CRISPR-Cas9 Editing of Myeloid Lectins Reveals Target-Dependent Transcriptional Remodeling in Tumor-Associated Macrophages

Tumor-associated macrophages (TAMs) integrate multiple microenvironmental signals, including lectin-mediated recognition of tumor-associated glycans. Although several myeloid lectins have been implicated in tumor-macrophage interactions, their contributions to immunosuppressive TAM phenotypes remain poorly defined. We implemented a CRISPR-Cas9 ribonucleoprotein (RNP) workflow in primary human monocytes to enable comparative loss-of-function interrogation of five potentially immunosuppressive lectins we previously reported. CRISPR-Cas9 RNP delivery conditions were benchmarked in freshly isolated monocytes using CD14 as a technical target, followed by sgRNA editing efficiency screening for CLEC4E, CLEC6A, CD209, SELL, and SIGLEC9 in HeLa S3 cells. Selected guides were applied to primary monocytes before co-culture in a 3D model with BT474 breast cancer cells. Editing was quantified by Sanger-sequencing deconvolution, while co-culture-derived CD45+ macrophages were isolated by fluorescence-activated cell sorting and characterized by bulk RNA sequencing. All five lectins were successfully perturbed, although gene editing efficiency remained target- and donor-dependent. Transcriptomic analysis revealed target-specific reprogramming and recurrent immunometabolic responses. mTORC1 signaling was consistently enriched, together with the remodeling of cholesterol homeostasis, fatty acid metabolism, and oxidative phosphorylation. CLEC6A deletion achieved attenuation of hypoxia-, angiogenesis-, and TNF/NF-kB-associated programs. CD209 and SIGLEC9 deletion similarly reduced hypoxia- and angiogenesis-associated signatures while promoting lipid and oxidative metabolism. CLEC4E perturbation induced broad remodeling of lipid and oxidative metabolism and stress-response, despite comparatively limited editing efficiency. Comparative lectin perturbation thus identified CLEC6A as the strongest candidate for further mechanistic investigation, while CLEC4E showed broad transcriptional remodeling despite lower editing efficiency, supporting its prioritization for improved genetic interrogation. Responses to CD209 and SIGLEC9 targeting further support tumor glycan-myeloid signaling as a regulator of TAM state. Overall, lectin perturbation redirected TAM transcriptional states through distinct immunometabolic programs rather than a uniform pro-inflammatory polarization, highlighting lectin-dependent immunometabolic regulation as a promising axis for further investigation.