The establishment of patient-derived pancreatic cancer organoid culture in recent years creates an exciting opportunity for researchers to perform a wide range of in vitro studies on a model that closely recapitulates the tumor. One of the outstanding question in pancreatic cancer biology is the causes and consequences of genomic heterogeneity observed in the disease. However, to use pancreatic cancer organoids as a model to study genomic variations, we need to first understand the degree of genomic heterogeneity and its stability within organoids. Here, we used single-cell whole-genome sequencing to investigate the genomic heterogeneity of two independent pancreatic cancer organoid lines, as well as their genomic stability with extended culture. Clonal populations with similar copy number profiles were observed within the organoids, and the proportion of these clones was shifted with extended culture, suggesting the growth advantage of some clones. However, sub-clonal genomic heterogeneity was also observed within each clonal population, indicating the genomic instability of the pancreatic cancer cells themselves. Furthermore, our transcriptomic analysis also revealed a positive correlation between copy number alterations and gene expression regulation, suggesting the “gene dosage” effect of these copy number alterations that translates to gene expression regulation.

The establishment of patient-derived pancreatic cancer organoid culture in recent years creates an exciting opportunity for researchers to perform a wide range of in vitro studies on a model that closely recapitulates the tumor. Among the outstanding questions in pancreatic cancer biology are the causes and consequences of genomic heterogeneity observed in the disease. However, to use pancreatic cancer organoids as a model to study genomic variations, we need to first understand the degree of genomic heterogeneity and its stability within organoids. Here, we used single-cell whole-genome sequencing to investigate the genomic heterogeneity of two independent pancreatic cancer organoids, as well as their genomic stability with extended culture. Clonal populations with similar copy number profiles were observed within the organoids, and the proportion of these clones was shifted with extended culture, suggesting the growth advantage of some clones. However, sub-clonal genomic heterogeneity was also observed within each clonal population, indicating the genomic instability of the pancreatic cancer cells themselves. Furthermore, our transcriptomic analysis also revealed a positive correlation between copy number alterations and gene expression regulation, suggesting the functionality of these copy number alterations.

Genomic heterogeneity in pancreatic cancer has been widely studied in recent years because of the development of next generation of genome-wide sequencing technology. However, little is known whether this genomic heterogeneity will drive phenotypic heterogeneity and the underlying mechanism that links genomic variations to phenotypic heterogeneity in pancreatic cancer. To study these questions, by using single-cell flow sorting, we established four clones from MiaPaCa-2, a cell line derived from human pancreatic primary tumors. Indeed, these four MiaPaCa-2 clones show heterogenous phenotypes including distinct cellular morphology and differential proliferation and migration rate. Further genomic DNA sequencing analysis of MiaPaCa-2 clones demonstrates genomic heterogeneity, suggesting heterogeneity of the population or genomic instability. Lastly, our mRNA-sequencing analysis of the four MiaPaCa-2 clones identified that ITGAV is necessary and sufficient for morphology change of MiaPaCa-2 subclones, suggesting that ITGAV might be a major regulator in controlling the heterogeneity in MiaPaCa-2 cell line. In conclusion, our study demonstrated the heterogeneity of pancreatic cancer cells at genomic, transcriptomic, and phenotypic levels.

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Pancreatic cancer adenocarcinoma (PDAC) has been reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and non-malignant cells such as inflammatory cells, cancer-associated fibroblasts (CAF), and lymphatic and blood vessels. Here, we decoupled the roles of the extracellular matrix on PDAC cell lines by investigating the effects of different ECM proteins on the cell lines. Our data showed that primary lines have different morphology that depends on the ECM proteins on which they are cultured, while metastatic PDAC lines’ morphology does not change with respect to different ECM proteins. Next, we examined how these ECM proteins affect the cell response to the gemcitabine’s cytotoxicity. Lastly, transcriptomics analysis of the cells cultured on different ECM reveals the regulation of various pathways, such as cell cycle, cell adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.