Tumor Associated Macrophages in Colorectal Liver Metastases: cell morphology correlates with single-cell diversity, metabolic profile, and prognosis.

Tumor-associated macrophages (TAMs) are key components of tumoral microenvironment and have been shown to impact prognosis in different cancer subtypes. Previously reported data from our group showed that TAMs’ morphology correlates with prognosis in colorectal liver metastases (CLMs), with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger ones (L-TAMs). L-TAMs display higher intracellular complexity than their S-TAM counterparts, with frequent presentation of intracellular vacuoles. Following this preliminary observation, we conducted retrospective validation on an external cohort (84 patients, resected for CLM between 2016-2017), that confirmed the association of TAM’s morphology and prognosis. Concurrently, we prospectively collected CLM tumoral and peritumoral tissue form patients operated at our institution. We were able to set up flow-cytometry panels for deep phenotyping studies and TAMs’ sorting and compared gene expression profiling by qRT-PCR on sorted and in vitro differentiated macrophages: while L-TAMs significantly overexpress M2 markers, S-TAMs displayed a mixed M1/M2 phenotype. Moreover, following our observations, we performed a targeted and untargeted metabolomic analysis on L- and S-TAMs and found a quantitative difference in the metabolite Riboflavin (Vitamin B2), that is implicated in the regulation of the protein LSD1, an enzyme recently linked to cancer aggressiveness and a promising therapeutic target. Interestingly, L- showed a significant over-expression of LSD1. Finally, we performed scRNA seq analysis on colorectal cancers (CRCs) samples and synchronous CLMs identifying differential genetic signatures, which allowed us to build specific genetic pathway maps for L- and S-TAMs. These included different pathways involved in T cells activation and regulation, such as PD-1 signaling, whose inhibition has shown efficacy in many cancers, including advanced CRCs. All these findings suggest that morphology of TAMs may represent a simple readout of TAM diversity in CLMs and pave the way to translating them from a strong prognostic indicator to a therapeutic target.