[CANCER RESEARCH 45, 2725-2731, June 1985]

Stability and Utility of the Unique Human Small Cell Carcinoma Line SHP-771

AuréliaM. C. Koros,2 Edwin C. Klein, Sylvia Pan, R. Wayne Atchison, Robert Lakomy, Alfred Bahnson, and

Carol Sherer

Departments of Infectious Diseases and Microbiology [A. M. C. K., ft W. A., ft L.¡,Radiation Health [S. P., C. S.], and Biostatistics [A. B.], Graduate School of PublicHealth, and Central Animal Facility [E. C. K.], University of Pittsburgh, Pittsburgh, Pennsylvania 15261

ABSTRACT

The human small cell (oat cell) carcinoma line, SHP-77, estab

lished by Fisher and Paulson in 1977 and originally described asa "large cell variant of oat cell cancer" has been evaluated by

several different parameters and shown even after more than200 passages to retain properties described for the original cellline. Karyotypic, histological, and biochemical features are retained, as well as tumorigenicity in nude mice. The originalauthors' suggestion that this is a propitious cell line for both in

vitro and in vivo studies is supported by this report. Modulationof growth characteristics in vivo (in xenografts) emphasizes theplasticity of this unique line which serves as a valuable model forbasic as well as therapeutic studies. SHP-77 can serve as an invitro target in 51Cr and 111lnrelease cytotoxicity assays as well

as in in vivo nude mouse assays for evaluating immune reactivityof cells and serum from lung cancer patients. The potentialhistological variability of SHP-77, despite its biochemical stability,

calls attention to the inadequacy of histological criteria for lungtumor classification.

INTRODUCTION

Small cell carcinoma (SCC)3 of the lung, comprising about 20

to 25% of the 110,000 new cases of lung cancer each year inthe United States, appears to be biologically distinct from otherforms of lung cancer (1). SCC is characterized by rapid metastaticspread and a poor prognosis. Surgical resection of the primarytumor is of limited value; by the time the patient becomessymptomatic, métastases(occult or obvious) are usually prevalent in the brain or other organs. Nevertheless, SCC is the celltype that has been most responsive to chemotherapy, and somecomplete remissions have been seen. These remissions, however, are not usually for more than 2 years, and recurrent tumorsare usually resistant to further therapy. There is a need, therefore, to understand the factors responsible for recurrence ofdisease and the biological relationships among different types oflung tumors (2). Although past evidence supported the distinctlyseparate origins for SCC and non-SCC types of lung cancer, the

possible common etiology as well as transitional relationshipsbetween different cell types has increasing support from autopsydata as well as in vitro studies (3-6).

Host immune factors play a significant role in the outcome ofneoplastic diseases in general, as evidenced from both animalmodels and human studies. In animal models, host immune

1Supported in part by The Cancer Federation, Inc." Recipient of support from Grant CA-30457 from the National Cancer Institute.

To whom requests for reprints should be addressed, at A449 Crabtree Hall,Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261.

3The abbreviations used are: SCC, small cell carcinoma; PBL, peripheral blood

lymphocytes; NK, natural killer cells.Received 8/6/84; revised 3/6/85; accepted 3/11/85.

factors may inhibit, enhance, or have no effect on tumor growthand métastases.Similarly in humans, host immune factors mayor may not be beneficial to the patient, depending on the particular type of cancer as well as the stage of disease. For example,adjuvant immunotherapy can stimulate existing host responsesto an early stage melanoma, but the use of nonspecific adjuvantsin later stages of the disease can cause havoc, since the majorityof these patients react to tissue antigens. The opposite seemsto be true in breast cancer patients, and the use of nonspecific,active immunotherapy in early stage breast cancer seems inappropriate (7).

The immune status of cancer patients, therefore, needs to beevaluated longitudinally in relation to disease progression duringtreatment. In considering alternatives to surgical removal aloneof neoplasms, especially in cases of SCC, there is a special needto evaluate the patient's immune status in order to select treat

ment modalities which will provide optimum benefit for eachtumor-host situation. Evidence from our studies as well as others' (3, 8-11) suggests that there is a spectrum among patients

with SCC not only with respect to heterogeneity of tumor types,but with respect to multiple parameters of immune reactivity.Immunoregulatory mechanisms need to be studied for individualSCC patients, using multiple parameters. Although the in vivoparameters provided from studies of human tumors grown inathymic (nude) mice may be useful for monitoring the effects ofpatient factors, i.e., PBL and serum on xenografts, such resultsmust be interpreted cautiously and in conjunction with other invitro parameters.

Identification of T-cell subsets in lung cancer patients, usingfluorescent-labeled monoclonal antibodies in conjunction withfunctional assays for NK cells using 51Cr cytotoxicity assays,

permits a rapid, sensitive evaluation of a limited category ofpatient immune factors which may be amenable to manipulationby appropriate treatment modalities. These parameters havebeen measured in some patients with lung cancer (10), but howthey may be interrelated, or how they are related to diseaseprogression is not clear. The elegant animal studies of Fidler eral. (12) emphasize the complexities of the tumor-host relation

ships which need to be identified on an individual basis. Recentdescriptions of the ever-expanding heterogeneity among lym

phocyte categories can now be used to approach a definition ofeach person's immune repertoire (13-15).

SHP-77 is a continuously cultured cell line, which has retainedimportant features of SCC even after long-term passage sinceits establishment in 1977 by Fisher and Paulson (21); it thereforeserves in both in vivo and in vitro assays of immunoreactivity ofpatients with SCC. This paper documents its stability as well asunique properties which render it especially useful as a modelfor elucidating immunoregulatory mechanisms in SCC.

MATERIALS AND METHODS

In Vitro Culture. SHP-77 cells obtained in their 66th passage in 1979from J. Paulson were grown in RPMI Medium 1640 (MA Bioproducts,

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Walkersville, MD) supplemented with 9% heat-inactivated fetal bovine

serum (HyClone; Sterile Systems, Logan, UT) without antibiotics. Cultures were passaged at least once a week by shaking the floating andloosely adherent clusters of cells without trypsinization. Cells wereseeded into 25-sq cm flasks (Corning). Cultures were kept sealed at37°C without CO2 and were screened periodically for Mycoplasma by

Hoechst (33258) staining and culture in agar. Any positive cultures werediscarded, and fresh cells were prepared from ampuls stored in liquidnitrogen. Early passage, 67, and late passage, >200, which have beenmaintained in our laboratory for 5 years were studied.

A growth curve was obtained on late-passage (>200) SHP-77 byseeding each of 5 flasks with 1.34 x 106 viable cells in 4 ml of complete

medium. Cells were 95% viable initially as determined by trypan blueexclusion. Samples were counted at 24-h intervals.

A mitotic index was estimated by counting the number of mitoses per500 cells from cultures prepared on glass-slide flasks (Flaskettes; Lab-

Tek) and stained with Giemsa after absolute methanol fixation.Chromosome Analyses. Cells in the log phase of growth were

arrested in metaphase by adding Colcemid, 0.1 ¿ig/ml,to the mediumand were dispersed by trypsinization with 0.5% trypsin:0.2% EDTA(Grand Island Biological Co., Grand Island, NY). Cells were swollen byexposure to 0.075 M KCI for 10 min, fixed in methanohglacial acetic acid(3:1), and spread on slides. Chromosome banding was done accordingto the G-banding method of Klinger (16) and interpreted according to

guidelines established by the Paris Conference (17).Growth of SHP-77 in Nude Mice. Virgin female BALB/c athymic nude

mice (HaríanSprague Dawley, Inc., Madison, Wl) certified pathogen freewere maintained in filter-capped sterile plastic cages with sterile food

and water ad libitum, and they were handled with sterile gloves in alaminar flow cabinet. They were 5 to 12 weeks old when used for in vivoassays of human immunocompetent cells (9, 10). SHP-77 cells were

inoculated s.c. on the abdomen; patient PBL or serum was inoculatedi.p.

Histology. Portions of tumors from nude mice as well as cell culturesof SHP-77 were fixed in either Bouin's fixative or buffered formalin andstained with hematoxylin:eosin or a modified Grimelius' stain (18).

"Cr and 111ln Release Cytotoxicity Assays Using SHP-77 as a

Target. SHP-77 was compared with other cultured cell line targets,K562, human erythroleukemia (19) (gift of Jun Minowada), NCI-H69 andNCI-H128, human SCC (4) (gift of Adi Gazdar), and YAC-1, murine

lymphoma (20) (gift of Howard Holden), in assays for NK cell activityamong PBL (9, 10).

RESULTS

Cultured Tumor Cells

SHP-77 has been maintained in continuous culture since Sep

tember 1979 after it was obtained from its founders in its 66thin vitro passage. Cells grow as loosely adherent grape-like

clusters (Fig. 6), if cultures are split 1:2 three times each weekwithout trypsinization. However, if cultures are left undisturbedfor 1 week, there is a transition to a monolayer as describedearlier (21). The doubling time currently is 96 h in the log phase;the mitotic index is 1%.

Cytogenetic Studies

Results of cytogenetic studies of passages 67 and >200 ofSHP-77 are shown in Tables 1 and 2. The modal chromosomenumber in the 2 lines was 46 (29 and 35% for the short-term-and long-term-passaged lines, respectively). Thirty and 20% of

cells were hyperdiploid, and 41 and 45% were hypodiploid forpassages 67 and >200, respectively. In comparison with theoriginal passage 19 (21) of SHP-77, the modal number has

shifted from 54 to 46; however, through the 140 to 150 passagesbetween short- and long-term lines, the modal number remained

stable.The most frequent structural anomalies in both passages 67

and >200 of SHP-77 were similar (Table 2), although a few more

chromosome rearrangements appeared to be present in theshort-term-passaged line, possibly due to cell sampling. Of the

14 markers, 7 (Markers A) were commonly present in both celllines, and the remainder (Markers B and Markers C) were different (Tables 1 and 2; Figs. 4 and 5). One Y chromosome wasseen in the cell karyotype shown fiorii the >200 passages (Fig.5), which is consistent with the original tumor (21).

The greatest changes in SHP-77 appear to have occurred

between passage 19 (21) and passage 67. Less change isevident between passages 67 and >200. Fisher and Paulson(21) described the lack of an intact chromosome 1. Table 2shows absence of both intact chromosomes 1. There is anabsence of one chromosome 3 both in Fisher and Paulson's

passage 19 as well as in the SHP-77 passages 67 and >200.This anomaly is consistent with the report of Whang-Peng ef al.

(22) identifying a deletion in at least one of the 3(p) chromosomesspecific for SCC.

SHP-77 Growth in Nude Mice

SHP-77 as Single Cell Suspension Inoculated s.c. Growthof the in w'fro-cultured SHP-77 cells in nude (athymic) mice has

been described (9, 10, 21). Tumors usually grow as circumscribed nodules without evidence of métastases(9,21) and afterabout 6 months, such xenografts can attain the size of the hostmouse (9). The tumor morphology in our studies is compatiblewith the description of Fisher and Paulson (21).

Histopathology. Tumors developing at the primary site of s.c.inoculation in nude mice were characterized microscopically bymultiple nests and clusters of closely packed, round to polyhedralcells with distinct eosinophilic cytoplasm, and large centrallylocated nuclei with dispersed dense accumulations of chromatin(Fig. 1) and prominent nucleoli. Cellular degeneration with markedpyknosis and karyorhexis was prominent, as were large multi-

focal areas of coagulative necrosis. Mitoses were frequent [3 to5/HPF (high power field)]. Scanty amounts of a delicate fibrovas-

cular stroma were present throughout the tumor, subdividing thecellular aggregates. The histological features observed in thesexenografts are consistent with those described in the originalneoplasm (6), and a modified Grimelius stain was strongly positive for numerous secretory cytoplasmic granules in many of thecells (Fig. 2).

SHP-77 xenografts appear to be neuroendocrine tumors or

carcinoids of the amine precursor uptake, decarboxylase (APUD),series (5). Their appearances are not always those of classicSCC (oat cell) of the lung (23), and histopathology can bemodulated (9).

Because of its unique features, as well as properties sharedwith other SCC (5), SHP-77 has been used to evaluate functionalactivity of small cell lung cancer patients' PBL and serum both

in vivo in nude mice (9, 10) as well as in vitro (10). In thecase of one patient (J. B.) who had extensive métastasestobrain and liver, as reported earlier (9), when PBL from J. B. wereinoculated i.p. into nude mice at the same time that SHP-77 was

inoculated s.c., the resulting xenografts appeared to metastasize

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Table 1Chromosome distribution in cell lines SHP-77, passage 67, and SHP-77, passage >200

Distribution ofchromosomesCell

lines 3435SHP-77,

passage 67 2SHP-77, passage >200 1 137

38 40 41 42431

2281 1 3 5 410Table

244

45468

18 298 1135Comparison

of chromosome alterations in short- and long-term passages of lineSHP-77 shown byG-bandingParticipating

chromosomesdel(1Xq21)

del(1Xp13)delt(1;?Xp13;?)Loss

of both intact 13q+Loss

of an intact3Lossof an intact46q+

ins(7;?Xq22;?)9p+Loss

of an intact 10del(11Xq23)t(12;15Xq11;q11)Loss

of an intact13Lossof an intact 18

Loss of one or both 21sLossof an intact22i(12q)15ps+Loss

of an intact 11Loss of an intact19Marker

A1234

567Marker

B12345Marker

C123

45Fr&QUGncv

(%)SHP-77,

passage SHP-77, passage67>20045.5

36.445.590.1

63.672.790.081.8

63.627.363.6

72.736.490.136.4

90.181.881.881.854.5

81.854.563^663.645.5

45.554.545.563.654.5

27.354.59.1000

0066.7

33.316.6100.0

58.391.666.766.7

50.025.083.3

58.358.375.058.3

100.091.6000

058.366.783.366.7

41.258.350.000o0025.041.716.7

33.325.047

48 49 51 54 55 57 70,78 79 83,81 84 85 89 9211214

6 3 2 1 1 11 173 11 311111abdomen

of one nude mouse after 18 months. Necropsy revealed a large, soft, indistinctly multilobulated tan mass in theposterior abdomen enveloping the uterine body and adherenttothe

colon. Microscopically, the mass consisted primarily ofhy-percellular

strands comprised of a pleomorphic population ofelongate, columnar-type cells with large heterochromaticnuclei.Individualization

of cells, with singular attachment tounderlyingbasementmembrane (tombstone-type appearance), was distinc

tive in many regions (Fig. 3). Massive areas of necrosiswerepresentthrough all sections evaluated. The location andgrossand

microscopic appearance of the tumor were originally suggestive of a papillary mesothelioma, but Grimelius stainingwasdecidedly

positive for cytoplasmic granules, leading to reconsideration that this neoplasm represented growth of a morphological variant of the original tumorinoculum.SHP-77

as a Target in 51Cr and 111lnCytotoxicity AssaysforKll^As»*ïuïfritixivActivityLongitudinal

studies have been done to evaluateimmunefunctionof small cell lung cancer patients' PBL in vitro aswellas

in vivo in nude mice (10). Studies thus far indicate thatmostnormalpersons and some patients with small cell lungcancerhave

highest NK cell activity against K562 target cells, lessonSHP-77,and least activity on YAC-1 . There is more heteroge

neity among patients than among normal persons. The heterogeneity in NK cell functional activity does not appear to berelatedto

characteristics of cells stained with fluorescentmonoclonalantibodiesto T-cell subsets. For example, there is nodirectcorrelation

between NK activity and percentages of PBLstainedwithanti-Leu-7 monoclonal antibody (10); anti-Leu-7 definesonlyone

subset of NK cells(13).Thereactivity of SHP-77 as a target in both 51Cr and111lnrelease

Cytotoxicity assays is greater than the reactivity of2otherSCC lines, NCI-H69 and NCI-H128, as shown in Tables3and

4 for the 3 SCC lines labeled with 51Cr. Data for 111lnandtheNCI-H69 and NCI-H128 are not shown, but those 2 linesdidnotlyse as well as SHP-77.

to regional nodes, liver, and spleen. This was seen in 2 experiments, done 2 months apart immediately preceding J. B.'s death.

No similar patient has been available for a repeat of this experiment.

The effects on growth of SHP-77 xenografts in nude mice ofseveral other SCC patients' as well as normal persons' PBL and

serum have been studied (10). There have been no significanteffects of either PBL or serum on the growth of SHP-77 instudies of 5 of 6 patients and 3 of 3 normal persons thus far.However, in the case of one SCC patient (N. P.), who appearedto be in remission when 3.6 x 106 of his PBL were injected i.p.into 4 nude mouse recipients of 1 x 106 SHP-77 cells s.c. into

each of 2 sites on the abdomen, a swelling appeared in the

DISCUSSION

SHP-77 has retained the unique features described by its

founders (21). After 6 years of passage in vitro, the only majorchange in the line appears to be in the modal number of 54 to amodal number of 46 chromosomes which occurred somewherebetween passages 19 and 67. There have been no other significant changes between passages 67 and >200. The change inmodal number, also, does not appear to have altered the unusualproperties of SHP-77 which distinguish it from numerous otherSCC lines established in other laboratories (4, 24). SHP-77displays a spectrum of features, ranging from those of carcinoidsto SCC, and under one rare circumstance, it was mistaken for

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Table 3Comparison of SHP-77 as a target with K562 and 2 other SCC lines. NCI-H69 and NCI-H128, in 51Crre/ease

cytotoxicity assays

Effector:targetratiosExperiment03/12/8307/13/83Effector

cellsNormal

female (A.K.)Normal

male (R.L.)Normal

male (D.T.)SCC

patient (W.B.)SCC

patient (A. D.)Target

cellsK562SHP-77NCI-H69NCI-H128K562NCI-H128K562SHP-77NCI-H69K562SHP-77NCI-H69K562SHP-77NCI-H69100:118.7±6.1"2.9

±12.6"10.7+9.9"-6.6±3.7e21

.8±6.21.0±5.0C66.7

±15.164.2±6.4"36.9

±10.7"68.1

±5.258.9±4.7*25.4±1.7"77.6

±5.940.9±3.8"0.4±3.3d50:113.1

±3.70.3±12.36.7±2.1-12.1

±4.123.0±3.15.7±6.572.8

±18.257.2±7.931

.7±9.558.2±3.647.1±6.321.0

±5.173.0±3.152.6±1.0-2.9

±17.525:14.8

±5.07.1±4.318.5±12.5-9.8

±5.117.4*0.97.0

±6.468.8

±8.447.3±2.923.5

±1.646.7±2.943.3+8.614.9

±2.544.6+15.546.8

±7-5.4±7.4

" Mean ±SE of triplicate samples.6 P > 0.05 (not significantly different),

other targets compared to K562.CP < 0.01 (significantlydifferent)."P < 0.001 (significantlydifferent).

with regard to the percentage of specific release of 5'Cr of

Table 4Comparison of SHP-77 as a target with K562 and YAC-1 in ln/n re/ease and "Cr release cytotoxicity assays

Effector: targetratiosExperiment

Effector cells Isotope Target cells 100:1 50:125:111/17/81SCC patient (N. P.) '"InK56251Cr

K562'"InSHP-7751CrSHP-77"'InYAC-151Cr

YAC-112/22/81

Normal mate (E. S.) '"InK56251CrK562"'InSHP-77slCrSHP-77'"InYAC-1"Cr

YAC-1"

Mean ±SE of triplicatesamples.:ig.

3). Its growth in nude mice can bemodu-of

PBL from patients with SCC. UnderthoseHP-77has been seen to sometimesspreadrapid

demise of the mice within 4 months (9)15.4

±3.2" 10.4 ±3.6 4.7 ±2.915.1±0.5 16.1 + 8.8 9.5 +4.439.2±30.0 5.4 ±9.2 14.5±6.730.6±7.5 31.8 ±7.8 30.0 +6.57.3±6.8 2.6 ±6.4 12.1 ±13.021.

3 ±6.4 30.6 ±1.9 20.0±14.840.3

±23.0 56.5 ±1.6 47.6 ±7.979.1±5.6 86.1 ±7.6 74.3 ±7.046.2±8.8 63.3 ±12.8 35.3 ±12.747.4±8.1 30.1 ±0.5 33.1 ±3.523.7±14.0 10.0 ±9.5 -5.6 ±0.41

5.6 + 17.0 -3.0 + 9.0 -1 4.4 + 10.7TabteSBiological

properties ofSHP-77Table

5 has been adapted from Refs. 1 and26.Biological

property Description

or remain dormant for 18 months in one case. The plasticity ofSHP-77 renders it unusually suitable as a model for studyingfunctional properties of patient immune factors, but SHP-77 can

also serve for evaluating mechanisms of action of immune modulators.

As described by Fisher and Paulson, "the histopathological

classification of the original tumor as well as those transplantedfrom SHP-77 into nude mice, from which it was found to be

morphologically indistinguishable, might be regarded as debatable if not difficult" (21). They reported that among the light

microscopic features were those of oat cell carcinoma, i.e.,marked anaplasia, frequent mitoses, and a tendency to formribbon-like aggregates. However, the relatively large cell size,frequent polygonal shape, and modest amount of acidophiliccytoplasm could place it with the undifferentiated, large, orpolygonal cell carcinomas, which is cited as the type of lungcancer which is often the source of interobserver disagreement(21). The detailed descriptions of electron microscopic evidence

L-dopa decarboxylaseNudearcytoplasmic ratioNucleoliDense core granulesCell culture growth and morphologyDoubling time (cell culture)Tumorigenicity in nude miceLeu-7 antigen

Present (high)8

HighProminentPresentSuspension aggregates or epithelioidLongPositivePresent

" Assays performed by Dr. Adi Gazdar, National Cancer Institute-Navy, Medical

Oncology Branch, Bethesda, MD.

for neurosecretory granules as well as biochemical evidence forL-dopa decarboxylase at levels ranging from 30 to 62 units/mgprotein on 3 separate samples4 in SHP-77 categorize it among

neuroendocrine (APUD) tumors (5, 25-27).Among its other features, SHP-77 has been found to react

specifically with anti-Leu-7 monoclonal antibody directed to a

subset of lymphocytes having NK activity (28, 29). This latestfinding, along with the features summarized in Table 5, suggests

4A. F. Gazdar, personal communication.

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anespeciallyvaluablerolefor SHP-77in elucidatingimmunoreg-ulatorymechanismsandhumanSCC.

ACKNOWLEDGMENTS

The authors thank Dr. Fisher and Dr. Paulson for SHP-77, Dr. Adi Gazdar forcell lines NCI-H69 and NCI-H128, Dr. Jun Minowada for K562, and Dr. HowardHolden for YAC-1. We thank Dr. Gazdar and Dr. Mary Matthews for reviewing ourslides. Special thanks are given to Doris La Pietra for histology. Dr. Meissler forallowing us to study his patients, and Judy Fossati and Elaine Rubinstein for typingthe manuscript.

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Fig. 1. Photomicrograph taken from SHP-77 tumor grown on a female BALB/c-nu/nu mouse. SHP-77 had been carried in culture, passaged at least once a week for3 years. Recipient mouse was 10 weeks old when given injections at 2 sites on the abdomen with 1 x 10s SHP-77 cells/site. Mouse was sacrificed 7 months afterinoculation. Tissue was fixed in Bouin's fluid. Light microscopic appearance of transplanted neoplasm. Large round to polygonal tumor cells are arranged in nests and

packets. Mitoses and individual cellular degeneration are frequent. General appearance and cellular morphology are consistent with the description of the original humantumor. H & E, x 250.

Fig. 2. Same tissue as described above but stained with a modified Grimelius stain. Silver stain demonstrates the presence of abundant neurosecretory granules, x400.

Fig. 3. Microscopic appearance of what was mistaken for a papillary "mesothelioma-like" tumor found in the abdomen (pelvic region) of a different 18-month-old

female BALB/c-nu/nu mouse (No. p2/2/3), demonstrating typical cellular pattern and morphology (tombstone appearance). Subsequent Grimelius staining was positive,confirming the neuroendocrine properties of the xenograft. H & E, x 500.

Fig. 4. Karyotype of SHP-77, passage 67.

Fig. 5. Karyotype of SHP-77, passage >200.

Fig. 6. Photomicrograph of SHP-77 cell culture, showing loosely adherent cell clusters, x 100.

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1985;45:2725-2731. Cancer Res   Aurelia M. C. Koros, Edwin C. Klein, Sylvia Pan, et al.   Line SHP-77Stability and Utility of the Unique Human Small Cell Carcinoma

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