RESUMEN
In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.
Asunto(s)
Alginatos , Química Clic , Matriz Extracelular , Hidrogeles , Maleimidas , Compuestos de Sulfhidrilo , Humanos , Alginatos/química , Adhesión Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados/química , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Hidrogeles/química , Hidrogeles/síntesis química , Maleimidas/química , Compuestos de Sulfhidrilo/químicaRESUMEN
The tumor microenvironment (TME) orchestrates cellular and extracellular matrix (ECM) interactions, playing a key role in tumorigenesis, tumor growth, and metastization. Investigating the interplay between stromal-epithelial cells within the TME is paramount for understanding cancer mechanisms but demands reliable biological models. 3D-models have emerged as powerful in vitro tools, but many fall short in replicating cell-cell/cell-matrix interactions. This study introduces a novel hybrid 3D-model of the breast TME, combining epithelial cells, cancer-associated fibroblasts (CAFs), and their ECM. To build the stromal compartment, porous 3D-printed alginate scaffolds were seeded with CAFs, which proliferated and produced ECM. The pores were infused with oxidized peptide-modified alginate hydrogel laden with MCF10A cells, forming the parenchymal compartment. The hybrid system supported epithelial morphogenesis into acini surrounded by fibroblasts and ECM, and could be readily solubilized to recover cells, their matrix, and sequestered soluble factors. Proteome profiling of the retrieved ECM showed upregulation of proteins associated with matrix assembly/remodeling, epithelial-to-mesenchymal transition (EMT), and cancer. The TME-like microenvironment induced a partial EMT in MCF10A cells, generating a hybrid population with epithelial and mesenchymal features, characteristic of aggressive phenotypes. Our model provided new insights into epithelial-stromal interactions within the TME, offering a valuable tool for cancer research in a physiologically-relevant 3D setting.
Asunto(s)
Alginatos , Neoplasias de la Mama , Células Epiteliales , Matriz Extracelular , Microambiente Tumoral , Humanos , Alginatos/química , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Femenino , Matriz Extracelular/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Fibroblastos Asociados al Cáncer/metabolismo , Hidrogeles/química , Impresión Tridimensional , Andamios del Tejido/química , Técnicas de Cultivo Tridimensional de Células/métodos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacosRESUMEN
The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regeneration, remaining a central challenge in tissue engineering. In this study, we present a novel (micro)vascularization strategy that explores the use of specialized "vascular units" (VUs) as building blocks to initiate blood vessel formation and create perfusable, stroma-embedded 3D microvascular networks from the bottom-up. We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels. This leads to the formation of VUs-derived capillaries, which fuse with adjacent capillaries to form stable microvascular beds within a supportive, extracellular matrix-rich fibroblastic microenvironment. Using a custom-designed biomimetic fibrin-based vessel-on-chip (VoC), we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused. Moreover, VUs can establish capillary bridges between channels, extending the microvascular network throughout the entire device. When VUs and intestinal organoids (IOs) are combined within the VoC, the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs. This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device. These findings underscore the remarkable potential of VUs as building blocks for engineering microvascular networks, with versatile applications spanning from regenerative medicine to advanced in vitro models.
RESUMEN
Successful strategies to promote neovascularization of ischemic tissues are still scarce, being a central priority in regenerative medicine. Microparticles harboring primitive vascular beds are appealing cell delivery candidates for minimally-invasive therapeutic vascularization. However, dynamic cellular alterations associated with in vitro vascular morphogenesis are still elusive. Here, bioengineered microgels guided the assembly of entrapped outgrowth endothelial cells (OEC) and mesenchymal stem cells (MSC) into cohesive vascularized microtissues. During in vitro maturation, OEC formed capillary-like networks enveloped in newly-formed extracellular matrix. Gene expression profiling showed that OEC acquired a mesenchymal-like phenotype, suggesting the occurrence of partial endothelial-to-mesenchymal transition (EndMT), while MSC remained transcriptionally stable. The secretome of entrapped cells became more pro-angiogenic, with no significant alterations of the inflammatory profile. Importantly, matured microgels showed improved cell survival/retention after transplantation in mice, with preservation of capillary-like networks and de novo formation of human vascular structures. These findings support that in vitro priming and morphogenesis of vessel-forming cells improves their vasculogenic/angiogenic potential, which is of therapeutic relevance, shedding some light on the associated mechanisms.
Asunto(s)
Células Madre Mesenquimatosas , Microgeles , Animales , Células Endoteliales , Ratones , Morfogénesis , Neovascularización Fisiológica , Ingeniería de TejidosRESUMEN
Efficient cell delivery strategies are urgently needed to improve the outcome of cell-based pro-angiogenic therapies. This study describes the design of an injectable cell delivery platform, based on biomaterial-guided morphogenesis principles. Soft high-mannuronic acid alginate microgels, oxidized and functionalized with integrin-binding peptides, provided adequate biochemical/biomechanical cues for the co-assembly of mesenchymal stem cells and outgrowth endothelial cells (OEC) into pre-vascularized microtissues. In vitro priming conditions regulated OEC tubulogenesis, which only occurred under normoxia (+O2) in the presence of angiogenic factors (+GF) and, importantly, did not revert in an ischemic-like environment. Primed (+O2+GF) microgel-entrapped cells secreted a large variety of angiogenesis-related proteins and produced endogenous extracellular-matrix, rich in fibronectin and collagen type I, fostering cell-cell/cell-matrix interactions and establishing a stable angiogenic niche. Extending the pre-culture time resulted in higher cell outward migration and in vivo angiogenic potential. Microgels partially disintegrated upon implantation in chick embryos, promoting interaction between pre-vascularized microtissues and the host. Preserved human vascular structures were still detected in vivo, and human cells showed the ability to migrate and integrate with the chick vasculature. Our results suggest that an integrated approach combining pro-angiogenic cells, cell-instructive microgels and adequate in vitro priming may provide the basis for successful therapeutic angiogenesis.
Asunto(s)
Geles/química , Morfogénesis , Neovascularización Patológica/terapia , Alginatos/química , Animales , Movimiento Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Embrión de Pollo , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Mesenquimatosas/citología , Neovascularización Patológica/patología , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/farmacología , Oxígeno/farmacologíaRESUMEN
Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Intermediate states, arising from inter-conversion between epithelial (E) and mesenchymal (M) states, are characterized by phenotypic heterogeneity combining E and M features and increased plasticity. Hybrid EMT states are highly relevant in metastatic contexts, but have been largely neglected, partially due to the lack of physiologically-relevant 3D platforms to study them. Here we propose a new in vitro model, combining mammary E cells with a bioengineered 3D matrix, to explore phenotypic and functional properties of cells in transition between E and M states. Optimized alginate-based 3D matrices provided adequate 3D microenvironments, where normal epithelial morphogenesis was recapitulated, with formation of acini-like structures, similar to those found in native mammary tissue. TGFß1-driven EMT in 3D could be successfully promoted, generating M-like cells. TGFß1 removal resulted in phenotypic switching to an intermediate state (RE cells), a hybrid cell population expressing both E and M markers at gene/protein levels. RE cells exhibited increased proliferative/clonogenic activity, as compared to M cells, being able to form large colonies containing cells with front-back polarity, suggesting a more aggressive phenotype. Our 3D model provides a powerful tool to investigate the role of the microenvironment on metastable EMT stages.