated tumors. The CD248 levels on cell surfaces of neovasculature pericytes were significantly reduced due to its internalization. This reduction of CD248 was also accompanied by reduced α-SMA expression, depolarization of pericytes and endothelium, and ultimately dysfunctional microvessels. These results suggest that MORAb-004 reduced CD248 on pericytes, impaired tumor microvasculature maturation and ultimately suppressed tumor development.

Keywords: CD248; MORAb-004; endosialin; tumor microvasculature; α-SMA.

Figure 1. Internalization of MORAb-004 and CD248 on human pericytes

A. IF images of human pericytes treated with MORAb-004; plasma membrane dye in green, AlexaFluor-555 conjugated anti-CD248 in red. B, C. Internalization assays of in vitro cultured human pericytes: (B) shows the percentage of internalized MORAb-004 (solid bars) or monoclonal IgG isotype control (hashed bars); (C) shows the percentage of reduction of surface CD248 upon MORAb-004 treatment for 12 hrs.

Figure 2. MORAb-004 inhibited B16-F10 tumor progression and lung colonization only in mice expressing human CD248

A. B16-F10 cells, at 5 × 10⁵, were injected s.c. into right flank of the huCD248 knock-in mice or syngeneic C57BL/6 wild type mice (n = 16). MORAb-004 or a monoclonal IgG isotype control antibody, were administered i.v. via tail vein at 50 mg/kg, 5 doses daily, starting on day 3 post tumor cell implantation. Tumor growth was monitored twice weekly starting on day 7 by three dimensional caliper measurement and presented by volume (mm³). Data presented as Mean ± SEM. B. B16-F10-L1 cells, at 1 × 10⁵, were injected i.v. via tail vein into the huCD248 knock-in mice (n = 16). MORAb-004 or a monoclonal IgG isotype control antibody was administered i.v. via tail vein at 50 mg/kg, 1 day prior to tumor cell injection and every other day post implantation for a total of 5 doses. At the end of the study (day 19 post tumor cell implantations) mouse lungs were harvested. Black-colored melanoma colonies were counted and colony numbers of every mouse were graphed as tumor burden. P = 0.012 (t-test).

Figure 3. Microfill perfusion and x-ray micro-CT angiography

The tumor bearing mice in either the MORAb-004 treated or control group were perfused with sodium nitroprusside containing solution followed by MICROFIL (Carver) injections. The tumors retaining polymerized latex were fixed in formalin then imaged with a X-ray micro-CT system performed by Numira Biosciences, and analyzed using an image analysis software package (Altaview, Numira). Microfill perfusion of animals show extensive neo-vascularization within the tumor of CTRL treated animals A. while MORAb-004 treatment B. reduced the amount of new vessels within the developing tumor.

Figure 4. Immunofluorescent staining and digital analysis of tumor microvessels

A. PBS treated (control) B16-F10 s.c. tumor section stained with Collagen IV (ColIV); B. MORAb-004 treated B16-F10 s.c. tumor section stained with ColIV; C. Comparison of digital counting of microvessels grouped by size. All images were captured at 20x using Panoramic Midi digital slide scanner.

Figure 5. Immunofluorescent staining and digital analysis of CD248 expression levels and other markers on tumor microvessels

A. IF images of B16-F10 tumor microvessels stained with CD31, CD248, and α-SMA (green), co-stained with ColIV (red). All images were captured at 60X using Olympus IX81confocal microscope; B. Comparison of relative expression levels of IF staining of markers on monoclonal isotype control and MORAb-004 treated microvessels.

Figure 6. MORAb-004 treatment caused internalization of CD248 and depolarization of pericytes and endothelium

IF images of control (PBS) or MORAb-004 treated B16-F10 tumor micro-vessels stained for CD31 (green) and CD248 (red). All images were captured at 60X using an Olympus IX81confocal microscope.

Figure 7. Induction of α-SMA in human pericytes and suppression effects of targeting CD248

A. Western blotting analysis of CD248, α-SMA expression on CD248 siRNA transfected human pericytes; B. Western blotting image quantification.