Researchers are using multiplex 3D imaging to conduct molecular and morphological evaluations of colorectal cancer


In a recent study published in cellIn this study, the researchers used multidimensional and 3D imaging with machine learning and spatial statistics to perform molecular and morphological assessments of colorectal cancer (CRC).

The study: a 3D multiplex atlas of state mutations and immune interaction in colorectal cancer.  Image credit: Anatomy Image/Shutterstock
Stady: A 3D multiplex atlas of state mutations and immune interaction in colorectal cancer. Image credit: Anatomy Image/Shutterstock

Solid tumors, in the advanced stage, are complex internalizations of tumor cells, stromal cells, and immune cells with significant histopathological differences within the tumor. Conventional histopathological analysis provides insufficient data for precision medicine and mechanistic studies. Spatial tumor atlases can build on the histological basis and contemporary tumor genetics by acquiring in-depth 3D morphological and molecular data.

about studying

In this study, the researchers performed a new, highly multi-disc 3D tissue imaging analysis to construct CRC atlases that provide data at subcellular resolution.

Images were segmented, and their fluorescence intensities were measured to generate data at the single-cell level on cellular type, status, and cell-cell interactions. 3D reconstructions of serial tissue sections were prepared and supervised through machine learning. Two methods were used to analyze the tumor cells, i.e., ‘bottom-up’ approach and ‘top-down’ approach.

The ‘bottom-up’ approach involved the use of spatial statistics to enumerate cell type, assess cellular interactions, establish local neighborhoods, and make use of tools used in single-cell analyzes such as cytometry and single-cell RNA sequencing (scRNA-seq). In contrast, a ‘top-down’ approach involved annotation of histological features or histological patterns related to disease states or outcomes, followed by multiplex data computations to identify underlying patterns at the molecular level.

High-array periodic immunofluorescence (CyCIF) analysis and hematoxylin and eosin (H&E)-stained CRC images obtained through histopathological analysis were combined with scRNA-seq analysis and microregion transcriptome analysis. Furthermore, CyCIF images were analyzed by t-SNE (t-distributed random neighbor embedding) and transcriptional microregions were analyzed by PCA (principal component analysis). In addition, a virtual tissue microarray analysis (vTMA) was performed.

results

Multiplex analysis showed mixed tumor morphologies and molecular gradients. The different cancer cellular features are observed as large, interconnected structures. Three-dimensional TLS (high lymphoid architecture) networks showed pattern differences within the TLS. The interaction of PD1 (programmed cell death protein 1)-PDL1 (programmed death ligand-1) was mainly observed between T lymphocytes and myeloid cells in the colorectal cancer cohort.

The analyzes showed frequent transitions between tumor morphology, a few of which were concurrent with broad gradients in epigenetic regulator and oncogene expression. At the invasive edges of the tumour, sites of competition between tumor cells, normal cells, and immune cells, the number of T lymphocytes, which include several cell types, was reduced. Apparently localized 2D CRC properties such as TLS were found to be correlated with molecular gradients and significantly larger in 3D analysis.

The tumor microenvironment (TME) showed a spatial organization spanning more than three to four orders of magnitude. The tumor showed invasive margins of the nascent type, mucosal invasive edges, and deeply extending invasive margins of the pusher type, extending the tumor into the underlying connective tissue and smooth muscle. Cell divisions in 75 WSI images showed that CK+ (cytokeratin positive) cells of normal and tumor epithelium were separated from a differentiation group 31+ (CD31+) of endothelial cells, primarily vascular, desmin-positive stroma cells, and CD45-positive immune cells.

The immune cells observed within the tumor consisted of CD8+ and apoptosis protein-1 expressing cytotoxic T-type lymphocytes, CD4-expressing T-type lymphocytes, macrophages expressing CD68 and/or CD163, and B lymphocytes expressing about CD20. In addition, subclasses such as CD4+FOXP3+ Tregs (regulatory T lymphocytes) were observed. Solid adenocarcinomas showed the highest percentage of cytokeratin-positive tumor cells (70.0%), while their adjacent normal epithelial tissues had the lowest number of cytokeratin-positive cells (25.0%) and showed abundance of immune cells and stromal cells.

In colorectal cancer (CRC) 1–17, the observed correlation lengths ranged from 80.0 mm for the 31-positive differentiation group and 400.0 mm for CD20- or keratin-positive. Lengths were associated with recurrent histological features, including small capillaries in size among the 31-positive differentiation group, tumor sheets for cytokeratin-positive cell types, and third lymphoid structures for the 20-cell-positive differentiation group.

The results of the hypothetical TMA and the real TMA were comparable, and the CyCIF results matched the theoretical data. Consistent with kNN-ranked modeling of the CyCIF analysis data, graded transitions from low-grade mucosal/glandular to high-grade budding/segmented tissues were observed between the neoplastic/epithelial and immune/stromal tissue compartments. CyCIF markers showed gradient intensities within the entire tumor or in association with local morphological gradients.

The results indicated that the mesothelial- and tuberculosis-like transitions of CRC1 were characterized by the formation of large fibrous structures that appear as small-sized buds in transverse sections at the distal periphery. The fibers can invade different environments, including mucin and stroma, which appear to be formed by the progressive rupture of cellular adhesion associated with graded epithelial and mesenchymal transitions. The team observed reduced tumor proliferation in buds and greater proliferation in the deep, invasive tumor margins, with varying levels of activation of the phosphotyrosine pathway, and immune suppression.

Overall, the results of the study showed that multiplex WSI analysis characterizes graded and mixed molecular and morphological features in human CRC samples, highlighting the significant and distinct structural features and differences within TLS in the spatial patterns of tumors.



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