Prolongation of survival of mice with glioma treated with semiallogeneic fibroblasts secreting interleukin-2.

OBJECT: The current prognosis for patients with malignant brain tumors remains poor, and new therapeutic options are urgently needed. We previously have shown that prolongation of survival can be achieved in C57BL/6 mice (H-2b) with a syngeneic intracerebral or subcutaneous glioma when treated with allogeneic mouse fibroblasts (H-2k) genetically engineered to secrete interleukin-2 (IL-2). Like other antigens, tumor-associated antigens are recognized by cytotoxic T lymphocytes in the context of determinants specified by the major histocompatibility complex class I locus. Because the rejection of allogeneic major histocompatibility complex determinants has the property of an immune adjuvant, immunotherapy of glioma with a cellular immunogen that combines the expression of both syngeneic class I determinants and allogeneic antigens could have advantages over an immunogen that expresses syngeneic or allogeneic determinants alone. METHODS: To investigate this question in a mouse glioma model, we further modified allogeneic mouse fibroblasts (H-2k), not only for IL-2 secretion, but also for the expression of H-2Kb class I determinants. We tested the immunotherapeutic properties of these semiallogeneic cells (LM-IL-2/H-2Kb) in C57BL/6 mice with Gl261 glioma in both subcutaneous and intracerebral glioma models. RESULTS: C57BL/6 mice with either a subcutaneous or intracerebral glioma treated solely by injection of these IL-2-secreting semiallogeneic cells had significantly prolonged survival rates compared with untreated mice or mice treated with cells secreting only IL-2 or cells lacking the H-2Kb determinants. In some instances, the mice treated with the semiallogeneic cells survived indefinitely, suggesting total eradication of the glioma. When a 51Cr release assay was used, the specific immunocytotoxicity measured by release of isotopes from labeled Gl261 glioma cells coincubated with spleen cells from mice immunized with the semiallogeneic IL-2-secreting cells was significantly higher than that of spleen cells from nonimmunized mice or mice immunized with allogeneic cells lacking syngeneic major histocompatibility complex determinants. In addition, antibody depletion studies using monoclonal antibodies against CD8+ and natural killer/lymphokine-activated killer cells demonstrated a specific CD8+ immunocytotoxic response in animals immunized with the semiallogeneic IL-2-secreting cells compared with only a natural killer/lymphokine-activated killer response in mice immunized with the allogeneic IL-2-secreting cells. CONCLUSION: The augmented immune response against glioma in mice treated with the semiallogeneic IL-2-secreting cells points toward a new form of immunotherapy, "immuno-gene therapy," for patients with malignant intracerebral glioma.     

Prolonged survival of mice with established intracerebral glioma receiving combined treatment with peroxisome proliferator-activated receptor-gamma thiazolidinedione agonists and interleukin-2-secreting syngeneic/allogeneic fibroblasts

OBJECT: In this study the authors explored the benefits of treating C57B1/6 mice with an established intracerebral glioma by combining immunotherapy with interleukin (IL)-2-secreting syngeneic/allogeneic fibroblasts administered into the tumor bed along with the chemotherapeutic agent pioglitazone, a thiazolidinedione (TZD). The TZDs are agonists of the peroxisome proliferator-activated receptor-gamma. They have been found to exert antiproliferative effects on several transformed cell lines. Data from prior studies by these authors have revealed the immunotherapeutic properties of the IL-2-secreting fibroblasts in treating intracerebral gliomas in mice. METHODS: The sensitivity of GL261 glioma cells and primary astrocytes to pioglitazone was determined in vitro by incubating the cells with increasing amounts of the drug. Viability was assessed by measuring lactate dehydrogenase release, and effects on metabolism were determined by measuring superoxide production and levels of superoxide dismutase. The GL261 cells were injected intracerebrally into C57B1/6 mice, followed by treatment with pioglitazone either orally or intracerebrally into the tumor bed. The effect of the combined therapy was determined by injecting C57B1/6 mice with an established intracerebral GL261 glioma with IL-2-secreting allogeneic fibroblasts and pioglitazone directly into the tumor bed through a unique cannula system. Pioglitazone was found to induce cell death in GL261 glioma cells grown in vitro while causing only modest damage to astrocytes. The application of pioglitazone also resulted in a significantly greater induction of cellular superoxide in glioma cells than in astrocytes, which can activate apoptotic pathways. Pioglitazone administered intracerebrally (p < 0.05) but not orally was found to prolong survival in mice harboring an intracerebral glioma. Synergistic effects of combination therapy on prolonging survival were found in mice receiving both pioglitazone and IL-2-secreting fibroblasts (p < 0.005, compared with untreated animals). Pioglitazone induces metabolic and oxidative stresses that are tolerated by astrocytes but not glioma cells, which could account for selective vulnerability and increased sensitivity to IL-2, suggesting potential for the use of this Food and Drug Administration-approved drug in the treatment of brain tumors. CONCLUSIONS: The data indicate the beneficial effects of combination therapy using pioglitazone and immunotherapy in mice harboring intracerebral glioma.     

Successful treatment of a malignant rat glioma with cytotoxic T lymphocytes.

Brain tumors are highly resistant to therapy. Their diffuse infiltrative nature and the relative inaccessibility of brain tissue to blood and lymph are barriers to surgical and cytotoxic treatments alike. The purpose of this study was to produce immune cells specifically reactive with an anaplastic rat glioma (RT2) and determine whether those cells could affect tumor progression in the brain. RT2-specific cytotoxic cells were prepared by priming rats in vivo with RT2 tumor cells and Corynebacterium parvum and stimulating the primed lymphocytes in vitro with irradiated RT2 tumor cells and interleukin-2 (IL-2). Cultured cells exhibited a high level of cytotoxicity against RT2, but not C6 (an allogeneic glioma), 3M2N (a syngeneic mammary tumor), or CSE (a syngeneic fibrosarcoma) tumor cells. To generate a model for therapy, rats were injected intracerebrally with RT2, generating progressing brain tumors, which killed untreated rats in approximately 2 weeks. To test the therapeutic potential of the effector cells, tumor-bearing rats were treated by intravenous injection of lymphocytes on Day 5 of tumor growth. Treated rats also received a 5-day course of systemic IL-2 beginning on Day 5. Treatment with IL-2 alone, RT2-primed spleen cells, or RT2-primed spleen cells stimulated in vitro with C6 did not affect rat survival. However, tumor-bearing rats treated with RT2-stimulated lymphocytes exhibited increased survival or were cured. Systemic IL-2 was an essential adjunct, because survival was not affected by treatment with effector cells alone. Therapy initiated on Day 8 of tumor progression lacked effect on survival.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human lung tumor cell secretion of interleukin-2 for protection against tumor engraftment

Background: Lung cancer continues to claim large numbers of human lives each year despite advances made in conventional therapies. The use of biologic response modifiers to modulate the immune system against human tumors is an alternate form of immunotherapy. Interleukin-2 (IL-2), or T-cell growth factor, is an important modulator of activated T cells. We show here that tumor cells transduced with human IL-2 cDNA provide protective immunity against engraftment of IL-2-secreting, as well as parental non-IL-2-secreting, tumor cellsin vivo. Methods: In an attempt to increase the antigen-induced proliferation and cytotoxicity T cells within the vicinity of tumor antigen, we have transduced human lung tumor cell lines (generated from whole tumor specimens obtained fresh from the operating room) with a vector containing the IL-2 gene. Cell lines secreting 0.5–20 Cetus units/ml of IL-2 were generated. Control cell lines were similarly established using the same retroviral vector containing the gene for adenosine deaminase (ADA). The growth of tumor xenografts of the vector-modified cell lines was observed in severe combined immunodeficient (scid) mice. Results: Using C.B-17scid mice, we have observed that the local secretion of IL-2 by these human lung tumor cell lines will prevent engraftment of that tumor intoscid mice. The parental tumor as well as the tumor containing the ADA gene grow aggressively in thescid mouse. Growth arrest also correlated strongly with the amount of IL-2 secreted by the tumor cells. The local secretion of IL-2 by the transduced cell line will abrogate the tumorigenicity of the parental cell line as well as an allogeneic tumor. The inhibition of growth occurs only when the tumors are placed in close proximity to each other. After gamma irradiation, transduced tumor cells will continue to secrete IL-2. Conclusion: These results indicate that (a) human lung tumor cell lines can be transduced with IL-2-containing retroviral vectors; (b) local and sustained release of IL-2 will induce an antitumor response by the host against the IL-2-secreting as well as the control tumor cells; (c) secretion of IL-2 continues after the cells are irradiated. This study suggests that cytokine-secreting human lung tumors may be used in vaccination protocols for cancer patients.Presented at the 46th Annual Cancer Symposium of The Society of Surgical Oncology, Los Angeles, California, March 18–21, 1993.     

Systemic overexpression of interleukin-10 fails to protect allogeneic islet transplants in nonobese diabetic mice

Interleukin (IL)-10 has proven effective in various allogeneic transplantation models and for preventing recurrent autoimmune rejection of syngeneic islets in NOD mice. Therefore, we evaluated systemic IL-10 overexpression on allogeneic islet graft survival. Diabetic NOD mice received a single injection of recombinant adeno-associated virus (rAAV) serotype 2 encoding murine IL-10 (rAAV-IL-10) four weeks prior to renal subcasular islet transplantation. In a model having both autoimmune and allogeneic responses, IL-10 failed to protect C57BL/6 islets in spontaneously diabetic NOD mice. In an allograft model (C57BL/6 islets into young male streptozotocin-induced diabetic NOD mice), long-term (i.e., >169 days) islet survival was only seen in 2 of 14 rAAV-IL-10 treated mice. These failures occurred despite in vivo IL-10 production at transplant previously associated with protection of syngeneic islet grafts in NOD mice. Thus, IL-10 appears insufficient in protecting transplanted islet cells from allogeneic rejection and suggests important mechanistic variances between alloreactivity and autoimmunity in terms islet graft loss.     

Adoptive immunotherapy of intracerebral metastases in mice

Lymphokine-activated killer (LAK) cells are a heterogeneous population of immune effector cells that nonspecifically destroy neoplastic cells but not normal cells. Although parenteral treatment with interleukin-2 (IL-2) alone or a combination of IL-2 and LAK cells reduces tumor load and prolongs survival in mice with pulmonary, peritoneal, or hepatic metastases, the effect of these treatments on brain metastases has not been studied. To determine in an animal model if intracerebral metastases would be protected by the immunologically privileged status of the brain, intracardiac and intravenous injections of 10(5) KHT sarcoma cells were performed in C3H mice to create brain and lung metastases, respectively. The mice were treated with adoptive immunotherapy to determine if efficacy seen in an extracerebral site could be reproduced in the brain, and if histological examination of these brains would reveal a significant degree of lymphocyte infiltration and cytolytic activity. Animals were treated with either parenteral IL-2 (7500 U three times daily on Days 3 to 7 after tumor injection), or IL-2 plus LAK cells (7500 U IL-2 times daily on Days 3 to 7, and 10(8) LAK cells intravenously on Days 3 and 6 after tumor injection), or IL-2 excipient (three times daily on Days 3 to 7 after tumor injection). As compared to control animals, pulmonary metastases on Day 14 after tumor injection were reduced or eliminated in animals treated with either IL-2 or IL-2 plus LAK cells (p less than 0.01). In these same animals, there was no reduction in the number of intracerebral metastases and no evidence of lymphocytic infiltration or cytolytic activity in the brain. This is the first study that reveals an organ-specific resistance to the treatment of metastases with adoptive immunotherapy, and affirms the concern that due to inadequate trafficking of endogenous or exogenous-activated lymphocytes or due to inadequate activation of in situ brain lymphoid precursors, there is no rejection of tumors in the brain. This information suggests that brain metastases in patients with systemic malignancies will not respond to intravenous treatment with LAK cells and IL-2, and that alternative forms of treatment are needed. Furthermore, this modification of a previously existing model of murine brain metastasis provides a method for concurrently evaluating the effectiveness of treatments for intra- and extracranial cancers.     

Antitumor activity against established intracerebral gliomas exhibited by cytotoxic T lymphocytes, but not by lymphokine-activated killer cells.

Specific immune responses against malignant brain tumors have been difficult to demonstrate. Moreover, immunotherapy has met with little success, despite using lymphocytes with high levels of cytotoxicity against brain tumor cells. Lymphokine-activated killer (LAK) cells that nonspecifically kill brain tumor cells are produced by stimulating resting precursors with high concentrations of interleukin-2 (IL-2). Cytotoxic T lymphocytes that specifically kill brain tumor cells are produced by stimulating antigen receptor-positive immune-cell precursors with tumor cells. In an attempt to gain insight into immune cell function against brain tumors, the present study compared the in vitro and in vivo activities of LAK cells and cytotoxic T lymphocytes produced against RT2, a fast-growing rat glioma cell line. Lymphokine-activated killer cells were produced by stimulating normal rat spleen cells with 1000 units of IL-2, and RT2-specific cytotoxic T lymphocytes were produced by priming them in vivo with RT2 and Corynebacterium parvum and restimulating primed spleen cells with RT2 in vitro. Lymphokine-activated killer cells were highly cytotoxic for a panel of syngeneic and allogeneic brain tumor and non-brain tumor target cells, including RT2, as measured in a 4-hour 51Cr release assay. Cytotoxic T lymphocytes were highly cytotoxic only for syngeneic brain tumor target cells. Lymphokine-activated killer cells and cytotoxic T lymphocytes were tested for in vivo antitumor activity against intracerebral RT2 by intravenous adoptive transfer of activated lymphocytes. Untreated rats died in approximately 2 weeks. Lymphokine-activated killer cells plus IL-2 failed to affect survival when treatment was initiated as early as 1 day following tumor inoculation. Cytotoxic T lymphocytes and IL-2 administered as late as Day 5 rejected progressing intracerebral tumor. Thus, although both cytotoxic T lymphocytes and LAK cells exhibited high levels of in vitro killing of glioma cells, only cytotoxic T lymphocytes rejected progressing intracerebral tumors.     

Treatment of murine hepatic metastases with vaccinia colon oncolysates and IL-2

To evaluate the feasibility and utility of vaccinia colon oncolysates (VCO) and low-dose interleukin-2 (IL-2) immunotherapy for advanced colon cancer, we have developed a murine model and tested the efficacy of combined treatment regimens. We employed intrasplenic injection of cultured colon adenocarcinomas (C-C36) in syngeneic Balb/c mice to produce experimental hepatic metastases. In the first set of experiments, animals were challenged with 5 X 10(5) tumor cells and sacrificed 14 days following tumor challenge. In the second set of experiments, animals were challenged with 2 X 10(5) tumor cells and followed for survival over the ensuing 90 days. In the first set of experiments, animals were treated prophylactically with VCO (40 micrograms, sc, 14 and 7 days prior to challenge) and/or therapeutically with IL-2 (25,000 u, Hoffmann-LaRoche rIL-2, ip BID, on Days 1-3 following challenge). In the second set of experiments, animals were treated with either the identical regimens or therapeutically with VCO (same dose, sc, 2 and 10 days following challenge) and/or IL-2 (same dose, ip BID, on Days 9-11 following challenge). Tumor burden data from sacrificed animals was in agreement with survival data and showed significant tumor burden reduction in the combined treatment group as assessed by liver weight and tumor nodule enumeration. Survival data demonstrated highly significant survival advantage for animals treated with the two biological response modifiers: VCO (P less than 0.0021), and IL-2 (P less than 0.0017). The data presented suggest a synergistic effect for these two agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

The therapeutic potential of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer

PurposeBacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy.Materials and methodsA tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-γ, TNF-α), and Th2 (IL-5, IL-6, IL-10, TGF-β) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival.ResultsBoth BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-α in the BCG treated group, as well as increases in TNF-α and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups.ConclusionsrBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG.     

An experimental approach to specific adoptive immunotherapy for malignant brain tumors

With the aid of interleukin 2 (IL-2), two phenotypically different cytotoxic T lymphocyte (CTL) clones were established with target specificity against syngeneic murine malignant brain tumor (a methylcholanthrene-induce ependymoblastoma of C57BL/6 mouse origin, 203-glioma). Furthermore, the cloned CTL lines were characterized in vitro, and their in vivo effectiveness was investigated by intracerebral (i.c.) tumor neutralization assay and adoptive immunotherapy with the clones for i.c. tumor-bearing mice. Each CTL clone retained an IL-2 dependency with a defined functional activity. G-CTLL 1 with a phenotype of Lyt-1-.2.3+ exhibited a target cytotoxicity against 2 kinds of murine glioma cells, syngeneic 203-glioma and allogeneic RSV-M glioma (Schmitt-Ruppin rous sarcoma virus-induced malignant astrocytoma). It is noted that G-CTLL 1 cells produced gamma interferon (IFN) by stimulation with glioma antigens. The spontaneous release of gamma IFN paralleled the amounts of exogenous IL-2 added into the cultures, but IL-2 had no synergistic effects on IFN release in the presence of tumor antigens. Furthermore, by adding anti-mouse gamma IFN antibody, the IFN production of G-CTLL 1 cells was inhibited but their lytic potential was hardly reduced in vitro. In contrast, G-CTLL 2 cells expressed a cell surface phenotype of Lyt-1+.2.3+ with more restricted target specificity against only syngeneic 203-glioma cells, although they showed a weaker cytotoxicity than G-CTLL 1 cells and no release of gamma IFN. The in vivo therapeutic efficacy using G-CTLL 1 cells was confirmed in both adoptive immunotherapy and tumor neutralization assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of operational tolerance to discordant dopaminergic porcine xenografts

BACKGROUND: Porcine embryonic neural tissue transplanted intracerebrally could potentially relieve the symptoms of Parkinson's disease if the immune response toward the graft could be overcome. However, conventional immunosuppressive treatments have proven inefficient in preventing rejection. An alternative is blocking the costimulatory signals for lymphocyte activation. Treatment with cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and anti-CD40L has been successful in preventing rejection of xenografts in some experimental studies, but not all. Lymphocyte function antigen (LFA)-1 is an important costimulatory molecule for CD8+ T cells, and we hypothesize that blockade with anti-LFA-1 may enhance the efficacy of CTLA4Ig and anti-CD40L therapy. METHODS: C57BL/6 mice received intracerebral transplants of ventral mesencephalic tissue from embryonic porcine donors. CTLA4Ig, anti-CD40L, and anti-LFA-1 were administered every other day on days 0 to 8, and the transplants were studied after 4 to 6 weeks. Grafts were histologically analyzed for size, survival of dopaminergic nerve cells, and immune responses. Recipients were challenged with cultured glia cells of donor origin or an allogeneic skin graft to evaluate tolerance induction. RESULTS: Mice treated with all three substances had large grafts containing high amounts of dopamine cells but a low degree of immune response. Grafts in recipients challenged with glial cells showed an increased immunologic activity but were not rejected. Triple-treated mice showed a normal rejection process of the allogeneic skin grafts. CONCLUSION: After a short course of costimulation blocking therapy, discordant neural xenografts demonstrate long-term survival, withstand immunologic challenge, yet maintain host-versus-graft reactivity. Anti-LFA-1 complements CTLA4Ig and anti-CD40L in the induction of operational tolerance to these xenografts.     

Interleukin-2 transfected prostate cancer cells generate a local antitumor effect in vivo

In an effort to stimulate host-mediated antitumor response against prostate cancer in an animal model, highly malignant Dunning MAT-LyLu rat prostate carcinoma cells were transfected with the interleukin-2 (IL-2) cDNA, resulting in their ability to secrete large amounts of biologically active IL-2. Although parental cells form lethal tumors when injected subcutaneously into syngeneic hosts at doses of ≥5,000, injections of IL-2 secreting cells initially formed tumors and regressed completely in each of over 200 animals at all doses tested (10⁴-8 × 10⁷ cells). Mixtures of parental and IL-2 transfected cells were similarly rejected, demonstrating the non-cell autonomous nature of the response. Histological analysis of regressing tumors revealed a vigorous, predominantly lymphocytic and macrophage infiltrate at day 2 and marked tumor necrosis by day 6. Immunohistochemical staining of infiltrating lymphocytes at this latter time point demonstrated numerous T cells bearing either CD4 or CD8 surface markers, suggesting these cells as possibly mediating the tumor rejection. The ability of athymic mice to reject the IL-2 secreting tumor cells, however, suggests a non-T-cell-mediated mechanism. Although splenic natural killer (NK) activity is increased following injection of IL2 secreting tumor cells, this activity appears to be unnecessary for tumor elimination since syngeneic animals injected with asialo-GM1 antiserum to decrease NK activity also rejected IL-2 transfected cells, albeit slightly less effectively than untreated animals. Immunization of animals with subcutaneous injections of IL-2 transfected cells protected animals against a subsequent challenge of 10⁴ wild-type cells 1 to 2 weeks later in 19 of 51 cases; however, immunization did not confer protection against larger doses of parental tumor. These studies indicate that high local concentrations of IL-2 stimulate the elimination of large local burdens of prostate cancer in this model system, and this elimination results in a weak, but detectable systemic immune response against wild-type prostate cancer cells. © 1994 Wiley-Liss, Inc.     

Intracerebral growth of a human glioma tumor line in athymic mice and treatment with procarbazine, 1,3-bis(2-chloroethyl)-1-nitrosourea, aziridinylbenzoquinone, and cis-platinum

Tumors derived from the established human glioma cell line D-54 MG were transplanted intracerebrally into athymic "nude" mice. All control mice developed tumors in the brain after the transplantation of 10(5) cells and died within 30 days. Tumor was purely intracerebral in the presymptomatic stages, including the time of drug treatment. The injection of serial cell dilutions reduced the tumor incidence and prolonged survival. Procarbazine (PCB), 1,3-bis(2-chloroethyl)-1-nitrosourea, diaziquone (aziridinylbenzoquinone), and cis-platinum each produced statistically significant increases in survival when administered on Day 10 after transplantation, and PCB alone cured most of the animals. These results extend the characterization of the human glioma line D-54 MG and confirm the value of the athymic mouse for the testing of chemotherapeutic agents of interest in brain tumor therapy.     

Tissue distribution and antitumor activity of topotecan delivered by intracerebral clysis in a rat glioma model

OBJECTIVE: Intracerebral clysis is a drug delivery technique that depends on convection-enhanced microinfusion to achieve therapeutic drug levels within the brain. In this study, brain tumor-bearing rats were treated with topotecan delivered systemically and by the intracerebral clysis method. Our objective was to determine the efficacy and tissue distribution of topotecan delivered by intracerebral clysis. METHODS: The C6/Wistar rat glioma model was used after a thymidine incorporation assay determined topotecan sensitivity of C6 cells in vitro. Long-term survival of animals provided objective measurements of efficacy; records of animal weight during treatment and neurological status served to approximate toxicity. Topotecan tissue penetration was measured in samples of ex vivo tumor and surrounding brain tissue with high-pressure liquid chromatography. RESULTS: Dose escalation demonstrated significant sensitivity of C6 glioma cells to topotecan (median lethal dose, 0.19 micromol/L). Eleven of 12 rats bearing established intracerebral C6 glioma and receiving topotecan by intracerebral clysis survived beyond the end point of 120 days; no untreated control or systemically treated animal survived beyond 26 days (n = 18; P < 0.005). Histopathological assessment of animals demonstrated significant tumor masses in the brains of intraperitoneally treated animals and untreated control animals. In contrast, no residual tumor was found in the brains of intracerebral clysis groups. Animal weights during treatment were markedly reduced by intraperitoneal dosing (n = 6) but not by low-dose intracerebral clysis (32 microg/kg/d for 5 d; n = 6). None of the low-dose intracerebral clysis-treated animals demonstrated neurological toxicity, and one high-dose intracerebral clysis-treated animal (160 microg/kg/d for 2 d; n = 6) died during follow-up. Topotecan was detected well beyond the boundaries of the tumor and even in the contralateral hemisphere in animals treated with intracerebral clysis. CONCLUSION: Topotecan delivered by the intracerebral clysis method is effective for treatment of brain tumors in the rat glioma model. These studies provide compelling justification for further preclinical testing to formally evaluate toxicity and efficacy with variable dosing schedules.     

Expansion of CD3+CD56+ lymphocytes correlates with induction of cytotoxicity by interleukin-2 gene transfer in human breast tumor cultures

Background: Mice immunized with murine mammary carcinoma cells genetically engineered to secrete interleukin-2 (IL-2) are rendered resistant to subsequent challenge with unmodified tumor cells, and in the case of mice bearing established tumors, the rate of development of pulmonary metastases is reduced. Despite these encouraging animal results, little is known about the induction of antitumor immunity by IL-2 gene transfer in human breast cancer. Methods: Adenovirally mediated IL-2 gene transfer was performed in 12 tumor fragment cultures established from seven primary breast cancers. Autologous tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) were cocultured with transduced tumor fragments, and changes in phenotype and cytotoxicity were measured. Results: IL-2 was never detectable in the untransduced cultures, but it peaked at 5.0—1,324.8 ng/ml in the transduced cultures. Lymphocyte counts declined in all untransduced cultures, but they increased two- to sevenfold in four transduced cultures. CD4:CD8 ratios decreased from a mean of 2.11 at baseline to 1.27 after stimulation in coculture (p=0.03). Expansion of lymphocytes expressing the natural killer cell phenotype (CD3⁻CD56⁺) occurred in only one culture, but the CD3⁺CD56⁺ population increased in four of six cultures. Lymphocytes from four of 10 cocultures generated significant cytotoxicity against allogeneic breast cancer cells. Induction of cytotoxicity correlated with expansion of the CD3⁺CD56⁺ phenotype (R²=0.805, p=0.02). Conclusions: IL-2 gene expression by human breast cancer causes expansion of CD3⁺CD56⁺ cytotoxic lymphocytes. This phenotype is consistent with that of a non-major histocompatibility complex (MHC)-restricted cytokine induced killer cell population previously described. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the U.S. Army. Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

Dendritic cells (DC) loaded with apoptotic, allogeneic melanoma cells reduce tumor progression and improve survival in a murine immunotherapy model

Introduction: DC loaded with apoptotic, syngeneic melanoma cells slow tumor growth and improve survival in a murine immunotherapy model. The use of allogeneic cells as a source of tumor antigen could increase the clinical applicability of this therapy, so we investigated the efficacy of DC loaded with apoptotic, allogeneic cells in this model. Methods: DC were cultured from bone marrow from C-57/BL-6 mice with GMCSF (10 ng/ml), IL-4 (5 ng/ml). Syngeneic B-16 or allogeneic K-1735 melanoma cells were irradiated (20 Gy) to induce apoptosis and cultured with DC at a 1:1 ratio for 48 hr to make DC/Apo vaccine. C-57/BL-6 mice (n = 6) were injected with 2.5 x 10⁵ B-16 SQ and treated with 1 x 10⁶ DC/Apo vaccine cells SQ or saline control weekly x4 starting 5 days after tumor implantation. In other experiments, splenocytes were harvested from control and immunized tumor-bearing animals at day 30 and stimulated weekly x2 with IFN-gamma treated, irradiated B-16 cells. Cytotoxic T lymphocyte (CTL) assays were performed with B-16 targets for 48 hr and % specific lysis calculated. Results: Summarized in table 1. Conclusions: Mice treated with DC loaded with apoptotic, syngeneic melanoma cells (DC/ApoB-16) or apoptotic, allogeneic melanoma cells (DC/ApoK-1735) had delayed tumor progression and improved survival compared to controls. CTL responses to B-16 cells were seen in both treatment groups, and there was no difference in tumor size, survival, or CTL responses among the treatment groups. We conclude that DC loaded with apoptotic allogeneic melanoma cells may be a practical and effective approach to polyvalent immunotherapy of melanoma. We are currently designing a clinical trial to evaluate the efficacy of this treatment in patients with high-risk disease.     

Neoadjuvant interleukin-12 immunogene therapy protects against cancer recurrence after liver resection in an animal model

OBJECTIVE: To evaluate the neoadjuvant use of a herpes simplex viral (HSV) amplicon vector expressing the murine interleukin-12 (IL-12) gene. SUMMARY BACKGROUND DATA: Surgery is the most effective therapy for hepatic malignancy. Recurrences, which are common, most often occur in the remnant liver and are due partly to growth of residual microscopic disease in the setting of postoperative host cellular immune dysfunction. The authors hypothesized that engineering tumors to secrete IL-12 in vivo would elicit an immune response directed at residual tumor and would reduce the incidence of recurrence after resection. METHODS: Solitary hepatomas were established in Buffalo rat livers and directly injected with 106 particles of HSV carrying the gene for IL-12, lacZ (beta-galactosidase) or with saline. One week after injection, the animals were challenged with an intraportal injection of 106 tumor cells, with subsequent resection of the hepatic lobe containing the previously established macroscopic tumor nodule, recreating the clinical scenario of residual microscopic cancer. RESULTS: Hepatoma cells transfected with HSV-IL-12 produced high levels of IL-12 in vitro and in vivo. A significant local immune response developed, as evidenced by a progressive increase in the number of CD4(+) and CD8(+) lymphocytes in the tumor. Treatment of established hepatomas with HSV-IL-12 protected against growth of microscopic residual cancer after hepatic resection. Sixty-four percent of the animals treated with HSV-IL-12 had zero or one tumors compared with 30% of HSVlac-treated and 24% of saline-treated animals. CONCLUSIONS: This neoadjuvant immune strategy may prove useful in reducing the incidence of cancer recurrence after hepatic resection.     

Cytokines as an adjuvant to tumor vaccines: Efficacy of local methods of delivery

Background: We examined alternative methods of delivering cytokines as an adjunct for priming lymph node (LN) cells draining sites of vaccine inoculation for the purpose of generating immune cells for adoptive immunotherapy. Methods: Using syngeneic murine tumors we examined the ability of IL-2, IL-4, or GM-CSF delivered locally to a site of tumor inoculum to induce antitumor reactive draining LN cells. Mice were inoculated subcutaneously with tumor cells transduced to secrete cytokine; tumor cells admixed with fibroblasts transduced to secrete cytokine; or intralesional inoculation of cytokine in established tumor to induce sensitized LN cells capable of mediating tumor regression in adoptive transfer. Results: Both IL-4 and GM-CSF cytokines were effective in enhancing the antitumor reactivity of vaccine-primed LN cells compared to IL-2, which was ineffective. The local delivery of GM-CSF by autocrine or paracrine secretion of genetically engineered cells, as well as direct intratumoral delivery was capable of upregulating LN sensitization compared to systemic administration, which did not. Conclusions: The local delivery of GM-CSF as an adjuvant for tumor vaccination can be accomplished by various methods, including direct injection, which avoids the need for gene transfer.     

癌相关成纤维细胞在乳腺癌侵袭转移及耐药中的作用研究进展

癌相关成纤维细胞(CAF)是肿瘤微环境中主要的基质细胞。CAF可在血小板源性生生长因子、成纤维细胞生长因子、白介素6及肝细胞生长因子等多种分泌因子作用下由正常成纤维细胞转化形成,也可由间充质干细胞、脂肪细胞等多种细胞可通过上皮间质转化(EMT)过程形成,还有部分由癌症干细胞转化而来。近来有研究显示,乳腺癌中的CAF可通过分泌多种细胞因子及外泌体、参与EMT及细胞外基质重塑,进而促进乳腺癌细胞侵袭转移,也可在肿瘤缺氧微环境下通过激活相关信号通路促进乳腺癌细胞生长和侵袭。此外,CAF通过增加了乳腺癌细胞的凋亡阈值、作为抗肿瘤药物的物理屏障、分泌的谷氨酰胺增加乳腺癌细胞的存活率、激活生长因子相关的信号通路或增加线粒体功能产生抗凋亡作用等多种途径介导乳腺癌化疗耐药、内分泌治疗耐药及多药耐药。笔者总结CAF的重要来源及其在乳腺癌侵袭转移与治疗耐药中的研究进展。     

乳腺癌组织中COX-2与IL-8表达的相关性及临床意义

目的：探讨乳腺癌组织中环氧化酶-2（COX-2）和白细胞介素-8（L-8）表达的相关性及临床意义。方法：采用免疫组织化学法对60例乳腺癌患者病理组织中的COX-2和IL-8进行检测，并与31例乳腺良性疾病对照，分析其相关性，以及二者与肿瘤大小、浸润程度、TNM分期、淋巴结转移及雌激素受体（estrogen receptor，ER）和孕激素受体（progesteronreceptor，PR）表达等生物学行为的关系。结果：乳腺癌组织中COX-2和IL-8阳性表达率分别为66．7％、60．0％均显著高于对照组（P=0．0006、P=0．0002），且二者表达存在明显正相关（r=0．433，P〈0．001）。COX-2和IL-8阳性细胞表达率与肿瘤大小、浸润程度、TNM分期、淋巴结转移情况均有明显差异（P均〈0．05），而与ER和PR的表达状态无明显相关（P均〉0．05）。结论：COX-2和IL-8在乳腺癌组织中过度表达且呈正相关，提示乳腺癌组织中COX-2和IL-8存在相互调控机制，共同促进肿瘤的发生和发展。