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Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer

Abstract

Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC1,2,3,4. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT)5,6. Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7, 8, 9, 10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear4,6, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.

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Figure 1: EMT inhibition does not alter primary tumour progression.
Figure 2: EMT inhibition does not alter invasion and metastasis.
Figure 3: EMT inhibition sensitizes tumours to gemcitabine in KPC mice.
Figure 4: EMT inhibition sensitizes tumours to gemcitabine in KTC mice.

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Gene Expression Omnibus

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Gene expression microarray data have been deposited in the Gene Expression Omnibus under accession number GSE66981.

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Acknowledgements

We wish to thank D. Lundy, S. Yang, Z. Xiao, R. Deliz-Aguirre, T. Miyake and S. Lovisa for technical support and K. M. Ramirez and R. Jewell in the South Campus Flow Cytometry Core Laboratory of MD Anderson Cancer Center for flow cytometry cell sorting and analyses (partly supported by NCI grant no. P30CA16672). We also wish to thank E. Chang for scanning slides of histopathological specimens. This study was primarily supported by the Cancer Prevention and Research Institute of Texas. The research in the LeBleu laboratory is supported by UT MDACC Khalifa Bin Zayed Al Nahya Foundation.

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Authors and Affiliations

Authors

Contributions

R.K. conceptually designed the strategy for this study and provided intellectual input. V.S.L. helped design experimental strategy, provided intellectual input, supervised the studies, performed immunohistochemistry and culture experiments, generated the figures and wrote the manuscript. X.Z. performed experiments to generate the genetically engineered mouse models and helped characterize the mouse phenotype, performed culture experiments, collected the tissue for analysis and contributed to the manuscript writing. J.L.C. characterized the mouse phenotype, analysed the data related to the genetically engineered mouse models, collected data, generated the figures and helped with manuscript writing and editing. H.S. performed experiments with mice and injected cancer cells and helped collect tissue, J.Ki., M.S., J.Ka., and C.-C.W. performed experiments and collected data. The data was analysed by J.L.C, V.S.L., X.Z., J.Ki, and C.-C.W.

Corresponding author

Correspondence to Raghu Kalluri.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 EMT inhibition is specific to tumour epithelium.

a, Representative images of haematoxylin and eosin-stained small intestine (SmInt), kidney, and heart (scale bar, 100 μm). b, Pancreatic mass of 29 (KPC), 13 (KPC;TwistcKO) and 28 (KPC;SnailcKO) mice, error bars represent s.d.; one-way ANOVA. c, Merge of Twist1 or Snai1 in situ hybridization (black) followed by CK8 (red) immunolabelling in tumours from KPC and KPC;TwistcKO or KPC;SnailcKO mice, respectively. White arrows highlight positive cells in the stroma, yellow arrows highlight negative epithelium (scale bar, 20 μm). d, Twist or Snail immunostaining in KPC and KPC;TwistcKO or KPC;SnailcKO tumours, respectively. Black arrows highlight positive cells in the stroma, red arrows highlight negative epithelium (scale bar, 20 μm). e, Channel separations of the representative images of αSMA immunolabelling in YFP lineage-traced tumours found in Fig. 1f (scale bar, 50 μm). f, EMT gene expression signature analysis in KPC, KPC;TwistcKO and KPC;SnailcKO cohorts (n = 3 mice). Red arrows indicate reduced Twist1 and Snai1 expression in KPC;TwistcKO and KPC;SnailcKO cohorts, respectively.

Extended Data Figure 2 General suppression of EMT markers does not affect desmoplasia.

a, E-cadherin immunolabelling and quantification of primary KPC (n = 5 mice), KPC;TwistcKO (n = 5 mice) and KPC;SnailcKO (n = 4 mice) (scale bar, 100 μm). b, Zeb2 immunolabelling and quantification of primary KPC (n = 6 mice), KPC;TwistcKO (n = 5 mice) and KPC;SnailcKO (n = 7 mice) (scale bar, 50 μm; inset scale bar, 20 μm). c, Sox4 immunolabelling and quantification of primary KPC (n = 7 mice), KPC;TwistcKO (n = 6 mice) and KPC;SnailcKO (n = 8 mice) (scale bar, 50 μm; inset scale bar, 20 μm). d, Slug immunolabelling and quantification of primary KPC (n = 4 mice), KPC;TwistcKO (n = 4 mice) and KPC; SnailcKO (n = 4 mice) tumours (scale bar, 50 μm; inset scale bar, 20 μm). e, Sirius Red staining and quantification of primary KPC (n = 21 mice), KPC;TwistcKO (n = 8 mice) and KPC;SnailcKO (n = 11 mice) (scale bar, 100 μm; error bars represent s.d.) f, αSMA immunolabelling and quantification of primary KPC (n = 5 mice), KPC;TwistcKO (n = 5 mice) and KPC;SnailcKO (n = 5 mice) (scale bar, 100 μm). g, CD31 immunolabelling and quantification of primary KPC (n = 4 mice), KPC;TwistcKO (n = 4 mice) and KPC;SnailcKO (n = 3 mice) (scale bar, 200 μm, inset scale bar, 100 μm). h, Pimonidazole staining and quantification of primary KPC (n = 4 mice), KPC;TwistcKO (n = 4 mice) and KPC;SnailcKO (n = 4 mice) (scale bar, 100 μm). i, CD3 immunolabelling and quantification of primary KPC (n = 5 mice), KPC;TwistcKO (n = 5 mice) and KPC;SnailcKO (n = 5 mice) (scale bar, 100 μm; inset scale bar, 25 μm). Unless otherwise indicated error bars represent s.e.m., and significance determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Extended Data Figure 3 EMT suppression does not alter epithelial characteristics of metastases.

a, Immunolabelling of primary tumours (n = 3 mice) for αSMA (red), CK8 (green), Ki67 (white) and DAPI (blue); yellow arrows indicate EMT+ cells (scale bar, 20 μm). b, Representative dot plots of circulating YFP+ cells. c, Images of serial sections of KPC;LSL-YFP lung and liver metastasis stained for haematoxylin and eosin or immunolabelled for CK19 or YFP. Yellow dashed box represents magnified areas in panel below (scale bar, 200 μm; magnification scale bar, 50 μm). d, KPC metastatic tumours stained for Twist and Snail (n = 3 mice; scale bar, 20 μm; inset scale bar, 10 μm). e, Zeb1 immunolabelling and quantification of metastatic KPC (n = 4 mice), KPC;TwistcKO (n = 3 mice) and KPC;SnailcKO (n = 4 mice) (scale bar, 50 μm; inset scale bar, 20 μm). f, αSMA immunolabelling and quantification of metastatic KPC (n = 3 mice), KPC;TwistcKO (n = 3 mice) and KPC;SnailcKO (n = 3 mice) (scale bar, 50 μm; inset scale bar, 20 μm). g, E-cadherin staining on serial sections of αSMA immunolabelling and quantification of metastatic KPC (n = 4 mice), KPC;TwistcKO (n = 3 mice) and KPC;SnailcKO (n = 4 mice) (scale bar, 50 μm; inset scale bar, 20 μm). h, Ki67 immunolabelling and quantification of metastatic KPC (n = 7 mice), KPC;TwistcKO (n = 3 mice) and KPC;SnailcKO (n = 3 mice) (scale bar, 50 μm; inset scale bar, 20 μm). Unless otherwise indicated error bars represent s.e.m., percentages indicated represent per cent decrease from control, and significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Extended Data Figure 4 EMT suppressed primary tumour cells have reduced mesenchymal markers and show resistance to chemotherapy in vitro.

a, Bright-field micrograph of cultured primary KPC, KPC;TwistcKO and KPC;SnailcKO cells (scale bar, 50 μm). b, EMT- and gemcitabine-transport-related gene expression shown by qPCR analysis in KPC (n = 3–4 cell lines), KPC;TwistcKO (n = 5 cell lines) and KPC;SnailcKO (n = 5–6 cell lines) (error bars represent s.d., one-tailed t-test, *P < 0.05, numbers list non-significant P values. nd, not detected, ns, not significant). c, MTT assay showing cell proliferation in KPC, KPC;TwistcKO and KPC;SnailcKO cells (n = 8, 8 and 8 biological replicates of a cell line for each genotype). d, Relative cell viability (MTT assay) in cultured KPC, KPC;TwistcKO and KPC;SnailcKO cells treated with gemcitabine or erlotinib (n = 8, 8 and 8 biological replicates of a cell line for each genotype). Unless otherwise indicated error bars represent s.e.m., significance was determined by one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Figure 5 EMT inhibition in KTC mice mirrors phenotype observed in KPC mice.

a, Representative images of haematoxylin and eosin-stained primary tumours (scale bar, 100 μm). b, Relative percentage of each histological tissue phenotype of KTC (n = 8 mice) and KTC;SnailcKO (n = 6 mice) primary tumours (error bars represent s.d.). c, Primary tumour invasiveness in KTC (n = 8 mice) and KTC;SnailcKO (n = 6 mice) (error bars represent s.d.). d, Pancreatic mass in KTC (n = 5 mice) and KTC;SnailcKO (n = 6 mice) (error bars represent s.d.). e, Immunolabelling and quantification of primary KTC (n = 5 mice), KTC;SnailcKO (n = 4 mice) for αSMA (red), CK8 (green) and DAPI (blue); white arrows indicate double-positive cells (scale bar, 20 μm), Zeb1 (scale bar, 50 μm; inset scale bar, 20 μm), cleaved caspase-3 (scale bar, 50 μm; n = 4 mice for both groups), Ki67 (scale bar, 100 μm), ENT2 (scale bar, 100 μm) and CNT3 (scale bar, 100 μm); error bars represent s.e.m. Significance was determined by two-tailed t-test. *P < 0.05, ***P < 0.001; ns, not significant.

Extended Data Figure 6 Desmoplasia is unaffected in EMT suppressed tumours with or without gemcitabine.

a, b, Staining and quantification of KTC (n = 5 or 6 mice), KTC;SnailcKO (n = 4 or 5 mice), KTC plus gemcitabine (+ GEM; n = 4 or 5 mice), KTC;SnailcKO + GEM (n = 5 mice) for Masson’s trichrome stain (MTS) (scale bars, 100 μm), Sirius Red staining (scale bars, 100 μm), and ENT1 (scale bars, 100 μm). Error bars represent s.d. (MTS and Sirius Red) or s.e.m. (ENT1), and significance was determined by two-tailed t-test. ns, not significant.

Extended Data Table 1 Pathological spectrum of primary disease and metastasis in KPC, KPC;TwistcKO and KPC;SnailcKO cohorts
Extended Data Table 2 Results of χ2 analysis of KPC cohorts in Extended Data Table 1
Extended Data Table 3 Survival and primary tumour burden determined by MRI in KPC, KPC;TwistcKO and KPC;SnailcKO cohorts treated with gemcitabine

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Zheng, X., Carstens, J., Kim, J. et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527, 525–530 (2015). https://doi.org/10.1038/nature16064

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