Expression Of Immune-related Genes Research Articles

OsMPK12 positively regulates rice blast resistance via OsEDC4-mediated transcriptional regulation of immune-related genes.

Mitogen-activated protein kinase (MAPK) signalling cascades are functionally important signalling modules in eukaryotes. Transcriptome reprogramming of immune-related genes is a key process in plant immunity. Emerging evidence shows that plant MAPK cascade is associated with processing (P)-body components and contributes to transcriptome reprogramming of immune-related genes. However, it remains largely unknown how this process is regulated. Here, we show that OsMPK12, which is induced by Magnaporthe oryzae infection, positively regulates rice blast resistance. Further analysis revealed that OsMPK12 directly interacts with enhancer of mRNA decapping protein 4 (OsEDC4), a P-body-located protein, and recruits OsEDC4 to where OsMPK12 is enriched. Importantly, OsEDC4 directly interacts with two decapping complex members OsDCP1 and OsDCP2, indicating that OsEDC4 is a subunit of the mRNA decapping complex. Additionally, we found that OsEDC4 positively regulates rice blast resistance by regulating expression of immune-related genes and maintaining proper mRNA levels of some negatively-regulated genes. And OsMPK12 and OsEDC4 are also involved in rice growth and development regulation. Taken together, our data demonstrate that OsMPK12 positively regulates rice blast resistance via OsEDC4-mediated mRNA decay of immune-related genes, providing new insight into not only the new role of the MAPK signalling cascade, but also posttranscriptional regulation of immune-related genes.

Prediction of prognosis and immunotherapy efficacy based on metabolic landscape in lung adenocarcinoma by bulk, single-cell RNA sequencing and Mendelian randomization analyses.

Immunotherapy has been a remarkable clinical advancement in cancer treatment, but only a few patients benefit from it. Metabolic reprogramming is tightly associated with immunotherapy efficacy and clinical outcomes. However, comprehensively analyzing their relationship is still lacking in lung adenocarcinoma (LUAD). Herein, we evaluated 84 metabolic pathways in TCGA-LUAD by ssGSEA. A matrix of metabolic pathway pairs was generated and a metabolic pathway-pair score (MPPS) model was established by univariable, LASSO, multivariable Cox regression analyses. The differences of metabolic reprogramming, tumor microenvironment (TME), tumor mutation burden and drug sensitivity in different MPPS groups were further explored. WGCNA and 117 machine learning algorithms were performed to identify MPPS-related genes. Single-cell RNA sequencing and in vitro experiments were used to explore the role of C1QTNF6 on TME. The results showed MPPS model accurately predicted prognosis and immunotherapy efficacy of LUAD patients regardless of sequencing platforms. High-MPPS group had worse prognosis, immunotherapy efficacy and lower immune cells infiltration, immune-related genes expression and cancer-immunity cycle scores than low-MPPS group. Seven MPPS-related genes were identified, of which C1QTNF6 was mainly expressed in fibroblasts. High C1QTNF6 expression in fibroblasts was associated with more infiltration of M2 macrophage, Treg cells and less infiltration of NK cells, memory CD8+ T cells. In vitro experiments validated silencing C1QTNF6 in fibroblasts could inhibit M2 macrophage polarization and migration. The study depicted the metabolic landscape of LUAD and constructed a MPPS model to accurately predict prognosis and immunotherapy efficacy. C1QTNF6 was a promising target to regulate M2 macrophage polarization and migration.

Exploring the molecular landscape of cancer of unknown primary: A comparative analysis with other metastatic cancers.

Cancer of unknown primary (CUP) tumors are biologically very heterogeneous, which complicates stratification of patients for treatment. Consequently, these patients face limited treatment options and a poor prognosis. With this study, we aim to expand on the current knowledge of CUP biology by analyzing two cohorts: a well-characterized cohort of 44 CUP patients, and 213 metastatic patients with known primary. These cohorts were treated at the same institution and characterized by identical molecular assessments. Through comparative analysis of genomic and transcriptomic data, we found that CUP tumors were characterized by high expression of immune-related genes and pathways compared to other metastatic tumors. Moreover, CUP tumors uniformly demonstrated high levels of tumor-infiltrating leukocytes and circulating Tcells, indicating a strong immune response. Finally, the genetic landscape of CUP tumors resembled that of other metastatic cancers and demonstrated mutations in established cancer genes. In conclusion, CUP tumors possess a distinct immunophenotype that distinguishes them from other metastatic cancers. These results may suggest an immune response in CUP that facilitates metastatic tumor growth while limiting growth of the primary tumor.

Enhancing Growth and Intestinal Health in Triploid Rainbow Trout Fed a Low-Fish-Meal Diet through Supplementation with Clostridium butyricum

This study evaluates the effects of dietary Clostridium butyricum on growth performance and intestinal health in triploid rainbow trout (Oncorhynchus mykiss). Administered in a 12-week trial, five isonitrogenous and isolipidic feeds contained different C. butyricum levels [G1 (0), G2 (1.6 × 106 CFU/g), G3 (1.2 × 107 CFU/g), G4 (1.1 × 108 CFU/g), and G5 (1.3 × 109 CFU/g)]. Significant enhancements in growth performance, including improved feed conversion ratios and specific growth rates in the G4 group, were indicative of enhanced nutrient utilization, corroborated by optimal digestive enzyme activity levels. Antioxidant capabilities were also enhanced in the G4 group, indicated by increased serum superoxide dismutase and catalase activities, along with a significant decrease in malondialdehyde levels. Gut microbiota analysis indicated a probiotic concentration-dependent modulation of microbial communities, with a marked enrichment in beneficial bacterial phyla like Firmicutes in the G4 group. This microbial shift correlated with significant downregulations in immune-related gene expressions, including interleukins (IL-1β, IL-8), NF-κB, MyD88, and TNF-α, highlighting an activated immune response. Correspondingly, serum lysozyme and immunoglobulin M contents were significantly elevated in the G4 group. Challenge tests with Aeromonas salmonicida exhibited the higher disease resistance of fish fed the G4 diet. In conclusion, the study shows the potential of dietary C. butyricum, especially at 1.1 × 108 CFU/g, in enhancing the growth, health, and disease resistance of triploid rainbow trout through modulating gut microflora and stimulating immune responses.

Effects of dietary β-sitosterol supplementation on growth performance, antioxidant ability, and disease resistance in largemouth bass Micropterus salmoides

β-sitosterol, as the most abundant phytosterol, has been shown to exert multiple biological roles in in vitro and in vivo animal studies. Largemouth bass (Micropterus salmoides) is an economical freshwater-farmed species widely cultivated in China due to its nutritious and delicious meat. This study investigated the effects of dietary β-sitosterol supplementation on largemouth bass farming. In the study, largemouth bass were fed with five levels of β-sitosterol (0, 20, 40, 80, and 160 mg/kg) supplementation in a commercial feed for four weeks. The growth performance, antioxidant ability, intestinal structure, immune-related gene expression, and resistance to pathogens such as Aeromonas hydrophila and largemouth bass virus (LMBRaV) were detected. The results revealed that β-sitosterol supplementation in feed increased the weight gain rate of largemouth bass, with 40 mg/kg supplementation displaying the best effect. The serum biochemical indices all showed an increase, including glucose and alkaline phosphatase. The intestinal villus length and muscularis propria thickness also increased, accompanied by elevated digestive enzyme activities, which promoted digestion and absorption of nutrients. Meanwhile, the levels of total cholesterol, aspartate aminotransferase, and alanine aminotransferase in serum decreased, indicating reduced liver damage. In addition, β-sitosterol supplementation effectively enhanced the antioxidant capacity of largemouth bass by decreasing malondialdehyde and elevating superoxide dismutase. Immune-related gene expression also changed. Pathogen infection tests revealed that β-sitosterol supplementation in feed had a certain protective effect against infection in largemouth bass, with the protection rate against A. hydrophila being higher than LMBRaV. The 40 mg/kg β-sitosterol supplementation group exhibited the best results. Collectively, the results revealed that the β-sitosterol can promote growth, improve enzyme activity, stimulate intestinal digestion, increase immune gene expression, and induce disease resistance in largemouth bass, with the optimal supplementation dosage being 40 mg/kg. β-sitosterol might be an excellent feed additive in aquaculture.

Effects of aflatoxin B1 on metabolism- and immunity-related gene expression in Hermetia illucens L. (Diptera: Stratiomyidae)

Contamination of food products with mycotoxins such as aflatoxin B1 (AFB1) poses a severe risk to human health. Larvae of the black soldier fly (BSFL), Hermetia illucens (Diptera: Stratiomyidae), can successfully metabolize AFB1 without any negative consequences on their survival or growth. Organic waste streams contaminated with mycotoxins can be upcycled into protein-rich BSFL as an alternative feed for livestock and the left-over feed residue into nutrient-rich crop fertilizers. However, the underlying mechanisms that allow BSFL to metabolize AFB1 are unknown. In this study, five-day-old BSFL were fed with either a control or an AFB1-spiked (20 μg/kg) diet to elucidate the underlying mechanisms. Larval samples were collected at three timepoints (6 h, 24 h and 72 h) and subjected to RNA-Seq analysis to determine gene expression patterns. Provision of an AFB1-spiked diet resulted in an up-regulation of 357 and a down-regulation of 929 unique genes. Upregulated genes include multiple genes involved in AFB1 metabolism in other (insect) species. Downregulated genes were generally involved in the insects' growth, development, and immunity. BSFL possesses a diverse genetic arsenal that encodes for enzymes capable of metabolizing AFB1 without trade-offs on larval survival. In conclusion, the adverse impact of AFB1 exposure on immunity-related processes is observed in the transcriptomic response, and is indicative of a trade-off between detoxification and immune responses.

New insights into resveratrol attenuates hepatotoxicity in emamectin benzoate-exposed grass carp (Ctenopharyngodon idella) via NO system/NF-κB signaling pathway

Emamectin benzoate (EMB) is extensively used as a crop protection agent. Overuse of EMB poses a serious threat to the quality of water and non-target organisms in the environment. Resveratrol (RES) is a natural phytoalexin with the function of anti-oxidation and anti-inflammation. Nonetheless, it is unclear whether EMB affects the expression of cytokines and induces autophagy, apoptosis, and necroptosis of hepatocytes (L8824 cell) in grass carp (Ctenopharyngodon idella), and whether RES has an attenuate function in this process. Therefore, we established the L8824 cells model of EMB exposure and treated it with RES. The results showed that compared with the control (CON) group, EMB exposure significantly increased the nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) activity, and the expression of iNOS and phosphorylated nuclear factor kappa B (p-NF-κB) (P < 0.05). In addition, compared with the CON group, the results of flow cytometry and dansylcadaverine (MDC) staining showed a significant increase in apoptosis and autophagy in the EMB-exposed group (P < 0.05) with the activation of the B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax)/cysteine-aspartic acid protease 3 (Caspase-3)/cysteine-aspartic acid protease 9 (Caspase-9) pathway and microtubule-associated protein light chain 3 (LC3)/sequestosome 1 (p62)/Beclin1 pathway. EMB exposure significantly increased the mRNA and protein expression of receptor-interacting protein 1 (RIPK1)/receptor-interacting protein 3 (RIPK3)/mixed the lineage kinase domain-like (MLKL) pathway (P < 0.05). Moreover, EMB exposure significantly increased the expression of genes related to immunity (immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin D (IgD), and antimicrobial peptide-related genes expression including β-defensin and hepcidin) (P < 0.05). The addition of RES significantly diminished autophagy, apoptosis, necroptosis, and immunity-related gene expression by inhibiting iNOS activity, NO content, and the protein expression of iNOS and p-NF-κB. In conclusion, RES attenuated autophagy, apoptosis, and necroptosis in EMB-exposed L8824 cells via suppression of the NO system/NF-κB signaling pathway.

Abstract PO1-06-02: Solti-1716 TATEN phase II trial: Targeting non-Luminal disease by PAM50 with pembrolizumab and paclitaxel in Hormone Receptor-positive/HER2-negative advanced breast cancer (ABC)

Abstract Background Within HR+/HER2-negative breast cancer, PAM50 non-luminal tumors (HER2-enriched [HER2-E] and Basal-like) have higher expression of proliferation and immune-related genes and tumor infiltrating lymphocytes and might benefit from immunotherapy. Here, we report the efficacy, safety, and correlative analysis data of the TATEN trial, the first study designed to evaluate pembrolizumab and paclitaxel in HR+/HER2-negative, PAM50 non-luminal, ABC. Methods TATEN trial (NCT04251169) is a single-arm, multicenter, phase II study evaluating pembrolizumab in combination with paclitaxel in patients with HR+/HER2-, PAM50 non-luminal ABC. Key inclusion criteria include progression to prior CDK4/6 inhibitors (CDK4/6i), presence of measurable disease, no prior chemotherapy for ABC, ECOG 0-1, and non-luminal metastatic disease by PAM50. Included patients received pembrolizumab at 200 mg every 3 weeks (beginning at cycle 1) in combination with weekly paclitaxel at 80 mg/m2 beginning at cycle 2. The primary endpoint was overall response rate (ORR) according to RECIST V1.1. in patients who received at least one dose of combination treatment and had a first, post-baseline tumor assessment (evaluable population). Secondary endpoints included progression-free survival (PFS), clinical benefit rate (CBR), safety, and predictive biomarkers. The study was based on a Simon two-stage design. Stage I of the trial would be considered successful if at least 6 of 15 patients achieved a partial response and/or complete response. In that case, the trial would recruit up to 46 evaluable patients for a target ORR ≥ 41%. Metastatic biopsies from patients enrolled in pre-screening were also evaluated for tumor infiltrating lymphocytes (TILs) and were further analyzed with an expression panel of 192 genes, including PDL1 and PD1. Results From July 2020 to December 2021, 132 tumors were screened, and 27 PAM50 non-luminal tumors were identified (20%). Non-luminal tumors trended to have higher PDL1 expression (p=0.090) and TILs (p=0.084) compared to luminal tumors, while no difference was observed for PD1 (p=0.850). Of 20 recruited patients in the study (stage I+II), 18 were evaluable for the primary endpoint. Baseline characteristics were as follows: median age 55 years, ECOG 0 55%, de novo MBC 22%, and visceral disease 72.2%. Eleven patients had received paclitaxel treatment in the adjuvant setting. Regarding PAM50 subtype, 2 patients had basal-like and 16 HER2-E tumors. At the time of data cut-off (June 2023), 13 patients (72.2%) had stopped their treatment because of progressive disease and 3 (16.6%) due to toxicity. Two patients (11.1%) were still on treatment. The ORR was 61.1 % (11 of 18, 95% CI 35.7-82.7). CBR was 88.9% (16 of 18, 95% CI 65.3-98.6), and median PFS was 8.3 months (95% CI 7.3 – 14.1). Treatment-related adverse events (TRAEs) of any grade (G) occurred in 19 patients (95%), while 45% of patients experienced G3 TRAEs. No G4 or G5 TRAEs were reported in the evaluable population. Gene expression analysis was successful for all recruited patients (n=20). High expression of the PAM50 luminal A signature (p=0.049), and the luminal genes PGR (p=0.028) and RRAGA (p=0.038) were associated with worse PFS (univariate analyses). The pan-leucocyte receptor CD84 (p=0.028) was associated with better PFS (univariate analysis). Conclusions Pembrolizumab in combination with paclitaxel is safe and exhibits promising efficacy outcomes in patients with CDK4/6i resistant HR+/HER2- ABC with a PAM50 non-luminal subtype. Although meeting the criteria for stage II, the enrollment was stopped prematurely due to the lack of funding and pembrolizumab supply. More correlative analyses will be presented at the conference. This study was funded in part by MSD. Citation Format: Aleix Prat, Benedetta Conte, Fara Brasó-Maristany, Nuria Chic, Montserrat Muñoz, Cristina Hernando, Manuel Alva, Silvia Vazquez, Salvador Blanch, Mafalda Oliveira, Esther Fernández, Oleguer Castillo, Patricia Galván, Ángela Aguirre, Esther Sanfeliu, Lorea Villanueva, Tomás Pascual, Eva Ciruelos. Solti-1716 TATEN phase II trial: Targeting non-Luminal disease by PAM50 with pembrolizumab and paclitaxel in Hormone Receptor-positive/HER2-negative advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-02.

Abstract PO3-14-07: Characterizing Immune Infiltration in Metastatic Hormone Receptor-Positive/HER2-Negative Breast Cancer: A Comprehensive Translational Analysis from four SOLTI Clinical Trials

Abstract Background: Hormone Receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (BC) is a highly heterogenous disease. Non-luminal subtypes within HR+/HER2- BC have been linked to poor survival in the metastatic setting. However, these subtypes might exhibit higher expression of immune-related genes, tumor-infiltrating lymphocytes (TILs), and APOBEC signatures. This study aimed to investigate the presence of tertiary lymphoid structures (TLS), TILs and immune gene expression (GE) across PAM50 intrinsic subtypes (IS) in metastatic samples from patients (pts) with HR+/HER2- BC. Methods: We included pts with HR+/HER2- metastatic BC who participated in the molecular prescreening of the SOLTI-1904-ACROPOLI (NCT04802876), SOLTI-1502-ARIANNA (NCT04142060), SOLTI-1718-NEREA (NCT04460430) and SOLTI-1716-TATEN (NCT04251169) studies, providing metastatic samples for analysis. The proportion of TILs was centrally evaluated by a single pathologist using standard criteria. GE analysis was performed on the same FFPE tumor block using a panel of 72 genes, including 10 immune genes and the PAM50 IS. In a subset of patients, we conducted additional GE analysis using a panel of 192 genes, including the 14-gene B-cell/IgG (IGG) signature, while also retrospectively assessing the presence of TLS. Descriptive statistics, significance analysis of microarrays (using False Discovery Rate [FDR]) and logistic regressions were used to investigate the association between TLS, TILs, immune GE and PAM50 IS. Results: A total of 401 pts were included. Distribution across IS was: 53.4% Luminal B (n=197), 27.1% Luminal A (n=100), 16% HER2-enriched (n=59), 2.7% basal-like (n=10) and 0.8% normal-like (n=3). TILs were evaluated in 375 samples (93.5%). Median (m) TILs proportion was 2% (IQR: 1-4, range 0-80). Non-luminal subtypes exhibited a higher enrichment of TILs compared to luminal subtypes (m=3% vs m=1%, p=0.002). Moreover, significant differences of TILs were also seen according to the site of metastasis (M1): m=6.5% in lymph nodes vs m=4.5% in lung M1 vs m=1% in liver M1 (p&amp;lt; 0.001). Immune GE was significantly higher in samples with a high proportion of TILs ( &amp;gt;10%), although the correlation between TILs and immune genes was moderate (e.g, coefficient=0.44, p&amp;lt; 0.001, between CD8A and TILs). TLS were evaluated in 147 samples and detected in 16 pts (10.9%). Compared to TLS-negative tumors, TILs were significantly higher in TLS-positive (TLS+) tumors (m=1.5% vs m=10%, p&amp;lt; 0.001). In addition, the presence of TLS was significantly higher in non-luminal subtypes compared to luminal subtypes (18% (11/61) vs 5.8% (5/86), p=0.019). TLS+ tumors had a significant enrichment of genes related to activated B-cells (CD19, CD79A), plasma cells (CD27), immunoglobulins (IGKC, IGLV), and T-cells (CD8A, PDCD1). Notably, the IGG signature as a continuous variable demonstrated a significant association with TLS presence, independently of the PAM50 IS and TILs (p=0.033, ROC AUC=0.817). Finally, higher expression of some immune genes was observed in non-luminal subtypes vs luminal subtypes (e.g., CD19 (p=0.016) and CD274/PDL1 (p&amp;lt; 0.001)). Conclusions: Our translational analysis reveals that non-luminal subtypes within metastatic HR+/HER2- BC exhibit a higher enrichment of TILs, immune GE, and presence of TLS. Importantly, we identified the IGG signature as a robust immune GE pattern that is strongly associated with TLS across PAM50 IS, independent of TILs. These findings highlight the potential significance of the IGG signature as a novel biomarker for precisely measuring TLS and selecting pts who might be more likely to derive benefit from immunotherapy. Citation Format: Elia Seguí, Esther Sanfeliu, Fara Brasó-Maristany, Blanca González-Farré, Fernando Salvador, Laura Angelats, Lorea Villanueva, Alejandra Espinosa, Patricia Galván, Esther Fernández, Xavier Gonzalez-Farré, Santiago Escrivá-de-Romaní, Núria Chic, Eva Ciruelos, Cristina Saura, Luis Manso, Mafalda Oliveira, Josep Tabernero, Aleix Prat, Tomás Pascual. Characterizing Immune Infiltration in Metastatic Hormone Receptor-Positive/HER2-Negative Breast Cancer: A Comprehensive Translational Analysis from four SOLTI Clinical Trials [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-07.

Abstract PO2-03-07: Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) tends to be more aggressive, and more likely to recur than other subtypes of breast cancer, and patients with metastatic TNBC have a poor prognosis. The recent approvals and data of pembrolizumab (anti-PD-1 inhibitor) in combination with chemotherapy in advanced-metastatic and early high-risk TNBC, and sacituzumab govitecan (an ADC that targets the tumor cell surface antigen TROP2 coupled with the irinotecan metabolite SN-38) in metastatic TNBC, has shed light on some of the immune and cancer signaling associated with TNBC. Although exciting progress has been made in the treatment of TNBC, there are still important gaps to fill to understand the biology of TNBC and identify additional therapies. Materials and Methods: We identified a total of 1500 patients whose tumors were negative for the expression of ER and HER2 receptors by gene array. PD-L1 and TROP2 expression data were available for 802 early TNBC patients, of whom 657 were treated with adjuvant chemotherapy. A comprehensive identification and analysis of immune-related genes (n= 1169) was conducted. Mann-Whitney test comparing the gene expression for all immune related-genes was evaluated in patients expressing PD-L1 and TROP2 at low and high expression levels. Immune gene modules resembling key immune gene signaling were analyzed in all subgroups identified. Fold changes and Mann-Whitney P values were calculated in each subgroup. Results: Patients were stratified based on high and low expression levels of PD-L1 and TROP-2 mRNA. PD-L1 highly expressing (PD-L1+) tumors showed a marked difference in immune-related genes as compared to PD-L1 low-level tumors (PD-L1-) with PD-L1+ tumors demonstrating a significantly higher proportion of correlated immune genes (CXCL9/10/11, IFN-γ, CCL5 and STAT1). TROP2 highly and low expressing tumors (TROP2+ and TROP2-, respectively) showed a relatively limited difference in terms of immune-related gene expression with key immune-related genes having statistical differential expression in the two subgroups. TROP2+ tumors showed a high presence of several Mucin family gene members (MUC1, -5AC, -4). In addition, CLDN 4, VTCN1, and MET were among the key immune genes significantly expressed in TROP2+ tumors (P = 5.73E-20; P = 6.2E-13, P = 2.1E-11). Overall, PD-L1+ tumors and TROP2+ tumors showed an immune suppressive genomic landscape but were potentially regulated by different signaling. Of note, tumors with very high expression of PD-L1 showed an inverse correlation with VTCN1 gene expression. Conclusions: This preliminary analysis confirmed that only a limited number of early TNBC highly expressed PD-L1 and that a different immune suppressive landscape for PD-L1± and TROP2± tumors exists. In addition, our data suggest that early TNBC cases lacking PD-L1 might escape immune surveillance by virtue of the upregulation of key alternate signaling. Ongoing efforts investigating the expression of such markers on stroma/epithelial tumor components will help to clarify their biological, clinical, and potential therapeutic role in TNBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-07.

Abstract PO5-16-02: Investigating survivin as a novel target for immunotherapy in diverse breast cancer patients

Abstract Background: The BIRC5 gene encodes survivin, a tumor-specific antigen associated with aggressive tumor features, Black race and poor prognosis in breast cancer (BC). Recent studies have shown that anti-survivin vaccines stimulate a strong anti-tumor immune response in multiple tumor types. However, the relationship between BIRC5/survivin and immune response in BC is poorly understood, particularly among diverse patients. In this analysis, we sought to investigate the relationship between BIRC5/survivin and the BC immune microenvironment across clinical tumor features and RNA-based risk scores for genomic instability. Methods: We leveraged the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled young (≤50 years) and Black women with invasive BC. NanoString mRNA expression profiling was used to evaluate breast tumor expression for 10 major immune cell types, adaptive and innate immune response, TP53 and homologous recombination deficiency (HRD) pathways, and BIRC5/survivin from 1952 BC patients, including 1,030 (53%) Black and 922 (47%) non-Black women from the CBCS, with additional comparison in The Cancer genome Atlas (TCGA) (n=1,095 breast tumors). BIRC5 was evaluated both as a continuous and categorical variable, using the third expression quartile as a cut point for BIRC5-high, with the lower three quartiles categorized as BIRC5-low. We used generalized linear models to estimate beta values and adjusted p-values as the measure of association between BIRC5 status and immune-related expression in strata defined by estrogen receptor (ER) status, tumor stage, TP53 and HRD status. All analyses were adjusted for multiple comparisons with the Benjamini-Hochberg procedure. Results: In an analysis of continuous BIRC5 expression and global classes of immune response, BIRC5 mRNA expression was highest among tumors with an adaptive-enriched immune class (p&amp;lt; 0.001) in both CBCS and TCGA. However, when investigating immune gene expression according to BIRC5 status (i.e., BIRC5-high relative to BIRC5-low), multivariate differential expression analysis revealed significantly decreased immune cell-related expression scores among BIRC5-high tumors relative to BIRC5-low, and this relationship persisted in analysis stratified on stage (I/II vs III/IV), ER status, TP53 and HRD status. Across all strata, the strongest relationship between BIRC5-high and the immune microenvironment was observed for adaptive immune response (Beta: -0.14; p=0.005), with decreased immune gene expression related to B cells (Beta: -0.16; p=0.01), T cells (Beta: -0.14; p=0.007), Cytotoxic cells (Beta: -0.14; p=0.005), and PD-L1 (Beta: -0.15; p=0.005), relative to BIRC5-low. Conclusion: High BIRC5/survivin expression is associated with decreased immune-related gene expression in both ER-positive and ER-negative disease, and across all tumor stages. Anti-survivin therapies may be beneficial for boosting immune response in breast tumors with low immunogenicity, offering promise for BC subtypes previously ineligible for immunotherapy. Citation Format: Alina Hamilton, Qichen Wang, Sarah Soppe, Melissa Troester, Yara Abdou. Investigating survivin as a novel target for immunotherapy in diverse breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-02.

Assessment of fish protein hydrolysate as a substitute for fish meal in white shrimp diets: Impact on growth, immune response, and resistance against Vibrio parahaemolyticus infection

This study investigated the effects of fish protein hydrolysate derived from barramundi on growth performance, muscle composition, immune response, disease resistance, histology and gene expression in white shrimp (Penaeus vannamei). In vitro studies demonstrated FPH enhanced mRNA expressions of key immune-related genes and stimulated reactive oxygen species (ROS) production and phagocytic activity in shrimp hemocytes. To evaluate the effects of substituting fish meal with FPH in vivo, four isoproteic (43 %), isolipidic (6 %), and isoenergetic diets (489 kcal/100 g) were formulated with fish meal substitution levels of 0 % (control), 30 % (FPH30), 65 % (FPH65), and 100 % (FPH100). After 8-week feeding, the growth performance of FPH65 and FPH100 were significantly lower than that of control and FPH30 (p < 0.05). Similarly, the midgut histological examination revealed the wall thickness and villi height of FPH100 were significantly lower than those of control (p < 0.05). The shrimps were received the challenge of AHPND + Vibrio parahaemolyticus at week 4 and 8. All FPH-fed groups significantly enhanced resistance against Vibrio parahaemolyticus at week 4 (p < 0.05). However, this protective effect diminished after long-period feeding. No significant difference of survival rate was observed among all groups at week 8 (p > 0.05). The expressions of immune-related genes were analyzed at week 4 before and after challenge. In control group, V. parahaemolyticus significantly elevated SOD in hepatopancreas and Muc 19, trypsin, Midline-fas, and GPx in foregut (p < 0.05). Moreover, hepatopancreatic SOD of FPH65 and FPH100 were significantly higher than that of control before challenge (p < 0.05). Immune parameters were measured at week 8. Compared with control, the phagocytic index of FPH 30 was significantly higher (p < 0.05). However, dietary FPH did not alter ROS production, phenoloxidase activity, phagocytic rate, and total hemocyte count (p > 0.05). These findings suggest that FPH30 holds promise as a feed without adverse impacts on growth performance while enhancing the immunological response of white shrimp.

Effects of in ovo feeding of xylobiose and xylotriose on growth performance, carcass traits, ileal histomorphometry, and immune-related gene expression in broiler chickens

Xylooligosaccharides (XOS) are known as prebiotics which promote beneficial gut microbes, improve gut health and the performance of broilers. Xylobiose (XOS2) and xylotriose (XOS3) are two compounds in the XOS group. In ovo feeding could deliver these prebiotics to the embryo, and this is considered as an effective method for studying the effects of the prebiotics during embryonic development and the post-hatch period. This study investigated the effects of in ovo feeding of XOS2 and XOS3 on growth performance, carcass traits, ileal histomorphometry, and immune-related gene expression in broilers. A total of 144 fertile eggs of Cobb 500 broilers were divided into three groups: (a) non-injected control (CON), (b) XOS2, and (c) XOS3. The eggs from XOS2 and XOS3 groups were injected with 0.5ml of 0.85% normal saline solution containing 3mg of XOS2 and 3mg of XOS3, respectively, into their amniotic sac on the 17th embryonic day. A total of 131 hatched chicks were raised in 18 replicate pens (5 or more chicks per pen) following standard management practices and fed a corn-soybean meal-based diet for 21 days. The feed intake and body weight (BW) data were recorded weekly. The chickens were euthanized on day 7, day 14, and day 21 to collect ileum sections, and the carcass traits were recorded on only day 21. Total RNA was extracted from ileum tissues, and qPCR was performed to measure the gene expression. All the statistical analyses considered the significance level at a P value <0.05. The result showed that in ovo treatments did not affect the hatchability of the eggs. The XOS2 group showed a trend to increase the BW on day 0, compared to CON (P=0.052), and the XOS3 group improved FCR than the XOS2 group in the first week. There was no difference (P>0.05) among the groups for the weight of the breast muscle, drumsticks, gizzard, proventriculus, and carcass weight at the age of 21 days. The histomorphometry of the ileum on day 7 revealed a significant reduction (P<0.05) of villi height (VH), villi width (VW), and crypt depth (CD) in XOS2, compared to the CON group. On day 7, a lower CD and a higher VH to CD ratio were recorded in XOS3, compared to CON. On day 14, XOS3 showed an increase in VW and villi surface area (VSA) compared to XOS2 birds. On day 7, the expression of IL10 in XOS2 was higher compared to CON and XOS3, and CD56 was significantly higher (P<0.05) in the XOS2 compared to CON group. On day 21, the XOS2 and XOS3 significantly lower the expression of IFNγ. Overall, XOS2 and XOS3 significantly increased immunity until day 21. In conclusion, in ovo XOS demonstrated the potential to modulate gut morphology and immunity of the chickens during the post-hatch days.

Dietary lipid sources affect growth performance, lipid deposition, antioxidant capacity and inflammatory response of largemouth bass (Micropterus salmoides)

The present study explored the effects of different lipid sources replacing fish oil on growth performance, lipid deposition, antioxidant capacity, inflammatory response and disease resistance of largemouth bass (Micropterus salmoides). Four isonitrogenous (crude protein 50.46%) and isolipidic (crude lipid 11.12%) diets were formulated to contain 7% of different oil sources including fish oil (FO) (control), soybean oil (SO), linseed oil (LO) and coconut oil (CO). Largemouth bass with initial body weight of 36.0 ± 0.2 g were randomly distributed into 12 tanks, with 30 fish per tank and 3 tanks per treatment. The fish were fed the experiment diets twice daily for 8 weeks. The results indicated that the weight gain of largemouth bass fed the FO diet was significantly higher than that of fish fed the LO and CO diets. The liver crude lipid content in FO group was significantly higher than other groups, while the highest liver triglyceride content was showed in SO group and the lowest was detected in LO group. At transcriptional level, expression of lipogenesis related genes (pparγ, srebp1, fas, acc, dgat1 and dgat2) in the SO and CO group were significantly higher than the FO group. However, the expression of lipolysis and fatty acids oxidation related genes (pparα, cpt1, and aco) in vegetable oils groups were significantly higher than the FO group. As to the antioxidant capacity, vegetable oils significantly reduced the malondialdehyde content of largemouth bass. Total antioxidant capacity in the SO and LO groups were significantly increased compared with the FO group. Catalase in the LO group was significantly increased compared with the FO group. Furthermore, the ER stress related genes, such as grp78, atf6α, atf6β, chop and xbp1 were significantly enhanced in the vegetable oil groups compared with the FO group. The activity of serum lysozyme in vegetable oil groups were significantly higher than in FO group. Additionally, the relative expression of non-specific immune related genes, including tlr2, mapk11, mapk13, mapk14, rela, tgf-β1, tnfα, 5lox, il-1β and il10, were all significantly increased in SO and CO groups compared to the other groups. In conclusion, based on the indexes including growth performance, lipid deposition, antioxidant capacity and inflammatory response, SO and LO could be alternative oil sources for largemouth bass.

Yarrowia lipolytica yeast as a dietary supplement for rainbow trout (Oncorhynchus mykiss): Effects on gut microbiota, health and immunity

This study evaluated the effect of whole and autolysed Yarrowia lipolytica CBS 7504 strain on growth performance, gut immunity and gut microbiota composition in juvenile rainbow trout (Oncorhynchus mykiss). Fish were fed diets containing 2% and 5% whole or autolysed Y. lipolytica (WY and AY) in a 45-day trial, with a no-yeast diet as control. Log2 fold change analysis of Y. lipolytica diets showed relative change in abundance and association of certain microbial taxa with respect to control such as a correlation between the relative abundance of Desulfovibrionaceae in diets with WY. Moreover, diets at 5% and 2% levels demonstrated a synergistic association with relative levels of Sphingobacteriaceae and Rhodobacteraceae, respectively. However, the overall analyses showed little/no impact of Y. lipolytica supplementation on overall intestinal microbial composition, growth parameters and body indices. Gene expression analysis of the intestine revealed significant elevation in expression of immune-related genes of the complement pathway (c3 and c-type lectin), membrane receptor pathway (tlr2 and tlr5), mucosal innate immune pathway (muc-2), cytokines (tnf-α) and adaptive immune pathway (igt and cd4) in 5% WY. In conclusion, it can be inferred that dietary Y. lipolytica can be a useful functional feed supplement for rainbow trout when used as whole yeast at a 5% inclusion level since it can modulate gut microbial communities and act as a potential immunostimulant for the host.

Extraction of lipopolysaccharide from fish-derived Aeromonas hydrophila and their immunostimulatory effect on Qihe crucian carp

Purified lipopolysaccharide (LPS) was first obtained from fish-derived Aeromonas hydrophila (A. hydrophila) by hot phenol-water combined with enzymatic hydrolysis. Subsequently, different concentrations of LPS (0, 0.2, 0.4, 0.8, and 1.2 mg/mL) were injected intraperitoneally into Qihe crucian carp (Carassius auratus var. Qihe) with a dose of 3 μL/g body weight to evaluated their immunostimulatory activity by detecting the expression of immune-related genes in the spleen, head kidney, and hepatopancreas. The results showed that the average extraction rate of LPS was about 1.063% based on the precipitation weight of wet bacteria, of which the polysaccharide content was 9.39%. The silver staining band of LPS after SDS-PAGE electrophoresis showed a ladder distribution typically characterized by smooth-type LPS. Protein and nucleic acid electrophoresis results showed very low amounts of residual protein and nucleic acid, and high-performance liquid chromatography (HPLC) results showed that the purity of the extracted A. hydrophila LPS was similar to that of high-purity commercial LPS. Pro-inflammatory cytokine genes such as IL-1, IL-8, TNF-α, and IL-6 were more sensitive to LPS stimulation than anti-inflammatory factors such as IL-10 and TGF-β. After 2 h of injection, their expression levels in the spleen, head kidney, and hepatopancreas were significantly increased (P < 0.05), with the 0.8 and 1.2 mg/mL injection groups showing the most significant increase. The gene expression of IL-10 and TGF-β showed slight fluctuations in response to A. hydrophila LPS stimulation (P < 0.05). In conclusion, high-purity LPS was extracted from A. hydrophila derived from fish by modified hot phenol-water method, it demonstrating that LPS product could significantly regulate the innate immunity of Qihe crucian carp, with the most effective vaccination dose being 2 mg/kg/BW. These findings provide useful information for future research on endotoxin vaccines.

Prognostic implications of STK11 with different mutation status and its relationship with tumor-infiltrating immune cells in non-small cell lung cancer.

Mutations in STK11 (STK11Mut) gene may present a negative impact on survival in Non-small Cell Lung Cancer (NSCLC) patients, however, its relationship with immune related genes remains unclear. This study is to unveil whether overexpressed- and mutated-STK11 impact survival in NSCLC and to explore whether immune related genes (IRGs) are involved in STK11 mutations. 188 NSCLC patients with intact formalin-fixed paraffin-embedded (FFPE) tissue available for detecting STK11 protein expression were included in the analysis. After immunohistochemical detection of STK11 protein, patients were divided into high STK11 expression group (STK11High) and low STK11 expression group (STK11Low), and then Kaplan-Meier survival analysis and COX proportional hazards model were used to compare the overall survival (OS) and progression-free survival (PFS) of the two groups of patients. In addition, the mutation data from the TCGA database was used to categorize the NSCLC population, namely STK11 Mutated (STK11Mut) and wild-type (STK11Wt) subgroups. The difference in OS between STK11Mut and STK11Wt was compared. Finally, bioinformatics analysis was used to compare the differences in IRGs expression between STK11Mut and STK11Wt populations. The median follow-up time was 51.0 months (range 3.0 - 120.0 months) for real-life cohort. At the end of follow-up, 64.36% (121/188) of patients experienced recurrence or metastasis. 64.89% (122/188) of patients ended up in cancer-related death. High expression of STK11 was a significant protective factor for NSCLC patients, both in terms of PFS [HR=0.42, 95% CI= (0.29-0.61), P<0.001] and OS [HR=0.36, 95% CI= (0.25, 0.53), P<0.001], which was consistent with the finding in TCGA cohorts [HR=0.76, 95%CI= (0.65, 0.88), P<0.001 HR=0.76, 95%CI= (0.65, 0.88), P<0.001]. In TCGA cohort, STK11 mutation was a significant risk factor for NSCLC in both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) histology in terms of OS [HR=6.81, 95%CI= (2.16, 21.53), P<0.001; HR=1.50, 95%CI= (1.00, 2.26), P=0.051, respectively]. Furthermore, 7 IRGs, namely CALCA, BMP6, S100P, THPO, CGA, PCSK1 and MUC5AC, were found significantly overexpressed in STK11-mutated NSCLC in both LUSC and LUAD histology. Low STK11 expression at protein level and presence of STK11 mutation were associated with poor prognosis in NSCLC, and mutated STK11 might probably alter the expression IRGs profiling.

Characterization, functional analysis, and expression of TNFR16 from Pinctada fucata martensii in response to exogenous stimuli and RNA interference

Marine pearls from Pinctada fucata martensii are valuable for decoration, skin care, and health care, but their quality and yield can be threatened by diseases, pearl nuclei transplantation rejection, and other external factors. The present study investigated the immune function of the tumor necrosis factor receptor (TNFR) gene in P. f. martensii to better understand immune responses related to nuclei rejection. The PmTNFR16 cDNA sequence obtained from cloning was 1675 bp in length and encoded 406 amino acid residues. Sequence analysis revealed that PmTNFR16 comprises one signal peptide domain, four TNFR domains, one transmembrane domain, and one death domain. Expression analysis revealed PmTNFR16 was highly expressed in the mantle and significantly up-regulated after exogenous stimuli. The effects of PmTNFR16 on immune effector molecules within the NF-κB signaling pathway were further explored using RNA interference probes for PmTNFR16. Pre-treatment of pearl oysters with dsRNA-PmTNFR before nucleus-insertion resulted in decreased expression of PmTNFR16 by 53 % at 24 h (p<0.01). Similarly, the expression of the NF-κB signaling pathway immune-related genes TRAF2, TRADD, IKK, NIK, and NF-κB significantly decreased at 24 h. The results indicated that PmTNFR16 was successfully silenced as were the expression of immune-related genes in the downstream NF-κB signaling pathway. Overall, these results indicate that PmTNFR16 may be involved in the immune response of the host organism by mediating the NF-κB signaling pathway. These observations provide a framework for future studies of the immune mechanisms of pearl oysters.

Synergistic Effect of Dietary Supplementation with Sodium Butyrate, β-Glucan and Vitamins on Growth Performance, Cortisol Level, Intestinal Microbiome and Expression of Immune-Related Genes in Juvenile African Catfish (Clarias gariepinus).

The effect of dietary supplementation with sodium butyrate, β-glucan and vitamins (A, D3, E, K, C) on breeding indicators and immune parameters of juvenile African catfish was examined. The fish were fed with unenriched (group C) and enriched feed with a variable proportion of sodium butyrate/β-glucan, and constant content of vitamins (W1-W3). After the experiment, blood and the middle gut were collected. The microbiome of the gut was determined using Next Generation Sequencing (NGS). Liver tissue was collected for determination of expression of immune-related genes (HSP70, IL-1β, TNFα). W2 and W3 were characterized by the most favorable values of breeding indicators (p < 0.05). The highest blood cortisol concentration was in group C (71.25 ± 10.45 ng/mL), and significantly the lowest in W1 (46.03 ± 7.01 ng/ mL) (p < 0.05). The dominance of Cetobacterium was observed in all study groups, with the largest share in W3 (65.25%) and W1 (61.44%). Gene expression showed an increased number of HSP70 genes in W1. IL-1β and TNFα genes peaked at W3. The W3 variant turns out to be the most beneficial supplementation, due to the improvement of breeding and immunological parameters. The data obtained can be used to create a preparation for commercial use in the breeding of this species.

Dietray artichoke (Cynara scolymus) extract ameliorated the growth performance, humoral immune parameters and resistance against Aeromonas hydrophila in goldfish (Carassius auratus)

Abstract This trial investigated the efficacy of artichoke (Cynara scolymus) extract (AE) on the growth performance, immunity, antioxidant parameters, and resistance against Aeromonas hydrophila of goldfish (Carassius auratus). For this purpose, a total number of 470 gold fish with initial weight 5.70±0.2 g fed with four experimental diets including 0 (T0), 100 (T1), 150 (T2), and 200 (T3) mg kg −1 diet AE for 8 weeks. At the end of feeding trial, growth performances, serum immune parameters, and mucus antioxidant enzymes were measured. Fish were challenged with A. hydrophila, and the antioxidant and immunity-related gene expression were investigated. Based on the results, the highest final weight (FW) and weight gain (WG) attained in T2 and T3 (P&lt;0.05). Immune factors including ACH50, lysozyme, and total immunoglobulin in T2 and T3 showed the highest values (P&lt;0.05). The expression of GR, IL1β, TNFα, HSC70, HSP70, and HSP90β genes in T1, T2, and T3 were higher than the control (P&lt;0.05). The GST expression was significantly enhanced in T2 (P&lt;0.05). The present study demonstrated that the administration of AE, especially at doses of 150 mg kg−1, could improve the growth, immunity, and antioxidant parameters, as well as enhance disease resistance against A. hydrophila in goldfish.