Abstract
An attempt has been made to investigate the toxicity of cancer immunotherapy based on the dendritic cells pulsed with lysate of allogenic melanoma cell, DM401. Dendritic cells pulsed with lysate of clone M3 were subcutaneously administered once a week eight times to C57BL/6 mice at 0, 2.5, 5, and 10 × 107 cells/kg. No changes attributable to the administration were observed in clinical signs and food and water consumption. The administration induced slight increases in body weights, white blood cells, total protein, total cholesterol, triglyceride, phospholipids, and absolute spleen weights, but a slight decrease in albumin/globulin ratio. Microscopic examinations revealed the infiltration of inflammatory cells in the lung, mainly in the pulmonary arteriole, in which the tunica media thickened, and in the pulmonary alveoli and alveolar space. Thickened tunica media of pulmonary arteriole was observed in both males and females at all selected doses. In addition, the subcutis at the test substance-application site showed inflammation and fibrosis. In conclusion, lung is a target organ of DM401, and most of the changes including the findings in lung are considered as the immunomodulatory functions of dendritic cells.
Melanoma is a common and malignancy that is increasingly common and highly metastatic. Early-stage melanoma can be cured by appropriate surgical therapy; however, for the metastasized melanoma, systematic therapies are necessary for the treatment. Although various kinds of treatments are employed for patients with metastasized melanoma, their survival rates have not yet been improved (Garbe 1993; Hansson 1997; Stoutenburg, Schrope, and Kaufman 2004; Terando, Sabel, and Sondak 2003). The resistance of melanoma cells to chemotherapy and disappointing results of standard therapies commonly used in other solid tumors led to a search for innovative therapy for melanoma, including immunotherapy (Komenaka, Hoerig, and Kaufman 2004; Terando, Sabel, and Sondak 2003). Although tumor cells are poorly immunogenic, knowledge of the immune system and its interaction with tumor cells has expanded dramatically over the last several decades (Faries and Czerniecki 2005). Many efforts have been directed to overcome the lack of T-cell response to tumor cells. Recent studies showed that the dendritic cells (DCs) have critical functions to elicit a T-cell response against tumor cells (Czerniecki et al. 2001; Schadendorf, Paschen, and Sun 2000; Steinman and Pope 2002).
DCs are specialized antigen-presenting cells derived from bone marrow that are distributed ubiquitously throughout the body (Banchereau et al. 2000; Steinman 1991). DCs phagocytize a diverse array of antigens and present them to T-cell as peptides bound to either major histocompatibility complex (MHC) class I or II (Mellman and Steinman 2001). These antigen-specific responses are critical for the resistance to tumors. DCs are also important in launching humoral and innate immunity (Banchereau and Palucka 2005). Numerous DC-based immunotherapeutic trials with antigen-loaded DCs have been performed for a wide range of tumors (Banchereau et al. 2001; Fong and Engleman 2000; Jenne et al. 2000) and showed effectiveness in some patients. The antigen-loading methods applied in most trials to achieve the MHC-restricted presentation of tumoral antigen were either peptide pulsing, involving immunodominant sequences of defined tumor-associated antigens (TAAs) (Dhodapkar et al. 1999; Gilboa 1999; Schuler-Thurner et al. 2000; Wang et al. 1999), or whole-tumor-cell lysate pulsing (Albert, Sauter, and Bhardwaj 1998; Berard et al. 2000; Neidhardt-Berard et al. 2004; Thumann et al. 2003).
Autologous tumor cells are preferred as a source of tumor antigen in the immunotherapy because those cells contain TAAs that are best matched to the tumor-bearing host. Clinically, however, difficulties are emerged in generating tumor cell lines successfully and reliably from individual patient (Lee et al. 2005a). Therefore, allogenic tumor cells were also considered as a tumor antigen sources that provide therapeutic effects. In our previous study, we found that the dendritic cells pulsed with lysates of allogenic cells, clone M3 or K1735, were more efficient than those pulsed with lysate of autologous cells, B16F10, in a pulmonary metastasis therapeutic model using C57BL/6 mice (Lee et al. 2005b). Compared to the efficacy, the potential toxicities of DC-based immunotherapy for melanoma have not been thoroughly studied yet.
DM401 is an immunotherapeutic agent that consists of mouse bone marrow-derived dendritic cells sensitized with lysate of melanoma cells, clone M3, which was originated from a BALB/c × DBA/2 F1 offspring mouse. In this study, we investigated the toxicity of DM401 administered subcutaneously to C57BL/6 mice. The results indicate that lung is the target organ of DM401 toxicity.
MATERIALS AND METHODS
Reagents
Earle’s Balanced Salt Solution (EBSS), propidium iodide, and 2-mercaptoethanol were purchased from Sigma (St. Louis, MO, USA). RPMI 1640 medium, penicillin-streptomycin solution, and HEPES were from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was from JRH (Lenexa, KS, USA). Lysis buffer, stain buffer, and antibodies against CD3, CD4, CD8, and CD45 were purchased from Pharmingen (San Diego, CA, USA). Live/dead cell–mediated cytotoxicity kit was acquired from Molecular Probe (Eugene, OR, USA).
DM401 Preparation
DM401 was prepared as described previously (Lee et al. 2005b). Briefly, bone marrow cells isolated from C57BL/6 mice were cultured in the presence of granuloctye-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 6 days. The lysate of clone M3 was made by fives times freezing-thawing and centrifugation at 1500 rpm for 15 min. DCs were incubated with 50
Animals and Housing Conditions
Four-week-old specific pathogen–free male and female C57BL/6 mice were purchased from Orient (Seoul, Korea) and used after 1 week of acclimation. Randomization was carried out using the Path/Tox System (Version 4.2.2; Xybion Medical Systems, USA). Males and females were separately housed in stainless steel cages (5 animals/cage, 175 (W) × 240 (L) × 145 (H) mm). Cages were changed once every 2 weeks. The animals were maintained at temperature of 23°C ± 3°C, relative humidity of 50% ± 10%, air ventilation of 10~20 times/h, and light intensity of 150~300 Lux with 12-h light/dark cycle. Pelleted food for experimental animals (PMI Nutrition International, Richmond, IN, USA) and ultraviolet (UV)-irradiated (Steritron SX-1; Daeyoung, Korea) and filtrated (1
Experimental Design
A single dose study of DM401 performed with five males and five females dosed up to at 1 × 108 cells/kg, which was 100-fold higher than the anticipated clinical dose, showed no clinical signs during the observation period of 2 weeks and no gross finding at necropsy. Therefore, 1 × 108, 5 × 107, and 2.5 × 107 cells/5 ml/kg were chosen as the high, middle, and low doses, respectively. DM401 was administered subcutaneously at the interscapular region, eight times at intervals of 1 week to cover the intended clinical administration route and regimen. In addition, saline was administered to the animals of the control group. One group consisted of 10 mice. All mice were terminated for examinations 13 days (male) or 14 days (female) later the final administration in consideration of the time delay between DC injection and cancer regression.
Clinical Observation and Mortality
Animals were monitored once a day during the study period. Clinical signs including mortality, moribundity, and general appearance and behavior were recorded with time and duration of the observation.
Body Weight Changes
Animals were weighed on arrival day, the day of group assignment, the first day of dosing (day 1), once a week thereafter, and at termination time.
Food and Water Consumption
Food and water consumption was recorded per cage once during the pretreatment period, at the initiation of treatment, and once a week thereafter. Measured amounts of food and water were supplied to each cage on the day of measurement and the remaining were weighed on the next day (about 24 h later) so as to calculate the daily consumption.
Necropsy
All animals were fasted overnight prior to necropsy and blood sampling. Blood samples were collected from the posterior vena cava of the animals under isoflurane anesthesia. After the blood collection, the animals were sacrificed by exsanguination from the aorta. Complete macroscopic examinations were performed on all terminated animals. The organs listed below taken from all animals were fixed with 10% neutral-buffered formalin solution except for eyes, testes, and epididymides: mammary glands, spleen, pancreas, jejunum, stomach, duodenum, ileum, cecum, colon, mesenteric lymph nodes, salivary glands, submaxillary lymph nodes, ovaries, uterus, vagina, urinary bladder, testes, epididymides, prostate, seminal vesicles, rectum, kidneys, adrenal glands, liver, sternum, thymus, heart, lungs, trachea, esophagus, thyroid (included parathyroid), tongue, arteriae aorta, sciatic nerves, skeletal muscles, femurs, spinal cord (mid-thoracic), skin, eyes, haderian glands, brain, and hypophysis. Eyes were preserved in Davidson’s fixative, and testes and epididymides were preserved in Bouin’s fixative.
Hematology
Blood samples collected into tubes containing EDTA-2K were analyzed by ADVIA102 Hematology system (Bayer, CA, USA) for white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), reticulocyte (RET), neutrophil (NEU), lymphocyte (LYM), monocyte (MON), eosinophil (EOS), basophil (BAS), large unstained cell (LUC).
Clinical Chemistry
Serum samples prepared by centrifugation at 3000 rpm for 10 min were analyzed by an autoanalyzer Shimadzu-CL-7200 (Shimadzu, Japan) and ion autoanalyzer (644; Ciba-Corning, USA) for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), glucose (GLU), total cholesterol (TCHO), albumin and globulin ratio (A/G), total protein (TP), albumin (ALB), creatine kinase (CK), triglyceride (TG), phospholipid (PL), total bilirubin (TBIL), calcium (CA), inorganic phosphorus (P), chloride (Cl), sodium (Na), and potassium (K).
Organ Weights
Absolute and relative (to body weight) organ weights were measured for organs, i.e., brain, hypophysis (pituitary gland), adrenal glands, liver, spleen, kidneys, heart, thymus, lungs, salivary glands, thyroid glands, testes, epididymides, seminal vesicles, prostate, ovaries, and uterus.
Histopathology
All organs checked on the gross findings were routinely processed, embedded in paraffin and sectioned. The sections were stained with hematoxylin-eosin (H&E) for histopathology. Histopathological examination was performed on all tissues from the animals in the vehicle-control and high-dose groups. If any lesion thought to be related to the DM401 treatment was found, the corresponding organs in the low and middle dose groups were also examined.
Statistical Analysis
Data collected during the study were analyzed with multiple comparison tests. Variance homogeneity was examined using the Bartlett’s test. Homogeneous data were analyzed with the analysis of variance (ANOVA) multiple comparison test (Dunnett’s
RESULTS
Mortalities and Clinical Signs
There were no unscheduled deaths. The loss of fur observed in many animals was not considered to be related with treatment but probably due to the stress from group housing, because it was observed in almost all groups including the control group.
Body Weight Changes
Slight increases in the body weight by the treatment were observed in both males and females. A significant increase in body weights compared with the vehicle control group was observed in the male 10 × 107 cells/kg group on day 57 (Figure 1A), the female 2.5 × 107 cells/kg group on days 15, 29, and 36, the female 5 × 107 cells/kg group on days 22, 29, 36, and 50, and the female 10 × 107 cells/kg group on days 15, 22, 29, and 50 (Figure 1B).
Food and Water Consumption
No significant treatment-related changes were observed.
Hematology
No statistically significant changes were noted in all male groups compared with the vehicle control group (Table 1). WBC increased 58% (
Clinical Chemistry
TP was increased in males treated with DM401 (
Necropsy
Discoloration of spleen and adrenal gland and enlarged kidneys were found in some mice (data not shown). But those findings were considered not to be related to the DM401 treatment due to the lack of dose dependency and the occurrence in the vehicle-treated animals.
Organ Weights
Absolute weights of spleen were increased 6% to 15% with the treatment of DM401 in males and females (Table 2). A decrease in relative weight of pituitary gland (30% to 45%) observed in males (data not shown) and decreases in relative weight of brain (4% to 8%) and heart (7% to 8%) observed in females (data not shown) were considered not to be related to the DM401 treatment.
Histopathology
Thickened medial wall of arteriole and infiltration of inflammatory cells (mostly eosinophils) in lungs were observed in both males and females at all dose levels (Table 3, Figure 2). The pulmonary arteries were preferentially infiltrated with inflammatory cells, showing thickened tunica media. The infiltration of inflammatory cells was also observed in the pulmonary alveoli and alveolar spaces of some animals (Table 3, Figure 2). The fibrous inflammation observed in the subcutis of the injection site was thought to be treatment related. Other histopathological findings including tubular basophilia and infiltration of inflammatory cells in the kidney, tension lipidosis in the liver, pigmentation in the spleen, lymphoid cell infiltration in the lung, lipogenic pigmentation of X-zone in the adrenal gland, cyst in the thyroid gland, spermatogenic granuloma in the testes, and aspermia in the epididymis were not considered to be treatment related because they are spontaneous-occurring lesions and noted in the minority mice. Although abnormalities such as inflammatory foci in the liver, subcapsular hyperplasia in the adrenal gland, mucosal vacuolation, submucosal inflammation and erosion in the stomach, and seminifeous tubular atrophy in the testes were found in many instances, no statistical significances were noted.
DISCUSSION
DM401 was administered subcutaneously to mice at doses of approximately up to 100-fold greater than the clinical dose and various toxicological parameters were evaluated. The slight increase in body weight observed in DM401-treated males and females is consistent with the clinical chemistry data, which showed increases in cholesterol, triglyceride, and phospholipid levels. There are ample data to support correlation of obesity with immune responses (or inflammation), especially with asthma (Flaherman and Rutherford 2006; Shaheen et al. 1999). Recently, a number of adipokines, which are signaling molecules secreted by adipose tissue, such as leptine, adiponectin, and resistin, were discovered. It has been reported that adipolkines have functions in immune system (Cancello et al. 2004; Hotamisligil 2003; Koerner, Kratzsch, and Kiess 2005; La Cava et al. 2003; Marti, Marcos, and Martinez 2001; Munoz, Mazure, and Culebras 2004; Rovin and Song 2006; Sanchez-Margalet et al. 2003). Therefore, the increases in body weight are considered to be related to the treatment of DM401. The decrease of 30%~45% relative organ weights observed in male pituitary gland was not supported by histopathological lesions nor observed in females. Decreases in relative weight of some organs are thought to be either secondary effects due to the increase in body weight or incidental. However, the increases in WBC and absolute spleen weight noted in females appear to be caused by DM401 treatment because they were also observed in males although statistically not significant. The increase in immune-related parameters such as WBC and spleen weights explains the decrease in A/G ratio in both males and females and the increase in TP in males. Antigen-sensitized dendritic cells, a test substance in this study, may stimulate immune responses such as lymphocyte proliferation and immunoglobulin production represented by increase in WBC and a decrease in A/G ratio, respectively. Although infiltration of inflammatory cells in the lung and thickening of tunica media of small pulmonary artery were observed in a few animals treated with vehicle and these are known to spontaneously occur in mice (Elwell and Mahler 1999), these lesions were noted in the DM401-treated animals in a more frequent and severe manner compared with the vehicle-treated animals, suggesting that the microscopic findings in the lungs were related to the treatment of test substance. However, in the absence of a DC control with no lysate pulsing, it is not clear if these lesions were due to the pulsing with lysate or the injection of DC itself.
The fact that the infiltration of eosinophils in the lung, which is a representative lesion in animal models for asthma (Lloyd et al. 2001), was the most significant observation in microscopic examination in this study may provide another clue on the relationship between the asthma and obesity. The administration of antigen-presenting cells pulsed with tumor lysate brought asthma-like lesion in the lung and resulted in high cholesterol and triglyceride levels as well as body weight increase. These might be two different aspects of tumor antigen–bearing DC injection. Recently, serum leptin level has been reported to increase during allergic reactions in the airways of BALB/c mice (Shore et al. 2005). Further research using general animal models with antigens are required to consolidate the relationship between asthma and obesity.
Taken together, the target organ of DM401 was found to be the lung, and most of the changes noted in this study are considered to result from pharmacological effects of the test substance, immunomodulatory functions of dendritic cells. Although the dose levels were much higher than anticipated clinical doses, present results suggest that asthma-like symptoms can be the biomarkers for the possible side effects of dendritic cells sensitized with allogenic tumor cell lysate in clinical trials.
Footnotes
Figures and Tables
This work was supported by KIT project for Implantation of Safety Evaluation System (grant no. 11200-461-1563-220-000) and Intramural Research Fund (DC-grant) in Midipost. The Authors thank Dr. S. V. S. Rana in Ch. Charan Singh University for his kind English correction.