NUF2 Promotes Breast Cancer Development as a New Tumor Stem Cell Indicator.

Multiple new subtypes of breast cancer (BRCA) are identified in women each year, rendering BRCA the most common and rapidly expanding form of cancer in females globally. NUF2 has been identified as a prognostic factor in various human cancers, regulating cell apoptosis and proliferation. However, its role in BRCA prognosis has not been clarified. This study explored the role of NUF2 in breast cancer development and prognosis using informatic analysis combined with in vivo intracellular studies. Through the online website TIMER, we evaluated the transcription profile of NUF2 across a variety of different cancer types and found that NUF2 mRNA was highly expressed in BRCA patients. Its transcription level was found to be related to the subtype, pathological stage, and prognosis of BRCA. The R program analysis showed a correlation of NUF2 with cell proliferation and tumor stemness in the BRCA patient samples. Subsequently, the association between the NUF2 expression level and immune cell infiltration was analyzed using the XIANTAO and TIMER tools. The results revealed that NUF2 expression was correlated with the responses of multiple immune cells. Furthermore, we observed the effect of NUF2 expression on tumor stemness in BRCA cell lines in vivo. The experimental results illuminated that the overexpression of NUF2 statistically upregulated the proliferation and tumor stemness ability of the BRCA cell lines MCF-7 and Hs-578T. Meanwhile, the knockdown of NUF2 inhibited the abilities of both cell lines, a finding which was verified by analyzing the subcutaneous tumorigenic ability in nude mice. In summary, this study suggests that NUF2 may play a key role in the development and progression of BRCA by affecting tumor stemness. As a stemness indicator, it has the potential to be one of the markers for the diagnosis of BRCA.

B cell deficiency promotes the initiation and progression of lung cancer.

Currently commercialized CAR-T cell therapies targeting CD19 and BCMA show great efficacy to cure B cell malignancies. However, intravenous infusion of these CAR-T cells severely destroys both transformed and normal B cells in most tissues and organs, in particular lung, leading to a critical question that what the impact of normal B cell depletion on pulmonary diseases and lung cancer is. Herein, we find that B cell frequency is remarkably reduced in both smoking carcinogen-treated lung tissues and lung tumors, which is associated with advanced cancer progression and worse patient survival. B cell depletion by anti-CD20 antibody significantly accelerates the initiation and progression of lung tumors, which is mediated by repressed tumor infiltration of T cells and macrophage elimination of tumor cells. These findings unveil the overall antitumor activity of B cells in lung cancer, providing novel insights into both mechanisms underlying lung cancer pathogenesis and clinical prevention post CAR-T cell therapy.

RIG-I contributes to dsDNA-induced innate immune activation in human brain microvascular endothelial cells.

Human brain microvascular epithelial cells (HBMECs) play a pivotal role in maintaining the stability of the blood-brain barrier (BBB), a potent physiological barrier to prevent the invasion of exotic pathogens. Our previous study indicated that polyI:C, an analog of double-stranded RNA (dsRNA), could initiate the TLR3/IFNs antiviral signaling pathway in HBMECs. However, the response of HBMECs to dsDNA remains unclear. In this study, we demonstrated that a dsDNA mimic, poly(dA:dT), could induce the production of a series of antiviral factors, including IFN-ß, IFN-λ1, and ISGs. Furthermore, the poly(dA:dT)-activated HBMECs could effectively restrain HSV-1 replication. In addition, we found that RIG-I rather than cGAS and IFI16 had a more crucial role in sensing poly(dA:dT). These observations indicate that HBMECs possess a dsDNA sensing system, and RIG-I may partly contribute to the dsDNA-induced antiviral innate immunity.

Dynamic Expression of EpCAM in Primary and Metastatic Lung Cancer Is Controlled by Both Genetic and Epigenetic Mechanisms.

Although great progress has been achieved in cancer treatment in the past decades, lung cancer remains the leading cause of cancer death, which is partially caused by the fact that most lung cancers are diagnosed at advanced stages. To improve the sensitivity and specificity of lung cancer diagnosis, the underlying mechanisms of current diagnosis methods are in urgent need to be explored. Herein, we find that the expression of EpCAM, the widely used molecular marker for tumor cell characterization and isolation, is strongly upregulated in primary lung tumors, which is caused by both gene amplification and promoter hypomethylation. In contrast, EpCAM expression is severely repressed in metastatic lung tumors, which can be reversed by epigenetic drugs, DNMT inhibitor 5-aza-dC and HDAC inhibitor MS-275. Moreover, tumor-associated macrophages (TAMs) impede EpCAM expression probably through TGFß-induced EMT signaling. These findings unveil the dynamic expression patterns of EpCAM and differential roles of epigenetic modification in EpCAM expression in primary and metastatic lung tumors, providing novel insights into tumor cell isolation and lung cancer diagnosis.

Induction of M-MDSCs with IL6/GM-CSF from adherence monocytes and inhibition by WP1066.

Peripheral blood monocytes acquire the phenotype of myeloid-derived suppressor cells (MDSCs) by induction of cytokine or co-culture with cancer cells and are widely used to model MDSCs for in vitro studies. However, the simplest method of plastic adhesive sorting is poorly described as the purity of monocyte resulting from this method is the lowest compared with flow cytometry cell-sorting and magnetic beads sorting. Therefore, the present study aimed at investigating the effect of the plastic adhesive monocyte isolation techniques on the resulting MDSCs phenotype. Monocytes were allowed to adhere for 1 h and cultured with IL6 and granulocyte-macrophage colony-stimulating factors (GM-CSF) for 7 days. Plastic adhesion sorting resulted in early low monocyte yield and purity, but high purity of MDSCs was obtained by refreshing the induction medium. The resulting MDSCs were the major subpopulation of CD33+CD11b+CD14+CD15-human leukocyte antigen (HLA)-/low cells and provided the potent capacity to suppress T cell proliferation and cytokine IFN-γ production. Moreover, the induced MDSCs were inhibited by STAT3 inhibitor WP1066, resulting in downregulation of phosphorylated-STAT3 and PD-L1 expression and upregulation of apoptosis respectively. In conclusion, the present study described the generation of monocytic MDSCs from adherence monocytes and the inhibition of STAT3 inhibitor WP1066 on the induced MDSCs. The present study contributed to the development of a new clinical drug, WP1066 targeting MDSC.

Establishment of a New Cryopreservation Solution for Chimeric Antigen Receptor T Cells.

Preservation and transportation are essential for the clinical application of chimeric antigen receptor T (CAR-T) cells. This study aimed to optimize a cryopreservation solution for CAR-T cells and evaluate the antitumor efficiency of CAR-T cells using this optimized solution in vitro and in vivo. First, the stability of the cryopreservation solution for CAR-T infusion was detected by the L27 (37) orthogonal experiment. Subsequently, osmolality and pH were analyzed for the preservation reagent. Additionally, apoptosis and CAR expression of CAR-T cells were measured by flow cytometry, and the cytotoxicity was determined by calcein-AM staining. The results showed that cryopreservation solutions used in this study demonstrated high chemical stability, which induced only 2% CAR-T cells apoptosis in optimal solutions, which were slightly lower than other commercial solutions. Moreover, the CAR expression was not significantly affected by preservation with these solutions. There were no significant differences in the cytotoxicity between fresh and thawed CAR-T cells cryopreserved in the cryopreservation solutions in vivo and in vitro. This study developed a new cryopreservation solution for CAR-T cells, and it was safe and also had negligible effects on the CAR-T cells antitumor activity.

MRTF-A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration.

Myocardin-related transcription factor-A/serum response factor (MRTF-A/SRF), a well-known transcriptional programme, has been proposed to play crucial roles in skeletal muscle development and function. However, whether MRTF-A participates in muscle regeneration and the molecular mechanisms are not completely understood. Here, we show that MRTF-A levels are highly correlated with myogenic genes using a RNA-seq assay, which reveal that MRTF-A knockdown in C2C12 cells significantly reduces PAX7 expression. Subsequent in vitro and in vivo data show that MRTF-A and PAX7 present identical expression patterns during myoblast differentiation and CTX-induced muscle injury and repair. Remarkably, MRTF-A overexpression promotes myoblast proliferation, while inhibiting cell differentiation and the expression of MyoD and MyoG. MRTF-A loss of function produces the opposite effect. Moreover, mice with lentivirus (MRTF-A) injection possesses more PAX7+ satellite cells, but less differentiating MyoD+ and MyoG+ cells, leading subsequently to diminished muscle regeneration. Our mechanistic results reveal that MRTF-A contributes to PAX7-mediated myoblast self-renewal, proliferation, and differentiation by binding to its distal CArG box. Overall, we propose that MRTF-A functions as a novel PAX7 regulator upon myoblast commitment to differentiation, which could provide pathways for dictating muscle stem cell fate and open new avenues to explore stem cell-based therapy for muscle degenerative diseases.

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy.

As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

The metabolic and immunological characteristics of pregnant women with COVID-19 and their neonates.

Our aim was to investigate whether SARS-CoV-2 infection raised high risks of late pregnancy complications, and posed health problems in fetuses and neonates. We analyzed the data of COVID-19 pregnant women with COVID-19 during late pregnancy and their neonates. Eleven out of 16 (69%) pregnant women with COVID-19 had ++ or +++ of ketone body in urine. The blood uric acid of pregnant patients was 334 µmol/L (IQR, 269-452). D-dimer and FDP in pregnant patients were 3.32 mg/L (IQR, 2.18-4.21) and 9.6 mg/L (IQR, 5.9-12.4). Results of blood samples collected at birth showed that 16 neonates had leukocytes (15.7 × 109/L (IQR, 13.7-17.2)), neutrophils (11.1 × 109/L (IQR, 9.2-13.2)), CK (401 U/L (IQR, 382-647)), and LDH (445 U/L (IQR, 417-559)). Twenty-four hours after birth, a neonate from COVID-19 woman had fever and positive of SARS-CoV-2 gene. Another woman had strongly positive for SARS-CoV-2 gene (+++) for 4 weeks, and delivered one neonate who had SARS-CoV-2 IgM (46 AU/mL) and IgG (140 AU/mL) on day 1 after birth. In the third trimester, COVID-19 infection in pregnant patients raised high risks of ketonuria, hypercoagulable state, and hyperfibrinolysis, which may lead to severe complications. COVID-19 increased the inflammatory responses of placenta, and fetuses and neonates had potential organ dysregulation and coagulation disorders. There was a potential intrauterine transmission while pregnant women had high titer of SARS-CoV-2, but it is necessary to detect SARS-CoV-2 in the blood cord, placenta, and amniotic fluid to further confirm intrauterine infection of fetuses.

The Metabolic Changes and Immune Profiles in Patients With COVID-19.

To explore the metabolic changes and immune profiles in patients with COVID-19, we analyzed the data of patients with mild and severe COVID-19 as well as young children with COVID-19. Of the leukocytes, 47% (IQR, 33-59) were lymphocytes [2.5 × 109/L (IQR, 2.2-3.3)], and monocytes were 0.51 × 109/L (IQR, 0.45-0.57) in young children with COVID-19. In 32 mild COVID-19 patients, circulating monocytes were 0.45 × 109/L (IQR, 0.36-0.64). Twenty-one severe patients had low PO2 [57 mmHg (IQR, 50-73)] and SO2 [90% (IQR, 86-93)] and high lactate dehydrogenase [580 U/L (IQR, 447-696)], cardiac troponin I [0.07 ng/mL (IQR, 0.02-0.30)], and pro-BNP [498 pg/mL (IQR, 241-1,726)]. Serum D-dimer and FDP were 9.89 mg/L (IQR, 3.62-22.85) and 32.7 mg/L (IQR, 12.8-81.9), and a large number of RBC (46/µL (IQR, 4-242) was presented in urine, a cue of disseminated intravascular coagulation (DIC) in severe patients. Three patients had comorbidity with diabetes, and 18 patients without diabetes also presented high blood glucose [7.4 mmol/L (IQR, 5.9-10.1)]. Fifteen of 21 (71%) severe cases had urine glucose +, and nine of 21 (43%) had urine ketone body +. The increased glucose was partially caused by reduced glucose consumption of cells. Severe cases had extraordinarily low serum uric acid [176 µmol/L (IQR, 131-256)]. In the late stage of COVID-19, severe cases had extremely low CD4+ T cells and CD8+ T cells, but unusually high neutrophils [6.5 × 109/L (IQR, 4.8-9.6)], procalcitonin [0.27 ng/mL (IQR, 0.14-1.94)], C-reactive protein [66 mg/L (IQR, 25-114)] and an extremely high level of interleukin-6. Four of 21 (19%) severe cases had co-infection with fungi, and two of 21 (9%) severe cases had bacterial infection. Our findings suggest that, severe cases had acute respiratory distress syndrome (ARDS) I-III, and metabolic disorders of glucose, lipid, uric acid, etc., even multiple organ dysfunction (MODS) and DIC. Increased neutrophils and severe inflammatory responses were involved in ARDS, MODS, and DIC. With the dramatical decrease of T-lymphocytes, severe cases were susceptible to co-infect with bacteria and fungi in the late stage of COVID-19. In young children, extremely high lymphocytes and monocytes might be associated with the low morbidity of COVID-19. The significantly increased monocytes might play an important role in the recovery of patients with mild COVID-19.

In Search of the E. coli Compounds that Change the Antibiotic Production Pattern of Streptomyces coelicolor During Inter-species Interaction.

The aim of this work was to investigate the interaction between E.coli and Streptomyces coelicolor A3 (2) for the increased production of undecylprodigiosin and identify the E. coli actives mediating this inter-species interaction. The antibiotics of interest were the red-pigmented undecylprodigiosin and blue-pigmented actinorhodin. Pure cultures of S. coelicolor in a defined medium produced higher concentrations of actinorhodin compared to those of undecylprodigiosin. The latter however, is more important due to its immunosuppressive and antitumor properties. As a strategy to increase undecylprodigiosin production, we added separately, live cells and heat-killed cells of E. coli C600, and the cell-free supernatant of E. coli culture to S. coelicolor cultures in shake flasks. The interaction with live cells of E. coli altered the antibiotic production pattern and undecylprodigiosin production was enhanced by 3.5-fold compared to the pure cultures of S. coelicolor and actinorhodin decreased by 15-fold. The heat-killed cells of E. coli however, had no effect on antibiotic production. In all cases, growth and glucose consumption of S. coelicolor remained almost the same as those observed in the pure culture indicating that the changes in antibiotic production were not due to nutritional stress. Results with cell-free supernatant of E. coli culture indicated that the interaction between S. coelicolor and E. coli was mediated via diffusible molecule(s). Using a set of extraction procedures and agar-well diffusion bioassays, we isolated and preliminarily identified a class of compounds. For the preliminary verification, we added the compound which was the common chemical structural moiety in this class of compounds to the pure S. coelicolor cultures. We observed similar effects on antibiotic production as with the live E. coli cells and their supernatant indicating that this class of compounds secreted by E. coli indeed could act as actives during interspecies interaction and increase the production of undecylprodigiosin.