MALDI imaging combined with two-photon microscopy reveals local differences in the heterogeneity of colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, accentuated by its heterogeneity and complex tumour microenvironment (TME). The role of TME on tumour pathophysiology is pivotal, especially the influence of components of the extracellular matrix (ECM), such as collagen. We introduce a novel multimodal imaging strategy to unravel the complex spatial heterogeneity of CRC by integrating the imaging features from two-photon laser scanning microscopy (2PLSM) and histology with proteomics signatures from matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI MSI). Our study is the first to correlate the structural coherence of collagen fibres and the nuclei distribution profile of tumour tissue with the peptide signatures, offering insights into the proteomic landscape of CRC within regions of high nuclei distribution (HND), as well as chaotic and organised regions of collagen. We use this approach to distinguish the patient tissues originating from left-sided colorectal cancer (LSCC) and from right-sided colorectal cancer (RSCC). This discriminative signature highlights the role of high nuclei distribution and collagen architecture in tumour progression. Complementary m/z values of several proteins associated to components of ECM, such as plectin, vinculin, vimentin, and myosin, have shown differentially intensity distributions between LSCC and RSCC. Our findings demonstrate the potential of combining structural information with peptide features to identify molecular signatures in different tumour regions and retrieve new insights into CRC pathophysiology.