Soft Agar Culture of Human Transitional Cell Carcinoma Colonies from Urine

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1 Sof Agar Culure of Human Transiional Cell Carcinoma Colonies from WM J. KRKELS, M.D., OLGA E. PELGRM, MBO, FRANS M. J. DEBRUYNE, M.D., PH.D., G. PETER VOOJS, M.D., PH.D., AND CHESTER J. HERMAN, M.D., PH.D. Sof-agar culure of ransiional cell carcinoma colonies from urine, bladder irrigaion fluid, and ransurehral resecion solid umor specimens demonsrae generally beer growh of umor cell colonies from urine han from irrigaion fluid. Growh of umor colonies from urine was adequae o evaluae presence of umor and o sudy growh parameers of he umor colonies in viro, alhough he number of colonies produced from urine was inadequae o evaluae sensiiviy or resisance o chemoherapeuic agens. Growh in sof agar of ransiional cell carcinoma colonies from urine may offer a simple, noninvasive mehod of evaluaing he effeciveness of herapy and he prognosis for umor progression. (Key words: sem cells; Sof-agar culure; Clonogenic cell culure; Transiional cell carcinoma; Bladder cancer) Am J Clin Pahol 982; 78: SNCE THE REPORT of Hamburger and Salmon 3 describing he culure echnics of umor "sem" cell colonies from humans in a double-layer sof-agar sysem, his cloning sysem (HTCS) of umors from humans has become a popular invesigaional ool o deermine resisance or sensiiviy of individual paiens' umors o chemoherapeuic agens in viro Preliminary daa sugges ha his mehod may prove very helpful in predicing paiens' response in vivo o chemoherapy regimens. 2 n addiion o deermining chemoherapy sensiiviy and resisance a leas wo oher poenial applicaions of his umor cell culure mehod have been suggesed (Herman and colleagues: Arch Pahol Lab Med, n Press). Growh of umor colonies in sof agar may provide a means of assessing he effeciveness of chemo- or radiaion herapy by deermining he presence of "viable," i.e., clonogenic, umor cells in pos-herapy biopsy specimens. n addiion, he parameers of growh in sof agar may be a predicor of umor behavior, i.e., prognosis, especially in hose umors, such as ovarian epihelial and papillary ransiional cell, where he borderline beween "benign" and malignan is no sharply discernible by rouine pahologic examinaion. Furher invesigaion and poenial rouine clinical applicaion of he cloning sysem of umors from humans in hese and oher problems in clinical oncology will be faciliaed considerably by he developmen of echnics for obaining qualiy umor cells for culure Received January 2, 982; acceped for publicaion April 3, 982. Address reprin requess o Dr. Herman: Deparmen of Anaomic Pahology, Radboud Hospial of Caholic Universiy, Nijmegen, Neherlands. Deparmens of Urology and Anaomic Pahology, Radboud Hospial of Caholic Universiy, Nijmegen, Neherlands wih minimal rauma o he paien. So far, mos repors dealing wih culure of umors from humans have used umor biopsies as a source of umor cells. ncreasing use has been repored of effusions and irrigaion fluids. ' 6 " The presen work repors he culure of ransiional cell carcinoma colonies from urine specimens of humans and compares he resuls of umor cell culure from urine wih resuls of culure of bladder biopsy and irrigaion specimens. Maerials and Mehods Tweny paiens admied o he Radboud Hospial for ransurehral resecion of a ransiional cell bladder umor (newly diagnosed or recurren) formed he populaion of he presen series. From hese 20 paiens, 8 solid umors, 2 urine samples, and 2 bladder irrigaion fluid samples were obained for HTCS. The urine was colleced in a serile urine bag before he resecion was sared, hrough he cysoscope sheah. Afer he urine collecion, he bladder was filled wih ml 5% glucose in waer. Then he bladder was genly lavaged wih a Thoomy syringe. This irrigaion fluid was also colleced in a serile urine bag. Following urine and irrigaion fluid collecion, he ransurehral resecion was sared. Approximaely -2 g umor issue was aken for HTCS afer ensuring ha adequae, represenaive maerial was aken for rouine hisopahologic sudy. For HTCS, he exophyic porion of he umor was aken in mos cases. Afer collecion, he urine and irrigaion fluid were brough o he Pahology laboraory and ransferred o serile 50-mL cenrifuge ubes. The samples were spun for 0 min a,500 rpm, he supernaan discarded, and 0-20 ml McCoy's wash was added o he cell pelle. Afer horough vorexing, he cell suspensions were combined ino one ube, urine and irrigaion fluid separaely. Afer anoher washing of he cells, McCoy's wash was added o he cell pelle, 2-5 ml according o he esimaed number of cells obained, and he specimens were processed furher as described below for he cell suspensions obained from solid umor. The solid umor was brough o he Pahology laboraory in serile 0.9% NaCl soluion and furher pro /82/00/0690 $0.05 American Sociey of Clinical Pahologiss Downloaded from hps://academic.oup.com/ajcp/aricle-absrac/78/5/690/79075 by gues on 05 Sepember

2 Vol. 78 No. 5 TUMOR COLONY CULTURE FROM URNE Table. Human Transiional Cell Carcinomas Culured in HTCS: Clinical and Pahologic Daa and Culure Resuls 69 Paien Number Age/Sex Commens Grade Pahologic Sage Culure Plaes Colony Coun per Plae Mean (Range) 3/m TUR Cysecomy (afer 2000 rad radiaion) Tlb-T2N0M0 7/3 37/6 4/3 nfecion nfecion No growh /m recurrence 65/m 53/m 70/m recurrence 70/f 56/m recurrence (afer adriamycin herapy) 69/m 64/m 76/m 53/m same paien as number 4, compleion of resecion 74/f 62/f 76/m 48/f 46/m 47/m 0 TO T2NXM+? pt2 TNXM0 TA-TNXM0 TNXM0 pt2 ptl T- T- rrigaion Fluid less han /0 3/3 5/6 0 78/6 5/6 0/6 78/6 5/3 9/5 43/4 2/6 5/3 2/2 6/6 8/6 /6 36/3 94/3 49/3 32/6 23/6 53/3 6/6 6/3 27/6 72/3 3/3 206/9 84/9 2? Growh posiive^ Growh posiive:): 28(23-40) 9(6-3) 77(6-05) Growh posiive:): 0(4-6) 9(3-29) 26(2-45) No couned, bloody background. No couned, bloody background 4 (0-8) 25(3-33) More han 00 More han 00 32(30-34) (0-3) 5 (2-6) 5(6-) 52(-04) 7(5-20) nfecion nfecion 75(7-78) 2? 7 (5-39) Downloaded from hps://academic.oup.com/ajcp/aricle-absrac/78/5/690/79075 by gues on 05 Sepember /f T3NXM+ 34/9 77(50-89) 9 76/f TNXM0 34/ /m 33/3 2 35/m 59/3 nfecion Recurrence during adriamycin herapy 5 X 0' large nucleaed cells per culure plae. Number of possible culures from he specimen/number of culure dishes acually se up. Mean and range of colony couns. % Growh clearly recognizable, colonies could no be couned because of bacerial or fungal infecion.

3 A.J.C.P. November 982 FG. 2 (righ). Colony of ransiional cell carcinoma from he same umor illusraed in Figure afer 2.5 weeks of growh in sof-agar culure. The cyologic and "hisologic" feaures of he colony reproduce hose of he umor cell nes in he biopsy maerial of Figure. PAS (X40). cessed as recenly described in deail.9 n brief, he issue was minced wih scissors for 0 min in McCoy's wash hrough a meal sieve wih 60 holes per inch. The suspension of cells was hen syringed once manually hrough a 25 g in needle and washed wice in McCoy's wash. Trypan-blue-excluding cells were deermined using 0.-mL cell suspension and 0. ml rypan blue. The cell concenraion was deermined in a Burker-Turk hemocyomeer. The concenraion of large nucleaed cells was hen adjused o 3 X 06 cells per ml McCoy's wash. Culure dishes (35 mm) were seeded wih 5 X 05 large nucleaed cells per dish in double-layer sof agar exacly as described elsewhere, 30 excep ha condiioned medium was no used. Culure dishes were examined wihin hours afer plaing o evaluae presence of clumps seeded ino culure and rule ou infecion of he culure. Colonies of umor cells were firs examined one week afer plaing. Par of he cell suspension no used for plaing was air-dried and sained by he Papanicolaou echnic o assess cell morphology and he ypes of cells brough ino culure. Deailed morphologic sudies of he colonies and oher srucures in culure were performed on agar layers mouned on sandard glass slides8 and sained wih he Periodic acid Schiff reacion. From seleced culures, paraffin secions were made of colonies, sained wih hemaoxylin and eosin,7 and he umor colony morphology was compared wih he umor hisology in he original biopsy specimens. Resuls Table summarizes he paien and culure daa in he presen series. Of he oal 42 samples culured, 25 showed growh in he sof-agar sysem, growh being defined as five or more colonies composed of 30 or more umor cells per culure plae. An addiional seven culures could no be evaluaed because of bacerial or fungal infecion of he culures or a markedly bloody background. The remaining 0 culures showed no growh. Of he 2 urines culured, eigh showed growh, which compares favorably wih he 2 of 8 umor samples which showed growh, and is noably beer han he five of 2 specimens from irrigaion fluid which showed evaluable growh. A leas one culure plae from each sample was evaluaed afer erminaion of he culure by examinaion Downloaded from hps://academic.oup.com/ajcp/aricle-absrac/78/5/690/79075 by gues on 05 Sepember 208 FG. (lef). Nes of ransiional cell carcinoma (Grade, Sage ptl) in bladder lamina propria of 52-year-old man. Hemaoxylin and eosin, (X40).

4 Vol. 78 No. 5 TUMOR COLONY CULTURE FROM URNE 693 of a PAS-sained agar layer conaining colonies. n all cases he morphologic feaures of he cells comprising he umor colonies in agar were clearly recognizable as being hose of ransiional cell carcinoma. n mos bu no all cases, he hiso- and cyopahologic grading of he umor as grown in agar agreed wih he grade of umor as seen in he hisologic maerial. Figure shows an invasive ransiional cell carcinoma in hemaoxylinand-eosin-sained, paraffin block maerial. Figqre 2 is a colony of umor cells from he same umor afer 2.5 weeks in agar culure. Figure 3 is a hemaoxylin-andeosin-sained, paraffin secion of a colony similar o ha shown in Figure 2. The cyologic and hisologic feaures of he cell ness are comparable. Table 2 shows he correlaion of colony growh in agar wih hisopahologic grading of he umor. Table 3 shows he correlaion of colony growh wih umor sage (pt) according o he UCC crieria. 4 These daa demonsrae ha, in general, more colonies are produced in agar from umor samples han from eiher urine or irrigaion fluid. However, in general, urine seems o produce evaluable growh more reliably han irrigaion fluid. Alhough he number of samples esed is small, he number of colonies per culure plae pro- FG. 3. 4ji secion of paraffin-embedded colony of ransiional cell carcinoma, similar o he inac colony shown in Figure 2. Hemaoxylin and eosin, (X50). Table 2. Correlaion of Grade wih Number of Colonies Produced in Sof Agar (Mean; Range) Grade rrigaion Fluid 0 2 (?) 9(3-29) owh negaive 25(3-33) 5(6-) 28 (23-40) 32 (30-40) 4 (8-0) 0(4-6) 52(-04) 9(6-3) (0-3). 26(2-45) 75(7-78) 7(5-39). 7(5-20) 77 (50-89) 77(6-05) 5 (2-6) No couned because of markedly bloody background in culure. No evaluable because of bacerial or fungal infecion in culure. $ More han five umor cell colonies clearly recognizable, bu reliable couning was no possible because of bacerial or fungal infecion. No growh afer rad radiaion o he bladder (same paien as he firs line of he group). duced from urine samples shows a rend oward correlaion wih grade and sage of umor, a rend no idenifiable in culures of irrigaion fluid or solid umor specimens. Discussion The presen daa demonsrae ha evaluable growh of human ransiional cell carcinoma colonies in doublelayer sof-agar culure can be obained from urine samples. The number of colonies per sample and he percenage of samples producing colonies compares favorably wih sof-agar culure of bladder irrigaion fluid, hough hese parameers of growh are inferior o culure of a solid umor obained by ransurehral resecion. The number of colonies produced from urine samples in sofagar culure ends o correlae wih he pahologic grade and sage of he umor. The evaluaion of sensiiviy or resisance of umors o chemoherapeuic agens in viro is he mos echnically demanding applicaion of he cloning sysem of umors from humans. n addiion o demanding low beween-plae variabiliy of number of colonies and low infecion raes, his applicaion requires growh of a leas colonies per conrol (non-drug-incubaed) culure plae o reproducibly recognize significan umor sensiiviy o a chemoherapeuic agen in viro. For his applicaion of HTCS, he use of urine samples as repored here is sill inadequae. Downloaded from hps://academic.oup.com/ajcp/aricle-absrac/78/5/690/79075 by gues on 05 Sepember 208

5 694 Table 3. Correlaion of Sage wih Number of Colonies Produced in Sof Agar (Mean; Range) Sage pto pta pta-ti ptl ptl-t2 PT2 rrigaion Fluid 9(3-29) 5 (6-) 28(23-40) 25(3-33) 32 (30-34) 0(4-6) 4 (0-8) 52(-04) 9(6-3) (0-3) 26(2-45) 7(5-20) 75(7-78) 7 (5-39) 77(6-05) 5(2-6) 77(50-89) Nol couned because of markedly bloody background in culure. No evaluable because of bacerial or fungal infecion in culure. % More han five umor cell colonies dearly recognizable, bu reliable couning was no possible because of bacerial or fungal infecion.. No growh afer 2,000 rad radiaion o he bladder (same paien as he firs line of he ptl-t2 group). However a second applicaion of umor cell culure, assessing he effecivenes of herapy by deermining he presence of "viable" or clonogenic umor cells afer herapy is clearly feasible using urine samples. A posiive sof-agar culure following herapy of a ransiional cell carcinoma clearly indicaes ha umor cells wih demonsrable proliferaive capaciy remain. How his informaion can be used o improve clinical managemen of paiens wih ransiional cell carcinoma mus be explored furher by parallel and prospecive clinical rials. The inerpreaion of a negaive pos-herapy sof-agar culure is made difficul by he relaively low growh raes of umors in sof agar. 9 Furher improvemen of he echnics of HTCS may provide a parial soluion o his problem. n addiion, he fac ha collecion of urine samples is non-invasive makes frequen culuring possible and may in par negae some of he problems of low growh rae in culure (comparable o blood culures insbe). The hird applicaion of sof-agar culure of ransiional cell carcinoma, evaluaion of prognosis, clearly deserves furher aenion. The presen daa sugges a correlaion of number of colonies produced in culure wih pahologic grade and sage of he umor. f in fac KRKELS T/4L. A.J.C.P. November 982 furher daa suppor his correlaion, he culuring of repeaed urine samples over ime may provide a relaively easy, non-invasive echnic for monioring paiens, especially hose wih low-grade papillary ransiional cell umors, for signs of progression, especially early invasion. For his purpose, he availabiliy of an auomaed umor colony couner 5 offers he poenial for more sophisicaed evaluaion of umor colony growh in culure. The availabiliy, from he auomaed couner, of oher parameers of colony growh, such as size disribuion of colonies, rae of change of colony size disribuion wih ime, and early rae of colony developmen, will provide objecive quaniaive measures of biologic behavior of umors in viro. These measures should provide a basis for comparison of in viro growh wih in vivo behavior ha is boh more varied and more reliable han manual observaion and couning. 5 The presen sudy shows ha he HTCS echnic is easily implemened in, and inegraed ino, he rouine. work flow of a pahology deparmen, requiring lile more in personnel and space han a very modes microbiology laboraory. The developmen of noninvasive echnics for obaining umor samples, as well as he rouine sof-agar culuring of umor samples obained for oher purposes, will allow his mehod o be evaluaed furher for poenial conribuions o solving a variey of paien managemen problems in clinical oncology. 0. References Buick RN: Developmen of an agar-mehylcellulose donogenic assay for cells in ransiional cell carcinoma of human bladder. Cancer Res 979; 39: Hamburger AW: Use of in viro ess in predicive cancer chemoherapy. J Nal Cancer ns 98; 66: Hamburger AW, Salmon SE: Primary bioassay of human umor sem cells. Science 977; 97: Harmer MH, ed: TNM classificaion of malignan umours. Geneva, UCC, 978 Kressner BE, Moron RRA, Marens AE, Salmon SE, Von Hoff DD, Soehhlein B: Use of an image analysis sysem o coun colonies in sem cell assays of human umors. Cloning of Human Sem Cells. Edied by Salmon SE. New York, Alan R Liss, 980, pp Ozols RF, Willson JKV, Grozinger KR, Young RC: Cloning of human ovarian cancer cells in sof agar from malignan effusions and perioneal washings. Cancer Res 980; 40: Pavelic ZP, Vaughan MM, Slocum HK, Rusum YM: Hisopahologic echnique for he morphological examinaion of fixed or frozen solid umor cells in sof agar. Sain Technol 98;56: Salmon SE: Preparaion of permanen slides of inac sof agar colony culures of hemaopoieic and umor sem cells. Cancer Res 979;39:33-36 Salmon SE, ed: Cloning of Human Sem Cells. New York, Alan R Liss, 980 Salmon SE, Hamburger AW, Soehnlein B, Durie BGM, Albers DS, Moon TE: Quaniaion of differenial sensiiviy of human-umor sem cells o anicancer drugs. N Engl J Med 978; 298: Sanisic TH: An in viro clonal assay for bladder cancer: clinical correlaion wih he saus of he urohelium in 33 paiens. J Urol 980; :30-33 Downloaded from hps://academic.oup.com/ajcp/aricle-absrac/78/5/690/79075 by gues on 05 Sepember 208