Automated brightfield layerwise evaluation in three-dimensional micropatterning via two-photon polymerization.

Two-photon polymerization (TPP) is an advanced 3D fabrication technique capable of creating features with submicron precision. A primary challenge in TPP lies in the facile and accurate characterization of fabrication quality, particularly for structures possessing complex internal features. In this study, we introduce an automated brightfield layerwise evaluation technique that enables a simple-to-implement approach for in situ monitoring and quality assessment of TPP-fabricated structures. Our approach relies on sequentially acquired brightfield images during the TPP writing process and using background subtraction and image processing to extract layered spatial features. We experimentally validate our method by printing a fibrous tissue scaffold and successfully achieve an overall system-adjusted fidelity of 87.5% in situ. Our method is readily adaptable in most TPP systems and can potentially facilitate high-quality TPP manufacturing of sophisticated microstructures.

Contributions of the distinct biophysical phenotype of polyploidal giant cancer cells to cancer progression.

Polyploid giant cancer cells (PGCCs) are a commonly observed histological feature of human tumors and are particularly prominent in late stage and drug resistant cancers. The chromosomal duplication conferred by their aneuploidy gives rise to DNA damage resistance and complex tumor cell karyotypes, a driving factor in chemotherapy resistance and disease relapse. Furthermore, PGCCs also exhibit key cytoskeletal features that give rise to a distinct biophysical phenotype, including increased density of polymerized actin and vimentin intermediate filaments, nuclear and cytoskeletal stiffening, increased traction force, and migratory persistence. Despite recent research highlighting the role PGCCs play in cancer progression, this population of tumor cells remains poorly characterized in terms of their biophysical properties. In this review, we will discuss the various aspects of their biomolecular phenotype, such as increased stemness as well as a mixed EMT signature. These features have been extensively associated with tumorigenesis and recurrence, and aggressive cancers. Additionally, we will also examine the distinct PGCC cytoskeletal features of actin and filamentous vimentin. Specifically, how the differential organization of these networks serve to support their increased size and drive migratory persistence. These findings could shed light on potential therapeutic strategies that allow for specific elimination or mitigation of the invasive potential of these polyploid cancer cells. Lastly, we will examine how the biophysical and molecular phenotype of PGCCs combine to tip the scale in favor of promoting cancer progression, presenting an important target in the clinical treatment of cancer.

Vimentin filaments drive migratory persistence in polyploidal cancer cells.

Polyploidal giant cancer cells (PGCCs) are multinucleated chemoresistant cancer cells found in heterogeneous solid tumors. Due in part to their apparent dormancy, the effect of PGCCs on cancer progression has remained largely unstudied. Recent studies have highlighted the critical role of PGCCs as aggressive and chemoresistant cancer cells, as well as their ability to undergo amitotic budding to escape dormancy. Our recent study demonstrated the unique biophysical properties of PGCCs, as well as their unusual migratory persistence. Here we unveil the critical function of vimentin intermediate filaments (VIFs) in maintaining the structural integrity of PGCCs and enhancing their migratory persistence. We performed in-depth single-cell analysis to examine the distribution of VIFs and their role in migratory persistence. We found that PGCCs rely heavily on their uniquely distributed and polarized VIF network to enhance their transition from a jammed to an unjammed state to allow for directional migration. Both the inhibition of VIFs with acrylamide and small interfering RNA knockdown of vimentin significantly decreased PGCC migration and resulted in a loss of PGCC volume. Because PGCCs rely on their VIF network to direct migration and to maintain their enlarged morphology, targeting vimentin or vimentin cross-linking proteins could provide a therapeutic approach to mitigate the impact of these chemoresistant cells in cancer progression and to improve patient outcomes with chemotherapy.

Senescent mesenchymal stem cells remodel extracellular matrix driving breast cancer cells to a more-invasive phenotype.

Mesenchymal stem cells (MSCs) are essential for the regenerative process; however, biological aging and environmental stress can induce senescence - an irreversible state of growth arrest - that not only affects the behavior of cells but also disrupts their ability to restore tissue integrity. While abnormal tissue properties, including increased extracellular matrix stiffness, are linked with the risk of developing breast cancer, the role and contribution of senescent MSCs to the disease progression to malignancy are not well understood. Here, we investigated senescence-associated biophysical changes in MSCs and how this influences cancer cell behavior in a 3D matrix interface model. Although senescent MSCs were far less motile than pre-senescent MSCs, they induced an invasive breast cancer phenotype, characterized by increased spheroid growth and cell invasion in collagen gels. Further analysis of collagen gels using second-harmonic generation showed increased collagen density when senescent MSCs were present, suggesting that senescent MSCs actively remodel the surrounding matrix. This study provides direct evidence of the pro-malignant effects of senescent MSCs in tumors.

Microenvironment Influences Cancer Cell Mechanics from Tumor Growth to Metastasis.

The microenvironment in a solid tumor includes a multitude of cell types, matrix proteins, and growth factors that profoundly influence cancer cell mechanics by providing both physical and chemical stimulation. This tumor microenvironment, which is both dynamic and heterogeneous in nature, plays a critical role in cancer progression from the growth of the primary tumor to the development of metastatic and drug-resistant tumors. This chapter provides an overview of the biophysical tools used to study cancer cell mechanics and mechanical changes in the tumor microenvironment at different stages of cancer progression, including growth of the primary tumor, local invasion, and metastasis. Quantitative single cell biophysical analysis of intracellular mechanics, cell traction forces, and cell motility can easily be combined with analysis of critical cell fate processes, including adhesion, proliferation, and drug resistance, to determine how changes in mechanics contribute to cancer progression. This biophysical approach can be used to systematically investigate the parameters in the tumor that control cancer cell interactions with the stroma and to identify specific conditions that induce tumor-promoting behavior, along with strategies for inhibiting these conditions to treat cancer. Increased understanding of the underlying biophysical mechanisms that drive cancer progression may provide insight into novel therapeutic approaches in the fight against cancer.

The malignancy of metastatic ovarian cancer cells is increased on soft matrices through a mechanosensitive Rho-ROCK pathway.

Although current treatments for localized ovarian cancer are highly effective, this cancer still remains the most lethal gynecological malignancy, largely owing to the fact that it is often detected only after tumor cells leave the primary tumor. Clinicians have long noted a clear predilection for ovarian cancer to metastasize to the soft omentum. Here, we show that this tropism is due not only to chemical signals but also mechanical cues. Metastatic ovarian cancer cells (OCCs) preferentially adhere to soft microenvironments and display an enhanced malignant phenotype, including increased migration, proliferation and chemoresistance. To understand the cell-matrix interactions that are used to sense the substrate rigidity, we utilized traction force microscopy (TFM) and found that, on soft substrates, human OCCs increased both the magnitude of traction forces as well as their degree of polarization. After culture on soft substrates, cells underwent morphological elongation characteristic of epithelial-to-mesenchymal transition (EMT), which was confirmed by molecular analysis. Consistent with the idea that mechanical cues are a key determinant in the spread of ovarian cancer, the observed mechanosensitivity was greatly decreased in less-metastatic OCCs. Finally, we demonstrate that this mechanical tropism is governed through a Rho-ROCK signaling pathway.

SNAIL-induced epithelial-to-mesenchymal transition produces concerted biophysical changes from altered cytoskeletal gene expression.

A growing body of evidence suggests that the developmental process of epithelial-to-mesenchymal transition (EMT) is co-opted by cancer cells to metastasize to distant sites. This transition is associated with morphologic elongation and loss of cell-cell adhesions, though little is known about how it alters cell biophysical properties critical for migration. Here, we use multiple-particle tracking (MPT) microrheology and traction force cytometry to probe how genetic induction of EMT in epithelial MCF7 breast cancer cells changes their intracellular stiffness and extracellular force exertion, respectively, relative to an empty vector control. This analysis demonstrated that EMT alone was sufficient to produce dramatic cytoskeletal softening coupled with increases in cell-exerted traction forces. Microarray analysis revealed that these changes corresponded with down-regulation of genes associated with actin cross-linking and up-regulation of genes associated with actomyosin contraction. Finally, we show that this loss of structural integrity to expedite migration could inhibit mesenchymal cell proliferation in a secondary tumor as it accumulates solid stress. This work demonstrates that not only does EMT enable escape from the primary tumor through loss of cell adhesions but it also induces a concerted series of biophysical changes enabling enhanced migration of cancer cells after detachment from the primary tumor.

Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress.

For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress.

Actomyosin tension as a determinant of metastatic cancer mechanical tropism.

Despite major advances in the characterization of molecular regulators of cancer growth and metastasis, patient survival rates have largely stagnated. Recent studies have shown that mechanical cues from the extracellular matrix can drive the transition to a malignant phenotype. Moreover, it is also known that the metastatic process, which results in over 90% of cancer-related deaths, is governed by intracellular mechanical forces. To better understand these processes, we identified metastatic tumor cells originating from different locations which undergo inverse responses to altered matrix elasticity: MDA-MB-231 breast cancer cells that prefer rigid matrices and SKOV-3 ovarian cancer cells that prefer compliant matrices as characterized by parameters such as tumor cell proliferation, chemoresistance, and migration. Transcriptomic analysis revealed higher expression of genes associated with cytoskeletal tension and contractility in cells that prefer stiff environments, both when comparing MDA-MB-231 to SKOV-3 cells as well as when comparing bone-metastatic to lung-metastatic MDA-MB-231 subclones. Using small molecule inhibitors, we found that blocking the activity of these pathways mitigated rigidity-dependent behavior in both cell lines. Probing the physical forces exerted by cells on the underlying substrates revealed that though force magnitude may not directly correlate with functional outcomes, other parameters such as force polarization do correlate directly with cell motility. Finally, this biophysical analysis demonstrates that intrinsic levels of cell contractility determine the matrix rigidity for maximal cell function, possibly influencing tissue sites for metastatic cancer cell engraftment during dissemination. By increasing our understanding of the physical interactions of cancer cells with their microenvironment, these studies may help develop novel therapeutic strategies.

VEGFR1-activity-independent metastasis formation.

Molecules such as vascular endothelial growth factor (VEGF) or placental growth factor-critical regulators of tumour angiogenesis-are also thought to mobilize into blood circulation bone marrow-derived cells (BMDCs), which may subsequently be recruited to tumours and facilitate tumour growth and metastasis. A study has suggested that BMDCs form 'metastatic niches' in lungs before arrival of cancer cells, and showed that pharmacological inhibition of VEGF receptor 1 (VEGFR1, also known as Flt1)-cognate receptor for VEGF and placental growth factor-prevented BMDC infiltration in lungs and 'metastatic niche' formation. Here we report that blockade of VEGFR1 activity does not affect the rate of spontaneous metastasis formation in a clinically relevant and widely used preclinical model. Therefore, alternative pathways probably mediate the priming of tissues for metastasis.

Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization.

Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.

Spatially coordinated changes in intracellular rheology and extracellular force exertion during mesenchymal stem cell differentiation.

The mechanical properties within the cell are regulated by the organization of the actin cytoskeleton, which is linked to the extracellular environment through focal adhesion proteins that transmit force. Chemical and mechanical stimuli alter the organization of cytoskeletal actin, which results in changes in cell shape, adhesion, and differentiation. By combining particle-tracking microrheology and traction force cytometry, we can monitor the mechanical properties of the actin meshwork and determine how changes in the intracellular network contribute to force generation. In this study, we investigated the effects of chemical (differentiation factors) and mechanical (substrate rigidity) stimuli important in mesenchymal stem cell (MSC) differentiation on the intracellular mechanics and traction stress generation. We found the presence of adipogenic factors resulted in stiffening of the actin meshwork regardless of substrate rigidity. In contrast, these factors increased traction stresses on hard substrates, which was associated with increased expression of contractility genes. Furthermore, MSCs cultured on hard substrates expressed both adipogenic and osteogenic markers indicative of mixed differentiation. On hard substrates, heterogeneity in the local elastic modulus-traction stress correlation was also increased in response to adipogenic factors, indicating that these mechanical properties may be reflective of differences in the level of MSC differentiation. These results suggest intracellular rheology and traction stress generation are spatially regulated and contribute insight into how single cell mechanical forces contribute to MSC differentiation.

Biomechanical analysis predicts decreased human mesenchymal stem cell function before molecular differences.

Multipotent human mesenchymal stem cells (hMSCs) are uniquely suited for the growing field of regenerative medicine due to their ease of isolation, expansion, and transplantation. However, during ex vivo expansion necessary to obtain clinically relevant cell quantities, hMSCs undergo fundamental changes culminating in cellular senescence. The molecular changes as hMSCs transition into senescence have been well characterized, but few studies have focused on the mechanical properties that govern many processes necessary for therapeutic efficacy. We show that before detectable differences in classical senescence markers emerge, single-cell mechanical and cytoskeletal properties reveal a subpopulation of 'non-functioning' hMSCs that appears after even limited expansion. This subpopulation, characterized by a loss of dynamic cytoskeletal stiffening and morphological flexibility in response to chemotactic signals grows with extended culture resulting in overall decreased hMSC motility and ability to contract collagen gels. These changes were mitigated with cytoskeletal inhibition. Finally, a xenographic wound healing model was used to demonstrate that these in vitro differences correlate with decreased ability of hMSCs to aid in wound closure in vivo. These data illustrate the importance of analyzing not only the molecular markers, but also mechanical markers of hMSCs as they are investigated for potential therapeutics.

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-ß 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.

Senescence-associated exosomes transfer miRNA-induced fibrosis to neighboring cells.

Radiation-induced fibrosis is a common side effect of radiotherapy, which is the most common treatment for cancer. However, radiation also causes p53-mediated cell cycle arrest, prolonged expression of p21, and the development of senescence in normal cells that reside in irradiated tissues. Bone marrow-derived mesenchymal stem cells (MSCs) accumulate in primary tumor sites because of their natural tropism for inflammatory and fibrotic tissues. MSCs are extremely sensitive to low doses of ionizing radiation and acquire senescence as a result of bystander radiation effects. Senescent cells remain metabolically active but develop a potent senescence-associated secretory phenotype (SASP) that correlates to hyperactive secretion of cytokines, pro-fibrotic growth factors, and exosomes (EXOs). Integrative pathway analysis highlighted that radiation-induced senescence significantly enriched cell-cycle, extracellular matrix, transforming growth factor-ß (TGF-ß) signaling, and vesicle-mediated transport genes in MSCs. EXOs are cell-secreted nanovesicles (a subclass of small extracellular vesicles) that contain biomaterials-proteins, RNAs, microRNAs (miRNAs)-that are critical in cell-cell communication. miRNA content analysis of secreted EXOs further revealed that radiation-induced senescence uniquely altered miRNA profiles. In fact, several of the standout miRNAs directly targeted TGF-ß or downstream genes. To examine bystander effects of radiation-induced senescence, we further treated normal MSCs with senescence-associated EXOs (SA-EXOs). These modulated genes related to TGF-ß pathway and elevated both alpha smooth muscle actin (protein increased in senescent, activated cells) and Ki-67 (proliferative marker) expression in SA-EXO treated MSCs compared to untreated MSCs. We revealed SA-EXOs possess unique miRNA content that influence myofibroblast phenotypes via TGF-ß pathway activation. This highlights that SA-EXOs are potent SASP factors that play a large role in cancer-related fibrosis.

Metronomic and single high-dose paclitaxel treatments produce distinct heterogenous chemoresistant cancer cell populations.

More than 75% of epithelial ovarian cancer (EOC) patients experience disease recurrence after initial treatment, highlighting our incomplete understanding of how chemoresistant populations evolve over the course of EOC progression post chemotherapy treatment. Here, we show how two paclitaxel (PTX) treatment methods- a single high dose and a weekly metronomic dose for four weeks, generate unique chemoresistant populations. Using mechanically relevant alginate microspheres and a combination of transcript profiling and heterogeneity analyses, we found that these PTX-treatment regimens produce distinct and resilient subpopulations that differ in metabolic reprogramming signatures, acquisition of resistance to PTX and anoikis, and the enrichment for cancer stem cells (CSCs) and polyploid giant cancer cells (PGCCs) with the ability to replenish bulk populations. We investigated the longevity of these metabolic reprogramming events using untargeted metabolomics and found that metabolites associated with stemness and therapy-induced senescence were uniquely abundant in populations enriched for CSCs and PGCCs. Predictive network analysis revealed that antioxidative mechanisms were likely to be differentially active dependent on both time and exposure to PTX. Our results illustrate how current standard chemotherapies contribute to the development of chemoresistant EOC subpopulations by either selecting for intrinsically resistant subpopulations or promoting the evolution of resistance mechanisms. Additionally, our work describes the unique phenotypic signatures in each of these distinct resistant subpopulations and thus highlights potential vulnerabilities that can be exploited for more effective treatment.

Spatial Heterogeneity in Cytoskeletal Mechanics Response to TGF-ß1 and Hypoxia Mediates Partial Epithelial-to-Meshenchymal Transition in Epithelial Ovarian Cancer Cells.

Metastatic progression of epithelial ovarian cancer (EOC) involves the partial epithelial-to-mesenchymal transition (EMT) of cancer cells in the primary tumor and dissemination into peritoneal fluid. In part to the high degree of heterogeneity in EOC cells, the identification of EMT in highly epithelial cells in response to differences in matrix mechanics, growth factor signaling, and tissue hypoxia is very difficult. We analyzed different degrees of EMT by tracking changes in cell and nuclear morphology, along with the organization of cytoskeletal proteins. In our analysis, we see a small percentage of individual cells that show dramatic response to TGF-ß1 and hypoxia treatment. We demonstrate that EOC cells are spatially aware of their surroundings, with a subpopulation of EOC cells at the periphery of a cell cluster in 2D environments exhibited a greater degree of EMT. These peripheral cancer cells underwent partial EMT, displaying a hybrid of mesenchymal and epithelial characteristics, which often included less cortical actin and more perinuclear cytokeratin expression. Collectively, these data show that tumor-promoting microenvironment conditions can mediate invasive cell behavior in a spatially regulated context in a small subpopulation of highly epithelial clustered cancer cells that maintain epithelial characteristics while also acquiring some mesenchymal traits through partial EMT.

Comparing the Secretomes of Chemorefractory and Chemoresistant Ovarian Cancer Cell Populations.

High-grade serous ovarian cancer (HGSOC) constitutes the majority of all ovarian cancer cases and has staggering rates of both refractory and recurrent disease. While most patients respond to the initial treatment with paclitaxel and platinum-based drugs, up to 25% do not, and of the remaining that do, 75% experience disease recurrence within the subsequent two years. Intrinsic resistance in refractory cases is driven by environmental stressors like tumor hypoxia which alter the tumor microenvironment to promote cancer progression and resistance to anticancer drugs. Recurrent disease describes the acquisition of chemoresistance whereby cancer cells survive the initial exposure to chemotherapy and develop adaptations to enhance their chances of surviving subsequent treatments. Of the environmental stressors cancer cells endure, exposure to hypoxia has been identified as a potent trigger and priming agent for the development of chemoresistance. Both in the presence of the stress of hypoxia or the therapeutic stress of chemotherapy, cancer cells manage to cope and develop adaptations which prime populations to survive in future stress. One adaptation is the modification in the secretome. Chemoresistance is associated with translational reprogramming for increased protein synthesis, ribosome biogenesis, and vesicle trafficking. This leads to increased production of soluble proteins and extracellular vesicles (EVs) involved in autocrine and paracrine signaling processes. Numerous studies have demonstrated that these factors are largely altered between the secretomes of chemosensitive and chemoresistant patients. Such factors include cytokines, growth factors, EVs, and EV-encapsulated microRNAs (miRNAs), which serve to induce invasive molecular, biophysical, and chemoresistant phenotypes in neighboring normal and cancer cells. This review examines the modifications in the secretome of distinct chemoresistant ovarian cancer cell populations and specific secreted factors, which may serve as candidate biomarkers for aggressive and chemoresistant cancers.

Force balancing ACT-IN the tumor microenvironment: Cytoskeletal modifications in cancer and stromal cells to promote malignancy.

The tumor microenvironment is a complex milieu that dictates the growth, invasion, and metastasis of cancer cells. Both cancer and stromal cells in the tumor tissue encounter and adapt to a variety of extracellular factors, and subsequently contribute and drive the progression of the disease to more advanced stages. As the disease progresses, a small population of cancer cells becomes more invasive through a complex process known as epithelial-mesenchymal transition, and nearby stromal cells assume a carcinoma associated fibroblast phenotype characterized by enhanced migration, cell contractility, and matrix secretion with the ability to reorganize extracellular matrices. As cells transition into more malignant phenotypes their biophysical properties, controlled by the organization of cytoskeletal proteins, are altered. Actin and its associated proteins are essential modulators and facilitators of these changes. As the cells respond to the cues in the microenvironment, actin driven mechanical forces inside and outside the cells also evolve. Recent advances in biophysical techniques have enabled us to probe these actin driven changes in cancer and stromal cells and demarcate their role in driving changes in the microenvironment. Understanding the underlying biophysical mechanisms that drive cancer progression could provide critical insight on novel therapeutic approaches in the fight against cancer.

Ovarian Cancer Exosomes Trigger Differential Biophysical Response in Tumor-Derived Fibroblasts.

Exosomes are cell-secreted microvesicles that play important roles in epithelial ovarian cancer (EOC) progression, as they are constantly secreted into ascites fluids. While cells spontaneously release exosomes, alterations in intracellular calcium or extracellular pH can release additional exosomes. Yet, little is known about how these exosomes compare to those that are continuously released without stimulation and how they mediate cellular activities important in cancer progression. Here, we demonstrate that chelation of extracellular calcium leads to release of chelation-induced exosomes (CI-exosomes) from OVCAR-3 EOC cells. CI-exosomes display a unique miRNA profile compared to naturally secreted exosomes (SEC-exosomes). Furthermore, treatment with CI- and SEC-exosomes leads to differential biophysical and functional changes including, adhesion and migration in EOC-derived fibroblasts that suggest the development of a malignant tumor microenvironment. This result highlights how tumor environmental factors contribute to heterogeneity in exosome populations and how different exosome populations mediate diversity in stromal cell behavior.