Bioengineered Submucosal Organoids for In Vitro Modeling of Colorectal Cancer

Abstract

The physical nature of the tumor microenvironment significantly impacts tumor growth, invasion, and response to drugs. Most in vitro tumor models are designed to study the effects of extracellular matrix (ECM) stiffness on tumor cells, while not addressing the effects of ECM's specific topography. In this study, we bioengineered submucosal organoids, using primary smooth muscle cells embedded in collagen I hydrogel, which produce aligned and parallel fiber topography similar to those found in vivo. The fiber organization in the submucosal organoids induced an epithelial phenotype in spheroids of colorectal carcinoma cells (HCT-116), which were embedded within the organoids. Conversely, unorganized fibers drove a mesenchymal phenotype in the tumor cells. HCT-116 cells in organoids with aligned fibers showed no WNT signaling activation, and conversely, WNT signaling activation was observed in organoids with disrupted fibers. Consequently, HCT-116 cells in the aligned condition exhibited decreased cellular proliferation and reduced sensitivity to 5-fluorouracil chemotherapeutic treatment compared to cells in the unorganized construct. Collectively, the results establish a unique colorectal tumor organoid model to study the effects of stromal topography on cancer cell phenotype, proliferation, and ultimately, chemotherapeutic susceptibility. In the future, such organoids can utilize patient-derived cells for precision medicine applications.

Keywords: collagen organization; colorectal cancer; epithelial-to-mesenchymal transition; extracellular matrix; tumor models.

FIG. 1.

Schematic of organoid fabrication. (A) A CAD program was used to produce a model of a six-well insert negative mold that was subsequently printed with a 3D printer. (B) PDMS was poured into the mold and allowed to cure. (C) After curing, the mold was removed, cleaned, and trimmed of any excess of PDMS. (D) The mold was placed in a six-well plate and cell-Col I solution was deposited into the microwells. (E) Culture media were added and the construct was allowed to crosslink and contract. (F) At predetermined times, constructs were harvested for analysis. Col I, collagen I; PDMS, polydimethylsiloxane. Color images available online at www.liebertpub.com/tea

FIG. 2.

Physical and histological characterization of submucosal organoids. (A–C) Immunohistochemical staining (red) of submucosal organoids for the indicated smooth muscle markers (nuclei stained blue with DAPI) (D–F) H&E staining shows cellular organization inside the submucosal organoids at days 1 (A), 3 (B), and 5 (C). (G) Size (diameter) measurements of submucosal organoids over 7 days in culture (^#p < 0.01, *p < 0.1). (A–C) Scale bar = 200 μm. (E–G) Scale bar = 50 μm. H&E, hematoxylin and eosin. Color images available online at www.liebertpub.com/tea

FIG. 3.

Collagen fiber properties in the submucosal organoids. Submucosal organoids (A–D) and bare collagen constructs (E–H) were stained with picrosirius red to highlight reticular (green) or bundled (red/orange) collagen (A' and E' are black and white conversions on A and E images). Images were analyzed with the CT-FIRE™ program. (B, F) Fiber angles (degrees vs. frequency). (C, G) Fiber length (pixels vs. frequency). (D, H) Fiber width (pixels vs. frequency). *p < 0.01, ^#p < 0.01, ^†p < 0.01. (A, E) Scale bar = 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 4.

Epithelial acini formation occurs in the submucosal organoid but not in unorganized collagen. (A–C) H&E images of CaCO2 cells grown in submucosal organoids (A), bare collagen (B), and in transwell inserts (with SMCs in the bottom well). The epithelial acini structures in the submucosal constructs were immunostained (red) for ZO-1 (D) and CK-18 (E). (A–C) Scale bar = 100 μm. (D, E) Scale bar = 25 μm. CK-18, cytokeratin-18; SMC, smooth muscle cell; ZO-1, zonula occludens-1. Color images available online at www.liebertpub.com/tea

FIG. 5.

Collagen fiber properties in submucosal organoids with embedded HCT-116 spheroids. (A) Submucosal organoids with embedded HCT-116 spheroids and Col I fibers (blue) were imaged using second-harmonic generation. Cellular autofluorescence (red) indicates cell bodies. Inset: a microscopic image of a 7-day organoid after fixation. A 3D reconstruction was generated in Imaris™ (A, bottom), and regions of interest, away from (B, B’) and near (C, C’) the tumor spheroid, were used to visualize the Col I fibers (white arrows in B denote elongated and aligned fibers). The regions of interest were analyzed with the CT-FIRE program. (B”, C”) Fiber angles (degrees vs. frequency). (B’”, C’”) Straightness (straightness vs. frequency). (A) Scale bar = 200 μm. (B, C) Scale bar = 20 μm. *p < 0.05, ^#p < 0.01. Color images available online at www.liebertpub.com/tea

FIG. 6.

EMT phenotype of HCT-116 submucosal tumor organoid. HCT-116 spheroids in submucosal organoids (A, C, E, G, I) and bare collagen (B, D, E, H, J) were harvested after 7 days in culture and stained for H&E (A, B) indicating cell membrane-associated proteins (C–H) and MMP (I, J). CK-18 staining was used to identify the HCT-116 cells inside the organoids. For each stain (.1), image is triple staining and (.2) image is the specific protein stain. (A, B) Scale bar = 100 μm. (C–J) Scale bar = 25 μm. EMT, epithelial-to-mesenchymal transition; MMP, matrix metalloproteinase. Color images available online at www.liebertpub.com/tea

FIG. 7.

The effects of collagen fiber organization on WNT pathway activation in HCT-116 submucosal tumor organoid. HCT-116 spheroids in submucosal organoids (A) and bare collagen (B) were harvested after 7 days in culture and stained for β-catenin, CK-18 (to identify HCT-116 cells), and DAPI. For each stain (A.1, B.1), images are triple staining and (A.2, B.2) images are β-catenin stain. (C, D) Spheroids of HCT-116 expressing GFP under control of WNT-activated promoter sequence (TOP-GFP) and constitutive nuclear mCherry were embedded in submucosal organoids (C) and bare collagen (D) and the ratio of signal intensity of GFP (green) and mCherry (red) was calculated after 7 days in culture (E, SMC—submucosal organoids; Col I—bare collagen). In parallel, these fluorescently labeled HCT-116 spheroids in submucosal organoids were incubated with WNT agonist, BIO (F), and WNT antagonist, XAV939 (G), as indicated, and signal intensity of GFP (green) and mCherry (red) was calculated after 7 days in culture (H). ^#p < 0.01, *p < 0.05. (A, B) Scale bar = 25 μm. (C, D, F, G) Scale bar = 150 μm. Color images available online at www.liebertpub.com/tea

FIG. 8.

The effects of collagen fiber organization on 5-FU response on HCT-116 CaCO2 and submucosal tumor organoids. (A, B) HCT-116 spheroids in submucosal organoids (A) and bare collagen (B) were harvested after 7 days in culture and stained for Ki-67 for proliferating cells CK-18 to identify HCT-116 cells. (C) The proportion of Ki-67-positive cells in submucosal organoids (SMC) and bare collagen (Col I) was calculated and graphed. Spheroids of HCT-116 (D, E) and CaCO2 (G, H) were embedded in submucosal organoids (D, G) and bare collagen (E, H) and cultured for 3 days and then exposed to 10 mM 5-FU for 3 days. The organoids were stained with a Live/Dead assay kit and the green to red fluorescence signal was calculated for HCT-116 (F) and CaCO2 (I). *p < 0.05. (A, B) Scale bar = 50 μm. (D, F–H) Scale bar = 150 μm. 5-FU, 5-fluorouracil. Color images available online at www.liebertpub.com/tea