Tissue harvesting and culture.
Patients consented for tissue donation in accordance with a protocol approved by the University of Michigan’s IRB (IRB no. HUM0009149). Fourteen different primary CA-MSC cell lines were generated. CA-MSC lines were isolated from Stage IIIc or Stage IV high-grade papillary-serous or poorly differentiated ovarian (n
= 10), fallopian tube (n
= 2), or primary peritoneal (n
= 2) carcinomas. Tumor samples were obtained from the operating room and immediately taken to the laboratory for processing. Tissue was maintained in RPMI/10% FBS throughout processing. A portion of the tumors were processed to single cell suspensions as previously described (57
). Briefly, a portion of each specimen was mechanically dissected and filtered, red cells were lysed with ACK buffer (Lonza Walkersville Inc.), and cells were then washed with media containing serum and plated in MEBM (Lonza) supplemented with 10% heat-inactivated FBS, 1X B27, 20 ng/ml EGF, 1 ng/ml hydrocortisone, 5 μg/ml insulin, 100 μM β-mercaptoethanol, 10 ng/ml β-FGF, 1% penicillin/streptomycin, and 20 μg/ml gentamicin. Alternatively, a fresh 4 × 4 × 2–mm tumor slice was rinsed several times to remove all loosely attached cells. The tissue was then placed in a tissue culture dish in a thin layer of MEBM and covered with a sterile cover glass to increase tissue contact with the dish. After 24 hours, the tissue was removed, and adherent cells were washed aggressively and cultured in MEBM. When CFU-F were apparent from either culture method, the cells were trypsinized and replated in limiting dilutions in MEBM. Individually arising CFU-F were then isolated and expanded in traditional mesenchymal media. Phenotypes of CFU-F from the 2 approaches were similar.
In order to isolate epithelial cells, whole tumor cell suspensions were plated in MesenCult MSC Basal Medium for Human Mesenchymal Stem Cells with Mesenchymal Stem Cell Stimulatory Supplements (StemCell Technologies). Under these conditions, tumor cells soon dominated the culture. Cells were then trypsinized and replated in limiting dilutions, and then individual epithelial clones were easily identified and isolated.
Ovarian cancer cell lines SKOV3, A2780, and Hey1 were obtained from ATCC and cultured in RPMI with 10% FBS (Invitrogen). As MSC-positive controls, normal healthy donor, adipose-derived MSCs were obtained (Invitrogen). BM-MSCs were provided by Max Wicha (University of Michigan).
The following antibodies were used for flow cytometric analysis of putative MSCs: anti-CD133/2-APC (Miltenyi Biotec); anti–CD44-PE and anti–CD44-FITC (BD Pharmingen); anti–hCD90-APC and anti–hCD90-PE (R&D Systems); anti–CD105-FITC and anti–CD105-APC (Abcam); anti–CD45-FITC, anti–CD45-APC, and anti–CD45-PE (BD Pharmingen); anti-CD34 (BD Pharmingen); anti-SH3 (BD Pharmingen); and anti-CD144 (VE-cadherin)-PE (eBioscience). The Aldefluor assay was performed as per the manufacturer’s instructions (StemCell Technologies). Flow cytometric analysis was performed on a BD Biosciences FACSCalibur through the University of Michigan Flow Cytometry Core, and data analysis was performed using FlowJo (Tree Star Inc.).
For bone differentiation, cells were plated at 5 × 104 cells/well of a 6-well plate in either StemPro Osteogenesis Differentiation Media (Invitrogen) or control media and then allowed to grow for approximately 14 days, with media changed twice per week. Cells were then rinsed with PBS, fixed with 3.7% formaldehyde, rinsed with water, stained with 2% Alizarin Red S (Sigma-Aldrich) solution (pH 4.2) for 2 minutes, and washed with distilled water. For cartilage differentiation, cells were plated in 5- to 10-μl micromass droplets onto a dry plate from a solution of 1.6 × 107 cells/ml, allowed to set for 2 hours, and then incubated in either StemPro Chondrogenesis Differentiation Media (Invitrogen) or control media. Cells were allowed to grow for approximately 14 days with media changed twice per week and then rinsed with PBS, fixed with 4% formaldehyde, rinsed with PBS, stained for 30 minutes with 1% Alcian Blue solution (Sigma-Aldrich) prepared in 0.1 N HCl, and washed with 0.1 N HCl. For adipose differentiation, cells were plated at 5 × 104 cells/well of a 6-well plate, in either differentiation media (StemCell Technologies) or control media, and the media was changed weekly. After 14 to 21 days, cells were fixed with 3.7% formaldehyde, stained with 0.3% Oil Red O (Fisher Scientific) for 1 hour, and washed with water.
CA-MSCs were cultured under conditions as described above, and then once they had reached 60%–70% confluence in the log phase of growth, they were fixed in buffer provided by Cell Line Genetics, to whom cells were then provided for commercial karyotyping using colcemid.
Isolation of single cell clones.
Cells were trypsinized and then resuspended in limiting dilutions of MesenCult media (~1 cell/50 μl, StemCell Technologies). Cells were then plated in 96-well dishes, and single cells were confirmed by microscopy. Clones were allowed to grow until they were confluent and were then trypsinized, passaged to a 24-well plate, allowed to grow to confluence, and passaged a second time. Only the colonies that successfully passaged twice were deemed true clones.
SKOV3-dsRed or A2780-dsRED tumor cells were plated with MSCs at 1:1 with a mixed medium (50:50, RPMI/MEBM), grown for 24 hours, and then treated with Noggin at 200 ng/ml or mock treated. Cultures were then observed daily under light microscopy. Cells were trypsinized and resuspended in PBS for FACS analysis performed with the antibodies and method as described above. Alternatively, spheres present in the supernatant were collected by 3 rounds of gentle washing, trypsinized, and then analyzed by FACS as above.
PCR array and RT-PCR.
MSCs were cultured with complete medium to 80% confluence and washed once with ice-cold PBS, before being homogenized in 3 ml TRizol reagent (Invitrogen). Total RNA was extracted with the PureLink Total RNA Purification System (Invitrogen) with on-column DNase treatment. RNA quality was examined on a Bioanalyzer 2100 (Agilent). We used a human MSC PCR array (SABiosciences), containing 84 target genes involved in MSC pluripotency, differentiation, and self-renewal. For the PCR array, first-strand cDNA was synthesized with the SABiosciences RT2
First Strand Kit; for all other real-time PCR experiments, first-strand cDNA was synthesized with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s recommendations. Two μg total RNA was used for each reaction. SYBR green–based array PCR was performed using the 7900 HT Sequence Detection System (Applied Biosystems); the other real-time PCR was performed using the StepOne System (Applied Biosystems) and the indicated primer (Supplemental Table 1). The comparative Ct method was used for data analysis described at RT2
Profiler PCR Array Data Analysis (SABiosciences;
To detect BMP2 protein expression in MSCs, cells were grown in 60-mm dishes to 80% confluence. Protein transport inhibitor GolgiPlug (BD Science) was added at 1 μl/ml for 4 hours. To detect phospho-SMAD 1/5 after BMP2 (R&D Systems) treatment, MSCs were grown in 60-mm dishes to 60% confluence with complete medium and then serum starved for 16 hours. BMP stimulation was carried out at 200 ng/ml for 30 minutes. SKOV3 cells were also stimulated with or without 50% MSC-conditioned medium for 30 minutes. Cells were washed 2 times with ice-cold PBS before lysis in 200 μl RIPA buffer (Invitrogen) with complete proteinase inhibitor and phosphatase inhibitor (Roche). Insoluble material was removed by centrifugation at 16,000 g at 4°C for 15 minutes. Protein concentrations were determined using the Bradford Protein Assay Kit (Bio-Rad). Lysates containing 10 μg total protein were separated on 4%–12% NuPAGE SDS gel (Invitrogen) and transferred onto a nitrocellulose membrane. Antibodies used for Western blot analysis include anti-BMP2 (1:800 dilution, Abcam), anti–p-SMAD 1/5 and anti-SMAD 5 (1:1,000 dilution, Cell Signaling Technology), and anti–β-actin (1:10,000 dilution, Sigma-Aldrich). Bands were visualized using the ECL Kit (Pierce, Thermo Scientific).
In vitro MSC-conditioned medium.
1 × 106 control MSCs or CA-MSCs were plated in 10-cm dishes with MEBM supplemented with 10% FBS as mentioned above, grown to 50% confluence, and then switched to a plain MEBM plus 5% charcoal-stripped FBS. After 2 days, the MSC medium was harvested, filtered (0.2-μm filter), mixed with RPMI 1640 supplemented with 10% FBS (1:1, v/v), and then plated at 40% confluence with the ovarian cancer tumor cells SKOV3, Hey1, or A2780 for 3 days. Tumor cells were washed, trypsinized, and resuspended in PBS for FACS analysis or lysed in TRIzol reagent for total RNA extraction.
In vitro tumor-conditioned medium.
5 × 105 SKOV3 or Hey1 cells were plated in 10-cm dishes, grown to 40% confluence in RPMI 1640 supplemented with 10% FBS, and then switched to RPMI 1640 containing 5% charcoal-stripped FBS. Two days later, the tumor medium was collected, filtered on a 0.2-μm filter, mixed with 50% complete MEBM growth medium, and then plated at 40% confluence of the MSCs for 3 days. MSCs were washed and lysed in TRIzol reagent for total RNA extraction.
Human tumor sphere assay.
1 × 106 freshly isolated human ovarian tumor cells were plated in ultralow-adherence plates in MEBM supplemented with 20 ng/ml EGF, 5 μg/ml insulin, and 10 ng/ml β-FGF. After 24 hours, cells replicates were treated with 100 ng Noggin (R&D Systems). After 72 hours, fresh media with or without Noggin was added, and then after an additional 72 hours, spheres were mechanically separated using a syringe and ultralow-dose trypsin (0.05%) and analyzed via flow cytometry as described above.
NOD/SCID mice were obtained from Charles River Laboratories. Animals were maintained in accordance with institutional policies, and all studies were performed with approval of the University Committee on Use and Care of Animals of the University of Michigan. To generate tumors, 1 × 106 tumor cells were injected in 300 μl of growth factor–reduced Matrigel (BD Biosciences) into the axillae of immunodeficient mice. 5 × 105 tumor cells with 5 × 105 MSCs were injected with Matrigel in the contralateral axilla. All studies were done using early-passage ovarian cancer MSCs (passage 3–8). Tumor growth was measured using calipers, and volumes were calculated based on the modified ellipsoid formula (L × W × W/2), where L represents length, and W represents width.
SKOV3 tumor cells were stably transduced with a luciferase-expressing lentivirus (plentiloxEV-Luc virus, provided by the vector core at the University of Michigan). The coinjection experiments were carried out with 1 × 106 SKOV3-Luc cells alone (n = 10) or a 1:1 mixture of control MSCs (n = 10) or CA-MSCs (n = 10). Bioluminescence optical imaging (Xenogen IVIS 2000, Caliper Life Sciences) was first obtained 7 days after tumor cell injection and then measured every other day thereafter for 10 days. Ten minutes prior to imaging, each mouse was given an i.p. injection with 100 μl coelenterazine in PBS at 40 mg/ml. During the imaging, general anesthesia was given with 2% isoflurane (IsoSol, Medeva Pharmaceuticals Inc.). Luminescence images were acquired for 3 seconds to 1 minute. The optical signal was expressed as radiance in units of photons/second/centimeter2 (p/s/cm2).
For the in vivo Noggin experiments, cells were pretreated with or without Noggin (R&D Systems) at 200 ng/ml for 60 minutes and injected into contralateral axillae with 200 ng Noggin. Mice were treated with PBS or Noggin (200 ng) subcutaneously every 48 hours for 5 doses. Mice were imaged as above starting at day 7 and every 48–72 hours until sacrifice. For Western blot analysis, a subset of tumors were treated with Noggin daily for 48 hours prior to harvesting tumors. Tumors were collected 4 hours after the final treatment, and a portion of each tumor was snap frozen and then lysed in RIPA buffer with phosphatase inhibitors and protease inhibitors.
A portion of each tumor was fixed in 10% formalin for 2 hours at room temperature and then transferred to 70% ethanol. Tumors were then paraffin embedded and stained at the histology core at the University of Michigan using EDTA-based antigen retrieval and mouse anti-ALDH antibody (BD Biosciences, clone 44/ALDH; 1:100) or anti-Ki67 antibody (Abcam no. 15580, 1:2,000). For stain quantification, 8–10 sections from 4 to 5 tumors per treatment group were analyzed. For ALDH, cells were counted from 10 high-power fields per section (×400). For Ki67, cells were counted from 4 low-power fields (×100) per section. Counts were then compared using a 2-sided Student’s t test.
P values of less than 0.05 were considered statistically significant.