Human Ovarian Tumor Research Articles

Endocannabinoid system expression in ovarian epithelial tumors according to the dualistic model of ovarian carcinogenesis.

Our study aimed to confirm the expression of the endocannabinoid system in the human epithelial ovarian tumors, assessing the immunohistochemical expression of Cannabinoid Receptor Type 1 and Fatty Acid Amide Hydrolase in benign, borderline and malignant tumors. Cannabinoid Receptor Type 1 and Fatty Acid Amide Hydrolase immunohistochemical expression was determined in 118 epithelial ovarian tumors sequentially treated during the last decade in our department: 36 benign, 34 borderline and 48 malignant neoplasms. Cannabinoid Receptor type 1 and Fatty Acid Amide Hydrolase expression resulted predominantly weak-moderate in the benign and borderline forms. concerning malignant tumors, Cannabinoid Receptor Type 1 expression resulted predominantly moderate-strong in Type I tumors and negative-weak in Type II tumors. Fatty Acid Amide Hydrolase expression resulted, instead, independent by the tumor types. Furthermore, there was no significant difference in the Cannabinoid Receptor Type 1 and Fatty Acid Amide Hydrolase expression relatively to the tumoral stages. The present study confirmed a variable expression of the endocannabinoid system in human ovarian tumors. Cannabinoid Receptor Type 1 expression was significantly different in malignant epithelial ovarian tumors according to dualistic model of ovarian carcinogenesis. Thus, in the most aggressive types II ovarian tumors, Cannabinoid Receptor Type 1 expression resulted predominantly negative or weak.

Abstract 325: Nascent proteome analysis of tumor cells and their microenvironment in cultured human tumor tissues

Abstract Solid tumors are often considered as abnormal organs composed of the cancerous cells and their surrounding tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. The heterotypic interactions between this diversity of cell types within the TME are maintained through a wide variety of secreted proteins, resulting in a favorable milieu for the progression of the malignancy. The interactions between tumor cells and TME are complex and remain poorly understood. Here we investigated this by developing a unique nascent proteomic approach in tumor tissues.Precision cut cancer tissue slices (PCCTS) maintain tissue heterogeneity with different cell types and preserved TME. Cultivation of PCCTS provides an ex vivo model for tumor tissues. We developed an approach for PCCTS's nascent proteome analysis, using pulsed-SILAC (stable isotope labeling with amino acids in cell culture) labeling combined with click-chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon applying a cellular perturbation. It is a powerful tool to selectively enrich secretory proteins from culture media even with presence of serum. Primary human ovarian tumors (phOVT) and patient derived xenografts (PDX) were used to produce the PCCTS with thickness of 150µm to 300µm. The different cell types and extracellular matrix of PCCTS make the depletion period of cells from the amino acids (methionine, lysine and arginine) prior the AHA-SILAC treatment difficult to define. The PCCTS need longer depletion periods than the 2D cell culture, the longer the depletion period the better depletion efficiency. Following, PCCTS were cultured in AHA-SILAC media and treated with cisplatin. PCCTS and culture media (containing secreted proteins) were harvested; newly synthesized proteins were enriched via click-chemistry and analyzed with mass spectrometry. The labeling time of 10 to 12h showed a good labeling efficiency of more than 60%, still further optimizations are needed. Different PCCTS showed various labeling efficiency indicating the patients heterogeneity. The nascent proteome analysis with cisplatin treatment demonstrated different protein regulations in patients suggesting different drug responses. STRING analysis can be applied to predict the protein-protein interactions. The immunohistochemical staining of the same PCCTS can be processed to further validate the results of the proteome analysis. In conclusion, we established a pulsed SILAC-AHA treatment approach for the PCCTS with the TME. This unique approach allows tracking the compositional and dynamic changes within the proteome and monitoring the direct proteome response at rapid time scale. It can be used to reveal a part of the proteome that has been poorly understood in the tumor tissues and contribute to studying cellular communication and finding new therapeutic targets. Citation Format: Meng Dong, Karim Aljakouch, Kathrin Böpple, Bernd Winkler, Julia Schüler, Frank Essmann, Hans-Georg Kopp, Jeroen Krijgsveld, Walter E. Aulitzky. Nascent proteome analysis of tumor cells and their microenvironment in cultured human tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 325.

Characterization of a novel mutation V136L in bone morphogenetic protein 15 identified in a woman affected by POI

BackgroundPremature ovarian insufficiency (POI) is an ovarian defect characterized by primary or secondary amenorrhea, hypergonadotropism and hypoestrogenism which occurs before the age of 40 years with a major genetic component. In this study we performed clinical evaluation and genetic analysis of a group of 18 patients with POI.The study involved 18 consecutive women with POI. Karyotiping and genetic analysis for research of mutations in GDF9 (Growth Differentation Factor 9) and BMP15 (Bone morphogentic protein 15) genes and FMR1 (Fragile X Mental Retardation 1) premutation were carried out. In vitro functional study of the novel BMP15 mutation was performed using COV434 (Human ovarian granulosa tumour cells 434) cells of ovarian granulosa, which consistently express BMP responsive element, and luciferase reporter assay.ResultsThree patients (17%) had a family history of POI. Ten patients (56%) had a family history of autoimmune diseases and nine patients (50%) showed a personal history of one or more autoimmune diseases. Of patients for whom morphological assessment was available, almost half (44%) had poor follicle assets or small ovaries’s size at pelvic US. Two patients (13%) showed reduced bone density at DEXA (Dual Energy X-ray Absorptiometry). All the women had normal female kariotype and no mutations in the GDF-9 gene or FMR1 premutations were found. A novel heterozygous mutation c.406G > C (V136L) of BMP15 gene was identified in one patient. After transfection in COV434 cells, BMP15 variant showed a significantly reduced luciferase activity compared to wild type.ConclusionsPOI is a multifactorial disease with several health implications. Autoimmunity and genetics represent the most common aetiology. We identified and characterized a novel BMP15 mutation, providing an additional elucidation of molecular basis of this complex disorder.

Fluoxetine affects cytosolic cAMP, ATP, Ca2+ responses to forskolin, and survival of human ovarian granulosa tumor COV434 cells.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor antidepressant, exhibits various other mechanisms of action in numerous cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The aim of this study was to determine the off-target effects of the anti-depressant drug FLX, on the human ovarian granulosa tumor COV434 cells stimulated by forskolin (FSK), by measuring the real-time kinetics of intracellular cyclic AMP (cAMP), ATP level, cytoplasmic calcium ([Ca2+]cyt) and survival of COV434 cells. We show that incubating COV434 cells with FLX (between 0.6 and 10 µM) induces a decrease in intracellular cAMP response to FSK, a drop in ATP content and stimulates cytoplasmic Ca2+ accumulation in COV434 cells. Only the highest concentrations of FLX (5–10 µM) diminished cell viability. The present report is the first to identify an action mechanism of FLX in human tumor ovarian cells COV434 cells and thus opening the way to potential use of fluoxetine as a complementary tool, in granulosa tumor treatments.

MiR-29c downregulates tumor-expressed B7-H3 to mediate the antitumor NK-cell functions in ovarian cancer

ObjectiveB7-H3 is a member of the B7 family of immune checkpoint molecule. Although B7-H3 has been shown to regulate T cell-mediated peripheral immune response, whether it also correlated with NK cell exhaustion in ovarian cancer remains unclear. The purpose of this study was to explore the mechanism of B7-H3 regulating NK-cell proliferation and function. Material and methodsTo investigate the relationship between B7-H3 expression and the NK-cell function in ovarian cancer, human ovarian tumor tissues and cell lines were first examined the protein and mRNA expression of B7-H3 by quantitative real-time PCR (qRT-PCR), Immunohistochemistry and Western-blot assays. Then we established B7-H3 knockout cell lines and measured the cytotoxicity of NK cells on these cells by LDH release assay and Flow Cytometry. In addition, we analyzed B7-H3 in the regulation of glycolysis and glycolysis-related proteins by Glycolysis Stress Test, Glucose Consumption Assay and Western-blot. Moreover, luciferase reporter assay was used to confirm the directly regulation of miR-29c to B7-H3. Finally, we carried out in vivo experiments. ResultsWe observed that tumor-expressed B7-H3 inhibits NK-cell function in vitro and in vivo, and is associated with glycolysis of ovarian cancer cell. Therapeutically, B7-H3 blockade prolonged the survival of SKOV3 tumor-bearing mice. In addition, miR-29c improved the anti-tumor efficacy of NK-cell by directly targeting B7-H3 in vitro were observed, but not in vivo. ConclusionOur results demonstrate that miR-29c downregulates B7-H3 to inhibit NK-cell exhaustion and associated with glycolysis, which suggest that NK cells may be a new target of anti-B7-H3 therapy in ovarian cancer patients.

C-myc promotes miR-92a-2-5p transcription in rat ovarian granulosa cells after cadmium exposure

Cadmium (Cd) can induce ovarian injury by microRNAs (miRNAs), however, the molecular mechanism of miRNAs after Cd exposure have not known. In this study, 56-day-old adult female Sprague-Dawley (SD) rats were injection with PMSG, after 48 h, ovarian granulosa cells (GCs) were extracted and cultured for 24 h, then treated with 0, 2.5, 5, 10 and 20 μM Cd for 24 h. The results showed that expression levels of miR-92a-2-5p (upregulated) and Bcl2 (downregulated) changed significantly after Cd exposure. The messenger RNA (mRNA) and protein expression levels of DNMT1, DNMT3A, and DNMT3B had changed, but no obvious differences were found in miR-92a-2-5p single site methylation. The transcription factors C-MYC (upregulated), E2F1 (downregulated), and SP1 (downregulated), which target miRNAs significantly changed after exposure to Cd. The human ovarian GC tumor line (COV434) was used to knocked down C-myc, and the expression of miR-92a-2-5p was downregulated in the COV434-C-myc + 10 μM Cd group compared with COV434 cells. The N6-methyladenosine (m6A) methylation modification levels of long noncoding RNA (lncRNA) MT1JP and lncRNA CDKN2B-AS, which regulate miR-92a-2-5p were detected. In the 10 μM Cd group, m6A methylation levels at MT1JP-84, CDKN2B-AS-257, and CDKN2B-AS-329 were reduced. In summary, after Cd exposure, expression of miR-92a-2-5p, which targets the antiapoptotic gene Bcl2, was upregulated, which may be primarily related to upregulation of C-myc. MiR-92a-2-5p promoter DNA methylation may has no obvious effect on miR-92a-2-5p. Otherwise, the role of m6A methylation modified lncRNA MT1JP and lncRNA CDKN2B-AS in the regulation of miR-92a-2-5p needs further study.

Molecular disruption of DNA polymerase \u03b2 for platinum sensitisation and synthetic lethality in epithelial ovarian cancers

Targeting PARP1 [Poly(ADP-Ribose) Polymerase 1] for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase β (Polβ), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polβ deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polβ depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polβ small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG [Poly(ADP-Ribose) Glycohydrolase] inhibitor (PDD00017273) [but not PARP1 inhibitor (Olaparib)] was synthetically lethal in polβ deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD+) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polβ-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polβ targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.

The role of miRNAs in regulating the effect of prenatal cadmium exposure on ovarian granulosa cells in a transgenerational manner in female rats

Cadmium (Cd) is known to affect ovarian granulosa cells (GCs), but no information on the transgenerational effects of Cd on GCs. In this study, pregnant Sprague-Dawley (SD) rats were orally dosed with Cd from gestation day 1 until birth. F1 or F2 female rats were mated with untreated males to produce the F2 or F3 generation. In the F1 generation, apoptotic cell bodies were observed in the Cd-treated group but not in the F2 generation. Moreover, significant changes in B-cell lymphoma 2 (Bcl2) expression were observed in both generations. Additionally, the expression of microRNAs (miRNAs) was significantly changed based on microarray analysis. Specifically, miR-16–5p and miR-181b-5p were upregulated in F1 and F2 rats, while miR-92a-2-5p demonstrated different expression patterns between the two generations. In F3 generation, miR-16–5p and miR-92a-2-5p were down-regulated. Further, another experiment was used to show that miR-16–5p and miR-92a-2-5p regulated the Bcl2-induced apoptotic effect of Cd on GCs by the Human ovarian GC tumor line (COV434 cell line) miRNA-knockdown model Overall, the results indicate that prenatal Cd exposure has epigenetic transgenerational effect on GCs, Moreover, the underlying mechanism may involve interference with miR-16–5p and miR-92a-2-5p-mediated regulation of Bcl2 genes in offspring.

Abstract PO043: Layer-by-layer nanoparticles for non-toxic delivery of interleukin 12 to disseminated syngeneic ovarian tumors

Abstract Ovarian cancer presents a notable challenge for cancer immunotherapy with less than a 10% objective response rate to checkpoint inhibitors (CPI). One strategy to augment the impact of checkpoint therapy is to initiate the infiltration of leukocytes into the tumor prior to CPI using pro-inflammatory cytokines. One particularly potent and promising cytokine is interleukin-12 (IL-12) – the hallmark Th-1 cytokine known to drive an influx of antigen presenting cells, expansion of cytotoxic T-cells, and NK-cell activation in a variety of solid tumors. Unfortunately, IL-12 has been hampered clinically by toxicity issues when administered systemically – a necessity for disseminated tumors such as advanced ovarian cancer. We have previously shown that nanoparticles engineered for cytokine delivery target human ovarian tumors in mouse models and that delivery of IL-12 using those particles enables the non-toxic delivery of IL-12 when given intratumorally to syngeneic tumors. In this study we demonstrate that these particles given systemically continue to limit IL-12 toxicity, accumulate within syngeneic, orthotopic models of ovarian tumors, induce tumor regressions, and reshape the immune microenvironment within those tumors. IL-12 was loaded onto the surface of negatively charged liposomes using the Ni-His interaction. The layer-by-layer process was then used to add layers of poly-L-arginine and poly-L-glutamine producing a final diameter of 120 nm and zeta potential of -60 mV. These layered nanoparticles (LNPs) preferentially accumulated in intraperitoneal HM-1 ovarian tumor nodules when given intravenously and intraperitoneally with a 30% increase in concentration over unlayered nanoparticles (ULNPs) as measured via fluorescence. IL-12 accumulation measured using ELISA was shown to correlate with NP accumulation for LNPs but not ULNPs. LNPs were non-toxic (via weight loss) at doses deemed toxic with ULNPs or free IL-12. Intraperitoneal treatment with LNPs extended survival and caused tumor regression in 30% of mice at a dose deemed toxic for free IL-12 or ULNPs (10 µg IL-12 given once daily for 5 days). Flow cytometry revealed a marked increase in memory T-cells and a decrease in granulocytes. The LNPs also induced substantial upregulation of exhaustion markers in the infiltrating T-cells – especially the TIM3+TIGIT+PD1+ highly exhausted phenotype suggesting potential for future combination therapies. Citation Format: Sean G. Smith, Antonio E. Barberio, Ivan S. Pires, Mariane Melo, Darrell J. Irvine, Paula T. Hammond. Layer-by-layer nanoparticles for non-toxic delivery of interleukin 12 to disseminated syngeneic ovarian tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO043.

TGF-β1 stimulates aromatase expression and estradiol production through SMAD2 and ERK1/2 signaling pathways in human granulosa-lutein cells.

Estradiol (E2), one of the main steroid hormones secreted by the ovaries, plays an important rolein maintaining normal female reproductive function. Ovarian granulosa cells are the main source of E2 production because these cells express aromatase, which is encoded by the CYP19A1 gene and catalyzes the final step in E2 biosynthesis from androgens. Transforming growth factor-beta 1 (TGF-β1) and its receptors are expressed in human granulosa cells, and TGF-β1 expression can be detected in human follicular fluid. To date, TGF-β1 has been shown to regulate various ovarian functions. However, whether aromatase can be regulated by TGF-β1 in human granulosa cells has not been determined. In the present study, we demonstrate that TGF-β1 stimulates aromatase expression in primary human granulosa-lutein cells and in the human ovarian granulose-like tumor cell line, KGN. We used pharmacological inhibitors and small interfering RNA-mediated knockdown approaches to reveal that the SMAD2 and ERK1/2 signaling pathways are involved in TGF-β1-induced aromatase expression and E2 production. These results not only provide important insights into the molecular mechanisms that mediate TGF-β1-induced aromatase expression and E2 production in human granulosa cells but also increase the understanding of the normal physiological roles of TGF-β1 in the ovary.

Resveratrol depolarizes the membrane potential in human granulosa cells and promotes mitochondrial biogenesis

To study the biological effects of resveratrol on the growth, electrophysiology, and mitochondrial function of human granulosa cells (h-GCs). Preclinical study. Electrophysiology laboratory and invitro fertilization unit. This study included h-GCs from seven infertile women undergoing assisted reproductive techniques. Human ovarian Granulosa Cell Tumor (GCT) cell line COV434 and h-GCs obtained after oocyte retrieval were cultured in the absence or presence of resveratrol. Granulosa cells were evaluated for cell viability and mitochondrial activity. Electrophysiological recordings and evaluation of potassium current (IKur) and Ca2+ concentration were also performed. Resveratrol induced mitochondrial activity in a bell-shaped, dose-effect-dependent manner. Specifically, resveratrol treatment (3 μM, 48 hours) increased ATP production and cell viability and promoted the induction of cellular differentiation. These biological changes were associated with mitochondrial biogenesis. Electrophysiological recordings showed that resveratrol reduced the functional expression of an ultra rapid activating, slow inactivating, delayed rectifier potassium current (IKur) that is associated with a plasma membrane depolarization and that promotes an increase in intracellular Ca2+. CONCLUSION(S): The effects of resveratrol on potassium current and mitochondrial biogenesis in h-GCs could explain the beneficial effects of this polyphenol on the physiology of the female reproductive system. These findings suggest there are therapeutic implications of resveratrol in a clinical setting.

Salvia miltiorrhiza solution and its active compounds ameliorate human granulosa cell damage induced by H2O2.

The dried roots or rhizomes of Salvia miltiorrhiza Bge are commonly used in Chinese medicine to promote blood circulation and regulate menstruation. Salvianic acid A and salvianolic acid B are the main active water-soluble compounds in Salvia miltiorrhiza solution. The present study investigated the protective effect of Salvia miltiorrhiza solution and its active compounds in H2O2-induced cell damage of the human ovarian granulosa tumor cell line (KGN) in vitro, as well as its underlying mechanism. Cell viability was detected using a Cell Counting Kit-8 assay. In addition, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and tumor necrosis factor-α (TNF-α) were measured. Western blotting was performed to detect the protein expression of cleaved caspase-3 and caspase-9. Furthermore, immunocytochemistry was used to detect the expression of TNF-α. It was demonstrated that Salvia miltiorrhiza solution, salvianic acid A and salvianolic acid B did not affect the viability of KGN cells. Additionally, salvianic acid A and salvianolic acid B significantly reduced the H2O2-induced increased MDA levels, and reversed the H2O2-induced suppression of SOD and GSH activities in KGN cells (P<0.05). Treatment with Salvia miltiorrhiza solution, salvianic acid A and salvianolic acid B significantly reduced the overexpression of cleaved caspase-3, cleaved caspase-9 and TNF-α compared with the H2O2-treated group (P<0.05). Therefore, the present results indicated that Salvia miltiorrhiza solution and its main water-soluble compounds, salvianic acid A and salvianolic acid B, ameliorated KGN cell damage induced by H2O2.

PGK1-AR axis: Benefits of a novel actor in PCOS pathology

PGK1-AR axis: Benefits of a novel actor in PCOS pathology

MiR‑132 is upregulated in polycystic ovarian syndrome and inhibits granulosa cells viability by targeting Foxa1.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine metabolic disorders characterized by hyperandrogenism, polycystic ovaries and ovulatory dysfunction. Several studies have suggested that the aberrant expression of microRNAs (miRNAs/miRs) plays an important role in the pathogenesis of PCOS; however, the role and underlying mechanisms of miR-132 in the development of PCOS remain unclear. In the present study, the expression of miR-132 in granulosa cells (GCs) derived from 26 patients with PCOS and 30 healthy controls was detected by reverse transcription-quantitative PCR (RT-qPCR). The apoptosis of GCs was examined using a TUNEL assay. The human ovarian granulosa-like tumor cell line, KGN, was cultured for Cell Counting Kit-8 assays following the overexpression or knockdown of miR-132. TargetScan was applied to identify the potential targets of miR-132, which was further verified by a luciferase assay, RT-qPCR and western blotting. The expression of miR-132 was decreased in GCs from patients with PCOS. Moreover, the GCs of patients with PCOS exhibited significantly increased apoptotic nuclei. Furthermore, the overexpression of miR-132 inhibited the viability of KGN cells. In addition, the results verified that miR-132 directly targeted forkhead box protein A1 (Foxa1), the knockdown of which suppressed KGN cell viability. On the whole, the findings of the present study demonstrated that miR-132 inhibited cell viability and induced apoptosis by directly interacting with Foxa1. Thus, miR-132 may be a potential target for the treatment of patients with PCOS.

Synergistic Anti-Tumor Activity by Targeting Multiple Signaling Pathways in Ovarian Cancer

Simple SummaryOvarian cancer remains the most lethal gynecological cancer in women. There is a critical need to develop novel strategies that can be used to improve the survival of patients with advanced and recurrent ovarian cancer. Preclinical and early clinical studies with single-targeted agents have shown limited antitumor activity in ovarian cancer. In this study, we found that combined treatment of several FDA-approved targeted drugs—sunitinib, dasatinib, and everolimus—results in simultaneous inhibition of multiple signaling pathways and a better anti-tumor activity than any single treatment. This combination also significantly improves efficacy of paclitaxel in human ovarian cancer. This study may provide a potential combination therapy for the treatment of advanced ovarian cancer.More effective therapy is needed to improve the survival of patients with advanced and recurrent ovarian cancer. Preclinical and early clinical studies with single molecular targeted agents have shown limited antitumor activity in ovarian cancer, likely due to compensation by alternative growth/survival pathways. An emerging strategy in overcoming resistance is to combine inhibitors targeting multiple pathways. In this study, we used a novel strategy of combining several FDA-approved targeted drugs, including sunitinib, dasatinib, and everolimus, in human ovarian cancers. Combination of the tyrosine kinase inhibitor sunitinib with the SRC inhibitor dasatinib showed synergistic anti-tumor activity in human ovarian cancer cells. The increased activity was associated with inhibition of the STAT3, SRC, and MAPK signaling pathways, but not AKT signaling. To inhibit the PI3K/AKT/mTOR pathway, we added the mTOR inhibitor everolimus, which further increased anti-tumor activity in cells. Combined treatment with sunitinib, dasatinib, and everolimus also resulted in greater inhibition of human ovarian tumor growth in mice. Furthermore, the triple combination also synergistically increased the anti-tumor activity of paclitaxel, both in vitro and in vivo. Taken together, our results demonstrate that simultaneous inhibition of several signaling pathways results in better anti-tumor activity compared to inhibiting any of these signaling pathways alone.

On the chemical composition of psammoma bodies microcalcifications in thyroid cancer tissues

Recently the knowledge of chemical composition of pathological mineralizations is an important topic extensively studied because it could give more in-depth information to understand pathologies themselves and to improve prevention methods. In this work, psammoma bodies (PBs) microcalcifications in thyroid cancer tissue are investigated by different and complementary analytical methods as: micro-Fourier transformed spectroscopy, X-ray fluorescence spectroscopy, Inductively Coupled plasma Optical Emission Spectroscopy (ICP-OES) and scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy imaging (EDX). For the first time the micro-FTIR analysis of the only inorganic phase isolated from PBs was reported. Signals of the recorded spectrum showed that the main component of the calcifications is the amorphous carbonated calcium phosphate, and the IR spectrum of thyroid PBs is strongly consistent with that of PBs in human ovarian tumors. The XRF and the ICP analysis detected also the presence of iron ad zinc in thyroid PBs. These results are validated by scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy imaging (EDX) carried out on tissue samples of the papillary thyroid carcinoma. By these analytical methods magnesium and sodium were detected within PBs while the presence of iron was confirmed by the Perls test. Summarizing the results of applied analytical methods, the main detected elements within the thyroid psammoma bodies are Ca, P, Mg, Na, Fe and Zn. Magnesium and sodium are found in malignant breast cancer microcalcifications, thus they seem correlated to neoplastic transformation. The Fe and Zn elements could give information about the origin of these pathological microcalcifications.

Abstract 1061: Increasing potency of anticancer drugs through Sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer

Abstract Rational : The development of personalized therapies against ovarian cancer remains highly challenging even in current modern oncology. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to peptide ligands that selectively target receptors abundantly and/or exclusively expressed in these tumor cells. Increased expression of Sortilin (SORT1), a scavenging receptor also known as neurotensin receptor-3, has been observed in invasive ovarian cancer biopsies. In addition, increased SORT1 expression correlated with breast tumor grades. Procedures : In light of these observations, we developed a peptide conjugation platform to target SORT1 and to increase cell selectivity and internalization of anticancer agents. As a proof-of-concept, doxorubicin and docetaxel were conjugated to a Sortilin-binding proprietary peptide (TH19P01). Results : In vitro, significant intracellular delivery of the doxorubicin-TH19P01 conjugate (TH1904) and docetaxel-TH19P01 conjugate (TH1902) were observed in a ES-2 ovarian cancer cell line model while preserving efficient doxorubicin or docetaxel cytotoxic mechanism. TH19P01 and drug conjugate uptake in ovarian cancer cells assays were reduced when SORT1 expression was specifically silenced using siRNA or upon competition with the SORT1 ligands neurotensin and progranulin. Importantly, drug-TH19P01 conjugates were found to bypass the P-glycoprotein (P-gp) efflux pump in MDCK-MDR1 cells overexpressing P-gp as the uptake of conjugates was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, TH1904 and TH1902 caused a more potent inhibition of human ovarian tumor xenografts grown in mice and were better tolerated than their unconjugated parent drugs. Conclusion : These results strongly support future clinical development of this targeted technology platform to generate novel personalized therapeutics with specific indications in Sortilin-positive cancers. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Larocque, Alain Zgheib, Sophie Kozelko, Richard Béliveau, Christian Marsolais, Borhane Annabi. Increasing potency of anticancer drugs through Sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1061.

Abstract 3946: HDL associated Paraoxonase 1 reduces the ovarian tumorigenesis by enhancing the HDL function

Abstract Background: Membrane lipids/cholesterol determines the fluidity, clustering of receptors and other protein interactions that delineate signaling pathways for cell cycle/proliferation. Emerging evidences suggest that highly proliferative cancer cells show a high lipid and cholesterol avidity and are considered as hallmarks of cancer aggressiveness including ovarian cancer. Paraoxonase 1 (PON1) is a high density lipoprotein (HDL) associated enzyme with a plethora of functions. PON1 deficiency aggravates HDL inflammatory index, systemic inflammation, and results in lipid retention associated diseases including atherosclerosis and hepatic steatosis. Meta-analysis studies documented that PON1 polymorphisms are associated with various cancers including ovarian cancer and PON1 activity is negatively correlated with human ovarian tumor size, providing clinical association between PON1 and ovarian cancers. Yet, the role and mechanism of action of PON1 in ovarian cancer development has not been elucidated. Hypothesis: We hypothesized that PON1 may inhibit the ovarian tumorigenesis by modulating HDL function. Methods and Results: In this report, in patients with ovarian cancer, we observed that plasma PON1 levels are higher, but with lower activity compared to healthy control. Immunoprecipitation of plasma PON1, followed by ELISA against oxidized lipids suggest that PON1 is oxidatively modified, specifically by 4-HNE. In addition, PON1 activity is negatively correlated with HDL pro-inflammatory index. Using a xenograft mouse model, we demonstrate that overexpression of PON1 prevents the development of ovarian cancer. HDL from xenograft transgenic mice shows less pro-inflammatory properties with increased cholesterol efflux efficiency from ovarian cancer cells. Mechanistically, PON1 impairs VEGF signaling in ovarian cancer cells by increasing cholesterol (Mitogenic factor) efflux, resulting in reduced dimerization of vascular endothelial growth factor receptor 2 (VEGF-2R) and VEGF-2R phosphorylation (angiogenic inducer) as well as downstream signaling pathways, including Akt and ERK1/2 which in turn reduce the cell proliferation and angiogenesis. Furthermore, cholesterol and VEGF levels are significantly lowered in the tumor microenvironment in PON1 transgenic mice but there are no changes in the gene expressions that are associated with either cholesterol synthesis or its degradation pathways. Conclusion: Taken together, we report for the first time that PON1 acts as a tumor suppressor of ovarian cancer by possibly reducing VEFG signaling and enhancing the HDL function, suggesting that activation of HDL associated PON1 might be a fruitful strategy to inhibit the ovarian tumor formation. Citation Format: Asokan Devarajan, Victor Grijalva, Dawoud Sulaiman, Feng Su, Ellen O'Connor, Robin Farias-Eisner, Srinivasa Reddy. HDL associated Paraoxonase 1 reduces the ovarian tumorigenesis by enhancing the HDL function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3946.

Abstract 5085: A preclinical model using perfusion air culture of tumor tissue slices for personalized medicine

Abstract For personalized medicine it is crucial that a preclinical model captures the complex tumor biology in vitro in order to individually predict in vivo therapy of tumors. Precision-cut tumor slices maintain tissue heterogeneity with regard to different cell types and preserved native microenvironment. To enable the use of tumor slices as preclinical model that fulfills these criteria we developed a perfusion air culture (PAC) system with continuous and precisely controlled oxygen, medium and drug supply. In the PAC system, precision-cut tumor slices are kept in-between two organotypic supports fixed in a special chamber and placed inside of a 50 mL tube with air exchange capacity housed in a standard CO2-incubator. To evaluate the PAC system, cultured tumor slices from mouse xenografts (MCF7, H1437) and primary human ovarian tumors (phOVT) were compared to in vivo source tissues using immunohistochemistry for morphology, proliferation, DNA damage, apoptosis, and transcriptional biomarkers for cellular stress response. Results show that viability and morphology of the tumor slices are preserved for more than 7 days in the PAC system. We also compared the PAC system with the commonly used static Millipore filter (MF) system which cultures slices on a filter support at an air-liquid interface and gives rise to intra-slice gradients. Both, mouse xenografts and 9 of 15 phOVT tissue slices showed a gradient of cell proliferation and biomarker expression in the MF system while no gradient was detected in slices cultured in the PAC system. Analysis of the culture media revealed lower glucose consumption and lactate production in the PAC system as compared to the MF system indicating more efficient oxygen supply. To analyze therapy response, Cisplatin was applied to phOVT tumor slices for 3 days. Cisplatin treatment was accompanied by minor increase of γ-H2AX in both MF and PAC systems while only in the PAC system strongly enhanced cleavage of caspase-3 was observed, indicating that the PAC system is suitable to assess functional response to drug treatment. To test whether the PAC system is also suitable for the detection of immune response in tumor slices, we analyzed the immune cells of tissue slices before and after cultivation. The patient specific immune cells and their composition is preserved throughout the culture period in the PAC system. In conclusion, cultivation of tumor tissue slices in the PAC system provides an ex vivo model that preserves tumor heterogeneity and native microenvironment. Because the PAC system facilitates homogenous and precisely controlled supply of oxygen, nutrients and drugs, it allows long-term culture of tumor tissue and analysis of therapy response - including immune therapy. We conclude that the newly developed PAC system is suitable to perform patient specific ex vivo tests and thus allows personalized therapy adaption. Citation Format: Meng Dong, Kathrin Böpple, Bernd Winkler, Markus Kleih, Julia Schüler, Emma Davies, Tauno Metsalu, Heike Walles, Hans-Georg Kopp, Frank Essmann, Walter E. Aulitzky. A preclinical model using perfusion air culture of tumor tissue slices for personalized medicine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5085.

OvCa-Chip microsystem recreates vascular endothelium–mediated platelet extravasation in ovarian cancer

AbstractIn ovarian cancer, platelet extravasation into the tumor and resulting metastasis is thought to be regulated mostly by the vascular endothelium. Because it is difficult to dissect complex underlying events in murine models, organ-on-a-chip methodology is applied to model vascular and platelet functions in ovarian cancer. This system (OvCa-Chip) consists of microfluidic chambers that are lined by human ovarian tumor cells interfaced with a 3-dimensional endothelialized lumen. Subsequent perfusion with human platelets within the device's vascular endothelial compartment under microvascular shear conditions for 5 days uncovered organ-to-molecular–level contributions of the endothelium to triggering platelet extravasation into tumors. Further, analysis of effluents available from the device's individual tumor and endothelial chambers revealed temporal dynamics of vascular disintegration caused by cancer cells, a differential increase in cytokine expression, and an alteration of barrier maintenance genes in endothelial cells. These events, when analyzed within the device over time, made the vascular tissue leaky and promoted platelet extravasation. Atorvastatin treatment of the endothelial cells within the OvCa-Chip revealed improved endothelial barrier function, reduction in inflammatory cytokines and, eventually, arrest of platelet extravasation. These data were validated through corresponding observations in patient-derived tumor samples. The OvCa-Chip provides a novel in vitro dissectible platform to model the mechanisms of the cancer-vascular-hematology nexus and the analyses of potential therapeutics.