3D Collagen-Nanocellulose Matrices Model the Tumour Microenvironment of Pancreatic Cancer (original) (raw)
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International Journal of Molecular Sciences, 2021
Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer (“tumouroid”) model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In...
Macromolecular Research
An emerging trend in cancer research is to develop engineered tumor models using bio-inspired biomaterials that can mimic the native tumor microenvironment. Although various bio-inspired hydrogels have been utilized, it is still challenging to develop advanced polymeric hydrogel materials that can more accurately reconstruct critical aspects of the native tumor microenvironment. Herein, we present interpenetrating polymer network (IPN) hydrogels composed of thiolated gelatin and tyramine-conjugated poly(ethylene glycol), which form IPN hydrogels via horseradish peroxidase-mediated dual cross-linking reactions. We demonstrate that the IPN hydrogels exhibit independently controllable physicochemical properties. Also, the IPN hydrogels show resistance to the proteolytic enzymes and cytocompatibility for long-term culture of human fibrosarcoma (HT1080) cells. Moreover, we utilize the engineered tumor construct as a platform to evaluate the effect of matrix stiffness on cancer cell proliferation and drug resistance against the anticancer drug 5-fluorouracil as a model drug. In conclusion, we suggest that our IPN hydrogel is a promising material to study cancer biology and to screen innovative therapeutic agents for better clinical outcomes.
Engineering a vascularised 3D in vitro model of cancer progression
Scientific Reports, 2017
The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis. Despite the significant advancements in early diagnostic and therapeutic regimens, the metastatic progression of tumours is the leading cause of mortality in colorectal cancer patients 1. Tumour progression is mediated by microenvironmental conditions that include oxygen gradients between tumour cells in spatially distinct regions, cell-cell and cell-extracellular matrix (ECM) interactions 2. Understanding the more complex mechanics of tumour cell migration within conventional 2D in vitro models has proved challenging and as a result, there has recently been an increase in tissue engineered solutions to address this problem 3,4. One avenue, not often explored within 3D in vitro tumour models, is the effect of the tumour stroma on cancer growth and invasion. ECM density and composition are factors that are often overlooked in cancer research but have increasingly been implicated as significant factors involved in cancer progression 5. Natural scaffolds are composed of ECM components that make up an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs) including collagens, fibrin and hyaluronic acid 6,7. They provide tissues and cells with mechanical stability and enable cell-matrix interactions to regulate normal tissue function. Natural scaffolds are also biologically active and promote excellent cell adhesion, growth and migration 8. When used for in vitro 3D cell culture, these scaffolds exist as cross-linked networks of ECM proteins known as hydrogels. Although one of their main disadvantages is their high water content (upwards of 99%), they are still extremely useful for mechanistic investigations as they are entirely malleable by cell behaviour and subject to cell mediated ECM degradation. Increasing the matrix density of these scaffolds can help recreate normal or pathological tissue function. We engineered tumouroids using colorectal cancer cells (HT29 or HCT116) and cultured them within collagen type I hydrogels. To increase the matrix density and mimic the dense nature of in situ tumours, the interstitial fluid within collagen hydrogels was removed using plastic compression (PC) 9. Tumouroids are spatially accurate and are based on a dense central artificial cancer mass (ACM) that contains the cancer cells, nested within a collagen hydrogel that represents the tumour stroma (Fig. 1a). The stromal compartment was populated with the basement membrane protein and attachment factor laminin, and stromal cells such as fibroblasts and endothelial cells (ECs). The effect of matrix density and composition on cancer invasion was investigated. The development of 'healthy' and 'tumourigenic' vascular networks in the stroma was also explored due to the presence of the
Biochemical and Biophysical Research Communications, 2012
Pancreatic cancer contains both fibrotic tissue and tumor cells with embedded vasculature. Therefore anti-cancer nanoparticles need to extravasate from tumor vasculature and permeate thick fibrotic tissue to target tumor cells. To date, permeation of drugs has been investigated in vitro using monolayer models. Since three-dimensional migration of nanoparticles cannot be analyzed in a monolayer model, we established a novel, three-dimensional, multilayered, in vitro model of tumor fibrotic tissue, using our hierarchical cell manipulation technique with K643f fibroblasts derived from a murine pancreatic tumor model. NIH3T3 normal fibroblasts were used in comparison. We analyzed the size-dependent effect of nanoparticles on permeation in this experimental model using fluorescent dextran molecules of different molecular weights. The system revealed permeation decreased as number of layers of cultured cells increased, or as molecule size increased. Furthermore, we showed changes in permeation depended on the source of the fibroblasts. Observations of this sort cannot be made in conventional monolayer culture systems. Thus our novel technique provides a promising in vitro means to investigate permeation of nanoparticles in fibrotic tissue, when both type and number of fibroblasts can be regulated.
Biomaterials Research
Background Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. Methods In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. Results The in vi...
Gelatine methacrylamide-based hydrogels: An alternative three-dimensional cancer cell culture system
Acta Biomaterialia, 2014
Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/ v) strongly influenced hydrogel stiffness (0.5 ± 0.2 kPa to 9.0 ± 1.8 kPa) but had little effect on solute diffusion. The diffusion coefficient of 70 kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4 kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.
2021
Three-dimensional (3D) cell culture systems mimic the structural complexity of the tissue microenvironment that includes the extracellular matrix (ECM) in addition to the cellular components Thus, 3D culture systems are increasingly important as they resemble the ECM-cell and cell-cell physical interactions occurring in vivo. So far, several scaffold-based culture systems and techniques have been proposed as valuable approaches for large-scale production of spheroids, but often suffering of poor reproducible conditions or high costs of production. In this work we present a reliable 3D culture system based on collagen I-blended agarose hydrogels and show how the variation of the agarose weight percentage affects the physical and mechanical properties of the resulting hydrogel, being that with a lower amount of agarose more permeable, softer and more prone to degradation compared to hydrogels with higher agarose concentrations. We have also evaluated the effect of the different physic...
Scientific Reports, 2016
Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150-4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150-200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.
Journal of The Royal Society Interface, 2012
Owing to the reduced co-relationship between conventional flat Petri dish culture (two-dimensional) and the tumour microenvironment, there has been a shift towards three-dimensional culture systems that show an improved analogy to the same. In this work, an extracellular matrix (ECM)-mimicking three-dimensional scaffold based on chitosan and gelatin was fabricated and explored for its potential as a tumour model for lung cancer. It was demonstrated that the chitosan–gelatin (CG) scaffolds supported the formation of tumoroids that were similar to tumours grownin vivofor factors involved in tumour-cell–ECM interaction, invasion and metastasis, and response to anti-cancer drugs. On the other hand, the two-dimensional Petri dish surfaces did not demonstrate gene-expression profiles similar to tumours grownin vivo. Further, the three-dimensional CG scaffolds supported the formation of tumoroids, using other types of cancer cells such as breast, cervix and bone, indicating a possible wide...
A 3D Fibrous Scaffold Inducing Tumoroids: A Platform for Anticancer Drug Development
The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and monomethoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.