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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.

Many biomedical applications require concentration measurements of biological compounds, which may be achieved using targeted fluorescent probes. It has been shown that the ratio between amyloid-Beta and tau protein in the cerebrospinal fluid (CSF) is a good indicator of incipient Alzheimer’s disease (AD). We have previously proposed a method that can accurately estimate the concentration ratio of these two proteins without the need to collect CSF samples: Fluorescent probes are injected to the blood and bind to the CSF biomarkers. A miniature needle with an optical fiber excites the fluorescent probes and collects the fluorescence emission. The concentration ratio between the proteins is estimated, and used for diagnosis of incipient AD. We present here the results of the method’s concentration ratio estimation during trials performed on mice. Miniature tubes containing two fluorescent probes in several concentration ratios were implanted in two locations in the mice: subcutaneously, and deeper in the abdomen. The fluorescent probes were excited and the fluorescence intensity was measured. The concentration ratios were estimated from the fluorescence intensities with an average error of less than 20%. This study is a step towards the demonstration of the method in in vivo studies in mice with markers in the bloodstream/CSF.

We developed (224)Ra-loaded wires, which release by recoil alpha emitting nuclei into solid tumors and cause tumor cell killing. This research examined if the major damage was inflicted by alpha particles emitted from these atoms or by direct gamma and beta emissions from the inserted wires. We also examined the efficacy of this treatment against colon cancer in combination with chemotherapy.Mouse colon carcinomas (CT-26 xenografts), treated by intra-tumoral radioactive wires loaded with (224)Ra atoms were monitored for effects on tumor growth, intratumoral tissue damage and distribution of alpha emitting atoms. The effects were compared with those of (224)Ra-loaded wires coated with poly methyl methacrylate (PMMA), which blocks atom recoil. Similar experiments were performed with radioactive wires combined with systemic 5-FU.(224)Ra-loaded wires inhibited tumor growth and formed necrotic areas inside the tumor. PMMA coated wires did not inhibit tumor growth, and caused minor intratumoral damage. Autoradiography images of tumors treated with (224)Ra-loaded wires revealed a spread of alpha emitters over several mm, whereas PMMA-coated wires showed no such spread. Injection of 5-FU with (224)Ra-loaded wires augmented tumor growth retardation and cure.(224)Ra-loaded wires ablate solid tumors by the release of alpha-particle emitting atoms inside the tissue, an effect that can be enhanced by combining this method with chemotherapy.

The current systemic anti-metastatic treatment is chemotherapy. Chemotherapy reacts mostly against replicating cells, which makes this therapy not specific. Moreover, resting cancer cells will not be destroyed. A better alternative is an engagement of the host immune system to react against tumor-associated antigens. An efficient immune-stimulating technique is an ablation of the tumor that results in the release of tumor antigens. Our ablation strategy is an innovative alpha-radiation-based technology, diffusing alpha-emitters radiation therapy (DaRT), which efficiently destroys local tumors and provides thereby an antigenic supply for antigen-presenting cells to stimulate T cells.Mice bearing weakly immunogenic DA3 adenocarcinoma or highly immunogenic CT26 colon carcinoma were treated by DaRT. Anti-tumor immune responses following tumor destruction were evaluated by (1) the resistance to a tumor challenge; (2) scanning by a CT imaging device for elimination of lung metastases; (3) improved tumor control when combining DaRT with an immunoadjuvant (CpG).CT26 model: 63-77 % of DaRT-treated mice became resistant to a re-inoculated tumor compared to 29-33 % resistant mice in the control. DA3 model: (1) The growth rate of challenge tumors was the lowest in mice which their primary tumor was treated by DaRT. (2) Most (93 %) mice in the control group developed lung metastases compared to 56 % in the DaRT group. (3) Combining DaRT with CpG resulted in a better control of the primary tumor. Our study offers a technique to eliminate local and distant malignant cells, regardless of their replication status, by stimulating specific anti-tumor immunity through the supply of tumor antigens from the destroyed tumor.

We developed 224 Ra-loaded wires that when inserted into solid tumors, release radioactive atoms that spread in the tumor and irradiate it effectively with alpha particles (diffusing alpha-emitters radiation therapy [DaRT]). In this study, we tested the ability of intratumoral 224 Ra-loaded wires to control the local growth of pancreatic tumors and the enhancement of this effect by chemotherapy. Pancreatic mouse tumors (Panc02) were treated with 224 Ra-loaded wire(s) with or without gemcitabine. The tumor size and survival were monitored, and autoradiography was performed to evaluate the spread of radioactive atoms inside the tumor. Mouse and human pancreatic cancer cells, irradiated in vitro by alpha particles with or without chemotherapy, were evaluated for cell growth inhibition. The insertion of 224 Ra-loaded wires into pancreatic tumors in combination with gemcitabine achieved significant local control and was superior to each treatment alone. A dosimetric analysis showed the spread of radioactive atoms in the tumor around the wires. Alpha particles combined with gemcitabine or 5-FU killed mouse and human cells in vitro better than each treatment alone. DaRT in combination with gemcitabine was proven effective against pancreatic tumors in vivo and in vitro , and the process may be applicable as a palliative treatment for patients with pancreatic cancer.

It has been demonstrated that aggressive in situ tumor destruction (ablation) could lead to the release of tumor antigens, which can stimulate anti-tumor immune responses. We developed an innovative method of tumor ablation based on intratumoral alpha-irradiation, diffusing alpha-emitters radiation therapy (DaRT), which efficiently ablates local tumors and enhances anti-tumor immunity. In this study, we investigated the anti-tumor potency of a treatment strategy, which combines DaRT tumor ablation with two approaches for the enhancement of anti-tumor reactivity: (1) neutralization of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and (2) boost the immune response by the immunoadjuvant CpG. Mice bearing DA3 mammary adenocarcinoma with metastases were treated with DaRT wires in combination with a MDSC inhibitor (sildenafil), Treg inhibitor (cyclophosphamide at low dose), and the immunostimulant, CpG. Combination of all four therapies led to a complete rejection of primary tumors (in 3 out of 20 tumor-bearing mice) and to the elimination of lung metastases. The treatment with DaRT and Treg or MDSC inhibitors (without CpG) also resulted in a significant reduction in tumor size, reduced the lung metastatic burden, and extended survival compared to the corresponding controls. We suggest that the therapy with DaRT combined with the inhibition of immunosuppressive cells and CpG reinforced both local and systemic anti-tumor immune responses and displayed a significant anti-tumor effect in tumor-bearing mice.

We developed a new method of brachytherapy, termed diffusing alpha-emitters radiation therapy (DaRT), based on the use of intratumoral (224)Ra-loaded wires, which release short-lived alpha-emitting atoms by recoil. Here, we examined their ability to destroy and control the development of several human-derived tumors implanted in athymic mice.The experiments were performed on athymic mice bearing malignant human-derived tumors including prostate (PC-3), glioblastoma (GBM, U87-MG), colon (HCT15), squamous cell carcinoma (FaDu) and melanoma (C32). One or more (224)Ra-loaded wires were inserted into the tumors, and mice were assessed for tumor growth rate and survival.In vivo studies showed that DaRT can effectively destroy the tumors, and in vitro tests confirmed the sensitivity of the studied cells to alpha particles. While the C32 cells were relatively resistant, other tumor types (e.g. HCT15) exhibited sensitivity in both measured aspects.DaRT could potentially be combined with chemotherapy or other treatment modalities to effectively treat non-resectable tumors.

The allele E4 of apolipoprotein E (apoE) is an important risk factor for Alzheimer's disease (AD) and the chronic brain inflammation which is associated with AD is more pronounced in subjects who carry this allele. In the present study, we employed mice transgenic for the human apoE isoforms apoE3 or apoE4 on a null mouse apoE background and intracerebroventricular injection of LPS to investigate the possibility that the regulation of brain inflammation is affected by the apoE genotype. LPS treatment of control mice resulted in activation of brain astrocytes and microglia whose extent decreased with age. LPS treatment of 6-month-old apoE transgenic and control mice resulted in marked activation of brain astrocytes in the control and apoE3 transgenic mice but had no effect on astrogliosis of age-matched apoE-deficient and apoE4 transgenic mice. In contrast, there were no significant differences between the levels of activated microglia of the apoE3 and apoE4 transgenic mice following LPS treatment. Immunoblot assays revealed that the apoE4 and apoE3 transgenic mice had the same levels of brain apoE, which were similarly increased following LPS treatment. These results show that LPS-induced astrogliosis in apoE transgenic mice is regulated isoform-specifically by apoE3 and not by apoE4 and suggest that similar mechanisms may mediate the phenotypic expression of the apoE4 genotype in AD and in other neurodegenerative diseases.

Genetic studies suggest that the neuropathology and etiology of Alzheimer’s disease (AD) are associated with several genotypes including mutations in the amyloid precursor protein (APP) gene and the allele E4 of apolipoprotein E (apoE). The present study investigated the possibility that cross talk interactions exist between APP and apoE and the extent to which they are affected by the apoE genotype. This was pursued by cell culture and immunoblot experiments utilizing neuroblastoma N2a cells in which the effects of distinct apoE isoforms on the levels of intracellular APP and of secreted APPs were determined. This revealed that treatment of the cells with apoE4, the AD risk factor, resulted in a marked increase in the levels of secreted APPs. This effect was dose dependent (ED50 ≅ 2.5μg/ml) and isoform specific in that apoE3 had virtually no effect on the secretion of APPs.

Murine B16 melanoma cells and metastasis variants of this tumor are resistant to NK activity mediated by normal splenocytes. The B16 cells are, however, sensitive to splenocyte-mediated cytostasis. Cytostasis was measured by a [125I]UDR incorporation-inhibition [(125I]UDR I-I) assay. The main characteristics of the [125I[UDR I-I assay and of the cells mediating it are as follows: The activity is mediated by splenocytes but not by thymocytes, it is not syngeneically restricted and it does not decrease with age. The presence of effector cells is required as splenocyte supernatants or supernatants of effector-target cell mixtures do not cause [125I]UDR I-I. The activity is probably mediated by at least 2 populations of non-phagocytic splenocytes. The first population adheres to plastic surfaces and to Sephadex G-10 columns while the other does not. The sensitivity to [125I]UDR I-I of the high metastasis B16 variant was similar to that of the low metastasis variant.

Three types of natural immune responses against malignant cells were studied in vitro: Cytotoxicity mediated by splenic NK cells; cytostasis mediated by splenocytes and binding of naturally occurring antibodies to various tumour targets. These responses were studied in untreated 3 and 12 month old mice and in mice of both age groups inoculated with B16 melanoma cells. The results showed that in normal mice NK activity decreases with age, cytostatic activity remains unchanged and the titre of natural antibodies increases. Twelve-month old mice were shown to be appreciably more resistant than 3 month old mice to the development of tumours from subthreshold numbers of B16 tumour cells. In mice injected with threshold amounts of the B16 tumour, there was no change in any of the responses in the tumour-free period, but there was a decrease in NK activity and an increase in cytostatic activity when a large tumour mass developed. An increase in the titre of natural antibodies in young mice injected with the tumour was also seen. The correlation between these changes and tumour appearance and development is discussed.

It is becoming increasingly clear that chemical, physical, and biologic carcinogens are immunosuppressive. Most of the available information on the immunosuppressive activity of oncogenic agents concerns adaptive immunity [17]. Recent studies have indicated that exposure of laboratory animals to various carcinogenic insults affected also certain expressions of innate, natural immunity such as natural killer (NK) activity (for review see [2]).

The NK activity of BALB/c mice bearing carcinomas induced by intragastric administration of dimethylbenzanthracene (DMBA) is severely depressed. The aim of the present study was to determine when, in relation to tumor appearance or to DMBA administration, was this depression first demonstrable.

Publisher Summary This chapter presents studies focusing on cytostatic activity mediated by murine splenocytes. It was found that the natural cytostatic activity (measured by the inhibition of 125IUDR incorporation (125IUDR I-I) into B16-F10 melanoma cells), unlike the natural killer (NK) activity does not decline with age. Further experiments confirmed this and showed that very young mice (10–12 days old) that have a low NK activity (even when assayed in an 18 h assay) have a fully expressed cytostatic activity similar to mature mice. These findings show that ontogenetically, the cytostatic cell develops earlier than the NK cell and that aged animals exhibit a high cytostatic activity. From this aspect, the cytostatic cell resembles the natural cytotoxic (NC) cell. These results also support the conclusion that the NK and the cytostatic activities are mediated largely by different cell populations. Another study examines effect of carrageenan and hydrocortisone acetate on natural cytostatic and cytotoxic activities. It is shown that in contrast to the decrease in NK activity, the cytostatic activity was not inhibited at all or even somewhat enhanced.

In this study we determined the effect of administering the chemical carcinogen dimethylbenzanthracene (DMBA) on NK activity mediated by murine splenocytes. The intragastric administration of six weekly l-mg doses of DMBA to 2- to 3-month-old mice either following a pituitary graft or not, resulted in a tumor incidence which was higher than 60 %. The cellularity of the spleens decreased significantly after the third DMBA feeding and the NK activity of these spleens was selectively suppressed. There was no spontaneous reconstitution of the NK activity following cessation of DMBA treatment and during the entire tumor-free period. The adoptive transfer of normal bone marrow, thymus or spleen cells did not restore NK activity in DMBA-treated mice. The NK activity of DMBA-fed mice could be augmented by interferon and by interferon inducers such as poly I:C and dsRNA from Us-tilago virus. The effect of these biological response modifiers on NK activity was short-lived but it was possible to restimulate this activity every 24h. Consecutive treatments of DMBA-fed mice with poly I:C maintained a high level of NK activity for at least 3 weeks.

Publisher Summary This chapter examines suppression of natural killer (NK) activity by the chemical carcinogen dimethylbenzanthracene (DBMA) and its restoration by interferon or poly I:C. In a study described in the chapter, normal BALB/c mice or pituitary isografted mice were fed with DMBA. Tumor incidence was 60% in the former and about 100% in the latter group. Untreated mice that were only given a pituitary isograft did not develop tumors. Mice belonging to these four groups were inspected regularly for tumor appearance and tested each week for NK activity. These assays demonstrated that pituitary isografted and DMBA fed mice or mice treated only with DMBA showed a significant decrease in the number of spleen cells and a significant decrease in the NK activity. The number of lytic units per spleen in these mice was 6–10 times lower than in control mice. This decrease was evident already after the third DMBA feeding and it lasted throughout the entire tumor induction period. The NK activity levels of mice that developed tumors during the 100-day observation period were as low as those of DMBA-fed mice that were still tumor-free. Pituitary isografted mice did not show any decrease in NK activity.

Several immunocyte populations are active in the natural cellular defense against tumors, Among these are macrophages [1], natural killer (NK) cells [2], cells mediating antibody-dependent cellular cytotoxicity [3], natural cytotoxic (NC) [4] cells, and a variety of tumor cells. The NK cells kill mainly lymphoid tumor cells, and the cytostatic activity is directed against adherent tumor cells which originate from solid tumors.