Local Tumor Irradiation Research Articles

SU-E-CAMPUS-J-01: Monitoring of 19F-Labeled Immune Cell Trafficking During Radiotherapy Using MRI

Purpose: Given the cross talk between cancer and the host immune system, there is a compelling need to monitor immunogenic responses in vivo during radiotherapy treatment. Over the last year our laboratory has been working toward (1) developing the nonradioactive isotope fluorine‐19 (19F), as a novel, clinically applicable tracking agent for immune cells using magnetic resonance imaging (MRI), and (2.) investigating if local irradiation of tumors augments expression of danger signals that recruit and activates immune cells using 19F MRI. Methods: Using a high‐resolution Varian 4.7 Tesla small animal MRI scanner several we have developed several imaging protocols to optimize a 19F MR imaging system that uses either a dedicated surface or volumetric fluorine coil. Given these protocols, human natural killer (NK) cells labeled with 19F‐PFPE were injected subcutaneously in the footpad of a mouse and scanned ∼1, 24, 48, 72, 120 hours after injection. Results: Initial experiments have demonstrated excellent SNR uniformity provided by the volume coil. Excellent signal linearity was demonstrated by acquiring images of phantom vials with increasing concentrations of 19F‐PFPE. We have also demonstrated the ability to monitor NK cells trafficking throughout the lymphatic system of a mouse. Conclusion: We are currently investigating the immunogenic response following irradiation of subcutaneous tumors in mice. The ability to monitor immune cell trafficking during therapy will help optimize the synergistic effects of radiation cytotoxicity and the host immune system. UW Carbone Cancer Center Pilot Grant

Effects of Tumor Irradiation on Host T-Regulatory Cells and Systemic Immunity in the Context of Adoptive T-Cell Therapy in Mice

In this study, we used a murine D5 melanoma model to study the effects of local tumor irradiation on the therapeutic efficacy of adoptive T-cell therapy. Tumor irradiation was delivered in 5 daily fractions (8.5 Gy) to subcutaneous tumors on days 7-11 after tumor inoculation. After the last radiation dose, activated tumor-draining lymph node cells were transferred intravenously followed by intraperitoneal IL-2 administration. Tumor irradiation alone had no significant effect on tumor growth; however, it synergistically enhanced the therapeutic efficacy of T-cell therapy. For 2 days after tumor irradiation there was a significant reduction in T cells, B cells, and CD11c(+) dendritic cells in both the tumor microenvironment and the systemic lymphoid compartments. By days 4-6 after irradiation, the relative reduction in the number of Treg cells within the tumor and the systemic compartments was greater than the reduction in conventional T cells. Furthermore, the suppressive function of the Tregs was significantly impaired in irradiated versus untreated mice. Using effector T cells derived from congenic mice, we found that local tumor irradiation resulted in increased proliferation of donor T cells within the tumor and the systemic lymphoid compartments. Radiation was associated with increased expression of the effector cytokines IFN-γ and TNF-α by donor and host CD4(+) and CD8(+) T cells. Altogether, our data indicate that local tumor irradiation has a distinct modulatory effect on Tregs and can enhance systemic antitumor immunity associated with adoptive T-cell therapy.

Abscopal Regression of Antigen Disparate Tumors by Antigen Cascade After Systemic Tumor Vaccination in Combination with Local Tumor Radiation

Radiation is a primary modality in cancer treatment. Radiation can also reduce tumor growth outside the treatment field, often referred to as the abscopal effect. The mechanisms and therapeutic potential of the abscopal effect have not been fully elucidated. We evaluated the role of vaccination directed against a tumor-associated antigen (TAA) in the induction and amplification of radiation induced abscopal effects. Active-specific immunotherapy with a TAA-specific vaccine regimen was used to induce and potentiate T-cell responses against carcinoembryonic antigen (CEA) in combination with local irradiation of subcutaneous tumors. We examined the potential synergy of a poxvirus-based CEA vaccine regimen in CEA-transgenic (Tg) mice in combination with either external beam radiation or brachytherapy of local tumors. The induction of CD8(+) T cells specific for multiple TAAs not encoded by the vaccine was observed after the combination therapy. In two tumor models, the antigen cascade responses induced by vaccine and local irradiation mediated the regression of antigen negative metastases at distal subcutaneous or pulmonary sites. Clinically, local control of the primary tumor is necessary and can sometimes prevent metastases; however, irradiation generally fails to control preexisting metastases. These studies suggest that by coupling tumor irradiation with immunotherapy, the abscopal effect can transcend from anecdotal observation to a defined mechanism that can be exploited for the treatment of systemic disease.

Role of T lymphocytes in tumor response to radiotherapy

Over thirty years ago, Helen Stone and colleagues compared the effects of local tumor irradiation in immunocompetent and T cell deficient mice, providing the first evidence that tumor response to radiotherapy is impaired in the absence of a normal T cell repertoire. In the following three decades there has been an exponential growth in understanding T cells and the complex molecular mechanisms that regulate their activation, migration to tumors and effector functions. We now also know that tumor progression is intrinsically linked to the development of multiple immunosuppressive mechanisms that allow cancer cells to escape immune control. Recent evidence about the role of T cells in determining the prognosis and outcome of patients at any clinical stages of cancer has been instrumental in re-directing the concept of immunosurveillance and immunoediting from the realm of preclinical models to the reality of clinical observations. Importantly, cell death induced by standard anti-cancer therapies like chemotherapy and radiation has been demonstrated to involve the immune system and, in certain specific settings, enable a specific immune response. It is, therefore, not surprising that the last few years have seen an increase in investigations exploring how to harness the ability of radiation to induce anti-tumor immune responses. We will review here the experimental evidence that anti-tumor T cells are key players in tumor control achieved by radiotherapy. The effects of radiation on the tumor that have been shown to enhance the priming and effector phases of anti-tumor immunity will be discussed. Finally, we will highlight promising combinations of immune response modifiers that enhance T cell function with radiotherapy which are being tested in the clinic.

Abstract 2669: Radiosensitizing effect of intratumoral mIL-12 electrogene therapy in murine sarcoma

Abstract Due to the limited success rate of conventional therapeutic approaches to soft tissue sarcomas, new treatment modalities, such as gene therapy, are being evaluated as adjuvant therapies to conventional methods. Electrogene therapy with interleukin-12 (IL-12) has been proved very effective due to the induction of adaptive immunity and innate resistance as well as anti-angiogenic action. The aim of our study was to evaluate the radiosensitizing effect of single and repetitive intratumoral (i.t.) mIL-12 electrogene therapy (mIL-12 EGT) in murine sarcoma. Solid SA-1 fibrosarcoma tumors were induced in the back of A/J mice by subcutaneous injection of tumor cells. The first therapeutic approach consisted of a single i.t. mIL-12 EGT (20 µg of plasmid DNA), followed by a single dose (10 Gy) or graded doses (2.5 – 70 Gy) of local tumor irradiation 24 h later. In the second therapeutic approach, tumor irradiation was followed by two additional i.t. mIL-12 EGT, on the third and fifth day. The tumor growth delay, complete response rates and local tumor control assay (TCD50 assay) were used as a measure of antitumor effectiveness of the therapies and skin damage in the irradiation field was also scored. Tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured using ELISA. Therapies with i.t. mIL-12 EGT demonstrated good antitumor effects in murine SA-1 sarcoma tumors and also a radiosensitizing effect when combined with irradiation. Both antitumor and radiosensitizing effects were more pronounced when using repetitive rather than single i.t. mIL-12 EGT. Single i.t. mIL-12 EGT resulted in 7% complete responses and, when combined with single dose irradiation, it produced a radiosensitizing effect resulting in 21% complete responses. In both cases we also detected significantly elevated levels of cytokines mIL-12 and mIFNγ in tumors but no significant increase of both cytokine levels in the serum. Repetitive i.t. mIL-12 EGT alone resulted in 75% complete responses and, when combined with single-dose irradiation, produced the most pronounced potentiation of the radiation response resulting in 100% complete responses of the treated tumors. The TCD50 value was 30 Gy for irradiation alone, and 13.8 Gy for i.t. mIL-12 EGT combined with irradiation. Therefore, the dose-modifying factor of a single i.t. mIL-12 EGT in murine SA-1 sarcoma was 2.17. At the same level of skin damage, dry desquamation, in combined therapy a 44% higher probability of local tumor control than with irradiation alone was observed. The results of our study demonstrate that i.t. mIL-12 EGT is an efficient treatment modality for treatment of soft tissue sarcoma alone and as adjuvant therapy to single dose irradiation. This is due to its pronounced radiosensitizing effect caused by boosting the immune response of the organism. The dose-modifying factor of a single i.t. mIL-12 EGT in murine SA-1 sarcoma was 2.17. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2669. doi:10.1158/1538-7445.AM2011-2669

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an "all in vivo" strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful "all in vivo" vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.

Abstract 2282: Inhibition of vasculogenesis, but not angiogenesis, reduces tumor recurrence following irradiation

Abstract Tumor blood vessels can derive from two sources: by angiogenesis, or the sprouting of endothelial cells from nearby blood vessels, and by vasculogenesis, which is produced by circulating cells. We have proposed that the radiation doses given in radiotherapy, will severely restrict local angiogenesis and thereby force tumor regrowth to rely on vasculogenesis. This involves the recruitment of proangiogenic circulating cells many of which are derived from the bone marrow. To investigate this hypothesis, we used the subcutaneously implanted FaDu human head and neck tumor and an orthotopic brain tumor using human U251 GBM cells. We found that local tumor irradiation induced the influx of bone marrow derived cells (BMDCs) in a dose-dependent manner into both tumor models with most of this increase reflecting influx of CD11b+ myelomonocytes. We postulated that this influx of BMDCs was stimulated by increased tumor hypoxia and HIF-1 levels caused by radiation damage to the tumor vasculature. To determine HIF-1 activity in real-time in our brain implanted U251 GBM, we stably expressed the HIF-1 reporter construct 5HRE-luc in U251 cells and showed that HIF-1 activity increased rapidly starting at about two weeks following 15 Gy, in parallel to the increase in tumor hypoxia that we observed at this time. To test our hypothesis that the increased HIF-1 levels were responsible for the increased influx of CD11b+ cells into the tumors, we used the HIF-1 inhibitor NSC-134754. When this was given daily for 2 weeks, starting immediately following irradiation, the increased tumor levels of CD11b+ monocytes observed after 15 Gy was abrogated. This treatment also prevented the regrowth of the irradiated tumors following irradiation. As a further test of our hypothesis we determined the effect of inhibiting the interaction of SDF-1 with its receptor CXCR4 using the clinically approved drug AMD3100, which we infused starting immediately following irradiation. This had no effect on the growth of unirradiated tumors in the brain but completely inhibited the recurrence of the irradiated tumors following either a single dose of 15 Gy or the more clinically relevant scheme of 5 daily doses of 2 Gy. Inhibition of angiogenesis with DC101 increased tumor growth delay but did not prevent tumor recurrences. We also showed that the recurrence of FaDu tumors locally irradiated with 20 Gy was prevented using a monoclonal neutralizing antibody against CD11b+ monocytes. This treatment did not increase the radiosensitivity of normal skin thereby providing a therapeutic gain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2282.

Retroperitoneal primitive neuroectodermal tumour (PNET). A case report and review of the literature

Purpose We report a clinical case and present a brief review of the literature of peripheral primitive neuroectodermal tumour (PNET) as a rare disease. We discuss the difficult clinical and pathological diagnosis and the multidisciplinary approach to treatment of PNET. We debate radiosensitivity of extracranial recurrent retroperitoneal PNET. Methods and materials External beam radiation therapy was applied for a non-resectable local recurrence of retroperitoneal PNET in a 74-year-old woman. There were no distant metastases and our patient has refused chemotherapy. Results Local tumour control (LTC) was achieved after administration of a total dose of 60 Gy in 30 fractions by external beam 60 Cobalt radiotherapy. Conclusions PNET is an aggressive malignant tumour infiltrating lymphatics and metastasizing haematogenously. It requires a multimodality treatment. Late local recurrence of extracranial retroperitoneal PNET has shown high radiosensitivity, so local tumour irradiation could be a radical treatment even in non-resectable cases.

Controlled systemic release of interleukin‐12 after gene electrotransfer to muscle for cancer gene therapy alone or in combination with ionizing radiation in murine sarcomas

The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer. Gene electrotransfer of the plasmid pORF-mIL12 was performed into the tibialis cranialis in A/J and C57BL/6 mice. Systemic release of the IL-12 was monitored in the serum of mice after carrying out two sets of intramuscular IL-12 gene electrotransfer of two different doses of plasmid DNA. The antitumor effectiveness of IL-12 gene electrotransfer alone or in combination with local tumor or lung irradiation with X-rays, was evaluated on subcutaneous SA-1 and LPB tumors, as well as on lung metastases. A synergistic antitumor effect of intramuscular gene electrotransfer combined with local tumor irradiation was observed as a result of the systemic distribution of IL-12. The gene electrotransfer resulted in up to 28% of complete responses of tumors. In combination with local tumor irradiation, the curability was increased by up to 100%. The same effect was observed for lung metastases, where a potentiating factor of 1.3-fold was determined. The amount of circulating IL-12 was controlled by the number of repeats of gene electrotransfer and by the amount of the injected plasmid. The present study demonstrates the feasibility of treatment by IL-12 gene electrotransfer combined with local tumor or lung metastases irradiation on sarcoma tumors for translation into the clinical setting.

Heat Shock Protein 27 as a New Therapeutic Target for Radiation Sensitization of Head and Neck Squamous Cell Carcinoma

In a wide range of human cancers, increased levels of heat shock protein 27 (Hsp27) are closely associated with tumorigenesis, metastasis, resistance to anticancer therapeutics, and thus poor prognosis. In this study, we evaluate the radiosensitizing effects of Hsp27 gene silencing using OGX-427, a second-generation antisense oligonucleotide (ASO), on the radioresistant head and neck squamous cell carcinoma (HNSCC) SQ20B cells. In vitro, the downregulation of Hsp27 significantly enhanced radiation-induced apoptotic and clonogenic death, and promoted Akt inactivation. In vivo, combining OGX-427 with local tumor irradiation (5 x 2 Gy) led to a significant regression of SQ20B tumors related to a high rate of apoptosis and decreased levels of glutathione antioxidant defenses. Increasing the total radiation dose (15 x 2 Gy) significantly amplified the radiosensitizing effect of OGX-427. Treatment of tumors with OGX-427 plus radiation resulted in a decrease in angiogenesis associated with a reduced activation of the Akt pathway. Furthermore, the combined treatment enhanced the survival of SQ20B-bearing mice and showed no signs of acute and delayed toxicity. Our findings demonstrate for the first time that Hsp27 knockdown enhances the cytotoxic effects of radiotherapy in vivo and provide preclinical proof of principle for clinical trials using Hsp27 antisense technology in the treatment of patients with HNSCC radioresistant cancers.

Radiotherapy Combined With Intratumoral Dendritic Cell Vaccination Enhances the Therapeutic Efficacy of Adoptive T-cell Transfer

Treatment of C57BL/6 mice with cyclophosphamide (100 mg/kg) and fludarabine (200 mg/kg) induced nonmyeloablative lymphodepletion without inhibiting D5 melanoma tumor growth. Using this model, we found that induction of lymphopenia before adoptive transfer of ex vivo anti-CD3/CD28 activated and interleukin-2 expanded D5-G6 tumor draining lymph node cells enhanced the antitumor efficacy of the infused cells in both pulmonary metastases and subcutaneous D5 bearing mice. However, induction of lymphopenia did not promote intratumoral or extratumoral proliferation or accumulation of the infused cells. We have previously shown that radiotherapy enhances the therapeutic efficacy of intratumoral unpulsed dendritic cell vaccination in subcutaneous murine tumor models by augmenting the induction of antitumor cellular immune responses. Here, we confirmed this finding in a murine metastatic melanoma liver tumor model. Furthermore, local tumor irradiation combined with intratumoral dendritic cell administration significantly enhanced the therapeutic efficacy of tumor-reactive T cell adoptive transfer in this lymphodepleted liver tumor model. This was evident by reduced liver tumor size, decreased incidence of spontaneous intra-abdominal metastasis, and prolonged survival, resulting in 46% of mice cured. This enhanced antitumor activity was associated with a selective increase in proliferation, accumulation, and function of CD4+ rather than CD8+ infused cells. This multimodality regimen may have translational applications for the treatment of human cancers.

Development of an intramuscular xenograft model of canine osteosarcoma in mice for evaluation of the effects of radiation therapy

To develop an IM xenograft model of canine osteosarcoma in mice for the purpose of evaluating effects of radiation therapy on tumors. 27 athymic nude mice. Mice were randomly assigned to 1 of 3 groups of 9 mice each: no treatment (control group), radiation at 10 Gy, or radiation at 15 Gy. Each mouse received 5 x 10(5) highly metastasizing parent osteosarcoma cells injected into the left gastrocnemius muscle. Maximum tumor diameter was determined with a metric circles template to generate a tumor growth curve. Conscious mice were restrained in customized plastic jigs allowing local tumor irradiation. The behavior and development of the tumor xenograft were assessed via evaluations of the interval required for tumor-bearing limbs to reach diameters of 8 and 13 mm, extent of tumor vasculature, histomorphology of tumors, degree of tumor necrosis, and existence of pulmonary metastasis and clinical disease in affected mice. Tumor-bearing limbs grew to a diameter of 8 mm (0.2-g tumor mass) in a mean +/- SEM interval of 7.0 +/- 0.2 days in all mice. Interval to grow from 8 to 13 mm was significantly prolonged for both radiation therapy groups, compared with that of the control group. Histologic evaluation revealed the induced tumors were highly vascular and had characteristics consistent with those of osteosarcoma. Pulmonary metastasis was not detected, and there was no significant difference in percentage of tumor necrosis between groups. A reliable, repeatable, and easily produced IM xenograft model was developed for in vivo assessment of canine osteosarcoma.

Mechanisms Involved in Radiation Enhancement of Intratumoral Dendritic Cell Therapy

We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4 or CD8 effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy.

Radiation-Induced IFN-γ Production within the Tumor Microenvironment Influences Antitumor Immunity

Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-gamma influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-gamma-deficient (IFN-gamma-/-) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-gamma-/- mice. Levels of the IFN-gamma-inducible chemokines MIG and IFN-gamma-inducible protein 10 were decreased in irradiated tumors from IFN-gamma-/- mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-gamma produced after irradiation. The role of IFN-gamma signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-gamma receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-gamma acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-gamma is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.

Analysis of Changes in Gene Expression Induced by Fusion of Dendritic Cells and Melanoma Cells.

Heterokaryons formed through electrofusion of Dendritic cells (DC) with tumor cells are highly effective vaccines for cancer immunotherapy. We have demonstrated in transplantable murine tumor models that active immunotherapy using a combination of DC-tumor fusion vaccine, local tumor irradiation, and adjuvant anti-CD134 mAb can mediate regression of advanced intracranial tumors. Due to their potency as tumor vaccines, and their unique physical attributes, consisting of multinucleated cells arising from partners with disparate genetic programs, we compared the gene expression profile of DC-melanoma fusion cells with DC-DC fusion cells and tumor-tumor fusion cells. Human DC were prepared by culturing PBMC obtained by leukapheresis for 7 days in medium supplemented with GM-CSF and IL-4 with addition of PGE-2 and TNF-α for the final 48 hrs. The human melanoma cell line 888 was irradiated (50Gy) and labeled with CFSE prior to use. DC and tumor cells were mixed at a 1:1 ratio at 2 × 107 cells/ml in electrofusion buffer [5% dextrose, 0.5 mM Mg(CH3COO)2, 0.1 mM Ca(CH3COO)2 pH 7.2]. Cells were subjected to dielectrophoresis using an ac current of 140V/cm × 10s to align the cells in a chain-like configuration between the electrodes, then pulsed with a 1500 V/cm × 25 μs to induce transient membrane breakdown. Heterokarons were purified through a combination of steps involving differential adherence and magnetic bead separation based on CD80 and CD86 expression resulting in 95–97% purity. Total RNA was isolated from DC-tumor fusion heterokaryons, or DC-DC fusions, or tumor-tumor fusion for 3 independent experiments and was analyzed by Affymetrix gene array. Bioinformatics analysis methods, including a relational database, were developed to identify and functionally classify genes with a robust change in expression. The criteria applied were:all 9 pairwise comparisons had to exhibit a Log ratio >1 increase or decrease,statistical significance had to be at least p<0.01, andsignals had to surpass a threshold expression of 500 units.Hierarchical cluster analysis using the entire set of transcripts (n=22,277) separated the samples into 3 distinct clusters with the DC-tumor cluster being closed to the tumor-tumor cells than the DC-DC cells. The DC-tumor fusion cells also displayed a higher Spearman rank correlation coefficient in pair-wise comparisons with the tumor-tumor samples. A relatively small number of genes (n=137) were expressed in the DC-tumor fusion cells but not in the DC-DC control. Among these were several well-characterized melanoma tumor antigens. Similarly, a small number of genes (n=116), primarily involved in antigen processing and immune response, were contributed to the DC-tumor fusion cell by the DC partner. These data support the hypothesis that ongoing expression of tumor antigens and immune function genes continue to be expressed after heterokaryons formation. More interestingly, we identified 83 DC genes that were specifically downregulated in DC-tumor fusion cells. Moreover, 28 genes were uniquely expressed at a significant level in the DC-tumor fusion cells relative to either the DC or tumor whereas only 1 gene was significantly repressed. Uniquely expressed genes are involved in transcriptional regulation, development, growth factor receptors, signal transduction, cell adhesion, and cytoskeletal organization and in physiologic membrane fusion processes. This suggests that transcription in fusion heterokaryons can be cross-regulated by transcription factors uniquely expressed in each of the component cells.

Cancer gene therapy: Combination with radiation therapy and the role of bystander cell killing in the anti-tumor effect

Current anti-cancer modalities such as surgery, chemo- and radiation therapies have only limited success in cancer treatment. Gene therapy is a promising new tool to improve outcomes. In this review, first we summarize the various strategies to kill tumor cells, and then focus on the bystander effect of gene therapy. A variety of strategies, such as gene-directed enzyme pro-drug therapy, activation of an anti-tumor immune attack, application of replication-competent and oncolytic viral vectors, tumor-specific as well as radiation- and hypoxiainduced gene expression, might be applied to target tumor cells. We put special emphasis on the combination of these approaches with local tumor irradiation. Using the available vector systems, only a small portion of cancer cells contains the therapeutic genes under clinical situations. However, cells directly targeted by gene therapy will transfer death signals to neighboring cancer cells. This bystander cell killing improves the efficiency of cancer gene therapy. Death signals are delivered by cell-to-cell communication through gap junction intercellular contacts, release of toxic metabolites into the neighborhood or to larger distances, phagocytosis of apoptotic bodies, and the activation of the immune system. Bystander cell killing can be enhanced by the introduction of gap junction proteins into cells, by further activating the immune system with immune-stimulatory molecules, or by introducing genes that help the transfer of cytotoxic genes and/or metabolites into bystander cells. In conclusion, although bystander cell killing can improve therapeutic effects, there should be additional developments in cancer gene therapy for a more efficient clinical application.

Detailed characterization of the mouse glioma 261 tumor model for experimental glioblastoma therapy

Mouse glioma 261 (Gl261) cells are used frequently in experimental glioblastoma therapy; however, no detailed description of the Gl261 tumor model is available. Here we present that Gl261 cells carry point mutations in the K-ras and p53 genes. Basal major histocompatibility complex (MHC)I, but not MHCII, expression was detected in Gl261 cells. The introduction of interferon-gamma-encoding genes increased expression of both MHCI and MHCII. A low amount of B7-1 and B7-2 RNA was detected in wild-type cells, but cytokine production did not change expression levels. Gl261 cells were transduced efficiently by adenoviral vectors; the infectivity of retroviral vectors was limited. Low numbers of transplanted Gl261 cells formed both subcutaneous and intracranial tumors in C57BL/6 mice. The cells were moderately immunogenic: prevaccination of mice with irradiated tumor cells 7 days before intracranial tumor challenge prevented tumor formation in approximately 90% of mice. When vaccination was carried out on the day or 3 days after tumor challenge, no surviving animals could be found. In vitro-growing cells were radiosensitive: less than 2 Gy was required to achieve 50% cell mortality. Local tumor irradiation with 4 Gy X-rays in brain tumor-bearing mice slowed down tumor progression, but none of the mice were cured off the tumor. In conclusion, the Gl261 brain tumor model might be efficiently used to study the antitumor effects of various therapeutic modalities, but the moderate immunogenicity of the cells should be considered.

Ultrasound-Enhanced Chemotherapy of Drug-Resistant Breast Cancer Tumors by Micellar-Encapsulated Paclitaxel

Currently, delivery of the poorly water-soluble chemotherapeutic agent paclitaxel is associated with a substantial array of systemic toxicities and results in low-efficiency tumor treatment. A novel on-demand delivery system based on paclitaxel encapsulated in polymeric micelles in conjunction with triggered release of the drug by local ultrasonic irradiation of the tumor was evaluated in vitro and in vivo using a drug-resistant MCF7/ADmt breast cancer human cell line. The effects of local ultrasonic tumor irradiation on cellular proliferation and intracellular drug uptake were compared for a developmental micellar paclitaxel formulation (SYP-PM) and a currently available clinical intravenous formulation of paclitaxel. Without ultrasound, the uptake of paclitaxel from the micellar formulation was significantly lower than that from the clinical formulation, which is advantageous for preventing unwanted drug interactions with healthy tissues in vivo. When micellar encapsulation was combined with ultrasonically triggered release, drug uptake from micellar paclitaxel was increased more than 20-fold and cellular proliferation was inhibited by nearly 90%. Without ultrasound, the clinical formulation of paclitaxel and SYP-PM manifested low efficacy in vivo, whereas injections of SYP-PM combined with ultrasound resulted in complete tumor resolution. The ability of micellar-encapsulated paclitaxel to exert a significant cytotoxic effect only when subjected to ultrasound proves promising for the development of a tumor-targeted ultrasound-enhanced paclitaxel delivery system for clinical application. This treatment modality could be successfully used for the therapy of both drug-sensitive and drug-resistant tumors. The major advantages of a micellar formulation of paclitaxel combined with local tumor sonication are the aqueous base of the drug formulation, reduced systemic toxicity, potential for tumor targeting, and on-demand delivery of drug to tumor cells.

Targeting Toll-like Receptor 9 with CpG Oligodeoxynucleotides Enhances Tumor Response to Fractionated Radiotherapy

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs detected by Toll-like receptor 9 of dendritic cells and B cells have potent immunomodulatory effects. CpG oligodeoxynucleotides induce cytokines, activate natural killer cells, and elicit T-cell responses leading to antitumor effects, including improved efficacy of chemotherapeutic agents and, as we reported recently, synergy between CpG oligodeoxynucleotide 1826 and single-dose radiotherapy of an immunogenic mouse fibrosarcoma. The present study extends this finding to the fractionated radiotherapy of the fibrosarcoma tumor and assesses the ability of CpG oligodeoxynucleotide 1826 to increase the radioresponse of a tumor (nonimmunogenic fibrosarcoma). The experiments used a murine immunogenic fibrosarcoma tumor, fibrosarcoma growing in the leg of mice, and response to radiotherapy was assessed by tumor growth delay and tumor cure rate (TCD50, radiation dose yielding 50% tumor cure). Multiple s.c. peritumoral or i.t. administrations of CpG oligodeoxynucleotide 1826 at a dose of 100 microg per mouse were given when established tumors were 6 mm in diameter. Local tumor irradiation was initiated when tumors grew to 8 mm in diameter; radiation was delivered in 1 to 9 Gy fractions given twice daily separated by 6 to 7 hours for 5 consecutive days to achieve a total dose of 10 to 90 Gy. CpG oligodeoxynucleotide 1826, given as a single agent, had only a small antitumor effect, but it dramatically enhanced fibrosarcoma response to radiotherapy. Although 83.1 (79.2-90.0) Gy total dose were needed to achieve tumor cures in 50% of mice treated with radiotherapy alone, only 23.0 (11.5-32.7) Gy total dose were needed in mice treated with both CpG oligodeoxynucleotide 1826 and radiotherapy. The magnitude of potentiation of tumor radioresponse at the TCD50 level was by a factor of 3.61, a much higher value than that (a factor of 1.93) that we reported for single-dose radiotherapy. Mice cured of their tumors by combined CpG oligodeoxynucleotide 1826 plus radiotherapy were highly resistant to s.c. tumor take or development of tumor nodules in the lung from i.v. injected tumor cells when rechallenged with fibrosarcoma cells 100 to 120 days after the treatment, suggesting the development of a memory response. CpG oligodeoxynucleotide 1826 also increased radioresponse of the nonimmunogenic fibrosarcoma tumor by a factor of 1.41 and 1.73 when CpG oligodeoxynucleotide 1826 was given s.c. and i.t., respectively. These findings show that CpG oligodeoxynucleotides are highly potent enhancers of tumor response to both single-dose and fractionated radiation and as such have potential to improve clinical radiotherapy.

Darbepoetin alfa Radiosensitizes Tumors Independent of Anemia or the Correction of Anemia.

Darbepoetin alfa (DA) is a FDA approved long acting erythropoietic protein. We hypothesized that correction of anemia in tumor-bearing mice by DA would secondarily increase the tumor pO2 and potentiate radiation-induced cell killing of tumor cells. To test this hypothesis, we used total body irradiation (TBI) to induce anemia in C3H mice. Murine squamous cell carcinoma tumor (SCC VII) and fibrosarcoma (RIF-1) models were used to study tumor responses to radiation in vivo. DA (30μg/kg) was administered i.p. either every two weeks or weekly. EPO-R RNA levels were measured in tumors from normal, anemic and DA treated mice in both tumor models. Tumors were locally irradiated with daily fractions of 250 cGy for 5 days. Following 500 cGy TBI, hemoglobin levels decreased and reached a nadir of 7.0 ± 0.9 gm/dL 14 days post TBI. Administration of DA reduced the depth and duration of anemia and improved the general health condition of anemic animals as evidenced by accelerated recovery of body weight following the TBI and maintenance of normal levels of activity compared to similarly irradiated animals not treated with DA. Mice treated with DA on the same day as the TBI had elevated hemoglobin levels with a nadir of 11.1 gm/dL on day 14 after TBI. Systemic administration of DA alone did not stimulate tumor growth in TBI-induced anemic mice. When combined with fractionated local tumor irradiation, administration of DA at any of the time points studied (18, 11, 4 and 0 days before initiation of local tumor irradiation) delayed tumor growth and increased the tumor growth delay time from 2.7 days for irradiation alone to 7.3 – 10.6 days for DA treated animals (p < 0.01). There was no statistically significant difference between tumor growth delay times for groups of mice treated with DA at various times before tumor irradiation. Although DA effectively corrected anemia in tumor-bearing mice and significantly decreased the number of hypoxic cells in the tumors as shown by EF5 staining, radiosensitization by DA was independent of the correction of anemia. EPOR RNA expression was barely detectible in tumors cultured in vitro. There were no differences in EPO-R RNA levels in tumors from anemic or DA treated mice (1–2 fold increase), although EPO-R transcription was upregulated in tumors grown in vivo compared to control tumors lines grown in vitro (40–80 fold increase). This may be due to hypoxic induction of EPO-R by tumors in vivo or expression of EPO-R by endothelial cells or infiltrating macrophages. Results from an experiment in non-anemic mice with RIF-1 tumors suggest that DA can sensitize tumor cells in non-anemic mice to radiation as well. These results support the idea that radiosensitization by DA is independent of hemoglobin and tumor pO2. It has long been assumed that anemia causes decreased tumor oxygenation and increased tumor radioresistance, and that correction of anemia would therefore increase tumor pO2, and result in enhanced radiosensitivity. However, the data presented here challenge this presumed relationship. These findings are promising and may have relevance to the treatment of patients with a variety of tumor types with radiation therapy.