Abstract 3360: Phototheranostics of cancer associated fibroblasts by targeting fibroblast activation protein-α

Abstract

Abstract Introduction: Stromal cells such as cancer associated fibroblasts (CAFs) mediate many of the aggressive characteristics of cancer1 and have an ever-replenishing supply that is largely left intact by our current therapeutic strategies.2 Fibroblast activation protein-α (FAP-α) is a target antigen that been reported to be selectively expressed in tumors by a subset of immunosuppressive stromal fibroblasts. Here, we extended our previous near-infrared photoimmunotherapy (NIR-PIT) study,3 to develop a FAP-α-targeted monoclonal antibody (mAb) photosensitizer conjugate. We developed FAP-α overexpressing MDA-MB-231 and HT-1080 cells to test the feasibility of targeting FAP-α expressing populations in cells and tumors in vivo, in terms of the specificity of the conjugate to bind to, detect, and eliminate FAP-α expressing populations. Method: FAP-α monoclonal antibody (AF3715) and its IgG isotype control were conjugated with a NIR phthalocyanine dye, IR700, to form FAP-α-IR700 and IgG-IR700. FAP-α overexpression was achieved by transducing MDA-MB-231 and HT-1080 cells with lentivirus encoding the gene for human FAP (Accession No. NM_004460.3) that was subcloned into lentiviral vector pMA3211 to obtain 231-FAP and HT-1080-FAP cells. Cell viability was measured by using CCK-8 assay. Bilateral tumor models were established by inoculating 1×106 MDA-MB-231 cells or 1×106 231-FAP cells in 0.05 ml of Hank's balanced salt solution on either side in the mammary fat pads of athymic Balb/c (nu/nu) female mice. A similar number of HT-1080 or HT-1080-FAP cells were inoculated bilaterally in the flank. 50 μg of FAP-α-IR700 or IgG-IR700 was injected i.v., and fluorescence images of IR700 in mice were obtained over a 24-h period (n = 3 per group). At 24 h post injection, mice were euthanized, and tumors were isolated for imaging. For PIT, tumor-bearing mice (n = 4 per group) received two i.v. injections of 100 μg of FAP-α-IR700 at a one-week interval, and tumors were exposed to NIR irradiation at 200 J/cm2 at 24h p.i. Tumor diameters were measured over 2 weeks. IgG-IR700 and PBS-injected mice were used as controls. Results and Discussion: We confirmed FAP-α overexpression in 231-FAP and HT-1080-FAP cells. FAP-α-IR700 was activated by NIR light, causing FAP-α-specific cell death. FAP-α-IR700-mediated phototoxicity was dependent on the conjugate concentration and NIR light dose; and it was inhibited by excess AF3715. We observed the preferential accumulation of FAP-α-IR700 in FAP-α-overexpressing 231-FAP and HT-1080-FAP tumors compared to their wild-type counterparts. The mean fluorescence intensity of FAP-α-IR700 in FAP-α-overexpressing tumors was approximately two to threefold higher than the wild type tumors. FAP-α-IR700 injection together with NIR light exposure resulted in the highest tumor growth delay in 231-FAP tumors and HT-1080-FAP tumors. Our results evaluate and confirm the ability of FAP-α-IR700 to target and eliminate FAP-α-overexpressing cell populations, providing novel opportunities to selectively deplete FAP-α high CAFs in cancers.