3D ECM Research Articles

Emerging technologies in regenerative medicine: The future of wound care and therapy.

Wound healing, an intricate biological process, comprises orderly phases of simple biological processed including hemostasis, inflammation, angiogenesis, cell proliferation, and ECM remodeling. The regulation of the shift in these phases can be influenced by systemic or environmental conditions. Any untimely transitions between these phases can lead to chronic wounds and scarring, imposing a significant socio-economic burden on patients. Current treatment modalities are largely supportive in nature and primarily involve the prevention of infection and controlling inflammation. This often results in delayed healing and wound complications. Recent strides in regenerative medicine and tissue engineering offer innovative and patient-specific solutions. Mesenchymal stem cells (MSCs) and their secretome have gained specific prominence in this regard. Additionally, technologies like tissue nano-transfection enable in situ gene editing, a need-specific approach without the requirement of complex laboratory procedures. Innovating approaches like 3D bioprinting and ECM bioscaffolds also hold the potential to address wounds at the molecular and cellular levels. These regenerative approaches target common healing obstacles, such as hyper-inflammation thereby promoting self-recovery through crucial signaling pathway stimulation. The rationale of this review is to examine the benefits and limitations of both current and emerging technologies in wound care and to offer insights into potential advancements in the field. The shift towards such patient-centric therapies reflects a paradigmatic change in wound care strategies.

Growth and organotypic branching of lung-specific microvascular cells on 2D and in 3D lung-derived matrices

Tissue-specific endothelial cells have vital roles in maintenance and functioning of native tissues with constant reciprocal crosstalk with resident cells. Three-dimensional (3D) physio-mimetic in vitro models which incorporate lung-specific microvasculature are needed to model lung-related diseases which involve modulation of endothelial cell behavior like cancer. In this study, we investigated the growth kinetics, morphological changes and responses to biological cues of lung microvasculature on two-dimensional (2D) and in lung matrix-derived 3D hydrogels. HUVEC and HULEC-5a cells were cultured on 2D and compared for their growth, morphologies, and responses to varying growth medium formulations. Brightfield and immunofluorescence imaging was performed to assess differences in morphology. For 3D cultures, native bovine lungs were decellularized, lyophilized, solubilized, and reconstituted into hydrogel form in which endothelial cells were embedded. Cell growth and organotypic branching was monitored in 3D hydrogels in the presence of varying biological cues including lung cancer cell secretome. HUVEC and HULEC-5a cells demonstrated comparable growth and morphology on 2D. However, in 3D lung-derived ECM hydrogels, tissue-specific HULEC-5a cells exhibited much better adaptation to their microenvironment, characterized by enhanced organotypic branching and longer branches. HULEC-5a growth was responsive to lung cancer cell-conditioned medium in both 2D and 3D conditions. In 3D, the concentration of ECM ligand significantly affected cell growth in long-term culture where molecular crowding had an inhibitory role. Our data reveals that HULEC-5a cells offer a reliable alternative to frequently pursued HUVECs with comparable growth and morphology. Due to their intrinsic program for cellular crosstalk with resident cells, the use of tissue-specific endothelium constitutes a vital aspect for modeling physiological and pathological processes. Furthermore, our study is the first demonstration of the synergy between lung-specific microvasculature with lung-specific ECM within a 3D in vitro model.

Abstract 5226: A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology

Abstract We developed a screening platform for immune oncology using an assay based on automated image guided injection of tumoroids in wells of multi-well plates preloaded with a collagen-rich ECM. The identical x-y-z position and size of the ECM-embedded tumoroids in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. The model is used to generate quantitative data for T cell recruitment to tumoroids and killing of tumoroids by T cells. We have applied this to screening a panel of CD3:Her2 bispecific antibodies (BsAb) binding with different affinities to CD3 or Her2 or displaying high affinity interactions with different epitopes on Her2. Exposure to fresh, non-activated peripheral blood mononuclear cell (PBMC) derived T cells shows an initial phase of random T cell movement throughout the ECM followed by a BsAb-dependent phase of active T cell recruitment to tumoroids (day 2-4) and a subsequent phase of tumoroid killing (day 4-6). We show that the wave of T cell recruitment following initial T cell-tumoroid contact involves chemotactic signaling. Decreased affinity at the Her2 or CD3 arm can be compensated for by increasing BsAb concentrations. However, we detect major differences in efficacy for different high affinity epitopes. I.e., of two BsAbs interacting with high affinity with distinct Her2 epitopes and each causing effective tumor cell killing in 2D co-culture, only one was able to trigger a wave of T cell recruitment and subsequent tumoroid killing in 3D. We have applied the same setup to testing the efficacy of T cells expressing engineered TCRs aimed at application in adoptive T-cell therapy. Kinetics of experiments using these activated engineered T cells are considerably shorter (24 hours instead of ~6 days) but show a similar pattern of T cell recruitment and subsequent tumoroid killing. We demonstrate successful generation of quantitative data for T cell recruitment and tumoroid killing for engineered T cells targeting tumor antigens expressed on TNBC and uveal melanoma tumoroids. Citation Format: Erik H. J. Danen. A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5226.

Flavonoid brachydin B decreases viability, proliferation, and migration in human metastatic prostate (DU145) cells grown in 2D and 3D culture models.

Brachydin B (BrB) is a unique dimeric flavonoid extracted from Fridericia platyphylla (Cham.) LG Lohmann with different biological activities. However, the antitumoral potential of this flavonoid is unclear. In our study, we evaluated the effects of the BrB flavonoid on cell viability (MTT, resazurin, and lactate dehydrogenase assays), proliferation (protein dosage and clonogenic assay), and migration/invasion (3D ECM gel, wound-healing, and transwell assays) of metastatic prostate (DU145) cells cultured both as traditional 2D monolayers and 3D tumor spheroids in vitro. The results showed that the BrB flavonoid promotes cytotoxic effects from ≥1.50μM after 24h of treatment in DU145 cells in monolayers. In 3D prostate tumor spheroids, BrB also induced cytotoxic effects at higher concentrations after longer treatment (48, 72, and 168h). Furthermore, BrB treatment is associated with reduced DU145 clonogenicity in 2D cultures, as well as decreased area/volume of 3D tumor spheroids. Finally, BrB (6μM) reduced cell migration/invasion in 2D monolayers and promoted antimigratory effects in DU145 tumor spheroids (≥30μM). In conclusion, the antitumoral and antimigratory effects observed in DU145 cells cultured in 2D and 3D models are promising results for future studies with BrB using in vivo models and confirm this molecule as a candidate for metastatic prostate cancer therapy.

Abstract 1880: High throughput screening of 30 PDX cell lines in a 3D ECM hydrogel platform, incorporating tumor, stroma and immune components to demonstrate simultaneous investigation of multiple anti-tumor modalities

Abstract High throughput screening offers tangible benefits towards rapidly testing various permutations of novel or existing therapeutic agents. In particular, tumor panels that cover a range of histotypes and molecular subtypes have been previously developed, such as the NCI-60, however they utilize cell lines and, in some cases, a 2D cell culture format, which limit their translatability to preclinical and clinical trials. Moreover, the biological complexity of the tumor microenvironment (TME) has revealed a need for more translatable 3D in vitro tumor models that reflect the in vivo physiological outcome to therapies, particularly with the explosion of immunotherapy programs in drug discovery which target the immune compartment of the TME. Here, we describe for the first time a 3D in vitro PDX panel comprising 30 distinct PDX models in coculture with fibroblasts and PBMCs in engineered extracellular matrix hydrogels that display distinct similarities to the three compartments of the TME - tumor, stroma, and immune cells. Histotypes in the panel include, amongst others, Non-small cell lung, colorectal, breast, pancreatic, gastric, melanoma, and renal cancers. The panel is constructed in a high throughput 96-well format and rapidly assays tumor growth delay and other endpoints such as apoptosis in a dose-dependent manner across various drug modalities such as small molecules, biologics and cell therapy. The panel has been tested against targeted therapy (Docetaxel, Cisplatin) and immunomodulatory agents (Pembrolizumab, Atezolizumab) and the results correlate to the corresponding PDX in vivo data. Interestingly, cisplatin treatment displayed an average size reduction of >30% for melanoma but a varied response in NSCLC tumors. And, several NSCLC responded more favorably to checkpoint inhibitors, however, the lung epidermoid PDX demonstrated no significant response. Moreover, subsequent cytokine analysis and immunofluorescence staining of several models revealed protein signatures of cancer-associated fibroblasts and CD3+ sequestration in the tumor stroma in some 3D models, suggesting the fibroblasts’ critical role in regulating the immune response. In short, the 30-PDX Panel described here represents a large step forward towards achieving translatable efficacy data at the earliest stages of drug discovery where little is known about the mechanism of action for a particular therapeutic agent or combination of agents. Citation Format: Kolin C. Hribar, Bin Xue, Christopher Harrod, Timothy Jensen, Julia Schüler. High throughput screening of 30 PDX cell lines in a 3D ECM hydrogel platform, incorporating tumor, stroma and immune components to demonstrate simultaneous investigation of multiple anti-tumor modalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1880.

Decellularized organ biomatrices facilitate quantifiable in vitro 3D cancer metastasis models.

Metastatic cancers are chemoresistant, involving complex interplay between disseminated cancer cell aggregates and the distant organ microenvironment (extracellular matrix and stromal cells). Conventional metastasis surrogates (scratch/wound healing, Transwell migration assays) lack 3D architecture and ECM presence. Metastasis studies can therefore significantly benefit from biomimetic 3D in vitro models recapitulating the complex cascade of distant organ invasion and colonization by collective clusters of cells. We aimed to engineer reproducible and quantifiable 3D models of highly therapy-resistant cancer processes: (i) colorectal cancer liver metastasis; and (ii) breast cancer lung metastasis. Metastatic seeds are engineered using 3D tumor spheroids to recapitulate the 3D aggregation of cancer cells both in the tumor and in circulation throughout the metastatic cascade of many cancers. Metastatic soil was engineered by decellularizing porcine livers and lungs to generate biomatrix scaffolds, followed by extensive materials characterization. HCT116 colorectal and MDA-MB-231 breast cancer spheroids were generated on hanging drop arrays to initiate clustered metastatic seeding into liver and lung biomatrix scaffolds, respectively. Between days 3-7, biomatrix cellular colonization was apparent with increased metabolic activity and the presence of cellular nests evaluated via multiphoton microscopy. HCT116 and MDA-MB-231 cells colonized liver and lung biomatrices, and at least 15% of the cells invaded more than 20 μm from the surface. Engineered metastases also expressed increased signatures of genes associated with the metastatic epithelial to mesenchymal transition (EMT). Importantly, inhibition of matrix metalloproteinase-9 inhibited metastatic invasion into the biomatrix. Furthermore, metastatic nests were significantly more chemoresistant (>3 times) to the anti-cancer drug oxaliplatin, compared to 3D spheroids. Together, our data indicated that HCT116 and MDA-MB-231 spheroids invade, colonize, and proliferate in livers and lungs establishing metastatic nests in 3D settings in vitro. The metastatic nature of these cells was confirmed with functional readouts regarding EMT and chemoresistance. Modeling the dynamic metastatic cascade in vitro has potential to identify therapeutic targets to treat or prevent metastatic progression in chemoresistant metastatic cancers.

Tissue engineering ECM-enriched controllable vascularized human microtissue for hair regenerative medicine using a biomimetic developmental approach

IntroductionRegenerative medicine is a promising approach for hair loss; however, its primary challenge is the inductivity of human dermal papilla cells (DPCs), which rapidly lose hair growth-inducing properties in 2D culture. Despite extensive research efforts to construct DPCs, current 3D microenvironments fabricated to restore hair inductivity remain insufficient. ObjectivesHere, we aimed to fabricate ECM-enriched controllable vascularized dermal papilla (DP) spheroids that highly mimic in vivo DPCs microenvironments to restore their hair inductivity. MethodsWe employed layer-by-layer (LbL) self-assembly using gelatin and alginate to construct nanoscale biomimetic ECM for DPCs, with Ca2+ as a cross-linking agent to create controllable DP spheroids. DPCs were also co-cultured with human umbilical vein endothelial cells to construct vascularized DP spheroids. Immunofluorescence staining and angiography was used to detect angiogenesis in vitro and in vivo. RNA sequencing and in vivo implantation were employed to investigate DPCs signature. ResultsLbL technology enabled DPCs to aggregate into controllable DP spheroids of size and cell numbers similar to those of primary DP. Vascularization prevented hypoxia-induced necrosis and functioned in association with host vessels post-transplantation. Compared with traditional 3D culture, nanoscale ECM and vascularization were found to restore the transcriptional signature of DPCs and triple hair induction efficiency following engraftment. ConclusionOur novel biomimetic developmental tissue engineering strategy is a crucial step toward the recovery of human DPC hair inductivity, which would enable the rapid clinical application of large-scale hair regeneration platforms.

Histological study of an experimentally constructed decellularized rat lung as a model of whole organ three-dimensional natural scaffold

Abstract Introduction With the increase of end stage lung diseases and the great problems facing lung transplantation tissue engineering become a promising solution. The first step in lung engineering is to obtain a 3D Extracellular matrix lung scaffold via decellularization. Decellularization aims to remove cells from tissue ultrastructure while preserving the mechanical and biological properties of the tissue. Intact ECM provides critical cues for differentiation and migration of cells that are seeded onto the organ scaffold. Objectives This study aimed to obtain an intact and well-preserved ECM lung scaffold by decellularization of rat lungs. Methods Decellularization of lungs of ten Wistar rats was achieved by perfusing detergents through the pulmonary artery. The resultant scaffolds were fixed and analyzed histologically. Results It was found that the decellularization process effectively removed the cellular and nuclear material while retaining native the 3D ECM of lung tissue. The architecture of the collagen and elastic fibers networks were preserved as comparable to the native lungs. Furthermore, the basement membranes of the bronchiolar and interalveolar septa were intact. Conclusions This methodology is expected to allow decellularization of human lung tissues and permits future scientific exploration in tissue engineering.

Microsphere Sensors for Charactering Stress Fields within Three-Dimensional Extracellular Matrix

Stress in the three-dimensional extracellular matrix is one of the key cues in regulating multiscale biological processes. Thus far, noticeable progress in methods and technologies (e.g., micropipette aspiration, AFM, and molecule probes) has been made to quantify forces/stresses in cell microenvironment at different length scales. Among them, the microsphere sensor-based method (MSS-based method) has emerged as an advantageous approach over conventional techniques in quantifying stress in situ and in vivo at the cellular and supra-cellular scales. This method is implemented by seven sequential steps, including fabrication, modification, characterization, cell adhesion, imaging, displacement field extraction, and stress calculation. Precise control of each step and inter-tunning between steps can provide quantitative characterization of stress field. However, detailed procedural information associated with each step and process has been scattered. This review aims to provide a comprehensive overview of MSS-based characterization of stress fields within 3D ECM, systematically summarizing the principles and research progresses. Firstly, the basic principles are introduced, and the specific experiment and calculation processes of the MSS-based method are presented in detail. Then, recent advances and applications of this method are summarized. Moreover, the selection of materials and techniques for the MSS-based method needs to be adjusted independently according to the research objectives and requirements (e.g., specific tissue, length scale, time scale and resolution). Finally, perspectives of the limitations and development trends of the MSS-based method are discussed. This specific and comprehensive review would provide a guideline for the widespread application of MSS-based method as an advantageous method for in situ and in vivo stress measurement over both cellular and supra-cellular scale within 3D ECM.

Dense Bone-Derived 3d Bioscaffold: Preparation, Characterization, and Assessment of Its Potential for Bone Marrow Mesenchymal Stem Cells (BMSCS) Growth and Differentiation

Background: Since bone defects can result in different disabilities, many efforts have been made to bone tissue engineering. In this case, scaffolds play an important role as a key element of tissue engineering in providing three-dimensional structure for cell growth in vitro Objective: The aim of the present study was to provide the three-dimensional biological bioscaffold from the bovine femur dense bone and investigate the possibility of its potential for application in tissue engineering as biological 3D ECM bioscaffold via mesenchymal stem cells seeding and differentiation toward bone tissue. Methods: For the preparation of bioscaffolds, after cutting bovine femur bone into small pieces, demineralization and decellularization were done. Bioscaffolds biocompatibility was evaluated using an MTT assay. The morphological and cell adhesion characteristics of Bone marrow mesenchymal stem cells (BMSCs) on the bioscaffolds were evaluated using Scanning Electron Microscopy (SEM) technique. Finally, the cells were treated with an osteogenic differentiation medium and then evaluated for differentiation. Results: Histological studies showed that the use of sodium dodecyl sulfate (2.5%) for 8 h eliminated the cells. Radiography and calcium oxalate test confirmed demineralization. MTT assay and SEM studies showed that the obtained bioscaffolds are biocompatible and could provide an optimum three-dimensional environment for cell adhesion and movement. Moreover, the Alizarin red staining showed a higher differentiation rate for BMSCs. Conclusion: In the present study, bone-derived 3D bioscaffold showed an important role in the growth and differentiation of BMSCs, due to the natural characteristics, cell adhesion properties, and potential to enhance differentiation toward bone tissue. It may have the potential for use as bioscaffold as supporting metrics for maintenance, growth in bone tissue engineering.

Collagen/kerateine multi-protein hydrogels as a thermally stable extracellular matrix for 3D in vitro models

Objective: To determine whether the addition of kerateine (reduced keratin) in rat tail collagen type I hydrogels increases thermal stability and changes material properties and supports cell growth for use in cellular hyperthermia studies for tumor treatment. Methods: Collagen type I extracted from rat tail tendon was combined with kerateine extracted from human hair fibers. Thermal, mechanical, and biocompatibility properties and cell behavior was assessed and compared to 100% collagen type I hydrogels to demonstrate their utility as a tissue model for 3D in vitro testing. Results: A combination (i.e., containing both collagen ‘C/KNT’) hydrogel was more thermally stable than pure collagen hydrogels and resisted thermal degradation when incubated at a hyperthermic temperature of 47°C for heating durations up to 60 min with a higher melting temperature measured by DSC. An increase in the storage modulus was only observed with an increased collagen concentration rather than an increased KTN concentration; however, a change in ECM structure was observed with greater fiber alignment and width with an increase in KTN concentration. The C/KTN hydrogels, specifically 50/50 C/KTN hydrogels, also supported the growth and of fibroblasts and MDA-MB-231 breast cancer cells similar to those seeded in 100% collagen hydrogels. Conclusion: This multi-protein C/KTN hydrogel shows promise for future studies involving thermal stress studies without compromising the 3D ECM environment or cell growth.

Role of interstitial flow in tumor migration through 3D ECM

Cancer invasion in tissue is simultaneously regulated by chemical and mechanical cues. Evidences suggest that interstitial flow plays a critical role in tumor metastasis. On one hand, the distribution of chemokines around cell is influenced by flow. On the other hand, interstitial flow may reconfigure the alignment of fiber matrix, which greatly changes the contact force between cell and extracellular matrix. In this study, we have upgraded a model by which we can quantitatively investigate the influence of flow on tumor cell migration. A hydrodynamic analysis of shear stress on a slender body is introduced to simulate the fiber realignment. Factors such as subtle flow and cell–matrix interaction which dominate tumor migration are integrated in this novel model. Simulation results show interstitial flow facilitates tumor cell migration in the flow direction. Moreover, the flow-related chemical and mechanical cues have a synergistic effect on the migration. This model provides better understanding on cancer metastasis and helps design vitro experiment precisely.

Evaluation of NV556, a Novel Cyclophilin Inhibitor, as a Potential Antifibrotic Compound for Liver Fibrosis.

Hepatic fibrosis can result as a pathological response to nonalcoholic steatohepatitis (NASH). Cirrhosis, the late stage of fibrosis, has been linked to poor survival and an increased risk of developing hepatocellular carcinoma, with limited treatment options available. Therefore, there is an unmet need for novel effective antifibrotic compounds. Cyclophilins are peptidyl-prolyl cis-trans isomerases that facilitate protein folding and conformational changes affecting the function of the targeted proteins. Due to their activity, cyclophilins have been presented as key factors in several stages of the fibrotic process. In this study, we investigated the antifibrotic effects of NV556, a novel potent sanglifehrin-based cyclophilin inhibitor, in vitro and in vivo. NV556 potential antifibrotic effect was evaluated in two well-established animal models of NASH, STAM, and methionine-choline-deficient (MCD) mice, as well as in an in vitro 3D human liver ECM culture of LX2 cells, a human hepatic stellate cell line. We demonstrate that NV556 decreased liver fibrosis in both STAM and MCD in vivo models and decreased collagen production in TGFβ1-activated hepatic stellate cells in vitro. Taken together, these results present NV556 as a potential candidate for the treatment of liver fibrosis.

Abstract 1028: Extracellular matrix dimensionality reduces cellular cortical tension to stimulate pro-survival signaling in mammary epithelial cells

Abstract Tumor dormancy is a clinical phenomenon in which disseminated tumor cells remain asymptomatic and undetectable over a long period of time. Dormant cells are able to maintain quiescence in hostile microenvironments, escape frontline cancer therapies and evade the immune system, as well as their propensity to reactivate from latency and cause metastatic relapse. Although tumor dormancy is an important problem in the treatment of cancer, the molecular mechanisms underlying this complex process remain unclear. Because dormant cells are frequently found surrounded by a laminin-rich ECM, we hypothesized that ECM dimensionality intrinsically affects cell behaviors that predispose to dormancy. Accordingly, we assessed the biophysical and biochemical response of mammary epithelial cells (MECs) to compliant polyacrylamide (PA) gels and micropatterned surfaces in which ligand presentation and ECM dimensionality were modulated to recapitulate different ECM landscapes. Using traction force microscopy, atomic force microscopy indentation, and laser ablation studies, we found that a 3D ECM led to a drop in cortical tension of MECs. Computational modeling predicted that reduced cortical tension should lead to an increase in the number and/or residence time of actin protrusions as well as a net increase in negative membrane curvature. To test the prediction, we ectopically expressed F-actin and plasma membrane markers in MECs to examine the plasma membrane topography and actin protrusion dynamics. Indeed, MECs in 3D ECM had longer and more stable actin protrusions and more negative membrane curvature inducing proteins, including Exo70, at the plasma membrane. The enrichment of Exo70 at the plasma membrane accompanied the activation of Arf6, which led to an increase in Rac/p38 pro-survival signaling pathway. Consistently, we found that the non-spread cells were able to survive in 3D ECM but the non-spread MECs plated on either a soft 2D PA gels or rigid micropatterned adhesive islands died. We next examined if reduced cortical tension is sufficient to activate pro-survival pathways in cells in 2D. Importantly, pharmacological inhibition of myosin in non-spread MECs on a 2D ECM, which causes loss of cortical tension, increased negative membrane curvature and cell viability. Conversely, genetic knockdown of the negative curvature-inducing protein Exo70 compromised MEC survival in a 3D ECM. Our results provide the first evidence demonstrating that ECM dimensionality alters the biophysical properties of cells to modulate plasma membrane curvature and activate pro-survival signaling pathways. Our findings also offer a unique perspective for why Arf6 and Rac GTPases have been implicated in cancer aggression and suggest that targeting the tissue ECM or cellular cortical tension may provide a novel therapeutic approach to target dormant cells. Citation Format: FuiBoon Kai, Guanqing Ou, Alexandra Long, Wei Guo, Richard Tourdot, Ravi Radhakrishnan, Christopher Chen, Sophie Dumont, Valerie M. Weaver. Extracellular matrix dimensionality reduces cellular cortical tension to stimulate pro-survival signaling in mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1028.

Soft Three Dimensional Stromal Matrix Confines Pancreatic Cancer Cells Through Induction of Autophagy Lysosome Flux

Pancreas is a soft orang. Malignancy of pancreatic ductal adenocarcinoma (PDAC) is intimately associated with pancreatic fibrogenesis and stiffness, generated by chronic pancreatitis, along with early oncogenetic transformation, indicating that soft stroma may confine tumor growth. Little is understood as to how soft three‐dimensional extracellular matrix (3D ECM) in the stroma can restain tumor progression even the cancer cells having acquired oncogenetic transformation. We noticed that embedded in soft 3D ECM, the pancreatic ductal adenocarcinoma cells (KRas/Trp53R172H) can be arrested at G1 phase, showing up‐regulation of p21 and Pten. In the soft 3D ECM, the cancer cells form tight junctions and epithelial ductal phenotypes. Conversely, on 2D stiff interface, the cancer cells are disseminated and exhibited EMT features. Among the 800 up‐regulated genes by the cancer cells in 3D ECM are the lysosomal genes, including cysteine cathepsins, acidic lipase, and v‐ATPase. Indeed, autophagy is highly active by the cells embedded in 3D ECM, featured by elevation of Atg8/LC3‐II, and turnover of p62, a cargo protein for auto‐lysosomal flux and substrate degradation. Lysosomal stress, induced by drugs of lipophilic amines, can inhibit the acidic proteinases, leading to accumulation of HDAC4 and Yap1, suggesting they are degraded through auto‐lysosome. On stiff interface (2D ECM), turnover of the cellular HDAC4 and is mostly operated by proteosomal pathway, wherein the auto‐lysosomal biogenesis is impaired, leading to HDAC4 and Yap1 accumulation. Animal experiments showed that pre‐programing of PDAC cells (Kras/Trp53) in soft 3D ECM could attenuate tumorigenicity. Conversely, existing liver fibrosis and stiffness can promote the tumorigenicity. Clinical studies showed that high levels of auto‐lysosoamal components such as cathepsins H and cathepsin L are related to better survival rate, indicating auto‐lysosomal flux in tumor suppression.Support or Funding InformationNational Science Foundation of China, and National Institutes of Health, USAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SAT-397-HIC-5 and GARP expression is upregulated by hydrogen peroxide and TGF beta in primary human hepatic stellate cells cultured on decellularized human liver 3D ECM scaffolds

SAT-397-HIC-5 and GARP expression is upregulated by hydrogen peroxide and TGF beta in primary human hepatic stellate cells cultured on decellularized human liver 3D ECM scaffolds

A Simple Three-dimensional Hydrogel Platform Enables Ex Vivo Cell Culture of Patient and PDX Tumors for Assaying Their Response to Clinically Relevant Therapies.

A cell culture platform that enables ex vivo tissue growth from patients or patient-derived xenograft (PDX) models and assesses sensitivity to approved therapies (e.g., temozolomide) in a clinically relevant time frame would be very useful in translational research and personalized medicine. Here, we present a novel three-dimensional (3D) ECM hydrogel system, VersaGel, for assaying ex vivo growth and therapeutic response with standard image microscopy. Specifically, multicellular spheroids deriving from either 5 patients with glioblastoma (GBM) or a renal cell carcinoma (RCC) PDX model were incorporated into VersaGel and treated with temozolomide and several other therapies, guided by the most recent advances in GBM treatment. RCC ex vivo tissue displayed invasive phenotypes in conditioned media. For the GBM patient tumor testing, all five clinical responses were predicted by the results of our 3D-temozolomide assay. In contrast, the MTT assay found no response to temozolomide regardless of the clinical outcome, and moreover, basement membrane extract failed to predict the 2 patient responders. Finally, 1 patient was tested with repurposed drugs currently being administered in GBM clinical trials. Interestingly, IC50s were lower than C max for crizotinib and chloroquine, but higher for sorafenib. In conclusion, a novel hydrogel platform, VersaGel, enables ex vivo tumor growth of patient and PDX tissue and offers insight into patient response to clinically relevant therapies. We propose a novel 3D hydrogel platform, VersaGel, to grow ex vivo tissue (patient and PDX) and assay therapeutic response using time-course image analysis.

Absorbing-active transition in a multi-cellular system regulated by a dynamic force network.

Collective cell migration in 3D extracellular matrix (ECM) is crucial to many physiological and pathological processes. Migrating cells can generate active pulling forces via actin filament contraction, which are transmitted to the ECM fibers and lead to a dynamically evolving force network in the system. Here, we elucidate the role of this force network in regulating collective cell behaviors using a minimal active-particle-on-network (APN) model, in which active particles can pull the fibers and hop between neighboring nodes of the network following local durotaxis. Our model reveals a dynamic transition as the particle number density approaches a critical value, from an "absorbing" state containing isolated stationary small particle clusters, to an "active" state containing a single large cluster undergoing constant dynamic reorganization. This reorganization is dominated by a subset of highly dynamic "radical" particles in the cluster, whose number also exhibits a transition at the same critical density. The transition is underlaid by the percolation of "influence spheres" due to the particle pulling forces. Our results suggest a robust mechanism based on ECM-mediated mechanical coupling for collective cell behaviors in 3D ECM.

Generating 3D artificial melanoma tumor tissues using CellMate™ hydrogels.

We have previously reported on the successful use of natural 3D ECM materials as artificial tissue scaffolds to generate tissue‐like constructs of melanoma using the ATCC cell lines B16F1and B16F10. In that earlier work we observed that 3D constructs like these undergo a progressive scaffold colonization, tissue development and maturation in‐vitro which closely parallels tumor progression in‐vivo. Now, we have extended that work by applying a new and unique 3D hydrogel matrix called CellMate™. This material is an embedding hydrogel comprised of hyaluronate and chitosan which crosslink to generate a matrix scaffolding for cell attachment and tissue development. In these new experiments, B16F1 and B16F10 cell lines were each seeded into CellMate™ at densities of 10 and 20 million cells per scaffold. The resulting structures where then maintained for 6 months of continuous culture with periodic new plates and regular media exchanges at 3 day intervals. This approach resulted in even more physiological artificial tumors, than previously developed on ECM scaffolds. As previously seen on natural scaffolds, these new artificial tumors develop two distinct cell populations which closely reflect the normal in‐vivo tumor structures seen in the clinical setting. These two cell types include pigmented cells with a relatively dendritic appearance and non‐pigmented cells with relatively less dendritic and more rounded cell bodies. These cellular phenotypes also gave rise to distinct tissue‐level structural phenotypes as well. Pigmented tissue regions tended to present with less regular surfaces and more tendency to produce branching structures that extend away from the construct. In contrast, non‐pigmented cell regions generated large rounded and bulging features which often resembled ridges of tissue and which had very smooth surface features. Additionally these structures also appeared to be highly friable and prone to peeling off the main pigmented tumor structure. Colonies and later cell‐strains were developed from these shed structures and these retained their non‐pigmented phenotypes through multiple passages in 2D monolayers. Collectively these studies illustrate the efficacy of using 3D artificial tissues to create better in‐vitro tumor models of melanoma.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FRI-107 - Primary human HSC cell phenotype is differently regulated by pro-fibrogenic and pro-inflammatory stimuli in cirrhotic and healthy human liver 3D ECM scaffolds

FRI-107 - Primary human HSC cell phenotype is differently regulated by pro-fibrogenic and pro-inflammatory stimuli in cirrhotic and healthy human liver 3D ECM scaffolds