Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Background: Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Methods: Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. Results: The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Conclusions: Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

Integrative analysis of DNA methylomes reveals novel cell-free biomarkers in lung adenocarcinoma.

Lung cancer is a leading cause of cancer-related deaths worldwide, with a low 5-year survival rate due in part to a lack of clinically useful biomarkers. Recent studies have identified DNA methylation changes as potential cancer biomarkers. The present study identified cancer-specific CpG methylation changes by comparing genome-wide methylation data of cfDNA from lung adenocarcinomas (LUAD) patients and healthy donors in the discovery cohort. A total of 725 cell-free CpGs associated with LUAD risk were identified. Then XGBoost algorithm was performed to identify seven CpGs associated with LUAD risk. In the training phase, the 7-CpGs methylation panel was established to classify two different prognostic subgroups and showed a significant association with overall survival (OS) in LUAD patients. We found that the methylation of cg02261780 was negatively correlated with the expression of its representing gene GNA11. The methylation and expression of GNA11 were significantly associated with LAUD prognosis. Based on bisulfite PCR, the methylation levels of five CpGs (cg02261780, cg09595050, cg20193802, cg15309457, and cg05726109) were further validated in tumor tissues and matched non-malignant tissues from 20 LUAD patients. Finally, validation of the seven CpGs with RRBS data of cfDNA methylation was conducted and further proved the reliability of the 7-CpGs methylation panel. In conclusion, our study identified seven novel methylation markers from cfDNA methylation data which may contribute to better prognosis for LUAD patients.

Baseline red blood cell distribution width and perforin, dynamic levels of interleukin 6 and lactate are predictors of mortality in patients with sepsis.

BACKGROUND: Sepsis is a critical illness often encountered in the intensive care unit. However, prognostic biomarkers for sepsis have limited sensitivity. This study aimed to identify more sensitive predictors of mortality through repeated monitoring of laboratory parameters. METHODS: Patients with sepsis (Sepsis 3.0 criteria met) were recruited and divided into the survivor and nonsurvivor groups after 28 days. Data on blood biochemistry, lymphocyte subsets, and cytokines were obtained on the first and seventh hospitalization days. Univariate and multivariate Cox regression analyses were performed to explore the correlation between these variables and patient mortality. RESULTS: Forty patients with sepsis were included. The mortality rate was 37.5%. Red blood cell distribution width-standard deviation (RDWSD) (hazard ratio [HR] = 1.107 [95% CI: 1.005-1.219], p = 0.040) and perforin level (HR = 1.001 [95% CI: 1-1.003], p = 0.035) on the first day, as well as lactate (HR = 112.064 [95% CI: 2.192-5729.629], p = 0.019) and interleukin 6 (IL-6) (HR = 1.005 [95% CI: 1.001-1.008], p = 0.014) levels on the seventh day, were independent risk factors of mortality. If the patients were divided into two groups based on RDWSD (normal: n = 31; increased: n = 9), the Kaplan-Meier curves showed that the group with increased RDWSD had a lower survival (p = 0.025). CONCLUSION: Baseline RDWSD and perforin, along with dynamic IL-6 and lactate levels, were independent predictors of mortality in patients with sepsis.

A GRIP-1-EZH2 switch binding to GATA-4 is linked to the genesis of rhabdomyosarcoma through miR-29a.

Terminal differentiation failure is an important cause of rhabdomyosarcoma genesis, however, little is known about the epigenetic regulation of aberrant myogenic differentiation. Here, we show that GATA-4 recruits polycomb group proteins such as EZH2 to negatively regulate miR-29a in undifferentiated C2C12 myoblast cells, whereas recruitment of GRIP-1 to GATA-4 proteins displaces EZH2, resulting in the activation of miR-29a during myogenic differentiation of C2C12 cells. Moreover, in poorly differentiated rhabdomyosarcoma cells, EZH2 still binds to the miR-29a promoter with GATA-4 to mediate transcriptional repression of miR-29a. Interestingly, once re-differentiation of rhabdomyosarcoma cells toward skeletal muscle, EZH2 was dispelled from miR-29a promoter which is similar to that in myogenic differentiation of C2C12 cells. Eventually, this expression of miR-29a results in limited rhabdomyosarcoma cell proliferation and promotes myogenic differentiation. We thus establish that GATA-4 can function as a molecular switch in the up- and downregulation of miR-29a expression. We also demonstrate that GATA-4 acts as a tumor suppressor in rhabdomyosarcoma partly via miR-29a, which thus provides a potential therapeutic target for rhabdomyosarcoma.

Ascitic Senescent T Cells Are Linked to Chemoresistance in Patients With Advanced High-Grade Serous Ovarian Cancer.

Senescent T cells are reported to be increased in patients with cancer and are poor prognostic indicators. However, the distribution of senescent T cells and their correlation with clinical features in high-grade serous ovarian cancer (HGSOC) is unknown. We detected the percentage of senescent T cells in the peripheral blood and ascites of patients with advanced HGSOC (n = 86) at diagnosis by flow cytometry. Compared with healthy donors, patients with HGSOC exhibited an accumulation of CD28-CD57+ (Tsen) CD8+ T cells in the peripheral blood and ascites. The frequency of Tsen CD8+ T cells in the peripheral blood was positively correlated with age and pretreatment serum CA125 and increased in patients with large volume ascites, whereas the frequency of Tsen CD8+ T cells in ascites was elevated in patients with lymph node metastasis. Patients with Tsen-high in ascites (>19.92%), but not in the peripheral blood, were more likely to be resistant to chemotherapy and had shorter progression-free survival. Tsen CD8+ T cells exhibited common senescence features including increased SA-ß-gal activity, declines in proliferation, loss of CD27 and gain of KLRG-1, and the production of cytokines. In ascites, the percentage of Tsen CD8+ T cells was positively correlated with levels of interleukin-10 and granzyme B. This study suggests the potential of ascitic Tsen CD8+ T cells at diagnosis as a prognostic biomarker in HGSOC.

Compromise or not? A case report of successful treatment of pembrolizumab-induced hepatitis in a patient with non-small cell lung cancer with low-dose methylprednisolone and bicyclol.

Pembrolizumab, an anti-programmed cell death protein 1 (PD-1) antibody, has been shown to improve survival in patients with non-small cell lung cancer (NSCLC) with high expression of programmed death-ligand 1 (PD-L1). Corticosteroids are the mainstay for most high-grade immune-related adverse events (irAEs) such as pembrolizumab-induced hepatitis. However, the dose and duration of corticosteroid therapy are not well defined. The objective of this case report was to describe a new treatment pattern for severe immune checkpoint inhibitor-associated hepatitis. Here, we report the case of a patient with metastatic lung adenocarcinoma who developed grade 3 immunotherapy-induced hepatitis after the first cycle of pembrolizumab. Alanine aminotransferase (ALT) levels peaked at 233 U/L. Hepatitis was alleviated after the administration of methylprednisolone. Therefore, we retreated the patient with pembrolizumab. However, aminotransferase levels increased again after the initiation of low-dose methylprednisolone or the reuse of pembrolizumab. Finally, hepatitis was controlled with low-dose methylprednisolone plus bicyclol, a Chinese hepatoprotective agent. Although the patient had been on low-dose methylprednisolone therapy for about six months, he showed a prompt response. During this period, we also found a dramatic decrease in the neutrophil-lymphocyte ratio (NLR), senescent T cells (CD8+ CD28- CD57+ ), and myeloid-derived suppressor cells (MDSCs) in the peripheral blood of the patient. To our knowledge, this is the first case report of successful management of grade 3 pembrolizumab-induced hepatitis with a combination of low-dose corticosteroids and bicyclol. The durable clinical response and changes in blood biomarkers indicate that low doses of corticosteroids do not compromise the efficacy of immune checkpoint inhibitors (ICIs). Therefore, this case may provide a new treatment pattern for severe immunotherapy-induced hepatitis.

Myeloid-derived suppressor cells: Roles in the tumor microenvironment and tumor radiotherapy.

Cancer progression is closely related to the tumor microenvironment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, signaling molecules and the extracellular matrix. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can impact the growth and evolution of cancerous cells. One of major cell components in the tumor microenvironment is myeloid-derived suppressor cells (MDSCs), which promote tumor growth and metastasis directly or indirectly by recognizing other immune cells, producing cytokines and exerting their immunosuppression functions. MDSCs have emerged as major regulators of immune responses in cancer and key targets for treating cancer. There are many limitations and side-effect in approaches of conventional cancer therapy, including radiotherapy. It has grown up to be a burgeoning field that a combination of radiotherapy and immunotherapy applied to cancer therapy. Therefore, it is fundamental to explore the immune mechanism in the process of cancer treatment. Here, we reviewed the recent progress of MDSCs in roles of the tumor microenvironment and tumor radiotherapy.

Histone methylation in DNA repair and clinical practice: new findings during the past 5-years.

DNA double-strand breaks (DSBs) are highly toxic lesions that can impair cellular homeostasis and genome stability to result in tumorigenesis for inappropriate repair. Although DSBs are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ), the related mechanisms are still incompletely unclear. Indeed, more and more evidences indicate that the methylation of histone lysine has an important role in choosing the pathways of DNA repair. For example, tri-methylated H3K36 is required for HR repair, while di-methylated H4K20 can recruit 53BP1 for NHEJ repair. Here, we reviewed the recent progress in the molecular mechanisms by which histone methylation functions in DNA double-strand breaks repair (DSBR). The insight into the mechanisms of histone methylation repairing DNA damage will supply important cues for clinical cancer treatment.