Effects of single-dose irradiation on bronchial epithelium: a comparison of BEAS 2B cell monolayers, human organ cultures, and Goettinger minipigs.

To assess the effects of radiation on bronchial epithelium, BEAS 2B cells cultured as monolayers and human bronchial epithelium cultured as organ cultures were exposed to single doses of 0, 10 and 30 Gy. The lactate dehydrogenase in the supernatant of the BEAS 2B cells increased markedly 24 h after irradiation, whereas in the organ cultures only a minor increase was found after 48 h. The nucleosomes in the supernatant of the BEAS 2B cells showed a massive increase in response to irradiation, whereas in the organ cultures no change could be seen. The number of BEAS 2B cells was dramatically diminished after 96 h, whereas in the organ cultures a smaller decrease was observed no earlier than 21 days after irradiation. To assess the effects of brachytherapy in bronchial epithelium in vivo, brachytherapy with 30 Gy was performed in Goettinger minipigs, and histological sections of the bronchi were analyzed for morphological alterations and cell numbers. After 2 weeks, only slight cell damage was observable, and after 3 weeks, moderate morphological changes and decreased cell numbers were found. However, after 8 weeks, the epithelium had nearly regained its normal structure. We conclude that the bronchial epithelium has a remarkably high radioresistance and that organ cultures, but not monolayers of BEAS 2B cells, reflect the effects of radiation in vivo.

The influence of autologous tumor fibroblasts on the radiosensitivity of squamous cell carcinoma megacolonies.

PURPOSE: To study the influence of tumor fibroblasts on radiosensitivity and stem cell fraction of tumor cells in squamous cell carcinoma megacolonies by determining colony cure and clonogen survival. METHODS AND MATERIALS: Murine squamous cell carcinoma cells (AT478c) grown as flat but multilayered megacolonies were co-cultured with pre-irradiated tumor fibroblasts derived from the same carcinoma, and irradiated with 1, 2, 4, or 8 fractions. Recurrent clones and their growth pattern in situ were recorded. From megacolony cure data and clonogen survival data, the clonogen number and the parameters of cellular radiosensitivity were calculated. RESULTS: The curability of the co-cultured megacolonies, as determined by TCD50 values, was significantly increased compared to the megacolonies without fibroblasts (p < 0.01). Both the megacolony cure and clonogen survival data suggested a decrease of the clonogen fraction in the co-cultured megacolonies. CONCLUSIONS: The presence of tumor fibroblasts increases megacolony radiosensitivity. This is due to a decrease in the fraction of clonogens in the tumor megacolony, apparently caused by a downregulation of the stem cell fraction of the tumor cells.

Tumour stem cells--the evidence and the ambiguity.

Progressive growth of malignant tumours, metastatic spread and local recurrence after treatment can only be explained by the presence of cells with unlimited proliferative ability. While this is generally accepted, the proportion of such cells and their organization in a hierarchical system of stem cells and non-stem cell progeny is still a matter of controversy. Results of quantitative transplantation and dose requirement of curative radiotherapy have indicated low stem cell fractions in human and early passage rodent tumours, but uncertainty is introduced by uncontrollable experimental or biological factors and the probabilistic nature of stem cell performance itself Studies using a particular mouse carcinoma (AT17) have given direct insight into the number and clonal expansion of stem cells in situ, strongly supporting the hierarchical concept. The implications are important and concern the relevance of predictive assays, possible mechanisms of accelerated repopulation, or the role of adjuvant treatment strategies.

Regrowth delay analysis of irradiated tumors in the curative dose region.

PURPOSE: To develop a method to analyze regrowth delay times, including cured tumors. METHODS AND MATERIALS: Regrowth delay measured in the in vitro squamous cell carcinoma megacolony system following fractionated irradiation was analyzed using two different approaches. A conventional regrowth delay analysis based on means or medians of dose groups was contrasted with a one-step procedure using the inverse of individual regrowth delay times, where cured tumors are represented by an infinite delay time. Two data sets with different proportions of cured tumors were compared in this way. In addition, for both approaches, confidence limits were also generated by the bootstrap technique. RESULTS: The parameter estimated was the alpha/beta ratio. The inverse analysis yielded similar results as the conventional analysis, but with much smaller confidence limits. When the dataset was stripped by stepwise removal of lower dose groups with no cures, the estimates of the alpha/beta ratio remained definitely more robust in inverse analysis. Results of the bootstrap procedure confirmed the accuracy of the calculated confidence intervals. CONCLUSION: Regrowth delay analysis using reciprocal regrowth delay times is a useful tool, especially when the data shows large variations, or a substantial fraction of the dataset falls into the curative dose region.

The radiosensitivity of recurrent clones of an irradiated murine squamous cell carcinoma in the in vitro megacolony system.

PURPOSE: To measure changes in spontaneous growth rate and radiation response in the progeny of irradiated squamous cell carcinoma cells. MATERIALS AND METHODS: Murine SCC cells of the line AT478 were grown as epithelial megacolonies in vitro, using both the original line and two subsequent passages derived from a clone that had recurred after a high radiation dose. Radiosensitivity was evaluated in terms of local control following single dose irradiation of standard size megacolonies (0.8 cm2). In addition, original megacolonies were given a priming dose of 20 Gy and the recurrent clones arising in situ were retreated at three dose levels for analysis of curability. RESULTS: A marked increase in radiosensitivity was observed in the megacolonies grown from irradiated progeny as compared to original megacolonies, reflected in a shift of the TCD50 from 24.5 to 16.5 Gy. Direct parameter estimation from the cure data suggested that the underlying change was a lowered number of clonogenic 'stem' cells rather than increased cellular sensitivity. A similar decrease in clonogen density was also apparent for the recurrent clones in situ. The change in megacolony curability was paralleled by a substantial growth retardation. CONCLUSION: The data demonstrate persistent changes in the progeny of irradiated SCC tumour cells that affect both growth and radiosensitivity and are compatible with the expression of delayed reproductive death.

Radiation-induced impairment of bone healing in the rat femur: effects of radiation dose, sequence and interval between surgery and irradiation.

BACKGROUND AND PURPOSE: Impairment of osseous healing in treatment combining surgery and radiotherapy is a frequent complication. Its dependence on sequence and interval was studied in a defined experimental model. MATERIALS AND METHODS: The effect of pre- and postoperative irradiation by single doses of X-rays on osseous closure of a 1.2 mm drill hole in the rat femur was measured 6 or 7 weeks after surgery in histological sections using morphometrical methods. RESULTS: Irradiation delivered between 1 day and 6 months before surgery resulted in a reduction of bone healing following very similar dose response relationships; there was no evidence of any slow repair of latent radiation damage. Radiosensitivity of bone healing during the first 3 days after surgery was not different from preoperative irradiation; however, irradiation 4 days or later after surgery failed to reduce osseous healing even after very high radiation doses. CONCLUSION: Tolerance increases enormously if radiotherapy is given later than 4 days after surgery. This has great implications for combined radiotherapy and surgery schedules involving bone reconstruction, but may be even more important for radiotherapy applied to prevent heterotopic ossification after total hip arthroplasty. Biologically, target cell regeneration alone is insufficient to account for the drastic rise in radiotolerance; it must be accompanied by an increase in cellular resistance due to differentiation.

[Combined intraoperative and external irradiation of the celiac artery in the rabbit: the effects on the blood flow of the gastric mucosa]. / Kombinierte intraoperative und externe Bestrahlung der Arteria coeliaca beim Kaninchen: Auswirkungen auf die Durchblutung der Magenschleimhaut.

AIM: To demonstrate changes in gastric mucosal blood flow caused by intraoperative radiotherapy of the celiac artery combined with external radiotherapy of the upper abdomen in a rabbit model. The study was designed to identify a possible correlation between a radiation-induced reduction in mucosal blood flow and the induction of gastric ulcer. MATERIAL AND METHOD: Intraoperative radiation doses of 0 or 30 Gy were given to the celiac artery in rabbits. After a delay of 14 days external radiotherapy of the upper abdomen with 3 x 4 Gy/week to a maximum total dose of 40 Gy was initiated. Gastric mucosal blood flow was assessed by intraventricular injection of radioactively-labelled microspheres (15 microns) followed by measurement of radioactivity in the mucosa. The injections were performed at various time intervals between 2 and 63 days after intraoperative radiation. RESULTS: Intraoperative radiotherapy, including sham-intraoperative radiation, resulted in a transitory reduction of mucosal blood flow by about 50% of the control value on day 7 (Figure 3). After a temporary recovery by day 14, a marked and permanent reduction in blood flow was assessed after week 6. This time corresponds to the time of development of gastric ulcer. CONCLUSIONS: A relationship between the time of ulcer development and of reduced gastric mucosal blood flow was observed after combined intraoperative and external radiotherapy. The mechanical component of intraoperative treatment has to be emphasized. Reduced blood flow was also seen after intraoperative radiotherapy alone, without an induction of ulcer by this treatment. Hence additional mucosal damage by external radiation must be present for the induction of gastric ulcer.

Complications of combined intraoperative radiation (IORT) and external radiation (ERT) of the upper abdomen: an experimental model.

An experimental model in the rabbit is presented which is suitable for analysis of clinically relevant, early side-effects of combined upper abdominal IORT and ERT. Fractionated ERT alone given through an upper abdominal a.-p. field including the entire stomach caused gastric ulcerations within < or = 58 days. Latent times decreased with increasing dose and the ED50 for occurrence of ulcers was 39 +/- 3.3 Gy. Single doses of IORT of 20-40 Gy alone administered through a 2-cm diameter field localized on the coeliac axis and carefully excluding any intestinal mucosa caused neither gastric ulcerations nor other clinical symptoms. When ERT with 40 Gy was preceded by IORT with 20-40 Gy or by sham IORT, 13 out of 15 animals developed ulcers after latent times which in a life-table analysis were shown to be significantly shorter than after ERT alone. However, a statistically significant IORT dose-dependence of latent time or incidence of ulcers could not be demonstrated in the present experiment. The most significant histological changes were observed in the areas of gastric ulcers. Already during ERT, the mucosal epithelium was depleted and regenerative activity was evident in spite of ongoing fractionated irradiation. However, profound irregularities in glandular structure and distribution, as well as number of proliferating epithelial cells were still present in healed ulcers at 80 days. In summary, IORT to the coeliac artery did precipitate the development of gastric ulcers induced by subsequent ERT. On the one hand, the data indicate that the surgical procedure of IORT did contribute to this effect. On the other hand, IORT to the coeliac artery could cause transient, functional alterations in blood supply to the depending organs, i.e. the stomach, and could thus precipitate the development of radiation-induced ulcers.

Effects of stimulated repopulation on oral mucositis during conventional radiotherapy.

The effect of local conditioning of human oral mucosa by silver nitrate solution (3%) on epithelial proliferation rates was tested in 11 healthy volunteers by in vitro labelling of biopsies with tritiated thymidine. Compared to control biopsies from 13 volunteers, stimulation over 3 days, 3 times per day, yielded a significant (p = 0.006) increase in the epithelial labelling index (LI) from 4.75 +/- 0.32% to 6.85 +/- 0.65%, i.e., by 44%. The increase in the absolute number of labelled cells per mm epithelial length was dependent on the overall cell density at the various intraoral sites and varied between 45% in the maxillary vestibule and 91% at the floor of the mouth. In an analysis of variance, stimulation turned out to be the most important source causing the effect (p = 0.011 for LI and 0.015 for labelled cells per mm). In a radiotherapy trial with conventional postoperative treatment with 5 x 2 Gy/week to a total dose of 60 Gy in 6 weeks, the left buccal mucosa in 10 patients with squamous cell carcinomas of the head and neck was conditioned (3% silver nitrate, 3 times per day, 5 days before and the first 2 days of radiotherapy) while the contralateral mucosa, receiving an identical dose, served as individual control. Mucositis scores according to the EORTC/RTOG or the Dische system showed that the time course and severity of the mucosal response was almost identical in both cheeks, which is in clear contrast to a previous clinical study (Maciejewski et al. Radiother. Oncol. 22, 7-11, 1991). Differences in radiation dose intensity, i.e., weekly dose, in these studies are discussed as a tentative explanation for the different clinical findings.

The proliferative capacity of mouse fibrosarcoma cells that survived x-irradiation.

Delayed reproductive death, the appearance of colonies with a reduced cell density (impaired colonies) and the number of giant cells per colony were investigated in murine fibrosarcoma cells after irradiation with 3 to 9 Gy of x-rays. Radiation survivors were replated after reaching confluence, which occurred after 13 to 15 doublings; this procedure was repeated three times. The replating efficiency decreased in a dose-dependent manner, the survivors of 9 Gy achieving only 30% of the plating efficiency of unirradiated cells. After the third replating, i.e. after 40 to 45 doublings, the plating efficiency of the survivors approached that of the controls. The median colony size of the survivors showed a similar dose-dependent decrease, which was pronounced after the first replating but still remained significant after the third replating. The fraction of impaired colonies was increased to more than 30% in 9-Gy survivors, and though abating, the increase was still significant even after the third replating. Evidence of residual damage was also provided by the presence of giant cells. For instance, after 6 Gy irradiation and 13 to 15 doublings, the proportion of colonies with giant cells was 60%, decreasing only to 45% after 40 to 45 doublings. The number of giant cells per colony was 1.4 in colonies arising immediately after 6 Gy, decreasing to 0.9 after the third replating. These results suggest that the proliferative capacity of surviving cells is depressed even longer than their clonogenic capacity.

Radiation-induced impairment of osseous healing: quantitative studies using a standard drilling defect in rat femur.

The femora of adult Wistar rats were locally irradiated with single doses of X rays and 1 day later were wounded by a standardized drilling defect that extended through the diaphyseal cortex into the marrow cavity. Healing of the lesion was followed over 30 weeks to assess the time course of osseous closure. In unirradiated bones healing was complete by week 7. Irradiation with doses up to 15 Gy imparted a dose-dependent delay in the formation of primary callus and its subsequent replacement by more mature bone, while after higher doses healing remained permanently compromised or even suppressed. Using histomorphometry, osseous closure was also measured quantitatively for healing periods of 7, 10, 16 and 30 weeks and the data were expressed as the percentage of responders with < or = 40% fractional closure. The resulting dose-response curves were steep, displaying a large threshold dose and ED50 values between 16.8 to 17.5 Gy (7 to 16 weeks) and 19.4 Gy (30 weeks), respectively.

Quantitative distinction of cisplatin-sensitive and -resistant mouse fibrosarcoma cells grown in multicell tumor spheroids.

As a suitable model to study the growth behavior and therapeutic response of drug-resistant and -sensitive cells in three-dimensional coculture we have established multicellular spheroids generated from both cisplatin-sensitive and -resistant cells of a murine fibrosarcoma cell line. A drug resistant clone was derived from the parent cisplatin-sensitive cells by intermittent drug exposure in vitro. As a prerequisite for analysis of differential growth and treatment response of spheroid subpopulations, two efficient methods to discriminate between the two morphologically indistinguishable subpopulations in mixed spheroids were established. In the cisplatin-resistant cell line chosen for the present study, resistance is mainly due to an increased cellular metallothionein content and is therefore associated with increased resistance to CdCl2. Exposure of colonies to high concentrations of CdCl2 thus allowed selective elimination of sensitive colonies. Permanent labeling of either resistant or sensitive cells was achieved by introduction of the Escherichia coli beta-galactosidase marker gene with a retroviral vector system. The transformation of an uncolored galactose derivative by this enzyme into an indigo stain allowed detection of cells carrying and expressing the marker gene. The marker gene and CdCl2 method led to identical results when used simultaneously to distinguish quantitatively between resistant and sensitive colonies grown from plated cells of untreated or irradiated mixed spheroids. The retroviral labeling method was also used successfully in the study of intact spheroids, showing that in 1:1 mixed spheroids, cisplatin-sensitive parent cells accumulate in the spheroid periphery, outgrowing resistant cells and displacing them into the metabolically restricted spheroid center. Only when sensitive and resistant cells are initially mixed at a ratio of 1:9 are the resulting spheroids composed of equal proportions of the 2 cell types throughout 10-20 days after spheroid initiation.

Proliferation equivalent of 'accelerated repopulation' in mouse oral mucosa.

The proliferation response and changes in cellularity of mouse tongue epithelium were studied after single doses of X-rays and during 3 weeks of daily irradiation. A single dose of 13 Gy resulted in minimum cellularity (70% of control values) on days 3-5 and complete restoration on day 7. Mitotic activity ceased for 1 day followed by normal-to-supranormal values until day 15. A wave of abnormal mitoses was observed with a peak at days 4-7. Daily irradiation with 3 or 4 Gy induced neither major structural nor visible cellular damage. Cellularity decreased to approximately 60% during week 1 and subsequently remained at 60-70%. The proliferation activity was reduced to approximately 8% by day 2. Mitotic activity during weeks 2 and 3 was subnormal-to-normal, with a dose-dependent increase to normal counts during the first weekend and a distinct overshoot over the second weekend respectively. A proliferation model is presented to explain the present findings and previous functional measurements of changes in tissue tolerance. Its major features are accelerated symmetrical stem cell divisions and abortive divisions of sterilized cells.

Cell kinetic analysis of murine squamous cell carcinomas: a comparison of single versus double labelling using flow cytometry and immunohistochemistry.

The study was originally set up to measure accurate cell kinetic parameters in two murine squamous cell carcinomas (scc) for comparison with radiobiological data on proliferation during radiotherapy. The tumours, AT84 and AT478, were both moderately well differentiated aneuploid scc. In the course of the study, several comparisons of techniques were made in two different centres. This paper reports on the results of those comparisons involving two different detection methods (flow cytometry and immunohistochemistry), single vs double labelling, and in vivo and in vitro labelling, the latter using tissue slices incubated under high pressure oxygen. Pulse labelling studies with bromodeoxyuridine (BrdUrd) showed that the labelling indices (LI) were not significantly different after in vitro or in vivo labelling. In addition, the flow cytometry (FCM) and immunohistochemistry (IHC) methods also gave labelling indices which were not significantly different. Only tumour cells were analysed in these studies by selecting cells on the basis of aneuploidy (FCM) or morphology (IHC). The DNA synthesis time of the tumour cells were analysed by both techniques. For FCM, the Relative Movement method was used (Begg et al., 1985). For IHC, a double labelling method was used, employing BrdUrd and triated thymidine (3H-TdR) administered several hours apart, detected simultaneously using immunoperoxidase and autoradiography, respectively. When both labels were administered in vivo, there was good agreement for Ts between the FCM and IHC methods. Attempts were also made to measure Ts in vitro using both techniques. With double labelling, it was found that cells did not take up the second label, implying a failure of cycle progression. This was confirmed by FCM results, showing no movement of labelled cells through the S-phase, despite an initially high uptake. This could not be influenced by lowering the DNA precursor concentration or by adding foetal calf serum. This indicates that DNA synthesis times are difficult or impossible to measure in vitro in fresh tumour explants. Finally, the double labelling IHC method allowed intratumoural variations of both LI and Ts to be studied. Both parameters were found to vary markedly throughout the tumour volume, particularly for larger tumours (600 mg), giving calculated local potential doubling time values (Tpot) ranging from 1-7 days.

Capacity and kinetics of SLD repair in mouse tongue epithelium.

The effect of dose fractionation on the radiation response of mouse tongue epithelium was quantified in fractionation protocols involving 1, 3, 4, 5 and 10 fractions, separated by at least 4 h. Fractionated irradiation was given either to the whole snout by 300 kV X-rays or locally to the tongue using 25 kV X-rays. Each protocol was terminated by a final local top-up dose (25 kV X-rays) of 5 Gy. The frequency of complete local denudation within the test area was used as the quantal end point. The kinetics of repair of sublethal damage was studied by snout irradiation with four equally spaced fractions, delivered at intervals of 35, 60, 90, 480 or 540 min, again followed by a local top-up dose of 5 Gy. The linear-quadratic model gave a satisfactory fit to the data with the exception of the four fraction/30-h data, suggesting cell cycle effects in this schedule. Analysis of the results with different two-step methods and with direct analysis yielded similar results. The alpha/beta ratio was determined to be approximately 11 GY (direct analysis: 11.6 Gy with 95% confidence limits of 8.1 and 16.4 Gy) and T1/2 was found to be 46 min (35-69 min). Both these values are in the range described for other acutely responding rodent tissues.

Increased radiation tolerance of mouse tongue epithelium after local conditioning.

The effect of local stimulation on mitotic activity and radiation tolerance was studied in mouse tongue mucosa. Silver nitrate solution (0.5-20%) was used for local conditioning. The most effective protocol comprised three daily treatments (days 0-2), yielding a delayed increase in 24 h mitotic counts by about 30% on days 5-7. The stimulating effect was independent of silver nitrate concentration. Sham treatment with saline or anaesthesia alone clearly depressed mitotic activity on days 2-4 without any subsequent overshoot. Radiation treatment was initiated on day 5 after three daily treatments with 3% silver nitrate solution. A top-up technique was employed, consisting of fractionated irradiation (300 kV X-rays) of the whole snout, followed by graded local test doses (25 kV X-rays) to induce denudation in a confined area of the inferior tongue surface. Silver nitrate conditioning did not alter the radiosensitivity of the epithelium to single local doses, but shortened the latency to denudation from 11 to 8 days. In contrast, a clear increase in tolerance to fractionated irradiation, delivering 5 x 2.5, 5 x 3.5, 5 x 4.5 Gy or 3 x 5.2 Gy in 7 days, was observed, equivalent to about four, two, one and two extra dose fractions. This approach may be a suitable way to increase radiation tolerance of oral mucosa in clinical radiotherapy.