Expression of GITR in MM patients.

<p>mRNA of GITR from 16 MM patients and 20 pooled normal bone marrow specimens were assessed by real time-PCR. 18S was considered as the internal control.</p

GITR expression correlates with NF-κB activation and sensitivity to NF-κB inhibitor.

<p>A. Sensitivity of MM cells to NF-κB inhibitor-BAY-11-7085 was assessed in five MM cell lines. Cell viability was determined by CellTiter-Glo assay. Veh indicating DMSO treated cells. Com indicates combination treatment of TNF with BAY-11-7085. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with Veh groups. B. MM cell lines were exposed to 10ng/ml TNF with/without BAY-11-7085. Cells were lysed after 12 hours incubation and subjected to Immunoblotting using anti-PARP1 and Actin antibodies.</p

Knockdown of GITR reduced the sensitivity to Bortezomib in RPMI cell line.

<p>A. Knockdown efficacy of GITR gene was assessed by real time-PCR. Scr (Scramble) indicates the non-targeting SiRNA control. B. Knockdown of GITR reduced sensitivity to Bortezomib in RPMI cell line. Cell viability was evaluated by CellTiter-Glo assay after 48 hours incubation. Data represent mean ± SD, * P<0.05, and **P<0.01. C. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-cleaved caspase-3 and Actin antibodies. D. Scr control and SiGITR-transfected RMPI cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by FACS based PI single staining and quantified by Flowjo software. Data represent mean ± SD, * P<0.05, and **P<0.01 compared with indicated groups.</p

Overexpression of GITR enhanced sensitivity to Bortezomib-induced apoptosis in MM1.S cells.

<p>A. Empty vector and GITR-transfected MM1.S cells were treated with Bortezomib for 48 hours. Cell viability was assessed by CellTiter-Glo assay. Data represent mean ± SD, **P<0.01 compared with indicated groups. B. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated overnight. Cells were lysed and subjected to Immunoblotting using anti-PARP1 and Actin antibodies. C. Empty control and GITR expressing MM1.S cells were exposed to different doses of Bortezomib and incubated for 24 hours. The number of dead cells were assessed by PI single staining and quantified by Flowjo software. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. D. NF-κB activity was evaluated in control and GITR expressing MM1.S cells. Nuclear protein lysates were subjected to western blot using anti-p65 and nucleolin antibodies. E. Empty vector and GITR-transfected MM1.S cells were treated with 50nM Bortezomib for 48 hours in co-cultured with or without BMSC. After 48 hours incubation, the cell viability was assessed by CellTiter-Glo assay. Data represent mean ± SD, *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups.</p

Overexpression of GITR impacts TNFα-induced non-canonical NF-κB activation.

<p>A. MM1.S, OPM1 (GITR low) and RPMI8226 (GITR high) cells were exposed to TNFα (10ng/ml) for 60 minutes. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with indicated groups. B. MM1.S, OPM1 and RPMI8226 cells were exposed to TNFα (10ng/ml) for 60 minutes. Nuclear and whole cell lysates were subjected to western blot using anti-p65, -p52 and Actin antibodies. C. Cells were exposed to TNFα (10ng/ml) for 15, 30, or 60 minutes. Nuclear protein and cytoplasmic extraction were subjected to western blot using anti-p52, -RelB and -nucleolin antibodies. D. pCMV-GITR and GITR-MM1.S cells were harvested at 24 hours after treatment with and without TNF-α (10ng/ml) for 60 minutes. Immunocytochemical analysis was assessed using anti-phospho-NF-κB-p52 antibody, with DAPI used to stain nuclei.</p

Overexpression of GITR enhanced Bortezomib induced tumor growth inhibition in MM1.S xenograft mice.

<p>A. Empty control and GITR expressing cells were isolated from MM1.S xenograft mice and subjected to mRNA extraction. The expression of GITR was examined by real time-PCR. GAPDH was considered as an internal control. B. CD138⁺ human plasma cells were isolated from femur of the four groups of investigated mice. The number of CD138⁺ cell was assessed by flow cytometry. Data represent mean ± SD, **P<0.01 and ***P<0.001 compared with indicated groups. C. NF-κB activity was evaluated by DNA binding ELISA assay. NF-κB p65 transcription factor binding to its consensus sequence on the plate-bound oligo nucleotide was examined from nuclear extracts. Data represent mean ± SD of triplicate experiments. ***P<0.001 compared with indicated groups.</p

Image_4_Evaluation of neoadjuvant immunotherapy in resectable gastric/gastroesophageal junction tumors: a meta-analysis and systematic review.tif

BackgroundNeoadjuvant therapy for resectable gastric cancer/gastroesophageal junction tumors is progressing slowly. Although immunotherapy for advanced gastric cancer/gastroesophageal junction tumors has made great progress, the efficacy and safety of neoadjuvant immunotherapy for locally resectable gastric cancer/gastroesophageal junction tumors have not been clearly demonstrated. Here, we conducted a systematic review and meta-analysis to assess the efficacy and safety of neoadjuvant immunotherapy and advance the current research.MethodsOriginal articles describing the safety and efficacy of neoadjuvant immunotherapy for resectable gastric cancer/gastroesophageal junction tumors published up until October 15, 2023 were retrieved from PubMed, Embase, the Cochrane Library, and other major databases. The odds ratios (OR) and 95% confidence intervals (CIs) were calculated for heterogeneity and subgroup analysis.ResultsA total of 1074 patients from 33 studies were included. The effectiveness of neoadjuvant immunotherapy was mainly evaluated using pathological complete remission (PCR), major pathological remission (MPR), and tumor regression grade (TRG). Among the included patients, 1015 underwent surgical treatment and 847 achieved R0 resection. Of the patients treated with neoadjuvant immunotherapy, 24% (95% CI: 19%–28%) achieved PCR and 49% (95% CI: 38%–61%) achieved MPR. Safety was assessed by a surgical resection rate of 0.89 (95% CI: 85%–93%), incidence of ≥ 3 treatment-related adverse events (TRAEs) of 28% (95% CI: 17%–40%), and incidence of ≥ 3 immune-related adverse events (irAEs) of 19% (95% CI: 11%–27%).ConclusionNeoadjuvant immunotherapy, especially neoadjuvant dual-immunotherapy combinations, is effective and safe for resectable gastric/gastroesophageal junction tumors in the short term. Nevertheless, further multicenter randomized trials are required to demonstrate which combination model is more beneficial.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=358752, identifier CRD42022358752.</p

Table_1_Efficacy and safety of neoadjuvant immunotherapy combined with chemotherapy in locally advanced esophageal cancer: A meta-analysis.docx

ObjectiveThe progress of neoadjuvant therapy for resectable locally advanced esophageal cancer has been stagnant. There has been much progress in immunotherapy for advanced esophageal cancer, but the efficacy and safety of neoadjuvant immunotherapy for resectable locally advanced esophageal cancer have not yet been definitively demonstrated.MethodsOriginal articles describing the safety and efficacy of neoadjuvant immunotherapy in resectable locally advanced esophagus published until July 2022 were retrieved from PubMed, Embase, and the Cochrane Library. The ratio (OR) and 95% confidence interval (CI) were calculated to conduct heterogeneity and subgroup analysis.ResultsIn total, 759 patients from 21 studies were enrolled. The effectiveness of neoadjuvant immunotherapy in combination with chemotherapy was evaluated using the major pathologic response (MPR) and pathologic complete response (PCR). In the enrolled patients, 677 were treated surgically and 664 achieved R0 resection. Major pathological remission was achieved in 52.0% (95% CI: 0.44–0.57) of patients on neoadjuvant immunotherapy combined with chemotherapy and complete pathological remission in 29.5% (95% CI: 0.25–0.32) of patients. The safety was primarily assessed by the incidence of treatment-related adverse events (TRAEs) and surgical resection rates. The incidence of TRAEs and the surgical resection rate combined ORs were 0.15 (95% CI: 0.09–0.22) and 0.86 (95% CI: 0.83–0.89), respectively.ConclusionNeoadjuvant immunotherapy combined with chemotherapy in locally advanced resectable esophageal cancer is effective and safe.</p