Adoptive transfer of bryostatin 1-activated T cells provides long-term protection from tumour metastases.

Treatment of human cancer with tumour-specific T lymphocytes is limited by the frequent unavailability of autologous tumour to stimulate T-cell growth and by the toxicity associated with high-dose interleukin-2 (IL-2) treatment. In the present study we demonstrate that Bryostatin 1 (B) plus ionomycin (I) can substitute for tumour antigen and activate tumour-bearing hosts' T-cells which provide long-term protection against tumour challenge after adoptive transfer. Lymphocytes obtained from the popliteal lymph nodes (DLN) draining an MCA-105 footpad sarcoma were stimulated with B/I, and then cultured for 7 days with 20 U ml-1 IL-2. This in vitro stimulation protocol consistently expanded cell numbers greater than 20-fold during 7 days. Mice given B/I-stimulated draining lymph node (DLN) cells were protected from specific i.v. tumour challenge for at least 15 weeks after adoptive transfer, even in the absence of IL-2 treatment. Tumour immunity conferred by B/I-activated DLN cells was systemic and independent of host T-cells. However, resistance to tumour challenge was lost when either CD4+ or CD8+ T-cells were depleted in vivo. These studies indicate that DLN cells activated with bryostatin 1 and ionomycin persist long-term in vivo as functional memory cells after adoptive transfer.     

Electroporation adopting trains of biphasic pulses enhances in vitro and in vivo the cytotoxic effect of doxorubicin on multidrug resistant colon adenocarcinoma cells (LoVo)

Few articles in the literature have focused on electroporation as a strategy to reverse multidrug resistance (MDR) of tumour cells and they are mostly limited to the improved efficacy of bleomycin. We tested the application of trains of biphasic pulses to cell suspensions and to murine xenografts as a strategy to increase the uptake of doxorubicin (DOX) and to enhance its cytotoxicity against chemoresistant cells. The human colon adenocarcinoma cell line LoVo DX, expressing MDR phenotype with high levels of P-glycoprotein (P-gp), has been used. The in vitro and in vivo studies gave the following results: (i) the application of the electric pulses to the cell suspension, immediately before DOX administration, induced a significant increase of drug retention; (ii) confocal microscopy observations showed a remarkable increase of intranuclear accumulation of DOX induced by electroporation; (iii) cell survival assay revealed a decrease of cell viability in the cultures treated with the combination of electroporation and doxorubicin; (iv) scanning electron microscopy observations revealed consistent morphological changes after the combined exposure to electroporation and doxorubicin; (v) in implanted mice the combined treatment induced an evident slowdown on the tumour growth when compared to treatment with DOX alone; (vi) histopathological analysis evidenced tumour destruction and its replacement by scar tissue in the tumours treated with the combination of doxorubicin and electroporation.     

Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF

Our previous study showed that DNA vaccination with a plasmid vector encoding a core peptide of mucin1 (PDTRP) provided modest protection against challenge with tumor cells that expressed mucin1 protein. We report here that a DNA vaccine comprising a modified PDTRP plasmid and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The increased protection was directly correlated with a stronger PDTRP-specific immune response induced by the GM-CSF fusion plasmid. The plasmid encoding GM-CSF and the target PDTRP antigen induced a greater PDTRP-specific Th proliferation, antibodies, and cytotoxicity. Interestingly, the modified plasmid vaccine predominantly enhanced the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice. In addition, protection against tumor challenge in vaccinated mice showed that there was no significant change in mice survival after in vivo CD8+CTL depletion, indicating that antitumor immunity augmented by plasmid encoding GM-CSF and target PDTRP gene vaccine was dominated by Th2 immune response.     

Flt3L therapy following localized tumor irradiation generates long-term protective immune response in metastatic lung cancer: its implication in designing a vaccination strategy

Flt3 ligand (Flt3L) therapy that expands dendritic cells in vivo in combination with local tumor radiotherapy (RT) significantly improved survival and induced a long-term tumor-specific immune response in a murine model of Lewis lung carcinoma (3LL). The irradiated tumor cells were able to significantly restimulate the splenocytes of the RT + Flt3L cohort in vitro. The restimulated splenocytes demonstrated increased cytotoxic response, lymphocytic proliferation and elevated levels of Th type I cytokines (IL-2, IL-12, IFN-gamma and TNF-alpha). The combination therapy of RT + Flt3L induced a long-term protective immunity in the disease-free animals. The protective effect was further enhanced when the disease-free animals were vaccinated with irradiated tumor cells. The vaccinated animals had significantly greater protection compared to the nonvaccinated group against subsequent challenge with 3LL cells. Taken together, these results indicate that the release of tumor antigens by irradiated dying tumors and concomitant administration of Flt3L was able to facilitate the generation of a tumor-specific long-term immune response against a poorly immunogenic tumor. This effect was further boosted by vaccination with irradiated tumor cells.     

DNA immunization using constant-current electroporation affords long-term protection from autochthonous mammary carcinomas in cancer-prone transgenic mice

A recently developed, adaptive constant-current electroporation technique was used to immunize mice with an intramuscular injection of plasmid coding for the extracellular and transmembrane domains of the product of the rat neu(664V-E) oncogene protein. In wild-type BALB/c mice, plasmid electroporation at lower current settings elicits higher antibody titers, a strong cytotoxic response and completely protects all mice vaccinated with 10, 25 and 50 microg of plasmid against a lethal challenge of rat neu+ carcinoma cells. BALB/c mice transgenic for the transforming rat neu(664V-E) (ErbB-2, Her-2/neu) oncogene (BALB-neuT(664V-E)) develop an invasive mammary gland carcinoma by 20 weeks of age. Remarkably, when transgenic BALB-neuT(664V-E) mice were vaccinated at a 10- week interval with 50 microg of plasmid with 0.2 A electroporation, mice remained tumor free for more than a year. A single administration of plasmid associated with electroporation was enough to markedly delay carcinogenesis progression in mice with multiple microscopic invasive carcinomas, and keep about 50% of mice tumor free at one year of age. Thus, vaccination using a clinically relevant dose of plasmid encoding the extracellular and transmembrane domains of the neu oncogene delivered by electroporation prevents long-term tumor formation. These improvements in the efficacy of this cancer vaccine regimen vastly increase its chances for clinical success.     

Properties of a herpesvirus-transformed hamster cell line: immunogenicity of sublines of high and low metastatic potential

The immunogenicity of a herpesvirus hominis type-2-transformed hamster cell line (HSV-2-333-2-26) of low spontaneous metastatic ability was compared with that of its two in vivo-derived sublines of increased metastatic potential. The parent (HSV-2-333-2-26) tumour was immunogenic as assessed by protection against tumour challenge afforded by implantation of irradiated tumour cells or tissue. In contrast, the two metastatic sublines, designated Met A and Met B, were non-immunogenic as defined by the above critera . However, the parent Met A and Met B tumours were shown to possess a common antigen(s), since immunization with irradiated parent tumour cells afforded protection to challenge with Met A or Met B. Immunization with the metastatic sublines, however, gave no protection to homologous or heterologous tumour challenge. Bacillus Calmette-Guérin (BCG) inoculated in admixture with irradiated tumour cells and followed 7 days later by one immunization with X-irradiated tumour cells alone, increased host immunocompetence to subsequent homologous or cross-tumour cell challenge with parent, Met A or Met B cells. The immunity raised by using BCG plus irradiated tumour cells was shown to be specific to antigens expressed on the HSV-2 parent cell line and its metastatic sublines. In addition, BCG admixed with live inocula of parent, Met A or Met B cells induced contact suppression of in vivo tumour growth of the parent cells, but not of Met A or Met B cells. It is suggested from these studies that the parent tumour possesses a tumour-specific transplantation antigen(s) ( TSTAs ) which is not functionally active on its metastatically derived sublines. Common antigens, shared between the parent and Met A and Met B cells, are detectable by cross-challenge experiments, but they themselves appear not to be immunologically offensive. The loss of immunogenicity is discussed as a possible mechanism for the in vivo selection of sublines with increased metastatic potential.     

An analysis of the factors allowing promotion (rather than inhibition) of tumour growth by corynebacterium parvum

The following factors were found to determine whether C. parvum (CP) treatment promoted rather than inhibited the growth of methylcholanthrene-induced fibrosarcoma cells injected into CBA mice. (1) The dose of tumour cells. Promotion occurred only with low doses, around the TD₅₀. (2) The route of injection of CP. Greater promotion was caused by intravenous (IV) than by subcutaneous (SC) administration. Addition of irradiated tumour cells to SC CP resulted in tumour inhibition. (3) The dose of CP. Promotion increased with increasing dose of either SC or IV CP. (4) The time of CP injection relative to tumour challenge. Promotion only occurred when CP was given before tumour cells, except when using IV CP and very few tumour cells. With increasing doses of tumour cells, first post-treatment with IV CP and then pre-treatment became inhibitory. The effect of CP on established immunity to tumour cells was also studied. Mice were immunized by tumour amputation. The resistance to tumour challenge thus generated could be abrogated by CP given before challenge, most effectively by a high dose IV. The data are interpreted according to the following hypothesis. (1) CP suppresses the expression of cell-mediated immunity to tumour antigens. (2) This is caused by trapping of anti-tumour effector cells at the site of CP deposition. (3) Promotion can only occur when CP is given before effector cells have reached the tumour site.     

Immune responses to naturally occurring rat sarcomas.

Attempts were made to induce immunity to 5 spontaneous rat sarcomas transplanted into syngeneic recipients. Rats were immunized by surgical removal of growing tumour transplants or by treatment with attenuated tumour, followed by challenge with tumour cells in suspension. Two tumours wee apparently not immunogenic, but a low level of immunity was induced against 2, and weak evidence of immunity was observed with another. Induced immunity was individually specific rather than cross-reactive. It is concluded that, contrary to some reports, some spontaneous animal tumours are immunogenic in the strain of origin.     

Trafficking of 'immune' CD4(+)/CD8(+)T-lymphocytes into the RENCA tumour microcirculation in vivo in mice

RENCA-IL-2 (Murine Renal Cell Carcinoma transfected with murine IL-2 gene) cells were rejected by immunocompetent (but not T-cell deficient) Balb/c mice, which developed 'immunity' to subsequent parental RENCA tumour cell challenge. Splenocytes adoptively transferred this immunity. CD4(+)and CD8(+)T-lymphocytes prepared from the spleens of 'tumour immune' mice were evaluated for their ability to traffic into the tumour environment using an in vivo model that enables visualization of events within the microvasculature. RENCA cells were implanted into the mouse cremaster muscle and the trafficking of syngeneic lymphocyte subpopulations, derived from naive and 'immune' animals, into both the RENCA tumour and the surrounding normal cremaster muscle microcirculation was measured by in vivo microscopy. Fluorescently labelled CD4(+)and CD8(+)T lymphocytes taken from the spleens of naive mice or mice previously immunized with RENCA-IL-2 were injected systemically into tumour-bearer mice. Naive effector cells migrated to, and flowed through both the tumour and the normal microcirculation, with negligible adhesion. However we observed the selective recruitment, localization and arrest of immune CD4(+)and CD8(+)T lymphocytes (P< 0.05) into the tumour microcirculation, and in some instances the subsequent extravasation of cells into the tumour interstitium. Lymphocyte rolling by 'immune' CD4(+)and CD8(+)T-cells in the tumour microcirculation was greatly reduced, suggesting impaired adhesion molecule expression on the tumour endothelium. This study clearly demonstrates, by direct in vivo microscopy assessment, the localization of effector cells, CD4(+)and CD8(+)lymphocytes into tumours.     

Improved antitumour immunity in murine neuroblastoma using a combination of IL-2 and IL-12

Neuroblastoma immunotherapy using cytokine-modified tumour cells has been tested in clinical trials. However, because of the complex nature of antitumour immune responses, a number of therapies may be required for complete tumour eradication and generation of systemic immunity. We report here the improved antitumour effect of two cytokines, interleukin-2 (IL-2) and interleukin-12 (IL-12), when coexpressed by neuroblastoma cell lines. Initially, transfection of human and mouse neuroblastoma cell lines resulted in high expression levels of biologically active IL-2 and IL-12 in vitro. These cytokines when expressed by transfected Neuro-2A cells completely abolished their in vivo tumorigenicity in a syngeneic neuroblastoma model. Vaccination of established tumours with IL-12-producing cells exhibited a clear effect with reduced tumour growth in the presence of IL-2. In vivo depletion studies showed that CD4(+) and CD8(+) T cells mediate the response against cytokine-producing cells. These results suggest that IL-2 and IL-12, when cotransfected in tumour cells, are effective against established disease and provide a promising immunotherapeutic approach for the treatment of neuroblastoma.     

Inhibition of RL male 1 tumor growth in BALB/c mice by introduction of the RLakt gene coding for antigen recognized by cytotoxic T-lymphocytes and the GM-CSF gene by in vivo electroporation

A DNA vaccine for inducing a tumor immune response was investigated using a well-characterized murine model tumor antigen. We demonstrated that in vivo electroporation augmented the induction of IFNγ enzyme-linked immunospot (ELISPOT) and cytotoxic T lymphocyte (CTL) generation against pRL1a peptide in BALB/c spleen cells upon immunization with RLakt plasmid. Immunization without in vivo electroporation resulted in only a marginal induction of IFNmG ELISPOT and CTL generation. Furthermore, co-injection of GM-CSF and RLakt plasmids significantly enhanced the induction of IFNmG ELISPOT and CTL generation compared to the injection of RLakt plasmid alone. Inhibition of RL male 1 tumor growth was observed by injecting BALB/c mice with GM-CSF and RLakt plasmids using in vivo electroporation, although no effect was observed against an established tumor using the same treatment. No growth inhibition was observed without in vivo electroporation. Immunization with either RLakt plasmid alone, or GM-CSF and pCIneo control plasmids using in vivo electroporation did not inhibit RL male 1 tumor growth.     

Active specific immunotherapy of mouse methylcholanthrene induced tumours with Corynebacterium parvum and irradiated tumour cells.

The relative efficiency of active nonspecific or specific immunotherapy of developing methylcholanthrene induced fibrosarcomata with C. parvum was compared. For nonspecific immunotherapy, mice were challenged with tumour cells s.c. or i.v., and 2 days later injected i.v. with dilutions of C. parvum. The only significant effect was a retardation of s.c. tumour growth by the highest concentration of C. parvum (350 mug). However, active specific immunotherapy, using mixtures of C. parvum and irradiated or living tumour cells in the footpads, suppressed tumour growth when given at 2 or 6, but not 10, days after tumour challenge. Successful therapy required: sufficient tumour cells (greater than or equal to 5 X 10(4)); an optimal dose of C. parvum (5-120 mug, increasing with the number of tumour cells); an intact T cell system; the same tumour cells for challenge and treatment. The specificity was confirmed in a protection system in which treatment was given 7 days before tumour challenge. No protective immunity could be achieved with mixtures of C. parvum and foetal cells. Thus in this system C. parvum potentiates protective immunity only to the tumour unique TSTA.     

Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model

Applications of nonviral vectors for gene transfer into tumors in vivo have been limited by the relatively low expression levels of the transferred gene. The aim of this study is to evaluate the efficacy of electroporation-mediated interleukin-12 (IL-12) gene therapy for hepatocellular carcinoma (HCC). First, we investigated the optimal conditions of electric pulses (voltage, pulsing duration, numbers of shocks) of in vivo electroporation for gene transfer into HCC established by s.c. implantation of MH134 cells to C3H mice. This process made use of plasmid DNA that express the luciferase gene. We concluded that the optimal conditions for the electric pulses are as follows: voltage at 150 V; pulsing duration at 50 ms; nonpulsing duration at 950 ms; and the number of shocks at 10. Second, we tried to treat s.c. HCC by electroporation using plasmid DNA that expresses the murine interleukin-12 (mlL-12) gene. Intratumoral administration of the mIL-12 vector elevated serum IL-12 and IFN-gamma and significantly inhibited the growth not only of HCC into which the mIL-12 vector had been directly transferred, but also of the distant HCC. In addition, intratumoral administration of the mIL-12 vector inhibited spontaneous lung metastasis and delayed establishment of HCC injected 3 days after mIL-12 gene therapy. The IL-12 gene therapy induced more lymphocyte infiltration by NK cells, CD3+ cells, and Mac-1 positive cells into the tumor and reduced the number of microvessels. Therefore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive tumor cells were found. These results demonstrate that gene therapy for HCC by electroporation in vivo using IL-12 is very efficient and is thus promising for further clinical trial.     

Corynebacterium parvum stimulation of adherent and non-adherent cytotoxic cells in mice.

Two naturally occurring cytotoxic cell populations have been identified in the peritoneal cavity of mice inoculated with C. parvum (CP), and are distinguishable on the basis of target-cell reactivity and intrinsic properties. The first effector cell was non-adherent to nylon wool and glass and non-phagocytic. These cells were selectively cytotoxic to the NK-sensitive target cell line K562, and present in the peritoneal cavity of mice 2 days after treatment with 700 micrograms of CP. The second cytotoxic effector cell was adherent to nylon wool and glass, and killed EL4 lymphoma cells derived from in vivo tumour transplants; these target cells are susceptible to phagocytic cell killing, but not NK-cell cytotoxicity in short-term (4h) assays. The adherent cytotoxic population of effector cells was present 4 days after inoculation of CP. In vivo studies showed that CP injected i.p. induced resistance to i.p. challenge with lymphoma EL4 cells, but no resistance was evident when the challenge dose was administered s.c. Adoptive-transfer studies showed that the effector cell type responsible for inhibiting tumour growth was nylon-wool adherent, probably CP-activated macrophages.     

Host antitumour immune responses to HIFU ablation.

The ideal cancer therapy not only induces the death of all localized tumour cells without damage to surrounding normal tissue, but also activates a systemic antitumour immunity. High-intensity focused ultrasound (HIFU) has the potential to be such a treatment, as it can non-invasively ablate a targeted tumour below the skin surface, and may subsequently augment host antitumour immunity. In addition to thermal and cavitation effects, which act directly and locally on the tumour, there is increasing evidence linking systemic anti-tumour immune response to HIFU ablation. This may provide micro-metastatic control and long-term tumour resistance for cancer patients. The goal of this article is to review the emerging pre-clinical and clinical results suggesting that HIFU ablation may enhance host anti-tumour immunity, and to discuss its potential mechanisms. It is concluded that the systemic immune response induced by thermal ablation may play an important role in local recurrence and metastasis control after HIFU treatment.     

Properties of bcr-abl-transformed mouse 12B1 cells secreting interleukin-2 and granulocyte-macrophage colony-stimulating factor: I. Derivation, genetic stability, oncogenicity and immunogenicity

The highly oncogenic bcr-abl-transformed mouse (Balb/c) 12B1 cells were transfected with plasmids carrying genes for either mouse interleukin-2 (IL?2) or the mouse granulocyte-macrophage colony-stimulating factor (GM?CSF) and the gene for blasticidine resistance. From the transduced cells several clones widely differing in the production of either cytokine were isolated. For further experiments, clones with the highest secretion of the cytokines were selected. When administered subcutaneously to mice, the IL-2-secreting cell line was approximately hundred times less pathogenic than the parental cells. A portion of animals developed small, spontaneously regressing tumours and most of them became resistant to challenge with the parental cells. Cell populations from either solid tumours or from organs infiltrated by the tumour cells predominantly consisted of cells which did not produce IL-2 and had lost resistance to blasticidine. This indicated that the IL-2 secreting cells were genetically unstable in the course of their propagation in vivo. On the other hand, the GM?CSF?secreting cells were more pathogenic than the parental cells, induced extensive organ damage and remained genetically stable in the course of their growth in vivo. The pathogenicity of different GM?CSF secreting clones directly depended on the magnitude of production of this cytokine. When used in the form of inactivated vaccines, the GM-CSF-secreting cells were more immunogenic than the IL-2-secreting cells. In comparative experiments, similar results were obtained with GM?CSF- and IL-2-secreting cells derived from B210 cells, another bcr-abl transformed cell line.     

Anti-HER-2 DNA vaccine protects Syrian hamsters against squamous cell carcinomas

This paper illustrates the efficacy of DNA vaccination through electroporation in the prevention of oral transplantable carcinoma in Syrian hamsters. At 21 and 7 days before tumour challenge, 19 hamsters were vaccinated with plasmids coding for the extracellular and transmembrane domains of rat HER-2 receptor (EC-TM plasmids), whereas 19 control hamsters were injected intramuscularly with the empty plasmid. Immediately following plasmid injection, hamsters of both groups received two square-wave 25 ms, 375 V cm(-1) electric pulses via two electrodes placed on the skin of the injection area. At day 0, all hamsters were challenged in the submucosa of the right cheek pouch with HER-2-positive HCPC I cells established in vitro from an 7,12-dimethylbenz[a]anthracene-induced oral carcinoma. This challenge gave rise to HER-2-positive buccal neoplastic lesions in 14 controls (73.37%), compared with only seven (36.8%, P<0.0027) vaccinated hamsters. In addition, the vaccinated hamsters displayed both a stronger proliferative and cytotoxic response than the controls and a significant anti-HER-2 antibody response. Most of the hamsters that rejected the challenge displayed the highest antibody titres. These findings suggest that DNA vaccination may have a future in the prevention of HER-2-positive human oral cancer.     

Distant metastasis facilitated by BCG: spread of tumour cells injected in the BCG-primed site.

Tumour metastasis in BCG-pretreated mice was studied using a methylcholanthrene-induced fibrosarcoma in C3H/He mice. When tumour cells were injected into the BCG-primed site, distant metastasis occurred in the lungs and the popliteal lymph node, through this tumour did not metastasize in normal mice. Such metastases were increased in proportion to the number of tumour cells injected into the BCG-primed site, and developed soon after tumour challenge. Concomitant immunity developed well in the mice bearing such metastases, but did not inhibit metastatic growth. Experiments using 125I-labelled SRBC or tumour cells revealed that such cells egressed rapidly from the BCG-primed site. When the tumour was inoculated into the contralateral foot to the BCG-primed site, the incidence and the number of metastases was reduced. Furthermore, BCG infection induced an increase of platelet count. I.v. injection of this tumour induced marked thrombocytopenia in normal mice. Administration of pentoxifylline, a methylxanthine derivative before tumour challenge reduced such metastases. These findings suggest that the changes in peripheral blood, such as increased platelet count and increased release of tumour cells from the injection site, facilitated distant metastasis in BCG-pretreated mice.     

Mechanisms of local immunosuppression in cutaneous melanoma

Cutaneous melanoma is highly immunogenic, yet primary melanomas and metastases develop successfully in otherwise immunocompetent patients. To investigate the local immunosuppressive microenvironment, we examined the presence of suppressor T lymphocytes and tolerising dendritic cells (DCs), the expression of immunosuppressive cytokines (IL-10, TGFbeta1 and TGFbeta2) and the enzyme indoleamine 2,3-dioxygenase (IDO) using qRT-PCR and immunohistochemistry in primary skin melanomas, negative and positive sentinel lymph nodes (SLN), and lymph nodes with advanced metastases. Our results indicate that tolerogenic DCs and suppressor T lymphocytes are present in melanoma at all stages of disease progression. They express transforming growth factor beta receptor 1 (TGFbetaR1), and are therefore susceptible to TGFbeta1 and TGFbeta2 specifically expressed by primary melanoma. We found that expression of IDO and interleukin 10 (IL-10) increased with melanoma progression, with the highest concentration in positive SLN. We suggest that negative SLN contain immunosuppressive cells and cytokines, due to preconditioning by tolerogenic DCs migrating from the primary melanoma site to the SLN. In primary melanoma, TGFbeta2 is likely to render peripheral DCs tolerogenic, while in lymph nodes IDO and TGFbeta1 may have a major effect. This mechanism of tumour-associated immunosuppression may inhibit the immune response to the tumour and may explain the discrepancy between the induction of systemic immunity by anti-melanoma vaccines and their poor performance in the clinic.