Cancer Res Treat.  2017 Apr;49(2):338-349. 10.4143/crt.2016.175.

Adipose Stromal Cells from Visceral and Subcutaneous Fat Facilitate Migration of Ovarian Cancer Cells via IL-6/JAK2/STAT3 Pathway

• Kim B
• Kim HS
• Kim S
• Haegeman G
• Tsang BK
• Dhanasekaran DN
• Song YS

• KMID: 2378106
• DOI: http://doi.org/10.4143/crt.2016.175

Abstract

PURPOSE
Adipose stromal cells (ASCs) play an important regulatory role in cancer progression and metastasis by regulating systemic inflammation and tissue metabolism. This study examined whether visceral and subcutaneous ASCs (V- and S-ASCs) facilitate the growth and migration of ovarian cancer cells.
MATERIALS AND METHODS
CD45- and CD31- double-negative ASCs were isolated from the subcutaneous and visceral fat using magnetic-activated cell sorting. Ovarian cancer cells were cultured in conditioned media (CM) obtained from ASCs to determine the cancer-promoting effects of ASCs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, Boyden chamber assay, and western blotting were performed to determine the proliferative activity, migration ability, and activation of the JAK2/STAT3 pathway, respectively.
RESULTS
CM from ASCs enhanced the migration of the ovarian cancer line, SKOV3, via activation of the JAK2/STAT3 signaling pathway. Interestingly, in response to ASC-CM, the ascites cells derived from an ovarian cancer patient showed an increase in growth and migration. The migration of ovarian cancer cells was suppressed by blocking the activation of JAK2 and STAT3 using a neutralizing antibody against interleukin 6, small molecular inhibitors (e.g., WP1066 and TG101348), and silencing of STAT3 using siRNA. Anatomical differences between S- and V-ASCs did not affect the growth and migration of the ovarian cancer cell line and ascites cells from the ovarian cancer patients.
CONCLUSION
ASCs may regulate the progression of ovarian cancer, and possibly provide a potential target for anticancer therapy.

Keyword

Ovarian neoplasms; Adipose tissue; Interleukin-6; Cell movement; Adipose stromal cells

MeSH Terms

Adipose Tissue
Antibodies, Neutralizing
Ascites
Blotting, Western
Cell Line
Cell Movement
Culture Media, Conditioned
Humans
Inflammation
Interleukin-6
Intra-Abdominal Fat
Metabolism
Neoplasm Metastasis
Ovarian Neoplasms*
RNA, Small Interfering
Stromal Cells*
Subcutaneous Fat*
Antibodies, Neutralizing
Culture Media, Conditioned
Interleukin-6
RNA, Small Interfering

Figure

• 

  Fig. 1. ASCs isolated from human subcutaneous and visceral fat. (A) The SVF was isolated from subcutaneous and visceral fat tissue. To remove the endothelial and hematopoietic cells from the SVF, CD31- and CD45-negative ASCs were isolated by MACS. (B) The morphologies of S- and V-ASCs were evaluated by an inverted phase-contrast microscope (×40 objective) at passage 2. (C) The S- and V-ASCs were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages under induction conditions. (D) One hundred S- and V-ASCs were cultured on a 100-mm plate for 7 days, and the number of clones was counted after staining with 0.5% crystal violet. ASC, adipose stromal cell; SVF, stromal vascular fraction; MACS, magnetic- activated cell sorting; S-ASC, subcutaneous adipose stromal cell; V-ASC, visceral adipose stromal cell. a)p < 0.05, S-ASC vs. V-ASC.

• 

  Fig. 2. S- and V-ASCs express specific surface markers of MSCs. The S- and V-ASCs were labeled with CD45, CD73, CD90, and CD166 antibodies conjugated to PE and with CD31, CD34, CD105, and HLA-DR antibodies conjugated to FITC at passage 3. The labeled ASCs were analyzed by flow cytometry. IgG1κ-conjugated to PE and to FITC were used as the isotype controls. Both S- and V-ASCs expressed CD90, CD166, CD73, and CD105, but did not express CD45, CD31, CD34, and HLA-DR. S-ASC, subcutaneous adipose stromal cell; V-ASC, visceral adipose stromal cell; MSC, mesenchymal stem cell; PE, phycoerythrin; FITC, fluorescein isothiocyana.

• 

  Fig. 3. Conditioned media from S- and V-ASCs promote the migration of ovarian cancer cells. (A) SKOV3 cells were cultured in S-ASC–CM and V-ASC–CM for 24, 48, 72, and 96 hours. The relative cell growth of the SKOV3 cells in S-ASC–CM and V-ASC–CM compared to the control medium was determined using an MTT assay. (B) The migration of SKOV3 cells was determined using a Boyden chamber assay. (C) The wound healing ability of SKOV3 cells was verified by measuring the wound closure distance in 12 hours. S-ASC, subcutaneous adipose stromal cell; V-ASC, visceral adipose stromal cell; CM, conditioned media; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; S, S-ASC–CM; V, V-ASC–CM. a)p < 0.05, control vs. S vs. V.

• 

  Fig. 4. S- and V-ASCs enhance the migration of ovarian cancer cells through an IL-6–mediated pathway. (A) Messenger RNA expression of adipokines including IL-6, TNF-α, adiponectin, and leptin was measured by RT-PCR. GAPDH was used as a reference gene. (B) An ELISA assay was conducted to detect the secretory IL-6 present in the control medium, S-ASC– CM, V-ASC–CM, and subcutaneous and visceral differentiated adipocytes (S- and V-adipo–CM). Complete medium was used as a control. (C) SKOV3 cells were cultured in S-ASC–CM and V-ASC–CM with or without anti–IL-6 Ab (500 ng/mL). The migration ability was determined using a Boyden chamber assay. Control medium with rh–IL-6 (50 ng/mL) was used as a positive control. S-ASC, subcutaneous adipose stromal cell; V-ASC, visceral adipose stromal cell; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; CM, conditioned media; rh, recombinant human; S, S-ASC–CM; V, V-ASC–CM; ns, not significant; Ctrl, control. a)p < 0.05.

• 

  Fig. 5. Activation of JAK2 and STAT3 in response to IL-6 from ASCs increases the migration of ovarian cancer cells. (A) The total JAK2, p-JAK2 (Y1007), total STAT3, and p-STAT3 (Y705) were determined by western blot analysis. (B) After the depletion of IL-6 using anti–IL-6 Ab (500 ng/mL), the total JAK2, p-JAK2, STAT3, and p-STAT3 were analyzed by western blot. The control medium with rh–IL-6 (50 ng/mL) was used as a positive control. (C) After the SKOV3 cells were incubated in S-ASC–CM and V-ASC–CM with the addition of 2 μM WP1066 or 1 μM TG101348 for 12 hours, a Boyden chamber assay was conducted. (D) Silencing of STAT3 using siRNA reduces the migration of SKOV3. SKOV3 was transfected with Scrb RNA (100 nM) and siRNA for STAT3 (STAT3-siRNA1 and 2, 100 nM), and cultured in S-ASC–CM and V-ASC–CM for the migration assay. The expression of p-STAT3 and total STAT3 was inhibited after the silencing of STAT3 using siRNA. (E) After the inhibition of STAT3 using siRNA2, the migratory cells were determined by a Boyden chamber assay. (F) After treating SKOV3 with S-ASC–CM and V-ASC–CM containing either 1 μM WP1066 or 2 μM TG101348, the inhibition of p- JAK2, total JAK2, p-STAT3, and total STAT3 protein levels was confirmed by western blot analysis. IL-6, interleukin 9; ASC, adipose stromal cell; rh, recombinant human; SASC, subcutaneous adipose stromal cell; V-ASC, visceral adipose stromal cell; CM, conditioned media; Scrb RNA, scrambled RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. a)p < 0.05, mock vs. WP vs. TG in S-ASC– CM, b)p < 0.05, in V-ASC–CM.

• 

  Fig. 6. ASCs enhance the proliferation and migration of ovarian cancer cells isolated from the ascites of an ovarian cancer patient through activation of the IL-6/JAK2/STAT3 pathway. (A) Ascites cells were cultured in S-ASC–CM and V-ASC– CM for 7 days. The proliferation of ascites cells was determined by an MTT assay. (B) Ascites cells were cultured in ASCCM with or without anti–IL-6 Ab (500 ng/mL), and in the control medium with rh–IL-6 (50 ng/mL) and/or anti–IL-6 Ab. After culturing for 24 hours, the number of migratory cells were counted. (C) The ascites cells cultured in ASC-CM for 24 hours were treated with WP1066 and TG101348. Migration was determined using a Boyden chamber assay. ASC, adipose stromal cell; IL-6, interleukin 9; S-ASC, subcutaneous adipose stromal cell; V-ASC, visceral adipose stromal cell; CM, conditioned media; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; rh, recombinant human; Ctrl, control; S, S-ASC–CM; V, V-ASC–CM. a)p < 0.05.

Reference

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