Multifractionated Irradiation Research Articles

Radiotherapy alters expression of molecular targets in prostate cancer in a fractionation- and time-dependent manner

The efficacy of molecular targeted therapy depends on expression and enzymatic activity of the target molecules. As radiotherapy modulates gene expression and protein phosphorylation dependent on dose and fractionation, we analyzed the long-term effects of irradiation on the post-radiation efficacy of molecular targeted drugs. We irradiated prostate cancer cells either with a single dose (SD) of 10 Gy x-ray or a multifractionated (MF) regimen with 10 fractions of 1 Gy. Whole genome arrays and reverse phase protein microarrays were used to determine gene expression and protein phosphorylation. Additionally, we evaluated radiation-induced pathway activation with the Ingenuity Pathway Analysis software. To measure cell survival and sensitivity to clinically used molecular targeted drugs, we performed colony formation assays. We found increased activation of several pathways regulating important cell functions such as cell migration and cell survival at 24 h after MF irradiation or at 2 months after SD irradiation. Further, cells which survived a SD of 10 Gy showed a long-term upregulation and increased activity of multiple molecular targets including AKT, IGF-1R, VEGFR2, or MET, while HDAC expression was decreased. In line with this, 10 Gy SD cells were more sensitive to target inhibition with Capivasertib or Ipatasertib (AKTi), BMS-754807 (IGF-1Ri), or Foretinib (VEGFR2/METi), but less sensitive to Panobinostat or Vorinostat (HDACi). In summary, understanding the molecular short- and long-term changes after irradiation can aid in optimizing the efficacy of multimodal radiation oncology in combination with post-irradiation molecularly-targeted drug treatment and improving the outcome of prostate cancer patients.

Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (−) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

53BP1/RIF1 signaling promotes cell survival after multifractionated radiotherapy

Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.

Differential expression of stress and immune response pathway transcripts and miRNAs in normal human endothelial cells subjected to fractionated or single-dose radiation.

Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy × 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed. Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy.

MRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy × 10, 1 Gy × 10, and 2 Gy × 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 × 10-73, 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 × 10-30) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.

A Mathematical Study to Select Fractionation Regimen Based on Physical Dose Distribution and the Linear–Quadratic Model

Hypofractionated irradiation is often used in precise radiotherapy instead of conventional multifractionated irradiation. We propose a novel mathematical method for selecting a hypofractionated or multifractionated irradiation regimen based on physical dose distribution adding to biologic consideration. The linear-quadratic model was used for the radiation effects on tumor and normal tissues, especially organs at risk (OARs). On the basis of the assumption that the OAR receives a fraction of the dose intended for the tumor, the minimization problem for the damage effect on the OAR was treated under the constraint that the radiation effect on the tumor is fixed. For an N-time fractionated irradiation regimen, the constraint of tumor lethality was described by an N-dimensional hypersphere. The total dose of the fractionated irradiations was considered for minimizing the damage effect on the OAR under the hypersphere condition. It was found that the advantage of hypofractionated or multifractionated irradiation therapies depends on the magnitude of the ratio of α/β parameters for the OAR and tumor in the linear-quadratic model and the ratio of the dose for the OAR and tumor. Our mathematical method shows that multifractionated irradiation with a constant dose is better if the ratio of α/β for the OAR and tumor is less than the ratio of the dose for the OAR and tumor, whereas hypofractionated irradiation is better otherwise.

Protective effects of insulin-like growth factor-I on the decrease in myogenic differentiation by ionizing radiation

Purpose: The aim of the work is to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the decrease in myotube formation induced by ionizing radiation.Materials and methods: We induced C2C12 cells to a myogenic linage following X-ray irradiation at 2 and 4 Gy. Myogenic differentiation was estimated using immnocytochemical staining with anti-myosin antibody, and the anti-myosin antibody positive areas, the total number of nuclei, the number of nuclei included in multinucleated myotubes per field, and the myotube formation ratio were analyzed.Results: In the myogenic differentiation in the presence of IGF-1, the decrease in anti-myosin antibody positive areas, the nuclei included in myotubes, and the myotube formation ratio induced by X-ray irradiation at 2 Gy was restored to control levels.Conclusions: The addition of IGF-1 protected against the decrease myotube formation induced by X-ray irradiation at 2 Gy. Since X-ray irradiation at 2 Gy is usually used for multi-fractionated irradiation in radiotherapy, our findings suggest that IGF-1 could be useful to protect against impairment of muscle repair induced by therapeutic dose radiation.

Fractionated Irradiation Augments Inter-Strain Variation of Skin Reactions among Three Strains of Mice

The multifraction regimens commonly used in conventional clinical radiotherapy are largely based on radiobiological experiments. However, no experimental reports on skin reactions focusing on inter-strain differences have displayed clinical relevance to the fractionated dose schedule. In this study, mice of inbred strains A/J, C57BL/6J, and C3H/HeMs were used to reveal inter-strain difference after multifractionated irradiation. Irradiation was performed daily at graded doses of 30-60 Gy total doses, with 10 fractions of 3-6 Gy. Acute skin reactions following irradiation were scored for 50 days after irradiation. Dividing a dose into a number of fractions obviously spared skin damage in the three strains of mice. No mouse exhibited a skin damage score more than 1.5, while single dose irradiation resulted in skin damage scores up to 3. The three different strains, however, showed varying susceptibility to fractionated irradiation within the range under 1.5. C3H/HeMs did not display any skin reaction after irradiation with 40 Gy total dose, while C57BL/6J and A/J demonstrated various skin reactions. Different latent periods of damage were also observed among the strains after irradiation at each dose. Our data suggest that genetic factors cause obvious variations in severity of damage and latent period after fractionated irradiation.

An in vitro evaluation of radiation effects of different fractionated regimens by absolute cell count beads

Recently, whole-body gamma-knife as a kind of radiotherapy equipment, characterized by precision of targeting irradiation, was applied in clinic. However, there is no conventional treatment regimen suitable for application to whole-body gamma-knife as yet. For providing reference to clinic to determine and optimize the treatment regimens for whole-body gamma-knife, a simple and quick bivariate flow cytometric approach was adopted to evaluate the killing effects of differently fractionated regimens of whole-body gamma-knife radiotherapy on the same cells, in which Annexin V-FITC (fluorescein-isothiocyanate) was used for discriminating apoptotic cells, propidium iodide (PI) for necrotic or dead cells and absolute cell count beads for absolute count of remnant surviving cells to examine the absolute killing effect. The results showed that the different survival rates of the same cells treated with different multifractionated irradiation regimens, relevant to clinical trial, might be sensitively, easily and rapidly evaluated by this approach. The evaluation of absolute killing effect, which is important to clinic, is very difficult to realize by a conventional clonogenic assay. For the approach adopted in the experiments, the cells in the treatment courses were not affected by non-treatment factors, such as cell migration, detachment, reattachment, bystander effect and medium factors encountered in a conventional clonogenic assay, and the conditions were more relevantly similar to cells in vivo. This approach might be used as a rapid cell survival assay especially in evaluating the absolute killing effect of a multifractionated treatment regimen, and also applied in the assessment in cytotoxic killing effect, such as chemotherapy.

Assessment of tolerance doses of multifractionated irradiation of lungs using the ellis and LQ models

1. The CTDI values as calculated from the Ellis model depend only on the scheme of dose multifractionation, whereas CTDI values calculated from the LQ model depend also on the number of RT days (CTDI value decreases with the increase in RT time). 2. The CTDI values as calculated from the Ellis model exceed those calculated from the LQ model.

758A damage-repair model for survival after multifractionated irradiation

758A damage-repair model for survival after multifractionated irradiation

COMBINATION THERAPY OF SINGLE OR FRACTIONATED X-RAYS AND SCHIZOPHYLLAN (SPG) FOR MURINE B-16 MELANOMA

B-16 melanoma transplanted into C57BL/6 mice was used to investigate the antitumor effect of Schizophyllan (SPG) combination with local irradiation by X-rays using 4 fractions of 3 Gy or 9 Gy and single fractions of 12, 19 and 26 Gy. In the groups which received a single fraction of 26 Gy and 4 fractions of 9 Gy, significant differences were found between the groups with/without SPG in terms of tumor volume change and lymph node and pulmonary metastases. In terms of survival, significant difference was found only in the 4 fraction of the 9 Gy group. SPG had no antitumor effect nor life-prolonging effect, when it was applied to tumors irradiated with single fractions of 12 and 19 Gy or 4 fractions of 3 Gy, except for some metastasis-suppressing effect in the group given 19 Gy. It seems that SPG has some adjuvant effect only where a limited number of tumor cells remain following rather large dose of X-irradiation. Furthermore, the gain produced by host-mediated immune response augmented by SPG seems to be larger in the 4 fraction group. Therefore, combination radiotherapy using SPG may be advantageous for patients with complete response or a good response after multifractionated irradiation.

Fraction size dependent sparing effects of clinically relevant fractionated radiotherapy protocols on human glioma multicellular spheroids

AbstractClinically relevant X‐ray irradiation protocols were chosen to evaluate the radiation response of human glioma cell lines, A1207 and U‐373 MG, grown as multicellular spheroids. The protocols included single‐dose fraction (SF), conventional fractionation (CF; 2 Gy per fraction, 24 hr interval), hyperfractionation (HF; 1.0 Gy per fraction, 12 hr interval), and accelerated hyperfractionation (AHF; 1.5 Gy per fraction, 6 hr interval). Spheroids from both cell lines are clearly more refractory to the cytotoxic effects of multifractionated irradiation than the single‐dose irradiation. Dose‐sparing effects of CF or HF were comparable in the two cell ilnes. More detailed studies of A1207 spheroids showing sparing effects are fraction size dependent. The smaller the dose size per fraction, the larger the sparing effect. In order to achieve the same biological effect using multifractionated irradiation, compared to single dose, a higher dose is needed to compensate for sublethal damage repair which occurred in the fraction intervals. The relative doses required for achieving isoeffect as SF were 145%, 160%, and 169% for CF, AHF, and HF, respectively, when a total dose of 10 Gy was administered. The isoeffective doses for AHF and HF compared to CF were 113% and 126%, respectively. There was also an increase in sparing effect with increasing total dose administered as multifraction. Our results suggest that in the design of multifractionated radiotherapy regimens, fraction size dependent sparing effects related to dose per fraction and total dose on human gliomas should be taken into consideration to optimize therapeutic outcome. © 1994 Wiley‐Liss, Inc.

Bone growth retardation induced by single and multifractionated irradiation

The right hind legs of 12- to 14-week-old mice were exposed to single and multifractionated courses of gamma-ray irradiation. The recovery curves, time-dose relationships, and fraction number-dose relationships were analyzed using femoral growth retardation as an endpoint. The bones grew 2 mm, about a 10% increase over their original lengths, in 12 months; and 63% and 85% in volume in 6 and 12 months, respectively. The ratio of the volume increase of the irradiated right leg per unit time, the actual growth, to the volume increase of the non-irradiated left leg, the potential growth, was calculated for each mouse. The doses necessary to produce a given degree of bone growth retardation in 50% of the tested animals were calculated for each treatment schedule. The isoeffect doses for a given degree of bone growth retardation after 2 and 4 equal fractionation exposure increased (peak at one day-interval) and decreased (trough at 2-day interval), and later increased additionally with increasing time intervals between doses.

Radiosensitivity of pre-irradiated mouse skin to second courses of single and multi-fractionated irradiation--skin shrinkage.

The hind legs of mice were re-irradiated with various gamma-ray doses in single or multi-fractionated exposures 6 or 12 months after conditioning irradiation was administered using a variety of treatment schedules. Dose-response relationships were evaluated to ascertain "residual injuries", permissible doses during the second treatments. Fifty to 60 days after the second treatments, the degrees of shrinkage of the skin on the dorsal aspects of the hind legs were measured and used as endpoints. The residual injuries were affected by the biologically effective doses, or by the treatment schedules of not only the first, but also the second exposure. The higher the biologically effective doses, the greater were the residual injuries. Compared to the previously untreated skin, the pre-irradiated skin was relatively radioresistant to the second course of treatments, regardless of the treatment schedule used. The response of the pre-treated skin to a given test dose (25 or 40 Gy) lessened with increasing dose for each treatment schedule used during the first treatment course.

Epilation in mice after single and multifractionated irradiation

The response of the resting (fully formed) hair follicle to irradiation was studied using an arbitrary 6 unit scale of epilation as an endpoint. Dose-response curves for single and multifractionated irradiations were analyzed in terms of the dose that gave a certain response in 50% of the mice (HRD 50). HRD 50 values increased with decrease in dose per fraction even when changing from 1.6 Gy to 1.15 Gy per fraction. The plots of the reciprocal of isoeffective doses versus dose per fraction are nonlinear suggesting either inappropriateness of the linear-quadratic model over the whole range of doses, or incompleteness of repair given as they were, at 3-h intervals. Allowing for incomplete repair [13], estimates for the alpha/beta ratio were 3.1 Gy and 1.7 Gy for the complete data set or for doses less than 7 Gy, respectively. The steep slope of the isoeffect curve plotting total dose versus dose per fraction was comparable to late responding normal tissues like lung, kidney and spinal cord. Such a response is consistent with slow proliferation of the matrix cells of resting follicles [10]. The same animals were kept to assess the effect of dose fractionation on lethal injury to the lung [17]. Since epilation occurs well before death from lung injury, the data for the two responses were correlated to determine whether epilation might help in predicting the probability of the later development of lung injury: no association was found.

Response of previously irradiated mouse skin to a second course of irradiation: early skin reaction and skin shrinkage

The responses of previously irradiated hind legs of mice to second courses of gamma ray irradiation were studied using early skin reaction and skin shrinkage as end points. The hind legs of mice were treated with various doses in single and multifractionated irradiation at one-day intervals, 12 months after a variety of doses in various treatment schedules and in a single exposure, respectively. The re-irradiated skin was relatively radioresistant compared to the previously non-irradiated skin, depending on the skin site used and the end point used to evaluate the skin's radiation sensitivity. Early skin reactions that occurred after test doses administered to pre-irradiated skin developed sooner, and remained longer, than skin reactions that developed in the previously non-irradiated controls, regardless of treatment regimens. The degree of early skin reaction to the test dose was greater in the pre-irradiated skin, when the test dose was relatively small, and lower, when the test dose was relatively large, regardless of treatment regimens with one exception: the ventral aspect of the mouse's hind leg responded more to multifractionated test doses in the pre-irradiated skin. The degree of skin shrinkage, assessed 50–60 days after the test dose, was less in the pre-irradiated skin than in the previously non-treated controls, for each dose in each test treatment schedule. The amount of skin shrinkage, resulting from a given test dose (25 or 40 Gy), decreased with increasing dose during the first course of each treatment schedule. The degree of skin shrinkage caused by the first and second exposure, however, increased when the total dose of the first course and the test exposure was increased.

Repair and repopulation in mouse skin during multifractionated irradiation.

To study the recovery from sublethal damage and the repopulation kinetics of skin during multifractionated exposures, mouse legs were exposed to 137Cs γ rays. Levels of skin shrinkage were measured 50 to 70 days after either single or multiple fractions (up to 32) with various intervals of time between doses. The higher the dose per fraction, the later the surviving cells started to repopulate after irradiation. When the dose per fraction was 370 to 490 rads, and the number of fractions 8 to 32, the regeneration rate per day was almost constant from day 7 to day 31 after the first exposure. It was calculated that the number of skin stem cells increased an average of 1.3 fold per day. When the dose per fraction was more than 1000 rads, the average repopulation rate between day 15 and day 31 was lower than that between day 6 and day 15.

Late effect in mouse skin following single and multifractionated irradiation

The time course of mouse skin contraction after single-dose and multifractionated gamma-irradiation using various time intervals was studied until day 540 after irradiation. Following an initial shrinkage of the skin, a period of relaxation was observed 20 to 30 days after irradiation. Thereafter there was a dose dependent period before a second wave of contraction began, which was earlier and more severe with increasing dose. Recovery from sub-effective injury, as measured by increase in dose for an isoeffect, was not complete within 12 hours. Constant exponents for N and T in an isoeffect formula are not appropriate for the data presented.

Single dose response curves of normal cells in situ.

Analysis of data on the response of normal tissues (skin, lung, spinal cord, intestine, testis) to single and multifractionated irradiation schedules revealed the characteristics of the single dose survival curves of target cells responsible for skin, lung, and spinal cord reactions, and of stem cells of colonic, jejunal, gastric mucosae, and testis exposed in situ to relatively low doses of x- or gamma-rays. The single dose survival curves of these cells seem to be complex (unlike those of cells studied in vitro), that is, simply exponential in the initial portion up to approximately 250 rads, and downward-bending thereafter in a curve which includes one or two linear portions on a semilogarithmic scale.