DOI: 10.5061/dryad.fn2z34v55
title: Differential Effects of Confinement on the Dynamics of Normal and Tumor-Derived Pancreatic Ductal Organoids

description: Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the epithelia
 comprising the ductal network of the pancreas. During disease progression,
 PDAC tumors recruit fibroblasts that promote fibrosis, increasing local
 tissue stiffness and subjecting epithelial cells to increased compressive
 forces. Previous in vitro studies have documented cytoskeletal and nuclear
 adaptation following compressive stresses in 2D and 3D. However,
 comparison of the responses of normal and tumor-derived ductal epithelia
 to physiologically relevant confinement remains underexplored, especially
 in 3D organoids. Here we control confinement with an engineered 3D
 microenvironment composed of Matrigel® mixed with a low yield stress
 granular microgel. Normal and tumor-derived murine pancreas organoids
 (normal and tumor) were cultured for 48 h within this composite 3D
 environment or in pure Matrigel® to investigate the effects of confinement
 on lumen morphogenesis. In confinement, tumor organoids (mT) formed lumen
 that expanded rapidly, whereas normal organoids (mN) expanded more slowly.
 Moreover, normal organoids in more confined conditions exhibited inverted
 apicobasal polarity compared to those in less confined conditions. Tumor
 organoids exhibited a collective “pulsing” behavior that increased in
 confinement. These pulses generated forces sufficient to locally overcome
 the yield stress of microgels in the direction of organoid expansion.
 Normal organoids more commonly exhibited unidirectional rotation. Our in
 vitro microgel confinement platform enabled the discovery of two distinct
 modes of collective force generation in organoids that may shed light on
 the mutual interactions between tumors and the microenvironment. These
 insights into in vitro dynamics may deepen our understanding of how
 confinement of healthy cells within a fibrotic tumor niche disrupts tissue
 organization and function in vivo.
description: # Differential Effects of Confinement on the Dynamics of Normal and
 Tumor-Derived Pancreatic Ductal Organoids
 [https://doi.org/10.5061/dryad.fn2z34v55](https://doi.org/10.5061/dryad.fn2z34v55) ## Description of the data and file structure Data from peer-reviewed article: Title: Differential Effects of Confinement on the Dynamics of Normal and Tumor-Derived Pancreatic Ductal Organoids Journal: ACS Applied BioMaterials DOI: 10.1021/acsabm.4c01301 Authors: Jonah M. Rosas,◊ Joseph P. Campanale,◊ Jacob L. Harwood, Lufei Li, Rachel Bae, Shujun Cheng, Julia M. Tsou, Kathi M. Kaiser, Dannielle D. Engle, Denise J. Montell,* and Angela A. Pitenis* ◊ Co-first author \* Corresponding authors: Denise Montell ([dmontell@ucsb.edu](mailto:dmontell@ucsb.edu)), Angela Pitenis ([apitenis@ucsb.edu](mailto:apitenis@ucsb.edu)) ### Files and variables #### File: 2024-ACSAppliedBioMaterialsRosasCampanale-Data.zip File List A) Figure 1c.csv B) Figure 1d.csv C) Figure 2c.csv D) Figure 2f.csv E) Figure 3c.csv F) Figure 4c.csv G) Figure 4d.csv H) Figure 4e.csv I) Figure 4f.csv J) Figure 5d.csv K) Figure 5e.csv L) Figure 6a.csv M) Figure 6b.csv N) Figure 6c.csv O) Figure 6d.csv P) Figure 6e.csv Q) Figure 6g.csv R) Figure 7e.csv S) Figure 7f.csv T) Supplementary Figure S1c.csv U) Supplementary Figure S3.csv V) Supplementary Figure S4a.csv W) Supplementary Figure S4b.csv X) Supplementary Figure S5c.csv Y)Supplementary Figure S6.csv Z)Supplementary Figure S7e.csv AA)Supplementary Figure S8a.csv AB)Supplementary Figure S8b.csv AC)Supplementary Figure S8c.csv AD)Supplementary Figure S8d.csv AE)Supplementary Figure S9.csv AF)Supplementary Figure S10.csv \------------------------------------------------------------------------------------------- FIGURE 1 Microgel composites increase confinement \------------------------------------------------------------------------------------------- Methods: Rheological properties of pure Matrigel® (“less confined”) and microgel-Matrigel® composites (“more confined”) were measured using a TA Instruments® ARES G2 strain-controlled rotational rheometer. All rheological testing used stainless steel parallel plate upper geometry (25 mm diameter), Advanced Peltier Stage (APS) standard flat plate (TA Instruments®, cat.#402619.901) lower geometry, and constant geometry gap of 400 µm. Sample temperature was maintained at 37ºC in a humidified (≈88% RH) and enclosed solvent trap for all experiments. Matrigel® gelation time was maintained at 300 s for all samples prior to experimentation, beyond the gelation time reported by Corning® (t=250 s). Average storage moduli (G’) were determined in the linear viscoelastic region (0.1 to 1% oscillatory strain) for each sample during amplitude sweeps conducted at a frequency f = 1 Hz to demonstrate the rheological tunability of microgel composite systems. Average yield stresses, σy, were calculated at the crossover point of storage and loss moduli intersect (G´ = G´´) and determined using a linear and cubic spline combination (Cubic/Linear (Orche) method in the Trios software that has been well described in rheological studies. A) Fig1c.csv: Average storage modulus (G') for Matrigel® (less confined, n=3) and 75 vol.% microgel, 25 vol.% Matrigel® composites (more confined, n=2) 75 vol.% microgel ("more confined") \- column 1: strain % \- column 2: storage modulus, G' [=] Pa \- column 3: loss modulus, G" [=] Pa \- column 4: strain % \- column 5: storage modulus, G' [=] Pa \- column 6: loss modulus, G" [=] Pa 0 vol.% microgel ("less confined") \- column 8: strain % \- column 9: storage modulus, G' [=] Pa \- column 10: loss modulus, G" [=] Pa \- column 11: strain % \- column 12: storage modulus, G' [=] Pa \- column 13: loss modulus, G" [=] Pa \- column 14: strain % \- column 15: storage modulus, G' [=] Pa \- column 16: loss modulus, G" [=] Pa Average Modulus Data 0 vol.% microgel ("less confined") \- column 18: 0 vol.% microgel ("less confined") loss modulus, G" [=] Pa \- column 19: 75 vol.% microgel ("less confined") loss modulus, G" [=] Pa \- row 3 and beyond: data B) Fig1d.csv: Average yield stress of less confined (n=3) and more confined (n=2) microenvironment. 0 vol.% microgel ("more confined") \- column 1: yield stress sigma_y [=] Pa 75 vol.% microgel ("less confined") \- column 2: yield stress sigma_y [=] Pa \- row 3 and beyond: data \------------------------------------------------------------------------------------------- FIGURE 2 Organoids exhibit genotype-dependent sensitivity to confinement \------------------------------------------------------------------------------------------- Methods: End-point brightfield images of organoids were taken at 48 h using a Nikon Ti2 Widefield microscope with a Plan Fluor 4✕PhL DL objective (NA = 0.13) at 5% CO2, 88% RH. Z-stacks spanned approximately 1.1 mm with a z-step of 20 µm. The widest region of each organoid was manually determined, and the organoid outer diameter was measured in FIJI by drawing a bisecting line across the equatorial plane of each organoid. A minimum of 300 organoids were measured per microenvironmental condition. C) Figure 2c.csv: ) Normal (mN) organoid diameter measurements in less confined and more confined environments. \- column 1: Normal organoid diameters in less confined environments [=] µm \- column 2: Normal organoid diameter in more confined environments [=] µm \- row 3 and beyond: data D) Figure 2f.csv: Tumor (mT) organoid diameter measurements in less confined and more confined environments. \- column 1: Tumor organoid diameters in "less confined" environments [=] µm \- column 2: Tumor organoid diameter in "more confined" environments [=] µm \- row 3 and beyond: data \------------------------------------------------------------------------------------------- FIGURE 3: Confinement disrupts cell polarity and inhibits columnar-to-squamous transition in normal organoids \------------------------------------------------------------------------------------------- E) Figure 3c.csv: Percentage of normal organoids exhibiting normal polarity with apical-inward aPKC staining toward the lumen, or with apical-outward aPKC staining membranes facing outward toward the extracellular environment \- column 1: microenvironment legends ("less confined" "more confined") \- column 2: normal polarity \- column 3: inverted polarity \- row 2 and beyond: data \------------------------------------------------------------------------------------------- FIGURE 4: Organoid projected area and expansion kinetics over time \------------------------------------------------------------------------------------------- Methods: Organoids cultured in Matrigel® (“less confined”) or microgel composites (“more confined”) were plated in Ibidi µ-Slide 8 Well plates with a #1.5 cover glass bottom (Idibi, cat. #80827) and maintained in 5% CO2, 88% RH, and 37ºC conditions for 15 min before overlaying with pre-warmed Mouse Complete Feed Media. Organoids were imaged using a Leica DMi8 widefield microscope with either a HC PL FLUOTAR 10✕ (NA = 0.30) or HC PL FLUOTAR L 20✕ Ph1 (NA = 0.40) objective at 5% CO2, 88% RH, and 37°C. Z-stacks were collected every 15 min for 48 h and spanned approximately 200 µm in z with a z-step of 20 µm. Organoid expansion movies were generated by manual identification of each organoid’s widest equatorial plane at each timepoint to create final single plane movies. We developed a semi-automated method for organoid area segmentation using built-in FIJI edge detection. Movies were segmented and converted to binary objects to determine area change over time. For movies where multiple organoids were in frame or edge detection did not provide high fidelity segmentation over 48 h, manual segmentation was performed by drawing organoid regions of interest over every time frame. Area data was analyzed and plotted using GraphPad Prism version 10.1.1 for Mac, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. F) Figure 4c.csv: Representative organoid area profiles for normal (mN) organoids in less confined and confined microenvironments \- column 1: time (h) with 15 minute intervals \- column 2: normal organoid area measurements over time in less confined microenvironments[=] µm^2 \- column 3: normal organoid area measurements over time in more confined microenvironments[=] µm^2 \- row 2 and beyond: data G) Figure 4d.csv: Representative organoid area profiles for tumor (mT) organoids in less confined and confined microenvironments \- column 1: time (h) \- column 2: normal organoid area measurements over time in less confined microenvironments[=] µm^2 \- column 3: normal organoid area measurements over time in more confined microenvironments[=] µm^2 \- row 2 and beyond: data H) Figure 4e.csv: Normalized organoid area profiles of normal (mN) organoids in less confined and confined microenvironments \- column 1: time (h) \- column 2: Normalized normal organoid area measurements over time in less confined microenvironments[=] µm^2 \- column 3: Normalized normal organoid area measurements over time in more confined microenvironments[=] µm^2 \- row 2 and beyond: data I) Figure 4f.csv: Normalized organoid area profiles of normal (mT) organoids in less confined and confined microenvironments \- column 1: time (h) \- column 2: normal organoid area measurements over time in less confined microenvironments[=] µm^2 \- column 3: normal organoid area measurements over time in more confined microenvironments[=] µm^2 \- row 2 and beyond: data \------------------------------------------------------------------------------------------- FIGURE 5: Particle tracking quantifies microgel displacement due to organoid movement. \------------------------------------------------------------------------------------------- Methods: Confocal imaging was conducted using a Nikon Confocal A1R Laser Scanning Microscope in Resonant scanning mode. Organoids were fluorescently labeled at the time of plating by incubating the organoid pellet in media solution at 1:500 InvitrogenTMCellTrackerTM CMFDA (ThermoFisher cat.#C34552) for 30 min prior to resuspension in Matrigel® (“less confined”) or microgel composites (“more confined”). At 24 h, organoids with lumen were identified and imaged every 30 min for an additional 24 h. The equatorial plane of each organoid was manually determined, and maximum intensity projections were generated in FIJI from volumes spanning ±16 µm in the z-dimension centered around the organoid equatorial plane. A 100 ✕ 100 µm ROI was drawn at the interface of the organoid membrane and adjacent microgel microparticle embedded with fluorescent nanoparticles (see Section 8.02). Nanoparticle displacement trajectories indicating organoid-induced microgel movements were determined using the Mosaic FIJI plugin. To account for nanoparticles moving in and out of the focal plane, trajectories that persisted for less than 5 h were excluded from analysis. J) Figure5d.csv: particle trajectory travel distances of tumor and normal organoids in more confined microenvironments \- column 1: particle trajectory identifier \- column 2: travel distance of particle trajectories generated by tumor organoids [=] µm \- column 3: travel distance of particle trajectories generated by normal organoids [=] µm \- row 3 and beyond: data K) Figure5e.csv: particle trajectory linearity indeces of tumor and normal organoids in more confined microenvironments \- column 1: particle trajectory identifier \- column 2: travel distance of particle trajectories generated by tumor organoids [=] µm \- column 3: travel distance of particle trajectories generated by normal organoids [=] µm \- row 3 and beyond: data \- row 3 and beyond: data \-------------------------------------------------------------------------------------- FIGURE 6: Tumor and normal organoid pulse profiles. \------------------------------------------------------------------------------------------- Methods: Organoid pulse profiles were generated by plotting the % area change every 15 minutes during extended live imaging experiments. % Change = (Area (tn) - Area (tn-1))/Area (tn). Area decreases 10% or greater were defined as a pulse. L) Figure6a.csv: normalized normal oraganoid area change vs. time (less confined) \- row 1: x and y axis, corresponding to time (x) and area change % (y) \- row 2 and beyond: data M) Figure6b.csv: normalized normal oraganoid area change vs. time (more confined) \- row 1: x and y axis, corresponding to time (x) and area change % (y) \- row 2 and beyond: data N) Figure6c.csv: normalized tumor oraganoid area change vs. time (less confined) \- row 1: x and y axis, corresponding to time (x) and area change % (y) \- row 2 and beyond: data O) Figure6d.csv: normalized tumor oraganoid area change vs. time (more confined) \- row 1: x and y axis, corresponding to time (x) and area change % (y) \- row 2 and beyond: data P) Figure6e.csv: organoid pulse rates \- row 1: x-label, corresponding to genotype and microenvironmental condition \- row 2: units for corresponding axes \- row 3 and beyond: data Q) Figure6g.csv: proportion of normal organoids per field of view pulsing in less confined and confined microenvironments \- row 1: x-lavel, corresponding to microenvironmental condition of normal organoids \- row 2: % of organoids in a given field of view observed to pulse when treated with DMSO \- row 3 % of organoids in a given field of view observed to pulse when treated with Forskolin \-------------------------------------------------------------------------------------- FIGURE 7: Increased hydrostatic pressure partially rescues normal organoid polarity but not diameter. \------------------------------------------------------------------------------------------- R) Figure7e.csv: quantification of percent normal organoids with apical membranes facing inward toward lumen. \- column 1: organoid genotype replicate \- column 2: % of normal organoids treated with DMSO in less confined microenvironments with apical inward polarity. \- column 3: % of normal organoids treated with forskolin in less confined microenvironments with apical inward polarity. \- column 4: % of normal organoids treated with DMSO in more confined microenvironments with apical inward polarity. \- column 5: % of normal organoids treated with forskolin in more confined microenvironments with apical inward polarity. \- row 2 and beyond: data S) Figure7f.csv: normal organoid diameter measurements at 48 h in less confined (circles) and confined (triangles) after treatment with DMSO or forskolin. \- row 1: x axis, corresponding to microenvironmental condition (less confined or more confined) and treatment condition (DMSO or FSK) \- row 2: normal organoid genotype tested \- row 3 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE S1: Microgel particle sorting produces a diverse range of particle sizes \-------------------------------------------------------------------------------------- Methods: Polyacrylamide hydrogels were synthesized at 3 wt.% AAm with a crosslinker concentration of 0.12 wt.% MBAm. Bulk hydrogels were mechanically processed and sorted by Stokes’ sedimentation to achieve an average microgel diameter of 104 ± 17 µm S.D. Microgels were imaged using a Nikon Ti2 Widefield Microscope with a Plan Fluor 4✕ (NA = 0.13) air objective and an S Plan Fluor 20✕ ELWD (NA = 0.45) air objective. Average microgel diameters were measured by defining three points at the edge of each microgel particle (n = 200) and fitting a circle that contained all three points. Microgel diameters were determined using the 3-point circle fitting tool in the NIS Element software. T) Supplementary Figure 1.csv \- column 1: microgel particle number \- column 2: image ID \- column 3: particle radius [=] µm \- column 4: particle diameter [=] µm \- row 2 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S3: Oscillatory strain rheology of less confined and confined microenvironments \-------------------------------------------------------------------------------------- U) Supplementary Figure 3.csv \- row 1: x and y axis, corresponding to strain percentage (x), storage modulus, G' (Y1), and loss modulus, G'' (Y2). \- row 2 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S4: Oscillatory strain rheology of 3 wt.% and 5 wt.% microgels mixed at different volume ratios of Matrigel® \-------------------------------------------------------------------------------------- V) Supplementary Figure 4a.csv: Storage Modulus \- row 1: microgel polymer content, corresponding to 3 wt.% and 5 wt.% polyacrylamide. \- row 2: x and y axis, corresponding the volume percent Matrigel, 0, 25, 50 vol%, (X), and storage modulus, G' [=] Pa (Y) \- row 3 and beyond: data W) Supplementary Figure 4b.csv: Yield Stress \- row 1: microgel polymer content, corresponding to 3 wt.% and 5 wt.% polyacrylamide. \- row 2: x and y axis, corresponding the volume percent Matrigel, 0, 25, 50 vol%, (X), and yield stress, sigma_y [=] Pa (Y) \- row 3 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S5: Interstitial space between microgel particles narrows with increasing microgel volume fraction \-------------------------------------------------------------------------------------- Methods: FITC-Matrigel® was mixed with nanoparticle-embedded microgels at 50 vol.% microgel or 75 vol.% microgel and equilibrated at 37ºC for at least 10 min prior to imaging. Confocal imaging was conducted using a Nikon A1R Laser Scanning Confocal Microscope with a PlanAPO lambda 10✕ air objective (NA: 0.45), 1 AU pinhole, 2.2 µm step size, and 156 µm in the z-dimension (71 z-slices). Matrigel® channel widths were determined for the 50 vol.% and 75 vol.% microgel composites using the built-in, calibrated local thickness tool in FIJI. X) Supplementary Figure 5c.csv Average Channel Width of Microgel Composites \- row 1: 3 wt.% micorgel composite labels corresponding to 50 and 75 vol% microgel composites. \- row 2: z-plane count, average channel width, and standard deviation of channel width as determined by local thickness analysis in ImageJ. \- row 3 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S6: Organoids exhibit cell-line-dependent sensitivity to confinement \-------------------------------------------------------------------------------------- Y)Supplementary Figure 6.csv: Organoid Diameter Measurements -row 1: Organoid genotype labels -row 2: microenvironment condition labels corresponding to "less confined" or "more confined" -row 3: data [=] µm \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S7: Cell division in normal and tumor organoids \-------------------------------------------------------------------------------------- Z)Supplementary Figure 7.csv: Organoid PH3 Counts -row 1: Labels corresponding to normal genotype tested (N10, N11, N12) -row 2: Labels corresponding to microenvironment tested (Matrigel, "less confined" or Microgel "more confined), Number of PH3+ cells encountered per organoid, total nuclei counted per organoid, and the average number of PH3 + cells calculated for all replicates within each microenvironment. -row 3–17: data -row 19: Labels corresponding to tumor genotypes tested (T8, T9, T69A) -row 20: microenvironment condition labels corresponding to "less confined" or "more confined" -row 21 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S8: Organoid growth kinetics \-------------------------------------------------------------------------------------- AA)Supplementary Figure 8a.csv: Less confined normal growth kinetics -row 1: organoid genotype labels -row 2: area units [=] µm^2 -row 3: x and y axes, corresponding to time in hours (x) and organoid area (y) [=] µm^2 -row 4 and beyond: data AB)Supplementary Figure 8b.csv: More confined growth kinetics -row 1: organoid genotype labels -row 2: area units [=] µm^2 -row 3: x and y axes, corresponding to time in hours (x) and organoid area (y) [=] µm^2 -row 4 and beyond: data AC)Supplementary Figure 8c.csv: Less confined tumor growth kinetics -row 1: organoid genotype labels -row 2: area units [=] µm^2 -row 3: x and y axes, corresponding to time in hours (x) and organoid area (y) [=] µm^2 -row 4 and beyond: data AD)Supplementary Figure 8d.csv: More confined tumor growth kinetics -row 1: organoid genotype labels -row 2: area units [=] µm^2 -row 3: x and y axes, corresponding to time in hours (x) and organoid area (y) [=] µm^2 -row 4 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S9: Percentage of tumor organoids pulsing when treated with 8 small molecules targeting cytoskeletal elements, ion channels, and signaling pathways \-------------------------------------------------------------------------------------- Methods: Organoids in pure Matrigel® (“less confined”) or in microgel composite (“more confined”) were plated in CellVis 96-Well Glass Bottom Plates with a #1.5 cover glass bottom (CellVis, cat.#P96-1.5H-N), overlaid with pre-warmed Mouse Complete Feed Media, and returned to 5% CO2, 88% RH, and 37ºC conditions for 24 h. After 24 h, organoids were treated with either dimethyl sulfoxide (SigmaAldrich, cat.# S-002-D, 20.5 mM) para-amino Blebbistatin (Cayman Chemicals, cat.#22699, 10 µM), Y-27637 Hydrochloride (Cayman Chemicals, cat.#1000583, 10.5 µM), Cytochalasin B (SigmaAldrich, cat.#C2743, 5 µM), CFTR(inh)-172 (MedChem Express, cat.#HY-16671, 10 µM), MRTX-1133 (MedChem Express, cat.#HY-134813, 5 µM), Wortmannin (SigmaAldrich, cat.#19545-26-7, 1 µM), Forskolin (MechChem Express, cat.#HY-15371, 5 µM), ETH-1864 (Cayman Chemicals, cat.#17258, 20 µM), or VX-11e (Selleckchem, cat.#S7709, 10 µM). Organoids were imaged using a Leica DMi8 widefield microscope with an HC PL FLUOTAR 10✕ (NA = 0.30) objective at 5% CO2, 88% RH, and 37°C. Z-stacks were collected every 30 min for 24 h and spanned 600 – 800 µm in z with a z-step of 25 µm. Each field of view was analyzed for the percent of organoids in view that exhibited one or more pulses over 24 h. Organoids were scored as either pulsing or non-pulsing with a minimum of 7 organoids averaged per field of view. AE)Supplementary Figure 9.csv -column 1: organoid genotype tested -column 2: drug treatment -column 3: File name -column 4: total number of organoids in field of view -column 5: total number of pulsing organoids per field of view -row 2 and beyond: data \-------------------------------------------------------------------------------------- SUPPLEMENTARY FIGURE S10: Forskolin induces pulsing in normal organoids \-------------------------------------------------------------------------------------- AF) Supplementary Figure 10.csv -column 1: organoid genotype tested -column 2: drug treatment -column 3: File name specifying Matrigel (less confined) or Microgel (more confined) growth conditions -column 4: total number of organoids in field of view -column 5: total number of organoids counted per drug treatment per microenvironment -column 6: total number of pulsing organoids per field of view -column 7: percentage of organoids pulsing -row 2 and beyond: data \--------------------------------------------------------------------------------------