Morphology-guided transcriptomic analysis of human pancreatic cancer organoids reveals microenvironmental signals that enhance invasion
Yea Ji Jeong, Hildur Knutsdottir, Fatemeh Shojaeian, Michael G. Lerner, Maria F. Wissler, Elodie Henriet, Tammy Ng, Shalini Datta, Bernat Navarro-Serer, Peter Chianchiano, Benedict Kinny-Köster, Jacquelyn W. Zimmerman, Genevieve Stein-O’Brien, Matthias M. Gaida, James R. Eshleman, Ming-Tseh Lin, Elana J. Fertig, Andrew J. Ewald, Joel S. Bader, Laura D. Wood
Yea Ji Jeong, Hildur Knutsdottir, Fatemeh Shojaeian, Michael G. Lerner, Maria F. Wissler, Elodie Henriet, Tammy Ng, Shalini Datta, Bernat Navarro-Serer, Peter Chianchiano, Benedict Kinny-Köster, Jacquelyn W. Zimmerman, Genevieve Stein-O’Brien, Matthias M. Gaida, James R. Eshleman, Ming-Tseh Lin, Elana J. Fertig, Andrew J. Ewald, Joel S. Bader, Laura D. Wood
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Research Article Gastroenterology Oncology

Morphology-guided transcriptomic analysis of human pancreatic cancer organoids reveals microenvironmental signals that enhance invasion

Abstract

Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared with matched noninvasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified 3 distinct transcriptomic groups in invasive organoids, 2 of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGF-β1, IL-6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.

Authors

Yea Ji Jeong, Hildur Knutsdottir, Fatemeh Shojaeian, Michael G. Lerner, Maria F. Wissler, Elodie Henriet, Tammy Ng, Shalini Datta, Bernat Navarro-Serer, Peter Chianchiano, Benedict Kinny-Köster, Jacquelyn W. Zimmerman, Genevieve Stein-O’Brien, Matthias M. Gaida, James R. Eshleman, Ming-Tseh Lin, Elana J. Fertig, Andrew J. Ewald, Joel S. Bader, Laura D. Wood

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Figure 6

Identification and experimental evaluation of distinct ligand-receptor interactions between fibroblasts and tumor cells belonging to tG1.

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Identification and experimental evaluation of distinct ligand-receptor i...
(A) A circos plot showing the 4 ligands that were tested experimentally and their target genes. (B) List of fibroblast ligand target genes and activation status in tG1 (n = 8), tG2 (n = 9), and neither (n = 7) group tumors. (C) Timeline of ligand treatment assay. (D) Representative images of the control, IL-6–, MMP9-, TGF-β1–, and CXCL12-treated collective and mesenchymal organoids after the ligand treatment assays. Scale bars: 50 μm. (E) Proportion of invasive (dark blue) and noninvasive (light blue) organoids per ligand condition (n = 12). (F) A metric of organoid invasive morphology (log2[1/circularity]) for control and ligand-treated mesenchymal (n = 9) and collective organoid cultures (n = 4). ****P ≤ 0.0001 by Mann-Whitney U test. Each dot represents a PDAC organoid; data are shown as box-and-whisker plots, with the box boundaries representing the 25th to 75th percentiles, horizontal lines representing medians, and whiskers that extend to 1.5 × IQR. Dashed line shows average shape metric across all 4 treatment groups. (G) qRT-PCR showing upregulation in mRNA levels of tG1 genes (MXRA8, SPARC, TGFBI) and downregulation in mRNA levels of tG2 genes (RND1, LGR4, CYP1A1) after ligand treatment assay (presented as mean ± SD for 12 patients). Day 0 samples and untreated controls showed no significant differences in mRNA expression levels of the genes (Student’s t test, P > 0.05); however, the differences between the control and all treated organoids were significant (Student’s t test, P < 0.01), except for the MXRA8 gene in MMP9-treated cultures (Student’s t test, P > 0.05). (H) A prediction of downstream genes in the IL-6 pathway