Delivery of chemotherapeutic agents using drug-loaded irradiated tumor cells to treat murine ovarian tumors
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Ovarian cancer is the leading cause of death among women with gynecologic malignancies in the United States. Advanced ovarian cancers are difficult to cure with the current available chemotherapy, which has many associated systemic side effects. Doxorubicin is one such chemotherapeutic agent that can cause cardiotoxicity. Novel methods of delivering chemotherapy without significant side effects are therefore of critical need.
In the current study, we generated an irradiated tumor cell-based drug delivery system which uses irradiated tumor cells loaded with the chemotherapeutic drug, doxorubicin.
We showed that incubation of murine ovarian cancer cells (MOSEC) with doxorubicin led to the intracellular uptake of the drug (MOSEC-dox cells) and the eventual death of the tumor cell. We then showed that doxorubicin loaded MOSEC-dox cells were able to deliver doxorubicin to MOSEC cells in vivo. Further characterization of the doxorubicin transfer revealed the involvement of cell contact. The irradiated form of the MOSEC-dox cells were capable of treating luciferase-expressing MOSEC tumor cells (MOSEC/luc) in C57BL/6 mice as well as in athymic nude mice resulting in improved survival compared to the non drug-loaded irradiated MOSEC cells. Furthermore, we showed that irradiated MOSEC-dox cells was more effective compared to an equivalent dose of doxorubicin in treating MOSEC/luc tumor-bearing mice.
Thus, the employment of drug-loaded irradiated tumor cells represents a potentially innovative approach for the delivery of chemotherapeutic drugs for the control of ovarian tumors.
KeywordsOvarian Cancer Doxorubicin Luciferase Activity Athymic Nude Mouse Tumor Cell Death
Ovarian cancer is the leading cause of death among women with gynecologic malignancies and is the eighth most common cancer in the United States [1, 2]. Most patients who are diagnosed with ovarian cancer are detected at an advanced stage (III/IV), often presenting with complications associated with intraperitoneal metastasis. Unfortunately, less than half of the women diagnosed with ovarian cancer survive 5 year post-diagnosis [1, 3]. Current chemotherapies are useful in the control of advanced stages of ovarian cancer but have many toxic side effects [4, 5, 6]. Thus, there is a critical need for alternative approaches to administer chemotherapeutic agents to control advanced stages of ovarian cancer without serious side effects.
Doxorubicin, which is part of the anthracyline family, has been successfully applied to treat a variety of tumors including ovarian cancer (for review see ). While doxorubicin is more effective than its structural precursor, daunorubicin, the major side effects of the drugs are similar. Studies have shown that the toxicity of doxorubicin can lead to chronic cardiomyopathy [8, 9, 10]. Thus, some attempts have been made to diminish the toxicity of doxorubicin. One currently administered form of doxorubicin is DOXIL®, whereby doxorubicin is encapsulated by lipids to prolong the circulation of the drug in the bloodstream . Although the liposome protects some cells from doxorubicin, they can reach systemic circulation and the drug can still reach heart tissue to cause damage.
In the current study, we hypothesized that local administration of doxorubicin delivered by irradiated tumor cells may reduce the dose required to treat murine ovarian cancer cells and decrease the systemic circulation of doxorubicin. We showed that preparation of murine ovarian cancer cells (MOSEC) with doxorubicin led to the intracellular uptake of the drug (MOSEC-dox cells). We then showed that doxorubicin loaded MOSEC-dox cells were able to deliver doxorubicin to MOSEC cells in vivo. Thus, local delivery of chemotherapeutic drugs by tumor may represent a potentially innovative approach for the control of ovarian tumors.
Materials and methods
Female C57BL/6 and athymic nude mice (6-8 wks) were acquired from the National Cancer Institute (Frederick, MD). All animals were maintained under specific pathogen-free conditions, and all procedures were done according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
Cell lines and reagents
The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously . In brief, HPV-16 E6, E7, and the ras oncogene were used to transform primary C57BL/6 mice lung epithelial cells to generate the TC-1 cell line. The MOSEC cell line was generated as described previously . The MOSEC cell line was originally derived from murine ovarian surface epithelial cells . MOSEC-luciferase (MOSEC/luc) cells were generated as described previously . MOSEC cells were transduced with a retrovirus containing luciferase. In order to generate a retrovirus containing luciferase, a pLuci-thy1.1 construct expressing both luciferase and thy1.1 was made. Firefly luciferase was amplified by PCR from pGL3-basic (Promega) using the 5' primer CGGAGATC TATGGAAGACGCCAAAAAC and the 3' primer CGGGTTAACTTACACGGCGATCTTTCC. The amplified luciferase cDNA was inserted into the Bgl II and Hpa I sites of the bicistronic vector pMIG-thy1.1. Both luciferase and thy1.1 cDNA are under the control of a single promoter element and separated by an internal ribosomal entry site (IRES). The pLuci-thy1.1 was transfected into Phoenix packaging cell line and the virion-containing supernatant was collected 48 h after transfection. The supernatant was immediately treated using a 0.45-mm cellulose acetate syringe filter (Nalgene, Rochester, NY, USA) and used to infect MOSEC cells in the presence of 8 mg/ml Polybrene (Sigma, St Louis, MO, USA). MOSEC/luc cells were sorted using preparative flow cytometry of stained cells with Thy1.1 antibody (BD, Franklin Lakes, NJ, USA). MOSEC-GFP cells were generated with a GFP-expressing lentivirus. Briefly, the lentiviral vector pCDH1-EF1-GFP was transfected into a Phoenix packaging cell line using lipofectamine (Invitrogen, Carlsbad, CA, USA) and the virion-containing supernatant was collected 48 hours after transfection. The supernatant was then filtered through a 0.45 mm cellulose acetate syringe filter (Nalgene, Rochester, NY, USA) and used to infect MOSEC cells in the presence of 8 mg/ml Polybrene (Sigma-Aldrich, St Louis, MO, USA). Transduced cells were isolated using preparative flow cytometry with GFP signal. The growth rate of all transduced cell lines was comparable with those of the parental, non-transduced cell lines (data not shown). Doxorubicin-HCL (D1515, Sigma-Aldrich, St Louis, MO, USA) was reconstituted with 0.9% NaCl normal saline and kept at 4°C for up to three weeks.
Determination of drug concentration inside doxorubicin-treated cells
MOSEC cells (1 × 106/ml) were cultured with complete media in the presence of different concentrations of doxorubicin (specifically 1, 10, 50, 100 μg/ml) for 2 hours at 37°C. The cells were then centrifuged at 10,000 rpm for 2 mins and the supernatant aspirated. The intracellular drug concentration was then determined within the remaining cell pellets. The cell pellets were lysed with protein extraction buffer (Pierce, Rockford, IL) and a 1:1 volume of DMSO was added. The concentration of drug was determined using a spectrophotometer at a 470 nm wavelength. Standard solutions of doxorubicin were made with media or extraction buffer with DMSO and used to generate a standard curve. Linear regression analysis was performed to generate the regression equation: y = 0.1607x -0.2143 with R2 = 0.9102.
Drug uptake, viability and proliferation of cells
MOSEC and MOSEC/luc cells (1 × 106/ml) were cultured in the presence of indicated doses of doxorubicin (specifically, 0.01, 0.1, 1, 10, 50, 100 μg/ml) at 37°C for 2 hours. Analysis was performed on a BD FACScan with CellQuest software (BD Biosciences Immunocytometry Systems, Mountain View, CA). After 2 hours of incubation with the drug, 5 × 104 cells/well of doxorubicin-treated MOSEC/luc cells were placed into 96-well plates with complete medium. D-Luciferin (potassium salt; Xenogen/Caliper Life Sciences, Alameda, CA) at a concentration of 150 μ g/ml was added to each well 7-8 minutes before imaging at 24 hrs. The imaging time was 30 seconds/plate. A MTT assay was performed with doxorubicin-treated MOSEC cells at 24 hours. The cells were then divided into 96-well plates. The MTT solution (30 μl of a 5 mg/ml solution) was added to the drug treated cancer cells and incubated for 4 hours. 100 μl DMSO was added to dissolve formazan crystals under vigorous shaking for 30 minutes which was followed by detection of absorption at OD 570 nm using a microplate reader (Molecular Probes, Invitrogen, Eugene, OR).
Drug transfer in vitro and in vivo
MOSEC cells pre-treated with doxorubicin (100 μg/ml) were mixed with MOSEC-GFP cells (5 × 105/well in 24-well plates) according to the indicated ratios (5 × 103 (100:1), 1 × 104 (50:1), 2 × 104 (25:1), or 5 × 104 (10:1)/well). After 24 hours, all the cells were collected and analyzed by flow cytometry. In order to confirm the necessity of cell to cell contact in the transfer of the drug, MOSEC-GFP cells (5 × 105/well in 24-well plates) were cultured in the bottom well of transwell plates (Corning Costar, Acton, MA) and MOSEC cells (5 × 104/well in 24-well plates) pre-treated with doxorubicin (100 μg/ml) were cultured in the upper chamber. After 24 hours, all of the cells in the bottom well were collected and analyzed by flow cytometry. For detecting transfer of drug in vivo, female C57BL/6 mice were inoculated with MOSEC-GFP (1 × 106/mouse) via the intraperitoneal route. After 24 hours, 2 × 104 (50:1) or 1 × 105 (10:1) MOSEC cells pre-treated with doxorubicin (100 μg/ml, 2 hrs) were injected into MOSEC-GFP tumor bearing mice. MOSEC-GFP cells or MOSEC-dox cells alone (1 × 106 /mouse) were injected into mice as controls. 24 hours after injecting the drug treated cells, mice from all groups were sacrificed with CO2 inhalation. Sterile PBS (10 ml) was injected into the peritoneum of each mouse to obtain peritoneal cells. Peritoneal cells (1 × 106 /mouse) were then analyzed by flow cytometry.
Characterization of tumor cell death by drug-treated tumor cells in vitro
MOSEC cells treated with a high dose (100 ug/ml) of doxorubicin were co-cultured with MOSEC/luc cells (5 × 104/well in 24-well plates) at different ratios (5 × 102(100:1), 1 × 103 (50:1), 2 × 103 (25:1), or 5 × 103 (10:1)/well). D-Luciferin (150 μg/ml) was added at different time points (just after mixing, on day1, and on day 2) and incubated for 7-8 min. An integration time of 30 seconds was used for luminescence image acquisition. Data was obtained on day 2.
Characterization of anti-tumor effects by drug-loaded tumor cells in C57BL/6 mice
Naïve female C57BL/6 mice were inoculated intraperitoneally with 5 × 105 live MOSEC/luc cells per mouse. On day 4 after tumor inoculation, tumor bearing mice were injected with low (2 × 105/mouse) or high (2 × 106/mouse) numbers of drug-treated, irradiated MOSEC cells. Tumor-bearing mice were also injected with 2 × 105 irradiated MOSEC cells as a control. For drug-treated, irradiated tumor cells, MOSEC cells were incubated for 2 hours with 100 μg/ml of doxorubicin and then subjected to 100,000 cGy/min for 10 minutes. Tumor growth was assessed with luminescence image acquisition on day 0 after treatment with drug treated cells and, subsequently, on a weekly basis. The mice were injected with 0.2 ml of 15 mg/ml D-luciferin. Detection of luminescence activity indicating relative tumor development was then performed using a Xenogen IVIS 200 Imaging System.
Characterization of anti-tumor effects of drug-loaded tumor cells in nude mice
Athymic nude mice (B6 background) were inoculated intraperitoneally with 2.5 × 105 live MOSEC/luc cells per mouse. On day 4, tumor bearing mice from each group (5mice/group) were treated with irradiated MOSEC cells (2 × 106/mouse) treated either with low (10 μg/ml) or high (100 μg/ml) doses of doxorubicin. Tumor growth was monitored on a weekly basis from the day of MOSEC/luc tumor challenge using the bioluminescence imaging method mentioned above.
Comparison of the different treatment regimens
The concentration of drug inside the doxorubicin-treated MOSEC cells was determined as described. Naïve female C57BL/6 mice were challenged intaperitoneally with 5 × 105 live MOSEC/luc cells per mouse. On day 4, tumor bearing mice were injected with 0.5 mg/kg (10 μg/mouse) of doxorubicin. To compare the effects of treatment on tumors, drug-loaded irradiated MOSEC cells (2 × 106/mouse, 100 μg/ml for 2 hrs) were injected into tumor-bearing mice. Tumor growth was monitored with luminescence activity on a weekly basis from the day of MOSEC/luc cells challenge.
All data expressed as mean ± SD are representative of at least two different experiments. Comparisons between individual data points were made using a Student's t test. Differences in survival between experimental groups were analyzed using the Kaplan-Meier approach. The statistical significance of group differences will be assessed using the log-rank test.
Doxorubicin is taken up by MOSEC tumor cells leading to tumor cell death
Transfer of doxorubicin from doxorubicin-loaded MOSEC cells to untreated MOSEC cells (MOSEC-GFP) is mediated through cells being in close vicinity of each other
MOSEC-luc cells incubated with MOSEC-dox cells are killed via transfer of doxorubicin
Doxorubicin is transferred from MOSEC-dox cells to MOSEC-GFP cells in vivo
Administration of irradiated MOSEC-dox tumor cells to MOSEC/luc tumor-bearing mice leads to decreased tumor burden
Administration of irradiated, pre-treated MOSEC cells with high levels of doxorubicin to MOSEC/luc tumor-bearing athymic nude mice leads to decreased tumor burden
Irradiated MOSEC-dox tumor cells are more effective than doxorubicin alone as treatment for MOSEC/luc tumors
In the current study, we generated a chemotherapeutic drug delivery system using irradiated MOSEC tumor cells which was capable of delivering the drug to other MOSEC tumor cells in tumor-bearing mice to result in potent therapeutic antitumor effects. Using the unique property of doxorubicin's red auto-fluoresence, we found that incubation of MOSEC cells with doxorubicin led to the intracellular uptake of the drug and the eventual death of the tumor cells. We also found that drug-loaded tumor cells were capable of transferring the drug to other non-drug-loaded tumor cells in close vicinity. In addition, we found that the use of irradiated MOSEC-dox cells to deliver doxorubicin is more effective in treating MOSEC/luc tumors than administration of a comparable dose of doxorubicin alone. Thus, our study suggests that local delivery of chemotherapeutic drugs by tumor cells may require significantly less amount of drug to control ovarian cancer. The success of the current study warrants further exploration of such a delivery approach using other chemotherapeutic drugs for the treatment of cancers.
Our study shows that irradiated tumor cells loaded with a chemotherapeutic drug can lead to the control of MOSEC tumors. We have revealed that this delivery system is capable of transferring doxorubicin to other tumor cells in vitro and in vivo resulting in tumor cell death. The mechanism of chemotherapeutic action of doxorubicin on cancer cells is through DNA intercalation and topoisomerase II enzyme inhibition . Through these two actions, doxorubicin can disrupt cellular processes involving DNA such as synthesis and transcription, leading to cell death. Thus, we can reason that the antitumor effects observed as a result of treatment with irradiated MOSEC-dox tumor cells can be partly attributed to doxorubicin-mediated tumor-cell killing. Other contributing factors for the observed therapeutic effects include chemotherapy-induced cell death and subsequent antitumor activity based on activation of the immune system. Our previous studies have shown that tumor cells treated with chemotherapy can lead to tumor cell death, resulting in activation of tumor-specific immunity [20, 21, 22].
The observed antitumor effects generated by doxorubicin-loaded tumor cells may also be contributed by tumor-specific immunity. Recent studies have shown that anthracycline drugs including doxorubicin induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface and result in improved processing of tumor cells by dendritic cells . Thus, the expression of CRT on the surface of tumor cells mediated by doxorubicin may play an important role in the generation of anticancer immune responses. Thus, doxorubicin-loaded tumor cells may generate antitumor effects through doxorubicin-mediated killing as well as tumor-specific immunity.
It is important to consider issues related to safety and feasibility for the use of this novel delivery system in clinics. While this delivery system is used to deliver the drug directly to tumor cells, it is possible that MOSEC-dox cells may also deliver doxorubicin to healthy fibroblasts and other cells. This raises concerns for toxicity. Nevertheless, we expect that normal cells such as fibroblasts would be less susceptible to the effects of the drug as compared to tumor cells at the same concentration of drug delivered by MOSEC-dox. This is generally true in the case of free form of doxorubicin. In addition, intraperitoneal mode of delivery of irradiated tumor cells loaded with drug would potentially have less systemic toxicity compared to intravenous drug delivery. The irradiation of drug-loaded tumor cells will further alleviate concerns for growth of the drug-loaded tumor cells following injection. Furthermore, the principle generated from the current study provides the rationale for further exploration of alternative options for drug delivery such as controlled release biodegradable polymers [24, 25] or non neoplastic cells from patients such as fibroblasts or PBMCs. It will be important to further test whether these kinds of reagents will be able to generate equivalent or better effects compared to the current approach.
In summary, our study demonstrates that the employment of drug-loaded irradiated tumor cells represents a potentially innovative approach for the delivery of chemotherapeutic drugs for the control of ovarian tumors. Further exploration in this area will create the opportunity for the development of innovative chemotherapy regimens for the control of ovarian cancer.
This work was supported by the NCDGG (1U19 CA113341-01), the American Cancer Society (ACS) and the 1 RO1 CA114425-01.
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