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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.

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.

Supplementary Figure 6 (A) Survival curve comparing patients with high expression of FLT3, the FLT3LG receptor, to patients with low expression of FLT3 expression. (B) Survival curve comparing patients with high expression of FLT3LG to patients with low expression of FLT3LG.

Supplementary Figure 5 (A) IL-15 amounts in serum of Balb/c mice treated with RT, anti-CD25, anti-asialoGM1 to deplete NK cells, and FLT3L; **** p{less than or equal to}0.0001; n=6. (B) Flow cytometric analysis of DCs MOC2 tumors after treatment with radiation therapy and anti-CD25; n=5. (C) growth curves of MOC2 tumors in individual mice treated with radiation therapy, anti-CD25, and anti-CD137.

Supplementary Figure 3 (A) MOC2 tumor volumes of mice treated with RT, anti-CD25, and pLEV vehicle control (black line); RT, anti-CD25, and pLEV FLT3L plasmid (green line), and RT + anti-CD25 (red line). n=7. (B) Flow Cytometry analysis of CD8 T cell populations in MOC2 tumors and lymph nodes (LN) of C57 BL/6 mice. MOC2 tumors were harvested 3 days after RT, NK cell depletion and FLT3L therapy; n=6.

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.

Supplementary Figure 1 (A) Flow cytometry analysis of NK cell population expression of NKG2A in MOC2 tumors 3 days after RT; n=3. (B). Flow cytometry analysis of NK cell populations in MOC2 tumors 3 days after RT and anti-NKG2A therapy; n=6. (C) Flow cytometry analysis of CD4 T cell populations in MOC2 tumors 3 days after RT, anti-NKG2A therapy and NK cell depletion with NK1.1 antibody; n=6. (D) MOC2 tumor volumes of C57BL/6 mice treated with RT and isotype control (red line), anti-CD137, anti-CD25, and anti-NKG2A (blue line), anti-CD17 and antiCD25 (black line); n=7.

Supplementary Figure from Targeting Treg-Expressed STAT3 Enhances NK-Mediated Surveillance of Metastasis and Improves Therapeutic Response in Pancreatic Adenocarcinoma

Supplementary Figure 4 (A) Gating strategy used in flow cytometry analysis analyzing NK46+ CD3-NK cells, CD11c+ MHCII+ DCs, CD3+ CD4+ T cells, and CD3+ CD8+ T cells. (B) Flow cytometry analysis of NK cell populations in blood from WT C57BL/6 mice treated with RT, anti-CD25, and anti-CD137; n=4. (C) Flow cytometry of CD4 T cell populations in tumors from WT C57BL/6 mice treated with RT, anti-CD25, and anti-CD137 and from DEREG mice treated with RT, anti-CD25, antiCD137, and diphtheria toxin (DT); *p{less than or equal to}0.05, ** p{less than or equal to}0.01; n=6. (D) FLT3L present in serum of C57BL6 treated with radiation therapy, anti-CD25, and anti-CD137, and DEREG mice treated with radiation therapy, anti-CD25, anti-CD137, and diphtheria toxin (DT); ** p{less than or equal to}0.01; n=6

Supplementary Figure 2 (A) Flow cytometry analysis of LY2 tumors analyzing DC populations; n=6. (B) Analysis of FLT3L present in the serum of LY2 bearing mice treated with radiation therapy and isotype antibodies, radiation therapy and anti-CD25 antibody, radiation therapy and anti-CD25 antibody and anti-asialoGM1 antibody to deplete NK cells, and radiation therapy, anti-CD25 antibody, anti-asialoGM1 antibody to deplete NK cells, and FLT3L therapy; n=5.

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.

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.

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.

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.

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.

BackgroundResistance 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.MethodsWe 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.ResultsIn 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.ConclusionsOur 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.