Mol Cell Biochem DOI 10.1007/s11010-015-2475-2
Cellular therapy in combination with cytokines improves survival in a xenograft mouse model of ovarian cancer Susan B. Ingersoll1,2 • Sarfraz Ahmad1,2,3 • Hasina C. McGann1 • Robert K. Banks3 Nicole M. Stavitzski1 • Milan Srivastava1 • Ghazanfar Ali1 • Neil J. Finkler1,2,3 • John R. Edwards3,4 • Robert W. Holloway1,2,3
•
Received: 30 January 2015 / Accepted: 30 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Studies have shown enhanced survival of ovarian cancer patients in which the tumors are infiltrated with tumor infiltrating lymphocytes and natural killer cells showing the importance of immune surveillance and recognition in ovarian cancer. Therefore, in this study, we tested cellular immunotherapy and varying combinations of cytokines (IL-2 and/or pegylated-IFNa-2b) in a xenograft mouse model of ovarian cancer. SKOV3-AF2 ovarian cancer cells were injected intra-peritoneally (IP) into athymic nude mice. On day 7 post-tumor cell injection, mice were injected IP with peripheral blood mononuclear cells (PBMC; 5 9 106 PBMC) and cytokine combinations [IL-2 ± pegylated-IFNa-2b (IFN)]. Cytokine injections were continued weekly for IFN (12,000 U/injection) and thrice weekly for IL-2 (4000 U/injection). Mice were
euthanized when they became moribund due to tumor burden at which time tumor and ascitic fluid were measured and collected. Treatment efficacy was measured by improved survival at 8 weeks and overall survival by Kaplan–Meier analysis. We observed that the mice tolerated all treatment combinations without significant weight loss or other apparent illness. Mice receiving PBMC plus IL-2 showed improved median survival (7.3 weeks) compared to mice with no treatment (4.2 weeks), IL-2 (3.5 weeks), PBMC (4.0 weeks), or PBMC plus IL-2 and IFN (4.3 weeks), although PBMC plus IL-2 was not statistically different than PBMC plus IFN (5.5 weeks, p [ 0.05). We demonstrate that cytokine-stimulated cellular immune therapy with PBMC and IL-2 was well tolerated and resulted in survival advantage compared to untreated controls and other cytokine combinations in the nude-mouse model.
Part of this study was presented as Featured Poster at the 44th Annual Meetings on Women’s CancerÒ of the Society of Gynecologic Oncology (SGO), in March 2013, at Los Angeles, CA; and the 104th Annual Meeting of the American Association of Cancer Research (AACR), in April 2013, at Washington, DC.
Keywords Cellular therapy Cytokines Ovarian cancer Xenograft mouse model Peripheral blood mononuclear cells
& Susan B. Ingersoll [email protected]
Introduction
& Sarfraz Ahmad [email protected] 1
Florida Hospital Gynecologic Oncology, Florida Hospital Cancer Institute, 2501 N. Orange Ave., Suite 786, Orlando, FL 32804, USA
2
Florida State University College of Medicine, Orlando, FL 32801, USA
3
University of Central Florida College of Medicine, Orlando, FL 32827, USA
4
Indiana Blood and Marrow Transplantation, Indianapolis, IN 46237, USA
Epithelial ovarian cancer is usually diagnosed in advanced stages with metastases, which requires both aggressive cytoreductive surgery and chemotherapy to control symptoms and prolong survival. Unfortunately, while median survival has improved significantly in recent decades due to these efforts, the majority of patients ultimately suffer symptomatic recurrence of disease and eventual death secondary to the development of chemotherapy-resistant tumor. In order to improve long-term survival for women with ovarian cancer, new therapeutic approaches that are
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more effective in the treatment of chemotherapy-resistance disease are needed. Immunotherapy has been proposed for the treatment of several malignancies including ovarian cancer. Early investigations using tumor infiltrating lymphocytes (TIL) for the treatment of ovarian cancer failed to demonstrate effectiveness, likely because of the inability to expand an adequate population of therapeutic cells [1]. Furthermore, specifics of the interplay between tumor micro-environment and many aspects of the host immune response were poorly described at that time. More recent advances in the understanding of immune environment modulation (such as the ability to down-regulate immunosuppressive cells through the use of antibodies), the processes of immune effector cell expansion, and the availability of commercially manufactured cellular therapy products (such as umbilical cord blood units or dendritic cells) may improve the efficacy of cellular therapy [2, 3]. Several lines of scientific evidence suggest that immunotherapy using stimulated immune cells is a rational approach for ovarian cancer therapy. Enhanced survival of patients with ovarian cancer whose tumors were infiltrated with TIL and natural killer (NK) cells has been observed, lending indirect evidence for this hypothesis. Zhang et al. [4] were the first to show that increased numbers of CD3? T-cell infiltrates were associated with improved progression-free and overall survival for patients with International Federation of Gynecology and Obstetrics (FIGO) stage III/IV ovarian cancer. The 5-year overall survival for patients with significant T-cell infiltrates was 38 % compared to only 4.5 % for those with fewer T-cell infiltrates, suggesting a possible relationship between a patient’s immune response and survival with ovarian cancer [4]. It has been hypothesized by others that patients who experience prolonged disease-free survival may have an augmented immunological response to their disease [5]. Furthermore, in another disease setting, Dudley et al. [6] found that patients with melanoma who received chemotherapy in combination with immunotherapy showed increased response rates compared to chemotherapy treated patients. Previously, we have shown that immune effector cells from healthy donors can elicit a significant cytotoxic response against ovarian cancer in vitro. Peripheral blood mononuclear cells (PBMC) in the presence of the cytokines [interleukin-2 (IL-2) and interferon a-2b (IFNa-2b)], elicit up to a 70 % cytotoxic response against ovarian cancer cells in vitro [7–10]. This cytotoxic response was not observed in the absence of IL-2 indicating that IL-2 is necessary for the PBMC to elicit the cytotoxic response [7, 10]. In addition, we have shown that IFNa-2b has an inhibitory effect on ovarian cancer cell growth [7]. Therefore, in this study, we proposed to further evaluate
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cellular therapy using different combinations of cytokines (IL-2 and IFNa-2b) in combination with PBMC in a xenograft murine model of ovarian cancer in order to study their effects in vivo.
Materials and methods Cell line culture SKOV3-AF2 cells were derived from SKOV-3 (ATCC, Manassas, VA) cells as recently described by Ingersoll et al. [7, 8]. Briefly, SKOV3-AF2 cells were derived by first stably transfecting SKOV-3 cells with red-fluorescent protein (SKOV3-RFP), so that the cells could be distinguished from the immune effector cells by fluorescence. Next, the cells were passaged through nude mice to make the cells more tumorigenic in a xenograft mouse model as described previously [7, 8]. Single-cell suspensions were made from solid tumor and ascitic fluid harvested from the parental SKOV3-RFP tumors and grown in culture. These cells were subsequently injected into the peritoneal cavities of nude mice and solid tumor was subsequently collected by necropsy. Single-cell suspensions from the harvested tumors were grown in culture. The line derived from this tumor suspension was called SKOV3-AF2. We have previously shown that these SKOV3-AF2 cells are more tumorigenic in nude mice, show increased resistance to IFNa-2b in vitro, and are sensitive to immune-mediated cytotoxicity in vitro [7, 8]. All the cells were grown in McCoy’s media (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS; ATCC) and 1 % penicillin and streptomycin (Invitrogen) in 5 % CO2 at 37 °C.
Mononuclear cell preparation LeukoPaks (Florida Blood Center, Orlando, FL) were diluted 1:1 with RPMI media (Invitrogen), and PBMC were isolated using a Ficoll-Paque (Invitrogen) density gradient. The PBMC were washed twice in phosphatebuffered saline (PBS; Invitrogen) and once in RPMI-1640 (Invitrogen) media at which point in time the cells were resuspended in cryopreservation media containing 10 % dimethyl-sulphoxide (DMSO; Sigma-Aldrich, St. Louis, MO), 20 % FBS, and 70 % RPMI, and stored in liquid nitrogen until the cells were to be prepared for the cytotoxicity experiments or injection into the mice. The PBMC were thawed at 37 °C and diluted with RPMI media, the PBMC were washed twice with PBS to remove the DMSO and re-suspended for use in the in vitro cytotoxicity assays or the in vivo mouse treatments as described below.
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In vitro cytotoxicity assays The cytotoxicity assay used in this study was similar to that we have recently reported [8, 10]. Briefly, percent cytotoxicity of ovarian cancer cells was measured by lactate dehydrogenase (LDH) release using the Cytotox Membrane Integrity Assay (Promega Corp., Madison, WI) [11, 12]. The LDH release assay has been shown to produce similar results to 51Cr release assays when experiments were run in parallel [11, 12]. PBMC were co-cultured with SKOV3-AF2 ovarian cancer cells in McCoy’s media supplemented with 5 % FBS at a ratio of 20:1 (effector cells:target cells) in the presence of IL-2 (25 ng/ml; PeproTech, Rocky Hill, NJ) and/or IFNa-2b (500 U/ml; R&D Systems, Minneapolis, MN) for 24 or 48 h at which point LDH release was measured by excitation at 530 nm and emission at 590 nm at 30 min using a Synergy 2 (BioTek, Winooski, VT) plate reader. Percent Cytotoxicity = 100 9 [(Target - Effector Cell Spontaneous)/(Maximum Release - Cell Spontaneous)]. All experimental conditions were performed in quadruplicate. The data shown are the mean cytotoxicity elicited by the PBMC isolated from two independent donor LeukoPaks. These were the same LeukoPaks that were used for the immune cell therapy in the in vivo mouse experiments. Xenograft mouse model for ovarian cancer Female athymic nude (nu/nu) mice (Harlan Sprague– Dawley, Inc., Indianapolis, IN) were housed at the University of Central Florida (Orlando, FL) Wild Animal Facility under specific pathogen-free conditions. This study was conducted following an Institutional Animal Care and Use Committee (IACUC) approved protocol. Female athymic nude (nu/nu) mice (7–9 weeks old; n = 90) were injected intra-peritoneally (IP) with 1 9 106 SKOV3-AF2 cells. The mice were weighed on day 0 and every 7 days until the time of euthanasia. The mice were euthanized when they became moribund (swollen abdomens, dehydration, difficult movement, hunched posture, and labored breathing) due to tumor burden. Solid tumor and ascitic fluid, when present, were collected from each animal, and samples were snap-frozen in liquid nitrogen.
Table 1 Survival of mice treated with cellular therapy and cytokines Treatment
Median survival (range), weeks
Percent survival at 8 weeks (%)
No treatment
4.2
17
(3.5–9.5) PBMC
4.0
15
(3.2–9.5) IL-2
3.5
0
(3.2–7.5) IFNa-2b PBMC ? IL-2
4.7 (3.2–9.5)
22
7.3
50
(3.5–16) PBMC ? IFNa-2b
5.5
PBMC ? IFNa-2b ? IL-2
4.3
11
(3.2–10.5) 10
(3.2–8.5) PBMC Peripheral blood mononuclear cells, IL-2 interleukin 2, IFNa2b pegylated-interferon a-2b
times a week, and the PEG-IFNa-2b injections were continued once a week. The PBMC dosage was based on our previous work to determine if the mice would tolerate 5 9 106 human immune cells injected IP (unpublished data). The cytokine dosages used were based on our previous optimization studies [7]. The in vivo cellular therapy experiments were performed in duplicate using two independent donor LeukoPaks. Statistical analyses Data are presented as percent and/or mean (±standard deviation, SD) in respective groups or time points. Statistical analysis was performed using SigmaPlot Version 11.0 (San Jose, CA). Student’s t test was performed when the data passed the equal variance test, and Mann–Whitney U test was used when the data failed the equal variance test. Statistical significance was declared when p \ 0.05 determined. Kaplan–Meier analysis was used for the survival determinations of the mice.
In vivo cell and cytokine treatments
Results
On day 7 post-tumor cell injection, the mice were randomized to seven groups (n = 10-15 per group; n = 90 total animals; treatment groups as shown in Table 1). The mice were injected IP with 5 9 106 PBMC isolated from a LeukoPak and cytokines [IL-2 (4000 U/injection) and PEG-IFNa-2b (12,000 U/injection, GenScript Corp. Piscataway, NJ). The IL-2 injections were continued three
Cryopreserved PBMC isolated from LeukoPaks elicit a significant cytotoxic response against ovarian cancer cells in vitro The cytotoxic response of cryopreserved PBMC isolated from LeukoPaks (multiple independent donors) in the presence of IL-2 and IFNa-2b against ovarian cancer cells
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(SKOV3-AF2) was determined. PBMC from LeukoPaks elicited a significant cytotoxic response in the presence of IL-2 plus IFNa-2b against SKOV3-AF2 cells (Fig. 1, left group). There was no significant response in the absence of cytokines or in the presence of cytokines alone (Fig. 1, right group). These experiments demonstrate that the response is PBMC-mediated and the greatest cytotoxicity was observed with combination IL-2 and INF-a2b (Fig. 1). The cryopreserved PBMC shown in these in vitro experiments were the same cells used as effector cells in the in vivo mouse experiments.
(7.3 weeks; p \ 0.05) showed a significant improvement in median survival compared to mice receiving IL-2 alone (3.5 weeks), PBMC alone (4.0 weeks), or no treatment (4.2 weeks; Table 1). Also, the Kaplan–Meier curve shows that the mice receiving combination treatment of PBMC plus IL-2 favored a shift to the right indicating improved overall survival (Fig. 2a). Mice receiving PBMC plus
A
Mice treated with PBMC Plus IL-2 have a survival advantage compared to untreated mice or those treated with PBMC or IL-2 alone
No Treatment IL-2 PBMC PBMC + IL-2
0.8
0.6
Survival
Immune cell therapy in combination with cytokines was tested in a xenograft mouse model of ovarian cancer using SKOV3-AF2 ovarian cancer cells. As demonstrated previously [7], SKOV3-AF2 cells produced tumors that are poorly differentiated surface epithelial carcinoma, growing in solid nests and sheets of large cells with a moderate amount of amphophilic or clear cytoplasm, and tumor cells were focally pleomorphic and multinucleated. The results shown are from multiple independent experiments using PBMC isolated from LuekoPaks from different donors. In these experiments, mice treated with PBMC plus IL-2
1.0
0.4
0.2
0.0 0
2
4
6
8
10
12
14
Weeks 40
No Cytokines
35
No Treatment IFN PMBC PBMC + IFN PBMC + IFN + IL-2
1.0
IL-2
30
IFN
25
0.8
IL-2 + IFN
20 15
0.6
10
Survival
Percent Cytotoxicity
B
5 0
0.4
-5 -10
20:1 PBMC:SKOV3-AF2
SKOV3-AF2 without PBMC
Fig. 1 In vitro cytotoxicity experiments: Cytotoxicity elicited by PBMC isolated from LeukoPaks against SKOV3-AF2 was assessed. The graph shows the mean percent cytotoxicity elicited plus the standard deviation. The left side of the graph shows the cytotoxicity elicited by PBMC at a ratio of 20:1 PBMC to SKOV3-AF2 cells. The right side of the graph shows the negative controls without PBMC. The PBMC elicited a significant cytotoxic response in the presence of IL-2 plus IFNa-2b compared to controls lacking effector cells (p \ 0.05, determined by t test). The IL-2 concentration was 25 ng/ ml, and the INFa-2b concentration was 500 U/ml. IL-2 Interleukin 2, IFN pegylated-interferon a-2b, PBMC peripheral blood mononuclear cells
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0.2
0.0 0
2
4
6
8
10
12
14
Weeks
Fig. 2 Kaplan–Meier survival analysis of mice treated with cell and/ or cytokine therapies. a Mice that were treated with PBMC plus IL-2 showed improved overall survival compared to the control groups. b Mice that were treated with PBMC plus IFN did not show improved survival compared to the control groups. IL-2 Interleukin 2, PBMC peripheral blood mononuclear cells, IFN pegylated-interferon a-2b
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IFNa-2b (median 5.5 weeks) or PBMC plus IFNa-2b and IL-2 (median 4.3 weeks) did not show improved survival (Fig. 2b; Table 1) compared to the control mice. In addition, there was no significant difference in the survival of mice treated with PBMC plus IFNa-2b compared to the mice treated with PBMC plus IFNa-2b and IL-2 (Fig. 2b; Table 1). As shown in Table 2, there were no significant differences in tumor weights or ascitic fluid volume at the time of necropsy, likely because the mice were euthanized when they became moribund due to tumor burden rather than a specific time point post-tumor cell injection. In addition, there was no apparent correlation with the presence or absence of ascites, and any of the experimental treatments. Also, as shown in Table 2, there were mice in each treatment group that did not produce ascitic fluid, while other mice in the same treatment groups had ascites at the time of euthanasia. In total, 51 % of the mice had ascites at the time of euthanasia.
Discussion In this study, we provide in vivo evidence that in a xenograft murine model of ovarian cancer, treatment with intraperitoneal PBMC (isolated from healthy donors) combined
Mice tolerate intra-peritoneal cytokine and cellular therapy treatment The mice tolerated all treatments as indicated by no significant weight loss (Fig. 3a), hunched posture, or labored breathing. Significant tumor growth was commonly observed by 3-week post-injection, as evidenced by hunched posture and abdominal distension. At necropsy, ovarian cancer was identified throughout the peritoneal cavity consisting of solid tumor masses on the peritoneal surface that easily dislodged, lymph node metastasis in the groins and bowel mesentery, and solid tumor masses that appear to invade the underlying tissues (Fig. 3b). Table 2 Tumor weights and ascites volume in mice treated with cellular therapy and cytokines
Fig. 3 a Graph showing the percent weight change of the mice in the various treatment groups. b Example of a harvested tumor from the mouse peritoneum. A 2.5-g tumor was harvested from a 25-g mouse. IL-2 Interleukin 2, IFN pegylated-interferon a-2b, PBMC peripheral blood mononuclear cells
Treatmenta
Mean tumor weight (range), g
Mean ascites volume (range), mL
No treatment
0.8 (0.5–1.3)
0.3 (0–1.7)
PBMC
1.1
0.4
(0.7–1.2)
(0–1.6)
IL-2
1.1
0.9
(0.6–1.8)
(0–3.3)
PBMC ? IL-2
1.2
0.98
(0.7–1.5)
(0–4.3)
PBMC ? IFNa-2b
1.0
0.5
(0.8–1.7)
(0–2.5)
PBMC ? IFNa-2b ? IL-2
1.5
0.8
(0.9–3.0)
(0–1.7)
PBMC Peripheral blood mononuclear cells, IL-2 interleukin 2, IFNa-2b pegylated-interferon a-2b a
No statistical difference between any of the treatments for mean tumor weight or volume of ascites (p [ 0.05)
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with intra-peritoneal IL-2 provided a survival advantage to the mice compared to the controls that were treated with IL-2 alone, PBMC alone, or no treatment (Fig. 2; Table 1). In addition, we have established a reliable intra-peritoneal mouse model for studying combination therapies against ovarian cancer. We found that the mice tolerated IP treatment with human PBMC plus cytokines, as the treatments did not result in significant weight loss or obvious graft versus host effects. These in vivo data are consistent with our previous in vitro findings that PBMC in combination with cytokines inhibit ovarian cancer cells [7, 8, 10]. We have previously shown that PBMC in combination with IL-2 and IFNa-2b elicit a significant cytotoxic response against the ovarian cancer cell lines, SKOV3-AF2, SKOV3-RFP cells (described in Ingersoll et al. [8] ), and ES-2 in vitro [7, 8, 10]. However, in the present mouse study, we found that the most effective combination was PBMC plus IL-2 alone, whereas in vitro PBMC plus IL-2 and INFa-2b was the most effective combination [8]. Mice that received PBMC plus IL-2 and PEG-IFNa-2b did not show improved overall survival advantage compared to the control mice. This could be secondary to the athymic nude mice, while they are immuno-compromised, they maintain functional myeloid and NK-cells. IFNa-2b is known to activate myeloidderived suppressor cells (MDSC), which could account for the lack of effectiveness observed in the treatment groups that received a combination of PBMC plus IFNa-2b. Depletion of MDSC in the mice by an anti-Gr1? antibody treatment may theoretically render cell therapy in combination with IFNa-2b more effective. Our observation that mice treated with low-dose IL-2 and PBMC had a survival advantage compared to mice treated with IL-2 or PBMC alone is consistent with other publications [13–15]. IL-2 is known to activate immune cells including cytotoxic T-cells, which could lead to augmented tumor cell killing [13]. In a phase II trial of single-agent intra-peritoneal IL-2 in patients with platinum-refractory ovarian cancer, an impressive overall response rate of 25 % was reported by Vlad et al. [14]. The median survival for this group of poor-prognosis patients was 2.1 years, and increased numbers of circulating IFNc producing CD8? T-cells was associated with improved survival [p = 0.05; hazard ratio (HR) = 0.971]. In the current study using athymic mice, we found that IL-2 treatment alone did not provide a survival advantage despite using similar IL-2 doses (Fig. 2a; Table 1). The dosages used were 6 9 105 IU/m2 in the phase II trial performed by Vlad et al. [14], which is comparable to the dosage used in our current study on mice. Our dose of 4000 U/injection is equivalent to 5.7 9 106 IU/m2 based on the conversion for mice as described by Reagan-Shaw et al. [15]. This finding may be explained by the fact that
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athymic nude mice lack functional T-cells and the therapeutic effects observed by Vlad et al. [14] in patients with ovarian cancer may be a result of IL-2 stimulation of endogenous T-cells. Therefore, our in vivo murine experiments as well as these clinical trial findings in patients with ovarian cancer both support the hypothesis that immune system activation may play key role(s) in ovarian cancer progression and survival. A recent phase I clinical trial performed by Wright et al. [16] reported that IP infusion of cytotoxic T-cells was well tolerated with only one of the seven patients affected with abdominal pain. Likewise, the mice in this study tolerated intra-peritoneal treatment using human PBMC without apparent indicators of pain. Wright et al. [16] did not demonstrate a clinical response by either a decrease in CA125 or tumor measurements. It is important to recognize that this phase I toxicity study did not include the use of cytokines which could modulate T-cell functions. Immune system modulation is likely necessary because of the immunosuppressive micro-environment supported by malignant tumors [17]. There are several mechanisms by which the solid malignancies might evade the immune system, including impaired mechanisms for antigen presentation [18], expression of immunosuppressive factors [19–21], down-regulation of adhesion molecules [22, 23], and peripheral tolerance [24]. Approaches that have been suggested to overcome the lack of efficacy observed in pilot cellular therapy trials include enhancers for immune stimulation such as cytokines and modification of the host immune environment [17, 25, 26]. Development of successful adaptive immunotherapy regimens that activate T-cell function with limited toxicity will be necessary to overcome these obstacles. Ovarian cancer is an ideal malignancy for intra-peritoneal cellular therapy treatment because the majority of cases present with carcinomatosis involving the peritoneal cavity. In addition, IP chemotherapy is routinely administered by gynecologic oncologists and consequently they are familiar with IP instillation technique and management of IP access devices [27]. Furthermore, IP cellular therapy treatments may escape or be minimally affected by the large number of immunosuppressive regulatory cells (such as MDSC and T-regulatory cells) normally found in the circulation of cancer patients. Thus, these pre-clinical investigations support our working hypothesis that cellular-based immunotherapy may be an effective treatment strategy for patients with ovarian cancer. These experiments indicate that PBMC in combination with IL-2 demonstrate the best efficacy in our murine xenograft model as evidenced by improved survival compared to mice treated with IL-2 or PBMC alone and likely also compared to IL-2 plus IFNa-2b treated mice. Similar to women with newly diagnosed ovarian cancer,
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the clinical presentation in our murine model was also quite variable with respect to ascites and tumor volume, despite the fact that mice were genetically similar and injected with the same batch of tumor cells at the same time. The next line of investigation should be the use of ex vivo expanded therapeutic cells from ovarian cancer patients in the murine model, as we have already shown that these expanded cells have an augmented anti-cancer effect in vitro [10]. As we consider the development of human clinical trials, use of autologous immune cells will be imperative to avoid the well-known toxicities associated with HLA mismatched treatments, and mononuclear cell population expansion will be necessary to produce the numbers of stimulated effector cells that are likely necessary for clinical efficacy. Nonetheless, this study provides the groundwork for further development of cellular immunotherapy in the treatment of ovarian cancer. Acknowledgments The authors would like to thank Mr. Brian Kirk for his technical help with a portion of the animal experiments. This research study was funded by the Bankhead-Coley Cancer Research Program (State of Florida Department of Health) and partial support from the Ovarian Cancer Alliance of Florida, the Gala Endowed Program for Oncologic Research, and the Wonderful Whacky Women of Florida, Redneck Riviera Chapter. Conflict of interest
None.
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Cellular therapy in combination with cytokines improves survival in a xenograft mouse model of ovarian cancer Susan B. Ingersoll1,2 • Sarfraz Ahmad1,2,3 • Hasina C. McGann1 • Robert K. Banks3 Nicole M. Stavitzski1 • Milan Srivastava1 • Ghazanfar Ali1 • Neil J. Finkler1,2,3 • John R. Edwards3,4 • Robert W. Holloway1,2,3
•
Received: 30 January 2015 / Accepted: 30 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Studies have shown enhanced survival of ovarian cancer patients in which the tumors are infiltrated with tumor infiltrating lymphocytes and natural killer cells showing the importance of immune surveillance and recognition in ovarian cancer. Therefore, in this study, we tested cellular immunotherapy and varying combinations of cytokines (IL-2 and/or pegylated-IFNa-2b) in a xenograft mouse model of ovarian cancer. SKOV3-AF2 ovarian cancer cells were injected intra-peritoneally (IP) into athymic nude mice. On day 7 post-tumor cell injection, mice were injected IP with peripheral blood mononuclear cells (PBMC; 5 9 106 PBMC) and cytokine combinations [IL-2 ± pegylated-IFNa-2b (IFN)]. Cytokine injections were continued weekly for IFN (12,000 U/injection) and thrice weekly for IL-2 (4000 U/injection). Mice were
euthanized when they became moribund due to tumor burden at which time tumor and ascitic fluid were measured and collected. Treatment efficacy was measured by improved survival at 8 weeks and overall survival by Kaplan–Meier analysis. We observed that the mice tolerated all treatment combinations without significant weight loss or other apparent illness. Mice receiving PBMC plus IL-2 showed improved median survival (7.3 weeks) compared to mice with no treatment (4.2 weeks), IL-2 (3.5 weeks), PBMC (4.0 weeks), or PBMC plus IL-2 and IFN (4.3 weeks), although PBMC plus IL-2 was not statistically different than PBMC plus IFN (5.5 weeks, p [ 0.05). We demonstrate that cytokine-stimulated cellular immune therapy with PBMC and IL-2 was well tolerated and resulted in survival advantage compared to untreated controls and other cytokine combinations in the nude-mouse model.
Part of this study was presented as Featured Poster at the 44th Annual Meetings on Women’s CancerÒ of the Society of Gynecologic Oncology (SGO), in March 2013, at Los Angeles, CA; and the 104th Annual Meeting of the American Association of Cancer Research (AACR), in April 2013, at Washington, DC.
Keywords Cellular therapy Cytokines Ovarian cancer Xenograft mouse model Peripheral blood mononuclear cells
& Susan B. Ingersoll [email protected]
Introduction
& Sarfraz Ahmad [email protected] 1
Florida Hospital Gynecologic Oncology, Florida Hospital Cancer Institute, 2501 N. Orange Ave., Suite 786, Orlando, FL 32804, USA
2
Florida State University College of Medicine, Orlando, FL 32801, USA
3
University of Central Florida College of Medicine, Orlando, FL 32827, USA
4
Indiana Blood and Marrow Transplantation, Indianapolis, IN 46237, USA
Epithelial ovarian cancer is usually diagnosed in advanced stages with metastases, which requires both aggressive cytoreductive surgery and chemotherapy to control symptoms and prolong survival. Unfortunately, while median survival has improved significantly in recent decades due to these efforts, the majority of patients ultimately suffer symptomatic recurrence of disease and eventual death secondary to the development of chemotherapy-resistant tumor. In order to improve long-term survival for women with ovarian cancer, new therapeutic approaches that are
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more effective in the treatment of chemotherapy-resistance disease are needed. Immunotherapy has been proposed for the treatment of several malignancies including ovarian cancer. Early investigations using tumor infiltrating lymphocytes (TIL) for the treatment of ovarian cancer failed to demonstrate effectiveness, likely because of the inability to expand an adequate population of therapeutic cells [1]. Furthermore, specifics of the interplay between tumor micro-environment and many aspects of the host immune response were poorly described at that time. More recent advances in the understanding of immune environment modulation (such as the ability to down-regulate immunosuppressive cells through the use of antibodies), the processes of immune effector cell expansion, and the availability of commercially manufactured cellular therapy products (such as umbilical cord blood units or dendritic cells) may improve the efficacy of cellular therapy [2, 3]. Several lines of scientific evidence suggest that immunotherapy using stimulated immune cells is a rational approach for ovarian cancer therapy. Enhanced survival of patients with ovarian cancer whose tumors were infiltrated with TIL and natural killer (NK) cells has been observed, lending indirect evidence for this hypothesis. Zhang et al. [4] were the first to show that increased numbers of CD3? T-cell infiltrates were associated with improved progression-free and overall survival for patients with International Federation of Gynecology and Obstetrics (FIGO) stage III/IV ovarian cancer. The 5-year overall survival for patients with significant T-cell infiltrates was 38 % compared to only 4.5 % for those with fewer T-cell infiltrates, suggesting a possible relationship between a patient’s immune response and survival with ovarian cancer [4]. It has been hypothesized by others that patients who experience prolonged disease-free survival may have an augmented immunological response to their disease [5]. Furthermore, in another disease setting, Dudley et al. [6] found that patients with melanoma who received chemotherapy in combination with immunotherapy showed increased response rates compared to chemotherapy treated patients. Previously, we have shown that immune effector cells from healthy donors can elicit a significant cytotoxic response against ovarian cancer in vitro. Peripheral blood mononuclear cells (PBMC) in the presence of the cytokines [interleukin-2 (IL-2) and interferon a-2b (IFNa-2b)], elicit up to a 70 % cytotoxic response against ovarian cancer cells in vitro [7–10]. This cytotoxic response was not observed in the absence of IL-2 indicating that IL-2 is necessary for the PBMC to elicit the cytotoxic response [7, 10]. In addition, we have shown that IFNa-2b has an inhibitory effect on ovarian cancer cell growth [7]. Therefore, in this study, we proposed to further evaluate
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cellular therapy using different combinations of cytokines (IL-2 and IFNa-2b) in combination with PBMC in a xenograft murine model of ovarian cancer in order to study their effects in vivo.
Materials and methods Cell line culture SKOV3-AF2 cells were derived from SKOV-3 (ATCC, Manassas, VA) cells as recently described by Ingersoll et al. [7, 8]. Briefly, SKOV3-AF2 cells were derived by first stably transfecting SKOV-3 cells with red-fluorescent protein (SKOV3-RFP), so that the cells could be distinguished from the immune effector cells by fluorescence. Next, the cells were passaged through nude mice to make the cells more tumorigenic in a xenograft mouse model as described previously [7, 8]. Single-cell suspensions were made from solid tumor and ascitic fluid harvested from the parental SKOV3-RFP tumors and grown in culture. These cells were subsequently injected into the peritoneal cavities of nude mice and solid tumor was subsequently collected by necropsy. Single-cell suspensions from the harvested tumors were grown in culture. The line derived from this tumor suspension was called SKOV3-AF2. We have previously shown that these SKOV3-AF2 cells are more tumorigenic in nude mice, show increased resistance to IFNa-2b in vitro, and are sensitive to immune-mediated cytotoxicity in vitro [7, 8]. All the cells were grown in McCoy’s media (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS; ATCC) and 1 % penicillin and streptomycin (Invitrogen) in 5 % CO2 at 37 °C.
Mononuclear cell preparation LeukoPaks (Florida Blood Center, Orlando, FL) were diluted 1:1 with RPMI media (Invitrogen), and PBMC were isolated using a Ficoll-Paque (Invitrogen) density gradient. The PBMC were washed twice in phosphatebuffered saline (PBS; Invitrogen) and once in RPMI-1640 (Invitrogen) media at which point in time the cells were resuspended in cryopreservation media containing 10 % dimethyl-sulphoxide (DMSO; Sigma-Aldrich, St. Louis, MO), 20 % FBS, and 70 % RPMI, and stored in liquid nitrogen until the cells were to be prepared for the cytotoxicity experiments or injection into the mice. The PBMC were thawed at 37 °C and diluted with RPMI media, the PBMC were washed twice with PBS to remove the DMSO and re-suspended for use in the in vitro cytotoxicity assays or the in vivo mouse treatments as described below.
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In vitro cytotoxicity assays The cytotoxicity assay used in this study was similar to that we have recently reported [8, 10]. Briefly, percent cytotoxicity of ovarian cancer cells was measured by lactate dehydrogenase (LDH) release using the Cytotox Membrane Integrity Assay (Promega Corp., Madison, WI) [11, 12]. The LDH release assay has been shown to produce similar results to 51Cr release assays when experiments were run in parallel [11, 12]. PBMC were co-cultured with SKOV3-AF2 ovarian cancer cells in McCoy’s media supplemented with 5 % FBS at a ratio of 20:1 (effector cells:target cells) in the presence of IL-2 (25 ng/ml; PeproTech, Rocky Hill, NJ) and/or IFNa-2b (500 U/ml; R&D Systems, Minneapolis, MN) for 24 or 48 h at which point LDH release was measured by excitation at 530 nm and emission at 590 nm at 30 min using a Synergy 2 (BioTek, Winooski, VT) plate reader. Percent Cytotoxicity = 100 9 [(Target - Effector Cell Spontaneous)/(Maximum Release - Cell Spontaneous)]. All experimental conditions were performed in quadruplicate. The data shown are the mean cytotoxicity elicited by the PBMC isolated from two independent donor LeukoPaks. These were the same LeukoPaks that were used for the immune cell therapy in the in vivo mouse experiments. Xenograft mouse model for ovarian cancer Female athymic nude (nu/nu) mice (Harlan Sprague– Dawley, Inc., Indianapolis, IN) were housed at the University of Central Florida (Orlando, FL) Wild Animal Facility under specific pathogen-free conditions. This study was conducted following an Institutional Animal Care and Use Committee (IACUC) approved protocol. Female athymic nude (nu/nu) mice (7–9 weeks old; n = 90) were injected intra-peritoneally (IP) with 1 9 106 SKOV3-AF2 cells. The mice were weighed on day 0 and every 7 days until the time of euthanasia. The mice were euthanized when they became moribund (swollen abdomens, dehydration, difficult movement, hunched posture, and labored breathing) due to tumor burden. Solid tumor and ascitic fluid, when present, were collected from each animal, and samples were snap-frozen in liquid nitrogen.
Table 1 Survival of mice treated with cellular therapy and cytokines Treatment
Median survival (range), weeks
Percent survival at 8 weeks (%)
No treatment
4.2
17
(3.5–9.5) PBMC
4.0
15
(3.2–9.5) IL-2
3.5
0
(3.2–7.5) IFNa-2b PBMC ? IL-2
4.7 (3.2–9.5)
22
7.3
50
(3.5–16) PBMC ? IFNa-2b
5.5
PBMC ? IFNa-2b ? IL-2
4.3
11
(3.2–10.5) 10
(3.2–8.5) PBMC Peripheral blood mononuclear cells, IL-2 interleukin 2, IFNa2b pegylated-interferon a-2b
times a week, and the PEG-IFNa-2b injections were continued once a week. The PBMC dosage was based on our previous work to determine if the mice would tolerate 5 9 106 human immune cells injected IP (unpublished data). The cytokine dosages used were based on our previous optimization studies [7]. The in vivo cellular therapy experiments were performed in duplicate using two independent donor LeukoPaks. Statistical analyses Data are presented as percent and/or mean (±standard deviation, SD) in respective groups or time points. Statistical analysis was performed using SigmaPlot Version 11.0 (San Jose, CA). Student’s t test was performed when the data passed the equal variance test, and Mann–Whitney U test was used when the data failed the equal variance test. Statistical significance was declared when p \ 0.05 determined. Kaplan–Meier analysis was used for the survival determinations of the mice.
In vivo cell and cytokine treatments
Results
On day 7 post-tumor cell injection, the mice were randomized to seven groups (n = 10-15 per group; n = 90 total animals; treatment groups as shown in Table 1). The mice were injected IP with 5 9 106 PBMC isolated from a LeukoPak and cytokines [IL-2 (4000 U/injection) and PEG-IFNa-2b (12,000 U/injection, GenScript Corp. Piscataway, NJ). The IL-2 injections were continued three
Cryopreserved PBMC isolated from LeukoPaks elicit a significant cytotoxic response against ovarian cancer cells in vitro The cytotoxic response of cryopreserved PBMC isolated from LeukoPaks (multiple independent donors) in the presence of IL-2 and IFNa-2b against ovarian cancer cells
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(SKOV3-AF2) was determined. PBMC from LeukoPaks elicited a significant cytotoxic response in the presence of IL-2 plus IFNa-2b against SKOV3-AF2 cells (Fig. 1, left group). There was no significant response in the absence of cytokines or in the presence of cytokines alone (Fig. 1, right group). These experiments demonstrate that the response is PBMC-mediated and the greatest cytotoxicity was observed with combination IL-2 and INF-a2b (Fig. 1). The cryopreserved PBMC shown in these in vitro experiments were the same cells used as effector cells in the in vivo mouse experiments.
(7.3 weeks; p \ 0.05) showed a significant improvement in median survival compared to mice receiving IL-2 alone (3.5 weeks), PBMC alone (4.0 weeks), or no treatment (4.2 weeks; Table 1). Also, the Kaplan–Meier curve shows that the mice receiving combination treatment of PBMC plus IL-2 favored a shift to the right indicating improved overall survival (Fig. 2a). Mice receiving PBMC plus
A
Mice treated with PBMC Plus IL-2 have a survival advantage compared to untreated mice or those treated with PBMC or IL-2 alone
No Treatment IL-2 PBMC PBMC + IL-2
0.8
0.6
Survival
Immune cell therapy in combination with cytokines was tested in a xenograft mouse model of ovarian cancer using SKOV3-AF2 ovarian cancer cells. As demonstrated previously [7], SKOV3-AF2 cells produced tumors that are poorly differentiated surface epithelial carcinoma, growing in solid nests and sheets of large cells with a moderate amount of amphophilic or clear cytoplasm, and tumor cells were focally pleomorphic and multinucleated. The results shown are from multiple independent experiments using PBMC isolated from LuekoPaks from different donors. In these experiments, mice treated with PBMC plus IL-2
1.0
0.4
0.2
0.0 0
2
4
6
8
10
12
14
Weeks 40
No Cytokines
35
No Treatment IFN PMBC PBMC + IFN PBMC + IFN + IL-2
1.0
IL-2
30
IFN
25
0.8
IL-2 + IFN
20 15
0.6
10
Survival
Percent Cytotoxicity
B
5 0
0.4
-5 -10
20:1 PBMC:SKOV3-AF2
SKOV3-AF2 without PBMC
Fig. 1 In vitro cytotoxicity experiments: Cytotoxicity elicited by PBMC isolated from LeukoPaks against SKOV3-AF2 was assessed. The graph shows the mean percent cytotoxicity elicited plus the standard deviation. The left side of the graph shows the cytotoxicity elicited by PBMC at a ratio of 20:1 PBMC to SKOV3-AF2 cells. The right side of the graph shows the negative controls without PBMC. The PBMC elicited a significant cytotoxic response in the presence of IL-2 plus IFNa-2b compared to controls lacking effector cells (p \ 0.05, determined by t test). The IL-2 concentration was 25 ng/ ml, and the INFa-2b concentration was 500 U/ml. IL-2 Interleukin 2, IFN pegylated-interferon a-2b, PBMC peripheral blood mononuclear cells
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0.2
0.0 0
2
4
6
8
10
12
14
Weeks
Fig. 2 Kaplan–Meier survival analysis of mice treated with cell and/ or cytokine therapies. a Mice that were treated with PBMC plus IL-2 showed improved overall survival compared to the control groups. b Mice that were treated with PBMC plus IFN did not show improved survival compared to the control groups. IL-2 Interleukin 2, PBMC peripheral blood mononuclear cells, IFN pegylated-interferon a-2b
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IFNa-2b (median 5.5 weeks) or PBMC plus IFNa-2b and IL-2 (median 4.3 weeks) did not show improved survival (Fig. 2b; Table 1) compared to the control mice. In addition, there was no significant difference in the survival of mice treated with PBMC plus IFNa-2b compared to the mice treated with PBMC plus IFNa-2b and IL-2 (Fig. 2b; Table 1). As shown in Table 2, there were no significant differences in tumor weights or ascitic fluid volume at the time of necropsy, likely because the mice were euthanized when they became moribund due to tumor burden rather than a specific time point post-tumor cell injection. In addition, there was no apparent correlation with the presence or absence of ascites, and any of the experimental treatments. Also, as shown in Table 2, there were mice in each treatment group that did not produce ascitic fluid, while other mice in the same treatment groups had ascites at the time of euthanasia. In total, 51 % of the mice had ascites at the time of euthanasia.
Discussion In this study, we provide in vivo evidence that in a xenograft murine model of ovarian cancer, treatment with intraperitoneal PBMC (isolated from healthy donors) combined
Mice tolerate intra-peritoneal cytokine and cellular therapy treatment The mice tolerated all treatments as indicated by no significant weight loss (Fig. 3a), hunched posture, or labored breathing. Significant tumor growth was commonly observed by 3-week post-injection, as evidenced by hunched posture and abdominal distension. At necropsy, ovarian cancer was identified throughout the peritoneal cavity consisting of solid tumor masses on the peritoneal surface that easily dislodged, lymph node metastasis in the groins and bowel mesentery, and solid tumor masses that appear to invade the underlying tissues (Fig. 3b). Table 2 Tumor weights and ascites volume in mice treated with cellular therapy and cytokines
Fig. 3 a Graph showing the percent weight change of the mice in the various treatment groups. b Example of a harvested tumor from the mouse peritoneum. A 2.5-g tumor was harvested from a 25-g mouse. IL-2 Interleukin 2, IFN pegylated-interferon a-2b, PBMC peripheral blood mononuclear cells
Treatmenta
Mean tumor weight (range), g
Mean ascites volume (range), mL
No treatment
0.8 (0.5–1.3)
0.3 (0–1.7)
PBMC
1.1
0.4
(0.7–1.2)
(0–1.6)
IL-2
1.1
0.9
(0.6–1.8)
(0–3.3)
PBMC ? IL-2
1.2
0.98
(0.7–1.5)
(0–4.3)
PBMC ? IFNa-2b
1.0
0.5
(0.8–1.7)
(0–2.5)
PBMC ? IFNa-2b ? IL-2
1.5
0.8
(0.9–3.0)
(0–1.7)
PBMC Peripheral blood mononuclear cells, IL-2 interleukin 2, IFNa-2b pegylated-interferon a-2b a
No statistical difference between any of the treatments for mean tumor weight or volume of ascites (p [ 0.05)
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with intra-peritoneal IL-2 provided a survival advantage to the mice compared to the controls that were treated with IL-2 alone, PBMC alone, or no treatment (Fig. 2; Table 1). In addition, we have established a reliable intra-peritoneal mouse model for studying combination therapies against ovarian cancer. We found that the mice tolerated IP treatment with human PBMC plus cytokines, as the treatments did not result in significant weight loss or obvious graft versus host effects. These in vivo data are consistent with our previous in vitro findings that PBMC in combination with cytokines inhibit ovarian cancer cells [7, 8, 10]. We have previously shown that PBMC in combination with IL-2 and IFNa-2b elicit a significant cytotoxic response against the ovarian cancer cell lines, SKOV3-AF2, SKOV3-RFP cells (described in Ingersoll et al. [8] ), and ES-2 in vitro [7, 8, 10]. However, in the present mouse study, we found that the most effective combination was PBMC plus IL-2 alone, whereas in vitro PBMC plus IL-2 and INFa-2b was the most effective combination [8]. Mice that received PBMC plus IL-2 and PEG-IFNa-2b did not show improved overall survival advantage compared to the control mice. This could be secondary to the athymic nude mice, while they are immuno-compromised, they maintain functional myeloid and NK-cells. IFNa-2b is known to activate myeloidderived suppressor cells (MDSC), which could account for the lack of effectiveness observed in the treatment groups that received a combination of PBMC plus IFNa-2b. Depletion of MDSC in the mice by an anti-Gr1? antibody treatment may theoretically render cell therapy in combination with IFNa-2b more effective. Our observation that mice treated with low-dose IL-2 and PBMC had a survival advantage compared to mice treated with IL-2 or PBMC alone is consistent with other publications [13–15]. IL-2 is known to activate immune cells including cytotoxic T-cells, which could lead to augmented tumor cell killing [13]. In a phase II trial of single-agent intra-peritoneal IL-2 in patients with platinum-refractory ovarian cancer, an impressive overall response rate of 25 % was reported by Vlad et al. [14]. The median survival for this group of poor-prognosis patients was 2.1 years, and increased numbers of circulating IFNc producing CD8? T-cells was associated with improved survival [p = 0.05; hazard ratio (HR) = 0.971]. In the current study using athymic mice, we found that IL-2 treatment alone did not provide a survival advantage despite using similar IL-2 doses (Fig. 2a; Table 1). The dosages used were 6 9 105 IU/m2 in the phase II trial performed by Vlad et al. [14], which is comparable to the dosage used in our current study on mice. Our dose of 4000 U/injection is equivalent to 5.7 9 106 IU/m2 based on the conversion for mice as described by Reagan-Shaw et al. [15]. This finding may be explained by the fact that
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athymic nude mice lack functional T-cells and the therapeutic effects observed by Vlad et al. [14] in patients with ovarian cancer may be a result of IL-2 stimulation of endogenous T-cells. Therefore, our in vivo murine experiments as well as these clinical trial findings in patients with ovarian cancer both support the hypothesis that immune system activation may play key role(s) in ovarian cancer progression and survival. A recent phase I clinical trial performed by Wright et al. [16] reported that IP infusion of cytotoxic T-cells was well tolerated with only one of the seven patients affected with abdominal pain. Likewise, the mice in this study tolerated intra-peritoneal treatment using human PBMC without apparent indicators of pain. Wright et al. [16] did not demonstrate a clinical response by either a decrease in CA125 or tumor measurements. It is important to recognize that this phase I toxicity study did not include the use of cytokines which could modulate T-cell functions. Immune system modulation is likely necessary because of the immunosuppressive micro-environment supported by malignant tumors [17]. There are several mechanisms by which the solid malignancies might evade the immune system, including impaired mechanisms for antigen presentation [18], expression of immunosuppressive factors [19–21], down-regulation of adhesion molecules [22, 23], and peripheral tolerance [24]. Approaches that have been suggested to overcome the lack of efficacy observed in pilot cellular therapy trials include enhancers for immune stimulation such as cytokines and modification of the host immune environment [17, 25, 26]. Development of successful adaptive immunotherapy regimens that activate T-cell function with limited toxicity will be necessary to overcome these obstacles. Ovarian cancer is an ideal malignancy for intra-peritoneal cellular therapy treatment because the majority of cases present with carcinomatosis involving the peritoneal cavity. In addition, IP chemotherapy is routinely administered by gynecologic oncologists and consequently they are familiar with IP instillation technique and management of IP access devices [27]. Furthermore, IP cellular therapy treatments may escape or be minimally affected by the large number of immunosuppressive regulatory cells (such as MDSC and T-regulatory cells) normally found in the circulation of cancer patients. Thus, these pre-clinical investigations support our working hypothesis that cellular-based immunotherapy may be an effective treatment strategy for patients with ovarian cancer. These experiments indicate that PBMC in combination with IL-2 demonstrate the best efficacy in our murine xenograft model as evidenced by improved survival compared to mice treated with IL-2 or PBMC alone and likely also compared to IL-2 plus IFNa-2b treated mice. Similar to women with newly diagnosed ovarian cancer,
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the clinical presentation in our murine model was also quite variable with respect to ascites and tumor volume, despite the fact that mice were genetically similar and injected with the same batch of tumor cells at the same time. The next line of investigation should be the use of ex vivo expanded therapeutic cells from ovarian cancer patients in the murine model, as we have already shown that these expanded cells have an augmented anti-cancer effect in vitro [10]. As we consider the development of human clinical trials, use of autologous immune cells will be imperative to avoid the well-known toxicities associated with HLA mismatched treatments, and mononuclear cell population expansion will be necessary to produce the numbers of stimulated effector cells that are likely necessary for clinical efficacy. Nonetheless, this study provides the groundwork for further development of cellular immunotherapy in the treatment of ovarian cancer. Acknowledgments The authors would like to thank Mr. Brian Kirk for his technical help with a portion of the animal experiments. This research study was funded by the Bankhead-Coley Cancer Research Program (State of Florida Department of Health) and partial support from the Ovarian Cancer Alliance of Florida, the Gala Endowed Program for Oncologic Research, and the Wonderful Whacky Women of Florida, Redneck Riviera Chapter. Conflict of interest
None.
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