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

Abstract

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.

Figure 1

Adipocytes promote homing of ovarian cancer cells to the omentum. (a) H&E staining of ovarian cancer (OvCa) tumor cells invading adipocytes in the human omentum. (b) In vivo homing assay. Fluorescently-labeled SKOV3ip1 human ovarian cancer cells were injected intraperitoneally into nude mice. Cancer cell localization was detected after 20 min (n = 6 mice). The mouse omentum is outlined in the bright-field image (left) and visible in the fluorescent image (right). (c,d) Migration (c) and invasion (d) of SKOV3ip1 cells toward serum-free medium (SFM), adipocyte-conditioned medium (CM) and primary human omental adipocytes (Adi). Bars report mean fold change ± s.e.m. One of three experiments, each using a different human subject samples, is shown. (e) Invasion of SKOV3ip1 cells comparing primary human omental (Adi-O) and subcutaneous (Adi-S) adipocytes from the same individual. Bars report mean fold change ± s.e.m. One of two experiments shown. (f) Cytokine expression in conditioned medium from primary human omental adipocytes. One of four arrays shown. (g) Fluorescence intensity of labeled SKOV3ip1 cells that homed to Matrigel plugs containing SFM or adipocytes in the presence or absence of inhibitory antibodies. Bars report means ± s.e.m. One of three experiments shown. (h) Fluorescence intensity of labeled SKOV3ip1 cells that adhered to sections of human omentum. SKOV3ip1 cells were pretreated with a CXCR1- or IL-6R–blocking antibody, or whole omentum sections were pretreated with a TIMP-1 inhibitory antibody (n = 5 sections per group).Bars report means ± s.e.m. from one of three experiments. (i) Immunoblot of total and phosphorylated (p) p38 (Thr180/Tyr182) and Stat3 (Ser727) in SKOV3ip1 cells cultured alone (−) or with (+) adipocytes from two human subject samples for 24 h. One of three experiments shown.

Figure 2

Ovarian cancer cells use adipocyte-derived lipids for tumor growth. (a) Lipid (green) accumulation in human omental metastatic ovarian cancer. The interface between ovarian cancer cells and adipocytes is indicated by a dashed line (H&E staining). Ovarian cancer cells (A) that do not interact with adipocytes (C) lack intracellular lipid staining. The ovarian cancer cells in contact with adipocytes (B) contain more intracellular lipids (nuclear counterstaining, blue). (b,c) Lipid (green) accumulation in SKOV3ip1 cells cultured alone or with primary human omental adipocytes (repeated with two additional human subject samples), as determined using confocal microscopy (b), or fixed and examined with transmission electron microscopy (c) (N, nucleus; L, lipid droplets). (d) Fluorsescently labeled fatty acids (FAs) were incubated with and taken up by adipocytes (left) or SKOV3ip1 cells (middle). The labeled adipocytes were cocultured with SKOV3ip1 cells, the adipocytes were removed, and the labeled FAs that were transferred from adipocytes to SKOV3ip1 cells were detected by confocal microscopy (right). Fluorescence quantification is in the right graph. Bars report means ± s.e.m. from one of three experiments, conducted with different human subject samples. (e) In vitro proliferation of SKOV3ip1 cells alone or cocultured with adipocytes over 4 d. Graph reports means ± s.e.m. from one of three experiments, completed using different human subject samples. (f) In vivo growth of subcutaneous tumors after injection of SKOV3ip1 cells with or without adipocytes in each flank of the same mouse. Graphs depict tumor volume measured over 24 d (left) and final tumor weight (right). Representative tumor images from one mouse are included (three or four mice per group). Graphs report means ± s.e.m. from one of three experiments, conducted using omental adipocytes from different human subject samples.

Figure 3

Cocultivation of ovarian cancer cells with adipocytes activates lipolysis in adipocytes and β-oxidation in cancer cells. (a,b) Free fatty acid (a) and glycerol release (b) are detected in primary human adipocytes cultured alone or with SKOV3ip1 cells alone. Bars report means ± s.e.m. from one of two experiments completed using omental adipocytes from different human subject samples. (c) Immunoblot for total and phosphorylated (p) HSL (Ser660) in adipocytes from three different human subject samples cultured with (+) and without (−) SKOV3ip1 cells. (d) Immunofluorescence using confocal microscopy for p-HSL (green) in SKOV3ip1 cells, adipocytes or a coculture of both. Arrowhead indicates an adipocyte (A) (nuclear counterstaining, blue). One of two experiments, conducted using different human subject samples, is shown. (e) Immunoblot for total and p-AMPK in SKOV3ip1 cells cocultured with (+) and without (−) adipocytes for 24 h from three different human subject samples. (f) Immunofluorescence using confocal microscopy for p-AMPK (Thr172, green) in SKOV3ip1 cells, adipocytes, or a coculture of both. Arrow points out a cancer cell (C) in the image (nuclear counterstaining, blue). One of two experiments, completed with different human subject samples, is shown. (g) β-oxidation rate in SKOV3ip1 cells cocultured with adipocytes (l-carnitine, positive control; etomoxir, negative control). Graph reports means at the indicated times ± s.e.m. One of three experiments, conducted with or without different human subject samples, is shown. (h) mRNA expression of the rate-limiting fatty acid oxidation enzymes carnitine palmitoyltransferase 1 (CPT) and acyl-CoA oxidase 1 (ACOX1) in SKOV3ip1 cells cultured alone or with adipocytes. Bars report mean fold change relative to glyceraldehyde 3-phosphate dehydrogenase expression ± s.e.m. One of three experiments, conducted with different human subject samples, is shown.

Figure 4

FABP4 has a key role in the interaction of cancer cells with adipocytes. (a) Representative immunohistochemical staining (bottom) for FABP4 in serial sections of primary ovarian tumor and corresponding omental metastatic tissues from a subject with stage IIIC advanced serous ovarian cancer (as classified by the International Federation of Gynecology and Obstetrics). H&E staining is in the top images. The graph on the right is a summary of FABP4 protein expression scoring in 20 subjects, as assessed by immunohistochemistry. The scoring (0, 1 or 2, corresponding to negative, weak or strong) was performed in different tissue compartments: Benign ovarian stroma (A), primary ovarian cancer in the ovary (B), ovarian cancer metastasis to the omentum (C), interface of ovarian cancer cells into adipocytes (Adi) (D) and adipocytes (E). Error bars, ± s.e.m. (b) Confocal microscopy images of lipid accumulation (green) in SKOV3ip1 cells cocultured with or without primary human omental adipocytes and a FABP4 inhibitor (nuclear counterstaining, blue). One of two experiments, conducted with different human subject samples, is shown. (c) Invasion assay of SKOV3ip1 cells toward adipocytes in the absence or presence of a FABP4 inhibitor. Bars report mean fold change ± s.e.m. One of two experiments, using two different human subject samples, is shown. (d) Metastatic tumor burden in Fabp4^−/− (n = 23) or WT (n = 28) mice 10 weeks after intraperitoneal injection of ID8 mouse ovarian cancer cells (5 × 10⁶). Bars report means ± s.e.m. (e) Metastatic tumor burden in Fabp4^−/− (n = 6) or WT (n = 7) mice 90 d after orthotopic injection of ID8 cells under the ovarian bursa. Bars report means ± s.e.m. (f) Images generated by confocal microscopy of intracellular lipid accumulation (green) in ID8 cells cocultured with visceral adipocytes extracted from Fabp4^−/− or WT mouse adipose tissue (nuclear counterstaining, blue). (g) Summary of metabolic changes that occur in interacting ovarian cancer cells and adipocytes as described in the text.