Biophysical Microenvironment Research Articles

Dense Collagen I as a Biomimetic Material to Track Matrix Remodelling in Renal Carcinomas.

Aims: Renal tissue is a dynamic biophysical microenvironment, regulating healthy function and influencing tumor development. Matrix remodelling is an iterative process and aberrant tissue repair is prominent in kidney fibrosis and cancer. Biomimetic 3D models recapitulating the collagen composition and mechanical fidelity of native renal tissue were developed to investigate cell-matrix interactions in renal carcinomas. Methods: Collagen I and laminin hydrogels were engineered with renal cancer cells (ACHN and 786-O), which underwent plastic compression to generate dense matrices. Mechanical properties were determined using shear rheology and qPCR determined the gene expression of matrix markers. Results: The shear modulus and phase angle of acellular dense collagen I gels (474 Pa and 10.7) are similar to human kidney samples (1410 Pa and 10.5). After 21 days, 786-O cells softened the dense matrix (∼155 Pa), with collagen IV downregulation and upregulation of matrix metalloproteinases (MMP7 and MMP8). ACHN cells were found to be less invasive and stiffened the matrix to ∼1.25 kPa, with gene upregulation of collagen IV and the cross-linking enzyme LOX. Conclusions: Renal cancer cells remodel their biophysical environment, altering the material properties of tissue stroma in 3D models. These models can generate physiologically relevant stiffness to investigate the different matrix remodelling mechanisms utilized by cancer cells.

Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations.

Somatic activating mutations in PIK3CA are common drivers of vascular and lymphatic malformations. Despite common biophysical signatures of tissues susceptible to lesion formation, including compliant extracellular matrix and low rates of perfusion, lesions vary in clinical presentation from localized cystic dilatation to diffuse and infiltrative vascular dysplasia. The mechanisms driving the differences in disease severity and variability in clinical presentation and the role of the biophysical microenvironment in potentiating progression are poorly understood. Here, we investigate the role of hemodynamic forces and the biophysical microenvironment in the pathophysiology of vascular malformations, and we identify hemodynamic shear stress and defective endothelial cell mechanotransduction as key regulators of lesion progression. We found that constitutive PI3K activation impaired flow-mediated endothelial cell alignment and barrier function. We show that defective shear stress sensing in PIK3CA E542K endothelial cells is associated with reduced myosin light chain phosphorylation, junctional instability, and defective recruitment of vinculin to cell-cell junctions. Using 3D microfluidic models of the vasculature, we demonstrate that PIK3CA E542K microvessels apply reduced traction forces and are unaffected by flow interruption. We further found that draining transmural flow resulted in increased sprouting and invasion responses in PIK3CA E542K microvessels. Mechanistically, constitutive PI3K activation decreased cellular and nuclear elasticity resulting in defective cellular tensional homeostasis in endothelial cells which may underlie vascular dilation, tissue hyperplasia, and hypersprouting in PIK3CA-driven venous and lymphatic malformations. Together, these results suggest that defective nuclear mechanics, impaired cellular mechanotransduction, and maladaptive hemodynamic responses contribute to the development and progression of PIK3CA-driven vascular malformations.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

Nanofiber-microwell cell culture system for spatially patterned differentiation of pluripotent stem cells in 3D

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.

Single-cell adhesive profiling in an optofluidic device elucidates CD8+ T lymphocyte phenotypes in inflamed vasculature-like microenvironments

Tissue infiltration by circulating leukocytes occurs via adhesive interactions with the local vasculature, but how the adhesive quality of circulating cells guides the homing of specific phenotypes to different vascular microenvironments remains undefined. We developed an optofluidic system enabling fluorescent labeling of photoactivatable cells based on their adhesive rolling velocity in an inflamed vasculature-mimicking microfluidic device under physiological fluid flow. In so doing, single-cell level multidimensional profiling of cellular characteristics could be characterized and related to the associated adhesive phenotype. When applied to CD8+ Tcells, ligand/receptor expression profiles and subtypes associated with adhesion were revealed, providing insight into inflamed tissue infiltration capabilities of specific CD8+ T lymphocyte subsets and how local vascular microenvironmental features may regulate the quality of cellular infiltration. This methodology facilitates rapid screening of cell populations for enhanced homing capabilities under defined biochemical and biophysical microenvironments, relevant to leukocyte homing modulation in multiple pathologies.

O146: Investigating the cell-matrix interplay in fibrotic remodelling using renal 3D in vitro models

Abstract Introduction The dynamic biophysical microenvironment of the renal extracellular matrix is integral in regulating tissue homeostasis. Matrix remodelling is an iterative process, which guides cell behaviour and pathological fibrosis occurs due to aberrant tissue repair. Fibrosis, characterised by the desmoplastic ECM deposition and tissue stiffening, is prominent in CKD and renal carcinomas. We engineered biomimetic 3D constructs to recapitulate cell-matrix interactions in fibrotic disease progression. Methods Soft collagen-I and laminin constructs (0.2%w/v) were engineered with renal cancer cells (786-O or ACHN) or renal fibroblasts (TK188). Plastic compression was used to generate dense collagen constructs, at 6%w/v collagen density. Mechanical properties were analysed using shear rheology. Gene expression of markers defining the healthy/diseased kidney matrix was determined using qPCR. Results Over 21 days, 786-O cancer cells softened the collagen matrix. Matrix degradation was supported by collagen-I downregulation and MMP7 and vimentin upregulation. Whereas metastatic cancer cells (ACHN) stiffened the dense matrix to ∼1.25kPa, with significant gene upregulation of collagen-I, collagen-IV and LOX. Fibrotic fibroblasts soften the dense matrix by ∼1kPa, with α-SMA and MMP9 upregulation. However, matrix stiffness significantly increases with cancer cell co-cultures. Discussion Renal cancer cells remodel their biophysical environment, altering the construct’s material properties. In our models, collagen density and stromal complexity influences cell behaviour, causing alterations in biomechanics and gene expression. These models can generate physiologically relevant stiffness, enabling investigation between key cell types and the physical environment. Drugs targeting MMPs and LOX will be used to determine if we can disrupt the fibrotic remodelling.

Investigating the impact of the interstitial fluid flow and hypoxia interface on cancer transcriptomes using a spheroid-on-chip perfusion system.

Solid tumours are complex and heterogeneous systems, which exist in a dynamic biophysical microenvironment. Conventional cancer research methods have long relied on two-dimensional (2D) static cultures which neglect the dynamic, three-dimensional (3D) nature of the biophysical tumour microenvironment (TME), especially the role and impact of interstitial fluid flow (IFF). To address this, we undertook a transcriptome-wide analysis of the impact of IFF-like perfusion flow using a spheroid-on-chip microfluidic platform, which allows 3D cancer spheroids to be integrated into extracellular matrices (ECM)-like hydrogels and exposed to continuous perfusion, to mimic IFF in the TME. Importantly, we have performed these studies both in experimental (normoxia) and pathophysiological (hypoxia) oxygen conditions. Our data indicated that gene expression was altered by flow when compared to static conditions, and for the first time showed that these gene expression patterns differed in different oxygen tensions, reflecting a differential role of spheroid perfusion in IFF-like flow in tumour-relevant hypoxic conditions in the biophysical TME. We were also able to identify factors primarily linked with IFF-like conditions which are linked with prognostic value in cancer patients and therefore could correspond to a potential novel biomarker of IFF in cancer. This study therefore highlights the need to consider relevant oxygen conditions when studying the impact of flow in cancer biology, as well as demonstrating the potential of microfluidic models of flow to identify IFF-relevant tumour biomarkers.

Magneto‐Mechano‐Electric Cascade Stimulation System Accelerates Wound Healing Constructed by Biodegradable Magnetoelectric Nanofibers

AbstractReconstructing the intricate biophysical microenvironment to provide multiple biologically beneficial stimulation is deemed a promising therapeutic strategy. However, developing multi‐biophysical stimuli with wireless integration and accurate management remains a formidable challenge. Herein, a novel design of a magneto‐mechano‐electric (MME) cascade stimulation system based on aligned magnetoelectric nanofibrous membranes (PLLA/CFO) is reported. Simply regulating the loading amount of CFO nanoparticles endows the PLLA/CFO membranes with excellent magnetism, enhanced crystallization, and resultant piezoelectric responsiveness, thus providing built‐in topographical guidance and nanoscale magnetic field as well as manageably remote stimulation. Specifically, assisted by an external magnetic field (EMF), the magnetic response in PLLA/CFO can be amplified to offer an apparent deformation, which then induces the output of mechanical and electrical cues. Benefiting from the slow degradability of PLLA and continuous EMF triggering, such a performance can be well modulated to match the practical wound healing process, demonstrated by the repair of a rat full‐thickness skin defect. More importantly, this work offers a new strategy to reconstruct the complex biophysical microenvironment in a wireless remote manner.

Targeting biophysical microenvironment for improved treatment of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is responsible for high disability rates, high death rates, and significant cost to health systems. Growing evidence in recent decades shows significant biophysical microenvironment changes in COPD, impacting lung tissues, cells, and treatment response. Furthermore, such biophysical changes have shown great potential as novel targets for improved therapeutic strategy of COPD, where both pharmacological and non-pharmacological therapies focusing on repairing the biophysical microenvironment of the lung have emerged. We present the first comprehensive review of four distinct biophysical hallmarks [i.e., extracellular matrix (ECM) microarchitecture, stiffness, fluid shear stress, and mechanical stretch] in COPD, the possible involvement of pathological changes, possible effects, and correlated in vitro models and sum up the emerging COPD treatments targeting these biophysical hallmarks.

SEMA7a primes integrin α5β1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5β1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Biophysical Parameters Can Induce Epithelial-to-Mesenchymal Phenotypic and Genotypic Changes in HT-29 Cells: A Preliminary Study.

Epithelial to mesenchymal transition (EMT) in cancer is the process described where cancer epithelial cells acquire mesenchymal properties which can lead to enhanced invasiveness. Three-dimensional cancer models often lack the relevant and biomimetic microenvironment parameters appropriate to the native tumour microenvironment thought to drive EMT. In this study, HT-29 epithelial colorectal cells were cultivated in different oxygen and collagen concentrations to investigate how these biophysical parameters influenced invasion patterns and EMT. Colorectal HT-29 cells were grown in physiological hypoxia (5% O2) and normoxia (21% O2) in 2D, 3D soft (60 Pa), and 3D stiff (4 kPa) collagen matrices. Physiological hypoxia was sufficient to trigger expression of markers of EMT in the HT-29 cells in 2D by day 7. This is in contrast to a control breast cancer cell line, MDA-MB-231, which expresses a mesenchymal phenotype regardless of the oxygen concentration. In 3D, HT-29 cells invaded more extensively in a stiff matrix environment with corresponding increases in the invasive genes MMP2 and RAE1. This demonstrates that the physiological environment can directly impact HT-29 cells in terms of EMT marker expression and invasion, compared to an established cell line, MDA-MB-231, which has already undergone EMT. This study highlights the importance of the biophysical microenvironment to cancer epithelial cells and how these factors can direct cell behaviour. In particular, that stiffness of the 3D matrix drives greater invasion in HT-29 cells regardless of hypoxia. It is also pertinent that some cell lines (already having undergone EMT) are not as sensitive to the biophysical features of their microenvironment.

A Magneto‐Responsive Hydrogel System for the Dynamic Mechano‐Modulation of Stem Cell Niche

AbstractThe biophysical microenvironment of cells dynamically evolves during embryonic development, leading to defined tissue specification. A versatile and highly adaptive magneto‐responsive hydrogel system composed of magnetic nanorods (MNRs) and a stress‐responsive polymeric matrix is developed to dynamically regulate the physical stem cell niche. The anisotropic magnetic/shape factor of nanorods is utilized to maximize the strains on the polymeric network, thus regulating the hydrogel modulus in a physiologically relevant range under a minimal magnitude of the applied magnetic fields below 4.5 mT. More significantly, the pre‐alignment of MNRs induces greater collective strains on the polymeric network, resulting in a superior stiffening range, over a 500% increase as compared to that with randomly oriented nanorods. The pre‐alignment of nanorods also enables a fast and reversible response under a magnetic field of the opposite polarity as well as spatially controlled heterogeneity of modulus within the hydrogel by applying anisotropic magnetic fields. The mechano‐modulative capability of this system is validated by a mechanotransduction model with human‐induced pluripotent stem cells where the locally controlled hydrogel modulus regulates the activation of mechano‐sensitive signaling mediators and subsequent stem cell differentiation. Therefore, this magneto‐responsive hydrogel system provides a platform to investigate various cellular behaviors under dynamic mechanical microenvironments.

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment.

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell's perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy.

Bioreactor expansion reconfigures metabolism and extracellular vesicle biogenesis of human adipose-derived stem cells in vitro

Human mesenchymal stem cells (hMSCs), including human adipose tissue-derived stem cells (hASCs), as well as the secreted extracellular vesicles (EVs), are promising therapeutics in treating inflammatory and neural degenerative diseases. However, prolonged expansion can lead to cellular senescence characterized by a gradual loss of self-renewal ability while altering secretome composition and EV generation. Additionally, hMSCs are highly sensitive to biophysical microenvironment in bioreactor systems utilized in scaling production. In this study, hASCs grown on Plastic Plus or Synthemax II microcarriers in a spinner flask bioreactor (SFB) system were compared to traditional 2D culture. The SFB microenvironment was found to increase the expression of genes associated with hASC stemness, nicotinamide adenine dinucleotide (NAD+) metabolism, glycolysis, and the pentose phosphate pathway as well as alter cytokine secretion (e.g., PGE2 and CXCL10). Elevated reactive oxidative species levels in hASCs of SFB culture were observed without increasing rates of cellular senescence. Expression levels of Sirtuins responsible for preventing cellular senescence through anti-oxidant and DNA repair mechanisms were also elevated in SFB cultures. In particular, the EV biogenesis genes were significantly upregulated (3–10 fold) and the EV production increased 40 % per cell in SFB cultures of hASCs. This study provides advanced understanding of hASC sensitivity to the bioreactor microenvironment for EV production and bio-manufacturing towards the applications in treating inflammatory and neural degenerative diseases.

Delineating cooperative effects of Notch and biomechanical signals on patterned liver differentiation

Controlled in vitro multicellular culture systems with defined biophysical microenvironment have been used to elucidate the role of Notch signaling in the spatiotemporal regulation of stem and progenitor cell differentiation. In addition, computational models incorporating features of Notch ligand-receptor interactions have provided important insights into Notch pathway signaling dynamics. However, the mechanistic relationship between Notch-mediated intercellular signaling and cooperative microenvironmental cues is less clear. Here, liver progenitor cell differentiation patterning was used as a model to systematically evaluate the complex interplay of cellular mechanics and Notch signaling along with identifying combinatorial mechanisms guiding progenitor fate. We present an integrated approach that pairs a computational intercellular signaling model with defined microscale culture configurations provided within a cell microarray platform. Specifically, the cell microarray-based experiments were used to validate and optimize parameters of the intercellular Notch signaling model. This model incorporated the experimentally established multicellular dimensions of the cellular microarray domains, mechanical stress-related activation parameters, and distinct Notch receptor-ligand interactions based on the roles of the Notch ligands Jagged-1 and Delta-like-1. Overall, these studies demonstrate the spatial control of mechanotransduction-associated components, key growth factor and Notch signaling interactions, and point towards a possible role of E-Cadherin in translating intercellular mechanical gradients to downstream Notch signaling.

Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery.

Organoids serve as a novel tool for disease modeling in three-dimensional multicellular contexts. Static organoids, however, lack the requisite biophysical microenvironment such as fluid flow, limiting their ability to faithfully recapitulate disease pathology. Here, we unite organoids with organ-on-a-chip technology to unravel disease pathology and develop therapies for autosomal recessive polycystic kidney disease. PKHD1-mutant organoids-on-a-chip are subjected to flow that induces clinically relevant phenotypes of distal nephron dilatation. Transcriptomics discover 229 signal pathways that are not identified by static models. Mechanosensing molecules, RAC1 and FOS, are identified as potential therapeutic targets and validated by patient kidney samples. On the basis of this insight, we tested two U.S. Food and Drug Administration–approved and one investigational new drugs that target RAC1 and FOS in our organoid-on-a-chip model, which suppressed cyst formation. Our observations highlight the vast potential of organoid-on-a-chip models to elucidate complex disease mechanisms for therapeutic testing and discovery.

Restoration of spinal cord biophysical microenvironment for enhancing tissue repair by injury-responsive smart hydrogel

Spinal cord injury (SCI) represents a central nervous system disaster, resulting in the destruction of spinal cord structure and function and the formation of an adverse microenvironment at the SCI site. Various biomaterial-based therapeutic strategies have been developed to repair SCI by bridging spinal cord lesions. However, constructing a favorable biophysical microenvironment with biomaterials for spinal cord regeneration remains challenging because of the unmatched mechanical and electrical transmission properties with native spinal cords and the supra- or subtherapeutic dose release of biological molecules independent of SCI activity. Herein, we developed a new hydrogel with mechanical properties and conductivities comparable to those of native spinal cords by controlling gelatin and PPy concentrations. To endow the hydrogel with a biological function, glutathione (GSH) was conjugated on the hydrogel through gelatin-derived amine groups and GSH-derived sulfhydryl groups to prepare an MMP-responsive hydrogel with a recombinant protein, GST-TIMP-bFGF. The MMP-responsive conductive hydrogel could release bFGF on-demand in response to the SCI microenvironment and provide a favorable biophysical microenvironment with comparable mechanical and electrical properties to native spinal cords. In SCI model rats, the MMP-responsive bionic mechanical and conductive hydrogel could inhibit MMPs levels, promote axon regeneration and angiogenesis, and improve locomotion function recovery after SCI.

Mechanical Cues Regulate Histone Modifications and Cell Behavior.

Change of biophysical factors in tissue microenvironment is an important step in a chronic disease development process. A mechanical and biochemical factor from cell living microniche can regulate cell epigenetic decoration and, therefore, further induce change of gene expression. In this review, we will emphasize the mechanism that biophysical microenvironment manipulates cell behavior including gene expression and protein decoration, through modifying histone amino acid residue modification. The influence given by different mechanical forces, including mechanical stretch, substrate surface stiffness, and shear stress, on cell fate and behavior during chronic disease development including tumorigenesis will also be teased out. Overall, the recent work summarized in this review culminates on the hypothesis that a mechanical factor stimulates the modification on histone which could facilitate disease detection and potential therapeutic target.

Reprogramming bone progenitor identity and potency through control of collagen density and oxygen tension

SummaryThe biophysical microenvironment of the cell is being increasingly used to control cell signaling and to direct cell function. Herein, engineered 3D tuneable biomimetic scaffolds are used to control the cell microenvironment of Adipose-derived Mesenchymal Stromal Cells (AMSC), which exhibit a collagen density-specific profile for early and late stage bone cell lineage status. Cell potency was enhanced when AMSCs were cultured within low collagen density environments in hypoxic conditions. A transitional culture containing varied collagen densities in hypoxic conditions directed differential cell fate responses. The early skeletal progenitor identity (PDPN+CD146−CD73+CD164+) was rescued in the cells which migrated into low collagen density gels, with cells continuously exposed to the high collagen density gels displaying a transitioned bone-cartilage-stromal phenotype (PDPN+CD146+CD73−CD164-). This study uncovers the significant contributions of the physical and physiological cell environment and highlights a chemically independent methodology for reprogramming and isolating skeletal progenitor cells from an adipose-derived cell population.

Integrins and Epithelial-Mesenchymal Cooperation in the Tumor Microenvironment of Muscle-Invasive Lethal Cancers.

Muscle-invasive lethal carcinomas traverse into and through this specialized biophysical and growth factor enriched microenvironment. We will highlight cancers that originate in organs surrounded by smooth muscle, which presents a barrier to dissemination, including prostate, bladder, esophageal, gastric, and colorectal cancers. We propose that the heterogeneity of cell-cell and cell-ECM adhesion receptors is an important driver of aggressive tumor networks with functional consequences for progression. Phenotype heterogeneity of the tumor provides a biophysical advantage for tumor network invasion through the tensile muscle and survival of the tumor network. We hypothesize that a functional epithelial-mesenchymal cooperation (EMC)exists within the tumor invasive network to facilitate tumor escape from the primary organ, invasion and traversing of muscle, and navigation to metastatic sites. Cooperation between specific epithelial cells within the tumor and stromal (mesenchymal) cells interacting with the tumor is illustrated using the examples of laminin-binding adhesion molecules—especially integrins—and their response to growth and inflammatory factors in the tumor microenvironment. The cooperation between cell-cell (E-cadherin, CDH1) and cell-ECM (α6 integrin, CD49f) expression and growth factor receptors is highlighted within poorly differentiated human tumors associated with aggressive disease. Cancer-associated fibroblasts are examined for their role in the tumor microenvironment in generating and organizing various growth factors. Cellular structural proteins are potential utility markers for future spatial profiling studies. We also examine the special characteristics of the smooth muscle microenvironment and how invasion by a primary tumor can alter this environment and contribute to tumor escape via cooperation between epithelial and stromal cells. This cooperative state allows the heterogenous tumor clusters to be shaped by various growth factors, co-opt or evade immune system response, adapt from hypoxic to normoxic conditions, adjust to varying energy sources, and survive radiation and chemotherapeutic interventions. Understanding the epithelial-mesenchymal cooperation in early tumor invasive networks holds potential for both identifying early biomarkers of the aggressive transition and identification of novel agents to prevent the epithelial-mesenchymal cooperation phenotype. Epithelial-mesenchymal cooperation is likely to unveil new tumor subtypes to aid in selection of appropriate therapeutic strategies.