Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. © 2012 Sieh et al.

A bioengineered 3D ovarian cancer model for the assessment of peptidase-mediated enhancement of spheroid growth and intraperitoneal spread

Cancer-associated proteases promote peritoneal dissemination and chemoresistance in malignant progression. In this study, kallikrein-related peptidases 4, 5, 6, and 7 (KLK4-7)-cotransfected OV-MZ-6 ovarian cancer cells were embedded in a bioengineered three-dimensional (3D) microenvironment that contains RGD motifs for integrin engagement to analyze their spheroid growth and survival after chemotreatment. KLK4-7-cotransfected cells formed larger spheroids and proliferated more than controls in 3D, particularly within RGD-functionalized matrices, which was reduced upon integrin inhibition. In contrast, KLK4-7-expressing cell monolayers proliferated less than controls, emphasizing the relevance of the 3D microenvironment and integrin engagement. In a spheroid-based animal model, KLK4-7-overexpression induced tumor growth after 4 weeks and intraperitoneal spread after 8 weeks. Upon paclitaxel administration, KLK4-7-expressing tumors declined in size by 91% (controls: 87%) and showed 90% less metastatic outgrowth (controls: 33%, P<0.001). KLK4-7-expressing spheroids showed 53% survival upon paclitaxel treatment (controls: 51%), accompanied by enhanced chemoresistance-related factors, and their survival was further reduced by combination treatment of paclitaxel with KLK4/5/7 (22%, P=0.007) or MAPK (6%, P=0.006) inhibition. The concomitant presence of KLK4-7 in ovarian cancer cells together with integrin activation drives spheroid formation and proliferation. Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient for late-stage disease than chemotherapeutics alone as these inhibitory regimens reduced cancer spheroid growth to a greater extent than paclitaxel alone.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis

Tumour microenvironment greatly influences the development and metastasis of cancer progression. The development of three dimensional (3D) culture models which mimic that displayed in vivo can improve cancer biology studies and accelerate novel anticancer drug screening. Inspired by a systems biology approach, we have formed 3D in vitro bioengineered tumour angiogenesis microenvironments within a glycosaminoglycan-based hydrogel culture system. This microenvironment model can routinely recreate breast and prostate tumour vascularisation. The multiple cell types cultured within this model were less sensitive to chemotherapy when compared with two dimensional (2D) cultures, and displayed comparative tumour regression to that displayed in vivo. These features highlight the use of our in vitro culture model as a complementary testing platform in conjunction with animal models, addressing key reduction and replacement goals of the future. We anticipate that this biomimetic model will provide a platform for the in-depth analysis of cancer development and the discovery of novel therapeutic targets.

Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

Tissue response and wound healing after placement of two types of bioengineered grafts containing vital cells in submucosal maxillary pouches: an experimental pilot study in rabbits

PURPOSE: This pilot study evaluated the wound healing and tissue response after placement of two different skin substitutes in subgingival mucosal pouches in rabbits. MATERIALS AND METHODS: Four rabbits were selected to receive a commercially available skin substitute consisting of a collagen matrix with fibroblasts and an epithelial layer (test membrane 1) and a prototype device consisting of a collagen matrix with fibroblasts only (test membrane 2). In each rabbit, two horizontal incisions were made in the buccal alveolar mucosa of the maxilla bilaterally to create submucosal pouches. Three pouches in each animal were filled with either the test 1 or test 2 membranes, and one pouch was left without a membrane (sham-operated control). All rabbits were sacrificed after a healing period of 4 weeks, and histologic samples were prepared and examined. RESULTS: After a healing period of 1 month, both tested membranes were still visible in the sections. Test membrane 1 was still bilayered, contained inflammatory cells in its center, and was encapsulated by a thick fibrous tissue. Numerous ectopic calcifications were evident in the collagenous part of the membrane and in association with some basal epithelial cells. Test membrane 2 was also encapsulated in fibrous tissue, with inflammatory cells present only between the fibrous encapsulation and the remnants of the membrane. For test membrane 2, no calcifications were visible. CONCLUSIONS: Test membrane 1 seemed to be more resistant to degradation, but there was also a more pronounced inflammatory reaction in comparison to test membrane 2, especially in the vicinity of the keratinocytes. The significance of the ectopic calcifications, along with that of the resorption or degradation processes of both tested membranes, must be evaluated in future experimental studies, with different time points after implantation examine

Bioengineered periodontal tissue formed on titanium dental implants

The ability to use autologous dental progenitor cells (DPCs) to form organized periodontal tissues on titanium implants would be a significant improvement over current implant therapies. Based on prior experimental results, we hypothesized that rat periodontal ligament (PDL)-derived DPCs can be used to bioengineer PDL tissues on titanium implants in a novel, in vivo rat maxillary molar implant model. Analyses of recovered implants revealed organized PDL tissues surrounding titanium implant surfaces in PDL-cell-seeded, and not in unseeded control, implants. Rat PDL DPCs also exhibited differentiative potential characteristic of stem cells. These proof-of-principle findings suggest that PDL DPCs can organize periodontal tissues in the jaw, at the site of previously lost teeth, indicating that this method holds potential as an alternative approach to osseointegrated dental implants. Further refinement of this approach will facilitate the development of clinically relevant methods for autologous PDL regeneration on titanium implants in humans.

Computer-assisted arthroplasty using bioengineered autografts

Recent advances in tissue-engineered cartilage open the door to new clinical treatments of joint lesions. Common to all therapies with in-vitro-engineered autografts is the need for optimal fit of the construct to allow screwless implantation and optimal integration into the live joint. Computer-assisted surgery (CAS) techniques are prime candidates to ensure the required accuracy, while at the same time simplifying the procedure. A pilot study has been conducted aiming at assembling a new set of methods to support ankle joint arthroplasty using bioengineered autografts. Computer assistance allows planning of the implant shape on a computed tomography (CT) image, manufacturing the construct according to the plan, and interoperatively navigating the surgical tools for implantation. A rotational symmetric model of the joint surface was used to avoid segmentation of the CT image; new software was developed to determine the joint axis and make the implant shape parameterizable. A complete cycle of treatment from planning to operation was conducted on a human cadaveric foot, thus proving the feasibility of computer-assisted arthroplasty using bioengineered autografts

Enhancement of titanium implants via bioengineered periodontal tissues

Osseointegration of titanium dental implants into the jaw bone, which is required for maintenance of the implant in the jaw, results in ankylosis. Dental implants are therefore very unlike natural teeth, which exhibit significant movement in response to mechanical forces. The ability to generate periodontal ligament (PDL) tissues onto dental implants would better mimic the functional characteristics of natural teeth, and would likely improve implant duration and function. OBJECTIVES: The objective of this study was to investigate the feasibility of bioengineering PDL tissues onto titanium implant surfaces. METHODS: Bilateral maxillary first and second molars of 8-week old rats were extracted and used to generate single cell suspensions of PDL tissues, which were expanded in culture. Immunohistochemistry and RT-PCR were used to identify putative PDL progenitor/stem cell populations and characterize stem cell properties, including self-renewal, multipotency and stem cell maker expression. Cultured rPDL cells were harvested at third passage, seeded onto Matrigel-coated titanium implants (1.75 mm x 1 mm), and placed into healed M1/M2 extraction sites. Non-cell seeded Matrigel-coated titanium implants served as negative controls. Implants were harvested after 8, 12, or 18 weeks. RESULTS: Cultured rPDL cells expressed the mesenchymal stem-cell marker STRO-1. Under defined culture conditions, PDL cells differentiated into adipogenic, neurogenic and osteogenic lineages. While control implants were largely surrounded by alveolar bone, experimental samples exhibited fibrous PDL-like tissues, and perhaps cementum, on the surface of experimental implants. CONCLUSIONS: PDL contains stem cells that can generate cementum/PDL-like tissue in vivo. Transplantation of these cells might hold promise as a therapeutic approach for the bioengineering of PDL tissues onto titanium implant. Further refinement of this method will likely result in improved dental implant strategies for use of autologous PDL tissue regeneration in humans. This research was supported by CIMIT, and NIH/NIDCR grant DE016132 (PCY), and TEACRS (YL).

α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α1-antitrypsin Portland (α1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged α1-PDX (α1-PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin’s active site, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358-↓ or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.