Systemic IL-1 and adjuvant treatment of an experimental tumor

Local adjuvant therapy of weakly immunogenic tumors protects against primary tumor challenge. However, this form of therapy does not produce long-lasting immunity to the tumor. In this study, local adjuvant therapy combined with systemic IL-1 administration produced not only primary tumor protection, but also long lasting immunity to the tumor. IL-1 and adjuvant protected animals resisted rechallenge with tumor as much as 180 days after initial tumor administration. Resistance to tumor rechallenge was IL-1 dose dependent. IL-1 and adjuvant protected animals also exhibited delayed type hypersensitivity reactions which were tumor-specific. Splenic and lymph node cell populations from IL-1 and adjuvant protected animals mounted tumor-specific lymphoproliferative responses. No such responses were observed in animals which had been administered either IL-1 or adjuvant alone. These results demonstrate that systemic IL-1 functions to augment specific immune protection when administered in conjunction with local adjuvant, resulting in long-lasting tumor immunity.     

Real-time visualization and quantitation of vascular permeability in vivo: implications for drug delivery

The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.     

Interleukin-2 in cancer treatment: Disappointing or (still) promising? A review

The central question to discuss in this review is whether the results of interleukin-2 (IL-2) treatment are still disappointing or again promising. Although in the (recent) past application of high doses of systemically applied rIL-2 has led to some success, the overall results are not as one had hoped. Considering these poor results it seems clear that the application of high systemic doses rIL-2 was not a good choice. IL-2 has been used more or less as a chemotherapeutic compound in the highest tolerable dose. This has led to a great number of unwanted toxic side-effects. In addition, these doses mainly stimulated nonspecific lymphokine-activated killer activity through low-affinity IL-2 receptors, which does not lead to systemic immunity. On the other hand, several groups have shown that application of intratumoral low doses of IL-2 can be highly effective against cancer and without toxic side-effects. Significant tumor loads constituting up to 6% of the total body weight of a mouse were eradicated after treatment with low-dose rIL-2 given locally. Furthermore local treatment can lead to eradication of a tumor at a distant site. This type of therapy is effective in many systems namely against different tumor types in mice, hepatocellular carcinoma in guinea-pigs and vulval papilloma and carcinoma and ocular carcinoma in cattle. Low-dose IL-2 is very effective in experimental animals if it is given relatively late after inoculation of the tumor cells. In other words, it seems necessary that some sort of immune reaction has started or is developing before low doses of rIL-2 effectively stimulate it. In fact there is strong evidence that T lymphocytes, both CD4⁺ and CD8⁺ cells, are directly involved in the process leading to induction of specific immunity. In our opinion rIL-2 therapy should therefore aim at the stimulation of such (originally weak) specific immune reaction. Under these conditions also systemic immunity can be induced. In conclusion, application of rIL-2 as a modality for cancer treatment is still promising. High priority should be given to a further delineation of the mechanisms involved after local application. The method of giving IL-2 systemically in the highest tolerable dose should be abandoned. Specific stimulation of the immune system by low-dose rIL-2 is a much more promising option.     

IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4(+) and CD8(+) cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4(+)-depleted, CD8(+)-depleted, and both CD4(+)- and CD8(+)-depleted C57BL/6J animals. Tumor-infiltrating CD8(+) T cells, but not CD4(+) T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8(+) T cells, because this did not occur in nude mice or in CD8(+)-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.     

Local interleukin 2 therapy is most effective against cancer when injected intratumourally

Local interleukin 2 (IL-2) therapy is more effective against systemic tumours than systemic IL-2 therapy, but it remains unclear whether IL-2 should be injected intratumourally or peritumourally. To investigate this question, we treated DBA/2 mice bearing a large subcutaneous syngeneic SL2 lymphoma with either intra or peritumoural IL-2 therapy. Both applications enhanced survival, but intratumourally injected IL-2 was more effective than peritumourally injected IL-2. Tumours started to regress 4 days after IL-2 injection. Tumour cells died at the IL-2 injection site, although IL-2 is not directly cytotoxic for SL2 cells in vitro. Tumour cell death correlated well with oedema and extravascular erythrocytes, but less with leukocyte infiltrates. In mice bearing two s.c. tumours, intratumoural application therapy of IL-2 in one tumour caused decrease in size of both tumours in 4–9 days after therapy. However, the IL-2 treated tumours regressed more strongly than the untreated tumours. We conclude that vascular leakage and/or tissue destruction inside the tumour may contribute to the enhanced effect of intratumoural IL-2 therapy compared to peritumoural IL-2 therapy. Hence, we recommend applying of intratumoural rather than peritumoural IL-2 therapy.     

Further development of local IL-2 therapy of cancer: multiple versus single IL-2 treatment of transplanted murine colon carcinoma

We have compared the effect of one and up to four local IL-2 treatments of transplanted MC38 colon carcinoma. A single IL-2 treatment prolonged the survival time (p=0.015), but no cure was obtained. One local IL-2 treatment inhibited tumor growth for about 1 week. After the start of tumor regrowth, a further IL-2 injection was given. After four IL-2 injections 6 out of 13 mice were cured. Histological studies show that IL-2 induced a local vascular leakage syndrome leading to massive peritumoral edema and subsequent necrosis of tumor tissue. IL-2 also attracted infiltrating cells, mainly macrophages. Subsequent IL-2 injections led to complete tumor regression. We believe that the combination of necrotic tumor debris and the IL-2–induced macrophage reaction enhanced a tumor-specific immune response. This local IL-2 application was not toxic.     

Delivery of interleukin 2 for immunotherapy

The local production of interleukin 2 (IL-2) by T lymphocytes acts to enhance the immune response by inducing growth and differentiation of a variety of immune cells. In clinical situations that require immunostimulation, such as vaccination and enhancement of tumor immunity, IL-2 therapy has been considered; however, the extraordinary toxicity of the drug inoculated systemically has greatly limited its application. Since the most serious toxic consequences of the drug are related to its systemic delivery, alternative strategies have been developed. Local delivery of the cytokine has been successfully used in some circumstances, and this form of delivery does not result in the life-threatening complications that limit systemic use. Liposome encapsulated IL-2 represents a mechanism to accentuate local delivery by causing a depot effect. Finally, the use of IL-2 has been predicated on the conception of the cytokine as an absolute monomer. Nevertheless, IL-2 spontaneously forms noncovalent and covalent self-associations. Because covalent dimers have been shown to initiate differential signalling in target cells, it is necessary to account for this property in devising and evaluating therapeutic protocols; moreover, it seems possible to use this property for modifying and regulating the therapeutic response.     

IL-13 receptor-targeted cytotoxin cancer therapy leads to complete eradication of tumors with the aid of phagocytic cells in nude mice model of human cancer

Tumor-directed therapeutic approaches require unique or overexpressed specific Ag or receptor as a target to achieve selective tumor killing. However, heterogeneous expression of these targets on tumor cells limits the efficacy of this form of therapy. In this study, we forced abundant expression of IL-13Ralpha2 chain by plasmid-mediated gene transfer in head and neck, as well as prostate tumors to provide a potential target. This was followed by successfully treating xenograft tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR). Although we did not observe an indirect cytotoxic bystander effect conveyed to nontransduced tumor cells in vitro, our approach in vivo led to a complete regression of established tumors transfected with IL-13Ralpha2 chain in most animals. We found that the tumor eradication was achieved in part by infiltration of macrophages and NK cells, assessed by immunohistochemistry. Moreover, head and neck tumors xenografted in macrophage-depleted nude mice were less sensitive to the antitumor effect of IL-13 cytotoxin. Because we did not observe vector-related toxicity in any vital organs, our novel combination strategy of gene transfer of IL-13Ralpha2 chain and receptor-directed cytotoxin therapy may be a useful approach for the treatment of localized cancer.     

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.     

Immunologic approaches to the treatment of human cancer based on a guinea pig model

Summary Local immunotherapy is a form of cancer treatment where exogenous antigen is introduced into the area of the tumor. Under favorable circumstances, the perfused tumor regresses, systemic tumor-specific transplantation immunity is augmented, and distant microscopic metastases regress. Successful local immunotherapy requires an immunologically competent host, small tumor burden, and tumor located usually in the skin. A wide variety of biologic agents are capable of promoting local immunotherapy. BCG has been most widely studied. The antitumor activity of two different preparations of the Tice substrain of BCG were compared. No significant differences in antitumor activity were found. Alternative approaches to intralesional injection were sought. Intradermal injection of BCG adjacent to dermal tumors, prior to surgery, led to eradication of axillary metastases and to the development of tumor-specific transplantation immunity.Successful local BCG immunotherapy is a by-product of the host response to BCG infection. Involved are lymphocytes specifically sensitized to mycobacterial antigens, lymphocyte mediators, and macrophages which develop the capacity to kill tumor cells. Tumor-cell killing may be mediated by exocytosis of macrophage lysosomes into tumor cells. Complete and permanent tumor eradication probably requires the development of tumor-specific transplantation immunity mediated by sensitized lymphocytes. Local infection of the tumor may augment the development of this tumor-specific immunity.     

Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice.

The systemic administration of human rIL-6 to mice resulted in the regression of established, 3-day pulmonary micrometastases from two weakly immunogenic tumors, but not from a nonimmunogenic tumor, in the absence of observable toxicity. Although IL-6 alone failed to have a significant therapeutic impact on advanced, 10-day pulmonary macrometastases from weakly immunogenic tumors, substantial cure rates of mice could be achieved when this cytokine was combined with cyclophosphamide. Histologic analysis of the lungs of mice receiving IL-6 revealed infiltration with lymphoid cells during the regression of pulmonary nodules from a weakly immunogenic tumor. IL-6-mediated tumor regression could be abrogated after selective in vivo depletion of either CD4 or CD8 T cell subsets by the systemic administration of specific mAb. In vivo generation of tumor-specific CTL, but not of lymphokine-activated killer cells, was detected in the lungs of IL-6-treated mice during regression of pulmonary metastases. Collectively, these findings demonstrate a role for IL-6 in the treatment of established solid tumors that have the capacity to elicit T cell responses in the host. Differences in host cellular mechanisms involved in tumor regression mediated by immunotherapy using IL-6 vs IL-2 are discussed.     

Pre-existing tumor-sensitized T cells are essential for eradication of established tumors by IL-12 and cyclophosphamide plus IL-12.

The antitumor immune response activated by IL-12, especially by a combination of cyclophosphamide and IL-12 (Cy+IL-12), is clinically significant in certain experimental tumor models, in that a number of well-established (10-20 mm in diameter) s.c. tumors are completely eradicated. Furthermore, Cy+IL-12 treatment is also able to eradicate well-established grossly detectable experimental lung metastases and advanced ascites tumors. Despite the dramatic antitumor effects seen in some tumor models, Cy+IL-12 fails to induce regression of other established tumors. Characterization of tumor immunogenicity shows that all tumors responding to IL-12 and Cy+IL-12 treatments are immunogenic tumors, in that an antitumor immune response is detectable in tumor-bearing hosts upon tumor establishment. In contrast, none of the nonimmunogenic tumor responds to IL-12 and Cy+IL-12 treatments. Analysis of cellular requirements for successful tumor rejection through an adoptive cell transfer approach reveals that the presence of tumor-sensitized, but not naive, T cells is essential for tumor rejection by IL-12 and Cy+IL-12. Transfer of these tumor-sensitized T cells must be conducted before, but not after, IL-12 treatment in order for tumor rejection to occur. The requirement of sensitized T cells is also tumor specific. In mice bearing immunogenic tumors, the presence of pre-existing tumor-sensitized T cells is demonstrated by adoptive cell transfer experiments using purified spleen T cells from these mice. Results from our study show that Cy+IL-12-based immunotherapy of cancer may be highly effective and that pre-existing tumor-sensitized T cells are essential for the success of the therapy.     

Tumor specific phage particles promote tumor regression in a mouse melanoma model

Within cancer research, phage display libraries have been widely used for the identification of tumor targeting peptides and antibodies. Additionally, phages are known to be highly immunogenic; therefore we evaluated the immunotherapeutic potential of tumor specific phages to treat established solid tumors in a mouse model of melanoma. We developed two tumor specific phages, one derived from a peptide phage display library and one Fab expressing phage with known specificity, for the treatment of mice bearing palpable B16-F10 or B16/A2K^b tumors. Therapy in B16-F10 tumor bearing mice with tumor specific phages was superior to treatment with non-tumor specific phages and lead to delayed tumor growth and increased survival. In B16/A2K^b tumor bearing mice, therapy with tumor specific phages resulted in complete tumor regression and long-term survival in 50% of the mice. Histological analysis of tumors undergoing treatment with tumor specific phages revealed that phage administration induced a massive infiltration of polymorphonuclear neutrophils. Furthermore, phages induced secretion of IL-12 (p70) and IFN-γ as measured in mouse splenocyte culture supernatants. These results demonstrate a novel, immunotherapeutic cancer treatment showing that tumor specific phages can promote regression of established tumors by recruitment of inflammatory cells and induction of Th1 cytokines.     

Treatment of stage III–IV nasopharyngeal carcinomas by external beam irradiation and local low doses of IL-2

The therapeutic effect of intratumoural application of Interleukin-2 (IL-2) was studied in patients with stage III–IV nasopharyngeal carcinoma (NPC) that received radiotherapy. Patients with stage III–IV NPC receiving a standard treatment of 7,000 cGy external beam irradiation have a mean disease-free survival of about 1.5 years. In this paper, we describe ten of these patients who were treated with additional peritumoural and intratumoural injections with 3×10⁴ U IL-2 on 5 days in weeks 2, 4, and 6 of the 7-weeks irradiation period. This combined treatment group was compared with a historical group of patients treated with standard irradiation alone. Local IL-2 therapy showed a marked clinical and statistical significant improvement of disease-free survival. After 5 years, 63% of the IL-2 treated patients were disease-free versus 8% of the control patients. These results suggest that the therapeutic results of radiotherapy can be significantly improved by combining it with local IL-2 treatment. To our knowledge, this is the first clinical report showing that local IL-2 therapy is effective against an infiltrative and locally metastasizing tumour in human patients.This study was presented as a poster at the Dutch Society for Immunology, December 2003, Noordwijkerhout, The Netherlands.     

IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice

Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of approximately 10-25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.     

DNA alkylating therapy induces tumor regression through an HMGB1-mediated activation of innate immunity

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. We have previously shown in tumor xenografts that DNA alkylating agents induce sporadic cell necrosis and regression of apoptosis-deficient tumors. Sporadic tumor cell necrosis is associated with extracellular release of cellular content such as the high mobility group box 1 (HMGB1) protein and subsequent recruitment of innate immune cells into the tumor tissue. It remained unclear whether HMGB1 and the activation of innate immunity played a role in tumor response to chemotherapy. In this study, we show that whereas DNA alkylating therapy leads to a complete tumor regression in an athymic mouse tumor xenograft model, it fails to do so in tumors deficient in HMGB1. The HMGB1-deficient tumors have an impaired ability to recruit innate immune cells including macrophages, neutrophils, and NK cells into the treated tumor tissue. Cytokine array analysis reveals that whereas DNA alkylating treatment leads to suppression of protumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using depleting Abs leads to a failure in tumor regression. Taken together, these results indicate that HMGB1 plays an essential role in activation of innate immunity and tumor clearance in response to DNA alkylating agents.     

Mechanical Disruption of Tumors by Iron Particles and Magnetic Field Application Results in Increased Anti-Tumor Immune Responses

The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor in situ. This was accomplished by intratumor injection of magneto-rheological fluid (MRF) consisting of iron microparticles, in Balb/c mice bearing orthotopic 4T1 breast cancer, followed by local application of a magnetic field resulting in immediate coalescence of the particles, tumor cell death, slower growth of primary tumors as well as decreased tumor progression in distant sites and metastatic spread. This treatment was associated with increased activation of DCs in the draining lymph nodes and recruitment of both DCs and CD8(+)T cells to the tumor. The particles remained within the tumor and no toxicities were observed. The immune induction observed was significantly greater compared to cryoablation. Further anti-tumor effects were observed when MRF/magnet therapy was combined with systemic low dose immunotherapy. Thus, mechanical disruption of the primary tumor with MRF/magnetic field application represents a novel means to induce systemic immune activation in cancer.     

Reversing tumor immune suppression with intratumoral IL-12: activation of tumor-associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8+ T effectors

A single intratumoral injection of IL-12 and GM-CSF-loaded slow-release microspheres induces T cell-dependent eradication of established primary and metastatic tumors in a murine lung tumor model. To determine how the delivery of cytokines directly to the microenvironment of a tumor nodule induces local and systemic antitumor T cell activity, we characterized therapy-induced phenotypic and functional changes in tumor-infiltrating T cell populations. Analysis of pretherapy tumors demonstrated that advanced primary tumors were infiltrated by CD4+ and CD8+ T cells with an effector/memory phenotype and CD4+CD25+Foxp3+ T suppressor cells. Tumor-associated effector memory CD8+ T cells displayed impaired cytotoxic function, whereas CD4+CD25+Foxp3+ cells effectively inhibited T cell proliferation demonstrating functional integrity. IL-12/GM-CSF treatment promoted a rapid up-regulation of CD43 and CD69 on CD8+ effector/memory T cells, augmented their ability to produce IFN-gamma, and restored granzyme B expression. Importantly, treatment also induced a concomitant and progressive loss of T suppressors from the tumor. Further analysis established that activation of pre-existing effector memory T cells was short-lived and that both the effector/memory and the suppressor T cells became apoptotic within 4 days of treatment. Apoptotic death of pre-existing effector/memory and suppressor T cells was followed by infiltration of the tumor with activated, nonapoptotic CD8+ effector T lymphocytes on day 7 posttherapy. Both CD8+ T cell activation and T suppressor cell purge were mediated primarily by IL-12 and required IFN-gamma. This study provides important insight into how local IL-12 therapy alters the immunosuppressive tumor milieu to one that is immunologically active, ultimately resulting in tumor regression.