Effects of a Unique Combination of the Whole-Body Low Dose Radiotherapy with Inactivation of Two Immune Checkpoints and/or a Heat Shock Protein on the Transplantable Lung Cancer in Mice.

Non-small cell lung cancer (NSCLC) continues to be the leading cause of cancer death worldwide. Recently, targeting molecules whose functions are associated with tumorigenesis has become a game changing adjunct to standard anti-cancer therapy. As evidenced by the results of preclinical and clinical investigations, whole-body irradiations (WBI) with X-rays at less than 0.1-0.2 Gy per fraction can induce remissions of various neoplasms without inciting adverse side effects of conventional chemo- and radiotherapy. In the present study, a murine model of human NSCLC was employed to evaluate for the first time the anti-neoplastic efficacy of WBI combined with inactivation of CTLA-4, PD-1, and/or HSP90. The results indicate that WBI alone and in conjunction with the inhibition of the function of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed death-1 (PD-1) receptor immune checkpoints (ICs) and/or heat shock protein 90 (HSP90) markedly reduced tumorigenesis in mice implanted by three different routes with the syngeneic Lewis lung cancer cells and suppressed clonogenic potential of Lewis lung carcinoma (LLC1) cells in vitro. These results were associated with the relevant changes in the profile of pro- and anti-neoplastic immune cells recruited to the growing tumors and the circulating anti- and pro-inflammatory cytokines. In contrast, inhibition of the tested molecular targets used either separately or in combination with each other did not exert notable anti-neoplastic effects. Moreover, no significant synergistic effects were detected when the inhibitors were applied concurrently with WBI. The obtained results supplemented with further mechanistic explanations provided by future investigations will help design the effective strategies of treatment of lung and other cancers based on inactivation of the immune checkpoint and/or heat shock molecules combined with low-dose radiotherapy.

A derivative of vitamin B3 applied several days after exposure reduces lethality of severely irradiated mice.

Most, if not all, of the hitherto tested substances exert more or less pronounced pro-survival effects when applied before or immediately after the exposure to high doses of ionizing radiation. In the present study we demonstrate for the first time that 1-methyl nicotinamide (MNA), a derivative of vitamin B3, significantly (1.6 to 1.9 times) prolonged survival of BALB/c mice irradiated at LD30/30 (6.5 Gy), LD50/30 (7.0 Gy) or LD80/30 (7.5 Gy) of γ-rays when the MNA administration started as late as 7 days post irradiation. A slightly less efficient and only after the highest dose (7.5 Gy) of γ-rays was another vitamin B3 derivative, 1-methyl-3-acetylpyridine (1,3-MAP) (1.4-fold prolonged survival). These pro-survival effects did not seem to be mediated by stimulation of haematopoiesis, but might be related to anti-inflammatory and/or anti-thrombotic properties of the vitamin B3 derivatives. Our results show that MNA may represent a prototype of a radioremedial agent capable of mitigating the severity and/or progression of radiation-induced injuries when applied several hours or days after exposure to high doses of ionizing radiation.

How fast does wasp venom immunotherapy affect a regulatory T cell subpopulation (CD4+ CD25+ Foxp3+) and the synthesis of interleukins 10, 21 and transforming growth factor ß1?

INTRODUCTION: The literature describes the influence of venom immunotherapy (VIT) on the subpopulation of T regulatory cells (CD4+ CD25+ Foxp3+) and the synthesis of IL-10, TGF-ß1 as well as many other cytokines at various times after immunotherapy. AIM: To assess changes in the percentage of cells of CD4+ and CD25+ in peripheral blood and serum concentrations of IL-10, IL-21 and TGF-ß1 in the early stages of VIT. MATERIAL AND METHODS: The study included 18 patients who were allergic to wasp venom and who in the past underwent systemic anaphylactic reaction after stinging, meeting the criteria to qualify for VIT. The immunoenzymatic method (ELISA) was used to assess concentrations of cytokines IL-10, IL-21 and TGF-ß1 and the surface antigens CD4 and CD25 on the cells. The concentrations were determined by flow cytometry method at baseline (before VIT) and after 2.5 and 24 h from the VIT starting point. RESULTS: The mean values of the activity of T lymphocytes CD4+ CD25+ FoxP3+ and concentrations of the cytokines IL-10, IL-21 and TGF-ß1 are shown in table. CONCLUSIONS: A 24-hour activation assessment of serum concentrations of cytokines IL-10, IL-21 and TGF-ß1 during the first day of the Hymenoptera venom immunotherapy by ultra-rush protocol does not show the significant dynamics of change of the examined parameters.

Effect of internal contamination with tritiated water on the neoplastic colonies in the lungs, innate anti-tumour reactions, cytokine profile, and haematopoietic system in radioresistant and radiosensitive mice.

Tritium is a potentially significant source of internal radiation exposure which, at high levels, can be carcinogenic. We evaluated whether single intraperitoneal injection of BALB/c and C57BL/6 mice with tritiated water (HTO) leading to exposure to low (0.01 or 0.1 Gy) and intermediate (1.0 Gy) cumulative whole-body doses of ß radiation is immunosuppressive, as judged by enhancement of artificial tumour metastases, functioning of NK lymphocytes and macrophages, circulating cytokine's levels, and numbers of bone marrow, spleen, and peripheral blood cells. We demonstrate that internal contamination of radiosensitive BALB/c and radioresistant C57BL/6 mice with HTO at all the absorbed doses tested did not affect the development of neoplastic colonies in the lungs caused by intravenous injection of syngeneic cancer cells. However, internal exposure of BALB/c and C57BL/6 mice to 0.1 and 0.01 Gy of ß radiation, respectively, up-regulated cytotoxic activity of and IFN-γ synthesis in NK lymphocytes and boosted macrophage secretion of nitric oxide. Internal contamination with HTO did not affect the serum levels of pro- (IL-1ß, IL-2, IL-6, TNF-α,) and anti-inflammatory (IL-1Ra, IL-4, IL-10) cytokines. In addition, exposure of mice of both strains to low and intermediate doses from the tritium-emitted ß-particles did not result in any significant changes in the numbers of bone marrow, spleen, and peripheral blood cells. Overall, our data indicate that internal tritium contamination of both radiosensitive and radioresistant mice leading to low and intermediate absorbed ß-radiation doses is not immunosuppressive but may enhance some but not all components of anticancer immunity.

Cancer immunotherapy: how low-level ionizing radiation can play a key role.

The cancer immunoediting hypothesis assumes that the immune system guards the host against the incipient cancer, but also "edits" the immunogenicity of surviving neoplastic cells and supports remodeling of tumor microenvironment towards an immunosuppressive and pro-neoplastic state. Local irradiation of tumors during standard radiotherapy, by killing neoplastic cells and generating inflammation, stimulates anti-cancer immunity and/or partially reverses cancer-promoting immunosuppression. These effects are induced by moderate (0.1-2.0 Gy) or high (>2 Gy) doses of ionizing radiation which can also harm normal tissues, impede immune functions, and increase the risk of secondary neoplasms. In contrast, such complications do not occur with exposures to low doses (≤0.1 Gy for acute irradiation or ≤0.1 mGy/min dose rate for chronic exposures) of low-LET ionizing radiation. Furthermore, considerable evidence indicates that such low-level radiation (LLR) exposures retard the development of neoplasms in humans and experimental animals. Here, we review immunosuppressive mechanisms induced by growing tumors as well as immunomodulatory effects of LLR evidently or likely associated with cancer-inhibiting outcomes of such exposures. We also offer suggestions how LLR may restore and/or stimulate effective anti-tumor immunity during the more advanced stages of carcinogenesis. We postulate that, based on epidemiological and experimental data amassed over the last few decades, whole- or half-body irradiations with LLR should be systematically examined for its potential to be a viable immunotherapeutic treatment option for patients with systemic cancer.

Effect of low doses of low-let radiation on the innate anti-tumor reactions in radioresistant and radiosensitive mice.

BALB/c and C57BL/6 mice differ in their Th1/Th2 lymphocyte and M1/M2 macrophage phenotypes, radiosensitivity, and post-irradiation tumor incidence. In this study we evaluated the effects of repeated low-level exposures to X-rays on the development of artificial tumor colonies in the lungs of animals from the two strains and cytotoxic activities of natural killer (NK) cells and macrophages obtained from these mice. After ten daily irradiations of BALB/c or C57BL/6 mice with 0.01, 0.02, and 0.1 Gy X-rays NK cell-enriched splenocytes collected from the animals demonstrated significant and comparable up-regulation of their anti-tumor cytotoxic function. Likewise, peritoneal macrophages collected from the two irradiated strains of mice exhibited the similarly stimulated cytotoxicities against susceptible tumor cells and produced significantly more nitric oxide. These results were accompanied by the significantly reduced numbers of the neoplastic colonies induced in the lungs by intravenous injection of syngeneic tumor cells. The obtained results indicate that ten low-level irradiations with X-rays stimulate the generally similar anti-tumor reactions in BALB/c and C57BL/6 mice.

Anti-neoplastic and immunostimulatory effects of low-dose X-ray fractions in mice.

PURPOSE: The exploration of immune mechanisms of the tumour-inhibitory effect of exposures to low-level fractions of X-rays. MATERIALS AND METHODS: BALB/c mice were exposed to whole-body daily irradiations with 0.01, 0.02, or 0.1 Gy X-rays per day for 5 days/week for two weeks. Then, mice were intravenously injected with L1 tumour cells, killed 14 days later, and neoplastic colonies were counted in the lungs. Natural killer (NK) cell-enriched splenocytes and activated peritoneal macrophages (Mϕ) were collected and cytotoxic activities of these cells against susceptible tumour targets were assayed. Concanamycin A (CMA) and antibody against the ligand for the Fas receptor (FasL) were used to inhibit the NK cell-mediated cytotoxicity. Production of nitric oxide (NO) was quantified using the Griess reagent. Secretion of interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), interleukin-12 (IL-12), and tumour necrosis factor-α (TNF-α) was measured using the enzyme-linked immunosorbent assays. RESULTS: All the exposures to X-rays significantly reduced the number of the induced tumour colonies and enhanced cytotoxic properties of the NK cell-enriched splenocytes and activated Mϕ. CONCLUSION: Suppression of the growth of pulmonary tumour colonies by irradiations of mice with low-dose fractions of X-rays may result from stimulation of anti-tumour reactions mediated by NK cells and/or cytotoxic macrophages.

Immunological mechanism of the low-dose radiation-induced suppression of cancer metastases in a mouse model.

According to the doctrine underlying the current radiation protection regulations each, no matter how small, exposure to ionizing radiation may be carcinogenic. However, numerous epidemiological observations demonstrate that cancer incidence and/or mortality are not elevated among inhabitants of the high- versus low-natural-background radiation areas and homes. Results of our own and other authors' studies described in this paper bear testimony to the possibility that stimulation of the anti-neoplastic immune surveillance mediated by NK lymphocytes and activated macrophages explains, at least partially, the accumulating epidemiological and experimental evidence indicating that low-level exposures to the low-linear energy transfer (LET) radiation inhibit the development of spontaneous and artificial metastases in humans and laboratory animals, respectively. The results presented also suggest the possibility of using low-level X- and gamma-ray exposures to cure cancer and to prevent cancer metastases. For a broader perspective, the results presented may help towards relaxing the current radiation protection regulations, especially as they apply to diagnostic and therapeutic exposures of patients to the indicated forms of radiation.

Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays.

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.

Enhanced cytotoxic activity of macrophages and suppressed tumor metastases in mice irradiated with low doses of X- rays.

We showed in our previous report that a single exposure of mice to 0.1 or 0.2 Gy X-rays led to the significant inhibition of the development of artificial tumor metastases in the lungs and that the effect was related to the enhanced activity of natural killer cells. In the present study, a possible involvement of cytotoxic macrophages in the anti-metastatic effect of the low-level X-ray exposures was investigated. We now demonstrate that irradiation of mice with either of the two low doses of X-rays significantly stimulates the macrophage-mediated cytolysis of the susceptible tumor targets and that the effect coincides with the enhanced production of nitric oxide in the collected effector cells. We also show that suppression of the in vivo function of macrophages by carrageenan eliminates the inhibitory effect of the two low doses of X-rays on the development of pulmonary tumor colonies as well as significantly suppresses the macrophage-mediated cytotoxicity and nitric oxide production. Finally, aminoguanidine added to the culture medium of the assayed macrophages not only shuts down the nitric oxide synthesis in these cells but also significantly suppresses their cytolytic activity. Overall, the obtained results indicate that inhibition of the tumor metastases by a single exposure of mice to 0.1 or 0.2 Gy X-rays results, to a large extent, from the radiation-induced stimulation of the cytocidal activity of macrophages which secrete enhanced amounts of nitric oxide.

Single low doses of X rays inhibit the development of experimental tumor metastases and trigger the activities of NK cells in mice.

There is evidence indicating that low-level exposures to low- LET radiation may inhibit the development of tumors, but the mechanism of this effect is virtually unknown. In the present study, BALB/c mice were irradiated with single doses of 0.1 or 0.2 Gy X rays and injected intravenously 2 h later with syngeneic L1 sarcoma cells. Compared to the values obtained for sham-irradiated control mice, the numbers of pulmonary tumor colonies were significantly reduced in the animals exposed to either 0.1 or 0.2 Gy X rays. Concurrently, a significant stimulation of NK cell-mediated cytotoxic activity was detected in splenocyte suspensions obtained from irradiated mice compared to sham-exposed mice. Intraperitoneal injection of the NK-suppressive anti-asialo GM1 antibody totally abrogated the tumor inhibitory effect of the exposures to 0.1 and 0.2 Gy X rays. These results indicate that single irradiations of mice with either 0.1 or 0.2 Gy X rays suppress the development of experimental tumor metastases primarily due to the stimulation of the cytolytic function of NK cells by radiation.