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STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies., (©2016 American Association for Cancer Research.)

The innate cellular and molecular components required to mediate effective vaccination against weak tumor-associated antigens remain unclear. In this study, we used polymeric cancer vaccines incorporating different classes of adjuvants to induce tumor protection, to identify dendritic cell (DC) subsets and cytokines critical to this efficacy. Three-dimensional, porous polymer matrices loaded with tumor lysates and presenting distinct combinations of granulocyte macrophage colony-stimulating factor (GM-CSF) and various Toll-like receptor (TLR) agonists affected 70% to 90% prophylactic tumor protection in B16-F10 melanoma models. In aggressive, therapeutic B16 models, the vaccine systems incorporating GM-CSF in combination with P(I:C) or CpG-ODN induced the complete regression of solid tumors (≤40 mm(2)), resulting in 33% long-term survival. Regression analysis revealed that the numbers of vaccine-resident CD8(+) DCs, plasmacytoid DCs (pDC), along with local interleukin (IL)-12, and granulocyte colony-stimulating factor (G-CSF) concentrations correlated strongly to vaccine efficacy regardless of adjuvant type. Furthermore, vaccine studies in Batf3(-/-) mice revealed that CD8(+) DCs are required to affect tumor protection, as vaccines in these mice were deficient in cytotoxic T lymphocytes priming and IL-12 induction in comparison with wild-type. These studies broadly demonstrate that three-dimensional polymeric vaccines provide a potent platform for prophylactic and therapeutic protection, and can be used as a tool to identify critical components of a desired immune response. Specifically, these results suggest that CD8(+) DCs, pDCs, IL-12, and G-CSF play important roles in priming effective antitumor responses with these vaccines., (©2014 AACR.)

The chemokine CXCL12 and its receptor CXCR4 are expressed widely in human cancers, including ovarian cancer, in which they are associated with disease progression at the levels of tumor cell proliferation, invasion, and angiogenesis. Here, we used an immunocompetent mouse model of intraperitoneal papillary epithelial ovarian cancer to show that modulation of the CXCL12/CXCR4 axis in ovarian cancer has multimodal effects on tumor pathogenesis associated with induction of antitumor immunity. siRNA-mediated knockdown of CXCL12 in BR5-1 cells that constitutively express CXCL12 and CXCR4 reduced cell proliferation in vitro, and tumor growth in vivo. Similarly, treatment of BR5-1-derived tumors with AMD3100, a selective CXCR4 antagonist, resulted in increased tumor apoptosis and necrosis, reduction in intraperitoneal dissemination, and selective reduction of intratumoral FoxP3(+) regulatory T cells (Treg). Compared with controls, CXCR4 blockade greatly increased T-cell-mediated antitumor immune responses, conferring a significant survival advantage to AMD3100-treated mice. In addition, the selective effect of CXCR4 antagonism on intratumoral Tregs was associated with both higher CXCR4 expression and increased chemotactic responses to CXCL12, a finding that was also confirmed in a melanoma model. Together, our findings reinforce the concept of a critical role for the CXCL12/CXCR4 axis in ovarian cancer pathogenesis, and they offer a definitive preclinical validation of CXCR4 as a therapeutic target in this disease.

The inhibition of VEGF signaling with antibodies or small molecules achieves clinical benefits in diverse solid malignancies. Nonetheless, therapeutic effects are usually not sustained, and most patients eventually succumb to progressive disease, indicating that antiangiogenic strategies require additional optimization. Vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) and antibody blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) trigger a tumor vasculopathy in some long-term responding subjects. These reactions are characterized by disrupted tumor blood vessels in association with lymphocyte and granulocyte infiltrates and zonal areas of ischemic tumor necrosis. However, the mechanisms underlying this immune-mediated destruction of the tumor vasculature remain to be clarified. Here, we show that GM-CSF-secreting tumor cell vaccines and CTLA-4 blockade elicit a functionally important humoral reaction against multiple angiogenic cytokines. Antibodies to angiopoietin-1 and angiopoietin-2 block Tie-2 binding, downstream signaling, endothelial cell tube formation, and macrophage chemotaxis. Antibodies to macrophage inhibitory factor (MIF) attenuate macrophage Tie-2 expression and matrix metalloproteinase-9 (MMP-9) production. Together, these results delineate an immunotherapy-induced host response that broadly targets the angiogenic network in the tumor microenvironment., (©2010 AACR.)

The pathogenesis of malignant melanoma involves the interplay of tumor cells with normal host elements, but the underlying mechanisms are incompletely understood. Here, we show that milk fat globule EGF-8 (MFG-E8), a secreted protein expressed at high levels in the vertical growth phase of melanoma, promotes disease progression through coordinated alpha(v)beta(3) integrin signaling in the tumor microenvironment. In a murine model of melanoma, MFG-E8 enhanced tumorigenicity and metastatic capacity through Akt-dependent and Twist-dependent pathways. MFG-E8 augmented melanoma cell resistance to apoptosis, triggered an epithelial-to-mesenchymal transition (EMT), and stimulated invasion and immune suppression. In human melanoma cells, MFG-E8 knockdown attenuated Akt and Twist signaling and thereby compromised tumor cell survival, EMT, and invasive ability. MFG-E8-deficient human melanoma cells also showed increased sensitivity to small molecule inhibitors of insulin-like growth factor I receptor and c-Met. Together, these findings delineate pleiotropic roles for MFG-E8 in the tumor microenvironment and raise the possibility that systemic MFG-E8 blockade might prove therapeutic for melanoma patients.

The classification of human tumors based on molecular criteria offers tremendous clinical potential; however, discerning critical and "druggable" effectors on a large scale will also require robust experimental models reflective of tumor genomic diversity. Here, we describe a comprehensive genomic analysis of 101 melanoma short-term cultures and cell lines. Using an analytic approach designed to enrich for putative "driver" events, we show that cultured melanoma cells encompass the spectrum of significant genomic alterations present in primary tumors. When annotated according to these lesions, melanomas cluster into subgroups suggestive of distinct oncogenic mechanisms. Integrating gene expression data suggests novel candidate effector genes linked to recurrent copy gains and losses, including both phosphatase and tensin homologue (PTEN)-dependent and PTEN-independent tumor suppressor mechanisms associated with chromosome 10 deletions. Finally, sample-matched pharmacologic data show that FGFR1 mutations and extracellular signal-regulated kinase (ERK) activation may modulate sensitivity to mitogen-activated protein kinase/ERK kinase inhibitors. Genetically defined cell culture collections therefore offer a rich framework for systematic functional studies in melanoma and other tumors.

Immunosuppression is frequently associated with malignancy and is particularly severe in patients with malignant glioma. Anergy and counterproductive shifts toward T(H)2 cytokine production are long-recognized T-cell defects in these patients whose etiology has remained elusive for >30 years. We show here that absolute counts of both CD4(+) T cells and CD4(+)CD25(+)FOXP3(+)CD45RO(+) T cells (T(regs)) are greatly diminished in patients with malignant glioma, but T(regs) frequently represent an increased fraction of the remaining CD4 compartment. This increased T(reg) fraction, despite reduced counts, correlates with and is sufficient to elicit the characteristic manifestations of impaired patient T-cell responsiveness in vitro. Furthermore, T(reg) removal eradicates T-cell proliferative defects and reverses T(H)2 cytokine shifts, allowing T cells from patients with malignant glioma to function in vitro at levels equivalent to those of normal, healthy controls. Such restored immune function may give license to physiologic antiglioma activity, as in vivo, T(reg) depletion proves permissive for spontaneous tumor rejection in a murine model of established intracranial glioma. These findings dramatically alter our understanding of depressed cellular immune function in patients with malignant glioma and advance a role for T(regs) in facilitating tumor immune evasion in the central nervous system.

Donor lymphocyte infusion (DLI) reliably induces durable remission in 75-80% of patients with relapsed chronic myelogenous leukemia (CML) after allogeneic hematopoietic stem cell transplantation. To identify immunological targets of the graft-versus-leukemia response (GVL) after DLI, we used CML post-DLI responder sera to screen a CML cDNA expression library. One of the antigens identified in this screen is a M(r) 28,000 protein, termed CML28. CML28 is identical to hRrp46p, a component of the human exosome, a multiprotein complex involved in the 3' processing of RNA. Components of the human exosome include known autoantigens, such as PMScl-100, an autoantibody target in patients with polymyositis, scleroderma, or polymyositis-scleroderma overlap syndrome. Recombinant CML28-GST fusion protein was purified, and used in Western blot and ELISA to demonstrate the development of a high-titer CML28-specific IgG antibody response in a patient with relapsed CML who responded to DLI. Northern blotting demonstrated that CML28 is highly expressed in a variety of hematopoietic and epithelial tumor cell lines, but not in normal hematopoietic tissues or other normal tissue, with the exception of testis. Purified recombinant CML28 was used to generate a CML28-specific murine monoclonal antibody. Western blotting with CML28 monoclonal antibody against whole-cell lysates derived from blood and marrow of normal donors and patients with leukemia revealed high expression of this antigen in tumor but not in normal samples. Because CML28 was highly expressed in epithelial tumor cell lines, anti-CML28 responses were also examined in patients with solid tumors. By ELISA, we found specific serological responses in 10-33% of patients with lung cancer, melanoma, and prostate cancer. Our studies suggest that immunogenicity of CML28 is likely because of overexpression of this antigen in tumor cells. Moreover, given its expression and immunogenicity in a wide variety of malignancies, CML28 merits additional evaluation as a target for antigen-specific immunotherapy.

Donor T cells play a critical role in mediating both harmful graft-versus-host disease (GVHD) and beneficial graft-versus-tumor effect after allogeneic bone marrow transplantation (BMT). We have recently demonstrated a novel treatment strategy to stimulate specific antitumor activity with preservation of tolerance to host antigens after T cell-depleted allogeneic BMT by vaccination of recipients with irradiated B16 melanoma cells engineered to secrete granulocyte-macrophage colony-stimulating factor. In this murine system, donor leukocyte infusion from a donor immunized with the recipient-derived B16 vaccines enhanced clinical activity of tumor vaccines without exacerbating GVHD. CD4(+) T cells are essential for this enhancement. In vitro analysis of splenocytes from donor leukocyte infusion donor mice demonstrated that immunization of donors with the recipient-derived B16 vaccines elicited potent T-cell proliferation and cytokine responses specific to B16 antigens. These results demonstrate that immunization of donors with recipient-derived tumor vaccines preferentially induces tumor-specific T-cell responses and that vaccination of both donors and recipients can generate potent antitumor immunity without exacerbating GVHD. This strategy has important implications to prevent recurrence of malignancies after BMT.

Allogeneic bone marrow transplantation (BMT) is currently restricted to hematological malignancies because of a lack of antitumor activity against solid cancers. We have tested a novel treatment strategy to stimulate specific antitumor activity against a solid tumor after BMT by vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). Using the B16 melanoma model, we found that vaccination elicited potent antitumor activity in recipients of syngeneic BMT in a time-dependent fashion, and that immune reconstitution was critical for the development of antitumor activity. Vaccination did not stimulate antitumor immunity after allogeneic BMT because of the post-BMT immunodeficiency associated with graft-versus-host disease (GVHD). Remarkably, vaccination was effective in stimulating potent and long-lasting antitumor activity in recipients of T-cell-depleted (TCD) allogeneic bone marrow. Recipients of TCD bone marrow who showed significant immune reconstitution by 6 weeks after BMT developed B16-specific T-cell-cytotoxic, proliferative, and cytokine responses as a function of vaccination. T cells derived from donor stem cells were, therefore, able to recognize tumor antigens, although they remained tolerant to host histocompatibility antigens. These results demonstrate that GM-CSF-based tumor cell vaccines after allogeneic TCD BMT can stimulate potent antitumor effects without the induction of GVHD, and this strategy has important implications for the treatment of patients with solid malignancies.

Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and flt3-ligand (FL) induce the development of dendritic cells (DCs). To compare the functional properties of DCs stimulated by these cytokines in vivo, we used retroviral-mediated gene transfer to generate murine tumor cells secreting high levels of each molecule. Injection of tumor cells expressing either GM-CSF or FL resulted in the dramatic increase of CD11c+ cells in the spleen and tumor infiltrate. However, vaccination with irradiated, GM-CSF-secreting tumor cells stimulated more potent antitumor immunity than vaccination with irradiated, FL-secreting tumor cells. The superior antitumor immunity elicited by GM-CSF involved a broad T cell cytokine response, in contrast to the limited Thl response elicited by FL. DCs generated by GM-CSF were CD8alpha- and expressed higher levels of B7-1 and CD1d than DCs cells generated by FL. Injection sites of metastatic melanoma patients vaccinated with irradiated, autologous tumor cells engineered to secrete GM-CSF demonstrated similar, dense infiltrates of DCs expressing high levels of B7-1. These findings reveal critical differences in the abilities of GM-CSF and FL to enhance the function of DCs in vivo and have important implications for the crafting of tumor vaccines.

Interactions between CD40 and its ligand, CD154 (CD40L, gp39), have been shown to play a central role in the regulation of humoral immunity. Recent evidence suggests that this ligand-receptor pair also plays an important role in the induction of cell-mediated immune responses, including those directed against viral pathogens, intracellular parasites, and alloantigens. The contribution of this ligand-receptor pair to the development of protective immunity against syngeneic tumors was evaluated by blocking the in vivo function of CD154 or by studying tumor resistance in mice genetically deficient in CD40 expression (CD40-/-). In the former case, anti-CD154 monoclonal antibody treatment inhibited the generation of protective immune responses after the administration of three potent tumor vaccines: irradiated MCA 105, MCA 105 admixed with Corynebacterium parvum adjuvant, and irradiated B16 melanoma cells transduced with the gene for granulocyte macrophage colony-stimulating factor. Confirmation of the role of CD40/CD154 interactions in tumor immunity was provided by the overt tumor susceptibility in CD40-deficient mice as compared to that in CD40+/+ mice. In this case, wild-type but not CD40-deficient mice could be readily protected against live TS/A tumor challenge by preimmunization with TS/A admixed with C. parvum. These findings suggest a critical role for CD40/CD154 interactions in the induction of cellular immunity by tumor vaccines and may have important implications for future approaches to cell-based cancer therapies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced, irradiated tumor vaccines induce potent, T-cell-mediated antitumor immune responses in preclinical models. We report the initial results of a Phase I trial evaluating this strategy for safety and the induction of immune responses in patients with metastatic renal cell carcinoma (RCC). Patients were treated in a randomized, double-blind dose-escalation study with equivalent doses of autologous, irradiated RCC vaccine cells with or without ex vivo human GM-CSF gene transfer. The replication-defective retroviral vector MFG was used for GM-CSF gene transfer. No dose-limiting toxicities were encountered in 16 fully evaluable patients. GM-CSF gene-transduced vaccines were equivalent in toxicity to nontransduced vaccines up to the feasible limits of autologous tumor vaccine yield. No evidence of autoimmune disease was observed. Biopsies of intradermal sites of injection with GM-CSF gene-transduced vaccines contained distinctive macrophage, dendritic cell, eosinophil, neutrophil, and T-cell infiltrates similar to those observed in preclinical models of efficacy. Histological analysis of delayed-type hypersensitivity responses in patients vaccinated with GM-CSF-transduced vaccines demonstrated an intense eosinophil infiltrate that was not observed in patients who received nontransduced vaccines. An objective partial response was observed in a patient treated with GM-CSF gene-transduced vaccine who displayed the largest delayed-type hypersensitivity conversion. No replication-competent retrovirus was detected in vaccinated patients. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous GM-CSF gene-transduced tumor vaccine for RCC patients.

Preclinical studies with murine tumor models have demonstrated that autologous tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. These results have engendered much interest in developing this strategy for gene therapy of human cancer. The major limitation to creating genetically modified autologous human tumor vaccines is efficient gene transfer into primary tumor explants, since the majority of human tumors fail to proliferate in long-term culture. Using the retroviral vector MFG in conjunction with short-term culture techniques, we have achieved, in the absence of selection, a mean transduction efficiency of 60% in primary renal, ovarian, and pancreatic tumor explants, and we have developed an autologous granulocyte-macrophage colony-stimulating factor secreting tumor vaccine for clinical trials.

Cellular supply of glutamine, an essential substrate for growth, is derived from extracellular fluid and de novo synthesis. We investigated the relative importance of these sources to the growth of six human anaplastic glioma- and one human medulloblastoma-derived permanent cell lines. Exogenous glutamine was limiting for the proliferation of glioma-derived lines D-54 MG, U-118 MG, and U-251 MG. In contrast, medulloblastoma-derived line TE-671 and glioma-derived lines U-373 MG, D-245 MG, and D-259 MG grew in the absence of supplemental glutamine. Two cell lines with contrasting glutamine requirements, D-54 MG and TE-671, were used to explore the pharmacological interference with glutamine metabolism. DL-alpha-Aminoadipic acid, a reported glutamic acid analogue with gliotoxic properties, significantly inhibited the growth of both lines. These effects were reversed by increasing glutamine, suggesting that the major action of DL-alpha-aminoadipic acid is as a glutamine antagonist. In contrast, the glutamine synthetase inhibitor delta-hydroxylysine demonstrated activity only against TE-671. Acivicin and 6-diazo-5-oxo-L-norleucine, glutamine analogues available for clinical use, reduced the proliferation of both cell lines at pharmacological concentrations. Methionine sulfoximine, a glutamine synthetase inhibitor previously used clinically, produced marked growth inhibition only against TE-671. These findings indicate that the synthesis and utilization of glutamine are potentially exploitable targets for the chemotherapy of some human gliomas and medulloblastomas.

The human glioma-derived cell line D-54 MG and the human medulloblastoma-derived cell line TE-671 have been shown to be sensitive in culture to the pharmacological interference with glutamine metabolism by acivicin, 6-diazo-5-oxo-L-norleucine, and methionine sulfoximine. Using as a guide the multiple contributions of glutamine to the biosynthesis of proteins, purines, and pyrimidines, we now have identified six additional antimetabolites active against these lines in vitro at clinically relevant concentrations. The 50% growth-inhibitory levels of the drugs against D-54 MG in 6-day continuous exposure experiments were: L-asparaginase, 0.057 IU/ml; 5-fluorouracil, 0.5 micrograms/ml; 6-mercaptopurine, 0.8 micrograms/ml; actinomycin D, 0.0007 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 2.3 micrograms/ml; and 5-azacytidine, 0.2 micrograms/ml (3-day exposure. The corresponding 50% growth-inhibitory values in TE-671 were: L-asparaginase, 0.54 IU/ml; 5-fluorouracil, 1.5 micrograms/ml; 6-mercaptopurine, 4.7 micrograms/ml; actinomycin D, 0.00044 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 4.5 micrograms/ml; and 5-azacytidine, 0.49 micrograms/ml. Dipyridamole up to 10 micrograms/ml was inactive against both lines. The isobologram method was used to evaluate the effectiveness of several two-drug combinations which were biochemically designed. The sums of the optimal fractional inhibitory concentrations for the pairs were: acivicin plus L-asparaginase, 0.14; acivicin plus methionine sulfoximine, 0.40; 6-diazo-5-oxo-L-norleucine plus methionine sulfoximine, 0.60; acivicin plus 6-mercaptopurine, 1.0, all in TE-671; and acivicin plus 5-fluorouracil, 0.79, in D-54 MG. Our findings suggest that an antimetabolite regimen exploiting glutamine sensitivity might improve the chemotherapy of some human gliomas and medulloblastomas.