Lethal and mutagenic interactions between γ-rays,cisplatin and etoposide at the cellular and molecular levels

Purpose:?We analysed the lethal and mutagenic interactions between γ-rays, cisplatin (Pt) and etoposide (E), three agents used in tumour chemoradiotherapy. Corresponding results at cellular and molecular levels could provide additional elements on involved mechanisms and, on antitumour activity and toxicity in combined cancer treatments.

Materials and methods:?The yeast Saccharomyces cerevisiae SC7K(lys2–3) (auxotrophic for lysine) was used as eukaryotic model. Exponential growing cells were exposed to the mentioned agents, as single and combined treatments. Lethal and mutation interaction equations were determined as a function of doses according to quantitative models. DNA double-strand breaks were evaluated immediately after treatments, through pulsed-field electrophoresis and laser densitometry.

Results:?All three agents induced significant mutant frequency. The γ?+Pt + E combination determined maximal lethal and mutagenic synergism, followed by γ?+?Pt and γ?+?E combinations. Meanwhile, Pt?+?E combination showed lethal additivity and very low mutagenic synergism. Pt?+?E double combination determined moderate DNA degradation. DNA degradation after γ-exposure, was similar to that of γ?+?Pt, γ?+?E and γ?+?Pt?+?E combinations.

Conclusions:?Synergistic lethal and mutagenic interactions indicate crosstalk between non-homologous end joining, homologous recombination and postreplicative repair pathways. Pt?+?E additivity indicate independence of involved repair pathways. Furthermore, the quantification of interactive events may be an additional suitable tool in tumour therapy planning.     

Three endpoints of in vivo tumour radiobiology and their statistical estimation

Purpose:?To review the existing endpoints of tumour growth delay assays in experimental radiobiology with an emphasis on their efficient estimation for statistically significant identification of the treatment effect. To mathematically define doubling time (DT), tumour-growth delay (TGD) and cancer-cell surviving fraction (SF) in?vivo using exponential growth and regrowth models with tumour volume measurements obtained from animal experiments.

Materials and methods:?A statistical model-based approach is used to define and efficiently estimate the three endpoints of tumour therapy in experimental cancer research.

Results:?The log scale is advocated for plotting the tumour volume data and the respective analysis. Therefore, the geometric mean should be used to display the mean tumour volume data, and the group comparison should be a t-test for the log volume to comply with the Gaussian-distribution assumption. The relationship between cancer-cell SF, TGD and rate of growth is rigorously established. The widespread formula for cell kill is corrected; it has been rigorously shown that TGD is the difference between DTs. The software for the tumour growth delay analysis based on the mixed modeling approach with a complete set of instructions and example can be found on the author's webpage.

Conclusions:?The existing practice for TGD data analysis from animal experiments suffers from imprecision and large standard errors that yield low power and statistically insignificant treatment effect. This practice should be replaced with a model-based statistical analysis on the log scale.     

Strategies for the analysis of in vitro radiation sensitivity and prediction of interaction with potential radiation modifying agents

Purpose:?To better predict radiation–drug interactions in in?vitro model systems, thorough assessment of the effects of in?vitro exposure is required. The aim of this article is to show that both clonogenic capacity and cellular proliferation, which represent important different elements of tumour conduct, can be considered when assessing in?vitro radio sensitisation.

Methods:?A model was designed that can predict radiation–drug interactions based on changes in clonogenic capacity and cell proliferation by radiation modifying agents.

Results:?Using this mechanistical model, the effect of combined exposure to radiation and potential drugs can be tested on both established cell lines and primary cells. In addition, we could obtain more information about the mechanisms underlying the radiation–drug interaction by assessing the results of in?vitro exposure on tumour cell proliferation and clonogenic capacity according to our model.

Conclusions:?The significance of our model is not to replace the clonogenic gold standard but to give additional information about the radiation–drug combination by determining cell proliferation. Moreover, the advantage is that the interaction can also be predicted in cases where a clonogenic assay is not possible. Additional research into the biological effect of potential radio-sensitisers is warranted for future (pre)clinical studies.     

HDR brachytherapy decreases proliferation rate and cellular progression of a radioresistant human squamous cell carcinoma in vitro

Purpose: To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters.

Materials and methods: An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method.

Results: Significant decreasing cellular proliferation rate (p?<?0.05) as well as reduced survival fraction (p?<?0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the G₂/M phase (32.3?±?1.5%) in the irradiated group as compared with untreated cells (8.22?±?1.2%) was detected. Also, a significant (p?<?0.0001) nuclei shrinking in irradiated cells without evidence of necrosis or apoptosis was found.

Conclusion: HDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G₂/M cell cycle arrest.     

There's something about ILK

Purpose:?Integrin-Linked Kinase (ILK) is associated with integrin and growth factor receptor signalling. As both signalling pathways contribute to cancer cell resistance, ILK seems well suited as a promising tumour target.

Material and methods:?Data were obtained by performing a PubMed database search and summarised with a focus on the function of ILK in cancer biology.

Results:?The findings on the catalytic function of ILK, on the putative substrates of ILK and on the expression of ILK in tumour and normal tissues are heterogeneous. In the context of cancer, two of these issues might be of importance. First, a variety of reports indicate a lack of ILK overexpression in tumours. Second, wild-type or overexpression of ILK has been found to considerably sensitise tumour cells to ionising irradiation as compared to ILK knockout or ILK knockdown conditions. In contrast, wild-type or overexpression of ILK has been shown to protect tumour cells from chemotherapy-induced cell death.

Conclusions:?Due to these conflicting data, it is difficult to evaluate if therapeutic targeting of ILK is a reasonable strategy in cancer therapy. A more comprehensive understanding of the molecular mechanisms controlled by ILK may help to answer this question.     

Irradiation of DNA loaded with platinum containing molecules by fast atomic ions C6+ and Fe26+

Purpose:?In order to study the role of the Linear Energy Transfer (LET) of fast atomic ions in platinum-DNA complexes inducing breaks, DNA Plasmids were irradiated by C⁶⁺ and Fe²⁶⁺ ions.

Material and methods:?DNA Plasmids (pBR322) loaded with different amounts of platinum contained in a terpyridine-platinum molecule (PtTC) were irradiated by C⁶⁺ ions and Fe²⁶⁺ ions. The LET values ranged between 13.4 keV/μm and 550 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added.

Results:?In all experiments, a significant increase in DNA strand breaks was observed when platinum was present. The yield of breaks induced per Gray decreased when the LET increased. The yield of single and double strand breaks per plasmid per track increased with the LET, indicating that the number of DNA breaks per Gray was related to the number of tracks through the medium.

Conclusions:?These findings show that more DNA breaks are induced by atomic ions when platinum is present. This effect increases for low LET heavy atoms. As DSB induction may induce cell death, these results could open new perspectives with the association of hadrontherapy and chemotherapy. Thus the therapeutic index might be improved by loading the tumour with platinum salts.     

Static electric fields interfere in the viability of cells exposed to ionising radiation

Purpose: The interference of electric fields (EF) with biological processes is an issue of considerable interest. No studies have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells.

Materials and methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the interval 0.5–5 kGy, using a ⁶⁰Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V · cm^?1 static electric field and viable cells were enumerated. Cultures of Candida albicans were incubated at 36°C for 20 h, gamma-irradiated with doses from 1–4 kGy, and submitted to an electric field of 180 V · cm^?1. Samples were examined under a fluorescence microscope and the number of unviable (red) and viable (apple green fluorescence) cells was determined. For crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric field of 1250 V/cm, incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a fluorescence microscope to quantify nuclei with γ-H2AX foci.

Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric field on the irradiated MRC5 cells the number of nuclei with γ-H2AX foci increased 40%, approximately.

Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation + EF suggests that EF likely inactivate cellular recovering processes. The result for the number of nuclei with γ-H2AX foci in MRC5 cells indicates that an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.     

Raman spectroscopy of tissue samples irradiated by protons

Purpose: Since the number of cancer patients treated by proton irradiation has increased in the last few years, it seems appropriate to study dose‐dependent effects of proton irradiation on mammalian tissues in more detail.

Materials and methods: Tissue samples of normal skin of mouse and swine, of a human tumour model xenograph, and of normal skin and a skin tumour (basal cell carcinoma) of a human patient of about 1?mm thickness were irradiated by 24?MeV protons (uniform delivered doses of 1, 7 and 50?Gy: skin of mouse and a human tumour model xenograph, and 0.5, 5 and 50?Gy: swine and human skin). Raman spectra of non‐irradiated and irradiated samples were recorded and analysed.

Results: Amide I, P=O and C‐O bond vibrations and aromatics were sensitive to the proton irradiation dose. In the C‐H stretching region, the irradiation‐mediated change of Raman spectra was significant only in the case of the skin tumour.

Conclusions: It has been shown that Raman spectroscopy is suited to assess the radiation damage done to biological samples by protons. Proteins of the human skin tumour seem to be more sensitive to proton irradiation than proteins of normal human skin.     

Differing effects of breast cancer 1, early onset (BRCA1) and ataxia‐telangiectasia mutated (ATM) mutations on cellular responses to ionizing radiation

Purpose: The ataxia‐telangiectasia mutated (ATM) gene encodes a protein kinase, the activation of which is an early event in the cellular response to ionizing radiation. One of the many substrates of ATM is BRCA1 (breast cancer 1, early onset gene), which has been associated with susceptibility to breast and ovarian cancer, and has been implicated in DNA repair processes. Various cellular responses to radiation were analysed in cells with mutations in ATM or BRCA1 in an attempt to clarify which effects of ATM can be mediated through BRCA1.

Materials and methods: The response to radiation of cells with mutations in ATM or BRCA1 was examined, as were BRCA1‐mutant tumour cells transfected with an exogenous wild‐type BRCA1 allele. Assays included cell‐survival curves, studies of potentially lethal damage repair, measurement of chromosomal aberrations and of G1 arrest, and Western blot analysis of lysates of irradiated cells to determine the phosphorylation of the product of the human Mdm2 gene (HDM2).

Results: Both ATM and BRCA1 mutations were associated with sensitivity to ionizing radiation, deficient repair of potentially lethal damage and markedly increased chromosomal aberrations. A BRCA1‐mutated tumour cell line HCC1937, like ATM mutant cells, did not exhibit a normal G1 arrest but, unlike ATM mutant cells, did exhibit phosphorylation of HDM2. Expression of wild‐type BRCA1 in HCC1937 cells partially restored radioresistance, restored repair of potentially lethal damage and markedly reduced radiation‐induced chromosomal aberrations. G1 arrest, however, was not restored by expression of BRCA1.

Conclusions: The results are consistent with a model in which ATM phosphorylation of BRCA1 regulates DNA repair functions, particularly those involved in potentially lethal damage repair and chromosomal integrity, but not other aspects of the cellular response to radiation such as G1 cell cycle arrest. To the authors' knowledge, this is the first demonstration of the ability of exogenously expressed BRCA1 to restore the ability to perform potentially lethal damage repair and maintain chromosomal integrity in irradiated cells.     

Enhanced transplantability of a cell line from a murine ovary granulosa cell tumour in syngeneic B6C3F1 mice continuously irradiated with low dose-rate gamma-rays

Purpose:?To understand the mechanisms of life-shortening due to early neoplastic death caused by chronic low dose-rate (LDR; 20 mGy/22 h/day) radiation which accumulates to a high dose (HD; 8 Gy) (LDR/HD) as reported previously.

Materials and methods:?Female B6C3F₁ mice were continuously exposed to LDR/HD gamma-rays under specific-pathogen-free (SPF) conditions for 400 days. OV3121 cells, which were derived from an ovarian granulosa cell tumour that arose in irradiated B6C3F₁ mice, were inoculated into LDR/HD irradiated and age-matched non-irradiated control mice. The transplantability of tumour cells as well as T cell subsets and the proliferative activities of T cells were compared between irradiated and non-irradiated mice.

Results:?We found that tumour formation of subcutaneously inoculated tumour cells occurred earlier in irradiated mice than in non-irradiated mice. Proliferative activity of draining lymph node lymphocytes against transplanted tumour cells as well as allogeneic mixed lymphocyte reactions were significantly reduced in irradiated mice compared to non-irradiated mice.

Conclusions:?These results suggest that decreased tumour-specific immune response due to LDR/HD irradiation may enhance tumorigenesis resulting in life-shortening of mice after chronic LDR/HD irradiation.     

Ultrafractionation in A7 human malignant glioma in nude mice

Purpose: Low‐dose hyperradiosensitivity (HRS) has been demonstrated in numerous cell lines in vitro, including a number of radioresistant human malignant glioma cell lines such as A7. The aim of our experiment was to show whether HRS can be exploited by using ultrafractionated irradiation (UF) to improve local control of A7 tumours growing in nude mice. Extrapolation of the in vitro results predict a 3.7‐fold difference in the efficacy of UF compared with conventional fractionation (CF).

Material and methods: Subcutaneuously growing A7 tumours were irradiated either with UF (126 fractions in 6 weeks, 0.4?Gy per fraction) or CF (30 fractions in 6 weeks, 1.68?Gy per fraction). The total dose was 50.4?Gy in both experimental arms. Fractionated irradiations were given under ambient conditions and followed by graded top‐up doses under clamp hypoxia. Endpoints were tumour growth delay and local tumour control 180 days after the end of treatment.

Results: UF resulted in a significant decrease of tumour growth delay and in a significant increase of the top‐up TCD₅₀ compared with CF (40.0?Gy [95% CI 29; 61?Gy] versus 28.3?Gy [24; 35?Gy], p=0.047).

Conclusions: Despite a pronounced HRS phenomenon in vitro, UF was significantly less effective than CF in A7 human malignant glioma in nude mice. These results neither disprove the existence of HRS nor do they exclude a possible clinical value of UF. The findings rather indicate that simplistic extrapolation from results obtained after single‐dose exposure or few fractions in vitro is not sufficient to predict outcome of UF in vivo and that comprehensive evaluation of novel treatment options in animal models continues to be an essential requirement for clinical translation.     

Cell Kinetics and Radiation Biology

Summary

The cell cycle, the growth fraction and cell loss influence the response of cells to radiation in many ways. The variation in radiosensitivity around the cell cycle, and the extent of radiation-induced delay in cell cycle progression have both been clearly demonstrated in vitro. This translates into a variable time of expression of radiation injury in different normal tissues, ranging from a few days in intestine to weeks, months or even years in slowly proliferating tissues like lung, kidney, bladder and spinal cord. The radiosensitivity of tumours, to single doses, is dominated by hypoxic cells which arise from the imbalance between tumour cell production and the proliferation and branching of the blood vessels needed to bring oxygen and other nutrients to each cell.

The response to fractionated radiation schedules is also influenced by the cell kinetic parameters of the cells comprising each tissue or tumour. This is described in terms of repair, redistribution, reoxygenation and repopulation. Slowly cycling cells show much more curved underlying cell survival curves, leading to more dramatic changes with fractionation, dose rate or l.e.t. Rapidly cycling cells redistribute around the cell cycle when the cells in sensitive phases have been killed, and experience less mitotic delay than slowly proliferating cells. Reoxygenation seems more effective in tumours with rapidly cycling cells and high natural cell loss rates. Compensatory repopulation within a treatment schedule may spare skin and mucosa but does not spare slowly proliferating tissues. Furthermore, tumour cell proliferation during fractionated radiotherapy may be an important factor limiting the overall success of treatment.     

The anti-oxidant capacity of tumour glycolysis

Purpose:?In this mini-review data are summarised which provide evidence for the biological and clinical significance of tumour glycolysis and of its relationship to the redox state of cancer cells.

Results:?Malignant transformation is associated with an overexpression of numerous glycolysis-related genes in the vast majority of human cancers. At the same time, glycolytic activity and glycolysis-linked metabolic milieu are often variable between individual tumours which induces large variations in treatment response and aggressiveness. Currently, there is no genetic or proteomic marker for the prediction of the therapeutic response for individual tumours, but the prognostic value of tumour lactate accumulation for the emergence of metastasis, for patient survival and for radioresistance has been documented in a number of studies.

Conclusions:?Transactivation of tumour glycolyis appears to generate a chemically reduced milieu associated with an inhibition of ROS (reactive oxygen species) -mediated fixation of DNA damage and induction of radioresistance. Furthermore, highly glycolytic cells enhance the antioxidant defense via glutathione, and pyruvate can be decarboxylated non-enzymatically upon reducing hydrogen peroxide. The summary of data given here emphasises the importance of further research efforts on the link between carbohydrate metabolism and redox state of cancer cells.     

Heterogeneity of tumour response to combined radiotherapy and EGFR inhibitors: Differences between antibodies and TK inhibitors

Purpose:?Clinical and preclinical data show a wide variability of tumour response to combined inhibition of the Epidermal Growth Factor Receptor (EGFR) and radiotherapy or chemotherapy. Differences are obvious not only between different tumour entities, but also between different combination schedules and different classes of drugs. The underlying reasons are currently not well understood.

Conclusions:?In light of the disappointing results of some phase III trials on combined EGFR tyrosine kinase (TK) inhibition and chemotherapy in non-small-cell lung cancer, but also of some early clinical trials on the triple combination of EGFR inhibitors and radio-chemotherapy, negative interactions between the components of the treatment cannot be ruled out. Also, there is increasing evidence for a differential activity of anti-EGFR antibodies and EGFR-TK inhibitors. Potential reasons are an immunogenic component of the cytotoxic effect of chimeric antibodies, alternative signal transduction pathways leading to acquired resistance against the drugs, different effects on tumour micromilieu or nutritional supply, differences in pharmacokinetics and intratumoural distribution or different effects on cancer stem cells. Clarifying these potential mechanisms will require further preclinical and clinical research effort but could in future enable us to individually tailor the use of molecular targeted drugs in order to fully utilise their high potential in cancer therapy.     

Extinction kinetics for metastatic cancer stem cells

Purpose:?Cancer Stem Cells (CSC) are hypothesised to influence tumour growth through their self-replication, cell loss, and differentiation into growth-limited cell types. A model for the random gain and loss of metastatic CSC is developed to investigate how the balance between these processes might affect metastatic efficiency, tumour involution and treatment response.

Materials and methods:?A stochastic birth-death model for metastasis was constructed for the replication and loss of CSC. The model was extended to account for single and sequential cancer treatments, with CSC repopulation.

Results:?If CSC losses exceed gains, the metastasis would become extinct. The resultant extinction probability was greatest during a period of stochastic susceptibility; treatment could extend, or reestablish, this period.

Conclusion:?Random CSC losses, with ‘seed and soil’ selection, provided a mechanistic explanation for the involution of metastases, as well as for metastatic inefficiency. With such background losses, and the growth limitations of differentiated cells, a metastasis could take years to reach macroscopic size. The susceptibility period could be protracted, providing for a window for therapeutic opportunity. Metastases with a high background CSC loss would be more responsive to treatment than stabler metastases. Modulation of this loss could enhance the efficacy of conventional cancer treatment.     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay,but not local control,after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

Purpose: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments.

Materials and methods: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5?µM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50?mg?kg^?1 body weight orally) or carrier. Tumour growth delay and dose–response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks.

Results: BIBX1382BS blocked radiation‐induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6?Gy (95% confidence interval 55; 73) for irradiation alone and 67.8?Gy (60; 77) for the combined treatment (p=0.5).

Conclusions: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

In vivo measurement of the hypoxia marker EF5 in Shionogi tumours using 19F magnetic resonance spectroscopy

Purpose:?¹⁹F magnetic resonance spectroscopy (MRS) was used to non-invasively detect EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] adducts in the Shionogi tumour model of prostate cancer to evaluate hypoxia.

Material and methods:?¹⁹F MRS signal of EF5 in Shionogi mouse tumours was acquired using a 2 cm diameter solenoid volume coil with a 7.05 T Bruker scanner. MRS signal was observed in mouse tumours longitudinally following intraperitoneal (IP) injection of EF5. Another mouse group was injected intravenously (IV) with EF5, and in vivo MRS signal was obtained two hours after injection. This data was compared with the ex vivo percentage of hypoxic cells present in the corresponding excised tumours, determined by flow cytometry of bound EF5.

Results:?Longitudinal ¹⁹F MRS signal attributable to EF5 began to decline within five hours of EF5 administration. Flow cytometry comparisons yielded an inverse correlation (p-value < 0.006) between the MRS signal and tumour hypoxic cell percentage. The tumours exhibited an average cell viability of 34 ± 26%.

Conclusions:?The results confirmed that MRS of EF5 in mice is an unsuitable technique for the determination of EF5 binding as a measure of tumour hypoxia.     

Radiation induces an antitumour immune response to mouse melanoma

Purpose:?Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response.

Materials and methods:?For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels.

Results:?Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC).

Conclusion:?The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.