TAMS in appendiceal cancer: Association with metabolic remodeling, prognosis, and response to intra-tumoral therapy.

3597 Background: Appendiceal cancer (AC) is a rare malignancy with a distinct immunobiology and propensity for peritoneal metastasis. Tumor-associated macrophages (TAMs) are recognized as influential immune cells in various cancers. In this study, we sought to define the role of TAMs in AC, given its unique immune landscape and peritoneal microenvironment. Methods: Immunohistochemistry-quantified M1-like (CD68+CD86+) and M2-like (CD68+CD206+) TAM densities in 112 archival AC specimens were compared against clinicopathologic features and overall survival (OS). In parallel, a comprehensive tissue proteomic analysis (LC-MS/MS) was performed. Gene set enrichment analysis identified proteomic signatures associated with M1 and M2 density. Independently prognostic proteins were identified via Cox regression. External validation was performed by correlating these proteomic signatures in independent tumor samples from the RIOT-1 clinical trial (NCT05751837), in which AC patients received intra-tumoral lipopolysaccharide (LPS), a TLR4 agonist. Results: M2-like TAMs outnumbered M1-like TAMs in AC (p = 0.0002), particularly in peritoneal metastases (p = 0.002), and were not associated with tumor grade or histology. Low M1 density ( < 18th percentile) correlated with worse OS (HR: 3.26, p = 0.03), while high M2 density ( > 52nd percentile) was associated with improved OS (HR: 0.1, p = 0.03). A combined high-M1/M2 percentile metric was highly prognostic of favorable OS (p < 0.001), independent of age, grade, and disease extent. On proteomic analysis, M1-rich tumors exhibited high bioenergetic demands, extensive metabolic reprogramming (isoprenoid/amino acid metabolism), retinol/xenobiotic metabolism, while M2-rich tumors emphasized ECM remodeling, profound amino acid metabolism (immunosuppressive), oxidoreductase activity, and complement activation, with suppressed cell motility. The combined high-M1/M2 phenotype correlated with intense amino acid/lipid metabolism and redox homeostasis. Five TAM-related, independently prognostic proteins were identified: GATD1 (favorable, HR 1.53x10-16), and AGR2, ENOPH1, RHOA, SERPINB6 (adverse, HRs 5.51-10.39). In RIOT-1, intra-tumoral LPS increased M1/M2 TAM infiltration and induced proteomic changes significantly correlated with M1-related (p = 0.0016), M2-related (p = 0.008), and combined TAM signatures (p = 0.047), supporting therapeutic tractability. Conclusions: AC harbors a TAM-rich immune microenvironment in which M2-like TAMs and a combined M1/M2 phenotype paradoxically associate with favorable prognosis. Distinct TAM-linked metabolic programs and validated prognostic proteins suggest clinically relevant biologic underpinnings of these findings, while modulation of the AC microenvironment via regional innate immune activation may represent a promising therapeutic strategy.