Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets
Ricardo Cruz-Acuña, … , Jason A. Burdick, Anil K. Rustgi
Ricardo Cruz-Acuña, … , Jason A. Burdick, Anil K. Rustgi
Published October 3, 2023
Citation Information: J Clin Invest. 2023;133(23):e168146. https://doi.org/10.1172/JCI168146.
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Research Article Cell biology Oncology

Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets

Abstract

Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying protumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM–mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform for identifying potential therapeutic targets to disrupt the contribution of protumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC and inform the development of therapeutics that target ECM stiffness in EAC.

Authors

Ricardo Cruz-Acuña, Secunda W. Kariuki, Kensuke Sugiura, Spyros Karaiskos, Eleanor M. Plaster, Claudia Loebel, Gizem Efe, Tatiana Karakasheva, Joel T. Gabre, Jianhua Hu, Jason A. Burdick, Anil K. Rustgi

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Figure 3

Engineered hydrogel stiffness modulates YAP activation in EAC PDOs.

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Engineered hydrogel stiffness modulates YAP activation in EAC PDOs. (A) ...
(A) Transcriptional expression and (B) representative fluorescence images of YAP and SOX9 in organoids within NorHA hydrogels of different stiffness at 14 days after encapsulation. Data are represented as mean ± SEM. n = 3 technical replicates, representative of 3 independent experiments. (A) One-way ANOVA with Tukey’s multiple comparisons test showed significant differences between 100 Pa and 1000 Pa, 350 Pa and 1000 Pa, and 1000 Pa and Matrigel. *P < 0.05; **P < 0.01; ***P < 0.001. RNA levels normalized to 100 Pa. Scale bars: 100 μm. Original magnification, ×5 (insets, 100 Pa and 350 Pa); ×3 (insets, 1,000 Pa). (C) Quantification and representative fluorescence microscopy images of CD44 expression in EAC PDOs cultured in NorHA hydrogels of different stiffness at 14 days after encapsulation. Data are represented as mean ± SEM. n = at least 20 organoids analyzed per group. Kruskal-Wallis test with Dunn’s multiple-comparisons test showed significant differences between 100 Pa and 1000 Pa, and 350 Pa and 1000 Pa. *P < 0.05; ****P < 0.0001. Scale bar: 100 μm. (D) Schematic of in vitro experiment of EAC PDOs within NorHA hydrogels being treated with YAP inhibitor verteporfin. Created with BioRender.com. (E) Representative immunohistochemistry microscopy images and quantification of Yap expression in EAC PDOs cultured in NorHA hydrogels of different stiffnesses at 7 days after encapsulation and treated with 5 nM verteporfin or DMSO. Data are represented as mean ± SEM. n = 4 hydrogels per group. One-way ANOVA with Tukey’s multiple-comparisons test showed significant differences between 1000 Pa+DMSO and every other group (**P < 0.01), and no significant differences among other groups (P > 0.05). Scale bar: 100 μm. (A–E) Three independent experiments were performed, and data are presented for 1 of the experiments. Every independent experiment was performed with 4 gel samples per experimental group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.