CVD phenotyping in oncologic disorders: cardio-miRNAs as a potential target to improve individual outcomes in revers cardio-oncology.

With the increase of aging population and prevalence of obesity, the incidence of cardiovascular disease (CVD) and cancer has also presented an increasing tendency. These two different diseases, which share some common risk factors. Relevant studies in the field of reversing Cardio-Oncology have shown that the phenotype of CVD has a significant adverse effect on tumor prognosis, which is mainly manifested by a positive correlation between CVD and malignant progression of concomitant tumors. This distal crosstalk and the link between different diseases makes us aware of the importance of diagnosis, prediction, management and personalized treatment of systemic diseases. The circulatory system bridges the interaction between CVD and cancer, which suggests that we need to fully consider the systemic and holistic characteristics of these two diseases in the process of clinical treatment. The circulating exosome-miRNAs has been intrinsically associated with CVD -related regulation, which has become one of the focuses on clinical and basic research (as biomarker). The changes in the expression profiles of cardiovascular disease-associated miRNAs (Cardio-miRNAs) may adversely affect concomitant tumors. In this article, we sorted and screened CVD and tumor-related miRNA data based on literature, then summarized their commonalities and characteristics (several important pathways), and further discussed the conclusions of Cardio-Oncology related experimental studies. We take a holistic approach to considering CVD as a risk factor for tumor malignancy, which provides an in-depth analysis of the various regulatory mechanisms or pathways involved in the dual attribute miRNAs (Cardio-/Onco-miRNAs). These mechanisms will be key to revealing the systemic effects of CVD on tumors and highlight the holistic nature of different diseases. Therefore, the Cardio-miRNAs should be given great attention from researchers in the field of CVD and tumors, which might become new targets for tumor treatment. Meanwhile, based on the principles of precision medicine (such as the predictive preventive personalized medicine, 3PM) and reverse Cardio-oncology to better improve individual outcomes, we should consider developing personalized medicine and systemic therapy for cancer from the perspective of protecting cardiovascular function.

Fusobacterium nucleatum Promotes Megakaryocyte Maturation in Patients with Gastric Cancer via Inducing the Production of Extracellular Vesicles Containing 14-3-3ε.

Fusobacterium nucleatum colonization contributes to the occurrence of portal vein thrombosis in patients with gastric cancer (GC). However, the underlying mechanism by which F. nucleatum promotes thrombosis remains unclear. In this study, we recruited a total of 91 patients with GC and examined the presence of F. nucleatum in tumor and adjacent non-tumor tissues by fluorescence in situ hybridization and quantitative PCR. Neutrophil extracellular traps (NETs) were detected by immunohistochemistry. Extracellular vesicles (EVs) were extracted from the peripheral blood and proteins in the EVs were identified by mass spectrometry (MS). HL-60 cells differentiated into neutrophils were used to package engineered EVs to imitate the EVs released from NETs. Hematopoietic progenitor cells (HPCs) and K562 cells were used for megakaryocyte (MK) in vitro differentiation and maturation to examine the function of EVs. We observed that F. nucleatum-positive patients had increased NET and platelet counts. EVs from F. nucleatum-positive patients could promote the differentiation and maturation of MKs and had upregulated 14-3-3 proteins, especially 14-3-3ε. 14-3-3ε upregulation promoted MK differentiation and maturation in vitro. HPCs and K562 cells could receive 14-3-3ε from the EVs, which interacted with GP1BA and 14-3-3ζ to trigger PI3K-Akt signaling. In conclusion, we identified for the first time that F. nucleatum infection promotes NET formation, which releases EVs containing 14-3-3ε. These EVs could deliver 14-3-3ε to HPCs and promote their differentiation into MKs via activation of PI3K-Akt signaling.

Association of hepatitis B virus DNA levels with overall survival for advanced hepatitis B virus-related hepatocellular carcinoma under immune checkpoint inhibitor therapy.

BACKGROUND: High hepatitis B virus (HBV) DNA level is an independent risk factor for postoperative HBV-associated liver cancer recurrence. We sought to examine whether HBV DNA level and antiviral therapy are associated with survival outcomes in patients with advanced hepatocellular carcinoma (HCC) treated with anti-programmed cell death protein 1 (PD-1)based immunotherapy. METHODS: This single-institution retrospective analysis included 217 patients with advanced HBV-related HCC treated from 1 June 2018, through 30 December 2020. Baseline information was compared between patients with low and high HBV DNA levels. Overall survival (OS) and progression-free survival (PFS) were compared, and univariate and multivariate analyses were applied to identify potential risk factors for oncologic outcomes. RESULTS: The 217 patients included in the analysis had a median survival time of 20.6 months. Of these HBV-associated HCC patients, 165 had known baseline HBV DNA levels. Baseline HBV DNA level was not significantly associated with OS (P = 0.59) or PFS (P = 0.098). Compared to patients who did not receive antiviral therapy, patients who received antiviral therapy had significantly better OS (20.6 vs 11.1 months, P = 0.020), regardless of HBV DNA levels. Moreover, antiviral status (adjusted HR = 0.24, 95% CI 0.094-0.63, P = 0.004) was an independent protective factor for OS in a multivariate analysis of patients with HBV-related HCC. CONCLUSIONS: HBV viral load does not compromise the clinical outcome of patients with HBV-related HCC treated with anti-PD-1-based immunotherapy. The use of antiviral therapy significantly improves survival time of HBV-related HCC patients.

The inter-link of ageing, cancer and immunity: findings from real-world retrospective study.

BACKGROUND: Although the concept of declined immune function associated with cancer has been accepted extensively, real-world clinical studies focusing on analysis of the peripheral blood immune changes underlying ageing, immunity and cancer are scarce. METHODS: In this case-control study, we retrospectively analysed 1375 cancer patients and enrolled 275 age and gender matched healthy individuals. Flow cytometry was conducted to assess the immune changes. Further analysis was examined by SPSS 17.0 and GraphPad Prism 9 software. RESULTS: Cancer patients showed obviously decreased CD3+ T, CD3+CD4+ Th, CD3+CD8+ CTL, CD19+ B, CD16+CD56+ NK cell counts and lower percentage of PD-1 (programmed cell death protein-1, PD-1) positive cells than healthy control (P < 0.0001). For cancer patients, the reference range of circulating percentage of PD-1+CD45+ cells, PD-1+CD3+ T cells, PD-1+CD3+CD4+ Th cells and PD-1+CD3+CD8+ CTL (Cytotoxic T Lymphocyte, CTL) were 11.2% (95% CI 10.8%-11.6%), 15.5% (95% CI 14.7%-16.0%), 15.4% (95% CI 14.9%-16.0%) and 14.5% (95% CI 14.0%-15.5%), respectively. Moreover, the reduction of CD3+ T, CD3+CD4+ Th, CD3+CD8+ CTL, CD19+ B cell counts accompanied with age and stage advancing (P < 0.05). CD16+CD56+ NK cells decreased with stage, but elevated in aged and male cancer patients (P < 0.05). Additionally, the percentage of PD-1 positive cells varied across cancer types, raised with age and stage. Head and neck, pancreatic, gynaecological and lung demonstrated a higher level of the percentage of PD-1 positive cells than melanoma, prostate, and breast cancer (P < 0.05). CONCLUSIONS: This study provides the reference range of the percentage of PD-1 positive cells on peripheral blood, confirms the decreased immune cells and a series of immune changes accompanying with cancer, expands our real world evidence to better understand the interactions of ageing, cancer and immunity. Moreover, the circulating percentage of PD-1 positive cells shows similar tumor type distribution with tumor mutational burden (TMB), supports that it maybe a potential predictive biomarker for immune checkpoint inhibitor therapy.

Immune-related pneumonitis requiring low-dose prednisone maintenance in one patient with durable complete response.

INTRODUCTION: Immune-related pneumonitis is an uncommon but potentially life-threatening adverse event associated with anti-programmed cell death protein-1 therapy, and has a higher recurrence rate than that of other pneumonitis. Glucocorticoids are the first treatment of choice for patients with immune-related pneumonitis over grade 1. Given the toxicity associated with glucocorticoids, they should be withdrawn gradually as soon as pneumonitis is controlled. However, low-dose glucocorticoids are maintained in some patients to prevent immune-related pneumonitis. CASE REPORT: We report a rare case of a patient with Hodgkin lymphoma who developed grade 2 immune-related pneumonitis, requiring long-term low-dose glucocorticoid maintenance therapy, during which pneumonitis disappeared, and complete response was achieved. MANAGEMENT AND OUTCOME: Tislelizumab treatment was stopped tentatively, and the patient was given prednisone at an initiating dose of 1 mg/kg/d. The cough symptoms were relieved significantly, and pneumonitis was reduced. The prednisone gradually dwindled, but the immune-related pneumonitis was recurrent, requiring prednisone 10 mg daily maintenance therapy. Subsequently, prednisone and tislelizumab were administered simultaneously, and at present, pneumonitis disappeared and the lesions are in complete remission. DISCUSSION: Low-dose glucocorticoids might play an important role in controlling the recurrence and development of immune-related pneumonitis. The dose and course of glucocorticoid in immune-related pneumonitis patients should be individualized to minimize the toxicity of glucocorticoid.

Efficacy and Safety of Apatinib in Patients with Recurrent or Refractory Melanoma.

BACKGROUND: The prognosis of patients with metastatic malignant melanoma is very poor and partly due to resistance to conventional chemotherapies. The study's objectives were to assess the activity and tolerability of apatinib, an oral small molecule anti-angiogenesis inhibitor, in patients with recurrent advanced melanoma. METHODS: This was a single-arm, single-center phase II trial. The primary endpoint was progression-free survival (PFS) and the secondary endpoints were objective response rate (ORR), disease control rate (DCR), and overall survival (OS). Eligible patients had received at least one first-line therapy for advanced melanoma and experienced recurrence. Apatinib (500 mg) was orally administered daily. RESULTS: Fifteen patients (V660E BRAF status: 2 mutation, 2 unknown, 11 wild type) were included in the analysis. The median PFS was 4.0 months. There were two major objective responses, for a 13.3% response rate. Eleven patients had stable disease, with a DCR of 86.7%. The median OS was 12.0 months. The most common treatment-related adverse events of any grade were hypertension (80.0%), mucositis oral (33.3%), hand-foot skin reaction (26.7%), and liver function abnormalities, hemorrhage, diarrhea (each 20%). The only grade ≥3 treatment-related adverse effects that occurred in 2 patients was hypertension (6.7%) and mucositis (6.7%). No treatment-related deaths occurred. CONCLUSION: Apatinib showed antitumor activity as a second- or above-line therapy in patients with malignant melanoma. The toxicity was manageable. CLINICALTRIALS.GOV IDENTIFIER: NCT03383237.

CELF2 suppresses non-small cell lung carcinoma growth by inhibiting the PREX2-PTEN interaction.

The phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway is important in the regulation of cell proliferation through its production of phosphatidylinositol 3,4,5-triphosphate (PIP3). Activation of this pathway is frequently observed in human cancers, including non-small cell lung carcinoma. The PI3-K/Akt pathway is negatively regulated by the dual-specificity phosphatase and tensin homolog (PTEN) protein. PTEN acts as a direct antagonist of PI3-K by dephosphorylating PIP3. Studies have shown that PTEN phosphatase activity is inhibited by PREX2, a guanine nucleotide exchanger factor (GEF). Multiple studies revealed that CELF2, an RNA binding protein, cooperates synergistically with PTEN as a tumor suppressor in multiple cancers. However, the underlying mechanism as to how CELF2 enhances PTEN activity remains unclear. Here, we report that CELF2 interacts with PREX2 and reduces the association of PREX2 with PTEN. Consistent with this observation, PTEN phosphatase activity is upregulated with CELF2 overexpression. In addition, overexpression of CELF2 represses both Akt phosphorylation and cell proliferation only in the presence of PTEN. In an ex vivo study, CELF2 gene delivery could significantly inhibit patient-derived xenografts (PDX) tumor growth. To further investigate the clinical relevance of this finding, we analyzed 87 paired clinical lung adenocarcinoma samples and the results showed that CELF2 protein expression is downregulated in tumor tissues and associated with poor prognosis. The CELF2 gene is located on the chromosome 10p arm, a region frequently lost in human cancers, including breast invasive carcinoma, low-grade glioma and glioblastoma. Analysis of TCGA datasets showed that CELF2 expression is also associated with shorter patient survival time in all these cancers. Overall, our work suggests that CELF2 plays a novel role in PI3-K signaling by antagonizing the oncogenic effect of PREX2.

Exploring a Tumor-Intrinsic PD-L1 Signal with Proximity-Dependent Biotin Identification in Lung Cancer Cells.

Blocking the PD-L1/PD-1 interaction with an antibody produces a durable response in patients with diverse advanced cancers. However, it remains elusive on whether the engagement of PD-L1 to PD-1 leads to tumor-intrinsic signaling. In this study, we aim to explore novel protein substrates participating in transducing this tumor-intrinsic PD-L1 signaling. To this end, we performed a BioID (proximity-dependent biotin identification) assay, in which we fused PD-L1 to BirA* (a promiscuous mutant of bacterial biotin ligase BirA) and overexpressed it in the lung adenocarcinoma A549 cell line. Through streptavidin affinity capture and mass spectrometry analysis, we identified 57 candidate proteins including 18 PD-L1/PD-1-interaction-dependent neighbors. In addition to this, 9 out of 57 candidates were involved in the EGFR signaling pathway, which is known to play a critical role in tumorigenesis and multiple therapeutic resistances of lung cancer. This study will provide a new insight in understanding tumor-intrinsic PD-L1-signaling effectors of lung cancer.

S-15 in combination of Akt inhibitor promotes the expansion of CD45RA-CCR7+ tumor infiltrating lymphocytes with high cytotoxic potential and downregulating PD-1+Tim-3+ cells as well as regulatory T cells.

BACKGROUND: Autologous tumor-infiltrating lymphocytes (Tils) immunotherapy is a promising treatment in patients with advanced hepatocellular cancer. Although Tils treatment has shown great promise, their persistence and the efficacy after adoptive-transfer are insufficient and remain a challenge. Studies have demonstrated that IL-15 and Akt inhibitor can regulate T cell differentiation and memory. Here, we constructed S-15 (Super human IL-15), a fusion protein consisting of human IL-15, the sushi domain of the IL-15 receptor α chain and human IgG-Fc. Herein we compared the effects of S-15 with IL-2 or in combination with Akti on the expansion and activation of Tils. METHODS: Hepatocellular cancer tissues were obtained from 6 patients, Tils were expanded using IL-2, IL-2/S-15, IL-2/Akti or in combination IL-2/S-15/Akti. At day 10, anti-CD3 antibody was added to the culture media and expanded to day 25. The composition, exhaustion and T-cell differentiation markers (CD45RA/CCR7) were analyzed by flow cytometry. RESULTS: We found that IL-2/S-15/Akti expanded Tils and showed the highest percentage of central memory CD45RA-CCR7+ phenotype prior to anti-CD3 antibody activation and after anti-CD3 antibody activation. T cells cultured with IL-2/S-15/Akti exhibited a mixture of CD4+, CD8+, and CD3+CD4-CD8- T cells; S-15 in combination with Akt inhibitor downregulated the expression of PD-1+Tim-3+ on Tils and decreased the Tregs in Tils. Additionally, the Tils expanded in the presence of the Akt inhibitor and S-15 showed enhanced antitumor activity as indicated by the increase in IFN-γ producing tumor infiltrating CD8+ T cells and without comprising the Tils expansion. CONCLUSION: Our study elucidates that IL-2/S-15/Akti expanded Tils and represent a viable source for the cellular therapy for patients with hepatocellular cancer.

TGFß1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFß1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFß1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFß1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFß1 exposure leads to an enrichment of a sub-population of CD44+ CD90+ cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFß1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFß1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFß1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target.

Establishment of lung cancer patient-derived xenograft models and primary cell lines for lung cancer study.

BACKGROUND: The overall 5-year survival rate of lung cancer is about 15% even with therapeutic drugs like tyrosine kinase inhibitors. Ideal models are urgently needed for exploring mechanisms and finding new drugs. Patient-derived xenografts (PDX) models and primary cells are both used to screen therapeutic regimens for cancer. However, PDX models and primary cells from the same patient are difficult to establish. Their consistency to the original tumor tissue is not well studied. METHODS: 31 lung cancer patient tissues were procured to establish the lung cancer PDX models and primary cell lines. Tumor growth measurements, histological and immunohistochemistry analysis, Western blotting, EGFR and K-RAS mutation detection and gefitinib sensitive assay were performed to evaluate the characteristic of established PDX models. Immunofluorescence analysis, anchorage-independent cell growth, Western blotting and gefitinib sensitive assay were performed to assay the characteristic of established primary cell lines. The whole-exome sequencing was used to compare the characteristic of the patient's tumor tissue, established PDX and primary cell line. RESULTS: Twenty-one lung cancer PDX models (67.74%, 21/31) and ten primary cell lines (32.25%, 10/31) were established from patients' tumor tissues. The histology and pathological immunohistochemistry of PDX xenografts are consistent with the patients' tumor samples. Various signal pathways were activated in different PDX models (n = 5) and primary cell lines (n = 2). EGFR mutation PDX model and primary cell line (LG1) were sensitive to gefitinib treatment. The expression of CK8/18, TTF1 and NapsinA in LG1 and LG50 primary cells were also positive. And the activated signal pathways were activated in LG1 and LG50 primary cell lines. Furthermore, the gene mutation in PDX tumor tissues and primary cell line (LG50) was consistent with the mutation in LG50 patient's tumor tissues. CONCLUSION: These data suggested that established lung cancer PDX models and primary cell lines reserved mostly molecular characteristics of primary lung cancer and could provide a new tool to further understand the mechanisms and explore new therapeutic strategies.

Pretreatment CD8 + PD-1 + to CD4 + PD-1 + ratio is associated with the prognosis of advanced melanoma patients treated with PD-1 inhibitors.

The aim of this study was to determine whether the pretreatment CD8 + PD-1 + to CD4 + PD-1 + (PERLS) ratio is an independent risk prognostic factor of advanced melanoma patients. We retrospectively analyzed the efficacy and flow cytometry data from advanced melanoma patients who received PD-1 inhibitor as monotherapy between January 1, 2018 and January 26, 2022. Fifty-nine patients were enrolled, the PERLS cutoff was 1.125. PERLS did not correlate with clinical characteristics but were significantly associated with baseline CD8 + , CD4 + , and CD8 + PD-1 + T cells. The mean overall survival and the progression-free survival were 45.8 and 17.1 months for the low PERLS group (n = 39), compared with 29.9 ( P  = 0.001) and 9.7 ( P  = 0.003) months for the high PERLS group ( n  = 20), respectively. Pretreatment PERLS might contribute to selecting patients before receiving anti-PD-1 therapy.

Transient-resting culture after activation enhances the generation of CD8+ stem cell-like memory T cells from peripheral blood mononuclear cells.

Adoptive cell therapy (ACT) has revolutionized the treatment of patients with cancer. The success of ACT depends largely on transferred T cell status, particularly their less-differentiated state with stem cell-like properties, which enhances ACT effectiveness. Stem cell-like memory T (TSCM) cells exhibit continuous self-renewal and multilineage differentiation similar to pluripotent stem cells. TSCM cells are promising candidates for cancer immunotherapies, whereas maintenance of a more stem-cell-like state before transfer is challenging. Here, we established a highly efficient protocol for generating CD8+ TSCM cells from peripheral blood mononuclear cells (PBMCs). The process involved activating PBMCs using anti-CD3 monoclonal antibody and RetroNectin, followed by a transient-resting culture period (24 h) and subsequent long-term expansion in vitro with interlukien-2. We report that this transient-resting culture after activation preserves CD8+ T cells in a stem memory phenotype (CD95+ CD45RA+ CCR7+) compared to the conventional culture method. Further, this approach reduces the expression of T cell immunoglobulin mucin-3, an exhaustion marker, and increases the expression of T cell factor-1, a master regulator of stemness even after long-term culture compared to the conventional culture method. In conclusion, our study presents a simplified and cost-effective method for generating and expanding CD8+ TSCM cells ex vivo. This approach streamlines the optimization of cancer immunotherapy using ACT.

Combination therapy using low-dose anlotinib and immune checkpoint inhibitors for extensive-stage small cell lung cancer.

Background: This study evaluated the efficacy and safety of low-dose anlotinib combined with immune checkpoint inhibitors as second-line or later treatment for extensive-stage small cell lung cancer (ES-SCLC). Methods: The study included 42 patients with ES-SCLC who were treated with low-dose anlotinib combined with programmed cell death protein 1/programmed cell death-ligand 1 inhibitors at Henan Cancer Hospital between March 2019 and August 2022. We retrospectively analyzed the efficacy and safety data for these patients. Indicators assessed included progression-free survival (PFS), overall survival (OS), the overall response rate (ORR), the disease control rate (DCR), and adverse events (AEs). Prognostic factors were identified in univariate and multivariate analyses. Results: Median PFS was 11.0 months (95% CI: 7.868-14.132) and median OS was 17.3 months (95% CI: 11.517-23.083). The ORR was 28.5% and the DCR was 95.2%. Treatment-related AEs were noted in 27 patients (64.3%), the most common of which was thyroid dysfunction (26.2%). Grade 3/4 treatment-related AEs were observed in two patients (4.8%). Conclusions: A combination of low-dose anlotinib and immune checkpoint inhibitors as second-line or later treatment for ES-SCLC may achieve longer PFS and OS and have manageable AEs.

AMPK-upregulated microRNA-708 plays as a suppressor of cellular senescence and aging via downregulating disabled-2 and mTORC1 activation.

Senescence-associated microRNAs (SA-miRNAs) are important molecules for aging regulation. While many aging-promoting SA-miRNAs have been identified, confirmed aging-suppressive SA-miRNAs are rare, that impeded our full understanding on aging regulation. In this study, we verified that miR-708 expression is decreased in senescent cells and aged tissues and revealed that miR-708 overexpression can alleviate cellular senescence and aging performance. About the molecular cascade carrying the aging suppressive action of miR-708, we unraveled that miR-708 directly targets the 3'UTR of the disabled 2 (Dab2) gene and inhibits the expression of DAB2. Interestingly, miR-708-caused DAB2 downregulation blocks the aberrant mammalian target of rapamycin complex 1 (mTORC1) activation, a driving metabolic event for senescence progression, and restores the impaired autophagy, a downstream event of aberrant mTORC1 activation. We also found that AMP-activated protein kinase (AMPK) activation can upregulate miR-708 via the elevation of DICER expression, and miR-708 inhibitor is able to blunt the antiaging effect of AMPK. In summary, this study characterized miR-708 as an aging-suppressive SA-miRNA for the first time and uncovered a new signaling cascade, in which miR-708 links the DAB2/mTOR axis and AMPK/DICER axis together. These findings not only demonstrate the potential role of miR-708 in aging regulation, but also expand the signaling network connecting AMPK and mTORC1.

Repurposing pentamidine for cancer immunotherapy by targeting the PD1/PD-L1 immune checkpoint.

Immunotherapy has emerged as an effective therapeutic approach to several cancer types. The reinvigoration of tumor-infiltrating lymphocyte-mediated immune responses via the blockade of immune checkpoint markers, such as program cell death-1 (PD-1) or its cognate ligand PD-L1, has been the basis for developing clinically effective anticancer therapies. We identified pentamidine, an FDA-approved antimicrobial agent, as a small-molecule antagonist of PD-L1. Pentamidine enhanced T-cell-mediated cytotoxicity against various cancer cells in vitro by increasing the secretion of IFN-γ, TNF-α, perforin, and granzyme B in the culture medium. Pentamidine promoted T-cell activation by blocking the PD-1/PD-L1 interaction. In vivo administration of pentamidine attenuated the tumor growth and prolonged the survival of tumor-bearing mice in PD-L1 humanized murine tumor cell allograft models. Histological analysis of tumor tissues showed an increased number of tumor-infiltrating lymphocytes in tissues derived from pentamidine-treated mice. In summary, our study suggests that pentamidine holds the potential to be repurposed as a novel PD-L1 antagonist that may overcome the limitations of monoclonal antibody therapy and can emerge as a small molecule cancer immunotherapy.

miR-146a impedes the anti-aging effect of AMPK via NAMPT suppression and NAD+/SIRT inactivation.

Nicotinamide adenine dinucleotide (NAD+) is indispensable for the anti-aging activity of the sirtuin (SIRT) family enzymes. AMP-activated protein kinase (AMPK) upregulates NAD+ synthesis and SIRT activity in a nicotinamide phosphoribosyltransferase (NAMPT)-dependent manner. However, the molecular mechanisms that affect AMPK-driven NAMPT expression and NAD+/SIRT activation remain unclear. In this study, we tried to identify senescence-associated microRNAs (miRNAs) that negatively regulate the cascade linking AMPK and NAMPT expression. miRNA-screening experiments showed that the expression of miR-146a increased in senescent cells but decreased following AMPK activation. Additionally, miR-146a overexpression weakened the metformin-mediated upregulation of NAMPT expression, NAD+ synthesis, SIRT activity, and senescence protection, whereas treatment with the miR-146a inhibitor reversed this effect. Importantly, these findings were observed both in vitro and in vivo. Mechanistically, miR-146a directly targeted the 3'-UTR of Nampt mRNA to reduce the expression of NAMPT. AMPK activators metformin and 5-aminoimidazole-4-carboxamide (AICAR) hindered miR-146a expression at the transcriptional level by promoting IκB kinase (IKK) phosphorylation to attenuate nuclear factor-kappaB (NF-κB) activity. These findings identified a novel cascade that negatively regulates the NAD+/SIRT pathway by suppressing miR-146a-mediated NAMPT downregulation. Furthermore, our results showed that miR-146a impedes the anti-aging effect of AMPK. This mutual inhibitory relationship between miR-146a and AMPK enriches our understanding of the molecular connections between AMPK and SIRT and provides new insight into miRNA-mediated NAD+/SIRT regulation and an intervention point for the prevention of aging and age-related diseases.

A high interferon gamma signature of CD8+ T cells predicts response to neoadjuvant immunotherapy plus chemotherapy in gastric cancer.

Background: While the tumor microenvironment (TME) affects immune checkpoint blockade (ICB) efficacy, ICB also reshapes the characteristics of TME. Thus far, studies have focused on the TME evolution during neoadjuvant or adjuvant ICB therapy in gastric cancer (GC). However, the interaction between TME characteristics and neoadjuvant immunotherapy plus chemotherapy remains to be elucidated. Methods: We performed single-cell RNA sequencing on ten GC specimens pre- and post-neoadjuvant camrelizumab plus mFOLFOX6 to determine the impact of the TME on the efficacy of the combination therapy and the remodeling of TME by the therapy. Results: A high baseline interferon gamma (IFN-γ) signature in CD8+ T cells predicts better responses to the combination therapy. We also observed that the IFN-γ signature significantly decreased in multiple cell types, and the exhausted signature of CD8+ T cells was significantly suppressed during the neoadjuvant therapy. Conclusions: Our data reveal interactions between the TME and neoadjuvant immunotherapy plus chemotherapy in GC. Importantly, it also highlights the signature of CD8+ T cells in predicting response to the combination therapy in GC.

In vivo growth of subclones derived from Lewis lung carcinoma is determined by the tumor microenvironment.

Heterogeneity is a fundamental feature of human tumors and plays a major role in drug resistance and disease progression. In the present study, we selected single-cell-derived cell lines (SCDCLs) derived from Lewis lung carcinoma (LLC1) cells to investigate tumorigenesis and heterogeneity. SCDCLs were generated using limiting dilution. Five SCDCLs were subcutaneously injected into wild-type C57BL/6N mice; however, they displayed significant differences in tumor growth. Subclone SCC1 grew the fastest in vivo, whereas it grew slower in vitro. The growth pattern of SCC2 was the opposite to that of SCC1. Genetic differences in these two subclones showed marked differences in cell adhesion and proliferation. Pathway enrichment results indicate that signal transduction and immune system responses were the most significantly altered functional categories in SCC2 cells compared to those in SCC1 cells in vitro. The number and activation of CD3+ and CD8+ T cells and NK cells in the tumor tissue of tumor-bearing mice inoculated with SCC2 were significantly higher, whereas those of myeloid cells were significantly lower, than those in the SCC1 and LLC1 groups. Our results suggest that the in vivo growth of two subclones derived from LLC1 was determined by the tumor microenvironment rather than their intrinsic proliferative cell characteristics.

Pan-mTOR inhibitors sensitize the senolytic activity of navitoclax via mTORC2 inhibition-mediated apoptotic signaling.

Compounds with senolysis activity are discovered in recent years, featuring by their capacity to specifically eliminate senescent cells in vitro or in vivo. These compounds, referring to as Senolytics, provide a new method for aging counteraction and probably for geriatric disease amelioration. However, their clinical application is unpractical still, mainly because of the safety issue. In fact, the effective dose range even of the most potent senolytic cannot guarantee the safety requirements application for human being. Here, we report a study which investigated the combinational application of one potential senolytic molecule navitoclax, a Bcl-2 inhibitor with several mTOR inhibitors, to assess the influence of this combination on the senolytic outcome. Our results reveal that pan-mTOR inhibitors can reduce the dosage or timespan of navitoclax necessary for reaching IC50 and LT50 in senescent cells, also extend the lifespan of premature-aged Drosophila and mitigate the aging-related phenotype. Our results also confirmed that mTOR inhibitor sensitized senolytic cell death is apoptotic and pan-mTOR inhibitors PP242 and AZD8055 works more effectively than mTORC1 inhibitor Rapamycin. Mechanically, we verified the crucial role of mTORC2 inhibition contributes sensitization by increasing the expression of the pro-apoptotic protein Bim. In summary, this study firstly exposes the sensitization effect of pan-mTOR inhibitors on navitoclax-induced senolytic apoptosis, therefore providing novel evidence to show the advantage of drug combination on setting senotherapy. It also provides an intriguing clue to demonstrate the value of mTORC2 inhibition for apoptotic death of senescent cells.