Type I interferon signaling promotes radioresistance in head and neck cancer.

Despite the promise of concurrent radiotherapy (RT) and immunotherapy in head and neck cancer (HNC), multiple randomized trials of this combination have had disappointing results. To evaluate potential immunologic mechanisms of RT resistance, we compared pre-treatment HNCs that developed RT resistance to a matched cohort that achieved curative status. Gene set enrichment analysis demonstrated that a pre-treatment pro-immunogenic tumor microenvironment (TME), including type II interferon [interferon gamma (IFNγ)] and tumor necrosis factor alpha (TNFα) signaling, predicted cure while type I interferon [interferon alpha (IFNα)] enrichment was associated with an immunosuppressive TME found in tumors that went on to recur. We then used immune deconvolution of RNA sequencing datasets to evaluate immunologic cell subset enrichment. This identified M2 macrophage signaling associated with type I IFN pathway expression in RT-recurrent disease. To further dissect mechanism, we then evaluated differential gene expression between pre-treatment and RT-resistant HNCs from sampled from the same patients at the same anatomical location in the oral cavity. Here, recurrent samples exhibited upregulation of type I IFN-stimulated genes (ISGs) including members of the IFN-induced protein with tetratricopeptide repeats (IFIT) and IFN-induced transmembrane (IFITM) gene families. While several ISGs were upregulated in each recurrent cancer, IFIT2 was significantly upregulated in all recurrent tumors when compared with the matched pre-RT specimens. Based on these observations, we hypothesized sustained type I IFN signaling through ISGs, such as IFIT2, may suppress the intra-tumoral immune response thereby promoting radiation resistance.

Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

BACKGROUND: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) remains a treatment-resistance disease with limited response to immunotherapy. While T cells in HNSCC are known to display phenotypic dysfunction, whether they retain rescuable functional capacity and tumor-killing capability remains unclear. METHODS: To investigate the functionality and tumor-specificity of tumor-infiltrating lymphocytes (TILs) across HNSCCs, malignant cell lines and TILs were derived from 31 HPV-negative HNSCCs at the time of standard surgical resection. T cell functional capacity was evaluated through ex vivo expansion, immunophenotyping, and IsoLight single-cell proteomics. Tumor-specificity was investigated through both bulk and single-cell tumor-TIL co-culture. RESULTS: TILs could be successfully generated from 24 patients (77%), including both previously untreated and radiation recurrent HNSCCs. We demonstrate that across HNSCCs, TILs express multiple exhaustion markers but maintain a predominantly effector memory phenotype. After ex vivo expansion, TILs retain immunogenic functionality even from radiation-resistant, exhausted, and T cell-depleted disease. We further demonstrate tumor-specificity of T cells across HNSCC patients through patient-matched malignant cell-T cell co-culture. Finally, we use optofluidic technology to establish an autologous single tumor cell-single T cell co-culture platform for HNSCC. Cells derived from three HNSCC patients underwent single-cell co-culture which enabled identification and visualization of individual tumor-killing TILs in real-time in all patients. CONCLUSIONS: These studies show that cancer-specific T cells exist across HNSCC patients with rescuable immunogenicity and can be identified on a single-cell level. These data lay the foundation for development of patient-specific T cell immunotherapies in HNSCC.

Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis.

Bone marrow vascular endothelial cells (BM EC) regulate multiple myeloma pathogenesis. Identification of the mechanisms underlying this interaction could lead to the development of improved strategies for treating multiple myeloma. Here, we performed a transcriptomic analysis of human ECs with high capacity to promote multiple myeloma growth, revealing overexpression of the receptor tyrosine kinases, EPHB1 and EPHB4, in multiple myeloma-supportive ECs. Expression of ephrin B2 (EFNB2), the binding partner for EPHB1 and EPHB4, was significantly increased in multiple myeloma cells. Silencing EPHB1 or EPHB4 in ECs suppressed multiple myeloma growth in coculture. Similarly, loss of EFNB2 in multiple myeloma cells blocked multiple myeloma proliferation and survival in vitro, abrogated multiple myeloma engraftment in immune-deficient mice, and increased multiple myeloma sensitivity to chemotherapy. Administration of an EFNB2-targeted single-chain variable fragment also suppressed multiple myeloma growth in vivo. In contrast, overexpression of EFNB2 in multiple myeloma cells increased STAT5 activation, increased multiple myeloma cell survival and proliferation, and decreased multiple myeloma sensitivity to chemotherapy. Conversely, expression of mutant EFNB2 lacking reverse signaling capacity in multiple myeloma cells increased multiple myeloma cell death and sensitivity to chemotherapy and abolished multiple myeloma growth in vivo. Complementary analysis of multiple myeloma patient data revealed that increased EFNB2 expression is associated with adverse-risk disease and decreased survival. This study suggests that EFNB2 reverse signaling controls multiple myeloma pathogenesis and can be therapeutically targeted to improve multiple myeloma outcomes. SIGNIFICANCE: Ephrin B2 reverse signaling mediated by endothelial cells directly regulates multiple myeloma progression and treatment resistance, which can be overcome through targeted inhibition of ephrin B2 to abolish myeloma.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

PURPOSE: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. PROCEDURES: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. RESULTS: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. CONCLUSIONS: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

X-ray and MR Contrast Bearing Nanoparticles Enhance the Therapeutic Response of Image-Guided Radiation Therapy for Oral Cancer.

INTRODUCTION: Radiation therapy for head and neck squamous cell carcinoma is constrained by radiotoxicity to normal tissue. We demonstrate 100 nm theranostic nanoparticles for image-guided radiation therapy planning and enhancement in rat head and neck squamous cell carcinoma models. METHODS: PEG conjugated theranostic nanoparticles comprising of Au nanorods coated with Gadolinium oxide layers were tested for radiation therapy enhancement in 2D cultures of OSC-19-GFP-luc cells, and orthotopic tongue xenografts in male immunocompromised Salt sensitive or SS rats via both intratumoral and intravenous delivery. The radiation therapy enhancement mechanism was investigated. RESULTS: Theranostic nanoparticles demonstrated both X-ray/magnetic resonance contrast in a dose-dependent manner. Magnetic resonance images depicted optimal tumor-to-background uptake at 4 h post injection. Theranostic nanoparticle + Radiation treated rats experienced reduced tumor growth compared to controls, and reduction in lung metastasis. CONCLUSIONS: Theranostic nanoparticles enable preprocedure radiotherapy planning, as well as enhance radiation treatment efficacy for head and neck tumors.

Photoactivated HPPH-Liposomal therapy for the treatment of HPV-Negative head and neck cancer.

OBJECTIVES: Human Papillomavirus (HPV)-negative head and neck cancer (HNC) is an aggressive malignancy with a poor prognosis. To improve outcomes, we developed a novel liposomal targeting system embedded with 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), a chlorin-based photosensitizer. Upon exposure to 660 nm light, HPPH phototriggering generates reactive oxygen species. The objective of this study was to evaluate biodistribution and test efficacy of HPPH-liposomal therapy in a patient-derived xenograft (PDX) model of chemoradioresistant HNC. MATERIALS AND METHODS: PDX models were developed from two surgically resected HNCs (P033 and P038) recurrent after chemoradiation. HPPH-liposomes were created including trace amounts of DiR (Ex/Em 785/830 nm), a near infrared lipid probe. Liposomes were injected via tail vein into PDX models. Biodistribution was assessed at serial timepoints in tumor and end-organs through in vivo DiR fluorescence. To evaluate efficacy, tumors were treated with a cw-diode 660 nm laser (90 mW/cm2, 5 min). This experimental arm was compared to appropriate controls, including HPPH-liposomes without laser or vehicle with laser alone. RESULTS: HPPH-liposomes delivered via tail vein exhibited selective tumor penetration, with a peak concentration at 4 h. No systemic toxicity was observed. Treatment with combined HPPH-liposomes and laser resulted in improved tumor control relative to either vehicle or laser alone. Histologically, this manifested as both increased cellular necrosis and decreased Ki-67 staining in the tumors treated with combined therapy. CONCLUSIONS: These data demonstrate tumor-specific anti-neoplastic efficacy of HPPH-liposomal treatment for HNC. Importantly, this platform can be leveraged in future studies for targeted delivery of immunotherapies which can be packaged within HPPH-liposomes.

Biological sex differences in renin angiotensin system enzymes ACE and ACE2 regulate normal tissue response to radiation injury.

Introduction: In experimental animal models, biological sex-differences in the manifestation and severity of normal tissue radiation injury have been well-documented. Previously we demonstrated male and female rats have differential and highly reproducible responses to high-dose partial body irradiation (PBI) with male rats having greater susceptibility to both gastrointestinal acute radiation syndrome (GI-ARS) and radiation pneumonitis than female rats. Methods: In the current study, we have investigated whether differential expression of the renin-angiotensin system (RAS) enzymes angiotensin converting enzyme (ACE) and ACE2 contribute to the observed sex-related differences in radiation response. Results: During the period of symptomatic pneumonitis, the relative ratio of ACE to ACE2 (ACE/ACE2) protein in the whole lung was significantly increased by radiation in male rats alone. Systemic treatment with small molecule ACE2 agonist diminazene aceturate (DIZE) increased lung ACE2 activity and reduced morbidity during radiation pneumonitis in both sexes. Notably DIZE treatment also abrogated morbidity in male rats during GI-ARS. We then evaluated the contribution of the irradiated bone marrow (BM) compartment on lung immune cell infiltration and ACE imbalance during pneumonitis. Transplantation of bone marrow from irradiated donors increased both ACE-expressing myeloid cell infiltration and immune ACE activity in the lung during pneumonitis compared to non-irradiated donors. Discussion: Together, these data demonstrate radiation induces a sex-dependent imbalance in the renin-angiotensin system enzymes ACE and ACE2. Additionally, these data suggest a role for ACE-expressing myeloid cells in the pathogenesis of radiation pneumonitis. Finally, the observed sex-differences underscore the need for consideration of sex as a biological variable in the development of medical countermeasures for radiation exposure.

Biomarkers to Predict Lethal Radiation Injury to the Rat Lung.

Currently, there are no biomarkers to predict lethal lung injury by radiation. Since it is not ethical to irradiate humans, animal models must be used to identify biomarkers. Injury to the female WAG/RijCmcr rat has been well-characterized after exposure to eight doses of whole thorax irradiation: 0-, 5-, 10-, 11-, 12-, 13-, 14- and 15-Gy. End points such as SPECT imaging of the lung using molecular probes, measurement of circulating blood cells and specific miRNA have been shown to change after radiation. Our goal was to use these changes to predict lethal lung injury in the rat model, 2 weeks post-irradiation, before any symptoms manifest and after which a countermeasure can be given to enhance survival. SPECT imaging with 99mTc-MAA identified a decrease in perfusion in the lung after irradiation. A decrease in circulating white blood cells and an increase in five specific miRNAs in whole blood were also tested. Univariate analyses were then conducted on the combined dataset. The results indicated that a combination of percent change in lymphocytes and monocytes, as well as pulmonary perfusion volume could predict survival from radiation to the lungs with 88.5% accuracy (95% confidence intervals of 77.8, 95.3) with a p-value of < 0.0001 versus no information rate. This study is one of the first to report a set of minimally invasive endpoints to predict lethal radiation injury in female rats. Lung-specific injury can be visualized by 99mTc-MAA as early as 2 weeks after radiation.

Analysis of the urinary metabolic profiles in irradiated rats treated with Activated Protein C (APC), a potential mitigator of radiation toxicity.

PURPOSE: The goal of the current study was to identify longitudinal changes in urinary metabolites following IR exposure and to determine potential alleviation of radiation toxicities by administration of recombinant APC formulations. MATERIALS AND METHODS: Female adult WAG/RijCmcr rats were irradiated with 13.0 Gy leg-out partial body X-rays; longitudinally collected urine samples were subject to LC-MS based metabolomic profiling. Sub-cohorts of rats were treated with three variants of recombinant APC namely, rat wildtype (WT) APC, rat 3K3A mutant form of APC, and human WT APC as two bolus injections at 24 and 48 hours post IR. RESULTS: Radiation induced robust changes in the urinary profiles leading to oxidative stress, severe dyslipidemia, and altered biosynthesis of PUFAs, glycerophospholipids, sphingolipids, and steroids. Alterations were observed in multiple metabolic pathways related to energy metabolism, nucleotide biosynthesis and metabolism that were indicative of disrupted mitochondrial function and DNA damage. On the other hand, sub-cohorts of rats that were treated with rat wildtype-APC showed alleviation of radiation toxicities, in part, at the 90-day time point, while rat 3K3A-APC showed partial alleviation of radiation induced metabolic alterations 14 days after irradiation. CONCLUSIONS: Taken together, these results show that augmenting the Protein C pathway and activity via administration of recombinant APC may be an effective approach for mitigation of radiation induced normal tissue toxicity.

Cord Blood-Derived Endothelial Progenitor Cells Promote In Vivo Regeneration of Human Hematopoietic Bone Marrow.

PURPOSE: Victims of acute radiation exposure are susceptible to hematopoietic toxicity due to bone marrow damage and loss of mature blood elements. Here, we evaluated cord blood-derived endothelial progenitor cells (CB-EPCs) as a potential cellular therapy for mitigation of hematologic acute radiation syndrome. CB-EPCs express endothelial cell markers and maintain their growth characteristics beyond 10+ passages without diminishing their doubling capacity. Further, CB-EPCs can be cryopreserved in vapor-phase liquid nitrogen and easily recovered for propagation, making them an attractive nonimmunogenic cellular therapy for off-the-shelf use. Importantly, we show CB-EPCs have the capacity to potently expand adult human bone marrow hematopoietic progenitor cells both in vitro and in vivo. METHODS AND MATERIALS: To demonstrate the role of CB-EPCs in promoting in vivo human immune reconstitution after irradiation, we employed a novel humanized mouse model established by transplant of CD34+ bone marrow cells from 9 unique adult organ donors into immunocompromised NSG-SGM3 mice. The response of the humanized immune system to ionizing irradiation was then tested by exposure to 1 Gy followed by subcutaneous treatment of CB-EPCs, Food and Drug Administration-approved growth factor pegfilgrastim (0.3 mg/kg), or saline. RESULTS: At day 7, total human bone marrow was decreased by 80% in irradiated controls. However, treatment with either growth factor pegfilgrastim or CB-EPCs increased recovery of total human bone marrow by 2.5-fold compared with saline. Notably, CB-EPCs also increased recovery of both human CD34+ progenitors by 5-fold and colony-forming capacity by 3-fold versus saline. Additionally, CB-EPCs promoted recovery of endogenous bone marrow endothelial cells as observed by both increased vessel area and length compared with saline. CONCLUSIONS: These findings indicate the feasibility of using humanized mice engrafted with adult bone marrow for radiation research and the development of CB-EPCs as an off-the-shelf cellular therapy for mitigation of hematologic acute radiation syndrome.

IPW-5371 mitigates the delayed effects of acute radiation exposure in WAG/RijCmcr rats when started 15 days after PBI with bone marrow sparing.

PURPOSE: To test IPW-5371 for the mitigation of the delayed effects of acute radiation exposure (DEARE). Survivors of acute radiation exposure are at risk for developing delayed multi-organ toxicities; however, there are no FDA-approved medical countermeasures (MCM) to mitigate DEARE. METHODS: WAG/RijCmcr female rat model of partial-body irradiation (PBI), by shielding part of one hind leg, was used to test IPW-5371 (7 and 20 mg kg-1 d-1) for mitigation of lung and kidney DEARE when started 15 d after PBI. Rats were fed known amounts of IPW-5371 using a syringe, instead of delivery by daily oral gavage, sparing exacerbation of esophageal injury by radiation. The primary endpoint, all-cause morbidity was assessed over 215 d. Secondary endpoints: body weight, breathing rate and blood urea nitrogen were also assessed. RESULTS: IPW-5371 enhanced survival (primary endpoint) as well as attenuated secondary endpoints of lung and kidney injuries by radiation. CONCLUSION: To provide a window for dosimetry and triage, as well as avoid oral delivery during the acute radiation syndrome (ARS), the drug regimen was started at 15 d after 13.5 Gy PBI. The experimental design to test mitigation of DEARE was customized for translation in humans, using an animal model of radiation that was designed to simulate a radiologic attack or accident. The results support advanced development of IPW-5371 to mitigate lethal lung and kidney injuries after irradiation of multiple organs.

Delayed renal injury in survivors of hematologic acute radiation syndrome.

PURPOSE: A mass casualty disaster involving radiological or nuclear agents continues to be a public health concern which requires consideration of both acute and late tissue toxicities in exposed victims. With the advent of advanced treatment options for the mitigation of hematological injuries, there are likely to be survivors of total body irradiation (TBI) exposures as high as 8-10 Gy. These survivors are at risk for a range of delayed multi-organ morbidities including progressive renal failure. MATERIAL AND METHODS: Here, we established the WAG/RijCmcr rat as an effective model for the evaluation of medical countermeasures (MCM) for acute hematologic radiation syndrome (H-ARS). The LD50/30 dose for adult and pediatric WAG/RijCmcr rats was determined for both sexes. We then confirmed the FDA-approved MCM pegfilgrastim (peg-GCSF, Neulasta®) mitigates H-ARS in adult male and female rats. Finally, we evaluated survival and renal dysfunction up to 300 d post-TBI in male and female adult rats. RESULTS: In the WAG/RijCmcr rat model, 87.5% and 100% of adult rats succumb to lethal hematopoietic acute radiation syndrome (H-ARS) at TBI doses of 8 and 8.5 Gy, respectively. A single dose of the hematopoietic growth factor peg-GCSF administered at 24 h post-TBI improved survival during H-ARS. Peg-GCSF treatment improved 30 d survival from 12.5% to 83% at 8 Gy and from 0% to 63% at 8.5 Gy. We then followed survivors of H-ARS through day 300. Rats exposed to TBI doses greater than 8 Gy had a 26% reduction in survival over days 30-300 compared to rats exposed to 7.75 Gy TBI. Concurrent with the reduction in long-term survival, a dose-dependent impairment of renal function as assessed by blood urea nitrogen (BUN) and urine protein to urine creatinine ratio (UP:UC) was observed. CONCLUSION: Together, these data show survivors of H-ARS are at risk for the development of delayed renal toxicity and emphasize the need for the development of medical countermeasures for delayed renal injury.

Organ-Specific Endothelial Dysfunction Following Total Body Irradiation Exposure.

As the single cell lining of the heart and all blood vessels, the vascular endothelium serves a critical role in maintaining homeostasis via control of vascular tone, immune cell recruitment, and macromolecular transit. For victims of acute high-dose radiation exposure, damage to the vascular endothelium may exacerbate the pathogenesis of acute and delayed multi-organ radiation toxicities. While commonalities exist between radiation-induced endothelial dysfunction in radiosensitive organs, the vascular endothelium is known to be highly heterogeneous as it is required to serve tissue and organ specific roles. In keeping with its organ and tissue specific functionality, the molecular and cellular response of the endothelium to radiation injury varies by organ. Therefore, in the development of medical countermeasures for multi-organ injury, it is necessary to consider organ and tissue-specific endothelial responses to both injury and candidate mitigators. The purpose of this review is to summarize the pathogenesis of endothelial dysfunction following total or near total body irradiation exposure at the level of individual radiosensitive organs.

Comparison of Absolute Expression and Turnover Number of COX-1 and COX-2 in Human and Rodent Cells and Tissues.

Objective: We aim to quantify the absolute protein expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in various cells and tissues to determine the relative contribution of COX-1 and COX-2 to PGE2 production. Methods: An LC-MS method was developed and validated, then used for quantifying the absolute amounts of COX-1 and COX-2 in recombinant human COX-1 and COX-2, lysates from different cells, tissue microsomes of rodents and humans, Pirc rat colonic polyps, and biopsy specimens from squamous cell carcinoma (SCC) patients. The COX-1 and COX-2 turnover numbers were subsequently calculated based on apparent formation rates of PGE2. Results: A robust LC-MS method for quantification of COX-1 and COX-2 was developed and validated and then used to calculate their apparent turnover numbers. The results showed that COX-1 expression levels were much higher than that of COX-2 in all the tested tissues including the colonic epithelium of F344 (28-fold) and Pirc rats (20-fold), colonic polyps of Pirc rats (8-fold), and biopsy specimens of SCC patients (11-17-fold). In addition, both COX-1 and COX-2 were higher in polyps when compared to adjacent mucosa of Pirc rats. The turnover number of recombinant human COX-2 was 14-fold higher than that of recombinant human COX-1. LPS stimulation increased COX-2 protein expression in three cell lines (Raw 264.7, SCC9 and EOMA) as expected but unexpectedly increased COX-1 protein expression (13.8-fold) in EOMA cells. Conclusion: In human oral cancer tissues and cells as well as Pirc rat colon, COX-1 plays an unexpectedly but more important role than COX-2 in abnormal PGE2 production since COX-1 expression is much higher than COX-2. In addition, COX-1 expression levels are inducible in cells, and higher in polyps than surrounding mucosa in Pirc rat colon. These results indicate that targeted suppression of local COX-1 should be considered to reduce colon-specific PGE2-mediated inflammation.

Mitigation of Multi-Organ Radiation Injury with ACE2 Agonist Diminazene Aceturate.

The renin-angiotensin system (RAS) is known to regulate the pathogenesis of radiation-induced injury as inhibitors of the RAS enzyme angiotensin converting enzyme (ACE) have established function as mitigators of multi-organ radiation injury. To further elucidate the role of RAS signaling during both the acute and delayed syndromes of radiation exposure, we have evaluated whether pharmacologic modulation of alternate RAS enzyme angiotensin converting enzyme 2 (ACE2) reduces the pathogenesis of multi-organ radiation-induced injuries. Here, we demonstrate pharmacologic ACE2 activation with the small molecule ACE2 agonist diminazene aceturate (DIZE) improves survival in rat models of both hematologic acute radiation syndrome (H-ARS) and multi-organ delayed effects of acute radiation exposure (DEARE). In the H-ARS model, DIZE treatment increased 30-day survival by 30% compared to vehicle control rats after a LD50/30 total-body irradiation (TBI) dose of 7.75 Gy. In the mitigation of DEARE, ACE2 agonism with DIZE increased median survival by 30 days, reduced breathing rate, and reduced blood urea nitrogen (BUN) levels compared to control rats after partial-body irradiation (PBI) of 13.5 Gy. DIZE treatment was observed to have systemic effects which may explain the multi-organ benefits observed including mobilization of hematopoietic progenitors to the circulation and a reduction in plasma TGF-beta levels. These data suggest the ACE2 enzyme plays a critical role in the RAS-mediated pathogenesis of radiation injury and may be a potential therapeutic target for the development of medical countermeasures for acute radiation exposure.

Chronic stress promotes an immunologic inflammatory state and head and neck cancer growth in a humanized murine model.

BACKGROUND: Despite the importance of immune response and environmental stress on head and neck cancer (HNC) outcomes, no current pre-clinical stress model includes a humanized immune system. METHODS: We investigated the effects of chronic stress induced by social isolation on tumor growth and human immune response in subcutaneous HNC tumors grown in NSG-SGM3 mice engrafted with a human immune system. RESULTS: Tumor growth (p < 0.0001) and lung metastases (p = 0.035) were increased in socially isolated versus control animals. Chronic stress increased intra-tumoral CD4+ T-cell infiltrate (p = 0.005), plasma SDF-1 (p < 0.0001) expression, and led to tumor cell dedifferentiation toward a cancer stem cell phenotype (CD44+ /ALDHhigh , p = 0.025). CONCLUSIONS: Chronic stress induced immunophenotypic changes, increased tumor growth, and metastasis in HNC in a murine model with a humanized immune system. This model system may provide further insight into the immunologic and oncologic impact of chronic stress on patients with HNC.

Pharmacologic ACE-Inhibition Mitigates Radiation-Induced Pneumonitis by Suppressing ACE-Expressing Lung Myeloid Cells.

PURPOSE: Radiation-induced lung injury is a major dose-limiting toxicity for thoracic radiation therapy patients. In experimental models, treatment with angiotensin converting enzyme (ACE) inhibitors mitigates radiation pneumonitis; however, the mechanism of action is not well understood. Here, we evaluate the direct role of ACE inhibition on lung immune cells. METHODS AND MATERIALS: ACE expression and activity were determined in the lung immune cell compartment of irradiated adult rats after either high dose fractionated radiation therapy to the right lung (5 fractions × 9 Gy) or a single dose of 13.5 Gy partial body irradiation. Mitigation of radiation-induced pneumonitis with the ACE-inhibitor lisinopril was evaluated in the 13.5 Gy rat partial body irradiation model. During pneumonitis, we characterized inflammation and immune cell content in the lungs and bronchoalveolar lavage fluid. In vitro mechanistic studies were performed using primary human monocytes and the human monocytic THP-1 cell line. RESULTS: In both the partial body irradiation and fractionated radiation therapy models, radiation increased ACE activity in lung immune cells. Treatment with lisinopril improved survival during radiation pneumonitis (P = .0004). Lisinopril abrogated radiation-induced increases in bronchoalveolar lavage fluid monocyte chemoattractant protein 1 (chemokine ligand 2) and MIP-1a cytokine levels (P < .0001). Treatment with lisinopril reduced both ACE expression (P = .006) and frequency of CD45+ CD11b+ lung myeloid cells (P = .004). In vitro, radiation injury acutely increased ACE activity (P = .045) and reactive oxygen species (ROS) generation (P = .004) in human monocytes, whereas treatment with lisinopril blocked radiation-induced increases in both ACE and ROS. Radiation-induced ROS generation was blocked by pharmacologic inhibition of either NADPH oxidase 2 (P = .012) or the type 1 angiotensin receptor (P = .013). CONCLUSIONS: These data demonstrate radiation-induced ACE activation within the immune compartment promotes the pathogenesis of radiation pneumonitis, while ACE inhibition suppresses activation of proinflammatory immune cell subsets. Mechanistically, our in vitro data demonstrate radiation directly activates the ACE/type 1 angiotensin receptor pathway in immune cells and promotes generation of ROS via NADPH oxidase 2.

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution.

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 - mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell - specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A - NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A - NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Brain-derived neurotrophic factor promotes immune reconstitution following radiation injury via activation of bone marrow mesenchymal stem cells.

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family which has been extensively studied for its roles in neural development, long-term memory, brain injury, and neurodegenerative diseases. BDNF signaling through tropomyosin receptor kinase B (TrkB) stimulates neuronal cell survival. For this reason, small molecule TrkB agonists are under pre-clinical develoment for the treatment of a range of neurodegenerative diseases and injuries. Our laboratory recently reported BDNF is secreted by pro-regenerative endothelial progenitor cells (EPCs) which support hematopoietic reconstitution following total body irradiation (TBI). Here we report BDNF-TrkB signaling plays a novel regenerative role in bone marrow and thymic regeneration following radiation injury. Exogenous administration of BDNF or TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following myelosuppressive radiation injury promoted faster recovery of mature blood cells and hematopoietic stem cells capable of multi-lineage reconstitution. BDNF promotes hematopoietic regeneration via activation of PDGFRα+ bone marrow mesenchymal stem cells (MSCs) which increase secretion of hematopoietic cytokines interleukin 6 (IL-6) and leukemia inhibitory factor (LIF) in response to TrkB activation. These data suggest pharmacologic activation of the BDNF pathway with either BDNF or 7,8-DHF may be beneficial for treatment of radiation or chemotherapy induced myelosuppression.