Andras Ladanyi

Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth

Intra-abdominal tumors, such as ovarian cancer, have a clear predilection for metastasis to the omentum, an organ primarily composed of adipocytes. Currently, it is unclear why tumor cells preferentially home to and proliferate in the omentum, yet omental metastases typically represent the largest tumor in the abdominal cavities of women with ovarian cancer. We show here that primary human omental adipocytes promote homing, migration and invasion of ovarian cancer cells, and that adipokines including interleukin-8 (IL-8) mediate these activities. Adipocyte–ovarian cancer cell coculture led to the direct transfer of lipids from adipocytes to ovarian cancer cells and promoted in vitro and in vivo tumor growth. Furthermore, coculture induced lipolysis in adipocytes and β-oxidation in cancer cells, suggesting adipocytes act as an energy source for the cancer cells. A protein array identified upregulation of fatty acid–binding protein 4 (FABP4, also known as aP2) in omental metastases as compared to primary ovarian tumors, and FABP4 expression was detected in ovarian cancer cells at the adipocyte-tumor cell interface. FABP4 deficiency substantially impaired metastatic tumor growth in mice, indicating that FABP4 has a key role in ovarian cancer metastasis. These data indicate adipocytes provide fatty acids for rapid tumor growth, identifying lipid metabolism and transport as new targets for the treatment of cancers where adipocytes are a major component of the microenvironment.

HOXA9 promotes ovarian cancer growth by stimulating cancer-associated fibroblasts

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-β2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

Reliable and Sensitive Identification of Occult Tumor Cells Using the Improved Rare Event Imaging System

Purpose: The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors. Experimental Design: We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis. Results: The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile. Conclusions: REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.

Targeting the Urokinase Plasminogen Activator Receptor Inhibits Ovarian Cancer Metastasis

Purpose: To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. Experimental Design: Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. Results: Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α5-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, β3-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo. Conclusions: This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α5-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication. Clin Cancer Res; 17(3); 459–71. ©2010 AACR.

Expression of the Homeobox Gene HOXA9 in Ovarian Cancer Induces Peritoneal Macrophages to Acquire an M2 Tumor-Promoting Phenotype

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8(+) tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages.

Laser microdissection in translational and clinical research

Laser microdissection (LMD) is now a well established method for isolating individual cells or subcellular structures from a heterogeneous cell population. In recent years, cell, DNA, RNA, and protein based techniques has been successfully coupled to LMD and important information has been gathered through the analysis of the genome, transcriptome, and more recently the proteome of individual microdissected cells.

Improved Indicators for Assessing the Reliability of Detection and Quantification by Kinetic PCR

Because of their low detection limits, PCR and the adapted kinetic (real-time quantitative) PCR have been used extensively for the detection and quantification of nucleic acids (1)(2)(3). However, at the detection limits of an assay, mathematical models (4)(5)(6) and experimental evidence (7)(8)(9)(10)(11)(12) have shown that nucleic acids are detected inconsistently and quantified imprecisely. With this potential unreliability, it is essential that indicators be used to identify the concentration at which analysis is occurring to ensure the accuracy of results. Conventional PCR indicators, however, currently provide only an indirect assessment of the concentration at which analysis occurs. The total nucleic acid content of a sample, commonly used to define the adequacy of sample loading, does not precisely reflect specific gene concentrations. Reference (control or housekeeping) genes, qualitative detection of which is often used to validate assay and sample integrity, are often present at concentrations different from those of the target genes of interest (13). In kinetic PCR, the inclusion of fluorescent probes allows the monitoring of reaction kinetics, which enables the measurement of a crossing point (CP), or cycle threshold, at a partial cycle number at which fluorescence becomes detectable above background signal (14). CP values are linearly proportional to gene copy number in an inverse logarithmic relationship (14)(15), and this correlation is central to algorithms for determining gene quantity (16)(17). In this study, we hypothesized that CP values might be more accurate indicators of the reliability of an analysis than conventional indicators because of their direct relationship with gene concentration. The results of this study provide experimental evidence to support this hypothesis; we also describe models for use of CP values as indicators for assessing the reliability of …

Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression data sets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in OvCa cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Abstract PR11: Ovarian cancer cells activate the mesothelium in the peritoneal microenvironment promoting fibronectin production and the early steps of metastasis

The main site of ovarian cancer (OvCa) metastasis is the omentum, which is covered by a single layer of mesothelial cells overlaying an extracellular matrix interspersed with fibroblasts (mesothelium). A 3D model of the human peritoneal surface (in vitro), fragments of human omentum (ex vivo), and a mouse xenograft model (in vivo) of OvCa were used to investigate the early effects of cancer cells on the host stromal microenvironment. Our results reveal that fibronectin is overexpressed in the stroma of more than 93% of OvCa omental metastases. OvCa cells induce fibronectin matrix assembly in omental stromal cells, and stimulate fibronectin expression in mesothelial cells. The fibronectin produced by human mesothelial stromal cells promotes OvCa cell adhesion, invasion and proliferation. Inhibition of fibronectin production in mesothelial cells or omental surface cells using a fibronectin specific siRNA decreases OvCa cell adhesion, invasion and proliferation. Genetic knock-down of fibronectin in vitro and in vivo, using fibronectinfl/fl mice, also impaired OvCa cell adhesion/invasion and metastasis. Re-analysis of an Australian OvCa study gene array data set suggested that OvCa, which highly express fibronectin, show co-activation of TGFβ1/Rac1 dependent signaling pathways. Indeed, OvCa cells increase phosphorylation of Smad2/3 in mesothelial cells upon co-culture. Inhibition of TGFβ1 in OvCa cells or inhibition of Smad 3, as well as TGFβRI or TGFβRII, in mesothelial cells resulted in decreased fibronectin production. Moreover, Rac 1 activity was increased upon the co-culture of OvCa and mesothelial cells. Subsequent inhibition of Rac 1 in mesothelial cells led to a decrease in fibronectin secretion. Our data suggest that OvCa cells regulate fibronectin expression in mesothelial cells through a TGFβ1/Rac1 dependent pathway. In vitro and in vivo therapy with a fibronectin inhibitor, ATN161, a peptide derived from the synergy region of fibronectin, reduced OvCa invasion, proliferation and metastasis. In summary, we show that abdominally metastasizing cancer cells induce fibronectin production in the peritoneal and omental microenvironment and that inhibition of this fibronectin-response prevents the first steps of peritoneal metastasis. By understanding the mechanism of OvCa metastasis we will be better able to target cells in the tumor microenvironment (i.e. mesothelial cells, fibroblasts and macrophages) during early ovarian cancer metastasis. This abstract is also presented as Poster A60. Citation Format: Hilary A. Kenny, Chun-Yi Chiang, Andras Ladanyi, Joshy George, David D. Bowtell, Katja Gwin, Andrew Mazar, Ernst Lengyel. Ovarian cancer cells activate the mesothelium in the peritoneal microenvironment promoting fibronectin production and the early steps of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR11.

Abstract IA30: Metabolic symbiosis: The contribution of adipocytes to visceral metastasis

Many tumor types (gastric, breast, colon, renal, and ovarian) grow in the anatomical vicinity of adipose tissue and are known to reprogram adjacent normal adipocytes into cancer-associated adipocytes (CAA). Clinical observations indicate that the most common, as well as the largest, site of ovarian cancer (OvCa) metastasis is the omentum, a large fat pad (20x12x3cm) positioned in front of the bowel. The omentum, which is primarily composed of adipocytes, provides energy storage and functions as an endocrine organ. Because cancer cell behavior is highly dependent on the microenvironment, and adipocytes are a major source of energy-dense fatty acids, we hypothesized that adipocytes promote OvCa omental metastasis and growth via altered lipid metabolism. To investigate this hypothesis, primary human visceral adipocytes were cultured from omental tissue specimens collected from patients who underwent surgical procedures for benign conditions. The rate of β-oxidation was assessed by a radioisotopic assay using 3H-palmitate. Sections of human serous ovarian carcinomas and corresponding omental metastases were utilized for immunohistochemistry, RT-PCR, and a reverse-phase protein array. Homozygous fatty acid binding protein 4 (FAPB4)-null mice or wild-type (WT) animals were inoculated orthotopically or intraperitoneally with mouse OvCa cells. Adipocytes were found to promote the invasion, migration, and proliferation of OvCa cells. The homing of OvCa towards adipocytes in vitro, or to the mouse omentum in vivo, was reduced by using inhibitory antibodies against interleukin (IL)-6,IL-8, or their receptors. Co-culture of OvCa cells with adipocytes resulted in cytoplasmic lipid accumulation in OvCa cells, which was a consequence of direct lipid transfer from the adipocytes. Co-injection of SKOV3ip1 OvCa cells with adipocytes enhanced tumor growth in mice, when compared to the injection of SKOV3ip1 cells alone. Moreover, lipolysis was activated in co-cultured adipocytes as evidenced by the secretion of free fatty acids and glycerol. FABP4 was found to be highly expressed in omental metastatic tissues as compared to the respective primary OvCa tumor tissue. When mouse OvCa cells were injected under the ovarian bursa of FABP4 knock-out mice the number of metastases was significantly lower than in WT mice. Treatment with a FABP4 inhibitor also induced apoptosis and inhibited migration and invasion of OvCa cells. The fatty acid translocase (FAT/CD36) was identified as the fatty acid transporter responsible for lipid accumulation in cancer cells co-cultured with adipocytes. Our results suggest that adipocytes advance the progression of OvCa through two major mechanisms: (1) They promote metastasis to the omentum via the secretion of cytokines, and (2) They support cancer cell proliferation by activating lipolysis, thereby mobilizing energy-dense fatty acids which are utilized by the cancer cells in an energy-yielding β-oxidation pathway through a FABP4 and FAT/CD36 dependent mechanism. By better understanding these mechanisms and, more generally, the biology of adipocytes in the tumor microenvironment, we will be able to identify metabolic targets and, ultimately, introduce new compounds for the treatment of cancer. Citation Format: Kristin Nieman, Andras Ladanyi, Ernst Lengyel. Metabolic symbiosis: The contribution of adipocytes to visceral metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA30.