Int. J. Cancer: 51,72-78 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer
IN VITRO RESPONSE OF HUMAN SMALL-CELL LUNG-CANCER CELL LINES TO CHEMOTHERAPEUTIC DRUGS; NO CORRELATION WITH CLINICAL DATA E.F. SMIT',~, E.G.E. DE VRIES'.~, H. TIMMER-BOSSCHA', L.F.H.M. DE LElJ3,J.W. 00STERHU1S4, R.J. SCHEPER', J.J. WEENING4, P.E. POSTMUS~ and N.H. MULDER' 'Dept. of Internal Medicine, Division of Medical Oncology, 2Dept.of Pulmonary Medicine, 3Dept. of Clinical Immunology, 4Dept.of Pathology, University Hospital Groningen; 5Dept.of Pathology, Free UniversityHospital Amsterdam, The Netherlands. Three cell lines derived from small-cell lungcarcinoma (SCLC) tumors of patients who had no clinical response after treatment with a multi-drugregimen were compared to 3 cell lines derived from tumors of patients who, upon treatment, showed a complete clinical response. These 2 groups of cell lines were considered to represent the in vitro counterparts of the 2 extremes of the clinical spectrum of sensitivity for chemotherapeutic drugs in small-cell lung cancer. To assess whether the in vivo (in)sensitivity of a tumor to a certain drug regimen is retained in vitro, the cell lines were tested for drug sensitivity using the microtiter-welltetrazolium assay and the results were compared with the in vivo data. No correlationwas found. Since in vitro models using cell lines are based on the assumption that a cell line reflects the properties of the tumor from which it is derived, several additional parameters such as MAb staining against different SCLC-associated antigens and DNA content were analyzed in the biopsies and the cell lines. The results showed that selection of discrete tumor-cell populationsin vitro occurs. Results of in vitro chemosensitivitytesting for individual SCLC patients should be interpreted with caution.
Small-cell lung cancer (SCLC) accounts for 20-25% of newly diagnosed cases of lung cancer (Ianuzzi and Scoggin, 1986). It is characterized by early and widespread occurrence of metastases and rapid progression of disease. Human SCLC provides a good model to study the problems of initially unresponsive tumors. The vast majority (about 90-95%) of patients with SCLC show major responses to chemotherapeutic agents of different classes (Hansen et al., 1985). However, 5 1 0 % of patients present with a tumor that is initially unresponsive to most commonly used cytotoxic agents, and invariably die within several weeks. Tumors of the latter group of patients may be termed intrinsically resistant according to the Goldie-Goldman hypothesis, as opposed to the initially sensitive tumors that are present in the former group (Goldie and Goldman, 1984). The aim of the present invcstigation was to study whether this in vivo phenomenon is also reflected in vitro. MATERIAL AND METHODS
Chemicals Doxorubicin was provided by Farmitalia Carlo Erba (Milan, Italy). Cisplatin and etoposide were obtained from Bristol Myers (Madrid, Spain), vincristine from E. Lilly (St. Cloud, France) and melphalan from Wellcome (London, UK). FCS was purchased from Flow (Irvine, UK). RPMI medium was obtained from Gibco (Paisley, UK). M 7 T was supplied by Sigma (St. Louis, MO). Cell lines Three cell lines GLC 3, GLC 12 and GLC 20, were established from patients who had no measurable response to chemotherapy. GLC 3 was derived from a pleural effusion of a 61-year-old male and has been described (de Leij etal., 1985a). Briefly, the clinical history of this patient was as follows: he received 2 courses of cyclophosphamide (750 mg/m2) on day 1, cisplatin (75 mg/m2) on day 1 and etoposide (100 mg/m2) on days 1, 3 , 5 over a 3-week period. Since no response was seen,
chemotherapy was changed to doxorubicin (60 mg/m2) on day 1, vincristine (1 mg/m2) on days 1and 8 and procarbazine (100 mg/m2) on days 1-10 over a 3-week period. After 2 courses of second-line chemotherapy, the tumor was still clearly progrcssive and at that time GLC 3 was established from the pleural effusion. The patient died 14weeks after the start of chemotherapy. GLC 12 was derived from a 64-year-old male who received 4 courses of cyclophosphamide (1,000 mg/m2) on day 1, doxorubicin (40 mg/m2) on day 1and vincristine (1 mg/m2) on days 1 and 8 over a 3-week period. The tumor showed no change on chest X-rays. A thoracotomy was performed and a lymph-node metastasis was found adherent to the pulmonary artery. From this lymph node GLC 12 was established. Radiotherapy (37.5 Gy) was administered to the left hilar region and mediastinum, which again induced no measurable tumor regression. This patient died with extensive liver metastases 33 weeks after the start of chemotherapy. GLC 20 was derived from a 67-year-old male after 6 courses of chemotherapy consisting of cyclophosphamide (1,000 mg/m2) on day 1, doxorubicin (40 mg/m2) on day 1 and vincristine (1 mg/m2) on days 1 and 8 over a 3-week period. The tumor showed no regression and became progressive within a few weeks after Chemotherapy had been stopped. A biopsy was taken of the primary tumor by bronchoscopy and the cell line established. The patient died 28 weeks after the diagnosis of SCLC. The cell lines GLC 14, GLC 22 and GLC 33 were established from patients who showed clinical complete remissions (CR) after chemotherapy. The patient history of GLC 14 has been described elsewhere (Berendsen et al., 1988a; de Vries et al., 1989). Briefly, GLC 14 was established from a supraclavicular lymph node of a 55-year-old female which was removed before Chemotherapy. After 5 courses of cyclophosphamide (1,000 mg/m2) on day 1, doxorubicin (45 mg/m2) on day 1 and etoposide (100 mg/m2) on days 1, 3, 5 over a 3-week period a clinical CR was achieved. The tumor relapsed after a 31-week disease-free interval and the patient died 86 weeks after the start of chemotherapy. GLC 22 was derived from the primary tumor site (right lower lobe) of a 50-year-old male prior to chemotherapy. This patient received the same chemotherapy regimen as the patient from whom GLC 14 was established. A clinical CR was achieved after 5 courses. This patient died from brain metastases 71 weeks after the diagnosis of SCLC. GLC 33 was established from a 62-year-old female with a ('To whom correspondence and reprint requests should be sent, at the Dept. of Internal Medicine, Division of Medical Oncology, University Hospital, Oostersingel59,9713 EZ Groningen, The Netherlands. Abbreviufionst SCLC, small-cell lung cancer; CR, complete response; FCS, fetal calf serum; MTA, MTT assay; MTT, 3 4 4 5 dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumhromide;PBS, phosphate-buffered saline (0.14 M MaC1,2.7 mM KCI, 6.4 mM NaZHPO4. 2H20 and 1.5 mM KHzPO4, pH 7.4); MAb, monoclonal antibody; MDR, multi-drug resistance; TBS, tumor biopsy specimen.
Received: June 20,1991 and in revised form December 16,1991
73
CLINICAL A N D CELL-LINE DRUG SENSITIVITY; NO CORRELATION
supraclavicular lymph-node metastasis present at the time of diagnosis. After 2 courses of chemotherapy as described under GLC 14, a CR was achieved. The patient received 3 further courses, but eventually died 47 weeks after the start of chemotherapy. The cell lines were maintained in 5% COz, 95% air at 37°C. GLC 3, GLC 20 and GLC 14 were kept in culture in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. The growth medium for GLC 12, GLC 22 and GLC 33 was SCLC-2 medium prepared according to the method of Minna et al. (1982) and supplemented with 3% FCS. This medium was chosen because it was impossible to culture these cell lines in RPMI 1640 medium containing 10% FCS. GLC 3, GLC 14 and GLC 33 grew partly attached and partly floating in aggregates. GLC 12, GLC 20 and GLC 22 grew exclusively floating in aggregates. The culture doubling time, as determined by the MTA under the conditions outlined for the MTA studies (see below), was 25,72 and 49 hr for GLC 3, GLC 12 and GLC 20, and 26,44 and 32 hr for GLC 14, GLC 22 and GLC 33. For all experiments, cells were incubated 1:l for 5 min with 0.02% EDTA solution (0.35 g NaHC03, 8 g NaCI, 0.4 g KCI, 1 g dextrose, 0.2 g EDTA, 1.0 1 HzO) to prepare a cell suspension containing small clumps with a viability, as determined by Trypan-blue exclusion, of > 75% for all cell lines. All cell lines were tested between passages 20 and 50.
MTT assay (MTA) The MTA, as described by Mossman (1983) and Carmichael et al. (1987), was used with some minor modifications (TimmerBosscha et al., 1989). Before the assays were performed, a linear relationship of cell number to MTT formazan crystal formation was checked. The relationship between seeding density, incubation volume and incubation period was established for each cell line separately after growth studies (Table I). In this way, all cell lines were cultured in the test plates for 2-3 cell divisions; they were in exponentional growth phase at the time of testing and produced an absorption of 1.0 at 520 nm (see below) at the end of the culture period. The appropriate number of cells was incubated in 96-well culture plates (Nunc, Gibco, Grand Island, NY) in culture medium to which one of the following agents was added for 1 hr: doxorubicin, cisplatin, vincristine, etoposide, melphalan. All stock solutions of drug were diluted with RPMI 1640 medium to reach the desired concentrations. Melphalan was used as a substitute for cyclophosphamide as melphalan does not require endogenous activation (Ruckdeschel et a[., 1987). For X-ray treatment (Philips X-ray source operated at 200 kV, 15 mA, 0.5 mm Cu/A filter) cells were placed in plastic tubes in a volume of 500 pl. After irradiation the cells were transferred to 96-well culture plates. After incubation, cells were washed 3 times with fresh medium by centrifugation of the microtiter plates at 180g for 10 min. They were again cultured for different time periods as determined earlier, then 20 pI of MTT solution (5 mg/ml PBS) were added to each well and the cells were incubated at 37°C for 165 min. Thereafter the plates were centrifuged at 180 g for 15 min and the supernatant was carefully removed without disturbing the formazan crystals. Cells grown in the SCLC-2 medium were washed once with PBS. This step was introduced because this medium caused a turbidity with DMSO, probably due to BSA. Subsequently, the formazan crystals were solubilized by adding 200 pl 100% DMSO. All plates were scanned at 520 nm immediately after resolubilizing of the formazan crystals using a scanning microtiter-well spectrophotometer (Titertek Multiscan, Flow, Irvine, UK). The percentage cell survival was calculated by the formula: mean of the test samplesimean of 3 untreated samples. Controls consisted of media without cells (background extinction) and cells incubated in wells with medium only. For each drug, drug combination and X-ray treatment at least 3 expcriments were performed, each in quadruplicate.
TABLE I - CELL-LINE GROWTH CHARACTERISTICS UNDER MTA CONDITIONS
Cell line
Number of cells plated
GLC 3 GLC 12 GLC 14 GLC 20 GLC 22 GLC 33
30,000 25,000 35,000 10,000
25,000 20,000
Volume (111)
100 250 100 100 250 250
Incubation Period (days)
5 16 4 11
22 8
Cellular doxorubicin content Cellular doxorubicin content was determined after 1 hr incubation in appropriate medium at 3 7 T , using at least 1 x lo6 cells. To correct for extracellular bound doxorubicin, control samples were incubated at 0°C for 5 min. The incubation period was terminated by washing the cells 3 times with ice-cold PBS, and extracted with 0.3 N HC1-50% overnight. After centrifugation the fluorescence was measured in the supernatant in a Kontron (Eching, Germany) spectrofluorometer at excitation and emission wavelengths of 474 and 549 nm, respectively (Bachur et al., 1970). Cellular doxorubicin content was calculated by subtracting the values measured at 0°C from the corresponding levels measured at 37°C. In order to correct for cell-size differences, cellular doxorubicin content was expressed as ng doxorubicin/mg cellular protein. Cellular protein determinations (Lowry et al., 1951) correlated significantly (r = 0.83, p < 0.05, results not shown) with tumor-cell size as determined by FACS analysis using a FACStar flow cytometer (Becton Dickinson, Sunnyvale, CA). Tumor biopsy specimen The original tumor biopsy specimens from which the cell lines were derived, except GLC 3 (which was derived from a pleural effusion), were split into 3 parts. One part was used for routine pathological procedures, one part for establishment of the cell line and the third was snap-frozen in OCT compound in liquid freon and kept at -80°C until use. This last part was used for immunohistological examination of the tumor cells with MAbs and DNA flow cytometry. Normal histological examination showed that the tumor biopsy specimen associated with GLC 12 and GLC 14 consisted of almost pure tumor material; the remaining tumor biopsy specimens were admixed with normal tissue, but at least 25% of their volume consisted of tumor cells. All tumor biopsy specimens were immunostained with MAbs, whereas DNA flow cytometric analysiswas possible with only 2 specimens, namely those of GLC 12 and GLC 22. Monoclonal antibodies Cryostat sections from frozen tumor biopsy specimens were made. Immunostaining was performed on these sections and on cytospin preparations of cell lines according to previously described methods (de Leij etal., 1984). MAbs directed against different SCLC-associated antigens were generated. These antibodies are reactive with antigens expressed in normal and malignant tissues with a neuroendocrine differentiation state (MOC-1, MOC-21, MOC-32, MOC-51, MOC-52) (de Leij et al., 1985a, 1986; Postmus et al., 1986) and an antibody reactive with tissues with an epithelial differentiation state (MOC-31). Three MAbs directed against intermediate-sized filament proteins were also included in the study: RGE 53, directed against cytokeratin 18 (Moll et al., 1982), Vim, directed against vimentin (Broers et al., 1985) and MNF, directed against the 210- and 68-kDa components of neurofilaments (Ramaekers et al., 1983; Kluk et al., 1984). Vim and MNF are non-reactive with normal bronchial epithelia. RGE 53 reacts with normal bronchial columnar epithelium and type-2 pneumocytes. Leu-7,
74
SMIT E T A L .
100
8
80
--
1
aoo
600
400
100
0001
1000
01
001
10
1
L
LL
X - - r a y (radj
Doxorubrcin (uM)
FIGURE 2
FIGURE 1
0
P
0001
001
U
01
100
10
1
1000
0
!!
0001
001
LL
Cisplatin (uM)
01
100
10
1
Etoposide (uM)
FIGURE 3
FIGURE 4
-
2
c.
1201 Lc
0
0
$
.-
a, 0
a,
v
100 l2OI
m
gcn Q
m
-
20
m
0
c-'
$
t
0.001
GO1
01
1
10
100
e
o
0
0.001
!z LL
0.01
0.1
1
10
100
Melphalan (uM)
Vicristine (iiM)
FIGURE 6
FIGURE 5
FIGURES 1-6 - Microtiter-well tetrazolium assay after X-ray treatment (Fig. 1) and 1 hr incubation with doxorubicin (Fig. 2), cisplatin (Fig. 3), etoposide (Fig. 4), vincristine (Fig. 5 ) and melphalan (Fig. 6). Each point represents the mean SE of 3 experiments, each performed in quadruplicate. 0 GLC3, A GLC 12, - GLC 14, D GLC 2 0 , O GLC 22, GLC 33.
+
*
75
CLINICAL AND CELL-LINE DRUG SENSITIVITY; NO CORRELATION TABLE I1 - X-RAY (RAD) AND DRUG CONCENTRATION RESULTING IN 50% GROWTH INHIBITION. NUMBERS ARE RELATIVE TO GLC 12
GLC
121
31
201
X-ray doxorubicin cisplatin etoposide vincristine melphalan
315 rad 0.1 nM 650 nM 380 nM 946 nM 131 nM
1.4 180 369.2 8.3 33.3 32.1
1.5 190 115.4 3.2 3.2 7.8
142
1.1
650 1538.5 > 138.9 67.0 178.5
222
332
0.7 33 12.3 0.6 1.0 1.9
1.1 35 17.2 2.1 1.4 0.9
'Post-treatment derived from non-responding patients.JPre-treatment derived from patients showing complete response after chemotherapy. an MAb directed against a natural-killer-cell-associated antigen (Ruff et al., 1986) was tested. The expression of the 170-kDa p-Glycoprotein was investigated using 2 antibodies, C-219 and JSB-1, as developed by Ling et al. (see Kartner et al., 1985) and Scheper et al. (1988), respectively.
DNAPow cytometiy Frozen biopsy specimens were processed using the detergenttrypsin method developed by VindeUv et al. (1983a,b). From the specimens which gave rise to GLC 12 and GLC 22 and from the 6 cell lines, nuclei were isolated and stained with propidium iodide. Flow cytometry was performed with an Ortho ICP flow cytometer. Trout red-blood cells were stained with propidium iodide and used as an internal reference for all samples. A cryostat section was made from the same biopsy to check cell composition of the samples histologically. Statistics The Mann-Whitney U-test was used for the correlations between the in vitro and in vivo response to cytostatic drugs. Student's t-test was applied to the results of cellular doxorubicin content. Onlyp levels < 0.05 were considered to indicate a significant difference. RESULTS
In vitro drug sensitivity Cell survival curves are shown in Figures 1-6 . In Table I1 the drug concentration and X-ray treatment resulting in 50% growth inhibition (ID5o)are calculated. In order to allow for a between the different direct comparison of the different IDSOs cell lines, figures are relative to the IDSOsof GLC 12 since this cell line proved to be the most sensitive for 3 out of 5 drugs tested. For all drugs tested there was a wide variation in sensitivity,e.g. for doxorubicin the IDS0scovered a 3-log range. MTA sensitivities did not significantly change with passage in culture for the different cell lines. Since, in this study, 2 types of culture medium were used and the cellular response to cytotoxic drugs may vary as a function of the type of medium used, 2 cell lines (GLC 3 and GLC 14) were cultured and tested in both media. There was no difference in IDSo,for the 5 drugs tested, between the culture media for GLC 3 and 14 (results not shown). There was no significant correlation for sensitivity of individual drugs with the clinical data. GLC 12 proved to be the most sensitive for 3 out of 5 drugs tested, although it originated from a patient who had progressive disease despite chemotherapy. In contrast, GLC 14, derived from a patient who achieved a clinical CR, was the most resistant for all drugs tested.
Cellular doxorubicin content Cellular doxorubicin content after 1 hr incubation with various concentrations is shown in Figure 7. GLC 22 had the lowest cellular doxorubicin content at all incubation concentrations, which was significant compared to all cell lines (p < 0.01). When cells were incubated with 1 pM doxorubicin, GLC 12, the cell line most sensitive to doxorubicin, had the highest
L
m 3 a, u
10 0.01
0.1
1
10
incubation concentration (uM)
RCURE 7 - Cellular doxorubicin content (ng doxorubicin/mg cellular protein) after 1 hr incubation with doxorubicin. Each point represents the mean k SE of 3 experiments, each performed in duplicate. Symbols as in Figures 1-6.
cellular doxorubicin content, which was statistically significant compared to GLC 14 (p < 0.05), GLC 20, 22 and 33 (each p < 0.01) but not compared to GLC 3. On the other hand, GLC 14, which was most resistant to doxorubicin in the MTA of this subset of cell lines, had a consistently higher cellular doxorubicin content than GLC 20, 22 and 33 at incubation concentrations ranging from 0.1 to 1.0 pM (p < 0.01). At these concentrations the cellular doxorubicin content was lower (p < 0.05) compared to GLC 3 and 12. When higher incubation concentrations (5.0 and 10.0 pM) were used, GLC 14 had the highest cellular doxorubicin content (p = NS compared to GLC 3, p < 0.01 compared to GLC 12, 20, 22, 33). Using rank order correlation (Kendall), there were no significant correlations between doxorubicin ICsOs(MTA) and cellular doxorubicin content at all incubation concentrations.
Zmmunohistochernistiy of tumor biopsy specimen and cell lines (Table ZZZ) Original tumor biopsy specimens were available for all corresponding cell lines except GLC 3. Immunohistochemical analysis revealed only minor differences between the tumor biopsy specimens and their corresponding cell lines. An antigen recognized by leu-7 was present on most tumor cells in culture, whereas it was invariably absent in the tumor biopsy specimens. Two MAbs reactive with intermediate filaments (MNF and Vim) showed positive staining with a small number of tumor cells in culture in all cell lines, with the exception of GLC 33 in which there was no staining with Vim. In contrast, in only 2 tumor biopsy specimens (20 and 14) did we find tumor cells reactive with these 2 MAbs. The neuroendocrine-related antigens recognized by MAbs MOC-1, MOC-21, MOC-32, MOC-51 and MOC-52 as well as the epithelial antigen
76
SMIT E T A L . TABLE I11 - IMMUNOSTAINING OF CELL LINES AND CORRESPONDING TUMOR BIOPSY SPECIMEN (TBS)
GLC.1
GLC12
TBS 12
GLC20
TBS20
GLC14
TBSt4
GLC22
TBS22
GLC33
< 1% < 1% < 10% < 1% +
Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer
IN VITRO RESPONSE OF HUMAN SMALL-CELL LUNG-CANCER CELL LINES TO CHEMOTHERAPEUTIC DRUGS; NO CORRELATION WITH CLINICAL DATA E.F. SMIT',~, E.G.E. DE VRIES'.~, H. TIMMER-BOSSCHA', L.F.H.M. DE LElJ3,J.W. 00STERHU1S4, R.J. SCHEPER', J.J. WEENING4, P.E. POSTMUS~ and N.H. MULDER' 'Dept. of Internal Medicine, Division of Medical Oncology, 2Dept.of Pulmonary Medicine, 3Dept. of Clinical Immunology, 4Dept.of Pathology, University Hospital Groningen; 5Dept.of Pathology, Free UniversityHospital Amsterdam, The Netherlands. Three cell lines derived from small-cell lungcarcinoma (SCLC) tumors of patients who had no clinical response after treatment with a multi-drugregimen were compared to 3 cell lines derived from tumors of patients who, upon treatment, showed a complete clinical response. These 2 groups of cell lines were considered to represent the in vitro counterparts of the 2 extremes of the clinical spectrum of sensitivity for chemotherapeutic drugs in small-cell lung cancer. To assess whether the in vivo (in)sensitivity of a tumor to a certain drug regimen is retained in vitro, the cell lines were tested for drug sensitivity using the microtiter-welltetrazolium assay and the results were compared with the in vivo data. No correlationwas found. Since in vitro models using cell lines are based on the assumption that a cell line reflects the properties of the tumor from which it is derived, several additional parameters such as MAb staining against different SCLC-associated antigens and DNA content were analyzed in the biopsies and the cell lines. The results showed that selection of discrete tumor-cell populationsin vitro occurs. Results of in vitro chemosensitivitytesting for individual SCLC patients should be interpreted with caution.
Small-cell lung cancer (SCLC) accounts for 20-25% of newly diagnosed cases of lung cancer (Ianuzzi and Scoggin, 1986). It is characterized by early and widespread occurrence of metastases and rapid progression of disease. Human SCLC provides a good model to study the problems of initially unresponsive tumors. The vast majority (about 90-95%) of patients with SCLC show major responses to chemotherapeutic agents of different classes (Hansen et al., 1985). However, 5 1 0 % of patients present with a tumor that is initially unresponsive to most commonly used cytotoxic agents, and invariably die within several weeks. Tumors of the latter group of patients may be termed intrinsically resistant according to the Goldie-Goldman hypothesis, as opposed to the initially sensitive tumors that are present in the former group (Goldie and Goldman, 1984). The aim of the present invcstigation was to study whether this in vivo phenomenon is also reflected in vitro. MATERIAL AND METHODS
Chemicals Doxorubicin was provided by Farmitalia Carlo Erba (Milan, Italy). Cisplatin and etoposide were obtained from Bristol Myers (Madrid, Spain), vincristine from E. Lilly (St. Cloud, France) and melphalan from Wellcome (London, UK). FCS was purchased from Flow (Irvine, UK). RPMI medium was obtained from Gibco (Paisley, UK). M 7 T was supplied by Sigma (St. Louis, MO). Cell lines Three cell lines GLC 3, GLC 12 and GLC 20, were established from patients who had no measurable response to chemotherapy. GLC 3 was derived from a pleural effusion of a 61-year-old male and has been described (de Leij etal., 1985a). Briefly, the clinical history of this patient was as follows: he received 2 courses of cyclophosphamide (750 mg/m2) on day 1, cisplatin (75 mg/m2) on day 1 and etoposide (100 mg/m2) on days 1, 3 , 5 over a 3-week period. Since no response was seen,
chemotherapy was changed to doxorubicin (60 mg/m2) on day 1, vincristine (1 mg/m2) on days 1and 8 and procarbazine (100 mg/m2) on days 1-10 over a 3-week period. After 2 courses of second-line chemotherapy, the tumor was still clearly progrcssive and at that time GLC 3 was established from the pleural effusion. The patient died 14weeks after the start of chemotherapy. GLC 12 was derived from a 64-year-old male who received 4 courses of cyclophosphamide (1,000 mg/m2) on day 1, doxorubicin (40 mg/m2) on day 1and vincristine (1 mg/m2) on days 1 and 8 over a 3-week period. The tumor showed no change on chest X-rays. A thoracotomy was performed and a lymph-node metastasis was found adherent to the pulmonary artery. From this lymph node GLC 12 was established. Radiotherapy (37.5 Gy) was administered to the left hilar region and mediastinum, which again induced no measurable tumor regression. This patient died with extensive liver metastases 33 weeks after the start of chemotherapy. GLC 20 was derived from a 67-year-old male after 6 courses of chemotherapy consisting of cyclophosphamide (1,000 mg/m2) on day 1, doxorubicin (40 mg/m2) on day 1 and vincristine (1 mg/m2) on days 1 and 8 over a 3-week period. The tumor showed no regression and became progressive within a few weeks after Chemotherapy had been stopped. A biopsy was taken of the primary tumor by bronchoscopy and the cell line established. The patient died 28 weeks after the diagnosis of SCLC. The cell lines GLC 14, GLC 22 and GLC 33 were established from patients who showed clinical complete remissions (CR) after chemotherapy. The patient history of GLC 14 has been described elsewhere (Berendsen et al., 1988a; de Vries et al., 1989). Briefly, GLC 14 was established from a supraclavicular lymph node of a 55-year-old female which was removed before Chemotherapy. After 5 courses of cyclophosphamide (1,000 mg/m2) on day 1, doxorubicin (45 mg/m2) on day 1 and etoposide (100 mg/m2) on days 1, 3, 5 over a 3-week period a clinical CR was achieved. The tumor relapsed after a 31-week disease-free interval and the patient died 86 weeks after the start of chemotherapy. GLC 22 was derived from the primary tumor site (right lower lobe) of a 50-year-old male prior to chemotherapy. This patient received the same chemotherapy regimen as the patient from whom GLC 14 was established. A clinical CR was achieved after 5 courses. This patient died from brain metastases 71 weeks after the diagnosis of SCLC. GLC 33 was established from a 62-year-old female with a ('To whom correspondence and reprint requests should be sent, at the Dept. of Internal Medicine, Division of Medical Oncology, University Hospital, Oostersingel59,9713 EZ Groningen, The Netherlands. Abbreviufionst SCLC, small-cell lung cancer; CR, complete response; FCS, fetal calf serum; MTA, MTT assay; MTT, 3 4 4 5 dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumhromide;PBS, phosphate-buffered saline (0.14 M MaC1,2.7 mM KCI, 6.4 mM NaZHPO4. 2H20 and 1.5 mM KHzPO4, pH 7.4); MAb, monoclonal antibody; MDR, multi-drug resistance; TBS, tumor biopsy specimen.
Received: June 20,1991 and in revised form December 16,1991
73
CLINICAL A N D CELL-LINE DRUG SENSITIVITY; NO CORRELATION
supraclavicular lymph-node metastasis present at the time of diagnosis. After 2 courses of chemotherapy as described under GLC 14, a CR was achieved. The patient received 3 further courses, but eventually died 47 weeks after the start of chemotherapy. The cell lines were maintained in 5% COz, 95% air at 37°C. GLC 3, GLC 20 and GLC 14 were kept in culture in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. The growth medium for GLC 12, GLC 22 and GLC 33 was SCLC-2 medium prepared according to the method of Minna et al. (1982) and supplemented with 3% FCS. This medium was chosen because it was impossible to culture these cell lines in RPMI 1640 medium containing 10% FCS. GLC 3, GLC 14 and GLC 33 grew partly attached and partly floating in aggregates. GLC 12, GLC 20 and GLC 22 grew exclusively floating in aggregates. The culture doubling time, as determined by the MTA under the conditions outlined for the MTA studies (see below), was 25,72 and 49 hr for GLC 3, GLC 12 and GLC 20, and 26,44 and 32 hr for GLC 14, GLC 22 and GLC 33. For all experiments, cells were incubated 1:l for 5 min with 0.02% EDTA solution (0.35 g NaHC03, 8 g NaCI, 0.4 g KCI, 1 g dextrose, 0.2 g EDTA, 1.0 1 HzO) to prepare a cell suspension containing small clumps with a viability, as determined by Trypan-blue exclusion, of > 75% for all cell lines. All cell lines were tested between passages 20 and 50.
MTT assay (MTA) The MTA, as described by Mossman (1983) and Carmichael et al. (1987), was used with some minor modifications (TimmerBosscha et al., 1989). Before the assays were performed, a linear relationship of cell number to MTT formazan crystal formation was checked. The relationship between seeding density, incubation volume and incubation period was established for each cell line separately after growth studies (Table I). In this way, all cell lines were cultured in the test plates for 2-3 cell divisions; they were in exponentional growth phase at the time of testing and produced an absorption of 1.0 at 520 nm (see below) at the end of the culture period. The appropriate number of cells was incubated in 96-well culture plates (Nunc, Gibco, Grand Island, NY) in culture medium to which one of the following agents was added for 1 hr: doxorubicin, cisplatin, vincristine, etoposide, melphalan. All stock solutions of drug were diluted with RPMI 1640 medium to reach the desired concentrations. Melphalan was used as a substitute for cyclophosphamide as melphalan does not require endogenous activation (Ruckdeschel et a[., 1987). For X-ray treatment (Philips X-ray source operated at 200 kV, 15 mA, 0.5 mm Cu/A filter) cells were placed in plastic tubes in a volume of 500 pl. After irradiation the cells were transferred to 96-well culture plates. After incubation, cells were washed 3 times with fresh medium by centrifugation of the microtiter plates at 180g for 10 min. They were again cultured for different time periods as determined earlier, then 20 pI of MTT solution (5 mg/ml PBS) were added to each well and the cells were incubated at 37°C for 165 min. Thereafter the plates were centrifuged at 180 g for 15 min and the supernatant was carefully removed without disturbing the formazan crystals. Cells grown in the SCLC-2 medium were washed once with PBS. This step was introduced because this medium caused a turbidity with DMSO, probably due to BSA. Subsequently, the formazan crystals were solubilized by adding 200 pl 100% DMSO. All plates were scanned at 520 nm immediately after resolubilizing of the formazan crystals using a scanning microtiter-well spectrophotometer (Titertek Multiscan, Flow, Irvine, UK). The percentage cell survival was calculated by the formula: mean of the test samplesimean of 3 untreated samples. Controls consisted of media without cells (background extinction) and cells incubated in wells with medium only. For each drug, drug combination and X-ray treatment at least 3 expcriments were performed, each in quadruplicate.
TABLE I - CELL-LINE GROWTH CHARACTERISTICS UNDER MTA CONDITIONS
Cell line
Number of cells plated
GLC 3 GLC 12 GLC 14 GLC 20 GLC 22 GLC 33
30,000 25,000 35,000 10,000
25,000 20,000
Volume (111)
100 250 100 100 250 250
Incubation Period (days)
5 16 4 11
22 8
Cellular doxorubicin content Cellular doxorubicin content was determined after 1 hr incubation in appropriate medium at 3 7 T , using at least 1 x lo6 cells. To correct for extracellular bound doxorubicin, control samples were incubated at 0°C for 5 min. The incubation period was terminated by washing the cells 3 times with ice-cold PBS, and extracted with 0.3 N HC1-50% overnight. After centrifugation the fluorescence was measured in the supernatant in a Kontron (Eching, Germany) spectrofluorometer at excitation and emission wavelengths of 474 and 549 nm, respectively (Bachur et al., 1970). Cellular doxorubicin content was calculated by subtracting the values measured at 0°C from the corresponding levels measured at 37°C. In order to correct for cell-size differences, cellular doxorubicin content was expressed as ng doxorubicin/mg cellular protein. Cellular protein determinations (Lowry et al., 1951) correlated significantly (r = 0.83, p < 0.05, results not shown) with tumor-cell size as determined by FACS analysis using a FACStar flow cytometer (Becton Dickinson, Sunnyvale, CA). Tumor biopsy specimen The original tumor biopsy specimens from which the cell lines were derived, except GLC 3 (which was derived from a pleural effusion), were split into 3 parts. One part was used for routine pathological procedures, one part for establishment of the cell line and the third was snap-frozen in OCT compound in liquid freon and kept at -80°C until use. This last part was used for immunohistological examination of the tumor cells with MAbs and DNA flow cytometry. Normal histological examination showed that the tumor biopsy specimen associated with GLC 12 and GLC 14 consisted of almost pure tumor material; the remaining tumor biopsy specimens were admixed with normal tissue, but at least 25% of their volume consisted of tumor cells. All tumor biopsy specimens were immunostained with MAbs, whereas DNA flow cytometric analysiswas possible with only 2 specimens, namely those of GLC 12 and GLC 22. Monoclonal antibodies Cryostat sections from frozen tumor biopsy specimens were made. Immunostaining was performed on these sections and on cytospin preparations of cell lines according to previously described methods (de Leij etal., 1984). MAbs directed against different SCLC-associated antigens were generated. These antibodies are reactive with antigens expressed in normal and malignant tissues with a neuroendocrine differentiation state (MOC-1, MOC-21, MOC-32, MOC-51, MOC-52) (de Leij et al., 1985a, 1986; Postmus et al., 1986) and an antibody reactive with tissues with an epithelial differentiation state (MOC-31). Three MAbs directed against intermediate-sized filament proteins were also included in the study: RGE 53, directed against cytokeratin 18 (Moll et al., 1982), Vim, directed against vimentin (Broers et al., 1985) and MNF, directed against the 210- and 68-kDa components of neurofilaments (Ramaekers et al., 1983; Kluk et al., 1984). Vim and MNF are non-reactive with normal bronchial epithelia. RGE 53 reacts with normal bronchial columnar epithelium and type-2 pneumocytes. Leu-7,
74
SMIT E T A L .
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400
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1000
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Doxorubrcin (uM)
FIGURE 2
FIGURE 1
0
P
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U
01
100
10
1
1000
0
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0001
001
LL
Cisplatin (uM)
01
100
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Etoposide (uM)
FIGURE 3
FIGURE 4
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Melphalan (uM)
Vicristine (iiM)
FIGURE 6
FIGURE 5
FIGURES 1-6 - Microtiter-well tetrazolium assay after X-ray treatment (Fig. 1) and 1 hr incubation with doxorubicin (Fig. 2), cisplatin (Fig. 3), etoposide (Fig. 4), vincristine (Fig. 5 ) and melphalan (Fig. 6). Each point represents the mean SE of 3 experiments, each performed in quadruplicate. 0 GLC3, A GLC 12, - GLC 14, D GLC 2 0 , O GLC 22, GLC 33.
+
*
75
CLINICAL AND CELL-LINE DRUG SENSITIVITY; NO CORRELATION TABLE I1 - X-RAY (RAD) AND DRUG CONCENTRATION RESULTING IN 50% GROWTH INHIBITION. NUMBERS ARE RELATIVE TO GLC 12
GLC
121
31
201
X-ray doxorubicin cisplatin etoposide vincristine melphalan
315 rad 0.1 nM 650 nM 380 nM 946 nM 131 nM
1.4 180 369.2 8.3 33.3 32.1
1.5 190 115.4 3.2 3.2 7.8
142
1.1
650 1538.5 > 138.9 67.0 178.5
222
332
0.7 33 12.3 0.6 1.0 1.9
1.1 35 17.2 2.1 1.4 0.9
'Post-treatment derived from non-responding patients.JPre-treatment derived from patients showing complete response after chemotherapy. an MAb directed against a natural-killer-cell-associated antigen (Ruff et al., 1986) was tested. The expression of the 170-kDa p-Glycoprotein was investigated using 2 antibodies, C-219 and JSB-1, as developed by Ling et al. (see Kartner et al., 1985) and Scheper et al. (1988), respectively.
DNAPow cytometiy Frozen biopsy specimens were processed using the detergenttrypsin method developed by VindeUv et al. (1983a,b). From the specimens which gave rise to GLC 12 and GLC 22 and from the 6 cell lines, nuclei were isolated and stained with propidium iodide. Flow cytometry was performed with an Ortho ICP flow cytometer. Trout red-blood cells were stained with propidium iodide and used as an internal reference for all samples. A cryostat section was made from the same biopsy to check cell composition of the samples histologically. Statistics The Mann-Whitney U-test was used for the correlations between the in vitro and in vivo response to cytostatic drugs. Student's t-test was applied to the results of cellular doxorubicin content. Onlyp levels < 0.05 were considered to indicate a significant difference. RESULTS
In vitro drug sensitivity Cell survival curves are shown in Figures 1-6 . In Table I1 the drug concentration and X-ray treatment resulting in 50% growth inhibition (ID5o)are calculated. In order to allow for a between the different direct comparison of the different IDSOs cell lines, figures are relative to the IDSOsof GLC 12 since this cell line proved to be the most sensitive for 3 out of 5 drugs tested. For all drugs tested there was a wide variation in sensitivity,e.g. for doxorubicin the IDS0scovered a 3-log range. MTA sensitivities did not significantly change with passage in culture for the different cell lines. Since, in this study, 2 types of culture medium were used and the cellular response to cytotoxic drugs may vary as a function of the type of medium used, 2 cell lines (GLC 3 and GLC 14) were cultured and tested in both media. There was no difference in IDSo,for the 5 drugs tested, between the culture media for GLC 3 and 14 (results not shown). There was no significant correlation for sensitivity of individual drugs with the clinical data. GLC 12 proved to be the most sensitive for 3 out of 5 drugs tested, although it originated from a patient who had progressive disease despite chemotherapy. In contrast, GLC 14, derived from a patient who achieved a clinical CR, was the most resistant for all drugs tested.
Cellular doxorubicin content Cellular doxorubicin content after 1 hr incubation with various concentrations is shown in Figure 7. GLC 22 had the lowest cellular doxorubicin content at all incubation concentrations, which was significant compared to all cell lines (p < 0.01). When cells were incubated with 1 pM doxorubicin, GLC 12, the cell line most sensitive to doxorubicin, had the highest
L
m 3 a, u
10 0.01
0.1
1
10
incubation concentration (uM)
RCURE 7 - Cellular doxorubicin content (ng doxorubicin/mg cellular protein) after 1 hr incubation with doxorubicin. Each point represents the mean k SE of 3 experiments, each performed in duplicate. Symbols as in Figures 1-6.
cellular doxorubicin content, which was statistically significant compared to GLC 14 (p < 0.05), GLC 20, 22 and 33 (each p < 0.01) but not compared to GLC 3. On the other hand, GLC 14, which was most resistant to doxorubicin in the MTA of this subset of cell lines, had a consistently higher cellular doxorubicin content than GLC 20, 22 and 33 at incubation concentrations ranging from 0.1 to 1.0 pM (p < 0.01). At these concentrations the cellular doxorubicin content was lower (p < 0.05) compared to GLC 3 and 12. When higher incubation concentrations (5.0 and 10.0 pM) were used, GLC 14 had the highest cellular doxorubicin content (p = NS compared to GLC 3, p < 0.01 compared to GLC 12, 20, 22, 33). Using rank order correlation (Kendall), there were no significant correlations between doxorubicin ICsOs(MTA) and cellular doxorubicin content at all incubation concentrations.
Zmmunohistochernistiy of tumor biopsy specimen and cell lines (Table ZZZ) Original tumor biopsy specimens were available for all corresponding cell lines except GLC 3. Immunohistochemical analysis revealed only minor differences between the tumor biopsy specimens and their corresponding cell lines. An antigen recognized by leu-7 was present on most tumor cells in culture, whereas it was invariably absent in the tumor biopsy specimens. Two MAbs reactive with intermediate filaments (MNF and Vim) showed positive staining with a small number of tumor cells in culture in all cell lines, with the exception of GLC 33 in which there was no staining with Vim. In contrast, in only 2 tumor biopsy specimens (20 and 14) did we find tumor cells reactive with these 2 MAbs. The neuroendocrine-related antigens recognized by MAbs MOC-1, MOC-21, MOC-32, MOC-51 and MOC-52 as well as the epithelial antigen
76
SMIT E T A L . TABLE I11 - IMMUNOSTAINING OF CELL LINES AND CORRESPONDING TUMOR BIOPSY SPECIMEN (TBS)
GLC.1
GLC12
TBS 12
GLC20
TBS20
GLC14
TBSt4
GLC22
TBS22
GLC33
< 1% < 1% < 10% < 1% +
