After more than a decade of controversy on the role of stromal cells in the tumor microenvironment, the emerging data shed light on pro-tumorigenic and potential anti-cancer factors, as well as on the roots of the discrepancies. We discuss the pro-tumorigenic effects of stromal cells, considering the effects of tumor drivers like hypoxia and tumor stiffness on these cells, as well as stromal cell-mediated adiposity and immunosuppression in the tumor microenvironment, and cancer initiating cells’ cellular senescence and adaptive metabolism. We summarize the emerging data supporting stromal cell therapeutic potential in cancer, discuss the possibility to reprogram stromal cells of the tumor microenvironment for anti-cancer effects, and explore some causes of discrepancies on the roles of stromal cells in cancer in the available literature. MSC differentiation potential includes various cell types of the mesodermal lineage, such as fibroblasts, adipocytes, endothelial cells, myocytes, chondrocytes, and osteoblasts1, while non-mesodermal differentiation, such as into neural, hepatic, pancreatic, and gastric cells, is still debated6. However, MSCs reside not only in the stroma of various tissues and organs, but also in the tumor microenvironment, where their role has been clarified recently8. Overall, a huge body of evidence supports that MSCs can promote tumorigenic processes, such as: (i) angiogenesis, neovascularization and formation of cancer stem cell (CSC) niche; (ii) malignant transformation, maintenance of cancer cells, and metastasis formation; as well as (iii) cancer cell stemness and chemoresistance to anti-cancer drugs10.Mesenchymal stromal cells (MSCs) are a heterogeneous mesenchymal cell population, commonly collected from the bone marrow (BM), fat, and other tissues, that includes multipotent stem cells capable of differentiating into a number of mesenchymal tissues, and thus can contribute to tissue repair. MSCs are positive for membranes CD105, CD73, and CD90, and negative for CD14, CD19, CD31, CD34, CD45, and HLA-DR13. Notably, MSC secretome includes numerous factors favoring tissue repair, such as angiopoietin-1, vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), granulocyte-colony stimulating factor (G-CSF)19, as well as other soluble factors, such as interleukin-6 (IL-6), IL-12, C-X-C motif chemokine 8 (CXCL8), CXCL9, CXCL16, C-C chemokine ligand 20 (CCL20), CCL25, and monocyte chemoattractant protein-3 (MCP-3)23.On the other hand, MSCs have become a key tool in tissue engineering and regenerative medicine, because they are easily collected and have the ability to migrate and home into damaged tissues. Here, they: (i) interact with the microenvironment to drive tissue repair; (ii) differentiate into the specific affected cell types to restore or replace damaged tissues; and (iii) rescue organ functions, thanks to their high proliferation, adhesion, migration, differentiation, and immunoregulatory propertiesin vivo, as well as data supporting the existence of stromal cells restraining cancer growth in the tumor microenvironment.Herein, we provide an overview of recent data suggesting that the pro-tumorigenic effects of MSCs as well as MSC-derived cancer-associated fibroblasts (CAFs) are the consequence of a process of cell reprogramming driven by the tumor microenvironment. We also discuss the emerging reports suggesting approaches to reprogram these cells to mediate anti-tumor effects 24. The authors reported the ability of prostate cancer-infiltrating MSCs to suppress T-cell proliferation through immunosuppressive properties comparable to canonical BM-derived MSCs. The suppression of proliferation mediated by prostate cancer-infiltrating MSCs was dose-dependent, and the expressions of programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2) were upregulated on T cells in the presence of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)24. In another study, the transcriptome analysis of MSCs from multiple myeloma (MM) patients revealed constitutive abnormalities in immune system activation, cell cycle progression, and osteoblastogenesis that were maintained even in the absence of tumors cells, thus strongly suggesting that MSCs may contribute to the immune evasion and bone lesions frequently found in MM25. MSCs shape the myelodysplastic syndrome microenvironment at least in part by inducing suppressive monocytes dampening natural killer (NK) cell function26. Moreover, MSCs participate in oral mucosa carcinogenesis by increasing immunosuppressive functions on T-cell proliferation; tumorigenesis of tumor-resident MSCs correlated with higher expression of cellular proliferative status indicator Ki6727. Interestingly and on the same hand, the CXCL8 supporting the survival and proliferation of acute myeloid leukemia (AML) cells via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in the affected BM microenvironment would be mainly secreted by MSCs28.It is well established that MSCs are major drivers of the typical immunomodulation observed in a solid tumor microenvironment. For instance, a recent study using MSCs expanded from BM and prostate cancer tissue from independent donors showed that tumor-infiltrating MSCs are major drivers of the immunosuppressive tumor microenvironment in prostate cancer30. For instance, gastric cancer cell-derived exosomes (extracellular vesicles) affect the immunomodulatory functions of MSCs by activating the nuclear factor-kappa B (NF-κB) signaling pathway, which in turn mediates support to tumor growth by maintaining the inflammatory environment and enhancing the ability of MSCs to activate immune cells31. AML blasts induce a senescence-associated secretory phenotype (SASP) in BM stromal cells through a p16INK4a-dependent mechanism, which encompasses the irreversible arrest of cell proliferation and the secretion of a set of chemokines, proinflammatory cytokines, and growth factors32. Similarly, some authors reported the alteration of cellular and immune-related properties of BM-derived MSCs (BM-MSCs) and macrophages through the release of exosomes from K562 chronic myeloid leukemia cell line; exosome concentration in BM-MSCs correlated with the enhanced expression of Dickkopf-related protein 1 (DKK1), wnt5a, CXCL12, IL-6, TGF-β, and TNF-α33. Furthermore, senescent breast luminal cells promoted carcinogenesis by activating CAFs through the inflammatory cytokine IL-834. BM stromal cells from patients with myelodysplastic syndrome display a senescence phenotype induced by S100A9-induced Toll-like receptor 4 (TLR4), NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, and IL-1β secretion35. TLR4 signaling was also reported to drive MSC commitment to promote tumor microenvironment transformation in MM36.The normal aging process and various age-related diseases, including some cancers, are marked by a chronic low-grade inflammation (“inflammaging”) and cellular senescence (“immunosenescence”). The role of MSC immunomodulation in shaping a senescent microenvironment in a broad spectrum of human malignancies, especially tumorigenesis, has been documented extensively37. Notably, the overexpression of O-GlcNAc transferase (OGT), an enzyme involved in tumor-initiating cell-mediated rewiring of energy metabolism, increases CSC populations and mammosphere formation in vitro and in vivo. The pharmacological or genetic inhibition of OGT induces a potent reduction of mammosphere formation, as well as CD44H/CD24L, ALDH+, and NANOG+ tumor-initiating cell populations in breast cancer cells38. These observations confirm that the inhibition of adaptive metabolic plasticity may serve as a therapeutic strategy to regulate tumor-initiating activity in breast cancer.Various authors have reported the involvement of microenvironmental stromal cells in cancer-associated metabolic changes supporting tumorigenic processes. Adaptive metabolic plasticity, i.e., tumor-initiating cell ability to switch between oxidative phosphorylation and glycolysis, depending on reactive oxygen species, hypoxia, and glucose availability in the tumor microenvironment, confers a survival advantage to malignant cells in breast cancer, thus representing a potential target for anti-cancer therapy39. A study in bevacizumab-resistant glioblastoma suggested that chemoresistance in cancer cells inside the hypoxic microenvironment occurs through: (i) metabolic reprogramming, with suppressed oxidative phosphorylation and upregulated glycolysis; (ii) perivascular invasiveness along remaining blood vessels in a VEGF- and neo-angiogenesis-independent manner; and (iii) enrichment of tumor-initiating stem cells residing in the perivascular niche close to residual blood vessels40. In addition, exosomes from glioma cells induced a tumor-like phenotype in MSCs by activating glycolysis41.Similarly, pancreatic cancer cells utilize “metabolic reprogramming”, through the enhancement of glycolysis with increased lactate production and glycolytic enzyme overexpression, to satisfy their energy demand and support malignant behaviors, despite a hypoxic and nutrient-deficient microenvironment42 showed that the expression of estrogen receptor (ER)-α, the target of endocrine therapies in breast cancer that is expressed by most metastatic breast cancer cells, is regulated by the BM microenvironment. In this study, the induction of estrogen receptor 1 (ESR1) mRNA and ER protein downregulation, through a mitogen- activated protein kinase (MAPK)-independent mechanism, was achieved by the treatment of breast cancer cells with conditioned culture media from either cancer-activated BM stromal cells or HS5 BM stromal cell line. In addition, thyroid hormones, which are well-established pro-tumorigenic players, may stimulate tumor growth and neovascularization in various solid cancers by activating MSCs through a non-classical integrin αvβ3 signaling43. Moreover, the EGF-like superfamily member EGFL6, playing an important role during embryonic development without any effect on wound healing, mediates a crosstalk between cancer and stromal cells to induce stemness and epithelial–mesenchymal transition (EMT)  in breast cancer cells in vitro, thus promoting tumor growth in vivo44.In 2019, a report from Lung and colleagues45. In addition, HSF1 stimulates tumor growth in nude mice and its expression significantly correlates with poor overall survival and prognosis in patients with oral squamous cell carcinoma46. Intracellular Notch1 signaling in CAFs inversely controls stromal regulation of the stemness and plasticity of CSCs in melanoma, acting as a molecular switch modulating tumor heterogeneity and aggressiveness47.CAFs play a pivotal role in cancer progression, partially through signaling molecules that may represent potential therapeutic strategies for cancer treatment. For instance, the overexpression of the potential prognostic factor, heat shock factor 1 (HSF1), promotes EMT, proliferation, migration, and invasion in Cal27 cells. The presence of CAFs expressing CD10 and GPR77 correlates with poor survival and chemoresistance in lung and breast cancer patients, and these CAFs supported cancer stemness and promoted cancer formation and chemoresistance in patient-derived xenograftsin situ hybridization to assess the role of stromal CAFs in the modulation of heterogeneity in pancreatic ductal adenocarcinoma (PDA) models48. The authors identified significant single-cell population shifts toward proliferative phenotypes and invasive EMT linked to MAPK and signal transducer and activator of transcription 3 (STAT3) signaling, which contributed to intratumoral heterogeneity in tumor glands and to differences in stromal abundance and clinical outcome. Furthermore, a study addressing the ability of mesenchymal HT1080 fibrosarcoma cell line to switch to amoeboid motility (migration plasticity) revealed that pharmacological or RNA interference (RNAi)-mediated downregulation of the Arp2/3 complex or decrease of adhesiveness to its substrate induced the transition from a lamellipodium-rich to a blebbing phenotype in fibrosarcoma cells, but not in normal subcutaneous fibroblasts49. Interestingly, still in this study, a significant fraction of fibrosarcoma cells expressing the blebbing phenotype exhibited stem cell-like features, such as increased efflux of Hoechst-33342 and CD133, Oct4, Sox2 and Nanog expression, and demonstrated an increased ability to switch to a bleb-rich amoeboid phenotype in three-dimensional (3D) collagen gels49.Strong evidence that stromal microenvironment shapes the intratumoral architecture in pancreatic tumors was shown by a study using single-cell RNA, protein analysis, and high-content digital imaging of RNA 51. For instance, human adipose-derived MSCs displayed repairing properties in damaged thymus following chemotherapy in mouse models of blood cancer52. Mice showed improvements in the thymic structure and functions, as shown by the proportion of circulating and splenic regulatory T (Treg) cells and the recovery of T-cell subpopulations.Various reports support the therapeutic potential of MSCs in cancer, also for repairing damaged tissues after chemotherapy53. Two microRNAs, i.e., small non-coding RNA molecules silencing post-transcriptional regulation of gene expression, associated with the capacity of MSCs to attenuate cancer growth were identified, namely microRNA 150 (miR-150) and miR-7. Similarly, human BM-MSC-derived exosomes overexpressing miR-34a inhibited glioblastoma development55. In another study, intra-BM but not systemic administration of BM-MSCs from healthy donors reduced tumor burden and prolonged survival of the leukemia-bearing mice54. In this study, the MSC senescence observed during disease progression was stopped and the BM microenvironment was restored, with functional recovery of host myelopoiesis and improvement of thrombopoiesis. Moreover, in a bioluminescence imaging study monitoring the effects of human umbilical cord-derived MSCs in mouse hepatoma tumor models with H7402 cell line, the MSC microenvironment effectively inhibited the growth of cancer cells56.In a study involving both human colorectal cancer cells and immunocompetent rat models of colorectal carcinogenesis, the treatment with BM-derived MSCs interfered with colon cancer progression. The effect was partially due to the modulation of the tumor microenvironmental immune effector cells, such as Tregs, CD8+ cells, and NK cells. In addition, there was evidence of Th17 cell activity restoration, macrophage reprogramming into regulatory cells performing phagocytosis with reduced production of proinflammatory cytokines, a 50% decrease in the infiltration rate of CD68+ cells, and a two-fold increase of CD3+ cells57. However, the identity of cancer-restraining CAFs remains poorly investigated, due to the lack of markers. Interestingly, a cell subpopulation with tumor inhibitory functions was isolated and characterized in a cancer metastasis microenvironment by using stromal cell lines derived from the central nervous system (CNS) metastasis of breast and lung cancer patients58. Interestingly, these cells were quite homogenous, expressed high levels of collagen, and displayed gene expression signatures of CAFs, MSCs, and EMT58. Mizutani and colleagues59 reported the glycosylphosphatidylinositol-anchored protein Meflin as a potential marker of cancer-restraining CAFs. These authors observed that the tissue infiltration of Meflin-positive CAFs correlated with a favorable patient outcome in PDA. By contrast, Meflin deficiency or low expression resulted in a markedly faster tumor progression in a PDA mouse model, and either the overexpression of Meflin in CAFs or the delivery of a Meflin-expressing lentivirus into the tumor stroma was sufficient to suppress the growth of xenograft tumors59. This new marker paves the way for isolation and further characterization of CAFs exerting anti-tumoral effects.Different studies, both clinical and in mouse models, suggest that there may exist at least two populations of MSC-derived CAFs, i.e., cancer-promoting CAFs, discussed already, and cancer-restraining CAFs61. Recently, Coffman and colleagues62 reported that ovarian carcinoma-associated MSCs, which are critical stromal progenitor cells promoting tumor cell growth, cancer stemness, and chemoresistance, arise from a process of tumor-mediated reprogramming of local tissue MSCs. This study also provided strong evidence that tumor-mediated MSC conversion is tissue- and cancer-type dependent, and requires tumor-secreted factors and hypoxia62. Breast tumor microenvironment transforms naive MSCs into tumor-forming cells in nude mice; in addition, MSCs pre-exposed to a conditioned medium or purified exosomes derived from breast cancer cells (MDA-MB-231) form a tumor-like mass rich in stromal tissue by 14 weeks when injected into mammary glands of nude mice63. Similarly, CCL5 secreted by classic Hodgkin lymphoma cells recruits MSCs and monocytes and enhances MSC proliferation and CCL5 secretion; conditioned medium from these MSCs increases tumor cell growth and monocyte migration64. Exosomes derived from chronic myeloid leukemia cells altered the cellular and immune-related properties of BM-MSCs and macrophages in vitro33. Moreover, the expression of gene signatures and mesenchymal shift in quiescent glioblastoma cells, a source of tumor recurrence in highly malignant glioblastoma, was observed following their interactions with niche microenvironment65. Reciprocal reprogramming of CSCs and associated MSCs may promote tumor progression in gastric cancer66. Consequently, unraveling the signaling molecules involved in pro-tumorigenic crosstalks between MSCs and tumor environment may lead to novel targets for inducing cancer regression and elimination.Early studies addressing the composition of the tumor microenvironment reported an atypical cellular and molecular microenvironment supporting carcinogenesis and chemoresistance67. MSCs promotes the progression of gastric cancer cells through the release of CXCL16, which activates STAT3-mediated expression of Ror1 in the cancer cells68. Dabbah and colleagues reported that microvesicles derived from BM-MSCs of MM patients increase the tumorigenicity of MM cells69. In this study, CD49d and CD29 integrin overexpression in MM-MSC microvesicles correlated with patient staging and response to treatment; the concomitant inhibition of these molecules resulted in reduced uptake of MM-MSC microvesicles , inhibition of MM cell signaling, expression of aggressiveness markers, and enhanced response to chemotherapy69. This study also suggested that the reciprocal interactions of malignant cells and MSCs in breast cancer microenvironment may result in the transformation of naive MSCs into cells capable of forming explants in nude mice. Notably, pre-metastatic niche in distant organs may be created, at least in part, by the transfer to stromal cells, such as peritoneal mesothelial cells (PMCs), fibroblasts, and endothelial cells, of tumor-derived extracellular vesicles secreted by tumor-associated macrophages (TAMs) into the blood70. STAT4 overexpression in gastric cancer cells makes normal fibroblasts acquire CAF-like features via activating the wnt/β-catenin pathway71. In addition, Guo and colleagues72 (in 2019) addressed the potential roles and mechanisms of long non-coding RNAs in CSC-like properties and EMT in non-small cell lung cancer (NSCLC) using Western blot, quantitative reverse transcription polymerase chain reaction (RT-PCR), colony formation, transwell migration, and wound healing assays in A549 and H1299 human NSCLC cell lines, L9981 and 95D highly metastatic cell lines, and NL9980 and 95C low-metastatic cell lines. These authors observed that knockdown of long non-coding RNA linc-ITGB1 inhibited the expression of various markers of cancer stemness and CSC formation by reducing the expression of the EMT-related transcription factor Snail. Overexpression of Snail reversed the inhibitory effects of linc-ITGB1 knockdown72.Interestingly, asporin, a factor secreted by MSCs following cellular interactions within the tumor microenvironment, alters the tumor microenvironment and inhibits MSC differentiation to drive metastatic progression through CD49d/CD29 signaling73 reported that even the decellularized ECMECM of BM-MSCs from MM patients was able to induce comparable pro-tumorigenic effects. A number of changes in microRNAs was shown affecting the MM phenotype and the activation of MAPK/TI, EMT, proliferation, and CXCR4, with a role for BM-MSC secretomes and microvesicles. On the other hand, the decellularized ECM of BM-MSCs from normal donors mediated anti-cancer effects, including a rapid and persistent decrease in MAPK/TI activation, proliferation, cell count, viability, migration, and invasion73. These authors also provided evidence for a synergism between the ECM and microvesicles in the modulation of MM cell response to chemotherapy as well as in the hierarchy and interdependence of MAPKs/TI/autophagy/phenotype cascade. In addition, extracellular vesicles released by monocytes from chronic myelomonocytic leukemia patients are sufficient to confer a procoagulant state through a tissue factor-dependent mechanism mediated by MSCs74.Emerging data strongly suggest that tumor extracellular matrix (ECM) and related factors contribute to the controversial role of stromal cells in the tumor microenvironment. For example, after showing that MM cells, cocultured with BM-MSCs, comodulated the phenotype of MM cells in an MAPKs/translation initiation (TI)-dependent manner, Ibraheem and colleaguesvia remodeling of the ECM of the BM microenvironment, and MMP-9 deficiency in the BM microenvironment reduces leukemia-initiating cells and prolongs survival of mice with BCR-ABL1-positive B-cell acute lymphoblastic leukemia (B-ALL)75. Similarly, senescent MSCs actively remodel the surrounding ECM to drive breast cancer cells to a more invasive phenotype76. Interestingly, 3D culture studies with cancer and stromal cells in ECM, incorporating multiplex quantitative analysis method, may reveal the major signaling molecules and mechanisms driving the pro- and anti-cancer interactions, providing new therapeutical targets78. Notably, in a recent study using a similar approach in hepatocellular carcinoma (HCC), cell repopulation of cirrhotic scaffolds showed a unique up-regulation of genes related to EMT and TGF-β signaling as well as a high concentration of endogenous TGF-β1 in comparison to healthy scaffolds and TGF-β1-induced phosphorylation of canonical proteins Smad2/379. This study characterized the inherent features of ECM micro-environment from human cirrhotic liver as key pro-carcinogenic components in HCC development. Similarly, MSCs cocultured with colorectal cancer cells showed increased invasiveness and proliferative abilities due to increased TGF-β1 and decreased p53 levels80. TGF-β1 promoted the migration and invasion of HCT116 and HT29 colorectal cancer cells, and induced the differentiation of MSCs into CAFs through a Janus kinase (JAK)/STAT3 signaling-dependent mechanism81. Long-term coculture of human MDA-MB-231 breast cancer cells with normal human MSCs was associated with the formation of 3D tumor spheroids in vitro, with a 14-fold enhanced expression of the breast tumor marker urokinase plasminogen activator (uPA)82.Matrix metalloproteinase-9 (MMP-9) produced by leukemia cells facilitates tumor progression 84. In addition, recently, stromal cells were reported as mediators of the pro-tumorigenic effects of hypoxia and tumor stiffness, which are known elements of the solid tumor microenvironment promoting tumor survival, progression, and metastasis. Osteopontin, a hypoxia-driven phosphorylated glycoprotein, may promote stem cell-like properties and EMT in pancreatic cancer cells by activating the integrin αvβ3-Akt/Erk-forkhead box protein M1 (FOXM1) signaling in a paracrine manner85. Microvesicles derived from human BM-MSCs support human osteosarcoma (U2OS) cell growth under hypoxia in vitro and in vivo through PI3K/AKT and HIF-1α-dependent mechanisms86. Similarly, hypoxic BM stromal cells-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT in an in vivo mouse syngeneic tumor model87. Moreover, exosomal miRNAs from hypoxic BM-MSCs promoted lung cancer cell metastasis via STAT3-induced EMT88 and hypoxia-conditioned MSCs promote HCC progression through yes-associated protein (YAP)-mediated lipogenesis reprogramming56, further suggesting that targeting the communication between MSC and cancer cells may be a potential target for anti-tumor therapy.Earlier reports suggested that hypoxia-inducible factor 1 (HIF-1) may link hypoxia, inflammation, and cancer89. A study addressing the end-stage myeloma cell mobilization from the BM into peripheral blood revealed that hypoxic BM niches, together with a pro-inflammatory microenvironment resulting from the interactions between tumor cells and BM stromal cells, induce an arrest in proliferation that forces tumor cells to circulate into the peripheral blood to seek other BM niches90. Hypoxia-induced EMT has been shown with a 17-gene panel aimed at assessing NSCLC prognosis91. Similarly, hypoxia-induced acquisition of CSC features in lung cancer cells occurs through CXCR4 activation92. In addition, the retention factor in the endoplasmic reticulum (RER1) enhances carcinogenesis and stemness of pancreatic cancer93. Finally, glioblastoma stem-like cell (GSC) phenotype, the worst prognostic marker of glioblastoma, persists partially due to the hypoxic microenvironment-dependent release of extracellular adenosine, thus promoting cell migration, invasion, and tumor recurrence through the activation of the A3 adenosine receptor (A3AR)95.Interactions of cancer cells and stromal cells in a hypoxic microenvironment drive EMT through NOTCH and c-MET signaling, and induce immunosuppressive response within the microenvironment in PDA, a fatal disease with limited response to both immunotherapy and cytotoxic chemoradiotherapy95. In addition, Saforo and colleagues96 described an in vitro cell culturing system incorporating elements of the in vivo lung environment, including physiological hypoxia (5% O2) and lung fibroblast-derived ECM. Through this culture system, a rapid expansion of stromal progenitors from patient’s lung tumor resections was achieved; these progenitor cells retained the secretion of factors associated with cancer progression, the expression of pluripotency markers, and the ability to enhance tumor cell growth and metastasis96.MSC-derived CAFs were reported as the link between biophysical forces and pro-metastatic signaling in colon cancer, as they respond to increased stiffness of the tumor microenvironment by the activation of the signaling mediated by TGF-β family members and activin A, a strong pro-metastatic cytokine99. Su and colleagues100 compared lean and obese mice grafted with prostate tumors and showed that obesity promotes EMT in cancer cells and tumor invasion into the surrounding fat tissue. In this study, adipose stromal cells induced EMT in prostate cancer cells and made them more migratory and chemo-resistant; by contrast, adipose stromal cell targeting suppressed both EMT and chemoresistance to docetaxel, cabazitaxel, and cisplatin chemotherapy in human prostate cancer cells100. Human adipose-derived MSCs promoted EMT in MCF7 breast cancer cells by cross-interacting with the TGF-β/Smad and PI3K/AKT signaling pathways, in a coculture system established to investigate the paracrine effects of MSCs on the migration and invasion potential of this aggressive breast cancer cell line101. In addition, a study in a xenograft model of early MM showed that bone niche switching toward a “fatty” marrow supports the development of malignant cells during carcinogenesis. In this study, MSCs mainly gave rise to adipocytes supporting tumor growth by increasing the survival and chemoresistance of malignant cells102.It is well established that fat tissue overgrowth in obesity promotes tumor progressionvia the recruitment of tumor-associated MSCs and the stimulation of angiogenesis pathways in endothelial cells through chemerin receptor 1 (CMKLR1), chemerin receptor 2 (GPR1), and CCLR2 signaling103.In addition, interestingly, various adipose-derived factors were reported to play a role in MSC-mediated pro-tumorigenic effects. For instance, adipokine chemerin is a major player in obesity-mediated support of cancer progression. This cell differentiation promoter and leukocyte chemoattractant factor was reported to promote the growth, proliferation migration, invasion, and metastasis of cancer cells 105.Early studies addressing the immunological hallmarks of MSCs in the tumor microenvironment revealed various molecular mechanisms through which MSCs modulate the immune response in the cancer microenvironment and indicated that it may be possible to convert the microenvironment from immunosuppressive to immunostimulantIn vitro studies support the anti-tumor effects of MSCs, but these effects can be markedly reduced in vivo by the tumor-trophic properties of these cells and the direct cell-to-cell integration with tumor stromal elements. A score of recent reports suggests promising strategies for reprogramming microenvironmental cells to mediate only anti-cancer effects. For instance, unlike conditioned medium from human adipose MSCs, eicosapentanoic acid-treated adipose MSCs reduce mRNA levels of the tumor-associated genes FASN, STAT3, and cIAP-2 in MDA-MB-231 and MCF-7 breast cancer cell lines, with marked decreases in their glycolysis, inflammation, and motility in vivo106.107 proposed the encapsulation of MSCs from the perinatal tissue with the sodium alginate biomaterial. The team isolated the 3D structure from the microenvironment and observed that the encapsulated MSCs displayed: (i) increased proliferation with expression enhancement of pluripotency genes, EMT, immune-modulation, and angiogenesis; (ii) increased secretion of VEGF, TGF-β, TNF-α, IFN-γ, IL-10 and IL-6, and IL-3β; (iii) and increased expression of the tumor invasion suppressor protein E-cadherin107. Furthermore, treatment of CSCs derived from MDA-MB-231 and MCF7 breast cancer cell lines with encapsulated MSCs lowered CSC viability and migration, with downregulation of markers related to angiogenesis, EMT and proliferation, and upregulation of Wnt antagonists secreted frizzled-related protein 4 (sFRP4) and DKK1107.Mandal and colleagues108. Engineered human placenta-derived MSCs, armed with a double fusion gene containing the herpes simplex virus truncated thymidine kinase and firefly luciferase, inhibited the tumorigenesis mediated by the HT29 colon cancer cell line in nude mice114. Treatment with 5-azacytidine restored IL-6-increased production in MSCs from myelodysplastic patients116.Prolonged culture of heterogeneous prostatic CAFs resulted in a marked decrease in the expression of proliferative endothelial cell surface marker endoglin (CD105), as compared to short-culture CAFs, and loss of their tumor expansion potential and heterogeneity in 3D cultures and patient-derived xenograft tissues117. In this in vitro study, low-dose photodynamic therapy: (i) induced the reorganization of MSC cytoskeleton, with a decrease in cell motility; (ii) induced the inhibition of glycogen synthase kinase-3 (GSK-3) and the activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) signaling in MSCs; (iii) significantly upregulated the secretion of VEGF-A, IL-8, plasminogen activator inhibitor-1 (PAI-1), MMP-9, and other proangiogenic factors by MSCs; (iv) dramatically inhibited the secretion of pro-tumorigenic macrophage infiltration marker CCL2 (MCP-1) by MSCs and decreased MSC viability and immunogenicity when cocultured with lymphocytes. In another study, MSCs loaded with a photosensitizer successfully shipped these nanoparticles into lung cancer tumor sites, enhancing the effects of photodynamic therapy in vivo110. In addition, irradiated endothelial cells decreased the malignancy of liver cancer cells in a coculture system using medium conditioned with endothelial cells, thus suggesting that irradiated endothelial cells are key players in the therapeutic effects of radiotherapy115. In addition, various flavonoids and non-flavonoid polyphenolic compounds from medicinal plants alleviate multidrug resistance in breast, prostate, lung, and colorectal cancer with survival benefits in patients, through their antioxidant capacity, the modulation of inflammatory responses, and the inactivation of oncogenes with the inhibition of survival, angiogenesis, proliferation, and metastasis118 (Table 1).A study addressing the response of human MSCs to low-dose photodynamic therapy revealed that this treatment may increase MSC immunogenicity and promote angiogenic potential119. MSCs in fact induced pro-tumorigenic effects in the presence of 5637 bladder cancer cell line and anti-cancer effects in the presence of HB-CLS-1 bladder cancer cell line, and thus the effect of crosstalk between MSCs and bladder cancer cells remains unclear. Similarly, in a study assessing how breast cancer cells from different stages of the metastatic cascade convert MSCs into tumor-associated MSCs, only MDA-MB-231 breast cancer secretomes, but not MCF-7 cells and sublines isolated from bone, lung, and brain metastases, converted MSCs into tumor-associated MSCs in bioengineered 3D microenvironments120. Altogether, these findings suggest that MSCs from the tumor microenvironment are pre-conditioned to mediate pro-tumorigenic effects on cancer cells, and that impeding this pre-conditioning or re-conditioning MSCs may warrant anti-cancer effects in the tumor microenvironment .In numerous reports, it is not clear whether the pro-tumorigenic rather than anti-cancer role of MSCs is dictated by a cell line-specific event. In a coculture study with bladder cancer cells displaying stem cell-like properties (CD133+) and adipose-derived MSCs, the latter cells produced soluble mediators that: (i) increased the phosphorylation of molecules involved in cancer progression and drug resistance, such as p70 S6K, ERK1/2, and AKT1/2/3 in CD133+ cells from 5637 cell line; but also (ii) decreased the phosphorylation of those PI3K/Akt and MAPK signaling molecules in CD133+ cells from HB-CLS-1 cell linein vitro and in vivo studies. Quach and colleagues121 reported that while the inhibition of the glypican-1 (GPC-1) prostate cancer biomarker decreases cell growth and migration in vitro of the aggressive prostate cancer cell line PC-3, quite surprisingly it increases the PC-3 tumor size in NCr nude mice xenografts. Also surprisingly, it increases cancer cell proliferation and migration in aggressive prostate cancer cell line DU-145 cells, suggesting that GPC-1 accounts for among the factors that drive a cell line-dependent response to stromal cells. In addition, in the same study, the decreased cell growth observed in GPC-1 knockdown PC-3 cells was rescued by coculturing the cells with MSCs and CAFs. Further, treatment of these stromal cells with tumor-conditioned media from PC-3 cells transfected with GPC-1 short hairpin RNA (shRNA) increased the expression of ECM components, endocrine and paracrine biomolecules, and migration markers121. Moreover, despite in vivo observations suggesting the ability of this signaling pathway to induce drug resistance and influence the ability to form metastasis via induction of EMT in pancreatic cancer, the activation of insulin-like growth factor (IGF)/IGF-I receptor (IGF-IR) signaling by stromal cells failed to induce EMT in cultures with MiaPaCa-2, AsPC-1, Capan-2, BxPC-3, and Panc1 pancreatic cancer cell lines122.Experimental evidence supports the idea that stromal cell effects and origin may explain the discrepancies amongst data from in vitro proliferation and migration of cancer cells through ERK signaling123. We hypothesize that these effects may be due to differences in the origin of MSCs, as in this study human umbilical cord MSCs, and not BM or adipose-derived MSCs were used. As a further support of this hypothesis, a comparative study of subcutaneous and visceral adipose-derived MSCs revealed various functional similarities and differences, despite similar surface markers124. Notably, visceral MSCs secreted higher levels of inflammatory cytokines  and had a more active sonic hedgehog pathway than subcutaneous MSCs. Fetal and adult lung MSCs possess lung-specific properties, unlike BM-MSCs22. However, a study profiling the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs), harvested at different passages and stimulated with or without inflammatory cytokines, revealed that UC-MSCs are a well-organized population with limited heterogeneity, as compared to other MSC types125.Considering that MSCs promoted anti-cancer effects in most reports, as discussed in the Cancer-restraining CAFs section, surprisingly, treatment of MDA-MB-231 and MCF-7 human breast cancer cells with medium containing extracellular vesicles promoted the in vitro and in vivo. These discrepancies should be taken into account in the design of future studies and interpretation of results.The available data clearly support that stromal cells normally have anti-cancer effects, and that reprogramming by cancer cells in the tumor microenvironment induces their switch to pro-tumorigenic activities, thus suggesting that targeting the tumor microenvironment could be a promising therapeutic strategy in cancer. A growing number of reports suggest the possibility to reprogram stromal cells to maintain or revert back to anti-cancer effects. Interestingly, cancer-restraining stromal cells have been identified in the microenvironment, and a marker was reported. Future studies characterizing the origin of these cells may provide clues to how they can be exploited for anti-cancer therapy. The emerging data shed light on the origin of previous controversies on the roles of stromal cells in the tumor microenvironment. Notably, MSCs have varying effects on cancer cell lines of different origins, and MSCs from different origins have different effects on cancer cell cocultures. On the other hand, the tumor microenvironment induces complex signals that affect how stromal and cancer cells respond to soluble factors


Colonic varices are rare among ectopic varices. A previous report demonstrated that once bleeding from colonic varices occurs, it can be fatal. Several treatments for colonic varices exist, including surgical, endoscopic, and endovascular treatments; however, management of colonic varices has not been standardized. For colonic varices, minimally invasive therapies would be desirable. Balloon-occluded retrograde transvenous obliteration (B-RTO) is one of the treatment options for colonic varices to prevent their rupture. Two cases of successful conventional B-RTO for these varices have already been reported. However, B-RTO using coil-assisted retrograde transvenous obliteration II (CARTO-II) procedure for these varices has not been reported.A 71-year-old male patient had liver cirrhosis caused by hepatitis C virus infection. A varix was located at the ascending colon, which was coincidentally found on colonic endoscopy. Contrast-enhanced computed tomography (CT) showed that the feeder vein was the ileocolic vein and that the main draining vein was the right renal vein. Physicians concluded that treatment was required to avoid the risk of death from massive bleeding due to varix rupture. However, endoscopic and surgical treatments were difficult due to the anatomical location of the varix and the high risk of operative compilations, respectively. This ascending colonic varix was treated by balloon-occluded retrograde transvenous obliteration (B-RTO) using coil-assisted retrograde transvenous obliteration II (CARTO-II) procedure via the right renal vein. There were no complications during the procedure and no recurrences for 36 months during long-term follow-up.CARTO-II can be one of the effective treatment techniques for ascending colonic varices. Ectopic varices are defined as large portal-systemic venous collaterals occurring anywhere in the intestinal tract other than the esophageal region  was inserted into the right renal vein via the right femoral vein under local anesthesia. Subsequently, a 3.3-Fr coaxial balloon catheter with a 10-mm balloon  was inserted into the draining vein near the varix via the right renal vein through the guiding sheath. Then, balloon-occluded retrograde transvenous venography (B-RTV) was performed. When B-RTV showed contrast medium retention in the ascending colonic varix Fig. . After fAfter CARTO-II, CT showed that obliteration was complete and successful without postoperative complications in this case Fig. d. There This is the first report of B-RTO using CARTO-II for an ascending colonic varix. B-RTO has been performed for various ectopic varices, such as duodenal, stomal, and small intestinal varices, as well as colonic varices   is one of the treatment options for varix therapy (Nakai et al. An endoscope may not always reach the ascending colon due to its anatomical location (Hitoshi and Fukuji In conclusion, this successful case suggests that B-RTO using CARTO-II can be one of the effective treatment techniques for ascending colonic varices.


The development of improved threading algorithms for remote homology modeling is a critical step forward in template-based protein structure prediction. We have recently demonstrated the utility of contact information to boost protein threading by developing a new contact-assisted threading method. However, the nature and extent to which the quality of a predicted contact map impacts the performance of contact-assisted threading remains elusive. Here, we systematically analyze and explore this interdependence by employing our newly-developed contact-assisted threading method over a large-scale benchmark dataset using predicted contact maps from four complementary methods including direct coupling analysis (mfDCA), sparse inverse covariance estimation (PSICOV), classical neural network-based meta approach (MetaPSICOV), and state-of-the-art ultra-deep learning model (RaptorX). Experimental results demonstrate that contact-assisted threading using high-quality contacts having the Matthews Correlation Coefficient (MCC) ≥ 0.5 improves threading performance in nearly 30% cases, while low-quality contacts with MCC <0.35 degrades the performance for 50% cases. This holds true even in CASP13 dataset, where threading using high-quality contacts (MCC ≥ 0.5) significantly improves the performance of 22 instances out of 29. Collectively, our study uncovers the mutual association between the quality of predicted contacts and its possible utility in boosting threading performance for improving low-homology protein modeling. Template based modeling, one of the most accurate approaches for structure prediction, utilizes homologous structural templates deposited in Protein Data Bank (PDB)2 to address this problem. In the absence of close homology, remote homology detection technique known as threading is one of the most reliable and robust strategies for predicting the 3D structure of a query protein5. Various threading methods have been developed during the last decade18 with noteworthy successes. Alongside, steady growth in sequence and structure2 databases in conjunction with the rapid development of statistical and computational methods for co-evolutionary sequence analysis coupled with deep learning have resulted in substantial progress in sequence based prediction of residue-residue contact information34. Consequently, residue-residue contact or distance has become valuable new information to explore in boosting the accuracy of protein threading. State-of-the-art threading methods such as EigenTHREADER35, map_align36 and DeepThreader37 recently revisit the idea of recognizing remote homology by incorporating inter-residue contact map or distance map information into threading. Specifically, Jones and coworkers developed EigenTHREADER that integrates contact information predicted by MetaPSICOV20 with a standard threading technique. Baker and coworkers developed map_align, which integrates co-evolutionary contacts (Gremlin38) with a threading-based method and subsequently use double dynamic programming39. Xu and coworkers developed DeepThreader, which integrates sequential features with inter-residue distance information.The problem of predicting the accurate three-dimensional (3D) structure of a protein from its amino acid sequence, known as the protein structure prediction problem, remains open40. Specifically, we have integrated residue-residue contact maps predicted by RaptorX43, one of the most accurate contact prediction methods, with structural and sequential information such as profiles, secondary structure, solvent accessibility, torsion angles (psi and phi), and hydrophobicity for contact-assisted threading. Experimental results have shown that the inclusion of contact information attains statistically significantly better performance compared to contact-free threading method when everything else remains the same, demonstrating that the inclusion of contact information in protein threading is a promising avenue for improving the performance of threading method. Furthermore, in a head-to-head performance comparison utilizing the same RaptorX-derived contact maps to guarantee a fair comparison, our method has successfully outperformed state-of-the-art contact-assisted threading methods EigenTHREADER and map_align, indicating our method as one of the best contact-assisted protein threading protocols. However, it is not clear how the quality of a predicted contact map affects contact-assisted threading. Nor it is clear whether contact-assisted threading with low-quality contact maps is as advantageous over pure threading as contact-assisted threading with high-quality contact maps such as those predicted from RaptorX. Finally, in the presence of competing contact maps of comparable qualities predicted by state-of-the-art contact predictors, is there any advantage of using one over the other in terms of improved threading performance? While assessing the efficacy of contact maps for low-homology protein modeling requires a head-to-head comparison between contact-assisted threading and contact-free pure threading, neither EigenTHREADER nor map_align can perform threading in a contact-free mode. Our method, on the other hand, can be seamlessly customized to perform contact-assisted or contact-free threading modes, enabling the evaluation of the utility of contact maps for remote homology modeling.Very recently, we have developed a new contact-assisted threading method by successfully integrating accurate residue-residue contact information for improved protein threadingTo evaluate the significance of contact maps in low-homology protein modeling, here we systematically investigate the impact of the quality of predicted contacts on the accuracy of contact-assisted threading by employing our newly developed contact-assisted threading method over several datasets. First, we analyze predicted contact maps from RaptorX and three other complementary methods having a wide range of qualities of their predicted contacts based on different contact map evaluation criteria to objectively evaluate how to select the most informative contact map. Then, we integrate the predicted contact maps from these contact predictors one by one into our contact-assisted threading method to examine the impact of each predicted contact map on the threading performance and compare them with a baseline threading algorithm that does not utilize contact information as well as RaptorX-assisted threading. Finally, we compare the performance of our contact-assisted threading by incorporating comparable-quality contact maps predicted by the top two officially ranked contact predictors from the recently concluded 13th Critical Assessment of protein Structure Prediction (CASP13) experiment to further study the impact of the quality of contacts in threading performance. Collectively, our study unravels the mutual association that exists between the quality of a contact map and the performance of contact-assisted threading.40, is an iterative query – template alignment approach where query-template alignments are performed by a Needleman-Wunsch global alignment algorithm44. The threading scoring function consists of close and distant sequence profiles, secondary structure, solvent accessibility, structure profile, torsion angles, and hydrophobicity match based on which a normalized alignment score or Zscore is calculated for ranking the templates.Our newly-developed contact-assisted threading method, described inCα or Cβ atoms of the residue pairs. Contact Map Overlap (CMO) finds the similarity between two contact maps, where the higher CMO score indicates that a higher similarity between the two comparing contact maps. Al-Eigen45, one of the state-of-the-art CMO methods, computes an overlap between two input contact maps and gives a score between  with higher score indicating better agreement of contact maps. We integrate CMO score returned from Al-Eigen into our threading method for selecting the best-fit template by formulating the final score as discussed in40. After identifying the best-fit template, the query-template alignment is used to copy the coordinate of the aligned residues from the template to build the final 3D model of the query protein. Please refer40 for further details about our method and its scoring function.Residue-residue contact map, which is a binary, square, symmetric matrix, is a two-dimensional representation of protein’s 3D structure. A contact indicates that the spatial distance between a pair of residues is less than a given distance threshold, typically set at 8 Å, between the https://zhanglab.ccmb.med.umich.edu/library/46.We use a representative non-redundant library of templates containing 70,670 templates, collected from: 19, which contains 150 single chain, single domain proteins. In order to test the impact of different types of contact maps in the performance of contact-assisted method, we choose predicted contact maps from four complementary methods having a wide range of qualities of predicted contacts including (i) mean field direct coupling analysis (mfDCA)23, (ii) sparse inverse covariance estimation method (PSICOV)19, (iii) classical neural network-based meta approach (MetaPSICOV)20, and (iv) state-of-the-art ultra-deep learning model (RaptorX)43. Here, we give a brief introduction of each contact predictor. mfDCA, an advanced formulation of direct coupling analysis (DCA), is a statistical inference framework used to infer direct co-evolutionary couplings between pair of residues in multiple sequence alignments. Another Evolutionary Coupling Analysis (ECA) technique, PSICOV, uses sparse inverse covariance estimation for contact prediction. Although ECA methods are useful for predicting long-range contacts in the presence of a large number of sequence homologs, their accuracy is substantially poor if the number of sequence homologs is low47. In recent years, machine learning or deep learning-based methods boost the accuracy of contacts. One such contact predictor, MetaPSICOV, a meta predictor, which uses a two-stage neural network by combining outputs of several ECA classifiers. It was ranked as one of the best contact predictors in CASP11 and CASP 1248. Another contact predictor powered by deep learning, RaptorX, incorporates the entire protein ‘image’ as a context for prediction by utilizing a Residual Convolutional Neural Network, or ResNet. It was ranked as one of the best contact predictors in CASP12 and CASP1328.Our first benchmark dataset is the PSICOV150 dataset22 to obtain contact-maps predicted by mfDCA. Since the contact likelihood scores of mfDCA predicted contact maps are not normalized in the range , we normalize contact likelihood scores by dividing each score by the maximum likelihood score of a given predicted contact map. PSICOV and MetaPSICOV contacts are obtained directly from the MetaPSICOV benchmark dataset20. RaptorX contacts are collected by submitting jobs to the RaptorX online server (http://raptorx.uchicago.edu/ContactMap/43). Residue pairs with contact likelihood scores <0.5 are excluded to reduce noise in all predicted contact maps. To make a fair performance comparison, we use the same template library for all competing methods by excluding templates with sequence identity >30% to the query protein to remove close homologs. It should be noted that, unlike other contact predictors, RaptorX fails to predict contacts for two targets namely: 1tqhA and 1hdoA. We, therefore, consider 148 targets for the current benchmarking.We use the FreeContact package49 to test the impact of using comparable-quality contact maps in the performance of our contact-assisted method. In CASP13, the contact prediction category is heavily dominated by the latest breakthroughs in deep learning technologies. For example, G498 (ranked 1) or RaptorX-Contact, developed by Xu and coworkers, has attained the top performance since CASP12. It predicts residue-residue contacts using an ultra-deep learning model. It is worth mentioning that we also use RaptorX predicted contacts for our previous study40 as well as for benchmarking on PSICOV150 dataset for this current work. The second-ranked contact predictor, G032 or TripletRes, developed by Zhang and coworkers34, is implemented by a deep residual fully convolutional neural network with evolutionary coupling features from deep multiple sequence alignment.Next, we benchmark on CASP13 dataset officially released in December 2018. We consider 20 full-length targets in a total of 32 domains for which CASP organizers released experimental structures so far. We consider the top two officially ranked contact predictors in CASP1340.For CASP13 benchmarking, the template library is curated before CASP13 started on May 1, 2018, which contains 69,041 template structures. For a fair comparison, we use the same template library for all competing methods. We have downloaded the predicted contact maps from the official website of CASP and subsequently exclude residue pairs with contact probability <0.5 from all predicted contact maps to reduce noise. It is also worth mentioning all residue pairs of a predicted contact map with contact likelihood of at least 0.5 with minimum sequence separation of 6 residues are considered for all experiments51. Precision is the percentage of correctly predicted contacts, c), ij represents the true distance of an incorrectly predicted pair of contacts. Spread is calculated by: c represents the number of true contacts, Ti is a true contact, and We use the following evaluation measures to evaluate predicted contact maps: precision, coverage, mean false positive error, spread, and Matthews correlation coefficient (MCC)52 is used to evaluate the quality of the predicted 3D structure of query proteins with respect to the native  structures. The value of TM-score lies in the range  for which RaptorX fails to predict contact maps. As shown in Table To objectively evaluate the most informative contact map, we compare the performance of each predicted contact map from different perspectives using various contact evaluation measuresAs a representative example, we present two case studies on targets 1aapA (56 residues) and 1dsxA (87 residues) to illustrate mutual comparisons between precision and MCC, and to substantiate how MCC is more balanced evaluation measure for predicted contacts. In Fig. RaptorX-assisted threading) and moderate-quality contacts from MetaPSICOV (referred to as rrMetaPSICOV-assisted threading) outperform contact-free pure threading method (referred to as pure threading) serving as a control in terms of the accuracy of the top ranked predicted models. Considering TM-score of top ranked models, RaptorX-assisted threading method delivers the best performance by achieving a mean TM-score of 0.66, which is 0.03 TM-score points more than that of baseline threading method, whereas the mean TM-score improvement reaches to 0.01 for MetaPSICOV-assisted threading method compared to baseline threading method. Moreover, 80.4% and 77.7% of the time RaptorX-assisted threading method and MetaPSICOV-assisted threading method predict the correct fold (TM-score >0.5), respectively, as opposed to 75.7% of baseline threading method. We also perform T-Test to examine whether the performance boost attained by contact-assisted threading work using high-quality RaptorX contacts and moderate-quality MetaPSICOV contacts over baseline threading method are statistically significantly better. Compared to baseline threading method, RaptorX-assisted threading method is statistically significantly better at 95% confidence level with a p-value of 0.0001. However, MetaPSICOV-assisted threading method improves the threading performance compared to baseline threading method, but the improvement is not statistically significant at 95% confidence level with a p-value of 0.07  performance with respect to pure threading method by 0.04 and 0.05 TM-score, respectively, in terms of the accuracy of top ranked predicted models. In finding correct overall folds, the performance of mfDCA-assisted threading method and PSICOV-assisted threading method drop by around 4% and 6%, respectively, compared to baseline threading method. The deterioration of performance of contact-assisted threading method using low-quality contact maps mfDCA and PSICOV are also statistically significant with p-values of 5.8e-08 and 2.9e-05, respectively with respect to baseline threading method  and RaptorX-assisted threading (referred to as rrRaptorX-assisted threading method) in terms of TM-score of top ranked model, where majority of the points (>70%) are below the diagonal line, RaptorX-assisted threading clearly outperforms mfDCA-assisted threading by a large margin. We see almost a similar trend in Fig. PSICOV) with RaptorX-assisted threading method. Around 67% points are below the diagonal line, which demonstrates the superior performance of RaptorX-assisted threading over PSICOV-assisted threading. In Fig. MetaPSICOV) while other using the high-quality RaptorX contacts. Around 28% more points are below the diagonal line, which illustrates the positive influence of higher-quality contact maps (RaptorX) for improved threading performance. In Fig. mfDCA-assisted threading and rrPSICOV-assisted threading respectively), the highest peak of RaptorX-assisted threading (referred to as rrRaptorX-assisted threading) is larger as well as skewed towards the higher TM-score side, indicating RaptorX-assisted threading finds more correct folds compared to others. Similarly, Fig. MetaPSICOV-assisted threading) and RaptorX-assisted threading, where the highest peak of RaptorX-assisted threading is still larger than MetaPSICOV-assisted threading as well as skewed towards the higher accuracy (right) side, illustrating the performance boost attained by contact-assisted threading method using the high-quality contacts (RaptorX) over moderate-quality contacts (MetaPSICOV). Overall, high-quality contacts predicted from RaptorX leads to statistically significantly better threading performance compared to that attained from inferior-quality contacts predicted from other methods.Since RaptorX-assisted threading method delivers the best threading performance we compare the performance of other three contact-assisted threading methods one by one against RaptorX-assisted threading acting as control. In Fig. Fig. 19 of 118 residues. Fig. A representative example sheds some light on the impact of diverse quality of contacts on threading performance, as shown in Fig. To further study the effect of the quality of contacts in threading performance over challenging CASP13 targets, we employ contact-assisted threading using the top two officially ranked contact maps on CASP13 dataset, consisting of 20 full-length targets (and 32 domains) officially released so far with native structures, the same template library and the same nr sequence database, curated before CASP13 started on May 1, 2018, are used by all competing methods. For each target, we make the prediction for the full sequence without utilizing any domain information. After the prediction phase, threading performance at the domain level is evaluated using the domain definitions provided by the official CASP13 assessors.p-value of 0.001 (and 0.0002) and 0.006 (and 0.0008), respectively, for full-length (and domain level) targets. Additionally, Supplementary Fig. Table 40. However, the nature of the interdependence between the quality of contact maps and contact-assisted threading performance remains elusive. Here, we present a large-scale analysis to study their mutual association by employing contact-assisted threading using contact maps of diverse qualities predicted from various contact predictors ranging from pure co-evolutionary methods (mfDCA and PSICOV) to hybrid approaches that combine sequence co-evolution and machine learning such as classical neural network (MetaPSICOV) and ultra-deep learning model (RaptorX). Experimental results demonstrate that contact-assisted threading method using high-quality RaptorX contacts and moderate-quality MetaPSICOV contacts outperform the baseline contact-free threading, whereas, low-quality contacts predicted from mfDCA and PSICOV deteriorate the threading performance compared to the baseline pure threading method. Contact-assisted threading with the best-quality contacts (RaptorX) delivers the best threading performance that is statistically significantly better compared to contact-free threading, demonstrating that accurate (MCC ≥ 0.5) residue-residue contact information is highly effective in boosting threading performance as opposed to low-quality (MCC < 0.35) contact information. This holds true even on the recently concluded CASP13 dataset, where contacts with MCC ≥ 0.5 lead to improved threading performance. Collectively, our study shows that contact-assisted threading is effective in the presence of high-quality (MCC ≥ 0.5) contact maps – indicating an evolving new direction for improved protein threading that is likely to mature further with future advancements in contact prediction methods.Protein threading represents one of the most successful approaches for modeling protein 3D structures from sequences, particularly when close homologous structural templates cannot be easily detected. Emerging methods in protein co-evolution coupled with deep learning have shown promise in sequence-based prediction of protein residue-residue contact maps, which are valuable source of information that can facilitate further progress in protein threading. Very recently, we have successfully incorporated contact maps to boost the accuracy of protein threading, demonstrating contact-assisted threading as a promising avenue for remote-homology protein modelingSupplementary Information.


YWHAZ is frequently up-regulated in multiple types of cancers and acts as an oncogene in a wide range of cell activities including cell growth, cell cycle, apoptosis, migration, and invasion. Moreover, YWHAZ was reported to be regulated by microRNAs (miRNAs) or long non-coding RNAs and exerted its malignant functions by targeting downstream molecules like protein kinase, apoptosis proteins, and metastasis-related molecules. Additionally, YWHAZ may be a potential biomarker of diagnosis, prognosis and chemoresistance in several cancers. Targeting YWHAZ by siRNA, shRNA or miRNA was reported to have great help in suppressing malignant properties of cancer cells. In this review, we perform literature and bioinformatics analysis to reveal the oncogenic role and molecular mechanism of YWHAZ in cancer, and discuss the potential clinical applications of YWHAZ concerning diagnosis, prognosis, and therapy in malignant tumors.YWHAZ  is a central hub protein for many signal transduction pathways and plays a significant role in tumor progression. Accumulating evidences have demonstrated that  Using UALCAN database YWHAZ in tumor tissues and adjacent tissues from nine types of high-morbidity cancers and observed that YWHAZ was significantly increased in breast carcinoma (BRCA), colon adenocarcinoma (COAD), esophagus carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous carcinoma (LUSC) and stomach adenocarcinoma (STAD) (p < 0.0001). However, in prostate adenocarcinoma (PRAD) and rectum adenocarcinoma (READ), there was no significant difference in the expression of YWHAZ between cancer tissues and adjacent tissues Figure .Growing researches have reported that YWHAZ in 53 pairs of hepatocellular carcinoma (HCC) tissues and adjacent tissues and found that YWHAZ was significantly up-regulated in HCC tissues YWHAZ mRNA expression was higher in eight liver cancer cell lines than normal liver cell line YWHAZ in 50 HCC tissues and 374 HCC tissues respectively from The Cancer Genome Atlas (TCGA) database We previously examined the mRNA level of HIF-1α) Previously, we performed gain- and loss-of-function experiments in liver cancer cells, demonstrating that YWHAZ silencing decreased cell proliferation, clonogenicity, migration/invasion and induced G2 arrest and apoptosis, while YWHAZ up-regulation led to the opposite MiR-451 was down-regulated in colon cancer, and its expression was inversely correlated with YWHAZ, which promoted cell growth through suppression of the nuclear accumulation of FoxO3 Li Y et al. observed that the mRNA and protein levels of YWHAZ were both increased in 46 colorectal cancer (CRC) tissues by qRT-PCR and IHC miR-375 and luciferase reporter indicated that miR-375 targets the 3′ UTR of YWHAZmiR-375-induced apoptosis by caspase-3/ caspase-7 activation and promoted autophagy by PI3K/AKT/mTOR signaling pathway Guo F et al. reported that the mRNA and protein level of YWHAZ were higher in four gastric cancer (GC) cell lines Deng Y et al. reported that YWHAZ mRNA and protein expression was significantly higher in 152 non-small cell lung cancer (NSCLC) tissues compared to 30 noncancerous lung tissues by qRT‐PCR and IHC In vitro, proliferation, migration/invasion and EMT were enhanced in lung cancer cells overexpressing YWHAZ in vivo, Chen CH et al. performed three approaches: 1) YWHAZ-cell and control-cell were subcutaneously implanted into the dorsal regions of severe combined immunodeficiency (SCID) mice; 2) YWHAZ-cell and control-cell were injected directly into the circulation of SCID mice to bypass the initial steps of local invasion and intravasation; 3) YWHAZ-cell and control-cell were orthotopically injected into one lobe of SCID mouse lung YWHAZ in cancer tissues compared with adjacent normal tissues YWHAZ protein expression was assessed by IHC in 139 BRCA tissues and was found to be higher in 45% of BRCA specimens in vivo studies, YWHAZ overexpression in FVB mice accelerated the progression of mammary tumors through EMT, angiogenesis promotion and apoptosis inhibition YWHAZ siRNA-treated cells compared with siRNA-control cells In BRCA, increased YWHAZ expression had been reported to induce anchorage-independent growth, malignant transformation of cancer cells, and resistance to apoptosis via inhibition of the mitochondrial apoptotic pathway YWHAZ mRNA levels showed consistency with protein level In prostate cancer, YWHAZ protein expression was observed to be significantly higher in tumorigenic/metastatic prostate cell lines compared with non-tumorigenic cell line and higher in 50 of 90 prostate cancer tissues than in benign prostate tissues YWHAZ by siRNA effectively reduced cell growth and proportion of cells in the S/G2 phases, while increasing the proportion of cells in the G0/G1 phase and enhancing sensitivity to topotecan in both drug-resistant and sensitive AML cells In acute myeloid leukemia (AML), YWHAZ protein expression was increased in 29 AML patients compared with 24 healthy donors YWHAZ is frequently up-regulated in various cancers, functioning as an oncogene by promoting the malignant phenotype of cancer cells, particularly through acceleration of migration and invasion.In summary, YWHAZ was a downstream target of miR-375 and YWHAZ expression was negatively correlated with miR-375 in HCC miR-375 resulted in decreased YWHAZ, subsequently accelerating caspase-related apoptosis in gastric carcinoma and repressing telomerase activity in HPV-associated cancers YWHAZ expression was enhanced by miR-451 down-regulation, subsequently regulating a series of cell activities, including cell proliferation, survival, apoptosis and endocrine chemoresistance miR-451/high YWHAZ expression was observed to promote cell proliferation and inhibit apoptosis through AKT targeting in AML and activate growth factor receptors and kinases  involved in endocrine resistance in breast cancer YWHAZ via high miR-451 expression greatly reduced cell proliferation and growth and induced cell-cycle arrest alongside apoptotic cascade in breast cancer YWHAZ expression via miR-451 activity inhibited cell growth in colorectal cancer through nuclear accumulation of FoxO3 miR-22 and was observed to subsequently reverse invasive phenotype of HCC cells through repression of YWHAZ-mediated AKT phosphorylation YWHAZ could be negatively regulated by miR-30c in cervical cancer, by miR-544 in breast cancer, and by miR-613 in HCC MiRNAs are small, non-coding RNAs of 20-22 nucleotides in length, which are considered to be vital components of gene regulation as important as transcription factors YWHAZ by targeting miRNAs LUCAT1 was negatively correlated with miR-134-5p and miR-134-5p was negatively related with YWHAZLUCAT1 inhibited YWHAZ expression, which can be reversed by miR-134-5p inhibitor SNHG14, acting as a miR-206 sponge and decreasing its expression, increased YWHAZ expression in cervical cancer LINC00858 regulated YWHAZ by inhibiting miR-22-3p in colorectal cancer Recently, long non-coding RNAs, more than 200nt and involved in multiple cell processes, are emerging as competing endogenous RNAs to regulate The 14-3-3 family interacted with a diverse range of cell signaling proteins by binding to an amphipathic helix and activating it through phosphorylation Pro-apoptotic proteins Caspase-3 and Bax were increased in YWHAZ-depleted liver cancer stem-like cells (CSCs) TGF-β) functions as a tumor suppressor in premalignant cells but, interestingly, as a metastasis promoter in cancer cells YWHAZ destabilized p53 and stabilizes Gli2, promoting TGF-β-induced bone metastasis TGF-β target genes and TGF-β1 in mesenchymal HCC cells, which is essentially required for Axl-mediated cell invasion ErbB2, a receptor tyrosine-protein kinase, was overexpressed in approximately 20%-30% of BRCA and played a vital role in the development and metastasis of BRCA Table 1. Importantly, three HCC RNA-seq datasets  from Gene Expression Omnibus (GEO) indicated that YWHAZ is a hub gene in HCC Figure .Taken together, some crucial upstream regulators and downstream targets of YWHAZ involving in cancer progression were summarized in YWHAZ, as well as HTR2B, CHL1, the ZNF family and FYN, were observed to be most obviously altered between 46 liver metastatic uveal melanoma samples and 45 non-metastatic uveal melanoma samples, derived from GEO database YWHAZ, PRDX2 and C1QBP were all related to inflammation and cell proliferation and could be regarded as candidate biomarkers for bladder cancer diagnosis In the past decades, the diagnostic potential of YWHAZ had aroused considerable interest. Liu M et al. detected that the prevalence of YWHAZ autoantibodies was 16.7% (28/168) in HCC, significantly higher than in liver cirrhosis, chronic hepatitis, and normal human sera by enzyme-linked immunosorbent assay (ELISA) analysis (P<0.01) YWHAZ expression and overall survival/disease-free survival at 60 months in BRCA, COAD, ESCA, LIHC, LUAD, LUSC, PRAD, READ, and STAD from the Cancer Genome Atlas (TCGA) database. As can be seen in Figure YWHAZ expression was significantly correlated with overall survival at 60 months in LIHC (p = 0.0197) and LUAD (p = 0.016), and with disease-free survival at 60 months in BRCA (p = 0.0279) and LUAD (p = 0.016). We also conducted Kaplan-Meier survival analysis in Gene Expression Profiling Interactive Analysis (GEPIA) database with larger samples YWHAZ expression  YWHAZ and low YWHAZ samples .Our team used Kaplan-Meier survival analysis to explore the relationship between ASH1, miR-375 and YWHAZ resulted in significant differences regarding overall survival at 50 months (p = 0.003), 60 months (p = 0.0096) and 100 months (p = 0.0158) In HCC, we observed that combination of s p = 0.08 15. FanLAPTM4B and YWHAZ gene sensitized tumor cells to anthracyclines, while overexpression of these genes induced drug resistance It is well known that barriers to chemotherapeutic agents during cancer therapy include intrinsic and acquired resistance, thus the effect of chemotherapy among cancer patients is still often sub-optimal. The anti-apoptosis ability exerted by YWHAZ may be responsible for chemoresistance. High levels of YWHAZ had been found in CHOP-resistant DLBCL cells and 9-nitrocamptothecin resistant prostate cancer cells, compared with chemo-sensitive cells YWHAZ-targeted therapies may be an attractive project in the future.Surgery, chemotherapy and radiotherapy have traditionally been the main therapeutic methods for human cancers. However, the prognosis of most cancer patients treated through these approaches still remain fairly poor. Given the oncogene role of YWHAZ in multiple cancers, the combination of traditional therapeutic methods and YWHAZ and si-YWHAZ/DOX using nanoliposomes (L) in established mouse HCC xenograft models, observing that tumor growth could be inhibited in the latter two groups compared with control in vivo by YWHAZ blockade YWHAZ siRNA using lipofectamine YWHAZ lung cancer cells using lipofectamine YWHAZ cells, which are derived from PVTT and prone to form PVTT, to establish orthotopic transplantation assays in nude mice YWHAZ cells or HCC- CSQT-2/sh-control cells into mice through the tail vein YWHAZ by shRNA suppressed lung metastases and formation of PVTT in vivoYWHAZ might also be worth exploring. Up-regulation of miRNA-451 by murine stem cell virus vector directly decreased YWHAZ expression and inhibited colon cancer growth in vitro and in vivoin vivoYWHAZ specifically are greatly needed in the future.Our team delivered si-NC, si-YWHAZ has been shown to be frequently up-regulated and function as an oncogene by regulating multiple signaling pathways in cancers (Table 1). YWHAZ overexpression is regulated by miRNAs or long non-coding RNAs and activates downstream molecules, including protein kinases, apoptosis proteins, and metastasis-related molecules, to facilitate the malignant potential of cancer cells. However, a comprehensive assessment of YWHAZ regulatory networks through bioinformatics analysis is warranted. Growing evidences suggested the potential role of YWHAZ in cancer diagnosis, prognosis and chemoresistance. However the specificity and sensibility of YWHAZ as an independent biomarker are limited. Combinations of YWHAZ with other cancer-specific molecules may have better ability to serve as biomarkers. At present, YWHAZ targeting therapy alone through siRNA, shRNA or miRNA to delay tumor development shows some preliminary results. Nevertheless, safer and more effective carriers for YWHAZ inhibitor delivery, or combinations of YWHAZ with other promising therapeutic targets are greatly needed. In summary, YWHAZ, acting as an important oncogene, is increasingly showing its potential as a biomarker for diagnosis, prognosis, chemoresistance and therapeutic target in a diverse range of malignancies.To date,


Our results showed that several HIV inhibitors such as lopinavir, ritonavir, and saquinavir produce strong interaction with the active site of SARS-CoV-2 main protease. Furthermore, broad library protease inhibitors obtained from PubChem and ZINC (www.zinc.docking.org) were evaluated. Our analysis revealed 20 compounds that could be clustered into three groups based on their chemical features. Then, these structures could serve as leading compounds to develop a series of derivatives optimizing their activity against SARS-CoV-2 and other coronaviruses. Altogether, the results presented in this work contribute to gain a deep understanding of the molecular pharmacology of SARS-CoV-2 treatment and validate the use of protease inhibitors against SARS-CoV-2.Direct-acting antivirals are effective tools to control viral infections. SARS-CoV-2 is a coronavirus associated with the epidemiological outbreak in late 2019. Previous reports showed that HIV-1 protease inhibitors could block SARS-CoV main protease. Based on that and using an  Lopinavir (LPV) and ritonavir (RTV) are protease inhibitors currently used in HIV therapy that could block SARS-CoV and MERS-CoV main proteases . Ribavirino 2005). The cot al 2020). Altoget al 2020). Thus, t al 2020). The onProtein sequences of the main protease were individually retrieved from GenBank , Vega Zhttp://www.swissadme.ch. A comprehensive analysis of physicochemical descriptors, as well as ADME parameters, pharmacokinetic properties, drug-like nature, and medicinal chemistry for the top 5 compounds obtained from the library virtually screened, was carried out by using SWISSADME tools. These tools were asset through the website at Rhinolophus affinis the probable ancestor of this new virus . The seThe SARS-CoV and SARS-CoV-2 proteins exhibit 96 % sequence identity to each other Figure 1. Figure n silico data demonstrated that the different protease inhibitors, used against HIV-1, could interact with the active site of SARS-CoV-2 protease producing an interaction with a binding energy lower than -6.9 Kcal/mol. However, the compound that produced the strongest interaction with the active site was saquinavir, with a binding energy of -9.6 Kcal/mol  and PubChem (over 200 compounds). After molecular docking, compounds were prioritized based on binding energies. The top 20 compounds were clustered using a hierarchical matrix and represented into a heat map Figure 5. These cin silico approach to validate the inhibition of SARS-CoV-2 protease by HIV-1 protease inhibitor. The results showed that the main protease inhibitors used in HIV-1 therapy could produce a decrease in the protease activity in coronavirus enzyme. The compound with the highest binding affinity was saquinavir. Preliminary reports are showing that patients infected with SARS-CoV-2 and treated with lopinavir could achieve viral clearance . Our stIn addition, to contribute to further development of antiviral drugs against SARS-CoV-2, in this work experimental compounds against the SARS-CoV-2 protease were evaluated by virtual screening. Some compounds showed comparable binding energy values to the SARS-CoV-2 protease than to those observed for SQV, LPV and RTV. ADME  parameters estimation for new drugs, significantly helps to reduce the pharmacokinetics-related failure in clinical phases. These results evidence that these compounds could be suitable for further medicinal chemistry optimization to produce the second generation of inhibitors more specific and potent against SARS-CoV-2.Other antivirals have been evaluated in SARS-CoV-2 as the polymerase inhibitor remdesivir, a nucleotide analog , alone or in combination with chloroquine, an inhibitor of lysosome acidification, with interesting results (Wang et al., 2020; de Wit


Human papillomavirus (HPV) has been the leading cause of cervical cancer for over 25 years. Approximately 5.5–11% of all cervical cancers are reported to be HPV-negative, which can be attributed to truly negative and false-negative results. The truly HPV-negative cervical cancers are almost all cervical adenocarcinomas with unclear etiology. False HPV negativity can arise from histological misclassification, latent HPV infection, disruption of the targeting fragment, non-high risk HPV infection, and HPV testing methods. HPV-negative cervical cancers are often diagnosed at an advanced FIGO stage and have a poor prognosis; thus, the management of these cases requires greater attention. Cervical cancer is the fourth most common malignancy among women worldwide, accounting for approximately 7% of all cancer cases in women , 2. Persvia HPV testing. The existence of cervical adenocarcinoma independent of HPV infection has been recognized by the majority of researchers  during cancer development . AlthougSeveral studies have reported the association between cervical cancer and infection with low-risk HPV types 6, 11, 42, 44, and 70 –33. WhetHPV testing can be divided into two categories, namely, nucleic and non-nucleic acid signal amplification Figure 1Non-nucleic acid signal amplification methods, including HC2™, Cervista™ HPV HR, and Cervista™ HPV16/18, have lower sensitivity but higher cut-off values than nucleic acid signal amplification methods, resulting in false-negative HPV test outcomes, especially when samples have low viral loads. The lack of an internal control in HC2™ also increases the false-negativity rate probably due to DNA contamination or degradation , 51. HC2In addition to the method used, HPV false-negative results are also related to sampling errors. Poor cell viability from necrotic and/or inflammatory sites often results in HPV false-negative outcomes. Faulty sample collection methods, including samples mixed with blood or lubricant, as well as fixation procedure can result in false negativity. Therefore, the accuracy of HPV testing in published studies should be questioned discreetly. Some studies employed HPV testing to investigate previously stored cervical cancer specimens, but it is unknown whether such specimens precisely reflect HPV infection in the patients. A retrospective study demonstrated that samples from elderly patients or those stored for a longer duration had lower HPV-positivity rates . The effIn summary, the most important reason for false-negative HPV testing results is the significant difference among HPV detection methods, which is not realized by all clinicians . Testingvia an HPV-independent mechanism  inhibitor repressed E6 activity to promote apoptosis in HPV-positive cervical cancer cells but caused G2 phase arrest in HPV-negative cervical cancer cells, while dehydroepiandrosterone caused apoptosis in HPV-positive cervical cancer cells and necrosis in HPV-negative cervical cancer cells , 62. Basp=0.001; disease-free survival: HR=0.362, p<0.001) (p=0.003), respectively, indicating that HPV infection was significantly correlated with poor OS of patients  . A meta-<0.001) . To date<0.001) –59. Nevepatients . FurtherHPV-negative cervical cancers are divided into truly negative and false-negative categories. Truly negative cervical cancers have an HPV-independent pathogenesis with specific pathological types, of which HPV vaccination and testing probably have little effect on their prevention. The diagnosis of truly negative cervical cancer mainly depends on cytological screening and observation of histological features, combined with cytological multiple staining. Further investigation of the pathways and biomarkers of the different pathological types is required to develop a basis for precise therapy. For false-negative cervical cancer, retesting should be considered using other HPV testing methods according to their characteristics after analyzing the reason for false-negative HPV results. HPV-positive results after retesting may be due to initial testing failures for hr-HPV or the inability of the standard HPV test to detect other HPV genotypes . RetestiCervical adenocarcinoma is the major pathological type of HPV-negative cervical cancer, most likely caused by mutations of PI3K-AKT or other pathways. Preclinical studies have demonstrated that tumorigenesis differs between HPV-positive and HPV-negative cervical cancers, which presents the possibility of developing targeted therapies for HPV-negative patients. This may provide a basis for cervical cancer treatment research in the future. Although studies in the past indicated that HPV-positive status was an independent risk factor that impacted cervical cancer prognosis , 72, resIn conclusion, we consulted studies involving HPV-negative cervical cancer, and gave a comprehensive review of HPV-negative cervical cancer of prevalence, etiology, clinical features, treatment and prognosis. Although HPV-negative cervical cancer reveals different characteristics from HPV-positive one, most studies ignore the HPV status of cervical cancer, which restricts a profound insights of HPV-negative cervical cancer. Clarifying the different categories of HPV-negative cervical cancers is crucial to the development of suitable treatments and to guide studies investigating HPV-negative cervical cancers. The presence of HPV-independent cervical cancers should not affect the promotion of HPV testing and vaccination. Clinicians should classify and treat HPV-negative cases cautiously, and consider the correlation between advanced stage and poor prognosis of cervical cancer to provide women with negative HPV testing better management and effective treatment.YZ and GW provided the concept and designed the study. BX and JG wrote the draft of the manuscript. YZ and YS gave critical revision of the manuscript. YZ and JG provided economic support to the study. The authors meet all the criteria set out in the journal’s authorship criteria. All authors contributed to the article and approved the submitted version.This study was funded by the National Natural Science Foundation of China (Grant Nos. 81974463 and 81672573).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chondrogenic progenitor cells (CPCs) have high self-renewal capacity and chondrogenic potential. Intra-articular delivery of purified mesenchymal stem cells (MSCs) from MRL/MpJ “superhealer” mice increased bone volume during repair and prevents post-traumatic arthritis. Recently, although extracellular vesicles released from MSCs have been used widely for treating OA, the application of extracellular vesicles secreted by CPCs from MRL/MpJ mice in OA therapy has never been reported. In this study, we evaluated the effects of extracellular vesicles secreted by CPCs from control CBA (CBA-EVs) and MRL/MpJ mice (MRL-EVs) on proliferation and migration of murine chondrocytes. We also determined here if weekly intra-articular injections of CBA-EVs and MRL-EVs would repair and regenerate surgically induced model in mice.CPC surface markers were detected by flow cytometry. CBA-EVs and MRL-EVs were isolated using an ultrafiltration method. Nanoparticle tracking analysis, transmission electron microscopy, and western blots were used to identify extracellular vesicles. CBA-EVs and MRL-EVs were injected intra-articularly in a mouse model of surgical destabilization of the medial meniscus (DMM)-induced OA, and histological and immunohistochemistry analyses were used to assess the efficacy of exosome injections. We used miRNA-seq analysis to analyze the expression profiles of exosomal miRNAs derived from CBA-EVs as well as MRL-EVs. Cell-counting and scratch assays were used to evaluate the effects of CBA-EVs and MRL-EVs on proliferation and migration of murine chondrocytes, respectively. Meanwhile, a specific RNA inhibitor assesses the roles of the candidate miRNAs in CPC-EV-induced regulation of function of chondrocytes.Both CBA-EVs and MRL-EVs stimulated chondrocyte proliferation and migration, but MRL-EVs exerted a stronger effect than CBA-EVs. The similar result was also observed in in vivo study, which indicated that injecting either CBA-EVs or MRL-EVs attenuated OA, but MRL-EVs showed a superior therapeutic effect in comparison with CBA-EVs. The results of bioinformatics analyses revealed that the differentially expressed exosomal miRNAs participated in multiple biological processes. We identified 80 significantly upregulated and 100 downregulated miRNAs. Moreover, we found that the top 20 differentially expressed exosomal miRNAs connected OA repair to processes such as AMPK signaling, regulation of autophagy, and insulin signaling. Notably, miRNA 221-3p were highly enriched in MRL-Exos and treatment with miR 221-3p inhibitor markedly decreased chondrocyte proliferation and migration induced by CBA-EVs or MRL-EVs in vitro.This is the first study to demonstrate MRL-EVs had a greater therapeutic effect on the treatment of OA than CBA-EVs. This study will hopefully provide new insight into the pathogenesis, prevention, and treatment of OA. The delivery of mesenchymal stem cells (MSCs) has been widely used as a regenerative therapy for various disease states. The progresses in stem cell transplantation therapy have become promising in the treatment of osteoarthritis (OA). In the past decade, adipose-derived MSCs (AMSCs) , 2 and bRecently, many studies have indicated that the activation of resident cells through a paracrine mechanism may be critical in progenitor cell-mediated tissue regeneration , 11. EviThe inbred MRL/MpJ mouse strain has shown remarkable regenerative capabilities after injury in different tissues including articular cartilage . Intra-a3/cm2) were transferred to 6 cm plastic dishes (10 μg/mL fibronectin coated overnight) and cultured in DMEM/F-12 with 15% FBS and 1% penicillin/streptomycin. Medium and non-adherent cells were removed after incubation for 20 min at 37 °C, and fresh culture medium was added to the dishes. The adherent cells were cultured until confluent for 7–10 days. Then, the colonies were collected and passaged two or three times prior to use in all experiments. At the same time, CPCs and chondrocytes were separated on the basis of CPCs’ differential adhesion to fibronectin, as described previously  in MRL-EVs was significantly increased  for the indicated times. Scratch wound assays revealed that both MRL-EVs and CBA-EVs led to a remarkable increase in chondrocyte migration in comparison with the control groups. Moreover, MRL-EVs showed more effective ability to increase the motility than CBA-EVs at 48 h, but the miR-221-3p inhibitor markedly decreased their upregulation induced by CBA-EVs or MRL-EVs  on chondrocyte function, chondrocytes were treated with CBA-EVs or MRL-EVs  and bone morphogenetic protein-7 (BMP-7) expression, and inhibits IL-1β expression in OA chondrocytes [Ppara, Max, and Arpc5. In another study, MSC exosomal miR-125b overexpression was found to suppress IL-1β-induced upregulation of ADAMTS-4 in human OA chondrocytes [Suv39h1, Tspan12, Bmf, and Bak1. Very little is known about the functions of these genes, and they deserved further investigation.After validation by qRT-PCR, three miRNAs  were observed to be significantly upregulated, while four miRNA  expression in MRL-EVs was decreased. Previous studies described miR-221 positively regulates chondrogenesis of chick limb MSCs . Many st pathway –46. We fdrocytes . AdditioOur results indicated that exosomal miRNAs may play a vital role in enhancing cartilage regeneration. Extracellular vesicles can be ingested more rapidly by homotypic cells, as opposed to other cells . We beliIn this study, we investigated the role and underlying mechanisms of CBA-EVs and MRL-EVs on chondrocyte proliferation and migration in vitro and on OA in vivo. Our data revealed that CPCs could produce amounts of extracellular vesicles, which exhibited the typical morphological features of extracellular vesicles. The CPC-EVs ameliorated the OA severity in vivo and significantly stimulated chondrocyte migration and proliferation in vitro. QRT-PCR results confirmed the validity of differentially expressed miRNAs identified by RNA sequence. There are three miRNAs  that were observed to be significantly upregulated while four miRNA  expression in MRL-EVs was decreased. MiR-221-3p was found to be highly enriched in MRL-EVs and served as a critical mediator in CPC-EV-induced regulation of function of chondrocytes in vitro. Our study provides evidence that intra-articular extracellular vesicles secreted by chondrogenic progenitor cells can prevent the development of OA. These results could help facilitate future research in the identification and development of novel approaches to treat OA.