Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the col-lective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4mutant PDAC organoids required exogenous TGFb, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFb signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFb-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss.Significance: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.

Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution

“…Transcripts upregulated in cell fate 1 showed enrichment of epithelial-mesenchymal transition, cell growth, reactive oxygen species (ROS) biosynthesis, and axon development, while transcripts in cell fate 2 were enriched for telomere maintenance, DNA replication, cell cycle, and hypoxia pathways (Figure 4C). To test whether there were genomic alterations accompanying the transcriptomic divergence, we inferred copy number aberrations from the scRNA-seq data using the inferCNV package [56], which revealed a chromosome 16p gain in the later passage PDO, and an additional chromosome 19q loss specifically in cell fate 2 (Figure 4D).…”

Organoids in cancer research: a review for pathologist‐scientists

“…(A) Organoids are particularly well‐suited to study phenotypes that are best appreciated in 3D, such as tumor cell invasion. Invasion in breast and pancreatic cancer organoids has been studied to identify molecular features of the most invasive cells and pharmacological and genetic perturbations that can alter the invasion phenotype [10,11]. (B) Genomic manipulation of organoids derived from normal pancreas or colon tissue investigated the phenotypic consequences of driver gene mutations, recapitulating multistep tumorigenesis in vitro [12–14].…”

The Small GTPase RAC1B: A Potent Negative Regulator of-and Useful Tool to Study-TGFβ Signaling

“…Organoids from cancers with somatic mutations in DPC4 exhibited collective invasion but nevertheless required exogenous TGFβ. Interestingly, RAC1 and CDC42 were identified as potential mediators of TGFβ-stimulated collective invasion in the DPC4 -mutant PDAC organoids [ 8 ].…”

“…RAC1-driven cancer cell invasion involves both single-cell and collective modes of migration. Single-cell migration is the predominant type of cell movement in poorly differentiated, mesenchymal tumors [ 6 ], whereas collective migration is more representative of differentiated epithelial tumors [ 7 , 8 ].…”