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Supplementary Figures S1-S4 from Accelerated Preclinical Testing Using Transplanted Tumors from Genetically Engineered Mouse Breast Cancer Models

Supplementary Table S1 from Accelerated Preclinical Testing Using Transplanted Tumors from Genetically Engineered Mouse Breast Cancer Models

Supplementary Figure Legend from Accelerated Preclinical Testing Using Transplanted Tumors from Genetically Engineered Mouse Breast Cancer Models

Purpose: The use of genetically engineered mouse (GEM) models for preclinical testing of anticancer therapies is hampered by variable tumor latency, incomplete penetrance, and complicated breeding schemes. Here, we describe and validate a transplantation strategy that circumvents some of these difficulties.Experimental Design: Tumor fragments from tumor-bearing MMTV-PyMT or cell suspensions from MMTV-PyMT, -Her2/neu, -wnt1, -wnt1/p53+/−, BRCA1/p53+/−, and C3(1)T-Ag mice were transplanted into the mammary fat pad or s.c. into naïve syngeneic or immunosuppressed mice. Tumor development was monitored and tissues were processed for histopathology and gene expression profiling. Metastasis was scored 60 days after the removal of transplanted tumors.Results: PyMT tumor fragments and cell suspensions from anterior glands grew faster than posterior tumors in serial passages regardless of the site of implantation. Microarray analysis revealed genetic differences between these tumors. The transplantation was reproducible using anterior tumors from multiple GEM, and tumor growth rate correlated with the number of transplanted cells. Similar morphologic appearances were observed in original and transplanted tumors. Metastasis developed in >90% of mice transplanted with PyMT, 40% with BRCA1/p53+/− and wnt1/p53+/−, and 15% with Her2/neu tumors. Expansion of PyMT and wnt1 tumors by serial transplantation for two passages did not lead to significant changes in gene expression. PyMT-transplanted tumors and anterior tumors of transgenic mice showed similar sensitivities to cyclophosphamide and paclitaxel.Conclusions: Transplantation of GEM tumors can provide a large cohort of mice bearing mammary tumors at the same stage of tumor development and with defined frequency of metastasis in a well-characterized molecular and genetic background.

Strenuous exercise increases gastrointestinal damage, but the dose–response relationship is yet to be elucidated. It is also commonly believed that running causes greater gastrointestinal damage than cycling. Two randomised, crossover studies aimed to 1) quantify gastrointestinal damage with increasing exercise intensity, and 2) determine if running was associated with greater gastrointestinal damage than cycling. Following a maximal oxygen uptake (V̇O2max) test, participants completed 3 cycling trials at different intensities (60 min at 40%, 60% and 80% V̇O2max; n = 10 (5 female, 5 male)) (INTENSITY), or 1 running and 1 cycling trial (45 min at 70% V̇O2max; n = 11 (3 female, 8 male)) (MODE). Venous blood samples were collected pre- and post-exercise to measure gastrointestinal damage via intestinal fatty acid binding protein (I-FABP). In INTENSITY, I-FABP magnitude of change was greater at 80% V̇O2max than 40% V̇O2max (p < 0.01). In MODE, I-FABP magnitude of change was greater with cycling (mean (SD)) (84.7 (133.2)% d = 1.07) compared with running (19.3 (33.1)%, d = 0.65) with a moderate effect (d = 0.68, p = 0.024). Rating of perceived exertion (RPE) and heart rate (HR) were higher during cycling (RPE p < 0.0001; HR p < 0.0001) but rectal temperature was not different between modes (p = 0.94). While gastrointestinal damage increases with increasing exercise intensity, running was not associated with greater gastrointestinal damage than cycling. Novelty: A fraction of the anaerobic threshold, rather than a fraction of V̇O2max, may be more predictive of intensity that results in exercise induced gastrointestinal damage. The mode of exercise may not be as important as intensity for inducing gastrointestinal damage. Improving anaerobic threshold may reduce susceptibility to gastrointestinal damage when exercising at high intensities.

Background The emergence of the novel coronavirus, SARS-CoV-2, created a crucial need for accurate tests for diagnosis, assessment of prior infection, and understanding its natural history. Serology assays play an important role in the assessment of anti-viral immune responses and previous infections. Evaluation of serology assays with well-characterized serum and/or plasma samples is critical to determine assay performance. CDC, FDA and NCI’s Frederick National Laboratory for Cancer Research (NCI-FNLCR) have established a collaborative network to independently evaluate commercial antibody tests prior to their authorization. Methods Positive (n=30) serum samples with a range of anti-SARS-CoV-2 antibody titers (Table) and negative (n=80) serum and/or plasma samples were selected to establish performance evaluation panels (PEVs). Three PEVs with similar overall antibody titer distribution have been created. Negative samples were collected prior to 2020, before the SARS-CoV-2 pandemic. Positive samples were from patients previously confirmed to have SARS-CoV-2 using a nucleic acid amplification test. Each sample was characterized at CDC and NCI-FNLCR for presence/absence of SARS-CoV-2 IgM and IgG antibodies using a SARS-CoV-2 spike enzyme linked immunosorbent assay (ELISA). NCI-FNLCR also performed a SARS-CoV-2 spike Receptor Binding Domain (RBD) IgG ELISA. Positive samples were assessed at multiple dilutions. Manufacturers submitted their serology assays for evaluation by this program. The sensitivity of each test was assessed for each antibody class (IgG and IgM) and in a combined manner, where a positive result for either antibody was considered as a positive result. For combined specificity, a negative result meant a sample was negative for both antibodies (IgG and IgM). Number of positive samples with anti-SARS-CoV-2 spike antibodies for each panel (n=30) Results To date, 53 serology assays have been evaluated. Sensitivity ranged from 30.0% to 100% for IgG, from 10.0% to 100% for IgM, and the combined specificity ranged from 57.5% to 100%. For 2 assays that measure total Ig, sensitivity was 96.7% and 100%. Conclusion This program completed over 50 performance evaluations with well-characterized PEVs. Results have been used to inform FDA regulatory decisions and are publicly available on FDA’s website. Disclosures Cristina Cassetti, PhD, Nothing to disclose

We develop a novel synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and encoded metabolites., For decades, fungi have been a source of U.S. Food and Drug Administration–approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

SUMMARYEmbryogenesis requires epigenetic information that allows each cell to respond appropriately to developmental cues. Histone modifications are core components of a cell’s epigenome, giving rise to chromatin states that modulate genome function. Here, we systematically profile histone modifications in a diverse panel of mouse tissues at 8 developmental stages from 10.5 days post conception until birth, performing a total of 1,128 ChIP-seq assays across 72 distinct tissue-stages. We combine these histone modification profiles into a unified set of chromatin state annotations, and track their activity across developmental time and space. Through integrative analysis we identify dynamic enhancers, reveal key transcriptional regulators, and characterize the role of chromatin-based repression in developmental gene regulation. We also leverage these data to link enhancers to putative target genes, revealing connections between coding and non-coding sequence variation in disease etiology. Our study provides a compendium of resources for biomedical researchers, and achieves the most comprehensive view of embryonic chromatin states to date.

TICs are characterized by their ability to self-renew, differentiate and initiate tumor formation. miRNAs are small noncoding RNAs that bind to mRNAs resulting in regulation of gene expression and biological functions. The role of miRNAs and TICs in cancer progression led us to hypothesize that miRNAs may regulate genes involved in TIC maintenance. Using whole genome miRNA and mRNA expression profiling of TICs from primary prostate cancer cells, we identified a set of up-regulated miRNAs and a set of genes down-regulated in PSs. Inhibition of these miRNAs results in a decrease of prostatosphere formation and an increase in target gene expression. This study uses genome-wide miRNA profiling to analyze expression in TICs. We connect aberrant miRNA expression and deregulated gene expression in TICs. These findings can contribute to a better understanding of the molecular mechanisms governing TIC development/maintenance and the role that miRNAs have in the fundamental biology of TICs.

Our previous studies have identified mechanisms by which cytokine production, blocked by Ly49G2 receptor cross-linking, can be overridden. In this study we analyzed the regulation of other ITAM-positive receptor signaling on NK, NKT, and T cells and characterized the biochemical pathways involved in this signaling. Our studies demonstrate that cross-linking of NKG2D and NK1.1 results in a synergistic NK IFN-γ response when combined with IL-12 or IL-18. Examination of NKT- and T-cell responses demonstrated that cross-linking of NKG2D and CD3 resulted in potent synergy when combined with IL-12 and, to a lesser degree, with IL-18. We have now found that both the p38 MAP kinase and the ERK-dependent signal transduction pathways are required for the synergistic response. Further mechanistic examination of the synergy indicated a potent up-regulation of total IFN-γ mRNA in the nuclear and the cytoplasmic compartment, but mRNA half-life was not affected. Fifteen minutes of IL-12 pretreatment was sufficient to result in maximal synergistic activation, indicating that the response of the cells to the IL-12 signal was rapid and immediate. Thus, our data demonstrate that multiple convergent signals maximize the innate immune response by triggering complementary biochemical signaling pathways.

PCR-based fluorescent detection assays for the relative and quantitative measurement of gene expression, such as Taq Man™, LUX™, and SYBR Green™, are currently in wide spread use due to their general applicability, low cost, reproducibility, accuracy, and ease of use. One current limitation of quantitative PCR (Q-PCR) is the lack of a fully integrated and high-throughput method for general genomic and diagnostic applications. Here we report a reliable and high-throughput system for the automated extraction of RNA, first-strand cDNA synthesis, quality control measures, consecutive real-time PCR amplification, and primary data analysis. As described, this procedure utilizes commonly available reagents and pre-packaged “kits” for RNA extraction, first strand cDNA synthesis, Q-PCR, liquid handling, and capillary electrophoresis that are generally applicable to a wide variety of robotic platforms. (JALA 2004;9:128-34)

A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY), and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs)/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found). A large number of focal copy number alterations (FCNAs) were detected, including homozygous deletions (HDs) targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements). Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.

Background: Polycyclic aromatic hydrocarbon (PAH) exposure is a risk factor for esophageal squamous cell carcinoma, and PAHs are ligands of the aryl hydrocarbon receptor (AhR). This study measured the expression of AhR and related genes in frozen esophageal cell samples from patients exposed to different levels of indoor air pollution, who did or did not have high-grade squamous dysplasia and who did or did not have a family history of upper gastrointestinal tract (UGI) cancer. Methods: 147 samples were evaluated, including 23 (16%) from patients with high-grade dysplasia and 48 (33%) from patients without dysplasia who heated their homes with coal, without a chimney (a “high” indoor air pollution group), and 27 (18%) from patients with high-grade dysplasia and 49 (33%) from patients without dysplasia who did not heat their homes at all (a “low” indoor air pollution group). Sixty-four (44%) had a family history of UGI cancer. RNA was extracted and quantitative PCR analysis was done. Results: AhR gene expression was detectable in 85 (58%) of the samples and was >9-fold higher in those with a family history of UGI cancer [median expression (interquartile range), −1,964 (−18,000, −610) versus −18,000 (−18,000, −1036); P = 0.02, Wilcoxon rank-sum test]. Heating status, dysplasia category, age, gender, and smoking were not associated with AhR expression (linear regression; all P values ≥ 0.1). Conclusion: AhR expression was higher in patients with a family history of UGI cancer. Such individuals may be more susceptible to the deleterious effects of PAH exposure, including PAH-induced cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(9):2391–6)

Innate immune responses provide the host with its first line of defense against infections. Signals generated by subsets of lymphocytes, including NK cells, NKT cells, and APC during this early host response determine the nature of downstream adaptive immune responses. In the present study, we have examined the role of innate NK cells in an autoimmune model through the use of primary immunization with the myelin oligodendrocyte glycoprotein peptide to induce experimental autoimmune encephalomyelitis (EAE). Our studies have shown that in vivo depletion of NK cells can affect the adaptive immune responses, because NK cells were found to regulate the degree of clinical paralysis and to alter immune adaptive responses to the myelin oligodendrocyte glycoprotein peptide. The requirement for NK cells was reflected by changes in the T cell responses and diminished clinical disease seen in mice treated with anti-NK1.1, anti-asialo GM1, and selected Ly49 subtype-depleted mice. In addition to alteration in T cell responses, the maturational status of dendritic cells in lymph nodes was altered both quantitatively and qualitatively. Finally, examination of TCR Vβ usage of the brain lymphocytes from EAE mice indicated a spectra-type change in receptor expression in NK- depleted mice as compared with non-NK-depleted EAE mice. These findings further establish a recently postulated link between NK cells and the generation of autoreactive T cells.

Purpose: The use of genetically engineered mouse (GEM) models for preclinical testing of anticancer therapies is hampered by variable tumor latency, incomplete penetrance, and complicated breeding schemes. Here, we describe and validate a transplantation strategy that circumvents some of these difficulties.Experimental Design: Tumor fragments from tumor-bearing MMTV-PyMT or cell suspensions from MMTV-PyMT, -Her2/neu, -wnt1, -wnt1/p53+/−, BRCA1/p53+/−, and C3(1)T-Ag mice were transplanted into the mammary fat pad or s.c. into naïve syngeneic or immunosuppressed mice. Tumor development was monitored and tissues were processed for histopathology and gene expression profiling. Metastasis was scored 60 days after the removal of transplanted tumors.Results: PyMT tumor fragments and cell suspensions from anterior glands grew faster than posterior tumors in serial passages regardless of the site of implantation. Microarray analysis revealed genetic differences between these tumors. The transplantation was reproducible using anterior tumors from multiple GEM, and tumor growth rate correlated with the number of transplanted cells. Similar morphologic appearances were observed in original and transplanted tumors. Metastasis developed in >90% of mice transplanted with PyMT, 40% with BRCA1/p53+/− and wnt1/p53+/−, and 15% with Her2/neu tumors. Expansion of PyMT and wnt1 tumors by serial transplantation for two passages did not lead to significant changes in gene expression. PyMT-transplanted tumors and anterior tumors of transgenic mice showed similar sensitivities to cyclophosphamide and paclitaxel.Conclusions: Transplantation of GEM tumors can provide a large cohort of mice bearing mammary tumors at the same stage of tumor development and with defined frequency of metastasis in a well-characterized molecular and genetic background.

Mucosal tolerance to E-selectin has been shown to prevent stroke and reduce brain infarcts in experimental stroke models. However, the effective E-selectin dose range required to achieve mucosal tolerance and the precise mechanisms of neuroprotection remain unclear. We sought to examine the mechanisms of cytoprotection using gene expression profiling of tissues in the setting of mucosal tolerance and inflammatory challenge. Using spontaneously hypertensive rats (SHRs), we achieved immune tolerance with 0.1 to 5 μg E-selectin per nasal instillation and observed a dose-related anti-E-selectin immunoglobulin G antibody production. We also show the distinct patterns of gene expression changes in the brain and spleen with the different tolerizing doses and lipopolysaccharide (LPS) exposure. Prominent differences were seen with such genes as insulin-like growth factors in the brain and downregulation of those encoding the major histocompatibility complex class I molecules in the spleen. In all, mucosal tolerance to E-selectin and subsequent exposure to LPS resulted in significant tissue changes. These changes, while giving an insight to the underlying mechanisms, serve as possible targets for future studies to facilitate translation to human clinical trials.

Ovarian cancer is an aggressive disease of poor prognostic when detected at advanced stage. It is widely accepted that the ovarian surface epithelium plays a central role in disease etiology, but little is known about disease progression at the molecular level. To identify genes involved in ovarian tumorigenesis, we carried out a genome-wide transcriptomic analysis of six spontaneously transformed mouse ovarian surface epithelial (MOSE) cell lines, an in vitro model for human ovarian carcinoma. Loess normalization followed by statistical analysis with control of multiple testing resulted in 509 differentially expressed genes using an adjusted P-value < or = 0.05 as cut-off. The top 20 differentially expressed genes included 10 genes (Spp1, Cyp1b1, Btg1, Cfh, Mt1, Mt2, Igfbp5, Gstm1, Gstm2, and Esr1) implicated in various aspects of ovarian carcinomas, and other 3 genes (Gsto1, Lcn7, and Alcam) associated to breast cancer. Upon functional analysis, the majority of alterations affected genes involved in glutathione metabolism and MAPK signaling pathways. Interestingly, over 20% of the aberrantly expressed genes were related to extracellular components, suggestive of potential markers of disease progression. In addition, we identified the genes Pura, Cnn3, Arpc1b, Map4k4, Tgfb1i4, and Crsp2 correlated to in vivo tumorigenic parameters previously reported for these cells. Taken together, our findings support the utility of MOSE cells in studying ovarian cancer biology and as a source of novel diagnostic and therapeutic targets.

Patents directed to naturally occurring genetic material, such as DNA, RNA, chromosomes, and genes, in an isolated or purified form have been granted in Australia for many years. This review provides scientists with a summary of the gene patent debate from an Australian perspective and specifically reviews how the various levels of the legal system as they apply to patents—the Australian Patent Office, Australian courts, and Australian government—have dealt with the issue of whether genetic material is proper subject matter for a patent.

We have established and characterized a new ovarian cancer cell line derived from stage IV serous ovarian adenocarcinoma metastatic tissue from a 47 year old Ashkenazi Jewish patient. The patient developed ovarian cancer at age 44 and died of the disease at age 48. The tissue was obtained after an extensive secondary cytoreductive intraperitoneal surgery. Prior to the surgery the patient underwent primary cytoreduction followed by several systemic therapies with recurrences. A culture of the metastatic tissue was transfected with Simian Virus 40 Large T-Antigen and telomerase. The cell line has been in culture on and off during 3 years. The identity of the cell line was confirmed by Short Tandem Repeat genotyping on genomic DNA extracted from early and late passages as well as from frozen metastatic tissue. The cell line was further characterized by array-Comparative Genomic Hybridization and gene expression microarray; and by Spectral Karyotyping and Fluorescent In Situ Hybridization. The results of these analyses were compared, when appropriate, with those of frozen metastatic tissue. A low level of genetic instability was detected in the cell line, but the karyotypes seem to be near diploid and relatively stable, after several years of cultivation. We believe that this novel ovarian cancer cell line could contribute to the understanding of the molecular genetic basis of the disease in this advanced stage, and be representative of the tumors of a great number of patients that become resistant to the majority if not all known cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3265.

Background The link between reproductive life history and incidence of ovarian tumors is well known. Periods of reduced ovulations may confer protection against ovarian cancer. Using phenotypic data available for mouse, a possible association between the ovarian transcriptome, reproductive records and spontaneous ovarian tumor rates was investigated in four mouse inbred strains. NIA15k-DNA microarrays were employed to obtain expression profiles of BalbC, C57BL6, FVB and SWR adult ovaries. Results Linear regression analysis with multiple-test control (adjusted p ≤ 0.05) resulted in ovarian tumor frequency (OTF) and number of litters (NL) as the top-correlated among five tested phenotypes. Moreover, nearly one-hundred genes were coincident between these two traits and were decomposed in 76 OTF(–) NL(+) and 20 OTF(+) NL(–) genes, where the plus/minus signs indicate the direction of correlation. Enriched functional categories were RNA-binding/mRNA-processing and protein folding in the OTF(–) NL(+) and the OTF(+) NL(–) subsets, respectively. In contrast, no associations were detected between OTF and litter size (LS), the latter a measure of ovulation events in a single estrous cycle. Conclusion Literature text-mining pointed to post-transcriptional control of ovarian processes including oocyte maturation, folliculogenesis and angiogenesis as possible causal relationships of observed tumor and reproductive phenotypes. We speculate that repetitive cycling instead of repetitive ovulations represent the actual link between ovarian tumorigenesis and reproductive records.