Tumor cell-released kynurenine biases MEP differentiation into megakaryocytes in individuals with cancer by activating AhR-RUNX1.

Tumor-derived factors are thought to regulate thrombocytosis and erythrocytopenia in individuals with cancer; however, such factors have not yet been identified. Here we show that tumor cell-released kynurenine (Kyn) biases megakaryocytic-erythroid progenitor cell (MEP) differentiation into megakaryocytes in individuals with cancer by activating the aryl hydrocarbon receptor-Runt-related transcription factor 1 (AhR-RUNX1) axis. During tumor growth, large amounts of Kyn from tumor cells are released into the periphery, where they are taken up by MEPs via the transporter SLC7A8. In the cytosol, Kyn binds to and activates AhR, leading to its translocation into the nucleus where AhR transactivates RUNX1, thus regulating MEP differentiation into megakaryocytes. In addition, activated AhR upregulates SLC7A8 in MEPs to induce positive feedback. Importantly, Kyn-AhR-RUNX1-regulated MEP differentiation was demonstrated in both humanized mice and individuals with cancer, providing potential strategies for the prevention of thrombocytosis and erythrocytopenia.

IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor.

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.

TCR activation directly stimulates PYGB-dependent glycogenolysis to fuel the early recall response in CD8+ memory T cells.

Glycolysis facilitates the rapid recall response of CD8+ memory T (Tm) cells. However, it remains unclear whether Tm cells uptake exogenous glucose or mobilize endogenous sugar to fuel glycolysis. Here, we show that intracellular glycogen rather than extracellular glucose acts as the major carbon source for the early recall response. Following antigenic stimulation, Tm cells exhibit high glycogen phosphorylase (brain form, PYGB) activity, leading to glycogenolysis and release of glucose-6-phosphate (G6P). Elevated G6P mainly flows to glycolysis but is also partially channeled to the pentose phosphate pathway, which maintains the antioxidant capacity necessary for later recall stages. Mechanistically, TCR signaling directly induces phosphorylation of PYGB by LCK-ZAP70. Functionally, the glycogenolysis-fueled early recall response of CD8+ Tm cells accelerates the clearance of OVA-Listeria monocytogenes in an infected mouse model. Thus, we uncover a specific dependency on glycogen for the initial activation of memory T cells, which may have therapeutic implications for adaptive immunity.

Sustained AhR activity programs memory fate of early effector CD8+ T cells.

Identification of mechanisms that program early effector T cells to either terminal effector T (Teff) or memory T (Tm) cells has important implications for protective immunity against infections and cancers. Here, we show that the cytosolic transcription factor aryl hydrocarbon receptor (AhR) is used by early Teff cells to program memory fate. Upon antigen engagement, AhR is rapidly up-regulated via reactive oxygen species signaling in early CD8+ Teff cells, which does not affect the effector response, but is required for memory formation. Mechanistically, activated CD8+ T cells up-regulate HIF-1α to compete with AhR for HIF-1ß, leading to the loss of AhR activity in HIF-1αhigh short-lived effector cells, but sustained in HIF-1αlow memory precursor effector cells (MPECs) with the help of autocrine IL-2. AhR then licenses CD8+ MPECs in a quiescent state for memory formation. These findings partially resolve the long-standing issue of how Teff cells are regulated to differentiate into memory cells.

Cell softness regulates tumorigenicity and stemness of cancer cells.

Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

[Systematic review of efficacy and safety of Angong Niuhuang Pills in adjuvant treatment of cerebral hemorrhage].

To systematically review the efficacy and safety of Angong Niuhuang Pills in adjuvant treatment of cerebral hemorrhage. CNKI, VIP, Wanfang, CBM, PubMed, EMbase, Cochrane Library were retrieved to collect the randomized controlled trial(RCT) from the time of database establishment to November 2020. Two researchers screened out the literatures and extracted the data according to the inclusion and exclusion criteria. RevMan 5.3 software was used for Meta-analysis. A total of 13 RCTs were included, involving 1 196 patients with cerebral hemorrhage, with 599 in the treatment group and 597 in the control group, and all of them were treated with internal medicine. The results of Meta-analysis showed that compared with conventional therapy, the combined administration with Angong Niuhuang Pills could improve the effective rate in patients with cerebral hemorrhage(RR=1.25, 95%CI[1.18, 1.34], P<0.000 01), the National Institutes of Health stroke scale(NIHSS)score(MD=-5.18, 95%CI[-8.12,-2.23], P=0.000 6) and Glasgow coma scale(GCS) score(MD=1.12, 95%CI[0.46, 1.78], P=0.000 9), activity of daily living(ADL)(MD=15.70, 95%CI[14.05, 17.36 ], P<0.000 01), reduce the malondialdehyde(MDA)(MD=-1.73,95%CI[-2.81,-0.64],P=0.002), but with no statistically significant difference in hematoma volume changes between the two groups. In terms of safety, the combined administration with Angong Niuhuang Pills reduced the incidence of adverse reactions compared with the single administration of conventional therapy(RR=0.40, 95%CI[0.28, 0.57], P<0.000 01), with no serious adverse events. The existing clinical study evidences show that Angong Niuhuang Pills had a good effect in adjuvant treatment of cerebral hemorrhage, and can improve the treatment efficacy, activity of daily living and symptoms of neurological deficits, and reduce oxidative stress, with a higher safety. However, the methodological quality of the included studies is not high, so the above conclusions still need to be verified with more high-quality studies.

Cell softness renders cytotoxic T lymphocytes and T leukemic cells resistant to perforin-mediated killing.

Mechanical force contributes to perforin pore formation at immune synapses, thus facilitating the cytotoxic T lymphocytes (CTL)-mediated killing of tumor cells in a unidirectional fashion. How such mechanical cues affect CTL evasion of perforin-mediated autolysis remains unclear. Here we show that activated CTLs use their softness to evade perforin-mediated autolysis, which, however, is shared by T leukemic cells to evade CTL killing. Downregulation of filamin A is identified to induce softness via ZAP70-mediated YAP Y357 phosphorylation and activation. Despite the requirements of YAP in both cell types for softness induction, CTLs are more resistant to YAP inhibitors than malignant T cells, potentially due to the higher expression of the drug-resistant transporter, MDR1, in CTLs. As a result, moderate inhibition of YAP stiffens malignant T cells but spares CTLs, thus allowing CTLs to cytolyze malignant cells without autolysis. Our findings thus hint a mechanical force-based immunotherapeutic strategy against T cell leukemia.

Hepatic glycogenesis antagonizes lipogenesis by blocking S1P via UDPG.

The identification of mechanisms to store glucose carbon in the form of glycogen rather than fat in hepatocytes has important implications for the prevention of nonalcoholic fatty liver disease (NAFLD) and other chronic metabolic diseases. In this work, we show that glycogenesis uses its intermediate metabolite uridine diphosphate glucose (UDPG) to antagonize lipogenesis, thus steering both mouse and human hepatocytes toward storing glucose carbon as glycogen. The underlying mechanism involves transport of UDPG to the Golgi apparatus, where it binds to site-1 protease (S1P) and inhibits S1P-mediated cleavage of sterol regulatory element-binding proteins (SREBPs), thereby inhibiting lipogenesis in hepatocytes. Consistent with this mechanism, UDPG administration is effective at treating NAFLD in a mouse model and human organoids. These findings indicate a potential opportunity to ameliorate disordered fat metabolism in the liver.

YAP Inactivation by Soft Mechanotransduction Relieves MAFG for Tumor Cell Dedifferentiation.

Solid tumor cells live in a highly dynamic mechanical microenvironment. How the extracellular-matrix-generated mechanotransduction regulates tumor cell development and differentiation remains an enigma. Here, we show that a low mechanical force generated from the soft matrix induces dedifferentiation of moderately stiff tumor cells to soft stem-cell-like cells. Mechanistically, integrin ß8 was identified to transduce mechano-signaling to trigger tumor cell dedifferentiation by recruiting RhoGDI1 to inactivate RhoA and subsequently Yes-associated protein (YAP). YAP inactivation relieved the inhibition of v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), allowing MAFG to transactivate the stemness genes NANOG, SOX2, and NESTIN. Inactivation also restored ß8 expression, thereby forming a closed mechanical loop. Importantly, MAFG expression is correlated with worse prognosis. Our findings provide mechanical insights into the regulation of tumor cell dedifferentiation, which has therapeutic implications for exploring innovative strategies to attack malignancies.

Epigenetic modification of CSDE1 locus dictates immune recognition of nascent tumorigenic cells.

Weak immunogenicity of tumor cells is a root cause for the ultimate failure of immunosurveillance and immunotherapy. Although tumor evolution can be shaped by immunoediting toward a less immunogenic phenotype, mechanisms governing the initial immunogenicity of primordial tumor cells or original cancer stem cells remain obscure. Here, using a single tumor-repopulating cell (TRC) to form tumors in immunodeficient or immunocompetent mice, we demonstrated that immunogenic heterogeneity is an inherent trait of tumorigenic cells defined by the activation status of signal transducer and activator of transcription 1 (STAT1) protein in the absence of immune pressure. Subsequent investigation identified that the RNA binding protein cold shock domain-containing protein E1 (CSDE1) can promote STAT1 dephosphorylation by stabilizing T cell protein tyrosine phosphatase (TCPTP). A methyltransferase SET and MYN domain-containing 3 (SMYD3) was further identified to mediate H3K4 trimethylation of CSDE1 locus, which was under the regulation of mechanotransduction by cell-matrix and cell-cell contacts. Thus, owing to the differential epigenetic modification and subsequent differential expression of CSDE1, nascent tumorigenic cells may exhibit either a high or low immunogenicity. This identified SMYD3-CSDE1 pathway represents a potential prognostic marker for cancer immunotherapy effectiveness that requires further investigation.

Aryl hydrocarbon receptor sulfenylation promotes glycogenolysis and rescues cancer chemoresistance.

Elevation of reactive oxygen species (ROS) levels is a general consequence of tumor cells' response to treatment and may cause tumor cell death. Mechanisms by which tumor cells clear fatal ROS, thereby rescuing redox balance and entering a chemoresistant state, remain unclear. Here, we show that cysteine sulfenylation by ROS confers on aryl hydrocarbon receptor (AHR) the ability to dissociate from the heat shock protein 90 complex but to bind to the PPP1R3 family member PPP1R3C of the glycogen complex in drug-treated tumor cells, thus activating glycogen phosphorylase to initiate glycogenolysis and the subsequent pentose phosphate pathway, leading to NADPH production for ROS clearance and chemoresistance formation. We found that basic ROS levels were higher in chemoresistant cells than in chemosensitive cells, guaranteeing the rapid induction of AHR sulfenylation for the clearance of excess ROS. These findings reveal that AHR can act as an ROS sensor to mediate chemoresistance, thus providing a potential strategy to reverse chemoresistance in patients with cancer.

Subtyping of microsatellite stability colorectal cancer reveals guanylate binding protein 2 (GBP2) as a potential immunotherapeutic target.

BACKGROUNDS: Proficient-mismatch-repair or microsatellite stability (pMMR/MSS) colorectal cancer (CRC) has limited efficacy for immune checkpoint blockade (ICB) therapy and its underlying mechanism remains unclear. Guanylate binding protein 2 (GBP2) is a member of the GTPase family and is crucial to host immunity against pathogens. However, the correlations between GBP2 and immunosurveillance and immunotherapy for pMMR/MSS CRC have not been reported. METHODS: Unsupervised clustering was employed to classify immune class and non-immune class in 1424 pMMR/MSS patients from six independent public datasets. This binary classification was validated using immune cells or response related signatures. The correlation between GBP2 and immune microenvironment was explored using well-established biological algorithms, multiplex immunohistochemistry (mIHC), in vitro and in vivo experiments. RESULTS: We classified 1424 pMMR/MSS CRC patients into two classes, 'immune' and 'non-immune', and GBP2 was identified as a gene of interest. We found that lower GBP2 expression was correlated with poor prognosis and metastasis. GBP2 expression was also upregulated in the immune class and highly associated with interferon-γ (IFN-γ) signaling pathway and CD8 +T cell infiltration using gene set enrichment analysis, gene ontology analysis, single-cell sequencing and mIHC. Moreover, reduced GBP2 expression inhibited the antigen processing and presentation machinery and CXCL10/11 expression in MSS CRC cells on IFN-γ stimulation. A Transwell assay revealed that deletion of GBP2 in murine MSS CRC cells reduced CD8 +T cell migration. Mechanistically, GBP2 promoted signal transducer and transcription activator 1 (STAT1) phosphorylation by competing with SHP1 for binding to STAT1 in MSS CRC cells. Finally, an unsupervised subclass mapping (SubMap) algorithm showed that pMMR/MSS patients with high GBP2 expression may correlate with a favorable response to anti-PD-1 therapy. We further confirmed that GBP2 knockout reduced CD8 +T cell infiltration and blunted the efficacy of PD-1 blockade in tumor-bearing mice. CONCLUSIONS: Our study reveals that pMMR/MSS CRC is immunogenically heterogeneous and that GBP2 is a promising target for combinatorial therapy with ICB.

SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages.

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1-mucin pathway.

Pancreatic ductal adenocarcinoma (PDAC) originates from normal pancreatic ducts where digestive juice is regularly produced. It remains unclear how PDAC can escape autodigestion by digestive enzymes. Here we show that human PDAC tumour cells use gasdermin E (GSDME), a pore-forming protein, to mediate digestive resistance. GSDME facilitates the tumour cells to express mucin 1 and mucin 13, which form a barrier to prevent chymotrypsin-mediated destruction. Inoculation of GSDME-/- PDAC cells results in subcutaneous but not orthotopic tumour formation in mice. Inhibition or knockout of mucin 1 or mucin 13 abrogates orthotopic PDAC growth in NOD-SCID mice. Mechanistically, GSDME interacts with and transports YBX1 into the nucleus where YBX1 directly promotes mucin expression. This GSDME-YBX1-mucin axis is also confirmed in patients with PDAC. These findings uncover a unique survival mechanism of PDAC cells in pancreatic microenvironments.

Metformin Use and Survival in Patients with Advanced Extrahepatic Cholangiocarcinoma: A Single-Center Cohort Study in Fuyang, China.

AIMS: Metformin is an oral antidiabetic agent that has been widely prescribed for the treatment of type II diabetes. In recent years, anticancer properties of metformin have been revealed for numerous human malignancies. However, there are few indications available regarding the feasibility and safety of these studies in an advanced extrahepatic cholangiocarcinoma (EHCC) population. This study is aimed at evaluating the feasibility, safety, and value of metformin use and survival in patients with advanced EHCC. METHODS: All patients with advanced EHCC observed at Fuyang People's Hospital between January 2015 and November 2020 were included in the study. Case data, clinical information, and imaging results were abstracted from the self-administered questionnaire and electronic medical record. All patients were divided into study subjects and control subjects, and the study subjects were given metformin, 0.5 g, three times a day, while control subjects were without metformin. The metformin use and survival time of the subjects were asked by telephone, out-patient, or door-to-door visit, after they left the hospital. RESULTS: One hundred and thirty-three study cases and 589 controls were included in the analysis. This study showed that metformin use cannot improve the overall survival rate of patients with advanced EHCC ([95% CI]: -17.05-0.375, t = -1.889, P value = 0.061), but the survival time of patients with drainage treatment from control group (n = 496) was significantly shorter than that of patients with drainage treatment from the study group (n = 113), and the difference was statistically significant (z = -2.230, P value = 0.026). There were significant differences between metformin used before or after the diagnosis of advanced EHCC (OR[95% CI], 3.432[2.617-4.502]; P value = 0.001) in survival time. And there was significant difference between the duration of metformin use and survival prognosis (OR[95% CI], 2.967[1.383-6.368]; P = 0.005). CONCLUSION: Metformin can improve the survival of advanced EHCC patients who underwent drainage treatment, especially for metformin use after diagnosis of advanced EHCC and long duration of metformin.

Hypoxia Promotes Breast Cancer Cell Growth by Activating a Glycogen Metabolic Program.

Hypoxia is known to be commonly present in breast tumor microenvironments. Stem-like cells that repopulate breast tumors, termed tumor-repopulating cells (TRC), thrive under hypoxic conditions, but the underlying mechanism remains unclear. Here, we show that hypoxia promotes the growth of breast TRCs through metabolic reprogramming. Hypoxia mobilized transcription factors HIF1α and FoxO1 and induced epigenetic reprogramming to upregulate cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme that initiates gluconeogenesis. PCK1 subsequently triggered retrograde carbon flow from gluconeogenesis to glycogenesis, glycogenolysis, and the pentose phosphate pathway. The resultant NADPH facilitated reduced glutathione production, leading to a moderate increase of reactive oxygen species that stimulated hypoxic breast TRC growth. Notably, this metabolic mechanism was absent in differentiated breast tumor cells. Targeting PCK1 synergized with paclitaxel to reduce the growth of triple-negative breast cancer (TNBC). These findings uncover an altered glycogen metabolic program in breast cancer, providing potential metabolic strategies to target hypoxic breast TRCs and TNBC. SIGNIFICANCE: Hypoxic breast cancer cells trigger self-growth through PCK1-mediated glycogen metabolism reprogramming that leads to NADPH production to maintain a moderate ROS level.

Cell Softness Prevents Cytolytic T-cell Killing of Tumor-Repopulating Cells.

Biomechanics is a fundamental feature of a cell. However, the manner by which actomysin tension affects tumor immune evasion remains unclear. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill soft tumor-repopulating cells (TRC). TRC softness prevented membrane pore formation caused by CTL-released perforin. Perforin interacting with nonmuscle myosin heavy-chain 9 transmitted forces to less F-actins in soft TRC, thus generating an inadequate contractile force for perforin pore formation. Stiffening TRC allowed perforin the ability to drill through the membrane, leading to CTL-mediated killing of TRC. Importantly, overcoming mechanical softness in human TRC also enhanced TRC cell death caused by human CTL, potentiating a mechanics-based immunotherapeutic strategy. These findings reveal a mechanics-mediated tumor immune evasion, thus potentially providing an alternative approach for tumor immunotherapy. SIGNIFICANCE: Tumor-repopulating cells evade CD8+ cytolytic T-cell killing through a mechanical softness mechanism, underlying the impediment of perforin pore formation at the immune synapse site.

Mucus production stimulated by IFN-AhR signaling triggers hypoxia of COVID-19.

Silent hypoxia has emerged as a unique feature of coronavirus disease 2019 (COVID-19). In this study, we show that mucins are accumulated in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients and are upregulated in the lungs of severe respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected mice and macaques. We find that induction of either interferon (IFN)-ß or IFN-γ upon SARS-CoV-2 infection results in activation of aryl hydrocarbon receptor (AhR) signaling through an IDO-Kyn-dependent pathway, leading to transcriptional upregulation of the expression of mucins, both the secreted and membrane-bound, in alveolar epithelial cells. Consequently, accumulated alveolar mucus affects the blood-gas barrier, thus inducing hypoxia and diminishing lung capacity, which can be reversed by blocking AhR activity. These findings potentially explain the silent hypoxia formation in COVID-19 patients, and suggest a possible intervention strategy by targeting the AhR pathway.

Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome.

Cytokine release syndrome (CRS) counteracts the effectiveness of chimeric antigen receptor (CAR) T cell therapy in cancer patients, but the mechanism underlying CRS remains unclear. Here, we show that tumor cell pyroptosis triggers CRS during CAR T cell therapy. We find that CAR T cells rapidly activate caspase 3 in target cells through release of granzyme B. The latter cleaves gasdermin E (GSDME), a pore-forming protein highly expressed in B leukemic and other target cells, which results in extensive pyroptosis. Consequently, pyroptosis-released factors activate caspase 1 for GSDMD cleavage in macrophages, which results in the release of cytokines and subsequent CRS. Knocking out GSDME, depleting macrophages, or inhibiting caspase 1 eliminates CRS occurrence in mouse models. In patients, GSDME and lactate dehydrogenase levels are correlated with the severity of CRS. Notably, we find that the quantity of perforin/granzyme B used by CAR T cells rather than existing CD8+ T cells is critical for CAR T cells to induce target cell pyroptosis.