Metastasis of breast cancer cells to the bone, lung, and lymph nodes promotes resistance to ionizing radiation.

BACKGROUND: Metastasis represents the leading cause of breast cancer deaths, necessitating strategies for its treatment. Although radiotherapy is employed for both primary and metastatic breast cancers, the difference in their ionizing radiation response remains incompletely understood. This study is the first to compare the radioresponse of a breast cancer cell line with its metastatic variants and report that such metastatic variants are more radioresistant. MATERIALS AND METHODS: A luciferase expressing cell line was established from human basal-like breast adenocarcinoma MDA-MB-231 and underwent in vivo selections, whereby a cycle of inoculations into the left cardiac ventricle or the mammary fat pad of athymic nude mice, isolation of metastases to the bone, lung and lymph nodes visualized with bioluminescence imaging, and expansion of obtained cells was repeated twice or three times. The established metastatic cell lines were assessed for cell proliferation, wound healing, invasion, clonogenic survival, and apoptosis. RESULTS: The established metastatic cell lines possessed an increased proliferative potential in vivo and were more chemotactic, invasive, and resistant to X­ray-induced clonogenic inactivation and apoptosis in vitro. CONCLUSION: Breast cancer metastasis to the bone, lung, and lymph nodes promotes radioresistance.

Optimized workflow and imaging protocols for whole-body oncologic PET/MRI.

Although PET/MRI has the advantages of a simultaneous acquisition of PET and MRI, high soft-tissue contrast of the MRI images, and reduction of radiation exposure, its low profitability and long acquisition time are significant problems in clinical settings. Thus, MRI protocols that meet oncological purposes need to be used in order to reduce examination time while securing detectability. Currently, half-Fourier acquisition single-shot turbo spin echo and 3D-T1 volumetric interpolated breath-hold examination may be the most commonly used sequences for whole-body imaging due to their shorter acquisition time and higher diagnostic accuracy. Although there have been several reports that adding diffusion weighted image (DWI) to PET/MRI protocol has had no effect on tumor detection to date, in cases of liver, kidney, bladder, and prostate cancer, the use of DWI may be beneficial in detecting lesions. Another possible option is to scan each region with different MRI sequences instead of scanning the whole body using one sequence continuously. We herein report a workflow and imaging protocols for whole-body oncologic PET/MRI using an integrated system in the clinical routine, designed for the detection, for example by cancer screening, of metastatic lesions, in order to help future users optimize their workflow and imaging protocols.

Comparison of integrated whole-body PET/MR and PET/CT: Is PET/MR alternative to PET/CT in routine clinical oncology?

PURPOSE: To compare the diagnostic accuracy of whole-body PET/CT and integrated PET/MR in relation to the total scan time durations. METHODS: One hundred and twenty-three (123) patients (40 males and 83 females; mean age 59.6 years; range 20-83 years) with confirmed primary cancer and clinical suspicion of metastatic disease underwent whole-body 18F-FDG-PET/CT and 18F-FDG-PET/MR. Data acquisition was done after intravenous administration of 110-301 MBq radioactivity of 18F-FDG, and PET/MR data were acquired after the PET/CT data acquisition. The mean uptake times for PET/CT and PET/MR acquisition were 68.0 ± 8.0 and 98.0 ± 14 min, respectively. Total scan time was 20.0 and 25.0 min for whole-body PET/CT and PET/MR imaging. RESULTS: The reconstructed PET/CT and PET/MR data detected 333/355 (93.8 %) common lesions in 111/123 (90.2 %) patients. PET/CT and PET/MR alone detected 348/355 and 340/355 lesions, respectively. No significant (p = 0.08) difference was observed for the overall detection efficiency between the two techniques. On the other hand, a significant difference was observed between the two techniques for the detection of lung (p = 0.003) and cerebrospinal (p = 0.007) lesions. The 15 lesions identified by PET/CT only included 8 lung, 3 lymph nodes, 2 bone, and 1 each of peritoneal and adrenal gland lesions. On the other hand, 7 (6 brain metastatic lesions and 1 bone lesion) were identified by PET/MR only. CONCLUSION: Integrated PET/MR is a feasible whole-body imaging modality and may score better than PET/CT for the detection of brain metastases. To further prove diagnostic utility, this technique requires further clinical validation.

A branching process model for the analysis of abortive colony size distributions in carbon ion-irradiated normal human fibroblasts.

A single cell can form a colony, and ionizing irradiation has long been known to reduce such a cellular clonogenic potential. Analysis of abortive colonies unable to continue to grow should provide important information on the reproductive cell death (RCD) following irradiation. Our previous analysis with a branching process model showed that the RCD in normal human fibroblasts can persist over 16 generations following irradiation with low linear energy transfer (LET) γ-rays. Here we further set out to evaluate the RCD persistency in abortive colonies arising from normal human fibroblasts exposed to high-LET carbon ions (18.3 MeV/u, 108 keV/µm). We found that the abortive colony size distribution determined by biological experiments follows a linear relationship on the log-log plot, and that the Monte Carlo simulation using the RCD probability estimated from such a linear relationship well simulates the experimentally determined surviving fraction and the relative biological effectiveness (RBE). We identified the short-term phase and long-term phase for the persistent RCD following carbon-ion irradiation, which were similar to those previously identified following γ-irradiation. Taken together, our results suggest that subsequent secondary or tertiary colony formation would be invaluable for understanding the long-lasting RCD. All together, our framework for analysis with a branching process model and a colony formation assay is applicable to determination of cellular responses to low- and high-LET radiation, and suggests that the long-lasting RCD is a pivotal determinant of the surviving fraction and the RBE.

A framework for analysis of abortive colony size distributions using a model of branching processes in irradiated normal human fibroblasts.

BACKGROUND: Clonogenicity gives important information about the cellular reproductive potential following ionizing irradiation, but an abortive colony that fails to continue to grow remains poorly characterized. It was recently reported that the fraction of abortive colonies increases with increasing dose. Thus, we set out to investigate the production kinetics of abortive colonies using a model of branching processes. METHODOLOGY/PRINCIPAL FINDINGS: We firstly plotted the experimentally determined colony size distribution of abortive colonies in irradiated normal human fibroblasts, and found the linear relationship on the log-linear or log-log plot. By applying the simple model of branching processes to the linear relationship, we found the persistent reproductive cell death (RCD) over several generations following irradiation. To verify the estimated probability of RCD, abortive colony size distribution (≤ 15 cells) and the surviving fraction were simulated by the Monte Carlo computational approach for colony expansion. Parameters estimated from the log-log fit demonstrated the good performance in both simulations than those from the log-linear fit. Radiation-induced RCD, i.e. excess probability, lasted over 16 generations and mainly consisted of two components in the early (<3 generations) and late phases. Intriguingly, the survival curve was sensitive to the excess probability over 5 generations, whereas abortive colony size distribution was robust against it. These results suggest that, whereas short-term RCD is critical to the abortive colony size distribution, long-lasting RCD is important for the dose response of the surviving fraction. CONCLUSIONS/SIGNIFICANCE: Our present model provides a single framework for understanding the behavior of primary cell colonies in culture following irradiation.

The suppression of hypoxia-inducible factor and vascular endothelial growth factor by siRNA does not affect the radiation sensitivity of multicellular tumor spheroids.

BACKGROUND: The hypoxic microenvironment is closely associated with the radiation resistance of tumor cells. Hypoxia induces several genes such as hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) to promote tumor cell growth and survival. The up-regulated expression levels of HIF-1 and VEGF in tumor cells also correlate with their resistance to radiation, suggesting that these genes are potential therapeutic targets for strategies designed to enhance radiation effects. To further investigate this possibility, we investigated the effects of suppressing these genes upon the radiation sensitivity of cancer cells. We conducted these experiments using multicellular spheroids as a three-dimensional in vitro tumor model and RNA interference as the method of gene suppression. MATERIAL AND METHODS: SQ5 human lung carcinoma cells were treated with HIF-1/VEGF siRNA and/or radiation. Reversed transfection methods were employed for the spheroids. Gene expression was analyzed using quantitative RT-PCR and western blotting. Cell toxicity was qualified by colony formation assay. RESULTS: Compared with monolayer cells, spheroids showed up-regulated expression of HIF-1 and increased radiation resistance. Hypoxic conditions elevated the expression of HIF-1 and VEGF and enhanced the surviving fraction of spheroids after exposure to radiation. However, when the expression of HIF-1 and VEGF was down-regulated by transfection of targeting siRNA, this did not influence the cytotoxic effects of the radiation under either normoxic or hypoxic conditions. CONCLUSIONS: We have established a method to transfect siRNA into spheroid cells. Our current data indicate that the functions of HIF-1 or VEGF are independent of radiation sensitivity in spheroids under either normoxic or hypoxic conditions.

Quantification of circulating cell-free DNA in the plasma of cancer patients during radiation therapy.

Cell-free plasma DNA is elevated in cancer patients and decreases in response to effective treatments. Consequently, these nucleic acids have potential as new tumor markers. In our current study, we investigated whether the plasma DNA concentrations in patients with cancer are altered during the course of radiation therapy. To first determine the origin of cell-free plasma DNA, plasma samples from mice bearing transplanted human tumors were analyzed for human-specific and mouse-specific cell-free DNA. Human-specific DNA was detectable only in plasma from tumor-bearing mice. However, mouse-specific plasma DNA was significantly higher in tumor-bearing mice than in normal mice, suggesting that cell-free plasma DNA originated from both tumor and normal cells. We measured the total cell-free plasma DNA levels by quantitative polymerase chain reaction in 15 cancer patients undergoing radiation therapy and compared these values with healthy control subjects. The cancer patients showed higher pretreatment plasma DNA concentrations than the healthy controls. Eleven of these patients showed a transient increase of up to eightfold in their cell-free plasma DNA concentrations during the first or second week of radiation therapy, followed by decreasing concentrations toward the end of treatment. In two other cancer patients, the cell-free plasma DNA concentrations only decreased over the course of the treatment. The total cell-free plasma DNA levels in cancer patients thus show dynamic changes associated with the progression of radiation therapy. Additional prospective studies will be required to elucidate the potential clinical utility and biological implications of dynamic changes in cell-free plasma DNA during radiation therapy.

The small-molecule Bcl-2 inhibitor HA14-1 sensitizes cervical cancer cells, but not normal fibroblasts, to heavy-ion radiation.

This is the first study to demonstrate that the small-molecule Bcl-2 inhibitor HA14-1 renders human cervical cancer cells and their Bcl-2 overexpressing radioresistant counterparts, but not normal fibroblasts, more susceptible to heavy ions. Thus, Bcl-2 may be an attractive target for improving the efficacy of heavy-ion therapy.

Flavopiridol potentiates the cytotoxic effects of radiation in radioresistant tumor cells in which p53 is mutated or Bcl-2 is overexpressed.

PURPOSE: Loss of the cell-cycle regulatory protein p53 or overexpression of the antiapoptotic protein Bcl-2 is associated with resistance to radiation in several types of cancer cells. Flavopiridol, a synthetic flavone, inhibits the growth of malignant tumors cells in vitro and in vivo through multiple mechanisms. The purpose of the present study is to clarify whether flavopiridol enhances the cytotoxic effects of radiation in tumor cells that contain dysfunction p53 or that overexpress Bcl-2. METHODS AND MATERIALS: A human glioma cell line (A172/mp53) stably transfected with a plasmid containing mutated p53 and a human cervical cancer cell line (HeLa/bcl-2) transfected with a bcl-2 expression plasmid were used. Cells were incubated with flavopiridol for 24 h after radiation, and then cell viability was determined by a colony formation assay. Foci of phosphorylated histone H2AX were also evaluated as a sensitive indicator of DNA double-strand breaks. RESULTS: Compared with the parental wild-type cells, both transfected cell lines were more resistant to radiation. Post-treatment with flavopiridol increased the cytotoxic effects of radiation in both transfected cell lines, but not in their parental wild-type cell lines. Post-treatment with flavopiridol inhibited sublethal damage repair as well as the repair of DNA double-strand breaks in response to radiation. CONCLUSIONS: Flavopiridol enhanced the cytotoxic effect of radiation in radioresistant tumor cells that harbor p53 dysfunction or Bcl-2 overexpression. A combination treatment of flavopiridol with radiation has the potential to conquer the radioresistance of malignant tumors induced by the genetic alteration of p53 or bcl-2.

The survival of heavy ion-irradiated Bcl-2 overexpressing radioresistant tumor cells and their progeny.

Here, we investigated the cell killing effectiveness of heavy-ion radiation in Bcl-2 overexpressing radioresistant tumor cells. First, irradiated cells underwent primary colony formation. Radioresistance decreased with increasing linear energy transfer (LET), indicating that heavy ions may be a promising therapeutic modality for Bcl-2 overexpressing tumors. Second, cells in primary colonies were reseeded for secondary colony formation. The incidence of delayed reproductive death increased with LET irrespective of Bcl-2 overexpression, suggesting that Bcl-2 overexpression may not facilitate heavy ion-induced genomic instability.

Energetic heavy ions overcome tumor radioresistance caused by overexpression of Bcl-2.

BACKGROUND AND PURPOSE: Overexpression of Bcl-2 is frequent in human cancers and has been associated with radioresistance. Here we investigated the potential impact of heavy ions on Bcl-2 overexpressing tumors. MATERIALS AND METHODS: Bcl-2 cells (Bcl-2 overexpressing HeLa cells) and Neo cells (neomycin resistant gene-expressing HeLa cells) exposed to gamma-rays or heavy ions were assessed for the clonogenic survival, apoptosis and cell cycle distribution. RESULTS: Whereas Bcl-2 cells were more resistant to gamma-rays (0.2keV/microm) and helium ions (16.2keV/microm) than Neo cells, heavy ions (76.3-1610keV/microm) yielded similar survival regardless of Bcl-2 overexpression. Carbon ions (108keV/microm) decreased the difference in the apoptotic incidence between Bcl-2 and Neo cells, and prolonged G(2)/M arrest that occurred more extensively in Bcl-2 cells than in Neo cells. CONCLUSIONS: High-LET heavy ions overcome tumor radioresistance caused by Bcl-2 overexpression, which may be explained at least in part by the enhanced apoptotic response and prolonged G(2)/M arrest. Thus, heavy-ion therapy may be a promising modality for Bcl-2 overexpressing radioresistant tumors.

Accelerator mass spectrometry analysis of background (14)C-concentrations in human blood: aiming at reference data for further microdosing studies.

OBJECTIVE: Phase 0 clinical studies, which are known as microdose trials, are expected to promote drug development and reduce development costs. The accelerator mass spectrometry (AMS) system is expected to play an important role in the microdosing tests, as it is a highly sensitive measurement system that can be used to determine the drug concentrations in these tests. Using the AMS system, we measured the background (14)C-concentration in human blood and evaluated the data for use as a reference in microdose studies that administer (14)C-labeled compounds in humans. METHODS: Blood samples of five healthy Japanese volunteers (three men, two women, median age 40.4 +/- 9.8 years) were collected around the same time and just prior to when the subjects ate a meal (between 12:00 noon and 2:00 pm). Centrifugal separations of blood that was allowed to clot and the plasma were performed at 503 g for 2 min at 4 degrees C. Background (14)C-concentration for each of the samples was measured using the AMS system. The Institute of Accelerator Analysis, which is the first contract research organization in Japan that is capable of providing AMS analysis services for carbon dating and bioanalysis work, performed the AMS analysis. RESULTS: The mean (14)C-concentration in blood was 1.613 +/- 0.125 dpm/ml (men 1.668 +/- 0.114 dpm/ml, women 1.514 +/- 0.076 dpm/ml), in clots 2.373 +/- 0.087 dpm/ml (men 2.381 +/- 0.101 dpm/ml, women 2.357 +/- 0.060 dpm/ ml), and in plasma 0.648 +/- 0.049 dpm/ml (men 0.647 +/- 0.059 dpm/ml, women 0.649 +/- 0.032 dpm/ml). The coefficient variation (CV) for blood was 7.8% (men 6.9%, women 5.0%), for clots 3.7% (men 4.3%, women 2.5%), and for plasma 7.6% (men 9.1%, women 4.9%). The (14)C-concentrations of the clot and blood were higher than those of plasma. The (14)C-concentrations in the blood and plasma were slightly different between individuals when compared with the values for the clot, although the differences were quite small, with a CV value less than 7.8%. CONCLUSIONS: Even though the (14)C-concentration differed only slightly between individuals, (14)C-concentrations of the clot and blood were higher than those of the plasma. Therefore, the variation and difference of the background data for blood and plasma might be of use as a reference for microdosing test evaluations.

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts.

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.

Vulnerability of feline T-lymphocytes to charged particles.

An analysis of ionizing radiation-induced damage in peripheral lymphocytes has been employed to predict the prognosis of radiotherapy in terms of toxicity in normal tissues. Therefore, understanding the sensitivity of lymphocytes to high linear energy transfer (LET)-charged particles would be indispensable for utilizing charged particle therapy in veterinary medicine. However, the availability of such information is very limited. This study aimed to compare the radiosensitivity of feline T lymphocytes to gamma-rays (0.2 keV/microm) and 4 different types of charged particles with LET values ranging from 2.8 to 114 keV/microm. It was observed that the relative biological effectiveness, inactivation cross-section, and isodose-induced apoptosis increased in an LET-dependent manner. On the other hand, no difference in apoptosis frequency was observed in the cells exposed to an isosurvival dose of all the radiation types tested. This is the first study that demonstrates the LET dependence of cell killing and apoptosis induction in feline T lymphocytes. Our results suggest that lymphocytes can be effectively used to predict the prognosis of charged-particle therapy in cat patients.

Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects.

A rapidly growing body of experimental evidence indicates that ionizing radiation induces biological effects in non-irradiated bystander cells that have received signals from adjacent or distant irradiated cells. This phenomenon, which has been termed the ionizing radiation-induced bystander effect, challenges the long-standing paradigm that radiation traversal through the nucleus of a cell is a prerequisite to elicit genetic damage or a biological response. Bystander effects have been observed in a number of experimental systems, and cells whose nucleus or cytoplasm is irradiated exert bystander responses. Bystander cells manifest a multitude of biological consequences, such as genetic and epigenetic changes, alterations in gene expression, activation of signal transduction pathways, and delayed effects in their progeny. Several mediating mechanisms have been proposed. These involve gap junction-mediated intercellular communication, secreted soluble factors, oxidative metabolism, plasma membrane-bound lipid rafts, and calcium fluxes. This paper reviews briefly the current knowledge of the bystander effect with a focus on proposed mechanisms. The potential benefit of bystander effects to cancer radiotherapy will also be discussed.

Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 overexpressing radioresistant tumor cells.

PURPOSE: Bcl-2, an inhibitor of apoptosis frequently shows elevated expression in human tumors, thus resulting in resistance to radiation therapy. Therefore, inhibiting Bcl-2 function may enhance the radiosensitivity of tumor cells. Tetrocarcin A (TC-A) and bcl-2 antisense oligonucleotides exhibit antitumor activity by inhibiting Bcl-2 function and transcription, respectively. We investigated whether these antitumor agents would enhance the cytotoxic effects of radiation in tumor cells overexpressing Bcl-2. METHODS AND MATERIALS: We used HeLa/bcl-2 cells, a stable Bcl-2-expressing cell line derived from wild-type HeLa (HeLa/wt) cells. Cells were incubated with TC-A and bcl-2 antisense oligonucleotides for 24 h after irradiation, and cell viability was then determined. Apoptotic cells were quantified by flow cytometric assay. RESULTS: The HeLa/bcl-2 cells were more resistant to radiation than HeLa/wt cells. At concentrations that are not inherently cytotoxic, both TC-A and bcl-2 antisense oligonucleotides increased the cytotoxic effects of radiation in HeLa/bcl-2 cells, but not in HeLa/wt cells. However, in HeLa/bcl-2 cells, additional treatment with TC-A in combination with radiation did not significantly increase apoptosis. CONCLUSIONS: The present results suggest that TC-A and bcl-2 antisense oligonucleotides reduce radioresistance of tumor cells overexpressing Bcl-2. Therefore, a combination of radiotherapy and Bcl-2 inhibitors may prove to be a useful therapeutic approach for treating tumors that overexpress Bcl-2.