scholarly journals Nutrigenomic Effects of Long-Term Grape Pomace Supplementation in Dairy Cows

Animals ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 714 ◽  
Author(s):  
Marianna Pauletto ◽  
Ramy Elgendy ◽  
Andrea Ianni ◽  
Elettra Marone ◽  
Mery Giantin ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Milk Production ◽  
Production Efficiency ◽  
Mitochondrial Fission ◽  
Enrichment Analysis ◽  
Basal Diet ◽  
Grape Pomace ◽  
Gene Set Enrichment Analysis ◽  
Genome Replication ◽  
The Impact

The increasing demand for more animal products put pressure on improving livestock production efficiency and sustainability. In this context, advanced animal nutrition studies appear indispensable. Here, the effect of grape pomace (GP), the polyphenol-rich agricultural by-product, was evaluated on Holstein-Friesian cows’ whole-blood transcriptome, milk production and composition. Two experimental groups were set up. The first one received a basal diet and served as a control, while the second one received a 7.5% GP-supplemented diet for a total of 60 days. Milk production and composition were not different between the group; however, the transcriptome analysis revealed a total of 40 genes significantly affected by GP supplementation. Among the most interesting down-regulated genes, we found the DnaJ heat-shock protein family member A1 (DNAJA1), the mitochondrial fission factor (MFF), and the impact RWD domain protein (IMPACT) genes. The gene set enrichment analysis evidenced the positive enrichment of ‘interferon alpha (IFN-α) and IFN-γ response’, ‘IL6-JAK-STAT3 signaling’ and ‘complement’ genes. Moreover, the functional analysis denoted positive enrichment of the ‘response to protozoan’ and ‘negative regulation of viral genome replication’ biological processes. Our data provide an overall view of the blood transcriptomic signature after a 60-day GP supplementation in dairy cows which mainly reflects a GP-induced immunomodulatory effect.

scholarly journals THE IMPACT OF BALANCED ENERGY AND PROTEIN SUPPLEMENTATION TO MILK PRODUCTION AND QUALITY IN EARLY LACTATING DAIRY COWS

2016 ◽  
Vol 41 (2) ◽  
pp. 83-90 ◽  
Author(s):  
W. P. Widyobroto ◽  
R. Rochijan ◽  
I. Ismaya ◽  
A. Adiarto ◽  
Y. Y. Suranindyah
Keyword(s):  
Dairy Cows ◽  
Milk Production ◽  
Milk Fat ◽  
Basal Diet ◽  
Total Solid ◽  
Protein Supplementation ◽  
Lactating Dairy Cows ◽  
Quality Of Milk ◽  
The Impact

This research was aimed to determine the impact of balanced energy and protein supplementation with high rumen undegraded protein (HRUP) to milk production and quality in early lactating dairy cows. Twelve early lactating Friesian Holstein cows were divided into two groups (control and HRUP). Both control and HRUP group were fed on a basal diet (forage to concentrate ratio was 60:40; DM basis), with rumen undegraded protein (RUP) levels were 27.47% and 32.78% for control and HRUP, respectively. The experimental diets were given to animals twice daily, morning and afternoon. Water was given by ad libitum. The observed parameters were nutrient intake, quantity and quality of milk production . Data were examined using t-test. Results showed that feed intake, milk production and 4% FCM, milk fat and lactose concentrations, and milk solid non-fat and total solid concentrations were not differed significantly between control and HRUP groups. However, milk protein concentration and production were differed (P

scholarly journals In Silico Identification of Contradictory Role of ADAMTS5 in Hepatocellular Carcinoma

2021 ◽  
Vol 20 ◽  
pp. 153303382098682
Author(s):  
Zhipeng Zhu ◽  
Jiuhua Xu ◽  
Xiaofang Wu ◽  
Sihao Lin ◽  
Lulu Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Subgroup Analysis ◽  
Prognostic Value ◽  
Molecular Mechanisms ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Clinicopathological Parameters ◽  
Free Survival ◽  
Cox Regression Analysis ◽  
The Impact

Background: ADAMTS5 has different roles in multiple types of cancers and participates in various molecular mechanisms. However, the prognostic value of ADAMTS5 in patients with hepatocellular carcinoma (HCC) still remains unclear. We carried the study to evaluate the prognostic value and identified underlying molecular mechanisms in HCC. Methods: Firstly, the association of ADAMTS5 expression and clinicopathological parameters was evaluated by in GSE14520. Next, ADAMTS5 expression in HCC was performed using GSE14520, GSE36376, GSE76427 and The Cancer Genome Atlas (TCGA) profile. Furthermore, Kaplan-Meier analysis, Univariate and Multivariate Cox regression analysis, subgroup analysis was performed to evaluate the prognostic value of ADAMTS5 in HCC. Finally, GO enrichment analysis, gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) were performed to revealed underlying molecular mechanisms. Result: The expression of ADAMTS5 was positively correlated with the development of HCC. Next, high ADAMTS5 expression was significantly associated with poorer survival (all P < 0.05) and the impact of ADAMTS5 on all overall survival (OS), disease-free survival (DFS), relapse-free survival (RFS), disease specific survival (DSS) and progression free interval (PFI) was specific for HCC among other 29 cancer types. Subgroup analysis showed that ADAMTS5 overexpression was significantly associated with poorer OS in patients with HCC. Finally, ADAMTS5 might participate in the status conversion from metabolic-dominant to extracellular matrix-dominant, and the activation of ECM-related biological process might contribute to high higher mortality risk for patients with HCC. Conclusion: ADAMTS5 may play an important role in the progression of HCC, and may be considered as a novel and effective biomarker for predicting prognosis for patients with HCC.

The efficacy of feeding a live probiotic yeast, Yea-Sacc®, on the performance of lactating dairy cows

2015 ◽  
Vol 3 ◽  
Author(s):  
D. Tristant ◽  
C. A. Moran
Keyword(s):  
Dairy Cows ◽  
Milk Production ◽  
Milk Fat ◽  
Milk Protein ◽  
Feed Additive ◽  
Control Group ◽  
Target Dose ◽  
Lactating Dairy Cows ◽  
Farm Model ◽  
The Impact

SummaryThe following trial was conducted to evaluate the impact of feeding Yea-Sacc® (YS; Alltech Inc, USA), a zootechnical feed additive based on a live probiotic strain of Saccharomyces cerevisiae, to lactating dairy cows over a 12 week period. Sixty-four primiparous and multiparous Holstein dairy cows, grouped to give similar range of parity, physiological and milk production stages, were selected for the study. Cows were equally allocated to either a control feed group or a diet supplemented with YS (32 cows per treatment). The test diet was formulated to include YS (Yea-Sacc® Farm Pak) incorporated in the total mixed ration (TMR), supplying a target dose of 5 × 107 CFU/kg feed dry matter (DM). This target dose delivered 1 × 109 CFU/cow/day, for a cow consuming 20 kg feed (DM basis) daily. Each cow was considered a replicate unit. Cows were fed a nutritionally adequate total TMR plus hay and a supplementary protein/energy concentrate (calculated according to milk yield) for 12 weeks, supplied once a day after the morning milking. Weigh backs of feed were recorded daily, with refusals being maintained at 3% of the total intake. During the 12 week study period, YS had significant beneficial effects on milk production (+0.8 kg/day; P = 0.003), energy corrected milk production (+1.4 kg/day; P < 0.0001), synthesis of milk protein (+36 g/day; P = 0.001), milk protein content (+0.3 g/kg; P = 0.009), and milk urea content (−0.09 mg/l; P = 0.004). The synthesis of milk fat was similar between treatments but milk fat content was lower for the YS group compared to the control group (−1.1 g/kg; P = 0.0002). Lactose content was always higher (+0.8 g/kg; P < 0.0001) for the YS group, indicating enhanced energy utilisation. In general, the effect of YS was higher during the first study period (one to seven weeks), when cows were in early lactation and the production potential was higher. YS cows produced significantly more milk during the study, and an additional 220 kg milk per cow was sold from this group from the output measured from the beginning of the study to two weeks post-trial. However, the statistical analysis including the post-study period did not show a significant effect. The 305-day simulated milk production was higher for the YS group (+400 kg/cow) but again the difference was not significant. In conclusion, YS at a target dose of 5 × 107 CFU/kg DM improved milk production and milk quality in healthy dairy cows. In addition, when the data were included in a whole-farm model, feeding YS reduced methane emissions by 4%, reduced the number of animals required for the desired milk production by 4% and increased overall farm margins by 1.4%.

scholarly journals Paternal Obesity and SGLT2 Inhibition Alter Expression of Placental Regulatory Genes

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A752-A753
Author(s):  
Lei Su ◽  
Soravis Osataphan ◽  
Jessica Desmond ◽  
Rui Fang ◽  
Jeremy Chimene-Weiss ◽  
...  
Keyword(s):  
Sglt2 Inhibitor ◽  
Enrichment Analysis ◽  
Placental Weight ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
Metabolic Effects ◽  
Altered Expression ◽  
Gene Sets ◽  
The Impact ◽  
Paternal Obesity

Abstract We previously demonstrated that paternal obesity is associated with offspring metabolic risk during later life, and that paternal SGLT2i treatment improves offspring metabolic phenotypes. Since the placenta is a key determinant of prenatal growth and development, we hypothesized the placenta could mediate the impact of paternal obesity and paternal SGLT2i treatment. Male C57BL/6J mice were fed standard chow (Purina 9F) or 60% high-fat diet (HFD, D12492, Research Diet), or 60% HFD plus the SGLT2 inhibitor canagliflozin (CANA, 25 mg/kg/d) for 4 weeks before mating with chow-fed females. Placenta were collected on E16.5, and RNA-seq was performed on placenta from male offspring (paternal chow, pChow, n=4, pHFD, n=5, and pHFD+CANA, n=4), and differentially expressed genes were identified using Limma. Placenta weight was significantly lower in pHFD (0.089±0.004 g, 7 litters from 6 fathers) vs. both pChow (0.108±0.011 g, 4 litters, 4 fathers) and pHFD+CANA (0.107±0.013 g, 5 litters, 5 fathers)(p&lt;0.05). Litter size, fetal or liver weight, or fetal/placental weight ratio did not differ between groups. No genes were differentially expressed in pHFD vs. pChow (FDR&lt;0.1). Gene set enrichment analysis (GSEA) identified significance (FDR&lt;0.05, NES&gt;1.8) for gene sets in steroid metabolic, drug catabolic, and protein-containing complex remodeling processes. Genes responsible for enrichment included cholesterol biosynthesis (Hmgcs1), transport (Apob, Apoa1/2/4, Apom, Apoc1, Vldlr, Pcsk9) and steroid hormone biosynthesis genes (Hsd3b1, Cyp11b1), all upregulated in pHFD by 1.5-3-fold. These results suggest pHFD could potentially affect maternal and fetal cholesterol homeostasis. pHFD+CANA altered expression of 154 genes vs. pHFD (7 up-, 147 down, FDR &lt;0.1, FC &gt;|1.5|); 18 gene sets were downregulated by pHFD+CANA (GSEA NES&lt;-1.8 and FDR&lt;0.05), including the 3 sets upregulated by pHFD. ChEA3 enrichment analysis (ENCODE library) predicted regulation by transcription factors important for cholesterol and sterol biosynthesis (Srebf1/2), embryonic development (Foxa2), & glucose homeostasis (Hnf4g), suggesting these pathways could mediate the “rescue” effect of pHFD+CANA (FDR&lt;0.05). Expression of Foxa2 was significantly downregulated (4-fold) in pHFD+CANA vs. pHFD. We independently analyzed expression of the 78 detected imprinted genes. None were significantly different in pHFD, but both paternally expressed (Nnat) and maternally expressed genes (H19, Phlda2, Meg3, Meg8) were downregulated in pHFD+CANA vs. pHFD by 1.4 to 3.8 fold in pHFD+CANA (p&lt;0.001,FDR&lt;0.1). In summary, paternal SGLT2i reversed the impact of pHFD on placental weight. Robust impact of both pHFD and pSGLT2i on the transcriptome suggests that the placenta is a key mediator of paternal metabolic effects on offspring development and metabolic disease risk, potentially via modification of lipid transport.

scholarly journals Importance of SNP Dependency Correction and Association Integration for Gene Set Analysis in Genome-Wide Association Studies

2021 ◽  
Vol 12 ◽  
Author(s):  
Michal Marczyk ◽  
Agnieszka Macioszek ◽  
Joanna Tobiasz ◽  
Joanna Polanska ◽  
Joanna Zyla
Keyword(s):  
Association Studies ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Genome Wide Association ◽  
Gene Set Analysis ◽  
Genome Wide Association Studies ◽  
Gene Set Enrichment ◽  
Gene Set ◽  
Genome Wide ◽  
The Impact

A typical genome-wide association study (GWAS) analyzes millions of single-nucleotide polymorphisms (SNPs), several of which are in a region of the same gene. To conduct gene set analysis (GSA), information from SNPs needs to be unified at the gene level. A widely used practice is to use only the most relevant SNP per gene; however, there are other methods of integration that could be applied here. Also, the problem of nonrandom association of alleles at two or more loci is often neglected. Here, we tested the impact of incorporation of different integrations and linkage disequilibrium (LD) correction on the performance of several GSA methods. Matched normal and breast cancer samples from The Cancer Genome Atlas database were used to evaluate the performance of six GSA algorithms: Coincident Extreme Ranks in Numerical Observations (CERNO), Gene Set Enrichment Analysis (GSEA), GSEA-SNP, improved GSEA for GWAS (i-GSEA4GWAS), Meta-Analysis Gene-set Enrichment of variaNT Associations (MAGENTA), and Over-Representation Analysis (ORA). Association of SNPs to phenotype was calculated using modified McNemar’s test. Results for SNPs mapped to the same gene were integrated using Fisher and Stouffer methods and compared with the minimum p-value method. Four common measures were used to quantify the performance of all combinations of methods. Results of GSA analysis on GWAS were compared to the one performed on gene expression data. Comparing all evaluation metrics across different GSA algorithms, integrations, and LD correction, we highlighted CERNO, and MAGENTA with Stouffer as the most efficient. Applying LD correction increased prioritization and specificity of enrichment outcomes for all tested algorithms. When Fisher or Stouffer were used with LD, sensitivity and reproducibility were also better. Using any integration method was beneficial in comparison with a minimum p-value method in specific combinations. The correlation between GSA results from genomic and transcriptomic level was the highest when Stouffer integration was combined with LD correction. We thoroughly evaluated different approaches to GSA in GWAS in terms of performance to guide others to select the most effective combinations. We showed that LD correction and Stouffer integration could increase the performance of enrichment analysis and encourage the usage of these techniques.

scholarly journals PI3K Pathway Inhibition with NVP-BEZ235 Hinders Glycolytic Metabolism in Glioblastoma Multiforme Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3065
Author(s):  
Shreya Udawant ◽  
Carl Litif ◽  
Alma Lopez ◽  
Bonnie Gunn ◽  
Erin Schuenzel ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Enrichment Analysis ◽  
Pi3k Pathway ◽  
Gene Set Enrichment Analysis ◽  
Metabolic Reprogramming ◽  
Clinical Samples ◽  
Rna Seq ◽  
Bioinformatics Analyses ◽  
Dual Inhibitor ◽  
The Impact

Glioblastoma (GBM) is the most lethal primary brain cancer that lacks effective molecular targeted therapies. The PI3K/AKT/mTOR pathway is activated in 90% of all Glioblastoma multiforme (GBM) tumors. To gain insight into the impact of the PI3K pathway on GBM metabolism, we treated U87MG GBM cells with NVP-BEZ235 (PI3K and mTOR a dual inhibitor) and identified differentially expressed genes with RNA-seq analysis. RNA-seq identified 7803 differentially regulated genes in response to NVP-BEZ235. Gene Set Enrichment Analysis (GSEA) identified two glycolysis-related gene sets that were significantly enriched (p < 0.05) in control samples compared to NVP-BEZ235-treated samples. We validated the inhibition of glycolytic genes by NVP-BEZ235 and examined the impact of the FOXO1 inhibitor (AS1842856) on these genes in a set of GBM cell lines. FOXO1 inhibition alone was associated with reduced LDHA expression, but not ENO1 or PKM2. Bioinformatics analyses revealed that PI3K-impacted glycolytic genes were over-expressed and co-expressed in GBM clinical samples. The elevated expression of PI3K-impacted glycolytic genes was associated with poor prognosis in GBM based on Kaplan–Meier survival analyses. Our results suggest novel insights into hallmark metabolic reprogramming associated with the PI3K-mTOR dual inhibition.

scholarly journals P3H4 Overexpression Serves as a Prognostic Factor in Lung Adenocarcinoma

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Xiangfeng Jin ◽  
Haiqing Zhou ◽  
Jianfang Song ◽  
Hong Cui ◽  
Yiren Luo ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Lung Adenocarcinoma ◽  
Differential Expression Analysis ◽  
Disease Free Survival ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
High Expression ◽  
Receptor Interaction ◽  
Hippo Signaling ◽  
The Impact

Background. The present study is aimed at evaluating the functional and clinical values of P3H4 in lung adenocarcinoma. Moreover, we also investigated the downstream pathways that P3H4 might participate in. Methods. The differential expression analysis was used to identify genes differentially expressed in lung adenocarcinoma tissues as compared with normal tissues. Survival analysis was used to test the association between P3H4 and survival time. Gene set enrichment analysis was conducted to explore the downstream pathways. CCK8 and transwell were employed to examine the impact of P3H4 on cell phenotypes. Results. P3H4 was highly upregulated in LUAD tissues at both RNA and protein levels. Moreover, the LUAD patients, who had high expression of P3H4, were also observed to have shorter disease-free survival and overall survival. These results demonstrated that P3H4 could be used as a prognostic biomarker for LUAD. Moreover, we also found that it was the copy number alterations (CNAs), not DNA methylation, that regulated the RNA expression of P3H4, indicating that its upregulation might be partially resulted from the CNAs. Furthermore, functional experiments revealed that the A549 and H1299 cells with siRNA treatment (siP3H4) exhibited significantly decreased cell proliferation after 24 hours, migratory ability, and invasiveness. Functionally, the upregulated proteins in the P3H4 high expression group were mainly enriched in tumor microenvironment-related pathways such as phagosome, focal adhesion, and ECM-receptor interaction and cancer-related pathways such as bladder cancer pathway, proteoglycans in cancer, and hippo signaling pathway. Conclusion. The present study systematically evaluated the functional and clinical values of P3H4 in LUAD, and explored the related biological pathways. P3H4 might promote LUAD progression through regulating tumor microenvironment-related pathways.

scholarly journals Diabetes Induces a Transcriptional Signature in Bone Marrow–Derived CD34+ Hematopoietic Stem Cells Predictive of Their Progeny Dysfunction

2021 ◽  
Vol 22 (3) ◽  
pp. 1423
Author(s):  
Yuri D’Alessandra ◽  
Mattia Chiesa ◽  
Vera Vigorelli ◽  
Veronica Ricci ◽  
Erica Rurali ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Coronary Bypass ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
P Value ◽  
Peptidase Activity ◽  
Biological Processes ◽  
Hematopoietic Stem ◽  
The Impact

Hematopoietic stem/progenitor cells (HSPCs) participate in cardiovascular (CV) homeostasis and generate different types of blood cells including lymphoid and myeloid cells. Diabetes mellitus (DM) is characterized by chronic increase of pro-inflammatory mediators, which play an important role in the development of CV disease, and increased susceptibility to infections. Here, we aimed to evaluate the impact of DM on the transcriptional profile of HSPCs derived from bone marrow (BM). Total RNA of BM-derived CD34+ stem cells purified from sternal biopsies of patients undergoing coronary bypass surgery with or without DM (CAD and CAD-DM patients) was sequenced. The results evidenced 10566 expressed genes whose 79% were protein-coding genes, and 21% non-coding RNA. We identified 139 differentially expressed genes (p-value < 0.05 and |log2 FC| > 0.5) between the two comparing groups of CAD and CAD-DM patients. Gene Set Enrichment Analysis (GSEA), based on Gene Ontology biological processes (GO-BP) terms, led to the identification of fourteen overrepresented biological categories in CAD-DM samples. Most of the biological processes were related to lymphocyte activation, chemotaxis, peptidase activity, and innate immune response. Specifically, HSPCs from CAD-DM patients displayed reduced expression of genes coding for proteins regulating antibacterial and antivirus host defense as well as macrophage differentiation and lymphocyte emigration, proliferation, and differentiation. However, within the same biological processes, a consistent number of inflammatory genes coding for chemokines and cytokines were up-regulated. Our findings suggest that DM induces transcriptional alterations in HSPCs, which are potentially responsible of progeny dysfunction.

scholarly journals Monitoring the Transition Period in Dairy Cows through 1H NMR-Based Untargeted Metabolomics

Dairy ◽  
2021 ◽  
Vol 2 (3) ◽  
pp. 356-366
Author(s):  
Andrés López Radcenco ◽  
María de Lourdes Adrien ◽  
Gretel Ruprechter ◽  
Elena de Torres ◽  
Ana Meikle ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Milk Production ◽  
1H Nmr ◽  
Metabolic Diseases ◽  
Transition Period ◽  
Principal Component ◽  
Partial Least Square ◽  
Least Square ◽  
Untargeted Metabolomics ◽  
The Impact

The metabolic alterations associated with the increase in milk production make the transition period critical to the health of dairy cows, usually leading to a higher incidence of disease in periparturient animals. In this manuscript, we describe the use of NMR-based untargeted metabolomics to follow how these changes impact the serum metabolome in a group of 28 transition dairy cows with no initial clinical diseases. Principal component analysis (PCA) of serum 1H NMR data from four weeks before calving to 8 weeks after parturition allowed us to clearly identify four stages during the transition period. Pairwise comparisons using orthogonal partial least square discriminant analysis (OPLS-DA) and univariate data analysis led to the identification of 18 metabolites that varied significantly through these stages. Species such as acetate, betaine, and creatine are observed early after calving, while other markers of metabolic stress, including acetone, β-hydroxybutyrate (BHB), and choline, accumulate significantly at the height of milk production. Furthermore, marked variations in the levels of lactate, allantoin, alanine, and other amino acids reveal the activation of different gluconeogenic pathways following parturition. Concomitant with a return to homeostasis, a gradual normalization of the serum metabolome occurs 8 weeks after calving. Correlations of metabolite levels with dietary and metabolic adaptations based on animal parity could also be identified. Overall, these results show that NMR-based chemometric methods are ideally suited to monitor manifestations of metabolic diseases throughout the transition period and to assess the impact of nutritional management schemes on the metabolism of dairy cows.

scholarly journals Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer

2021 ◽  
Vol 9 (3) ◽  
pp. e001717
Author(s):  
Anna Wilkins ◽  
Elisa Fontana ◽  
Gift Nyamundanda ◽  
Chanthirika Ragulan ◽  
Yatish Patil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rectal Cancer ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Poor Responders ◽  
Type I ◽  
Immune Gene ◽  
Neoadjuvant Radiotherapy ◽  
The Impact ◽  
Crt Response

BackgroundRectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer.MethodsWe generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses.ResultsΔTCD scores ranged from 12.4% to −47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders.ConclusionThis study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune “cold” towards an immunologically “hot” phenotype on treatment with radiotherapy.

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