ECM Architecture Research Articles

Mechanotransduction alterations in tissue-engineered tumor models for new drug interventions

Mechanotransduction is a collection of pathways in which the cells reprogram themselves by sensing mechanical stimuli. Cells use biological cues to interpret the physiological stresses and respond to changing conditions by modifying the cellular and ECM architecture. This feedback loop regulates a variety of cellular processes, including migration, growth, differentiation, and death, which is essential for the network stability to work together in a coordinated manner. The effect of stress on cancer progression and the role of mechanics as a critical inducer in determining the cancer cell fate has been studied. This review discusses the progression of cancer cells to epithelial to mesenchymal transitions. It examines tumor microenvironment models, such as spheroids, bio-printing, and microfluidics, and how they recapitulate the tumor microenvironment. These offer certain benefits and help replicate the fundamental behavior in vivo conditions. We further discuss mechanosensing, the associated signaling molecules, and how it modulates the cancer drug resistance and transduction pathways that implicate cancer treatment. The difficulties with the existing methods and the prospects for additional study that may be applied in this area are discussed, and how they allow for new therapeutic development.

Abstract We036: Recapitulating Cardiac Microenvironment: Elucidating the Role of ECM Components and Architecture on Cardiac Differentiation

The discovery of iPSCs was revolutionary for regenerative medicine, especially as a source to replace a cell population with limited regeneration capability, like cardiomyocytes (CMs). However, the exact mechanism(s) governing how the microenvironment influences cardiac differentiation at a cellular scale is still widely unclear. To bridge this gap, we created an in vitro fibrous model from a decellularized porcine cardiac extracellular matrix. The aim is to assess the impact of the microenvironment, specifically the effects of protein composition and micro-architecture, on the cardiac differentiation of iPSCs. To study the role of protein composition, atrial and ventricular regions of the porcine heart are manually identified and separated as their protein composition is observed to be different. To test this, the material was subjected to proteomics analysis with LS-Mass Spectrometry where about 1000 proteins were identified in the ventricular region and only 300 proteins in atria. The decellularized tissue was tested for complete removal of nuclei by staining and further validated with DNA quantification. The dECM was blended with synthetic polymer (polycaprolactone) for electrospinning. The average collected fiber diameter was 14.8 ± 3.9 μm. The fibrous scaffolds were seeded with iPSCs and differentiated towards cardiac lineage. dECM samples show comparable signs of biocompatibility as laminin-521 coated TCPS, forming clusters throughout the differentiation process indicating that the scaffolds encourage cardiac differentiation. Further analysis is being conducted to compare the cellular expressions of differentiation markers including mesoderm formation, cardiac specification, and cardiomyocyte structural and functional markers. The effects of the ECM composition will be checked with myosin light and heavy chain expressions, as their expressions vary in the atrial and ventricular regions of the heart.

Fibroblast senescence-associated extracellular matrix promotes heterogeneous lung niche

Senescent cell accumulation in the pulmonary niche is associated with heightened susceptibility to age-related disease, tissue alterations, and ultimately a decline in lung function. Our current knowledge of senescent cell-extracellular matrix (ECM) dynamics is limited, and our understanding of how senescent cells influence spatial ECM architecture changes over time is incomplete. Herein is the design of an in vitro model of senescence-associated extracellular matrix (SA-ECM) remodeling using a senescent lung fibroblast-derived matrix that captures the spatiotemporal dynamics of an evolving senescent ECM architecture. Multiphoton second-harmonic generation microscopy was utilized to examine the spatial and temporal dynamics of fibroblast SA-ECM remodeling, which revealed a biphasic process that established a disordered and heterogeneous architecture. Additionally, we observed that inhibition of transforming growth factor-β signaling during SA-ECM remodeling led to improved local collagen fiber organization. Finally, we examined patient samples diagnosed with pulmonary fibrosis to further tie our results of the in vitro model to clinical outcomes. Moreover, we observed that the senescence marker p16 is correlated with local collagen fiber disorder. By elucidating the temporal dynamics of SA-ECM remodeling, we provide further insight on the role of senescent cells and their contributions to pathological ECM remodeling.

An inspired microenvironment of cell replicas to induce stem cells into keratocyte-like dendritic cells for corneal regeneration

Corneal stromal disorders due to the loss of keratocytes can affect visual impairment and blindness. Corneal cell therapy is a promising therapeutic strategy for healing corneal tissue or even enhancing corneal function upon advanced disorders, however, the sources of corneal keratocytes are limited for clinical applications. Here, the capacity of cell-imprinted substrates fabricated by molding human keratocyte templates to induce differentiation of human adipose-derived stem cells (hADSCs) into keratocytes, is presented. Keratocytes are isolated from human corneal stroma and grown to transmit their ECM architecture and cell-like topographies to a PDMS substrate. The hADSCs are then seeded on cell-imprinted substrates and their differentiation to keratocytes in DMEM/F12 (with and without chemical factors) are evaluated by real-time PCR and immunocytochemistry. The mesenchymal stem cells grown on patterned substrates present gene and protein expression profiles similar to corneal keratocytes. In contrast, a negligible expression of myofibroblast marker in the hADSCs cultivated on the imprinted substrates, is observed. Microscopic analysis reveals dendritic morphology and ellipsoid nuclei similar to primary keratocytes. Overall, it is demonstrated that biomimetic imprinted substrates would be a sufficient driver to solely direct the stem cell fate toward target cells which is a significant achievement toward corneal regeneration.

Chondroitin sulfate is required for follicle epithelial integrity and organ shape maintenance in Drosophila.

Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture.

Nitric oxide-releasing gel accelerates healing in a diabetic murine splinted excisional wound model.

According to the American Diabetes Association (ADA), 9-12 million patients suffer from chronic ulceration each year, costing the healthcare system over USD $25 billion annually. There is a significant unmet need for new and efficacious therapies to accelerate closure of non-healing wounds. Nitric Oxide (NO) levels typically increase rapidly after skin injury in the inflammatory phase and gradually diminish as wound healing progresses. The effect of increased NO concentration on promoting re-epithelization and wound closure has yet to be described in the context of diabetic wound healing. In this study, we investigated the effects of local administration of an NO-releasing gel on excisional wound healing in diabetic mice. The excisional wounds of each mouse received either NO-releasing gel or a control phosphate-buffered saline (PBS)-releasing gel treatment twice daily until complete wound closure. Topical administration of NO-gel significantly accelerated the rate of wound healing as compared with PBS-gel-treated mice during the later stages of healing. The treatment also promoted a more regenerative ECM architecture resulting in shorter, less dense, and more randomly aligned collagen fibers within the healed scars, similar to that of unwounded skin. Wound healing promoting factors fibronectin, TGF-β1, CD31, and VEGF were significantly elevated in NO vs. PBS-gel-treated wounds. The results of this work may have important clinical implications for the management of patients with non-healing wounds.

Fibrous Matrix Architecture-Dependent Activation of Fibroblasts with a Cancer-Associated Fibroblast-like Phenotype.

Cancer-associated fibroblasts (CAFs) are one of the most prevalent cell types within the tumor microenvironment (TME). While several physicochemical cues from the TME, including growth factors, cytokines, and ECM specificity, have been identified as essential factors for CAF activation, the precise mechanism of how the ECM architecture regulates CAF initiation remains elusive. Using a gelatin-based electrospun fiber mesh, we examined the effect of matrix fiber density on CAF activation induced by MCF-7 conditioned media (CM). A less dense (3D) gelatin mesh matrix facilitated better activation of dermal fibroblasts into a CAF-like phenotype in the CM than a highly dense (3D) gelatin mesh matrix. In addition, it was discovered that CAF activation on the less dense (LD) matrix is dependent on the cell size-related AKT/mTOR signaling cascade, accompanied by an increase in intracellular tension within the well-spread fibroblasts.

Self-assembly of mesoscale collagen architectures and applications in 3D cell migration

3D in vitro tumor models have recently been investigated as they can recapitulate key features in the tumor microenvironment. Reconstruction of a biomimetic scaffold is critical in these models. However, most current methods focus on modulating local properties, e.g. micro- and nano-scaled topographies, without capturing the global millimeter or intermediate mesoscale features. Here we introduced a method for modulating the collagen I-based extracellular matrix structure by disruption of fibrillogenesis and the gelation process through mechanical agitation. With this method, we generated collagen scaffolds that are thickened and wavy at a larger scale while featuring global softness. Thickened collagen patches were interconnected with loose collagen networks, highly resembling collagen architecture in the tumor stroma. This thickened collagen network promoted tumor cell dissemination. In addition, this novel modified scaffold triggered differences in morphology and migratory behaviors of tumor cells. Altogether, our method for altered collagen architecture paves new ways for studying in detail cell behavior in physiologically relevant biological processes. Statement of significanceTumor progression usually involves chronic tissue damage and repair processes. Hallmarks of tumors are highly overlapped with those of wound healing. To mimic the tumor milieu, collagen-based scaffolds are widely used. These scaffolds focus on modulating microscale topographies and mechanics, lacking global architecture similarity compared with in vivo architecture. Here we introduced one type of thick collagen bundles that mimics ECM architecture in human skin scars. These thickened collagen bundles are long and wavy while featuring global softness. This collagen architecture imposes fewer steric restraints and promotes tumor cell dissemination. Our findings demonstrate a distinct picture of cell behaviors and intercellular interactions, highlighting the importance of collagen architecture and spatial heterogeneity of the tumor microenvironment.

Noncanonical Wnt/Ror2 signaling regulates cell-matrix adhesion to prompt directional tumor cell invasion in breast cancer.

Cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression, yet how particular signal-transduction factors prompt the conversion of tumor cells into migratory populations capable of systemic spread during metastasis remains elusive. We demonstrate that the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling and invasion in breast cancer. Ror2 loss-of-function (LOF) triggers the disruption of E-cadherin within tumor cells, accompanied by an increase in tumor cell invasion and collagen realignment in three-dimensional cultures. RNA sequencing of Ror2-deficient organoids further uncovered alterations in actin cytoskeleton, cell adhesion, and collagen cross-linking gene expression programs. Spatially, we pinpoint the up-regulation and redistribution of α5 and β3 integrins together with the production of fibronectin in areas of invasion downstream of Ror2 loss. Wnt/β-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of tumor cell-driven ECM exchanges during cancer invasion and metastasis.

Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture.

Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 μm2 was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 μm2 (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.

P–699 Multi-scale study of the architecture, topography and mechanics of the human ovary from prepuberty to menopause: a blueprint for next-generation bioengineering and diagnosis

Abstract Study question Does the ovarian ECM have a precise and unique biophysical phenotype, specific to each age, from prepuberty to menopause? Summary answer Differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. What is known already Ovarian engineering has recently emerged to respond to patient needs and offer reliable models for basic research. It has relied on synthetic and natural biomaterials and microfluidics. However, these techniques were designed based on knowledge acquired from 2D cell culture and animal models. Our lack of information on the human ovary hampers our ability to mimic the main features of this organ, for clinical applications. The complex composition and hierarchical structure of its ECM complicates the design of truly biomimetic constructs, notably: fiber morphology, interstitial and perifollicular fiber orientation, porosity, topography, and viscoelasticity, which all play a role in mechanotransduction. Study design, size, duration Ovarian biopsies were taken from prepubertal (mean age [±SD]=7±3 years, n = 21), reproductive age (mean age [±SD]=27±5, n = 26 ) and menopausal (mean age [±SD]=61±6 years, n = 29) patients after obtaining their informed consent. All participating adult subjects were undergoing laparoscopic surgery for benign gynecological diseases not affecting the ovaries. Prepubertal tissue was derived from young cancer patients scheduled for ovarian cortex cryopreservation as a fertility preservation strategy, before being subjected to acute gonadotoxic cancer treatments. Participants/materials, setting, methods All samples were cryopreserved by slow freezing and kept frozen until the day of their analysis. Tissues provided from the same patients (n = 5 per age group) were investigated by scanning electron microscopy (SEM) (fiber, pore and topography analyses) and atomic force microscopy (AFM). A larger number of paraffin-fixed biopsies (prepubertal, n = 16, reproductive-age, n = 21, and menopausal, n = 24) obtained from the biobank of St-Luc’s Hospital were used to conduct computed fiber orientation analysis. Main results and the role of chance Our results revealed a unique ECM architecture at reproductive age, where fibers of intermediate diameter are assembled into thickest bundles compared to prepubertal and menopausal tissues(p &amp;lt; 0.0001). Indeed, during prepuberty the bundles assemble into a tight network with high number of small pores while reproductive-age ovary gain more porosity(p &amp;lt; 0.0001). However, at menopause tissue pore number and area change significantly(p &amp;lt; 0.001). These pore geometry and distribution changes contribute to diffusion and access of key molecules to/from cells, which can be translated into changes in permeability and molecule selectivity with age. Fiber directionality around follicle borders at preantral stages revealed that before and after puberty, secondary follicles appear to modify their microenvironment arrangement locally compared to follicles at earlier stages of development (p &amp;lt; 0.01), by reorienting the majority of collagen fibers below 50°.This could indicate that follicles at this stage require higher fiber contact and adhesion signaling to complete their development and maturation towards ovulation. AFM evidenced a relatively rigid ovarian tissue at prepuberty, softening significantly at reproductive age, then stiffening considerably upon menopause. These differences(p &amp;lt; 0.01) are not only structure-dependent, but also related to biochemical differences in ECM composition, as previously demonstrated in our follow-up of variations in elastic matrisome components from prepuberty to menopause. Limitations, reasons for caution The samples represent single time points from each age group which could present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. Wider implications of the findings: Our study provides the first conclusive proof of a link between ECM biophysics and fertility by comparing different stages of ovarian transformation related to a woman’s reproductive life, which will oriente new strategies for infertility prognoses based on ECM biophysics and may become a blueprint for designing functional engineered ovaries. Trial registration number Not applicable

Abstract 239: Integrated computational image analysis of cellular and acellular tissue components as a method for detailed tumor tissue mapping and structural patterns recognition

Abstract Tumor microenvironment (TME) represents an integrated system that affects cancer cell behavior and contributes directly to disease outcome. Systemic approach to analysis of TME should uncover its complexity and facilitate discovery of mechanisms orchestrating tumor development and metastasis. Multiplex fluorescence tissue staining followed by spatial analysis of tumor tissue architecture can provide insights to pivotal interactions of cellular and acellular components of TME. Extracellular matrix (ECM is represented mainly by collagen deposition. Number of reports indicates that ECM contribution to TME state not only depends upon amount of accumulated collagen but its geometrical features and spatial orientation of fibers. These characteristics of collagen fibers contribute directly to physical and mechanical properties of tissue and can change tumor growth and metastasis. Current methods of computational image analysis of tissue implement assessment of cellular or acellular components separately. The goal of current work was to develop a new computational tool to perform integrated analysis of fibrous and cellular components of tumor tissue in spatial dependent manner to achieve detailed tumor tissue mapping and structural patterns recognition. To pursue this goal, we generated images of human lung adenocarcinoma tissue characterized by indolent and aggressive behavior. We performed multiplex immunofluorescence staining for following markers: CD3 - marker of T-lymphocytes, PanCytokeratin - marker of epithelial/tumor cells, collagen hybridizing peptide (3Helix) - marker of collagen, DAPI - nuclear counterstain. To develop image analysis pipeline, we utilized an open source graphical interface analytical platform KNIME, where we generated modular workflow. For ECM analysis, we integrated Python written code into KNIME node. Segmentation of collagen fibers was performed using skeletonization with subsequent calculation of geometrical properties (length, alignment, widths) and orientation of each fiber. Data, collected from single cell analysis and ECM architecture assessment, were combined and forwarded to downstream spatial analysis, where distances from cell to cell or cell to ECM were computed and neighborhood analysis was performed. We demonstrated that tumor cells in aggressive adenocarcinoma samples were co-localized with a smaller number of collagen fibers. In addition, length of that fibers was less in comparison to indolent group. Correlation analysis revealed positive correlation between length of collagen fibers and number of tumor cells in indolent group, but we did not observe this phenomenon in indolent group. Developed computational method provides additional dimensionality to tissue image analysis and can reveal underrecognized structural patterns of the tumor microenvironment. Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Maria-Fernanda Senosain, Anna Menshikh, Andries Zijlstra, Sergey Novitskiy, Pierre Massion. Integrated computational image analysis of cellular and acellular tissue components as a method for detailed tumor tissue mapping and structural patterns recognition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 239.

A Computational Framework to Predict and Understand in situ Heart Valve Tissue Engineering

Objective: It is widely accepted that there is a strong relation between mechanical factors due to hemodynamic loading and tissue adaptation in native and tissue-engineered heart valves (TEHVs)[1,2]. Yet, this process remains poorly understood due to complex interactions between cell-mediated tissue growth and remodeling (G&R) and the hemodynamic environment. Computational modeling can aid in understanding valve adaptation. Yet, existing models have limitations that do not allow investigating simultaneous tissue formation and scaffold degradation, which is instrumental for an in situ TEHV. Hypothesizing that G&R occurs to (re-)establish mechanical homeostasis, we aimed to develop a computational framework that enables simulation, and ultimately understanding, of in situ TEHV development under complex hemodynamic loading conditions. Methods: A valve mechanical model [3] was enriched with a G&R algorithm [4] to compute the turnover of individual constituents following [5]. This model predicts the evolution of the tissue volume (growth), composition, and microstructure (remodeling) due to changes in hemodynamic loading. Results: To demonstrate the concept, increased blood pressure is modeled by increasing tissue stretch 25% and computing the adaptation of the tissue. The elevated tissue production leads to increased tissue volume and a relative increase in collagen content (Figure 1). Both these mechanisms improve the load-bearing capacity of the tissue and enable the tissue to return to its homeostatic mechanical state. Conclusions: Computational models of heart valve G&R provide valuable insights into interactions between hemodynamics and evolving tissue properties. The presented framework allows, for the first time, simulations of evolving in situ TEHVs, which will aid in understanding experimental outcomes and rationally improving valve designs. Additionally, it enables prediction of morphology and ECM architecture after certain clinical interventions, like the Ross-Yacoub procedure.

3D Scaffolds to Model the Hematopoietic Stem Cell Niche: Applications and Perspectives.

Hematopoietic stem cells (HSC) are responsible for the production of blood and immune cells during life. HSC fate decisions are dependent on signals from specialized microenvironments in the bone marrow, termed niches. The HSC niche is a tridimensional environment that comprises cellular, chemical, and physical elements. Introductorily, we will revise the current knowledge of some relevant elements of the niche. Despite the importance of the niche in HSC function, most experimental approaches to study human HSCs use bidimensional models. Probably, this contributes to the failure in translating many in vitro findings into a clinical setting. Recreating the complexity of the bone marrow microenvironment in vitro would provide a powerful tool to achieve in vitro production of HSCs for transplantation, develop more effective therapies for hematologic malignancies and provide deeper insight into the HSC niche. We previously demonstrated that an optimized decellularization method can preserve with striking detail the ECM architecture of the bone marrow niche and support HSC culture. We will discuss the potential of this decellularized scaffold as HSC niche model. Besides decellularized scaffolds, several other methods have been reported to mimic some characteristics of the HSC niche. In this review, we will examine these models and their applications, advantages, and limitations.

Hierarchical fibrous guiding cues at different scales influence linear neurite extension

Surface topographies at micro- and nanoscales can influence different cellular behavior, such as their growth rate and directionality. While different techniques have been established to fabricate 2-dimensional flat substrates with nano- and microscale topographies, most of them are prone to high costs and long preparation times. The 2.5-dimensional fiber platform presented here provides knowledge on the effect of the combination of fiber alignment, inter-fiber distance (IFD), and fiber surface topography on contact guidance to direct neurite behavior from dorsal root ganglia (DRGs) or dissociated primary neurons. For the first time, the interplay of the micro-/nanoscale topography and IFD is studied to induce linear nerve growth, while controlling branching. The results demonstrate that grooved fibers promote a higher percentage of aligned neurite extension, compensating the adverse effect of increased IFD. Accordingly, maximum neurite extension from primary neurons is achieved on grooved fibers separated by an IFD of 30 μm, with a higher percentage of aligned neurons on grooved fibers at a large IFD compared to porous fibers with the smallest IFD of 10 µm. We further demonstrate that the neurite “decision-making” behavior on whether to cross a fiber or grow along it is not only dependent on the IFD but also on the fiber surface topography. In addition, axons growing in between the fibers seem to have a memory after leaving grooved fibers, resulting in higher linear growth and higher IFDs lead to more branching. Such information is of great importance for new material development for several tissue engineering applications. Statement of SignificanceOne of the key aspects of tissue engineering is controlling cell behavior using hierarchical structures. Compared to 2D surfaces, fibers are an important class of materials, which can emulate the native ECM architecture of tissues. Despite the importance of both fiber surface topography and alignment to direct growing neurons, the current state of the art did not yet study the synergy between both scales of guidance. To achieve this, we established a solvent assisted spinning process to combine these two crucial features and control neuron growth, alignment, and branching. Rational design of new platforms for various tissue engineering and drug discovery applications can benefit from such information as it allows for fabrication of functional materials, which selectively influence neurite behavior.

Dynamic interactions between the extracellular matrix and estrogen activity in progression of ER+ breast cancer.

Metastatic, anti-estrogen resistant estrogen receptor α positive (ER+) breast cancer is the leading cause of breast cancer deaths in U.S. women. While studies have demonstrated the importance of the stromal tumor microenvironment in cancer progression and therapeutic responses, effects on the responses of ER+ cancers to estrogen and anti-estrogens are poorly understood, particularly in the complex in vivo environment. In this study, we used an estrogen responsive syngeneic mouse model to interrogate how a COL1A1-enriched fibrotic ECM modulates integrated hormonal responses in cancer progression. We orthotopically transplanted the ER+ TC11 cell line into wild-type (WT) or collagen-dense (Col1a1tm1Jae/+, mCol1a1) syngeneic FVB/N female mice. Once tumors were established, recipients were supplemented with 17β-estradiol (E2), tamoxifen, or left untreated. Although the dense/stiff environment in mCol1a1 recipients did not alter the rate of E2-induced proliferation of the primary tumor, it fostered the agonist activity of tamoxifen to increase proliferation and AP-1 activity. Manipulation of estrogen activity did not alter the incidence of lung lesions in either WT or mCol1a1 hosts. However, the mCol1a1 environment enabled tamoxifen-stimulated growth of pulmonary metastases and further fueled estrogen-driven growth. Moreover, E2 remodeled peritumoral ECM architecture in WT animals, modifying alignment of collagen fibers and altering synthesis of ECM components associated with increased alignment and stiffness, and increasing FN1 and POSTN expression in the pulmonary metastatic niche. These studies demonstrate dynamic interactions between ECM properties and estrogen activity in progression of ER+ breast cancer, and support the need for therapeutics that target both ER and the tumor microenvironment.

Abstract 1883: Fibrillar 1D tumor microenvironment is the key driver associating high-speed tumor cell motility with nuclear shape in breast cancer

Abstract Unlike normal breast stroma, which contains curly collagen fibrils, aligned collagen fibers oriented perpendicular to blood vessels are seen in both human breast tumors and mouse models of breast cancer. These linear collagen fibers provide “highways” for tumor cells to migrate toward blood vessels in a directional migration mode known as tumor cell streaming. Streaming is characterized by tumor cell migration at high speed and directional persistence on 1D collagen fibers. Previous studies utilizing linear ECM substrates have shown that tumor cells adopt elongated morphology and display increased speeds on linear 1D substrates compared to their 2D motility. However, the relationship between 1D geometry of ECM fibers in breast tumor microenvironment and the underlying mechanotransduction mechanism regulating high-speed migration of tumor cells is not well understood. Here, we analyzed in vivo ECM architecture by SHG intravital imaging and found a narrow peak of fiber diameters falling in the range 2-3 µm. These fibers were composed of collagen I and fibronectin. Based on these findings, we developed a high fidelity in vitro nanofiber system to study the molecular mechanisms underlying tumor cell streaming migration. Breast carcinoma cells plated on 2 µm ECM-coated fibers showed enhanced motility matching in vivo velocities averaging 1.2 µm/min. We varied 1D fiber diameter (0.7-20 µm) and found that tumor cells move the fastest with highest persistence on smaller fibers within a narrow range of diameters from 0.7-3 µm. High tumor cell speeds correlated with enhanced alignments of F-actin and focal adhesions along the fiber axial dimension. Unexpectedly, we also observed nuclear deformation during carcinoma cell migration on narrow fibers in vitro, similar to nuclear deformation observed in vivo. This was a surprising finding because nuclear deformation in vivo was assumed to be caused by squeezing through ECM pores. Thus, we hypothesized that actomyosin forces not only regulate cell motility parameters, but also nuclear deformation independently of ECM pore size. To test this hypothesis, we disrupted the transmission of cytoskeletal forces to the nucleus by knocking down LINC complex proteins - SUN1 and SUN2, and found increased nuclear elongation and cell motility parameters, through the upregulation of actin polymerization. These results indicate that in carcinoma cells, F-actin associated forces are shared between the leading edge (to maintain cell speed) and the nucleus (to dynamically regulate nuclear shape). LINC complex disruption releases F-actin forces acting on the nucleus to the cell front, leading to higher tumor cell motility speeds. In summary, our results provide new insights into the interplay between actomyosin contractility and the LINC complex in the regulation of nuclear shape and high-speed tumor cell motility during carcinoma cell metastasis. Citation Format: Ved P. Sharma, James Williams, Edison Leung, Joseph Sanders, Robert Eddy, James Castracane, John Condeelis. Fibrillar 1D tumor microenvironment is the key driver associating high-speed tumor cell motility with nuclear shape in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1883. doi:10.1158/1538-7445.AM2017-1883

Implantation of a Poly-l-Lactide GCSF-Functionalized Scaffold in a Model of Chronic Myocardial Infarction

A previously developed poly-l-lactide scaffold releasing granulocyte colony-stimulating factor (PLLA/GCSF) was tested in a rabbit chronic model of myocardial infarction (MI) as a ventricular patch. Control groups were constituted by healthy, chronic MI and nonfunctionalized PLLA scaffold. PLLA-based electrospun scaffold efficiently integrated into a chronic infarcted myocardium. Functionalization of the biopolymer with GCSF led to increased fibroblast-like vimentin-positive cellular colonization and reduced inflammatory cell infiltration within the micrometric fiber mesh in comparison to nonfunctionalized scaffold; PLLA/GCSF polymer induced an angiogenetic process with a statistically significant increase in the number of neovessels compared to the nonfunctionalized scaffold; PLLA/GCSF implanted at the infarcted zone induced a reorganization of the ECM architecture leading to connective tissue deposition and scar remodeling. These findings were coupled with a reduction in end-systolic and end-diastolic volumes, indicating a preventive effect of the scaffold on ventricular dilation, and an improvement in cardiac performance.

Influence of 3D porous galactose containing PVA/gelatin hydrogel scaffolds on three-dimensional spheroidal morphology of hepatocytes.

Three-dimensional liver scaffolds are temporary framework that mimics native ECM architecture and positively influence hepatocyte lodging, proliferation with retention of metabolic activities. The aim of the current study is to develop galactose containing physical cross-linked polyvinyl alcohol/gelatin (P/G 8:2 and 9:1) hydrogel scaffolds via freeze/thaw technique. The 8:2 and 9:1 P/G hydrogels exhibited comparable pore size and porosity (P > 0.05). The tensile strength of the fabricated 8:2 and 9:1 P/G hydrogel scaffolds were found to be in accordance with native human liver. Pore interconnectivity of both the P/G hydrogel scaffolds was confirmed by scanning electron micrographs and liquid displacement method. Further galactose containing hydrogel promoted cell-cell and cell-hydrogel interaction, aiding cellular aggregation leading to spheroids formation compared to void P/G hydrogel by 7 days. Hence, galactose containing P/G hydrogel could be more promising substrate as it showed significantly higher cell proliferation and albumin secretion for 21 days when compared to non-galactose P/G hydrogels (P < 0.05).

Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium

AbstractExtracellular matrix (ECM) derived by tissue decellularization has applications as a tissue engineering scaffold and for support of cellular regeneration. Myocardial ECM from animals has been produced by whole‐organ perfusion or immersion processes, but methods for preparation of human myocardial ECM for therapy and research have not been compared in detail, yet. We analyzed the impact of decellularization processes on human myocardial ECM, and tested its ability to serve as a scaffold for cell seeding. Sodium dodecyl sulfate (SDS)‐based decellularization, but not treatments based on Triton X‐100, deoxycholate or hypo/hypertonic incubations, removed cells satisfactorily, and incubation with fetal bovine serum (FBS) eliminated residual DNA. ECM architecture was best preserved by a protocol consisting of 2 h lysis, 6 h SDS, and 3 h FBS, but age and pathology of the donor tissue are highly important for producing reproducible, high‐quality scaffolds. We also studied ECM repopulation with mesenchymal stem cells (CB‐MSC), cardiomyocytes derived from induced pluripotent stem cells (iPS‐CM), and naïve neonatal mouse cardiomyocytes. Cells attached to the matrix and proliferated and displayed higher viability than in standard culture. We conclude that human cardiac ECM sheets may be suitable scaffold for cell‐matrix interaction studies and as a biomaterial for tissue regeneration and engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3263–3272, 2014