Cell-Matrix Interactions Contribute to Barrier Function in Human Colon Organoids

Abstract

The importance of cell-matrix adhesion to barrier control in the colon is unclear. The goals of the present study were to: (i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control measurement and (ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes can mitigate the effects of cell-matrix disruption. Human colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions and in the presence of Aquamin®, a multi-mineral product. A function-blocking antibody directed at the C-terminal region of the laminin α chain was used in parallel. The effects of Aquamin® on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. Aquamin® increased the expression of multiple basement membrane, hemidesmosomal and focal adhesion proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. The blocking antibody reduced TEER values under both conditions but was most effective in the absence of Aquamin®, where expression of cell-matrix adhesion proteins was lower to begin with. These findings provide evidence that cell-matrix interactions contribute to barrier control in the colon.

Keywords: Aquamin®; basement membrane; cell-cell junction; cell-matrix adhesion; colonoid; gut barrier; laminin; proteomics.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Varani, McClintock and Aslam.

Figures

Figure 1

Effects of Aquamin® on expression of proteins involved in cell-matrix interactions: Proteomic screen. Organoid cultures were established with colon biopsies from four healthy subjects and three with ulcerative colitis in remission. Organoids were cultured in the presence or absence of Aquamin® as described in the Materials and Methods section. At the end of the incubation period, protein was isolated from the cultures and assessed in the proteomic screen. Values from respective controls were set at 1.0 and the values from Aquamin®-treated samples reflect a ratio (increase or decrease fold-change) relative to respective control. Values shown reflect mean (±SD) fold-change from respective control in colon organoids from normal subjects (N = 4) and subjects with UC in remission (N = 3). Methods for generating the data sets were described in detail in past reports (22, 24). Data for individual proteins were compared for statistical differences using the student t-test. Asterisk (*) indicates a difference from respective control at p < 0.05.

Figure 2

Effects of Aquamin® on expression of Laminin β1: Western blotting. Protein isolated from control and Aquamin®-treated (healthy normal subjects) colon organoid-derived monolayer cells was assessed for laminin β1 expression by Western blotting as described in the Materials and Methods section. 10 μg of protein from each condition was used. β-actin was assessed in parallel (as a loading control). Band quantitation was done using ImageJ software. Relative band density is presented for laminin β1 and β-actin.

Figure 3

Transepithelial electrical resistance in differentiation medium (Preliminary assessment). (A) Time-dependent changes in TEER values. Values shown are means and standard deviations based on four separate experiments with four samples (individual transwell insert filters seeded with healthy colon organoid-derived monolayer cells) per data point at each time-point in each experiment. Insert: Confocal fluorescent microscopic (max-projected) images of organoids and organoid-derived cells on transwell inserts stained after the day-2 and day-5 readings with antibody to occludin and with a combination of antibody to occludin and DAPI. Scale bars = 50 μm. (B) Effects of anti-laminin antibody on TEER values. Values shown are means and standard deviations based on two separate experiments with 4 samples (individual transwell insert filters seeded with healthy colon organoid-derived monolayer cells) per data point at each time-point in each experiment. Insert: hematoxylin and eosin-stained images of the cell monolayers still attached to the transwell inserts from IgG-treated and anti-laminin-treated wells. Arrows in the anti-laminin-treated image show areas where cell detachment from the underlying transwell insert was visible. Scale bar = 100 μm (small) and 50 μm (Large).

Figure 4

Transepithelial electrical resistance in KGM Gold-growth medium with or without Aquamin® and with or without anti-laminin antibody. TEER values shown are means and standard deviations based on three separate experiments with four samples (individual transwell insert filters seeded with healthy colon organoid-derived monolayer cells) per data point in each experiment. Data were compared for statistical differences using ANOVA followed by unpaired-group comparisons. Asterisk (*) above the open Aquamin® bar indicates a difference from control at p < 0.05. Asterisks (*) above the closed bars indicates difference from respective IgG control at p < 0.05.

Figure 5

Colon organoid cohesion in KGM Gold-growth medium mix: Effect of anti-laminin antibody. Colon organoids were maintained for 7 days in KGM Gold-growth medium with either IgG or anti-laminin. At the end of the incubation period, organoid cohesion was assessed as described in the Materials and Methods section. Values shown represent the change in organoid size (i.e., mean surface area ± SD of individual colon organoids based on two separate experiments with a minimum of 53-104 colon or-ganoids assessed individually per treatment group in both pre- and post-harvest cultures. Data were compared for statistical differences using ANOVA followed by unpaired-group comparisons. While the decrease in organoid size between post-harvest and pre-harvest organoids were statistically significant with either IgG or anti-laminin, the differences between anti-laminin and IgG were not different. Inset: Representative examples of organoid appearance immediately prior to harvest (upper) and 1 day after the harvested organoids had been reestablished in culture. Scale bar = 200 μm.

Figure 6

Aquamin®-responsive cell-matrix adhesion structures in the colon. A cartoon depicting structures important to cell-matrix adhesion in the colon and components of those structures that are responsive to Aquamin® (shown in red). Based on the profile of proteins that are induced by Aquamin®, cell-matrix adhesion through both focal adhesions and hemidesmosomes could be affected. Laminin-binding integrins did not alter with Aquamin®.