Down-regulation of KLRB1 is associated with increased cell growth, metastasis, poor prognosis, as well as a dysfunctional immune microenvironment in LUAD.

Killer cell lectin-like receptor B1 (KLRB1) is implicated in cancer progression and immunity. In this study, we aimed to evaluate the expression levels of KLRB1 in lung adenocarcinoma (LUAD) and analyze the relationship between KLRB1 expression levels, LUAD progression, and the tumor immune microenvironment. KLRB1 levels in LUAD were analyzed using data from the TCGA and XENA databases. Additionally, the diagnostic values of KLRB1 were analyzed in patients with LUAD. Survival and meta-analyses were employed to investigate the relationship between KLRB1 levels and other prognostic factors in patients with LUAD. Bioinformatics and cellular experiments were used to understand the functions and mechanisms of KLRB1. In addition, correlation analysis was used to investigate the relationship between KLRB1 levels and the immune microenvironment in LUAD. Reduced KLRB1 expression in LUAD was found to positively correlate with tumor size, distant metastasis, pathological stage, age, overall survival, diagnostic value, and disease-specific survival in patients with LUAD (P < 0.05). Conversely, increased KLRB1 expression was found to positively correlate with the overall survival and disease-specific survival in patients with LUAD (P < 0.05). We also found that the overexpression of KLRB1 can inhibit the proliferation, migration, and invasion of LUAD cells and promote apoptosis. KLRB1 was involved in immune cell differentiation, NF-kB, PD-L1, and PD-1 checkpoint pathways and others. Additionally, KLRB1 expression was linked to tumor purity, stromal, immune, and estimate scores, the levels of immune cells including B cells, CD8+ T cells, and CD4+ T cells, and immune cell markers in LUAD. Reduced KLRB1 expression has a significant positive correlation with diagnosis, poor prognosis, and immunity to cancer in patients with LUAD. KLRB1 inhibited cell proliferation and migration in patients with LUAD. These results suggest that KLRB1 may serve as a potential therapeutic target in patients with LUAD.

Respiratory failure as first presentation of myasthenia gravis: a case report.

Myasthenia gravis (MG) is often complicated by respiratory failure, an exacerbation known as myasthenic crisis. However, most patients with MG develop respiratory symptoms during the late course of the disease. Respiratory failure as an exclusive initial and primary complaint in patients with MG is rare and seldom reported. We herein describe a woman in her late 50s who presented with respiratory failure and was diagnosed with obesity hypoventilation syndrome at a local hospital. Her condition gradually worsened during the next 4 months and became accompanied by dysphagia. After 1 year of medical investigation, she was diagnosed in our hospital. A high level of anti-muscle-specific receptor tyrosine kinase antibody was found in her serum, and stimulation and electromyography results suggested MG. The patient's symptoms were improved by intravenous immunoglobulin and hormone therapy. This case reminds physicians to consider MG when encountering a patient who initially presents with respiratory failure.

Identification and genetic characterization of a recently identified enterovirus C116 in China.

Enterovirus C116 (EV-C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV-C116 in clinical samples worldwide, there is currently limited information available. In this study, two EV-C-positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta-transcriptome sequencing. Based on the prototypes of several EV-Cs, two lineages were observed. Lineage 1 included many types that could not cause EV-like cytopathic effect in cell culture. Three genogroups of EV-C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV-C116 still diffuses worldwide. Obvious inter-type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV-Cs biodiversity. The EV-C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV-like cytopathic effects were not observed. HEp-2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV-C116 growth. These results challenge the traditional recognition of the uncultured nature of EV-C116 strains and explain the difficulty of clinical detection.

Synergetic association between coxsackievirus A16 genotype evolution and recombinant form shifts.

Coxsackievirus A16 (CVA16) is a major pathogen that causes hand, foot, and mouth disease (HFMD). The recombination form (RF) shifts and global transmission dynamics of CVA16 remain unknown. In this retrospective study, global sequences of CVA16 were retrieved from the GenBank database and analyzed using comprehensive phylogenetic inference, RF surveys, and population structure. A total of 1,663 sequences were collected, forming a 442-sequences dataset for VP1 coding region analysis and a 345-sequences dataset for RF identification. Based on the VP1 coding region used for serotyping, three genotypes (A, B, and D), two subgenotypes of genotype B (B1 and B2), and three clusters of subgenotype B1 (B1a, B1b, and B1c) were identified. Cluster B1b has dominated the global epidemics, B2 disappeared in 2000, and D is an emerging genotype dating back to August 2002. Globally, four oscillation phases of CVA16 evolution, with a peak in 2013, and three migration pathways were identified. Europe, China, and Japan have served as the seeds for the global transmission of CVA16. Based on the 3D coding region of the RFs, five clusters of RFs (RF-A to -E) were identified. The shift in RFs from RF-B and RF-C to RF-D was accompanied by a change in genotype from B2 to B1a and B1c and then to B1b. In conclusion, the evolution and population dynamics of CVA16, especially the coevolution of 3D and VP1 genes, revealed that genotype evolution and RF replacement were synergistic rather than stochastic.

Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention.

Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.

SLC7A8 overexpression inhibits the growth and metastasis of lung adenocarcinoma and is correlated with a dismal prognosis.

BACKGROUND: Overexpression of solute carrier family 7 member 8 (SLC7A8) has been shown to relate to the survival time and tumor progression in cancer patients. However, the role of SLC7A8 in lung adenocarcinoma (LUAD) is still obscure. METHOD: The relationships between SLC7A8 expression in LUAD tissues and clinical values as well as immune infiltration were explored through bioinformatics. The functions and pathways of SLC7A8 in LUAD were investigated using Kyoto Encyclopedia of Genes and Genomes enrichment analysis, Gene Set Enrichment Analysis, Western blotting, and other methods. RESULTS: We found that the expression of SLC7A8 was decreased significantly in LUAD tissues compared with normal tissues, which was related to the dismal survival time and disease progression. Moreover, it carried diagnostic value in LUAD and was a risk factor for dismal prognosis. Receiver operating characteristic curve analysis indicated that the expression level of SLC7A8 carried significant diagnostic value in LUAD. Overexpression of SLC7A8 inhibited the proliferation, invasion, and migration of LUAD cells, likely through a mechanism involving the cell cycle. SLC7A8 expression in LUAD was significantly correlated with the infiltration of immune cells, especially B cells, interstitial dendritic cells, mast cells, CD56 bright cells, natural killer cells, plasmacytoid dendritic cells, T follicular helper cells, T helper 2 and 17 cells, and immune factors. CONCLUSION: The downregulation of SLC7A8 was related to a dismal prognosis and immune cell infiltration in LUAD. Increasing the expression of SLC7A8 inhibited the growth and migration of LUAD cells, thereby improving the prognosis of patients.

High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) is treated with transurethral resection of bladder tumor (TURBT) followed by intravesical instillation of chemotherapy or Bacillus Calmette-Guérin therapy. However, these treatments have a high recurrence rate and side effects, emphasizing the need for alternative instillations. Previously, we revealed that expanded allogeneic human natural killer (NK) cells from peripheral blood are a promising cellular therapy for prostate cancer. However, whether NK cells exhibit a similar killing effect in bladder cancer (BCa) remains unknown. METHODS: Expansion, activation, and cryopreservation of allogeneic human NK cells obtained from peripheral blood were performed as we previously described. In vitro cytotoxicity was evaluated using the cell counting kit-8. The levels of perforin, granzyme B, interferon-γ, tumor necrosis factor-α, and chemokines (C-C-motif ligand [CCL]1, CCL2, CCL20, CCL3L1, and CCL4; C-X-C-motif ligand [CXCL]1, CXCL16, CXCL2, CXCL3, and CXCL8; and X-motif ligand 1 and 2) were determined using enzyme-linked immunosorbent assay. The expression of CD107a, major histocompatibility complex class I (MHC-I), MHC-I polypeptide-related sequences A and B (MICA/B), cytomegalovirus UL16-binding protein-2/5/6 (ULBP-2/5/6), B7-H6, CD56, CD69, CD25, killer cell Ig-like receptors (KIR)2DL1, KIRD3DL1, NKG2D, NKp30, NKp46, and CD16 of NK cells or BCa and normal urothelial cells were detected using flow cytometry. Cytotoxicity was evaluated using lactate dehydrogenase assay in patient-derived organoid models. BCa growth was monitored in vivo using calipers in male NOD-scid IL2rg-/- mice subcutaneously injected with 5637 and NK cells. Differential gene expressions were investigated using RNA sequence analysis. The chemotaxis of T cells was evaluated using transwell migration assays. RESULTS: We revealed that the NK cells possess higher cytotoxicity against BCa lines with more production of cytokines than normal urothelial cells counterparts in vitro, demonstrated by upregulation of degranulation marker CD107a and increased interferon-γ secretion, by MICA/B/NKG2D and B7H6/NKp30-mediated activation. Furthermore, NK cells demonstrated antitumor effects against BCa in patient-derived organoids and BCa xenograft mouse models. NK cells secreted chemokines, including CCL1/2/20, to induce T-cell chemotaxis when encountering BCa cells. CONCLUSIONS: The expanded NK cells exhibit potent cytotoxicity against BCa cells, with few toxic side effects on normal urothelial cells. In addition, NK cells recruit T cells by secreting a panel of chemokines, which supports the translational application of NK cell intravesical instillation after TURBT from bench to bedside for NMIBC treatment.

Analysis of risk factors for positive surgical margin after laparoscopic radical prostatectomy with and without neoadjuvant hormonal therapy.

Background: Positive surgical margins (PSM) is not only an independent risk factor for recurrence, metastasis, and prognosis, but also an important indicator of adjuvant therapy for prostate cancer (PCa) patients treated with radical prostatectomy (RP). At present, there are few reports analyzing risk factors of PSM in laparoscopic RP (LRP), especially for those PCa cases who accepted neoadjuvant hormonal therapy (NHT). Hence, the aim of the current study was to explore risk factors for PSM after LRP in PCa patients with and without NHT. Methods: The clinicopathological data of patients who underwent LRP from January 2012 to July 2020 was retrospectively analyzed. Risk factors for PSM after LRP in NHT and non-NHT groups were respectively explored. Results: The overall PSM rate was 33.3% (90/270), PSM rate was 39.3% (64/163) in patients without NHT and 24.3% (26/107) in those with NHT. The apex was the most common location of PSM in non-NHT group (68.8%, 44/64), while the fundus was the most common location of PSM in NHT group (57.7%, 15/26). Multiple logistic regression revealed that body mass index (BMI), PSA, ISUP grade after LRP, pathological stage T (pT) and pathological lymph node status (pN) were independent factors affecting the PSM for patients without NHT (OR=1.160, 95%CI:1.034-1.301, p=0.011; OR=3.385, 95%CI:1.386-8.268, p=0.007; OR=3.541, 95%CI:1.008-12.444, p=0.049; OR=4.577, 95%CI:2.163-9.686, p<0.001; OR=3.572, 95%CI:1.124-11.347, p=0.031), while pT, pN, and lymphovascular invasion (LVI) were independent risk factors affecting PSM for patients with NHT (OR=18.434, 95%CI:4.976-68.297, p<0.001; OR=7.181, 95%CI:2.089-24.689, p=0.002; OR=3.545, 95%CI:1.109-11.327, p=0.033). Conclusions: The apex was the most common location in NHT group, and BMI, PSA, ISUP after LRP, pT and pN were independent risk factors affecting PSM for NHT patients; while the fundus was the most common location in non-NHT group, and pT, pN, and LVI were independent risk factors affecting PSM for non-NHT patients.

TIGIT immune checkpoint blockade enhances immunity of human peripheral blood NK cells against castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) patients have a 14-month median survival, emphasizing the need for alternative treatments. Previously, we demonstrated that expanded high-dose natural killer (NK) cells derived from human peripheral blood exhibit therapeutic efficacy against CRPC. However, which immune checkpoint blockade promotes NK cell antitumor immunity against CRPC remains unknown. Here, we explored immune checkpoint molecule expression in NK and CRPC cells during their interactions, and identified that the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) monoclonal antibody (mAb), vibostolimab, significantly enhanced NK cell cytotoxicity against CRPC cells and cytokine production in vitro, demonstrated by upregulation of degranulation marker CD107a and Fas-ligand (Fas-L) and increased interferon-gamma (IFN-γ) and tumor necrosis factor-alpha secretion. TIGIT blockade increased Fas-L expression and IFN-γ production via the NF-κB signaling pathway and restored degranulation via the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase) kinase/ERK pathway in activated NK cells. Vibostolimab significantly enhanced NK cell antitumor effects against CRPC in two xenograft mouse models. Vibostolimab also increased T cell chemotaxis induced by activated NK cells in vitro and in vivo. Overall, blocking TIGIT/CD155 signaling enhances the antitumor effect of expanded NK cells against CRPC; this finding supports the translational application of TIGIT mAb and NK cell combination strategies from bench to bedside for CRPC treatment.

Realizing near white and warm light emission of cuprous iodide complexes by alkyl-isomerization of ligands.

Four novel all-in-one structured cuprous iodide hybrid materials are presented. Isomerization of the alkyl chain on the ligand improved material thermal stability and regulated their luminescence to warm and near-white light emission, with the internal quantum yield increasing from 5% to 83%. This provides a reasonable route for designing white light emitting cuprous iodide materials for solid-state lighting in future.

The changed endemic pattern of human adenovirus from species B to C among pediatric patients under the pressure of non-pharmaceutical interventions against COVID-19 in Beijing, China.

BACKGROUND: Under the pressure of non-pharmaceutical interventions (NPIs) targeting severe acute respiratory syndrome coronavirus 2, the prevalence of human adenovirus (HAdV) was monitored before and after NPIs launched on Jan 24, 2020 in pediatric patients in Beijing, China. METHODS: Respiratory samples collected from children hospitalized with acute respiratory infections from Jan 2015 to Dec 2021 were screened by direct immunofluorescence test or capillary electrophoresis-based multiplex PCR assay. The hexon, penton base, and fiber genes were amplified from HAdV positive specimens, then sequenced. For HAdV typing, phylogenetic trees were built by MEGA X. Then clinical data of HAdV positive cases were collected. All data were evaluated using SPSS Statistics 22.0 software. RESULTS: A total of 16,097 children were enrolled and 466 (2.89%, 466/16,097) were HAdV-positive. The positive rates of HAdV varied, ranging from 4.39% (151/3,438) in 2018 to1.25% (26/2,081) in 2021, dropped from 3.19% (428/13,408) to 1.41% (38/2,689) from before to after NPIs launched (P < 0.001). There were 350 cases typed into nine types of species B, C, or E and 34 recorded as undetermined. Among them, HAdV-B3 (51.56%, 198/384) was the most prevalent types from 2015 to 2017, and HAdV-B7 (29.17%, 112/384) co-circulated with HAdV-B3 from 2018 to 2019. After NPIs launched, HAdV-B3 and B7 decreased sharply with HAdV-B7 undetected in 2021, while HAdV-C1 became the dominant one and the undetermined were more. CONCLUSIONS: The endemic pattern of HAdV changed in Beijing because of the NPIs launched for COVID-19. Especially, the dominant types changed from HAdV-B to HAdV-C.

Comparative risk of acute kidney injury among cancer patients treated with immune checkpoint inhibitors.

With the development and introduction of immune checkpoint inhibitors (ICIs) in cancer patients, immune-related side effects have increasingly attracted attention. However, the risks of immune-related renal toxicity are poorly characterized. In this study, we performed a network meta-analysis (NMA) of ICI-related randomized clinical trials (RCTs) to elucidate the comparative risk of acute kidney injury (AKI) in cancer patients receiving different ICIs. We also sought to identify other factors potentially affecting the risk of AKI. PubMed and EMBASE were searched for peer-reviewed trial reports published between January 2000 and May 2021. Eligible studies were RCTs studying ICIs in cancer patients and reporting AKI data. We performed a frequentist NMA to evaluate the risk ratios for grade 1-5 and grade 3-5 AKI between the treatment groups. We also assessed the absolute incidence of AKI in the ICI-containing arm using traditional direct meta-analysis. Once significant heterogeneity was detected in a traditional direct meta-analysis, multivariable meta-regression analysis was applied to identify factors that significantly affected the absolute incidence of AKI. A total of 85 RCTs were included in this study. In the NMA for the risk of grade 1-5 and 3-5 AKI, ipilimumab showed a significantly higher risk than avelumab and durvalumab, whereas 1 mg/kg nivolumab plus 3 mg/kg ipilimumab (N1I3) showed a significantly higher risk than other groups. In terms of treatment ranking, durvalumab ± low-dose tremelimumab and avelumab were consistently among the top three safest treatments for grade 1-5 or 3-5 AKI, whereas N1I3, ipilimumab and tremelimumab were consistently among the top three treatments with the highest risk for grade 1-5 or 3-5 AKI. Compared with other cancers, renal cell carcinoma and urothelial carcinoma showed a significantly higher risk of AKI. The incidence of AKI was significantly higher with ICI+chemotherapy than with ICI monotherapy. In this NMA involving large-scale up-to-date ICI trials, we demonstrated the comparative safety of existing ICI drugs for grade 1-5 and grade 3-5 AKI. Based on data from the ICI arms of these trials, we also revealed several potential risk factors for immune-related AKI, including tumor type and treatment paradigm.

Preoperative red cell distribution width is associated with postoperative lymphovascular invasion in prostate cancer patients treated with radical prostatectomy: A retrospective study.

Purpose: To investigate the relationship between baseline clinicopathological and laboratory variables especially hematological parameters and lymphovascular invasion (LVI) in patients who underwent radical prostatectomy (RP). Methods: We retrospectively evaluated 348 prostate cancer (PCa) patients who underwent RP in our center between May 2018 and June 2021. We divided them into non-LVI and LVI groups based on LVI status, and compared clinicopathological characteristics between non-LVI and LVI groups. Clinicopathological parameters including age, body mass index (BMI), history of hypertension and diabetes mellitus, neoadjuvant hormonal therapy (NHT), pathological stage T (pT) and lymph node status (pN), ISUP (international society of urological pathology) grade, positive surgical margin (PSM) rate, and hematological parameters containing prostate-specific antigen (PSA), whole blood parameters and inflammatory indexes were collected. The association between the clinicopathological parameters and the presence of LVI was identified by multivariate logistic regression analysis. Results: The pathological results of the RP specimen consisted of 53 (15.2%) patients with LVI and 295 (84.8%) cases without LVI. The level of PSA, percentages of advanced pT and grade, pN1, and PSM were significantly higher in the LVI group when compared with the non-LVI counterpart (p<0.001, p<0.001, p<0.001, p<0.001, p=0.007, respectively). Among the whole blood parameters, only red cell distribution width (RDW) was significantly different (41.2 ± 2.5 vs. 42.1 ± 3.1, p=0.035). Multivariate regression analysis demonstrated that RDW and NHT were negatively correlated with the presence of LVI (OR = 0.870, p=0.024; OR = 0.410, p=0.025), while PSA, ISUP, and pT were positively correlated with the presence of LVI (OR=1.013, p=0.005; OR =1.589, p=0.001; OR=1.655, p=0.008) after adjusting for confounding factors. Conclusions: RDW rather than other whole blood parameters was independently and negatively associated with the presence of LVI in PCa patients, suggesting that RDW might play an essential role in PCa invasion.

Evaluation and multi-institutional validation of a novel urine biomarker lncRNA546 to improve the diagnostic specificity of prostate cancer in PSA gray-zone.

Background and objectives: Prostate specific antigen (PSA) is currently the most commonly used biomarker for prostate cancer diagnosis. However, when PSA is in the gray area of 4-10 ng/ml, the diagnostic specificity of prostate cancer is extremely low, leading to overdiagnosis in many clinically false-positive patients. This study was trying to discover and evaluate a novel urine biomarker long non-coding RNA (lncRNA546) to improve the diagnostic accuracy of prostate cancer in PSA gray-zone. Methods: A cohort study including consecutive 440 participants with suspected prostate cancer was retrospectively conducted in multi-urology centers. LncRNA546 scores were calculated with quantitative real-time polymerase chain reaction. The area under the receiver operating characteristic curve (AUROC), decision curve analysis (DCA) and a biopsy-specific nomogram were utilized to evaluate the potential for clinical application. Logistic regression model was constructed to confirm the predictive power of lncRNA546. Results: LncRNA546 scores were sufficient to discriminate positive and negative biopsies. ROC analysis showed a higher AUC for lncRNA546 scores than prostate cancer antigen 3 (PCA3) scores (0.78 vs. 0.66, p<0.01) in the overall cohort. More importantly, the AUC of lncRNA546 (0.80) was significantly higher than the AUCs of total PSA (0.57, p=0.02), percentage of free PSA (%fPSA) (0.64, p=0.04) and PCA3 (0.63, p<0.01) in the PSA 4-10 ng/ml cohort. A base model constructed by multiple logistic regression analysis plus lncRNA546 scores improved the predictive accuracy (PA) from 79.8% to 86.3% and improved AUC results from 0.862 to 0.915. DCA showed that the base model plus lncRNA546 displayed greater net benefit at threshold probabilities beyond 15% in the PSA 4-10 ng/ml cohort. Conclusion: LncRNA546 is a promising novel biomarker for the early detection of prostate cancer, especially in the PSA 4-10 ng/ml cohort.

Preoperative Platelet Count Correlates With Postoperative Perineural Invasion on Specimen in Patients Treated With Radical Prostatectomy.

Objective: It has been reported that perineural invasion (PNI) after radical prostatectomy (RP) is associated with unfavorable prostate cancer (PCa) prognosis. However, the clinicopathological factors especially hematological parameters that influenced PNI remain unknown. Our aim was to explore the relationship between clinicopathological parameters and PNI in patients who underwent RP. Methods: A total of 348 patients with PCa who underwent RP at our center between 2018 and 2021 were consecutively collected. We divided them into non-PNI and PNI groups based on PNI status and compared clinicopathological characteristics including hematological parameters between non-PNI and PNI groups. The association of clinicopathological parameters including whole blood parameters, age, body mass index (BMI), hypertension, diabetes mellitus, prostate-specific antigen (PSA), ISUP (International Society of Urological Pathology) grade, pathological stage T (pT), and neoadjuvant hormonal therapy (NHT) with PNI was determined by univariate and multivariate logistic regression analyses. Results: The pathological results of the RP specimen consisted of 254 (73.0%) patients with PNI and 94 (27.0%) cases without PNI. The level of PSA, percentages of advanced pT and grade, positive surgical margin rate, and vessel carcinoma embolus rate were significantly higher in the PNI group when compared with non-PNI counterpart (p = 0.007, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively). Among the whole blood parameters, only platelet count and plateletcrit were significantly different [216 (178.8-252.0) vs. 200.5 (173.5-236.5), p = 0.04; 0.0021 (0.0018-0.0025) vs. 0.0020 (0.0017-0.0023), p = 0.008, respectively]. Univariate logistic regression analysis demonstrated that platelet, ISUP, and pT were all positively correlated with the presence of PNI (T3 vs. T1, odds ratio (OR) = 2.029, p = 0.020; OR = 1.697, p < 0.001; OR = 3.836, p < 0.001). In the stepwise multivariate regression analysis, the association between platelet and PNI remained significant (T2 vs. T1, OR = 2.171, 95% CI: 1.082-4.354, p = 0.029; T3 vs. T1, OR = 2.595, 95% CI: 1.259-5.349, p = 0.010) after adjusting for confounding factors including age, BMI, hypertension, diabetes mellitus, PSA, ISUP, pT, and NHT. Conclusions: The study first revealed that platelet count rather than other whole blood parameters was independently associated with the presence of PNI in patients with PCa, suggesting that platelets might play an essential role in PCa aggressiveness.

The establishment of polypeptide PSMA-targeted chimeric antigen receptor-engineered natural killer cells for castration-resistant prostate cancer and the induction of ferroptosis-related cell death.

BACKGROUND: The mortality of castration-resistant prostate cancer (CRPC) is high due to lack of an effective treatment. Chimeric antigen receptor (CAR)-based therapy is a promising immunotherapeutic strategy. Here, we aimed to design a novel CAR-natural killer (NK) cells with a clinically significant tumoricidal effect on CRPC. METHODS: We constructed novel CAR-NK92MI cells with a CD244-based recombinant lentiviral vector. Different intracellular segments (CD244, NKG2D, or CD3ζ) were screened to identify the best candidate according to cell lysis assay and CD107a expression levels. To enhance the affinity of the CAR to the tumor antigen, we compared an antibody specific for prostate-specific membrane antigen (anti-PSMA) with PSMA-targeted polypeptide (p-PSMA), which was screened by phage display combinatorial library. Then, CAR-NK92MI cells with both a high affinity for PSMA and a strong tumoricidal capacity were generated. In addition, we verified their tumor-killing effect in vitro and in vivo. The release of cytokine by NK92MI cells was compared with that by CAR-NK92MI cells through flow cytometry and enzyme-linked immunosorbent assay. Moreover, ferroptosis-related cell death was explored as a possible underlying mechanism. RESULTS: Three different CAR intracellular regions CAR1 (CD244), CAR2 (CD244, NKG2D) and CAR3 (CD244, NKG2D, and CD3ζ) were constructed. CAR2 was chosen to confer a stronger tumoricidal ability on CAR-NK92MI cells. Compared with anti-PSMA, p-PSMA exhibited enhanced affinity for the tumor antigen. Thus, p-PSMA-CAR-NK92MI cells, which expressed CAR with a polypeptide-based antigen-binding region, an intracellular CD244 and a NKG2D costimulatory domain, were generated. They could selectively and successfully kill PSMA+ target cells and exhibited specific lysis rate of 73.19% for PSMA-positive C4-2 cells and 33.04% for PSMA-negative PC3 cells. Additionally, p-PSMA-CAR-NK92MI cells had significantly higher concentrations of IFN-γ, TNF-α and granzyme B than NK92MI cells. In a CRPC cancer xenograft model, p-PSMA-CAR-NK92MI cells significantly inhibited tumor growth and exerted a more consistent killing effect than NK92MI cells. Moreover, ferroptosis is a potential mechanism through which CAR-NK92MI cells attack cancer cells, and is triggered by IFN-γ. CONCLUSIONS: p-PSMA-CAR-NK92MI cells can effectively kill CRPCPSMA+ cells in vitro and in vivo. This strategy may provide additional treatment options for patients with CRPC.

Combined treatment with anti-PSMA CAR NK-92 cell and anti-PD-L1 monoclonal antibody enhances the antitumour efficacy against castration-resistant prostate cancer.

BACKGROUND: The chimeric antigen receptor NK-92 (CAR NK-92) cell targeting the prostate-specific membrane antigen (PSMA) has shown antitumour effects in castration-resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD-L1) and its mechanisms on CAR NK-92 and CRPC cells and the effect of the anti-PD-L1 monoclonal antibody (mAb) on PD-L1 expressed on CAR NK-92 cells remain unknown. METHODS: Human dendritic cells and CD8+ T cells were acquired from blood samples of healthy donors and cocultured with C4-2 cells. Changes in PD-L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD-L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit-8 assay and the bioluminescent intensity (BLI) of green fluorescent protein-labelled C4-2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4-2 cells and treated intravenously with anti-PD-L1/PD-1 mAb, CAR NK-92 or cocultured CD8+ T cells. RESULTS: Significantly upregulated expression of PD-L1k was observed in cocultured C4-2 and CAR NK-92 cells. In addition, upregulation of PD-L1 expression was dependent on interferon-γ in C4-2 cells, while it was dependent on direct cell-to-cell interaction via the NK group 2 member D/ phosphatidylinositol 3-kinase/AKT pathway in CAR NK-92 cells. The anti-PD-L1 mAb directly acted on PD-L1 expressed on CAR NK-92 cells and augmented the cytotoxicity of CAR NK-92 cells against C4-2 and CRPC cells from one patient in vitro. Anti-PD-L1 mAb significantly enhanced the antitumour effect of CAR NK-92 cells against CRPC cells in vivo when compared to treatment with CAR NK-92 cells or combined with anti-PD-1 mAb in the absence or presence of cocultured CD8+ T cells. CONCLUSION: Combined treatment with CAR NK-92 and anti-PD-L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.

Allogeneic Expanded Human Peripheral NK Cells Control Prostate Cancer Growth in a Preclinical Mouse Model of Castration-Resistant Prostate Cancer.

Adoptive allogeneic natural killer (NK) cell therapy has shown promise in treating castration-resistant prostate cancer (CRPC), which is the terminal stage of prostate cancer (PCa) and incurable. Thus, we employed an efficient manufacturing method for the large-scale ex vivo expansion of high-quality NK cells from peripheral blood of healthy donors. In the present study, we evaluated the in vitro cytotoxicity of NK cells against human PCa cell lines and in vivo antitumor activity in a preclinical mouse model of CRPC. CCK-8 results demonstrated that the NK cells exerted potent cytotoxicity against all PCa cell lines in vitro. The NK cells were activated when cocultured with PCa C4-2 cells, evidenced by upregulation of the degranulation marker CD107a and secretion of cytokines (TNF-α and IFN-γ). In a xenograft mouse model of CRPC, the caliper, CT, and ultrasonography examination results showed that the size of tumors treated with NK cells was significantly smaller than that in the control group. Moreover, ultrasonography examination also indicated that the NK cell treatment evidently reduced the blood supply of the tumors and HE staining results demonstrated that the NK treatment increased the proportion of necrosis in the tumor specimen compared to PBS treatment. Meanwhile, the NK cell treatment did not cause significant serum IL-6 elevation. Therefore, our study suggested that the expanded NK cells exhibited significant cytotoxicity against PCa cell lines in vitro and excellent therapeutic efficacy against CRPC in a xenograft mouse model, which was of great value for the clinical treatment of CRPC.

Comparative Analysis of Differentially Mutated Genes in Non-Muscle and Muscle-Invasive Bladder Cancer in the Chinese Population by Whole Exome Sequencing.

Objective: To characterize the spectra of mutations in non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) in the Chinese population to identify any mutational features and find potential therapeutic targets. Materials and methods: We collected fresh bladder tumor samples from NMIBC (n = 9) and MIBC patients (n = 11) along with adjacent normal bladder tissue specimen and peripheral blood sample. Using whole exome sequencing (WES), we analyzed the mutation spectra of those NMIBC and MIBC bladder cancer (BCa) specimen. Results: Our results demonstrated that 95% of BCa patients (19/20) had varying degrees of driver gene mutations, FGFR3 (45%), KMT2D (40%), PIK3CA (35%), ARID1A (20%), EP300 (20%), KDM6A (20%), KMT2C (20%), and STAG2 (20%) were the most frequently mutated genes in BCa patients. NMIBC and MIBC exhibited different genomic alterations. FGFR3 (67%), PIK3CA (56%), and RHOB (44%) were the most frequently mutated genes in NMIBC patients. Of note, RHOB mutation only occurred in NMIBC, whereas mutations of KMT2D (55%), TP53 (36%) and KMT2B (27%) were frequently detected in MIBC, and TP53 and KMT2B mutation only occurred in MIBC. The frequency of mutations in DNA-damage repair (DDR) gene was higher in MIBC than that in NMIBC (91 vs 78%, 6.2 vs 2.4 gene mutations per patient). Copy number alterations (CNAs) occurred at more diverse chromosomal locations in NMIBC, but the CNA burden was higher in MIBC [9.01 (2.07-31.51) vs 4.98 (0.99-9.73) mutations/Mb]., the trend of which was consistent with the tumor mutation burden (TMB) [8.26 (4.63-21.84) vs 5.58 (3.87-9.58) mutations/Mb]. Among the current set of single-base substitution (SBS) signatures including SBS 1, 2, 5, 13, and 40, we identified one differently expressed signature between NMIBC and MIBC patients: SBS13. Conclusions: There were different gene mutational characteristics and signatures between NMIBC and MIBC in the Chinese population. Frequency of DDR, CNA burden and TMB were higher in MIBC. Our analysis revealed that several genes in NMIBC did not overlap with those reported in MIBC, suggesting that a fraction of NMIBC and MIBC likely developed secondary to different precursor lesions.

Surgical Maneuver and Tactics of Using the Inferior Phrenic Vein as a Landmark in Retroperitoneal Laparoscopic Left Adrenalectomy.

Identification and control of the central adrenal vein (CAV) are key steps in laparoscopic adrenalectomy. However, the retroperitoneal laparoscopic left adrenalectomy (RLLA) lacks identifiable anatomical landmarks and does not have advantage of quickly exposing CAV. Here, we developed surgical maneuver and tactics of using the inferior phrenic vein (IPV) as a landmark in RLLA. During operations, we searched for the IPV between superior margin of renal artery and anterior aspect of psoas major muscle, and then the left IPV was followed and applied as an anatomical landmark to identify the CAV. Moreover, our study showed that variations in the left adrenal venous anatomy occurred in cases with pheochromocytomas. The application of left IPV as a landmark to search for CAV has important clinical significance in RLLA.