Alos Diallo

Violin plot; overall abundance

Protein abundance by compartment

Figure 4 from Spatially Resolved, Multiregion Proteomics for Prediction of Immunotherapy Outcome in Deficient Mismatch Repair Metastatic Colorectal Cancer

Purpose:Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer.Experimental Design:Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression.Results:Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts.Conclusions:Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

The complex interplay between metal abundance, transport mechanisms, cell distribution, and tumor progression-related biological pathways (e.g. metabolism, collagen remodeling) remains poorly understood. Traditionally, genes and metals have been studied in isolation, limiting insights into their interactions. Recent advances in spatial transcriptomics and elemental profiling now enable comprehensive exploration of tissue-wide metal-gene interactions, though integration remains challenging. In this proof-of-concept study, we investigated metal-dependent signaling within the tumor microenvironment of a unique colorectal cancer (CRC) tumor. We implemented a spatial multimodal workflow which integrated elemental imaging, gene expression, cellular composition, and histopathological features to uncover metals-related pathways through spatially resolved gene expression correlation analyses. Preliminary findings revealed significant associations, for instance: elevated iron correlated with mesenchymal phenotypes located at the tumor’s proliferative front, correlating with expression of genes involved in the epithelial-to-mesenchymal transition pathways, and extracellular matrix remodeling. Preliminary observations from this single sample revealed that high copper concentrations were localized to regions of active tumor growth and were associated with increased expression of immune response genes. This proof-of-concept workflow demonstrates the feasibility of integrating elemental imaging with spatial transcriptomics to identify metals-based gene correlates. Future application of this workflow to larger patient cohorts will pave the way for expansive comparisons across the metallome and transcriptome, ultimately identifying novel targets for tumor progression biomarkers and therapeutic interventions.

Protein abundance by compartment

Violin plot; overall abundance