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

Image_1_Significant difference in Th1/Th2 paradigm induced by tuberculosis-specific antigens between IGRA-positive and IGRA-negative patients.tif

False negative interferon-γ release assay (IGRA) results constitute the major dilemma for the diagnosis of tuberculosis (TB) infections. Herein, we conducted a cohort study to compare the host immunological response to TB-specific antigens between active TB patients with positive and negative IGRA results and control groups. A total of 274 laboratory-confirmed TB patients were included in our analysis, consisting of 221 were IGRA positive and 53 were IGRA negative. Patients with the elderly were identified as an independent risk factor for negative IGRA results. In addition, the elevated level of IL-4 and the decreased levels of IFN-γ, IL-2, IL-6, IL-1β, and IL-12 in IGRA negative TB relative to IGRA positive TB group, demonstrating a significant difference in Th1/Th2 paradigm between two groups. The IFN-γ&IL-2 based assay could correctly identify 247 out of 307 MTB-infected individuals [271 TB patients and 36 individuals with latent TB infection (LTBI)], demonstrating a sensitivity of 80.5%. Then the IFN-γ and IL-4 were applied to distinguish healthy control and IGRA-negative group. When using the stepwise algorithm, the sensitivity for detecting Mycobacterium tuberculosis (MTB) infections was significantly increased from 80.5% to 89.6%. Additionally, patients with negative IGRA results had a conversion to culture-negative status longer than those with positive IGRA results. In conclusion, a stepwise algorithm outperforms IGRA assays to accurately identify MTB infections by the combination IFN-γ, IL-2, and IL-4. Further study is needed to evaluate the accuracy of our diagnostic algorithm in the LTBI population.</p

Ade decreases cell viability and induces cell death.

<p>(A) A549, MCF7, and Hela cells were exposed to Ade as indicated in normal culture medium for 72 h. Cell viability was detected using the MTS assay. Each column represents the average of five replicated experiements. Mean ±SD (n = 5). p<0.05, vs. vehicle control. (B) Ana-1 cells were treated with Ade for 72 h, cell viability was detected as in (A). Mean ±SD (n = 5). *p<0.05, **p<0.01, vs. vehicle control. (C, D, E) Ana-1 cells were incubated with Ade in normal culture medium for 12 h, then cell apoptosis was detected by either flow cytometry (FACScan; BD Biosciences) or Western blot. Representative cell death image and cell death data in Ana-1 cells are shown in (C, D). Mean ±SD (n = 3). *p<0.05, **p<0.01 vs. vehicle control. PARP cleavage is shown in (E). GAPDH was used as a loading control. (F, G) Thymus lymphocytes were incubated with Ade as indicated for 12 h, cell death was detected. Cell death images by PI staining in living cells under an inverted fluorescence microscope were shown in (F) and cell death data by flow cytometry are summarized in (G). **p<0.01 <i>vs.</i> vehicle control. Mean ±SD (n = 3).</p

Adenosine (Ade) increases intracellular ATP contents in multiple cells.

<p>(A) Primary thymocytes were exposed to either vehicle DMSO (DM) or Ade in normal culture medium for 4 h and cells were collected for total ATP assay by HPLC (LC-6AD; Shimadzu). ATP contents (µM) of equal number of cells (2×10⁵) were compared (n = 4). Each column represents the average of independent repeated experiments. Mean ±SD. *p<0.05 compared to controls. (B) K562 cells were treated with indicated doses of Ade or oligomycin (Oli; 1 µg/ml) in the absence of d-glucose in RPMI 1640 medium for 6 h. d-glucose (2 g/L) was used as a positive control. Mean ±SD (n = 4). *p<0.05 vs. control; ^#p<0.05 vs. Oli treatment alone. (C) K562 was exposed to 2 mM of Ade for 0.5, 2, and 6 h in the absence of d-glucose in the culture medium. Mean ±SD. *p<0.05 vs. 0.5 h treatment. (D) Increase of ATP in multiple cell lines: A549, MCF7, and Hela cells were exposed to either DMSO or 2 mM of Ade for 6 h in the absence (G-) or presence of d-glucose (2 g/L, G+) in the culture medium. ATP contents were detected by HPLC (n = 4) and the increase of ATP after Ade treatment was calculated as: Ade-treated/vehicle-treated. All controls were set as 1.0.</p

Oligomycin decreases proteasome inhibition-induced cell death in the presence of Ade.

<p>(A, B) K562 cells cultured in d-glucose-free medium were exposed to Ade (0.2 and 2 mM), MG132 (5 µM, or MG262 (1 µM) and their combinations; PI staining was dynamically recorded under a fluorescent microscope, typical images at 12 h are shown in (A) and (B). (C, D) K562 cells were treated with Oli (1 µM), MG132 (5 µM), or MG262 (1 µM) and their combinations in the absence or presence of Ade (2 mM) for 12 h; cell apoptosis was detected using Annexin V/PI staining. Typical images are shown in (C) and a summary of cell death is shown in (D). Mean +SD (n = 3). *p<0.05 <i>vs.</i> proteasome inhibitor treatment alone.</p

Ade increases cell viability in low ATP states and rescued cell death induced by ATP-depletion.

<p>(A, B) K562 cells were exposed to 2 mM of Ade cultured in RPMI 1640 medium with or without d-glucose for different time points. Cell density was imaged using an inverted microscope (Axio Obsever Z1; Zeiss, Germany). Typical images of cell density were selected from cells treated with Ade in d-glucose-free medium for 24 h (A) or in d-glucose-containing medium for 72 h (B). Scale bar  = 50 µm. (C) K562 cells were exposed to Ade for 24 h (left) or 48 h (right) with or without d-glucose; absolute cell numbers were counted using a cell counter. Mean ±SD (n = 3). *p<0.05 vs. d-glucose-containing cells. (D) K562 cells were incubated with Ade with or without d-glucose for 36 h. Cell viability was detected using the MTS assay. Mean ±SD (n = 3). *p<0.05 <i>vs.</i> glucose-containing cells. (E, F) K562 cells were treated with Oli with or without Ade (2 mM) in the d-glucose-free RPMI 1640 medium for 6 h, then cell apoptosis was detected by flow cytometry. Typical flow images are shown in (E) and cell death in (F). Mean ±SD (n = 3). *p<0.05 vs. Ade-treated cells. (G) K562 cells were treated with Oli (1 µg/ml) and Ade (2 mM) for 18 h in the glucose-free medium, and cells were then stained with PI and dynamically recorded under an inverted epi-fluorescent microscope. A typical image is shown. Scale bar  = 50 µM.</p

Ade transportation, but not Ade receptors, is required for Ade to exert its cytotoxic or cytoprotective effects.

<p>(A) K562 cells cultured in glucose-free culture medium were treated with Oli (0.5 µg/ml), Ade (2 mM) and DP (10 µM) for 3 h, followed by HPLC ATP assay. Mean ±SD (n = 3). *p<0.05 vs. Oli+Ade treatment. (B) K562 cells were cultured and treated in the d-glucose-free medium as indicated for 18 h, and cells were then stained with Annexin V/PI followed by flow cytometry. Viable cells are shown. Mean ±SD (n = 3). *p<0.05 each compared with Oli treatment alone; #p<0.05 each compared with Oli+Ade combination treatment. (C) K562 cells were cultured in normal medium and exposed to 2 mM Ade and DP (10 µM) for 18 h, cell numbers were counted using a cell counter. Mean ±SD (n = 3). *p<0.05 compared with vehicle control; ^#p<0.05 compared with Ade treatment alone. (D) As treated in (B), typical flow images are shown (Ade: 2 mM, Oli: 1.0 µg/ml, DP: 10 µM). (E) K562 cells were cultured in d-glucose-free medium and treated with the agents as indicated (Oli: 1.0 µg/ml, Ade: 2 mM, 8-SPT: 10 µM) for 4 h followed by ATP assay. Mean ±SD (n = 3). (F) K562 cells were cultured in normal glucose-containing medium and treated as indicated (Ade: 2 mM, 8-SPT: 10 µM) for 18 h, cell numbers were counted and summarized. Mean ±SD (n = 3). (G) K562 cells were cultured in glucose-free medium and treated as indicated (Oli: 1.0 µg/ml, 8-SPT: 10 µM) for 15 h, cell death was detected by flow cytometry. Mean ±SD (n = 3).</p

LC treatment selectively induces expression of p21^cip1 gene, mRNA and protein in cancer cells.

<p>(<b>a</b>) LC induces p21^cip1 gene expression but not p27 and GAPDH. HepG2 cells were treated with LC (2.5, 5.0, 10 mM) for 24 h; cells were collected for gene expression profile analysis. In the gene chip, there are 2 probes for p21^cip1 and 1 probe for p27^kip1. All the fold increases of p21^cip1 and p27^kip1 gene expression <i>versus</i> control were shown. (<b>b</b>) LC dose-dependently induces p21^cip1 mRNA expression but not p27^kip1 in HepG2 cells. HepG2 cells were incubated with different concentrations of LC (2.5, 5, 10 mM) for either 12 h or 24 h; the cells were collected for mRNA assay of p21^cip1 and p27^kip1 by real-time PCR. Fold increase of the LC-treated <i>versus</i> control was shown. Mean+SD (n = 3). *<i>P</i><0.01, **<i>P</i><0.05, compared with control. (<b>c</b>) LC dose-dependently and time-dependently induces p21^cip1 protein accumulation in HepG2 cancer cells. HepG2 and SMMC7721 cells were treated with various doses of LC for 48 h or HepG2 cells were exposed to 5 mM of LC for 12, 24, 36, 48 h; p21 and p27 proteins were detected by Western blot. (<b>d</b>) LC dose-dependently decreases Rb phosphorylation. HepG2 cells were treated with LC for 48 h; Rb and phosphorylated Rb were dectected by Western blot. Typical Western images were shown (left) and band intensity was quantified (right).</p

L-carnitine treatment fails to increase ATP concentration in cancer cells.

<p>(<b>a</b>) Cancer cells are resistant to oligomycin in the presence of D-glucose (2 g/L) but not L-glucose (2 g/L). Human HepG2 cancer cells were cultured in the presence or absence of either D-glucose or L-glucose in the culture medium and treated with various doses of oligomycin (0.1, 0.25, 0.5. 1.0 µg/ml) for 6 h and ATP content was assessed. Mean+SD (n = 3). *<i>P</i><0.01, <i>versus</i> control. DM: DMSO. (<b>b</b>) LC does not increase intracellular ATP content in cancer cells. Human hepatic HepG2 and SMMC-7721 cells were cultured in the normal culture medium respectively and treated with different doses of LC for 6 h, ATP content was detected. LC: L-carnitine. (<b>c</b>) Thymotytes are sensitive to oligomycin in the presence of D-glucose (2 g/L). Mouse thymocytes were treated with oligomycin (1 mg/ml) for different time points (1, 3, 6, 9 h), total ATP content was detected. (<b>d</b>) LC efficiently increases cellular ATP content. Mouse thymocytes were treated with LC (1 mM) for various times, cellular ATP content was assassed. Veh: vehicle.</p

Computional molecular docking of LC with HDAC.

<p>(<b>a</b>) The chemical structure of LC was shown. (<b>b</b>) The docking model of L-carnitine in active site of HDAC.</p