Tunable hybrid hydrogels with multicellular spheroids for modeling desmoplastic pancreatic cancer

Abstract

The tumor microenvironment consists of diverse, complex etiological factors. The matrix component of pancreatic ductal adenocarcinoma (PDAC) plays an important role not only in physical properties such as tissue rigidity but also in cancer progression and therapeutic responsiveness. Although significant efforts have been made to model desmoplastic PDAC, existing models could not fully recapitulate the etiology to mimic and understand the progression of PDAC. Here, two major components in desmoplastic pancreatic matrices, hyaluronic acid- and gelatin-based hydrogels, are engineered to provide matrices for tumor spheroids composed of PDAC and cancer-associated fibroblasts (CAF). Shape analysis profiles reveals that incorporating CAF contributes to a more compact tissue formation. Higher expression levels of markers associated with proliferation, epithelial to mesenchymal transition, mechanotransduction, and progression are observed for cancer-CAF spheroids cultured in hyper desmoplastic matrix-mimicking hydrogels, while the trend can be observed when those are cultured in desmoplastic matrix-mimicking hydrogels with the presence of transforming growth factor-β1 (TGF-β1). The proposed multicellular pancreatic tumor model, in combination with proper mechanical properties and TGF-β1 supplement, makes strides in developing advanced pancreatic models for resembling and monitoring the progression of pancreatic tumors, which could be potentially applicable for realizing personalized medicine and drug testing applications.

Keywords: Desmoplasia; Extracellular matrix; Fibrosis; Pancreatic cancer; Tumor microenvironment.

Graphical abstract

Fig. 1

Schematic of pancreatic cancer development and cancer spheroid production. A- PDAC development with cancer microenvironment alterations. B- Cancer only and Cancer + CAFs were formed into spheroids in Aggrewell400 microplates (cancer: green, CAF: orange; F-actin: Alexa Fluor 488-Phalloidin, Nucleus: DAPI). C- Spheroids were stained for live/dead dye and imaged for days 1, 3, and 7 (Green: Calcein-live, Red: Ethidium homodimer-dead, scale bar: 500 μm, insert scale bar: 100 μm). D- Size analysis of the spheroids from micrographs for days 1, 2, 3, 4, and 7 (Two-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). E− Shape analysis of the spheroids from micrographs for days 1, 2, 3, 4, and 7 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Fig. 2

HAMA and GelMA hydrogel synthesis and characterization. A- Schematic of HAMA, GelMA, and HAMA/GelMA. B- Analysis of HAMA/GelMA storage modulus using different UV power outputs during real-time crosslinking (n = 3). C- Compressive modulus of HAMA/GelMA hydrogels (1% HAMA, 10% GelMA composition) irradiated with 1 W/cm²s for 2, 5 and 10 s (n = 3, One-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). D- Rheological analysis (change of storage modulus during frequency sweeps) of uncrosslinked (0 s), 2, 5, and 10 s crosslinked HAMA/GelMA hydrogels (1% HAMA, 10% GelMA composition) irradiated with 1 W/cm²s of UV (n = 3). E− Rheological analysis (temperature sweeps) of uncrosslinked (0 s), 2, 5, and 10 s crosslinked HAMA/GelMA hydrogels (1% HAMA, 10% GelMA composition) irradiated with 1 W/cm²s of UV (n = 3). F- SEM images of HAMA/GelMA crosslinked for 2, 5, and 10s (Scale bar: 100 μm, blue arrows: macropores, orange arrows: micropores). G- Pore size distribution of 2, 5, and 10s HAMA/GelMA hydrogels measured from SEM images after freeze-drying (n ≥ 5, One-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Fig. 3

Analysis of the cancer microtissue models prepared using Cancer + CAFs spheroids embedded into HAMA/GelMA hydrogels with tuned stiffness. A- Calcein staining of the spheroids in HAMA/GelMA hydrogels and PrestoBlue proliferation analysis of the samples (n ≥ 2, Two-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). B– F-actin staining of the micro-cancer tissue models (Red: Alexa fluor 488 Phalloidin, scale bar: 100 μm). C- Spheroid size and shape analysis in HAMA/GelMA 2, 5, 10s for days 3, 5 and 7 of micro-cancer tissue preparation (One-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). D- Cell tracker staining of the micro-cancer tissue models (Cancer: green, CAF: red). E− CAF to cancer ratio per spheroid within different hydrogel stiffnesses for a 7-day period. (One-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Fig. 4

Immunostaining and RT-qPCR analysis of the micro-cancer tissue models. A- E-CAD and VIM staining of Cancer + CAFs micro-cancer tissue models (red: VIM, green: E-CAD, blue: DAPI, scale bar: 40 μm). B- α-SMA and YAP staining Cancer + CAFs micro-cancer tissue models (red: α-SMA, green: YAP, blue: DAPI, scale bar: 40 μm). C- qRT PCR analysis and heatmap of the genes for micro-cancer tissue models prepared by 3, 15, 36 kPa compressive modulus of HAMA/GelMA on days 3 and 14 (red: maximal change, green: minimal change). D- Fold changes of the selected genes (VIM, E-Cad, YAP, Nanog, PDGFRB1, and VEGFR2) (Two-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Fig. 5

Analysis of the TGF-β treated micro-cancer tissue models for cell tracker, shape, area ratio, immunohistochemistry, and qRT-PCR. A- Cell tracker staining of the TGF-β treated micro-cancer tissue models (Cancer: green, CAF: red). B- Spheroid size and shape analysis in TGF-β treated HAMA/GelMA 3, 15, 36 kPa of compressive modulus for days 3, 5 and 7 of micro-cancer tissue model preparation (One-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). C- CAF to cancer ratio per spheroid treated with TGF-β within different hydrogel stiffnesses for a 7-day period. (One-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). D- E-CAD and VIM staining of Cancer + CAF micro-cancer tissue model treated with the TGF-β (red: VIM, green: E-CAD, blue: DAPI, scale bar: 40 μm). E− α-SMA and YAP staining Cancer + CAF micro-cancer tissue model (red: α-SMA, green: YAP, blue: DAPI, scale bar: 40 μm). F- qRT PCR analysis and heatmap of the genes for the TGF-β treated micro-cancer tissue model prepared by 3, 15, 36 kPa compressive modulus of HAMA/GelMA at days 3 and 14 (red: maximal change, green: minimal change). G- Fold changes of the selected genes (VIM, E-CAD, and YAP) for the TGF-β treated micro-cancer tissue model (Two-Way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).