Diffuse large B-cell lymphoma: the significance of CD8+ tumor-infiltrating lymphocytes exhaustion mediated by TIM3/Galectin-9 pathway.

BACKGROUND: Overexpression of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is related to the exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) in diffuse large B-cell lymphoma (DLBCL). However, the mechanism of TIM3-mediated CD8+TILs exhaustion in DLBCL remains poorly understood. Therefore, we aimed to clarify the potential pathway involved in TIM3-mediated CD8+TILs exhaustion and its significance in DLBCL. METHODS: The expression of TIM3 and its correlation with CD8+TILs exhaustion, the key ligand of TIM3, and the potential pathway of TIM3-mediated CD8+TILs exhaustion in DLBCL were analyzed using single-cell RNA sequencing and validated by RNA sequencing. The biological significance of TIM3-related pathway in DLBCL was investigated based on RNA sequencing, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction data. Finally, the possible regulatory mechanism of TIM3-related pathway in DLBCL was explored using single-cell RNA sequencing and RNA sequencing. RESULTS: Our results demonstrated that CD8+TILs, especially the terminally exhausted state, were the major clusters that expressed TIM3 in DLBCL. Galectin-9, mainly expressed in M2 macrophages, is the key ligand of TIM3 and can induce the exhaustion of CD8+TILs through TIM3/Galectin-9 pathway. Meanwhile, high TIM3/Galectin-9 enrichment is related to immunosuppressive tumor microenvironment, severe clinical manifestations, inferior prognosis, and poor response to CHOP-based chemotherapy, and can predict the clinical efficacy of immune checkpoint blockade therapy in DLBCL. Furthermore, the TIM3/Galectin-9 enrichment in DLBCL may be regulated by the IFN-γ signaling pathway. CONCLUSIONS: Our study highlights that TIM3/Galectin-9 pathway plays a crucial role in CD8+TILs exhaustion and the immune escape of DLBCL, which facilitates further functional studies and could provide a theoretical basis for the development of novel immunotherapy in DLBCL.

Pararespiratory and paradigestive lymph node metastases in esophageal squamous cell carcinoma: predicting survival and refining the N staging system.

BACKGROUND: The site of lymph node metastasis (LNM) may affect the prognosis of patients with esophageal squamous cell carcinoma (ESCC). To investigate the prognoses of pararespiratory and paradigestive LNM and to propose a novel N (nN) staging system that integrates both the LNM site and count. METHODS: This study was a multicenter, large-sample, retrospective cohort study that included ESCC patients with LNM between January 2014 and December 2019 from three Chinese institutes. Patients were set into training (two institutes) and external validation (one institute) cohorts. The primary outcomes were survival differences in LNM site and the development of novel nodal staging system. The overall survival (OS) of patients with pararespiratory LNM only (Group A), paradigestive LNM only (Group B), and both sites (Group C) was evaluated by Kaplan-Meier. Cox proportional hazards models were used to identify the independent prognostic factors. An nN staging system considering both the LNM site and count was developed and evaluated by the area under the receiver operating characteristic curve (AUC). RESULTS: In total, 1313 patients were included and split into training (n = 1033) and external validation (n = 280) cohorts. There were 342 (26.0%), 568 (43.3%) and 403 (30.7%) patients in groups A, B and C, respectively. The OS of patients with pararespiratory and patients with paradigestive LNM presented significant differences in the training and validation cohorts (P < 0.050). In the training cohort, LNM site was an independent prognostic factor (hazard ratio: 1.58, 95% confidence intervals: 1.41-1.77, P < 0.001). The nN staging definition: nN1 (1-2 positive pararespiratory/paradigestive LNs), nN2 (3-6 pararespiratory LNs or 1 pararespiratory with 1paradigestive LN), nN3 (3-6 LNs with ≥ 1 paradigestive LN), nN4 (≥ 7 LNs). Subsets of patients with different nN stages showed significant differences in OS (P < 0.050). The prognostic model of the nN staging system presented higher performance in the training and validation cohorts at 3-year OS (AUC, 0.725 and 0.751, respectively) and 5-year OS (AUC, 0.740 and 0.793, respectively) than the current N staging systems. CONCLUSIONS: Compared to pararespiratory LNM, the presence of paradigestive LNM is associated with worse OS. The nN staging system revealed superior prognostic ability than current N staging systems.

Metformin attenuates chronic lung allograft dysfunction: evidence in rat models.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) directly causes an abysmal long-term prognosis after lung transplantation (LTx), but effective and safe drugs are not available. Metformin exhibits high therapeutic potential due to its antifibrotic and immunomodulatory effects; however, it is unclear whether metformin exerts a therapeutic effect in CLAD. We sought to investigate the effect of metformin on CLAD based on rat models. METHODS: Allogeneic LTx rats were treated with Cyclosporin A (CsA) in the first week, followed by metformin, CsA, or vehicle treatment. Syngeneic LTx rats received only vehicles. All rats were sacrificed on post-transplant week 4. Pathology of lung graft, spleen, and thymus, extent of lung fibrosis, activity of profibrotic cytokines and signaling pathway, adaptive immunity, and AMPK activity were then studied. RESULTS: Allogeneic recipients without maintenance CsA treatment manifested CLAD pathological characteristics, but these changes were not observed in rats treated with metformin. For the antifibrotic effect, metformin suppressed the fibrosis extent and profibrotic cytokine expression in lung grafts. Regarding immunomodulatory effect, metformin reduced T- and B-cell infiltration in lung grafts, spleen and thymus weights, the T- and B-cell zone areas in the spleen, and the thymic medullary area. In addition, metformin activated AMPK in lung allografts and in α-SMA+ cells and T cells in the lung grafts. CONCLUSIONS: Metformin attenuates CLAD in rat models, which could be attributed to the antifibrotic and immunomodulatory effects. AMPK activation suggests the potential molecular mechanism. Our study provides an experimental rationale for further clinical trials.

Suspension culture strategies to enrich colon cancer stem cells.

How to efficiently obtain high-purity cancer stem cells (CSCs) has been the basis of CSC research, but the optimal conditions for serum-free suspension culture of CSCs are still unclear. The present study aimed to define the optimal culture medium composition and culture time for the enrichment of colon CSCs via suspension culture. Suspension cell cultures of colon cancer DLD-1 cells were prepared using serum-free medium (SFM) containing variable concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce spheroids. Culture times were set at 10, 20 and 30 days. A total of nine different concentrations of EGF and bFGF were added to SFM to generate nine experimental groups. The proportions of CD44+, CD133+, and CD44+CD133+ double-positive spheroid cells were detected via flow cytometry. mRNA expression of stemness-, epithelial-mesenchymal transition- and Wnt/ß-catenin pathway-associated genes was determined via reverse transcription-quantitative PCR. Self-renewal ability was evaluated by a sphere-forming assay. Tumorigenesis was studied in vitro using a colony formation assay and in vivo via subcutaneous cell injection in nude mice. It was found that the highest expression proportions of CD133+ and CD44+ spheroid cells were observed in group (G)9 (20 ng/ml EGF + 20 ng/ml bFGF) at 30 days (F=123.554 and 99.528, respectively, P<0.001), CD133+CD44+ cells were also observed in G9 at 30 days (and at 10 days in G3 and 20 days in G6; F=57.897, P<0.001). G9 at 30 days also displayed the highest expression of Krüppel-like factor 4, leucine-rich repeat-containing G protein-coupled receptor 5, CD44, CD133, Vimentin and Wnt-3a (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001) and the lowest expression of E-cadherin (F=10.851, P<0.001). G9 at 30 days produced the highest yield of cell spheroids, as determined by a sphere forming assay (F=19.147, P<0.001); colony formation assays also exhibited the greatest number of colonies derived from G9 spheroids at 30 days (F=60.767, P<0.01), which also generated the largest mean tumor volume in the subcutaneous tumorigenesis xenograft model (F=12.539, P<0.01). In conclusion, 20 ng/ml EGF + 20 ng/ml bFGF effectively enriched colon CSCs when added to suspension culture for 30 days, and conferred the highest efficiency compared with other combinations.

Noninvasive evaluation of early diabetic nephropathy using diffusion kurtosis imaging: an experimental study.

OBJECTIVES: To evaluate the value of renal diffusion kurtosis imaging (DKI) in the diagnosis of early diabetic nephropathy (DN) in a rat model. MATERIALS AND METHODS: Forty male Zucker diabetic fatty rats that spontaneously developed type 2 diabetes mellitus (DM) and 20 age-matched nondiabetic lean Zucker rats were included. Renal DKI scans and histological examinations were performed on the rats in batches at the end of the 4th, 8th, 12th, 16th, and 20th week after DM model was built. Based on renal histopathological appearance, included animals were divided into three groups: a nondiabetic control group, a DM group without DN, and an early DN group. Mean kurtosis (MK) and mean diffusivity (MD) values of renal cortex and medulla were analyzed statistically. RESULTS: MK values of renal cortex and medulla tended to increase from the control group to the early DN group, respectively, while MD values tended to decrease. The cutoff MD and MK values of renal cortex and medulla showed different values in discriminating early DN from controls. Among them, cutoff MK value of medulla of 0.62 was the best parameter (sensitivity, 93.9%; specificity, 96.4%; and area under the curve, 0.95). For discriminate early DN from DM without DN and DM without DN from controls, cutoff MK value of renal cortex or medulla achieved an area under the curve of 0.76-0.85. CONCLUSIONS: MR DKI may be valuable for the noninvasive detection of early DN, and MK value might serve as a more sensitive biomarker of early DN than MD value. KEY POINTS: • In this article, diffusion kurtosis imaging (DKI) was used to detect the changes in the kidneys due to early diabetic nephropathy (DN). • MR DKI may be valuable for the noninvasive detection of early DN. • The mean kurtosis values of renal cortex and medulla might serve as a more sensitive biomarker of early DN than the mean diffusivity values.

Clinical nomogram for lymph node metastasis in pathological T1 esophageal squamous cell carcinoma: a multicenter retrospective study.

BACKGROUND: Endoscopic resection is increasingly used to treat pathological T1 (pT1) esophageal cancer (EC) patients. However, the procedures are limited by lymph node metastasis (LNM) and remain controversial. We aimed to construct a nomogram to predict the risk of LNM in patients with pT1 esophageal squamous cell carcinoma (ESCC). METHODS: A total of 243 patients with pT1 ESCC who underwent esophagectomy and lymph node dissection at two different institutes between February 2013 and June 2019 were analyzed retrospectively. Patients were categorized into the negative group and the positive group according to whether there was LNM. Risk factors for LNM were evaluated by univariate and multivariate analyses. The nomogram was used to estimate the individual risk of LNM. RESULTS: Forty-six (18.9%) of the 243 patients with pT1 ESCC exhibited LNM. The LNM rate in patients with stage T1a disease was 5.7% (5/88), and the rate in patients with stage T1b disease was 26.5% (41/155). Multivariable logistic regression analysis showed that tumor differentiation [odds ratio (OR) =1.942, 95% confidence interval (CI): 1.067-3.536, P=0.030], the T1 sub-stage (OR =4.750, 95% CI: 1.658-13.611, P=0.004), the preoperative alanine aminotransferase/aspartate aminotransferase ratio (LSR) (OR =5.371, 95% CI: 1.676-17.210, P=0.005), and the high-density lipoprotein cholesterol (HDL-C) level (OR =5.894, 95% CI: 1.917-18.124, P=0.002) were independent risk factors for LNM. The nomogram had relatively high accuracy, with an area under the receiver operating characteristic curve (AUC) of 0.803 (95% CI: 0.732-0.873). The calibration curve showed that the predicted probability of LNM was in good agreement with the actual probability. CONCLUSIONS: Clinicopathological and hematological parameters of tumor differentiation, the T1 sub-stage, the preoperative LSR, and the HDL-C level may predict the risk of LNM in T1 ESCC. The risk of LNM can be predicted by the nomogram.