Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Combining large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers for promoting protective melanosome autophagy via the PI3K/Akt/mTOR signalling pathway for the treatment of melasma.

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma-like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16-F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana-Masson staining, and melanin particles were evaluated in B16-F10 cells. Real-time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy-related messenger ribonucleic acid (mRNA) and proteins. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin-synthesizing enzymes (TYR, TRP-1 and MITF). This combination also led to increased expression of the autophagy-related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y-P, increased TYR, TRP-1, MITF, p-PI3K, p-AKT, p-mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content.

The Impact of Dose Rate on the Tumor Microenvironment Using Flattening-filter-free Beams.

AIMS: Recently, dose delivery technology has rapidly evolved with flattening filter-free beams (FFF), and the biological effects of high dose rates are a matter of interest. We hypothesized that FFF beams at different dose rates obtained with modern linear accelerators have different effects on the TME. MATERIALS AND METHODS: The B16-F10 melanoma syngeneic tumor model was established, and mice were randomized to 2 different doses (2 Gy and 10 Gy) and 3 different dose rates (1 Gy/min, 6 Gy/min, and 14 Gy/min) along with the control group. Euthanasia was performed on the seventh day after RT, and intracardiac blood was collected for a comet assay. Tumors were harvested and examined histomorphologically and immunohistochemically. Statistical analyses were performed using SPSS software version 23 (SPSS Inc., Chicago, IL, USA). RESULTS: The daily growth rate was uniform, and no difference was observed between tumor volumes across all three dose rates for each dose. Deoxyribonucleic acid (DNA) damage in blood mononuclear cells was not affected by dose or dose rate. In the TME histomorphological examination, the number of mitosis is less in the 10 Gy arm, whereas the pleomorphism score was greater. Nevertheless, varying dose rates had no effect on the number of mitosis or the pleomorphism score. The severity of the inflammation, cell densities in the TME, and expression of immunohistochemical markers were comparable across all doses and dose rates. CONCLUSION: In our study involving the B16-F10 syngeneic tumor model, varying dose rates obtained with FFF beams had no effect on tumor volume, blood mononuclear cell DNA damage, or TME parameters. However, in order to fully understand the biological impacts of novel techniques, our study should be validated with alternative preclinical setups.

Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

Background: Hypofractionated radiotherapy (hRT) can induce a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, clinically, this effect is still rare, and ICB-mediated adverse events are common. Lenalidomide (lena) is an anti-angiogenic and immunomodulatory drug used in the treatment of hematologic malignancies. We here investigated in solid tumor models whether lena can enhance the abscopal effect in double combination with hRT. Methods: In two syngeneic bilateral tumor models (B16-CD133 melanoma and MC38 colon carcinoma), the primary tumor was treated with hRT. Lena was given daily for 3 weeks. Besides tumor size and survival, the dependence of the antitumor effects on CD8+ cells, type-I IFN signaling, and T cell costimulation was determined with depleting or blocking antibodies. Tumor-specific CD8+ T cells were quantified, and their differentiation and effector status were characterized by multicolor flow cytometry using MHC-I tetramers and various antibodies. In addition, dendritic cell (DC)-mediated tumor antigen cross-presentation in vitro and directly ex vivo and the composition of tumor-associated vascular endothelial cells were investigated. Results: In both tumor models, the hRT/lena double combination induced a significant abscopal effect. Control of the non-irradiated secondary tumor and survival were considerably better than with the respective monotherapies. The abscopal effect was strongly dependent on CD8+ cells and associated with an increase in tumor-specific CD8+ T cells in the non-irradiated tumor and its draining lymph nodes. Additionally, we found more tumor-specific T cells with a stem-like (TCF1+ TIM3- PD1+) and a transitory (TCF1- TIM3+ CD101- PD1+) exhausted phenotype and more expressing effector molecules such as GzmB, IFNγ, and TNFα. Moreover, in the non-irradiated tumor, hRT/lena treatment also increased DCs cross-presenting a tumor model antigen. Blocking type-I IFN signaling, which is essential for cross-presentation, completely abrogated the abscopal effect. A gene expression analysis of bone marrow-derived DCs revealed that lena augmented the expression of IFN response genes and genes associated with differentiation, maturation (including CD70, CD83, and CD86), migration to lymph nodes, and T cell activation. Flow cytometry confirmed an increase in CD70+ CD83+ CD86+ DCs in both irradiated and abscopal tumors. Moreover, the hRT/lena-induced abscopal effect was diminished when these costimulatory molecules were blocked simultaneously using antibodies. In line with the enhanced infiltration by DCs and tumor-specific CD8+ T cells, including more stem-like cells, hRT/lena also increased tumor-associated high endothelial cells (TA-HECs) in the non-irradiated tumor. Conclusions: We demonstrate that lena can augment the hRT-induced abscopal effect in mouse solid tumor models in a CD8 T cell- and IFN-I-dependent manner, correlating with enhanced anti-tumor CD8 T cell immunity, DC cross-presentation, and TA-HEC numbers. Our findings may be helpful for the planning of clinical trials in (oligo)metastatic patients.

Combination low-dose cyclophosphamide with check-point blockade and ionizing radiation promote an abscopal effect in mouse models of melanoma.

PURPOSE: The complex strategy of hypo-fractionated radiotherapy (HFRT) in combination with an immune checkpoint inhibitor (ICI) can stimulate a potential systemic antitumor response; however, the abscopal effect is always precluded by the tumor microenvironment, which may limit sufficient T-cell infiltration of distant nonirradiated tumors for certain kinds of inhibitory factors, such as regulatory T-cells (Tregs). Additionally, low-dose cyclophosphamide (LD-CYC) can specifically kill regulatory Tregs and strongly synergize antigen-specific immune responses, which could promote an abscopal effect. MATERIALS AND METHODS: We explored whether a triple regimen consisting of HFRT, ICI, and LD-CYC could achieve a better systemic antitumor response in bilateral mouse tumor models. RESULT: Our data demonstrate that LD-CYC combined with HFRT and antiprogrammed cell death ligand 1 (PDL-1) therapy could enhance the abscopal effect than only HFRT/antiPDL-1 or HFRT alone. Surprisingly, repeat CYC doses cannot further restrain tumor proliferation but can prolong murine overall survival, as revealed by the major pathologic responses. These results are associated with increased CD8 + effector T-cell infiltration, although LD-CYC did not upregulate PDL-1 expression in the tumor. CONCLUSIONS: Compared with traditional strategies, for the first time, we demonstrated that a triple treatment strategy remarkably increased the number of radiation-induced tumor-infiltrating CD8 + T-cells, effectively decreasing infiltrating Tregs, and promoting an abscopal effect. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.

Evaluation of the therapeutic efficacy of 213Bi-labelled DOTA-conjugated alpha-melanocyte stimulating hormone peptide analogues in melanocortin-1 receptor positive preclinical melanoma model.

Melanocortin-1 receptor (MC1-R) targeting alpha-melanocyte stimulating hormone-analogue (α-MSH) biomolecules labelled with α-emitting radiometal seem to be valuable in the targeted radionuclide therapy of MC1-R positive melanoma malignum (MM). Herein is reported the anti-tumor in vivo therapeutic evaluation of MC1-R-affine [213Bi]Bi-DOTA-NAPamide and HOLDamide treatment in MC1-R positive B16-F10 melanoma tumor-bearing C57BL/6J mice. On the 6th, 8th and 10th days post tumor cell inoculation; the treated groups of mice were intravenously injected with approximately 5 MBq of both amide derivatives. Beyond body weight and tumor volume assessment, [68Ga]Ga-DOTA-HOLDamide and NAPamide-based PET/MRI scans, and ex vivo biodistribution studies were executed 30,- and 90 min postinjection. In the PET/MRI imaging studies the B16-F10 tumors were clearly visualized with both 68Ga-labelled tracers, however, significantly lower tumor-to-muscle (T/M) ratios were observed by using [68Ga]Ga-DOTA-HOLDamide. After alpha-radiotherapy treatment the tumor size of the control group was larger relative to both treated cohorts, while the smallest tumor volumes were observed in the NAPamide-treated subclass on the 10th day. Relatively higher [213Bi]Bi-DOTA-NAPamide accumulation in the B16-F10 tumors (%ID/g: 2.71 ± 0.15) with discrete background activity led to excellent T/M ratios, particularly 90 min postinjection. Overall, the therapeutic application of receptor selective [213Bi]Bi-DOTA-NAPamide seems to be feasible in MC1-R positive MM management.

Targeted α-Therapy Using 225Ac Radiolabeled Single-Domain Antibodies Induces Antigen-Specific Immune Responses and Instills Immunomodulation Both Systemically and at the Tumor Microenvironment.

Targeted radionuclide therapy (TRT) using targeting moieties labeled with α-particle-emitting radionuclides (α-TRT) is an intensely investigated treatment approach as the short range of α-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of α-TRT is lacking in literature. Methods: Using flow cytometry of tumors, splenocyte restimulation, and multiplex analysis of blood serum, we studied immunologic responses ensuing from TRT with an antihuman CD20 single-domain antibody radiolabeled with 225Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with α-TRT and increased blood levels of various cytokines such as interferon-γ, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on α-TRT. At the tumor site, α-TRT modulated the cold tumor microenvironment (TME) to a more hospitable and hot habitat for antitumoral immune cells, characterized by a decrease in protumoral alternatively activated macrophages and an increase in antitumoral macrophages and dendritic cells. We also showed that α-TRT increased the percentage of programmed death-ligand 1 (PD-L1)-positive (PD-L1pos) immune cells in the TME. To circumvent this immunosuppressive countermeasure we applied immune checkpoint blockade of the programmed cell death protein 1-PD-L1 axis. Combination of α-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A long-term toxicity study revealed severe kidney damage ensuing from α-TRT. Conclusion: These data suggest that α-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of α-TRT. However, further optimization is warranted to avoid adverse events.

Contrast-enhanced ultrasound for evaluation of tumor perfusion and outcome following treatment in a murine melanoma model.

Due to a lack of data on predictors of electroporation-based treatment outcomes, we investigated the potential predictive role of contrast-enhanced harmonic ultrasound (CEUS) in mice B16F10 melanoma treated by gene electrotransfer (GET) to silence melanoma cell adhesion molecule (MCAM) and radiotherapy, which has not been evaluated yet. CEUS evaluation was verified by tumor histological analysis. Mice bearing subcutaneous tumors were treated with GET to silence MCAM, irradiation or the combination of GET to silence MCAM and irradiation (combined treatment). CEUS of the tumors used to evaluate tumor perfusion was performed before and up to 10 days after the beginning of the experiment, and the CEUS results were compared with tumor growth and the number of blood vessels analyzed in the histological tumor sections. CEUS revealed a decrease in tumor perfusion in the combined therapy groups compared with the control groups and correlated with tumor histological analyses, which showed a decreased vascular density. In this study a trend of inverse correlation was observed between tumor perfusion and treatment efficacy. The greater the perfusion of the tumor, the shorter the expected doubling time. Furthermore, decreased perfusion showed a trend to correlate with higher antitumor efficacy. Thus, CEUS could be used to predict tumoral vascular density and treatment effectiveness.

Therapeutic Efficacy of Carbon Ion Irradiation Enhanced by 11-MUA-Capped Gold Nanoparticles: An in vitro and in vivo Study.

PURPOSE: Gold nanoparticles (AuNPs) are widely studied as radiosensitizers, but their radiosensitization in carbon ion radiotherapy is unsatisfactory. There is a lack of in vivo data on the radiosensitization of AuNPs under carbon ion irradiation. This study focused on the radiosensitization effect of AuNPs in the mouse melanoma cell line B16-F10 in vitro and in vivo. MATERIALS AND METHODS: 11-mercaptoundecanoic acid (11-MUA)-coated gold (Au) nanoparticles (mAuNPs) formulations were prepared and characterized. To verify the radiosensitization effect of mAuNPs, hydroxyl radicals were generated in aqueous solution, and the detection of intracellular reactive oxygen species (ROS) and clone survival were carried out in vitro. The tumor growth rate (TGR) and survival of mice were analyzed to verify the radiosensitization effect of mAuNPs in vivo. The apoptosis of tumor cells was detected, and the expression of key proteins in the apoptosis pathway was verified by immunohistochemistry. RESULTS: The intracellular ROS level in B16-F10 cells was enhanced by mAuNPs under carbon ion irradiation. The sensitization rate of mAuNPs was 1.22 with a 10% cell survival rate. Compared with irradiation alone, the inhibitory effect of mAuNPs combined with carbon ion irradiation on tumor growth was 1.94-fold higher, the survival time of mice was prolonged by 1.75-fold, and the number of apoptotic cells was increased by 1.43-fold. The ratio of key proteins Bax and Bcl2 in the apoptosis pathway was up-regulated, and the expression of caspase-3, a key executor of the apoptosis pathway, was up-regulated. CONCLUSION: In in vivo and in vitro experiments, mAuNPs showed radiosensitivity to carbon ion irradiation. The sensitization effect of mAuNPs on mice tumor may be achieved by activating the mitochondrial apoptosis pathway and increasing tumor tissue apoptosis. To our best knowledge, the present study is the first in vivo evidence for radiosensitization of mAuNPs in tumor-bearing mice exposed to carbon ion irradiation.

Calcium Carbonate Core-Shell Particles for Incorporation of 225Ac and Their Application in Local α-Radionuclide Therapy.

Actinium-225 (225Ac) radiolabeled submicrometric core-shell particles (SPs) made of calcium carbonate (CaCO3) coated with biocompatible polymers [tannic acid-human serum albumin (TA/HSA)] have been developed to improve the efficiency of local α-radionuclide therapy in melanoma models (B16-F10 tumor-bearing mice). The developed 225Ac-SPs possess radiochemical stability and demonstrate effective retention of 225Ac and its daughter isotopes. The SPs have been additionally labeled with zirconium-89 (89Zr) to perform the biodistribution studies using positron emission tomography-computerized tomography (PET/CT) imaging for 14 days after intratumoral injection. According to the PET/CT analysis, a significant accumulation of 89Zr-SPs in the tumor area is revealed for the whole investigation period, which correlates with the direct radiometry analysis after intratumoral administration of 225Ac-SPs. The histological analysis has revealed no abnormal changes in healthy tissue organs after treatment with 225Ac-SPs (e.g., no acute pathologic findings are detected in the liver and kidneys). At the same time, the inhibition of tumor growth has been observed as compared with control samples [nonradiolabeled SPs and phosphate-buffered saline (PBS)]. The treatment of mice with 225Ac-SPs has resulted in prolonged survival compared to the control samples. Thus, our study validates the application of 225Ac-doped core-shell submicron CaCO3 particles for local α-radionuclide therapy.

Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy.

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 90Y 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 90Y-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.

Involvement of TRPM8 Channel in Radiation-Induced DNA Damage Repair Mechanism Contributing to Radioresistance of B16 Melanoma.

Radiation is an effective cancer treatment, but cancer cells can acquire radioresistance, which is associated with increased DNA damage response and enhanced proliferative capacity, and therefore, it is important to understand the intracellular biochemical responses to γ-irradiation. The transient receptor potential melastatin 8 (TRPM8) channel plays roles in the development and progression of tumors, but it is unclear whether it is involved in the DNA damage response induced by γ-irradiation. Here, we show that a TRPM8 channel inhibitor suppresses the DNA damage response (phosphorylated histone variant H2AX-p53-binding protein 1 (γH2AX-53BP1) focus formation) and colony formation of B16 melanoma cells. Furthermore, the TRPM8 channel-specific agonist WS-12 enhanced the DNA damage response and increased the survival fraction after γ-irradiation. We found that the TRPM8 channel inhibitor enhanced G2/M phase arrest after γ-irradiation. Phosphorylation of ataxia telangiectasia mutated and p53, which both contribute to the DNA damage response was also suppressed after γ-irradiation. In addition, the TRPM8 channel inhibitor enhanced the γ-irradiation-induced suppression of tumor growth in vivo. We conclude that the TRPM8 channel is involved in radiation-induced DNA damage repair and contributes to the radioresistance of B16 melanoma cells. TRPM8 channel inhibitors might be clinically useful as radiosensitizers to enhance radiation therapy of melanoma.

Influence of the particle size of gadolinium-loaded chitosan nanoparticles on their tumor-killing effect in neutron capture therapy in vitro.

Neutron capture therapy using 157Gd (Gd-NCT) is currently under development as a cancer radiotherapy. Melanoma cells were treated with gadolinium-loaded chitosan nanoparticles (Gd-nanoCPs) for Gd-NCT. Smaller Gd-nanoCPs had higher Gd content and better cellular association of Gd and thereby made the tumor-killing effect more efficient in comparison to larger Gd-nanoCPs. This indicates that Gd-nanoCP size reduction is an efficient method for improving the cellular affinity of Gd-nanoCPs and for enhancing the tumor-killing effect of Gd-NCT.

Evaluation of [225Ac]Ac-DOTA-anti-VLA-4 for targeted alpha therapy of metastatic melanoma.

Very late antigen 4 (VLA-4; also called integrin α4ß1) is overexpressed in melanoma tumor cells with an active role in tumor growth, angiogenesis, and metastasis, making VLA-4 a potential target for targeted alpha therapy (TAT). METHODS: An anti-VLA-4 antibody was conjugated to DOTA for [225Ac]Ac-labeling and DTPA for [111In]In-labeling. The resulting agents, [225Ac]Ac- or [111In]In-labeled anti-VLA-4 were evaluated in vitro, including binding affinity, internalization, and colony formation assays as well as in vivo biodistribution studies. In addition, the therapeutic efficacy of [225Ac]Ac-DOTA-anti-VLA-4 was evaluated in a disseminated disease mouse model of melanoma. RESULTS: [111In]In-DTPA-anti-VLA-4 demonstrated high affinity for VLA-4 (Kd = 5.2 ± 1.6 nM). [225Ac]Ac-DOTA-anti-VLA-4 was labeled with an apparent molar activity of 3.5 MBq/nmol and > 95% radiochemical purity. Colony formation assays demonstrated a decrease in the surviving fraction of B16F10 cells treated with [225Ac]Ac-DOTA-anti-VLA-4 compared to control. Biodistribution studies demonstrated accumulation in the VLA-4-positive tumor and VLA-4 rich organs. Therapeutic efficacy studies demonstrated a significant increase in survival in mice treated with [225Ac]Ac-DOTA-anti-VLA-4 as compared to controls. CONCLUSION: The work presented here demonstrated that [225Ac]Ac-DOTA-anti-VLA-4 was effective as a treatment in mice with disseminated disease, but potentially has dose limiting hematopoietic toxicity. Preliminary studies presented here also supported the potential to overcome this limitation by exploring a pre-loading or blocking dose strategy, to optimize the targeting vector to help minimize the absorbed dose to VLA-4 rich organs while maximizing the dose delivered to VLA-4-positive melanoma tumor cells.

Brachytherapy in a Single Dose of 10Gy as an "in situ" Vaccination.

Radiotherapy (RT) is one of the major methods of cancer treatment. RT destroys cancer cells, but also affects the tumor microenvironment (TME). The delicate balance between immunomodulation processes in TME is dependent, among other things, on a specific radiation dose. Despite many studies, the optimal dose has not been clearly determined. Here, we demonstrate that brachytherapy (contact radiotherapy) inhibits melanoma tumor growth in a dose-dependent manner. Doses of 10Gy and 15Gy cause the most effective tumor growth inhibition compared to the control group. Brachytherapy, at a single dose of ≥ 5Gy, resulted in reduced tumor blood vessel density. Only a dose of 10Gy had the greatest impact on changes in the levels of tumor-infiltrating immune cells. It most effectively reduced the accumulation of protumorogenic M2 tumor-associated macrophages and increased the infiltration of cytotoxic CD8+ T lymphocytes. To summarize, more knowledge about the effects of irradiation doses in anticancer therapy is needed. It may help in the optimization of RT treatment. Our results indicate that a single dose of 10Gy leads to the development of a robust immune response. It seems that it is able to convert a tumor microenvironment into an "in situ" vaccine and lead to a significant inhibition of tumor growth.

Exosome-Containing Preparations From Postirradiated Mouse Melanoma Cells Delay Melanoma Growth In Vivo by a Natural Killer Cell-Dependent Mechanism.

PURPOSE: To investigate the ability of radiation to stimulate exosome release from melanoma cells and to characterize the resulting exosome-containing vesicle preparations for their ability to promote a host antitumor immune response. MATERIALS AND METHODS: Cultured B16F10 murine melanoma cells or tumors were irradiated, and secreted extracellular vesicles were isolated and characterized. The exosome-containing vesicle preparations were injected into fresh tumors in syngeneic mice, and tumor growth and infiltrating T cells and natural killer (NK) cells were characterized. RESULTS: Irradiation stimulated exosome release from B16F10 murine melanoma cells. Exosome preparations from irradiated cell culture supernatants were biologically active, as demonstrated by uptake into recipient cells and by the ability to induce dendritic cell maturation and activation in vitro. Intratumoral injection significantly delayed tumor growth in vivo, whereas injection of similar preparations from non irradiated cells had no effect. The antitumor effect was correlated to an increase in interferon gamma-producing tumor-infiltrating NK cells. Pretreatment of the host mice with anti-NK cell antibodies abolished the effect, whereas pretreatment with anti-CD8+ T-cell antibodies did not. CONCLUSION: Exosomes from irradiated cells, or synthetic mimics, might provide an effective strategy for potentiation of NK cell-mediated host antitumor immunity.

Combination of CHEK1/2 inhibition and ionizing radiation results in abscopal tumor response through increased micronuclei formation.

We explore a novel strategy of activating immune signaling through increased micronuclei formation utilizing a cell cycle checkpoint inhibitor to drive cell cycle progression following ionizing radiation. The Chk1/2 inhibitor AZD7762 is used to abrogate radiation therapy (RT)-induced G2/M cell cycle arrest in multiple cell lines and, we find that this therapeutic combination promotes increased micronuclei formation in vitro and subsequently drives increased type I interferon signaling and cytotoxic T-cell activation. In vivo studies using B16-F10 melanoma cancer cells implanted in C57/BL6 mice demonstrate improved rates of tumor control at the abscopal (unirradiated) site, located outside of the radiation field, only in the AZD7762 + RT group, with a corresponding reduction in mean tumor volume, increase in the CD8 T-cell population, and immune activated gene signaling. Our results demonstrate that targeted inhibition of cell cycle checkpoint activation following ionizing radiation drives increased production of immunogenic micronuclei, leading to systemic tumor response with potential future clinical benefit.

Combination of Gas Plasma and Radiotherapy Has Immunostimulatory Potential and Additive Toxicity in Murine Melanoma Cells in Vitro.

Despite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFα, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further.

Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced in response to endoplasmic reticulum (ER) stress induced by various stressors, but its involvement in the radioresistance of tumor cells is unknown. Here, we found that MANF is released after γ-irradiation (2 Gy and 4 Gy) of B16 melanoma cells, and its release was suppressed by 4-phenylbutyric acid, an ER stress inhibitor. MANF was not released after low-dose (1 Gy) γ-irradiation, but pretreatment of 1 Gy-irradiated cells with recombinant MANF enhanced the cellular DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the DNA damage response and p53 activation by γ-irradiation (2 Gy) were suppressed, and reproductive cell death was increased. MANF also activated the ERK signaling pathway. Our findings raise the possibility that MANF could be a new target for overcoming radioresistance.

A Genomic Profile of Local Immunity in the Melanoma Microenvironment Following Treatment with α Particle-Emitting Ultrasmall Silica Nanoparticles.

An α particle-emitting nanodrug that is a potent and specific antitumor agent and also prompts significant remodeling of local immunity in the tumor microenvironment (TME) has been developed and may impact the treatment of melanoma. Biocompatible ultrasmall fluorescent core-shell silica nanoparticles (C' dots, diameter ∼6.0 nm) have been engineered to target the melanocortin-1 receptor expressed on melanoma through α melanocyte-stimulating hormone peptides attached to the C' dot surface. Actinium-225 is also bound to the nanoparticle to deliver a densely ionizing dose of high-energy α particles to cancer. Nanodrug pharmacokinetic properties are optimal for targeted radionuclide therapy as they exhibit rapid blood clearance, tumor-specific accumulation, minimal off-target localization, and renal elimination. Potent and specific tumor control, arising from the α particles, was observed in a syngeneic animal model of melanoma. Surprisingly, the C' dot component of this drug initiates a favorable pseudopathogenic response in the TME generating distinct changes in the fractions of naive and activated CD8 T cells, Th1 and regulatory T cells, immature dendritic cells, monocytes, MΦ and M1 macrophages, and activated natural killer cells. Concomitant upregulation of the inflammatory cytokine genome and adaptive immune pathways each describes a macrophage-initiated pseudoresponse to a viral-shaped pathogen. This study suggests that therapeutic α-particle irradiation of melanoma using ultrasmall functionalized core-shell silica nanoparticles potently kills tumor cells, and at the same time initiates a distinct immune response in the TME.