Functional Characterization of CD47 Splice-Variants

Cancer cells overexpress CD47, a surface protein that acts as an anti-phagocytic (‘don’t eat me’) signal by binding to its SIRPα receptor on macrophages. Antibodies that bind to CD47 and block this interaction enable the macrophages to phagocytose and kill the cancer cells to which the macrophages had attached. However, immunotherapy based on CD47 blockade elicits variable efficacy and response across different tumor lines. CD47 is a cell surface molecule encoded by several distinct mRNA isoforms that result from alternative splicing of the same CD47 pre-mRNA transcript. I wished to test the hypothesis that the variation in efficacy may be a result of variation in the expression ratios of CD47 splice-variants across tumor types. I proposed that different CD47 splice-variants may have distinct functions which may confer different capacities to the tumor cells in evading immune surveillance. In this study, I sought to functionally characterize two prominent CD47 splice-variants—the full-length isoform and the shortest isoform that lacks the cytoplasmic tail—by stably expressing them individually in colorectal cancer lines where I first excised the endogenous CD47 gene. Using these engineered cancer lines, I performed in-vitro phagocytosis assays and monitored the growth of the tumor after injecting these cells in an immunodeficient mouse model. Based on in vitro phagocytosis assays I found that 1) complete knock-out (KO) of CD47 results in a cancer cell line that shows increased phagocytosis when opsonized by an unrelated antibody that binds to a highly expressed cell-surface protein and which has an isotype that binds to the macrophage high affinity Fc receptor; 2) CD47 isoform 1 (the long-form with a cytoplasmic tail) is a stronger ‘don’t eat-me’ signal in the context of colorectal cancer cell line derived from the primary tumor—DLD1. 3) In the context of cancer cell lines derived from the metastatic site of a colorectal cancer, SW620, it appears that both the long and short (without cytoplasmic tail) CD47 isoforms are required to function as the effective ‘don’t eat-me’ signal, indicating a function—perhaps as a heterodimer consisting of long and the short isoform—in the tumor cell bound by antibodies; and 4) SIRPα receptor may interact with some other ligand on the cancer cell-surface—exemplified by the fact that even the DLD1 cells that are devoid of CD47 get phagocytosed by macrophages at dramatically high levels when SIRPα receptor is blocked by an antibody (KWAR) against it in the context where the cancer cells were opsonized by an unrelated antibody (Cetuximab), especially when blockade of SIRPα by KWAR alone had no effect on phagocytosis. I am attempting to confirm my findings in vivo by injecting graded numbers of these cancer cells into immunodeficient NSG mice. Our lab has now titrated the cell numbers necessary for injection—from a couple of million to 50,000—in NSG mice to decipher the efficacy of a CD47-blockade based immunotherapy regimen on tumors derived from the battery of engineered primary and metastatic lines. My findings have implications with respect to cancer initiation, metastasis, and possible immunotherapy as the expression ratio of CD47 splice-variants may serve as a biomarker in screening and predicting patients’ responses to the anti-CD47 antibody treatment.

Keywords:  Department of Biology
Biology
CD47
cancer
immunotherapy
splicing
isoform
Thesis
Advisor: Weissman Irving
Jones Patricia