Chemotherapy of lung cancer

The potential for substantial improvement in the outcome of patients with carcinoma of the lung seem most likely to develop in the field of chemotherapy. In the past decade, striking advances in the management of small cell carcinoma have yielded response rates and longer survival. While the greatest improvement can be predicted for patients whose disease is limited in extent, combination chemotherapy and combined modality therapy generally are effective in causing tumor regression for the majority of patients. About 20 percent of patients with disease limited to the thorax and lymph nodes will survive two years. In non-small cell tumors, response rates are improved with intensive drug combinations, although the majority of cases are unresponsive to present regimens. Careful staging and evaluation of patients indicates that patients with good performance status and limited extent of disease appear to obtain the most benefit from intensive treatment. The considerable morbidity of some treatments often influences the choice for or against chemotherapy in patients with non-small cell carcinomas. For the future, problems of particular interest will be in investigation of factors-biologic, pharmacokinetic, immunologic-that are related to the failure to cure small cell carcinoma, the most therapeutically responsive pulmonary tumor. Additionally, in the non-small cell tumors, more effective therapies as well as clarification of the basis for relative resistance to cytotoxic agents are areas for intensive investigation.     

Sulforaphane-induced apoptosis involves p53 and p38 in melanoma cells

In malignant melanoma complex reprogramming of cell death and survival pathways leads to increased chemoresistance and poor longer-term survival. Sulforaphane (SF) is a promising isothiocyanate compound occurring in cruciferous plants with reported antiproliferative and proapoptotic activity in several tumor cell lines including melanoma. In this work we investigated the effects of SF in several melanoma cell lines and fresh melanoma cultivates. We found that SF is cytotoxic and induces mitochondrial, caspase-dependent apoptosis in our study model, however with lower efficiency in fresh melanoma cultivates. Moreover, our results indicate that in melanoma cell lines and fresh melanoma cultivates SF induces multiple signaling including oxidative stress-mediated activation of DNA-damage response pathway, changes in p38 kinase activity and enhanced expression of Bax and Puma proapoptotic proteins. In addition, in SF-exposed p53-mutant melanoma cells Puma expression seem to be under p38 control and acts as a compensatory proapoptotic mechanism. Conversely, decreased apoptosis in SF-exposed melanoma cultivates might be attributed to Akt-mediated suppression of p38 as well as p53 activity. Together, our results suggest that SF inhibits growth and proliferation and induces mitochondrial apoptosis both in melanoma cell lines as well as in fresh melanoma cultivates. This proapoptotic effect might be enhanced in combination with Akt inhibitors, in particular in melanoma samples. SF is thus commendable for further preclinical testing, both as a single agent as well as in combination regimens.     

Targeting the NF-κB Pathway as a Combination Therapy for Advanced Thyroid Cancer

NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα) or pharmacologic (proteasome inhibitor bortezomib and IKKβ inhibitor GO-Y030) inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic approach.     

Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment

The components of the apoptotic program are targets for anticancer therapy. Bcl-2 protein inhibits apoptosis and confers resistance to treatment with traditional cytotoxic chemotherapy, radiotherapy, and monoclonal antibodies (mAb). Oblimersen sodium (G3139, Genasense, Genta Inc., Berkeley Heights, NJ) is an antisense oligonucleotide (AS-ON) compound designed to specifically bind to the first 6 codons of the human bcl-2 mRNA sequence, resulting in degradation of bcl-2 mRNA and subsequent decrease in Bcl-2 protein translation. Oblimersen is the first oligonucleotide to demonstrate proof of principle of an antisense effect in human tumors by the documented downregulation of the target Bcl-2 protein. A growing body of preclinical and clinical evidence suggests that oblimersen synergizes with many cytotoxic and biologic/immunotherapeutic agents against a variety of hematologic malignancies and solid tumors. Randomized clinical trials are currently underway to evaluate the efficacy and tolerability of oblimersen in combination with cytotoxic chemotherapy in chronic lymphocytic leukemia, multiple myeloma, malignant melanoma, and non-small cell lung cancer. In addition, nonrandomized trials are under way to evaluate oblimersen in non-Hodgkin's lymphoma, acute myeloid leukemia, and hormone-refractory prostate cancer. Preclinical data also support the clinical evaluation of oblimersen in additional tumor types, including chronic myelogenous leukemia and breast, small cell lung, gastric, colon, bladder, and Merkel cell cancers. Enhancement of the efficacy of anticancer treatments with oblimersen Bcl-2 antisense therapy represents a promising new apoptosis-modulating strategy, and ongoing clinical trials will test this therapeutic approach.     

Suppression of the proinflammatory response of metastatic melanoma cells increases TRAIL-induced apoptosis

Melanoma is the most lethal form of human skin cancer. However, only limited chemotherapy is currently available for the metastatic stage of the disease. Since chemotherapy, radiation and sodium arsenite treatment operate mainly through induction of the intrinsic mitochondrial pathway, a strongly decreased mitochondrial function in metastatic melanoma cells, could be responsible for low efficacy of the conventional therapy of melanoma. Another feature of metastatic melanoma cells is their proinflammatory phenotype, linked to endogenous expression of the inflammatory cytokines, such as TNFα IL6 and IL8, their receptors, and constitutive NF-κB- and STAT3-dependent gene expression, including cyclooxygenase-2 (PTGS2/COX2). In the present study, we treated melanoma cells with immunological (monoclonal antibody against TNFα or IL6), pharmacological (small molecular inhibitors of IKKβ-NF-κB and JAK2-STAT3) or genetic (specific RNAi for COX-2) agents that suppressed the inflammatory response in combination with induction of apoptosis via TRAIL. As a result of these combined treatments, exogenous TRAIL via interactions with TRAIL-R2/R1 strongly increased levels of apoptosis in resistant melanoma cells. The present study provides new understanding of the regulation of TRAIL-mediated apoptosis in melanoma and will serve as the foundation for the potential development of a novel approach for a therapy of resistant melanomas.     

Topoisomerase inhibitors modulate expression of melanocytic antigens and enhance T cell recognition of tumor cells

While there are many obstacles to immune destruction of autologous tumors, there is mounting evidence that tumor antigen recognition does occur. Unfortunately, immune recognition rarely controls clinically significant tumors. Even the most effective immune response will fail if tumors fail to express target antigens. Importantly, reduced tumor antigen expression often results from changes in gene regulation rather than irrevocable loss of genetic information. Such perturbations are often reversible by specific compounds or biological mediators, prompting a search for agents with improved antigen-enhancing properties. Some recent findings have suggested that certain conventional chemotherapeutic agents may have beneficial properties for cancer treatment beyond their direct cytotoxicities against tumor cells. Accordingly, we screened an important subset of these agents, topoisomerase inhibitors, for their effects on antigen levels in tumor cells. Our analyses demonstrate upregulation of antigen expression in a variety of melanoma cell lines and gliomas in response to nanomolar levels of certain specific topoisomerase inhibitors. To demonstrate the ability of CD8+ T cells to recognize tumors, we assayed cytokine secretion in T cells transfected with T cell receptors directed against Melan-A/MART-1 antigen. Three days of daunorubicin treatment resulted in enhanced antigen expression by tumor cells, in turn inducing co-cultured antigen-specific T cells to secrete Interleukin-2 and Interferon-γ. These results demonstrate that specific topoisomerase inhibitors can augment melanoma antigen production, suggesting that a combination of chemotherapy and immunotherapy may be of potential value in the treatment of otherwise insensitive cancers.     

Combined Anti-Angiogenic Therapy Targeting PDGF and VEGF Receptors Lowers the Interstitial Fluid Pressure in a Murine Experimental Carcinoma

Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half‐life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4‐Nerolidylcatechol (4‐NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4‐NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl‐1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4‐NC is time‐dependent upon the pro‐apoptotic protein Noxa, which is able to bind and neutralize Mcl‐1. We demonstrate the role of 4‐NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4‐NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.     

RNA Silencing of Mcl-1 Enhances ABT-737-Mediated Apoptosis in Melanoma: Role for a Caspase-8-Dependent Pathway

Background

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma''s striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance.

Methodology/Principal Findings

Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity.

Conclusions/Significance

Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.     

Combination of vasostatin gene therapy with cyclophosphamide inhibits growth of B16(F10) melanoma tumours

Angiogenesis, i.e. formation of new blood vessels out of pre-existing capillaries, is essential to the development of tumour vasculature. The discovery of specific antiangiogenic inhibitors has important therapeutic implications for the development of novel cancer treatments. Vasostatin, the N-terminal domain of calreticulin, is a potent endogenous inhibitor of angiogenesis and tumour growth. In our study, using B16(F10) murine melanoma model and electroporation we attempted intramuscular transfer of human vasostatin gene. The gene therapy was combined with antiangiogenic drug dosing schedule of a known chemotherapeutic (cyclophosphamide). The combination of vasostatin gene therapy and cyclophosphamide administration improved therapeutic effects in melanoma tumours. We observed both significant inhibition of tumour growth and extended survival of treated mice. To our knowledge, this is one of the first reports showing antitumour efficacy of electroporation-mediated vasostatin gene therapy combined with antiangiogenic chemotherapy.     

Preclinical evaluation of the proteasome inhibitor bortezomib in cancer therapy

Bortezomib is a highly selective, reversible inhibitor of the 26S proteasome that is indicated for single-agent use in the treatment of patients with multiple myeloma who have received at least 2 prior therapies and are progressing on their most recent therapy. Clinical investigations have been completed or are under way to evaluate the safety and efficacy of bortezomib alone or in combination with chemotherapy in multiple myeloma, both at relapse and presentation, as well as in other cancer types. The antiproliferative, proapoptotic, antiangiogenic, and antitumor activities of bortezomib result from proteasome inhibition and depend on the altered degradation of a host of regulatory proteins. Exposure to bortezomib has been shown to stabilize p21, p27, and p53, as well as the proapoptotic Bid and Bax proteins, caveolin-1, and inhibitor κB-α, which prevents activation of nuclear factor κB-induced cell survival pathways. Bortezomib also promoted the activation of the proapoptotic c-Jun-NH₂ terminal kinase, as well as the endoplasmic reticulum stress response. The anticancer effects of bortezomib as a single agent have been demonstrated in xenograft models of multiple myeloma, adult T-cell leukemia, lung, breast, prostate, pancreatic, head and neck, and colon cancer, and in melanoma. In these preclinical in vivo studies, bortezomib treatment resulted in decreased tumor growth, angiogenesis, and metastasis, as well as increased survival and tumor apoptosis. In several in vitro and/or in vivo cancer models, bortezomib has also been shown to enhance the antitumor properties of several antineoplastic treatments. Importantly, bortezomib was generally well tolerated and did not appear to produce additive toxicities when combined with other therapies in the dosing regimens used in these preclinical in vivo investigations. These findings provide a rationale for further clinical trials using bortezomib alone or in combination regimens with chemotherapy, radiation therapy, immunotherapy, or novel agents in patients with hematologic malignancies or solid tumors.     

Imaging and Therapy of Pancreatic Cancer with Phosphatidylserine-Targeted Nanovesicles

Pancreatic cancer remains one of the most intractable cancers, with a dismal prognosis reflected by a 5-year survival of ~ 6%. Since early disease symptoms are undefined and specific biomarkers are lacking, about 80% of patients present with advanced, inoperable tumors that represent a daunting challenge. Despite many clinical trials, no single chemotherapy agent has been reliably associated with objective response rates above 10% or median survival longer than 5 to 7 months. Although combination chemotherapy regimens have in recent years provided some improvement, overall survival (8-11 months) remains very poor. There is therefore a critical need for novel therapies that can improve outcomes for pancreatic cancer patients. Here, we present a summary of the current therapies used in the management of advanced pancreatic cancer and review novel therapeutic strategies that target tumor biomarkers. We also describe our recent research using phosphatidylserine-targeted saposin C–coupled dioleoylphosphatidylserine nanovesicles for imaging and therapy of pancreatic cancer.     

The role of ferroptosis in melanoma

Melanoma is the deadliest form of skin cancer. Although treatment with targeted therapies and immune checkpoint inhibitors has dramatically improved survival in advanced melanoma, many patients do not benefit from these therapies or relapse after an initial period of response. Thus, future outcomes in these categories of melanoma patients will depend on the identification of novel therapeutic targets and methods to enhance existing targeted therapy and immunotherapy regimens. Ferroptosis is a newly identified form of iron-dependent regulated cell death that is morphologically, biochemically, and genetically distinct from apoptosis, autophagy, pyroptosis, and necroptosis. Dysregulation of ferroptosis has been linked to the development of several forms of cancer. This review examines ferroptosis in the context of melanoma. It presents an overview of ferroptosis biology, summarizes and interprets the current literature, and poses several outstanding questions and areas of future direction.     

Showering c-MET-dependent cancers with drugs

The receptor tyrosine kinase, c-MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) have become leading candidates for targeted cancer therapies. Inappropriate c-MET signaling through autocrine, paracrine, amplification, and mutational activation occurs in virtually all types of solid tumors (http://www.vai.org/met), contributing to one or a combination of proliferative, invasive, survival, or angiogenic cancer phenotypes. c-MET and HGF/SF participate in all stages of malignant progression and represent promising drug targets in a variety of cancer types, including carcinomas, sarcomas, and brain tumors. While many are in pre-clinical testing, a few inhibitors have entered clinical trials. With hundreds of thousands of potential responding cancers that express c-MET, the interest in this molecule as a drug target is not surprising. However, the cognate c-MET diagnostic tests lag behind. In addition, despite the great enthusiasm based on response rates in phase I trials, there is a need for caution. It is almost without question that combination therapies with c-MET-HGF/SF inhibitors will be required for most cancers to achieve a cytotoxic tumor response.     

Pharmacologic modulation of serine/threonine phosphorylation highly sensitizes PHEO in a MPC cell and mouse model to conventional chemotherapy

Background

The failure of cytotoxic cancer regimens to cure the most drug-resistant, well-differentiated solid tumors has been attributed to the heterogeneity of cell types that differ in their capacities for growth, differentiation, and metastases. We investigated the effect of LB1, a small molecule inhibitor of serine/threonine protein phosphatase 2A (PP2A), on its ability to inhibit a low growth fraction and highly drug-resistant solid neuroendocrine tumor, such as metastatic pheochromocytoma (PHEO). Subsequently, we evaluated the increased efficacy of chemotherapy combined with LB1.

Methodology/Principal Findings

The effect of LB1 and temozolomide (TMZ), a standard chemotherapeutic agent that alone only transiently suppressed the growth and regression of metastatic PHEO, was evaluated in vitro on a single PHEO cell line and in vivo on mouse model of metastatic PHEO. In the present study, we show that metastatic PHEO, for which there is currently no cure, can be eliminated by combining LB1, thereby inhibiting PP2A, with TMZ. This new treatment approach resulted in long term, disease-free survival of up to 40% of animals bearing multiple intrahepatic metastases, a disease state that the majority of patients die from. Inhibition of PP2A was associated with prevention of G1/S phase arrest by p53 and of mitotic arrest mediated by polo-like kinase 1 (Plk-1).

Conclusions/Significance

The elimination of DNA damage-induced defense mechanisms, through transient pharmacologic inhibition of PP2A, is proposed as a new approach for enhancing the efficacy of non-specific cancer chemotherapy regimens against a broad spectrum of low growth fraction tumors very commonly resistant to cytotoxic drugs.     

Novel therapeutic approaches in GEP-NETs based on genetic and epigenetic alterations

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous malignancies with distinct prognosis based on primary tumor localization, grade, stage and functionality. Surgery remains the only curative option in localized tumors, but systemic therapy is the mainstay of treatment for patients with advanced disease. For decades, the therapeutic landscape of GEP-NETs was limited to chemotherapy regimens with low response rates. The arrival of novel agents such as somatostatin analogues, peptide receptor radionuclide therapy, tyrosine kinase inhibitors or mTOR-targeted drugs, has changed the therapeutic paradigm of GEP-NETs. However, the efficacy of these agents is limited in time and there is scarce knowledge of optimal treatment sequencing. In recent years, massive parallel sequencing techniques have started to unravel the genomic intricacies of these tumors, allowing us to better understand the mechanisms of resistance to current treatments and to develop new targeted agents that will hopefully start an era for personalized treatment in NETs. In this review we aim to summarize the most relevant genomic aberrations and signaling pathways underlying GEP-NET tumorigenesis and potential therapeutic strategies derived from them.     

Cyclin-dependent kinase inhibitors sensitize tumor cells to nutlin-induced apoptosis: a potent drug combination

Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose- and time-dependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser(15) on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage-mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy.     

Combinations of anticancer drugs and immunotherapy

Immunotherapy (biological therapy) comprises such things as active specific immunotherapy ("cancer vaccines"), nonspecific immunostimulation with cytokines, and the inhibition of suppressor influences exerted or elicited by the tumor. Just as cancer chemotherapy began with the use of single agents and evolved into combination therapy, so immunotherapeutic agents have been combined with each other and with chemotherapy. The alkylating agent cyclophosphamide (Cytoxan; CYC) has been used for many years to inhibit tumor-derived suppressor influences in rodents, and has been exploited for the same use in humans. Combinations of CYC and cancer vaccines such as autologous tumor cells, Melacine, large multivalent immunogen (LMI), and Theratope have been tested with some success in humans for more than a decade. In this use, the CYC is a biological response modifier rather than an antitumor agent, intended to inhibit suppressor influences. CYC and low- to moderate-dose IL-2 has also been a useful regimen in treating human melanomas. IL-2 is itself a useful component of combination immunotherapy, such as with melanoma peptide vaccines, or with interferon -2b, (IFN-), as a dual combination or part of a biochemotherapy regimen. Several different combinations of drugs and biological agents have been used as biochemotherapy for melanoma, but although there are higher response (regression) rates the long-range survival benefits have been marginal, not justifying the severe toxicity. Combinations of 5-fluorouracil (5-FU) and IFN- or levamisole have had efficacy in colon and head and neck cancers, but here the biological agents have been biochemical modulators, not immunotherapy. Although experience with combinations of monoclonal antibodies and chemotherapy has been limited, it appears that trastuzumab (Herceptin) potentiates antitumor therapy in breast cancer but also increases the cardiotoxicity of those regimens.This article forms part of the Symposium in Writing on "Cellular immunity for cancer chemoimmunotherapy" in Volume 52 (2003)     

Focal Radiation Therapy Combined with 4-1BB Activation and CTLA-4 Blockade Yields Long-Term Survival and a Protective Antigen-Specific Memory Response in a Murine Glioma Model

Background

Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4) blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137) is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model.

Methods

GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors.

Results

Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4⁺ and CD8⁺ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4⁺ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8⁺ T cells had no effect on the treatment response.

Conclusion

Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse model of glioma by a CD4⁺ T cell dependent mechanism and generates antigen-specific memory.