Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.     

Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia

A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89. A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdU + 3HAra-C slides of leukemic patients. Their implications are discussed in the text. Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S-phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of S-phase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.     

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques

Abstract. A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([³H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase-anti-alkaline-phosphatase technique, the peroxidase-anti-perox-idase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a GdG, peak nor a G₂+ M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.     

Intratumoral heterogeneity of S phase transition in solid tumours determined by bromodeoxyuridine labelling and flow cytometry

Abstract. Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5–14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti-BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log-normal distributions for S phase transition (T_s). The log-normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric T_s distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (T_pot) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.     

Immediate bromodeoxyuridine labelling of unseparated human bone marrow cells ex vivo is superior to labelling after routine laboratory processing

It is important to evaluate the proliferation of bone marrow cells in several disease conditions and during treatment of patients with for example cytokines. Labelling with bromodeoxyuridine (BrdUrd), immunocytochemical staining with anti-BrdUrd antibody and analysis by flow cytometry provides a reliable and reproducible technique for estimation of the fraction of cells that incorporated BrdUrd into DNA during S-phase. We have compared immediate BrdUrd labelling of unseparated bone marrow cells with the previously used labelling in the laboratory after routine separation of the mononuclear cells. Bone marrow aspirates from seven lymphoma patients without bone marrow involvement were studied with these two methods. We found higher BrdUrd labelling indices (LI) in the mononuclear cells, when cells were labelled immediately. A large variation in LI was found between patients. Our results suggest that ex vivo BrdUrd labelling of bone marrow cells should be performed immediately after aspiration and before separation, because these data are closer to values reported from in vivo labelling with BrdUrd.     

A method to measure the duration of DNA synthesis and the potential doubling time from a single sample

A method is described whereby the DNA synthesis time, Ts, can be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC-labelled anti-BrdUrd-DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd-labelled cells through the S phase can be quantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, Ts can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of Ts for these tissues.     

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and T_pot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's (<10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. T_pot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

Optimal conditions for immunohistochemical determination of the in vitro DNA synthesis labelling index with bromodeoxyuridine in head and neck cancer

The in vitro DNA synthesis labelling index was assessed immunohistochemically in 24 freshly obtained specimens of head and neck cancer using bromodeoxyuridine (BrdUrd) as the DNA precursor to determine the influence of BrdUrd concentration on labelling index (LI). Initially, tumour fragments were incubated in varying concentrations of BrdUrd from 2 to 100 microM for 2 h, and BrdUrd was detected with an anti-BrdUrd monoclonal antibody using immunoperoxidase labelling. There was a dose-response gradient with mean LI varying from 1.6% at 2 microM BrdUrd to 8.8% at 100 microM. The concentration-response gradient best fit a quadratic model when LI was plotted against log BrdUrd concentration (r = 0.65, P less than 0.0001). Eleven additional tumours were then studied to determine whether LI increased for BrdUrd concentrations above 100 microM. The mean LI at 125 microM and at 150 microM in these 11 tumours did not differ from the value at 100 microM, suggesting a plateau at this level. The gradient effect accounted for 17% of the variance in LI, while 60% of the variance was explained by between tumour differences. Within individual tumours, three response patterns were observed: (i) LI rose at a constant rate to the highest concentration tested (n = 8), (ii) the LI plateaued or declined at high BrdUrd concentrations (n = 6); and (iii) there was a biphasic slope slope in which the rate of rise in the LI increased at the higher BrdUrd concentrations (n = 2). The data show that BrdUrd concentration is an important variable in the immunohistochemical assessment of the in vitro LI in head and neck cancer.     

IN VITRO AND IN VIVO LABELLING OF ANIMAL TUMOURS WITH TRITIATED THYMIDINE

The labelling indices obtained by incubating tumour specimens with tritiated thymidine in vitro under hyperbaric oxygen have been compared with those obtained by labelling a matched tumour in vivo. The correspondence between these individual labelling indices is sometimes very poor; however, the average labelling index derived from groups of tumours labelled in the two ways does not differ significantly. There was a large variation from field to field within any tumour, and considerable variation from one tumour to another within each tumour type. The mitotic index was also compared in the matched tumour preparations; the mitotic index in vitro was almost always considerably lower than the values observed in vivo.     

Ki-67 expression and BrdUrd incorporation as markers of proliferative activity in human prostate tumour models

Abstract. The validity of the use of the monoclonal antibodies Ki-67 and anti-BrdUrd to evaluate proliferative activity of human prostate tumour models was studied. Growth of the transplantable PC-82 and PC-EW prostate tumours, as assessed by tumour volume measurements, was significantly correlated with the proliferative activity as reflected by BrdUrd incorporation into DNA ( r = 0.64 and r = 0.78, respectively). The proliferative activity of PC-82 tumours detected by Ki-67 antigen expression paralleled the pattern observed with BrdUrd ( r = 0.51) and a significant correlation ( r = 0.60) between the results obtained with both markers was found. In growing PC-82 and PC-EW tumours only small variations in the Ki-67 and BrdUrd indices were observed. In contrast, Ki-67 expression in regressing PC-82 tumours varied considerably (2.7 ± 2.2%). The BrdUrd index in regressing PC-32 tumours showed less variation (1.3 ± 0.2%), but part of the BrdUrd-positive cells were found in the stromal (murine) part of the regressing tissue. It is concluded that the Ki-67 and BrdUrd proliferation markers are reliable parameters to monitor changes in growth of prostate tumour lines, but that in slow growing or regressing tumours Ki-67 and BrdUrd data should be interpreted with caution.     

Modelling of cell proliferation: nuclear versus membrane labelling

Cell proliferation is a fundamental process involved in growth, development and oncogenesis. Monitoring and quantification of proliferation are essential to analyse the behaviour of cells drug-treated or not. Flow cytometry assessment of cell proliferation requires mathematical models to extract information of interest from fluorescence distributions. Various methods are available for cell cycle analysis, including estimation of cell phase durations and doubling time. In this context, we compare widely used flow cytometric methods based on nuclear labelling (using BrdUrd incorporation in combination with DNA content) to membrane labelling (using intercalating dyes PKH).     

Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy response in solid human tumors

In vitro bromodeoxyuridine (BrdUrd) incubated single-cell suspensions obtained from solid tumors were fixed on slides for subsequent sample processing. As dispersal of nuclei largely was avoided, only small amounts of cells were needed for examination. The sensitivity of detecting even low BrdUrd incorporation rates could be improved by treatment with intense DNA denaturation conditions. This technique was applied to monitor cytokinetic response to chemotherapy and radiation in oral carcinomas by analysing biopsies taken consecutively in the course of treatment. By combining BrdUrd labeling and DNA flow cytometry, cells arrested in S phase easily could be distinguished from cells showing continuous proliferation.     

High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.     

Observations regarding DNA replication sites in human cells in vivo following infusions of iododeoxyuridine and bromodeoxyuridine

In studies using bromodeoxyuridine (BrdUrd) and/or iododeoxyuridine (IdUrd) to label S phase cells in cancer patients, several unique observations were made regarding DNA replication sites and the organization of newly synthesized DNA in post-mitotic cells. While the majority of tumour specimens removed at the end of infusions demonstrated concentration of replication sites around the nuclear membrane, biopsies obtained in leukaemic patients 1 week later demonstrated several distinct patterns of labelling. For example, one, two or all lobes of granulocytes were labelled. Scavenger macrophages bearing labelled leukaemic cells in their cytoplasm were also seen. Sequential IdUrd/BrdUrd labelling of solid tumours showed various patterns of nuclear/nucleolar/membrane labelling, allowing more precise localization of early versus late replication sites.     

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.     

Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine.

Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.     

Cell cycle effects of hypertonic stress on various human cells in culture

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE. cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immuno-labelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protien band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G_0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however, differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.     

BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.

The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.     

Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay

This report describes a rapid and sensitive procedure for detection of cytosine arabinoside- (Ara-C) resistant mouse leukemia cells (L1210) in a predominantly Ara-C-sensitive population. L1210 cell lines sensitive or resistant to Ara-C were grown and treated with Ara-C in vitro or in vivo. Ara-C-resistant cells were detected as those cells with S-phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara-C. The BrdUrd incorporation ability of the S-phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara-C-resistant cells was accurately estimated at frequencies approaching 10(-3).