Analysis of cell surface proteins delineates a differentiation pathway linking endocrine and nonendocrine human

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1 Poc. NatL Acad. Sci. USA Vol. 8, pp , June 1983 Medical Sciences Analysis of cell suface poteins delineates a diffeentiation pathway linking endocine and nonendocine human lung cances [small (oat) cell cance/lage cell cance/two-dimensional electophoesis] GREGORY GOODWIN*, JOEL H. SHAPER*t, MARTIN D. ABELOFF*1, GEOFFREY MENDELSOHN, AND STEPHEN B. BAYLIN*t *Oncology Cente, and Depatments of WMedicine, Pathology, and tphamacology and Expeimental Theapeutics, The Johns Hopkins Univesity School of Medicine, Baltimoe, Mayland 2125 Communicated by Victo A. McKusick, Mach 14, 1983 ABSTRACT We have peviously detemined that the cell suface potein phenotype distinguishes human small cell lung cacinoma (SCC), a neoplasm with endocine popeties, fom non- SCC in cultue. We now demonstate that cloned cell cultues of human lage cell undiffeentiated lung cacinoma, established diectly fom a patient with mixed SCC and non-scc, simultaneously expess suface poteins chaacteistic of SCC and non- SCC lung cance cells. Hence, SCC and a fom of lage cell cacinoma appea linked though a continuum of diffeentiation events that: (i) may explain clinically impotant tansitions that occu between the majo types of human lung cance and (ii) povide evidence fo a common cellula oigin of endocine and nonendocine cells in the bonchial mucosa. Thee majo histologic foms of diffeentiated lung cance aise in the complex bonchial epithelium of man: squamous, adeno-, and small cell cacinomas (1). A fouth majo categoy, lage cell undiffeentiated cacinoma, encompasses a goup of lung neoplasms that do not exhibit mophologic diffeentiation featues of the othe thee (1). Evidence has accumulated to suggest that these majo foms of human lung cance may be linked along a diffeentiation continuum (2, 3). Such a postulation is of paticula biologic and clinical impotance with espect to the elationship between small cell cacinoma (SCC) and the othe histologic foms of lung cance and fo soting out the pecise cell lineages of lage cell undiffeentiated cacinomas. SCC is a viulent cance that usually exhibits neuoendocine popeties, including the synthesis of biologically active peptide homones (4), capacity fo biogenic amine synthesis (3, 5, 6), pesence of neuon-specific enolase (7, 8), and the pesence of cytoplasmic neuosecetoy ganules (9, 1). These neual popeties initially led to the speculation that SCC aises fom lung endocine cells that ae embyologic deivatives of the neual cest (11), wheeas non- SCC cances aise fom endodemally deived nonendocine bonchial mucosal cells. Howeve, ecent data have demonstated that neuoendocine featues can be found in non-scc lung cances as well. Caeful analyses of neuoendocine-elated biochemical popeties in all the majo histologic foms of lung cances eveal the pesence of immunoeactive peptide homones and amine synthesis capacity in non-scc lung cances, although concentations ae usually less than those detected in SCC (12). Futhemoe, mophological tansitions between SCC and non-scc lung cances may occu. In some patients, with an initial di- The publication costs of this aticle wee defayed in pat by page chage payment. This aticle must theefoe be heeby maked "advetisement" in accodance with 18 U.S.C solely to indicate this fact. 387 agnosis of SCC, a complete change fom SCC to the othe diffeentiated foms of non-scc histology has been documented (13, 14). In established SCC cell cultues, Gazda et al. have obseved a time-dependent change in which SCC cells lose thei endocine featues and, in nude mouse heteotansplants, change fom SCC histology to typical lage cell undiffeentiated cacinoma (3). In egad to the above cell cultue changes descibed by Gazda et al (3), it is then of paamount biologic and clinical impotance to detemine whethe: (i) the lage undiffeentiated cacinoma cells in SCC cultues ae simply selected fo with time and have no diect lineage elationship to SCC; (ii) these cells ae linked diectly to SCC cells though a pocess of diffeentiation; and (iii) such cells ae capable of pluipotent expession with espect to the entie spectum of diffeentiated human lung cance cell types. In the pesent wok, we addess these questions by analyzing the cell suface poteins of a unique human cell cultue line ecently established in ou laboatoy. We have ecently epoted that a distinct cell suface potein phenotype distinguishes SCC fom the non-scc lung cacinomas in cultue (15). We now epot that such analysis of suface poteins may: (i) define diffeentiation pathways that may mediate clinically impotant tansitions between the majo foms of human lung cance; and (ii) claify the histogenesis of nonneoplastic endocine cells esiding in pedominantly nonendocine epithelial systems. We thus pesent evidence at a molecula level that SCC is indeed diectly elated to a fom of lage undiffeentiated tumo cell that may have pluipotent diffeentiation capabilities fo the majo foms of diffeentiated lung cance. MATERIALS AND METHODS Cell Cultues. Line OH-2 was established fom the pleual effusion of a patient diagnosed as having SCC. The pleual fluid, obtained by thoacentesis, was hepainized, tansfeed unde steile conditions to 5-ml plastic centifuge tubes, and centifuged at 2 X g fo 5 min. Cell pellets wee then pooled and washed twice in phosphate-buffeed saline and finally esuspended in gowth media containing the following ingedients: RPMI 164 medium, 16% fetal calf seum (B & B Reseach, Fiskeville, RI), 2 mm glutamine, penicillin at 6 units/ml, and steptomycin at 6 ttg/ml (GIBCO). Cells wee gown at 37C in 5% CO2 in ai. All othe SCC and non-scc cultues wee maintained and chaacteized as descibed (3, 6, 15). Line OH-2 was found to be fee of mycoplasma (test pefomed by Micobiological Associates) and HeLa cell contam- Abbeviation: SCC, small cell cacinoma.

2 388 Medical Sciences: Goodwin et al. ination as detemined by glucose-6-phosphate dehydogenase isoenzyme studies (16). Cell Cloning. Single cell suspensions wee obtained by teating confluent flasks of OH-2 cells with.25% typsin. In steile Costa Peti dishes (35 X 1 mm), 1 X 13 viable cells wee plated in 1 ml of.85% SeaKem 15/45 aga (Maine Colloids, Rockland, ME) (clones 1-5) o.3% GIBCO aga (clones 6-19) made up with RPMI 164 medium and 16% fetal calf seum (B & B Reseach), 2 mm glutamine, penicillin at 6 units/ml, steptomycin at 6,ug/ml, and 25% conditioned medium fom OH-2 cells plus human placental extact pepaed exactly as descibed (17) at.1 ml pe plate. Clones, consisting of >5 cells, wee caefully emoved fom aga unde phase-contast micoscopy by using steile plastic Pipetman tips (2-2 Al; Rainin) attached to a Pi Pump (2 ml; Glasfin). Individual explanted clones wee then placed in 96-well cultue plates (Costa) with.5 ml of medium until cells eached confluency. Cells wee then tansfeed to 24-well Costa plates and, when confluent, to 75-cm2 flasks fo stock cultues. Analysis of Cell Suface Poteins. Mateials and expeimental pocedues ae as descibed (15). Biefly, the cell suface poteins of intact viable stationay-phase cells wee adioiodinated (Na125I, caie-fee, Amesham/Seale) by the pocedue of Fake and Speck (18) as modified by Makwell and Fox (19). Metabolic labeling of cells was accomplished by Poc. Natl. Acad. Sci. USA 8 (1983) incubating 5 x 16 cells fo 12 h in 2 ml of seum-fee medium containing 25-5 AQCi (1 Ci = 3.7 X 1' Bq) of [35S]methionine (1,1 Ci/mmol; Amesham) as the only souce of methionine. Tichlooacetic-acid pecipitable potein of both 1"Iand [3S]methionine-labeled cells was then detemined and aliquots wee analyzed by the two-dimensional polyacylamide gel electophoesis pocedue of O'Faell (2). Labeled poteins wee then visualized by adioautogaphy of the seconddimension slab gels (15). nude Mice Heteotansplants. nude mice heteotansplants wee obtained by inoculating cultued OH-2 cells (2-5 X 16 viable cells) into the ea flanks of BALB/c athymic nude mice. Tumos wee excised afte 3-4 weeks, potions wee immediately fixed in 1% phosphate-buffeed fomalin fo hematoxylin and eosin histologic analysis, and potions wee homogenized in.1 M sodium phosphate buffe, ph 6.8, fo subsequent biochemical assays (12). Histaminase and L-Dopa Decaboxylase Radioassays and Calcitonin and fi-endophin Radioimmunoassays. All assays wee conducted on cell sonicates and tumo tissue homogenates exactly as descibed in ef. 12. RESULTS Chaacteization of Cultue Line OH-2. The cells taken fom the pleual fluid adapted eadily to cell cultue, showing evi- A*Wa. i,i n 3 %*HeGof At 'f.m of S jt *:*- w a d + 1.po. - Y*4 46;. 9 *a a k., A.' ~~~, *A i to,,.' -4~ *~,' 4C 14 a' ~- 4.>i,-4 t- f kaa At- : - AW3N F' Pi.. *,n '* 9' ta 4't-;^.1I a -t :, d * I!. 1, ' _... :.Y * t*. * d a İ 6 v 6 I,. " i... FIG. 1. Biopsy, autopsy, and heteotansplanted nude mouse tumo tissues fom a patient with an initial diagnosis of SCC ofthe lung. (A) Oiginal biopsy of lung mass showing SCC. Note the chaacteistic appeaance of the cells, including small hypechomatic nuclei and scant cytoplasm. (B) Autopsy tissues obtained 7 months afte the oiginal biopsy again evealed SCC histology in both pimay and metastatic tissues. (C) Caeful examinations of multiple histological sections at autopsy evealed foci of lage, undiffeentiated cells chaacteized by lage vesicula nuclei with occasional nucleoli and modeately abundant cytoplasm. (D) Infequent aeas of tumo tissues demonstated featues of squamous cell cacinoma diffeentiation with well-developed keatin peals (aows). (E) Appeaance of cells cultued fom the pleual effusion of this patient obtained 4 weeks pio to autopsy. Note the compaison of these cells to the moe typical suspended aggegate gowth of SCC shown in F (line OH-1). Note also the lage nuclei and nucleoli simila to those of the lage undiffeentiated cells in autopsy mateial (C) and nude mouse explants descibed below (G). (G) Histology of nude mice heteotansplants fom the paent and five of five OH-2 cloned cultues, evealing lage cell undiffeentiated cacinoma mophology, chaacteized by lage vesicula nuclei, pominent nucleoli, and abundant cytoplasm. (A, B, C, D, and G, hematoxylin and eosin, x55; E and F, phase contast, x 1,2.)

3 dence of gowth within 1-2 weeks. Afte seveal days, these cultued cells failed to demonstate the suspended cell aggegate gowth patten chaacteistic of SCC in vito (3, 6); but athe, they gew as an anchoage-dependent monolaye (Fig. 1 E and F). Futhemoe, OH-2 cells completely lacked the high levels of L-dopa decaboxylase activity that typically distinguish SCC cells in vito fom non-scc lung cance (5, 6). In nude mouse heteotansplants, both ealy (passage 6) and late (paskda l l Medical Sciences: do. H 'i. i I Goodwin et al M i....;vm--4 A * ' t K M N 7. Poc. Nati Acad. Sci. USA 8 (1983) 389 sage 37) passages of OH-2 cells gew typical lage cell undiffeentiated lung cacinomas (Fig. 1G). Analysis of Autopsy Tissues. Fou weeks afte these cells wee obtained, the patient expied and autopsy evealed pedominantly SCC histology as shown in Fig. LB. Howeve, a caeful eexamination of multiple sections of pimay and metastatic tumo evealed small foci of cells with histologic popeties vey simila to those seen in the nude mouse explants, ph.- U,- 4-U H. RS T A l c D%..m. J * L--Z-.-.- m a R s T P 7 M~~~~~~ f d -1 %- CD~~~~~-..E D( C D 44 E F FIG. 2. Two-dimensional polyacylamide gels of lmi-labeled suface poteins and [3S]methionine-labeled total cellula poteins fom cultues of SCC, non-scc, and paent and cloned cultues ofline OH-2. kda, Kilodaltons; ph values ae shown acoss the top. (A andb) The suface labeling patten typical of the paent (A) and cloned cells (B) of line OH-2 exhibited all of the SCC poteins (15) that distinguish SCC (backeted egions in C) fom non-scc lung cances (D). These SCC poteins ae in egions E, F, G, U, I, Q, R, S, and T. Some suface poteins chaacteistic of non- SCC lung cance cells (D) ae also visualized on OH-2 cells (A andb) and include the high molecula weight poteins seen above the A egion and poteins numbeed 8 and 9. (C) The "WI suface labeling patten fom line OH-1 (2), which is chaacteistic of the patten found fo six established lines of SCC lung cance. (D) The gel shown fo the suface poteins of non-scc lung cances is that fo line NCI-HUT 125 of lung adenocacinoma. This patten is identical to that fo one additional line each of lung adenocacinoma and squamous cacinoma. Note the absence of the SCC-specific poteins fom the backeted aeas and the pesence of poteins 1-1 and the high molecula weight poteins above potein A. (E-H) The [35S]methionine-labeled potein synthesis pattens fo SCC, non-scc lung cance cells, and OH-2 cultued cells ae shown. The egions shown ae esticted to potein egions E and F of the second-dimension gel. Note the pesence of potein E (aow) in the potein synthesis pool of SCC (E), its absence fom non-scc lung cances (F), and its pesence in intemediate amounts in both paent and cloned cultues of OH-2 (G and H). The intemediate levels of E coelated with a moe vaiable intensity of 'ml labeling fo thipotein on the suface of OH-2 cells (A andb) thanon typical SCC cells (C). G I _b H

4 381 Medical Sciences: Goodwin et al. including lage vesicula nuclei and abundant cytoplasm (Fig. 1C). Impotantly, foci of pooly diffeentiated squamous cell cacinoma (Fig. 1D) with well-developed squamous peals wee also seen. Multiple biochemical analyses of the autopsy tumo (most of which contained typical SCC histology) failed to eveal significant concentations of fou biochemical makes (L-dopa decaboxylase activity, diamine oxidase activity, /3-endophin, calcitonin), one o moe of which ae chaacteistically pesent in high quantities in SCC tissues (12). Cell Suface Potein Analysis of OH-2 Paent Cells and Clones. In ode to assess the pecise elationship of the cultued OH-2 cells to the tumo histologies obseved in the patient, we obtained and expanded 19 clones fom single cultued cells gown in soft aga. Five of five tested clones maintained the lage cell undiffeentiated histology shown in Fig. 1G when gown as heteotansplants in the nude mouse. Multiple passages (5 though 44) of the paent cells (Fig. 2A) and all 19 OH- 2 clones (Fig. 2B) expessed the complete cell suface potein phenotype that chaacteizes SCC (Fig. 2C). Impotantly, each clone of OH-2 cells and the paent line also expessed, simultaneously, some of the suface poteins chaacteistic of cultued non-scc lung cances (15). Seveal poteins of molecula weight >1,, which ae heavily labeled on non-scc lung cacinoma cells (above the A egion in Fig. 2D) but not on SCC cells (Fig. 2C), wee consistently demonstated on the OH-2 cells (Fig. 2 A and B). Non-SCC poteins (designated as 8 and 9),wee also visualized on cloned and paent OH-2 cultues (Fig. 2 A and B). In ou initial studies (15), we had detemined that at least one suface potein, designated E, may be a unique gene poduct fo SCC, as analyzed by metabolic labeling with [3S]methionine (Fig. 2E). Potein E was not detected in non-scc lung cance cells o human fiboblasts (Fig. 2F). In the cuent study, potein E was pesent in amounts intemediate to those fo SCC and non-scc cells in the electophoetic pattens of poteins fom paent cultues (Fig. 2G) and fou of fou OH-2 clones incubated with [3S]methionine (Fig. 2H). Potein E was also detected by silve stain (21) in two-dimensional gels of all othe OH-2 clones examined to date (eight of eight). DISCUSSION The data fo cultue line OH-2, paticulaly the clonal etention of SCC suface poteins, stongly suggest that a fom of lage cell undiffeentiated lung cance is diectly elated to SCC though events in cell diffeentiation. This conclusion is based on the assumption that the suface potein phenotype peviously descibed fo SCC cells (15) is diectly detemined by the diffeentiation lineage of these cells, athe than simply eflecting the gowth chaacteistics, such as the ound cell shape and lack of anchoage dependency, typical fo SCC cells in cultue. Fom the data in ou cuent study, we favo the concept that diffeentiation events ae the most impotant deteminants because OH-2 cells ae geneally flat, gow as anchoage-dependent monolayes, and yet exhibit all of the suface poteins that constitute the unique SCC suface potein phenotype peviously demonstated (15). The concept that a fom of lage cell undiffeentiated cacinoma is linked along a diffeentiation continuum with SCC aises the question whethe this association is limited to the cell cultue envionment in vito o whethe the pevious obsevations (3) of a time-dependent tansition, in cultue, of SCC to a fom of lage cell undiffeentiated tumo also occus in vivo. Two featues of ou OH-2 cells now suggest stongly that this tansition can occu in the human host. Fist, the OH-2 cells gew immediately, in cultue and in nude mice heteotans- Poc. Natl. Acad. Sci. USA 8 (1983) plants, as lage cell undiffeentiated tumos. Second, undiffeentiated lage tumo cells with mophologic similaities to the cultued and nude mouse heteotansplanted OH-2 cells wee found in the autopsy tissues of the patient fom whom the cells wee obtained. We must then conclude that the time-dependent tansitions fom SCC to lage cell tumos, obseved in cultue by Gazda et al (3), also occu in vivo. Ou data also suggest, although moe indiectly, a diffeentiation link between SCC and squamous cell lung cacinoma. The simultaneous expession of non-scc and SCC suface poteins in single clones of OH-2 cells (taken fom a patient with both SCC and squamous cacinoma) suppots such a possibility. The close elationships demonstated in this study between histologically distinct bonchial neoplasms pobably eflect ongoing pathways of diffeentiation in nomal bonchial epithelial cells. Thee is inceasing evidence that tumos epesent clonal expansions of nomal stages of diffeentiation in the cell systems fom which they aise. This conclusion has been best illustated in the elationships between nomal and neoplastic lymphoid cells. Biochemical analysis of these neoplasms, including studies of cell suface antigens, has shown that the diffeentiation status of vaious lymphocytic tumos epesents specific stages of nomal lymphocyte development (22-24). In tems of the human bonchus, McDowell et al. have poposed, fom mophological studies of cellula egeneation afte mechanical damage of nonneoplastic bonchial epithelium (25) and fom ultastuctual studies of human lung cances (26), that a pluipotent lage "indiffeent" cell is linked to both the endocine and nonendocine cellula elements of the bonchial mucosa. Ou data demonstating that clones of lage undiffeentiated neoplastic cells simultaneously expess both SCC and non-scc cell suface poteins futhe suppot the notion that endocine and nonendocine bonchial epithelial cells ae elated though such a totipotent "indiffeent" cell of endodemal oigin. The potential fo tansitions between SCC and non-scc lung cances has significant clinical implications that emphasize ou need to ecognize moe pecisely cell populations in given lung tumos. SCC tumos ae geneally quite sensitive to adiation theapy o chemotheapy (27), in contast to squamous, adeno-, and lage cell lung cacinomas. Mixtues of SCC and non-scc histology can occu in the same tumo lesion (28, 29) and, moeove, some patients with an initial diagnosis of SCC may, at autopsy, have only non-scc histology evident (13, 14, 29). A change fom SCC towads non-scc diffeentiation could then be an integal featue of the pogessive theapeutic esistance that develops with time in vitually all patients with SCC (27). Indeed, we have ecently epoted that a loss of endocine featues fom cultues of SCC, even in the absence of a goss mophological change in the cultued cells o nude mouse explants, can accompany the emegence of adiation esistance (3). Immunohistochemical studies using antibodies to the cell suface poteins we have demonstated could pove extemely valuable fo identifying, in lung cances fom individual patients, cell populations at the specific stages of diffeentiation that may detemine thei theapeutic sensitivities. Note Added in Poof. In ef. 15, we inadvetently labeled the ph gadients fo figues 1 and 2 in the evese diection. The coected pi values fo the majo distinguishing poteins fo SCC and non-scc lung cance cells, as detemined fom the gadient in Fig. 2 of the pesent manuscipt, ae as follows. (i) SCC poteins: E, pi = 4.5; F, pi = 5.2; G, p1 = 5.3; I, pi = <4.; egion, p1 = ; P egion, pi = ; Q, R, S, and T, pi = ; and U, pi = (ii) Non- SCC poteins: 1-6, pi = ; 7, pi = ; 8, pi = 5.3; 9, pi = 5.; and the high molecula weight poteins above potein A, pi =

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