# Assessment of Tumor Margin and Heterogeneity of Colorectal Cancer Using Imaging Mass Spectrometry and Image Segmentation

## TL;DR

This study uses imaging mass spectrometry and image segmentation to better understand the molecular and morphological heterogeneity of colorectal cancer.

## Contribution

The study demonstrates that low mass range IMS with image segmentation can depict tumor margins and heterogeneity in CRC.

## Key findings

- IMS in the 800–1000 Da range with binary segmentation accurately identified tumor margins.
- Denary segmentation revealed detailed molecular heterogeneity within and between CRC patients.
- Most m/z signals were patient-specific, highlighting interindividual molecular variation in CRC.

## Abstract

There is an increasing need for methods that provide improved insight into the molecular basis of colorectal cancer (CRC) and, in conjunctions with that, a better understanding of its morphological heterogeneity. Hematoxylin and eosin staining does not provide detailed information about the intratumor proteome and metabolome differences that may affect diagnostic evaluation and treatment. Imaging mass spectrometry (IMS) represents a novel technology well-suited for the analysis of tumor tissue that enables phenotyping of malignant diseases. This study aimed to assess whether the greater image information content provided by low mass range IMS can depict CRC heterogeneity and tumor margins.

Background/Objectives: There is an increasing need for methods that provide improved insight into the molecular basis of colorectal cancer (CRC) and thus a better understanding of its morphological heterogeneity. The objectives of this study were to evaluate the use of imaging mass spectrometry (IMS) to examine tumor margins and gain insight into the molecular heterogeneity of CRC. Methods: An observational study involving 10 cases was conducted. Native tissue samples were collected during the subject’s surgery, and consecutively taken tissue sections were immediately prepared for light microscopic and IMS analysis. IMS was performed across the 200–1000 Da mass range, divided into four sub-ranges, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) in both negative and positive modes of ionization. For tumor margin and tissue heterogeneity assessment, image segmentation was used. Segmented MS images were analyzed against the respective light microscopy images of hematoxylin-eosin-stained tissue sections. Results: Quantitative analysis of the sample collection indicated that IMS enabled correct recognition of tumor margin in the 800–1000 Da range using binary segmentation. Denary image segmentation depicted tissue heterogeneity in greater detail. The strongest m/z signals specific to tumor, peritumor, and margins were identified and tentatively annotated: aside from dCTP, all other compounds were patient-specific, indicating interindividual variations in the molecular composition of CRC. Conclusions: IMS provides new insights into the morphological and biochemical properties of CRC: binary segmented MS images can clearly depict the tumor margin in the 800–1000 Da range, while denary segmented MS images depict intra- and inter-individual molecular heterogeneity of CRC.