Pyra-Metho-Carnil Suppresses CD73-associated Macrophage Infiltration in KRAS-mutant Colorectal Cancer.

We previously reported that Pyra-Metho-Carnil (PMC) suppresses macrophage differentiation in co-culture with mutant (mt) Kirsten rat sarcoma (KRAS) tumors. In this study, we used integrative proteomics and identified a KRAS-regulated membrane and exosomal protein, 5'-nucleotidase ecto (NT5E)/CD73, as a key candidate linking KRAS signaling to macrophage-associated tumor immune microenvironment (TIME) remodeling. We investigated whether PMC modulates CD73-associated pathways during macrophage differentiation. THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and treated with PMC or a CD73 inhibitor (CD73i).

Three-dimensional (3D) co-culture assays were performed using HKe3 wild-type (wt) KRAS or HKe3 mt KRAS spheroids. Macrophage infiltration into spheroids was evaluated by live-cell imaging and immunohistochemistry (IHC) of co-cultured spheroid sections. Using clinical colorectal cancer (CRC) specimens, the co-localization of CD73+ tumor cells and CD68+ macrophages was examined. IHC of co-cultured spheroid sections revealed that PMC treatment reduced THP-1-derived macrophage infiltration into mt-KRAS spheroids.

Notably, while CD73i reproduced only the effect on macrophage infiltration, PMC demonstrated a more comprehensive antitumor effect by suppressing both CD73-mediated infiltration and direct tumor cell proliferation. Analysis of clinical mt-KRAS CRC tissues revealed significant clustering of CD73+ tumor cells and CD68+ macrophages specifically at the invasive fronts. In contrast, such infiltration was minimal in non-invasive regions or wt-KRAS tissues.

These findings suggest that PMC suppresses macrophage infiltration and promotes TIME reprogramming through mechanisms associated with CD73 regulation.

Therefore, by targeting tumor growth and immune evasion, PMC offers superior therapeutic value over conventional CD73i in mt-KRAS CRC, where CD73 expression correlates with clinical macrophage clustering.