Background Targeting “don’t eat me” signaling has achieved profound responses in clinal trials for hematologic malignancies, highlighting the potential therapeutic value of macrophage-mediated phagocytosis for cancer treatment. In recent years, CRISPR-Cas9 based in vitro co-culture screens have significantly expanded our knowledge of the key tumor-intrinsic mechanisms mediating cytotoxic lymphocyte killing. However, the interactions between tumor cells and macrophages remain elusive. To illuminate the tumor-intrinsic biology governing the interactions between tumor cells and macrophages, we developed both positive and negative screening strategies, and deployed multiple primary bone marrow-derived macrophages (BMDM) or macrophage cell line-based co-culture screens.

Methods DLD1 and MDA-MB-231 cancer lines were infected with a whole-genome CRISPR-Cas9 library and co-cultured with Raw264.7 or J774A.1 in the presence or absence of the therapeutic antibodies magrolimab or cetuximab. MC38 syngeneic cancer cells were infected with a library of 3000 immune-related genes and co-cultured with primary BMDM. For negative selection, the macrophages were selectively killed off post co-culture and the tumor cells were collected for next generation sequencing (NGS) and statistical analysis. For positive selection, macrophages and tumor cells were fully dissociated from the plate post co-culture, and the mixture of cells was subjected to a CD11b positive selection column. Macrophages and tumor cells were enriched separately for downstream analysis.

Results Bioinformatic analysis revealed that CD47 knockout in tumor cells strongly sensitized macrophage-mediated clearance of target cells both from naïve and cetuximab-treated conditions. Furthermore, CD47 or EGFR knockout conveyed growth advantage in co-culture upon treatment with magrolimab or cetuximab, respectively. For the positive screen, we developed a method to sequence bar-coded DNA from tumor cells engulfed by macrophages. Consistently, CD47 sgRNAs were significantly enriched inside macrophages in the naïve condition and were depleted upon treatment with magrolimab. Together, these findings demonstrated the robustness of our phagocytosis-based CRISPR screening platform. In additional to validating the role of CD47 in this system, discovered and validated a group of novel tumor intrinsic regulators of macrophage-mediated phagocytosis.

Conclusions We successfully established and implemented multiple macrophage and tumor cell co-culture screening platforms, and systematically revealed tumor intrinsic regulators of phagocytosis, uncovering multiple known and novel therapeutic targets regulating vulnerability of tumor cells to macrophage-mediated phagocytosis. Our proprietary high-throughput phagocytosis CRISPR screening platform provides an unbiased and rapid solution to study macrophage and tumor cell interactions and discover novel targets for cancer therapy.