Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.

Bioprinted anisotropic scaffolds with fast stress relaxation bioink for engineering 3D skeletal muscle and repairing volumetric muscle loss.

The creation of in vitro three-dimensional cellular model systems (in vitro 3D cultures) is a fast-growing leading-edge segment of the biotechnological industry. We have examined in this work the key 80 articles published after 2008, and focused on applications of in vitro 3D culture in translational oncology. We described a broad range of 3D culture systems, including models with and without extracellular matrix (ECM). 3D culture models based on decellularized ECM were discussed in more detail. The role of ECM in pathogeneis of malignant neoplasms, in particular, in the phenomenon of the tumor resistance to chemotherapy, was evaluated. 2D and 3D culture systems were compared, and natural and synthetic ECM were described, as well as the model creation based on 3D bioprinting. Particular attention was paid to in vitro models of various cancers, including those at the metastatic stage, based on 3D cell cultures, which maximally mimic the in vivo tumor behavior. The prospects of the practical application of 3D cell culture models in preclinical drag screening and in personalized therapy were discussed. We also presented our data on in vitro 2D and 3D culturing of human cells on various substrates. 3D cellular models, 3D bioprinting, biotechnology, extracellular matrix, cancer, translational medicine, personalized medicine, drag development, in vitro, ex vivo, oncology The authors are grateful to Dr. E. Shabalina for providing part of the experimental data and to OKA-Biotech Company for the samples of recombinant Funding-The work was supported by a Grant from the Russian Science Foundation (no. 18-15-00391). doi: 10.21519/0234-2758-2020-36-3-3-15

Traumatic injuries to the central nervous system, such as traumatic brain injury, spinal cord injury and stroke, have a high prevalence, enormous financial costs and lack clinical treatments that restore neurological function (Ma et al., 2014). These injuries trigger a series of secondary biochemical and cellular responses that ultimately lead to cellular death and the maintenance of an unsupportive extracellular matrix (ECM) for tissue regeneration (Silva et al., 2014). Artificial ECM or scaffolds represent a way to alter this unsupportive environment to improve the efficacy of stem cell therapies and enhance neural tissue regeneration (Figure 1). Scaffold use could lead to greater improvements in neurological function (such as improved bladder control, increased dexterity and body control, etc.) than observed with the implantation of cells alone. To date, the inclusion of basic scaffolds with stem cell therapy treatments have shown increased efficacy in rodent models (Yasuda et al., 2010). More advanced scaffolds could better mimic the chemical, physical and mechanical properties of the ECM to promote cellular survival, adhesion, proliferation and differentiation. Altering the injured ECM to mitigate the barriers to axon invasion, myelination and cellular maturation further and lead to even greater gains in neurological function.

During bone formation, osteoblasts are embedded in a collagen-rich osteoid tissue and differentiate into an extensive 3D osteocyte network throughout the mineralizing matrix. However, how these cells dynamically remodel the matrix and undergo 3D morphogenesis remains poorly understood. Although previous reports investigated the impact of matrix stiffness in osteocyte morphogenesis, the role of matrix viscoelasticity is often overlooked. Here, we report a viscoelastic alginate-collagen interpenetrating network (IPN) hydrogel for 3D culture of murine osteocyte-like IDG-SW3 cells. The IPN hydrogels consist of an ionically crosslinked alginate network to tune stress relaxation as well as a permissive collagen network to promote cell adhesion and matrix remodeling. Two IPN hydrogels were developed with comparable stiffnesses (4.4-4.7 kPa) but varying stress relaxation times (t1/2, 1.5 s and 14.4 s). IDG-SW3 cells were pre-differentiated in 2D under osteogenic conditions for 14 days to drive osteoblast-to-osteocyte transition. Cellular mechanosensitivity to fluid shear stress (2 Pa) was confirmed by live-cell calcium imaging. After embedding in the IPN hydrogels, cells remained highly viable following 7 days of 3D culture. After 24 h, osteocytes in the fast-relaxing hydrogels showed the largest cell area and long dendritic processes. However, a significantly larger increase of some osteogenic markers (ALP, Dmp1, hydroxyapatite) as well as intercellular connections via gap junctions were observed in slow-relaxing hydrogels on day 14. Our results imply that fast-relaxing IPN hydrogels promote early cell spreading, whereas slow relaxation favors osteogenic differentiation. These findings may advance the development of 3D in vivo-like osteocyte models to better understand bone mechanobiology.

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.

Cell-based biosensors use living cells or tissues as sensing element to monitor physiological and functional changes induced by external stimuli. They have become an important pillar of drug discovery process, to provide a simple, fast and cost-effective tool to avoid large-scale and cost-intensive animal testing. The sensing element---cultured cells, is the most critical part of a cell-based biosensor. To date, almost all cell-based biosensors use traditional 2 dimensional (2D) monolayer cells cultured on flat and rigid substrate as the sensing element. Although the time-honored 2D cell culture has proven to be a valuable method for cell-based studies, its limitations have been increasingly recognized. In in vivo environment, almost all cells are surrounded by other cells and extracellular matrix (ECM) in a 3D fashion. As a result, 2D cell culture tests sometimes give unsatisfactorily misleading and non-predictive data for in vivo responses. On the other hand, 3D cell culture provides a more physiologically relevant environment for cells and allows the study of cellular responses in a setting that resembles in vivo environments. The 3D structure not only influences the spatial organization of the cell surface receptors engaged in interactions with surrounding cells, but also induce the physical constraint to cells. These spatial and physical aspects in 3D affect the signal transduction from the outside to the inside of cells, and ultimately influence on gene expression and cellular behaviors. Compared to 2D cell culture, 3D culture replicates more accurately the actual microenvironment where cells reside in tissue and therefore the behavior of cells in 3D culture reflects closely the in vivo responses.This study focused on the adoption of 3D cell cultures to cell-based biosensors, aiming to provide more in vivo-like experimental results for drug discovery. In this study, we established the conditions for growing cancer cells into 3D spheroids on Matrigel for two different cancer cell lines: oral cancer cell line CAL 27 and prostate cancer cell line DU 145. We systematically investigated the cellular responses to different anticancer drugs in 3D culture in comparison to those of 2D culture, within the same cell line and between different cell lines. We also examined the difference in expressions of drug-action related factors in 3D and 2D cultures. The results showed that the cell proliferation rate in 3D culture on Matrigel in comparison to 2D culture was cell line dependent, as we observed the proliferation of CAL27 cells was enhanced in 3D whereas the proliferation DU 145 cells we reduced in 3D. Sensitivity of cellular responses to drugs in 3D relative to 2D was drug-action and cell line dependent. For DU 145 cells, 3D cell culture model was more resistant to Docetaxel, but less resistant to Rapamycin as compared to 2D cell culture model. And surprisingly, for CAL 27 cells, 3D cell culture model was less resistant to both bleomycin and erlotinib as compared to 2D cell culture. Further examination of drug-action related factors indicated that the cellular responses of 3D culture were correlated with the expression of drug action related biomarkers as compared to 2D cell culture. Acknowledgement: The research is supported by NSF (CBET #1159871)

Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

Heparan sulfate proteoglycans and integrin signaling in articular cartilage homeostasis

Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. Here, we show that tyrosine phosphorylation of a focal adhesion protein, paxillin, crucially participates in these regulations. We found that tyrosine phosphorylation of paxillin was a prominent event upon integrin activation during epithelial-mesenchymal trans-differentiation and cell migration. Four major tyrosine phosphorylation sites were identified, and two of them were highly inducible upon integrin activation. Paxillin exhibits three distinct subcellular localizations as follows: localization along the cell periphery colocalized with circumferential actin meshworks, macroaggregation at focal adhesions connected to actin stress fibers, and diffuse cytoplasmic distribution. Tyrosine phosphorylation of paxillin localized at the cell periphery and focal adhesions was shown using phosphorylation site-specific antibodies. Mutations in the phosphorylation sites affected the peripheral localization of paxillin and paxillin-containing focal adhesion formation during cell migration and cell-cell collision, accompanied by altered actin organizations. Our analysis indicates that phosphorylation of multiple tyrosines in paxillin α is necessary for the proper function of paxillin and is involved in the temporospatial regulation of focal adhesion formation and actin cytoskeletal organization in motile cells.

During malignant invasion tumor cells establish contact with extracellular matrix proteins, including fibrillar collagen. In addition to providing a physical barrier against invasion, fibrillar collagen also restricts cell proliferation. It has been assumed that the growth regulatory activity of fibrillar collagen is the result of an indirect restrictive effect on cell spreading and cytoskeletal organization. Here we provide evidence for a direct inhibitory effect of fibrillar collagen on proliferation of human melanoma and fibrosarcoma cells that involves activation of the tyrosine kinase discoidin domain receptor 2 and is independent of effects on cell spreading. Cells plated in the presence of fibrillar collagen were growth arrested in the G0/G1 phase of the cell cycle. However treatment with the tyrosine kinase inhibitor genistein, down-regulation of discoidin domain receptor 2, or collagen deglycosylation that prevents discoidin domain receptor 2 activation allowed cells to enter the cell cycle in the presence of fibrillar collagen without a requirement for spreading and actin organization. Our data provide evidence for a novel direct mechanism by which cell contact with fibrillar collagen restricts proliferation.

Background: Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. 3D modeling of cancer is of particular importance in drug development where predicting in vivo effectiveness is challenging. Not only is the 3D structure important for proper modeling of cancer but the response from the surrounding microenvironment, including the extracellular matrix (ECM) and fibroblasts, is also necessary to accurately predict drug response. A major hindrance to 3D culture is loss of cell viability due to nutrient limitation. Herein, we demonstrate the ability of our novel bioreactor system to prolong viability of 3D cultures and the importance of ECM composition in breast cancer modeling. Methods: To gain further understanding of the effect of different ECM on the 3D arrangement of breast cancer cells and breast fibroblasts, three different variations of ECM were tested: 1) 100% basement membrane (BM, reduced growth factor Matrigel) diluted to 9-12 μg/ml, 2) an equal volume of BM and Collagen I (50% BM + 50% Collagen I), and 3) 10% BM in Collagen I. MDA-MB-231 (231) breast cancer cells were grown in each ECM in monoculture or co-culture with breast fibroblasts (ratio of 2:1) for 3 or 7 days. The formation of cell aggregates, as seen in most infiltrating carcinomas of the breast, was assessed by image analysis. To improve viability, 250 μM channels penetrated the 3D co-cultures (consisting of 231 cells and fibroblasts (2:1) mixed into 10% BM/Collagen I) in our perfusion bioreactor system. Proliferation, measured by Ki-67 immunostaining, was compared over time in solid co-cultures and perfused and non-perfused co-cultures after 3 or 7 days. Results: In 3D monocultures, significantly greater cell aggregation was seen with 100% BM compared to 50% and 10% BM at both 3 and 7 days (p<0.002, ANOVA). A similar result was seen in 3D co-cultures with fibroblasts (p<0.002, ANOVA). 3D cultures without channels (solid) demonstrated a reduced Ki-67 labeling index over time (65% at 1 day, 35% at 3 days, and 8.5% at 7 days). Whereas, 3D co-cultures with channels, both perfused and non-perfused, had a more constant Ki-67 labeling index over time (49.6% at 3 days and 37.3% at 7 days with perfusion and 37.4% at 3 days and 34.4% at 7 days without perfusion). Conclusions: Using 3D co-culture with fibroblasts and ECM to model breast cancer recapitulates in vivo tumor-stromal interactions in breast carcinomas better than monocultures in 2D. The formulation of ECM affected cell arrangement, with the presence of BM promoting cell aggregation. The use of our perfusion bioreactor system improved cell proliferation in comparison to solid 3D cultures, which did not sustain growth over time. We anticipate that further refinement of our 3D culture system will allow more accurate investigation of tumor-stromal interactions and drug testing in breast cancer. Citation Format: Kayla F. Goliwas, Lauren E. Marshall, Kun Yuan, Joel Berry, Andra R. Frost. Importance of ECM and media permeation in 3D modeling of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2022. doi:10.1158/1538-7445.AM2014-2022

Engineering the Extracellular Matrix to Model the Evolving Tumor Microenvironment.

Introduction: In conventional 2D monolayer cell culture systems cell-matrix adhesions are well known and studied protein aggregations with functions in cell-adhesion, mechanotransduction, cell-signaling, invasion and migration. However, to date little is known about focal adhesion formation in mesenchymal cells growing in 3D cell culture environments and in vivo, where cells are completely embedded in an extracellular matrix. 3D cell culture models more closely resemble the physiological in vivo condition. Currently, it is a hot debate and ongoing controversy whether cells in such a 3D matrix would form adhesions at all. Attempts to visualize subcellular structures (like focal adhesions) in 3D cell culture models bear several microscopic limitations such as low working distances, lack of high resolution, high fluorescent background and weak antibody stainings. Therefore, we are interested in establishing 3D cell culture models and improving microscopic techniques for studying subcellular structures such as focal adhesions and cytoskeletal proteins in lung fibroblasts and their impacts on 3D migration and invasion. New findings will help to understand the underlying mechanisms of chronic lung diseases like fibrosis or pulmonary metastases.

Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands