Biphasic reinforcement of nascent adhesions by vinculin.

Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens.

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.

Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity.

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response.

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1ß and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1ß and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

Estrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

The Mechanics of Mitotic Cell Rounding.

When animal cells enter mitosis, they round up to become spherical. This shape change is accompanied by changes in mechanical properties. Multiple studies using different measurement methods have revealed that cell surface tension, intracellular pressure and cortical stiffness increase upon entry into mitosis. These cell-scale, biophysical changes are driven by alterations in the composition and architecture of the contractile acto-myosin cortex together with osmotic swelling and enable a mitotic cell to exert force against the environment. When the ability of cells to round is limited, for example by physical confinement, cells suffer severe defects in spindle assembly and cell division. The requirement to push against the environment to create space for spindle formation is especially important for cells dividing in tissues. Here we summarize the evidence and the tools used to show that cells exert rounding forces in mitosis in vitro and in vivo, review the molecular basis for this force generation and discuss its function for ensuring successful cell division in single cells and for cells dividing in normal or diseased tissues.

Oncogenic Signaling Alters Cell Shape and Mechanics to Facilitate Cell Division under Confinement.

To divide in a tissue, both normal and cancer cells become spherical and mechanically stiffen as they enter mitosis. We investigated the effect of oncogene activation on this process in normal epithelial cells. We found that short-term induction of oncogenic RasV12 activates downstream mitogen-activated protein kinase (MEK-ERK) signaling to alter cell mechanics and enhance mitotic rounding, so that RasV12-expressing cells are softer in interphase but stiffen more upon entry into mitosis. These RasV12-dependent changes allow cells to round up and divide faithfully when confined underneath a stiff hydrogel, conditions in which normal cells and cells with reduced levels of Ras-ERK signaling suffer multiple spindle assembly and chromosome segregation errors. Thus, by promoting cell rounding and stiffening in mitosis, oncogenic RasV12 enables cells to proliferate under conditions of mechanical confinement like those experienced by cells in crowded tumors.

Zebrafish Spinal Cord Repair Is Accompanied by Transient Tissue Stiffening.

Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glial-fibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes axon regrowth and is accompanied by neurogenesis. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ atomic force microscopy-enabled nanoindentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter before returning to baseline values after the completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. This work constitutes the first quantitative mapping of the spatiotemporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.

nanite: using machine learning to assess the quality of atomic force microscopy-enabled nano-indentation data.

BACKGROUND: Atomic force microscopy (AFM) allows the mechanical characterization of single cells and live tissue by quantifying force-distance (FD) data in nano-indentation experiments. One of the main problems when dealing with biological tissue is the fact that the measured FD curves can be disturbed. These disturbances are caused, for instance, by passive cell movement, adhesive forces between the AFM probe and the cell, or insufficient attachment of the tissue to the supporting cover slide. In practice, the resulting artifacts are easily spotted by an experimenter who then manually sorts out curves before proceeding with data evaluation. However, this manual sorting step becomes increasingly cumbersome for studies that involve numerous measurements or for quantitative imaging based on FD maps. RESULTS: We introduce the Python package nanite, which automates all basic aspects of FD data analysis, including data import, tip-sample separation, base line correction, contact point retrieval, and model fitting. In addition, nanite enables the automation of the sorting step using supervised learning. This learning approach relates subjective ratings to predefined features extracted from FD curves. For ratings ranging from 0 to 10, our approach achieves a mean squared error below 1.0 rating points and a classification accuracy between good and poor curves that is above 87%. We showcase our approach by quantifying Young's moduli of the zebrafish spinal cord at different classification thresholds and by introducing data quality as a new dimension for quantitative AFM image analysis. CONCLUSION: The addition of quality-based sorting using supervised learning enables a fully automated and reproducible FD data analysis pipeline for biological samples in AFM.

3D Microenvironment Stiffness Regulates Tumor Spheroid Growth and Mechanics via p21 and ROCK.

The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment is revealed here. Thus, the findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments.

Engineering a humanized bone organ model in mice to study bone metastases.

Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4-6 months. Additive biomanufactured scaffolds seeded and cultured with human bone-forming cells are implanted ectopically in combination with osteogenic factors into mice to generate a physiological bone 'organ', which is partially humanized. The model comprises human bone cells and secreted extracellular matrix (ECM); however, other components of the engineered tissue, such as the vasculature, are of murine origin. The model can be further humanized through the engraftment of human hematopoietic stem cells (HSCs) that can lead to human hematopoiesis within the murine host. The humanized organ bone model has been well characterized and validated and allows dissection of some of the mechanisms of the bone metastatic processes in prostate and breast cancer.

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments.

UNLABELLED: Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14days, cancer spheroids of 100-200µm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process. STATEMENT OF SIGNIFICANCE: Threedimensional in vitro cancer models have generated great interest over the past decade. However, most models are not suitable to systematically study the effects of environmental cues on cancer development and progression. To overcome this limitation, we have developed an innovative hydrogel platform to study the interactions between breast and prostate cancer cells and extracellular matrix ligands relevant to the tumour microenvironment. Our results show that hydrogels with laminin- and collagen-derived adhesive peptides induce a malignant phenotype in a cell-line specific manner. Thus, we have identified a method to control the incorporation of biochemical cues within a three dimensional culture model and anticipate that it will help us in better understanding the effects of the tumour microenvironment on cancer progression.

New mechanistic insights of integrin ß1 in breast cancer bone colonization.

Bone metastasis is a frequent and life-threatening complication of breast cancer. The molecular mechanisms supporting the establishment of breast cancer cells in the skeleton are still not fully understood, which may be attributed to the lack of suitable models that interrogate interactions between human breast cancer cells and the bone microenvironment. Although it is well-known that integrins mediate adhesion of malignant cells to bone extracellular matrix, their role during bone colonization remains unclear. Here, the role of ß1 integrins in bone colonization was investigated using tissue-engineered humanized in vitro and in vivo bone models. In vitro, bone-metastatic breast cancer cells with suppressed integrin ß1 expression showed reduced attachment, spreading, and migration within human bone matrix compared to control cells. Cell proliferation in vitro was not affected by ß1 integrin knockdown, yet tumor growth in vivo within humanized bone microenvironments was significantly inhibited upon ß1 integrin suppression, as revealed by quantitative in/ex vivo fluorescence imaging and histological analysis. Tumor cells invaded bone marrow spaces in the humanized bone and formed osteolytic lesions; osteoclastic bone resorption was, however, not reduced by ß1 integrin knockdown. Taken together, we demonstrate that ß1 integrins have a pivotal role in bone colonization using unique tissue-engineered humanized bone models.

In vitro microenvironments to study breast cancer bone colonisation.

Bone metastasis occurs frequently in patients with advanced breast cancer and is a major cause of morbidity and mortality in these patients. In order to advance current therapies, the mechanisms leading to the formation of bone metastases and their pathophysiology have to be better understood. Several in vitro models have been developed for systematic studies of interactions between breast cancer cells and the bone microenvironment. Such models can provide insights into the molecular basis of bone metastatic colonisation and also may provide a useful platform to design more physiologically relevant drug testing assays. This review describes different in vitro approaches and discusses their advantages and disadvantages.

Mimicking breast cancer-induced bone metastasis in vivo: current transplantation models and advanced humanized strategies.

Bone metastasis is a complication that occurs in 80 % of women with advanced breast cancer. Despite the prevalence of bone metastatic disease, the avenues for its clinical management are still restricted to palliative treatment options. In fact, the underlying mechanisms of breast cancer osteotropism have not yet been fully elucidated due to a lack of suitable in vivo models that are able to recapitulate the human disease. In this work, we review the current transplantation-based models to investigate breast cancer-induced bone metastasis and delineate the strengths and limitations of the use of different grafting techniques, tissue sources, and hosts. We further show that humanized xenograft models incorporating human cells or tissue grafts at the primary tumor site or the metastatic site mimic more closely the human disease. Tissue-engineered constructs are emerging as a reproducible alternative to recapitulate functional humanized tissues in these murine models. The development of advanced humanized animal models may provide better platforms to investigate the mutual interactions between human cancer cells and their microenvironment and ultimately improve the translation of preclinical drug trials to the clinic.

A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone.

The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact 'organ' bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis.

As microenvironmental factors such as three-dimensionality and cell-matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor ß1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Single-cell force spectroscopy, an emerging tool to quantify cell adhesion to biomaterials.

Cell adhesion receptors play a central role in sensing and integrating signals provided by the cellular environment. Thus, understanding adhesive interactions at the cell-biomaterial interface is essential to improve the design of implants that should emulate certain characteristics of the cell's natural environment. Numerous cell adhesion assays have been developed; among these, atomic force microscopy-based single-cell force spectroscopy (AFM-SCFS) provides a versatile tool to quantify cell adhesion at physiological conditions. Here we discuss how AFM-SCFS can be used to quantify the adhesion of living cells to biomaterials and give examples of using AFM-SCFS in tissue engineering and regenerative medicine. We anticipate that in the near future, AFM-SCFS will be established in the biomaterial field as an important technique to quantify cell-biomaterial interactions and thereby will contribute to the optimization of implants, scaffolds, and medical devices.

A bioengineered microenvironment to quantitatively measure the tumorigenic properties of cancer-associated fibroblasts in human prostate cancer.

Stromal-epithelial cell interactions play an important role in cancer and the tumor stroma is regarded as a therapeutic target. In vivo xenografting is commonly used to study cellular interactions not mimicked or quantified in conventional 2D culture systems. To interrogate the effects of tumor stroma (cancer-associated fibroblasts or CAFs) on epithelia, we created a bioengineered microenvironment using human prostatic tissues. Patient-matched CAFs and non-malignant prostatic fibroblasts (NPFs) from men with moderate (Gleason 7) and aggressive (Gleason 8-9 or castrate-resistant) prostate cancer were cultured with non-tumorigenic BPH-1 epithelial cells. Changes in the morphology, motility and phenotype of BPH-1 cells in response to CAFs and NPFs were analyzed using immunofluorescence and quantitative cell morphometric analyses. The matrix protein gene expression of CAFs, with proven tumorigenicity in vivo, had a significantly different gene expression profile of matrix proteins compared to patient matched NPFs. In co-culture with CAFs (but not NPFs), BPH-1 cells had a more invasive, elongated phenotype with increased motility and a more directed pattern of cell migration. CAFs from more aggressive tumors (Gleason 8-9 or CRPC) were not quantitatively different to moderate grade CAFs. Overall, our bioengineered microenvironment provides a novel 3D in vitro platform to systematically investigate the effects of tumor stroma on prostate cancer progression.