Diffusing Alpha-Emitters Radiation Therapy Promotes a Proimmunogenic Tumor Microenvironment and Synergizes With Programmed Cell Death Protein 1 Blockade.

PURPOSE: Diffusing alpha-emitters Radiation Therapy (DaRT) releases alpha-emitting atoms into the tumor microenvironment. The treatment effectively ablates human and mice xenografts and shows 100% response rates in skin or head and neck squamous cell carcinoma patients. DaRT induces specific and systemic antitumor immune activation and synergizes with immune stimulation and modulation in mice. Here, the transcriptional profile activated by DaRT, and its potential to enhance responsiveness to immune checkpoint inhibition by programmed cell death protein 1 (PD-1) blockade were studied. METHODS AND MATERIALS: Squamous cell carcinoma tumor- bearing BALB/C mice were treated with DaRT or inert seeds in combination with anti-PD-1 (aPD-1) or IgG control antibody. Sixteen days after seed insertion, tumors and spleens were subjected to immunophenotyping and immunohistochemical staining. Combination of DaRT and aPD-1 was tested for efficacy. Gene expression analysis was performed on mRNA extracted from tumors 7 days after DaRT or inert insertion using Nanostring PanCancer-IO-360 panel, and tumors and spleens were subjected to flow cytometry analysis. RESULTS: DaRT in combination with aPD-1 delayed tumor development, induced CD3 and CD8 lymphocytes infiltration more efficiently than either monotherapy. The combined treatment reduced splenic polymorphonuclear myeloid derived suppressor cells more than aPD-1 therapy or control. Granzyme B release in the tumor was increased only in the combinational treatment and was correlated with T-lymphocyte infiltration. Gene expression and gene set enrichment analysis of mRNA levels 7 days after DaRT insertion indicated that DaRT upregulated apoptosis, p53 signaling, G1/S-related arrest, interferon signaling and myeloid related transcription, while downregulating DNA repair, cell proliferation, and notch-related transcription. Flow cytometry showed that DaRT increased dendritic cells activation and led to changes in MDSCs distribution. CONCLUSIONS: DaRT promotes a "hot" tumor microenvironment and changes in immune suppression that lead to a potentiation of aPD-1 blockade induced effector T cell function and improved treatment efficacy. This study provides rationale for investigating DaRT and aPD-1 combination in patients with squamous cell carcinoma.

Diffusing alpha-emitters radiation therapy in combination with temozolomide or bevacizumab in human glioblastoma multiforme xenografts.

Glioblastoma multiforme (GBM) is at present an incurable disease with a 5-year survival rate of 5.5%, despite improvements in treatment modalities such as surgery, radiation therapy, chemotherapy [e.g., temozolomide (TMZ)], and targeted therapy [e.g., the antiangiogenic agent bevacizumab (BEV)]. Diffusing alpha-emitters radiation therapy (DaRT) is a new modality that employs radium-224-loaded seeds that disperse alpha-emitting atoms inside the tumor. This treatment was shown to be effective in mice bearing human-derived GBM tumors. Here, the effect of DaRT in combination with standard-of-care therapies such as TMZ or BEV was investigated. In a viability assay, the combination of alpha radiation with TMZ doubled the cytotoxic effect of each of the treatments alone in U87 cultured cells. A colony formation assay demonstrated that the surviving fraction of U87 cells treated by TMZ in combination with alpha irradiation was lower than was achieved by alpha- or x-ray irradiation as monotherapies, or by x-ray combined with TMZ. The treatment of U87-bearing mice with DaRT and TMZ delayed tumor development more than the monotherapies. Unlike other radiation types, alpha radiation did not increase VEGF secretion from U87 cells in culture. BEV treatment introduced several days after DaRT implantation improved tumor control, compared to BEV or DaRT as monotherapies. The combination was also shown to be superior when starting BEV administration prior to DaRT implantation in large tumors relative to the seed size. BEV induced a decrease in CD31 staining under DaRT treatment, increased the diffusive spread of 224Ra progeny atoms in the tumor tissue, and decreased their clearance from the tumor through the blood. Taken together, the combinations of DaRT with standard-of-care chemotherapy or antiangiogenic therapy are promising approaches, which may improve the treatment of GBM patients.

RIG-1-Like Receptor Activation Synergizes With Intratumoral Alpha Radiation to Induce Pancreatic Tumor Rejection, Triple-Negative Breast Metastases Clearance, and Antitumor Immune Memory in Mice.

Diffusing alpha-emitting radiation therapy (DaRT) employs intratumoral Ra-224-coated seeds that efficiently destroy solid tumors by slowly releasing alpha-emitting atoms inside the tumor. In immunogenic tumor models, DaRT was shown to activate systemic antitumor immunity. Agonists of the membrane-bound toll-like receptors (TLRs) enhanced these effects and led to tumor rejection. Here, we examined the combination of DaRT with agents that activate a different type of pattern recognition receptors, the cytoplasmatic RIG1-like receptors (RLRs). In response to cytoplasmatic viral dsRNA, RLRs activate an antiviral immune response that includes the elevation of antigen presentation. Thus, it was postulated that in low-immunogenic tumor models, RLR activation in tumor cells prior to the induction of their death by DaRT will be superior compared to TLR activation. Intratumoral cytoplasmatic delivery of the dsRNA mimic polyIC by polyethylenimine (PEI), was used to activate RLR, while polyIC without PEI was used to activate TLR. PolyIC(PEI) prior to DaRT synergistically retarded 4T1 triple-negative breast tumors and metastasis development more efficiently than polyIC and rejected panc02 pancreatic tumors in some of the treated mice. Splenocytes from treated mice, adoptively transferred to naive mice in combination with 4T1 tumor cells, delayed tumor development compared to naïve splenocytes. Low-dose cyclophosphamide, known to reduce T regulatory cell number, enhanced the effect of DaRT and polyIC(PEI) and led to high long-term survival rates under neoadjuvant settings, which confirmed metastasis clearance. The epigenetic drug decitabine, known to activate RLR in low doses, was given intraperitoneally prior to DaRT and caused tumor growth retardation, similar to local polyIC(PEI). The systemic and/or local administration of RLR activators was also tested in the squamous cell carcinoma (SCC) tumor model SQ2, in which a delay in tumor re-challenge development was demonstrated. We conclude that RIG-I-like activation prior to intratumoral alpha radiation may serve as a potent combination technique to reduce both tumor growth and the spread of distant metastases in low-immunogenic and metastatic tumor models.

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long-lived and cancer-resistant subterranean wild rodent, Spalax.

The blind mole rat (Spalax) is a wild, long-lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence-associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide-induced senescence (EIS), evidenced by an increased activity of senescence-associated beta-galactosidase (SA-ß-Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well-known SASP factors: interleukin-6 (IL6), IL8, IL1α, growth-related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM-1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro-inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA-MB-231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF-κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory-related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age-related chronic inflammation supporting healthy aging and longevity.

Combining alpha radiation-based brachytherapy with immunomodulators promotes complete tumor regression in mice via tumor-specific long-term immune response.

Diffusing alpha-emitters radiation therapy (DaRT) is the only known method for treating solid tumors with highly destructive alpha radiation. More importantly, as a monotherapy, DaRT has been shown to induce a systemic antitumor immune response following tumor ablation. Here, immunomodulatory strategies to boost the antitumor immune response induced by DaRT, and the response specificity, were investigated in the colon cancer CT26 mouse model. Local treatment prior to DaRT, with the TLR3 agonist poly I:C, was sufficient to inhibit tumor growth relative to poly I:C or DaRT alone. DaRT used in combination with the TLR9 agonist CpG, or with the TLR1/2 agonist XS15 retarded tumor growth and increased tumor-rejection rates, compared to DaRT alone, curing 41% and 20% of the mice, respectively. DaRT in combination with CpG, the Treg inhibitor cyclophosphamide, and the MDSC inhibitor sildenafil, cured 51% of the animals, compared to only 6% and 0% cure when immunomodulation or DaRT was used alone, respectively. Challenge and Winn assays revealed that these high cure rates involved a specific immunological memory against CT26 antigens. We suggest that DaRT acts in synergy with immunomodulation to induce a specific and systemic antitumor immune response. This strategy may serve as a safe and efficient method not only for tumor ablation, but also for in situ vaccination of cancer patients.

Reduced calcium influx in the hypoxia-tolerant Spalax: The role of the erythropoietin receptor.

Tissue hypoxia is a condition that induces calcium influx into living cells. Calcium is a major player in maintaining cell signaling and homeostasis, and mediates the regulation of gene transcription and cell proliferation; however, acute and aggressive calcium influx induced by hypoxia eventually leads to programmed cell death. The blind mole rat, Spalax, is a wild-spread burrowing mammal adapted to hypoxic environments. A tyrosine -to- phenylalanine (F481 in Spalax corresponding to Y485 in human full-length receptor; Y460 in human mature form) substitution is found in the erythropoietin receptor of Spalax and other species, which was previously shown to be strongly involved in the calcium channels activation and subsequent calcium influx. The current work aimed to explore the dynamics of calcium transport across Spalax nonhematopoietic cells' membrane compared to above ground rat and mouse, and the role of the erythropoietin receptor of Spalax in the regulation of calcium influx under hypoxia. We show here that Epo-induced calcium influx in HEK293 cells transfected with Spalax EpoR is significantly lower than that of mouse; in hypoxia this difference was even more pronounced. Western blots confirmed a significant increase of Erk phosphorylation after stimulation with erythropoietin under hypoxia in cells transfected with mouse full length erythropoietin receptor compared to Spalax. Native primary fibroblasts showed lower cytosolic calcium concentrations in Spalax cells when compared to those of rats under normoxic and hypoxic conditions. Spalax EpoR appears to play an important role in preventing deleterious consequences of hypoxia and maintaining cellular homeostasis under stress.

Resistance to DNA damage and enhanced DNA repair capacity in the hypoxia-tolerant blind mole rat Spalax carmeli.

Blind mole rats of the genus Spalax are the only mammalian species to date for which spontaneous cancer has never been reported and resistance to carcinogen-induced cancers has been demonstrated. However, the underlying mechanisms are still poorly understood. The fact that Spalax spp. are also hypoxia-tolerant and long-lived species implies the presence of molecular adaptations to prevent genomic instability, which underlies both cancer and aging. We previously demonstrated the upregulation of transcripts related to DNA replication and repair pathways in Spalax Yet, to date, no direct experimental evidence for improved genomic maintenance has been demonstrated for this genus. Here, we show that compared with skin fibroblasts of the above-ground rat, Spalax carmeli skin fibroblasts in culture resist several types of genotoxic insult, accumulate fewer genotoxic lesions and exhibit an enhanced DNA repair capacity. Our results strongly support that this species has evolved efficient mechanisms to maintain DNA integrity as an adaptation to the stressful conditions in the subterranean habitat.

Genome maintenance and bioenergetics of the long-lived hypoxia-tolerant and cancer-resistant blind mole rat, Spalax: a cross-species analysis of brain transcriptome.

The subterranean blind mole rat, Spalax, experiences acute hypoxia-reoxygenation cycles in its natural subterranean habitat. At the cellular level, these conditions are known to promote genomic instability, which underlies both cancer and aging. However, Spalax is a long-lived animal and is resistant to both spontaneous and induced cancers. To study this apparent paradox we utilized a computational procedure that allows detecting differences in transcript abundance between Spalax and the closely related above-ground Rattus norvegicus in individuals of different ages. Functional enrichment analysis showed that Spalax whole brain tissues maintain significantly higher normoxic mRNA levels of genes associated with DNA damage repair and DNA metabolism, yet keep significantly lower mRNA levels of genes involved in bioenergetics. Many of the genes that showed higher transcript abundance in Spalax are involved in DNA repair and metabolic pathways that, in other species, were shown to be downregulated under hypoxia, yet are required for overcoming replication- and oxidative-stress during the subsequent reoxygenation. We suggest that these differentially expressed genes may prevent the accumulation of DNA damage in mitotic and post-mitotic cells and defective resumption of replication in mitotic cells, thus maintaining genome integrity as an adaptation to acute hypoxia-reoxygenation cycles.

Adaptive patterns in the p53 protein sequence of the hypoxia- and cancer-tolerant blind mole rat Spalax.

BACKGROUND: The subterranean blind mole rat, Spalax (genus Nannospalax) endures extreme hypoxic conditions and fluctuations in oxygen levels that threaten DNA integrity. Nevertheless, Spalax is long-lived, does not develop spontaneous cancer, and exhibits an outstanding resistance to carcinogenesis in vivo, as well as anti-cancer capabilities in vitro. We hypothesized that adaptations to similar extreme environmental conditions involve common mechanisms for overcoming stress-induced DNA damage. Therefore, we aimed to identify shared features among species that are adapted to hypoxic stress in the sequence of the tumor-suppressor protein p53, a master regulator of the DNA-damage response (DDR). RESULTS: We found that the sequences of p53 transactivation subdomain 2 (TAD2) and tetramerization and regulatory domains (TD and RD) are more similar among hypoxia-tolerant species than expected from phylogeny. Specific positions in these domains composed patterns that are more frequent in hypoxia-tolerant species and have proven to be good predictors of species' classification into stress-related categories. Some of these positions, which are known to be involved in the interactions between p53 and critical DDR proteins, were identified as positively selected. By 3D modeling of p53 interactions with the coactivator p300 and the DNA repair protein RPA70, we demonstrated that, compared to humans, these substitutions potentially reduce the binding of these proteins to Spalax p53. CONCLUSIONS: We conclude that extreme hypoxic conditions may have led to convergent evolutionary adaptations of the DDR via TAD2 and TD/RD domains of p53.

Immune perturbations in patients along the perioperative period: alterations in cell surface markers and leukocyte subtypes before and after surgery.

BACKGROUND: Surgery renders patients susceptible to life-threatening complications, including infections, multiple organ failure, and presumably cancer metastases. Surgery-induced immune perturbations were suggested to contribute to such deleterious effects, but also to facilitate post-injury healing. Preoperative psychological and physiological stress responses may contribute to these immune perturbations, and could thus jeopardize patients even before surgery. The current study assessed the effects of various operations on an array of immune indices during the perioperative period. To qualify immune changes before surgery, patients' immune status was also compared to that of healthy controls. METHODS: A total of 81 subjects (operated patients and healthy controls) provided up to five daily blood samples during the perioperative period, for assessment of leukocyte subtypes (granulocytes, monocytes, Tc, Th, NK, NKT, CD4+CD25+, CD8(bright)CD4(dim), and B cells) and their surface markers (HLA-DR and LFA-1). RESULTS: Even before surgery patients displayed immune perturbations, including reduced lymphocyte HLA-DR expression and increased monocyte LFA-1 expression. Following surgery, we recorded a reduction in lymphocyte numbers that was subtype specific, increased granulocyte numbers, and reduced expression of HLA-DR by lymphocytes and monocytes. Finally, no significant associations were found between alteration in leukocyte numbers and cell surface markers (although these indices showed high correlations with other variables), implying differential mediating mechanisms. CONCLUSION: Several immune alterations are manifested prior to surgery, and contribute to the marked postoperative changes, which are commonly interpreted as immune suppression. We discuss the possible adaptive and maladaptive nature of these perturbations in the context of natural injury, stress, and surgery.

Metastatic-promoting effects of LPS: sexual dimorphism and mediation by catecholamines and prostaglandins.

Inflammation is implicated in several medical conditions that are sexually dimorphic, including depression, cardiovascular diseases, autoimmunity, and presumably cancer progression. Here we studied the effects of the proinflammatory agent, LPS, on MADB106 lung tumor retention (LTR), and sought to elucidate underlying mechanisms and sexual dimorphism. F344 male and female rats were administered with LPS (0.001-1mg/kg i.v.) simultaneously with tumor cell inoculation, and treated with a beta-blocker (nadolol, 0.2-0.3mg/kg s.c.), a COX inhibitor (indomethacin, 4mg/kg s.c.) or both drugs. To study the role of NK cells, numbers and cytotoxicity of marginating-pulmonary NK cells were studied, and selective in vivo NK-depletion was employed. Serum levels of corticosterone, IL-6, and TNF-alpha were also assessed. The findings indicated that LPS increased LTR in both sexes, but 10-fold higher doses were needed in females to reach the increase evident in males. Additionally, nadolol and indomethacin reduced the effects of LPS, more so in males. In vivo NK-depletion and ex vivo NK activity studies suggested that LPS affected LTR through both NK-independent and NK-dependent mechanisms, the latter mediated through prostaglandin release in males. Corticosterone, IL-6, and TNF-alpha responses to LPS were sexually dimorphic, but were not associated with LPS or drugs' impacts on LTR. Overall, our findings demonstrate sexual dimorphism in LPS-induced elevated susceptibility to MADB106 experimental metastasis, and in potential humoral underlying mechanisms. Further studies are needed to elucidate additional immunological and non-immunological mediators of these dimorphisms, as well as to assess their involvement in other sexually dimorphic pathologies that are associated with inflammation.