Cell Killing Function Research Articles

A microphysiological assay for studying T-cell chemotaxis, trafficking and tumor killing

Tumors in patients non-responsive to immunotherapy harbor a series of barriers that impede the efficacy of effector T-cells. Consequently, therapeutically modulating the chemotaxis machinery to enable effector T cell infiltration and function in the tumor could result in more successful therapeutic outcomes. Complexin-vitromodels allow re-creation ofin-vivotumor complexities in anin-vitrosetting, allowing improved translatability to patient biology at the laboratory scale. We identified a gap in available industrial scale microphysiological (MPS) assays for faster validation of targets and strategies that enable T-cell chemotaxis and effector function within tumor microenvironments. Using a commercially available, 96-chip 2-lane microfluidic assay system, we present a novel, scalable, complexin vitroMPS assay to study 3D T-cell chemotaxis and function within native, extracellular matrix (ECM)-rich multicellular tumor environments. Activated or naïve CD3+ T-cells stained with far-red nuclear stain responded to the chemokine gradients generated within the matrigel-collagen ECM by migrating into the microfluidic channel (∼5 mm horizontal window), in a concentration- and cell type-dependent manner. Furthermore, we observed and tracked chemotaxis and cancer cell killing function of antigen-specific CD4.CD8. chimeric antigen receptor (CAR)-T cells that responded to CXCR3 agonist gradient built through the expansive 5 mm of cancer cell colony containing stroma. The 2-lane assay system yielded useful information regarding donor and dose-dependent differences in CAR-T cell chemotaxis and tumor killing. The scalable assay system allows a granular window into immune cell migration and function in tissue spaces beyond endothelium, addressing a missing gap in studying tissue-specific immune cell chemotaxis and function to bring forward advancements in cancer immunotherapy.

TLR9 agonism differentially impacts human NK cell-mediated direct killing and antibody-dependent cell-mediated cytotoxicity

There are two known mechanisms by which natural killer (NK) cells recognize and kill diseased targets: (i) direct killing and (ii) antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated an indirect NK cell activation strategy for the enhancement of human NK cell killing function. We did this by leveraging the fact that toll-like receptor 9 (TLR9) agonism within pools of human peripheral blood mononuclear cells (PBMCs) results in a robust interferon signaling cascade that leads to NK cell activation. After TLR9 agonist stimulation, NK cells were enriched and incorporated into assays to assess their ability to kill tumor cell line targets. Notably, differential impacts of TLR9 agonism were observed—direct killing was enhanced while ADCC was not increased. To ensure that the observed differential effects were not attributable to differences between human donors, we recapitulated the observation using our Natural Killer—Simultaneous ADCC and Direct Killing Assay (NK-SADKA) that controls for human-to-human differences. Next, we observed a treatment-induced decrease in NK cell surface CD16—known to be shed by NK cells post-activation. Given the essential role of CD16 in ADCC, such shedding could account for the observed differential impact of TLR9 agonism on NK cell-mediated killing capacity.

Abstract 3962: A CCR6+Th1-like CD4+T-cell cluster involved in HEV formation predicted lung cancer patients requiring anti-CTLA-4 therapy

Abstract Background: CD4+ T cells are known to help in cytotoxic T-cell priming, clonal proliferation, migratory and infiltrative capacity, and target cell killing function by licensing dendritic cells. Although CXCR3+ CCR6- classical Th1 CD4+ T cells are thought to be essential for acute cellular immunity, such as antiviral responses, the details of Th1-type CD4+ T cells required for anti-tumor immunity as a long-term chronic T cell immune responses over years remain unclear. We reported the Th7R, a CCR6 expressing Th1-like CD4+ T cell cluster predicted anti-PD-1 antibody therapy efficacy and postoperative recurrence-free survival. Objective: In this study, we focus on two Th1-like CD4+ T cell clusters, Th1 and Th7R, to determine their functions and roles in long-lasting anti-tumor immune phenomena. Methods: We studied patients with advanced stage lung cancer treated with immune checkpoint inhibitors (Pembrolizumab monotherapy n=63, Ipilimumab + Nivolumab n=148) and stage I-II lung cancer patients undergoing surgery (n=54). Mass cytometry, single cell RNA sequencing, and immunohistochemical staining were performed using peripheral blood, tumor-infiltrating lymphocytes and lymph node T cells. These studies were approved by the IRB of Saitama Medical University International Medical Center, and all patients gave written consent. Results: Unlike classical Th1, which highly expresses PRF1, GZMB, GNLY, and NKG7, Th7R expressed LTβ, CXCR6, CTLA-4, CD28, and CXCL13. Analysis of HEV index (MECA79+/CD31+ vessels) by MECA79 and CD31 staining of tumor tissues showed that Th7R in peripheral blood correlated with tumor tissue HEV index, suggesting that LTβ and CXCL13 expressed by Th7R are involved in HEV formation.Th1 mainly expressed PD-1 and did not express CTLA-4, whereas Th7R expressed both PD-1 and CTLA-4. When pre-treatment peripheral Th7R quartile was used to analyze OS after Ipilimumab + Nivolumab treatment, both the top 25% and intermediate 50% patients showed favorable prognosis, but the lower 25% had significantly poor OS. In contrast, only the top 25% Th7R patients but not the intermediate 50% and lower 25% patients who were treated with Pembrolizumab had favorable OS. Thus, it is likely that the Th7R intermediate 50% patients required anti-CTLA-4 therapy to achieve antitumor immune responses. These results may be useful for stratification of patients requiring anti-CTLA-4 antibody therapy as well as for predicting the therapeutic response of Ipilimumab + Nivolumab. Conclusion: Th7R exhibits LTβ and CXCL13 expression, which is absent in classical Th1, and may help maintain sustained T-cell immunity through HEV/TLS formation. Th7R may be a biomarker to select the patients who benefit from anti-CTLA-4 treatment. Citation Format: Hiroshi Kagamu, Satoshi Yamasaki, Atsuhito Mouri, Ou Yamaguchi, Ayako Shiono, Fuyumi Nishihara, Yu Miura, Kosuke Hashimoto, Hisao Imai, Kyoichi Kaira, Kunihiko Koabayashi, Katsuhisa Horimoto. A CCR6+Th1-like CD4+T-cell cluster involved in HEV formation predicted lung cancer patients requiring anti-CTLA-4 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3962.

The Three-dimensional Environment of Type Ⅰ Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells

To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional (3D) culture environments of the gels on the morphology, free migration ability, and cell killing function of natural killer (NK) cells. Type Ⅰ collagen was isolated from the tails of Sprague Dawley (SD) rats and collagen gels with different levels of stiffnesses were prepared accordingly. The microstructure of the collagen gels was observed by laser confocal microscopy. The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer. NK-92MI cells were cultured in collagen gels with different levels of stiffness. The morphology of NK-92MI cells was observed by inverted microscope. High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance. NK-92MI cells were cultured with type Ⅰ collagen gels with different levels of stiffness for 24 h and 48 h and, then, co-cultured with human colorectal DLD-1, a human adenocarcinoma epithelial cell line. CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells. Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰ collagen gel with the respective stiffness of (10.970±2.10) Pa and (114.50±3.40) Pa were successfully prepared. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape (P<0.05), the mean area of the cells was reduced ([69.88±26.97] μm2 vs. [46.59±21.62] μm2, P<0.05), the roundness of the cells decreased (0.82±0.12 vs. 0.78±0.18, P<0.05), cell migration speed decreased ([2.50±0.91] μm/min vs. [1.70±0.72] μm/min, P<0.001) and the migration distance was shortened ([147.10±53.74] μm vs. [98.03± 40.95] μm, P<0.0001), with all the differences being statistically significant. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation, with the proliferation rate being (46.39±12.79)% vs. (65.87±4.45)% (P<0.05) and reduce the cell killing ability. Comparison of the cells cultured for 48 h led to similar results, with the proliferation rates being (31.36±2.88)% vs. (74.57±2.16)% (P<0.05), and the differences were all statistically significant. The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology, migration ability, and killing function of NK-92MI cells. This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells, as well as laying the theoretical foundation for optimizing immunotherapy protocols.

Umbilical cord blood-derived neutrophils possess higher viability than peripheral blood derived neutrophils.

Neutrophils, a primary type of immune cell, play critical roles in numerous biological processes. Both umbilical cord blood (UCB) and peripheral blood are rich in neutrophils. UCB is more abundant than peripheral blood, with cells generally at a more immature stage. However, comparative data between these two cell sources is lacking. This study aims to elucidate differences between UCB-derived neutrophils (UCBN) and peripheral blood-derived neutrophils (PBN). UCBN and PBN were isolated from fresh human umbilical cord blood and peripheral blood, respectively. Transcriptomic profiling was performed and compared against neutrophil RNA from three different donors. Bioinformatics analysis was employed to compare cell phenotypes. A cytokine cocktail (IFN-β, IFN-γ, and LPS) was used to activate UCBN and PBN in vitro. A united multi-omic approach, combining transcriptomic and proteomic analysis, was followed by experimental validation through flow cytometry, cell killing assays, and proteome profiler array to verify cell functions. Transcriptomic analysis revealed that the most upregulated genes in freshly isolated umbilical cord blood neutrophils (UCBN) compared to peripheral blood neutrophils (PBN) predominantly involve neutrophil activation and cell-killing functions. Validation through flow cytometry and cell-killing experiments demonstrated that highly viable UCBN exhibited significantly stronger ovarian tumor cell-killing activity in vitro compared to PBN. Both transcriptomic and proteomic analyses indicated that the primary upregulated genes in activated UCBN are chiefly involved in biological processes related to the regulation of cytokine secretion. Integrative multi-omic analysis, including a proteome profiler array, confirmed that UCBN indeed secrete elevated levels of cytokines. In conclusion: UCBN shows higher viability and cellular activity compared with PBN, particularly in tumor cell-killing and cytokine secretion.

Magnetic silk fibroin nanospheres loaded with amphiphilic polypeptides and antibiotics for biofilm eradication.

The eradication of established biofilms is a highly challenging task, due to the protective barrier effect of extracellular polymeric substances (EPS) and the presence of persister cells. Both increased drug permeability and elimination of persister cells are essential for the eradication of biofilms. Here, magnetic silk fibroin nanospheres loaded with antibiotics and host defense peptide (HDP) mimics (MPSN/S@P) were developed to demonstrate a new strategy for biofilm eradication. As an HDP mimic, an amphiphilic polypeptide containing 90% L-lysine and 10% L-valine (Lys90Val10) was selected for loading onto magnetic silk fibroin nanospheres via electrostatic interactions. Lys90Val10 exhibited excellent antibacterial activities against both planktonic and persister cells of Staphylococcus aureus (S. aureus). As a representative of the hydrophobic drug, spiramycin (SPM) was conveniently embedded into the β-sheet domain during the self-assembly process of silk fibroin. The sustained release of SPM during biofilm eradication enhanced the antibacterial efficacy of MPSN/S@P. The antibacterial test demonstrated that the extract from the MPSN/S@P suspension can kill both planktonic and persister cells of S. aureus, as well as inhibiting biofilm formation. Importantly, with the assistance of magnetic guidance and photothermal effects derived from Fe3O4 nanoparticles (Fe3O4 NPs), over 92% of bacteria in the biofilm were killed by MPSN/S@P, indicating the successful eradication of mature biofilms. The simple preparation method, integration of photothermal and magnetic responsiveness, and persister cell killing functions of MPSN/S@P provide an accessible strategy and illustrative paradigm for efficient biofilm eradication.

Altered NCR3 Splice Variants May Result in Deficient NK Cell Function in Renal Cell Carcinoma Patients.

The natural killer (NK) cell function of patients with malignant tumours may be suppressed by deficiency, and the poor prognosis of renal cell carcinoma (RCC) patients may be due to escape from NK cell cytotoxicity, especially with respect to natural cytotoxicity receptors (NCRs) on the NK cell surface. However, the specific mechanism remains unclear. Therefore, in this study, we sought to explore the role of NCR, especially NCR3 splice variants, in the process of NK cell deficiency in RCC patients. We used flow cytometry to analyse the phenotype of NK cells from the peripheral blood and kidney tumour tissue of RCC patients. The NKp30-mediated NK cell killing function was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) in NK and RCC cell coincubation. We extracted RNA from the peripheral blood mononuclear cells (PBMCs) of RCC patients and renal carcinoma tissue and carried out real-time quantitative PCR to detect the mRNA levels of NKp30a, NKp30b and NKp30c. mRNA expression levels of cytokines (IL-6, IL-8, IL-10, IL-18 and TGF-β) based on RNA extracted from renal carcinoma tissue and adjacent normal kidney tissues were also measured by real-time quantitative PCR. Regarding the phenotype of NK cells in RCC patients, the proportion of NK cells in tumour tissue was significantly reduced, with changes in the NK cell proportion being most obvious in NKp30+ NK cells. Furthermore, the results of the ADCC function assay showed limited NKp30+ NK cell-mediated cytotoxicity in RCC patients. Through real-time quantitative PCR, we found lower expression of NKp30a and NKp30b, the immunostimulatory splice variants of NCR3 encoding NKp30, in RCC patients. Moreover, expression of activating cytokines (IL-6 and IL-8) in renal cancer tissue was decreased, though inhibitory cytokine (TGF-β) expression remained unchanged, which may result in an immunosuppressive cytokine microenvironment. Decreased expression of immunostimulatory NCR3 splice variants and the inhibitory cytokine microenvironment in RCC patients may contribute to deficient NK cell cytotoxicity and renal carcinoma cell immune escape from NK cell killing, which may provide a theoretical basis for finding new immunotherapeutic targets for RCC.

The brief introduction of CAR T-cell: From mechanism to application

Chimeric antigen receptor T-cell (CAR-T) gene therapy is a newly developed solution to fight tumor cells. The therapy develops from the combination of the immunity and cell bioengineering, taking advantage of the cell killing function of T-cells, scientists modify them with a bioengineering method to target specific cells. In recent days, this therapy shows great results in several trials especially in hematological malignancies. However, there are still many obstacles, such as toxicities, that need to be overcome. Taking the promising potentials of CAR T-cell gene therapy, it is still a revolutionary step in the improvement of immunotherapy. In this paper, we focus on the structure and target antigens of CAR T-cell, summarize the generations of CAR T-cell gene therapies, as well as list out the FDA approved treatments using CAR. Most importantly, we will discuss the past advancements, recent innovations, and the existing factors that still prevent CAR T-cells from becoming the first line treatment for cancers.

Single-Cell Analyses Reveal Necroptosis's Potential Role in Neuron Degeneration and Show Enhanced Neuron-Immune Cell Interaction in Parkinson's Disease Progression.

Parkinson's disease (PD) is a common neuron degenerative disease among the old, characterized by uncontrollable movements and an impaired posture. Although widely investigated on its pathology and treatment, the disease remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) has been applied to the area of PD, providing valuable data for related research. However, few works have taken deeper insights into the causes of neuron death and cell-cell interaction between the cell types in the brain. Our bioinformatics analyses revealed necroptosis-related genes (NRGs) enrichment in neuron degeneration and selecting the cells by NRGs levels showed two subtypes within the main degenerative cell types in the midbrain. NRG-low subtype was largely replaced by NRG-high subtype in the patients, indicating the striking change of cell state related to necroptosis in PD progression. Moreover, we carried out cell-cell interaction analyses between cell types and found that microglia (MG)'s interaction strength with glutamatergic neuron (GLU), GABAergic neuron (GABA), and dopaminergic neuron (DA) was significantly upregulated in PD. Also, MG show much stronger interaction with NRG-high subtypes and a stronger cell killing function in PD samples. Additionally, we identified CLDN11 as a novel interaction pattern specific to necroptosis neurons and MG. We also found LEF1 and TCF4 as key transcriptional regulators in neuron degeneration. These findings suggest that MG were significantly overactivated in PD patients to clear abnormal neurons, especially the NRG-high cells, explaining the neuron inflammation in PD. Our analyses provide insights into the causes of neuron death and inflammation in PD from single-cell resolution, which could be seriously considered in clinical trials.

Mathematical model predicts tumor control patterns induced by fast and slow cytotoxic T lymphocyte killing mechanisms

Immunotherapy has dramatically transformed the cancer treatment landscape largely due to the efficacy of immune checkpoint inhibitors (ICIs). Although ICIs have shown promising results for many patients, the low response rates in many cancers highlight the ongoing challenges in cancer treatment. Cytotoxic T lymphocytes (CTLs) execute their cell-killing function via two distinct mechanisms: a fast-acting, perforin-mediated process and a slower, Fas ligand (FasL)-driven pathway. Evidence also suggests that the preferred killing mechanism of CTLs depends on the antigenicity of tumor cells. To determine the critical factors affecting responses to ICIs, we construct an ordinary differential equation model describing in vivo tumor-immune dynamics in the presence of active or blocked PD-1/PD-L1 immune checkpoint. Specifically, we identify important aspects of the tumor-immune landscape that affect tumor size and composition in the short and long term. We also generate a virtual cohort of mice with diverse tumor and immune attributes to simulate the outcomes of immune checkpoint blockade in a heterogeneous population. By identifying key tumor and immune characteristics associated with tumor elimination, dormancy, and escape, we predict which fraction of a population potentially responds well to ICIs and ways to enhance therapeutic outcomes with combination therapy.

The intrinsic defects of T cells impact the efficacy of CAR-T therapy in patients with diffuse large B-cell lymphoma

CAR-T cell therapy did not achieve the desired efficacy in some patients with diffuse large B-cell lymphoma (DLBCL). We conducted single-cell RNA and TCR sequencing as well as methylation chip profiling of peripheral blood samples in DLBCL patients. Patients who achieved complete remission (CR) showed an upward trend in T-cell levels, especially CD8-effector T cells. The responders exhibited T-cell clone expansion, more active T-cell transformation, and frequent cell communication. Highly expressed genes in the CR group were enriched in functions like leukocyte-mediated cytotoxicity and activation of immune response, while the non-CR group was enriched in pathways related to DNA damage and P53-mediated intrinsic apoptotic. More differentially methylated probes (DMPs) were identified in the baseline of the non-CR group (779 vs 350). GSEA analysis revealed that the genes annotated by DMPs were associated with cellular immune functions in T cells, including the generation of chemokines, leukocyte-mediated cytotoxicity, and cell-killing functions. The genes with low expression in the non-CR group exhibited a high methylation status. There is heterogeneity in the cellular, molecular, and epigenetic characteristics of host T cells in patients with different clinical outcomes. Intrinsic defects in T cells are important factors leading to poor efficacy of CAR-T therapy.

Influence of immunosuppressive drugs on natural killer cells in therapeutic drug exposure in liver transplantation.

Natural killer (NK) cells are enriched in the liver and are the main regulators in liver transplantation regarding rejection or tolerance, viral infection, or tumor recurrence. Immunosuppression consists of a triple drug standard regimen comprising tacrolimus (TAC) and corticosteroids (CS) with either mycophenolate mofetil (MMF) or sirolimus (SIR)/everolimus (EVE). The aim of this study was to evaluate the impact of trough levels of these regimens under clinical conditions and exposure on human NK-cell activity and function in order to better understand the antiviral and anti-tumor effects of mammalian target of rapamycin inhibitor (mTORI). Peripheral blood mononuclear cells (PBMCs) were collected from liver transplant recipients and healthy controls. Number and phenotypes of NK cells in vivo were analyzed by flow cytometry. In this study we simulated the immunosuppressive microenvironment in vitro. PBMCs were cultured at the clinically effective plasma concentration of drugs for 3 d to detect the effect of immunosuppressants on NK cells. Drug type and concentration: single drug [EVE, 5 ng/mL; SIR, 5 ng/mL; TAC, 5 ng/mL; cyclosporine A (CSA), 125 ng/mL; MMF, 15 µg/mL; CS, 0.5 µg/mL] and combined immunosuppressants (Group 1: TAC, 5 ng/mL + MMF, 15 µg/mL + CS, 0.5 µg/mL; Group 2: TAC, 5 ng/mL + SIR, 5 ng/mL + CS, 0.5 µg/mL; Group 3: TAC, 5 ng/mL + EVE, 5 ng/mL + CS, 0.5 µg/mL). In addition, NK cells were sorted from PBMCs and treated under the above conditions to detect NK cell killing function and RNA transcription characteristics. CS significantly impaired the cytolytic activity of NK cells, followed by MMF and SIR/EVE. CS and TAC/CSA significantly decreased the secretion of IFN-γ and CD107a. NK cell function in liver transplant recipients was most pronouncedly inhibited by a triple immunosuppressive regimen, with CS playing the most prominent role compared with the other drugs. The MMF-containing regimen demonstrated a significant increase in the expression of suppressive genes, especially of the Siglec7/9 family. The SIR group had stronger NK cell activity compared with that of the MMF group, although liver transplantation patients have lower NK cell activity and function. Despite an overall comparable immunosuppressive efficiency in terms of prevention of acute rejection, a mTORIs-including regimen might be considered as having less impact on NK cell function.

Dynamic changes of phenotype and function of natural killer cells in peripheral blood before and after thermal ablation of hepatitis B associated hepatocellular carcinoma and their correlation with tumor recurrence

BackgroundThermal therapy induces an immune response in patients with hepatocellular carcinoma (HCC), but the dynamic characteristics of the natural killer (NK) cell immune response post-thermal ablation remain unclear. We conducted a prospective longitudinal cohort study to observe the dynamic changes of phenotype and function of NK cells in peripheral blood before and after thermal ablation of hepatitis B-associated HCC and their correlation with tumor recurrence.MethodsFifty-six patients clinically and pathologically confirmed with hepatitis B-associated HCC were selected for thermal ablation. Peripheral blood was collected on day 0, day 7, and month 1. NK cell subsets, receptors, and killing function were detected by flow cytometry, and the LDH levels were examined. Overall recurrence and associated variables were estimated using Kaplan–Meier, log-rank, and Cox proportional-hazards analyses.ResultsThe frequency of CD3−CD56+ cells was increased on day 7 (P < 0.01) without significant differences between D0 and M1. NKG2D, NKp44, NKp30, CD159a, and CD158a expression was increased on M1 (all P < 0.05). The granzyme B and IFN-γ expression in NK cells were higher on M1 vs. D0 (P < 0.05). On day 7, the NK cell lysis activity of the target K562 cells was increased (P < 0.01) but decreased on M1 (P < 0.05). Survival analysis showed that CD158a expression and IFN-γ and perforin release on day 0 were associated with the risk of HCC recurrence. Cox regression analysis showed that the expression changes in CD56, NKp46, granzyme B, and perforin (D7-D0) induced by thermal ablation were associated with recurrence-free survival (RFS) of patients with HCC.ConclusionThermal ablation increased the frequency and function of CD3−CD56+ NK cells in the peripheral blood of patients with HCC. These cells tended to be more differentiated and activated. Notably, expression levels of NK cell receptors NKp46, perforin, and granzyme B were associated with RFS.

Comparison of NK cell subsets, receptors and functions induced by radiofrequency ablation and microwave ablation in HBV-associated primary hepatocellular carcinoma.

Topical therapy has been shown to induce an immune response in patients with hepatocellular carcinoma (HCC). In this study, a prospective parallel group control experiment was conducted to compare the differences between radiofrequency ablation and microwave ablation in inducing the immune regulation of NK cells. Sixty patients with clinically and pathologically confirmed hepatitis B-associated hepatocellular carcinoma (HCC) were selected for thermal ablation. Patients were randomly assigned into the MWA group (n = 30) and the RFA group (n = 30). Patient's peripheral blood was isolated on days D0, D7, and month M1. NK cell subsets, receptors, and killing function were detected by flow cytometry and LDH. Student t test and rank sum test were used to compare the statistical differences between the RFA (radio frequency) and MWA (microwave) groups. The Kaplan-Meier curve and log-rank test were used to calculate the difference between the two survival curves. Comparison of the frequency of CD3-CD56+ and CD3-CD56+CD16+ in NK cells between the RFA and WMA groups showed that there was no difference in the D0, D7, M1, D7-D0, M1-D0, and M1-D7 groups. The changes of the inhibitory NK cell receptor CD159A were significantly different at D7 (P<0.05). CD107a were compared between the RFA and WMA groups, indicating that CD107a changes induced by NK cells were significantly different at D7-D0 (P<0.05). Comparison of NK cell lysis activity of target K562 cells between the RFA and WMA groups showed that there was no difference at D0, D7, D7-D0. There was no difference in recurrence-free survival (RFS) between the RFA and WMA groups (P=0.11). The difference between MWA and RFA-induced NK cell changes was mainly manifested in the inhibitory receptors CD159a and CD107a 1 week after surgery, with microwave-induced changes being more severe. Comparison of the NK cell lysis activity of the target K562 cells between the RFA and WMA groups showed that there was no difference in D0, D7, D7- D0. Survival analysis showed that these differences did not affect the recurrence-free survival (RFS) in the two groups.

Identification of a novel necroptosis-related classifier to predict prognosis and guide immunotherapy in breast invasive carcinoma.

BackgroundNecroptosis plays a crucial function in the progression of breast invasive carcinoma (BRCA). It may be triggered in cancer therapy to enhance anti-tumor immunity. However, the functions of necroptosis in tumors and its relationship with the tumor microenvironment (TME) remain largely unclear.MethodsNecroptosis-related genes (NRGs) were collated from high-quality literature reviews. A robust risk model was constructed to systematically evaluate the clinical value, functional status, effects exerted by the risk model on the TME, and the genomic variations based on the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) meta-cohorts.ResultsA risk model was constructed which comprised of six NRGs, including TNF receptor-associated factor 5 (TRAF5), Toll-like receptor 3 (TLR3), a riboflavin kinase (RFK), Fas ligand (FASLG), Fas-associated protein with death domain (FADD), and baculoviral IAP repeat-containing 3 (BIRC3). The stability and accuracy of the risk model were demonstrated for both the training and validation cohorts and its utility as an independent prognostic model for BRCA was verified. Patients in the low-risk group exhibited “hot” tumors having active immune and cell killing functions, while those in the high-risk group showed “cold” tumors having active tumor proliferation and immunosuppression. Moreover, patients in the high-risk group had a greater number of CNV events in their genome, while the somatic mutations were fewer. Furthermore, patients in the low-risk group showed high sensitivity toward immunotherapy and chemotherapy.ConclusionA reliable risk model based on NRGs to assess patient prognoses and guide clinical decision-making was constructed and validated. Our findings may contribute to the understanding of necroptosis and aid clinical management, along with precision treatment in BRCA.

Selectively targeting the cancer cells by reactivating the cell killing function of p53 protein

Selectively targeting the cancer cells by reactivating the cell killing function of p53 protein - IJPCA- Print ISSN No: - 2394-2789 Online ISSN No:- 2394-2797 Article DOI No:- 10.18231/j.ijpca.2022.010, International Journal of Pharmaceutical Chemistry and Analysis-Int J Pharm Chem Anal

The cross-talk of cancer-associated fibroblasts assist in prognosis and immunotherapy in patients with breast carcinoma.

The association between cancer-associated fibroblasts (CAFs) and tumor microenvironment (TME) is a key factor in promoting tumor progression. However, the correlation between CAFs and TME in breast carcinoma has not been elucidated. Thus, further study about the cross-effect between CAFs and TME can provide novel strategies for breast carcinoma treatment, particularly targeted immunotherapy. First, we systematically analyzed cell communication in a single-cell dataset and identified the interacted genes between CAFs and TME components. Then, a robust fibroblast-related score (FRS) model was developed using the LASSO algorithm. The FRS can be a reliable adverse prognostic factor in three cohorts with breast carcinoma. Functional enrichment analysis and single-sample Gene Set Enrichment Analysis showed that patients with a high FRS had cold tumors with active proliferation and immunosuppression. Patients with a low FRS presented with hot tumors with active immune and cell-killing functions. Genomic variation analysis revealed that patients with a low FRS had a higher somatic mutation load and copy number variation burden. Finally, patients with a low FRS were more sensitive to chemotherapy and immunotherapy, particularly anti-PD-1 therapy. In conclusion, a reliable FRS model was constructed not only reliable for predicting prognosis but also competent to estimate clinical immunotherapy and chemotherapy response for patients with BRCA, which might provide significant clinical implications for guiding clinical decision-making for patients with BRCA.

Development and validation of a robust necroptosis related classifier for colon adenocarcinoma.

Background: Necroptosis, a novel form of apoptosis, plays a crucial function in the progression of colon adenocarcinoma (COAD) and is expected to be triggered in cancer therapy for enhancing anti-tumor immunity. However, the function of necroptosis in tumors and its relationship with the tumor microenvironment (TME) remains largely unclear. Methods: Necroptosis-related genes (NRGs) were collected from high-quality literature. Using The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov) and the Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/geo) meta-cohorts, a robust risk model was constructed to systematically examine the clinical value, functional status, the role of TME based on the risk model, as also the genomic variations. Results: A risk model containing nine NRGs, including TNF receptor-associated factor (TRAF2), TNF receptor 1 associated via death domain (TRADD), ubiquitin carboxyl-terminal hydrolase 21 (USP21), TNF receptor superfamily, member 6 (FAS), tumor necrosis factor receptor superfamily 10B (TNFRSF10B), mitogen-activated protein kinase 8 (MAPK8), mixed lineage kinase domain-like (MLKL), TNF receptor-associated factor 5 (TRAF5), and recombinant receptor-interacting serine-threonine kinase 3 (RIPK3), was constructed. The risk model’s stability and accuracy were demonstrated in training, as also the validation cohorts; it was verified as an independent prognostic model for COAD. High-risk group patients developed “cold” tumors having active tumor proliferation and immunosuppression, while those in the low-risk group developed “hot” tumors with active immune and cell killing functions. Moreover, a higher number of copy number variations in the genome and fewer somatic mutations were found in high-risk group patients. Furthermore, higher sensitivity towards immunotherapy and chemotherapy was seen in patients of the low-risk group. Conclusion: A reliable risk model based on NRGs to assess patient prognosis and guide clinical decision-making was constructed and validated. Our findings may contribute to the understanding of necroptosis and are expected to aid clinical management and guide precision treatment for patients with COAD.

Vγ9Vδ2 T cells expressing a BCMA-Specific chimeric antigen receptor inhibit multiple myeloma xenograft growth.

Vγ9Vδ2 T cells are immune effector cells capable of killing multiple myeloma (MM) cells and have been tested in clinical trials to treat MM patients. To enhance the MM cell killing function of Vγ9Vδ2 T cells, we introduced a BCMA-specific CAR into ex vivo expanded Vγ9Vδ2 T cells through electroporation of the CAR-encoding mRNA. The modified Vγ9Vδ2 T cells displayed a high cytolytic activity against BCMA-expressing MM cell lines in vitro, while sparing BCMA-negative cells, including normal B cells and monocytes. Subsequently, we intravenously injected KMS-11 human MM cells to generate a xenograft mouse model. The treatment of the tumor-bearing mice with Zometa and anti-BCMA CAR- Vγ9Vδ2 T cells resulted in a significant reduction of tumor burden in the femur region, as well as the overall tumor burden. In association with the decrease in tumor burden, the survival of the MM cell-inoculated mice was markedly prolonged. Considering the potential of Vγ9Vδ2 T cells to be used as off-the-shelf products, the modification of these cells with a BCMA-specific CAR could be an attractive option for cancer immunotherapy against bone marrow cancer MM.

High resolution microfluidic assay and probabilistic modeling reveal cooperation between T cells in tumor killing

Cytotoxic T cells are important components of natural anti-tumor immunity and are harnessed in tumor immunotherapies. Immune responses to tumors and immune therapy outcomes largely vary among individuals, but very few studies examine the contribution of intrinsic behavior of the T cells to this heterogeneity. Here we show the development of a microfluidic-based in vitro method to track the outcome of antigen-specific T cell activity on many individual cancer spheroids simultaneously at high spatiotemporal resolution, which we call Multiscale Immuno-Oncology on-Chip System (MIOCS). By combining parallel measurements of T cell behaviors and tumor fates with probabilistic modeling, we establish that the first recruited T cells initiate a positive feedback loop to accelerate further recruitment to the spheroid. We also provide evidence that cooperation between T cells on the spheroid during the killing phase facilitates tumor destruction. Thus, we propose that both T cell accumulation and killing function rely on collective behaviors rather than simply reflecting the sum of individual T cell activities, and the possibility to track many replicates of immune cell-tumor interactions with the level of detail our system provides may contribute to our understanding of immune response heterogeneity.