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Head and neck squamous cell carcinoma (HNSCC) is diagnosed in more than 71,000 patients each year in the United States, with nearly 16,000 associated deaths. One significant hurdle in the treatment of HNSCC is acquired and intrinsic resistance to existing therapeutic agents. Over the past several decades, the University of Wisconsin has formed a multidisciplinary team to move basic scientific discovery along the translational spectrum to impact the lives of HNSCC patients. In this review, we outline key discoveries made throughout the years at the University of Wisconsin to deepen our understanding of therapeutic resistance in HNSCC and how a strong, interdisciplinary team can make significant advances toward improving the lives of these patients by combatting resistance to established therapeutic modalities. We are profoundly grateful to the many scientific teams worldwide whose groundbreaking discoveries, alongside evolving clinical paradigms in head and neck oncology, have been instrumental in making our work possible.

Therapies against cell-surface targets (CSTs) represent an emerging treatment class in solid malignancies. However, high-throughput investigations of CST expression across cancer types have been reliant on data sets of mostly primary tumors, despite therapeutic use most commonly in metastatic disease. We identified a total of 818 clinical trials of CST therapies with 78 CSTs. We assembled a data set spanning RNA-seq and microarrays in 7,927 benign samples, 16,866 primary tumor samples, and 6,124 metastatic tumor samples. We also utilized single-cell RNA-seq data from 36 benign tissues and 558 primary and metastatic tumor samples, and matched RNA versus protein expression in 29 benign tissue samples, 1,075 tumor samples, and 942 cell lines. High RNA expression accurately predicted high protein expression across CST therapies in benign tissues, tumor samples, and cell lines. We compared metastatic versus primary tumor expression, identified potential opportunities for repositioning, and matched cell lines to tumor types based on CST and global RNA expression. We evaluated single-cell heterogeneity across tumors, and identified rare normal cell subpopulations that may contribute to toxicity. Finally, we identified combinations of CST therapies for which bispecific approaches could improve tumor specificity. This study helps better define the landscape of CST expression in metastatic and primary cancers.

Radiopharmaceutical therapies (RPT) activate a type I interferon (IFN1) response in tumor cells. We hypothesized that the timing and amplitude of this response varies by isotope. We compared equal doses delivered by 90 Y, 177 Lu, and 225 Ac in vitro as unbound radionuclides and in vivo when chelated to NM600, a tumor-selective alkylphosphocholine. Response in murine MOC2 head and neck carcinoma and B78 melanoma was evaluated by qPCR and flow cytometry. Therapeutic response to 225 Ac-NM600+anti-CTLA4+anti-PD-L1 immune checkpoint inhibition (ICI) was evaluated in wild-type and stimulator of interferon genes knockout (STING KO) B78. The timing and magnitude of IFN1 response correlated with radionuclide half-life and linear energy transfer. CD8 + /Treg ratios increased in tumors 7 days after 90 Y- and 177 Lu-NM600 and day 21 after 225 Ac-NM600. 225 Ac-NM600+ICI improved survival in mice with WT but not with STING KO tumors, relative to monotherapies. Immunomodulatory effects of RPT vary with radioisotope and promote STING-dependent enhanced response to ICIs in murine models., Teaser: This study describes the time course and nature of tumor immunomodulation by radiopharmaceuticals with differing physical properties.

Background: The majority of experimental approaches for cancer immunotherapy are tested against relatively small tumors in tumor-bearing mice, because in most cases advanced cancers are resistant to the treatments. In this study, we asked if even late-stage mouse tumors can be eradicated by a rationally designed combined radio-immunotherapy (CRI) regimen., Methods: CRI consisted of local radiotherapy, intratumoral IL-12, slow-release systemic IL-2 and anti- CTLA-4 antibody. Therapeutic effects of CRI against several weakly immunogenic and immunogenic mouse tumors including B78 melanoma, MC38 and CT26 colon carcinomas and 9464D neuroblastoma were evaluated. Immune cell depletion and flow cytometric analysis were performed to determine the mechanisms of the antitumor effects., Results: Tumors with volumes of 2,000 mm 3 or larger were eradicated by CRI. Flow analyses of the tumors revealed reduction of T regulatory (Treg) cells and increase of CD8/Treg ratios following CRI. Rapid shrinkage of the treated tumors did not require T cells, whereas T cells were involved in the systemic effect against the distant tumors. Cured mice developed immunological memory., Conclusions: These findings underscore that rationally designed combination immunotherapy regimens can be effective even against large, late-stage tumors., Competing Interests: Author WO was formerly employed by the company Nektar Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rakhmilevich, Tsarovsky, Felder, Zaborek, Moram, Erbe, Pieper, Spiegelman, Cheng, Witt, Overwijk, Morris and Sondel.)

By reconstructing and comparing the sequence of ontogenetic (embryonic development and post-natal growth) events across species, developmental biologists have gained unique insights into the key processes underlying the evolution of modern lineages and their extinct relatives. However, despite the importance of intraspecific variation to evolutionary transformation and lineage divergence, variation in the sequence of developmental events is seldom acknowledged. Thus, how much variation or variability should be expected during ontogeny remains poorly understood and it is an open question to what extent it impacts interspecific comparisons of developmental patterns. To address this crucial question, we studied the skeletal development of the important biomedical and developmental model organism, Monodelphis domestica . We investigated cranial, forelimb, and hindlimb elements using ontogenetic sequence analysis (OSA) to quantify and assess the full range of variation and variability in the sequence of ossification. Our study documented that previously unrecognized variation exists during M. domestica ontogeny-with over 5000 sequences for the full 92 event analysis. Further, OSA revealed unexpectedly high variability (i.e., the propensity to express variation) in the sequence of ossification for the skull and across the entire skeleton. Reconstructed modal sequences were generally in agreement with previously recognized patterns, including earlier ossification of the facial skeleton and a slight offset between forelimb and hindlimb development. However, the full range of variation shows that the majority of specimens in our analysis followed developmental trajectories distinct from those recovered by prior studies. This level of variation is quite remarkable and demonstrates the importance of assessing intraspecific ontogenetic variation. By quantifying sequence polymorphism and studying how developmental variation and variability differ among species, we can clarify more precisely how developmental patterns differ among species and gain insights into how ontogeny itself evolves., Competing Interests: The authors declare no competing or conflicting interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Rationale: Androgen deprivation therapy (ADT) is pivotal in treating recurrent prostate cancer and is often combined with external beam radiation therapy (EBRT) for localized disease. However, for metastatic castration-resistant prostate cancer, EBRT is typically only used in the palliative setting, because of the inability to radiate all sites of disease. Systemic radiation treatments that preferentially irradiate cancer cells, known as radiopharmaceutical therapy or targeted radionuclide therapy (TRT), have demonstrable benefits for treating metastatic prostate cancer. Here, we explored the use of a novel TRT, 90 Y-NM600, specifically in combination with ADT, in murine prostate tumor models., Methods: 6-week-old male FVB mice were implanted subcutaneously with Myc-CaP tumor cells and given a single intravenous injection of 90 Y-NM600, in combination with ADT (degarelix). The combination and sequence of administration were evaluated for effect on tumor growth and infiltrating immune populations were analyzed by flow cytometry. Sera were assessed to determine treatment effects on cytokine profiles., Results: ADT delivered prior to TRT (ADT→TRT) resulted in significantly greater antitumor response and overall survival than if delivered after TRT (TRT→ADT). Studies conducted in immunodeficient NRG mice failed to show a difference in treatment sequence, suggesting an immunological mechanism. Myeloid-derived suppressor cells (MDSCs) significantly accumulated in tumors following TRT→ADT treatment and retained immune suppressive function. However, CD4+ and CD8+ T cells with an activated and memory phenotype were more prevalent in the ADT→TRT group. Depletion of Gr1+MDSCs led to greater antitumor response following either treatment sequence. Chemotaxis assays suggested that tumor cells secreted chemokines that recruited MDSCs, notably CXCL1 and CXCL2. The use of a selective CXCR2 antagonist, reparixin, further improved antitumor responses and overall survival when used in tumor-bearing mice treated with TRT→ADT., Conclusion: The combination of ADT and TRT improved antitumor responses in murine models of prostate cancer, however, this was dependent on the order of administration. This was found to be associated with one treatment sequence leading to an increase in infiltrating MDSCs. Combining treatment with a CXCR2 antagonist improved the antitumor effect of this combination, suggesting a possible approach for treating advanced human prostate cancer., Competing Interests: Competing interests: JPW is a co-founder and Senior Science Advisor for Archeus Technologies, which holds the license rights to NM600-related technologies. ZSM and RH have financial interest in Archeus Technologies. HCR has served as a consultant for Archeus Technologies. ZSM is a member of the Scientific Advisory Boards for Archeus Technologies, Seneca Therapeutics, and NorthStar Medical Isotopes. ZSM is an inventor on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to immunotherapies and the interaction of targeted radionuclide therapies and immunotherapies. The other authors have no relevant potential conflicts of interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Radiation therapy (RT) is a pillar of cancer therapy used by more than half of all cancer patients. Clinically, RT is mostly delivered as external beam radiation therapy (EBRT). However, the scope of EBRT is limited in the metastatic setting, where all sites of disease need to be irradiated. Such a limitation is attributed to radiation-induced toxicities, for example on bone marrow and hematologic toxicities, resulting from a large EBRT field. Radiopharmaceutical therapy (RPT) has emerged as an alternative to EBRT for the irradiation of all sites of metastatic disease. While RPT can reduce tumor burden, it can also impact the immune system and anti-tumor immunity. Understanding these effects is crucial for predicting and managing treatment-related hematological toxicities and optimizing their integration with other therapeutic modalities, such as immunotherapies. Here, we review the immunomodulatory effects of α- and β-particle emitter-based RPT on various immune cell lines, such as CD8+ and CD4+ T cells, natural killer (NK) cells, and regulatory T (Treg) cells. We briefly discuss Auger electron-emitter (AEE)-based RPT, and finally, we highlight the combination of RPT with immune checkpoint inhibitors, which may offer potential therapeutic synergies for patients with metastatic cancers., Competing Interests: RH received patent royalties from the Wisconsin Alumni Research Foundation and consulting fees from Archeus Technologies Inc. and Monopar Therapeutics. ZSM is a member of the Scientific Advisory Board for Archeus Technologies, Seneca Therapeutics, and NorthStar Medical Isotopes; received royalties from patents held by the Wisconsin Alumni Research Foundation; received stock/stock options from the Archeus Technologies Scientific Advisory Board and the Seneca Therapeutics Scientific Advisory Board; received research support from Point Biopharma, Telix Pharmaceuticals, and XRD Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. The author(s) declared that they were editorial board members of Frontiers at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Shea, Idrissou, Torres, Chen, Hernandez, Morris and Sodji.)

Background and Purpose: Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) can elicit immunostimulatory effects. However, TRT has never been combined with CAR T cells against solid tumors in a clinical setting. This study investigated the effects of radiation delivered by Lutetium-177 ( 177 Lu) and Actinium-225 ( 225 Ac) on the viability and effector function of CAR T cells in vitro to evaluate the feasibility of such therapeutic combinations. After the irradiation of anti-GD2 CAR T cells with various doses of radiation delivered by 177 Lu or 225 Ac, their viability and cytotoxic activity against GD2-expressing human CHLA-20 neuroblastoma and melanoma M21 cells were determined by flow cytometry. The expression of the exhaustion marker PD-1, activation marker CD69 and the activating receptor NKG2D was measured on the irradiated anti-GD2 CAR T cells. Both 177 Lu and 225 Ac displayed a dose-dependent toxicity on anti-GD2 CAR T cells. However, radiation enhanced the cytotoxic activity of these CAR T cells against CHLA-20 and M21 irrespective of the dose tested and the type of radionuclide. No significant changes in the expression of PD-1, CD69 and NKG2D was noted on the CAR T cells following irradiation. Given a lower CAR T cell viability at equal doses and an enhancement of cytotoxic activity irrespective of the radionuclide type, 177 Lu-based TRT may be preferred over 225 Ac-based TRT when evaluating a potential synergism between these therapies in vivo against solid tumors.

We report an in situ vaccination, adaptable to nearly any type of cancer, that combines radiotherapy targeting one tumor and intratumoral injection of this site with tumor-specific antibody and interleukin-2 (IL-2; 3xTx). In a phase I clinical trial, administration of 3xTx (with an immunocytokine fusion of tumor-specific antibody and IL-2, hu14.18-IL2) to subjects with metastatic melanoma increases peripheral CD8 + T cell effector polyfunctionality. This suggests the potential for 3xTx to promote antitumor immunity against metastatic tumors. In poorly immunogenic syngeneic murine melanoma or head and neck carcinoma models, 3xTx stimulates CD8 + T cell-mediated antitumor responses at targeted and non-targeted tumors. During 3xTx treatment, natural killer (NK) cells promote CTLA4 + regulatory T cell (T reg ) apoptosis in non-targeted tumors. This is dependent on NK cell expression of CD86, which is upregulated downstream of KLRK1. NK cell depletion increases T reg infiltration, diminishing CD8 + T cell-dependent antitumor response. These findings demonstrate that NK cells sustain and propagate CD8 + T cell immunity following 3xTx., Competing Interests: Declaration of interests Z.S.M. is a member of the scientific advisory boards for Archeus Technologies, NorthStar Medical Isotopes, and Seneca Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group., Competing Interests: RSP, BG & JP are all employees of Nimble Therapeutics, the producer of the high-density peptide arrays used for this research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hoefges, McIlwain, Erbe, Mathers, Xu, Melby, Tetreault, Le, Kim, Pinapati, Garcia, Patel, Heck, Feils, Tsarovsky, Hank, Morris, Ong and Sondel.)

The Immuno-Oncology Translational Network (IOTN) was established in 2018 as part of the Cancer Moonshot. In 2022, President Joe Biden set new goals to reduce the cancer death rate by half within 25 years and improve the lives of people with cancer and cancer survivors. The IOTN is focused on accelerating translation of cancer immunology research, from bench to bedside, and improving immunotherapy outcomes across a wide array of cancers in the adult population. The unique structure and team science approach of the IOTN is designed to accelerate discovery and evaluation of novel immune-based therapeutic and prevention strategies. In this article, we describe IOTN progress to date, including new initiatives and the development of a robust set of resources to advance cancer immunology research. We summarize new insights by IOTN researchers, some of which are ripe for translation for several types of cancers. Looking to the future, we identify barriers to the translation of immuno-oncology concepts into clinical trials and key areas for action and improvements that are suitable for high-yield investments. Based on these experiences, we recommend novel National Institutes of Health funding mechanisms and development of new resources to address these barriers., (© The Author(s) 2023. Published by Oxford University Press.)

Radiation is commonly used as a treatment intended to cure or palliate cancer patients. Despite remarkable advances in the precision of radiotherapy delivery, even the most advanced forms inevitably expose some healthy tissues surrounding the target site to radiation. On rare occasions, this results in the development of radiation-associated secondary malignancies (RASM). RASM are typically high-grade and carry a poorer prognosis than their non-radiated counterparts. RASM are characterized by a high mutation burden, increased T cell infiltration, and a microenvironment that bears unique inflammatory signatures of prior radiation, including increased expression of various cytokines (e.g., TGF-β, TNF-α, IL4, and IL10). Interestingly, these cytokines have been shown to up-regulate the expression of PD-1 and/or PD-L1-an immune checkpoint receptor/ligand pair that is commonly targeted by immune checkpoint blocking immunotherapies. Here, we review the current understanding of the tumor-immune interactions in RASM, highlight the distinct clinical and molecular characteristics of RASM that may render them immunologically "hot," and propose a rationale for the formal testing of immune checkpoint blockade as a treatment approach for patients with RASM., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: We have previously shown that an intratumoral (IT) injection of the hu14.18-IL2 immunocytokine (IC), an anti-GD2 antibody linked to interleukin 2, can serve as an in situ vaccine and synergize with local radiotherapy (RT) to induce T cell-mediated antitumor effects. We hypothesized that cyclophosphamide (CY), a chemotherapeutic agent capable of depleting T regulatory cells (Tregs), would augment in situ vaccination. GD2 + B78 mouse melanoma cells were injected intradermally in syngeneic C57BL/6 mice., Methods: Treatments with RT (12Gy) and/or CY (100 mg/kg i.p.) started when tumors reached 100-300 mm 3 (day 0 of treatment), followed by five daily injections of IT-IC (25 mcg) on days 5-9. Tumor growth and survival were followed. In addition, tumors were analyzed by flow cytometry., Results: Similar to RT, CY enhanced the antitumor effect of IC. The strongest antitumor effect was achieved when CY, RT and IC were combined, as compared to combinations of IC+RT or IC+CY. Flow cytometric analyses showed that the combined treatment with CY, RT and IC decreased Tregs and increased the ratio of CD8+ cells/Tregs within the tumors. Moreover, in mice bearing two separate tumors, the combination of RT and IT-IC delivered to one tumor, together with systemic CY, led to a systemic antitumor effect detected as shrinkage of the tumor not treated directly with RT and IT-IC. Cured mice developed immunological memory as they were able to reject B78 tumor rechallenge., Conclusion: Taken together, these preclinical results show that CY can augment the antitumor efficacy of IT- IC, given alone or in combination with local RT, suggesting potential benefit in clinical testing of these combinations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tsarovsky, Felder, Heck, Slowinski, Rasmussen, VandenHeuvel, Zaborek, Morris, Erbe, Sondel and Rakhmilevich.)

Background: Radiation therapy (RT) elicits DNA double-strand breaks, resulting in tumor cytotoxicity and a type I interferon (IFN) response via stimulator of interferon genes (STING) activation. We investigated whether combining RT with an ataxia-telangiectasia mutated inhibitor promoted these effects and amplified tumor immunity., Methods: Mice-bearing syngeneic flank tumors (MOC2 head and neck squamous cell carcinoma or B78 melanoma) were treated with tumor-directed RT and oral administration of AZD0156. Specific immune cell depletion, type 1 interferon receptor 1 knock-out mice (IFNAR1-KO), and STING-deficient tumor cells were used to investigate tumor-immune crosstalk following RT and AZD0156 treatment., Results: Combining RT and AZD0156 reduced tumor growth compared with RT or AZD0156 alone in mice bearing MOC2 or B78 tumors. Low-dose AZD0156 (1-100 nM) alone did not affect tumor cell proliferation but suppressed tumor cell clonogenicity in combination with RT. Low-dose AZD0156 with RT synergistically increased IFN-β, major histocompatibility complex (MHC)-I, and programmed death-ligand 1 (PD-L1) expression in tumor cells. In contrast to wild-type mice, IFNAR1-KO mice showed reduced CD8+T cell tumor infiltration and poor survival following RT+AZD0156 treatment. CD8+T cell depletion reduced antitumor response during RT+AZD0156 treatment. STING-deficient MOC2 (MOC2-STING+/-) or B78 (B78-STING-/-) tumors eliminated the effects of RT+AZD0156 on the expression of IFN-β, MHC-I, and PD-L1, and reduced CD8+T cell infiltration and migration. Additional anti-PD-L1 therapy promoted antitumor response by elevation of tumor-MHC-I and lymphocyte activation., Conclusions: Combined radiation and AZD0156 increase STING-dependent antitumor response. Tumor-derived cell-autonomous IFN-β amplification drives both MHC-I and PD-L1 induction at the tumor cell surface, which is required by anti-PD-L1 therapy to promote antitumor immune response following RT and AZD0156 combination therapy., Competing Interests: Competing interests: ZSM is a member of the Scientific Advisory Boards for Archeus Technologies, Seneca Therapeutics, and NorthStar Medical Isotopes., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The in-situ vaccine using CpG oligodeoxynucleotide combined with OX40 agonist antibody (CpG + OX40) has been shown to be an effective therapy activating an anti-tumor T cell response in certain settings. The roles of tumor volume, tumor model, and the addition of checkpoint blockade in the efficacy of CpG + OX40 in-situ vaccination remains unknown., Methods: Mice bearing flank tumors (B78 melanoma or A20 lymphoma) were treated with combinations of CpG, OX40, and anti-CTLA-4. Tumor growth and survival were monitored. In vivo T cell depletion, tumor cell phenotype, and tumor infiltrating lymphocyte (TIL) studies were performed. Tumor cell sensitivity to CpG and macrophages were evaluated in vitro., Results: As tumor volumes increased in the B78 (one-tumor) and A20 (one-tumor or two-tumor) models, the anti-tumor efficacy of the in-situ vaccine decreased. In vitro, CpG had a direct effect on A20 proliferation and phenotype and an indirect effect on B78 proliferation via macrophage activation. As A20 tumors progressed in vivo, tumor cell phenotype changed, and T cells became more involved in the local CpG + OX40 mediated anti-tumor response. In mice with larger tumors that were poorly responsive to CpG + OX40, the addition of anti-CTLA-4 enhanced the anti-tumor efficacy in the A20 but not B78 models., Conclusions: Increased tumor volume negatively impacts the anti-tumor capability of CpG + OX40 in-situ vaccine. The addition of checkpoint blockade augmented the efficacy of CpG + OX40 in the A20 but not B78 model. These results highlight the importance of considering multiple preclinical model conditions when assessing the efficacy of cancer immunotherapy regimens and their translation to clinical testing., (© 2023. The Author(s).)

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference., Competing Interests: MHBH is or has have been a recipient of research grants paid to UCSF or in-kind resources from Roche-Genentech, Varian Medical Systems, Eli Lilly, Pathway Innovations and has received fees for consulting from EMD-Serono, Varian Medical Systems, Genentech, Pathway Innovation, Scholar Rock. KHhas Honoraria: Arch Oncology (Inst), AstraZeneca (Inst), BMS (Inst), Boehringer Ingelheim (Inst), Codiak Biosciences (Inst), F-Star Therapeutics (Inst), Inzen Therapeutics (Inst), Merck Serono (Inst), MSD (Inst), Oncolys Biopharma (Inst), Pfizer (Inst), Replimune (Inst), VacV Biotherapeutics (Inst); Consulting or Advisory Role: Arch Oncology (Inst), AstraZeneca (Inst), BMS (Inst), Boehringer Ingelheim (Inst), Inzen Therapeutics (Inst), Merck Serono (Inst), MSD (Inst), Oncolys BioPharma (Inst), Replimune (Inst); Speakers’ Bureau: BMS (Inst), Merck Serono (Inst), MSD (Inst); Research Funding: AstraZeneca (Inst), Boehringer Ingelheim (Inst), Merck Sharp & Dohme (Inst), Replimune (Inst). FGH received Grant or Research Support Companies from Accuray inc, Bioprotect, Bristol-Myers Squibb, Roche-ImFlame/ImCore, Nanobiotix, AstraZeneca, Debio Pharmaceuticals, Seagen, Eisai, MSD; Grant or Research Support Foundations from Prostate Cancer Foundation, San Salvatore Foundation; Investigator or Co-Investigator Clinical Trials in Bristol-Myers Squibb; Consultations: Johnson & Johnson; Academic Collaborations: EORTC chairman Gynecology Cancer Group, ESMO Scientific Committee member for drug development, ASTRO Scientific Committee Annual Meeting. TH has Consulting: Synthetic Biologics, Novocure, Boston Scientific, Inivata, Merck, GSK; Scientific Advisory Board: PanTher Therapeutics (Equity), Lustgarten; Research Funding (Clinical Trials): Taiho, AstraZeneca, BMS, GSK, IntraOp, Ipsen. EJ reports other support from Abmeta and Adventris, personal fees from Achilles, Dragonfly, Mestag, The Medical Home Group, and Surgtx, other support from Parker Institute, grants and other support from the Lustgarten Foundation, Genentech, BMS, and Break Through Cancer outside the submitted work. SDK receives clinical funding from AstraZeneca, Genentech, and Ionis; she also receives preclinical research funding from Roche. KS is founder and consultant for Faeth Therapeutics and Transomic Technologies. CK is the co-recipient of a Sponsored Research Agreement from Ion Beam Applications (IBA). AM is funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC IG 2018 Id.21763 and AIRC Programma di ricerca 5 per Mille 2019 Id.22737). MM declare grants from Boehringer Ingelheim, AC Biosciences and MSD outside the submitted work. ZSM has Scientific Advisory Board roles and equity options with Archeus Technologies and Seneca Therapeutics. JS owns stock and is a member of the Scientific Advisory Board of Surface Oncology, and is a member of the Scientific Advisory Board of Domain Therapeutics. RT has research grants to his institution from: Varian Medical Systems, Elekta Oncology, Accuray, Inc; scientific advisory board member for: Reflexion Medical, ImmuneSensor Therapeutics. AW acknowledge funding from AstraZeneca and imCORE. RW has stock and other ownership interests with Boost Therapeutics, Immvira LLC, Reflexion Pharmaceuticals, Coordination Pharmaceuticals Inc., Magi Therapeutics, Oncosenescence, Aqualung Therapeutics Corporation, and Cyntegron; he has served in a consulting or advisory role for Aettis Inc., AstraZeneca, Coordination Pharmaceuticals, Genus, Merck Serono S.A., Nano Proteagen, NKGen Biotech, Shuttle Pharmaceuticals, Highlight Therapeutics, S.L., Aqualung Therapeutics Corporation; he has research grants with Varian and Regeneron. RZ is scientific advisory board member of iTeos Therapeutics, receives research grant support from Bristol Myers Squibb and AstraZeneca, and is inventor on patent applications related to work on GITR, CTLA-4, and PD-1 (patent numbers: US20180244793A1; US10323091B2; WO2018106864A1; WO2019094352A1). SD has received compensation for consultant/advisory services from Lytix Biopharma, Mersana Therapeutics, EMD Serono, Ono Pharmaceutical, and Genentech, and research support from Lytix Biopharma and Boehringer-Ingelheim for unrelated projects. LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. ED reports grants and personal fees from Roche Genentech; grants from Servier; grants from AstraZeneca; grants and personal fees from Merck-Serono; grants from BMS; and grants from MSD outside the submitted work. SCF has Consultant: Bayer, Bristol Myers Squibb, Varian, ViewRay, Accuray, Elekta, Janssen, Regeneron, GlaxoSmithKline, Eisai, Astra Zeneca, MedImmune, Merck US, EMD Serono/Merck, Genentech/ROCHE, Boehringer Ingelheim, Nanobiotix and Grant/Research: support from: Bristol Myers Squibb, Varian, Regeneron, Merck, Celldex. All other authors have no conflict of interest to declare., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Most patients with estrogen receptor positive (ER+) breast cancer do not respond to immune checkpoint inhibition (ICI); the tumor microenvironment (TME) of these cancers is generally immunosuppressive and contains few tumor-infiltrating lymphocytes. Radiation therapy (RT) can increase tumor inflammation and infiltration by lymphocytes but does not improve responses to ICIs in these patients. This may result, in part, from additional effects of RT that suppress anti-tumor immunity, including increased tumor infiltration by myeloid-derived suppressor cells and regulatory T cells. We hypothesized that anti-estrogens, which are a standard of care for ER+ breast cancer, may ameliorate these detrimental effects of RT by reducing the recruitment/ activation of suppressive immune populations in the radiated TME, increasing anti-tumor immunity and responsiveness to ICIs., Methods: To interrogate the effect of the selective estrogen receptor downregulator, fulvestrant, on the irradiated TME in the absence of confounding growth inhibition by fulvestrant on tumor cells, we used the TC11 murine model of anti-estrogen resistant ER+ breast cancer. Tumors were orthotopically transplanted into immunocompetent syngeneic mice. Once tumors were established, we initiated treatment with fulvestrant or vehicle, followed by external beam RT one week later. We examined the number and activity of tumor infiltrating immune cells using flow cytometry, microscopy, transcript levels, and cytokine profiles. We tested whether fulvestrant improved tumor response and animal survival when added to the combination of RT and ICI., Results: Despite resistance of TC11 tumors to anti-estrogen therapy alone, fulvestrant slowed tumor regrowth following RT, and significantly altered multiple immune populations in the irradiated TME. Fulvestrant reduced the influx of Ly6C+Ly6G+ cells, increased markers of pro-inflammatory myeloid cells and activated T cells, and augmented the ratio of CD8+: FOXP3+ T cells. In contrast to the minimal effects of ICIs when co-treated with either fulvestrant or RT alone, combinatorial treatment with fulvestrant, RT and ICIs significantly reduced tumor growth and prolonged survival., Conclusions: A combination of RT and fulvestrant can overcome the immunosuppressive TME in a preclinical model of ER+ breast cancer, enhancing the anti-tumor response and increasing the response to ICIs, even when growth of tumor cells is no longer estrogen sensitive., (© 2023. The Author(s).)

Purpose: The recently reported FLAME trial demonstrated a biochemical disease-free survival benefit to using a focal intraprostatic boost to multiparametric magnetic resonance imaging (mpMRI)-identified lesions in men with localized prostate cancer treated with definitive radiation therapy. Prostate-specific membrane antigen (PSMA)-directed positron emission tomography (PET) may identify additional areas of disease. In this work, we investigated using both PSMA PET and mpMRI in planning focal intraprostatic boosts using stereotactic body radiation therapy (SBRT)., Methods and Materials: We evaluated a cohort of patients (n = 13) with localized prostate cancer who were imaged with 2-(3-(1-carboxy-5-[(6-[18F]fluoro-pyridine-2-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid ( 18 F-DCFPyL) PET/MRI on a prospective imaging trial before undergoing definitive therapy. The number of lesions concordant (overlapping) and discordant (no overlap) on PET and MRI was assessed. Overlap between concordant lesions was evaluated using the Dice and Jaccard similarity coefficients. Prostate SBRT plans were created fusing the PET/MRI imaging to computed tomography scans acquired the same day. Plans were created using only MRI-identified lesions, only PET-identified lesions, and the combined PET/MRI lesions. Coverage of the intraprostatic lesions and doses to the rectum and urethra were assessed for each of these plans., Results: The majority of lesions (21/39, 53.8%) were discordant between MRI and PET, with more lesions seen by PET alone (12) than MRI alone (9). Of lesions that were concordant between PET and MRI, there were still areas that did not overlap between scans (average Dice coefficient, 0.34). Prostate SBRT planning using all lesions to define a focal intraprostatic boost provided the best coverage of all lesions without compromising constraints on the rectum and urethra., Conclusions: Using both mpMRI and PSMA-directed PET may better identify all areas of gross disease within the prostate. Using both imaging modalities could improve the planning of focal intraprostatic boosts., Competing Interests: Dr Morris reports the following conflicts of interest: Archeus Technologies, scientific advisory board; Seneca Therapeutics, scientific advisory board. Dr Wells reports the following conflicts of interest: Ethicon, consultant. Dr Cho reports the following conflicts of interest: Progenics Pharmaceuticals, consultant. No other disclosures were reported., (© 2023 The Authors.)

Background: The antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with intratumoral anti-GD2 hu14.18-IL2 immunocytokine (IC) generates an effective in situ vaccine (ISV) against GD2-positive murine tumors. This ISV is effective in eradicating single tumors with sustained immune memory; however, it does not generate an adequate abscopal response against macroscopic distant tumors. Given the immune-stimulatory capacity of radiation therapy (RT), we hypothesized that delivering RT to all sites of disease would augment systemic antitumor responses to ISV., Methods: We used a syngeneic B78 murine melanoma model consisting of a 'primary' flank tumor and a contralateral smaller 'secondary' flank tumor, treated with 12 Gy EBRT and intratumoral IC immunotherapy to the primary and additional EBRT to the secondary tumor. As a means of delivering RT to all sites of disease, both known and occult, we also used a novel alkylphosphocholine analog, NM600, conjugated to 90 Y as a targeted radionuclide therapy (TRT). Tumor growth, overall survival, and cause of death were measured. Flow cytometry was used to evaluate immune population changes in both tumors., Results: Abscopal effects of local ISV were amplified by delivering as little as 2-6 Gy of EBRT to the secondary tumor. When the primary tumor ISV regimen was delivered in mice receiving 12 Gy EBRT to the secondary tumor, we observed improved overall survival and more disease-free mice with immune memory compared with either ISV or 12 Gy EBRT alone. Similarly, TRT combined with ISV resulted in improved overall survival and a trend towards reduced tumor growth rates when compared with either treatment alone. Using flow cytometry, we identified an influx of CD8 + T cells with a less exhausted phenotype in both the ISV-targeted primary and the distant secondary tumor following the combination of secondary tumor EBRT or TRT with primary tumor ISV., Conclusions: We report a novel use for low-dose RT, not as a direct antitumor modality but as an immunomodulator capable of driving and expanding antitumor immunity against metastatic tumor sites following ISV., Competing Interests: Competing interests: ZSM, JPW, RH, and JG have financial interests in Archeus Technologies. ZSM is a member of the Scientific Advisory Boards for Archeus Technologies and for Seneca Therapeutics. PMS is an unpaid medical advisor for Invenra. JPW is a cofounder, CSO, and director of Archeus Technologies, which holds the licence rights to NM600-related technologies. BPB and JG are cofounders of Voximetry, and BPB is the CSO. The following patents have been applied for or filed by the University of Wisconsin Alumni Research Foundation: US Patent 10,736,949, 'Radiohalogenated agents for in situ immune modulated cancer vaccination', with ZSM, PMS, JPW, and BPB as inventors; US Patent 10,751,430, 'Targeted radiotherapy chelates for in situ immune modulated cancer vaccination' with ZSM, PMS, JPW, BPB, and PMC as inventors; application no. 15/809,427, 'Using targeted radiotherapy to drive anti-tumor immune response to immunotherapies', ZSM, PMS, JPW, PMC, JG, RBP, and RH as inventors; and US 2011/0060602, 'A1 treatment planning system for radiopharmaceuticals', with BPB and JG as inventors., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Targeted radionuclide therapy (TRT) and immunotherapy are rapidly growing classes of cancer treatments. Basic, translational, and clinical research are now investigating therapeutic combinations of these agents. In comparison to external beam radiation therapy (EBRT), TRT has the unique advantage of treating all disease sites following intravenous injection and selective tumor uptake and retention-a particularly beneficial property in metastatic disease settings. The therapeutic value of combining radiation therapy with immune checkpoint blockade to treat metastases has been demonstrated in preclinical studies, whereas results of clinical studies have been mixed. Several clinical trials combining TRT and immune checkpoint blockade have been initiated based on preclinical studies combining these with EBRT and/or TRT. Despite the interest in translation of TRT and immunotherapy combinations, many questions remain surrounding the mechanisms of interaction and the optimal approach to clinical implementation of these combinations. This review highlights the mechanisms of interaction between anti-tumor immunity and radiation therapy and the status of basic and translational research and clinical trials investigating combinations of TRT and immunotherapies.

Background: Radiation therapy (RT) has been demonstrated to generate an in situ vaccination (ISV) effect in murine models and in patients with cancer; however, this has not routinely translated into enhanced clinical response to immune checkpoint inhibition (ICI). We investigated whether the commonly used vaccine adjuvant, monophosphoryl lipid A (MPL) could augment the ISV regimen consisting of combination RT and ICI., Materials/methods: We used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). Tumor-bearing mice received either RT (12 Gy, day 1), RT+anti-CTLA-4 (C4, day 3, 6, 9), MPL (20 µg IT injection days 5, 7, 9), RT+C4+MPL, or PBS control. To evaluate the effect of MPL on the irradiated tumor microenvironment, primary tumor with tumor draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling, and serum was collected for analysis of antitumor antibody populations., Results: Combination RT+C4+MPL significantly reduced tumor growth, increased survival and complete response rate compared with RT+C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor-immune microenvironment toward M1 macrophage and Th1 TBET + CD4 + T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1-associated and M1-associated proinflammatory cytokines. In co-culture models, MPL-stimulated macrophages directly activated CD8 T cells and polarized CD4 cells toward Th1 phenotype. MPL treatment significantly increased production of Th1-associated, IgG2c antitumor antibodies, which were required for and predictive of antitumor response to RT+C4+MPL, and enabled macrophage-mediated antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In metastatic models, RT and MPL generated a systemic antitumor immune response that augmented response to ICIs. This was dependent on macrophages and CD4 + but not CD8+T cells., Conclusions: We report the potential for MPL to augment the ISV effect of combination RT+C4 through FcγR, macrophage, and TBET + CD4 + Th1 cell dependent mechanisms. To our knowledge, this is the first report describing generation of a CD8 + T cell-independent, Th1 polarized, systemic antitumor immune response with subsequent generation of immunologic memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches., Competing Interests: Competing interests: ZSM has financial interest in Archeus Technologies. ZSM is a member of the Scientific Advisory Boards for Archeus Technologies and for Seneca Therapeutics. Based on the results presented herein, ZSM and JCJ are inventors on a filed patent that is managed by the Wisconsin Alumni Research Foundation relating to the use of MPL as an adjuvant for in situ vaccines. ZSM and PMS are inventors on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to mAb-related or nanoparticle immunotherapies and the interaction of targeted radionuclide therapies and immunotherapies., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Radiation therapy (RT) activates an in situ vaccine effect when combined with immune checkpoint blockade (ICB), yet this effect may be limited because RT does not fully optimize tumor antigen presentation or fully overcome suppressive mechanisms in the tumor-immune microenvironment. To overcome this, we develop a multifunctional nanoparticle composed of polylysine, iron oxide, and CpG (PIC) to increase tumor antigen presentation, increase the ratio of M1:M2 tumor-associated macrophages, and enhance stimulation of a type I interferon response in conjunction with RT. In syngeneic immunologically "cold" murine tumor models, the combination of RT, PIC, and ICB significantly improves tumor response and overall survival resulting in cure of many mice and consistent activation of tumor-specific immune memory. Combining RT with PIC to elicit a robust in situ vaccine effect presents a simple and readily translatable strategy to potentiate adaptive anti-tumor immunity and augment response to ICB or potentially other immunotherapies., (© 2022. The Author(s).)

Background: Most pediatric cancers are considered immunologically cold with relatively few responding to immune checkpoint inhibition. We recently described an effective combination radio-immunotherapy treatment regimen ( c ombination a daptive- i nnate immunotherapy r egimen (CAIR)) targeting adaptive and innate immunity in 9464D-GD2, an immunologically cold model of neuroblastoma. Here, we characterize the mechanism of CAIR and the role of major histocompatibility complex class I (MHC-I) in the treatment response., Methods: Mice bearing GD2-expressing 9464D-GD2 tumors were treated with CAIR (external beam radiotherapy, hu14.18-IL2 immunocytokine, CpG, anti-CD40, and anti-CTLA4) and tumor growth and survival were tracked. Depletion of specific immune cell lineages, as well as testing in immunodeficient R2G2 mice, were used to determine the populations necessary for treatment efficacy. Induction of MHC-I expression in 9464D-GD2 cells in response to interferon-γ (IFN-γ) and CAIR was measured in vitro and in vivo , respectively, by flow cytometry and quantitative real-time PCR. A cell line with IFN-γ-inducible MHC-I expression (9464D-GD2-I) was generated by transfecting a subclone of the parental cell line capable of expressing MHC-I with GD2 synthase and was used in vivo to assess the impact of MHC-I expression on responsiveness to CAIR., Results: CAIR cures some mice bearing small (50 mm 3 ) but not larger (100 mm 3 ) 9464D-GD2 tumors and these cured mice develop weak memory responses against tumor rechallenge. Early suppression of 9464D-GD2 tumors by CAIR does not require T or natural killer (NK) cells, but eventual tumor cures are NK cell dependent. Unlike the parental 9464D cell line, 9464D-GD2 cells have uniformly very low MHC-I expression at baseline and fail to upregulate expression in response to IFN-γ. In contrast, 9464D-GD2-I upregulates MHC-I in response to IFN-γ and is less responsive to CAIR., Conclusion: Treatment with CAIR cures 9464D-GD2 tumors in a NK cell dependent manner and induction of MHC-I by tumors cells was associated with decreased efficacy. These results demonstrate that the early tumor response to this regimen is T and NK cell independent, but that NK cells have a role in generating lasting cures in the absence of MHC-I expression by tumor cells. Further strategies to better inhibit tumor outgrowth in this setting may require further NK activation or the ability to engage alternative immune effector cells., Competing Interests: Competing interests: SDG declares employment and ownership interests in Provenance Biopharmaceuticals., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Soft tissue sarcomas (STS) are a rare class of tumors that originate from mesenchymal tissues and occur most frequently in the extremities, trunk, and retroperitoneum. Surgical resection with R0 margins is the primary curative treatment for most localized STS. In this setting, radiation therapy is used either pre-operatively or post-operatively to reduce the rate of local recurrence. Modern pre- or post-operative radiation therapy rely on the use of MRI sequences to guide target delineation during treatment planning. MRI-guided radiotherapy also offers unique advantages over CT-guided approaches in differentiating STS from surrounding normal soft tissues and enabling better identification of target volumes on daily imaging. For patients with unresectable STS, radiation therapy may offer the best chance for local tumor control. However, most STS are relatively radioresistant with modest rates of local control achieved using conventionally fractionated radiation. Specialized techniques such as hypofractionated radiation may allow for dose intensification and may increase rates of local control for STS. In these settings, MRI becomes even more critical for the delineation of targets and organs at risk and management of tumor and organ at risk motion during and between radiotherapy treatment fractions.

Introduction: Combining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically "cold" tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically "cold" tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically "cold" tumor models., Methods: Mice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment., Results: An in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the "cold" B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen., Conclusion: RT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically "cold", solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression., Competing Interests: ZM is a member of the scientific advisory board for Archeus Technologies and Seneca Therapeutics and received equity options for these companies. ZM is an inventor on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to the interaction of targeted radionuclide therapies and immunotherapies, nanoparticles designed to augment the anti-tumor immune response following radiation therapy, and the development of a brachytherapy catheter capable of delivering intra-tumor injectables. PS is an inventor on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to mAb-related immunotherapies and the interaction of targeted radionuclide therapies and immunotherapies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WS declared a shared affiliation with one of the authors, RP, to the handling editor at the time of the review., (Copyright © 2021 Pieper, Zangl, Speigelman, Feils, Hoefges, Jagodinsky, Felder, Tsarovsky, Arthur, Brown, Birstler, Le, Carlson, Bates, Hank, Rakhmilevich, Erbe, Sondel, Patel and Morris.)

Rationale: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer., Methods: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed., Results: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT., Conclusions: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following compeXng interests: ZM – Scientific Advisory Board Member and equity options, Archeus Technologies, Scientific Advisory Board Member and equity options, Seneca Therapeutics, Research Material support (reagents) from Bristol-Myers Squibb, AstraZeneca, Nektar Therapeutics, Apeiron Biologics, XRAd Therapeutics. BB and JG has ownership interest in Voximetry Inc (Middleton, WI). MRA - Research collaborations through the University of Wisconsin with Bristol Myers Squibb (Redwood City, CA and Princeton, NJ) and with Apeiron Biologics (Vienna, Austria). JW is a co-founder and CSO of Archeus Technologies, Inc (Madison, WI). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Radiotherapy can facilitate the immune recognition of immunologically "cold" tumors and enhance the efficacy of anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors (ICIs) in melanoma. Systemic administration of receptor-targeted radionuclide therapy has the potential to selectively deliver radionuclides to multiple tumors throughout the body in metastatic settings. By triggering immunologic cell death and increasing the immune susceptibility of surviving tumor cells in these locations, targeted radionuclide therapies may overcome resistance to ICIs and render immunologically "cold" tumors throughout the body responsive to ICIs and immunologically "hot". Here, we show the anti-tumor cooperation of targeted α-particle radionuclide therapy (α-TRT) and ICIs in preclinical models of melanoma. Melanocortin 1 receptor (MC1R)-targeted radiopeptide [ 212 Pb]VMT01 was employed to deliver α-radiation to melanoma tumors in mice. A single injection of 4.1 MBq [ 212 Pb]VMT01 significantly slowed the tumor growth of B16-F10 melanoma and the combination of [ 212 Pb]VMT01 and ICIs induced a cooperative anti-tumor effect leading to 43% complete tumor response with no sign of malignancy on autopsy. Animals with complete response developed anti-tumor immunity to reject further tumor inoculations. This therapeutic cooperation was completely abolished in RAG1 KO mice, which are deficient in T-cell maturation. In addition, the anti-tumor cooperation was compromised when fractionated [ 212 Pb]VMT01 was used in the combination. We also demonstrated that [ 212 Pb]VMT01 induced immunogenic cell death in tumor vaccination assays and in vitro exposure to [ 212 Pb]VMT01 sensitized immunotolerant melanoma to ICIs treatment in vivo. Enhanced tumor infiltrating CD3 + , CD4 + , CD8 + lymphocytes were observed following injection of 1.4 MBq [ 212 Pb]VMT01. Overall, we demonstrated anti-tumor cooperation between α-TRT and ICIs in melanoma that is mediated by tumor specific immunity.

Background: Current clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential IL-2 pathway agonist with prolonged in vivo half-life and preferential intratumoral expansion of T effector cells over T regulatory cells. BEMPEG has shown encouraging safety and efficacy in clinical trials when used in combination with PD-1 checkpoint blockade. In this study, we investigated the antitumor effect of local RT combined with BEMPEG in multiple immunologically 'cold' tumor models. Additionally, we asked if ICB could further enhance the local and distant antitumor effect of RT+BEMPEG in the setting of advanced solid tumors or metastatic disease., Methods: Mice bearing flank tumors (B78 melanoma, 4T1 breast cancer, or MOC2 head and neck squamous cell carcinoma) were treated with combinations of RT and immunotherapy (including BEMPEG, high-dose IL-2, anti(α)-CTLA-4, and α-PD-L1). Mice bearing B78 flank tumors were injected intravenously with B16 melanoma cells to mimic metastatic disease and were subsequently treated with RT and/or immunotherapy. Tumor growth and survival were monitored. Peripheral T cells and tumor-infiltrating lymphocytes were assessed via flow cytometry., Results: A cooperative antitumor effect was observed in all models when RT was combined with BEMPEG, and RT increased IL-2 receptor expression on peripheral T cells. This cooperative interaction was associated with increased IL-2 receptor expression on peripheral T cells following RT. In the B78 melanoma model, RT+BEMPEG resulted in complete tumor regression in the majority of mice with a single ~400 mm 3 tumor. This antitumor response was T-cell dependent and supported by long-lasting immune memory. Adding ICB to RT+BEMPEG strengthened the antitumor response and cured the majority of mice with a single ~1000 mm 3 B78 tumor. In models with disseminated metastasis (B78 primary with B16 metastasis, 4T1, and MOC2), the triple combination of RT, BEMPEG, and ICB significantly improved primary tumor response and survival., Conclusion: The combination of local RT, BEMPEG, and ICB cured mice with advanced, immunologically cold tumors and distant metastasis in a T cell-dependent manner, suggesting this triple combination warrants clinical testing., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend their biological functions in various physiological and pathological processes. Herein, we report a novel approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for localization of the C[double bond, length as m-dash]C bond in unsaturated FAs, which enables both quantification and spatial visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the C[double bond, length as m-dash]C positions were produced upon fragmentation of the FA epoxides derived from either in-solution or on-tissue PAA epoxidation of free FAs. The performance of the proposed approach was evaluated by analysis of FAs in human cell lines as well as mapping the FA isomers from cancer tissue samples with MALDI-TOF/TOF-MS. Merits of the newly developed method include high sensitivity, simplicity, high reaction efficiency, and capability of spatial characterization of FA isomers in tissue samples., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Surgical resection or hypo-fractionated radiation therapy (RT) in early-stage non-small cell lung cancer (NSCLC) achieves local tumor control, but metastatic relapse remains a challenge. We hypothesized that immunotherapy with anti-CTLA-4 and bempegaldesleukin (BEMPEG; NKTR-214), a CD122-preferential IL2 pathway agonist, after primary tumor RT or resection would reduce metastases in a syngeneic murine NSCLC model. Mice bearing Lewis Lung Carcinoma (LLC) tumors were treated with combinations of BEMPEG, anti-CTLA-4, and primary tumor treatment (surgical resection or RT). Primary tumor size, mouse survival, and metastatic disease at the time of death were assessed. Flow cytometry, qRT-PCR, and cytokine analyses were performed on tumor specimens. All mice treated with RT or surgical resection of primary tumor alone succumbed to metastatic disease, and all mice treated with BEMPEG and/or anti-CTLA-4 succumbed to primary tumor local progression. The combination of primary tumor RT or resection and BEMPEG and anti-CTLA-4 reduced spontaneous metastasis and improved survival without any noted toxicity. Flow cytometric immunoprofiling of primary tumors revealed increased CD8 T and NK cells and decreased T-regulatory cells with the combination of BEMPEG, anti-CTLA-4, and RT compared to RT alone. Increased expression of genes associated with tumor cell immune susceptibility, immune cell recruitment, and cytotoxic T lymphocyte activation were observed in tumors of mice treated with BEMPEG, anti-CTLA-4, and RT. The combination of BEMPEG and anti-CTLA-4 with primary tumor RT or resection enabled effective control of local and metastatic disease in a preclinical murine NSCLC model. This therapeutic combination has important translational potential for patients with early-stage NSCLC and other cancers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bates, Brown, Pieper, Zangl, Arthur, Carlson, Le, Sosa, Clark, Sriramaneni, Kim, Patel and Morris.)

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90 Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90 Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies., Competing Interests: Competing Interests: JPW is a cofounder of Archeus Technologies which owns licensing rights to NM600. ZSM, RH, and JJG have financial interest in Archeus Technologies. A patent has been filed by the University of Wisconsin Alumni Research Foundation with RBP, RH, PMC, JJG, ZSM, and JPW listed as inventors., (© The author(s).)

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically 'cold' tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190-230 mm 3 , they were grouped into a 'wave' and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the 'mobile' or 'fixed' phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical 'fixed' versus 'mobile' characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In head and neck squamous cell carcinoma (HNSCC) tumors that over-expresses huEGFR, the anti-EGFR antibody, cetuximab, antagonizes tumor cell viability and sensitizes to radiation therapy. However, the immunologic interactions between cetuximab and radiation therapy are not well understood. We transduced two syngeneic murine HNSCC tumor cell lines to express human EGFR (MOC1- and MOC2-huEGFR) in order to facilitate evaluation of the immunologic interactions between radiation and cetuximab. Cetuximab was capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in MOC1- and MOC2-huEGFR cells but showed no effect on the viability or radiosensitivity of these tumor cells, which also express muEGFR that is not targeted by cetuximab. Radiation enhanced the susceptibility of MOC1- and MOC2-huEGFR to ADCC, eliciting a type I interferon response and increasing expression of NKG2D ligands on these tumor cells. Co-culture of splenocytes with cetuximab and MOC2-huEGFR cells resulted in increased expression of IFNγ in not only NK cells but also in CD8+ T cells, and this was dependent upon splenocyte expression of FcγR. In MOC2-huEGFR tumors, combining radiation and cetuximab induced tumor growth delay that required NK cells, EGFR expression, and FcγR on host immune cells. Combination of radiation and cetuximab increased tumor infiltration with NK and CD8+ T cells but not regulatory T cells. Expression of PD-L1 was increased in MOC2-huEGFR tumors following treatment with radiation and cetuximab. Delivering anti-PD-L1 antibody with radiation and cetuximab improved survival and resulted in durable tumor regression in some mice. Notably, these cured mice showed evidence of an adaptive memory response that was not specifically directed against huEGFR. These findings suggest an opportunity to improve the treatment of HNSCC by combining radiation and cetuximab to engage an innate anti-tumor immune response that may prime an effective adaptive immune response when combined with immune checkpoint blockade. It is possible that this approach could be extended to any immunologically cold tumor that does not respond to immune checkpoint blockade alone and for which a tumor-specific antibody exists or could be developed., (Copyright © 2020 Jin, Erbe, Schwarz, Jaquish, Anderson, Sriramaneni, Jagodinsky, Bates, Clark, Le, Lan, Chen, Kim and Morris.)

Background: Some patients with cancer treated with anticancer monoclonal antibodies (mAbs) develop antidrug antibodies (ADAs) that recognize and bind the therapeutic antibody. This response may neutralize the therapeutic mAb, interfere with mAb effector function or cause toxicities. We investigated the potential influence of ADA to modify the tumor-binding capability of a tumor-reactive 'immunocytokine' (IC), namely, a fusion protein (hu14.18-IL2) consisting of a humanized, tumor-reactive, anti-GD2 mAb genetically linked to interleukin 2. We characterize the role of treatment delivery of IC (intravenous vs intratumoral) on the impact of ADA on therapeutic outcome following IC treatments in an established antimelanoma (MEL) regimen involving radiotherapy (RT) +IC., Methods: C57BL/6 mice were injected with human IgG or the hu14.18-IL2 IC to develop a mouse anti-human antibody (MAHA) response (MAHA + ). In vitro assays were performed to assess ADA binding to IC using sera from MAHA + and MAHA - mice. In vivo experiments assessed the levels of IC bound to tumor in MAHA + and MAHA - mice, and the influence of IC route of delivery on its ability to bind to B78 (GD2+) MEL tumors., Results: MAHA is inducible in C57BL/6 mice. In vitro assays show that MAHA is capable of inhibiting the binding of IC to GD2 antigen on B78 cells, resulting in impaired ADCC mediated by IC. When B78-bearing mice are injected intravenously with IC, less IC binds to B78-MEL tumors in MAHA + mice than in MAHA - mice. In contrast, when IC is injected intratumorally in tumor-bearing mice, the presence of MAHA does not detectibly impact IC binding to the tumor. Combination therapy with RT+IT-IC showed improved tumor regression compared with RT alone in MAHA + mice. If given intratumorally, IC could be safely readministered in tumor-bearing MAHA + mice, while intravenous injections of IC in MAHA + mice caused severe toxicity. Histamine levels were elevated in MAHA + mice compared with MAHA - mice after reintroduction of IC., Conclusions: Intratumoral injection may be a means of overcoming ADA neutralization of therapeutic activity of tumor-reactive mAbs or ICs and may reduce systemic toxicity, which could have significant translational relevance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In a syngeneic murine melanoma (MEL) model, we recently reported an in situ vaccination response to combined radiation (RT) and intra-tumoral (IT) injection of anti-GD2 hu14. 18-IL2 immunocytokine (IC). This combined treatment resulted in 71% complete and durable regression of 5-week tumors, a tumor-specific memory T cell response, and augmented response to systemic anti-CTLA-4 antibody checkpoint blockade. While the ability of radiation to diversify anti-tumor T cell response has been reported, we hypothesize that mice rendered disease-free (DF) by a RT-based ISV might also exhibit a heightened B cell response. C57BL/6 mice were engrafted with 2 × 10 6 GD2+ B78 MEL and treated at a target tumor size of ~200 mm 3 with 12 Gy RT, IT-IC on day (D)6-D10, and anti-CTLA-4 on D3, 6, and 9. Serum was collected via facial vein before tumor injection, before treatment, during treatment, after becoming DF, and following rejection of subcutaneous 2 × 10 6 B78 MEL re-challenge on D90. Flow cytometry demonstrated the presence of tumor-specific IgG in sera from mice rendered DF and rejecting re-challenge with B78 MEL at D90 after starting treatment. Consistent with an adaptive endogenous anti-tumor humoral memory response, these anti-tumor antibodies bound to B78 cells and parental B16 cells (GD2-), but not to the unrelated syngeneic Panc02 or Panc02 GD2+ cell lines. We evaluated the kinetics of this response and observed that tumor-specific IgG was consistently detected by D22 after initiation of treatment, corresponding to a time of rapid tumor regression. The amount of tumor-specific antibody binding to tumor cells (as measured by flow MFI) did not correlate with host animal prognosis. Incubation of B16 MEL cells in DF serum, vs. naïve serum, prior to IV injection, did not delay engraftment of B16 metastases and showed similar overall survival rates. B cell depletion using anti-CD20 or anti-CD19 and anti-B220 did not impact the efficacy of ISV treatment. Thus, treatment with RT + IC + anti-CTLA-4 results in adaptive anti-tumor humoral memory response. This endogenous tumor-specific antibody response does not appear to have therapeutic efficacy but may serve as a biomarker for an anti-tumor T cell response., (Copyright © 2020 Baniel, Heinze, Hoefges, Sumiec, Hank, Carlson, Jin, Patel, Sriramaneni, Gillies, Erbe, Schwarz, Pieper, Rakhmilevich, Sondel and Morris.)

Background: Immune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model., Methods: B78 (GD2 + ) melanoma 'primary' tumors were engrafted on the right flank of C57BL/6 mice. After 3-4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6-10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay., Results: ISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8 + and CD4 + T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8 + infiltration and CD8 + :FOXP3 + ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or Mhc-1 . Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts., Conclusion: ISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Purpose: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, to identify predictive immune and/or tumor biomarkers in patients with melanoma at high risk for recurrence., Experimental Design: Patients were randomly assigned to receive the first of three monthly courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known diseases. Tumors were evaluated by histology and whole transcriptome sequencing., Results: Tumor-infiltrating lymphocyte (TIL) levels directly associated with relapse-free survival (RFS) and overall survival (OS) in resected tumors from Group A, where early responses to the immunotherapy agent could be assessed. TIL levels directly associated with a previously reported immune signature, which associated with RFS and OS, particularly in Group A tumors. In Group A tumors, there were decreased cell-cycling gene RNA transcripts, but increased RNA transcripts for repair and growth genes. We found that outcome (RFS and OS) was directly associated with several immune signatures and immune-related RNA transcripts and inversely associated with several tumor growth-associated transcripts, particularly in Group A tumors. Most of these associations were not seen in Group B tumors., Conclusions: We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNA-sequencing analyses detected shortly after initiation of hu14.18-IL2 therapy, are associated with long-term survival and could potentially be used as prognostic biomarkers in tumor resection specimens obtained after initiating neoadjuvant immunotherapy., (©2020 American Association for Cancer Research.)

Background: Unlike some adult cancers, most pediatric cancers are considered immunologically cold and generally less responsive to immunotherapy. While immunotherapy has already been incorporated into standard of care treatment for pediatric patients with high-risk neuroblastoma, overall survival remains poor. In a mouse melanoma model, we found that radiation and tumor-specific immunocytokine generate an in situ vaccination response in syngeneic mice bearing large tumors. Here, we tested whether a novel immunotherapeutic approach utilizing radiation and immunocytokine together with innate immune stimulation could generate a potent antitumor response with immunologic memory against syngeneic murine neuroblastoma., Methods: Mice bearing disialoganglioside (GD2)-expressing neuroblastoma tumors (either NXS2 or 9464D-GD2) were treated with radiation and immunotherapy (including anti-GD2 immunocytokine with or without anti-CTLA-4, CpG and anti-CD40 monoclonal antibody). Tumor growth, animal survival and immune cell infiltrate were analyzed in the tumor microenvironment in response to various treatment regimens., Results: NXS2 had a moderate tumor mutation burden (TMB) while N-MYC driven 9464D-GD2 had a low TMB, therefore the latter served as a better model for high-risk neuroblastoma (an immunologically cold tumor). Radiation and immunocytokine induced a potent in situ vaccination response against NXS2 tumors, but not in the 9464D-GD2 tumor model. Addition of checkpoint blockade with anti-CTLA-4 was not effective alone against 9464D-GD2 tumors; inclusion of CpG and anti-CD40 achieved a potent antitumor response with decreased T regulatory cells within the tumors and induction of immunologic memory., Conclusions: These data suggest that a combined innate and adaptive immunotherapeutic approach can be effective against immunologically cold syngeneic murine neuroblastoma. Further testing is needed to determine how these concepts might translate into development of more effective immunotherapeutic approaches for the treatment of clinically high-risk neuroblastoma.

The distinctive anatomy of the crocodylian skull is intimately linked with dietary ecology, resulting in repeated convergence on blunt- and slender-snouted ecomorphs. These evolutionary shifts depend upon modifications of the developmental processes which direct growth and morphogenesis. Here we examine the evolution of cranial ontogenetic trajectories to shed light on the mechanisms underlying convergent snout evolution. We use geometric morphometrics to quantify skeletogenesis in an evolutionary context and reconstruct ancestral patterns of ontogenetic allometry to understand the developmental drivers of craniofacial diversity within Crocodylia. Our analyses uncovered a conserved embryonic region of morphospace (CER) shared by all non-gavialid crocodylians regardless of their eventual adult ecomorph. This observation suggests the presence of conserved developmental processes during early development (before Ferguson stage 20) across most of Crocodylia. Ancestral state reconstruction of ontogenetic trajectories revealed heterochrony, developmental constraint, and developmental systems drift have all played essential roles in the evolution of ecomorphs. Based on these observations, we conclude that two separate, but interconnected, developmental programmes controlling craniofacial morphogenesis and growth enabled the evolutionary plasticity of skull shape in crocodylians.

Finding improved therapeutic strategies against T-cell Non-Hodgkin's Lymphoma (NHL) remains an unmet clinical need. We implemented a theranostic approach employing a tumor-targeting alkylphosphocholine (NM600) radiolabeled with 86 Y for positron emission tomography (PET) imaging and 90 Y for targeted radionuclide therapy (TRT) of T-cell NHL. PET imaging and biodistribution performed in mouse models of T-cell NHL showed in vivo selective tumor uptake and retention of 86 Y-NM600. An initial toxicity assessment examining complete blood counts, blood chemistry, and histopathology of major organs established 90 Y-NM600 safety. Mice bearing T-cell NHL tumors treated with 90 Y-NM600 experienced tumor growth inhibition, extended survival, and a high degree of cure with immune memory toward tumor reestablishment. 90 Y-NM600 treatment was also effective against disseminated tumors, improving survival and cure rates. Finally, we observed a key role for the adaptive immune system in potentiating a durable anti-tumor response to TRT, especially in the presence of microscopic disease., Competing Interests: J.P.W. is a co-founder of Archeus Technologies, LLC, of which Z.S.M. is a member of the Scientific Advisory Board, and R.H. and J.J.G. are consultants. The remaining authors declare no competing interests.

Purpose: Complementary and alternative medicines (CAMs) are widely used by patients with cancer. However, little is known about the extent to which these potential remedies are used internationally to treat the most common toxicities of radiation therapy. We report on the results of an international survey that assessed the use of CAMs., Methods and Materials: Surveys were distributed to 1174 practicing radiation oncologists. Questions evaluated the perceptions of CAMs and specific practice patterns for the use of CAM remedies in the treatment of common radiation-induced toxicities (eg, skin, fatigue, nausea, diarrhea, and mucositis/xerostomia). The responses were compared between the groups using the χ 2 test and stratified on the basis of provider location, number of years in practice, and perception of CAMs., Results: A total of 114 radiation oncologists from 29 different countries completed the survey, with a balanced distribution between North American (n = 56) and non-North American (n = 58) providers. Among the responding clinicians, 63% recommended CAMs in their practice. The proportion of clinicians who recommend CAMs for radiation toxicities did not significantly vary when stratified by provider's number of years in practice ( P = .23) or location (United States/Canada vs other; P = .74). Overall, providers reported that 29.4% of their patients use CAMs, and 87.7% reported that their practice encouraged or was neutral on CAM use, whereas 12.3% recommended stopping CAMs. The most common sources of patient information on CAMs were the Internet (75.4%), friends (60.5%), and family (58.8%). Clinicians reported the highest use of CAMs for radiation skin toxicity at 66.7%, followed by 48.2% for fatigue, 40.4% for nausea, and 36.8% for mucositis/xerostomia., Conclusions: Nearly two-thirds of the surveyed radiation oncologists recommend CAMs for radiation-related toxicities; however, they estimated that less than one third of patients use CAMs for this purpose. This suggests a need for further investigation and perhaps greater patient education on the roles of CAMs in treating radiation toxicities.

The architectural and biochemical features of the plasma membrane are governed by its intimate association with the underlying cortical cytoskeleton. The neurofibromatosis type 2 (NF2) tumor suppressor merlin and closely related membrane:cytoskeleton-linking protein ezrin organize the membrane:cytoskeleton interface, a critical cellular compartment that both regulates and is regulated by growth factor receptors. An example of this poorly understood interrelationship is macropinocytosis, an ancient process of nutrient uptake and membrane remodeling that can both be triggered by growth factors and manage receptor availability. We show that merlin deficiency primes the membrane:cytoskeleton interface for epidermal growth factor (EGF)-induced macropinocytosis via a mechanism involving increased cortical ezrin, altered actomyosin, and stabilized cholesterol-rich membranes. These changes profoundly alter EGF receptor (EGFR) trafficking in merlin-deficient cells, favoring increased membrane levels of its heterodimerization partner, ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that the macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in NF2 mutant and other tumors., (© 2018 Chiasson-MacKenzie et al.; Published by Cold Spring Harbor Laboratory Press.)

Interest in combining radiotherapy and immune checkpoint therapy is growing rapidly. In this study, we explored a novel combination of this type to augment antitumor immune responses in preclinical murine models of melanoma, neuroblastoma, and head and neck squamous cell carcinoma. Cooperative effects were observed with local radiotherapy and intratumoral injection of tumor-specific antibodies, arising in part from enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We could improve this response by combining radiation with intratumoral injection of an IL2-linked tumor-specific antibody (termed here an immunocytokine), resulting in complete regression of established tumors in most animals associated with a tumor-specific memory T-cell response. Given the T-cell response elicited by combined local radiation and intratumoral immunocytokine, we tested the potential benefit of adding this treatment to immune checkpoint blockade. In mice bearing large primary tumors or disseminated metastases, the triple-combination of intratumoral immunocytokine, radiation, and systemic anti-CTLA-4 improved primary tumor response and animal survival compared with combinations of any two of these three interventions. Taken together, our results show how combining radiation and intratumoral immunocytokine in murine tumor models can eradicate large tumors and metastases, eliciting an in situ vaccination effect that can be leveraged further by T-cell checkpoint blockade, with immediate implications for clinical evaluation. Cancer Res; 76(13); 3929-41. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Aberrant regulation of the EGF receptor family (EGFR, HER2, HER3, HER4) contributes to tumorigenesis and metastasis in epithelial cancers. Pan-HER represents a novel molecular targeted therapeutic composed of a mixture of six monoclonal antibodies against EGFR, HER2, and HER3., Experimental Design: In the current study, we examine the capacity of Pan-HER to augment radiation response across a series of human lung and head and neck cancers, including EGFR inhibitor-resistant cell lines and xenografts., Results: Pan-HER demonstrates superior antiproliferative and radiosensitizing impact when compared with cetuximab. The mechanisms underlying these effects appear to involve attenuation of DNA damage repair, enhancement of programmed cell death, cell-cycle redistribution, and induction of cellular senescence. Combined treatment of Pan-HER with single or fractionated radiation in human tumor xenografts reveals a potent antitumor and regrowth delay impact compared with Pan-HER or radiation treatment alone., Conclusions: These data highlight the capacity of Pan-HER to augment radiation response in lung and head and neck cancer models and support investigation of Pan-HER combined with radiation as a promising clinical therapeutic strategy., (©2015 American Association for Cancer Research.)

Background: End-of-life care policy has a focus on enabling patients to die in their preferred place; this is believed for most to be home. This review assesses patient preferences for place of death examining: the extent of unreported preferences, the importance of patient factors (place of care and health diagnosis) and who reports preferences., Methods and Findings: Systematic literature review of 7 electronic databases, grey literature, backwards citations from included studies and Palliative Medicine hand search. Included studies published between 2000-2015, reporting original, quantifiable results of adult UK preferences for place of death. Of 10826 articles reviewed, 61 met the inclusion criteria. Summary charts present preferences for place of death by health diagnosis, where patients were asked and who reported the preference. These charts are recalculated to include 'missing data,' the views of those whose preferences were not asked, expressed or reported or absent in studies. Missing data were common. Across all health conditions when missing data were excluded the majority preference was for home: when missing data were included, it was not known what proportion of patients with cancer, non-cancer or multiple conditions preferred home. Patients, family proxies and public all expressed a majority preference for home when missing data were excluded: when included, it was not known what proportion of patients or family proxies preferred home. Where patients wished to die was related to where they were asked their preference. Missing data calculations are limited to 'reported' data., Conclusions: It is unknown what proportion of patients prefers to die at home or elsewhere. Reported preferences for place of death often exclude the views of those with no preference or not asked: when 'missing data' are included, they supress the proportion of preferences for all locations. Caution should be exercised if asserting that most patients prefer to die at home.

Introduction: Contralateral lung tumors in non-small-cell lung cancer (NSCLC) are classified as stage M1a yet may represent hematogenous metastases or synchronous primary tumors. The impact of these tumors on overall survival (OS) is poorly understood. Here, we aim to determine whether NSCLC patients with M1a disease due only to a contralateral tumor nodule exhibit a favorable prognosis relative to other M1a or M1b patients., Methods: Retrospective evaluation of the impact of contralateral tumor nodules on OS in NSCLC stratified by primary tumor size and N stage attained from Surveillance, Epidemiology, and End Results database., Results: Of 173,640 patients, 5161 M1a-contra patients were identified. Median and 3-year OS for these patients exceeded that of patients with M1b (p < 0.0001) or other M1a disease (p < 0.0001). Primary tumor size and N stage were strongly associated with OS in M1a-contra patients. Three-year OS demonstrated a delayed convergence between M1a-contra and other M1a patients with primary tumors greater than or equal to 3 cm or mediastinal lymph node involvement. Proportional hazard modeling indicated that T1-2N0-1M1a-contra patients exhibit OS not significantly different (p = 0.258) from that predicted with comparable T and N stage disease plus a second early-stage primary., Conclusions: Contralateral tumors in NSCLC carry a more favorable prognosis than other M1a or M1b disease. Primary tumor size and N stage may help distinguish M1a-contra patients with hematogenous metastasis from those with a synchronous, second primary.

The proliferation of normal cells is inhibited at confluence, but the molecular basis of this phenomenon, known as contact-dependent inhibition of proliferation, is unclear. We previously identified the neurofibromatosis type 2 (NF2) tumor suppressor Merlin as a critical mediator of contact-dependent inhibition of proliferation and specifically found that Merlin inhibits the internalization of, and signaling from, the epidermal growth factor receptor (EGFR) in response to cell contact. Merlin is closely related to the membrane-cytoskeleton linking proteins Ezrin, Radixin, and Moesin, and localization of Merlin to the cortical cytoskeleton is required for contact-dependent regulation of EGFR. We show that Merlin and Ezrin are essential components of a mechanism whereby mechanical forces associated with the establishment of cell-cell junctions are transduced across the cell cortex via the cortical actomyosin cytoskeleton to control the lateral mobility and activity of EGFR, providing novel insight into how cells inhibit mitogenic signaling in response to cell contact., (© 2015 Chiassson-MacKenzie et al.)

Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be "specifically activated" through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies.