Fig. 5: Immunohistochemical (IHC) immuno-stromal profiling of the three main cohorts and xCell-based immune cell type enrichment analysis.

IHC analysis of stromal markers showing increased COL1 deposition after neoadjuvant treatments (a) while αSMA expression remained unchanged (b). c–f No significant differences in IHC expression were observed among the three groups for CD3+ (c), CD8+ (d). However, expression of CD4+ (e) and CD20+ cells (f) was significantly decreased in the FFX + SBRT group compared to No_NAT. No significant differences in IHC expression was observed for CD68+ (g) and CCR2 cells (h) across the three groups. i No significant differences in IHC expression was observed for FOXP3+ cells across the three groups. PD-L1 IHC expression significantly increased but remained scarce in the FFX + SBRT group (j) while PD-1 IHC expression was significantly decreased (k). No significant differences in IHC expression was observed for CD68+ (j) and CCR2 cells (k) across the three groups. l The median number of intra-tumoral tertiary lymphoid structures (TLS) did not differ significantly between groups. A representative mIHC image illustrates the rare presence of TLS adjacent to tumour glands in a No_NAT sample. m Immune cell type enrichment analysis using xCell deconvolution revealed no difference in CD4 + Th1 T cells (m(a)), but a significantly lower enrichment of CD4 + Th2 T cells (p = 0.029) (m(b)) and myeloid dendritic cells (p = 0.032) (m(e)) in the FFX + SBRT group compared to FFX. A significant enrichment of M1 tumour-associated macrophages (TAMs) (p = 0.0045) (m(c)) and M2-TAMs (p = 0.024) (m(d)) was also observed in FFX + SBRT vs FFX group. No_NAT no neoadjuvant treatment group, FFX FOLFIRINOX, SBRT stereotactic body radiotherapy, NS non-significant, IHC immunohistochemistry, mIHC multiplex immunohistochemistry, TLS tertiary lymphoid structure.