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1.
Nat Commun ; 15(1): 3503, 2024 Apr 25.
Article En | MEDLINE | ID: mdl-38664372

The assembly of chiral molecules with multiple stereogenic elements is challenging, and, despite of indisputable advances, largely limited to toxic, cost-intensive and precious metal catalysts. In sharp contrast, we herein disclose a versatile C-H alkylation using a non-toxic, low-cost iron catalyst for the synthesis of substituted indoles with two chiral elements. The key for achieving excellent diastereo- and enantioselectivity was substitution on a chiral N-heterocyclic carbene ligand providing steric hindrance and extra represented by noncovalent interaction for the concomitant generation of C-N axial chirality and C-stereogenic center. Experimental and computational mechanistic studies have unraveled the origin of the catalytic efficacy and stereoselectivity.

3.
J Immunother Cancer ; 11(3)2023 03.
Article En | MEDLINE | ID: mdl-36990508

BACKGROUND: High-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined. METHODS: To understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles. RESULTS: We identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted. CONCLUSIONS: These data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.


Head and Neck Neoplasms , Oropharyngeal Neoplasms , Papillomavirus Infections , Humans , Human papillomavirus 16 , Tumor Microenvironment
5.
Nat Cancer ; 3(11): 1300-1317, 2022 11.
Article En | MEDLINE | ID: mdl-36434392

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.


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/therapy
6.
Nat Commun ; 13(1): 3535, 2022 06 20.
Article En | MEDLINE | ID: mdl-35725568

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.


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 Microenvironment
7.
Clin Cancer Res ; 28(5): 1013-1026, 2022 Mar 01.
Article En | MEDLINE | ID: mdl-34862244

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.


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 Neoplasms
8.
Mol Ther ; 30(3): 1149-1162, 2022 03 02.
Article En | MEDLINE | ID: mdl-34793974

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.


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/genetics
9.
Cancer Immunol Immunother ; 71(5): 1049-1061, 2022 May.
Article En | MEDLINE | ID: mdl-34559306

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.


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 Microenvironment
10.
Chem Commun (Camb) ; 57(95): 12784-12787, 2021 Nov 30.
Article En | MEDLINE | ID: mdl-34782896

A mechanistic study is performed on the reaction method for iron-catalyzed C-H methylation with AlMe3 reagent, previously proposed to involve cyclometalated iron(III) intermediates and an iron(III)/(I) reaction cycle. Detailed spectroscopic studies (57Fe Mössbauer, EPR) during catalysis and in stoichiometric reactions identify iron(II) complexes, including cyclometalated iron(II) intermediates, as the major iron species formed in situ under catalytic reaction conditions. Reaction studies identify a cyclometalated iron(II)-methyl species as the key intermediate leading to C-H methylated product upon reaction with oxidant, consistent with a previously proposed iron(II)/iron(III)/iron(I) reaction manifold for C-H arylation.

11.
Clin Cancer Res ; 27(22): 6235-6249, 2021 11 15.
Article En | MEDLINE | ID: mdl-34518311

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.


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 Microenvironment
12.
J Immunother Cancer ; 9(4)2021 04.
Article En | MEDLINE | ID: mdl-33883256

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.


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/metabolism
13.
J Immunother Cancer ; 9(3)2021 03.
Article En | MEDLINE | ID: mdl-33789881

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.


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 effects
14.
Cancer Res ; 81(12): 3255-3269, 2021 06 15.
Article En | MEDLINE | ID: mdl-33526513

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.


ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Carcinoma, Pancreatic Ductal/radiotherapy , Epithelial-Mesenchymal Transition , Fibrosis/pathology , Gamma Rays/adverse effects , Membrane Proteins/metabolism , Pancreatic Neoplasms/radiotherapy , Radiation Injuries/pathology , ADAM10 Protein/antagonists & inhibitors , ADAM10 Protein/genetics , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Animals , Antifibrotic Agents/therapeutic use , Apoptosis , Carcinoma, Pancreatic Ductal/pathology , Cell Movement , Cell Proliferation , Ephrin-B2/blood , Female , Fibrosis/drug therapy , Fibrosis/etiology , Fibrosis/metabolism , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/pathology , Prognosis , Radiation Injuries/drug therapy , Radiation Injuries/etiology , Radiation Injuries/metabolism , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
15.
Cancer Immunol Immunother ; 70(4): 989-1000, 2021 Apr.
Article En | MEDLINE | ID: mdl-33097963

Pancreatic ductal adenocarcinoma (PDAC) has a heterogeneous tumor microenvironment (TME) comprised of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages, neutrophils, regulatory T cells, and myofibroblasts. The precise mechanisms that regulate the composition of the TME and how they contribute to radiotherapy (RT) response remain poorly understood. In this study, we analyze changes in immune cell populations and circulating chemokines in patient samples and animal models of pancreatic cancer to characterize the immune response to radiotherapy. Further, we identify STAT3 as a key mediator of immunosuppression post-RT. We found granulocytic MDSCs (G-MDSCs) and neutrophils to be increased in response to RT in murine and human PDAC samples. We also found that RT-induced STAT3 phosphorylation correlated with increased MDSC infiltration and proliferation. Targeting STAT3 using an anti-sense oligonucleotide in combination with RT circumvented RT-induced MDSC infiltration, enhanced the proportion of effector T cells, and improved response to RT. In addition, STAT3 inhibition contributed to the remodeling of the PDAC extracellular matrix when combined with RT, resulting in decreased collagen deposition and fibrotic tissue formation. Collectively, our data provide evidence that targeting STAT3 in combination with RT can mitigate the pro-tumorigenic effects of RT and improve tumor response.


Carcinoma, Pancreatic Ductal/radiotherapy , Gamma Rays , Myeloid-Derived Suppressor Cells/immunology , Oligonucleotides, Antisense/genetics , Pancreatic Neoplasms/radiotherapy , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Apoptosis , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation , Female , Humans , Immunosuppression Therapy , Mice , Mice, Inbred C57BL , Mice, Nude , Myeloid-Derived Suppressor Cells/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Prognosis , STAT3 Transcription Factor/genetics , T-Lymphocytes, Regulatory/immunology , Tumor Cells, Cultured , Tumor Microenvironment
16.
Mol Carcinog ; 59(9): 1064-1075, 2020 09.
Article En | MEDLINE | ID: mdl-32567728

The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.


Biomarkers, Tumor/metabolism , Cell Proliferation , Ephrin-B2/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Receptor, EphB4/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Movement , Ephrin-B2/genetics , Female , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Phosphorylation , Prognosis , Receptor, EphB4/genetics , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
17.
J Vis Exp ; (146)2019 04 22.
Article En | MEDLINE | ID: mdl-31058896

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.


Head and Neck Neoplasms , Neoplasms, Experimental , Squamous Cell Carcinoma of Head and Neck , Animals , Cell Line, Tumor , Feasibility Studies , Head and Neck Neoplasms/immunology , Humans , Lymph Nodes/pathology , Mice , Mice, Inbred BALB C , Mouth Mucosa/pathology , Neoplasm Transplantation , Squamous Cell Carcinoma of Head and Neck/immunology , Tumor Microenvironment
18.
Clin Cancer Res ; 25(11): 3352-3365, 2019 06 01.
Article En | MEDLINE | ID: mdl-30944125

PURPOSE: A driving factor in pancreatic ductal adenocarcinoma (PDAC) treatment resistance is the tumor microenvironment, which is highly immunosuppressive. One potent immunologic adjuvant is radiotherapy. Radiation, however, has also been shown to induce immunosuppressive factors, which can contribute to tumor progression and formation of fibrotic tumor stroma. To capitalize on the immunogenic effects of radiation and obtain a durable tumor response, radiation must be rationally combined with targeted therapies to mitigate the influx of immunosuppressive cells and fibrosis. One such target is ephrinB2, which is overexpressed in PDAC and correlates negatively with prognosis.Experimental Design: On the basis of previous studies of ephrinB2 ligand-EphB4 receptor signaling, we hypothesized that inhibition of ephrinB2-EphB4 combined with radiation can regulate the microenvironment response postradiation, leading to increased tumor control in PDAC. This hypothesis was explored using both cell lines and in vivo human and mouse tumor models. RESULTS: Our data show this treatment regimen significantly reduces regulatory T-cell, macrophage, and neutrophil infiltration and stromal fibrosis, enhances effector T-cell activation, and decreases tumor growth. Furthermore, our data show that depletion of regulatory T cells in combination with radiation reduces tumor growth and fibrosis. CONCLUSIONS: These are the first findings to suggest that in PDAC, ephrinB2-EphB4 interaction has a profibrotic, protumorigenic role, presenting a novel and promising therapeutic target.


Ephrin-B2/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Receptor, EphB4/metabolism , Tumor Microenvironment/drug effects , Tumor Microenvironment/radiation effects , Animals , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Biomarkers , Cell Line, Tumor , Disease Models, Animal , Ephrin-B2/antagonists & inhibitors , Ephrin-B2/genetics , Female , Flow Cytometry , Gene Expression , Gene Knockdown Techniques , Humans , Immunohistochemistry , Mice , Molecular Targeted Therapy/adverse effects , Molecular Targeted Therapy/methods , Neutrophils/immunology , Neutrophils/metabolism , Pancreatic Neoplasms/therapy , Radiotherapy/adverse effects , Radiotherapy/methods , Receptor, EphB4/antagonists & inhibitors , Receptor, EphB4/genetics , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Xenograft Model Antitumor Assays
19.
Cancer Res ; 79(10): 2722-2735, 2019 05 15.
Article En | MEDLINE | ID: mdl-30894369

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact, resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.


Ephrin-B2/metabolism , Head and Neck Neoplasms/metabolism , Receptor, EphB4/metabolism , Signal Transduction , Squamous Cell Carcinoma of Head and Neck/metabolism , Tumor Microenvironment/immunology , Chemoradiotherapy , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/therapy , Heterografts , Humans , Macrophages/immunology , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/therapy
20.
J Natl Cancer Inst ; 111(12): 1339-1349, 2019 12 01.
Article En | MEDLINE | ID: mdl-30863843

BACKGROUND: Radioresistance represents a major problem in the treatment of head and neck cancer (HNC) patients. To improve response, understanding tumor microenvironmental factors that contribute to radiation resistance is important. Regulatory T cells (Tregs) are enriched in numerous cancers and can dampen the response to radiation by creating an immune-inhibitory microenvironment. The purpose of this study was to investigate mechanisms of Treg modulation by radiation in HNC. METHODS: We utilized an orthotopic mouse model of HNC. Anti-CD25 was used for Treg depletion. Image-guided radiation was delivered to a dose of 10 Gy. Flow cytometry was used to analyze abundance and function of intratumoral immune cells. Enzyme-linked immunosorbent assay was performed to assess secreted factors. For immune-modulating therapies, anti-PD-L1, anti-CTLA-4, and STAT3 antisense oligonucleotide (ASO) were used. All statistical tests were two-sided. RESULTS: Treatment with anti-CD25 and radiation led to tumor eradication (57.1%, n = 4 of 7 mice), enhanced T-cell cytotoxicity compared with RT alone (CD4 effector T cells [Teff]: RT group mean = 5.37 [ 0.58] vs RT + αCD25 group mean =10.71 [0.67], P = .005; CD8 Teff: RT group mean = 9.98 [0.81] vs RT + αCD25 group mean =16.88 [2.49], P = .01) and induced tumor antigen-specific memory response (100.0%, n = 4 mice). In contrast, radiation alone or when combined with anti-CTLA4 did not lead to durable tumor control (0.0%, n = 7 mice). STAT3 inhibition in combination with radiation, but not as a single agent, improved tumor growth delay, decreased Tregs, myeloid-derived suppressor cells, and M2 macrophages and enhanced effector T cells and M1 macrophages. Experiments in nude mice inhibited the benefit of STAT3 ASO and radiation. CONCLUSION: We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving radiation.


Head and Neck Neoplasms/radiotherapy , Lymphocyte Depletion , Radioimmunotherapy/methods , STAT3 Transcription Factor/antagonists & inhibitors , Squamous Cell Carcinoma of Head and Neck/radiotherapy , T-Lymphocytes, Regulatory/radiation effects , Analysis of Variance , Animals , Cytotoxicity, Immunologic , Female , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/immunology , Humans , Immunologic Memory , Interleukin-2 Receptor alpha Subunit/genetics , Lymphocyte Depletion/methods , Macrophages/radiation effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Myeloid-Derived Suppressor Cells/radiation effects , Radiation Tolerance , Radiotherapy, Image-Guided , STAT3 Transcription Factor/metabolism , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta1/genetics , Tumor Microenvironment
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