J Neurosurg 73:254-258, 1990

Cell kinetics of rat 9L brain tumors determined by double labeling with iodo- and bromodeoxyuridine AKIO ASAI, M.D., D.M.Sc., SOICHIRO SHIBU1, M.D., D.M.Sc., MARVIN BARKER, M.S., MARTIN VANDERLAAN,PH.D., JOE W. GRAY, PH.D., AND TAKAO HOSHINO, M.D., D.M.Sc.

Brain Tumor Research Center, Department of Neurological Surgery, School of Medicine, University of California, San Francisco, and the Biochemical Science Division, Lawrence Livermore National Laboratory, Livermore, California t/ Rats with 9L brain tumors received intraperitoneal injections of iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) to estimate the duration of the deoxyribonucleic acid (DNA) synthesis phase (Ts) and the potential doubling time (Tp) of individual tumors. Different sequences and intervals (2 or 3 hours) of IUdR and BUdR administration were evaluated. After denaturation, tumor sections were reacted with Br-3, a monoclonal antibody that identifies only BUdR, and then were stained immunohistochemically by the avidinbiotin complex method. An antibody that recognizes both IUdR and BUdR, IU-4, was applied to the sections and identified by the alkaline phosphatase-antialkaline phosphatase method. Nuclei labeled only with IUdR stained blue, while those labeled with BUdR or with BUdR and IUdR stained brown. The fraction of cells that either left or entered the S-phase during the time between administration of IUdR and BUdR was measured to calculate Ts and Tp, assuming that the labeled cohort completed the DNA synthesis at a constant rate. The Ts was 8.8 hours (coefficient of variation (cv) = 0.05) and the Tp was 64.2 hours (cv = 0.08). The sequence and interval of administration of IUdR and BUdR had a minimal effect on Ts and Tp. In studies of 9L cells in monolayer culture, the Ts was 9.6 hours (cv = 0.08) and the Tp was 30.6 hours (cv = 0.06). Double labeling with BUdR and IUdR allows the duration of the S-phase and potential doubling time of individual brain tumors to be estimated in situ from a single biopsy specimen. KEY WORDS cell kinetics 9 brain neoplasm 9 bromodeoxyuridine iododeoxyuridine 9 monoclonal antibody immunohistochemistry "

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ELL kinetics studies were greatly facilitated by the development of a monoclonal antibody against halogenated pyrimidines. 4'8""~'~7 Although two thymidine analogues, iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR), are available, this antibody alone cannot be used for double-labeling studies because it reacts equally with BUdR and IUdR. Recently, more specific monoclonal antibodies against halogenated pyrimidines have been created. The monoclonal antibody Br-3 identifies only BUdR, 3 whereas the IU-4 antibody cross-reacts with both BUdR and IUdR. 2~ Theoretically, all cell kinetics studies that previously used radioactive thymidine 5`6,j4'~s are now feasible using these agents without the hazard or pollution involved in the use of radioactive thymidine. To take advantage of these two antibodies, we have developed a double-labeling method with BUdR and IUdR.~9 This report describes the use of that method to estimate the 254

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duration of the deoxyribonucleic acid (DNA) synthesis phase (Ts) and the potential doubling time (Tp) of rat 9L brain tumors in situ. Materials and Methods

In Vitro Studies For the in vitro studies, rat 9L gliosarcoma cells (4 • l04 cells/0.5 ml of medium) growing in monolayer culture in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum were transferred to each chamber of tissue culture/chamber slides,* The cells were incubated at 37~ in a 100% humidified atmosphere with 95% air/5% CO> To estimate the BUdR labeling index (LI), cells were exposed to 10 uM * LabTek tissue culture/chamber slides manufactured by Miles Scientific, Naperville, Illinois.

J. Neurosurg. / Volume 73/August, 1990

Double labeling for cell kinetics studies Definitions of Abbreviations

BSA = BUdR = cv = DNA = IUdR = LI = PBS = TBS = Tp = Ts =

bovine serum albumin bromodeoxyuridine coefficient of variation deoxyribonucleic acid iododeoxyuridine labeling index phosphate-buffered saline Tris-buffered saline potential doubling time duration of tumor S-phase

BUdR in 0.5 ml of medium for 30 minutes at 37~ in a 100% humidified atmosphere. For the single-labeling study, the ceils were fixed in 4~ 70% ethanol for 30 minutes, denatured with 4 N HC1, and stained with Br-3 by the avidin-biotin complex (ABC) method. For double-labeling studies, the cells were incubated first with 10 uM IUdR for 2 hours and then with 10 uM BUdR for 30 minutes. After fixation in chilled 70% ethanol, the cells were denatured with 4 N HC1. In Situ Studies Eighteen male Fisher 344 rats, each weighing about 200 gm, were injected intracerebrally with approximately 4 x 104 9L gliosarcoma cells in 10 ul of complete medium using a standard stereotactic technique. Near the terminal stage, when motor palsy or pigmentation of the eyelids was evident (approximately 16 to 21 days after implantation), the rats were separated into four groups. Groups 1 and 2 received intraperitoneal injections of IUdR and BUdR (80 mg/kg each) at intervals of 2 and 3 hours, respectively. Groups 3 and 4 received equal doses of the same agents in reverse order at intervals of 2 and 3 hours, respectively. All rats were sacrificed 1 hour after the second injection, and the tumors were excised immediately. All of the tumors were approximately 6 m m in diameter and weighed more than 200 mg. Each specimen was fixed in chilled 70% ethanol for 12 hours, embedded in paraffin, and cut into sections 5 um thick. The sections were deparaffinized in xylene and rehydrated in 100% ethanol, 95% ethanol, and phosphatebuffered saline (PBS). The slides were incubated for 15 minutes in 0.3% H202 to block endogenous peroxidase and for 10 minutes in 4 N HC1 to denature DNA.

Imrnunohistochernistry After DNA denaturation, the cells in the LabTek slides and tissue sections were rinsed three times for 5 minutes each in PBS and incubated with Br-3 diluted 1:250 in PBS containing 1% bovine serum albumin (BSA) and 0.5% Tween 20 for 1 hour at room temperature in a 100% humidified atmosphere. The slides were rinsed with PBS and covered with a 1:200 dilution ofbiotinylated antimouse immunoglobulin G. After 30 minutes, they were rinsed in PBS, incubated with a 1:100 dilution of avidin D H and biotinylated horseradJ. Neurosurg. / Volume 73/August, 1990

ish peroxide H complex (ABC)t for 30 minutes, 16 and then reacted for 5 to 10 minutes with 0.5 mg/ml of diaminobenzidine and 0.01% H202 in 0.05 M Trisbuffered saline (TBS). To measure the BUdR LI, the slides were counterstained with hematoxylin. For double staining with BUdR and IUdR, the slides were left overnight in 5% acetic acid to inactivate immunoglobulin G and incubated with IU-4 diluted 1:5000 in PBS containing 1% BSA and 0.5% Tween 20 for 1 hour at room temperature in a 100% humidified atmosphere. The slides were rinsed in TBS and covered with rabbit antimouse immunoglobulin for 30 minutes, rinsed again in TBS, and reacted with alkaline phosphatase-antialkaline phosphatase complex$ for 30 minutes at room temperature. The slides were then incubated for 5 to 10 minutes at room temperature in filtered substrate solution containing 5 mg of naphthol AS MX phosphate dissolved in 250 ul of dimethyl sulfoxide, 10 mg of fast blue BB base dissolved in 250 ul of 2 N HC1 and 250 izl of 4% sodium nitrite, and 10 mg of levamisole in 40 ml of 0.1 M Tris-HC1 buffer (pH 8.5). The BUdR-labeled cells contained brown reaction product, the IUdR-labeled cells stained blue, and double-labeled cells contained brown reaction product against a blue background (Fig. 1). To calculate the BUdR LI, approximately 3000 cells from viable areas in which the labeled cells were evenly distributed were examined in each tissue section. Approximately 1000 cells from monolayer cultures were examined.

Calculation of Ts and Tp The duration of the S-phase (Ts) was estimated using the equation Ts = t/dS, where "t" is the time interval between administration of I U d R and BUdR and dS is the fraction of S-phase cells that entered or left the Sphase during time "t." The fraction of cells that entered or left the S-phase (dS) was estimated as the ratio of cells labeled only with IU-4 (blue) to those labeled only with Br-3 or with both Br-3 and IU-4 (brown or mixed). Approximately 1000 labeled cells in each section or culture slide chamber were analyzed. The potential doubling time (Tp), which is the time required for a tumor to double its size in the absence of cell loss, was calculated as k x 100 x Ts/LI, where k is approximately 0.7 if the expected doubling time is over 50 hours and 0.8 if it is 30 to 50 hours? ~ Results

In Vitro Studies Double-labeling studies showed a d S value (dynamic fraction of the S-phase) of 0.21 (coefficient of variation t Vectastain ABC kit manufactured by Vector Laboratories, Budingame, California. $ Universal alkaline phosphatase-antialkaline phosphatase complex kit manufactured by DAKO Corp., Santa Barbara, California.

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FIG. 1. Photomicrographs showing the results of the double-staining technique in cultured 9L cells (left) and in a rat 9L tumor section (right). Nuclei stained blue are labeled only with iododeoxyuridine. Nuclei stained brown are labeled with bromodeoxyuridine only. Nuclei with brown reaction products against a blue background are double-labeled with both iodo- and bromodeoxyuridine. • 240.

TABLE 1 CellMn~wparam~e~ ~rat9Lbraintumo~ determin~ ~ ~ub& & b ~

w~hiod~ andbromodeoxyuridine*

Mode of Administration No. of Time Dynamic Fraction Labeling Duration of PotentialDoubling Group Studies Sequence Interval of S-Phase Index (%) S-Phase(hrs) Time (hrs) (hrs) 1 5 IUdR, BUdR 2 0.242 (0.16) 9.6 (0.27) 8.3 (0.16) 60.6 (0.31) 2 6 IUdR, BUdR 3 0.317 (0.18) 9.8 (0.21) 9.5 (0.18) 67.8 (0.28) 3 4 BUdR, IUdR 2 0.234 (0.18) 9.6 (0.28) 8.5 (0.18) 62.0 (0.33) 4 3 BUdR, IUdR 3 0.332 (0.15) 9.5 (0.20) 9.0 (0.16) 66.3 (0.25) * Values in parentheses represent the coefficient of variation. IUdR = iododeoxyuridine;BUdR = bromodeoxyuridine.

(cv) = 0.08) and an average LI of 22% (cv = 0.1). The Ts was calculated as 9.6 hours (cv = 0.01), and the Tp was estimated as 34.9 hours, assuming that k is 0.8. In Situ Studies Table 1 shows cell kinetic parameters in each group o f rats. The Ts was fairly constant, approximately 8.8 hours, despite the different sequences and time intervals in the administration o f I U d R and BUdR. There were no statistically significant differences in the Ts between groups. The average LI was approximately 9.6% _+ 0.1% (+ standard error o f the mean). The mean Tp was approximately 64.2 + 3.0 hours, assuming that k is 0.7.

Discussion The fraction of S-phase cells, the LI, and the growth fraction o f various tumors have been estimated immunohistochemically utilizing monoclonal antibodies that identify I U d R or B U d R incorporated into cellular D N A during D N A synthesis. 9,23 Using new, more spe256

cific antibodies against these halogenated pyrimidines, Shibui, et al., 19 developed a double-staining technique for analyzing cell cycle progression from a single specimen labeled with I U d R and B U d R at an appropriate interval. We have used this technique to measure the Ts and Tp o f the rat 9L brain-tumor model. The Ts, measured by the various combinations o f sequence and interval (2 to 3 hours) o f I U d R and B U d R administration in situ, yielded a fairly constant value (8.8 hours). This value was reasonably close to that o f 9L cells in vitro (9.6 hours), but was slightly higher than previously found in vitro (8.2 hours), 22 perhaps because the cells were almost confluent in our study. Thus, the Ts appears to be fairly constant whether cells are grown in situ or in vitro. The Ts determined in situ was also similar to that o f 9L tumors determined autoradiographically (7.6 to 10 hours). 1'7'13 Although the fraction cells entering or leaving the S-phase (dS) estimated by administering I U d R first followed by B U d R theoretically should be smaller than that estimated by administering these two agents in the reverse sequence (because o f the cell cycle age distribution o f exponentially

J. Neurosurg. / Volume 73/August, 1990

Double labeling for cell kinetics studies growing population), 2~ this slight difference was not detected in the current study. The Tp determined in situ in the present study, 64.2 hours, was higher than that measured earlier in this tumor model (42 hours). ~One reason for this difference is that the tumor LI in the current study was approximately 10%, whereas that of the earlier study was 18 %. There are two possible explanations for the lower LI: growth retardation due to biological changes in cells during the long interval between the two studies and a decreased growth fraction and/or a decreased regional blood flow in a tumor at its terminal stage. The calculated Tp (64.2 hours) seemed to contradict an actual doubling time (40 hours) reported previouslf ~because, theoretically, the Tp should be smaller than the actual doubling time. One explanation for this contradiction is that the actual doubling time was measured from an extrapolation of the growth curves (3 to 100 mg of tumor weight) 8 to 15 days after tumor implantation. After this period, the growth rate may become much slower. Because all tumors used in our study were excised 16 to 21 days after tumor implantation, the actual doubling time at this stage might be larger than at an earlier stage. The Ts determined by the double-labeling method was also fairly stable. Because it is calculated independent of heterogeneity in labeling or LI, it is not affected by the heterogeneity of tissue within a tumor. The LI itself does not directly indicate the rate of tumor growth, although it is important in estimating the Tp. Further accumulation of data regarding a relationship between the LI and tumor growth from various kinds of tumors is certainly required for a better understanding of the Ts. On the other hand, the Tp is a direct indicator of tumor growth. Although the Tp is fairly variable because it depends on the LI, which varies from site to site and at different times, the smallest and largest Tp's can be estimated from the labeled areas showing the greatest and least amount of labeling, and the average Tp could be calculated. We believe this double-labeling method provides improved characterization of tumor cell kinetics at any specified stage of tumor growth from a single biopsy specimen. Determining the proliferative potential of individual tumors is extremely important in predicting the prognosis precisely and in designing treatment modalities for individual patients with brain tumors. In the past several years, BUdR LI has been measured in many types of brain tumors. The BUdR studies appear to predict the proliferative potential of individual tumors more precisely than does histopathological diagnosis and may even correlate with survival better than with histology.2' 11,12The BUdR LI, however, does not predict the actual rate of tumor growth or other important cell kinetic parameters. Double-labeling studies with BUdR and IUdR allow some of these parameters to be determined very quickly from a single biopsy specimen. Such studies may help us to understand further the biological as well as the clinical nature of individual tumors. J. Neurosurg. / Volume 73/August, 1990

Acknowledgments

The authors thank Cheryl Christensen for manuscript preparation and Stephen Ordway for editorial assistance.

References

1. Barker M, Hoshino T, Gurcay O, et al: Development of an animal brain tumor model and its response to therapy with 1,3-bis (2-ehlorethyl)-l-nitrosourea. Cancer Res 33: 976-986, 1973 2. Cho KG, Hoshino T, Nagashima T, et al: Prediction of tumor doubling time in recurrent meningiomas. Cell kinetics studies with bromodeoxyuridine labeling. J Neurosurg 65:790-794, 1986 3. Dolbeare F, Kuo WL, Vanderlaan M, et al: Cell cycle analysis by flow cytometric analysis of the incorporation of iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd). Proc Am Assoc Cancer Res 29:477, 1988 (Abstract) 4. Gratzner HG: Monoclonal antibody to 5-bromo- and 5iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474-475, 1982 5. Hoshino T, Barker M, Wilson CB, et al: Cell kinetics of human gliomas. J Neurosurg 37:15-26, 1972 6. Hoshino T, Barker M, Wilson CB: The kinetics of cultured human glioma cells. Autoradiographic studies. Acta Neuropathol 32:235-244, 1975 7. Hoshino T, Gray JW, Nomura K: Flow cytometry of isolated nuclei prepared from 9L rat brain tumor. Lab Invest 41:72-76, 1979 8. Hoshino T, Nagashima T, Cho KG: S-phase fraction of human brain tumors in situ measured by uptake of bromodeoxyuridine. Int J Cancer 38:369-374, 1986 9. Hoshino T, Nagashima T, Murovic JA: Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine. Cytometry 6:627-632, 1985 10. Hoshino T, Nagashima T, Murovic JA, et al: In situ cell kinetics studies on human neuroectodermal tumors with bromodeoxyuridine labeling, d Neurosurg 64:453-459, 1986 11. Hoshino T, Prados M, Wilson CB, et al: Prognostic implications of the bromodeoxyuridine labeling index of human gliomas. J Neurosurg 71:335-341, 1989 12. Hoshino T, Rodriguez LA, Cho KG, et al: Prognostic implications of the proliferative potential of low-grade astrocytomas. J Neurosurg 69:839-842, 1988 13. Hoshino T, Tel E, Barker M, et al: Therapeutic challenges to a rat brain tumor model. Neuroi Med Chit 16: 201-206, 1976 14. Hoshino T, Townsend J J, Muraoka I, et al: An autoradiographic study of human gliomas: growth kinetics of anaplastic astrocytoma and glioblastoma multiforme. Brain 103:967-984, 1980 15. Hoshino T, Wilson CB, Ellis WG: Gemistocytic astrocytes in gliomas. An autoradiographic study. J Neuropathol Exp Neurol 34:263-281, 1975 16. Hsu SM, Raine L, Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, d Histochcm Cytochem 29: 577-580, 1981 17. Nagashima T, Hoshino T, Cho KG, et al: Comparison of bromodeoxyuridine labeling indices obtained from tissue sections and flow cytometry of brain tumors. J Neurosurg 68:388-392, 1988 18. Rosenblum ML, Knebel KD, Vasquez DA, et al: Brain257

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23. Yoshii Y, Maki Y, Tsuboi K, et al: Estimation of growth fraction with bromodeoxyuridine in human central nervous system tumors, J Nenrosurg 65:659-663, 1986 Manuscript received October 3, 1989, This study was supported in part by Grant PDT-159 from the American Cancer Society and by Grants CA 50210 and CA 13525 from the National Cancer Institute. Address reprint requests to: Takao Hoshino, M.D., Department of Neurological Surgery, c/o The Editorial Office, 1360 Ninth Avenue, Suite 210, San Francisco, California 94122.

J. Neurosurg. / Volume 73/August, 1990

Cell kinetics of rat 9L brain tumors determined by double labeling with iodo- and bromodeoxyuridine.

Rats with 9L brain tumors received intraperitoneal injections of iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) to estimate the duration of the ...
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