Three-dimensional Collagen Research Articles

Three-Dimensional Hierarchical Nanofibrous Collagen Scaffold Fabricated Using Fibrillated Collagen and Pluronic F-127 for Regenerating Bone Tissue.

It is well known that a nanoscale fibrous structure can provide a unique stage for encouraging reasonable cell activities including attachment and proliferation owing to its similar topological structure to the extracellular matrix. Hence, the structure has been widely applied in tissue regeneration. Type-I collagen has been typically used as a typical tissue regenerative material owing to its biocompatibility and abundance, although it has potential for antigenicity. In particular, collagen has been fabricated in two different forms, porous spongy and nanofibers. However, although the structures provided outstanding cellular activities, they exhibit disadvantages such as low cell migration capabilities in a spongy scaffold owing to the low degree of interconnected macropores and low processability in fabricating three-dimensional (3D) structures in an electrospun collagen scaffold. Hence, the fabrication of 3D nanofibrous collagen structures with interconnected macropores can be extremely challenging. In this work, we developed a 3D collagen scaffold consisting of multilayered nanofibrous struts fabricated using a 3D printing process and pluronic F-127 (PF-127), which is a thermoreversible polymer. After optimizing various processing conditions, we successfully achieved the 3D nanofibrous collagen mesh structure with fully interconnected macropores. A 3D-printed collagen scaffold that was fabricated using a low-temperature printing process was applied as a control. Through various analyses using physical properties (surface morphology, fibronectin absorption, mechanical properties, etc.) and cell activities using preosteoblasts (MC3T3-E1), we are convinced that the newly designed 3D nanofibrous collagen scaffold can be a new promising scaffold for bone tissue engineering.

A versatile perfusion bioreactor and endothelializable photo cross-linked tubes of gelatin methacryloyl as promising tools in tissue engineering.

Size and function of bioartificial tissue models are still limited due to the lack of blood vessels and dynamic perfusion for nutrient supply. In this study, we evaluated the use of cytocompatible methacryl-modified gelatin for the fabrication of a hydrogel-based tube by dip-coating and subsequent photo-initiated cross-linking. The wall thickness of the tubes and the diameter were tuned by the degree of gelatin methacryl-modification and the number of dipping cycles. The dipping temperature of the gelatin solution was adjusted to achieve low viscous fluids of approximately 0.1 Pa s and was different for gelatin derivatives with different modification degrees. A versatile perfusion bioreactor for the supply of surrounding tissue models was developed, which can be adapted to several geometries and sizes of blood-vessel mimicking tubes. The manufactured bendable gelatin tubes were permeable for water and dissolved substances, like Nile Blue and serum albumin. As a proof of concept, human fibroblasts in a three-dimensional collagen tissue model were successfully supplied with nutrients via the central gelatin tube under dynamic conditions for 2 days. Moreover, the tubes could be used as scaffolds to build-up a functional and viable endothelial layer. Hence, the presented tools can contribute to solving current challenges in tissue engineering.

Rehabilitative exercise and spatially patterned nanofibrillar scaffolds enhance vascularization and innervation following volumetric muscle loss

Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.

Simple and cost-effective assay for isolating invasive living cells.

A modified invasion assay using a three-dimensional collagen gel was developed that enables isolation of invasive living cells; it was named the invading cell trapping (iCT) assay. A small cell strainer consisting of a nylon mesh with 40-μm2 pores was used, and collagen gel layers formed across the membrane. Test cells were seeded in the lower gel layer and invasive cells were attracted upward and trapped in the upper gel. After incubation, the collagen gel layers in cell strainers were easily separated and living cells in the gel were counted and analyzed. An advantage of the iCT assay is that it can capture living invasive cells in the upper gel while leaving noninvasive ones in the lower layer. Further enrichment of the two cell populations can be achieved by repeating the assay. Thus, the iCT assay allows comparative analysis of invasive versus noninvasive cells.

Liprin-\u03b11 modulates cancer cell signaling by transmembrane protein CD82 in adhesive membrane domains linked to cytoskeleton

BackgroundPPFIA1 is located at the 11q13 region commonly amplified in cancer. The protein liprin-α1 encoded by PPF1A1 contributes to the adhesive and invasive structures of cytoskeletal elements and is located at the invadosomes in cancer cells. However, the precise mechanism of liprin-α1 function in cancer progression has remained elusive.MethodsInvasion regulating activity of liprin-α1 was examined by analyzing the functions of squamous cell carcinoma of head and neck (HNSCC) cell lines in three-dimensional collagen I after RNAi mediated gene knockdown. Transcriptome profiling and Gene Set Enrichment Analysis from HNSCC and breast cancer cells were used to identify expression changes relevant to specific cellular localizations, biological processes and signaling pathways after PPFIA1 knockdown. The significance of the results was assessed by relevant statistical methods (Wald and Benjamini-Hochberg). Localization of proteins associated to liprin-α1 was studied by immunofluorescence in 2D and 3D conditions. The association of PPFIA1 amplification to HNSCC patient survival was explored using The Cancer Genome Atlas data.ResultsIn this study, we show that liprin-α1 regulates biological processes related to membrane microdomains in breast carcinoma, as well as protein trafficking, cell-cell and cell-substrate contacts in HNSCC cell lines cultured in three-dimensional matrix. Importantly, we show that in all these cancer cells liprin-α1 knockdown leads to the upregulation of transmembrane protein CD82, which is a suppressor of metastasis in several solid tumors.ConclusionsOur results provide novel information regarding the function of liprin-α1 in biological processes essential in cancer progression. The results reveal liprin-α1 as a novel regulator of CD82, linking liprin-α1 to the cancer cell invasion and metastasis pathways.

Abstract 4492: Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment

Abstract Clinical studies indicate that obesity is correlated with poor outcomes in breast cancer patients. Obese individuals have a high proportion of adipose tissue, which is abundant in stem and progenitor cells. Human adipose-derived stromal cells (hADSCs) secrete several cytokines including CCL5, an inflammatory chemokine shown to increase tumor metastatic ability in vitro. We previously identified that CCL5 is secreted into the microenvironment when hADSCs are co-cultured with breast tumor cells. When the clinical relevance of CCL5 was assessed using cBioPortal, an open-source genomics database, we found that CCL5 mRNA overexpression was correlated with increased overall survival in breast cancer patients, which counters what we and others have observed in vitro. In this study, we compared the expression of CCL5 and Nodal, a potent embryonic morphogen re-expressed by aggressive cancer cells, in either hADSC or MDA-MB-231 breast cancer cells in response to three-dimensional collagen matrices conditioned by hADSCs or breast cancer cells. After preconditioning a three-dimensional collagen matrix with either hADSCs or MDA-MB-231 cells (and following removal of the cells), the denuded three-dimensional matrices were reseeded with the opposite cell type. We found that the expression of both CCL5 and Nodal was significantly reduced in the breast cancer cells plated on the hADSC-preconditioned matrix. However, hADSCs did not express Nodal or alter CCL5 protein expression when grown on a MDA-MB-231-preconditioned matrix. MDA-MB-231 cells plated onto a hADSC-preconditioned matrix appeared more mesenchymal and stem-like, while the adipose stem cells placed into a tumor-preconditioned matrix were induced into a more aggressive cancer cell phenotype by engaging in vasculogenic mimicry. Furthermore, we found there was no change in cell proliferation of any cells grown under these different conditions. These observations suggest a relationship between hADSCs and the breast tumor microenvironment where human adipose-derived stem cells appear to inhibit the expression of CCL5 and Nodal by the aggressive tumor cells without altering tumor cell proliferation. Citation Format: Daniel Berrebi, Abhishek Yadav, Eric Lundstrom, Nicole Werwie, Dominic Muttillo, Richard E. Seftor, Elisabeth A. Seftor, Linda Vona-Davis. Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4492.

Assessing fluorescence detection and effective photothermal therapy of near-infrared polymer nanoparticles using alginate tissue phantoms.

Photothermal therapy (PTT) uses light absorbing materials to generate heat for treatment of diseases, like cancer. The advantages of using PTT components that absorb in the near-infrared (NIR) include reduced tissue auto-fluorescence and higher penetration depths. However, NIR laser light can still be scattered and absorbed by biological tissues, thus decreasing the amount of the energy reaching the PTT agents. We have developed two distinct formulations of NIR-absorbing nanoparticles, one which can be utilized for PTT only, and another for both PTT and fluorescence imaging of colorectal cancer. In this work, the fluorescence detection limit and the PTT heating potential of the two nanoparticle types were determined using alginate tissue phantoms. The objective of this study was to determine the PTT efficiency and theranostic potential of the nanoparticles by irradiating 3D collagen tumor spheroids, containing nanoparticles and CT26 mouse colorectal cancer cells, through increasing tissue phantom thicknesses and then quantifying cell death. Materials and Methods Our lab has previously developed nanoparticles based on the semiconducting, conjugated polymer poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe). We have also made a hybrid nanoparticle that heats and fluoresces by combining PCPDTBSe polymer with the fluorescent poly[(9,9-dihexylfluorene)-co-2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBTDBT10) polymer to yield nanoparticles termed Hybrid Donor-Acceptor Polymer Particles (H-DAPPs). H-DAPPs and PCPDTBSe nanoparticles were added to three-dimensional collagen gel tumor spheroids in order to represent nanoparticles in a tumor. Alginate tissue phantoms, comprised of an optical scattering agent (Intralipid) and an optical absorbing material (hemoglobin) in order to mirror biological tissue scattering effects, were used to simulate increasing tissue thickness between the nanoparticles and the PTT energy source. Fluorescence from the H-DAPPs was detectable through 6 mm of tissue phantoms. It was found that less than 10% of the laser energy could penetrate through 9 mm of tissue phantoms and only 60% of the laser energy passed through the 1.5 mm phantoms, regardless of laser power. PTT experiments, using 800 nm light at 2.2 W/cm2 for 60 s through tissue phantoms to stimulate nanoparticle-doped tumor spheroids, showed 55% cell death through 3 mm of tissue phantoms using H-DAPPs. Results from using the PCPDTBSe nanoparticles showed 72% cell death through 3 mm and over 50% cell death through 6 mm of tissue phantoms. The results of this work validated the heating potential and fluorescence detection limitations of two theranostic polymer nanoparticles by utilizing alginate tissue phantoms and 3D tumor spheroids. H-DAPPs and PCPDTBSe polymer nanoparticles can be utilized as effective PTT agents by exploiting their absorption of NIR light and H-DAPPs have advantageous fluorescence for imaging colorectal cancer. The data generated from this study design can allow for other NIR absorbing and fluorescing nanoparticle formulations to be evaluated prior to in vivo experimentation. Lasers Surg. Med. 50:1040-1049, 2018. © 2018 Wiley Periodicals, Inc.

Promising effects of silver tungstate microcrystals on fibroblast human cells and three dimensional collagen matrix models: A novel non-cytotoxic material to fight oral disease

Silver tungstate (α-Ag2WO4) microcrystals have shown encouraging results regarding their antimicrobial activity. However, in addition to the promising outcomes in fighting oral disease, cytotoxic tests are mandatory for screening new materials for biological applications. Here, we developed a better understanding of the effects of microcrystals on the behavior of both human gingival fibroblast (HGF) cells and three-dimensional (3D) collagen matrices. To perform these experiments, the lowest concentration of α-Ag2WO4 capable of preventing the visible growth of Candida albicans (C. albicans) planktonic cells was defined as the test concentration, and it ranged from 0.781 (C1) to 7.81 (C2) to 78.1 (C3) μg/mL. Complete medium and lysis buffer (LB) served as negative (C-) and positive (C+) controls, respectively. The effect of the microcrystal concentration on the morphology, remodeling and proliferation of HGF cells was evaluated by different approaches. Quantitative and qualitative assessments demonstrated that α-Ag2WO4 did not affect the mitochondrial enzymatic activity of HGF cells cultured in a monolayer or the cell viability within 3D collagen matrices. These experiments showed that α-Ag2WO4 at the C2 concentration did not damage the genomic DNA. The development of new materials is attractive for the possible treatment of diseases and for avoiding indiscriminate prescribing of antibiotics. These findings provide information on the effect of α-Ag2WO4 on cell behavior and reveal that these microcrystals are non-cytotoxic against human gingival cells over a sufficient period to measure the hazard potential.

Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.

Transient Scleraxis Overexpression Combined with Cyclic Strain Enhances Ligament Cell Differentiation.

Efforts to generate tissue-engineered anterior cruciate ligament replacements are limited by a lack of methods to derive mature ligament cells. Viral overexpression of the tendon/ligament marker scleraxis (Scx) can drive cell differentiation; however, the use of viral vectors hampers translation to clinical use. In this study, C3H10T1/2 cells were transiently transfected with expression vectors containing the full-length murine Scx cDNA and cultured in three-dimensional collagen hydrogels under static or cyclic strain for up to 14 days. β-galactosidase (LacZ) transfected cells served as controls. Cell morphology and gene expression for ligament-related genes, in addition to contraction (hydrogel width), mechanical properties, and glycosaminoglycan (GAG) and DNA content of hydrogels, were quantified and compared over time, between Scx and LacZ groups, and between static and cyclically strained constructs. Increased Scx expression was maintained for the entire 14-day study in both static and cyclically strained constructs. In static culture, overexpression of Scx resulted in greater cell elongation and construct contraction compared to LacZ controls. There were no differences in gene expression, DNA, or GAG content between Scx and LacZ constructs cultured under static conditions and no differences in DNA content between Scx and LacZ constructs. When exposed to cyclic strain, Scx-overexpressing cells maintained the elongated phenotype exhibited in static constructs, increased GAG production compared to static culture, and increased expression of the ligament-related genes collagen type I, decorin, and tenascin-C compared to strained LacZ controls. Cyclically strained constructs containing Scx-overexpressing cells had increased maximum load and stiffness compared to LacZ controls. The maintenance of increased Scx expression throughout the 14 day study and subsequent increases in ligament marker gene expression and mechanical properties with cyclic, but not static strain, suggest that transient transfection may be a viable alternative to viral transduction of Scx for ligament engineering studies and support a synergistic effect of Scx and mechanical strain on driving early ligament cell differentiation.

Evaluation of Tissue Behavior on Three-dimensional Collagen Scaffold Coated with Carbon Nanotubes and β-tricalcium Phosphate Nanoparticles

Evaluation of Tissue Behavior on Three-dimensional Collagen Scaffold Coated with Carbon Nanotubes and β-tricalcium Phosphate Nanoparticles

Cancer cells growing on perfused 3D collagen model produced higher reactive oxygen species level and were more resistant to cisplatin compared to the 2D model.

Three-dimensional (3D) collagen scaffold models, due to their ability to mimic the tissue and organ structure in vivo, have received increasing interest in drug discovery and toxicity evaluation. In this study, we developed a perfused 3D model and studied cellular response to cytotoxic drugs in comparison with traditional 2D cell cultures as evaluated by cancer drug cisplatin. Cancer cells grown in perfused 3D environments showed increased levels of reactive oxygen species (ROS) production compared to the 2D culture. As determined by growth analysis, cells in the 3D culture, after forming a spheroid, were more resistant to the cancer drug cisplatin compared to that of the 2D cell culture. In addition, 3D culturing cells showed elevated level of ROS, indicating a physiological change or the formation of a microenvironment that resembles tumor cells in vivo. These data revealed that cellular response to drugs for cells growing in 3D environments are dramatically different from that of 2D cultured cells. Thus, the perfused 3D collagen scaffold model we report here might be a potentially very useful tool for drug analysis.

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

Effects of three-dimensional collagen scaffolds on the expression profiles and biological functions of glioma cells.

Three-dimensional (3D) culture has been increasingly used to investigate tumor cell biology for improved simulation of the natural developing environment. However, the way in which 3D culture affects the gene expression and biological functions of glioma cells remains to be fully elucidated. In the present study, 3D culture environments were established using collagen scaffolds with different pore sizes, followed by the comparison of gene expression profiles and associated biological functions of glioma cells, including the U87, U251 and HS683 cell lines, in 3D collagen scaffolds with conventional two-dimensional (2D) cultured cells. Finally, the possible signaling pathways regulating these differences were investigated. It was found that the 3D collagen scaffold culture upregulated the expression of genes associated with stemness, cell cycle, apoptosis, epithelia-mesenchymal transition, migration, invasion and glioma malignancy, and induced the corresponding functional changes. Apoptotic pathways, the Wnt pathway, Sonic Hedgehog pathway and Notch pathway, may be involved in the regulation of these changes. The aperture size of the collagen-scaffold did not appear to affect the gene expression or functions of the glioma cells. The results of the study suggested that the 3D collagen scaffold enhanced the malignancy of glioma cells and may be a promising in vitro platform for investigations of glioma.

Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells

BackgroundVascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities.MethodsThe effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines.ResultsIn vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects.ConclusionsAltogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin.

Early Effects of Ionizing Radiation on the Collagen Hierarchical Structure of Bladder and Rectum Visualized by Atomic Force Microscopy.

Radiation therapy, widely used in the treatment of a variety of malignancies in the pelvic area, is associated with inevitable damage to the surrounding healthy tissues. We have applied atomic force microscopy (AFM) to track the early damaging effects of ionizing radiation on the collagen structures in the experimental animals' bladder and rectum. The first signs of the low-dose radiation (2 Gy) effect were detected by AFM as early as 1 week postirradiation. The observed changes were consistent with initial radiation destruction of the protein matrix. The alterations in the collagen fibers' packing 1 month postirradiation were indicative of the onset of fibrotic processes. The destructive effect of higher radiation doses was probed 1 day posttreatment. The severity of the radiation damage was proportional to the dose, from relatively minor changes in the collagen packing at 8 Gy to the growing collagen matrix destruction at higher doses and complete three-dimensional collagen network restructuring towards fibrotic-type architecture at the dose of 22 Gy. The AFM study appeared superior to the optical microscopy-based studies in its sensitivity to early radiation damage of tissues, providing valuable additional information on the onset and development of the collagen matrix destruction and remodeling.

Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of\xa0stroma-mediated cell motility and drug resistance

BackgroundPancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance.MethodsA 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay.ResultsPANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of α-SMA. Expression of EMT-related markers, such as vimentin and TGF-β, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs.ConclusionsWe demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.

Atomic force microscopy nano-characterization of 3D collagen gels with tunable stiffness

As extracellular matrix (ECM) nano-characteristics play a crucial role in cell behavior, including cancer development and metastasis, several ECM in vitro models have been used in order to study cells behavior under different biochemical and mechanical conditions. Among the ECM constituents, collagen (especially collagen type I) has been extensively used as an essential component of ECM models, since it is one of the most abundant ECM protein. Use of three-dimensional (3D) collagen gels provides the advantage of allowing the cells to grow in a 3D environment that bears strong similarities to their natural, in vivo setting. Thus, the ability to form collagen gels with tunable stiffness and well defined naturally occurring nano-characteristics is crucial for these studies. Atomic Force Microscopy (AFM) is a unique tool that is ideal for the complete characterization of such models, in terms of morphology and mechanical properties without destroying the collagen fiber structure. In this protocol, the development and the AFM nano-scale characterization of 3D collagen type I gels is presented. The protocol includes:•The formation of 3D collagen type I gels with tunable stiffness•The preparation of histological sections from collagen gels•The AFM-based morphological and mechanical nano-characterization of the gels

Establishment of a three-dimensional model to study human uterine angiogenesis.

Can primary human uterine microvascular endothelial cells (UtMVECs) be used as a model to study uterine angiogenic responses in vitro that are relevant in pregnancy? UtMVECs demonstrated angiogenic responses when stimulated with proangiogenic factors, including sphingosine 1-phosphate (S1P), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), physiological levels of wall shear stress (WSS), human chorionic gonadotropin (hCG) and various combinations of estrogen and progesterone. During sprouting angiogenesis, signaling from growth factors and cytokines induces a monolayer of quiescent endothelial cells (ECs) lining the vasculature to degrade the extracellular matrix and invade the surrounding tissue to form new capillaries. During pregnancy and the female reproductive cycle, the uterine endothelium becomes activated and undergoes sprouting angiogenesis to increase the size and number of blood vessels in the endometrium. The study was designed to examine the angiogenic potential of primary human UtMVECs using the well-characterized human umbilical vein EC (HUVEC) line as a control to compare angiogenic potential. ECs were seeded onto three-dimensional (3D) collagen matrices, supplemented with known proangiogenic stimuli relevant to pregnancy and allowed to invade for 24 h. Sprouting responses were analyzed using manual and automated methods for quantification. RT-PCR, Western blot analysis and immunostaining were used to characterize UtMVECs. Angiogenic responses were examined using 3D invasion assays. Western blotting was used to confirm signaling responses after proangiogenic lipid, pharmacological inhibitor, and recombinant lentiviral treatments. All experiments were repeated at least three times. After ensuring that UtMVECs expressed the proper endothelial markers, we found that UtMVECs invade 3D collagen matrices dose-dependently in response to known proangiogenic stimuli (e.g. S1P, VEGF, bFGF, hCG, estrogen, progesterone and WSS) present during early pregnancy. Invasion responses were positively correlated with phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p42/p44 mitogen-activated protein kinase (ERK). Inhibition of these second messengers significantly impaired sprouting (P < 0.01). Gene silencing of membrane type 1-matrix metalloproteinase using multiple approaches completely abrogated sprouting (P < 0.001). Finally, UtMVECs displayed a unique ability to undergo sprouting in response to hCG, and combined estrogen and progesterone treatment. Not applicable. The study of uterine angiogenesis in vitro has limitations and any findings many not fully represent the in vivo state. However, these experiments do provide evidence for the ability of UtMVECs to be used in functional sprouting assays in a 3D environment, stimulated by physiological factors that are produced locally within the uterus during early pregnancy. We show that UtMVECs can be used reliably to investigate how growth factors, hormones, lipids and other factors, such as flow, affect angiogenesis in the uterus. This work was supported by NIH award HL095786 to K.J.B. The authors have no conflicts of interest.

Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8+ T Cell Responses.

Dendritic cell (DC) trafficking from peripheral tissues to lymph nodes (LNs) is a key step required to initiate T cell responses against pathogens as well as tumors. In this context, cellular membrane protrusions and the actin cytoskeleton are essential to guide DC migration towards chemotactic signals. Caveolin-1 (CAV1) is a scaffolding protein that modulates signaling pathways leading to remodeling of the actin cytoskeleton and enhanced migration of cancer cells. However, whether CAV1 is relevant for DC function and specifically for DC migration to LNs is unknown. Here, we show that CAV1 expression is upregulated in DCs upon LPS- and TNF-α-induced maturation. CAV1 deficiency did not affect differentiation, maturation, or the ability of DCs to activate CD8+ T cells in vitro. However, CAV1-deficient (CAV1−/−) DCs displayed reduced in vivo trafficking to draining LNs in control and inflammatory conditions. In vitro, CAV1−/− DCs showed reduced directional migration in CCL21 gradients in transwell assays without affecting migration velocity in confined microchannels or three-dimensional collagen matrices. In addition, CAV1−/− DCs displayed reduced activation of the small GTPase Rac1, a regulator of actin cytoskeletal remodeling, and lower numbers of F-actin-forming protrusions. Furthermore, mice adoptively transferred with peptide-pulsed CAV1−/− DCs showed reduced CD8+ T cell responses and antitumor protection. Our results suggest that CAV1 promotes the activation of Rac1 and the formation of membrane protrusions that favor DC chemotactic trafficking toward LNs where they can initiate cytotoxic T cell responses.