Abstract 134: Integrating spatial transcriptomics and second harmonic generation imaging of collagen identifies genes associated with discoidin domain receptor 1 and collagen alignment in colorectal cancer

Abstract Immune checkpoint blockade (ICB) has been shown to be an effective treatment for colorectal cancers (CRCs) highly infiltrated by CD8+ T cells. However, there is a paucity of CD8+ T-cell infiltration in the majority of CRCs. Tumors with highly aligned collagen fibers have been shown to have reduced CD8+ T-cell infiltration. We previously identified a four-gene signature associated with T-cell exclusion in CRC. One of the genes, Transforming Growth Factor Beta Induced (TGFBi), is known to be involved in extracellular matrix regulation. Another, Discoidin Domain Receptor 1 (DDR1), promotes collagen fiber alignment in breast cancer. To determine if TGFBi and DDR1 are associated with collagen alignment in CRC, as well as to identify additional genes involved in collagen alignment, we have developed a method to spatially resolve collagen fiber alignment and integrate it with spatial transcriptomics. To do this, 30 μm thick CRC sections are imaged using a Nikon AX multiphoton microscope by second harmonic generation imaging, which permits label-free imaging of collagen fibers. Spatial heatmaps of the collagen alignment are generated from the resulting images. Spatial transcriptomics is performed on a serial tumor section using the Visium v2 platform and the gene expression matrix is integrated with the collagen alignment heatmap data. As proof-of-principle, two CRC tumors have been analyzed using this method. Highly aligned regions were found to be associated with expression of genes involved in extracellular matrix organization, keloidal collagen and specific fibroblast subtypes. The genes that correlate with collagen alignment will be confirmed at the protein level by multiplexed immunofluorescence on serial sections. This method will provide a rich dataset permitting elucidation of the complex interactions between T-cell exclusion, gene expression and collagen fiber alignment and may support the discovery of new strategies to overcome immune exclusion in CRC. Citation Format: Maxwell Stuart Hamilton, Harsimran Kaur, Alan J. Simmons, Ken S. Lau, Ambra Pozzi, Robery J. Coffey. Integrating spatial transcriptomics and second harmonic generation imaging of collagen identifies genes associated with discoidin domain receptor 1 and collagen alignment in colorectal cancer [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 134.