Galunisertib plus neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer: a single-arm, phase 2 trial.

BACKGROUND: TGF-ß is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-ß blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-ß type I receptor kinase inhibitor galunisertib to neoadjuvant chemoradiotherapy would improve pathological complete response rates in patients with locally advanced rectal cancer. METHODS: This was an investigator-initiated, single-arm, phase 2 study done in two medical centres in Portland (OR, USA). Eligible patients had previously untreated, locally advanced, rectal adenocarcinoma, stage IIA-IIIC or IV as per the American Joint Committee on Cancer; Eastern Cooperative Oncology Group status 0-2; and were aged 18 years or older. Participants completed two 14-day courses of oral galunisertib 150 mg twice daily, before and during fluorouracil-based chemoradiotherapy (intravenous fluorouracil 225 mg/m2 over 24 h daily 7 days per week during radiotherapy or oral capecitabine 825 mg/m2 twice per day 5 days per week during radiotherapy; radiotherapy consisted of 50·4-54·0 Gy in 28-30 fractions). 5-9 weeks later, patients underwent response assessment. Patients with a complete response could opt for non-operative management and proceed to modified FOLFOX6 (intravenous leucovorin 400 mg/m2 on day 1, intravenous fluorouracil 400 mg/m2 on day 1 then 2400 mg/m2 over 46 h, and intravenous oxaliplatin 85 mg/m2 on day 1 delivered every 2 weeks for eight cycles) or CAPEOX (intravenous oxaliplatin 130 mg/m2 on day 1 and oral capecitabine 1000 mg/m2 twice daily for 14 days every 3 weeks for four cycles). Patients with less than complete response underwent surgical resection. The primary endpoint was complete response rate, which was a composite of pathological complete response in patients who proceeded to surgery, or clinical complete response maintained at 1 year after last therapy in patients with non-operative management. Safety was a coprimary endpoint. Both endpoints were assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02688712, and is active but not recruiting. FINDINGS: Between Oct 19, 2016, and Aug 31, 2020, 38 participants were enrolled. 25 (71%) of the 35 patients who completed chemoradiotherapy proceeded to total mesorectal excision surgery, five (20%) of whom had pathological complete responses. Ten (29%) patients had non-operative management, three (30%) of whom ultimately chose to have total mesorectal excision. Two (67%) of those three patients had pathological complete responses. Of the remaining seven patients in the non-operative management group, five (71%) had clinical complete responses at 1 year after their last modified FOLFOX6 infusion. In total, 12 (32% [one-sided 95% CI ≥19%]) of 38 patients had a complete response. Common grade 3 adverse events during treatment included diarrhoea in six (16%) of 38 patients, and haematological toxicity in seven (18%) patients. Two (5%) patients had grade 4 adverse events, one related to chemoradiotherapy-induced diarrhoea and dehydration, and the other an intraoperative ischaemic event. No treatment-related deaths occurred. INTERPRETATION: The addition of galunisertib to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer improved the complete response rate to 32%, was well tolerated, and warrants further assessment in randomised trials. FUNDING: Eli Lilly via ExIST program, The Providence Foundation.

Five-Year Outcomes of a Randomized Trial of Treatments for Varicose Veins.

BACKGROUND: Endovenous laser ablation and ultrasound-guided foam sclerotherapy are recommended alternatives to surgery for the treatment of primary varicose veins, but their long-term comparative effectiveness remains uncertain. METHODS: In a randomized, controlled trial involving 798 participants with primary varicose veins at 11 centers in the United Kingdom, we compared the outcomes of laser ablation, foam sclerotherapy, and surgery. Primary outcomes at 5 years were disease-specific quality of life and generic quality of life, as well as cost-effectiveness based on models of expected costs and quality-adjusted life-years (QALYs) gained that used data on participants' treatment costs and scores on the EuroQol EQ-5D questionnaire. RESULTS: Quality-of-life questionnaires were completed by 595 (75%) of the 798 trial participants. After adjustment for baseline scores and other covariates, scores on the Aberdeen Varicose Vein Questionnaire (on which scores range from 0 to 100, with lower scores indicating a better quality of life) were lower among patients who underwent laser ablation or surgery than among those who underwent foam sclerotherapy (effect size [adjusted differences between groups] for laser ablation vs. foam sclerotherapy, -2.86; 95% confidence interval [CI], -4.49 to -1.22; P<0.001; and for surgery vs. foam sclerotherapy, -2.60; 95% CI, -3.99 to -1.22; P<0.001). Generic quality-of-life measures did not differ among treatment groups. At a threshold willingness-to-pay ratio of £20,000 ($28,433 in U.S. dollars) per QALY, 77.2% of the cost-effectiveness model iterations favored laser ablation. In a two-way comparison between foam sclerotherapy and surgery, 54.5% of the model iterations favored surgery. CONCLUSIONS: In a randomized trial of treatments for varicose veins, disease-specific quality of life 5 years after treatment was better after laser ablation or surgery than after foam sclerotherapy. The majority of the probabilistic cost-effectiveness model iterations favored laser ablation at a willingness-to-pay ratio of £20,000 ($28,433) per QALY. (Funded by the National Institute for Health Research; CLASS Current Controlled Trials number, ISRCTN51995477.).

Dendritic Cell Maturation Defines Immunological Responsiveness of Tumors to Radiation Therapy.

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8+ T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.

Information Dark Energy Can Resolve the Hubble Tension and Is Falsifiable by Experiment.

We consider the role information energy can play as a source of dark energy. Firstly, we note that if stars and structure had not formed in the universe, elemental bits of information describing the attributes of particles would have exhibited properties similar to the cosmological constant. The Landauer equivalent energy of such elemental bits would be defined in form and value identical to the characteristic energy of the cosmological constant. However, with the formation of stars and structure, stellar heated gas and dust now provide the dominant contribution to information energy with the characteristics of a dynamic, transitional, dark energy. At low redshift, z < ~1.35, this dark energy emulates the cosmological constant with a near-constant energy density, w = −1.03 ± 0.05, and an energy total similar to the mc2 of the universe's ∼1053 kg of baryons. At earlier times, z > ~1.35, information energy was phantom, differing from the cosmological constant, Λ, with a CPL parameter difference of ∆wo = −0.03 ± 0.05 and ∆wa = −0.79 ± 0.08, values sufficient to account for the H0 tension. Information dark energy agrees with most phenomena as well as Λ, while exhibiting characteristics that resolve many tensions and problems of ΛCDM: the cosmological constant problem; the cosmological coincidence problem; the H0 tension, and the σ8 tension. As this proposed dark energy source is not usually considered, we identify the expected signature in H(a) that will enable the role of information dark energy to be falsified by experimental observation.

Response to radiotherapy in pancreatic ductal adenocarcinoma is enhanced by inhibition of myeloid-derived suppressor cells using STAT3 anti-sense oligonucleotide.

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.

Amplifying IFN-γ Signaling in Dendritic Cells by CD11c-Specific Loss of SOCS1 Increases Innate Immunity to Infection while Decreasing Adaptive Immunity.

Although prophylactic vaccines provide protective humoral immunity against infectious agents, vaccines that elicit potent CD8 T cell responses are valuable tools to shape and drive cellular immunity against cancer and intracellular infection. In particular, IFN-γ-polarized cytotoxic CD8 T cell immunity is considered optimal for protective immunity against intracellular Ags. Suppressor of cytokine signaling (SOCS)1 is a cross-functional negative regulator of TLR and cytokine receptor signaling via degradation of the receptor-signaling complex. We hypothesized that loss of SOCS1 in dendritic cells (DCs) would improve T cell responses by accentuating IFN-γ-directed immune responses. We tested this hypothesis using a recombinant Listeria monocytogenes vaccine platform that targets CD11c+ DCs in mice in which SOCS1 is selectively deleted in all CD11c+ cells. Unexpectedly, in mice lacking SOCS1 expression in CD11c+ cells, we observed a decrease in CD8+ T cell response to the L. monocytogenes vaccine. NK cell responses were also decreased in mice lacking SOCS1 expression in CD11c+ cells but did not explain the defect in CD8+ T cell immunity. We found that DCs lacking SOCS1 expression were functional in driving Ag-specific CD8+ T cell expansion in vitro but that this process was defective following infection in vivo. Instead, monocyte-derived innate TNF-α and inducible NO synthase-producing DCs dominated the antibacterial response. Thus, loss of SOCS1 in CD11c+ cells skewed the balance of immune response to infection by increasing innate responses while decreasing Ag-specific adaptive responses to infectious Ags.

The Stanford Hall consensus statement for post-COVID-19 rehabilitation.

The highly infectious and pathogenic novel coronavirus (CoV), severe acute respiratory syndrome (SARS)-CoV-2, has emerged causing a global pandemic. Although COVID-19 predominantly affects the respiratory system, evidence indicates a multisystem disease which is frequently severe and often results in death. Long-term sequelae of COVID-19 are unknown, but evidence from previous CoV outbreaks demonstrates impaired pulmonary and physical function, reduced quality of life and emotional distress. Many COVID-19 survivors who require critical care may develop psychological, physical and cognitive impairments. There is a clear need for guidance on the rehabilitation of COVID-19 survivors. This consensus statement was developed by an expert panel in the fields of rehabilitation, sport and exercise medicine (SEM), rheumatology, psychiatry, general practice, psychology and specialist pain, working at the Defence Medical Rehabilitation Centre, Stanford Hall, UK. Seven teams appraised evidence for the following domains relating to COVID-19 rehabilitation requirements: pulmonary, cardiac, SEM, psychological, musculoskeletal, neurorehabilitation and general medical. A chair combined recommendations generated within teams. A writing committee prepared the consensus statement in accordance with the appraisal of guidelines research and evaluation criteria, grading all recommendations with levels of evidence. Authors scored their level of agreement with each recommendation on a scale of 0-10. Substantial agreement (range 7.5-10) was reached for 36 recommendations following a chaired agreement meeting that was attended by all authors. This consensus statement provides an overarching framework assimilating evidence and likely requirements of multidisciplinary rehabilitation post COVID-19 illness, for a target population of active individuals, including military personnel and athletes.

Methionine Metabolites in Patients With Sepsis.

OBJECTIVE: Sepsis is characterized by microvascular dysfunction and thrombophilia. Several methionine metabolites may be relevant to this sepsis pathophysiology. S-adenosylmethionine (SAM) serves as the methyl donor for trans-methylation reactions. S-adenosylhomocysteine (SAH) is the by-product of these reactions and serves as the precursor to homocysteine. Relationships between plasma total homocysteine concentrations (tHcy) and vascular disease and thrombosis are firmly established. We hypothesized that SAM, SAH, and tHcy levels are elevated in patients with sepsis and associated with mortality. METHODS: This was a combined case-control and prospective cohort study consisting of 109 patients with sepsis and 50 control participants without acute illness. The study was conducted in the medical and surgical intensive care units of the University of Rochester Medical Center. Methionine, SAM, SAH, and tHcy concentrations were compared in patients with sepsis versus control participants and in sepsis survivors versus nonsurvivors. RESULTS: Patients with sepsis had significantly higher plasma SAM and SAH concentrations than control participants (SAM: 164 [107-227] vs73 [59-87 nM], P < .001; SAH: 99 [60-165] vs 35 [28-45] nM, P < .001). In contrast, plasma tHcy concentrations were lower in sepsis patients compared to healthy control participants (4 [2-6]) vs 7 [5-9] µM; P = .04). In multivariable analysis, quartiles of SAM, SAH, and tHcy were independently associated with sepsis ( P = .006, P = .05, and P < .001, respectively). Sepsis nonsurvivors had significantly higher plasma SAM and SAH concentrations than survivors (SAM: 223 [125-260] vs 136 [96-187] nM; P = .01; SAH: 139 [81-197] vs 86 [55-130] nM, P = .006). Plasma tHcy levels were similar in survivors vs nonsurvivors. The associations between SAM or SAH and hospital mortality were no longer significant after adjusting for renal dysfunction. CONCLUSIONS: Methionine metabolite concentrations are abnormal in sepsis and linked with clinical outcomes. Further study is required to determine whether these abnormalities have pathophysiologic significance.

A randomized trial comparing treatments for varicose veins.

BACKGROUND: Ultrasound-guided foam sclerotherapy and endovenous laser ablation are widely used alternatives to surgery for the treatment of varicose veins, but their comparative effectiveness and safety remain uncertain. METHODS: In a randomized trial involving 798 participants with primary varicose veins at 11 centers in the United Kingdom, we compared the outcomes of foam, laser, and surgical treatments. Primary outcomes at 6 months were disease-specific quality of life and generic quality of life, as measured on several scales. Secondary outcomes included complications and measures of clinical success. RESULTS: After adjustment for baseline scores and other covariates, the mean disease-specific quality of life was slightly worse after treatment with foam than after surgery (P=0.006) but was similar in the laser and surgery groups. There were no significant differences between the surgery group and the foam or the laser group in measures of generic quality of life. The frequency of procedural complications was similar in the foam group (6%) and the surgery group (7%) but was lower in the laser group (1%) than in the surgery group (P<0.001); the frequency of serious adverse events (approximately 3%) was similar among the groups. Measures of clinical success were similar among the groups, but successful ablation of the main trunks of the saphenous vein was less common in the foam group than in the surgery group (P<0.001). CONCLUSIONS: Quality-of-life measures were generally similar among the study groups, with the exception of a slightly worse disease-specific quality of life in the foam group than in the surgery group. All treatments had similar clinical efficacy, but complications were less frequent after laser treatment and ablation rates were lower after foam treatment. (Funded by the Health Technology Assessment Programme of the National Institute for Health Research; Current Controlled Trials number, ISRCTN51995477.).

"Take-home" box trainers are an effective alternative to virtual reality simulators.

BACKGROUND: Practice on virtual reality simulators (VRSs) has been shown to improve surgical performance. However, VRSs are expensive and usually housed in surgical skills centers that may be inaccessible at times convenient for surgical trainees to practice. Conversely, box trainers (BT) are inexpensive and can be used anywhere at anytime. This study assesses "take-home" BTs as an alternative to VRS. METHODS: After baseline assessments (two simulated laparoscopic cholecystectomies, one on a VRS and one on a BT), 25 surgical trainees were randomized to two groups. Trainees were asked to practice three basic laparoscopic tasks for 6 wk (BT group using a "take-home" box trainer; VR group using VRS in clinical skills centers). After the practice period, all performed two laparoscopic cholecystectomy, one on a VRS and one on a BT; (i.e., posttraining assessment). VRS provided metrics (total time [TT], number of movements instrument tip path length), and expert video assessment of cholecystectomy in a BT (Global Operative Assessment of Laparoscopic Skills [GOALS] score) were recorded. Performance during pretraining and posttraining assessment was compared. RESULTS: The BT group showed a significant improvement for all VRS metrics (P = 0.008) and the efficiency category of GOALS score (P = 0.03). Only TT improved in the VRS group, and none of the GOALS categories demonstrated a statistically significant improvement after training. Finally, the improvement in VRS metrics in the BT group was significantly greater than in the VR group (TT P = 0.005, number of movements P = 0.042, path length P = 0.031), although there were no differences in the GOALS scores between the groups. CONCLUSIONS: This study suggests that a basic "take-home" BT is a suitable alternative to VRS.

Programmed cell death-1 blockade enhances response to stereotactic radiation in an orthotopic murine model of hepatocellular carcinoma.

AIM: Small, solitary hepatocellular carcinoma is curable with stereotactic radiation or other methods of tumor ablation, however, regional and systemic tumor recurrence occurs in over 70% of patients. Here we describe the ability of immunoradiotherapy to induce an antitumor immune response and delay the growth of tumors in immunocompetent mice. METHODS: A syngeneic hepatocellular carcinoma cell line (Hep-55.1c) was injected directly into the livers of C57BL/6 mice using ultrasound guidance, then tumors were treated with stereotactic radiation using a Small Animal Radiation Research Platform with computed tomography guidance. RESULTS: Delivery of three doses of 250 µg anti-programmed cell death protein-1 (αPD-1) antibody concurrently with 30 Gy stereotactic body radiation therapy in three fractions reduced the growth rate of tumors and improved survival (P < 0.05). Combined treatment was associated with increased CD8+ cytotoxic T cells in the tumor; depletion of CD8 T cells eliminated the efficacy of combined treatment. Combined treatment also induced expression of programmed cell death-1 ligand expression on tumor-infiltrating macrophages, and the tumors grew rapidly after αPD-1 treatment was discontinued. CONCLUSIONS: Tumor response to stereotactic radiation can be augmented by concurrent treatment with αPD-1. The efficacy of this combination therapy was transient, however, and treatment induced markers of adaptive immune resistance. These data are promising, but also indicate that mechanisms of immune resistance will need to be durably overcome for this combination to generate lasting immunity to protect against tumor recurrence.

Bioavailable estradiol concentrations are elevated and predict mortality in septic patients: a prospective cohort study.

BACKGROUND: Experimental studies demonstrate beneficial immunological and hemodynamic effects of estradiol in animal models of sepsis. This raises the question whether estradiol contributes to sex differences in the incidence and outcomes of sepsis in humans. Yet, total estradiol levels are elevated in sepsis patients, particularly nonsurvivors. Bioavailable estradiol concentrations have not previously been reported in septic patients. The bioavailable estradiol concentration accounts for aberrations in estradiol carrier protein concentrations that could produce discrepancies between total and bioavailable estradiol levels. We hypothesized that bioavailable estradiol levels are low in septic patients and sepsis nonsurvivors. METHODS: We conducted a combined case-control and prospective cohort study. Venous blood samples were obtained from 131 critically ill septic patients in the medical and surgical intensive care units at the University of Rochester Medical Center and 51 control subjects without acute illness. Serum bioavailable estradiol concentrations were calculated using measurements of total estradiol, sex hormone-binding globulin, and albumin. Comparisons were made between patients with severe sepsis and control subjects and between hospital survivors and nonsurvivors. Multivariable logistic regression analysis was also performed. RESULTS: Bioavailable estradiol concentrations were significantly higher in sepsis patients than in control subjects (211 [78-675] pM vs. 100 [78-142] pM, p < 0.01) and in sepsis nonsurvivors than in survivors (312 [164-918] pM vs. 167 [70-566] pM, p = 0.04). After adjustment for age and comorbidities, patients with bioavailable estradiol levels above the median value had significantly higher risk of hospital mortality (OR 4.27, 95 % CI 1.65-11.06, p = 0.003). Bioavailable estradiol levels were directly correlated with severity of illness and did not differ between men and women. CONCLUSIONS: Contrary to our hypothesis, bioavailable estradiol levels were elevated in sepsis patients, particularly nonsurvivors, and were independently associated with mortality. Whether estradiol's effects are harmful, beneficial, or neutral in septic patients remains unknown, but our findings raise caution about estradiol's therapeutic potential in this setting. Our findings do not provide an explanation for sex-based differences in sepsis incidence and outcomes.

The ataxia telangiectasia mutated kinase pathway regulates IL-23 expression by human dendritic cells.

Little is known of the regulation of IL-23 secretion in dendritic cells (DC) despite its importance for human Th17 responses. In this study, we show for first time, to our knowledge, that the ataxia telangiectasia mutated (ATM) pathway, involved in DNA damage sensing, acts as an IL-23 repressor. Inhibition of ATM with the highly selective antagonist KU55933 markedly increased IL-23 secretion in human monocyte-derived DC and freshly isolated myeloid DC. In contrast, inhibiting the closely related mammalian target of rapamycin had no effect on IL-23. Priming naive CD4(+) T cells with ATM-inhibited DC increased Th17 responses over and above those obtained with mature DC. Although ATM blockade increased the abundance of p19, p35, and p40 mRNA, IL-12p70 secretion was unaffected. To further examine a role for ATM in IL-23 regulation, we exposed DC to low doses of ionizing radiation. Exposure of DC to x-rays resulted in ATM phosphorylation and a corresponding depression of IL-23. Importantly, ATM inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of x-rays to suppress IL-23. To explore how ATM repressed IL-23, we examined a role for endoplasmic reticulum stress responses by measuring generation of the spliced form of X-box protein-1, a key endoplasmic reticulum stress transcription factor. Inhibition of ATM increased the abundance of X-box protein-1 mRNA, and this was followed 3 h later by increased peak p19 transcription and IL-23 release. In summary, ATM activation or inhibition, respectively, inhibited or augmented IL-23 release. This novel role of the ATM pathway represents a new therapeutic target in autoimmunity and vaccine development.

Transient removal of CD46 is safe and increases B-cell depletion by rituximab in CD46 transgenic mice and macaques.

We have developed a technology that depletes the complement regulatory protein (CRP) CD46 from the cell surface, and thereby sensitizes tumor cells to complement-dependent cytotoxicity triggered by therapeutic monoclonal antibodies (mAbs). This technology is based on a small recombinant protein, Ad35K++, which induces the internalization and subsequent degradation of CD46. In preliminary studies, we had demonstrated the utility of the combination of Ad35K++ and several commercially available mAbs such as rituximab, alemtuzumab, and trastuzumab in enhancing cell killing in vitro as well as in vivo in murine xenograft and syngeneic tumor models. We have completed scaled manufacturing of Ad35K++ protein in Escherichia coli for studies in nonhuman primates (NHPs). In macaques, we first defined a dose of the CD20-targeting mAb rituximab that did not deplete CD20-positive peripheral blood cells. Using this dose of rituximab, we then demonstrated that pretreatment with Ad35K++ reconstituted near complete elimination of B cells. Further studies demonstrated that the treatment was well tolerated and safe. These findings in a relevant large animal model provide the rationale for moving this therapy forward into clinical trials in patients with CD20-positive B-cell malignancies.

Fluorescence tracking demonstrates T cell recirculation is transiently impaired by radiation therapy to the tumor.

T cells recirculate through tissues and lymphatic organs to scan for their cognate antigen. Radiation therapy provides site-specific cytotoxicity to kill cancer cells but also has the potential to eliminate the tumor-specific T cells in field. To dynamically study the effect of radiation on CD8 T cell recirculation, we used the Kaede mouse model to photoconvert tumor-infiltrating cells and monitor their movement out of the field of radiation. We demonstrate that radiation results in loss of CD8 T cell recirculation from the tumor to the lymph node and to distant sites. Using scRNASeq, we see decreased proliferating CD8 T cells in the tumor following radiation therapy resulting in a proportional enrichment in exhausted phenotypes. By contrast, 5 days following radiation increased recirculation of T cells from the tumor to the tumor draining lymph node corresponds with increased immunosurveillance of the treated tumor. These data demonstrate that tumor radiation therapy transiently impairs systemic T cell recirculation from the treatment site to the draining lymph node and distant untreated tumors. This may inform timing therapies to improve systemic T cell-mediated tumor immunity.

The impact of the myeloid response to radiation therapy.

Radiation therapy is showing potential as a partner for immunotherapies in preclinical cancer models and early clinical studies. As has been discussed elsewhere, radiation provides debulking, antigen and adjuvant release, and inflammatory targeting of effector cells to the treatment site, thereby assisting multiple critical checkpoints in antitumor adaptive immunity. Adaptive immunity is terminated by inflammatory resolution, an active process which ensures that inflammatory damage is repaired and tissue function is restored. We discuss how radiation therapy similarly triggers inflammation followed by repair, the consequences to adaptive immune responses in the treatment site, and how the myeloid response to radiation may impact immunotherapies designed to improve control of residual cancer cells.

Ex vivo analysis of radiation effects on tumor infiltrating immune cells using tumor explants.

The response to radiation therapy incorporates both the direct impacts of radiation on cancer cells as well as the immune consequences that can help or hinder control of residual disease. Understanding the response of an individual patient's cancer to radiation, and the impact of radiation on the immune cell subsets present in the tumor prior to radiation therapy, can help identify potential predictors of outcome. Here, we describe a methodological approach to using an explant tumor model to characterize and study the immune cell subsets in murine tumors following exposure to ex vivo radiation treatment. The broader tumor environment incorporates distinct microenvironments consisting of tumor stroma and cancer cell nests, with limited interchange between these zones. Ex vivo analysis of tumor explants ensures that these environments remain intact and allows patient-specific response assessments with a broader range of treatment conditions to find optimal conditions and immunotherapy combinations. While this protocol describes the treatment of murine tumors, with minor variations the same protocol can be used to study and characterize various immune populations following radiation therapy in human tumors.

The role of dendritic cells in radiation-induced immune responses.

Dendritic cells perform critical functions in bridging innate and adaptive immunity. Their ability to sense adjuvant signals in their environment, migrate on maturation, and cross-present cell-associated antigens enables these cells to carry antigen from tissue sites to lymph nodes, and thereby prime naïve T cells that cannot enter tissues. Despite being an infrequent cell type in tumors, we discuss how dendritic cells impact the immune environment of tumors and their response to cancer therapies. We review how radiation therapy of tumors can impact dendritic cells, through transfer of cell associated antigens to dendritic cells and the release of endogenous adjuvants, resulting in increased antigen presentation in the tumor-draining lymph nodes. We explore how tumor specific factors can result in negative regulation of dendritic cell function in the tumor, and the impact of direct radiation exposure to dendritic cells in the treatment field. These data suggest an important role for dendritic cell subpopulations in activating new T cell responses and boosting existing T cell responses to tumor associated antigens in tumor draining lymph nodes following radiation therapy. It further justifies a focus on the needs of the lymph node T cells to improve systemic anti-immunity following radiation therapy.

The immunogenic radiation and new players in immunotherapy and targeted therapy for head and neck cancer.

Although treatment modalities for head and neck cancer have evolved considerably over the past decades, survival rates have plateaued. The treatment options remained limited to definitive surgery, surgery followed by fractionated radiotherapy with optional chemotherapy, and a definitive combination of fractionated radiotherapy and chemotherapy. Lately, immunotherapy has been introduced as the fourth modality of treatment, mainly administered as a single checkpoint inhibitor for recurrent or metastatic disease. While other regimens and combinations of immunotherapy and targeted therapy are being tested in clinical trials, adapting the appropriate regimens to patients and predicting their outcomes have yet to reach the clinical setting. Radiotherapy is mainly regarded as a means to target cancer cells while minimizing the unwanted peripheral effect. Radiotherapy regimens and fractionation are designed to serve this purpose, while the systemic effect of radiation on the immune response is rarely considered a factor while designing treatment. To bridge this gap, this review will highlight the effect of radiotherapy on the tumor microenvironment locally, and the immune response systemically. We will review the methodology to identify potential targets for therapy in the tumor microenvironment and the scientific basis for combining targeted therapy and radiotherapy. We will describe a current experience in preclinical models to test these combinations and propose how challenges in this realm may be faced. We will review new players in targeted therapy and their utilization to drive immunogenic response against head and neck cancer. We will outline the factors contributing to head and neck cancer heterogeneity and their effect on the response to radiotherapy. We will review in-silico methods to decipher intertumoral and intratumoral heterogeneity and how these algorithms can predict treatment outcomes. We propose that (a) the sequence of surgery, radiotherapy, chemotherapy, and targeted therapy should be designed not only to annul cancer directly, but to prime the immune response. (b) Fractionation of radiotherapy and the extent of the irradiated field should facilitate systemic immunity to develop. (c) New players in targeted therapy should be evaluated in translational studies toward clinical trials. (d) Head and neck cancer treatment should be personalized according to patients and tumor-specific factors.