12/28/2023 0 Comments Tams analyzer tutorialFACS allows for single-cell analysis and is highly quantitative however, it requires a large number of cells and, due to its reliance on fluorescent tags, issues including autofluorescence and spectral spillover can lead to loss of resolution. Although IHC can provide spatial information on the cells within a tumor section, it is low throughput and not quantitative. Both techniques require high-quality antibodies against known cell type-specific marker proteins. Traditionally, measuring and phenotyping of cells in a solid tissue is done using immunohistochemical staining (IHC) and/or flow cytometry. The accurate initial quantitative measurement of these components and the on-going effects of these stoma-targeted agents are critical to their successful development and therapeutic optimization. The rationale behind the development of these agents is that they will modulate the TME to a less fibrotic and/or less immunosuppressive state and thus lead to improved drug penetration and/or antitumor T cell infiltration. Those agents either target the non-cellular components such as extracellular proteins (e.g., recombinant hyaluronidase that degrades hyaluronan) or aim to modulate the activity of certain stromal cell types such as cancer-associated fibroblasts and immune cells. Recently, many drug development programs focus on developing stroma-remodeling agents for PDAC. The highly reactive and dense stroma contributes to the aggressiveness and drug resistance of PDAC, hence leading to the high mortality rate of the disease. Pancreatic ductal adenocarcinoma (PDAC), which accounts for > 90% of all pancreatic cancer cases, is one of the solid tumor types known to have a highly inflammatory and desmoplastic TME. The non-cellular components of TME include the extracellular matrix (ECM) and the signaling molecules produced by the cancer cells and stromal cells. The cellular components of TME (i.e., stromal cells) usually contain cells of hematopoietic origin (e.g., immune cells) and cells of mesenchymal origin (e.g., fibroblasts). The tumor microenvironment (TME) which includes cellular and non-cellular components plays an important role in the progression, metastasis, and drug resistance of the tumors. These findings provide valuable insights on the PDAC microenvironment and could potentially inform the management of PDAC patients. Furthermore, the cellular composition was an important factor in defining PDAC subtypes and significantly correlated with patient outcome. Taken together, our work identifies significant heterogeneity in cellular compositions of PDAC tumors and between primary tumors and metastatic lesions. Furthermore, the expression levels of cell type-specific markers for EMT + cancer cells, activated CAFs, and endothelial cells significantly associated with patient survival. We found that the tumor cellular composition was an important factor in defining the PDAC subtypes. ![]() Immune infiltration varies significantly from patient to patient with majority of the immune cells being macrophages and exhausted lymphocytes. The cancer cells showed high inter-patient heterogeneity, whereas the stromal cells were more homogenous across patients. Our single-cell RNA sequencing analysis revealed distinct cell types in primary and metastatic PDAC tissues including tumor cells, endothelial cells, cancer-associated fibroblasts (CAFs), and immune cells. The expressions of the cell type-specific signature genes were also correlated with patient survival using public datasets. The expression signatures of the different cell types were then compared between primary tumors and metastatic biopsies. ![]() Unsupervised clustering analysis as well as a new supervised classification algorithm, SuperCT, was used to identify the different cell types within the tumor tissues. In this study, we employed a single-cell RNA sequencing technology to profile the transcriptomes of individual cells from dissociated primary tumors or metastatic biopsies obtained from patients with PDAC. However, almost all of those studies used primary tumor tissues. Single-cell transcriptomics has been used to study the cellular composition of different solid tumor types including PDAC. ![]() Detailed characterization of the cellular composition of the tumor microenvironment is critical to the understanding of the disease and treatment of the patient. Solid tumors such as pancreatic ductal adenocarcinoma (PDAC) comprise not just tumor cells but also a microenvironment with which the tumor cells constantly interact.
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