Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Obesity-associated Adipose Stromal Cells Promote Breast Cancer Invasion Through Direct Cell Contact and ECM Remodeling.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS.

Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, we have utilized a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. Our results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increased their glycocalyx thickness while enhancing resistance to attack by Natural Killer (NK) cells. These changes were functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, our results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Phosphoproteomic Changes Induced by Cell-Derived Matrix and Their Effect on Tumor Cell Migration and Cytoskeleton Remodeling.

Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling. Here, we combine a cell-derived matrix (CDM) model with phosphotyrosine phosphoproteomic analysis to investigate tumor cell migration on fibrotic ECM relative to standard tissue culture plastic (TCP). Our results suggest that tumor cells cultured on CDMs migrate faster and in a more directional manner than their counterparts on TCP. These changes in migration correlate with decreased cell spreading and increased cell elongation. While the formation of phosphorylated focal adhesion kinase (pFAK)+ adhesion complexes did not vary between TCP and CDMs, time-dependent phosphoproteomic analysis identified that the SRC family kinase LYN may be differentially regulated. Pharmacological inhibition of LYN decreased tumor cell migration and cytoskeletal rearrangement on CDMs and also on TCP, suggesting that LYN regulates tumor cell migration on CDMs in combination with other mechanisms. These data highlight how the combination of physicochemically complex in vitro systems with phosphoproteomics can help identify signaling mechanisms by which the fibrotic ECM regulates tumor cell migration.

Bone-matrix mineralization dampens integrin-mediated mechanosignalling and metastatic progression in breast cancer.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization.

Convergent Approaches to Delineate the Metabolic Regulation of Tumor Invasion by Hyaluronic Acid Biosynthesis.

Metastasis is the leading cause of breast cancer-related deaths and is often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion are associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process, remains unclear due to the lack of convergent approaches integrating engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis, and bioinformatic analysis of patient data, the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis are investigated. These results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induces a metabolic phenotype that promotes cancer cell stemness and invasion in vitro and upregulates a transcriptomic signature predictive of increased invasion and worse patient survival. This study suggests that HA overproduction leads to metabolic adaptations to satisfy the energy demands for 3D invasion of breast CSCs highlighting the importance of engineered model systems and multidisciplinary approaches in cancer research.

Disruption of Bcr-Abl coiled coil oligomerization by design.

Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

Engineered ECM models: Opportunities to advance understanding of tumor heterogeneity.

Intratumoral heterogeneity is a negative prognostic factor for cancer and commonly attributed to microenvironment-driven genetic mutations and/or the emergence of cancer stem-like cells. How aberrant extracellular matrix (ECM) remodeling regulates the phenotypic diversity of tumor cells, however, remains poorly understood due in part to a lack of model systems that allow isolating the physicochemical heterogeneity of malignancy-associated ECM for mechanistic studies. Here, we review the compositional, microarchitectural, and mechanical hallmarks of cancer-associated ECM and highlight biomaterials and engineering approaches to recapitulate these properties for in vitro and in vivo studies. Subsequently, we describe how such engineered platforms may be explored to define the spatiotemporal dynamics through which cancer-associated ECM remodeling regulates intratumoral heterogeneity and the cancer stem-like cell phenotype. Finally, we highlight future opportunities and technological advances to further elucidate the relationship between tumor-associated ECM dynamics and intratumoral heterogeneity.

The Learning Curve for Magnetic Resonance Imaging/Ultrasound Fusion-guided Prostate Biopsy.

BACKGROUND: Magnetic resonance imaging/ultrasound-guided fusion biopsy (FBx) is more accurate at detecting clinically significant prostate cancer than conventional transrectal ultrasound-guided systematic biopsy. However, learning curves for attaining accuracy may limit the generalizability of published outcomes. OBJECTIVE: To delineate and quantify the learning curve for FBx by assessing the targeted biopsy accuracy and pathological quality of systematic biopsy over time. DESIGN, SETTING, AND PARTICIPANTS: We carried out a retrospective analysis of 173 consecutive men who underwent Artemis FBx with computer-template systematic sampling between July 2015 and May 2017. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The accuracy of targeted biopsy was determined by calculating the distance between planned and actual core trajectories stored on Artemis. Systematic sampling proficiency was assessed via pathological analysis of fibromuscular tissue in all cores and then comparing pathology elements from individual cores from men in the first and last tertiles. Polynomial linear regression models, change-point analysis, and piecewise linear regression were used to quantify the learning curve. RESULTS AND LIMITATION: A significant improvement in targeted biopsy accuracy occurred up to 98 cases (p<0.01). There was a significant decrease in fibromuscular tissue in the systematic biopsy cores up to 84 cases (p<0.01) and an improvement in pathological quality when comparing systematic cores from the first and third tertiles. Use of a different fusion platform may limit the generalizability of our results. CONCLUSIONS: There is a significant learning curve for targeted and systemic biopsy using the Artemis platform. Improvements in accuracy of targeted biopsy and better sampling for systematic biopsy can be achieved with greater experience. PATIENT SUMMARY: We define the learning curve for magnetic resonance imaging/ultrasound-guided fusion biopsy (FBx) using targeted biopsy accuracy and systematic core sampling quality as measures. Our findings underscore the importance of overcoming learning curves inherent to FBx to minimize patient discomfort and biopsy risk and improve the quality of care for accurate risk stratification, active surveillance, and treatment selection.

Adoption of the Q Transcriptional System for Regulating Gene Expression in Stem Cells.

The field of mammalian synthetic biology seeks to engineer enabling technologies to create novel approaches for programming cells to probe, perturb, and regulate gene expression with unprecedented precision. To accomplish this, new genetic parts continue to be identified that can be used to build novel genetic circuits to re-engineer cells to perform specific functions. Here, we establish a new transcription-based genetic circuit that combines genes from the quinic acid sensing metabolism of Neorospora crassa and the bacterial Lac repressor system to create a new orthogonal genetic tool to be used in mammalian cells. This work establishes a novel genetic tool, called LacQ, that functions to regulate gene expression in Chinese hamster ovarian (CHO) cells, human embryonic kidney 293 (HEK293) cells, and in mouse embryonic stem (ES) cells.