ABSTRACT
The EphB4-ephrinB2 signaling axis has been heavily implicated in metastasis across numerous cancer types. Our emerging understanding of the dichotomous roles that EphB4 and ephrinB2 play in head and neck squamous cell carcinoma (HNSCC) poses a significant challenge to rational drug design. We find that EphB4 knockdown in cancer cells enhances metastasis in preclinical HNSCC models by augmenting immunosuppressive cells like T regulatory cells (Tregs) within the tumor microenvironment. EphB4 inhibition in cancer cells also amplifies their ability to metastasize through increased expression of genes associated with epithelial mesenchymal transition and hallmark pathways of metastasis. In contrast, vascular ephrinB2 knockout coupled with radiation therapy (RT) enhances anti-tumor immunity, reduces Treg accumulation into the tumor, and decreases metastasis. Notably, targeting the EphB4-ephrinB2 signaling axis with the engineered EphB4 ligands EFNB2-Fc-His and Fc-TNYL-RAW-GS reduces local tumor growth and distant metastasis in a preclinical model of HNSCC. Our data suggest that targeted inhibition of vascular ephrinB2 while avoiding inhibition of EphB4 in cancer cells could be a promising strategy to mitigate HNSCC metastasis.
ABSTRACT
Clinically approved head and neck squamous cell carcinoma (HNSCC) immunotherapies manipulate the immune checkpoint blockade (ICB) axis but have had limited success outside of recurrent/metastatic disease. Interleukin-7 (IL7) has been shown to be essential for effector T-cell survival, activation, and proliferation. Here, we show that IL7 in combination with radiotherapy (RT) is effective in activating CD8 + T-cells for reducing tumor growth. Our studies were conducted using both human papillomavirus related and unrelated orthotopic HNSCC murine models. Immune populations from the tumor, draining lymph nodes, and blood were compared between treatment groups and controls using flow cytometry, proteomics, immunofluorescence staining, and RNA sequencing. Treatment with RT and IL7 (RT + IL7) resulted in significant tumor growth reduction, high CD8 T-cell tumor infiltration, and increased proliferation of T-cell progenitors in the bone marrow. IL7 also expanded a memory-like subpopulation of CD8 T-cells. These results indicate that IL7 in combination with RT can serve as an effective immunotherapy strategy outside of the conventional ICB axis to drive the antitumor activity of CD8 T-cells.
Subject(s)
Head and Neck Neoplasms , Interleukin-7 , Humans , Mice , Animals , Squamous Cell Carcinoma of Head and Neck/radiotherapy , Memory T Cells , CD8-Positive T-Lymphocytes , Head and Neck Neoplasms/radiotherapy , Tumor MicroenvironmentABSTRACT
PURPOSE: Head and neck cancer (HNC) improvements are stagnant, even with advances in immunotherapy. Our previous clinical trial data show that altered fatty acid (FA) metabolism correlates with outcome. We hypothesized that pharmacologic and dietary modulation of FA catabolism will affect therapeutic efficacy. EXPERIMENTAL DESIGN: We performed in vivo and in vitro experiments using PPARα agonism with fenofibrate (FF) or high oleic acid diets (OAD) with radiotherapy, generating metabolomic, proteomic, stable isotope tracing, extracellular flux analysis, and flow-cytometric data to investigate these alterations. RESULTS: FF improved antitumor efficacy of high dose per fraction radiotherapy in HNC murine models, whereas the OAD reversed this effect. FF-treated mice on the control diet had evidence of increased FA catabolism. Stable isotope tracing showed less glycolytic utilization by ex vivo CD8+ T cells. Improved efficacy correlated with intratumoral alterations in eicosanoid metabolism and downregulated mTOR and CD36. CONCLUSIONS: Metabolic intervention with increased FA catabolism improves the efficacy of HNC therapy and enhances antitumoral immune response.
Subject(s)
Head and Neck Neoplasms , Oleic Acid , PPAR alpha , Animals , PPAR alpha/agonists , Mice , Oleic Acid/pharmacology , Humans , Head and Neck Neoplasms/immunology , Fenofibrate/pharmacology , Cell Line, Tumor , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Fatty Acids/metabolism , Disease Models, AnimalABSTRACT
BACKGROUND: Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. METHODS: We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. FINDINGS: Calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 T cell activity in vivo. CONCLUSIONS: Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 T cells and activated CD8 T cells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. FUNDING: 1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).
Subject(s)
Calcitonin Gene-Related Peptide , Head and Neck Neoplasms , Animals , Humans , Mice , Calcitonin Gene-Related Peptide/metabolism , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck , Tumor MicroenvironmentABSTRACT
Surgical removal of lymph nodes (LNs) to prevent metastatic recurrence, including sentinel lymph node biopsy (SLNB) and completion lymph node dissection (CLND), are performed in routine practice. However, it remains controversial whether removing LNs which are critical for adaptive immune responses impairs immune checkpoint blockade (ICB) efficacy. Here, our retrospective analysis demonstrated that stage III melanoma patients retain robust response to anti-PD1 inhibition after CLND. Using orthotopic murine mammary carcinoma and melanoma models, we show that responses to ICB persist in mice after TDLN resection. Mechanistically, after TDLN resection, antigen can be re-directed to distant LNs, which extends the responsiveness to ICB. Strikingly, by evaluating head and neck cancer patients treated by neoadjuvant durvalumab and irradiation, we show that distant LNs (metastases-free) remain reactive in ICB responders after tumor and disease-related LN resection, hence, persistent anti-cancer immune reactions in distant LNs. Additionally, after TDLN dissection in murine models, ICB delivered to distant LNs generated greater survival benefit, compared to systemic administration. In complete responders, anti-tumor immune memory induced by ICB was systemic rather than confined within lymphoid organs. Based on these findings, we constructed a computational model to predict free antigen trafficking in patients that will undergo LN dissection.
ABSTRACT
The implementation of cancer immunotherapies has seen limited clinical success in head and neck squamous cell carcinoma (HNSCC). Interleukin-2 (IL-2), which modulates the survival and functionality of lymphocytes, is an attractive target for new immunotherapies but one that is limited by presence of regulatory T cells (Tregs) expressing the high-affinity IL-2Rα. The bispecific immunocytokine PD1-IL2v preferentially delivers IL-2 signaling through IL-2Rßγ on PD-1-expressing cells. Selectively targeting the intermediate-affinity IL-2Rßγ can be leveraged to induce anti-tumor immune responses in effector T cells and natural killer (NK) cells while limiting the negative regulation of IL-2Rα activation on Tregs. Using radiation therapy (RT) in combination with PD1-IL2v improves local tumor control and survival, and controls metastatic spread in orthotopic HNSCC tumor models. PD1-IL2v drives systemic activation and expansion of circulating and tumor-infiltrating cytotoxic T cells and NK cells while limiting Treg-mediated immunosuppression. These data show that PD1-L2v induces durable systemic tumor control in HNSCC.
Subject(s)
Head and Neck Neoplasms , Interleukin-2 , Humans , Squamous Cell Carcinoma of Head and Neck/radiotherapy , Interleukin-2/pharmacology , Interleukin-2/therapeutic use , Interleukin-2 Receptor alpha Subunit , T-Lymphocytes, Cytotoxic , Head and Neck Neoplasms/radiotherapyABSTRACT
In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rß and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , CD8-Positive T-Lymphocytes , Programmed Cell Death 1 Receptor , Interleukin-2 Receptor alpha Subunit/therapeutic use , Interleukin-2/pharmacology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/radiotherapy , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/radiotherapy , Carcinoma, Pancreatic Ductal/drug therapy , ImmunotherapyABSTRACT
Five-year survival for human papilloma virus-unrelated head and neck squamous cell carcinomas remain below 50%. We assessed the safety of administering combination hypofractionated stereotactic body radiation therapy with single-dose durvalumab (anti-PD-L1) neoadjuvantly (n = 21) ( NCT03635164 ). The primary endpoint of the study was safety, which was met. Secondary endpoints included radiographic, pathologic and objective response; locoregional control; progression-free survival; and overall survival. Among evaluable patients at an early median follow-up of 16 months (448 d or 64 weeks), overall survival was 80.1% with 95% confidence interval (95% CI) (62.0%, 100.0%), locoregional control and progression-free survival were 75.8% with 95% CI (57.5%, 99.8%), and major pathological response or complete response was 75% with 95% exact CI (51.6%, 100.0%). For patients treated with 24 Gy, 89% with 95% CI (57.1%, 100.0%) had MPR or CR. Using high-dimensional multi-omics and spatial data as well as biological correlatives, we show that responders had: (1) an increase in effector T cells; (2) a decrease in immunosuppressive cells; and (3) an increase in antigen presentation post-treatment.
Subject(s)
Head and Neck Neoplasms , Papillomavirus Infections , Radiosurgery , Humans , Head and Neck Neoplasms/therapy , Neoadjuvant Therapy/adverse effects , Papillomavirus Infections/complications , Radiosurgery/adverse effects , Squamous Cell Carcinoma of Head and Neck/therapyABSTRACT
In the setting of conventional radiation therapy, even when combined with immunotherapy, head and neck cancer often recurs locally and regionally. Elective nodal irradiation (ENI) is commonly employed to decrease regional recurrence. Given our developing understanding that immune cells are radio-sensitive, and that T cell priming occurs in the draining lymph nodes (DLNs), we hypothesize that radiation therapy directed at the primary tumor only will increase the effectiveness of immunotherapies. We find that ENI increases local, distant, and metastatic tumor growth. Multi-compartmental analysis of the primary/distant tumor, the DLNs, and the blood shows that ENI decreases the immune response systemically. Additionally, we find that ENI decreases antigen-specific T cells and epitope spreading. Treating the primary tumor with radiation and immunotherapy, however, fails to reduce regional recurrence, but this is reversed by either concurrent sentinel lymph node resection or irradiation. Our data support using lymphatic sparing radiation therapy for head and neck cancer.
Subject(s)
Head and Neck Neoplasms , Sentinel Lymph Node , Humans , Head and Neck Neoplasms/radiotherapy , Combined Modality Therapy , Lymph Node Excision , ImmunotherapyABSTRACT
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
PURPOSE: Metastasis remains a major hurdle in treating aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). Improving response to treatment, therefore, requires a more detailed characterization of the cellular populations involved in controlling metastatic burden. EXPERIMENTAL DESIGN: PDAC patient tissue samples were subjected to RNA sequencing analysis to identify changes in immune infiltration following radiotherapy. Genetically engineered mouse strains in combination with orthotopic tumor models of PDAC were used to characterize disease progression. Flow cytometry was used to analyze tumor infiltrating, circulating, and nodal immune populations. RESULTS: We demonstrate that although radiotherapy increases the infiltration and activation of dendritic cells (DC), it also increases the infiltration of regulatory T cells (Treg) while failing to recruit natural killer (NK) and CD8 T cells in PDAC patient tissue samples. In murine orthotopic tumor models, we show that genetic and pharmacologic depletion of Tregs and NK cells enhances and attenuates response to radiotherapy, respectively. We further demonstrate that targeted inhibition of STAT3 on Tregs results in improved control of local and distant disease progression and enhanced NK-mediated immunosurveillance of metastasis. Moreover, combination treatment of STAT3 antisense oligonucleotide (ASO) and radiotherapy invigorated systemic immune activation and conferred a survival advantage in orthotopic and metastatic tumor models. Finally, we show the response to STAT3 ASO + radiotherapy treatment is dependent on NK and DC subsets. CONCLUSIONS: Our results suggest targeting Treg-mediated immunosuppression is a critical step in mediating a response to treatment, and identifying NK cells as not only a prognostic marker of improved survival, but also as an effector population that functions to combat metastasis.
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/therapy , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/therapy , Disease Progression , Humans , Mice , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/therapy , STAT3 Transcription Factor/genetics , T-Lymphocytes, Regulatory , Pancreatic NeoplasmsABSTRACT
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
Resistance to radiation therapy (RT) remains an obstacle in HPV-negative head and neck squamous cell carcinomas (HNSCCs)-even with a combined RT-immunotherapy approach. Jak-Stat proteins have long been studied for both their immune regulatory role in the host immune response as well as their cancer cell signaling role in shaping the tumor microenvironment (TME). Here, we identify STAT1 as a mediator of radioresistance in HPV-negative preclinical mouse models of HNSCC, by which knockout of STAT1 in the cancer cell (STAT1 KO)-but not in the host-resulted in decreased tumor growth alongside increased immune activation. We show that RT increases STAT1/pSTAT1 expression, which may act as a marker of radioresistance. Whereas RT increased JAK-STAT and interferon (IFN) signaling, transcriptomic analysis revealed that STAT1 KO in the cancer cell resulted in decreased expression of IFN-associated genes of resistance. In vitro experiments showed that STAT1 KO increased T cell chemoattraction and decreased baseline growth. These results indicate that STAT1 may serve a tumor-promoting role in the cancer cell and will inform biomarker development and treatment regimens for HNSCC incorporating RT.
Subject(s)
Head and Neck Neoplasms , Papillomavirus Infections , Animals , Cell Line, Tumor , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/radiotherapy , Humans , Immunotherapy , Mice , STAT1 Transcription Factor/genetics , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/radiotherapy , T-Lymphocytes , Tumor MicroenvironmentABSTRACT
Presentation of tumor antigens is a critical step in producing a robust antitumor immune response. Classically tumor antigens are thought to be presented to both CD8 and CD4 T cells by professional antigen-presenting cells (pAPCs) like dendritic cells using major histocompatibility complexes (MHC) I and II. But recent evidence suggests that in the tumor microenvironment (TME) cells other than pAPCs are capable of presenting tumor antigens on both MHC I and II. The evidence currently available on tumor antigen presentation by epithelial cells, vascular endothelial cells (VECs), fibroblasts, and cancer cells is reviewed herein. We refer to these cell types in the TME as "amateur" APCs (aAPCs). These aAPCs greatly outnumber pAPCs in the TME and could, potentially, play a significant role in priming an antitumor immune response. This new evidence supports a different perspective on antigen presentation and suggests new approaches that can be taken in designing immunotherapies to increase T cell priming.
Subject(s)
Endothelial Cells , Tumor Microenvironment , Antigen Presentation , Antigen-Presenting Cells , Antigens, Neoplasm/metabolism , Dendritic Cells , HumansABSTRACT
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
Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.