An aqueous extract of Prunella vulgaris L. ameliorates cadmium-induced bone loss by promoting osteogenic differentiation in female rats.

Cadmium (Cd) causes bone loss, concerning inhibiting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Prunella vulgaris L. (PV) has the potential for promoting osteogenic differentiation, but its influence on Cd-induced bone loss is unclear. This study investigated the effect of PV aqueous extract (PVE) on Cd-induced bone loss and its underlying mechanisms. Eight-week-old female SD rats were randomly assigned into four groups and treated for 16 weeks: Control, Cd (50 mg/L of Cd chloride), Cd + PV Low (125 mg/kg bw of PVE), and Cd + PV High (250 mg/kg bw of PVE). PV ameliorated femoral bone loss in Cd-treated rats manifested as increases in bone mineral density, bone volume, trabecular thickness, number, and area, and decreases in trabecular separation. Compared with Cd group, PV-treatment groups had higher serum levels of bone formation markers (ALP, BGP). Additionally, in PV-treatment groups, expressions of bone formation markers (Osterix, Runx2) and molecules involved in osteogenic differentiation signal pathway BMP/Smad (BMP4, Smad1/5/9) in the tibia of rats and isolated rat primary BMSCs were upregulated. These results suggest that PV alleviates Cd-induced bone loss by promoting osteogenic differentiation, which is likely associated with BMP/Smad pathway.

Cadmium contributes to atherosclerosis by affecting macrophage polarization.

Chronic cadmium (Cd) exposure contributes to the progression of atherosclerosis, but the direct role of Cd and its mechanisms in atherosclerosis remains incompletely understood. Atherosclerosis is a chronic inflammatory disease promoting macrophage polarization to M1 phenotype and producing pro-inflammations that are vital in regulating the inflammatory response. Herein, through a case-control study, we found that Cd exposure may promote the occurrence of carotid plaque via inflammation, where interleukin-6 (IL-6) may play an important role. We also combined in vivo and in vitro experiments to explore the underlying mechanism of Cd-promoted plaque formation and the production of IL-6. With or without cadmium chloride (CdCl2) fed ApoE-/- mouse and treated RAW264.7 cells, we found Cd accumulated in the aortas which significantly increased the plaque area in atherosclerotic mice, macrophage accumulation, and lipid accumulation, and Cd promoted M1 phenotype macrophage polarization reflected by the increased expression of CD86 which produced tumor necrosis factor-α (TNF-α) and IL-6. However, the influences on M2 phenotype and anti-inflammatory cytokines interleukin-4 (IL-4) and interferon-γ (IFN-γ) were non-significant. Moreover, we found that JAK2/STAT3 pathway was greatly activated in the plaques and CdCl2-treated macrophages. The inhibition of JAK2/STAT3 substantially reversed the Cd-stimulated macrophage M1 phenotype macrophage polarization and the expression of pro-inflammatory cytokines including TNF-α and IL-6. Altogether, Cd intensifies atherosclerosis by modulating macrophage polarization via JAK2/STAT3 to up-regulated the expression of IL-6.

Bone morphogenetic protein 4 is involved in cadmium-associated bone damage.

Cadmium (Cd) is a well-characterized bone toxic agent and can induce bone damage via inhibiting osteogenic differentiation. Bone morphogenetic protein (BMP)/SMAD signaling pathway can mediate osteogenic differentiation, but the association between Cd and BMP/SMAD signaling pathway is yet to be illuminated. To understand what elements of BMPs and SMADs are affected by Cd to influence osteogenic differentiation and if BMPs can be the biomarkers of which Cd-induced osteoporosis, human bone marrow mesenchymal stem cells (hBMSCs) were treated with cadmium chloride (CdCl2) in vitro to detect the expression of BMPs and SMADs, and 134 subjects were enrolled to explore if the BMPs can be potential biomarkers of Cd-associated bone damage. Our results showed that Cd exposure significantly promoted the adipogenic differentiation of hBMSCs and inhibited its osteogenic differentiation by inhibiting the expression of BMP-2/4, SMAD4, and p-SMAD1/5/9 complex. And mediation analyses yielded that BMP-4 mediated 39.32% (95% confidence interval 7.47, 85.00) of the total association between the Cd and the risk of Cd-associated bone damage. Moreover, during differentiation, BMP-4 had the potential to enhance mineralization compared with CdCl2 only group. These results reveal that BMP-4 can be a diagnostic biomarker and therapeutic target for Cd-associated bone damage.

MYCBP2 expression correlated with inflammatory cell infiltration and prognosis immunotherapy in thyroid cancer patients.

Introduction: Immune checkpoint inhibitors (ICIs) have shown promising results for the treatment of multiple cancers. ICIs and related therapies may also be useful for the treatment of thyroid cancer (TC). In TC, Myc binding protein 2 (MYCBP2) is correlated with inflammatory cell infiltration and cancer prognosis. However, the relationship between MYCBP2 expression and ICI efficacy in TC patients is unclear. Methods: We downloaded data from two TC cohorts, including transcriptomic data and clinical prognosis data. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to predict the efficacy of ICIs in TC patients. MCPcounter, xCell, and quanTIseq were used to calculate immune cell infiltration scores. Gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) were used to evaluate signaling pathway scores. Immunohistochemical (IHC) analysis and clinical follow up was used to identify the MYCBP2 protein expression status in patients and associated with clinical outcome. Results: A higher proportion of MYCBP2-high TC patients were predicted ICI responders than MYCBP2-low patients. MYCBP2-high patients also had significantly increased infiltration of CD8+ T cells, cytotoxic lymphocytes (CTLs), B cells, natural killer (NK) cells and dendritic cells (DC)s. Compared with MYCBP2-low patients, MYCBP2-high patients had higher expression of genes associated with B cells, CD8+ T cells, macrophages, plasmacytoid dendritic cells (pDCs), antigen processing and presentation, inflammatory stimulation, and interferon (IFN) responses. GSEA and ssGSEA also showed that MYCBP2-high patients had significantly increased activity of inflammatory factors and signaling pathways associated with immune responses.In addiation, Patients in our local cohort with high MYCBP2 expression always had a better prognosis and greater sensitivity to therapy while compared to patients with low MYCBP2 expression after six months clinic follow up. Conclusions: In this study, we found that MYCBP2 may be a predictive biomarker for ICI efficacy in TC patients. High MYCBP2 expression was associated with significantly enriched immune cell infiltration. MYCBP2 may also be involved in the regulation of signaling pathways associated with anti-tumor immune responses or the production of inflammatory factors.

MCM7 supports the stemness of bladder cancer stem-like cells by enhancing autophagic flux.

Autophagy plays critical roles in the pluripotent stemness of cancer stem cells (CSCs). However, how CSCs maintain the elevated autophagy to support stemness remains elusive. Here, we demonstrate that bladder cancer stem-like cells (BCSLCs) are at slow-cycling state with enhanced autophagy and mitophagy. In these slow-cycling BCSLCs, the DNA replication initiator MCM7 is required for autophagy and stemness. MCM7 knockdown inhibits autophagic flux and reduces the stemness of BCSLCs. MCM7 can facilitate autolysosome formation through binding with dynein to promote autophagic flux. The enhanced autophagy/mitophagy helps BCSLCs to maintain mitochondrial respiration, thus inhibiting AMPK activation. AMPK activation can trigger switch from autophagy to apoptosis, through increasing BCL2/BECLIN1 interaction and inducing P53 accumulation. In summary, we find that MCM7 can promote autophagic flux to support.

Low-dose cadmium exposure promotes osteoclastogenesis by enhancing autophagy via inhibiting the mTOR/p70S6K1 signaling pathway.

Cadmium (Cd)-induced bone damage may be mediated through activating osteoclastogenesis. However, the underlying mechanism is unknown. The purpose of this study was to explore the effect and possible mechanism of CdCl2-induced osteoclastogenesis in RAW264.7 cells. We found that a low concentration of CdCl2 (0.025 and 0.050 µM) did not affect the viability of RAW264.7 cells, but promoted osteoclastogenesis. A low concentration of CdCl2 increased the mRNA and protein expression of osteoclastogenesis-related genes. TRAP staining and transmission electron microscopy (TEM) also demonstrated that CdCl2 promoted osteoclastogenesis. A low concentration of CdCl2 upregulated the levels of LC3-II and Beclin-1, and decreased p62 expression. TEM showed relatively abundant autophagic vacuoles (autophagosomes) after CdCl2 exposure. A low concentration of CdCl2 downregulated the expression levels of Mtor and p70S6K1, and the relative protein expression ratios of p-mTOR/mTOR and p-p70S6K1/p70S6K1. When cells were treated with the autophagy inhibitor chloroquine (CQ) or mTOR activator MHY1485 combined with CdCl2, the expressions of osteoclastogenesis related-genes were decreased and autophagy was attenuated compared with cells treated with CdCl2 alone. Deficiencies in autophagosomes and osteoclasts were also observed. Taken together, the results indicate that a low concentration of CdCl2 promotes osteoclastogenesis by enhancing autophagy via inhibiting the mTOR/p70S6K1 signaling pathway.

Norcantharidin Induces Immunogenic Cell Death of Bladder Cancer Cells through Promoting Autophagy in Acidic Culture.

The acidic tumor microenvironment stands as a major obstacle to the efficient elimination of tumor cells. Norcantharidin (NCTD) is a powerful antitumor agent with multiple bioactivities. However, the effect of NCTD under acidic conditions is still unclear. Here, we report that NCTD can efficiently kill bladder cancer (BC) cells in acidic culture, and more intriguingly, NCTD can induce immunogenic cell death (ICD), thereby promoting antitumor immunity. In NCTD-treated BC cells, the surface-exposed calreticulin (ecto-CALR) was significantly increased. Consistently, co-culture with these cells promoted dendritic cell (DC) maturation. The NCTD-induced ICD is autophagy dependent, as autophagy inhibition completely blocked the NCTD-induced ecto-CALR and DC maturation. In addition, the DC showed a distinct maturation phenotype (CD80high CD86low) in acidic culture, as compared to that in physiological pH (CD80 high CD86high). Finally, the NCTD-induced ICD was validated in a mouse model. NCTD treatment significantly increased the tumor-infiltrating T lymphocytes in MB49 bladder cancer mice. Immunizing mice with NCTD-treated MB49 cells significantly increased tumor-free survival as compared to control. These findings demonstrate that NCTD could induce ICD in an acidic environment and suggest the feasibility to combine NCTD with anticancer immunotherapy to treat BC.

Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

Shikonin suppresses the epithelial-to-mesenchymal transition by downregulating NHE1 in bladder cancer cells.

Shikonin (SK) is the major bioactive component extracted from the roots of Lithospermum erythrorhizon with anticancer activity. SK could inhibit the epithelial-to-mesenchymal transition (EMT) of cancer cells. However, the underlying mechanism is elusive. In the present study, the inhibitory activities of SK on proliferation, invasion and migration were examined in bladder cancer (BC) cells. SK potently decreased the viabilities of BC cells but showed less cytotoxicity to normal bladder epithelial cells. Moreover, SK reversed the EMT, suppressed the migration and invasion of BC cells. Intriguingly, NHE1, the major proton efflux pump, was dramatically down-regulated by SK. The EMT-inhibitory effect of SK was mediated by NHE1 down-regulation, as NHE1-overexpress alleviated while Cariporide (NHE1 inhibitor) enhanced this effect. Further, enforced alkalinization of intracellular pH (pHi) reversed the EMT-inhibitory effect of SK, indicating a key role of acidic pHi in this process. Finally, elevated NHE1 expression was observed in human bladder cancer tissues. Collectively, this research reveals a supportive effect of NHE1 and alkaline pHi on EMT. SK can suppress EMT through inhibiting NHE1 and hence inducing an acidic pHi.

Cadmium induces renal inflammation by activating the NLRP3 inflammasome through ROS/MAPK/NF-κB pathway in vitro and in vivo.

Cadmium (Cd) has been reported to induce kidney damage by triggering oxidative stress and inflammation. The NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome has been implicated a role in the pathogenesis of inflammation. However, the connection between Cd and NLRP3 inflammasome in the development of renal inflammation remains unknown. In this study, in vitro experiments based on the telomerase-immortalized human renal proximal-tubule epithelial cell line (RPTEC/TERT1) were carried out. Results revealed that CdCl2 (2-8 µM) increased ROS production and activated NLRP3, thereby enhancing secretion of IL-1ß and IL-18 (P < 0.05). Knock-down of NLRP3 rescued the RPTEC/TERT1 cells from Cd-induced inflammatory damage. Cd activated the MAPK/NF-κB signaling pathway in RPTEC/TERT1 cells (P < 0.05). In addition, treatment with N-acetylcysteine (NAC) improved inflammation and blocked the upregulation of the MAPK/NF-κB signaling pathway. Pre-treatment with MAPK and NF-κB inhibitors also suppressed NLRP3 inflammasome activation (P < 0.05). Moreover, CdCl2 (25-00 mg/L) stimulated the MAPK/NF-κB signaling pathway, activated the NLRP3 inflammasome, and increased inflammatory response (P < 0.05) leading to renal injury in rats. Exposure to cadmium elevated serum levels of NLRP3 and IL-1ß in populations (P < 0.05). Further analysis found that serum NLRP3 and IL-1ß levels were positively correlated with urine cadmium (UCd) and urine N-acetyl-ß-D-glucosaminidase (UNAG). Overall, Cd induced renal inflammation through the ROS/MAPK/NF-κB signaling pathway by activating the NLRP3 inflammasome. Our research thus provides new insights into the molecular mechanism that NLRP3 contributes to Cd-induced kidney damage.

Sequential administration of anti-PD-1 and anti-Tim-3 combined with an SA-GM-CSF-anchored vaccine overcomes adaptive immune resistance to reject established bladder cancer.

Program death receptor-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) play an important role in tumor immune evasion. PD-1 blockade could produce an effective anti-tumor effect but the response rate was low due to lacking of tumor infiltrating lymphocytes (TILs) and existing of other negative regulatory pathways. Streptavidin(SA)-GM-CSF surface-anchored tumor cells vaccine could induce specific anti-tumor immune response. However, this vaccine failed to induce regression of established tumor because it also up-regulated PD-1 expression on tumor cells dependent on IFNγ and up-regulated PD-1/Tim-3 expression on CD8+ TILs. Subsets of CD8+ TILs assay showed that PD-1 expression was closely associated with CD8+ TILs exhaustion, and Tim-3 expression was closely correlated with secretion function but not proliferation of CD8+ TILs. Sequential administration of anti-PD-1 and anti-Tim-3 could further improve the efficacy of SA-GM-CSF-anchored vaccine therapy, and tumor regression was noted in over 50%. This triple therapy improves the specific cytotoxic activity and decreased the apoptosis of CD8+ TILs. These findings indicated that this triple therapy could induce a more robust anti-tumor immune response.

Circadian clock protein CRY1 prevents paclitaxel­induced senescence of bladder cancer cells by promoting p53 degradation.

Bladder cancer is a common tumor type of the urinary system, which has high levels of morbidity and mortality. The first­line treatment is cisplatin­based combination chemotherapy, but a significant proportion of patients relapse due to the development of drug resistance. Therapy­induced senescence can act as a 'back­up' response to chemotherapy in cancer types that are resistant to apoptosis­based anticancer therapies. The circadian clock serves an important role in drug resistance and cellular senescence. The aim of the present study was to investigate the regulatory effect of the circadian clock on paclitaxel (PTX)­induced senescence in cisplatin­resistant bladder cancer cells. Cisplatin­resistant bladder cancer cells were established via long­term cisplatin incubation. PTX induced apparent senescence in bladder cancer cells as demonstrated via SA­ß­Gal staining, but this was not observed in the cisplatin­resistant cells. The cisplatin­resistant cells entered into a quiescent state with prolonged circadian rhythm under acute PTX stress. It was identified that the circadian protein cryptochrome1 (CRY1) accumulated in these quiescent cisplatin­resistant cells, and that CRY1 knockdown restored PTX­induced senescence. Mechanistically, CRY1 promoted p53 degradation via increasing the binding of p53 with its ubiquitin E3 ligase MDM2 proto­oncogene. These data suggested that the accumulated CRY1 in cisplatin­resistant cells could prevent PTX­induced senescence by promoting p53 degradation.

Cancer Cell enters reversible quiescence through Intracellular Acidification to resist Paclitaxel Cytotoxicity.

Cancer cells can enter quiescent or dormant state to resist anticancer agents while maintaining the potential of reactivation. However, the molecular mechanism underlying quiescence entry and reactivation remains largely unknown. In this paper, cancer cells eventually entered a reversible quiescent state to resist long-term paclitaxel (PTX) stress. The quiescent cells were characterized with Na+/H+ exchanger 1 (NHE1) downregulation and showed acidic intracellular pH (pHi). Accordingly, decreasing pHi by NHE1 inhibitor could induce cell enter quiescence. Further, acidic pHi could activate the ubiquitin-proteasome system and inhibiting proteasome activity by MG132 prevented cells entering quiescence. In addition, we show that after partial release, the key G1-S transcription factor E2F1 protein level was not recovered, while MCM7 protein returned to normal level in the reactivated cells. More importantly, MCM7 knockdown inhibited G1/S genes transcription and inhibited the reactivated proliferation. Taken together, this study demonstrates a regulatory function of intracellular acidification and subsequent protein ubiquitination on quiescence entry, and reveals a supportive effect of MCM7 on the quiescence-reactivated proliferation.

Phenotypic transition of tumor cells between epithelial- and mesenchymal-like state during adaptation to acidosis.

Acidic microenvironment is an important feature of solid tumors that contributes to malignant transformation. Low extracellular pH could promote epithelial-mesenchymal transition (EMT) thereby endowing tumor cells with higher invasive capability. However, the relation between EMT and tumor cell proliferation under long-term acidic condition is still not fully understood. Here, we show that tumor cells have undergone a phenotypic transition from EMT to mesenchymal-epithelial transition (MET) during adaptation to acidosis, and is closely related with cell proliferative state. Under early stage of acidic stress, tumor cells entered a non-cycling quiescent state with mesenchymal phenotype and expressed high level of stemness genes. Whereas, after long-term acid culture (2 months), acid-adapted cells resumed proliferating but lost mesenchymal phenotype. Further, our results show that the acid-adapted cells have distinct proliferative mechanism from non-acid cells, as the G1-S transcriptional factor E2F1 protein was not recovered in the adapted cells. Meanwhile, mini-chromosome maintenance 7 (MCM7) is shown to regulate the EMT to MET phenotypic transition, and is required for proliferation of the adapted cells under acidic condition. MCM7 Knockdown promoted mesenchymal phenotype and inhibited proliferation of the acid-adapted cells. While, MCM7 overexpression inhibited acid-induced EMT and supported tumor cell proliferation under acidic condition. Thus, our study provides evidence that tumor cells display phenotypic plasticity that allows them to survive acid stress.

PD-1 Blockade Overcomes Adaptive Immune Resistance in Treatment with Anchored-GM-CSF Bladder Cancer Cells Vaccine.

Purpose: To investigate whether PD-L1 mediated adaptive resistance could occur in treatment with Anchored-GM-CSF vaccine and whether PD-1 blockade combined with Anchored-GM-CSF vaccine could induce a greater anti-tumor immune response than either immunotherapy alone. Materials and Methods: After establishing long-established subcutaneous metastasis bladder cancer models, mice were treated with Anchored-GM-CSF vaccine and/or anti-PD-1 antibody. T-lymphocyte-cytotoxicity, flow cytometric analysis, immunohistochemical, immunofluorescence staining, CD8+ -T cell apoptosis and enzyme-linked immunosorbent assays were performed to evaluate the efficacy of combination therapy with anchored-GM-CSF vaccine and PD-1 blockade and to explore the related mechanism. Results: Anchored-GM-CSF vaccine could significantly increase the number of mature DCs and up-regulate PD-L1 expression dependent on IFN-γ released from CD8+ T cells. Anchored-GM-CSF vaccine combined with anti-PD-1 antibody could effectively inhibit tumor growth and even cause regression of the established tumor. More CD4+ and CD8+ T cells appeared at tumor sites and in peripheral blood in the combination therapy group than in the control groups. Splenocytes from mice of the combination therapy group exhibited the most potent cytotoxicity to MB49 cells. Apoptotic assays showed that PD-1 blockade could significantly reduce CD8+ T cells apoptosis. Conclusions: Anchored-GM-CSF vaccines and anti-PD-1 antibodies have synergistic effects in metastatic bladder cancer treatment. PD-1 blockade can overcome immune resistance in treatment with the Anchored-GM-CSF vaccine, while Anchored-GM-CSF vaccine can enhance the efficacy of PD-1 blockade therapy.

Exosome-packaged miR-1246 contributes to bystander DNA damage by targeting LIG4.

BACKGROUND: An increasing number of studies have recently reported that microRNAs packaged in exosomes contribute to multiple biological processes such as cancer progression; however, little is known about their role in the development of radiation-induced bystander effects. METHODS: The exosomes were isolated from the culture medium of BEP2D cells with or without γ-ray irradiation by ultracentrifugation. To monitor DNA damage and repair efficiency, the DNA double-strand break biomarker 53BP1 foci, comet, micronuclei, expression of DNA repair genes and NHEJ repair activity were detected. The miR-1246 targeting sequence of the DNA ligase 4 (LIG4) mRNA 3'UTR was assessed by luciferase reporter vectors. RESULTS: miR-1246 was increased in exosomes secreted from 2 Gy-irradiated BEP2D cells and inhibited the proliferation of nonirradiated cells. The miR-1246 mimic, exosomes from irradiated cells, and radiation-conditioned cell culture medium increased the yields of 53BP1 foci, comet tail and micronuclei in nonirradiated cells, and decreased NHEJ efficiency. miR-1246 downregulated LIG4 expression by directly targeting its 3'UTR. CONCLUSIONS: Our findings demonstrate that miR-1246 packaged in exosomes could act as a transfer messenger and contribute to DNA damage by directly repressing the LIG4 gene. Exosomal miR-1246 may be a critical predictor of and player in radiation-induced bystander DNA damage.

Norcantharidin enhances antitumor immunity of GM-CSF prostate cancer cells vaccine by inducing apoptosis of regulatory T cells.

Norcantharidin (NCTD) is a promising antitumor drug with low toxicity. It was reported to be able to regulate immunity, but the mechanism is not yet clear. Here we explored whether NCTD could enhance the antitumor immunity induced by prostate cancer cell vaccine. The results of the in vitro study showed that NCTD induced apoptosis and inhibited proliferation of regulatory T cells (Tregs). Mechanistic research showed that NCTD inhibited Akt activation and activated FOXO1 transcription, resulting in a pro-apoptotic effect. The results of the in vivo study showed that more tumor-infiltrating Tregs existed within peripheral blood and tumor tissue after treatment with the vaccine. Adding NCTD to vaccine treatment could decrease the number of tumor-infiltrating Tregs and increase the number of CD4+ and CD8+ T cells. Combination therapy with NCTD and vaccine was more effective in inhibiting tumor growth than the vaccine alone. In general, this is the first report that NCTD could induce apoptosis of Tregs and enhance the vaccine-induced immunity.

Evaluation of the clinical value of hematological parameters in patients with urothelial carcinoma of the bladder.

This study aimed to evaluate the clinical significance of pretreatment red cell distribution width (RDW), monocyte/lymphocyte ratio (MLR), neutrophil/lymphocyte ratio (NLR), and platelet/lymphocyte ratio (PLR) in patients with urothelial carcinoma of the bladder (UCB).Hematological parameters of 127 consecutive patients with UCB and 162 healthy controls were retrospectively analyzed. Receiver operating characteristic curve was plotted to determine the optimal cut-off value of RDW, MLR, NLR, and PLR to predict UCB. Whether these parameters could be independent predictors of UCB and had an association with the demographics and clinical characteristics of patients were also assessed.Patients with UCB had higher pretreatment RDW, MLR, NLR, and PLR compared with the healthy controls. With the tumor progression, MLR, NLR, and PLR rose consistently, whereas no significant difference was observed in RDW across tumor stages. NLR and PLR were associated with tumor size and tumor grade, while MLR was correlated with tumor size only. The best threshold of RDW, MLR, NLR, and PLR to predict UCB was 13.50%, 0.26, 2.16, and 128.46, respectively. Multivariate logistic regression model identified NLR ≥ 2.16 (odds ratio [OR] = 2.914; P < .001) and PLR ≥ 128.46 (OR = 2.761; P < .001) as independent predictors of UCB. High NLR and PLR were also associated with tumor markers, such as carcinoembryonic antigen and α-fetoprotein.Pretreatment NLR and PLR could be significant independent predictors of UCB. These simple and readily available inflammatory markers therefore might be used to manage the disease.

PD-1 blockade enhances the antitumor efficacy of GM-CSF surface-modified bladder cancer stem cells vaccine.

Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin-granulocyte-macrophage-colony stimulating factor (SA-GM-CSF) surface-modified bladder CSCs vaccine previously developed using our protein-anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor-1 (PD-1)/program death ligand 1 (PD-L1) signaling in tumor microenvironment results in tumor-adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8+ T cells, a part of these CD8+ T cells expressed PD-1. Moreover, the CSCs vaccine upregulated the PD-L1 expression of tumor cells, resulting in immune resistance. Adding PD-1 blockade to the CSCs vaccine therapy increased the population of CD4+ , CD8+ and CD8+ IFN-γ+ but not CD4+ Foxp3+ T cells and induced the highest production of IFN-γ. PD-1 blockade could effectively enhance the functions of tumor-specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM-1 cell challenge. These results indicate that PD-1 blockade combined with the GM-CSF-modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.