Multimodal Imaging Study of the Tumor Microenvironment in Colorectal Cancer: Revealing Spatial Heterogeneity

Academic Background

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, with its complexity and heterogeneity posing significant challenges for treatment and prognosis prediction. The tumor microenvironment (TME) plays a crucial role in cancer progression, metastasis, and treatment response, particularly the influence of collagen in the extracellular matrix (ECM) on tumor pathophysiology. However, traditional histological, colonoscopic, and molecular screening methods cannot fully characterize the spatial complexity of tumor tissues, such as the interplay between the cancer proteome, collagen structure, and nuclear distribution.

To gain a deeper understanding of the heterogeneity of CRC, this study proposes a multimodal imaging strategy that combines two-photon laser scanning microscopy (2PLSM), histology, and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to reveal the spatial heterogeneity of the CRC tumor microenvironment. This research is the first to correlate the structural coherence of collagen fibers and the nuclear distribution profile of tumor tissue with peptide signatures, providing new insights into the pathophysiology of CRC.

Source of the Paper

This paper was jointly completed by a research team from the Max Planck Institute for Multidisciplinary Sciences in Germany, the Institute of Pathology at the University Medical Center Göttingen, and the Berlin Institute of Health. The main authors include Bharti Arora, Ajinkya Kulkarni, Andrea M. Markus, and others. The paper was published in 2024 in the journal npj Imaging.

Research Process

1. Sample Collection and Processing

The study included tumor tissue samples from 14 CRC patients, with detailed records of the anatomical location, tumor stage, and morphological characteristics. All samples were collected and used in accordance with the approval and regulations of the Ethics Committee of the University Medical Center Göttingen.

2. Label-Free Imaging

Two-photon laser scanning microscopy (2PLSM) was used to image tissue sections, capturing the second harmonic generation (SHG) signal from collagen fibers and the two-photon excitation autofluorescence (2PEF) signal from the tissue. These signals allowed researchers to visualize the collagen fiber structure within the tissue at sub-micron resolution without the need for exogenous fluorescent dyes.

3. MALDI-MSI Sample Preparation

Tissue samples were fixed and paraffin-embedded, then cut into 5 µm thick sections and mounted on conductive glass slides. After 2PLSM imaging, the samples were prepared for MALDI-MSI analysis. The samples underwent deparaffinization, antigen retrieval, and tryptic digestion before MALDI-MSI imaging.

4. MALDI-MSI Imaging

MALDI-MSI imaging was performed using the Rapiflex® MALDI TissueTyper® from Bruker Daltonik GmbH, with a detection range of 800-3200 m/z, 500 laser shots per spot, a sampling rate of 1.25 GS/s, and a raster width of 50 µm.

5. Protein Identification

Protein identification was performed on adjacent tissue sections using nano-liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). Peptides were separated using a C18 column and detected using a mass spectrometer. Peptide identification was conducted by searching the human Swiss-Prot database.

6. Histological Analysis

After MALDI-MSI imaging, tissue sections were stained with hematoxylin and eosin (H&E) and scanned using a whole-section scanner. H&E images were used to annotate tumor regions in QuPath software.

7. Image Processing

The SHG signal was extracted from 2PLSM images, and texture analysis was performed to generate a heatmap of collagen fiber coherence. Nuclei were segmented from H&E images, and a heatmap of nuclear distribution was generated. These heatmaps were used for further analysis of proteomic features in different regions.

Main Results

1. Proteomic Signature Differences Between Left- and Right-Sided Colorectal Cancer

Univariate analysis of MALDI-MSI data revealed differentially distributed peptides between left-sided (LSCC) and right-sided (RSCC) colorectal cancer tissues. A total of 520 m/z peaks were identified, of which 203 m/z values corresponded to 83 proteins. Receiver operating characteristic (ROC) analysis identified 76 m/z values with significant differences between LSCC and RSCC.

2. Proteomic Features in High Nuclear Density Regions

The study found that high nuclear density (HND) regions occupied a significantly smaller area compared to low nuclear density (LND) regions in both LSCC and RSCC tissues. ROC analysis identified 14 m/z values with significant differences between HND and LND regions, corresponding to 7 proteins.

3. Proteomic Features in Collagen Fiber Organization

Analysis of collagen fiber organization using 2PLSM revealed that the percentage of chaotic collagen regions was significantly higher than organized regions in both LSCC and RSCC tissues. ROC analysis of MALDI-MSI data identified 43 m/z values with significant differences between chaotic collagen regions in LSCC and RSCC, corresponding to 19 proteins.

Conclusion

This study developed a novel multimodal imaging approach that combines 2PLSM, histology, and MALDI-MSI to characterize the spatial heterogeneity of the CRC tumor microenvironment. By quantifying peptide and imaging features in LSCC, RSCC, and healthy tissues, the research revealed the heterogeneity of the TME and distinguished peptide features in spatially distinct regions of LSCC and RSCC. This study provides new insights into the progression, metastasis, and treatment response of CRC and lays the foundation for future development of novel therapeutic targets.

Research Highlights

1. Multimodal Imaging Strategy: For the first time, 2PLSM, histology, and MALDI-MSI were combined to comprehensively characterize the spatial heterogeneity of the CRC tumor microenvironment.
2. Proteomic Signature Differences: Revealed differences in proteomic signatures between LSCC and RSCC tumor tissues, providing potential targets for personalized therapy.
3. Impact of Collagen Fiber Structure: The study highlighted the important role of collagen fiber organization in tumor pathophysiology, offering new directions for future research.

Research Significance

This study not only provides new insights into the pathophysiology of CRC but also lays the foundation for the future development of personalized therapeutic strategies based on the tumor microenvironment. By combining multiple imaging techniques, researchers can gain a more comprehensive understanding of tumor heterogeneity, thereby offering more precise guidance for clinical diagnosis and treatment.