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Localized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, this effect is still clinically rare and improvements are highly desirable. We here compared two triple therapy regimens (RT+⍺PD1+⍺CTLA4 vs. RT+⍺PD1+IL2/⍺IL2 complexes [IL2c]) for their efficacy to improve systemic (abscopal) anti-tumor effects. In mice bearing bilateral subcutaneous C51 colon carcinoma, the primary tumor was irradiated with two fractions of 8 Gy. ⍺PD1 and ⍺CTLA4 were given concomitantly and weekly thereafter; IL2c was given i.p. for 3 consecutive days. Besides tumor size and survival, tumor-specific CD8+ T cells were determined flow cytometrically using MHC-I tetramers and various antibodies. In addition, isolated TILs were cultured with PMA, ionomycin, and Brefeldin A in vitro to assess polyfunctionality (based on cytokine production) of CD8+ and CD4+ TILs. The abscopal effect was significantly stronger in mice treated with the ⍺CTLA4-containing triple combination (n = 13) than in mice treated with the IL2c-containing triple combination (n = 9) (p<0,05), and compared to mice treated with RT+⍺PD1 (n = 8) (p<0,05) or RT+⍺CTLA4 (n = 10) (p<0,05), respectively. The IL2c triple combination induced the abscopal effect better than RT+⍺PD1 (p<0,05). The ⍺CTLA4 triple therapy improved survival and resulted in complete cures of 8/13 mice. In mice treated with ⍺CTLA4-containing triple therapy, control of the irradiated tumor was partially dependent and that of the non-irradiated tumor was strongly dependent on T cells, primarily CD8+ T cells but also CD4+ T cells. In mice treated with the IL-2c triple combination, tumor control was more dependent on CD8+ T cells. With ⍺CTLA4 triple treatment (n = 9), the frequency and absolute numbers of polyfunctional TNF-⍺+IFN-g+ tumor-specific CD8+ T cells, TNF-⍺+IFN-g+ CD4+ and IFN-g+IL2+ CD4+ effector T cells were higher than with the IL2c triple combination (n = 9) (p<0,05), particularly in the non-irradiated tumor. We also found that ⍺CTLA-4 triple treatment induced higher CD80+CD86+ expression on dendritic cells than IL-2c triple treatment. hRT+⍺PD1+⍺CTLA4 triple treatment induced more cytotoxic effector TILs than hRT+⍺PD1+IL2c triple treatment, enhancing the abscopal effect and inducing a high abscopal cure rate. [ABSTRACT FROM AUTHOR]

Background. Clear cell renal cell carcinoma (ccRCC) is the most common subtype detected in 70-80 % of patients with renal cell carcinoma (Lopez-Beltran, 2006). However, due to complicated diagnostics, in 40-50 % of patients ccRCC is found after the metastases generation (Shinmei, 2013). Hence, there is an urgent need to develop novel, more effective methods for the ccRCC diagnostics. Many studies have shown that miRNAs take part in several processes of tumorigenesis, involving proliferation, migration, invasion, and metastasis (Fendler, 2011), which suggests potential values of miRNAs as the disease biomarkers. As biomarker candidates two miRNAs were chosen: miR-200c-3p which is known to play crucial roles in epithelial-mesenchymal transition, cell invasion, proliferation, metastasis, and therapy resistance in several cancer types (Humphries, 2015, Mutlu, 2015), and miR-30a-5p, the cancerogenic role of which has been explored in various cancer types including ccRCC (Sun, 2019). Aim. Here we investigated the possibility to use the miR-200c-3p and miR-30a-5p expression level in blood plasma samples as a diagnostic biomarker. Methods. Tumor surgical specimens and control samples (non-malignant tissues) of kidney and blood plasma (taken before the surgery) were obtained from the Institute of Urology, NAMS of Ukraine. Total RNA from the tissues samples was isolated with TRI Reagent (Sigma, USA) and reverse transcribed by the High-Specificity miRNA 1st-Strand cDNA Synthesis Kit (Agilent Technologies, USA) for quantitative real-time PCR determination of the miR-200c-3p and miR-30a-5р expression. Results. By comparison of tumor to control samples we found 5-fold downregulation of miR-30a-5p and 5.8-fold of miR-200c-3p with AUC=0.879 and AUC=0.865 respectively. Interestingly, [the] miR-200c-3p expression level in blood plasma was 7.9-fold lower than in tumor and 8.7-fold lower than in plasma of healthy donors. [The] miR-30а-5p expression level in blood plasma was 9.8-fold lower than in RCC tissues and 6.4- fold lower than the plasma levels of healthy donors. AUC of miR-30а-5p and miR-200c-3p in the blood plasma of the patients compared to controls was 0.969 and 0.973 indicating a highly significant diagnostic potential of this microRNA. Conclusions. Based on these results, we consider miR-30a-5p and miR-200c-3p as a promising marker of ccRCC early diagnostics. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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 IFNβ1 markedly depended on the cGAS/STING pathway. Doxorubicin-treated/Doxil-treated tumor cells depleted of mtDNA secreted less IFNβ1, of the related T cell-recruiting chemokine CXCL10, and ATP; coincubation with mtDNA-depleted tumor cells strongly reduced IFNβ1 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., Competing Interests: Competing interests: None declared., (© 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.)

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., (© 2023. The Author(s).)

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., (©2022 American Association for Cancer Research.)

Aim: To analyze the expression levels of hsa-miR-30c-5p and hsa-miR-138-1 in tumors of patients with renal cell carcinoma to determine whether they could be used as diagnostic markers., Materials and Methods: The relative expression of hsa-miR-30c-5p and hsa-miR-138-1 was compared in the paired samples of kidney tumor tissue and conventionally normal tissue adjacent to the tumor., Results: We found a significant decrease in miR-30c-5p and miR-138-1 levels in tumor tissues even in the cases of early stage cancer. In addition, miR-138-1 expression was lower in renal cell carcinoma Fuhrman grade G3 + G4 as compared to Fuhrman grade G2. However, we found no association between miR-30c-5p and miR-138-1 expression in the tumors and the major clinical and pathological characteristics of renal cell carcinoma patients., Conclusions: A significant reduction in the expression levels of hsa-miR-30c-5p and hsa-miR-138-1 in renal cell carcinoma indicates the feasibility of further studies on the probable diagnostic utility of these markers.

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., Competing Interests: The authors declare that they have no competing interests.