Possible Involvement of Antitumor Immunity in the Eradication of Colon 26 Induced by Low-Voltage Electrochemotherapy with Bleomycin

Purpose: The antitumor efficiency of electrochemotherapy using chemotherapeutic agents and high-voltage electric pulse has been reported. This study was done to define the precise nature of the involvement of antitumor immunity in the regression of tumor nodules in electrochemotherapy, and to evaluate the effectiveness of using low-voltage electroporation. Methods: Balb/c mice and Balb/c nu/nu nude mice were inoculated subcutaneously with Colon 26 cells or Meth A cells. Electrochemotherapy using bleomycin and low-voltage electroporation (CUY21) was performed as a treatment against tumor nodules. Results: Colon 26 tumors were eradicated in the mice given an intratumor (i.t.) injection of 500 μg bleomycin followed by treatment with electric fields ranging from 50 to 150 V/cm, with complete response rates ranging from 80% to 100%. The mice rejected inoculations of rechallenged Colon 26 cells, but not Meth A cells. In the Balb/c nu/nu nude mice, complete regression of the tumor was not seen after electrochemotherapy under the same therapeutic conditions that resulted in almost complete cure in the Balb/c mice. Conclusion: Our results suggest that the generation of T-cell-dependent, tumor-specific protective immunity might be involved in the process of tumor nodule regression in low-voltage electrochemotherapy. Received: February 14, 2002 / Accepted: July 2, 2002 Reprint requests to: Y. Gunji     

Dendritic Cells Armed With Anti-CD3 mAbs Reduce Pulmonary Metastases, Prolong Survival, and Engender Antitumor Effector Cells Demonstrable by Adoptive Transfer

Background: Dendritic cells (DCs) pulsed with tumor cells or peptides are effective antitumor agents in a number of tumor models. In light of our earlier demonstration that T-cell signaling via the CD3 proteins induces cytolytic activity and constrains tumor progression, we equipped DCs pulsed with tumor cells with anti-CD3 mAbs and tested their antitumor efficacy in a murine renal cell cancer pulmonary metastasis model.Methods: We investigated the antitumor efficacy of DCs pulsed with whole irradiated tumor cells (DC/R) or DCs pulsed with irradiated tumor cells and armed with anti-CD3 mAbs (DC/R/anti-CD3 mAbs). Experimental end points included the number of pulmonary metastases and survival of tumor-inoculated mice.Results: Our studies demonstrate that arming tumor-pulsed DCs with anti-CD3 mAbs results in a superior outcome compared to that from tumor-pulsed DCs alone in terms of reduction in the number of pulmonary metastases and survival times. Furthermore, adoptive transfer experiments revealed that the splenocytes from DC/R/anti-CD3 mAbs-treated mice are superior to splenocytes from DC/R-treated mice in reducing renal cancer pulmonary metastases in severe combined immunodeficient (SCID) beige mice.Conclusion: Our data suggest that the therapeutic efficacy of DCs pulsed with tumor cells can be augmented by arming them with anti-CD3 mAbs. DC-based treatment regimens that currently are being pursued in clinical trials might be improved by equipping such cells with anti-CD3 mAbs.     

Bupivacaine and Lidocaine Induce Apoptosis in Osteosarcoma Tumor Cells

BackgroundOsteosarcoma is the most common type of bone cancer in adolescents. There have been no significant improvements in outcomes since chemotherapy was first introduced. Bupivacaine and lidocaine have been shown to be toxic to certain malignancies. This study evaluates the effect of these medications on two osteosarcoma cell lines.Questions/purposes(1) Does incubation of osteosarcoma cells with bupivacaine or lidocaine result in cell death? (2) Does this result from an apoptotic mechanism? (3) Is a specific apoptotic pathway implicated?MethodsTwo cell lines were chosen to account for the inherent heterogeneity of osteosarcoma. UMR-108 is a transplantable cell line that has been used in multiple studies as a primary tumor. MNNG/HOS has a high metastatic rate in vivo. Both cell lines were exposed bupivacaine (0.27, 0.54, 1.08, 2.16, 4.33 and 8.66 mM) and lidocaine (0.66, 1.33, 5.33, 10.66, 21.32 and 42.64 mM) for 24 hours, 48 hours, and 72 hours. These concentrations were determined by preliminary experiments that found the median effective dose was 1.4 mM for bupivacaine and 7.0 mM for lidocaine in both cell lines. Microculture tetrazolium and colony formation assay determined whether cell death occurred. Apoptosis induction was evaluated by phase-contrast micrographs, flow cytometry, DNA fragmentation and reactive oxygen species (ROS). The underlying pathways were analyzed by protein electrophoresis and Western blot. All testing was performed in triplicate and compared with pH-adjusted controls. Quantitative results were analyzed without blinding.ResultsBoth medications caused cell death in a dose- and time-dependent manner. Exposure to bupivacaine for 24 hours reduced viability of UMR-108 cells by 6 ± 0.75% (95% CI 2.9 to 9.11; p = 0.01) at 1.08 mM and 89.67 ± 1.5% (95% CI 82.2 to 95.5; p < 0.001) at 2.16 mM. Under the same conditions, MNNG/HOS viability was decreased in a similar fashion. After 24 hours, the viability of UMR-108 and MNNG/HOS cells exposed to 5.33 mM of lidocaine decreased by 25.33 ± 8.3% (95% CI 2.1 to 48.49; p = 0.03) and 39.33 ± 3.19% (95% CI 30.46 to 48.21; p < 0.001), respectively, and by 90.67 ± 0.66% (95% CI 88.82 to 92.52; p < 0.001) and 81.6 ± 0.47% (95% CI 79.69 to 82.31; p < 0.001) at 10.66 mM, respectively. After 72 hours, the viability of both cell lines was further reduced. Cell death was consistent with apoptosis based on cell morphology, total number of apoptotic cells and DNA fragmentation. The percentage increase of apoptotic UMR-108 and MNNG/HOS cells confirmed by Annexin-V positivity compared with controls was 21.3 ± 2.82 (95% CI 16.25 to 26.48; p < 0.001) and 21.23 ± 3.23% (95% CI 12.2 to 30.2; p = 0.003) for bupivacaine at 1.08 mM and 25.15 ± 4.38 (95% CI 12.9 to 37.3; p = 0.004) and 9.11 ± 1.74 (95% CI 4.35 to 13.87; p = 0.006) for lidocaine at 5.33 mM. The intrinsic apoptotic pathway was involved as the expression of Bcl-2 and survivin were down-regulated, and Bax, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase-1 were increased. ROS production increased in the UMR-108 cells but was decreased in the MNNG/HOS cells.ConclusionThese findings provide a basis for evaluating these medications in the in vivo setting. Studies should be performed in small animals to determine if clinically relevant doses have a similar effect in vivo. In humans, biopsies could be performed with standard doses of these medications to see if there is a difference in biopsy tract contamination on definitive resection.Clinical RelevanceBupivacaine and lidocaine could potentially be used for their ability to induce and enhance apoptosis in local osteosarcoma treatment. Outcome data when these medications are used routinely during osteosarcoma treatment can be evaluated compared with controls. Further small animal studies should be performed to determine if injection into the tumor, isolated limb perfusion, or other modalities of treatment are viable.     

Tumor specific cytotoxicity and telomerase down-regulation in prostate cancer by autologous dendritic cells loaded with whole tumor cell antigens

ObjectivesWe investigated the efficacy of cytotoxic activity of whole tumor cell-antigen loaded dendritic cells in the treatment of hormone refractory prostate cancer.Materials and methodsFrom 10 patients with HRPC, peripheral blood samples were obtained and cultured with GM-CSF and IL-4 to provide differentiation of peripheral blood mononuclear cells (PBMs) into dendritic cells (DCs). DC phenotype was confirmed by flow cytometry (MHC class II HLA-DR, CD80, CD86, CD83, CD14 expression analysis). Subsequently, whole tumor cell lysates of LNCaP, DU-145, and PC-3 lines were incubated with DCs. Direct antitumoral activity of induced DCs and activation of PBM cells by these DCs was assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Post-treatment changes in the telomerase gene expression of tumor cells were investigated by real time RT-PCR analysis.ResultsLDH activity was highest in the PC-3 cell line (9.5%) and lowest in the DU-145 line (3.2%). Co-incubation of PBMs with activated DCs resulted in a significant increase at the levels of cytotoxicity in all cell lines. Likewise, incubation of tumor cells with activated DCs caused significant down-regulation of telomerase gene expression in all cell lines. Most pronounced suppression was in the LNCaP cell line (decrease by 97.1%). The decrease in the level of telomerase gene expression in DU-145 and PC-3 cell lines was 80% and 70%, respectively.ConclusionsCytotoxic immune response to prostate cancer-associated antigens can be elicited in vitro in patients with HRPC using an allogeneic tumor cell-based strategy. DC-based active immunotherapy appears as an effective treatment method in the pre-clinical setting and further phase I/II trials are warranted.     

Synergistic Effect of a Granulocyte-Macrophage Colony-Stimulating Factor–Transduced Tumor Vaccine and Systemic Interleukin-2 in the Treatment of Murine Colorectal Cancer Hepatic Metastases

Background: Granulocyte-macrophage colony-stimulating factor–transduced tumor cell vaccines are less effective against cancer as the interval between metastasis and the initial vaccination increases.Methods: Hepatic metastases were generated in BALB/c mice by using a syngeneic colorectal cancer line (CT26) with a splenic injection model. Irradiated CT26 cells transduced to secrete granulocyte-macrophage colony-stimulating factor were used as vaccine. Treatment groups received vaccine, systemic interleukin (IL-2), or both. Livers were examined for gross metastases 21 days after tumor challenge. Splenocytes were analyzed for in vitro activity against CT26 by using an enzyme-linked immunospot assay and a cytotoxic T lymphocyte assay.Results: Eighty-eight percent of mice treated with vaccines and IL-2 were tumor free on day 21 (P .001 vs. control). Treatment with vaccines or IL-2 alone did not result in a significant treatment effect. Splenocytes from mice treated with both vaccines and IL-2 showed greater CT26 lysis than splenocytes from mice treated with vaccines alone at effector:target ratios of 100, 30, and 10 (P < .05 for all). More splenocytes from these mice released interferon- in response to stimulation with the CT26 tumor antigen AH1 compared with mice treated with vaccines alone (P = .05).Conclusions: Systemic IL-2 augments tumor vaccine efficacy in the treatment of microscopic murine colorectal hepatic metastases.     

Survival of mitomycin C-treated pancreatic islet xenografts is mediated by increased expression of transforming growth factor-beta

BACKGROUND: Mitomycin C (MMC) can trigger various intracellular signals. The authors previously showed that pretreatment of highly immunogenic crude pancreatic islets with MMC improved their survival in a rat-to-mouse transplantation model. The aim of this study was to investigate the role of transforming growth factor (TGF)-beta in mediating MMC-induced survival of islet xenografts. METHODS.: Collagenase-digested islets obtained from WS rats (RT1k) were incubated for 30 min with 10 microg/mL MMC and then transplanted into streptozotocin-induced diabetic C57BL/6 (H-2b) mice after 20 hr of culture at 37 degrees C. RESULTS: Survival of xenografts was enhanced by pretreatment of islets with MMC. MMC-treated xenografts showed a mild inflammatory cell response and significantly minimal infiltration of macrophages, CD4 T cells, and CD8 T cells compared with untreated grafts. TGF-beta mRNA was increased at 20 hr after MMC treatment, and TGF-beta protein expression was also increased compared with untreated islet xenografts. TGF-beta concentration in blebs formed around the xenografts (but not in the serum) was higher in animals that underwent transplantation with MMC-treated islets than with untreated islets. Simultaneous transplantation of MMC-treated and untreated islets separately in each kidney of recipient mice showed that protection was only found in MMC-treated islets. Treatment of islets before transplantation by neutralizing anti-TGF-beta antibody suppressed the MMC-protective effects on graft survival, whereas no such effect was noted with isotype-matched immunoglobulin. CONCLUSIONS.: The authors' results indicate that MMC treatment effectively reduces local inflammatory response and that such effects are mediated by increase of TGF-beta during the early period of islet transplantation.     

Immunological memory induced by genetically transduced tumor cells

Background: Recent studies have demonstrated the usefulness of gene-modified tumor cells for immunotherapy. Using the tumorigenic murine fibrosarcoma, MCA 106, we investigated the effects of localized interferon-γ (IFNg) secretion on tumorigenicity and on long-term memory. Methods: The murine IFNg (MuIFNg) gene was introduced into tumor cells. High and low IFNg-secreting clones were isolated. C57BL/6 mice were injected subcutaneously (s.c.) with either parental (P), high or low IFNg-secreting (H- or L-IFNg) cells, and tumor growth was assessed weekly. Spleens were harvested on different days postinjection (p.i.) to assess in vitro cytolytic activity. In parallel, tissues from injection sites were stained with macrophage-, CD4-, and CD8-detecting antibodies. Mice were injected s.c. with H-IFNg MCA106 tumor. After 150 days the animals were rechallenged s.c. with MCA106P in one leg and with irrelevant syngeneic tumor in the other. Results: Both P- and L-IFNg cells had similar growth, whereas the H-IFNg cells never grew. Only splenocytes from the H-IFNg animals showed in vitro CTL activity persisting until day 30 p.i. Histological data revealed a macrophage and CD4⁺ infiltrate much earlier in the H-IFNg group compared with the P group. Only the irrelevant, syngeneic tumor grew in animals previously injected with H-IFNg cells, whereas both P and irrelevant syngeneic tumors grew in controls. Conclusions: Transduction of MCA106 cells with the MuIFNg gene diminished in vivo tumorigenicity in proportion to the amount of IFNg secreted. Immunization with H-IFNg cells elicited a host response characterized by macrophages and CD4⁺ cells. Long-term tumor-specific memory was seen after immunization with H-IFNg cells.     

Intraperitoneal hyperthermic perfusion with mitomycin C for colorectal cancer with peritoneal metastases

Background: Intraperitoneal (i.p.) metastases pose a special problem for surgical treatment because of their multiplicity and microscopic size. This study was designed to examine the feasibility and safety of i.p. hyperthermic perfusion (IPHP) with mitomycin C (MMC) for treating recurrent colorectal cancer. Methods: Fifteen patients with metastatic colon cancer were treated. All patients underwent cytoreductive procedures leaving only residual i.p. metastases <1 cm in diameter. All patients had received prior systemic chemotherapy, but their disease had progressed. Intraperitoneal chemotherapy was administered through three large catheters (28 French) using a closed system of two pumps, a heat exchanger, and two filters. After the patient’s abdominal temperature reached 41°C, 45–60 mg of MMC was circulated intraperitoneally for 1 h. Results: The majority of patients had various anastomoses: small bowel (n=11), large bowel (n=5), and urologic (n=5). No anastomotic complications occurred in any of the patients. One patient experienced severe systemic MMC toxicity, which caused cytopenia and respiratory depression. In all patients the carcinoembryonic antigen (CEA) level decreased after surgery and IPHP. Median follow-up was 10 months, and recurrence was defined as an elevation in CEA level. Disease recurred in three patients within 5 months, and disease recurred in seven other patients over the next 3 months; one patient remains clinically free of disease after 8 months. Conclusion: Our data suggest that IPHP is a safe palliative method of treatment for patients with peritoneal carcinomatosis. The median patient response duration of 6 months may warrant consideration of a repeat IPHP procedure at that time. Presented at the 46th Annual Cancer Symposium of The Society of Surgical Oncology, Los Angeles, March 18–21, 1993.     

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model.MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS).ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α⁺ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α⁻ DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS.ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.     

Disseminated Epithelial Tumor Cells in Bone Marrow of Patients with Esophageal Cancer: Detection and Prognostic Significance

p < 0.01). It was concluded that although bone marrow is not a preferential site of overt metastasis of esophageal cancer, the frequent occurrence of isolated tumor cells at this distant site indicates that hematogenous dissemination of viable malignant cells occurs early in tumor progression.     

Permanent acceptance of mitomycin C-treated islet allograft

BACKGROUND: Mitomycin C (MMC) treatment produces genotoxic stress and exerts various biologic effects on cell function. This study determines the feasibility of MMC pretreatment of islet grafts as a sole immunomodulatory regimen to protect murine crude-digested islet allografts. METHODS: Collagenase-digested BALB/c (H-2d) islets were incubated for 30 min with MMC at different doses (0, 3.2, 10, 32, and 100 microg/mL; n=20, 15, 55, 15, 15, respectively), cultured for 20 hr, and transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (B6; H-2b) mice. RESULTS: All mice that received MMC-treated islets showed restoration of normoglycemia within 5 days postgrafting, which was maintained until rejection. All untreated islets were acutely rejected with a mean survival time of 15.1+/-3.5 days. Significant prolongation of graft survival was noted in mice undergoing transplantation with islets treated with 10 microg/mL MMC compared with untreated islets (58.9+/-37.7 days, P<0.01). Notably, the grafts of 24 of 55 animals (43%) that received islets treated with 10 microg/mL MMC survived more than 100 days without any other treatment. Furthermore, antigen-specific prolongation of graft survival of secondary untreated islets was observed in mice bearing long-term functioning islet grafts. CONCLUSIONS: Our results indicate that pretreatment of islets with MMC alone protects the graft against rejection and produces long-term graft survival with normal blood glucose levels, and that pretreatment with MMC offers a new strategy for allogeneic islet transplantation.     

Macrophage Inflammatory Protein-2 Promotes Angiogenesis, Cell Migration, and Tumor Growth in Hepatic Metastasis

Background In a mouse model of hepatic metastasis, we herein analyzed whether the CXC chemokine macrophage inflammatory protein (MIP)-2, a functional analogue of the human interleukin 8, stimulates tumor cell migration in vitro and angiogenesis and tumor growth in vivo. Methods By using chemotaxis chambers, CT26.WT colorectal tumor cell adhesion and migration were studied under stimulation with different concentrations of MIP-2. To evaluate angiogenesis and tumor growth in vivo, 1 × 10⁵ CT26.WT cells were implanted into the left liver lobe of syngeneic BALB/c mice, and 10, 100, and 1000 nM of MIP-2 or phosphate-buffered saline (controls) was injected into the peritumoral area. After 7 days, angiogenesis, proliferation, tumor growth, apoptosis, cleaved caspase 3, and CXCR-2 expression were analyzed by using intravital fluorescence microscopy, histology, immunohistochemistry, and fluorescence-activated cell sorting. Results In vitro, 98.8% of unstimulated CT26.WT cells showed CXCR-2 receptor expression. In the chemotaxis assays, MIP-2 provoked a dose-dependent increase of cell migration and a most pronounced cell adhesion at a dose of 100 nM. In vivo, MIP-2, in particular in a dose of 100 or 1000 nM, induced a significant increase of tumor capillary density and a marked widening of the angiogenic front at the tumor margin. Capillaries of the angiogenic front, but not of the tumor center, showed significant dilation, thus indicating a pronounced action of vascular endothelial growth factor. Tumor volume was significantly increased, in particular after 100 nM of MIP-2 stimulation, when compared with phosphate-buffered saline–treated controls, whereas only 1000 nM of MIP-2–treated animals additionally showed a higher frequency of apoptotic cell death within the tumor margin. Conclusions Our study indicates for the first time that the CXC chemokine MIP-2 promotes angiogenesis and growth of colorectal CT26.WT hepatic metastasis.     

Hydrostatic pressure enhances mitomycin C induced apoptosis in urothelial carcinoma cells

ObjectivesUrothelial carcinoma (UC) of the bladder is the second most common cancer of the genitourinary system. Clinical UC treatment usually involves transurethral resection of the bladder tumor followed by adjuvant intravesical immunotherapy or chemotherapy to prevent recurrence. Intravesical chemotherapy induces fewer side effects than immunotherapy but is less effective at preventing tumor recurrence. Improvement to intravesical chemotherapy is, therefore, needed.Methods and MaterialsCellular effects of mitomycin C (MMC) and hydrostatic pressure on UC BFTC905 cells were assessed. The viability of the UC cells was determined using cellular proliferation assay. Changes in apoptotic function were evaluated by caspase 3/7 activities, expression of FasL, and loss of mitochondrial membrane potential.ResultsReduced cell viability was associated with increasing hydrostatic pressure. Caspase 3/7 activities were increased following treatment of the UC cells with MMC or hydrostatic pressure. In combination with 10 kPa hydrostatic pressure, MMC treatment induced increasing FasL expression. The mitochondria of UC cells displayed increasingly impaired membrane potentials following a combined treatment with 10 μg/ml MMC and 10 kPa hydrostatic pressure.ConclusionsBoth MMC and hydrostatic pressure can induce apoptosis in UC cells through an extrinsic pathway. Hydrostatic pressure specifically increases MMC-induced apoptosis and might minimize the side effects of the chemotherapy by reducing the concentration of the chemical agent. This study provides a new and alternative approach for treatment of patients with UC following transurethral resection of the bladder tumor.     

7-Hydroxystaurosporine-induced apoptosis in 9L glioma cells provides an effective antigen source for dendritic cells and yields a potent vaccine strategy in an intracranial glioma model

OBJECTIVE: On the basis of recent studies indicating that tumoral apoptotic bodies may provide a potent source of antigen for delivery to antigen-presenting cells, as well as observations that signal transduction modulation may constitute a promising approach for inducing glioma cell apoptosis, we explored the efficacy of vaccination with glioma apoptotic body-pulsed dendritic cells (DCs) for inhibiting tumor growth in the syngeneic 9L glioma/Fischer rat model. METHODS: For induction of apoptosis, 7-hydroxystaurosporine (UCN-01) (200-300 ng/ml), a selective protein kinase C inhibitor, was co-incubated with 9L cells in vitro for 72 or 96 hours. After this pretreatment period, glioma cells and DCs were mixed, and the interaction between DCs and apoptotic 9L tumor cells was assessed using two-color flow cytometry. In a series of experiments, the efficacy of vaccination strategies using DCs co-cultured with apoptotic 9L cells was then examined in animals harboring intracranial tumors. RESULTS: Pretreatment of 9L cells with UCN-01 resulted in approximately 50% of cells' being observed to undergo apoptosis as compared with less than 3% of controls. After subsequent co-culture, two-color flow cytometry demonstrated a time-dependent physical association of DCs with the apoptotic glioma cells. Survival in animals harboring intracranial tumors was significantly longer for the animals treated with a glioma apoptotic body-pulsed DC vaccine than in the animals that received apoptotic glioma cells and DCs alone or vehicle (i.e., the controls), especially those that underwent a sequential vaccination strategy (P < 0.0001). Long-term survival (>90 d) was demonstrated in 6 (75%) of 8 animals that underwent this vaccination approach versus 0 (0%) of 16 controls. In contrast, no survival benefit was observed in animals that received DCs that were co-cultured with vehicle-treated (non-apoptotic) 9L cells. Three of four long-term survivors that were rechallenged intracranially with tumor cells also survived over the long term. CONCLUSION: These studies suggest that induction of apoptosis in glioma cells by use of UCN-01 may promote the uptake of tumor antigens by DCs. This finding is important because apoptotic body-stimulated DCs may hold promise in promoting a host response against an established intracranial glioma, particularly if the parameters for apoptotic induction, duration of co-culture, and vaccination can be optimized.     

Interleukin-12 Gene Transfer Results in CD8-Dependent Regression of Murine CT26 Liver Tumors

Background: Interleukin (IL)-12 has potent antitumor effects in animal models. We hypothesized that direct transfer of the IL-12 gene to established tumors would result in tumor regression without significant toxicity.Methods: Liver tumors were established by direct injection of CT26, a murine adenocarcinoma, into the livers of BALB/c mice, followed by three transfections with either murine IL-12, murine granulocyte-macrophage colony-stimulating factor, or luciferase cDNA using particle-mediated gene transfer. To assess the mechanism of this effect, immunohistochemical staining and depletion experiments with anti-CD4 or -CD8 antibodies were performed.Results: Progressive growth of primary tumors and carcinomatosis were present by day 16 after transfection with luciferase or murine granulocyte-macrophage colony-stimulating factor. At 50 days, complete regression of tumor was evident in seven of eight IL-12-treated mice (P < .001). In IL-12-transfected livers, immunohistochemical staining revealed an increase in CD8⁺ T cells. Selective depletion of CD4⁺ or CD8⁺ T cells was performed before and during transfection with murine IL-12. At 50 days, 75% of control mice were tumor-free. Only 46% of CD4⁺ cell-depleted mice (P = .143) and 7% of CD8⁺ cell-depleted mice (P < .001) were tumor-free.Conclusions: IL-12 gene transfer using particle-mediated gene transfer results in complete regression of established CT26 liver tumors in 88% of mice; this effect is dependent on CD8⁺ T cells.Presented at the 1998 meeting of The Society of Surgical Oncology     

Transduction of murine and human tumors using recombinant adenovirus vectors

Background: Most cytokine-based cancer gene therapy clinical trials have used labor-intensive, retrovirus-mediated strategies resulting in unpredictable gene expression. Recombinant AdV vectors were evaluated for easier, more reproducible gene transfer into 12 human melanoma, 2 murine fibrosarcomas, and 8 other tumor cell lines. Methods: AdV vectors contained a reporter (Escherichia coli β-galactosidase or firefly luciferase) or cytokine gene (human interleukin-2 [IL-2] or IL-7). Transduction efficiencies and expression levels were assessed by histochemical staining, flow cytometry, polymerase chain reaction, fluorometry, and enzyme-linked immunosorbent assay. Tumorigenicity was determined by subcutaneous injection of cells into syngeneic mice. Results: All cell lines studied were transduced with AdV. Most cell lines exhibited 100% transduction efficiencies (by flow cytometry) at multiplicities of infection (MOI) ε10. Gene expression correlated linearly with MOI, but a cytopathic effect was observed at MOI >100 with all vectors. Nanogram gene expression levels were routinely achieved. Irradiation (30 Gy) minimally affected expression levels. Tumorigenicity of AdV-IL-2-transduced fibrosarcoma cells in mice was inversely related to IL-2 production. A majority of mice that rejected their tumor challenge were immune to tumor rechallenge. Conclusions: E1-deleted AdV vectors may prove useful in generating tumor vaccines ex vivo with high, transient cytokine expression levels. Results of this study were presented in part at the 45th Annual Meeting of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Generation of tolerogenic dendritic cells by treatment with mitomycin C: inhibition of allogeneic T-cell response is mediated by downregulation of ICAM-1, CD80, and CD86

BACKGROUND: Deliberately generated tolerogenic dendritic cells (DC) might be a useful tool for induction of donor-specific tolerance in transplantation. In this article, the authors study the effect of mitomycin C (MMC)-treated DC on rat T cells and delineate the mechanism of their conversion into tolerogenic cells. METHODS: The influence of MMC treatment on the capacity of DC to activate allogeneic T cells was tested in vitro, and the expression of cell surface molecules was studied by flow cytometry. RESULTS: MMC-treated DC lose their allostimulatory capacity, and this cannot be attributed to cell death or release of MMC. Interestingly, suppressed T cells cannot be restimulated, indicating that MMC-treated DC induce tolerance. MMC treatment selectively decreases adhesion (intercellular adhesion molecule [ICAM]-1) and co-stimulatory (CD80, CD86) molecules. Functional blocking of these molecules with specific antibodies confers to DC the same T-cell-suppressive properties as treatment with MMC. CONCLUSIONS: MMC treatment converts rat DC into tolerogenic cells. This mechanism is mediated by decrease of ICAM-1, CD80, and CD86.     

Induction of unresponsiveness to islet xenograft by MMC treatment of graft and blockage of LFA-1/ICAM-1 pathway

BACKGROUND: Induction of unresponsiveness to graft is one of major interest in xenotransplantation. Two different modalities [direct graft treatment by mitomycin C (MMC) and blockage of the lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) pathway in recipients by species-specific mAbs] were tested for their ability to produce unresponsiveness to secondary islet xenografts. METHODS: Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 min with MMC 10 microg/ml, cultured for 20 hr, and transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipient mice were divided into experimental groups according to anti-rat ICAM-1 and/or anti-mouse LFA-1 mAb treatment and transplantation of MMC-treated or nontreated islets. RESULTS: MMC pretreatment alone prolonged graft survival, with a mean survival time (MST) of 23.0+/-7.4 days, compared with that of cultured islets (12.4+/-2.7 days; P<0.01). MMC treatment of islets significantly augmented graft survival, compared with that of crude islet grafts under treatment with anti-donor ICAM-1 mAb (MST: >41.3+/-30 vs. 16.6+/-5.4 days, P<0.01), anti-recipient LFA-mAb (MST: >70.3+/-28.9 vs. 30.4+/-10.4 days, P<0.001), or both mAbs (MST: >88.1+/-24.1 vs. 23+/-7.4 days, P<0.0001). One of six, four of nine, and six of eight animals accepted MMC-treated islet xenografts over 100 days after treatment with anti-rat ICAM-1, anti-mouse LFA-1, or both mAbs treatments, respectively, whereas none of the animals accepted nontreated islets under the same treatment. When the mice bearing long-term functioning xenografts were challenged with the secondary graft from the original donor strain, the animals previously treated with anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept it than animals given anti-recipient LFA-1 mAb alone (MST: 55.8+/-25.7 vs. 15+/-2.4 respectively; P<0.001), although they rejected the third-party xenograft and allograft acutely. CONCLUSIONS: In the xenogeneic islet transplantation model, MMC graft pretreatment and blockage of the ICAM-1/LFA-1 pathway constitute a potent protocol for inducing unresponsiveness to islet xenografts.     

Mitomycin-C treatment followed by culture produces long-term survival of islet xenografts in a rat-to mouse model

One of the goals of islet transplantation is to transplant viable islets without host immunosuppression. The present study was designed to determine whether pretreatment of islets with mitomycin-C (MMC) followed by culture enhances islet survival in a rat-to-mouse xenogeneic combination. WS(RT1k) rat islets pretreated with various concentrations of MMC (0, 3.2, 10, 32, 100, 320, and 1000 microg/ml) were tested for viability by in vitro insulin secretory capacity and vital staining of islets. The MMC-treated islets (10 microg/ml) cultured for various periods (4, 20, or 40 h, 3 or 7 days) were transplanted into the renal subcapsular space of STZ-induced diabetic C57BL/6 (B6: H-2b) mice. MMC-treated or nontreated islets were subjected to microarray gene analysis and immunohistological study. Evaluation of in vitro insulin secretory capacity and vital staining of islets indicated that MMC at a dose < or =32 microg/ml is nontoxic and preserves islet function. Marked prolongation of graft survival was noted with half of islet grafts surviving indefinitely (>100 days) when 10 microg/ml of MMC-treated islets was transplanted after 40 h or 3 days in culture, but not when they were transplanted within 4 h following treatment or at 7 days following treatment, indicating that there is a critical culture period necessary for successful islet graft survival. Microarray analysis suggested possible genes for this prolongation with TGF-beta highly expressed in MMC-treated islets subjected to culture for 3 days. Our results indicate that MMC treatment followed by a critical culture period induces marked prolongation of rat islet xenograft survival in nonimmunosuppressed recipient mice, offering a strategy for islet transplantation without immunosuppression.     

Enhancement of antitumor immune response in glioma models in mice by genetically modified dendritic cells pulsed with Semliki forest virus-mediated complementary DNA

OBJECT: The aim of this study was to further investigate dendritic cell (DC)-based immunotherapy for malignant glioma to improve its therapeutic efficacy. METHODS: Dendritic cells were isolated from the bone marrow and pulsed with phosphate-buffered saline, tumor RNA, tumor lysate, Semliki Forest virus (SFV)-LacZ, SFV-mediated B16 complementary (c)DNA, or SFV-mediated 203 glioma cDNA, respectively, to treat mice bearing tumors of the 203 glioma cell line. The results indicated that pre-immunization with DCs pulsed with the same type of cDNA as in the tumor by a self-replicating RNA vector (that is, SFV) protected mice from tumor challenge, and that therapeutic immunization prolonged the survival of mice with established tumors. The SFV induced apoptosis in DCs and their death facilitated the uptake of apoptotic cells by other DCs, thus providing a potential mechanism for enhanced immunogenicity. CONCLUSIONS: Therapy with DCs that have been pulsed with SFV-mediated tumor cDNA may be an excellent procedure for the development of new cancer vaccines.