Abstract 5322: Spatial profiling of the tumor microenvironment in colorectal cancer: Insights into immune, tumor, and stromal interactions using high-dimensional protein analysis

Abstract Introduction: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths globally, claiming over 900, 000 lives annually. In the US, CRC disproportionately affects certain populations, with higher prevalence among racial and ethnic minorities. The tumor microenvironment (TME) plays a pivotal role in CRC progression, therapeutic response, and overall patient outcomes. However, the spatially intricate interactions within the TME that distinguish effective from ineffective tumor control remain poorly understood. Addressing these gaps is essential for enhancing our understanding of spatial heterogeneity and advancing immunotherapy development. Methodology: To investigate these dynamics, we employed a multi-modal approach using re-engineered co-detection by indexing (CODEX) on publicly available data (Schürch et al., 2020) from 35 late-stage CRC patients. Our systematic workflow classified cells, analyzed protein expression, and spatially represented the data. Cells were grouped into three main classes—stroma, tumor, and immune—based on structural characteristics and functional roles. Protein expression analysis was conducted by integrating data for individual surface protein expression, enabling direct comparisons between the stroma, tumor, and immune classes. Through this framework, 56 protein markers were profiled to identify conserved cellular neighborhoods (CNs) that characterize the spatial and functional organization within the CRC TME. Results: Protein expression analysis revealed distinct patterns across the stroma, tumor, and immune classes for the biomarkers profiled. These markers exhibited differential expression profiles across the three classes, highlighting their unique roles within the TME. For instance, immune markers such as CD3, CD8, CD68, and PD-1 showed significant variation between the tumor and immune classes, underscoring their spatial organization and functional importance within the TME. Similarly, functional markers such as MMP9 and Ki-67 demonstrated distinct expression dynamics, reflecting their roles in tumor progression and immune responses. Auxiliary markers, including cytokeratins, highlighted structural differences unique to the stroma class. These results provided a comprehensive understanding of the spatial and functional interplay between immune, tumor, and stromal components in the CRC TME. Conclusion: This study provides a deeper understanding of protein expression within the CRC TME, enhancing the classification and spatial characterization of TME. These results provide key insights into spatial heterogeneity within the TME through distinct protein expression profiles. These findings provide valuable guidance for future research in drug discovery, with the potential to significantly advance the field of precision oncology. Citation Format: Enrique Velazquez Villarreal, Ei-Wen Yang, Fernando C. Diaz, Maria Ninova. Spatial profiling of the tumor microenvironment in colorectal cancer: Insights into immune, tumor, and stromal interactions using high-dimensional protein analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5322.