Volume-dependent dose-response of the intestinal stem cell niche and lymphoid tissue.

BACKGROUND AND PURPOSE: Plasticity of the intestinal stem cell compartment in response to radiation injury is regulated by a stem cell niche. We present here the first experimental observations of a dose-volume effect of the intestinal stem cell niche and of the solitary intestinal lymphoid tissues (SILT). MATERIALS AND METHODS: Regeneration of intestinal crypts in mice was studied following irradiation of millimetre-size jejunal sections with single doses of 6 to 24 Gy and compared to total body irradiation (TBI). The statistical distribution of cells per crypt was scored and regressed to a biomathematical model. The number of SILTs was scored for different doses and field sizes and crypt regeneration was correlated with SILT proximity. RESULTS: We observed a differential dose-response of the intestinal stem cell niche at the centres of the irradiated sections, but only for field sizes below 10 mm. Irradiation of 5 mm jejunum results in an increase in crypt survival by up to an order of magnitude, compared to TBI. Distributions of cell-per-crypt numbers and comparison to biomathematical modelling suggest that these observations stem from a field size-dependent regeneration rate. The density of SILTs also exhibits a volume-dependent dose-response and increased crypt survival correlates with a proximity to SILTs. CONCLUSION: Our findings present the first observation of a field-size dependent dose-response of the intestinal stem cell niche. Its regeneration process does apparently not rely on distant radiation-sensitive resources of the organism, such as the bone marrow. Yet, our observations suggest that the niche interacts with intact tissue in millimetres distance, leading to faster crypt regeneration. The field-size dependent dose-response of SILTs posits a role of the immune system on the dose-volume effect.

Reserve stem cell population in intestinal crypts found to be consistently small by analysis of in vivo clonogenic assays with a biomathematical dynamic model.

BACKGROUND AND PURPOSE: The high plasticity of the intestinal epithelium is maintained by a resilient reserve stem cell population, whose extent and biology are a matter of ongoing debate. The in vivo clonogenic assay (IVCA), presents a well established and efficient analysis of radiation insult to the intestinal crypts. However, we found that inadequate mathematical analysis over the last four decades led to systematic errors and contradictory results in estimates of radio-sensitivity and size of the reserve stem cell pool. MATERIAL AND METHODS: We devised a refinement of the IVCA via development of a biomathematical model that delivers a full statistical dynamic description of epithelial radiation injury and subsequent regeneration. We validated the model against cellular and crypt distribution statistics obtained from IVCA experiments and through systematic re-analysis of experimental data from 27 publications. RESULTS: A full dynamic description of the evolution of stem cell niche population statistics is obtained. A systematic re-analysis reveals a consistent clonogenic content of the crypt of 31±6 cells. The stem cell reserve manifests to be, contrary to prior predictions, radio-resistant: α=(0.22±0.04) Gy-1. CONCLUSION: We established a precision tool for the quantitative analysis of radiation insult to the intestinal crypts, which we employ to show that the reserve stem cell population is small, radio-resistant, and remarkably immutable against a large variety of interventions. The increased resolution of the model allows not only a reduction of the number of animals by about 75%, but also to quantify experimentally the influence of additional agents on damage and on regeneration of the stem cell niche.

Different dose rate-dependent responses of human melanoma cells and fibroblasts to low dose fast neutrons.

PURPOSE: To analyze the dose rate influence in hyper-radiosensitivity (HRS) of human melanoma cells to very low doses of fast neutrons and to compare to the behaviour of normal human skin fibroblasts. MATERIALS AND METHODS: We explored different neutron dose rates as well as possible implication of DNA double-strand breaks (DSB), apoptosis, and energy-provider adenosine-triphosphate (ATP) levels during HRS. RESULTS: HRS in melanoma cells appears only at a very low dose rate (VLDR), while a high dose rate (HDR) induces an initial cell-radioresistance (ICRR). HRS does not seem to be due either to DSB or to apoptosis. Both phenomena (HRS and ICRR) appear to be related to ATP availability for triggering cell repair. Fibroblast survival after neutron irradiation is also dose rate-dependent but without HRS. CONCLUSIONS: Melanoma cells or fibroblasts exert their own survival behaviour at very low doses of neutrons, suggesting that in some cases there is a differential between cancer and normal cells radiation responses. Only the survival of fibroblasts at HDR fits the linear no-threshold model. This new insight into human cell responses to very low doses of neutrons, concerns natural radiations, surroundings of accelerators, proton-therapy devices, flights at high altitude. Furthermore, ATP inhibitors could increase HRS during high-linear energy transfer (high-LET) irradiation.

Combination of the mTOR inhibitor RAD001 with temozolomide and radiation effectively inhibits the growth of glioblastoma cells in culture.

The present in vitro study aimed to assess the effects of combining the mTOR inhibitor RAD001 and temozolomide (TMZ) together with irradiation by either low-linear energy transfer (LET) radiation (γ-rays) or high-LET radiation (fast neutrons) on the growth and cell survival of the human glioblastoma cell line U-87. We observed a strong decrease in cell proliferation along with a concomitant increase in cell death as a function of the radiation dose. As expected, high-LET radiation was more effective and induced more sustained damage to DNA than low-LET radiation. While RAD001 in association with TMZ induced autophagic cell death, additional combination with either type of radiation did not further increase its occurrence. On the contrary, apoptosis remained at a low level in all experimental groups.

The mTOR inhibitor RAD001 augments radiation-induced growth inhibition in a hepatocellular carcinoma cell line by increasing autophagy.

Treatment of hepatocellular carcinoma (HCC) is a major concern for physicians as its response to chemotherapy and radiotherapy remains generally poor, due, in part, to intrinsic resistance to either form of treatment. We previously reported that an irradiation with fast neutrons, which are high-linear energy transfer (LET) particles, massively induced autophagic cell death in the human HCC SK-Hep1 cell line. In the present study, we tested the capacity of the mammalian target of rapamycin (mTOR) inhibitor RAD001 to augment the cytotoxicity of low and high-LET radiation in these cells. As mTOR is a key component in a series of pathways involved in tumor growth and development, it represents a potential molecular target for cancer treatment. Results indicate that RAD001, at clinically relevant nanomolar concentrations, enhances the efficacy of both high- and low-LET radiation in SK-Hep1 cells, and that the induction of autophagy may account for this effect. However, fast neutrons were found to be more efficient at reducing tumor cell growth than low-LET radiation.

Cell death after high-LET irradiation in orthotopic human hepatocellular carcinoma in vivo.

Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and a major health problem since the choice of treatment is limited due to chemo- and radio-resistance. It was previously reported that high linear energy transfer (LET) radiation induced massive autophagic cell death in the human HCC SK-Hep1 cell line in vitro. This study analyzed the effects of high-LET radiation on the same HCC tumor model, orthotopically transplanted into nude mice. For this purpose, after surgical xenograft in the liver, animals were irradiated with fast neutrons and cell death occurring in the tumors was assessed with various techniques, including electron microscopy and probe-based confocal laser endomicroscopy. Results indicate that considerable autophagy and only limited apoptosis took place in the tumor xenografts after high-LET irradiation. These data confirm the previous in vitro results, suggesting that autophagy may act as a predominant mode of cell death in the efficacy of high-LET radiation.

In vivo radiobiological characterization of proton beam at the National Cancer Center in Korea: effect of the Chk2 mutation.

PURPOSE: The relative biological effectiveness (RBE) in the presence or absence of CHK2 was estimated at the Korean National Cancer Center Proton Therapy Center (NCCPTC). METHODS AND MATERIALS: The proton beam was fixed at 210 MeV with 6-cm spread-out Bragg peaks (SOBPs) because this is expected to be the most frequently used clinical setting. X-rays were obtained using a 6-MV conventional linear accelerator. The RBE was estimated from the survival of jejunal crypt in C3H/He and Chk2(-/-) mice. RESULTS: The estimated RBEs of the NCCPTC at the middle of the SOBP were 1.10 and 1.05 in the presence and absence of CHK2, respectively. The doses that reduced the number of regenerated crypt per jejunal circumference to 20 (D(20)) in C3H/He mice were 14.8 Gy (95% confidence interval [CI], 13.7-15.9) for X-rays and 13.5 Gy (95% CI, 14.5-15.5) for protons. By contrast, the doses of D(20) in Chk2(-/-) mice were 15.7 Gy (95% CI, 15.0-16.4) and 14.9 Gy (95% CI, 14.0-15.8) for X-rays and protons, respectively. CONCLUSIONS: The RBE of the NCCPTC is clearly within the range of RBEs determined at other facilities and is consistent with the generic RBE value of 1.10 for 150- to 250-MeV beams. The mutation of Chk2 gave rise to radioresistance but exhibited similar RBE.

Pharmacological enhancement of autophagy induced in a hepatocellular carcinoma cell line by high-LET radiation.

The aim of the present study was to determine the cytotoxic consequences of high-linear energy transfer (LET) irradiation in the presence of oxaliplatin on hepatocellular carcinoma (HCC) cells in vitro. We attempted to correlate the induction of apoptosis and autophagy with the formation of DNA double-strand breaks (DSBs). SK-Hep1 cells were irradiated by 65 MeV neutrons in the presence of oxaliplatin and/or the poly(ADP-ribose) polymerase (PARP) inhibitor PJ34. DSBs were measured by the formation of gammaH2AX foci. Results show that in SK-Hep1 cells exposed to fast neutrons in the presence of oxaliplatin, DSBs occurred and persisted with time after irradiation. While apoptosis remained low in co-treated cells, autophagy was considerably increased after irradiation and augmented by the addition of oxaliplatin. Thus, autophagic cell death appears to play a prominent role in the cytotoxicity of the combined treatment and may be linked to the generation of heavy damage to DNA.

High-LET radiation combined with oxaliplatin induce autophagy in U-87 glioblastoma cells.

Modern protocols of concomitant chemo/radiotherapy provide a very effective strategy to treat certain types of tumors. High-linear energy transfer (LET) radiations, on the other hand, have an increased efficacy against cancer with low radiosensibility and critical localization. We previously reported that oxaliplatin, a third generation platinum drug, was able to reinforce the cytotoxicity of an irradiation by fast neutrons towards human glioblastoma U-87 cells in culture. We show here that such a combination has the capacity to enhance the number of double strand breaks in DNA and to induce autophagy in these cells. Xenografts experiments were further performed in nude mice subcutaneously transplanted with U-87 cells. When injected shortly before a single irradiation by fast neutrons, oxaliplatin causes a marked reduction of tumor growth compared with the irradiation alone. Overall, our data indicate the unique cytotoxic mechanism of a combined high-LET irradiation and oxaliplatin treatment modality and suggest its potential application in anticancer therapy.

Comparison of the Methods of Specifying Carbon Ion Doses at NIRS and GSI.

Due to the RBE variations, the carbon-ion doses (in Gy) are no longer sufficient to monitor adequately the biological effect of these radiations. Therefore, "RBE dose weighting factors" - W(RBE) - allowing for the RBE variations with energy, dose and biological system have to be introduced in the treatment plans in order to provide the physician with interpretable information. This paper compares the methods employed for this purpose at NIRS and GSI, which are specific of the beam delivery system of these institutions. NIRS has a "passive" beam delivery system where the dose distribution in the SOBP is determined by a Ridge filter. The dose distribution - and thus, the shaping of the filter - is chosen according to the clinical situation and determined with respect to W(RBE) factors in order to yield a biologically iso-effective SOBP. W(RBE )factors in the SOBP are at first derived from a RBE/LET function for HSG cells, then normalized to 3 at a LET of 80 keV/mum. The latter value of 3 corresponds to the clinical RBE of NIRS-neutrons, which were found to exhibit the same radiobiological properties as 80 keV/mum carbon-ions. GSI has a "dynamic" beam delivery system ("spot" or "voxel" scanning) making it possible to irradiate irregular volumes and to modulate the radiation intensity according to the radiosensitivity of different tissues and/or different sub-volumes. Due to the "power" and the resulting complexity of the system, W(RBE )factors are determined through an integrated calculation code allowing iterative interaction of both physical and radiobiological parameters. The "Local Effect Model" (LEM) was developed in this view with the aim of deriving carbon-ion W(RBE )factors from the parameters determining the response to photons. Advantages and weaknesses of the respective methods will be discussed.

The cytotoxicity of high-linear energy transfer radiation is reinforced by oxaliplatin in human glioblastoma cells.

The combination of high-linear energy transfer (LET) radiation with chemotherapeutic agents may offer new perspectives in cancer treatment. We therefore assessed the consequences of a treatment in which U-87 human glioblastoma cells were irradiated with p(65)+Be neutrons in the presence of oxaliplatin, a third generation platinum anticancer drug having higher apoptosis-inducing activity than cisplatin. Cell survival, apoptosis, cell cycle progression as well as p21 and p53 protein expressions were analyzed. Results show that an enhanced cytotoxic effect was obtained when the two treatments were combined and that, unlike what we previously observed with cisplatin, this was not due to a reinforcement of apoptosis. Altogether, our results also indicate the potential of oxaliplatin for use in association with high-LET radiation against tumors refractory to conventional photon radiotherapy.

Cell death induced in a human glioblastoma cell line by p(65)+Be neutrons combined with cisplatin.

High linear energy transfer (LET) radiation have the ability to kill cancer cells resistant to conventional radiotherapy. On the other hand, protocols combining radiotherapy and chemotherapy are effective in eradicating certain inoperable cancers. In this study, we investigated the cytotoxicity of a co-treatment with fast neutrons and cisplatin in a human glioblastoma cell line, U-87. Cells cultured in vitro were irradiated with p(65)+Be neutrons in the presence of cisplatin. Cell survival and the induction of apoptosis and premature senescence were assessed at different time intervals thereafter, using a variety of methods. A marked reinforcement of the cytotoxicity was obtained when irradiation and cisplatin were associated. This reflected both an amplification of the apoptotic process and the induction of premature cell senescence. The efficiency of a combination between fast neutrons and cisplatin in inducing cell death in U-87 is more than additive. The present data concur with those we previously reported in a mouse lymphoma and suggest the potential utility of platinum compounds as adjuncts to future cancer therapy protocols using high-LET radiation.

Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells.

We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis.

Relative biologic effectiveness determination in mouse intestine for scanning proton beam at Paul Scherrer Institute, Switzerland. Influence of motion.

PURPOSE: To determine the relative biologic effectiveness (RBE) of the Paul Scherrer Institute (PSI) scanning proton beam in reference conditions and to evaluate the influence of intestine motion on the proton dose homogeneity. METHODS AND MATERIALS: First, RBE was determined for crypt regeneration in mice after irradiation in a single fraction. Irradiation was performed at the middle of a 7-cm spread out Bragg peak (SOBP; reference position), as well as in the proximal part of the plateau and at the distal end of the SOBP. Control gamma-irradiation was randomized with proton irradiation and performed simultaneously. Second, motion of mouse intestine was determined by radiographs after copper wire markers had been placed on the jejunum and intestinal wall. RESULTS: Proton RBE (reference (60)Co gamma) was equal to 1.16 for irradiation at the middle of the SOBP and to 1.11 and 1.21 for irradiation in the initial plateau and end of the SOBP, respectively. The confidence intervals for these RBE values were much larger than those obtained in the other proton beams we have tested so far. They exceeded +/-0.20 (compared with the usual value of +/-0.07), which resulted from the unusually large dispersion of the individual proton data. The instantaneous positions of the mice intestines varied by +/-2 mm in the course of irradiation. CONCLUSION: The results of this study have shown that the RBE of the PSI proton beam is in total accordance with the RBE obtained at the other centers. This experiment has corroborated that proton RBE at the middle of the SOBP is slightly larger than the generic value of 1.10 and that there is a slight tendency for the RBE to increase close to the end of the SOBP. Also, excessive dispersion of individual proton data may be considered to result from intestine motion, taking into account that irradiation at the PSI is delivered dynamically by scanning the target volume with a pencil proton beam ("spot scanning"). Because 2-mm movements resulted in significant variations in local dose depositions, this should be considered for moving targets. Strategies to reduce this effect for the spot scanning technique have been developed at the PSI for radiotherapy of humans.

Plasma citrulline concentration: a surrogate end point for radiation-induced mucosal atrophy of the small bowel. A feasibility study in 23 patients.

PURPOSE: Plasma citrulline, a nitrogen end product of glutamine metabolism in small-bowel enterocytes, was suggested as a marker of radiation-induced small-bowel epithelial cell loss in mice after single-dose whole-body irradiation. Our objective was to evaluate the feasibility of citrulline as a marker for radiation-induced small-intestinal mucosal atrophy in patients during and after abdominal fractionated radiotherapy. METHODS AND MATERIALS: Twenty-three patients were studied weekly during treatment and at intervals of 2 weeks and 3 and 6 months after treatment by postabsorptive plasma citrulline concentration and clinical toxicity grading. The interrelationship between these variables and the correlation with small-bowel dose and volume parameters were investigated. RESULTS: During fractionated radiotherapy, citrulline concentration significantly decreased as a function of the radiation dose (p < 0.001) and the volume of small bowel treated (p = 0.001). The plasma citrulline concentration correlated with clinical toxicity during the last 3 weeks of treatment. As a whole, citrulline concentration correlated better with radiation dose and volume parameters than clinical toxicity grading. CONCLUSIONS: In patients treated with fractionated radiation therapy for abdominal or pelvic cancer sites, plasma citrulline concentration may be a simple objective marker for monitoring epithelial cell loss, a major event in acute radiation-induced small-bowel toxicity.

Radiobiological rationale and patient selection for high-LET radiation in cancer therapy.

The rationale for introducing ion beams in cancer therapy is the high level of physical selectivity that can be achieved with ions, equal or even better than with proton beams or modern photon techniques, as well as the potential advantage of high-LET radiations for some tumour types and sites. The radiobiological arguments for high-LET radiation in cancer therapy are reviewed: reduction of OER in the case of hypoxic and poorly-reoxygenating tumours, and the lesser importance of repair phenomena which are a problem in controlling repair-proficient photon-resistant tumours. Fast neutrons were the first type of high-LET radiation used clinically, and were often applied under suboptimal technical conditions. Nevertheless, useful clinical information was derived from the neutron experience. A greater benefit from neutrons than from conventional radiotherapy was found for several tumour sites. The present discussion is limited to the results for salivary gland tumours and prostatic adenocarcinoma. Based on the fast neutron experience, radiobiological arguments, and the added benefit of excellent physical selectivity of ion beams, the potential clinical indications for high-LET ions are discussed: hypoxic, slowly growing and well-differentiated photon-resistant tumours. One of the main remaining issues is the selection of individual patients for high- or low-LET radiation. Since the physical selectivity of ions now matches that obtained with other techniques, the selection of patients will be based only on the radiobiological characteristics of the tumour.

Intestinal crypt regeneration in mice: a biological system for quality assurance in non-conventional radiation therapy.

BACKGROUND AND PURPOSE: The Relative Biological Effectiveness (RBE) of 8 fast-neutron beams, 5 proton beams and 1 carbonion beam was determined using as biological criterion intestinal crypt regeneration in mice, i.e. an in vivo system. These beams are used or planned for clinical cancer therapy applications. In addition, the RBE of 6 epithermal neutron beams, used or planned for Boron Neutron Capture Therapy (BNCT), was determined; no boron was administered. The goal of the program was to improve the exchange of information between the centers, facilitate the interpretation of the results and increase the safety of the clinical applications. MATERIALS AND METHODS: In all visited centers, the same technique was applied in the same conditions by the same radiobiology team. The number of regenerating crypts per circumference was scored 3.5 days after single fraction total body irradiation. The control irradiations were performed locally using cobalt-60 units. The mice were randomized according to radiation quality and dose level. RESULTS: (1) For fast neutron beams, the RBE (Ref. cobalt-60 gamma rays) increases with decreasing energy (from approximately 1.7 for p(65)+Be neutrons to approximately 2.4 for d(14.5)+Be neutrons). In addition, it is specific to each facility and depends on the nuclear reaction (p or d + Be), target and collimation type. (2) For proton beams, the RBEs (Ref cobalt-60 gamma rays) at the reference position (middle of a 7-cm Spread Out Bragg Peak, SOBP) range between 1.08 and 1.18. They might differ by approximately 6-8% according to the mode of beam production or delivery. The RBEs at the end of the SOBP are always 5-10 % higher than at the middle of the SOBP. (3) For the carbon ion beam studied at NIRS in Chiba, Japan, the RBE significantly increases with depth. Relative to gamma rays, it ranges from 1.3 in the initial plateau, 1.6 at the beginning, 1.7 at the middle and 1.9 at the end of a 6-cm SOBP. 4) In BNCT beams, the radiation quality (in particular the relative contribution of the different dose components) varies rapidly with depth and depends strongly on the arrangement of the irradiation set-up (e.g. presence or not of back scattering material). Moreover, the (total) dose rates are highly variable (from 0.05 to approximately 0.5 Gy/min) according to the power of the reactors. Wide range of RBE values (Ref. gamma rays) was thus obtained (RBE = 1.4 - 2.2) at shallow depths of 1.5 - 2.5 cm. DISCUSSION AND CONCLUSION: Intestinal crypt regeneration in mice is an in vivo system perfectly suitable to perform intercomparisons between centers applying different types of non-conventional radiation qualities. It was proven to be reproducible, reliable and accurate, and becomes progressively recognized worldwide as part of the Quality Control (QA) procedures for new beams. It should be stressed that the observed RBE for intestinal crypt cells after a single high dose provide some radiobiological characterization of the radiation quality but cannot be used as the RBE weighting factor in clinical prescriptions.

Citrulline: a physiologic marker enabling quantitation and monitoring of epithelial radiation-induced small bowel damage.

PURPOSE: Small bowel irradiation results in epithelial cell loss and consequently impairs function and metabolism. We investigated whether citrulline, a metabolic end product of small bowel enterocytes, can be used for quantifying radiation-induced epithelial cell loss. METHODS AND MATERIALS: NMRI mice were subjected to single-dose whole body irradiation (WBI). The time course of citrullinemia was assessed up to 11 days after WBI. A dose-response relationship was determined at 84 h after WBI. In addition, citrullinemia was correlated with morphologic parameters at this time point and used to calculate the dose-modifying factor (DMF) of glutamine and amifostine on acute small bowel radiation damage. RESULTS: After WBI, a time- and dose-dependent decrease in plasma citrulline level was observed with a significant dose-response relationship at 84 h. At this time point, citrullinemia significantly correlated with jejunal crypt regeneration (p < 0.001) and epithelial surface lining (p = 0.001). A DMF of 1.0 and 1.5 was computed at the effective dose 50 (ED50) level for glutamine and amifostine, respectively. CONCLUSIONS: Citrullinemia can be used to quantify acute small bowel epithelial radiation damage after single-dose WBI. Radiation-induced changes in citrullinemia are most pronounced at 3 1/2 to 4 days postirradiation. At this time point, citrullinemia correlates with morphologic endpoints for epithelial radiation damage.

Caspase 8-mediated cleavage of the pro-apoptotic BCL-2 family member BID in p53-dependent apoptosis.

The objective of this study was to characterize the apoptotic pathways activated by fast neutrons in the human lymphoblastoid cell line TK6 and in its p53 -/- derivative. Our results demonstrate that while p53 is not required for neutron-induced apoptosis, as previously shown, it does affect the kinetics of apoptosis and the molecular pathways leading to the activation of effector caspases. Indeed, rapid p53-dependent apoptosis was associated with the activation of caspase 9, 8, 3, and 7 and the cleavage of BID by caspase 8. In contrast, the slow-occurring p53-independent apoptotic process, mediated by caspase 7, took place without BID cleavage and loss of transmembrane mitochondrial potential. Altogether, our findings highlight an essential role for caspase 8-mediated BID cleavage, in the course of p53-dependent apoptosis triggered by fast neutrons in lymphoid cells. They also demonstrate that this mechanism is not involved in p53-independent apoptosis.