A single photodynamic priming protocol augments delivery of ⍺-PD-L1 mAbs and induces immunogenic cell death in head and neck tumors.

Photodynamic priming (PDP) leverages the photobiological effects of subtherapeutic photodynamic therapy (PDT) regimens to modulate the tumor vasculature and stroma. PDP also sensitizes tumors to secondary therapies, such as immunotherapy by inducing a cascade of molecular events, including immunogenic cell death (ICD). We and others have shown that PDP improves the delivery of antibodies, among other theranostic agents. However, it is not known whether a single PDP protocol is capable of both inducing ICD in vivo and augmenting the delivery of immune checkpoint inhibitors. In this rapid communication, we show for the first time that a single PDP protocol using liposomal benzoporphyrin derivative (Lipo-BPD, 0.25 mg/kg) with 690 nm light (75 J/cm2 , 100 mW/cm2 ) simultaneously doubles the delivery of ⍺-PD-L1 antibodies in murine AT-84 head and neck tumors and induces ICD in vivo. ICD was observed as a 3-11 fold increase in tumor cell exposure of damage-associated molecular patterns (Calreticulin, HMGB1, and HSP70). These findings suggest that this single, highly translatable PDP protocol using clinically relevant Lipo-BPD holds potential for improving immunotherapy outcomes in head and neck cancer. It can do so by simultaneously overcoming physical barriers to the delivery of immune checkpoint inhibitors, and biochemical barriers that contribute to immunosuppression.

Biomimetic Human Lung Alveolar Interstitium Chip with Extended Longevity.

Determining the mechanistic causes of lung diseases, developing new treatments thereof, and assessing toxicity whether from chemical exposures or engineered nanomaterials would benefit significantly from a preclinical human lung alveolar interstitium model of physiological relevance. The existing preclinical models have limitations because they fail to replicate the key anatomical and physiological characteristics of human alveoli. Thus, a human lung alveolar interstitium chip was developed to imitate key alveolar microenvironmental factors including an electrospun nanofibrous membrane as the analogue of the basement membrane for co-culture of epithelial cells with fibroblasts embedded in 3D collagenous gels, physiologically relevant interstitial matrix stiffness, interstitial fluid flow, and 3D breathing-like mechanical stretch. The biomimetic chip substantially improved the epithelial barrier function compared to transwell models. Moreover, the chip having a gel made of a collagen I-fibrin blend as the interstitial matrix sustained the interstitium integrity and further enhanced the epithelial barrier, resulting in a longevity that extended beyond eight weeks. The assessment of multiwalled carbon nanotube toxicity on the chip was in line with the animal study.

Orthologs of human circulating miRNAs associated with hepatocellular carcinoma are elevated in mouse plasma months before tumour detection.

Research examining the potential for circulating miRNA to serve as markers for preneoplastic lesions or early-stage hepatocellular carcinoma (HCC) is hindered by the difficulties of obtaining samples from asymptomatic individuals. As a surrogate for human samples, we identified hub miRNAs in gene co-expression networks using HCC-bearing C3H mice. We confirmed 38 hub miRNAs as associated with HCC in F2 hybrid mice derived from radiogenic HCC susceptible and resistant founders. When compared to a panel of 12 circulating miRNAs associated with human HCC, two had no mouse ortholog and 7 of the remaining 10 miRNAs overlapped with the 38 mouse HCC hub miRNAs. Using small RNA sequencing data generated from serially collected plasma samples in F2 mice, we examined the temporal levels of these 7 circulating miRNAs and found that the levels of 4 human circulating markers, miR-122-5p, miR-100-5p, miR-34a-5p and miR-365-3p increased linearly as the time approaching HCC detection neared, suggesting a correlation of miRNA levels with oncogenic progression. Estimation of change points in the kinetics of the 4 circulating miRNAs suggested the changes started 17.5 to 6.8 months prior to HCC detection. These data establish these 4 circulating miRNAs as potential sentinels for preneoplastic lesions or early-stage HCC.

Evaluation of the Response of HNSCC Cell Lines to γ-Rays and 12C Ions: Can Radioresistant Tumors Be Identified and Selected for 12C Ion Radiotherapy?

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of 12C radiotherapy including enhanced cell killing, a decreased oxygen enhancement ratio, generation of complex DNA damage, and the potential to overcome immune suppression make its application well suited to the treatment of HNSCC. We examined the 12C radioresponse of five HNSCC cell lines, whose surviving fraction at 3.5 Gy ranged from average to resistant when compared with a larger panel of 38 cell lines to determine if 12C irradiation can overcome X-ray radioresistance and to identify biomarkers predictive of 12C radioresponse. Cells were irradiated with 12C using a SOBP with an average LET of 80 keV/µm (CNAO: Pavia, Italy). RBE values varied depending upon endpoint used. A 37 gene signature was able to place cells in their respective radiosensitivity cohort with an accuracy of 86%. Radioresistant cells were characterized by an enrichment of genes associated with radioresistance and survival mechanisms including but not limited to G2/M Checkpoint MTORC1, HIF1α, and PI3K/AKT/MTOR signaling. These data were used in conjunction with an in silico-based modeling approach to evaluate tumor control probability after 12C irradiation that compared clinically used treatment schedules with fixed RBE values vs. the RBEs determined for each cell line. Based on the above analysis, we present the framework of a strategy to utilize biological markers to predict which HNSCC patients would benefit the most from 12C radiotherapy.

Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex.

Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP's interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

Facile and direct detection of human papillomavirus (HPV) DNA in cells using loop-mediated isothermal amplification (LAMP).

Human papillomavirus (HPV)-mediated cancers, particularly cervical and oropharyngeal cancer, lead to hundreds of thousands of deaths worldwide each year. Simple, straightforward, and cost-effective detection of HPV DNA from patients with these malignancies or at risk for developing cancer can improve outcomes for patients, serving as a tool for early detection, monitoring treatment response, and assessment of cancer recurrence. Loop-mediated isothermal amplification (LAMP) is a simple and robust method for the detection and amplification of DNA in a single tube, utilizing the Bst strand-displacing DNA polymerase. We developed a workflow utilizing LAMP for the visual detection of HPV DNA in oral rinses. We demonstrate that LAMP is able to easily discriminate between two of the high-risk HPV subtypes, HPV16 and HPV18. We then utilized LAMP to visually detect HPV DNA directly from cells in oral rinses, mimicking a clinical inspired scenario of detecting HPV DNA in clinical samples. Our results suggest that LAMP is a robust, colorimetric assay method for the detection of HPV DNA in complex cellular samples, and further development is warranted to bring LAMP into the clinic.

Transcriptomic analysis links hepatocellular carcinoma (HCC) in HZE ion irradiated mice to a human HCC subtype with favorable outcomes.

High-charge, high-energy ion particle (HZE) radiations are extraterrestrial in origin and characterized by high linear energy transfer (high-LET), which causes more severe cell damage than low-LET radiations like γ-rays or photons. High-LET radiation poses potential cancer risks for astronauts on deep space missions, but the studies of its carcinogenic effects have relied heavily on animal models. It remains uncertain whether such data are applicable to human disease. Here, we used genomics approaches to directly compare high-LET radiation-induced, low-LET radiation-induced and spontaneous hepatocellular carcinoma (HCC) in mice with a human HCC cohort from The Cancer Genome Atlas (TCGA). We identified common molecular pathways between mouse and human HCC and discovered a subset of orthologous genes (mR-HCC) that associated high-LET radiation-induced mouse HCC with a subgroup (mrHCC2) of the TCGA cohort. The mrHCC2 TCGA cohort was more enriched with tumor-suppressing immune cells and showed a better prognostic outcome than other patient subgroups.

Associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation.

The space radiation environment consists of multiple species of charged particles, including 28Si ions, that may impact brain function during and following missions. To develop biomarkers of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with 28Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following 28Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation could be used for the development of biomarker of the space radiation response.

Avasopasem manganese synergizes with hypofractionated radiation to ablate tumors through the generation of hydrogen peroxide.

Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine- and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide-dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974).

An overview of potential novel mechanisms of action underlying Tumor Treating Fields-induced cancer cell death and their clinical implications.

Traditional cancer therapy choices for clinicians are surgery, chemotherapy, radiation and immune therapy which are used either standalone therapies or in various combinations. Other physical modalities beyond ionizing radiation include photodynamic therapy and heating and the more recent approach referred to as Tumor Treating Fields (TTFields). TTFields are intermediate frequency, low-intensity, alternating electric fields that are applied to tumor regions and cells using noninvasive arrays. TTFields have revolutionized the treatment of newly diagnosed and recurrent glioblastoma (GBM) and unresectable and locally advanced malignant pleural mesothelioma (MPM). TTFields are thought to kill tumor cells predominantly by disrupting mitosis; however it has been shown that TTFields increase efficacy of different classes of drugs, which directly target mitosis, replication stress and DNA damage pathways. Hence, a detailed understanding of TTFields' mechanisms of action is needed to use this therapy effectively in the clinic. Recent findings implicate TTFields' role in different important pathways such as DNA damage response and replication stress, ER stress, membrane permeability, autophagy, and immune response. This review focuses on potentially novel mechanisms of TTFields anti-tumor action and their implications in completed and ongoing clinical trials and pre-clinical studies. Moreover, the review discusses advantages and strategies using chemotherapy agents and radiation therapy in combination with TTFields for future clinical use.

Dose rate determination for preclinical total body irradiation.

The accuracy of delivered radiation dose and the reproducibility of employed radiotherapy methods are key factors for preclinical radiobiology applications and research studies. In this work, ionization chamber (IC) measurements and Monte Carlo (MC) simulations were used to accurately determine the dose rate for total body irradiation (TBI), a classic radiobiologic and immunologic experimental method. Several phantom configurations, including large solid water slab, small water box and rodentomorphic mouse and rat phantoms were simulated and measured for TBI setup utilizing a preclinical irradiator XRad320. The irradiator calibration and the phantom measurements were performed using an ADCL calibrated IC N31010 following the AAPM TG-61 protocol. The MC simulations were carried out using Geant4/GATE to compute absorbed dose distributions for all phantom configurations. All simulated and measured geometries had favorable agreement. On average, the relative dose rate difference was 2.3%. However, the study indicated large dose rate deviations, if calibration conditions are assumed for a given experimental setup as commonly done for a quick determination of irradiation times utilizing lookup tables and hand calculations. In a TBI setting, the reference calibration geometry at an extended source-to-surface distance and a large reference field size is likely to overestimate true photon scatter. Consequently, the measured and hand calculated dose rates, for TBI geometries in this study, had large discrepancies: 16% for a large solid water slab, 27% for a small water box, and 31%, 36%, and 30% for mouse phantom, rat phantom, and mouse phantom in a pie cage, respectively. Small changes in TBI experimental setup could result in large dose rate variations. MC simulations and the corresponding measurements specific to a designed experimental setup are vital for accurate preclinical dosimetry and reproducibility of radiobiological findings. This study supports the well-recognized need for physics consultation for all radiobiological investigations.

The Long and Short of It: The Emerging Roles of Non-Coding RNA in Small Extracellular Vesicles.

Small extracellular vesicles (EVs) play a significant role in intercellular communication through their non-coding RNA (ncRNA) cargo. While the initial examination of EV cargo identified both mRNA and miRNA, later studies revealed a wealth of other types of EV-related non-randomly packed ncRNAs, including tRNA and tRNA fragments, Y RNA, piRNA, rRNA, and lncRNA. A number of potential roles for these ncRNA species were suggested, with strong evidence provided in some cases, whereas the role for other ncRNA is more speculative. For example, long non-coding RNA might be used as a potential diagnostic tool but might also mediate resistance to certain cancer-specific chemotherapy agents. piRNAs, on the other hand, have a significant role in genome integrity, however, no role has yet been defined for the piRNAs found in EVs. While our knowledgebase for the function of ncRNA-containing EVs is still modest, the potential role that these EV-ensconced ncRNA might play is promising. This review summarizes the ncRNA content of EVs and describes the function where known, or the potential utility of EVs that harbor specific types of ncRNA.

MicroRNA Profile of Exosomes and Parental Cells is Differently Affected by Ionizing Radiation.

Exosomes are key mediators of cell-to-cell communication involved in different aspects of the response to ionizing radiation. The functional role of exosomes depends on their molecular cargo, including protein and miRNA content. In this work, we compared the miRNA profile of cells exposed to a high-dose of radiation and the exosomes released by those cells. FaDu cells (derived from human head and neck cancer) were exposed to 2 and 8 Gy doses, exosomes were purified from culture media at 36 h postirradiation using a combination of differential centrifugation, ultrafiltration and precipitation, then microRNA was analyzed using the RNA-seq approach. There were 439 miRNA species quantified, and significant differences in their relative abundance were observed between the cells and exosomes; several low-abundance miRNAs were over-represented while high-abundance miRNA were under-represented in exosomes. There were a few miRNA species markedly affected in irradiated cells and in exosomes released by these cells. However, markedly different radiation-induced effects were observed in both miRNA sets, which could be exemplified by miR-3168 significantly downregulated in cells and upregulated in exosomes. On the other hand, both 2 and 8 Gy radiation doses induced similar effects. Radiation-affected miRNA species present in exosomes are linked to genes involved in the DNA damage and cytokine-mediated response, which may suggest their hypothetical role in the exosome-mediated radiation-induced bystander effect reported elsewhere.

Tumor treating fields cause replication stress and interfere with DNA replication fork maintenance: Implications for cancer therapy.

Tumor treating fields (TTFields) is a noninvasive physical modality of cancer therapy that applies low-intensity, intermediate frequency, and alternating electric fields to a tumor. Interference with mitosis was the first mechanism describing the effects of TTFields on cancer cells; however, TTFields was shown to not only reduce the rejoining of radiation-induced DNA double-strand breaks (DSBs), but to also induce DNA DSBs. The mechanism(s) by which TTFields generates DNA DSBs is related to the generation of replication stress including reduced expression of the DNA replication complex genes MCM6 and MCM10 and the Fanconi's Anemia pathway genes. When markers of DNA replication stress as a result of TTFields exposure were examined, newly replicated DNA length was reduced with TTFields exposure time and there was increased R-loop formation. Furthermore, as cells were exposed to TTFields a conditional vulnerability environment developed which rendered cells more susceptible to DNA damaging agents or agents that interfere with DNA repair or replication fork maintenance. The effect of TTFields exposure with concomitant exposure to cisplatin or PARP inhibition, the combination of TTFields plus concomitant PARP inhibition followed by radiation, or radiation alone at the end of a TTFields exposure were all synergistic. Finally, gene expression analysis of 47 key mitosis regulator genes suggested that TTFields-induced mitotic aberrations and DNA damage/replication stress events, although intimately linked to one another, are likely initiated independently of one another. This suggests that enhanced replication stress and reduced DNA repair capacity are also major mechanisms of TTFields effects, effects for which there are therapeutic implications.

miR-125a-5p Functions as Tumor Suppressor microRNA And Is a Marker of Locoregional Recurrence And Poor prognosis in Head And Neck Cancer.

MicroRNAs (miRNAs) are short single-stranded RNAs, measuring 21 to 23 nucleotides in length and regulate gene expression at the post-transcriptional level through mRNA destabilization or repressing protein synthesis. Dysregulation of miRNAs can lead to tumorigenesis through changes in regulation of key cellular processes such as cell proliferation, cell survival, and apoptosis. miR-125a-5p has been implicated as a tumor suppressor miRNA in malignancies such as non-small cell lung cancer and colon cancer. However, the role of miR-125a-5p has not been fully investigated in head and neck squamous cell carcinoma (HNSCC). We performed microRNA microarray profiling of HNSCC tumor samples obtained from a prospective clinical trial evaluating the role of postoperative radiotherapy in head and neck cancer. We also mined through The Cancer Genome Atlas to evaluate expression and survival data. Biological experiments, including cell proliferation, flow cytometry, cell migration and invasion, clonogenic survival, and fluorescent microscopy, were conducted using HN5 and UM-SCC-22B cell lines. miR-125a-5p downregulation was associated with recurrent disease in a panel of high-risk HNSCC and then confirmed poor survival associated with low expression in HNSCC via the Cancer Genome Atlas, suggesting that miR-125a-5p acts as a tumor suppressor miRNA. We then demonstrated that miR-125a-5p regulates cell proliferation through cell cycle regulation at the G1/S transition. We also show that miR-125a-5p can alter cell migration and modulate sensitivity to ionizing radiation. Finally, we identified putative mRNA targets of miR-125a-5p, including ERBB2, EIF4EBP1, and TXNRD1, which support the tumor suppressive mechanism of miR-125a-5p. Functional validation of ERBB2 suggests that miR-125a-5p affects cell proliferation and sensitivity to ionizing radiation, in part, through ERBB2. Our data suggests that miR-125a-5p acts as a tumor suppressor miRNA, has potential as a diagnostic tool and may be a potential therapeutic target for the management and treatment of squamous cell carcinoma of the head and neck.

Radiation-Induced DNA Damage Cooperates with Heterozygosity of TP53 and PTEN to Generate High-Grade Gliomas.

Glioblastomas are lethal brain tumors that are treated with conventional radiation (X-rays and gamma rays) or particle radiation (protons and carbon ions). Paradoxically, radiation is also a risk factor for GBM development, raising the possibility that radiotherapy of brain tumors could promote tumor recurrence or trigger secondary gliomas. In this study, we determined whether tumor suppressor losses commonly displayed by patients with GBM confer susceptibility to radiation-induced glioma. Mice with Nestin-Cre-driven deletions of Trp53 and Pten alleles were intracranially irradiated with X-rays or charged particles of increasing atomic number and linear energy transfer (LET). Mice with loss of one allele each of Trp53 and Pten did not develop spontaneous gliomas, but were highly susceptible to radiation-induced gliomagenesis. Tumor development frequency after exposure to high-LET particle radiation was significantly higher compared with X-rays, in accordance with the irreparability of DNA double-strand breaks (DSB) induced by high-LET radiation. All resultant gliomas, regardless of radiation quality, presented histopathologic features of grade IV lesions and harbored populations of cancer stem-like cells with tumor-propagating properties. Furthermore, all tumors displayed concomitant loss of heterozygosity of Trp53 and Pten along with frequent amplification of the Met receptor tyrosine kinase, which conferred a stem cell phenotype to tumor cells. Our results demonstrate that radiation-induced DSBs cooperate with preexisting tumor suppressor losses to generate high-grade gliomas. Moreover, our mouse model can be used for studies on radiation-induced development of GBM and therapeutic strategies. SIGNIFICANCE: This study uncovers mechanisms by which ionizing radiation, especially particle radiation, promote GBM development or recurrence.

Ionizing radiation affects the composition of the proteome of extracellular vesicles released by head-and-neck cancer cells in vitro.

Exosomes and other extracellular vesicles are key players in cell-to-cell communication, and it has been proposed that they are involved in different aspects of the response to ionizing radiation, including transmitting the radiation-induced bystander effect and mediating radioresistance. The functional role of exosomes depends on their molecular cargo, including proteome content. Here we aimed to establish the proteome profile of exosomes released in vitro by irradiated UM-SCC6 cells derived from human head-and-neck cancer and to identify processes associated with radiation-affected proteins. Exosomes and other small extracellular vesicles were purified by size-exclusion chromatography from cell culture media collected 24 h after irradiation of cells with a single 2, 4 or 8 Gy dose, and then proteins were identified using a shotgun LC-MS/MS approach. Exosome-specific proteins encoded by 1217 unique genes were identified. There were 472 proteins whose abundance in exosomes was significantly affected by radiation (at any dose), including 425 upregulated and 47 downregulated species. The largest group of proteins affected by radiation (369 species) included those with increased abundance at all radiation doses (≥2 Gy). Several gene ontology terms were associated with radiation-affected exosome proteins. Among overrepresented processes were those involved in the response to radiation, the metabolism of radical oxygen species, DNA repair, chromatin packaging, and protein folding. Hence, the protein content of exosomes released by irradiated cells indicates their actual role in mediating the response to ionizing radiation.

The Reciprocity between Radiotherapy and Cancer Immunotherapy.

The clinical success of immune checkpoint inhibitors in treating metastatic and refractory cancers has generated significant interest in investigating their role in treating locally advanced diseases, thus requiring them to be combined with standard treatments in the hope of producing synergistic antitumor responses. Radiotherapy, in particular, has long been hypothesized to have actions complementary to those of immune checkpoint blockade, and a growing body of evidence indicates that cancer immunotherapy may also have radiosensitizing effects, which would provide unique benefit for locoregional treatments. Recent studies have demonstrated that when immune cells are activated by immunotherapeutics, they can reprogram the tumor microenvironment in ways that may potentially increase the radiosensitivity of the tumor. In this review, we highlight the evidence that supports reciprocal interactions between cancer immunotherapy and radiotherapy, where in addition to the traditional notion that radiation serves to enhance the activation of antitumor immunity, an alternative scenario also exists in which T-cell activation by cancer immunotherapy may sensitize tumors to radiation treatment through mechanisms that include normalization of the tumor vasculature and tissue hypoxia. We describe the empirical observations from preclinical models that support such effects and discuss their implications for future research and trial design.

Developing Predictive or Prognostic Biomarkers for Charged Particle Radiotherapy.

The response to radiotherapy can vary greatly among individuals, even though advances in technology allow for the highly localized placement of therapeutic doses of radiation to a tumor. This variability in patient response to radiation is biologically driven, but the individuality of tumor and healthy tissue biology are not used to create individual treatment plans. Biomarkers of radiosensitivity, whether intrinsic or from hypoxia, would move radiation oncology from precision medicine to precise, personalized medicine. Charged particle radiotherapy allows for even greater dose conformity, but the biological advantages of charged particle radiotherapy have not yet been cultivated. The development of biomarkers that would drive biologically based clinical trials, identify patients for whom charged particles are most appropriate, or aid in particle-selection strategies could be envisioned with appropriate biomarkers. Initially, biomarkers for low-linear energy transfer (LET) radiation responses should be tested against charged particles. Biomarkers of tumor radioresistance to low-LET radiations could be used to identify patients for whom the enhanced relative biological effectiveness (RBE) of charged particles would be more effective compared with low-LET radiations and those for whom specific DNA-repair inhibitors, in combination with charged particles, may also be appropriate. Furthermore, heavy charged particles can overcome the radioresistance of hypoxic tumors when used at the appropriate LET. Biomarkers for hypoxia could identify hypoxic tumors and, in combination with imaging, define hypoxic regions of a tumor for specific ion selection. Moreover, because of the enhanced RBE for charged particles, the risk for adverse healthy tissue effects may be greater, even though charged particles have greater tumor conformality. There are many validated healthy-tissue biomarkers available to test against charged particle exposures. Lastly, newer biological techniques, as well as newer bioinformatic and computational methods, are rapidly changing the landscape for biomarker identification, validation, and clinical trial design.