Intra-abdominal tumors, such as ovarian cancer1,2, 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.

IntroductionOvarian cancer (OvCa) has the highest mortality rate of all gynecologic tumors and is the fifth leading cause of cancer death among US women (1). It is predominantly confined within the abdominal cavity and, unlike breast, colon, or lung cancer, rarely metastasizes hematogenously. Once an ovarian epithelial cell undergoes neoplastic transformation, it freely disseminates throughout the peritoneal cavity, carried by peritoneal fluid that facilitates attachment to peritoneum and omentum. The omentum is a large fat pad (approximately 12 × 12 cm) located inferior to the stomach and draped over the small bowel. It is the most common metastatic site (80%) for OvCa cells (2) followed by implants on the abdominal peritoneum. Identification of cofactors regulating OvCa cell attachment to omentum and/or peritoneum would have tremendous clinical utility, by enabling identification of cellular or molecular targets that could be pursued therapeutically and thus, enabling blockade of a critical step necessary for OvCa metastasis within the peritoneal cavity.A role for MMPs in OvCa development has been postulated based upon the observation that several members of the MMP family are upregulated during OvCa neoplastic progression (3). When MMPs were first characterized (4), it was hypothesized that their major contribution to cancer development was merely to

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of A 5 -integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an A 5 B 1 -integrin-blocking antibody. When E-cadherin is silenced, A 5 -integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogenactivated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/B-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of A 5 -integrin, i.p. treatment with an A 5 B 1 -integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). A 5 -Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had A 5 -integrin overexpression, and 39% had some level of A 5 -integrin expression. The median survival for patients with high A 5 -integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified A 5 -integrin upregulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

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 datasets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in ovarian cancer cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Ovarian cancer preferentially metastasizes to the omentum, a fatty tissue characterized by immune structures called milky spots, but the cellular dynamics that direct this tropism are unknown. Here, we identified that neutrophil influx into the omentum is a prerequisite premetastatic step in orthotopic ovarian cancer models. Ovarian tumor–derived inflammatory factors stimulated neutrophils to mobilize and extrude chromatin webs called neutrophil extracellular traps (NETs). NETs were detected in the omentum of ovarian tumor–bearing mice before metastasis and of women with early-stage ovarian cancer. NETs, in turn, bound ovarian cancer cells and promoted metastasis. Omental metastasis was decreased in mice with neutrophil-specific deficiency of peptidylarginine deiminase 4 (PAD4), an enzyme that is essential for NET formation. Blockade of NET formation using a PAD4 pharmacologic inhibitor also decreased omental colonization. Our findings implicate NET formation in rendering the premetastatic omental niche conducive for implantation of ovarian cancer cells and raise the possibility that blockade of NET formation prevents omental metastasis.