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.

Adding liposomal doxorubicin enhances the abscopal effect induced by radiation/αPD1 therapy depending on tumor cell mitochondrial DNA and cGAS/STING.

BACKGROUND: Localized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade. However, this effect is still clinically rare and improvements are highly desirable. We investigated whether triple combination with a low dose of clinically approved liposomal doxorubicin (Doxil) could augment abscopal responses compared with RT/αPD-1 and Doxil/αPD-1. We also investigated whether the enhanced abscopal responses depended on the mitochondrial DNA (mtDNA)/cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)/IFN-I pathway. MATERIALS/METHODS: We used Doxil in combination with RT and αPD-1 in two tumor models (B16-CD133 melanoma and MC38 colon carcinoma) with mice bearing two tumors, only one of which was irradiated. Mechanistic studies on the role of the mtDNA/cGAS/STING/IFN-I axis were performed using inhibitors and knockout cells in vitro as well as in mice. RESULTS: Addition of a single low dose of Doxil to RT and αPD-1 strongly enhanced the RT/αPD-1-induced abscopal effect in both models. Complete cures of non-irradiated tumors were mainly observed in triple-treated mice. Triple therapy induced more cross-presenting dendritic cells (DCs) and more tumor-specific CD8+ T cells than RT/αPD-1 and Doxil/αPD-1, particularly in non-irradiated tumors. Coincubation of Doxil-treated and/or RT-treated tumor cells with DCs enhanced DC antigen cross-presentation which is crucial for inducing CD8+ T cells. CD8+ T cell depletion or implantation of cGAS-deficient or STING-deficient tumor cells abolished the abscopal effect. Doxorubicin-induced/Doxil-induced IFNß1 markedly depended on the cGAS/STING pathway. Doxorubicin-treated/Doxil-treated tumor cells depleted of mtDNA secreted less IFNß1, of the related T cell-recruiting chemokine CXCL10, and ATP; coincubation with mtDNA-depleted tumor cells strongly reduced IFNß1 secretion by DCs. Implantation of mtDNA-depleted tumor cells, particularly at the non-irradiated/abscopal site, substantially diminished the Doxil-enhanced abscopal effect and tumor infiltration by tumor-specific CD8+ T cells. CONCLUSIONS: These data show that single low-dose Doxil can substantially enhance the RT/αPD-1-induced abscopal effect, with a strong increase in cross-presenting DCs and CD8+ tumor-specific T cells particularly in abscopal tumors compared with RT/αPD-1 and Doxil/αPD-1. Moreover, they indicate that the mtDNA/cGAS/STING/IFN-I axis is important for the immunogenic/immunomodulatory doxorubicin effects. Our findings may be helpful for the planning of clinical radiochemoimmunotherapy trials in (oligo)metastatic patients.

Expansion of circulating stem-like CD8+ T cells by adding CD122-directed IL-2 complexes to radiation and anti-PD1 therapies in mice.

Combination of radiation therapy (RT) with immune checkpoint blockade can enhance systemic anti-tumor T cell responses. Here, using two mouse tumor models, we demonstrate that adding long-acting CD122-directed IL-2 complexes (IL-2c) to RT/anti-PD1 further increases tumor-specific CD8+ T cell numbers. The highest increase (>50-fold) is found in the blood circulation. Compartmental analysis of exhausted T cell subsets shows that primarily undifferentiated, stem-like, tumor-specific CD8+ T cells expand in the blood; these cells express the chemokine receptor CXCR3, which is required for migration into tumors. In tumor tissue, effector-like but not terminally differentiated exhausted CD8+ T cells increase. Consistent with the surge in tumor-specific CD8+ T cells in blood that are migration and proliferation competent, we observe a CD8-dependent and CXCR3-dependent enhancement of the abscopal effect against distant/non-irradiated tumors and find that CD8+ T cells isolated from blood after RT/anti-PD1/IL-2c triple treatment can be a rich source of tumor-specific T cells for adoptive transfers.

Necroptosis-dependent Immunogenicity of Cisplatin: Implications for Enhancing the Radiation-induced Abscopal Effect.

PURPOSE: Cisplatin is increasingly used in chemoimmunotherapy and may enhance the T cell-dependent radiation-induced abscopal effect, but how it promotes antitumor immunity is poorly understood. We investigated whether and why cisplatin is immunogenic, and the implications for the cisplatin-enhanced abscopal effect. EXPERIMENTAL DESIGN: Cisplatin, carboplatin, and the well-known immunogenic cell death (ICD) inducer oxaliplatin were compared for their potency to enhance the abscopal effect and induce type I IFN (IFN-I) and extracellular ATP, danger signals of ICD. The hypothetical role of necroptosis and associated damage-associated molecular patterns for cisplatin-induced ICD was investigated by inhibitors and knockout cells in vitro and in two tumor models in mice. A novel necroptosis signature for tumor immune cell infiltration and therapy response was developed. RESULTS: Cisplatin enhanced the abscopal effect more strongly than oxaliplatin or carboplatin. This correlated with higher induction of IFN-I and extracellular ATP by cisplatin, in a necroptosis-dependent manner. Cisplatin triggered receptor-interacting protein kinase 3 (RIPK3)-dependent tumor cell necroptosis causing cytosolic mitochondrial DNA (mtDNA) release, initiating the cyclic GMP-AMP synthase-stimulator of interferon genes pathway and IFN-I secretion promoting T-cell cross-priming by dendritic cells (DC). Accordingly, tumor cell RIPK3 or mtDNA deficiency and loss of IFN-I or ATP signaling diminished the cisplatin-enhanced abscopal effect. Cisplatin-treated tumor cells were immunogenic in vaccination experiments, depending on RIPK3 and mtDNA. In human tumor transcriptome analysis, necroptotic features correlated with abundant CD8+ T cells/DCs, sparse immunosuppressive cells, and immunotherapy response. CONCLUSIONS: Cisplatin induces antitumor immunity through necroptosis-mediated ICD. Our findings may help explain the benefits of cisplatin in chemo(radio)immunotherapies and develop clinical trials to investigate whether cisplatin enhances the abscopal effect in patients.

Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer.

The critical point for successful treatment of cancer is diagnosis at early stages of tumor development. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA (cfDNA) circulating in the blood is a convenient tumor-associated DNA marker. Therefore methylated cfDNA can be used as a minimally invasive diagnostic marker. We analysed the concentration of plasma cfDNA and methylation of six tumor suppressor genes in samples of 27 patients with renal cancer and 15 healthy donors as controls. The cfDNA concentrations in samples from cancer patients and healthy donors was measured using two different methods, the SYBR Green I fluorescence test and quantitative real-time PCR. Both methods revealed a statistically significant increase of cfDNA concentrations in cancer patients. Hypermethylation on cfDNA was detected for the LRRC3B (74.1%), APC (51.9%), FHIT (55.6%), and RASSF1 (62.9%) genes in patients with renal cancer. Promoter methylation of VHL and ITGA9 genes was not found on cfDNA. Our results confirmed that the cfDNA level and methylation of CpG islands of RASSF1A, FHIT, and APC genes in blood plasma can be used as noninvasive diagnostic markers of cancer.