Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

doi:10.1038/nm.4082

. 2016 May;22(5):497-505.

doi: 10.1038/nm.4082. Epub 2016 Apr 18.

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Hanane Laklai¹, Yekaterina A Miroshnikova¹, Michael W Pickup¹, Eric A Collisson², Grace E Kim³, Alex S Barrett⁴, Ryan C Hill⁴, Johnathon N Lakins¹, David D Schlaepfer⁵, Janna K Mouw¹, Valerie S LeBleu⁶, Nilotpal Roy⁷, Sergey V Novitskiy⁸, Julia S Johansen⁹, Valeria Poli¹⁰, Raghu Kalluri⁶, Christine A Iacobuzio-Donahue¹¹, Laura D Wood¹², Matthias Hebrok⁷, Kirk Hansen⁴, Harold L Moses⁸, Valerie M Weaver^{1

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Affiliations

¹ Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California, San Francisco, San Francisco, California, USA.
² Department of Medicine, University of California, San Francisco, San Francisco, California, USA.
³ Department of Pathology, University of California, San Francisco, San Francisco, California, USA.
⁴ Department of Biochemistry and Molecular Genetics, University of Colorado, Denver, Aurora, Colorado, USA.
⁵ Department of Reproductive Medicine, University of California, San Diego Moores Cancer Center, La Jolla, California, USA.
⁶ Department of Cancer Biology, Metastasis Research Center, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
⁷ Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, California, USA.
⁸ Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
⁹ Department of Oncology, Herlev Hospital, Copenhagen University Hospital, Copenhagen, Denmark.
¹⁰ Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy.
¹¹ Department of Pathology, David Rubenstein Center for Pancreatic Cancer Research, Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
¹² Gastrointestinal and Liver Pathology Department, Johns Hopkins University, Baltimore, Maryland, USA.
¹³ Department of Anatomy, University of California, San Francisco, San Francisco, California, USA.
¹⁴ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.
¹⁵ Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, USA.
¹⁶ Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.

PMID: 27089513
PMCID: PMC4860133
DOI: 10.1038/nm.4082

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Hanane Laklai et al. Nat Med. 2016 May.

. 2016 May;22(5):497-505.

doi: 10.1038/nm.4082. Epub 2016 Apr 18.

Authors

Affiliations

¹ Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California, San Francisco, San Francisco, California, USA.
² Department of Medicine, University of California, San Francisco, San Francisco, California, USA.
³ Department of Pathology, University of California, San Francisco, San Francisco, California, USA.
⁴ Department of Biochemistry and Molecular Genetics, University of Colorado, Denver, Aurora, Colorado, USA.
⁵ Department of Reproductive Medicine, University of California, San Diego Moores Cancer Center, La Jolla, California, USA.
⁶ Department of Cancer Biology, Metastasis Research Center, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
⁷ Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, California, USA.
⁸ Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
⁹ Department of Oncology, Herlev Hospital, Copenhagen University Hospital, Copenhagen, Denmark.
¹⁰ Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy.
¹¹ Department of Pathology, David Rubenstein Center for Pancreatic Cancer Research, Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
¹² Gastrointestinal and Liver Pathology Department, Johns Hopkins University, Baltimore, Maryland, USA.
¹³ Department of Anatomy, University of California, San Francisco, San Francisco, California, USA.
¹⁴ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.
¹⁵ Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, USA.
¹⁶ Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.

PMID: 27089513
PMCID: PMC4860133
DOI: 10.1038/nm.4082

Erratum in

Author Correction: Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.
Laklai H, Miroshnikova YA, Pickup MW, Collisson EA, Kim GE, Barrett AS, Hill RC, Lakins JN, Schlaepfer DD, Mouw JK, LeBleu VS, Roy N, Novitskiy SV, Johansen JS, Poli V, Kalluri R, Iacobuzio-Donahue CA, Wood LD, Hebrok M, Hansen K, Moses HL, Weaver VM. Laklai H, et al. Nat Med. 2024 Mar;30(3):908. doi: 10.1038/s41591-023-02694-w. Nat Med. 2024. PMID: 38017076 No abstract available.

Abstract

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

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Figures

**Figure 1. Tissue tension and collagen thickness linked to PDAC prognosis**
(a) H&E and Trichrome (top insert) staining of PDAC tissue arrays for well (n = 19), moderately (n = 23) or poorly differentiated (n = 26) tumors; Scale bar, 100 µm (main) and 50 µm (insert). Immunofluorescence images and quantifications of α-SMA, p-MLC2, p-SMAD2 and DAPI; Scale bar, 75 µm (main) and 40 µm (insert). Polarized light images of picrosirius red (PR) within the main stroma (MS) (3^rd) and periductal stroma (DS) (3^rd insert); Scale bar, 50 µm (main) and 40 µm (insert). Second harmonic generation (SHG) of periductal collagen and diameter color-coded SHG; Scale bar, 75 µm. (b) H&E and Trichrome (top insert) staining of tissue from PDAC patients with a median short survival of 11–289 days (n = 29) and median long survival of 1090–3298 days (n = 28); Scale bar, 100 µm. Immunofluorescence and quantification of tissue stained for α-SMA, p-MLC2, p-SMAD2 and DAPI; Scale bar, 75 µm. PR staining within the main stroma (MS) and periductal stroma (DS) (insert); Scale bar, 50µm. Periductal SHG and diameter color-coded SHG; Scale bar, 75 µm. (c) H&E staining of PDAC tissue from wild type SMAD4 (n = 10) and mutant SMAD4 (n = 10) patients. Scale bar, 100 µm. PR staining and quantification within the main stroma (MS) and periductal stroma (DS); Scale bar, 50 µm. Force maps and quantification of the main stroma (MS) (insert) and periductal stroma (DS) (insert). SHG of periductal collagen and diameter color-coded SHG; Scale bar, 75 µm. Immunofluorescence staining and quantification for p-MLC2 and DAPI; Scale bar, 75 µm. Results are presented as the mean +/− SEM. Subsequent statistical analysis was performed with either unpaired two-sided student t-tests, one-way ANOVA with Tukey’s method for multiple comparisons. (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” not significant).

**Figure 2. PDAC genotype tunes epithelial tension to regulate fibrosis**
(a) SHG images from 20 week KC, KPC or KTC transgenic pancreatic tissues (top); Scale bar, 75 µm. Force maps from AFM PDAC ECM (top insert). Immunofluorescence images of p-Mlc2, p-MyPT1 (insert), β1-integrin and p-Ptk2, Yap1 and DAPI; Scale bar, 50 µm. Tenascin C, Fibronectin (insert), Collagen type XII alpha1 and DAPI; Scale bar, 75 µm. (b) Quantification of SHG fibril thickness and distribution around PDAC lesions. (c) Distribution of PDAC ECM stiffness measured by AFM. (d) Quantification of Tenascin C, Fibronectin and Collagen type XII alpha 1 images shown in (a). (e) Quantification of traction force KC, KPC and KTC cells on 2300 Pa polyacrylamide gels. (f) Quantification of mean collagen fiber diameter in three-dimensional collagen gels with KC, KPC or KTC cells or with KTC cells treated with vehicle or ROCK inhibitor Y27632 at 24 hours. (g) PR staining of tissue excised from nude mice 3 weeks after injection with KC, KPC, or KTC cells expressing either a control shRNA or an shRNA to Rock1 (top); Scale bar, 75 µm. Immunofluorescence of p-Mlc2, p-MyPT1 (insert), Tenascin C, Yap1 and DAPI; Scale bar, 50 µm. (h) Quantification of stiffness of tissue in (e). (i) Quantification of total levels of fibrillar collagen. For *in vitro* bar graphs, 3 technical replicates were performed and results are the mean +/− SEM of 3 independent experiments. For *in vivo* experiments, n = 5 mice per group. Subsequent statistical analysis was performed with either unpaired two-sided student t-tests, one-way ANOVA with Tukey’s method for multiple comparisons. For survival analysis, the log-rank (Mantel-Cox) test was used. (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” not significant).

**Figure 3. JAK-Stat3 signaling drives ECM remodeling and stiffening**
(a) Immunofluorescence images and quantification of epithelial and stromal staining in 20 week old KC, KPC and KTC mice stained for pStat3 and DAPI; Scale bar, 50 µm. (b) Immunofluorescence images of KC, KPC and KTC tumor cells stained for p-Stat3 and DAPI; Scale bar, 25 µm. (c) Quantification of collagen contraction as measured by total collagen gel area in three dimensional collagen gels with KTC tumor cells for 24 hours either with vehicle or the Jak inhibitor Ruxolitinib. (d) Polarized light images and quantification of PR stained color-coded fibrillar collagen diameter on gels from (c); Scale bar 75 µm. (e) Immunoblot and relative quantification of Stat3 activation (pStat3) in KC cells following exposure to KTC conditioned media (CM). (f) Quantification of collagen contraction measured by total collagen gel area of gels with KTC vehicle or Ruxolitinib CM treated KC tumor cells for 48 hours. (g) Polarized light images and quantification of PS, color-coded fibrillar collagen diameter; Scale bar 75 µm. (h) Immunoblot and relative quantification of total Stat3 and p-Stat3 levels in KC tumor cells cultured on soft or stiff polyacrylamide substrates. (i) Quantification of cytokine levels in the CM of KC cells cultured on soft or stiff polyacrylamide substrates. For *in vitro* quantification, results represent 3 technical replicates for and are the mean +/− SEM of 3 independent experiments. For *in vivo* experiments, n = 5 mice per group. Subsequent statistical analysis was performed with either unpaired two-sided student t-tests, one-way ANOVA with Tukey’s method for multiple comparisons. For survival analysis, the log-rank (Mantel-Cox) test was used. (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” not significant).

**Figure 4. Tumor cell tension accelerates PDAC progression in mice**
(a) Cartoon of mouse manipulations used to study the impact of increasing pancreatic epithelial cell mechanosignaling using β1-V737N on Kras-induced pancreatic malignancy. (b) Immunofluorescence images and quantification of pancreatic tissues from 3 month old KC and KC/β1-V737N mice stained for p397-Ptk2 and p-Mlc2, Tenascin C, Yap1, p-Stat3; Scale bar, 50 µm, CD68 and DAPI; Scale bar, 100 µm. Polarized light images of PS staining; Scale bar, 75 µm. Force maps of ECM stiffness. (c) Alcian blue H&E images of KC and KC/β1-V737N tissue; Scale bar, 100µm. (d) Quantification of histopathologic phenotypes present in KC and KC+β1-V737N mice. (e) Polarized light images and quantification of PS staining of pancreatic tissues in nude mice injected with KTC tumor cells expressing either a control shRNA or a FAK shRNA; Scale bar, 75 µm. Immunofluorescence images and quantification of Tenascin C (scale bar 75 µm), Yap1 (75 µm), p-Stat3 (scale bar, 25 µm) and DAPI. (f) Quantification of average elastic modulus (Pa). For *in vivo* experiments, n = 5 mice per group. Subsequent statistical analysis was performed with unpaired two-sided student t-tests.. (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” not significant).

**Figure 5. Stat3 induces fibrosis and accelerates PDAC**
(a) Cartoon depicting the mouse crosses used for the activated Stat3C manipulations. (b) Immunofluorescence images and quantification of pancreatic tissues from 5 week old KC and KC/Stat3C mice stained for p-Stat3, scale bar, 75 µm, p397-Ptk2 and p-Myl2, scale bar, 50 µm, CD68, scale bar, 100 µm, and DAPI. Polarized light images and quantification of PS staining; Scale bar, 75 µm and H&E staining; Scale bar, 100 µm. (c) Kaplan-Meier graph showing survival of KC and KC/Stat3C mice. (d) Quantification of ECM stiffness in (b). (e) Cartoon depicting the mouse crosses used for the Stat3 knock studies. (f) Immunofluorescence images and quantification of pancreatic tissues from 5 week old homozygous KTC (Control) and KTC+Stat3^−/− mice (KTC Stat3^−/−) stained for p-Stat3, scale bar, 75 µm, p397-Ptk2 and p-Myl2, scale bar, 50 µm, Tenascin C, CD68, scale bar, 100 µm, Yap1 scale bar, 50 µm, and DAPI. Polarized light images and quantification of PS staining; Scale bar, 75 µm, and H&E staining. Scale bar, 100 µm. (g) Kaplan-Meier graph showing survival of KTC and KTC/Stat3 KO mice. (h) Quantification of ECM stiffness on tissue in (f). For *in vivo* experiments, n = 5 mice per group. Subsequent statistical analysis was performed with unpaired two-sided student t-tests. (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” not significant).

**Figure 6. STAT3 enhances epithelial contractility to induce PDAC fibrosis and aggression**
(a) H&E images of PDAC tumors from patients with a median short survival of 11–289 days (n = 29) and median long survival of 1090–3298 days (n = 28). Scale bar, 100 µm. SHG images and quantification of extracellular collagen architecture in the pancreatic tissue around the epithelial ductal region in tissue described above. Scale bar, 75 µm (main). Immunofluorescence images and quantification of Tenascin C, Fibronectin, Collagen XII alpha1, p-STAT3, YAP1 and DAPI. Scale bar, 75 µm and 10 µm (insert). (b) H&E images of PDAC tumors from patients with WT SMAD4 (n = 10) or mutant SMAD4 (n = 10); Scale bar, 100 µm. SHG images and quantification of periductal collagen. Scale bar, 75 µm. Immunofluorescence images and quantification of Tenascin C, Fibronectin, Collagen XII alpha1, p-STAT3, YAP1 and DAPI. Scale bar, 75 µm. Results are presented as the mean +/− SEM. Subsequent statistical analysis was performed with unpaired two-sided student t-tests. (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, “ns” not significant).

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Comment in

Regulation of pancreatic cancer aggressiveness by stromal stiffening.
Rath N, Olson MF. Rath N, et al. Nat Med. 2016 May 5;22(5):462-3. doi: 10.1038/nm.4099. Nat Med. 2016. PMID: 27149218 No abstract available.
Building up the tension between the epithelial and stromal compartment in pancreatic ductal adenocarcinoma.
Biffi G, Öhlund D, Tuveson D. Biffi G, et al. Cell Death Differ. 2016 Aug;23(8):1265-6. doi: 10.1038/cdd.2016.50. Epub 2016 Jun 10. Cell Death Differ. 2016. PMID: 27285108 Free PMC article. No abstract available.

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References

1. Chauhan VP, et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat. Commun. 2013;4:2516. - PMC - PubMed
1. Swartz MA, Lund AW. Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity. Nature Reviews Cancer. 2012;12:210–219. - PubMed
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[1] Chauhan VP, et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat. Commun. 2013;4:2516. - PMC - PubMed

[2] Chauhan VP, et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat. Commun. 2013;4:2516. - PMC - PubMed

[3] Swartz MA, Lund AW. Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity. Nature Reviews Cancer. 2012;12:210–219. - PubMed

[4] Swartz MA, Lund AW. Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity. Nature Reviews Cancer. 2012;12:210–219. - PubMed

[5] Yu M, Tannock IF. Targeting Tumor Architecture to Favor Drug Penetration: A New Weapon to Combat Chemoresistance in Pancreatic Cancer? Cancer Cell. 2012;21:327–329. - PubMed

[6] Yu M, Tannock IF. Targeting Tumor Architecture to Favor Drug Penetration: A New Weapon to Combat Chemoresistance in Pancreatic Cancer? Cancer Cell. 2012;21:327–329. - PubMed

[7] Provenzano PP, Hingorani SR. Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer. Br. J. Cancer. 2013;108:1–8. - PMC - PubMed

[8] Provenzano PP, Hingorani SR. Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer. Br. J. Cancer. 2013;108:1–8. - PMC - PubMed

[9] Olive KP, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science. 2009;324:1457–1461. - PMC - PubMed

[10] Olive KP, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science. 2009;324:1457–1461. - PMC - PubMed

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Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

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Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

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