Nichole Peterson

Placental growth factor deficiency is associated with impaired cerebral vascular development in mice

STUDY HYPOTHESIS Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning. STUDY FINDING PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development. WHAT IS KNOWN ALREADY PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf  (-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf  (-/-) mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf  (-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf  (-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay. MAIN RESULTS AND THE ROLE OF CHANCE Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgf  (-/-) brains. At E14.5, Pgf  (-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgf  (-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not. LIMITATIONS, REASONS FOR CAUTION Since Pgf  (-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring. WIDER IMPLICATIONS OF THE FINDINGS These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments. LARGE SCALE DATA N/A. STUDY FUNDING AND COMPETING INTERESTS This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queens University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests.

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator‐activated receptor (PPAR)γ(+/−) mice, we showed normal expression of PPARγ was critical to stop 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE‐specific loss of PPARγ was hypothesized to enhance DMBA‐mediated breast tumorigenesis. To test this, MSE cell‐specific PPARγ knockout (PPARγ‐MSE KO) and control (PPARγ‐WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ‐WT, n = 15; PPARγ‐MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ‐WT, n = 17; PPARγ‐MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ‐MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ‐WT controls. PPARγ activation significantly reduced DMBA‐mediated malignant mammary tumor volumes irrespective of genotype. MSE‐specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down‐regulating Cox‐1, Cox‐2 and cyclin D1. Collectively, these studies highlight a protective role of MSE‐specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.

Stromal Adipocyte PPARγ Protects Against Breast Tumorigenesis

Peroxisome proliferator-activated receptor (PPAR)γ regulates the expression of genes essential for fat storage, primarily through its activity in adipocytes. It also has a role in carcinogenesis. PPARγ normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumours as revealed with PPARγ haploinsufficient mice. Since many cell types associated with the mammary gland express PPARγ, each with unique signal patterns, this study aimed to define which tissues are required for PPARγ-dependent antitumour effects. Accordingly, adipocyte-specific PPARγ knockout (PPARγ-A KO) mice and their wild-type (PPARγ-WT) controls were generated, and treated with DMBA for 6 weeks to initiate breast tumorigenesis. On week 7, mice were randomized to continue on normal chow diet or one supplemented with rosiglitazone (ROSI), and followed for 25 weeks for tumour outcomes. In PPARγ-A KO versus PPARγ-WT mice, malignant mammary tumour incidence was significantly higher and mammary tumour latency was decreased. DMBA + ROSI treatment reduced average mammary tumour volumes by 50%. Gene expression analyses of mammary glands by quantitative real-time polymerase chain reaction and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared with PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. Together, these data are the first to suggest that in vivo PPARγ expression in mammary stromal adipocytes attenuates breast tumorigenesis through BRCA1 upregulation and decreased leptin secretion. This study supports a protective effect of activating PPARγ as a novel chemopreventive therapy for breast cancer.

STAT1‐associated intratumoural TH1 immunity predicts chemotherapy resistance in high‐grade serous ovarian cancer

High‐grade serous ovarian carcinoma (HGSC) accounts for 70% of all epithelial ovarian cancers but clinical management is challenged by a lack of accurate prognostic and predictive biomarkers of chemotherapy response. This study evaluated the role of Signal Transducer and Activator of Transcription 1 (STAT1) as an independent prognostic and predictive biomarker and its correlation with intratumoural CD8+ T cells in a second independent biomarker validation study. Tumour STAT1 expression and intratumoural CD8+ T cell infiltration were assessed by immunohistochemistry as a multicentre validation study conducted on 734 chemotherapy‐naïve HGSCs. NanoString‐based profiling was performed to correlate expression of STAT1 target genes CXCL9, CXCL10 and CXCL11 with CD8A transcript expression in 143 primary tumours. Multiplexed cytokine analysis of pre‐treatment plasma from resistant and sensitive patients was performed to assess systemic levels of STAT1‐induced cytokines. STAT1 was validated as a prognostic and predictive biomarker in both univariate and multivariate models and its expression correlated significantly with intra‐epithelial CD8+ T cell infiltration in HGSC. STAT1 levels increased the prognostic and predictive value of intratumoural CD8+ T cells, confirming their synergistic role as biomarkers in HGSC. In addition, expression of STAT1 target genes (CXCL9, CXCL10 and CXCL11) correlated significantly with levels of, and CD8A transcripts from intratumoural CD8+ T cells within the resistant and sensitive tumours. Our findings provide compelling evidence that high levels of STAT1, STAT1‐induced chemokines and CD8+ T cells correlate with improved chemotherapy response in HGSC. These results identify STAT1 and its target genes as novel biomarkers of chemosensitivity in HGSC. These findings provide new translational opportunities for patient stratification for immunotherapies based on emerging biomarkers of inflammation in HGSC. An improved understanding of the role of interferon‐inducible genes will be foundational for developing immunomodulatory therapies in ovarian cancer.

CXCL10 alters the tumour immune microenvironment and disease progression in a syngeneic murine model of high-grade serous ovarian cancer

OBJECTIVE We recently established that high STAT1 expression and associated T helper type I tumour immune microenvironment (TME) are prognostic and chemotherapy response predictive biomarkers in high-grade serous ovarian cancer (HGSC). STAT1 induced chemokine CXCL10 is key to the recruitment of lymphocytes in the TME and is significantly highly expressed in the tumours from patients with longer survival. In the current study we therefore aimed to elucidate the role CXCL10 in disease progression and tumour immune transcriptomic alterations using the ID8 syngeneic murine model of HGSC. METHODS ID8 ovarian cancer cells were engineered for stable knockdown (KD) and overexpression (OX) of CXCL10. The OX and KD cell line derivatives, along with their respective vector controls, were implanted in immunocompetent C57BL/6 mice via intra-peritoneal injections. At end point, immune transcriptomic profiling of tumour tissues and multiplex cytokine profiling of ascites, was performed. Effect of CXCL10 expression on the tumour vasculature and tumour cell proliferation was evaluated by CD31 and Ki67 immunostaining, respectively. RESULTS Increased CXCL10 expression led to decreased tumour burden and malignant ascites accumulation in the ID8 syngeneic murine model of HGSC. The ascites levels of IL-6 and VEGF were significantly reduced in OX mice compared to the vector controls. The OX tumours also showed reduced vasculature (CD31) and proliferative index (Ki67) compared to the control tumours. Significantly higher expression of genes associated with antigen processing, apoptosis and T cell function was observed in OX tumours compared to the controls. Reduced CXCL10 expression in tumours from KD mice led to increased ascites accumulation and disease progression compared to the controls. CONCLUSION CXCL10 is a positive determinant of anti-tumour immune responses in HGSC TME and disease progression. These findings are foundational for future translational studies aimed at improving treatment response and survival in HGSC patients, via exploiting the TME.

Maternal hypertension programs increased cerebral tissue damage following stroke in adult offspring.

The maternal system is challenged with many physiological changes throughout pregnancy to prepare the body to meet the metabolic needs of the fetus and for delivery. Many pregnancies, however, are faced with pathological stressors or complications that significantly impact maternal health. A shift in this paradigm is now beginning to investigate the implication of pregnancy complications on the fetus and their continued influence on offspring disease risk into adulthood. In this investigation, we sought to determine whether maternal hypertension during pregnancy alters the cerebral response of adult offspring to acute ischemic stroke. Atrial natriuretic peptide gene-disrupted (ANP−/−) mothers exhibit chronic hypertension that escalates during pregnancy. Through comparison of heterozygote offspring born from either normotensive (ANP+/−WT) or hypertensive (ANP+/−KO) mothers, we have demonstrated that offspring exposed to maternal hypertension exhibit larger cerebral infarct volumes following middle cerebral artery occlusion. Observation of equal baseline cardiovascular measures, cerebrovascular structure, and cerebral blood volumes between heterozygote offspring suggests no added influences on offspring that would contribute to adverse cerebral response post-stroke. Cerebral mRNA expression of endothelin and nitric oxide synthase vasoactive systems demonstrated up-regulation of Et-1 and Nos3 in ANP+/−KO mice and thus an enhanced acute vascular response compared to ANP+/−WT counterparts. Gene expression of Na+/K+ ATPase channel isoforms, Atp1a1, Atp1a3, and Atp1b1, displayed no significant differences. These investigations are the first to demonstrate a fetal programming effect between maternal hypertension and adult offspring stroke outcome. Further mechanistic studies are required to complement epidemiological evidence of this phenomenon in the literature.

STING agonist therapy in combination with PD-1 immune checkpoint blockade enhances response to carboplatin chemotherapy in high-grade serous ovarian cancer

BackgroundHigh-grade serous carcinoma (HGSC) of the ovary is predominantly diagnosed at late stages and primarily treated with debulking surgery followed by platinum/taxane-based chemotherapy. Although certain patients benefit significantly from currently used chemotherapy, there are patients who either do not respond or have an inadequate duration of response. We previously showed that tumours from chemoresistant patients have an immunosuppressed pre-existing tumour immune microenvironment with decreased expression of Type I Interferon (IFN1) genes.MethodsEfficacy of a ‘STimulator of INterferon Genes’ agonist was evaluated in combination with carboplatin chemotherapy and PD-1 immune checkpoint blockade therapy in the ID8-Trp53−/− immunocompetent murine model of HGSC.ResultsTreatment with STING agonist led to decreased ascites accumulation and decreased tumour burden. Survival of mice treated with a combination of carboplatin, STING agonist and anti-PD-1 antibody was the longest. Tumour immune transcriptomic profiling revealed higher IFN response, antigen presentation and MHC II genes in tumours from STING agonist-treated mice compared to vehicle controls. Flow cytometry analysis revealed significantly higher intra-tumoural PD-1+ and CD69+CD62L−, CD8+ T cells in STING agonist-treated mice.ConclusionsThese findings will enable rational design of clinical trials aimed at combinatorial approaches to improve chemotherapy response and survival in HGSC patients.

Abstract TMEM-036: INTERFERON INDUCED STAT1 ASSOCIATES WITH DIFFERENTIAL CHEMOTHERAPY RESPONSE IN HIGH-GRADE SEROUS OVARIAN CANCER

PURPOSE: High-grade serous ovarian cancer (HGSC) is the most prevalent and fatal histological subtype of ovarian cancer. Unfortunately, 70% of HGSC patients show resistance to chemotherapeutic drugs, and clinical management is challenged by a lack of accurate prognostic and predictive biomarkers of chemotherapy response. It is now established that immune cells within the tumour microenvironment (TME) significantly contribute to tumor cell death or survival following exposure to chemotherapy. We previously reported the presence of a distinct, pre-existing T-helper Type I associated immune TME, mediated by Signal Transducer and Activator of Transcription Factor 1 (STAT1), that predicted chemotherapy response in HGSC. Current work builds on our previous findings and investigates the mechanisms linking STAT1 to a pre-existing inflammatory TME and differential response to chemotherapy. EXPERIMENTAL PROCEDURES: Tumour STAT1 expression and intra-epithelial CD8+ T-cells were confirmed as prognostic and predictive biomarkers in a second independent biomarker validation study. Immunohistochemistry and a custom NanoString platform, composed of 34-target genes of STAT1 and immune phenotypic markers, were used on fresh frozen chemo-naive HGSC tumour samples. Based on the findings from these profiling studies, a subset of samples with high and low STAT1 expression levels were subjected to further gene profiling analysis to correlate the immune transcriptomic landscape and its association with chemotherapy response. Stable silencing of the STAT1 gene was performed by lentiviral transduction of murine ovarian cancer cells (ID8) with STAT1 short hairpin RNA (shRNA) to create STAT1-knockdown (KD) cells and STAT1-non targeting (NT) control cells. Ongoing in vivo experiments will define the contribution of STAT1 in modulating; the TME, tumor progression, the recruitment of tumor infiltrating lymphocytes, and response to chemotherapy in HGSC. RESULTS: Gene expression analysis revealed significant differential expression of genes involved in the cellular Type I Interferon pathway, notably STAT1 and its target genes (such as ISG15, DDX58, IFIT1, CXCL10, CXCL11), between chemosensitive and resistant groups. Preliminary in vitro analysis has revealed proliferative and migratory advantages to STAT1-KD cells compared to the STAT1-NT cells; indicating that STAT1 plays a role in the regulation of cell survival and proliferation in the absence of the immune TME. CONCLUSION: STAT1 expression significantly associates with progression free survival and response to chemotherapy in HGSC. High levels of STAT1 and its target genes potentially contribute to CD8+ T-cell recruitment and immune mediated chemosensitivity in HGSC. Ongoing in vitro and in vivo studies will confirm the mechanisms underlying variations in the interferon induced STAT1 pathways in the TME of HGSC. Elucidating the mechanisms underlying differential STAT1 expression in HGSC primary tumours will contribute to better patient stratification for informed biomarker guided immunotherapies. Citation Format: Gillian Reid-Schachter, Peter Truesdell, Nichole Peterson, Charles Graham, Julie-Ann Francis, Andrew Craig, Madhuri Koti. INTERFERON INDUCED STAT1 ASSOCIATES WITH DIFFERENTIAL CHEMOTHERAPY RESPONSE IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-036.