Volumetric Compression Induces Intracellular Crowding to Control Intestinal Organoid Growth via Wnt/β-Catenin Signaling

Abstract

Enormous amounts of essential intracellular events are crowdedly packed inside picoliter-sized cellular space. However, the significance of the physical properties of cells remains underappreciated because of a lack of evidence of how they affect cellular functionalities. Here, we show that volumetric compression regulates the growth of intestinal organoids by modifying intracellular crowding and elevating Wnt/β-catenin signaling. Intracellular crowding varies upon stimulation by different types of extracellular physical/mechanical cues and leads to significant enhancement of Wnt/β-catenin signaling by stabilizing the LRP6 signalosome. By enhancing intracellular crowding using osmotic and mechanical compression, we show that expansion of intestinal organoids was facilitated through elevated Wnt/β-catenin signaling and greater intestinal stem cell (ISC) self-renewal. Our results provide an entry point for understanding how intracellular crowdedness functions as a physical regulator linking extracellular physical cues with intracellular signaling and potentially facilitate the design of engineering approaches for expansion of stem cells and organoids.

Keywords: LRP6 signalosome; Wnt/β-catenin signaling; biophysics; cell mechanics; cell volume; mechanobiology; molecular crowding; organoid; stem cell; volumetric compression.

Fig. 1.. Distinct physical properties of Lgr5⁺ stem cells in intestinal crypts correlate with their Lgr5 expression levels.

(A) Schematic illustration of an intestinal crypt. (B) Immunofluorescent images of an intestinal crypt. Scale bar, 15 μm. (C-D) Quantitative analysis shows the differences of cell volume (C) and cellular aspect ratio (D) among four different types of cells (ISCs, Paneth cells, TA cells, and +4 cells). n=72 cells from 3 independent experiments for each condition in (C), n=42 cells from 3 independent experiments for each condition in (D). (E) Immunofluorescent images of intestinal crypts reveal highly deformed nuclei in Lgr5⁺ stem cells, while nuclei in Lgr5⁻ cells remain a round shape. Scale baris 10 μm in the image and 5 μm in the inset. (F-G) Quantitative analysis shows the differences of the nucleus to cell ratio (F) and the nuclear aspect ratio (G) among four different types of cells (ISCs, Paneth cells, TA cells, and +4 cells). n=25 cells from 3 independent experiments for each condition in (F), n=29 cells from 3 independent experiments for each condition in (G). (H) Immunofluorescent images of intestinal crypts under uniaxial stretch or without stretch. Scale bar, 15 μm. (I) Quantitative analysis shows different resulted engineering strains among four types of cells (ISCs, Paneth cells, TA cells, and +4 cells) under an applied bulk engineering strain of 70%. n=25 cells for each condition. (J) Quantitative analysis shows that different resulted volumetric strains under extreme osmotic compression (100% PEG 300) among four types of cells (ISCs, Paneth cells, TA cells, and +4 cells). n=50 cells from 3 independent experiments for each condition. (K) Lgr5⁺ stem cells can be distinguished from Lgr5⁻ cells in the crypt based on their smaller volume and lager aspect ratio. n=71. (L) The Lgr5 RNA expression level, indicated by Lgr5-GFP intensity, of Lgr5⁺ stem cells is inversely correlated with the cell volume. n=57. (M) Immunofluorescent images of intestinal crypts show that Lgr5⁺ stem cells accumulate more cytosolic β-catenin than Lgr5⁻ cells. Scale bar, 10 μm. (N) The amount of nuclear β-catenin in Lgr5⁺ stem cells is inversely correlated with the cell volume. n=74. (O) Quantitative analysis shows different amount of nuclear β-catenin among four types of cells (ISCs, Paneth cells, TA cells, and +4 cells). n=30 cells from 3 independent experiments for each condition. (P-Q) Amount of nuclear β-catenin plotted against cell volume (P) and cell aspect ratio (Q), in all four types of cells in intestinal crypts. n=30 cells from 3 independent experiments for each condition. (R) Ex vivo cultured intestinal crypts are treated with IWP-2 and Wnt3A ligand. Quantitative analysis shows the different amount of nuclear β-catenin among four types of cells (ISCs, Paneth cells, TA cells, and +4 cells). n=30 cells from 3 independent experiments for each condition. (S-T) Amount of nuclear β-catenin plotted against cell volume (S) and cell aspect ratio (T) in all four types of cells in intestinal crypts treated with IWP-2 and Wnt3A ligand. The accumulated nuclear β-catenin is negatively correlated with cell volume, while positively correlated with cell aspect ratio. n=30 cells from 3 independent experiments for each condition.

Fig. 2.. Physical properties of the cell modulate Wnt/β-catenin signaling.

(A) 3D images of RKO cells under various levels of osmotic compression. Scale bar represents 10 μm. The color scale indicates cell height. (B) Volume of RKO cells decreases under osmotic compression. n=16 cells. (C) Stiffness of the cytoplasm of RKO cells increases under osmotic compression, as a result of the increasing crowdedness. n=16 cells. (D) Fluorescence recovery after photobleaching of Dendra II in RKO cells reveals a significantly increased recovery time under osmotic compression. n=16 cells. (E-G) Wnt/β-catenin signaling is negatively correlated with cell volume (E), positively correlated with cytoplasm modulus (G) and volume fraction (F). n=16 cells for each condition. (H) Correlation of multiple physical properties of cells with Wnt/β-catenin signaling. Intracellular crowdedness may serve as a mechanoresponsive regulator of Wnt/β-catenin signaling. (I) The volume of RKO cells negatively correlates with Wnt/β-catenin signaling under combinations of osmotic compression and substrate stiffness. The substrates were made of polyacrylamide hydrogel, whose stiffness was varied by changing the crosslinker bis-acrylamide concentration. (J) Schematics of a 3D cell compression device (left). The volume of RKO cells negatively correlates with Wnt/β-catenin signaling under different levels of mechanical compressions (right). (K) Schematics of a 2D cell stretching device (left). The volume of RKO cells negatively correlates with Wnt/β-catenin signaling under biaxial stretch (right). n=3 for reporter assays, and n=25 for cell volume measurements in (M-O). Individual data points of both TOP-Flash signal and cell volume are derived from n=3 independent experiments, and each of cell volume data contains n=20 cells in (I-K). (L) Top flash assays of RKO cells with (left) or without Wnt3a ligand (middle), and DLD1 cells with Wnt3a ligand (right) under various levels of osmotic compression. n=3 independent experiments for all conditions. (M) Western blotting of β-catenin in RKO cells (left without Wnt3a ligand, middle with Wnt3a), and DLD1 cells with Wnt3a ligand (right) under various conditions of compression. (N) Immunostaining of β-catenin in RKO cells under various conditions of compression. Scale bar, 5 μm. (O) The level of total β-catenin per cell (left), and the ratio of nuclear β-catenin to total β-catenin (right); both increase under osmotic compression. n=30 cells derived from 3 independent experiments for each condition.

Fig. 3.. Intracellular crowding stabilizes β-catenin by promoting formation of the LRP6 receptor signaling complex.

(A) Time dependent accumulation of cytosolic β-catenin and phosphorylation of LRP6 in RKO cells. (B) Quantifications show that the cytosolic β-catenin accumulates more and faster in cells with osmotic compression. n=3. The relative amount of β-catenin was normalized to GAPDH intensity. (C) Quantifications show the faster and stronger phosphorylation of LRP6 in cells with compression. n=3. The relative amount of phosphorylated LRP6 was normalized to total LRP6 intensity. (D) Time-dependent images of LRP6 signalosome formation in RKO cells. Red signal indicates mCherry labeled Axin1, while the green signal indicates the EGFP tagged LRP6. Yellow dots indicate the formation of the LRP6 signalosome. Scale bar, 10 μm. (E) More LRP6 signalosomes are formed per cell under osmotic compression. n=15 cells derived from 3 independent experiments for each condition. (F) Lysates of cells with or without osmotic compression were immunoprecipitated with Axin1-specific antibody followed by western blotting analysis of LRP6. (G) Cells were transfected with FLAG-hLRP6, Mesd, mFz8, hAxin, and GSK3b and treated with control (Con), Wnt3a, or Wnt3a+PEG for 3 hours. Sucrose gradient sedimentation analysis of Triton X-100 lysates of cells. Total and phosphorylated (Tp1479) LRP6 amounts were analyzed by immunoblot.

Fig. 4.. Compression elevates intracellular crowding, and influences morphology, function and fate of ISCs and expansion of intestinal organoids.

(A) Apparent diffusion of EGFP in the cytoplasm of organoids was measured by FRAP, reflecting the degree of intracellular crowding in the conditions of varying osmotic stress. For each condition, the result is an average of 20 independent experiments. (B) Images show the typical morphology of organoids cultured with or without compression over 5 passages. Scale bar, 100 μm. (C-D) Morphology analysis of organoids shows that organoids are becoming rounder and larger under compression over passages. n=30 organoids derived from 3 independent experiments for each condition. (E) Morphology analysis shows that the portion of organoids transiting to ISC colony increases as organoids are passaged under compression over time. n=3 independent experiments for each condition. (F) Bright-field images of organoids cultured in petri-dishes with or without compression. Wild field images of fields were directly obtained using tile scan function of confocal microscopy. Scale bar, 4 mm (left), 500 μm (right). (G-H) Images (G) and quantification (H) indicated by Lgr5-eGFP show that the ratio of intestinal stem cells (ISCs) in organoids increases as organoids are passaged under compression. (I-J) Images (I) and quantification (J) indicated by EdU staining show that the ratio of cells proliferating in organoids increases over passages under compression. (K-L) Images (K) and quantification (L) indicated by Ki67 staining show that the ratio of cells proliferating in organoids increases over passages under compression. n=15 organoids derived from 3 independent experiments for each condition in H, J and L. Scale bar: 80 μm in F, H and G, 50 μm in insets.

Fig. 5.. Compression-mediated intracellular crowding regulates intestinal organoids via Wnt/β-catenin signaling.

(A) qRT-PCR analysis of organoids collected from the 1^st, 3^rd and 5^th passages of intestinal organoids cultured under prolonged compression indicate the elevated Wnt/β-catenin target genes (Axin2, Lgr5 and Sox9). n=3 independent experiments for each condition. (B) Immunostaining of β-catenin in intestinal organoids cultured in hypertonic medium and isotonic medium respectively. Scale bar, 300 μm in top panels, 20 μm in bottom panels. (C) Quantification shows more β-catenin high expression cells in intestinal organoids cultured in hypertonic medium than in isotonic medium. n=6 independent experiments for each conditions. (D) Quantification shows more total β-catenin accumulated in intestinal organoids cultured in hypertonic medium than in isotonic medium. n=6 independent experiments for each conditions. (E) Quantification shows more nuclear β-catenin accumulated in intestinal organoids cultured in hypertonic medium than in isotonic medium. n=6 independent experiments for each conditions. (F-H) ISCs with Apc depletion exhibit comparable colony forming capabilities (F), comparable sizes (G) and similar stem cell marker Lgr5 expression (H) with or without compression. Images were obtained using tile scan function of confocal microscopy. Scale bar, 600 μm. n=105 organoids derived from 3 independent experiments for each conditions (G); n=3 independent experiments for each condition in (H). (I) Inhibition of Porcupine (endogenous Wnt ligand production) using IWP2 suppresses the growth of intestinal organoids, while increasing intracellular crowding using osmotic compression does not rescue normal organoids growth. As a comparison, IWP2 treatment does not affect the growth of cystic organoids with APC depletion. Scale bar, 100 μm. (J) Inhibition of Porcupine (endogenous Wnt ligand production) using shWls suppresses the growth of intestinal organoids, while increasing intracellular crowding using osmotic compression does not rescue normal organoids growth. As a comparison, shWls treatment does not affect the growth of cystic organoids with APC depletion. Scale bar, 100 μm.

Fig. 6.. Volumetric compression modulates Wnt/β-catenin signaling and LRP6-Axin signalosome formation in 2D enteroid monolayers

(A) Volumetric compression increases the cytoplasmic stiffness of cells in 2D enteroid monolayers. n= 26 cells from 3 independent experiments for each condition. (B-C) Images (B) and quantifications (C) show that the osmotic compression increases the number of signalosome formed per cell in enteroid monolayers. n=7 independent experiments for each condition, each contains n=35 cells measurements. (D) Osmotic compression increases the active β-catenin accumulation, and the phosphorylation of LRP6 in 2D enteroid monolayers. (E) Osmotic compression increases the crowdedness of cytosolic Axin, as shown by the decreased recovery efficiency of cytosolic Axin after bleaching (Left); osmotic compression stabilizes the formation of Axin in signalosome, as shown by the decreased recovery efficiency of Axin in signalosome after bleaching. n=15 independent experiments for each condition. (F) Images show the recovery of Axin in signalosome after photo-bleaching. Scale bar, 4 μm.

Fig. 7.

A working hypothesis and schematic illustration of how cell interior crowding enhances formation of the LRP6 receptor signaling complex/signalosome.