Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

A first-in-class pan-lysyl oxidase inhibitor impairs stromal remodeling and enhances gemcitabine response and survival in pancreatic cancer.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis.

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow­induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

Towards engineering heart tissues from bioprinted cardiac spheroids.

Currentin vivoandin vitromodels fail to accurately recapitulate the human heart microenvironment for biomedical applications. This study explores the use of cardiac spheroids (CSs) to biofabricate advancedin vitromodels of the human heart. CSs were created from human cardiac myocytes, fibroblasts and endothelial cells (ECs), mixed within optimal alginate/gelatin hydrogels and then bioprinted on a microelectrode plate for drug testing. Bioprinted CSs maintained their structure and viability for at least 30 d after printing. Vascular endothelial growth factor (VEGF) promoted EC branching from CSs within hydrogels. Alginate/gelatin-based hydrogels enabled spheroids fusion, which was further facilitated by addition of VEGF. Bioprinted CSs contracted spontaneously and under stimulation, allowing to record contractile and electrical signals on the microelectrode plates for industrial applications. Taken together, our findings indicate that bioprinted CSs can be used to biofabricate human heart tissues for long termin vitrotesting. This has the potential to be used to study biochemical, physiological and pharmacological features of human heart tissue.

In Vitro 3D Models of Tunable Stiffness.

Three-dimensional models of spheroid formation have been routinely used in the cancer field to test the colony forming capacity of malignant cells in an in vitro setting. Use of such a model provides a robust surrogate for in vivo testing, enabling large-scale interrogation into the effect of certain treatment conditions. This adapted protocol describes a high throughput and readily accessible composite alginate hydrogel system for spheroid formation, within a biomechanically tunable three-dimensional environment. This model therefore allows users to examine the effect of certain treatment conditions while cells are embedded within a hydrogel of defined stiffness. This is particularly important in the context of cancer where cells experience a wide range of mechanical properties within their microenvironment, driven by widespread changes in the extracellular matrix composition and architecture.This protocol describes a high-throughput method which results in homogeneous interpenetrating polymer networks of collagen and alginate. We show that this network readily supports single-cell spheroid formation in numerous malignant cell lines (breast cancer, lung cancer, and melanoma) and that these can be robustly analyzed for colony formation measures such as spheroid size, spheroid number, and overall cell viability; therefore, allowing users to undertake high-throughput, in vitro screening against a controlled biomechanical background.

Plasma polymerized nanoparticles effectively deliver dual siRNA and drug therapy in vivo.

Multifunctional nanocarriers (MNCs) promise to improve therapeutic outcomes by combining multiple classes of molecules into a single nanostructure, enhancing active targeting of therapeutic agents and facilitating new combination therapies. However, nanocarrier platforms currently approved for clinical use can still only carry a single therapeutic agent. The complexity and escalating costs associated with the synthesis of more complex MNCs have been major technological roadblocks in the pathway for clinical translation. Here, we show that plasma polymerized nanoparticles (PPNs), synthesised in reactive gas discharges, can bind and effectively deliver multiple therapeutic cargo in a facile and cost-effective process compatible with up scaled commercial production. Delivery of siRNA against vascular endothelial growth factor (siVEGF) at extremely low concentrations (0.04 nM), significantly reduced VEGF expression in hard-to-transfect cells when compared with commercial platforms carrying higher siRNA doses (6.25 nM). PPNs carrying a combination of siVEGF and standard of care Paclitaxel (PPN-Dual) at reduced doses (< 100 µg/kg) synergistically modulated the microenvironment of orthotopic breast tumors in mice, and significantly reduced tumor growth. We propose PPNs as a new nanomaterial for delivery of therapeutics, which can be easily functionalised in any laboratory setting without the need for additional wet-chemistry and purification steps.

The Mini-Organo: A rapid high-throughput 3D coculture organotypic assay for oncology screening and drug development.

BACKGROUND: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. AIM: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. METHODS AND RESULTS: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. CONCLUSIONS: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner.

Altered processing enhances the efficacy of small-diameter silk fibroin vascular grafts.

Current synthetic vascular grafts are not suitable for use in low-diameter applications. Silk fibroin is a promising natural graft material which may be an effective alternative. In this study, we compared two electrospun silk grafts with different manufacturing processes, using either water or hexafluoroisopropanol (HFIP) as solvent. This resulted in markedly different Young's modulus, ultimate tensile strength and burst pressure, with HFIP spun grafts observed to have thicker fibres, and greater stiffness and strength relative to water spun. Assessment in a rat abdominal aorta grafting model showed significantly faster endothelialisation of the HFIP spun graft relative to water spun. Neointimal hyperplasia in the HFIP graft also stabilised significantly earlier, correlated with an earlier SMC phenotype switch from synthetic to contractile, increasing extracellular matrix protein density. An initial examination of the macrophage response showed that HFIP spun conduits promoted an anti-inflammatory M2 phenotype at early timepoints while reducing the pro-inflammatory M1 phenotype relative to water spun grafts. These observations demonstrate the important role of the manufacturing process and physical graft properties in determining the physiological response. Our study is the first to comprehensively study these differences for silk in a long-term rodent model.

Bioactive Materials Facilitating Targeted Local Modulation of Inflammation.

Cardiovascular disease is an inflammatory disorder that may benefit from appropriate modulation of inflammation. Systemic treatments lower cardiac events but have serious adverse effects. Localized modulation of inflammation in current standard treatments such as bypass grafting may more effectively treat CAD. The present study investigated a bioactive vascular graft coated with the macrophage polarizing cytokine interleukin-4. These grafts repolarize macrophages to anti-inflammatory phenotypes, leading to modulation of the pro-inflammatory microenvironment and ultimately to a reduction of foreign body encapsulation and inhibition of neointimal hyperplasia development. These resulting functional improvements have significant implications for the next generation of synthetic vascular grafts.

Recent advances in understanding the complexities of metastasis.

Tumour metastasis is a dynamic and systemic process. It is no longer seen as a tumour cell-autonomous program but as a multifaceted and complex series of events, which is influenced by the intrinsic cellular mutational burden of cancer cells and the numerous bidirectional interactions between malignant and non-malignant cells and fine-tuned by the various extrinsic cues of the extracellular matrix. In cancer biology, metastasis as a process is one of the most technically challenging aspects of cancer biology to study. As a result, new platforms and technologies are continually being developed to better understand this process. In this review, we discuss some of the recent advances in metastasis and how the information gleaned is re-shaping our understanding of metastatic dissemination.

Rapid Endothelialization of Off-the-Shelf Small Diameter Silk Vascular Grafts.

Synthetic vascular grafts for small diameter revascularization are lacking. Clinically available conduits expanded polytetrafluorethylene and Dacron fail acutely due to thrombosis and in the longer term from neointimal hyperplasia. We report the bioengineering of a cell-free, silk-based vascular graft. In vitro we demonstrate strong, elastic silk conduits that support rapid endothelial cell attachment and spreading while simultaneously resisting blood clot and fibrin network formation. In vivo rat studies show complete graft patency at all time points, rapid endothelialization, and stabilization and contraction of neointimal hyperplasia. These studies show the potential of silk as an off-the-shelf small diameter vascular graft.

Charting the unexplored extracellular matrix in cancer.

The extracellular matrix (ECM) is present in all solid tissues and considered a master regulator of cell behaviour and phenotype. The importance of maintaining the correct biochemical and biophysical properties of the ECM, and the subsequent regulation of cell and tissue homeostasis, is illustrated by the simple fact that the ECM is highly dysregulated in many different types of disease, especially cancer. The loss of tissue ECM homeostasis and integrity is seen as one of the hallmarks of cancer and typically defines transitional events in progression and metastasis. The vast majority of cancer studies place an emphasis on exploring the behaviour and intrinsic signalling pathways of tumour cells. Their goal was to identify ways to target intracellular pathways regulating cancer. Cancer progression and metastasis are powerfully influenced by the ECM and thus present a vast, unexplored repository of anticancer targets that we are only just beginning to tap into. Deconstructing the complexity of the tumour ECM landscape and identifying the interactions between the many cell types, soluble factors and extracellular-matrix proteins have proved challenging. Here, we discuss some of the emerging tools and platforms being used to catalogue and chart the ECM in cancer.

Plasma mediated protein immobilisation enhances the vascular compatibility of polyurethane with tissue matched mechanical properties.

Polyurethanes are a diverse class of polymers, with independently tunable mechanical and biodegradation properties making them a versatile platform material for biomedical implants. Previous iterations have failed to adequately embody appropriate mechanical and biological properties, particularly for vascular medicine where strength, compliance and multifaceted biocompatibility are required. We have synthesized a new polyurethane formulation with finely tuned mechanical properties, combining high strength and extensibility with a low Young's modulus. Additional cross-linking during synthesis enhanced stability and limits leaching. Under cyclic testing, hysteresis was minimal following completion of the initial cycles, indicating the robustness of the material. Building on this platform, we used plasma immersion ion implantation to activate the polymer surface and functionalized it with recombinant human tropoelastin. With tropoelastin covalently bound to the surface, human coronary endothelial cells showed improved attachment and proliferation. In the presence of heparinized whole blood, tropoelastin-coated polyurethane showed very low thrombogenicity in both static and flow conditions. Using this formulation, we synthesized robust, elastic prototype conduits which easily retained multiple sutures and were successfully implanted in a pilot rat aortic interposition model. We have thus created an elastic, strong biomaterial platform, functionalized with an important regulator of vascular biology, with the potential for further evaluation as a new synthetic graft material.

Mechanically Robust Plasma-Activated Interfaces Optimized for Vascular Stent Applications.

The long-term performance of many medical implants is limited by the use of inherently incompatible and bioinert materials. Metallic alloys, ceramics, and polymers commonly used in cardiovascular devices encourage clot formation and fail to promote the appropriate molecular signaling required for complete implant integration. Surface coating strategies have been proposed for these materials, but coronary stents are particularly problematic as the large surface deformations they experience in deployment require a mechanically robust coating interface. Here, we demonstrate a single-step ion-assisted plasma deposition process to tailor plasma-activated interfaces to meet current clinical demands for vascular implants. Using a process control-feedback strategy which predicts crucial coating growth mechanisms by adopting a suitable macroscopic plasma description in combination with noninvasive plasma diagnostics, we describe the optimal conditions to generate highly reproducible, industry-scalable stent coatings. These interfaces are mechanically robust, resisting delamination even upon plastic deformation of the underlying material, and were developed in consideration of the need for hemocompatibility and the capacity for biomolecule immobilization. Our optimized coating conditions combine the best mechanical properties with strong covalent attachment capacity and excellent blood compatibility in initial testing with plasma and whole blood, demonstrating the potential for improved vascular stent coatings.

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVß3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVß3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVß3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.