Small Extracellular Vesicles Promote Stiffness-mediated Metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiologic matrix stiffness affects the quantity and protein cargo of small extracellular vesicles (EV) produced by cancer cells, which in turn aid cancer cell dissemination. Primary patient breast tissue released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα2ß1, ITGα6ß4, ITGα6ß1, CD44) compared with EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix proteins including collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer-associated fibroblast phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. SIGNIFICANCE: Here we show that the quantity, cargo, and function of breast cancer-derived EVs vary with mechanical properties of the extracellular microenvironment.

Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment.

CD8+ T cell dysfunction impedes antitumor immunity in solid cancers, but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional coactivator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend on oncogene-mediated ECM composition and remodeling.

Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma.

Melanoma, the most lethal form of skin cancer, often has worse outcomes in older patients. We previously demonstrated that an age-related decrease in the secreted extracellular matrix (ECM) protein HAPLN1 has a role in slowing melanoma progression. Here we show that HAPLN1 in the dermal ECM is sufficient to maintain the integrity of melanoma-associated blood vessels, as indicated by increased collagen and VE-cadherin expression. Specifically, we show that HAPLN1 in the ECM increases hyaluronic acid and decreases endothelial cell expression of ICAM1. ICAM1 phosphorylates and internalizes VE-cadherin, a critical determinant of vascular integrity, resulting in permeable blood vessels. We found that blocking ICAM1 reduces tumor size and metastasis in older mice. These results suggest that HAPLN1 alters endothelial ICAM1expression in an indirect, matrix-dependent manner. Targeting ICAM1 could be a potential treatment strategy for older patients with melanoma, emphasizing the role of aging in tumorigenesis.

Sarcoma Cells Secrete Hypoxia-Modified Collagen VI to Weaken the Lung Endothelial Barrier and Promote Metastasis.

Intratumoral hypoxia correlates with metastasis and poor survival in patients with sarcoma. Using an impedance sensing assay and a zebrafish intravital microinjection model, we demonstrated here that the hypoxia-inducible collagen-modifying enzyme lysyl hydroxylase PLOD2 and its substrate collagen type VI (COLVI) weaken the lung endothelial barrier and promote transendothelial migration. Mechanistically, hypoxia-induced PLOD2 in sarcoma cells modified COLVI, which was then secreted into the vasculature. Upon reaching the apical surface of lung endothelial cells, modified COLVI from tumor cells activated integrin ß1 (ITGß1). Furthermore, activated ITGß1 colocalized with Kindlin2, initiating their interaction with F-actin and prompting its polymerization. Polymerized F-actin disrupted endothelial adherens junctions and induced barrier dysfunction. Consistently, modified and secreted COLVI was required for the late stages of lung metastasis in vivo. Analysis of patient gene expression and survival data from The Cancer Genome Atlas (TCGA) revealed an association between the expression of both PLOD2 and COLVI and patient survival. Furthermore, high levels of COLVI were detected in surgically resected sarcoma metastases from patient lungs and in the blood of tumor-bearing mice. Together, these data identify a mechanism of sarcoma lung metastasis, revealing opportunities for therapeutic intervention. SIGNIFICANCE: Collagen type VI modified by hypoxia-induced PLOD2 is secreted by sarcoma cells and binds to integrin ß1 on endothelial cells to induce barrier dysfunction, which promotes sarcoma vascular dissemination and metastasis.

Small extracellular vesicles promote stiffness-mediated metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα 2 ß 1 , ITGα 6 ß 4 , ITGα 6 ß 1 , CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment.

Comparative oncology reveals DNMT3B as a molecular vulnerability in undifferentiated pleomorphic sarcoma.

PURPOSE: Undifferentiated pleomorphic sarcoma (UPS), an aggressive subtype of soft-tissue sarcoma (STS), is exceedingly rare in humans and lacks effective, well-tolerated therapies. In contrast, STS are relatively common in canine companion animals. Thus, incorporation of veterinary patients into studies of UPS offers an exciting opportunity to develop novel therapeutic strategies for this rare human disease. Genome-wide studies have demonstrated that UPS is characterized by aberrant patterns of DNA methylation. However, the mechanisms and impact of this epigenetic modification on UPS biology and clinical behavior are poorly understood. METHODS: DNA methylation in mammalian cells is catalyzed by the canonical DNA methyltransferases DNMT1, DNMT3A and DNMT3B. Therefore, we leveraged cell lines and tissue specimens from human and canine patients, together with an orthotopic murine model, to probe the functional and clinical significance of DNMTs in UPS. RESULTS: We found that the DNA methyltransferase DNMT3B is overexpressed in UPS relative to normal mesenchymal tissues and is associated with a poor prognosis. Consistent with these findings, genetic DNMT3B depletion strongly inhibited UPS cell proliferation and tumor progression. However, existing hypomethylating agents, including the clinically approved drug 5-aza-2'-deoxycytidine (DAC) and the DNMT3B-inhibiting tool compound nanaomycin A, were ineffective in UPS due to cellular uptake and toxicity issues. CONCLUSIONS: DNMT3B represents a promising molecular susceptibility in UPS, but further development of DNMT3B-targeting strategies for these patients is required.

Virtual screening for small-molecule pathway regulators by image-profile matching.

Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.

Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

Tumor-Derived Retinoic Acid Regulates Intratumoral Monocyte Differentiation to Promote Immune Suppression.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor-associated macrophages (TAMs) rather than immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we find that the TME induces tumor cells to produce retinoic acid (RA), which polarizes intratumoral monocyte differentiation toward TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production in tumor cells or pharmacologic inhibition of RA signaling within TME increases stimulatory monocyte-derived cells, enhances T cell-dependent anti-tumor immunity, and synergizes with immune checkpoint blockade. Furthermore, an RA-responsive gene signature in human monocytes correlates with an immunosuppressive TME in multiple human tumors. RA has been considered as an anti-cancer agent, whereas our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

TGFß and Hippo Pathways Cooperate to Enhance Sarcomagenesis and Metastasis through the Hyaluronan-Mediated Motility Receptor (HMMR).

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFß signaling. Therefore, we investigated crosstalk between YAP1 and TGFß resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFß and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFß-YAP1-RHAMM axis prevents vascular migration of tumor cells to distant sites. IMPLICATIONS: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFß cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

Hypoxia and the Tumor Secretome.

Metastasis remains the leading cause of cancer-related deaths. To date, there are no specific treatments targeting disseminated disease. New therapeutic options will become available only if we enhance our understanding of mechanisms underlying metastatic spread. A large body of literature shows that the metastatic potential of tumor cells is strongly influenced by microenvironmental cues such as low oxygen (hypoxia). Clinically, hypoxia is a hallmark of most solid tumors and is associated with increased metastasis and poor survival in a variety of cancer types. Mechanistically, hypoxia influences multiple steps within the metastatic cascade and particularly impacts the interactions between tumor cells and host stroma at both primary and secondary sites. Here we review current evidence for a hypoxia-induced tumor secretome and its impact on metastatic progression. These studies have identified potential biomarkers and therapeutic targets that could be integrated into strategies for preventing and treating metastatic disease.

A Feedback Loop between Hypoxia and Matrix Stress Relaxation Increases Oxygen-Axis Migration and Metastasis in Sarcoma.

Upregulation of collagen matrix crosslinking directly increases its ability to relieve stress under the constant strain imposed by solid tumor, a matrix property termed stress relaxation. However, it is unknown how rapid stress relaxation in response to increased strain impacts disease progression in a hypoxic environment. Previously, it has been demonstrated that hypoxia-induced expression of the crosslinker procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), in sarcomas has resulted in increased lung metastasis. Here, we show that short stress relaxation times led to increased cell migration along a hypoxic gradient in 3D collagen matrices, and rapid stress relaxation upregulated PLOD2 expression via TGFß-SMAD2 signaling, forming a feedback loop between hypoxia and the matrix. Inhibition of this pathway led to a decrease in migration along the hypoxic gradients. In vivo, sarcoma primed in a hypoxic matrix with short stress relaxation time enhanced collagen fiber size and tumor density and increased lung metastasis. High expression of PLOD2 correlated with decreased overall survival in patients with sarcoma. Using a patient-derived sarcoma cell line, we developed a predictive platform for future personalized studies and therapeutics. Overall, these data show that the interplay between hypoxia and matrix stress relaxation amplifies PLOD2, which in turn accelerates sarcoma cell motility and metastasis. SIGNIFICANCE: These findings demonstrate that mechanical (stress relaxation) and chemical (hypoxia) properties of the tumor microenvironment jointly accelerate sarcoma motility and metastasis via increased expression of collagen matrix crosslinker PLOD2.

YAP1 enhances NF-κB-dependent and independent effects on clock-mediated unfolded protein responses and autophagy in sarcoma.

Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy.

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

YAP1-Mediated Suppression of USP31 Enhances NFκB Activity to Promote Sarcomagenesis.

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705-20. ©2018 AACR.

The Bad, the Good and eIF3e/INT6.

Recent research on translation and protein synthesis in several pathologies, including cancer, peripheral artery disease, and wound healing, demonstrates the key role played by translational factors in tumorigenic and angiogenic processes. This review will focus on one specific translational factor, eIF3e also called INT6, the "e" subunit of the translation initiation factor eIF3. INT6/eIF3e has recently been described as a multifunction protein playing a role in translation, protein degradation, DNA repair, nonsense-mediated mRNA decay, cell cycle and control of cell response to low oxygen (hypoxia or ischemia) through modulation of the Hypoxia Inducible Factors (HIFs). Interestingly, INT6/eIF3e is a double-edged sword that has both oncogenic and tumor suppressive abilities. In addition to its role in tumorigenesis, its silencing has recently been suggested as a potential therapeutic strategy to improve cell survival and function after ischemic injuries. Although a deeper understanding of the molecular mechanisms involved in these pathophysiological functions is essential, particularly to transform the in vitro/in vivo findings into clinical applications, INT6/eIF3e modulation could provide therapeutic benefit for a variety of human diseases such as cancer or vascular diseases.

Intratumoral oxygen gradients mediate sarcoma cell invasion.

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm(3)) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth.

In soft tissue sarcomas (STS), low intratumoural O2 (hypoxia) is a poor prognostic indicator. HIF-1α mediates key transcriptional responses to hypoxia, and promotes STS metastasis; however, the role of the related HIF-2α protein is unknown. Surprisingly, here we show that HIF-2α inhibits high-grade STS cell growth in vivo, as loss of HIF-2α promotes sarcoma proliferation and increases calcium and mTORC1 signalling in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. We find that most human STS have lower levels of EPAS1 (the gene encoding HIF-2α) expression relative to normal tissue. Many cancers, including STS, contain altered epigenetics, and our findings define an epigenetic mechanism whereby EPAS1 is silenced during sarcoma progression. The clinically approved HDAC inhibitor Vorinostat specifically increases HIF-2α, but not HIF-1α, accumulation in multiple STS subtypes. Vorinostat inhibits STS tumour growth, an effect ameliorated by HIF-2α deletion, implicating HIF-2α as a biomarker for Vorinostat efficacy in STS.

Targeting the Hippo pathway: Clinical implications and therapeutics.

The Hippo pathway plays a critical role in tissue and organ size regulation by restraining cell proliferation and apoptosis under homeostatic conditions. Deregulation of this pathway can promote tumorigenesis in multiple malignant human tumor types, including sarcoma, breast, lung and liver cancers. In this review, we summarize the current understanding of Hippo pathway function, it's role in human cancer, and address the potential of Hippo pathway member proteins as therapeutic targets for a variety of tumors.