Upregulation Of Ki-67 Research Articles

Multi-parametric atlas of the pre-metastatic liver for prediction of metastatic outcome in early-stage pancreatic cancer.

Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.

Prognostic and clinicopathological value of Ki-67 in patients with oesophageal squamous cell carcinoma: a systematic review and meta-analysis

ObjectivesThe relationship between Ki-67 expression and the prognosis of patients with oesophageal squamous cell carcinoma (ESCC) has been extensively studied. However, their findings were inconsistent. Consequently, the present meta-analysis was...

Ex Vivo Analysis of Cell Differentiation, Oxidative Stress, Inflammation, and DNA Damage on Cutaneous Field Cancerization.

Cutaneous field cancerization (CFC) refers to a skin region containing mutated cells' clones, predominantly arising from chronic exposure to ultraviolet radiation (UVR), which exhibits an elevated risk of developing precancerous and neoplastic lesions. Despite extensive research, many molecular aspects of CFC still need to be better understood. In this study, we conducted ex vivo assessment of cell differentiation, oxidative stress, inflammation, and DNA damage in CFC samples. We collected perilesional skin from 41 patients with skin cancer and non-photoexposed skin from 25 healthy control individuals. These biopsies were either paraffin-embedded for indirect immunofluorescence and immunohistochemistry stain or processed for proteins and mRNA extraction from the epidermidis. Our findings indicate a downregulation of p53 expression and an upregulation of Ki67 and p16 in CFC tissues. Additionally, there were alterations in keratinocyte differentiation markers, disrupted cell differentiation, increased expression of iNOS and proinflammatory cytokines IL-6 and IL-8, along with evidence of oxidative DNA damage. Collectively, our results suggest that despite its outwardly normal appearance, CFC tissue shows early signs of DNA damage, an active inflammatory state, oxidative stress, abnormal cell proliferation and differentiation.

Long-Term Ex Situ Normothermic Kidney Perfusion

Abstract Background Marginal grafts are increasingly considered for transplantation due to the widening gap of available donor grafts and waiting recipients. Graft assessment and repair prior to implantation are therefore essential to minimize the risk of failure. Normothermic perfusion has not been widely evaluated for kidney grafts, with existing perfusions limited to 2 days. Aims The aim of this study was to develop a long-term (&amp;gt;3 days) normothermic ex situ swine perfusion model in kidney grafts, while maintaining organ viability. We also wanted to investigate possible regenerative processes of the kidney during this period outside the donor animal. Methods Swine kidneys were retrieved with minimal injury. After cannulation, organs were perfused at 37°C for up to several days in a newly designed normothermic ex situ device with automated blood gas and glucose regulation, addition of nutrition, antibiotics, and vasodilators. Kidney biopsies, urine and perfusate samples were taken daily for analysis as well as markers of viability. Results In total 17 kidneys were perfused. Four kidneys were perfused for 3 or more days. Viability was maintained up to 4 days without histological evidence of glomerular and minimal to moderate tubular necrosis. We observed upregulation of ki67 and pH3 in tubular cells with mean values of up to 14.2% and 2.2% respectively. This expression of ki67, a proliferation associated protein, correlated with the duration of ex situ perfusion. Conclusion This is the first report of normothermic kidney perfusion for up to 4 days with unprecedented observation of upregulated ki67 and pH3 after multiple days of perfusion. These preclinical results open the door for development of long-term perfusion protocols for discarded human grafts while also hinting at limitations and challenges.

Di-(2-ethylhexyl) phthalate aggravates psoriasis-like skin lesions: In vitro and in vivo evaluation

Di-(2-ethylhexyl) phthalate (DEHP), which is a widely used phthalate (PAE), has recently received public attention owing to it causing health problems. The aim of this study was to elucidate the aggravating effects of DEHP on psoriasis and skin toxicity. Human keratinocyte (HaCaT) cells were treated with gradient concentrations of DEHP, and mice with imiquimod (IMQ)-induced psoriasiform dermatitis were hypodermically injected with 40 μg/kg/day of DEHP for seven consecutive days. The skin condition was assessed based on the psoriasis area and severity index score, which indicated the deterioration of IMQ-induced psoriasis-like skin lesions after DEHP exposure. To further analyze the effect of DEHP on psoriasis, the proliferation, inflammation, and tight junction (TJ) damage were examined, which correlated with the development and severity of psoriasis. The results showed that DEHP promoted proliferation both in vivo and in vitro, which manifested as epidermal thickening; an increase in cell viability; upregulation of Ki67, CDK2, cyclinD1, and proliferating cell nuclear antigen; and downregulation of p21. An excessive inflammatory response is an important factor that exacerbates psoriasis, and our results showed that DEHP can trigger the release of inflammatory cytokines as well as the infiltration of T cells. TJ disorders were found in mice and cells after DEHP treatment. Additionally, p38 mitogen-activated protein kinase (MAPK) was strongly activated during this process, which may have contributed to skin toxicity caused by DEHP. In conclusion, DEHP treatment promotes proliferation, inflammation, TJ disruption, and p38 MAPK activation in HaCaT cells and psoriasis-like skin lesions.

Tissue Resistance Decrease during Irreversible Electroporation of Pancreatic Cancer as a Biomarker for the Adaptive Immune Response and Survival

PurposeTo correlate irreversible electroporation (IRE) procedural resistance changes with survival outcomes and the IRE-induced systemic immune response in patients with locally advanced pancreatic cancer (LAPC). Materials and MethodsData on IRE procedural tissue resistance (R) features and survival outcomes were collected from patients with LAPC treated within the context of 2 prospective clinical trials in a single tertiary center. Preprocedural and postprocedural peripheral blood samples were prospectively collected for immune monitoring. The change (ie, decrease) in R during the first 10 test pulses (ΔR10p) and during the total procedure (ΔRtotal) were calculated. Patients were divided in 2 groups on the basis of the median change in R (large ΔR vs small ΔR) and compared for differences in overall survival (OS) and progression-free survival and immune cell subsets. ResultsA total of 54 patients were included; of these, 20 underwent immune monitoring. Linear regression modeling showed that the first 10 test pulses reflected the change in tissue resistance during the total procedure appropriately (P < .001; R2 = 0.91). A large change in tissue resistance significantly correlated with a better OS (P = .026) and longer time to disease progression (P = .045). Furthermore, a large change in tissue resistance was associated with CD8+ T cell activation through significant upregulation of Ki-67+ (P = .02) and PD-1+ (P = .047). Additionally, this subgroup demonstrated significantly increased expression of CD80 on conventional dendritic cells (cDC1; P = .027) and PD-L1 on immunosuppressive myeloid-derived suppressor cells (P = .039). ConclusionsIRE procedural resistance changes may serve as a biomarker for survival and IRE-induced systemic CD8+ T cell and cDC1 activation.

A single administration of hIL-7-hyFc induces long-lasting T-cell expansion with maintained effector functions

AbstractInterleukin-7 (IL-7) is an essential cytokine for T-cell homeostatic proliferation and maintenance. Clinical studies have shown the potential benefits of IL-7 therapy in various diseases associated with lymphopenia. However, the kinetics of the T-cell response to a single administration of IL-7 in humans have not been fully elucidated. Here, we investigated the effects of Fc-fused long-acting recombinant human IL-7 (hIL-7-hyFc, efineptakin alfa) on lymphocytes in healthy adults after a single subcutaneous or intramuscular administration. Administration of hIL-7-hyFc increased the CD8+ and CD4+ T-cell numbers up to 2.5-fold, with corresponding upregulation of Ki-67 and Bcl-2 expression, peaking at day 3 or 7. Regulatory T cells (Tregs) did not expand. Among CD8+ and CD4+ T cells, all T-cell subsets (TN, TEM, TCM, TEMRA, and TSCM) increased for 56 days. The T-cell receptor repertoire diversity of naive CD8+ and CD4+ T cells was increased by hIL-7-hyFc, whereas the memory T-cell subsets did not differ between day 56 and day 0. Transcriptomic analysis revealed that hIL-7-hyFc induced robust T-cell expansion without changes in gene expression profiles associated with T-cell functions or genes related to T-cell exhaustion, senescence, and anergy. The effector functions of antigen-specific CD8+ T cells were preserved after hIL-7-hyFc administration. Our results suggest that hIL-7-hyFc administration induced a sustained increase in the numbers of CD8+ and CD4+ T cells, but not Tregs, without qualitative changes. These results support the potential of hIL-7-hyFc as a treatment for patients with compromised T-cell immunity or as a vaccine adjuvant.

Increased expression of ST2 on regulatory T cells is associated with cancer associated fibroblast-derived IL-33 in laryngeal cancer

Interleukin (IL)− 33 plays an essential role in regulatory T cell (Treg)-mediated immunosuppression in cancers and underlies the crosstalk between Tregs and the tumor microenvironment. However, the phenotypic characteristics of subset Tregs modulated by IL-33 and its association with the tumor microenvironment are not fully understood. This study aimed to examine the expression of ST2, the receptor of IL-33, on Tregs in tumors and to evaluate their association with cancer associated fibroblasts (CAFs) and reciprocal influences on the prognosis of laryngeal cancer. Our results showed that increased numbers of Tregs were found in laryngeal tumor tissues. Tregs in stromal IL-33-positive tumor tissues demonstrated significantly higher expression of ST2 than those in IL-33- or adjacent nontumor tissues. ST2-expressing Tregs exhibited upregulation of Ki67 and CTLA4 compared with their ST2- negative counterparts. Furthermore, IL-33 in the tumor microenvironment was mainly derived from fibroblasts. ST2 expression on Tregs was correlated with the number of IL-33-positive CAFs. High ST2 expression on Tregs, combined high ST2 on Tregs and the presence of IL-33 expressing CAFs was associated with worse survival outcomes in laryngeal cancer. This study indicated that increased expression of ST2 on Tregs is associated with microenvironmental IL-33 signaling derived from CAFs in laryngeal cancer, unraveling the special role of Tregs and fibroblasts in modulating IL-33/ST2 involved immune-evasive tumor microenvironment.

The generation of stem cell-like memory cells early after BNT162b2 vaccination is associated with durability of memory CD8+ Tcell responses.

COVID-19 vaccines elicit humoral and cellular immune responses. Durable maintenance of vaccine-induced immunity is required for long-term protection of the host. Here, we examine activation and differentiation of vaccine-induced CD8+ T cells using MHC class I (MHC-I) multimers and correlations between early differentiation and the durability of CD8+ T cell responses among healthcare workers immunized with two doses of BNT162b2. The frequency of MHC-I multimer+ cells is robustly increased by BNT162b2 but decreases 6 months post-second vaccination to 2.4%–65.6% (23.0% on average) of the peak. MHC-I multimer+ cells dominantly exhibit phenotypes of activated effector cells 1–2 weeks post-second vaccination and gradually acquire phenotypes of long-term memory cells, including stem cell-like memory T (TSCM) cells. Importantly, the frequency of TSCM cells 1–2 weeks post-second vaccination significantly correlates with the 6-month durability of CD8+ T cells, indicating that early generation of TSCM cells determines the longevity of vaccine-induced memory CD8+ T cell responses.

Tim-3 deletion on Treg increases virus-specific T cell response and reduces viral burden in chronic LCMV infection

Abstract Tim-3, a surface protein, is upregulated on Treg during chronic viral infections. During HCV infection in humans there is an increase in frequency of Tim-3+ Treg, which have increased proliferation, survival, and expression of immunosuppressive cytokines like IL-10. Similarly, it’s been recently reported that there is an increase in Tim-3+ Treg in people with HIV (PWH). However, the role of regulatory T cells (Treg) during chronic viral infection has not been fully defined. We hypothesize that Tim-3 promotes an effector phenotype on Treg during chronic viral infection, which limits the virus-specific T cell response and impairs viral clearance. Using an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) model, we found a significant decrease in morbidity during LCMV chronic infection. Tim-3 Treg KO mice mounted a higher virus-specific T cell response and had lower viral burden in blood, liver, and kidney at 30dpi. Lack of Tim-3 expression on Treg limited their ability to upregulate IL-10, TGFb, CD39, PD-1, CD25, CTLA-4 and CD44. Similarly, in PWH Tim-3+ Treg have increased expression of IL-10 compared to HIV(−). In addition, Tim-3+ Treg from PWH show significant upregulation of Ki67+, CD73+, Gzmb+ and TGFb+ compared to their Tim-3− Treg counterpart. Our findings demonstrate that modulation of a surface protein in Treg can lead to a reduction in viral burden and enhance LCMV-specific T cell response. We found that Tim-3+ Treg have an increased suppressive phenotype, compared with Tim-3− Treg in PWH, regardless of viral suppression. Elucidation of the mechanism of Tim-3 function in Treg may provide insights into the biology of chronic infections and for developing novel potential strategies to achieve HIV remission off ART. Supported by grants from NIH (T32GM008208, R01AI138504, U01AI131285)

Tissue resident lung natural killer (NK) cells demonstrate enhanced responses to IL-15 compared to peripheral populations

Abstract Tissue resident NK cells with unique phenotype and function have been identified in organs such as the lung and liver. We have shown that inhaled (IH) rhIL-15 can lead to durable clinical responses in dogs with naturally occurring osteosarcoma or melanoma lung metastases, suggesting that lung NK populations can be targeted therapeutically. Thus, we set out to evaluate the phenotype of resting and rhIL-15-stimulated lung NK cells isolated from murine and human lung. C57BL/6 and BALB/c mice were exposed to IH or systemic IL-15 at escalating doses, and NK cells were isolated by negative selection from lungs and spleen of wild type mice for co-culture. In human patients undergoing surgery for malignancy, non-tumor lung tissue was isolated for flow cytometry and ex vivo stimulation with IL-15. Using mouse spleen or human blood as controls, we observed a higher proportion of lung NK cells per live CD45+ cells in baseline mouse and human samples. Lung NK also had higher expression of activation markers CD69 and HLA-DR (human), but lower expression of the inhibitory receptor TIGIT. Ex vivo culture with IL-15 also resulted in greater upregulation of Ki67 on lung NK cells. Compared to systemic delivery of IL-15, IH IL-15 resulted in greater upregulation of Ki67 and lower upregulation of TIGIT in lung NK cells. Subset analysis of CD49a+ lung NK cells showed similar results in both mouse and human models for activation marker and Ki67 expression after IL-15. Taken together, our results suggest that lung NK cells possess a more active baseline phenotype and exhibit preferential stimulation in response to rhIL-15 compared to circulating NK cells. Ultimately, targeting of lung resident NK cells may be a viable therapeutic strategy in patients with lung metastases. Supported by NIH/ NCI 1R03CA252793 NIH/NCI U01 CA224166-01 NIH/NCI T32 CA251007-01

Accelerated Wound Healing and Keratinocyte Proliferation through PI3K/Akt/pS6 and VEGFR2 Signaling by Topical Use of Pleural Fluid.

Impaired wound healing is an ongoing issue that cancer patients undergoing chemotherapy or radiotherapy face. Our previous study regarding lung-cancer-associated pleural fluid (LCPF) demonstrated its propensity to promote endothelial proliferation, migration, and angiogenesis, which are crucial features during cutaneous wound healing. Therefore, the current study aimed to investigate the effect of pleural fluid on cutaneous wound closure in vitro and in vivo using HaCaT keratinocytes and a full-thickness skin wound model, respectively. Both heart-failure-associated pleural fluid (HFPF) and LCPF were sequentially centrifuged and filtered to obtain a cell-free status. Treatment with HFPF and LCPF homogeneously induced HaCaT proliferation with cell cycle progression, migration, and MMP2 upregulation. Western blotting revealed increased PI3K/Akt phosphorylation and VEGFR2/VEGFA expression in HaCaT cells. When treated with the PI3K inhibitor, LCPF-induced keratinocyte proliferation was attenuated with decreased pS6 levels. By applying the VEGFR2 inhibitor, LCPF-induced keratinocyte proliferation was ameliorated by pS6 and MMP2 downregulation. The effect of LCPF-induced cell junction rearrangement was disrupted by co-treatment with a VEGFR2 inhibitor. Compared with a 0.9% saline dressing, LCPF significantly accelerated wound closure and re-epithelization when used as a dressing material in a full-thickness wound model. Histological analysis revealed increased neo-epidermis thickness and dermis collagen synthesis in the LCPF-treated group. Furthermore, LCPF treatment activated basal keratinocytes at the wound edge with the upregulation of Ki-67, VEGFA, and MMP2. Our preliminaries provided the benefit of wet dressing with pleural fluid to improve cutaneous wound closure through enhanced re-epithelization and disclosed future autologous application in cancer wound treatment.

Dexmedetomidine inhibits the growth and metastasis of esophageal cancer cells by down-regulation of lncRNA MALAT1.

This study aims to evaluate the effect of dexmedetomidine (DEX)-on esophageal cancer (EC) via regulating long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The effect of DEX on MALAT1 expression and EC cell viability was detected. EC cells were divided into Blank, DEX, scrambled/MALAT1 siRNA, and DEX + control/MALAT1 groups, followed by a series of experiments including quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), Annexin V-FITC/PI staining, wound healing, and Transwell assays. Additionally, mice were subjected to the subcutaneous injection of Eca109 cells transfected by control/MALAT1 activation lentiviral vector to construct EC models with the DEX treatment, and then the tumor volume and the expression of Ki-67 and active caspase-3 were determined. DEX reduced the expression of MALAT1 in EC cells in a dose-dependent manner. DEX inhibited the viability of EC cells, but increased the cell apoptosis, which, however, was reversed by MALAT1 overexpression. Moreover, MALAT1 overexpression abolished the inhibitory effect of DEX on the epithelial-mesenchymal transition (EMT) of EC cells, with enhanced migration and invasion. Furthermore, DEX succeeded in decreasing the tumor volume with the down-regulation of MALAT1. In comparison with the DEX group, mice in the DEX + MALAT1 group had larger tumors, with the up-regulation of Ki-67 and the down-regulation of active caspase-3. DEX can reduce the expression of MALAT1 in EC cells, thereby inhibiting the proliferation, invasion and migration, as well as EMT, and promoting the apoptosis of EC cells.

Can we use Ki67 expression to predict prostate cancer aggressiveness?

specialists have an urge for biomarkers that can discriminate indolent prostate cancer from aggressive tumors. Ki67 is a proliferation marker, and its expression is associated with the aggressiveness of several cancers. analyze the expression of Ki67 in prostate cancer samples correlating with the aggressiveness of the disease. Ki67 mRNA levels were determined utilizing data from a TCGA cohort (Tumor(n)=492 and control(n)=52). The protein expression was determined on 94 biopsies from patients by immunohistochemical assay. in mRNA, the Ki67 upregulation is associated with cancer tissue (p<0.0001) and worst disease-free survival (p=0.035). The protein upregulation is associated with increase of the ISUP score (p<0.0001), cancer stage (p=0.05), biochemical recurrence (p=0.0006) and metastasis (p<0.0001). We also show a positive correlation between Ki67 expression and ISUP score (r=0.5112, p<0.0001) and disease risk stratification (r=0.3388, p=0.0009). Ki67 expression is a factor independently associated with biochemical recurrence (p=0.002) and metastasis (p<0.0001). Finally, the patients with high Ki67expression shows better survival regarding biochemical recurrence (p=0.008) and metastasis (p=0.056). Patients with high Ki67 expression are 2.62 times more likely to develop biochemical recurrence (p=0.036). Ki67 upregulation is associated with prostate cancer aggressiveness.

The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation.

Butyrophilin 1A1 (BTN1A1) is implicated in the secretion of lipid droplets from mammary epithelial cells as a membrane receptor, which forms a secretion complex with the redox enzyme, xanthine oxidoreductase (XDH). The first evidence that BTN1A1 functions in this process was the generation of Btn1a1 −/− mouse lines, in which lipid secretion was disrupted and large unstable droplets were released into alveolar spaces with fragmented surface membranes. We have revisited one of these mutant mouse lines using RNAseq and proteomic analysis to assess the consequences of ablating the Btn1a1 gene on the expression of other genes and proteins. Disruption of intact Btn1a1 protein expression led to a large build‐up of Xdh in the cytoplasm, induction of acute phase response genes and Lif‐activation of Stat3 phosphorylation. At peak lactation, approx. 10% of the cells were dying, as assessed by TUNEL‐analysis of nuclear DNA. Possible cell death pathways included expression of caspase 8 and activated caspase 3, autophagy, Slc5a8‐mediated inactivation of survivin (Birc5), and pStat3‐mediated lysosomal lysis, the latter of which is the principal death route in involuting wild type cells. Milk secretion was prolonged by renewal of the secretory epithelium, as evidenced by the upregulation of Ki67 in approx. 10% of cell nuclei and expression of cyclins and Fos/Jun. These data highlight the plasticity of the mammary epithelium and the importance of functional BTN1A1 expression for maintenance of terminally differentiated secretory cells and optimal milk production throughout lactation.

Abstract 514: Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade

Abstract Using HER2-targeted antibody drug conjugates (ADC), we tested the therapeutic efficacy, direct impact on cancer stem cells (CSCs), and potential added benefit when combined with PD-L1 blockade in immunocompetent mouse tumor models. Two murine mammary tumor models were used in this study: D2F2/E2, a cell line resulting from stable transfection of parental D2F2 murine mammary tumor cells with wild-type human HER2, and 4T1-HER2, derived from parental 4T1 by stable transduction with wild-type human HER2. We demonstrate that HER2-targeted ADC treatment induced both cellular and humoral anti-HER2 adaptive immune responses which effectively targeted both bulk tumor and cancer stem cells. We recently investigated induction of host immunity using a tumor re-challenge strategy in tumor free animals subjected to ADC or ADC + anti-PD-L1 therapy. D2F2/E2 tumor-bearing mice were treated with ADC or ADC + anti-PD-L1 to generate tumor free mice. These tumor free animals were re-challenged with the relevant tumor D2F2/E2 or irrelevant tumor WT 4T1 respectively. While all the tumor free animals re-challenged with WT 4T1 showed aggressive tumor growth, ADC-treated tumor free animals re-challenged with D2F2/E2 demonstrated significantly (p&amp;lt;0.0001) slower tumor growth compared with the age-matched naive mice, and this protection was significantly (p=0.0078) enhanced in ADC + anti-PD-L1-treated tumor free animals, resulting in completely rejection of the re-challenge and thus documenting immunological memory formation. B cell activation was obvious by the significant upregulation of Ki67 and GL7 CD86 on both CD45+CD19+ tumor-infiltration lymphocytes (TILs) and splenocytes. B cell differentiation was evident by the upregulation of GL7 on both TILs and splenocytes, as well as the expression of CD138 on splenotytes. We found that IgG1 was the predominant isotype in treated mice vs. IgG2a, IgG2b, IgG3, and ADC + anti-PD-L1 dual treatment drastically increased the amount of IgG1 in serum. In addition, ADC+ anti-PD-L1 dual treatment significantly increased the frequency of CD8+IFNγ+ cells in TILs. Both ADC alone (p=0.0004) and ADC+anti-PD-L1 dual treatment (p=0.01) significantly decreased CD4+FoxP3+ regulatory T cells. These results provide evidence that HER2-targeted ADC + anti-PD-L1 immunotherapy-induced B cell activation and differentiation along with the modulation of T cell subsets could confer host anti-HER2 immunity. Citation Format: You Qin, Hannah E. Dobson, Frank I. Comer, Alfred E. Chang, Max S. Wicha, Qiao Li. Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 514.

Cold Atmospheric Plasma Promotes Regeneration-Associated Cell Functions of Murine Cementoblasts In Vitro.

The aim of the study was to examine the efficacy of cold atmospheric plasma (CAP) on the mineralization and cell proliferation of murine dental cementoblasts. Cells were treated with CAP and enamel matrix derivates (EMD). Gene expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), periostin (POSTN), osteopontin (OPN), osterix (OSX), collagen type I alpha 1 chain (COL1A1), dentin matrix acidic phosphoprotein (DMP)1, RUNX family transcription factor (RUNX)2, and marker of proliferation Ki-67 (KI67) was quantified by real-time PCR. Protein expression was analyzed by immunocytochemistry and ELISA. ALP activity was determined by ALP assay. Von Kossa and alizarin red staining were used to display mineralization. Cell viability was analyzed by XTT assay, and morphological characterization was performed by DAPI/phalloidin staining. Cell migration was quantified with an established scratch assay. CAP and EMD upregulated both mRNA and protein synthesis of ALP, POSTN, and OPN. Additionally, DMP1 and COL1A1 were upregulated at both gene and protein levels. In addition to upregulated RUNX2 mRNA levels, treated cells mineralized more intensively. Moreover, CAP treatment resulted in an upregulation of KI67, higher cell viability, and improved cell migration. Our study shows that CAP appears to have stimulatory effects on regeneration-associated cell functions in cementoblasts.

SLC6A8-mediated intracellular creatine accumulation enhances hypoxic breast cancer cell survival via ameliorating oxidative stress

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia.MethodsRNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo.ResultsSLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3.ConclusionsOur study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.

Overexpression of DJ-1 alleviates autosomal dominant polycystic kidney disease by regulating cell proliferation, apoptosis, and mitochondrial metabolism in vitro and in vivo.

BackgroundDJ-1 is critical for the mitochondrial function associated with autosomal dominant polycystic kidney disease (ADPKD). We aimed to investigate DJ-1’s function in the pathogenesis of ADPKD.MethodsDJ-1 was knocked-down in IMCD3 cells to evaluate the effects of DJ-1 on cell phenotype and mitochondrial function in vitro. Furthermore, we generated three groups of mice with different expression levels of DJ-1 within an established ADPKD model: ADPKD, ADPKDpcDNA, and ADPKDpcDNA-DJ-1.ResultsDJ-1 knock-down significantly increased oxidative stress as well as the proliferation and apoptosis rate of IMCD3 cells, along with Bcl-2 down-regulation and the up-regulation of Ki67, PCNA, Bax, cleaved caspase-3, and cleaved caspase-9. DJ-1 knock-down suppressed the cellular respiration, Ca2+ absorption, and mitochondrial complex I activity in mitochondria. In vivo, we verified that DJ-1 was down-regulated in ADPKD models, and its overexpression attenuated the renal dysfunction in ADPKD models. The transgenic mice had a significantly smaller renal cyst and less interstitial fibrosis than control, accompanied byα-SMA, fibronectin, and TGF-β1 up-regulation. Moreover, in vivo results confirmed DJ-1 overexpression inhibited the proliferation and apoptosis of tubular epithelial cells along with down-regulation of Ki67, PCNA, p53, intracellular Cyt c, cleaved caspase-3, and cleaved caspase-9 and the up-regulation of Bcl-2.ConclusionsDJ-1 was down-regulated in ADPKD models, and its overexpression may attenuate the renal dysfunction and pathological damage by regulating the proliferation, apoptosis, oxidative stress and mitochondrial metabolism, which may be mediated by the p53 signaling pathway.

Abstract 1044: Frequency of circulating CX3CR1+ CD8+ T cells to predict response to immune checkpoint inhibitor therapy

Abstract While immune checkpoint inhibitors (ICI) have revolutionized treatment for various cancers including non-small cell lung cancer (NSCLC), only a subset of patients receives durable clinical benefit. Discovery of blood-based biomarkers that reflect dynamic change of tumor microenvironment, and predict response to ICI will markedly improve current treatment regimens. Emerging evidence has shown that the frequency of peripheral blood (PB) CD8+ T cells expressing CX3CR1, a marker of effector T-cell differentiation increases in patients receiving ICI. However, no studies have rigorously evaluated the utility of CX3CR1 as a PB biomarker to predict response to ICI, and identify responders and non-responders. Here, we evaluated the utility of circulating T-cell biomarkers to predict response to ICI therapy in preclinical models and NSCLC patients (n=35). We found successful treatment with ICI significantly increases the frequency of PB CX3CR1+ CD8+ T-cell subsets that enrich neoantigen- as well as shared tumor-associated antigen-specific T cells in mice bearing MC38 and CT26 colon adenocarcinoma. Significantly increased expression of IFN-γ and Ki67 suggests that these subsets are highly proliferative effector CD8+ T cells. Interestingly, upregulation of Ki67 was transient; however, expression of CX3CR1 remained high on tumor-specific tetramer+ CD8+ T cells. Moreover, high-throughput sequencing of the T-cell receptor (TCR) identified significantly higher clonality and overlapping TCR repertoires in between peripheral CX3CR1+ CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs), suggesting the potential utility as a dynamic biomarker early on-treatment. Most importantly, changes in the frequency of PB CX3CR1+ CD8+ T cells associate with response to ICI therapy in individual mice. Phenotypic analysis of PB CD8+ T cells from 35 NSCLC patients undergoing PD-1/PD-L1 blockade revealed that the maximum percent change of these T-cell subsets from baseline identified responders and non-responders as early as 3 weeks from the initiation of ICI therapy with higher sensitivity, specificity, positive and negative predictive value compared to PD-L1 expression in the tumor, tumor mutational burden, and pre-existing TILs. Importantly, TCR sequencing confirmed clonally-expanding TCR repertoires within the T-cell subsets that were also found in TILs. Collectively, the frequency of circulating CX3CR1+ CD8+ T cells correlates with response to ICI therapy, and identifies responders vs non-responders early on-treatment. These findings provide a solid foundation for future development of clinical trials with large cohorts of patients undergoing ICI therapy not only for NSCLC, but also for a wide variety of malignancies. Citation Format: Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Kristopher Attwood, Sebastiano Battaglia, Igor Puzanov, Hongbin Chen, Grace Dy, Brahm Segal, Marc Ernstoff, Fumito Ito. Frequency of circulating CX3CR1+ CD8+ T cells to predict response to immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1044.