Gotowa bibliografia na temat „4AC 1 CELL TINE”

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P<0.01) and the number of oocytes developing to the blastocyst stage (P<0.04). There was a temperature x S1P interaction for cleavage rate (P<0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

Abstract Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3΄-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl2) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8+ and CD4+ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8+ and CD4+ T-cell exhaustion. Analysis of CD8+ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8+ and CD4+ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8+ (tumors and spleen) and CD4+ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes. Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 236.

BackgroundColorectal cancer (CRC) is a highly complex disease with multiple risk factors and both genetic and environmental components contribute to disease incidence.1 2 Cancer immunotherapy using immune-checkpoint blockades represents a major advance in treatment strategy.3 4 The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in lung, colon, liver and breast cancers and in Rhabdomyosarcoma and is a negative prognostic factor for cancer patient survival.5–8 Previous studies in breast cancer cells showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter. Genome-wide studies have identified NR4A1 as a key mediator of T-cell dysfunction and NR4A1 also plays an important role in regulating genes which are involved in tumor-induced T-cell exhaustion.9 Bis-indole derived NR4A1 ligand that act as receptor antagonists have been developed in this laboratory and these compounds block pro-oncogenic NR4A1-regulated genes/pathways.MethodsImmune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population.ResultsTwo compounds: 1,1-bis(3′-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and 3,5-dichlorophenyl analog (DIM-3,5-Cl2) inhibited tumor growth and weight at doses of 2.5 and 7.5mg/kg/day (figure 1). Tumor CD8+ T-cells isolated from mice treated with DIM-3,5-Cl2 and DIM-3-Br-5-OCF3 exhibited decreased mRNA expression of NR4A1 and high mobility group – box transcription factors NFAT, TOX and TOX2 and increased mRNA levels of Interferon-γ (IFNγ), granzyme β (GzB) and perforin compared to control animals (figure 2). As TOX and TOX2 cooperate with NR4A1 to modulate CD8+ T-cell exhaustion, we investigated the expression of several inhibitory receptors of T-cell exhaustion in CD8+ TILs, including PD-1, 2B4, TIGIT and TIM3. Following treatment with DIM-3,5-Cl2 or DIM-3-Br-5-OCF3, there was a significant decrease in the percentage of PD1 and 2B4 cells and a decrease in TIGIT and TIM3 (figure 3). These results indicate that NR4A1 antagonists reverses T-cell exhaustion.ConclusionsNR4A1 plays a critical role in T-cell dysfunction, and this includes T-cell exhaustion.10 11 Our results demonstrate that the NR4A1 antagonists reverse many markers of T-cell exhaustion including activation of cytokines. The combined effects of NR4A1 antagonists in both tumors and T-cells result in inhibition of colon tumorigenesis by targeting pathways/genes in tumor cells and by enhancing immune surveillance via reversal of T-cell exhaustion.ReferencesAhmed M. Colon cancer: a Clinician's perspective in 2019. Gastroenterology Res 2020;13(1):1–10. Epub 2020/02/26. doi: 10.14740/gr1239.Keum N, Giovannucci E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol 2019;16(12):713–32. Epub 2019/08/29. doi: 10.1038/s41575-019-0189-8.Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12(4):252–64. Epub 2012/03/23. doi: 10.1038/nrc3239.Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 2018;359(6382):1350–1355.Yang et al. Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis. Genome Biol 2019;21(1):2. doi: 10.1186/s13059-019-1921-y.Safe S, Karki K. The paradoxical roles of orphan nuclear receptor 4A (NR4A) in cancer. Mol Can Res 2020. Epub 2020/10/28. doi: 10.1158/1541-7786.MCR-20-0707.Lee SO, et al. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells. Molecular Endocrinology. 2014;28(10):1729–39. doi: 10.1210/me.2014-1102.Hedrick E, et al. The nuclear orphan receptor NR4A1 regulates β1-integrin expression in pancreatic and colon cancer cells and can be targeted by NR4A1 antagonists. Mol Carcinog 2017;56(9):2066–2075.Liu X, et al. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. Nature 2019;567(7749):525–9. Epub 2019/03/01. doi: 10.1038/s41586-019-0979-8.Chen J et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019;567(7749):530–4. Epub 2019/03/01. doi: 10.1038/s41586-019-0985-x. PubMed PMID: 30814732; PMCID: PMC6546093.Seo H, et al. TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8(+) T cell exhaustion. Proc Natl Acad Sci USA 2019;116(25):12410–5.Ethics ApprovalAll animal studies were carried out according to the ethical procedures approved by the Texas A&M University Institutional Animal Care and Use Committee. Approval number is 2020-0138.Abstract 732 Figure 1CDIM/NR4A1 antagonists inhibit colon tumor growth. Model for regulation of genes with GC-rich promoters by NR4A1/SP1 (A). C57BL/6 mice bearing MC-38 cells as xenografts were treated for 21 days with corn oil (control), DIM-3-Br-5-OCF3 (2.5 and 7.5 mg/kg/d), DIM-3,5-Cl2 (2.5 and 7.5 mg/kg/d) and effects on tumor volume (B), and tumor weights (C) were determined.Abstract 732 Figure 2CDIM analogs alter transcription factors expression. FACS analysis and CD4 and CD8 – specific antibodies were used to determine T-cell population in tumors of mice treated with corn oil (control), DIM-3-Br-5-OCF3 (2.5 and 7.5 mg/kg/d) and DIM-3,5-Cl2 (2.5 and 7.5 mg/kg/d) (A). Real time PCR was used to determine expression of nuclear factors (B) and cytokine (C) mRNA levels in CD8+ T-cells isolated from tumors. Results are expressed as means ± SD replicates from each treatment group and significant (p<0.05) induction or inhibition is indicated (*)Abstract 732 Figure 3NR4A1 ligands decreases T-cell exhaustion markers. FACS analysis in tumors derived from mice treated with corn oil (control), DIM-3-Br-5-OCF3 (2.5 and 7.5 mg/kg/d) and DIM-3,5-Cl2 (2.5 and 7.5 mg/kg/d) using specific antibodies was carried out to determine percentage of CD8+ T-cells expressing T-cell exhaustion markers - PD1 (A), 2B4 (B), TIGIT (C) and TIM3 (D). Significant (p<0.05) induction or inhibition is indicated (*) and results are expressed as means ± SD for at least 4 separate mice per treatment group.

11562 Background: Afami-cel is an autologous, HLA-A*02-restricted, specific peptide enhanced affinity receptor, T-cell therapy engineered to target MAGE-A4+ solid tumors. The pivotal, 2-cohort, single-arm, Phase 2, SPEARHEAD-1 trial (NCT04044768) with afami-cel met its primary endpoint based on Cohort 1 data. As of September 1, 2021, in 47 patients (pts) with metastatic synovial sarcoma (SyS) or myxoid/round cell liposarcoma (MRCLS), the overall response rate (ORR) per independent review was 34% with encouraging durability (Van Tine, et al. Paper 30: CTOS 2021; Virtual). To identify potential stratification factors for response and assess whether response is a proxy for progression-free survival (PFS), we present pooled analyses using data from the prior Phase 1 trial (NCT03132922) and Cohort 1 of the SPEARHEAD-1 trial. Methods: Eligible pts (16–75 years) were HLA-A*02+ with MAGE-A4+ tumors. Pts received afami-cel after lymphodepleting chemotherapy. The pooled analyses evaluated ORR per RECIST v1.1 by investigator review, stratified by 7 factors, and safety. Results: In the pooled data, 69 pts received afami-cel (2.12–9.99×109 transduced T-cells) and were evaluable for response (Phase 1, n = 18; Phase 2, n = 51); all expressed one eligible HLA-A*02 allele. Median (range) for: age was 42 years (19–76), number of prior lines of therapy was 2 (1–12), and tumor MAGE-A4 H-score was 230 (60–300). Median (range) H-score was higher in SyS (256 [60–300]) than in MRCLS (180 [112–230]). The pooled investigator-assessed ORR was 36.2% (40.7% in SyS; 10.0% in MRCLS). Responses occurred across a wide MAGE-A4 H-score range (134–300). Median (range) duration of response was 52 weeks (8.29–75.14). Response rate was higher in the 59 pts with SyS: with ≤2 vs ≥3 prior lines of therapy (55.2% vs 26.7%), baseline target lesion sum of longest diameters <10cm vs ≥10cm (53.1% vs 25.9%), MAGE-A4 H-score ≥200 vs <200 (46.3% vs 27.8%), without vs with bridging therapy (48.6% vs 29.2%), who were female vs male (46.4% vs 35.5%), aged ≥40 vs <40 years (45.7% vs 33.3%), and from North America vs Europe (42.6% vs 33.3%). In responders vs non-responders with SyS, respectively, median PFS was 58.3 vs 11. 0 weeks (log-rank p-value <0.0001); the probability of being progression-free at 24 weeks was 0.8 vs 0.2. The pooled benefit:risk profile of afami-cel was similar to that in the SPEARHEAD-1 trial (Van Tine, et al. Paper 30: CTOS 2021; Virtual.). Conclusions: We show that baseline tumor burden, prior systemic treatment history, and MAGE-A4 tumor expression levels are potential factors associated with response to afami-cel, although their true predictive value for response status awaits confirmation. Our findings will inform the ongoing clinical development of afami-cel in sarcoma, especially for prognostic studies with PFS or overall survival endpoints. Clinical trial information: NCT04044768, NCT03132922.

11562 Background: Afami-cel is an autologous, HLA-A*02-restricted, specific peptide enhanced affinity receptor, T-cell therapy engineered to target MAGE-A4+ solid tumors. The pivotal, 2-cohort, single-arm, Phase 2, SPEARHEAD-1 trial (NCT04044768) with afami-cel met its primary endpoint based on Cohort 1 data. As of September 1, 2021, in 47 patients (pts) with metastatic synovial sarcoma (SyS) or myxoid/round cell liposarcoma (MRCLS), the overall response rate (ORR) per independent review was 34% with encouraging durability (Van Tine, et al. Paper 30: CTOS 2021; Virtual). To identify potential stratification factors for response and assess whether response is a proxy for progression-free survival (PFS), we present pooled analyses using data from the prior Phase 1 trial (NCT03132922) and Cohort 1 of the SPEARHEAD-1 trial. Methods: Eligible pts (16–75 years) were HLA-A*02+ with MAGE-A4+ tumors. Pts received afami-cel after lymphodepleting chemotherapy. The pooled analyses evaluated ORR per RECIST v1.1 by investigator review, stratified by 7 factors, and safety. Results: In the pooled data, 69 pts received afami-cel (2.12–9.99×109 transduced T-cells) and were evaluable for response (Phase 1, n = 18; Phase 2, n = 51); all expressed one eligible HLA-A*02 allele. Median (range) for: age was 42 years (19–76), number of prior lines of therapy was 2 (1–12), and tumor MAGE-A4 H-score was 230 (60–300). Median (range) H-score was higher in SyS (256 [60–300]) than in MRCLS (180 [112–230]). The pooled investigator-assessed ORR was 36.2% (40.7% in SyS; 10.0% in MRCLS). Responses occurred across a wide MAGE-A4 H-score range (134–300). Median (range) duration of response was 52 weeks (8.29–75.14). Response rate was higher in the 59 pts with SyS: with ≤2 vs ≥3 prior lines of therapy (55.2% vs 26.7%), baseline target lesion sum of longest diameters <10cm vs ≥10cm (53.1% vs 25.9%), MAGE-A4 H-score ≥200 vs <200 (46.3% vs 27.8%), without vs with bridging therapy (48.6% vs 29.2%), who were female vs male (46.4% vs 35.5%), aged ≥40 vs <40 years (45.7% vs 33.3%), and from North America vs Europe (42.6% vs 33.3%). In responders vs non-responders with SyS, respectively, median PFS was 58.3 vs 11. 0 weeks (log-rank p-value <0.0001); the probability of being progression-free at 24 weeks was 0.8 vs 0.2. The pooled benefit:risk profile of afami-cel was similar to that in the SPEARHEAD-1 trial (Van Tine, et al. Paper 30: CTOS 2021; Virtual.). Conclusions: We show that baseline tumor burden, prior systemic treatment history, and MAGE-A4 tumor expression levels are potential factors associated with response to afami-cel, although their true predictive value for response status awaits confirmation. Our findings will inform the ongoing clinical development of afami-cel in sarcoma, especially for prognostic studies with PFS or overall survival endpoints. Clinical trial information: NCT04044768, NCT03132922.

Abstract Background: Afami-cel is an autologous, specific peptide enhanced affinity receptor T-cell therapy genetically engineered to target MAGE-A4+ solid tumors in HLA-A*02+ patients. SPEARHEAD-1 (NCT04044768) is a Phase 2, two-cohort, single-arm, open-label trial evaluating afami-cel in patients with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma (MRCLS) and is the largest trial in metastatic synovial sarcoma to date. Preliminary data from Cohort 1 in 47 heavily pre-treated patients aged 16–75 years from 22 centers in North America and Europe, showed an overall response rate (ORR) per independent review of 34.0% (14/39 [35.9%] in synovial sarcoma; 2/8 [25%] in MRCLS) and a favorable benefit–risk profile with mainly low-grade cytokine release syndrome and tolerable/reversible hematologic toxicities.1 The reported ORR in synovial sarcoma in Cohort 1 was higher than reported ORRs for current standard-of-care therapies, such as pazopanib and trabectedin, in the second-line metastatic setting.2 As synovial sarcoma is the most common malignant nonrhabdomyosarcoma soft-tissue sarcoma in children and adolescents with few treatment options, especially for recurrent disease, the trial opened a second cohort to allow enrollment of pediatric patients with MAGE-A4+ synovial sarcoma who experienced disease progression post first-line chemotherapy, and to better understand MAGE-A4 tumor expression in children. Methods: Cohort 2 of the SPEARHEAD-1 trial is enrolling patients with advanced synovial sarcoma who are at least 10 years old and weigh at least 40 kg. The planned enrollment is 45 patients, including up to 13 children, to enable a pooled analysis of ORR in &gt;90 patients across Cohorts 1 and 2. HLA and MAGE-A4+ screening in Cohort 2 is conducted at a central laboratory using the same method as Cohort 1; MAGE-A4 testing is done using a clinical trial assay. All patients enrolled in Cohort 2 undergo apheresis and their isolated T-cells are then transduced with the MAGE-A4c1032 TCR using a lentivirus vector, followed by ex vivo expansion. Prior to afami-cel infusion of 1–10 × 109 transduced T-cells, patients will receive lymphodepleting chemotherapy consisting of fludarabine (30 mg/m2/day for 4 days) and cyclophosphamide (600 mg/m2/day for 3 days). Disease will be assessed by independent review per RECIST v1.1 using computerized tomography or magnetic resonance imaging at weeks 4, 8, 12, 16, 24, and every 2 months thereafter until confirmed disease progression. Patients enter long-term follow-up for 15 years. 1. Van Tine BA, et al. Paper 30: CTOS 2021; Virtual 2. Carroll C, et al. Cancer Res 2021;81(13_Suppl): Abstract nr 2630. Citation Format: Colin Lunt, Sandra P. D’Angelo, Albiruni Ryan Abdul Razak, Michael J. Wagner, Brian A. Van Tine, Kristen Ganjoo, Jean-Yves Blay, Dejka M. Araujo, Mark Agulnik, John W. Glod, Erin Van Winkle, Erica Elefant, Swethajit Biswas, Dennis Williams, Axel Le Cesne. Enrollment of pediatric and adolescent patients with MAGE-A4+ advanced synovial sarcoma into cohort 2 of SPEARHEAD-1: a phase 2 trial of afamitresgene autoleucel (“afami-cel” [formerly ADP-A2M4]) [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A038.

3622 Background: Human β-defensin 1 (hBD-1) encoded by the DEFB1 gene is an antimicrobial peptide involved in the innate immune response and is expressed in epithelial cells, including the colon. hBD-1 has been shown to have tumor suppressor functions in urothelial cancer models. We tested whether 4 germline single nucleotide polymorphisms (SNPs) in DEFB1 could predict time to tumor recurrence (TTR) in stage II and III colon cancer (CC) patients. We then sought to demonstrate if hBD-1 has tumor suppressor functions in CC models. Methods: A total of 234 patients, 105 stage II and 129 stage III, treated with 5-FU-based chemotherapy at the University of Southern California were included. The median follow-up time was 4.4 yrs. SNPs were analyzed from whole blood samples using direct DNA-sequencing. To gain insight into hBD-1’s function, hBD-1 expression in CC cell lines was determined by qRT-PCR and Western blotting. hBD-1 expression was induced ectopically and CC cells and viability, membrane permeability, cell-cycle and apoptosis analyzed. Results: Stage III patients with rs1800972 DEFB1 -44G containing genotypes had longer TTR than patients with C/C genotype (11.3 mo vs 2.7 mo; p=.008). In contrast in stage II, patients with rs1799946 DEFB1 -52A containing genotypes, had significantly longer TTR than patients with G/G genotype (16.8 mo vs 5.9 mo; p=.017). In the multivariate analysis, both DEFB1 -44C/G and DEFB1 -52G/A SNPs remained significant, respectively in stage III (HR=.419; 95%CI .201-.87; p=.020) and in stage II (HR=.360; 95%CI .152-.853; p=.020). Haplotype of all four SNPs was significantly associated with stage-specific TTR (to be presented at the meeting). In CC cell line models, there was a loss of hBD-1 expression, and induction of hBD-1 expression resulted in a loss of cell viability, membrane permeability and the induction of apoptosis. Conclusions: The results demonstrate for the first time evidence of hBD-1’s potential influence on the microenvironment and its role as a tumor suppressor in CC. Further studies need to be conducted to better understand the role of hBD-1 in CC development and progression and evaluate the potential utility of hBD-1 as a therapeutic strategy.

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induceβ-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3βinhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested thatβ-catenin/TCF1 complex directly regulated the expression ofSox1during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

Abstract This study aimed to examine whether nuclear receptor 4a1 (NR4A1) is involved in inhibiting hepatic stellate cell (HSC) activation and liver fibrosis through the epithelial–mesenchymal transition (EMT). HSC-T6 cells were divided into the control group, the acetaldehyde (200 μM, an EMT activator) group, and the NR4A1 activation group (Cytosporone B; 1 μM). The expression levels of the epithelial marker E-cadherin, the mesenchymal markers fibronectin (FN), vimentin, smooth muscle alpha-actin (α-SMA), and fibroblast-specific protein 1 (FSP-1), and the components of the transforming growth factor (TGF)-β pathway were detected by real-time polymerase chain reaction and western blotting. Compared with the control group, E-cadherin in the acetaldehyde group was downregulated, whereas FN, FSP-1, vimentin, α-SMA, and COL1A1/COL1A2 were upregulated (P < 0.05). Compared with the acetaldehyde group, NR4A1 agonist upregulated E-cadherin and downregulated FN, FSP-1, vimentin, α-SMA, and COL1A1/COL1A2 (P < 0.05). After acetaldehyde stimulation, TGF-β, Smad2/3/4, and zinc finger E-box-binding homeobox (ZEB) were upregulated, while Smad7 mRNA levels were downregulated (all P < 0.05). Compared with acetaldehyde alone, NR4A1 agonist increased Smad7 mRNA levels and reduced TGF-β, Smad2/3/4, and ZEB mRNA levels (all P < 0.05). NR4A1 activation suppresses acetaldehyde-induced EMT, as shown by epithelial and mesenchymal marker expression. The inhibition of the TGF-β–Smad2/3/4–ZEB signaling during HSC activation might be involved.

BackgroundAutologous cell therapies with an engineered T-cell receptor targeting MAGE-A4 have shown responses in patients with synovial sarcoma1 with additional responses in myxoid/round cell liposarcoma (MRCLS), head and neck, lung, esophagogastric junction, and melanoma cancers.2 3 Low-dose radiation may control tumor growth locally and modulate stroma of solid tumors,4 potentially facilitating T-cell infiltration into tumors and antitumor activity.MethodsSub-study designed to assess safety, tolerability, and efficacy in up to 10 patients with low-dose radiation in combination with lymphodepleting chemotherapy, followed by afami-cel (an autologous TCR cell T-cell therapy targeting MAGE-A4). Eligible patients are HLA-A*02^+ with MAGE-A4 expressing tumors including urothelial, melanoma, head and neck, ovarian, non-small cell lung, esophageal, gastric, synovial sarcoma, and MRCLS cancers. Patients receive afami-cel by infusion following low-dose radiation and lymphodepleting chemotherapy. Radiation was 4.2–7 Gy per lesion or isocenter (maximum of 5). Lymphodepleting regimen was IV fludarabine 30 mg/m^2/day for 4 days (−7 to −4) and cyclophosphamide 600 mg/m^2/day for 3 days (−7 to −5). Afami-cel doses ranged from 1.2 x 10^9 to 10 x 10^9 transduced cells. Pts receive afami-cel infusion on Day 1.ResultsAs of Dec 27, 2020, a total of 8 patients, including 4 patients (1 male) with melanoma (2), HNSCC (1), or ovarian (1) cancers received low-dose radiation and afami-cel. Most frequently reported AEs (4/4 pts) were leukopenia/decreased white blood cell count, lymphopenia/decreased lymphocyte count, and neutropenia/decreased neutrophil count; all of which were related to the lymphodepletion regimen. The most commonly (>1 patient) reported AEs considered related to T-cell infusion were cytokine release syndrome (2/4 pts) and fatigue (2/4 pts). Two patients had a total of 5 SAEs: adrenal insufficiency, hyperglycemia, neurotoxicity, pneumonia aspiration, and pneumothorax. The only SAE considered to be related to treatment was Grade 3 neurotoxicity. Best overall responses per RECIST 1.1: 1 partial response (melanoma, −42% in target lesions), 2 stable diseases (ovarian cancer, −23%; HNSCC, no change), and 1 patient did not have post-baseline scans yet. Translational analyses showed peripheral persistence and serum cytokine response profiles consistent with that of afami-cel monotherapy, whilst a relatively greater T cell infiltration in tumor biopsies was evident.ConclusionsAfami-cel with low-dose radiation has had an acceptable safety profile. Most AEs were consistent with those typically experienced by cancer patients undergoing lymphodepletion cytotoxic chemotherapy and cellular therapy. Infused T-cells were observed in tumor biopsies at high frequency, and one patient exhibited a clinical partial response.Trial RegistrationNCT03132922ReferencesVan Tine BA, et al. CTOS 2020.Hong DS, et al. ASCO 2020.Hong DS, et al. SITC 2020.De Selm C, et al. Mol Ther 2018;26(11):2542–2552.

The meniscus is a fibrous tissue essential to healthy knee mechanics. It functions to redirect vertical forces laterally, converting compressive into tensile loads which are taken up by an array of highly organized collagen fibers. Load transmission is not only the operative mode of the meniscus, but is also required for normal development and homeostatic maintenance [1]. With injury, disruption of the aligned collagen fiber architecture impairs function, altering joint loading and initiating osteoarthritis. Toward engineering replacement meniscus tissue, we have investigated scaffolds of aligned electrospun nanofibers that direct cell orientation and provide a suitable microenvironment for the deposition of organized extracellular matrix (ECM) (Fig 1) [2]. In previous work, human meniscus fibrochondrocytes (MFCs) seeded on such scaffolds formed robust ECM with commensurate increases in tensile properties [3]. After 10-weeks of static, free-floating culture, however, mechanical properties still fell short of native tissue values. Over this time course, full-thickness MFC colonization was not observed due to the tight packing of nanofibers, although better infiltrated constructs revealed larger improvements in tensile properties. To accelerate cell ingress, we next explored composite scaffolds containing slow eroding poly(e-caprolactone) (PCL) fibers and water-soluble poly(ethylene oxide) (PEO) fibers that augment pore size when removed (Fig 4A-D) [4]. Based on this precedent, the current study explored two strategies for improving the maturation of MFC-laden nanofibrous constructs: dynamic tensile loading mimicking the in vivo mechanical environment and inclusion of sacrificial PEO fibers to enhance cell infiltration. We hypothesized that dynamic control of the mechanical and material microenvironment would improve matrix production and lead to enhanced mechanical properties.