ABSTRACT
Differential outcomes of EphB4-ephrinB2 signaling offers formidable challenge for the development of cancer therapeutics. Here, we interrogate the effects of targeting EphB4 and ephrinB2 in head and neck squamous cell carcinoma (HNSCC) and within its microenvironment using genetically engineered mice, recombinant constructs, pharmacologic agonists and antagonists. We observe that manipulating the EphB4 intracellular domain on cancer cells accelerates tumor growth and angiogenesis. EphB4 cancer cell loss also triggers compensatory upregulation of EphA4 and T regulatory cells (Tregs) influx and their targeting results in reversal of accelerated tumor growth mediated by EphB4 knockdown. EphrinB2 knockout on cancer cells and vasculature, on the other hand, results in maximal tumor reduction and vascular normalization. We report that EphB4 agonism provides no additional anti-tumoral benefit in the absence of ephrinB2. These results identify ephrinB2 as a tumor promoter and its receptor, EphB4, as a tumor suppressor in HNSCC, presenting opportunities for rational drug design.
Subject(s)
Ephrin-B2 , Head and Neck Neoplasms , Receptor, EphB4 , Squamous Cell Carcinoma of Head and Neck , Animals , Ephrin-B2/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Mice , Receptor, EphB4/genetics , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Tumor MicroenvironmentABSTRACT
STAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs.
Subject(s)
Head and Neck Neoplasms , STAT3 Transcription Factor/metabolism , Animals , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/therapy , Mice , Mice, Knockout , STAT3 Transcription Factor/genetics , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/therapy , T-Lymphocytes, Regulatory , Tumor Microenvironment/geneticsABSTRACT
PURPOSE: Natural killer (NK) cells are type I innate lymphoid cells that are known for their role in killing virally infected cells or cancer cells through direct cytotoxicity. In addition to direct tumor cell killing, NK cells are known to play fundamental roles in the tumor microenvironment through secretion of key cytokines, such as FMS-like tyrosine kinase 3 ligand (FLT3L). Although radiotherapy is the mainstay treatment in most cancers, the role of radiotherapy on NK cells is not well characterized. EXPERIMENTAL DESIGN: This study combines radiation, immunotherapies, genetic mouse models, and antibody depletion experiments to identify the role of NK cells in overcoming resistance to radiotherapy in orthotopic models of head and neck squamous cell carcinoma. RESULTS: We have found that NK cells are a crucial component in the development of an antitumor response, as depleting them removes efficacy of the previously successful combination treatment of radiotherapy, anti-CD25, and anti-CD137. However, in the absence of NK cells, the effect can be rescued through treatment with FLT3L. But neither radiotherapy with FLT3L therapy alone nor radiotherapy with anti-NKG2A yields any meaningful tumor growth delay. We also identify a role for IL2 in activating NK cells to secrete FLT3L. This activity, we show, is mediated through CD122, the intermediate affinity IL2 receptor, and can be targeted with anti-CD25 therapy. CONCLUSIONS: These findings highlight the complexity of using radio-immunotherapies to activate NK cells within the tumor microenvironment, and the importance of NK cells in activating dendritic cells for increased tumor surveillance.
Subject(s)
Head and Neck Neoplasms , Radioimmunotherapy , Animals , Head and Neck Neoplasms/radiotherapy , Humans , Immunity, Innate , Killer Cells, Natural , Membrane Proteins , Mice , Squamous Cell Carcinoma of Head and Neck/radiotherapy , Tumor MicroenvironmentABSTRACT
BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Dendritic Cells/drug effects , Drug Resistance, Neoplasm , Head and Neck Neoplasms/therapy , Immune Checkpoint Inhibitors/pharmacology , Immunotherapy , Radiation Dose Hypofractionation , Radiation Tolerance , Squamous Cell Carcinoma of Head and Neck/therapy , T-Lymphocytes, Regulatory/drug effects , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Line, Tumor , Combined Modality Therapy , Dendritic Cells/immunology , Dendritic Cells/metabolism , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Interleukin-2 Receptor alpha Subunit/antagonists & inhibitors , Interleukin-2 Receptor alpha Subunit/metabolism , Lymphocyte Depletion , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Repressor Proteins/genetics , Repressor Proteins/metabolism , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tumor Burden , Tumor Microenvironment , Tumor Necrosis Factor Receptor Superfamily, Member 9/antagonists & inhibitors , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolismABSTRACT
BACKGROUND: Resistance to therapy is a major problem in treating head and neck squamous cell carcinomas (HNSCC). Complement system inhibition has been shown to reduce tumor growth, metastasis, and therapeutic resistance in other tumor models, but has yet to be explored in the context of HNSCC. Here, we tested the effects of complement inhibition and its therapeutic potential in HNSCC. METHODS: We conducted our studies using two Human Papilloma Virus (HPV)-negative HNSCC orthotopic mouse models. Complement C3aR and C5aR1 receptor antagonists were paired with radiation therapy (RT). Tumor growth was measured and immune populations from tumor, lymph node, and peripheral blood were compared among various treatment groups. Genetically engineered mouse models DEREG and C3-/- were used in addition to standard wild type models. Flow cytometry, clinical gene sets, and in vitro assays were used to evaluate the role complement receptor blockade has on the immunological makeup of the tumor microenvironment. RESULTS: In contrast to established literature, inhibition of complement C3a and C5a signaling using receptor antagonists accelerated tumor growth in multiple HNSCC cell lines and corresponded with increased frequency of regulatory T cell (Treg) populations. Local C3a and C5a signaling has importance for CD4 T cell homeostasis and eventual development into effector phenotypes. Interruption of this signaling axis drives a phenotypic conversion of CD4+ T cells into Tregs, characterized by enhanced expression of Foxp3. Depletion of Tregs reversed tumor growth, and combination of Treg depletion and C3a and C5a receptor inhibition decreased tumor growth below that of the control groups. Complete knockout of C3 does not harbor the expected effect on tumor growth, indicating a still undetermined compensatory mechanism. Dexamethasone is frequently prescribed to patients undergoing RT and inhibits complement activation. We report no deleterious effects associated with dexamethasone due to complement inhibition. CONCLUSIONS: Our data establish Tregs as a pro-tumorigenic driver during complement inhibition and provide evidence that targeted C3a and C5a receptor inhibition may add therapeutic advantage when coupled with anti-Treg therapy.
Subject(s)
Complement Inactivating Agents/toxicity , Head and Neck Neoplasms/metabolism , Lymphocytes, Tumor-Infiltrating/drug effects , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Receptors, Complement/antagonists & inhibitors , Squamous Cell Carcinoma of Head and Neck/metabolism , T-Lymphocytes, Regulatory/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Complement C3/genetics , Complement C3/metabolism , Dexamethasone/toxicity , Forkhead Transcription Factors/metabolism , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/pathology , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Receptor, Anaphylatoxin C5a/metabolism , Receptors, Complement/metabolism , Signal Transduction , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Time Factors , Tumor Burden/drug effectsABSTRACT
Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.See related commentary by Garcia Garcia et al., p. 3158 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.