Jichi Medical University and others develop new fluorescence technology to visualize disease causing cell-cell interactions

A research team led by Associate Professor Takahiro Kuchimaru, Professor Norihiko Takeda, and Professor Satoshi Nishimura of the Center for Molecular Medicine at Jichi Medical University, in collaboration with PhD Student Misa Minegishi of the School of Life Science and Technology at Tokyo Institute of Technology (at the time of the research, currently a JSPS postdoctoral fellow at the RIKEN Cluster for Pioneering Research), Visiting Professor Shinae Kizaka-Kondoh, and Team Leader Hirofumi Shintaku of the RIKEN Cluster for Pioneering Research (RIKEN Hakubi Research), developed a new analytical method for identifying, from out of several thousand candidate molecules, the proteins that mediate possible disease-causing cell-cell interactions.

This analytical method combines the technique of optical labeling of cells in the vicinity of a specific cell and single-cell omics analysis and can be widely applied to the analysis of disease mechanisms involving cell-cell interactions. The optical labeling was based on the extracellular reconstitution of green fluorescent protein (GFP). Using this technology in a mouse model for reproducing the cancer metastasis process, the researchers found that the protein galectin-3 may mediate the interaction between cancer cells and hepatic parenchymal cells to promote liver metastasis (the spread of cancer to the liver). The results have been published in Nature Communications.

Cells in the human body maintain tissue homeostasis (balance/stability) by interacting with each other via proteins presented on the cell membrane surface and humoral factors secreted outside the cell. A breakdown of these cell-cell interactions can result in the development of diseases and their malignant transformation. Clarifying the function of proteins that mediate cell-cell interactions is expected to lead to the development of new therapeutic methods. However, there are thousands of candidate protein molecules that mediate cell-cell interactions, and it is very difficult to identify the important ones among them.

To perform an omics analysis of cell-cell interactions in deep biological tissues, the research group used an approach of optical labeling of cells whereby the target cells that they approach and interact with are highlighted. For example, if it is possible to place an in situ fluorescent marker on a cell in the immediate vicinity of cancer cells in living tissue, the interaction partner can be located in the fractured tissue via this marker. Single-cell omics analysis of the identified target cells will enable the discovery of cell-cell interaction factors through detailed characterization of each individual cell.

To achieve this, the researchers focused on GRAPHIC (an acronym for "glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections"), which fluorescently labels cells in contact with each other (split-GFP fragments are presented on the surface of the interacting cell membrane via peptide chains, and GFP is reconstituted and fluoresces when the cells come into close contact). Although the possibility of using GRAPHIC to detect cancer cell-related cell-cell interactions was investigated, it was found that the cells could not be sufficiently fluorescently labeled.

The researchers speculated that this was because cancer cells are less likely to adopt a close interaction state with neighboring cells, making it difficult for the split-GFP fragments presented on the cell membrane to associate with them.

With this in mind, a new secreted GRAPHIC (sGRAPHIC) system was constructed to secrete one of the split fragments to the outside of the cell. In cultured cell experiments, sGRAPHIC was able to fluoresce only cells that were relatively proximal to cells secreting GFP fragments.

To demonstrate the potential of sGRAPHIC in analyzing the mechanisms of cell-cell interactions mediated by secreted proteins, an experiment was performed in a mouse disease model. Specifically, sGRAPHIC was used in a mouse model of liver metastasis to analyze the interaction between mouse cancer cell lines and liver parenchymal cells. Signals from GFP reconstituted through the interaction between cancer cells and hepatic parenchymal cells were identified around metastatic foci formed in the liver of mice.

Cancer cells and hepatic parenchymal cells were isolated from a fractured liver tissue. The differences in the expression patterns of approximately 3000 different genes expressed in hepatic parenchymal cells at sites close to cancer cells (GFP signal positive) and those at distant sites (GFP signal negative) were compared and analyzed by means of single-cell RNA sequencing. The results showed that the expression of the gene coding for galectin-3, a secreted sugar-binding protein, was upregulated in hepatic parenchymal cells interacting with cancer cells. In fact, through immunohistochemical staining, the presence of galectin-3 protein was also confirmed at the site of contact between cancer cells and hepatic parenchymal cells in mice with liver metastasis.

These results suggest that cell-cell interaction between cancer cells and hepatic parenchymal cells is mediated through galectin-3 and contributes to the formation of metastatic foci. The use of sGRAPHIC is expected to advance our understanding of the cell-cell interaction mechanisms involved in various diseases, including cancer. The research group is proceeding with the implementation of sGRAPHIC technology with the goal of establishing treatment methods based on pathological analysis.

Journal Information
Publication: Nature Communications
Title: Secretory GFP reconstitution labeling of neighboring cells interrogates cell−cell interactions in metastatic niches
DOI: 10.1038/s41467-023-43855-2