(F-226) In Vitro Dynamics of Pancreatic Cancer Organoids in 3D

Introduction:: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths globally with a 5-year median survival rate less than 10%. Progression from healthy tissue through intraepithelial neoplasia (PanIN) to advanced PDAC results in tissue level changes in the microenvironment. Tissue stiffening due to fibrosis is a hallmark of disease progression. Tissue stiffness changes from 1 kPa (healthy) to 4 kPa (PDAC) confine functional communities of islet and acinar cells in an evolving extracellular matrix (ECM) rich in collagen-type 1. Significant work has focused on both the biochemical contributions of the ECM in PDAC as a source of scavenged nutrients as well as how activated fibroblast communities remodel and stiffen the ECM. A renewed focus has been placed on the role of the microenvironment in disease progress.



Herein, we sought to decouple the biochemical and mechanical contributions of the ECM in PDAC using in vitro pancreas organoids derived from normal and tumorous ducts. We designed hydrogel composite systems that rheologically match existing MatrigelTM culture conditions of pancreas duct organoid models. We identified specific healthy and tumor dependencies on ECM abundance and microenvironmental confinement. We further identified a characteristic “pulse” behavior exhibited by cancer epithelia that persists in highly confined microenvironments. Our data on ECM abundance and confinement provide new insight into the mechanisms cancer cells might use to generate mechanical forces to thrive in a complex 3D microenvironment.

Materials and Methods:: Polyacrylamide hydrogels were polymerized using 3 wt% acrylamide and crossling linker of 0.12 wt% N,N’-methylenebisacrylamide. Bulk hydrogels were mechanically processed and sorted by sedimentation to achieve a final microgel particle size distribution of 100 ± 10 µm. Prior to experimentation, microgels equilibrated in organoid media for 24 hours. Microgels were mixed at 50:50 and 75:25 (PAAm:Matrigel volume ratios) with High Concentration GFR Corning Matrigel. Composites created encapsulation environments with tunable rheological properties to support the continuous organoid culture. Murine tissue-derived pancreas ductal organoids were generously gifted by Dr. Danielle Engle at the Salk Institute.



Rheological properties of the PAAm-Matrigel composite systems were measured using a parallel plate rheometer (TA Instruments ARES G2). A 40 mm stainless steel parallel plate upper geometry and an Advanced Peltier Stage (APS) standard flat plate (402619.901) lower geometry were used within a humidified and enclosed solvent trap. Sample temperature was maintained at 37ºC. Strain-controlled experiments were performed with a constant geometry gap of 500 µm. Storage moduli (G’) of the PAAm-Matrigel composites were determined based on the linear viscoelastic region of each sample during amplitude sweeps conducted at a frequency, f = 1 Hz.



Organoids were fluorescently labeled using CMFDA CellTracker Green at 20nM, grown in PAAm-Matrigel composites and imaged every 30 minutes for 24 hours using a Nikon A1R HD confocal laser scanning microscope.



FIJI image analysis software was used to generate binary masks from projected z-stacks to visualize changes in organoid cross-sectional area over time.


Results, Conclusions, and Discussions:: Rheological analysis of the PAAm-Matrigel composites showed no significant change in storage modulus (G’) from that of Matrigel at 9.0 mg/mL protein content (Figure 1). The stiffness of both PAAm: Matrigel composites was identified in the linear viscoelastic region of strain measurements and found to be approximately 100 Pa. Image analysis of fluorescently labeled PAAm-Matrigel composites showed that increasing microgel content increased particle packing (Figure 2).



We cultured three independent normal and tumor organoid lines in 100% Matrigel alone and in PAAm-Matrigel composites and measured organoid outer diameter. Tumor organoids formed to outer diameters between 130 µm and 160 µm with no significant decrease in organoid diameter across all microenvironments. In contrast, normal organoids grew in all conditions, but were significantly smaller in 75 PAAm-Matrigel environments compared to Matrigel alone (Figure 3). Normal organoids formed fewer lumen successfully under highly confining 75 PAAm-Matrigel environments, whereas tumor organoids formed similar sized lumen even under high confinement.



Tumor organoids exhibited a periodic pulsing behavior where a change in organoid area >10% was considered a unique “pulse” event (Figure 4 a,b). Normal organoids did not pulse in either pure Matrigel or 75 PAAm-Matrigel microenvironment (Figure 4). In highly confined microenvironments, tumors pulsed with sufficient force to displace adjacent, non-degradable hydrogel particles (Figure 4 c,d). Treatment with both ROCK inhibitor and a selective KRASG12Dinhibitor (MRTX-1133) were insufficient to fully suppress pulsing behavior.




Healthy organoids rely on greater than 50% of the ECM in their surroundings for biochemical cell-ECM signaling. In contrast, tumor organoids exhibited greater ECM independence and formed large lumen in ECM scarce environments, suggesting the biochemical role of the ECM is less important for tumor organoids than the mechanical support the ECM provides.



Tumor and healthy organoids exhibit distinct abilities to overcome confining microenvironments in vitro. While healthy organoids form smaller and more compact lumen after 48 hours, tumor organoids can generate forces sufficient to displace non-degradable hydrogels in highly confined environments (75 PAAm-Matrigel) that may enable larger lumen size. This demonstrates tumor organoids can plastically deform their surrounds, transmitting forces multiple organoid diameters away (Figure 4).

Acknowledgements (Optional): :

References (Optional): :