Gynecologic Oncology 132 (2014) 203–210
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Monoclonal antibody-based immunotherapy of ovarian cancer: Targeting ovarian cancer cells with the B7-H3-specific mAb 376.96☆,☆☆ Janelle M. Fauci a,⁎,1, Francesco Sabbatino b,1, YangYang Wang b, Angelina I. Londoño-Joshi c, J. Michael Straughn Jr. a, Charles N. Landen a, Soldano Ferrone b, Donald J. Buchsbaum d a
University of Alabama at Birmingham, Department of Obstetrics and Gynecology, USA Massachusetts General Hospital, Harvard Medical School, Department of Surgery, USA University of Alabama at Birmingham, Division of Molecular and Cellular Pathology, USA d University of Alabama at Birmingham, Department of Radiation Oncology, USA b c
H I G H L I G H T S • mAb 376.96 defined B7-H3 epitope is expressed by chemosensitive and chemoresistant ovarian cancer cells. • mAb 376.96 enhances cytotoxicity of Sunitinib in vitro.
a r t i c l e
i n f o
Article history: Received 7 August 2013 Accepted 31 October 2013 Available online 9 November 2013 Keywords: Ovarian cancer Immunotherapy Chemoresistance Cancer initiating cells B7-H3 Sunitinib
a b s t r a c t Objective. The high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs. Methods. Eight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs. Results. The mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs. Conclusion. The mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted. © 2013 Elsevier Inc. All rights reserved.
Introduction ☆ Data presented at the University of Pittsburgh Cancer Institute's Ovarian Cancer Symposium (May, 2012). ☆☆ Grant support: This work was supported by PHS grants RO1CA138188 and RO1CA110249 awarded by the National Cancer Institute and by a Susan Komen Post Doctoral Fellowship KG111486 awarded by the Susan G. Komen for the Cure Foundation. ⁎ Corresponding author at: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Alabama at Birmingham, 1700 6th Avenue South, Birmingham, AL 35233, USA. Fax: +1 205 975 6174. E-mail address: [email protected] (J.M. Fauci). 1 Contributed equally to this work. 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.10.038
Over 15,000 women will die of ovarian cancer in 2013 [1]. The majority of patients have advanced stage disease at diagnosis, and although modern surgical intervention and chemotherapy protocols allow most ovarian cancer patients to achieve clinical remission, most tumors will recur [2–4]. One proposed mechanism for this pattern is the survival of subpopulations of chemoresistant cancer cells, known as cancer initiating cells (CICs) [5]. CICs represent the most tumorigenic and treatment resistant subpopulation of tumor cells [6,7]. Several cell surface markers have been
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used to identify CICs, including CD133 and CD44. Aldehyde dehydrogenase-1A1 (ALDH) has also been reported to be a valid CIC marker [5,6,8–14]. ALDH positive CICs form 3-dimensional spheres more readily than their ALDH negative counterparts, and are more tumorigenic in murine xenograft models [15,16]. Additionally, CICs are more resistant to standard chemotherapy treatment, in both in vitro and in vivo studies [5]. The chemoresistance of CICs is likely to be responsible for the high relapse rate in ovarian cancer patients. This warrants the investigation of novel therapeutics to be used in combination with standard chemotherapy to increase cytotoxic activity against chemoresistant cells. One potential therapeutic agent is represented by B7-H3-specific monoclonal antibodies (mAb). B7-H3 is a member of the B7superfamily of ligands, and is believed to play a role in down-regulation of T-cells involved in tumor immunity [17,18]. It is highly expressed in a variety of cancer types, including breast, pancreatic, and ovarian cancer [19–25]. High B7-H3 expression correlates with poor survival in ovarian cancer patients. In a study by Zang and colleagues, 93% of the 77 ovarian carcinomas studied expressed B7-H3 and was associated with increased rates of recurrence and mortality. Furthermore, B7-H3 expression in tumor vasculature also correlated with poor survival, suggesting that this ligand may play a role in tumor cell migration [24]. Sunitinib is a potent tyrosine kinase inhibitor that has proven its efficacy in renal cell carcinoma, and is approved by the Food and Drug Administration (FDA) for use in renal cell carcinoma and for treatment of gastrointestinal stromal tumors (GIST). Recent data have shown that some types of ovarian cancer, such as ovarian clear cell carcinoma, show a remarkable similarity in gene expression profiles across organs such as kidney, lung, and ovary [26]. Currently, Sunitinib is being investigated as part of a large, phase II randomized trial by the Gynecologic Oncology Group (GOG) in the treatment of patients with clear cell ovarian cancer [27]. The objective of the present study was to evaluate the effect of the mAb 376.96 defined B7-H3 epitope in combination with chemotherapy and the tyrosine kinase inhibitor Sunitinib on the chemosensitive and chemoresistant ovarian cancer cells and CICs. Materials and methods Monoclonal and polyclonal antibodies The B7-H3-specific mouse mAb 376.96, an IgG2a, was developed and characterized as described [28]. The specificity of mAb 376.96 for B7-H3 was proven by its specific reactivity with purified B7-H3 in binding assays and by mass spectrometry analysis of the molecule immunoprecipitated by this mAb from cultured human melanoma cells. The isotype matched anti-idiotypic mAb F3-C25 was developed and characterized as described [29]. mAb was purified from ascitic fluid by affinity chromatography on Protein A. The activity and purity of mAb preparations were assessed by reactivity with the corresponding antigens in binding assays and by SDS-PAGE analysis, respectively. Isotype IgG2a κ murine myeloma control and conjugated goat antimouse IgG antibodies (1 μg/mL) were purchased from Sigma-Aldrich (St. Louis, MO). PE-labeled anti-mouse IgG xenoantibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Drugs and reagents Carboplatin, paclitaxel and docetaxel were purchased from SigmaAldrich. Carboplatin was prepared as a 25 mM stock solution in sterile H2O, while paclitaxel and docetaxel were prepared in DMSO as a 10 ηM and a 12.5 ηM stock solution, respectively. Sunitinib was purchased from Sigma-Aldrich and prepared as a 25 mM stock solution in DMSO. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich. ALDEFLUOR kits including ALDEFLUOR-488 and diethylaminobenzaldehyde (DEAB) negative
control were obtained from StemCell Technologies (Durham, NC). Alexa fluor 488 goat anti-rabbit IgG antibody was purchased from Invitrogen (Carlsbad, CA). Cells and cell culture Ovarian cancer cell lines ES-2, OVCAR3, CaOV3, HEY-A8, SKOV3.ip1 and A2780 were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to the supplier's directions. Taxane-resistant SCOV3TRip2 and platinum-resistant A2780cp20 cells were obtained from Dr. Anil Sood (M.D., Anderson Cancer Center, Houston, TX). SKOV3TRip2 cells were developed from SKOV3TR cells obtained from Michael Sieden (M.D., Anderson Cancer Center, Houston, TX) by culturing selected tumors growing in mice to select highly tumorigenic cells [30]. SCOV3TRip2 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (HyClone, Logan, UT) and paclitaxel 150 ηmol/L. A2780cp20 cells were maintained in the same conditions as chemosensitive A2780 cells. All cell lines were maintained in antibiotic-free medium at 37 °C in a 5% CO2 atmosphere and routinely screened for Mycoplasma contamination. Ovarian cancer tumor specimens Ovarian cancer tumors were obtained from ten patients undergoing primary debulking surgery for advanced ovarian cancer by surgeons within the Gynecologic Oncology division at the University of Alabama at Birmingham. All tumors were papillary serous histology from patients with stage 3 disease. Immunohistochemical staining of frozen ovarian tumor tissue sections with mAb 376.96 Frozen sections of ovarian cancer tumors from 10 patients were stained with the B7-H3-specific mAb 376.96. Tissue sections were post-fixed in 4% paraformaldehyde (PFA), treated with 3% hydrogen peroxide, 1% bovine serum albumin (BSA) (Invitrogen), and 5% normal horse serum in Tris-buffered saline (25 mM Tris [pH 7.4], 150 mM NaCl) and incubated in a closed humid chamber overnight at 4 °C with the B7-H3-specific mAb 376.96 (5 μg/mL). Immunohistochemical (IHC) signals were generated with EnVision1System-HRP (Dako North America, Inc) and substrate diaminobenzidine (Dako North America, Inc). Tissue sections were counterstained with Mayer Hematoxylin (Sigma-Aldrich), dehydrated with ethyl alcohol, incubated in 100% xylene, and mounted in Canada balsam (Sigma-Aldrich). Frozen sections derived from the cell lines MV3 (B7-H3+) and Raji (B7-H3−) were simultaneously stained as positive and negative controls respectively, to determine the specificity of the staining obtained with mAb 376.96 (data not shown). Cytoplasmic membrane staining was considered positive for mAb 376.96 defined B7-H3 epitope. The staining intensity was graded negative, weak and strong by semiquantitative analysis. Flow cytometry analysis of cell lines stained with mAb Chemosensitive ovarian cancer cell lines ES-2, Hey-A8, OVCAR3, CAOV3, SKOV3.ip1, and A2780 and chemoresistant ovarian cell lines SCOV3TRip2 and A2780cp20 were harvested at 80% confluency using Cell Stripper (Mediatech, Manassas, VA) to prevent cleavage of the B7H3 epitope. Tubes containing 2.1 × 106 cells/mL were washed in PBS supplemented with 1% BSA and azide (PBS/BSA + azide), and centrifuged at 1000 rpm for 5 min. The cell pellets were resuspended in 100 μL PBS/BSA + azide. Isotype control antibody IgG2a κ, murine myeloma (0.5 μg/mL) was added to the control tube, and mAb 376.96 (5 μg/mL) was added to the sample tube and incubated on ice for 30 min. Cells were washed in PBS/BSA + azide and centrifuged at 1000 rpm for 5 min. Cells from isotype control, secondary antibody control and sample tubes were incubated with secondary antibody,
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R-Phycoerythrin (RPE) labeled goat anti-mouse IgG antibodies (Zenon, Eugene, OR) (5 μg/mL) in a dark room on ice for 30 min. Cell suspensions were then washed and resuspended in 0.5 mL PBS/BSA + azide. All samples were analyzed on a LSRII flow cytometer (BD Biosciences, San Jose, CA). Data was evaluated using FlowJo software (Tree Star, Inc., Ashland, OR).
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Growth inhibition assays using MTT assay Chemoresistant cell lines A2780cp20 and SCOV3TRip2 and chemosensitive cell line SKOV3.ip1 were seeded in 96-well tissue culture-treated plates (Costar) at 2500 cells/100 μL RPMI 1640 medium with 2% FCS. A2780cp20 and SCOV3TRip2 cells were treated
Fig. 1. B7-H3 expression on chemosensitive and chemoresistant ovarian cancer cell lines. (A) Ovarian cancer cell lines were stained with mAb 376.96 and analyzed by flow cytometry. Cells expressing B7-H3 bound by mAb 376.96 are depicted as the bold line to right, as compared to isotype control antibody, represented as the thin line to the left. (B) Similar testing for expression of B7-H3 was confirmed on chemosensitive and chemoresistant subclones. (C) Chemosensitive and resistant cell lines were treated with chemotherapy or Sunitinib and then analyzed for B7-H3 expression.
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with 0.25, 0.5 or 1 mg/mL mAb 376.96 and SKOV3.ip1 cells were treated with mAb 376.96 (0.25 or 0.5 mg/mL) ± Sunitinib (1.5 μM) for 3 days at 37 °C in a 5% CO2 atmosphere. mAb F3-C25 (0.5 mg/mL) was used as an isotype control. DMSO vehicle concentration of Sunitinib was maintained at 0.02% in all wells. Cell proliferation was evaluated by MTT assay. Data are expressed as percent of growth inhibition of treated cells compared with the untreated control cells as reference for 100% cell growth.
Supplementary Fig. 1 (panels A and B, respectively). The staining is specific, since no staining of the ovarian cancer lesions by the isotype control mAb F3-C25 was detected. A representative example is shown in Supplementary Fig. 1 (panel C).
A
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Cell viability assays using ATPLite assay ATP (% control)
Cell viability was determined by measurement of cellular ATP levels, using the ATPLite luminescence-based assay (Perkin Elmer Biosciences, Meriden, CT) as described elsewhere [31]. Cells were lysed using 25 μL/well mammalian cell lysis solution and 25 μL/well of lyophilized substrate. Plates were sealed, gently mixed and luminescence was measured using a TopCount NXT (Perkin Elmer Biosciences).
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Identification of CICs utilizing high ALDH expression
SKOV3.ip1 cells were incubated with ALDEFLUOR ± DEAB inhibitor and stained with mAb 376.96 and PE-labeled anti-mouse IgG xenoantibody to identify cells expressing high levels of ALDH activity (ALDHbright) and mAb 376.96 defined B7-H3 epitope. mAb F3-C25 was used as an isotype control. ALDHbright cells were identified as those ALDH+ cells with twice the mean fluorescence intensity (MFI) of the ALDH+ cell population.
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To exclude that the detection of the mAb 376.96 defined B7-H3 epitope on ovarian cancer cell lines does not represent an in vitro artifact, but reflects the phenotype of ovarian cancer tumors in vivo, frozen sections of ovarian cancer lesions from 10 patients were stained with mAb 376.96. All of the lesions were positive. The staining intensity was strong in 7 and weak in 3. Representative examples are shown in
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mAb 376.96 defined B7-H3 epitope expression in surgically removed ovarian cancer tumors
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Results
Flow cytometry analysis of ovarian cancer lines CAOV3, ES-2, Hey-A8 and OVCAR3, and chemoresistant subclones A2780cp20 and SCOV3TRip2 sequentially incubated with the mAb 376.96 defined B7-H3 epitope and PE-labeled anti-mouse IgG xenoantibody detected a strong, homogeneous staining of all cell lines. The staining is specific, since no staining was detected in lines incubated with the isotype matched anti-idiotypic mAb F3-C25. These results indicate that the mAb 376.96 defined B7-H3 epitope is highly expressed on both chemosensitive and chemoresistant ovarian cancer cell lines (Fig. 1).
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B7-H3 expression on ovarian CICs by flow cytometry
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Chemosensitive ovarian cancer cell lines ES-2, OVCAR3, CaOV3, HEY-A8, SKOV3.ip1 and A2780 and chemoresistant ovarian cell lines A2780cp20 and SCOV3TRip2 were harvested with 1 mL of PBS/ ethylenediaminetetraacetic acid (EDTA) collected by centrifugation at 1000 rpm for 5 min, and resuspended in Hank's balanced salt solution (HBSS). ALDEFLUOR assay (StemCell Technologies, Vancouver, British Columbia) was employed and used according to the manufacturer's protocol. As a control, cells were resuspended in ALDEFLUOR assay buffer containing diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH activity. All samples were analyzed on an LSRII flow cytometer and data were evaluated using FlowJo software.
carboplatin carboplatin paclitaxel 25µM 100µM 2.5 nM
carbopaltin carboplatin paclitaxel 25µM 100µM 2.5 nM
Fig. 2. Chemosensitivity of parental and chemoresistant cell lines. (A) SKOV3ip1, and taxane-resistant SCOV3TRip2 cells were plated in 96-well plates at 2500 cells/well in attached conditions on Day 0. Carboplatin (range, 0–100 μM) or paclitaxel (range, 0–300 nM) were added on Day 1. ATP assay was used to assess cell viability on Day 4, and data is presented as a mean of 8 replicates. Error bars denote SE. (B) A2780 and platinum-resistant A2780cp20 cells were plated in 96-well plates at 2500 cells/ well in attached conditions on Day 0. Carboplatin (range, 0–100 μM) or paclitaxel (range, 0–300 nM) were added on Day 1. ATP assay was used to assess cell viability on Day 4, and data is presented as a mean of 8 replicates. Error bars denote SE.
Modulation by chemotherapy of mAb 376.96 defined B7-H3 epitope expression on ovarian cancer cell lines To test the effect of chemotherapeutic agents on mAb 376.96 defined B7-H3 epitope expression on ovarian cancer cell lines, the SKOV3.ip1 and SCOV3TRip2 cells (1.0 × 106/well) were incubated with Sunitinib (5 μM) or paclitaxel (25 nM) for 3 days at 37 °C in a 5% CO2 atmosphere. Cells were then isolated, stained with mAb 376.96 and analyzed using flow cytometry. The MFI was examined (Fig. 1C) and found to be highest after treatment with paclitaxel. Treatment of SKOV3.ip1 cells with the same dose of paclitaxel resulted in a 1.7-fold increase in mAb 376.96 defined B7-H3 epitope MFI, while treatment of taxaneresistant SCOV3TRip2 cells with 100 nM paclitaxel resulted in a 0.8fold decrease in the MFI. Similar results were obtained when SKOV3.ip1 and SCOV3TRip2 cells as well as A2780 and A2780cp20 cells were treated with carboplatin (25 μM) for 3 days at 37 °C (data not shown). Anti-proliferative effect of mAb 376.96 and Sunitinib on the chemoresistant cell lines A2780cp20 and SCOV3TRip2 Platinum-resistant A2780cp20 cells and the parental cells A2780, as well as the taxane-resistant SCOV3TRip2 cells and the parental cells SKOV3.ip1 were plated in 96-well plates at 2500 cells/well. Cells were treated with various doses of carboplatin (range, 0–100 μM) or paclitaxel (range, 0–300 nM) on day 1. Following a 3-day incubation at 37 °C in a 5% CO2 atmosphere, cell viability was determined by measurement of cellular ATP levels. A2780cp20 cells were resistant to carboplatin treatment, even at doses as high as 100 μM (60.3% viable, compared to A2780: 20.3% viable at 100 μM). Additionally, SCOV3TRip2 cells were strongly resistant to taxane treatment, as there was no cell death after treatment with 300 nM of paclitaxel, while only 31.8% of SKOV3.ip1 cells were viable after treatment with 100 nM paclitaxel (Fig. 2). Chemoresistant SCOV3TRip2 and A2780cp20 cells were treated with mAb 376.96 at various doses (range, 0.25–1.0 mg/mL) and compared to treatment with isotype control mAb F3-C25 (Fig. 3). Monotherapy with mAb 376.96 (1 mg/mL) resulted in nearly 50% growth inhibition of SCOV3TRip2 cells, and over 30% inhibition of A2780cp20 cell growth. Monotherapy with Sunitinib resulted in a 10% inhibition of SKOV3.ip1 cell growth, while combination treatment with Sunitinib and mAb 376.96 resulted in greater than 20% inhibition of cell growth (Fig. 4).
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Inhibition of cell growth (%)
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0.25
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Fig. 4. mAb 376.96 enhances the ability of Sunitinib to inhibit growth of SKOV3.ip1 ovarian cancer cells in vitro. SKOV3.ip1 cells (2500/well) were seeded in a 96-well plate and treated with mAb 376.96 and/or Sunitinib (1.5 μM) for 3 days at 37 °C in a 5% CO2 atmosphere. Cells were then tested by MTT assay. The results are expressed as % inhibition of cell growth, utilizing the values obtained for untreated cells, as a reference. Triplicates were performed in each condition.
or 100 μM (A2780cp20), Sunitinib (5 μM) and/or mAb 376.96 (0.5 mg/mL) for 3 days at 37 °C. Cells were harvested and six 10 μL aliquots were removed and plated at 10 μL/well in a 96-well plate for analysis with ATPLite luminescence-based assay. Monotherapy with mAb 376.96 resulted in decreased cell viability compared to controls; this effect was most pronounced in the platinum resistant A2780cp20 cell line. The effect of mAb 376.96 on cell viability was not enhanced by Sunitinib and carboplatin (Fig. 5). Content of CICs in ovarian cancer cell lines Ovarian cancer cell lines ES-2, OVCAR3, CaOV3, HEY-A8, SKOV3.ip1 and A2780 and chemoresistant cell lines A2780cp20 and SCOV3TRip2
Effect of mAb 376.96 in combination with carboplatin and/or Sunitinib on chemosensitive and chemoresistant cells
Inhibition of cell growth (%)
Chemosensitive A2780 cells and platinum-resistant A2780cp20 cells (1 × 106 cells/well) were incubated with carboplatin 25 μM (A2780)
Fig. 3. mAb 376.96 inhibits growth of chemoresistant ovarian cancer cells in vitro. Cells were seeded in 96-well plates at 2500 cells/well and incubated with various concentrations of mAb 376.96 for 3 days. Monotherapy with mAb 376.96 resulted in inhibition of cell growth, compared to control mAb F4-C25.
Fig. 5. Cytotoxicity of ovarian cancer cells. 1 × 106 cells/well were plated in a 6-well plate with 2 mL media on Day 0. On Day 1, carboplatin at 25 μM (A2780) or 100 μM (A2780cp20) ± Sunitinib (5 μM) ± mAb 376.96 (0.5 mg/mL) were added. On Day 3, cells were harvested and six 10 μL aliquots were removed and plated in a 96-well plate for immediate analysis with ATPLite. Data represent mean of 6 replicates, and error bars denote SE.
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A DEAB + ALDEFLUOR
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Fig. 6. mAb 376.96 enhances ability of Sunitinib to decrease proportion of CIC in A2780 and SKOV3.ip1 cells in vitro. (A) SKOV3.ip1 cells were incubated with ALDEFLUOR per the above protocol and stained with mAb 376.96 to evaluate the presence of the mAb 376.96-recognized epitope for B7-H3 on CICs. Results indicate that approximately 90% of SKOV3.ip1 CICs express B7-H3. (B) 1 × 106 A2780 cells/well were plated in a 6-well plate with 2 mL media on Day 0. On Day 1, carboplatin (25 μM) ± Sunitinib (5 μM) ± mAb 376.96 (0.5 mg/mL) were added. On Day 3, cells were harvested using 2 mL Cell Stripper and cells were analyzed for ALDH expression using ALDEFLUOR assay and flow cytometry. Treatment with carboplatin increased the CIC population from 1.1% to 7.4% while treatment with mAb 376.96 decreased the CIC population by 50%. (C) Experiment was repeated on SKOV3.ip1 cells using mAb 376.96, Sunitinib, or control antibody. Treatment of A2780 cells or SKOV3.ip1 cells with Sunitinib alone had no effect on the proportion of CICs, while the addition of mAb 376.96 to Sunitinib decreased proportion of ALDHbright cells compared to treatment with Sunitinib alone.
cells were analyzed for the content of CICs. The latter were identified on the basis of ALDH expression, measured with the ALDEFLUOR assay. The highest ALDH expressing cell line was ES2 (37.6 ± 8.2) followed by OVCAR-3 (24.5 ± 3.2). Chemoresistant cell line SCOV3TRip2 had a higher content of ALDHbright cells (13.3 ± 9.1%) than its chemosensitive parental cell line (0.9 ± 0.6%). However, this pattern was not observed in the A2780 (2.6 ± 1.7%) and A2780cp20 (1.1 ± 0.3%) cell lines (Supplementary Table 1).
mAb 376.96 defined B7-H3 epitope expression on CICs in ovarian cancer cell lines To test whether the mAb 376.96 defined B7-H3 epitope is expressed on CICs in ovarian cancer cell lines, SKOV3.ip1 cells were incubated with ALDEFLUOR and then stained with mAb 376.96. As shown in Fig. 6A, the
mAb 376.96 defined B7-H3 epitope is expressed on approximately 90% of SKOV3.ip1 CICs defined as ALDHbright cells. Effect of treatment with mAb 376.96, chemotherapy, and/or Sunitinib on the content of CICs in ovarian cancer cell lines A2780 cells were treated with Sunitinib (5 μM), carboplatin (25 μM) or mAb 376.96 (0.5 mg/mL) for 3 days at 37 °C and the content of CICs was assessed via ALDEFLUOR staining for ALDHbright population. Treatment of A2780 cells with carboplatin resulted in a 7-fold increase in the content of CICs, while treatment with mAb 376.96 decreased the population by 50%. Treatment of A2780 cells with Sunitinib had no effect on the content of CICs (Fig. 6B). Additionally, SKOV3.ip1 cells were treated with mAb 376.96 (0.5 mg/mL) and/or Sunitinib (5 μM) at 37 °C for 7 days, and compared to cells treated with isotype control mAb F3-C25. Although mAb 376.96 had no marked effect on the content
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of CICs in the SKOV3.ip1 cell line, it enhanced the effect of Sunitinib on the content of CICs (Fig. 6C).
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Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2013.10.038.
Discussion Interest in finding novel therapeutics to target CICs is fueled by data suggesting that this subpopulation of cancer cells is resistant to chemotherapy [32–38]. Because CICs are likely responsible for the high rate of recurrence seen in ovarian cancer patients, they are appropriate targets for development of new therapeutics. B7-H3 is a type-I membrane protein expressed on stimulated antigen-presenting cells and B cells, natural killer cells, CD4+ and CD8+ T-cells. It is upregulated in a number of cancer types [19–25], and has been associated with poor prognosis in ovarian cancer [24,39]. The commercially available polyclonal goat B7H3 antiserum recognizes the B7-H3 ligand of both malignant and healthy tissue cells, decreasing its utility for cancer therapeutics [25]. The different antigenic profiles of B7-H3 expressed on malignant cells compared to normal cells is likely to result from differential glycosylation patterns by the two tissue types. Therefore, we propose that the increased selectivity of mAb 376.96 to B7-H3 expressed on malignant cells renders it a potential agent for immunotherapy-based treatment of ovarian cancer. The mAb 376.96 defined B7-H3 epitope is expressed by chemosensitive and chemoresistant ovarian cancer cell lines, as well as tumors from 10 ovarian cancer patients. Furthermore, treatment of cancer cell lines with paclitaxel increased the expression of B7-H3, while carboplatin and Sunitinib had minimal effect on expression, confirming a stable target despite cytotoxic therapy. In vitro treatment of chemoresistant ovarian cancer cells with mAb 376.96 alone resulted in significant inhibition of cell growth. Therefore, we evaluated the effect of mAb 376.96 in combination with Sunitinib, a tyrosine kinase inhibitor. Sunitinib alone produced approximately 10% inhibition of growth of SKOV3.ip1 cells in vitro, whereas combination treatment with mAb 376.96 and Sunitinib produced 20–25% growth inhibition as compared to lower levels with mAb 376.96 alone. Additionally, we aimed to investigate the level of ALDH expression in ovarian cancer cell lines and study the effect of both chemotherapy and mAb 376.96 alone and in combination with Sunitinib on the content of CICs in vitro. Treatment with carboplatin increased the content of CICs 7-fold in A2780 cells, while treatment with mAb 376.96 reduced the content by one-half. Furthermore, studies on SKOV3.ip1 cells comparing treatment with Sunitinib to Sunitinib + mAb 376.96 showed that addition of the mAb 376.96 enhanced the reduction of CICs. Given the promising effect of mAb 376.96 treatment on CICs, we evaluated concurrent treatment with mAb 376.96 in combination with Sunitinib and chemotherapy on A2780 and platinum-resistant A2780cp20 cells. Cell viability revealed little cytotoxicity of mAb 376.96 alone, but did show substantial cell kill when used in combination with Sunitinib and carboplatin. These data show that B7-H3 is widely expressed in ovarian cancer cell lines and in tumor cells from patients with ovarian cancer. Because mAb 376.96 recognizes a B7-H3 epitope selectively expressed on malignant cells, it is a valid target for novel immunotherapy. Further studies are warranted on both cell lines and patient samples.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments The authors would like to acknowledge Jeffrey Sellers, Patsy Oliver, PhD, Debbie Della Manna, and Laura Aristizabal for their skillful technical assistance and support for this project.
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Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Monoclonal antibody-based immunotherapy of ovarian cancer: Targeting ovarian cancer cells with the B7-H3-specific mAb 376.96☆,☆☆ Janelle M. Fauci a,⁎,1, Francesco Sabbatino b,1, YangYang Wang b, Angelina I. Londoño-Joshi c, J. Michael Straughn Jr. a, Charles N. Landen a, Soldano Ferrone b, Donald J. Buchsbaum d a
University of Alabama at Birmingham, Department of Obstetrics and Gynecology, USA Massachusetts General Hospital, Harvard Medical School, Department of Surgery, USA University of Alabama at Birmingham, Division of Molecular and Cellular Pathology, USA d University of Alabama at Birmingham, Department of Radiation Oncology, USA b c
H I G H L I G H T S • mAb 376.96 defined B7-H3 epitope is expressed by chemosensitive and chemoresistant ovarian cancer cells. • mAb 376.96 enhances cytotoxicity of Sunitinib in vitro.
a r t i c l e
i n f o
Article history: Received 7 August 2013 Accepted 31 October 2013 Available online 9 November 2013 Keywords: Ovarian cancer Immunotherapy Chemoresistance Cancer initiating cells B7-H3 Sunitinib
a b s t r a c t Objective. The high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs. Methods. Eight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs. Results. The mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs. Conclusion. The mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted. © 2013 Elsevier Inc. All rights reserved.
Introduction ☆ Data presented at the University of Pittsburgh Cancer Institute's Ovarian Cancer Symposium (May, 2012). ☆☆ Grant support: This work was supported by PHS grants RO1CA138188 and RO1CA110249 awarded by the National Cancer Institute and by a Susan Komen Post Doctoral Fellowship KG111486 awarded by the Susan G. Komen for the Cure Foundation. ⁎ Corresponding author at: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Alabama at Birmingham, 1700 6th Avenue South, Birmingham, AL 35233, USA. Fax: +1 205 975 6174. E-mail address: [email protected] (J.M. Fauci). 1 Contributed equally to this work. 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.10.038
Over 15,000 women will die of ovarian cancer in 2013 [1]. The majority of patients have advanced stage disease at diagnosis, and although modern surgical intervention and chemotherapy protocols allow most ovarian cancer patients to achieve clinical remission, most tumors will recur [2–4]. One proposed mechanism for this pattern is the survival of subpopulations of chemoresistant cancer cells, known as cancer initiating cells (CICs) [5]. CICs represent the most tumorigenic and treatment resistant subpopulation of tumor cells [6,7]. Several cell surface markers have been
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used to identify CICs, including CD133 and CD44. Aldehyde dehydrogenase-1A1 (ALDH) has also been reported to be a valid CIC marker [5,6,8–14]. ALDH positive CICs form 3-dimensional spheres more readily than their ALDH negative counterparts, and are more tumorigenic in murine xenograft models [15,16]. Additionally, CICs are more resistant to standard chemotherapy treatment, in both in vitro and in vivo studies [5]. The chemoresistance of CICs is likely to be responsible for the high relapse rate in ovarian cancer patients. This warrants the investigation of novel therapeutics to be used in combination with standard chemotherapy to increase cytotoxic activity against chemoresistant cells. One potential therapeutic agent is represented by B7-H3-specific monoclonal antibodies (mAb). B7-H3 is a member of the B7superfamily of ligands, and is believed to play a role in down-regulation of T-cells involved in tumor immunity [17,18]. It is highly expressed in a variety of cancer types, including breast, pancreatic, and ovarian cancer [19–25]. High B7-H3 expression correlates with poor survival in ovarian cancer patients. In a study by Zang and colleagues, 93% of the 77 ovarian carcinomas studied expressed B7-H3 and was associated with increased rates of recurrence and mortality. Furthermore, B7-H3 expression in tumor vasculature also correlated with poor survival, suggesting that this ligand may play a role in tumor cell migration [24]. Sunitinib is a potent tyrosine kinase inhibitor that has proven its efficacy in renal cell carcinoma, and is approved by the Food and Drug Administration (FDA) for use in renal cell carcinoma and for treatment of gastrointestinal stromal tumors (GIST). Recent data have shown that some types of ovarian cancer, such as ovarian clear cell carcinoma, show a remarkable similarity in gene expression profiles across organs such as kidney, lung, and ovary [26]. Currently, Sunitinib is being investigated as part of a large, phase II randomized trial by the Gynecologic Oncology Group (GOG) in the treatment of patients with clear cell ovarian cancer [27]. The objective of the present study was to evaluate the effect of the mAb 376.96 defined B7-H3 epitope in combination with chemotherapy and the tyrosine kinase inhibitor Sunitinib on the chemosensitive and chemoresistant ovarian cancer cells and CICs. Materials and methods Monoclonal and polyclonal antibodies The B7-H3-specific mouse mAb 376.96, an IgG2a, was developed and characterized as described [28]. The specificity of mAb 376.96 for B7-H3 was proven by its specific reactivity with purified B7-H3 in binding assays and by mass spectrometry analysis of the molecule immunoprecipitated by this mAb from cultured human melanoma cells. The isotype matched anti-idiotypic mAb F3-C25 was developed and characterized as described [29]. mAb was purified from ascitic fluid by affinity chromatography on Protein A. The activity and purity of mAb preparations were assessed by reactivity with the corresponding antigens in binding assays and by SDS-PAGE analysis, respectively. Isotype IgG2a κ murine myeloma control and conjugated goat antimouse IgG antibodies (1 μg/mL) were purchased from Sigma-Aldrich (St. Louis, MO). PE-labeled anti-mouse IgG xenoantibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Drugs and reagents Carboplatin, paclitaxel and docetaxel were purchased from SigmaAldrich. Carboplatin was prepared as a 25 mM stock solution in sterile H2O, while paclitaxel and docetaxel were prepared in DMSO as a 10 ηM and a 12.5 ηM stock solution, respectively. Sunitinib was purchased from Sigma-Aldrich and prepared as a 25 mM stock solution in DMSO. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich. ALDEFLUOR kits including ALDEFLUOR-488 and diethylaminobenzaldehyde (DEAB) negative
control were obtained from StemCell Technologies (Durham, NC). Alexa fluor 488 goat anti-rabbit IgG antibody was purchased from Invitrogen (Carlsbad, CA). Cells and cell culture Ovarian cancer cell lines ES-2, OVCAR3, CaOV3, HEY-A8, SKOV3.ip1 and A2780 were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to the supplier's directions. Taxane-resistant SCOV3TRip2 and platinum-resistant A2780cp20 cells were obtained from Dr. Anil Sood (M.D., Anderson Cancer Center, Houston, TX). SKOV3TRip2 cells were developed from SKOV3TR cells obtained from Michael Sieden (M.D., Anderson Cancer Center, Houston, TX) by culturing selected tumors growing in mice to select highly tumorigenic cells [30]. SCOV3TRip2 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (HyClone, Logan, UT) and paclitaxel 150 ηmol/L. A2780cp20 cells were maintained in the same conditions as chemosensitive A2780 cells. All cell lines were maintained in antibiotic-free medium at 37 °C in a 5% CO2 atmosphere and routinely screened for Mycoplasma contamination. Ovarian cancer tumor specimens Ovarian cancer tumors were obtained from ten patients undergoing primary debulking surgery for advanced ovarian cancer by surgeons within the Gynecologic Oncology division at the University of Alabama at Birmingham. All tumors were papillary serous histology from patients with stage 3 disease. Immunohistochemical staining of frozen ovarian tumor tissue sections with mAb 376.96 Frozen sections of ovarian cancer tumors from 10 patients were stained with the B7-H3-specific mAb 376.96. Tissue sections were post-fixed in 4% paraformaldehyde (PFA), treated with 3% hydrogen peroxide, 1% bovine serum albumin (BSA) (Invitrogen), and 5% normal horse serum in Tris-buffered saline (25 mM Tris [pH 7.4], 150 mM NaCl) and incubated in a closed humid chamber overnight at 4 °C with the B7-H3-specific mAb 376.96 (5 μg/mL). Immunohistochemical (IHC) signals were generated with EnVision1System-HRP (Dako North America, Inc) and substrate diaminobenzidine (Dako North America, Inc). Tissue sections were counterstained with Mayer Hematoxylin (Sigma-Aldrich), dehydrated with ethyl alcohol, incubated in 100% xylene, and mounted in Canada balsam (Sigma-Aldrich). Frozen sections derived from the cell lines MV3 (B7-H3+) and Raji (B7-H3−) were simultaneously stained as positive and negative controls respectively, to determine the specificity of the staining obtained with mAb 376.96 (data not shown). Cytoplasmic membrane staining was considered positive for mAb 376.96 defined B7-H3 epitope. The staining intensity was graded negative, weak and strong by semiquantitative analysis. Flow cytometry analysis of cell lines stained with mAb Chemosensitive ovarian cancer cell lines ES-2, Hey-A8, OVCAR3, CAOV3, SKOV3.ip1, and A2780 and chemoresistant ovarian cell lines SCOV3TRip2 and A2780cp20 were harvested at 80% confluency using Cell Stripper (Mediatech, Manassas, VA) to prevent cleavage of the B7H3 epitope. Tubes containing 2.1 × 106 cells/mL were washed in PBS supplemented with 1% BSA and azide (PBS/BSA + azide), and centrifuged at 1000 rpm for 5 min. The cell pellets were resuspended in 100 μL PBS/BSA + azide. Isotype control antibody IgG2a κ, murine myeloma (0.5 μg/mL) was added to the control tube, and mAb 376.96 (5 μg/mL) was added to the sample tube and incubated on ice for 30 min. Cells were washed in PBS/BSA + azide and centrifuged at 1000 rpm for 5 min. Cells from isotype control, secondary antibody control and sample tubes were incubated with secondary antibody,
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R-Phycoerythrin (RPE) labeled goat anti-mouse IgG antibodies (Zenon, Eugene, OR) (5 μg/mL) in a dark room on ice for 30 min. Cell suspensions were then washed and resuspended in 0.5 mL PBS/BSA + azide. All samples were analyzed on a LSRII flow cytometer (BD Biosciences, San Jose, CA). Data was evaluated using FlowJo software (Tree Star, Inc., Ashland, OR).
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Growth inhibition assays using MTT assay Chemoresistant cell lines A2780cp20 and SCOV3TRip2 and chemosensitive cell line SKOV3.ip1 were seeded in 96-well tissue culture-treated plates (Costar) at 2500 cells/100 μL RPMI 1640 medium with 2% FCS. A2780cp20 and SCOV3TRip2 cells were treated
Fig. 1. B7-H3 expression on chemosensitive and chemoresistant ovarian cancer cell lines. (A) Ovarian cancer cell lines were stained with mAb 376.96 and analyzed by flow cytometry. Cells expressing B7-H3 bound by mAb 376.96 are depicted as the bold line to right, as compared to isotype control antibody, represented as the thin line to the left. (B) Similar testing for expression of B7-H3 was confirmed on chemosensitive and chemoresistant subclones. (C) Chemosensitive and resistant cell lines were treated with chemotherapy or Sunitinib and then analyzed for B7-H3 expression.
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with 0.25, 0.5 or 1 mg/mL mAb 376.96 and SKOV3.ip1 cells were treated with mAb 376.96 (0.25 or 0.5 mg/mL) ± Sunitinib (1.5 μM) for 3 days at 37 °C in a 5% CO2 atmosphere. mAb F3-C25 (0.5 mg/mL) was used as an isotype control. DMSO vehicle concentration of Sunitinib was maintained at 0.02% in all wells. Cell proliferation was evaluated by MTT assay. Data are expressed as percent of growth inhibition of treated cells compared with the untreated control cells as reference for 100% cell growth.
Supplementary Fig. 1 (panels A and B, respectively). The staining is specific, since no staining of the ovarian cancer lesions by the isotype control mAb F3-C25 was detected. A representative example is shown in Supplementary Fig. 1 (panel C).
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Cell viability was determined by measurement of cellular ATP levels, using the ATPLite luminescence-based assay (Perkin Elmer Biosciences, Meriden, CT) as described elsewhere [31]. Cells were lysed using 25 μL/well mammalian cell lysis solution and 25 μL/well of lyophilized substrate. Plates were sealed, gently mixed and luminescence was measured using a TopCount NXT (Perkin Elmer Biosciences).
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Identification of CICs utilizing high ALDH expression
SKOV3.ip1 cells were incubated with ALDEFLUOR ± DEAB inhibitor and stained with mAb 376.96 and PE-labeled anti-mouse IgG xenoantibody to identify cells expressing high levels of ALDH activity (ALDHbright) and mAb 376.96 defined B7-H3 epitope. mAb F3-C25 was used as an isotype control. ALDHbright cells were identified as those ALDH+ cells with twice the mean fluorescence intensity (MFI) of the ALDH+ cell population.
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To exclude that the detection of the mAb 376.96 defined B7-H3 epitope on ovarian cancer cell lines does not represent an in vitro artifact, but reflects the phenotype of ovarian cancer tumors in vivo, frozen sections of ovarian cancer lesions from 10 patients were stained with mAb 376.96. All of the lesions were positive. The staining intensity was strong in 7 and weak in 3. Representative examples are shown in
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mAb 376.96 defined B7-H3 epitope expression in surgically removed ovarian cancer tumors
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Results
Flow cytometry analysis of ovarian cancer lines CAOV3, ES-2, Hey-A8 and OVCAR3, and chemoresistant subclones A2780cp20 and SCOV3TRip2 sequentially incubated with the mAb 376.96 defined B7-H3 epitope and PE-labeled anti-mouse IgG xenoantibody detected a strong, homogeneous staining of all cell lines. The staining is specific, since no staining was detected in lines incubated with the isotype matched anti-idiotypic mAb F3-C25. These results indicate that the mAb 376.96 defined B7-H3 epitope is highly expressed on both chemosensitive and chemoresistant ovarian cancer cell lines (Fig. 1).
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B7-H3 expression on ovarian CICs by flow cytometry
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Chemosensitive ovarian cancer cell lines ES-2, OVCAR3, CaOV3, HEY-A8, SKOV3.ip1 and A2780 and chemoresistant ovarian cell lines A2780cp20 and SCOV3TRip2 were harvested with 1 mL of PBS/ ethylenediaminetetraacetic acid (EDTA) collected by centrifugation at 1000 rpm for 5 min, and resuspended in Hank's balanced salt solution (HBSS). ALDEFLUOR assay (StemCell Technologies, Vancouver, British Columbia) was employed and used according to the manufacturer's protocol. As a control, cells were resuspended in ALDEFLUOR assay buffer containing diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH activity. All samples were analyzed on an LSRII flow cytometer and data were evaluated using FlowJo software.
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carbopaltin carboplatin paclitaxel 25µM 100µM 2.5 nM
Fig. 2. Chemosensitivity of parental and chemoresistant cell lines. (A) SKOV3ip1, and taxane-resistant SCOV3TRip2 cells were plated in 96-well plates at 2500 cells/well in attached conditions on Day 0. Carboplatin (range, 0–100 μM) or paclitaxel (range, 0–300 nM) were added on Day 1. ATP assay was used to assess cell viability on Day 4, and data is presented as a mean of 8 replicates. Error bars denote SE. (B) A2780 and platinum-resistant A2780cp20 cells were plated in 96-well plates at 2500 cells/ well in attached conditions on Day 0. Carboplatin (range, 0–100 μM) or paclitaxel (range, 0–300 nM) were added on Day 1. ATP assay was used to assess cell viability on Day 4, and data is presented as a mean of 8 replicates. Error bars denote SE.
Modulation by chemotherapy of mAb 376.96 defined B7-H3 epitope expression on ovarian cancer cell lines To test the effect of chemotherapeutic agents on mAb 376.96 defined B7-H3 epitope expression on ovarian cancer cell lines, the SKOV3.ip1 and SCOV3TRip2 cells (1.0 × 106/well) were incubated with Sunitinib (5 μM) or paclitaxel (25 nM) for 3 days at 37 °C in a 5% CO2 atmosphere. Cells were then isolated, stained with mAb 376.96 and analyzed using flow cytometry. The MFI was examined (Fig. 1C) and found to be highest after treatment with paclitaxel. Treatment of SKOV3.ip1 cells with the same dose of paclitaxel resulted in a 1.7-fold increase in mAb 376.96 defined B7-H3 epitope MFI, while treatment of taxaneresistant SCOV3TRip2 cells with 100 nM paclitaxel resulted in a 0.8fold decrease in the MFI. Similar results were obtained when SKOV3.ip1 and SCOV3TRip2 cells as well as A2780 and A2780cp20 cells were treated with carboplatin (25 μM) for 3 days at 37 °C (data not shown). Anti-proliferative effect of mAb 376.96 and Sunitinib on the chemoresistant cell lines A2780cp20 and SCOV3TRip2 Platinum-resistant A2780cp20 cells and the parental cells A2780, as well as the taxane-resistant SCOV3TRip2 cells and the parental cells SKOV3.ip1 were plated in 96-well plates at 2500 cells/well. Cells were treated with various doses of carboplatin (range, 0–100 μM) or paclitaxel (range, 0–300 nM) on day 1. Following a 3-day incubation at 37 °C in a 5% CO2 atmosphere, cell viability was determined by measurement of cellular ATP levels. A2780cp20 cells were resistant to carboplatin treatment, even at doses as high as 100 μM (60.3% viable, compared to A2780: 20.3% viable at 100 μM). Additionally, SCOV3TRip2 cells were strongly resistant to taxane treatment, as there was no cell death after treatment with 300 nM of paclitaxel, while only 31.8% of SKOV3.ip1 cells were viable after treatment with 100 nM paclitaxel (Fig. 2). Chemoresistant SCOV3TRip2 and A2780cp20 cells were treated with mAb 376.96 at various doses (range, 0.25–1.0 mg/mL) and compared to treatment with isotype control mAb F3-C25 (Fig. 3). Monotherapy with mAb 376.96 (1 mg/mL) resulted in nearly 50% growth inhibition of SCOV3TRip2 cells, and over 30% inhibition of A2780cp20 cell growth. Monotherapy with Sunitinib resulted in a 10% inhibition of SKOV3.ip1 cell growth, while combination treatment with Sunitinib and mAb 376.96 resulted in greater than 20% inhibition of cell growth (Fig. 4).
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0.25
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Fig. 4. mAb 376.96 enhances the ability of Sunitinib to inhibit growth of SKOV3.ip1 ovarian cancer cells in vitro. SKOV3.ip1 cells (2500/well) were seeded in a 96-well plate and treated with mAb 376.96 and/or Sunitinib (1.5 μM) for 3 days at 37 °C in a 5% CO2 atmosphere. Cells were then tested by MTT assay. The results are expressed as % inhibition of cell growth, utilizing the values obtained for untreated cells, as a reference. Triplicates were performed in each condition.
or 100 μM (A2780cp20), Sunitinib (5 μM) and/or mAb 376.96 (0.5 mg/mL) for 3 days at 37 °C. Cells were harvested and six 10 μL aliquots were removed and plated at 10 μL/well in a 96-well plate for analysis with ATPLite luminescence-based assay. Monotherapy with mAb 376.96 resulted in decreased cell viability compared to controls; this effect was most pronounced in the platinum resistant A2780cp20 cell line. The effect of mAb 376.96 on cell viability was not enhanced by Sunitinib and carboplatin (Fig. 5). Content of CICs in ovarian cancer cell lines Ovarian cancer cell lines ES-2, OVCAR3, CaOV3, HEY-A8, SKOV3.ip1 and A2780 and chemoresistant cell lines A2780cp20 and SCOV3TRip2
Effect of mAb 376.96 in combination with carboplatin and/or Sunitinib on chemosensitive and chemoresistant cells
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Chemosensitive A2780 cells and platinum-resistant A2780cp20 cells (1 × 106 cells/well) were incubated with carboplatin 25 μM (A2780)
Fig. 3. mAb 376.96 inhibits growth of chemoresistant ovarian cancer cells in vitro. Cells were seeded in 96-well plates at 2500 cells/well and incubated with various concentrations of mAb 376.96 for 3 days. Monotherapy with mAb 376.96 resulted in inhibition of cell growth, compared to control mAb F4-C25.
Fig. 5. Cytotoxicity of ovarian cancer cells. 1 × 106 cells/well were plated in a 6-well plate with 2 mL media on Day 0. On Day 1, carboplatin at 25 μM (A2780) or 100 μM (A2780cp20) ± Sunitinib (5 μM) ± mAb 376.96 (0.5 mg/mL) were added. On Day 3, cells were harvested and six 10 μL aliquots were removed and plated in a 96-well plate for immediate analysis with ATPLite. Data represent mean of 6 replicates, and error bars denote SE.
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A DEAB + ALDEFLUOR
mAb 376.96
mAb F3-C25
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Fig. 6. mAb 376.96 enhances ability of Sunitinib to decrease proportion of CIC in A2780 and SKOV3.ip1 cells in vitro. (A) SKOV3.ip1 cells were incubated with ALDEFLUOR per the above protocol and stained with mAb 376.96 to evaluate the presence of the mAb 376.96-recognized epitope for B7-H3 on CICs. Results indicate that approximately 90% of SKOV3.ip1 CICs express B7-H3. (B) 1 × 106 A2780 cells/well were plated in a 6-well plate with 2 mL media on Day 0. On Day 1, carboplatin (25 μM) ± Sunitinib (5 μM) ± mAb 376.96 (0.5 mg/mL) were added. On Day 3, cells were harvested using 2 mL Cell Stripper and cells were analyzed for ALDH expression using ALDEFLUOR assay and flow cytometry. Treatment with carboplatin increased the CIC population from 1.1% to 7.4% while treatment with mAb 376.96 decreased the CIC population by 50%. (C) Experiment was repeated on SKOV3.ip1 cells using mAb 376.96, Sunitinib, or control antibody. Treatment of A2780 cells or SKOV3.ip1 cells with Sunitinib alone had no effect on the proportion of CICs, while the addition of mAb 376.96 to Sunitinib decreased proportion of ALDHbright cells compared to treatment with Sunitinib alone.
cells were analyzed for the content of CICs. The latter were identified on the basis of ALDH expression, measured with the ALDEFLUOR assay. The highest ALDH expressing cell line was ES2 (37.6 ± 8.2) followed by OVCAR-3 (24.5 ± 3.2). Chemoresistant cell line SCOV3TRip2 had a higher content of ALDHbright cells (13.3 ± 9.1%) than its chemosensitive parental cell line (0.9 ± 0.6%). However, this pattern was not observed in the A2780 (2.6 ± 1.7%) and A2780cp20 (1.1 ± 0.3%) cell lines (Supplementary Table 1).
mAb 376.96 defined B7-H3 epitope expression on CICs in ovarian cancer cell lines To test whether the mAb 376.96 defined B7-H3 epitope is expressed on CICs in ovarian cancer cell lines, SKOV3.ip1 cells were incubated with ALDEFLUOR and then stained with mAb 376.96. As shown in Fig. 6A, the
mAb 376.96 defined B7-H3 epitope is expressed on approximately 90% of SKOV3.ip1 CICs defined as ALDHbright cells. Effect of treatment with mAb 376.96, chemotherapy, and/or Sunitinib on the content of CICs in ovarian cancer cell lines A2780 cells were treated with Sunitinib (5 μM), carboplatin (25 μM) or mAb 376.96 (0.5 mg/mL) for 3 days at 37 °C and the content of CICs was assessed via ALDEFLUOR staining for ALDHbright population. Treatment of A2780 cells with carboplatin resulted in a 7-fold increase in the content of CICs, while treatment with mAb 376.96 decreased the population by 50%. Treatment of A2780 cells with Sunitinib had no effect on the content of CICs (Fig. 6B). Additionally, SKOV3.ip1 cells were treated with mAb 376.96 (0.5 mg/mL) and/or Sunitinib (5 μM) at 37 °C for 7 days, and compared to cells treated with isotype control mAb F3-C25. Although mAb 376.96 had no marked effect on the content
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of CICs in the SKOV3.ip1 cell line, it enhanced the effect of Sunitinib on the content of CICs (Fig. 6C).
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Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2013.10.038.
Discussion Interest in finding novel therapeutics to target CICs is fueled by data suggesting that this subpopulation of cancer cells is resistant to chemotherapy [32–38]. Because CICs are likely responsible for the high rate of recurrence seen in ovarian cancer patients, they are appropriate targets for development of new therapeutics. B7-H3 is a type-I membrane protein expressed on stimulated antigen-presenting cells and B cells, natural killer cells, CD4+ and CD8+ T-cells. It is upregulated in a number of cancer types [19–25], and has been associated with poor prognosis in ovarian cancer [24,39]. The commercially available polyclonal goat B7H3 antiserum recognizes the B7-H3 ligand of both malignant and healthy tissue cells, decreasing its utility for cancer therapeutics [25]. The different antigenic profiles of B7-H3 expressed on malignant cells compared to normal cells is likely to result from differential glycosylation patterns by the two tissue types. Therefore, we propose that the increased selectivity of mAb 376.96 to B7-H3 expressed on malignant cells renders it a potential agent for immunotherapy-based treatment of ovarian cancer. The mAb 376.96 defined B7-H3 epitope is expressed by chemosensitive and chemoresistant ovarian cancer cell lines, as well as tumors from 10 ovarian cancer patients. Furthermore, treatment of cancer cell lines with paclitaxel increased the expression of B7-H3, while carboplatin and Sunitinib had minimal effect on expression, confirming a stable target despite cytotoxic therapy. In vitro treatment of chemoresistant ovarian cancer cells with mAb 376.96 alone resulted in significant inhibition of cell growth. Therefore, we evaluated the effect of mAb 376.96 in combination with Sunitinib, a tyrosine kinase inhibitor. Sunitinib alone produced approximately 10% inhibition of growth of SKOV3.ip1 cells in vitro, whereas combination treatment with mAb 376.96 and Sunitinib produced 20–25% growth inhibition as compared to lower levels with mAb 376.96 alone. Additionally, we aimed to investigate the level of ALDH expression in ovarian cancer cell lines and study the effect of both chemotherapy and mAb 376.96 alone and in combination with Sunitinib on the content of CICs in vitro. Treatment with carboplatin increased the content of CICs 7-fold in A2780 cells, while treatment with mAb 376.96 reduced the content by one-half. Furthermore, studies on SKOV3.ip1 cells comparing treatment with Sunitinib to Sunitinib + mAb 376.96 showed that addition of the mAb 376.96 enhanced the reduction of CICs. Given the promising effect of mAb 376.96 treatment on CICs, we evaluated concurrent treatment with mAb 376.96 in combination with Sunitinib and chemotherapy on A2780 and platinum-resistant A2780cp20 cells. Cell viability revealed little cytotoxicity of mAb 376.96 alone, but did show substantial cell kill when used in combination with Sunitinib and carboplatin. These data show that B7-H3 is widely expressed in ovarian cancer cell lines and in tumor cells from patients with ovarian cancer. Because mAb 376.96 recognizes a B7-H3 epitope selectively expressed on malignant cells, it is a valid target for novel immunotherapy. Further studies are warranted on both cell lines and patient samples.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments The authors would like to acknowledge Jeffrey Sellers, Patsy Oliver, PhD, Debbie Della Manna, and Laura Aristizabal for their skillful technical assistance and support for this project.
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