Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity.

T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8+ tumor-infiltrating lymphocytes, the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life-extended interleukin-10-Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8+ tumor-infiltrating lymphocytes by promoting oxidative phosphorylation, a process that was independent of the progenitor exhausted T cells. Interleukin-10-Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent oxidative phosphorylation can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy.

Cyclin D1 silencing impairs DNA double strand break repair, sensitizes BRCA1 wildtype ovarian cancer cells to olaparib.

OBJECTIVE: Poly(ADP-ribose) polymerase inhibitors (PARPi) are active in cancer cells that have impaired repair of DNA by the homologous recombination (HR) pathway. Strategies that disrupt HR may sensitize HR-proficient tumors to PARP inhibition. As a component of the core cell cycle machinery, cyclin D1 has unexpected function in DNA repair, suggesting that targeting cyclin D1 may represent a plausible strategy for expanding the utility of PARPi in ovarian cancer. METHODS: BRCA1 wildtype ovarian cancer cells (A2780 and SKOV3) were treated with a combination of CCND1 siRNA and olaparib in vitro. Cell viability was assessed by MTT. The effects of the combined treatment on DNA damage repair and cell cycle progression were examined to dissect molecular mechanisms. In vivo studies were performed in an orthotopic ovarian cancer mouse model. Animals were treated with a combination of lentivirus-mediated CCND1 shRNA and olaparib or olaparib plus scrambled shRNA. Molecular downstream effects were examined by immunohistochemistry. RESULTS: Silencing of cyclin D1 sensitized ovarian cancer cells to olaparib through interfering with RAD51 accumulation and inducing cell cycle G0/G1 arrest. Treatment of lentivirus-mediated CCND1-shRNA in nude mice statistically significantly augmented the olaparib response (mean tumor weight ±â€¯SD, CCND1-shRNA plus olaparib vs scrambled shRNA plus olaparib: 0.172 ±â€¯0.070 g vs 0.324 ±â€¯0.044 g, P< 0.05). CONCLUSIONS: Silencing of cyclin D1 combined with olaparib may lead to substantial benefit for ovarian cancer management by mimicking a BRCAness phenotype, and induction of G0/G1 cell cycle arrest.

Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

OBJECTIVE: Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. APPROACH AND RESULTS: To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/ß-arr1 (ß-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. CONCLUSIONS: Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

RhoA-stimulated intra-capillary morphology switch facilitates the arrest of individual circulating tumor cells.

Metastasis is the primary cause of death for most cancer patients. Hematogenous arrest of circulating tumor cells (CTCs) is an essential prerequisite for metastases formation. Using transparent transgenic zebrafish (kdrl:eGFP; Casper), together with resonant laser scanning confocal microscopy, we tracked the fate of CTCs in vivo in the blood circulation for days. We found the intra-capillary morphology-switch (ICMS) of individual CTCs from strip to sphere was necessary for their intravascular arrests. Further genetic and pharmacological inhibition experiments indicated that the RhoA signaling was necessary for ICMS and the arrest of CTCs. At last, we demonstrated that early treatment by a clinically approved RhoA/ROCK inhibitor, Fasudil, could efficiently inhibit the initial arrest of individual CTCs and reduce the incidence of tumor metastasis in both zebrafish and mouse models. These results together indicate that RhoA-stimulated ICMS represents a mechanism for the arrest of individual CTCs, providing a potential target for future treatments of hematogenous metastatic disease.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Zebrafish functional xenograft vasculature platform identifies PF-502 as a durable vasculature normalization drug.

Tumor vasculature often exhibits disorder and inefficiency. Vascular normalization offers potential for alleviating hypoxia and optimizing drug delivery in tumors. However, identifying effective agents is hindered by a lack of robust screening. We aimed to establish a comprehensive method using the zebrafish functional xenograft vasculature platform (zFXVP) to visualize and quantify tumor vasculature changes. Employing zFXVP, we systematically screened compounds, identifying PF-502 as a robust vascular normalization agent. Mechanistic studies showed PF-502 induces endothelial cell-cycle arrest, streamlines vasculature, and activates Notch1 signaling, enhancing stability and hemodynamics. In murine models, PF-502 exhibited pronounced vascular normalization and improved drug delivery at a sub-maximum tolerated dose. These findings highlight zFXVP's utility and suggest PF-502 as a promising adjunctive for vascular normalization in clinical settings.

Mitochondrial UQCC3 controls embryonic and tumor angiogenesis by regulating VEGF expression.

Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5-10.5 days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

EpCAM-targeting CAR-T cell immunotherapy is safe and efficacious for epithelial tumors.

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.

Taurine protects methamphetamine-induced developmental angiogenesis defect through antioxidant mechanism.

Investigations have characterized addictive drug-induced developmental cardiovascular malformation in human, non-human primate and rodent. However, the underlying mechanism of malformation caused by drugs during pregnancy is still largely unknown, and preventive and therapeutic measures have been lacking. Using 1H NMR spectroscopy, we profiled the metabolites from human embryo endothelial cells exposed to methamphetamine (METH) and quantified a total of 226 peaks. We identified 11 metabolites modified robustly and found that taurine markedly increased. We then validated the hypothesis that this dramatic increase in taurine could attribute to its effect in inhibiting METH-induced developmental angiogenesis defect. Taurine supplement showed a more significant potential than other metabolites in protecting against METH-induced injury in endothelial cells. Taurine strongly attenuated METH-induced inhibition of proliferation and migration in endothelial cells. Furthermore, death rate and vessel abnormality of zebrafish embryos treated with METH were greatly reversed by taurine. In addition, taurine supplement caused a rapid decrease in reactive oxygen species generation and strongly attenuated the excitable arise of antioxidase activities in the beginning of METH exposure prophase. Dysregulations of NF-κB, p-ERK as well as Bax, which reflect apoptosis, cell cycle arrest and oxidative stress in vascular endothelium, were blocked by taurine. Our results provide the first evidence that taurine prevents METH-caused developmental angiogenesis defect through antioxidant mechanism. Taurine could serve as a potential therapeutic or preventive intervention of developmental vascular malformation for the pregnant women with drug use.

Stat5-/- CD4+ T cells elicit anti-melanoma effect by CD4+ T cell remolding and Notch1 activation.

Signal transducers and activators of transcription 5 (Stat5) is known to engage in regulating the differentiation and effector function of various subsets of T helper cells. However, how Stat5 regulates the antitumor activity of tumor-infiltrating CD4+ T cells is largely unknown. Here, we showed that mice with specific deletion of Stat5 in CD4+ T cells were less susceptible to developing subcutaneous and lung metastatic B16 melanoma with CD4+ tumor-infiltrating lymphocytes (TILs) remolding. Especially, we confirmed that Stat5-deficient CD4+ naïve T cells were prone to polarization of two subtypes of Th17 cells: IFN-γ+ and IFN-γ- Th17 cells, which exhibited increased anti-melanoma activity through enhanced activation of Notch1 pathway compared with wild type Th17 cells. Our study therefore revealed a novel function of Stat5 in regulating tumor-specific Th17 cell differentiation and function in melanoma. This study also provided a new possibility for targeting Stat5 and other Th17-associated pathways to develop novel immunotherapies for melanoma patients.

The inhibition of protein translation promotes tumor angiogenic switch.

The 'angiogenic switch' is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study, we established mammal xenografts in zebrafish to visually investigate the first vessel growth (angiogenic switch) in real-time, by inoculating tumor cells into the perivitelline space of live optically transparent Transgenic (flk1:EGFP) zebrafish larvae. Using this model, we found that hypoxia and hypoxia-inducible factor (HIF) signaling were unnecessary for the angiogenic switch, whereas vascular endothelial growth factor A gene (Vegfa) played a crucial role. Mechanistically, transcriptome analysis showed that the angiogenic switch was characterized by inhibition of translation, but not hypoxia. Phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α) and the expression of Vegfa were increased in the angiogenic switch microtumors, and 3D tumor spheroids, and puromycin-treated tumor cells. Vegfa overexpression promoted early onset of the angiogenic switch, whereas Vegfa knockout prevented the first tumor vessel from sprouting. Pretreatment of tumor cells with puromycin promoted the angiogenic switch in vivo similarly to Vegfa overexpression, whereas Vegfa knockdown suppressed the increase. This study provides direc and dynamic in vivo evidences that inhibition of translation, but not hypoxia or HIF signaling promotes the angiogenic switch in tumor by increasing Vegfa transcription.

Acquired semi-squamatization during chemotherapy suggests differentiation as a therapeutic strategy for bladder cancer.

Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.

KMT2C deficiency promotes small cell lung cancer metastasis through DNMT3A-mediated epigenetic reprogramming.

Small cell lung cancer (SCLC) is notorious for its early and frequent metastases, which contribute to it as a recalcitrant malignancy. To understand the molecular mechanisms underlying SCLC metastasis, we generated SCLC mouse models with orthotopically transplanted genome-edited lung organoids and performed multiomics analyses. We found that a deficiency of KMT2C, a histone H3 lysine 4 methyltransferase frequently mutated in extensive-stage SCLC, promoted multiple-organ metastases in mice. Metastatic and KMT2C-deficient SCLC displayed both histone and DNA hypomethylation. Mechanistically, KMT2C directly regulated the expression of DNMT3A, a de novo DNA methyltransferase, through histone methylation. Forced DNMT3A expression restrained metastasis of KMT2C-deficient SCLC through repressing metastasis-promoting MEIS/HOX genes. Further, S-(5'-adenosyl)-L-methionine, the common cofactor of histone and DNA methyltransferases, inhibited SCLC metastasis. Thus, our study revealed a concerted epigenetic reprogramming of KMT2C- and DNMT3A-mediated histone and DNA hypomethylation underlying SCLC metastasis, which suggested a potential epigenetic therapeutic vulnerability.

Distinct contributions of angiogenesis and vascular co-option during the initiation of primary microtumors and micrometastases.

Primary tumors and metastases have been thought to initiate avascularly as multicellular aggregates and later induce angiogenesis or initiate vascularly by co-opting pre-existing host blood vessels without inducing angiogenesis. These two distinct concepts of microtumor vascularization have raised significant controversies. To clarify intratumoral vascularization and tumor cell behaviors at single-cell level during the earliest stage of microtumor initiation, we established primary and metastatic microtumor models in Tg(flk1:EGFP) transgenic zebrafish. We found that tumor cells preferred to initiate avascularly as multicellular aggregates and only later (50-100 cells in size) induced angiogenesis in blood-supply-sufficient microenvironments. In blood-supply-deficient microenvironments, less tumor cells (20-30 cells per fish) managed to co-opt and migrate along host vessels, whereas more tumor cells (100-300 cells per fish) could immediately induce angiogenesis without obvious cell migration. In a metastatic model, we clearly observed that tumor cells co-opted, migrated along and proliferated on the surface of host vessels at an early stage after they extravasated from host vessels and induced angiogenesis later when micromatastases comprised only 15-30 tumor cells. Moreover, the inducement of neovessels accelerated the growth of micromatastases in size, meanwhile, decreased the migration of tumor cells on the surface of host vessels. These results suggest that vessel co-option and angiogenesis have distinct contributions during the initiation of microtumors. Microtumors initiated reasonably through co-opting host vessels or inducing angiogenesis, depending on the differences of local microenvironments and cell numbers in microtumors. The results in this study may have important implications for the therapeutic application of antiangiogenic strategies.

Kzp controls canonical Wnt8 signaling to modulate dorsoventral patterning during zebrafish gastrulation.

During vertebrate embryonic development, the body axis formation requires the action of Wnt signals and their antagonists. Zygotic canonical wnt8 expression appears exclusively at the ventrolateral margin and mediates Wnt/ß-catenin activities to promote posterior and ventral cell fate. However, the mechanisms involved in the initiation of zygotic wnt8 signals are poorly understood. Here, we identify a novel, maternally derived transcription factor, Kzp (Kaiso zinc finger-containing protein), as an important determinant for the initiation of zygotic Wnt signals in zebrafish. Kzp is a DNA-binding transcription factor that recognizes specific consensus DNA sequences, 5'-(t/a/g)t(a/t/g)nctgcca-3', through zinc fingers and controls the initiation of zygotic wnt8 expression by directly binding to the wnt8 promoter during zebrafish embryonic development. Depletion of Kzp strongly dorsalized embryos, which was characterized by the expansion of dorsal gene expression. Overexpression of Kzp caused posteriorization. These phenotypes were highly similar to ones induced by wnt8 depletion or overexpression and were rescued by alteration of wnt8 activity. Thus, our results provide the first insight into the mechanism involved in the initiation of zygotic canonical Wnt signals by a maternally derived transcription factor.

Mmp23b promotes liver development and hepatocyte proliferation through the tumor necrosis factor pathway in zebrafish.

The matrix metalloproteinase (MMP) family of proteins degrades extracellular matrix (ECM) components as well as processes cytokines and growth factors. MMPs are involved in regulating ECM homeostasis in both normal physiology and disease pathophysiology. Here we report the critical roles of mmp23b in normal zebrafish liver development. Mmp23b was initially identified as a gene linked to the genomic locus of an enhancer trap transgenic zebrafish line in which green fluorescent protein (GFP) expression was restricted to the developing liver. Follow-up analysis of mmp23b messenger RNA (mRNA) expression confirmed its liver-specific expression pattern. Morpholino knockdown of mmp23b resulted in defective hepatocyte proliferation, causing a reduction in liver size while maintaining relatively normal pancreas and gut development. Genetically, we showed that mmp23b functions through the tumor necrosis factor (TNF) signaling pathway. Antisense knockdown of tnfa or tnfb in zebrafish caused similar reductions of liver size, whereas overexpression of tnfa or tnfb rescued liver defects in mmp23b morphants but not vice versa. Biochemically, MMP23B, the human ortholog of Mmp23b, directly interacts with TNF and mediates its release from the cell membrane in a cell culture system. Because mmp23b/MMP23B is highly conserved, our findings in zebrafish warrant further investigation of its role in regulating liver development in mammals.

[Antitumor effect by combination of shRNA interfering plasmid targeting PKM2 with recombinant endostatin].

OBJECTIVE: To investigate the enhancement effect of the combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin in the treatment of lung cancer. METHODS: Twenty five BABL/nu/nu mice bearing A549 lung cancer were divided into 5 groups (NS control, psh-Control, psh-PKM2 treated group, Endostar treated group, psh-PKM2+Endostar treated group) and treated with shRNA interfering plasmid targeting PKM2 and recombinant Endostatin respectively or in combination. The expression of PKM2 in A549 detected with immunofluorescent assay. The interference effect of psh-PKM2 was determined by Western blot. The tumor volume, microvessel density (MVD), apoptosis index (AI) and side effects were observed. RESULTS: The combination treatment of RNA interfering plasmid targeting PKM2 with recombinant Endostatin inhibited tumor growth obviously (P < 0.05); The combination group revealed a decreased MVD and an increased AI (P < 0.05). CONCLUSION: The combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin might enhance anti-tumor effect by increasing the apoptosis of the cancer cell.

[The molecular mechanism of extravasation in mouse melanoma lung metastasis model].

OBJECTIVE: To study molecular mechanisms underlying the extravasation of mice melanoma cells during lung metastasis. METHODS: B16-RED melanoma cell line was established which stably express the red fluorescent protein. B16-RED cells were compared with B16 cells in ability of proliferation and lung metastasis. A mouse lung metastasis model was established with B16-RED melanoma cells. FITC-dextran was injected i.v. and CD31 indirect immunoflourescence (IIF) staining was made to identify the location of the tumor cells and the time of tumor cell extravasation. Finally, at 48 hours post cell injection, the lung and a normal lung were removed and used for 32K mice microarray analysis. RESULTS: B16-RED was consistent with B16 in cell shape and ability of proliferation and lung metastasis. 52.7% of B16-RED melanoma cells completed the extravasation within 48 hours in mouse lung metastasis model. Many important signal pathways were involved during lung metastasis, including leukocyte transendothelial migration, MAPK signaling pathway, neuroactive ligand-receptor interaction, focal adhesion, cytokine-cytokine receptor interaction, regulation of actin cytoskeleton, axon guidance, calcium signaling pathway, tight junction, etc. CONCLUSION: The extravasation during metastasis is a complex and multiple-steps process, in which many important signal pathways in host tissues were involved.

Enhancing the sensitivity of ovarian cancer cells to olaparib via microRNA-20b-mediated cyclin D1 targeting.

We previously reported that cyclin D1 silencing interferes with RAD51 accumulation and increases the sensitivity of BRCA1 wild-type ovarian cancer cells to olaparib. However, the mechanisms associated with cyclin D1 overexpression in ovarian cancer are not fully understood. TargetScan predicted the potential binding sites for microRNA-20b (miR-20b) and the 3'-untranslated region of cyclin D1 mRNA; thus, we used luciferase reporter assay to verify those binding sites. The Kaplan-Meier method and log-rank test were used to examine the relationship between miR-20b and progression-free survival of ovarian cancer patients in The Cancer Genome Atlas (n = 367) dataset. In vitro experiments were performed to evaluate the effects of miR-20b on cyclin D1 expression, cell cycle and response to olaparib. A peritoneal cavity metastasis model of ovarian cancer was established to determine the effect of miR-20b on the sensitivity of olaparib. Immunohistochemistry was performed to evaluate molecular mechanisms. In this work, we demonstrated that miR-20b down-regulates cyclin D1, increases the sensitivity of ovarian cancer cells to olaparib, reduces the expression of RAD51, and induces cell cycle arrest in G0/G1 phase. Ovarian cancer patients with higher expression of miR-20b had significantly longer progression-free survival. These results indicate that miR-20b may be a potential clinical indicator for the sensitivity of ovarian cancer to olaparib and the survival of ovarian cancer patients. Our findings suggest that miR-20b may have therapeutic value in combination with olaparib treatment for ovarian cancer.

A flow-cytometry-based protocol for detection of mitochondrial ROS production under hypoxia.

Hypoxia is known to stimulate mitochondrial reactive oxygen species (mROS) in cells. Here, we present a detailed protocol to detect mROS using MitoSOX staining in live cells under normoxia and hypoxia. Flow cytometry allows sensitive and reliable quantification of mROS by FlowJo software. We optimized several aspects of the procedure including hypoxic treatment, working concentrations of the staining buffer, and quantitative analyses. Here, we use HepG2 cells, but the protocol can be applied to other cell lines. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).