Influence of neoadjuvant therapy on tumor-vascular-immune stromal networks in colorectal cancer liver metastases.

3586 Background: Neoadjuvant therapy's effect on vascular, tumor, and immune compartments, and their co-regulation, in colorectal liver metastases (CRLMs) remains poorly understood. We performed multiplex-based tissue profiling to quantify how treatment reshapes stromal compartment and co-occurrence networks, defined by inside-case correlations between features. Methods: We analyzed 76 CRLM cases (27 untreated, 49 neoadjuvant-treated [43 chemotherapy, 6 chemo + VEGF-/EGFR-targeted]). Tissue microarrays (1-3 1-mm cores/tumor) underwent multiplex immunofluorescence to quantify immune/tumor subsets (CD3/CD4/CD8/PD-1/FOXP3/TIM-3/Ki67/CDX2), vessels (claudin-5), and stromal perivascular compartments (αSMA/PDGFRβ). Statistical analysis included Mann-Whitney U tests and Spearman correlations. Results: Neoadjuvant-treated CRLMs had 42% fewer proliferating tumor cells (CDX2⁺KI67⁺density, p=0.006) and 33% lower CDX2⁺ density (p=0.031) compared with untreated CRLMs. This was accompanied by a 32% reduction in proliferating cytotoxic T-cells (CD3⁺CD8⁺Ki67⁺ density, p=0.029). A reduction in vessel size was also observed in treated CRLMs (vessel size, -12%, p=0.077). Analyses suggested different effects by type of neoadjuvant treatment. Chemotherapy alone reduced proliferating tumor cells (CDX2⁺KI67⁺ density, -37%, p=0.016) and suppressed cytotoxic T-cell proliferation (CD3⁺CD8⁺Ki67⁺ density, -40%, p=0.017). The addition of VEGF/EGFR-targeted agents produced the most pronounced reduction in tumor burden (CDX2⁺ density, -73%, p=0.012; CDX2⁺KI67⁺ density, -76%, p=0.010) vs. untreated. CRLM co-occurrence networks were also affected by treatment. Treatment abolished positive correlations between tumor cells (CDX2⁺ and CDX2⁺KI67⁺ densities) and αSMA⁺PDGFRβ⁺ perivascular fraction/vessel size (r=+0.42 – +0.60, p<0.05), and negative correlations between tumor cells and αSMA⁺PDGFRβ⁻ perivascular fraction/vessel density (r=-0.47 – -0.40, p<0.05) in untreated (vs +0.05 – +0.27 and +0.09 – +0.21, p=n.s., in treated). Treatment also eliminated positive correlations between vessel size and exhausted (CD8⁺PD-1⁺) and regulatory (CD4⁺FOXP3⁺) T-cell densities (untreated r=+0.40 – +0.47, p<0.05). In parallel, new positive links emerged between αSMA⁻PDGFRβ⁺ perivascular fraction and CD4⁺ and CD8⁺ T cells (r=+0.41 – +0.42, p<0.05). Conclusions: This exploratory study indicates that neoadjuvant treatment alters the CRLM microenvironment, affecting tumor, vascular, and immune compartments. Additionally, analyses suggest that treatment induces a restructuring of their interdependence networks, defined by the loss of specific tumor-stromal-vascular links seen in untreated CRLMs, while establishing new ones. These findings motivate continued mechanistic studies and analyses on how these changes are related to benefits of treatment.