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 4

Engineered hydrogel stiffness-dependent growth of EAC PDOs in in vivo xenograft model.

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Engineered hydrogel stiffness-dependent growth of EAC PDOs in in vivo xe...
(A) Schematic of in vivo transplantation experiment of EAC PDOs within NorHA hydrogels into mouse subcutaneous pockets. Created with BioRender.com. (B) Representative fluorescence microcopy images of EAC PDOs within NorHA hydrogels stained for MUC5ac, E-cad, and CK8 at 28 days after encapsulation and in vivo transplantation. Scale bar: 100 μm. (C) Histological (H&E) microcopy images and quantification of PDO size (area) as a function of matrix stiffness at 28 days after encapsulation and in vivo transplantation. Data are represented as mean ± SEM. n = at least 5 organoids analyzed per group. Welch’s t test with 2-tailed comparison showed significant differences between 100 Pa and 1000 Pa. **P < 0.01. Scale bar: 100 μm. (D) Quantification and representative fluorescence microscopy images of percentages of proliferating cells (%Ki67+) per EAC PDOs as a function of matrix stiffness at 28 days after encapsulation and in vivo transplantation. Data are represented as mean ± SEM. n = at least 6 organoids analyzed per group. Mann-Whitney U test showed significant differences between 100 Pa and 1000 Pa. ***P < 0.001. Scale bar: 100 μm. (E) Representative fluorescence microcopy images of EAC PDOs within NorHA hydrogels stained for Sox9 and Yap at 28 days after encapsulation and in vivo transplantation. Quantification of percentage of nuclear Yap+ cells (%Yap+) per EAC PDO as a function of matrix stiffness. Data are represented as mean ± SEM. n = 6 organoids analyzed per group. Welch’s t test with 2-tailed comparison showed significant differences between 100 Pa and 1000 Pa. ***P < 0.001. Scale bar: 100 μm. Original magnification, ×5 (insets). (A–E) Two independent experiments were performed, and data are presented for 1 of the experiments. Every independent experiment was performed with 2 gels per mouse and 5 mice per experimental group.