Abstract 2709: Mass spectrometry imaging for the assessment of intratumor molecular heterogeneity in canine metastatic mammary carcinomas: A comparative approach for biomarker discovery
Authors: Cordeiro Yonara G. , Heijs Bram P.A.M , Mulder Leandra M. , van Zeijl René J. , van Veelen Peter , de Ru Arnoud , Strefezzi Ricardo F. , Fukumasu Heidge 
Published: September 2020

Abstract
A reliable animal model that captures the key features of human cancer is essential to bring new insights for oncology research. In this context, spontaneously occurring canine cancers are excellent models to study tumor development and progression, since dogs share the same environment as humans and present many similarities regarding anatomic, histological, molecular and clinical features. Here, we aimed to identify and characterize intratumor heterogeneity using mass spectrometry imaging (MSI) of proteolytic peptides in order to simultaneously detect morphological and molecular alterations whilst preserving tissue integrity, directing the search for cancer biomarkers. Samples were obtained from five dogs diagnosed with metastatic disease, and MALDI-FT-ICR-MSI data of 17 primary tumors were co-registered to hematoxylin and eosin stained slides. Unsupervised clustering of tumor spectra was performed using the bisecting k-means method in SCiLS Lab (Bruker Daltonics, DE) and two molecularly distinct clusters (CL1 and CL2) were selected regardless morphological presentation. We also performed a supervised analysis, by annotating (FlexImagingTM, Bruker Daltonics, DE) and classifying tumor regions of interest into low- and high-grade populations according to previously established morphological criteria. LC/MS-MS of extracted peptides was used for protein identity assignment based on mass matching, and functional enrichment was performed using a human protein-coding database. Discriminative peaks were identified using ROC and Wilcoxon methods. M/z signals presenting AUC &gt; 0.7 and FDR &lt; 0.05, and biological functions with FDR &lt; 0.05 were considered significant. We found 266 features discriminating low- and high-grade tumor populations, resulting in 44 protein IDs, and 185 features significantly different between CL1 and CL2, with 35 IDs retrieved. CANX, RSPA5 and PDIA3, found down-regulated in high-grade compared to low-grade regions, enriched for protein folding in endoplasmic reticulum process, suggesting that morphology changes may have arisen from protein misfolding due to reticular stress. Both protein sets enriched for cell adhesion molecule binding, cadherin binding and structural molecule activity. HMGN3, HNRNPC and histones H2AFY and HIST1H1C, resulting from the comparison of clusters CL1 and CL2, also enriched for nucleosome binding and chromatin DNA binding, alterations that could have not been detected only by conventional histopathological evaluation. Ongoing analyses aim to further characterize the intratumor molecular heterogeneity of microscopically indistinct populations and its association to cancer progression and metastasis.
Citation Format: Yonara G. Cordeiro, Bram P.A.M Heijs, Leandra M. Mulder, René J. van Zeijl, Peter van Veelen, Arnoud de Ru, Ricardo F. Strefezzi, Heidge Fukumasu. Mass spectrometry imaging for the assessment of intratumor molecular heterogeneity in canine metastatic mammary carcinomas: A comparative approach for biomarker discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2709.