The extracellular matrix (ECM) plays multifaceted tumor-promoting and tumor-restraining roles in breast can-cer progression. Extracellular peroxidases represent a critical yet underexplored component of stromal re-modeling machinery. Peroxidasin (PXDN), an extracellular peroxidase with established roles in collagen IV crosslinking, has been associated with poor outcomes in several cancers, but its role in breast cancer re-mains unclear. Through temporal proteomic analysis of dysregulated ECM proteins, we identified PXDN as upregulated during early tumor development in mouse models. In human breast cancers, PXDN expres-sion shows compartment-specific associations with patient outcome, with high stromal PXDN, predomi-nantly cancer-associated fibroblast (CAF)-derived, correlating with poor prognosis. We demonstrate that PXDN regulates CAF behavior, with subsequent matrix remodeling affecting cancer cell behavior. Reduction of CAF-derived PXDN in vivo slows tumor development, while pharmacological inhibition of extracellular per-oxidases improves overall survival. These findings establish PXDN as a mediator of breast tumor progression and a promising future therapeutic target.

Biofabrication of cardiac patches is a challenging strategy proposed as an alternative to transplantation for end-stage heart failure patients. The optimization of the bioink used for this strategy can be limited by costs, properties, and biocompatibility of its building blocks. Lately, sericin has emerged within a wide range of natural proteins, thanks to its bioadhesive and biocompatibility potential. In this study, we assessed for the first time the effects of adding silk sericin on alginate-gelatin hydrogels, proposed for cardiac applications. To this aim, we first biofabricated sericincontaining hydrogels with increasing protein concentrations.Thus, we characterized hydrogels' mechanical behavior, porosity and structure through rheology, Brillouin microspectroscopy, and scanning electron microscopy. Then, we bioprinted the formulated hydrogels and evaluated their effects on human cardiac spheroids (CSs) in vitro. Our mechanical characterization demonstrated that adding sericin significantly enhanced the elasticity and the viscosity of alginate-gelatin hydrogels. Sericin also modified hydrogels' swelling behavior and their pore size, increasing by 20%, 62%, and 92% in Ser1%, Ser2%, and Ser3%, respectively. Although Ser1% did not exhibit significant effects on CSs, Ser2%and Ser3%enhanced cardiac cell viability for up to 14 days compared to the sericin-free hydrogel by acting on the fibroblast population. Sericin-based bioinks showed better printability and durability with +33% and +28% intact patches after 28 days of culture at 37 degrees C compared to alginate-gelatin. Taken together, our results validated the use of sericin as a promising component for the optimization of bioink intended for cardiac applications.

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 β1 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.

Microalgae have emerged as promising photosynthetic microorganisms for biofabricating advanced tissue constructs, with improved oxygenation and reduced reactive oxygen species (ROS) production. However, their use in the engineering of human tissues has been limited due to their intrinsic growth requirements, which are not compatible with human cells. In this study, we first formulated alginate-gelatin (AlgGel) hydrogels with increasing densities of Chlorella vulgaris. Then, we characterised their mechanical properties and pore size. Finally, we evaluated their effects on cardiac spheroid (CS) pathophysiological response under control and ischemia/reperfusion (I/R) conditions. Our results showed that the addition of Chlorella did not affect AlgGel mechanical properties, while the mean pore size significantly decreased by 35% in the presence of the 10(7) cells ml(-1) microalgae density. Under normoxic conditions, the addition of 10(7)Chlorella cells ml(-1) significantly reduced CS viability starting from 14 d in. No changes in pore size nor CS viability were measured for hydrogels containing 10(5) and 10(6)Chlorella cells ml(-1). In our I/R model, all Chlorella-enriched hydrogels reduced cardiac cell sensitivity to hypoxic conditions with a corresponding reduction in ROS production, as well as protected against I/R-induced reduction in cell viability. Altogether, our results support a promising use of Chlorella-enriched Alg-Gel hydrogels for cardiovascular tissue engineering.

Silk fibroin (SF) is a natural protein extracted from Bombyx mori silkworm thread. From its common use in the textile industry, it emerged as a biomaterial with promising biochemical and mechanical properties for applications in the field of tissue engineering and regenerative medicine. In this study, we evaluate for the first time the effects of SF on cardiac bioink formulations containing cardiac spheroids (CSs). First, we evaluate if the SF addition plays a role in the structural and elastic properties of hydrogels containing alginate (Alg) and gelatin (Gel). Then, we test the printability and durability of bioprinted SF-containing hydrogels. Finally, we evaluate whether the addition of SF controls cell viability and function of CSs in Alg-Gel hydrogels. Our findings show that the addition of 1% (w/v) SF to Alg-Gel hydrogels makes them more elastic without affecting cell viability. However, fractional shortening (FS%) of CSs in SF-Alg-Gel hydrogels increases without affecting their contraction frequency, suggesting an improvement in contractile function in the 3D cultures. Altogether, our findings support a promising pathway to bioengineer bioinks containing SF for cardiac applications, with the ability to control mechanical and cellular features in cardiac bioinks.

Peroxidasin is a heme-containing peroxidase enzyme that plays a vital role in the cross-linking of collagen IV molecules in basement membranes. Collagen IV cross-links are essential for providing structure and mechanical stability throughout tissue development, homeostasis, and wound healing. During cancer progression, the basement membrane is degraded, and proteins typically found in the basement membrane, including peroxidasin and collagen IV, can be found spread throughout the tumour microenvironment where they interact with cancer cells and alter cell behaviour. Whilst peroxidasin is reported to be up-regulated in a number of different cancers, the role that it plays in disease progression and metastasis has only recently begun to be studied. This review highlights the current literature exploring the known roles of peroxidasin in normal tissues and cancer progression, regulators of peroxidasin expression, and the reported relationships between peroxidasin expression and patient outcome in 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.

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

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.

The lysyl oxidase (LOX) family of enzymes are a major driver in the biogenesis of desmoplastic matrix at the primary tumour and secondary metastatic sites. With the increasing interest in and development of anti-stromal therapies aimed at improving clinical outcomes of cancer patients, the Lox family has emerged as a potentially powerful clinical target. This review examines how lysyl oxidase family dysregulation in solid cancers contributes to disease progression and poor patient outcomes, as well as an evaluation of the preclinical landscape of LOX family targeting therapeutics. We also discuss the suitability of the LOX family as a diagnostic and/or prognostic marker in solid tumours.