Problem Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells ‘educate’ monocytes/macrophages to create an adequate environment that promote trophoblast survival. Method of study CD14 + monocytes were isolated from peripheral blood using magnetic beads. Co‐culture experiments were conducted using a two‐chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. Results Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro‐inflammatory cytokines [interleukin‐6 (IL‐6), IL‐8, tumor necrosis factor‐ α ] and chemokines (growth‐related oncogen‐ α , monocyte chemoattractant protein‐1, macrophage inflammatory protein‐1 β , RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre‐exposed to trophoblast cells. Conclusion The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro‐inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.
5580 Background: Emerging data suggests that key differences exist between BRCA1 and BRCA2 associated OC, including response to therapy and survival. The purpose of this study was to identify the gene expression profiles, interacting pathways and immune microenvironment of BRCA1 mutant (BRCA1mut), BRCA2 mutant (BRCA2mut) and homologous recombination wild-type (HRwt) associated high grade serous OC (HGSOC). Methods: Next-generation sequencing (592, NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ) were performed in 8196 OC tumors classified into 3 groups: BRCA1mt; BRCA2mt; and HRwt. BRCA mutations were defined as variants that result in loss-of-function of the BRCA protein and HRwt was defined as samples negative for aberrations in both BRCA1 and BRCA2, as well as for 28 other homologous recombination genes Microsatellite instability (MSI) was tested by fragment analysis, IHC and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations (TMB-H: >10 mutations/MB). LOH cut-off >16%. Immune cell infiltrates were calculated by XCell. Differential gene expression was calculated using Limma. Significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons (q-value < 0.05). Results: We identified 677 BRCA1mt, 439 BRCA2mt, and 7080 HRwt OC tumors. HGSOC made up the largest portion of BRCA1mt (523; 77%), BRCA2mt (306; 70%), and HRwt (4281; 60%) tumors. TP53 was most commonly mutated gene in all three groups. LOH (>16%) was highest in BRCA1mt (86.8%) compared to BRCA2mt (74.8%) and HRwt (38.4%). TMB-H was highest in BRCA2mt (6.29%) than in BRCA1mt (1.35%) and HRwt (0.91%) HGSOC (all q < 0.05). Expression of immune checkpoint genes CD80, CD86, CD274, CTLA4, HAVCR2/TIM3, IFNG, IDO1, LAG3, PDCD1 and PDCD1LG2 were significantly higher in BRCA1 and BRCA2 mt compared to HRwt HGSOC (FC: 1.12-1.59, q < 0.05). HRwt tumors had decreased infiltration of Activated Dendritic cells compared to BRCA1mt, and lower Macrophage M1 compared to both BRCA1mt and BRCA2mt (all q < 0.05). Additionally, T-inflamed score was higher in BRCA1mt compared to HRwt, while IFN score was higher in BRCA1mt compared to both BRCA2mt and HRwt (all q < 0.05). From 17,408 genes with measured expression. 522 (3.0%) differentially expressed genes (DEG) were found between BRCA2mt vs BRCA1mt; 1487 (8.54%) between BRCA2mt vs HRwt; and 9297 (53.4%) between BRCA1mt and HRwt HGSOC. Pathway analysis identified Fatty Acid Metabolism, Myc targets, ROS pathway, Oxidative Phosphorylation, and Wnt B-catenin signaling pathways as differentially regulated between the 3 groups. Conclusions: We describe the genomic, pathway and immunologic analyses in the largest cohort of BRCA1 and 2 mutated HGSOC to date. Both metabolic and immune response pathways are differentially regulated between the groups. Results can potentially inform targeted therapeutic studies based on unique BRCA genotype.
<p>Transcriptomic differences between patients with <i>BRCA1</i>mt, <i>BRCA2</i>mt, and HRwt ovarian cancer. RNA-seq was performed on patients with HRwt (<i>n</i> = 375), <i>BRCA1</i>mt (<i>n</i> = 16), and <i>BRCA2</i>mt (<i>n</i> = 15) ovarian cancer. <b>A,</b> Volcano plots showing the distribution of DEGs (<i>P</i> = 0.05; FC > 1.5) in the compared groups as indicated. <b>B,</b> Venn diagram of DEGs in each of the groups compared showing 163 genes unique to <i>BRCA2</i>mt tumors. <b>C,</b> Venn diagram of differentially regulated pathways in each of the groups compared showing six pathways unique to <i>BRCA2</i>mt tumors compared with <i>BRCA1</i>mt and HRwt; these six pathways are shown in <b>D</b>.</p>
<p>Wnt signaling is predicted to be inhibited in <i>BRCA2</i>mt ovarian tumors. <b>A,</b> DEGs comparing <i>BRCA2</i>mt vs. <i>BRCA1</i>mt and (<b>B</b>) <i>BRCA2</i>mt vs. HRwt reported as logFC; red bars represent upregulated, and blue bars represent downregulated; note that several Wnt inhibitors are upregulated in <i>BRCA2</i>mt tumors (red asterisk). <b>C,</b> Perturbation analysis of the Wnt signaling pathway comparing <i>BRCA2</i>mt and <i>BRCA1</i>mt tumors; red genes are predicted to be upregulated, and blue genes are predicted to be downregulated; the dashed circle shows β-catenin is predicted to be downregulated. Red arrows indicate upregulation of several Wnt signaling inhibitors at the level of the receptor.</p>
Current immunotherapies have proven effective in strengthening anti-tumor immune responses but constant opposing signals from tumor cells and surrounding microenvironment eventually lead to immune escape. We hypothesize that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system will provide a robust and long-term anti-tumor effect by creating immunological memory against the tumor. To achieve this, we developed CARG-2020, a virus-like-vesicle (VLV). It is a genetically modified and self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three transgenes: 1 ) the pleiotropic antitumor cytokine IL-12 in which the subunits (p35 and p40) are tethered together; 2) the extracellular domain (ECD) of the pro- tumor IL-17RA, which can serve as a dominant negative antagonist; and 3) shRNA for PD-L1. Using a mouse model of ovarian cancer, we demonstrate the oncolytic effect and immune modulatory capacities of CARG-2020. By enhancing IL-12 and blocking IL-17 and PD-L1, CARG-2020 successfully reactivates immune surveillance by promoting M1 instead of M2 macrophage differentiation, inhibiting MDSC expansion, and establishing a potent CD8+ T cell mediated anti-tumoral response. Furthermore, we demonstrate that this therapeutic approach provides tumor-specific and long-term protection preventing the establishment of new tumors. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance the anti-tumor response and to prevent recurrence.
Abstract Purpose: Early detection would significantly decrease the mortality rate of ovarian cancer. In this study, we characterize and validate the combination of six serum biomarkers that discriminate between disease-free and ovarian cancer patients with high efficiency. Experimental Design: We analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All six markers were evaluated in a training set (181 samples from the control group and 113 samples from OC patients) and a test set (181 sample control group and 43 ovarian cancer). Results: Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves were good enough to differentiate healthy versus cancer cells. However, the combination of the six markers provided a better differentiation than CA-125. Four models with <2% classification error in training sets all had significant improvement (sensitivity 84%-98% at specificity 95%) over CA-125 (sensitivity 72% at specificity 95%) in the test set. The chosen model correctly classified 221 out of 224 specimens in the test set, with a classification accuracy of 98.7%. Conclusions: We describe the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. This novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer.
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