American Journal of Pathology, Vol. 139, No. 4, October 1991 Copyright © American Association of Pathologists

Serum Response Heterogeneity Among Nonsmall Cell Lung Cancer Cell Lines Kelly T. Goldsmith,* Catherine M. Listinsky,t and Robert 1. Garver, Jr.* From the Department of Medicine,* Division of Pulmonary and Critical Care Medicine, and the Department of

Pathology, t Division of Surgical Pathology, University of Alabama School ofMedicine, Birmingham, Alabama

This study examined the morphology, in vitro growth, and two genetic responses to serum stimulation in the nonsmall cell lung cancer (NSCLC) cell lines SKLu-I, SK-MES-1, A427, and A549. Morphologically, all four were NSCLC: SK-Lu- I was undifferentiated the remainder were adenocarcinoma variants. SKLu-i and SK-MES-1 were slow growing with lowanchorage independent growth capacity; the A427 and A549 lines were fast growing with highanchorage independent growth capacity. All of the lines expressed basicfibroblastgrowth factor (bFGF) as a dominant 7.1 kb transcript at amounts significantly lower than in control human lung fibroblasts. bFGF expression could be upregulated by serum exposure in several nontransformed human cell lines, but only the SK-Lu-I NSCLC cells increased bFGF after serum exposure (482%) compared with a peak increase of 1222% in the fibroblast controls. All of the NSCLC cell lines increased c-fos in response to the same serum stimulations. These results show that growth-factor gene e-xpression can be modulated in NSCLC, and that significant differences e-xist among NSCLC cell lines commonly used as laboratory correlates of human disease. (Am J Pathol 1991,

139:939-947)

Nonsmall cell lung cancer (NSCLC) remains a leading cause of cancer deaths in both men and women.1 The major variants of NSCLC include squamous, adenosquamous, adenocarcinoma, bronchoalveolar, large cell, and undifferentiated variants although additional subcategories are well described.2 Morphologic criteria can also be used to assess the state of differentiation of the cancer.3 Differences do exist in the clinical characteristics of the different NSCLC subtypes, perhaps best exemplified by data from multiple groups showing a modest but consis-

tent survival advantage in individuals with the squamous cell variant relative to the other variants.45 In contrast, the significance of the state of relative differentiation with respect to prognosis is not clear.

The molecular pathology of NSCLC is in the process of being defined, but two significant phenomena have been well documented. First, activating mutations of proto-oncogenes and inactivating mutations of antioncogenes can be demonstrated in many cases of NSCLC. The proto-oncogene k-ras-2 is activated predominantly by codon 12 or 13 point mutations in 21 to 27% of individuals with the adenocarcinoma variant of NSCLC.6'7 Activation of other proto-oncogenes has been observed in a smaller percentage of NSCLC tissues.-9 With respect to anti-oncogenes, p53 mutations have been found in a substantial proportion of NSCLC.1011 Second, growth factors are produced by NSCLC and likely modulate NSCLC growth. Many studies have found a variety of growth factor transcripts or proteins in NSCLC cell lines or resected/autopsy specimens, including basic fibroblast growth factor,12 gastrin-releasing peptide,13 insulinlike growth factor-I (IGF-1),14 IGF-I1,15 plateletderived growth factor A and B chains,'1516 transforming growth factor a (TGFa),1918 and TGFP.'516'19 NSCLC cell lines have been extensively used as laboratory models of NSCLC. In the context of growth-factor effects, some growth factors, such as epidermal growth factor (EGF) and TGFa, stimulate NSCLC cell-line growth.oc021 NSCLC cells have also been found to produce growth-stimulating TGFa and its cognate receptor,21 raising the possibility that some growth factors may stimulate NSCLC growth via an autocrine mechanism. At least one growth factor, TGFP, retards NSCLC cell-line growth.22'23 Most of these studies have utilized only 1 or 2 cell lines; little is known about possible growth factor Presented at the National Amencan Federation of Clinical Research Meeting, Seattle, Washington, May 6, 1991, and published in abstract form in Clinical Research 1991, 39:356A. Supported in part by a research grant from the American Lung Association of Alabama and grant IN-66-30 from the Amercan Cancer Society. Dr. Robert Garver is a recipient of an American Cancer Society Clinical Oncology Career Development Award. Accepted for publication June 12, 1991. Address reprint requests to Dr. Robert I. Garver, Jr., University of Alabama School of Medicine, LHRB 337, University Station, Birmingham, AL 35294.

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response heterogeneity among a larger number of NSCLC cell lines. As an initial approach to the question of growth-factor response heterogeneity among NSCLC cell lines, we examined the expression of the basic fibroblast growth factor (bFGF) gene in NSCLC tissues. bFGF is a growth factor previously found to be expressed in one NSCLC cell line,12 but little else was known about its possible role in NSCLC. bFGF is a 16-18 kDa protein, and a member of the heparin-binding growth-factor family that includes the growth factors acidic FGF24 and keratinocyte growth factor,25 as well as the proto-oncogenes/oncogenes int2,26 hst/KFGF,27 FGF-528 and FGF-6.-9 The protein is the product of a single copy, 3 exon gene that is expressed in almost all tissues examined.30 It affects many cell types as a mitogenic agent,31 stimulates angiogenesis32 and likely plays a major role in differentiation.33 We hypothesized that bFGF might be differently expressed or regulated among several NSCLC cell lines and that genetic responses might correlate with other growth or morphologic characteristics. The expression of bFGF in 4 NSCLC cell lines was compared to that in human lung fibroblasts under normal culture conditions. Serum-starved cultures were stimulated with serum to determine whether bFGF expression could be upregulated in the normal or NSCLC tissues. The results showed that bFGF expression responded differently in the majority of the NSCLC cell lines compared with normal tissues, and that this heterogeneous response correlated with state of differentiation.

Methods Morphologic Examination The NSCLC cell lines were examined in a blinded manner by C.L. Cytopathologic analysis was performed on cells detached by a 5-minute incubation with 0.25% trypsin that were either directly applied to slides ("cytospin preps") for Papanicolou staining, or as cell blocks embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Cell-to-cell relationships were examined in scrapings from the culture dishes that were fixed in formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin, or fixed in Carson's fixative, epoxy-embedded, and sectioned for electron microscopy. Paraffin-embedded sections were stained for mucin with both standard mucicarmine and periodic acid schiff with diastase stains.

Population Doubling Times and Soft Agar Cloning Efficiency The four NSCLC were plated (5 x 1 05/10 cm plate) for 48 hours, after which the cells were detached with 0.25%

trypsin and counted via a hemocytometer. The population doubling time (PDT) was calculated by the formula: PDT = 48 hr/(1og2 #cells at 48 hr) - (1092 #cells at 0 hr) The ability of NSCLC and the normal adult human lung fibroblast cell lines to form colonies under anchorage independent conditions was evaluated by a modification of Macpherson's technique.34 Trypsinized cells (103-105/35 mm dish) in 1 ml of complete medium with 3% molten noble agar (Difco) were poured on top of hardened feeder layers (complete media with 5% noble agar). Plates were allowed to harden, then incubated in a water-saturated, 5% CO2 and air atmosphere for 2 weeks. Preliminary studies showed that 1 week was too short for adequate colony development in all of the cell lines. After 2 weeks, colonies (¢40-50 cells) were manually counted.

Tissue Sources and Culture Conditions The normal adult human lung fibroblast (CCD-8Lu), human liver epithelium (Chang liver), human intestinal smooth muscle (HISM), human embryonic palatal mesenchyme (HEPM) and NSCLC cell lines (SK-Lu-1, SKMES-1, A427, A549) were obtained directly from the American Type Culture Collection. All cultures were maintained in "complete media" (1:1 mixture of F12 and Dulbecco's modified essential media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and penicillin/streptomycin) at 37°C in a water-saturated atmosphere of 5% CO2 and air. Serum stimulations were performed by growing cells to confluence, followed by a 48-hour period in serumfree media (same as complete media without FCS). Viability of cell lines by trypan blue exclusion was >95% in all of the NSCLC cell lines after the serum starvation period. After the serum starvation period, the media was replaced with either fresh serumfree media (controls), or complete media for varying lengths of time as indicated in the text, after which total cellular RNA was extracted.

RNA Extractions Total cellular RNA was extracted from the cell cultures by guanidine lysis followed by cesium chloride gradient centrifugation. Cells were lysed with 4 M guanidine isothiocyanate, 20 mM sodium acetate, 0.1 mM dithiothreitol, 0.5% sarkosyl solution. The RNA was isolated by centrifugation through a 5.6 M cesium chloride, 100 mM ethylenediaminetetraacetic acid cushion and concentration determined by ultraviolet spectroscopy.

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DNA Probes and Radiolabelling The pHFL1 partial human bFGF cDNA probe was provided by Judith Abraham (California Biotechnology, Mountain View, CA).30 This 800-base pair (bp) probe includes most of exons 2 and 3 of the human bFGF cDNA. The complete gamma actin cDNA probe was provided by L. Kedes (Stanford University, Palo Alto, CA).35 The c-fos probe was a 490 bp Apa fragment of the pcfos(human)-1 (ATCC #41042 [36]) genomic c-fos clone, and included 50 bp of intron 3 and 440 bp of exon 4 of the human c-fos proto-oncogene. Probes were radiolabelled with [a32P]dCTP by the random primer tech-

nique.37

complete removal of the labelled bFGF probe. The slot blots were rehybridized with oligo(dT) (12-1 8mers, Pharmacia), labelled with [y32P]ATP by polynucleotide kinase, according to the technique described by Harwell.3 Integrated autoradiogram signals of slot blots hybridized with the bFGF probe and oligo (dT) were quantified with an LKB Ultrascan XL Laser Densitometer using the Gelscan XL software. Corrections for the variations in mRNA amounts among the total cellular RNA samples were performed by multiplying the averaged bFGF signal for any specimen "n" by the averaged oligo-dT signal for the serum-free control RNA specimen divided by the oligo-dT signal from specimen "n." For all corrected data points, correction values ranged from 0.8 to 1.2. Final data is expressed as the mean ± SEM.

Gene Expression Analysis Qualitative evaluation of bFGF expression was performed by hybridizing northern blots with a radiolabelled human bFGF probe. Total cellular RNA (10 ,ug) was electrophoresed in agarose under denaturing conditions, then immobilized on nylon membranes (Gene Screen, NEN/DuPont) by capillary transfer and ultraviolet light fixation. The prehybridization and hybridization conditions used were those recommended by the Gene Screen manufacturer. The labelled membranes were exposed to Kodak X-AR2 film with intensifying screens at - 70'C for 24 to 48 hours. Quantitative evaluation of bFGF and c-fos expression was performed on serially diluted total cellular RNA hybridized with the corresponding radiolabelled probes. Slot blots were made by applying duplicate serial dilutions of RNA (5, 2.5, 1.25 ,ug) to nitrocellulose membranes via a Minifold 11 apparatus (Schleicher & Schuell) under conditions recommended by the manufacturer and fixed by baking in vacuo at 800C for 2 hours. The prehybridization mixture (50% deionized formamide, 5X Denhardt's solution, 0.5% sodium dodecyl sulfate [SDS], 5X: 0.3M sodium chloride/0.02M sodium phosphate/ 1 mM EDTA [SSPE]) bathed the membranes for 12 to 24 hours at 42°C. The radiolabelled probe was added directly to the mixture and incubated at the same temperature with agitation for 20 to 24 hours. The filters were washed as follows: (2) 10-minute washes in 2X SSPE/ 0.1% SDS at room temperature, (2) 10-minute washes in 0.1X SSPE/0.1 % SDS at room temperature, (1) 60-minute wash in 0.1 X SSPE/O.1 % SDS at 58°C. Autoradiography was performed as described earlier. The relative amount of polyadenylated RNA, i.e., mRNA, in each total cellular RNA specimen was determined by oligo(dT) rehybridization of all slot blots. The slot blots were stripped by boiling in 0.1X SSPE/0.1% SDS followed by overnight autoradiography to ensure

Results Morphologic Characteristics of NSCLC Cell Lines Light microscopic examination of the NSCLC cell lines showed features consistent with bronchogenic carcinoma. The SK-Lu-1 cells (Figure 1 a) exhibited no specific features of epithelial differentiation. The SK-MES-1 cells (Figure 1 b) demonstrated villous-like cytoplasmic projections, occasional groupings resembling gland formation, and rare vacuoles. Both the SK-Lu-1 and SK-MES-1 cells were mucin-negative. A427 cells (Figure 1 c) contained frequent intracytoplasmic vacuoles that stained positive for mucin and occasional signet ring cells. A549 cells (Figure 1 d) had occasional clumps of columnar cells and were mucin-positive. Electron microscopy confirmed the light microscopy morphologic evaluations. All of the cell lines had distinct morphologic features, but all were consistent with NSCLC.

Growth Characteristics of the NSCLC Cell Lines The NSCLC cell lines differed considerably in the context of in vitro growth characteristics (Table 1). The SK-Lu-1 and SK-MES-1 cells were slow growing, as evidenced by population doubling times of 79.2 ± 0.4 hours and 57.3 + 14.1 hours, respectively. Both the A427 and A549 cells doubled significantly (P < 0.05) more quickly than the SK-Lu-1, but the SK-MES-1 doubling time difference was not statistically significant. The soft agar cloning efficiency results paralleled the population doubling times (Table 1). The SK-Lu-1 and SK-MES-1 cells had a much lower soft agar cloning efficiency than the A427 and A549

Goldsmith, Listinsky, and Garver

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Figure 1. Morphologic evaluation of the NSCLC cell lines. Shouw are photomicrographs of cells detached by trypsin and applied directly to slides beforefixation and Papanicolou staining (526X). A: SK-Lu-1 cells. B: SK-MES-1 cells. Inset: One glandlike grouping of cells. C: A427 cells. Inset: 'Signet-ring" cell. D: A549 cells. Inset: One group of columnar cells resembling cells adjacent to a gland lumen.

cells, a difference that was statistically significant (P < 0.05). These studies demonstrated that population doubling times correlated with the efficiency of anchorage independent growth.

Steady-State Expression of bFGF in NSCLC Qualitative gene expression analysis showed that bFGF was expressed under normal culture conditions in all of the NSCLC cell lines (Figure 2a). A dominant 7.1 kb transcript was detected in the NSCLC cell lines SK-Lu-1, SKMES-1, A427, and A549. The normal adult human lung fibroblast line, CCD-8Lu, showed a 3.7 kb transcript in Table 1. Growth Characteristics of 4 NSCLCLines Anchorage-

Population doubling time (hr) CCD-8Lu SK-Lu-1 SK-MES-1 A427 A549

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addition to the 7.1 kb transcript seen in all of the lung specimens. The two dominant transcripts observed in the fibroblast RNA specimen are similar to that reported by others in a variety of other tissues.30"9 Quantitative evaluation of bFGF expression in the same cell lines demonstrated significantly lower amounts of bFGF mRNA in the NSCLC cell lines relative to the human lung fibroblasts (Figure 2b). Additional aliquots of the RNA used for the northern blots were analyzed by slot blots hybridized with the bFGF probe, and the autoradiograms quantified by densitometry. The expression in the CCD-8Lu fibroblasts was arbitrarily assigned a relative expression value of 1; all of the NSCLC cell lines had substantially less bFGF expression. Thus, although bFGF was expressed under normal culture conditions in all of the NSCLC cell lines, the relative expression was less than that in normal adult human lung fibroblasts.

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Modulation of bFGF Expression by Serum Serum starvation reduced bFGF expression, and subsequent serum stimulation increased bFGF expression, in several human tissues (Figure 3). A 48-hour serum star-

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Basic Fibroblast Growth Factor Expression 943 AJP October 1991, Vol. 139, No. 4

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Figure 2. bFGF gene expression in NSCLC cell lines under normal culture conditions. A: Qualitative evaluation of bFGF gene expression with northern blots. Shown are autoradiograms of RNA from the normal adult human lung fibroblast line CCD-8Lu and the NSCLC cell lines hybridized with the bFGF cDNAprobe. B: Quantitative evaluation of bFGF expression with slot blots. The RNA used for the northerns was also applied to slot blots hybridized with the bFGF probe. Results of densitometry autoradiogram signal quantification are shown here.

vation reduced bFGF expression to undetectable or low levels in all four human cell lines examined. A subsequent 4-hour serum stimulation produced marked increases in bFGF expression in all of the cell lines. These experiments suggested the bFGF gene was serum-responsive in multiple tissues, including liver epithelium, palatal mesenchyme, smooth muscle, and lung fibroblasts.

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The response of bFGF expression to serum stimulation was heterogeneous among the 4 NSCLC cell lines (Figure 4). The CCD-8Lu fibroblasts had a peak 1222% increase in bFGF expression 4 hours after the addition of serum, followed by a gradual decline over the next 16 hours. The SK-Lu-1 NSCLC cell line had a qualitatively similar response, showing a peak bFGF expression in-

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crease of 482% 4 hours after serum addition. In contrast, the other 3 NSCLC cell lines had no significant increase in bFGF expression over 24 hours of serum stimulation. Thus, one of the four NSCLC cell lines regulated the bFGF gene similar to that observed in normal tissues, whereas the bFGF gene was not serum-responsive in the other three lines.

c-fos Serum Response in NSCLC Cell Lines In contrast to the bFGF gene, the c-fos gene was serum responsive in all of the NSCLC cell lines (Figure 5). The same RNA specimens from the serum stimulated NSCLC and fibroblast cell lines were hybridized with the human c-fos probe. In all cell lines, c-fos expression was minimal to undetectable in the serum-starved cells before the ad-

4

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dition of serum. The c-fos expression markedly increased with a peak expression 30 minutes after serum exposure in both the lung fibroblasts and the NSCLC cell lines. The expression of c-fos in all of the cells exposed to serum for longer periods (4, 8, 24 hr) reverted to barely detectable amounts (not shown). Changes in c-fos expression could not be accurately quantified by densitometry because of the undetectable expression in the serum-starved cells not exposed to serum. These results show that the serum-starvation conditions were adequate to reduce the expression of c-fos to basal amounts, but not so severe as to impair the ability of the cells to respond to the serum stimulation.

Discussion This study demonstrated important differences between four NSCLC cell lines in the context of morphology,

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Figure 5. c-fos serum response in the NSCLC cell lines. Shown are autoradiograms of slot blots made with some of the RNA usedfor Figure 3 hybridized with the c-fosprobe. Serum-starved cells (-), cells serum-stimulated 30 min (+). Allpaired specimens shown werefrom the same experiment and autoradiogram.

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growth, and genetic responses. All four lines had light and electron microscopic features consistent with NSCLC. Half of the cell lines (SK-Lu-1, SK-MES-1) were slow growing with a low soft agar cloning efficiency, the remaining two lines (A427, A549) were rapid growers with a high soft agar cloning efficiency. All four lines expressed bFGF in a qualitatively similar manner under normal culture conditions. Exposure of serum-starved cells to fetal calf serum produced a large increase in bFGF expression among several nontransformed cell lines. In contrast, serum stimulation of NSCLC cell lines showed only one (SK-Lu-1) to respond with increased bFGF expression. Another serum responsive gene, c-fos, responded with marked serum-mediated increases in expression in all of the NSCLC cell lines. Four NSCLC cell lines used in many previous studies of lung cancer were used in these experiments. The A427 and A549 cell lines were isolated from tumor explant cultures by Giard and colleagues using tissue only identified as lung carcinoma.40 An electron microscopy study of A549 suggested the presence of Type 11 cell features,41 but biochemical characterization by the American Type Culture Collection did not confirm Type 11 cell features. Our morphologic evaluation of the A427 and A549 cell lines demonstrated features consistent with adenocarcinoma. The SK-MES-1 cell line was derived from a malignant pleural effusion in a patient with squamous cell carcinoma.42 The morphologic evaluation performed in this study found SK-MES-1 to be most consistent with an adenocarcinoma. The apparent disparity in our morphologic evaluation and tissue origin may be due to either an incorrect initial diagnosis, or from changes in the cell line produced by in vitro growth. The SK-Lu-1 cell line was isolated from a primary tumor identified as a poorly differentiated adenocarcinoma.43 Our morphologic evaluation confirmed the poorly differentiated nature of this cell line, but could not demonstrate any convincing adenocarcinoma features. Although morphologic and in vitro growth differences among NSCLC cell lines have been well documented in earlier studies, much less is known about possible differences in growth factor responses. Growth factor response heterogeneity was examined by using fetal calf serum as a source of multiple growth factors. Serum has been shown to stimulate the expression of multiple genes, such as immediate early genes like c-fos444 or growth factors like basic fibroblast growth factor (bFGF).12'46 Since serum responses can be duplicated by defined growth factors,47'8 it is believed that growth factors in serum produce serum responses. In this context, serum responses can be considered the equivalent of undefined growth-factor responses. Among the NSCLC cell lines, only the SK-Lu-1 cells

demonstrated a bFGF serum response that was similarly demonstrated in human lung fibroblasts, embryonic palatal mesenchyme, liver epithelial cells, and intestinal smooth muscle cells. These results extend the observation by Sternfeld and colleagues12 that a 4-hour stimulation with 5% FCS produced an 8-fold increase in bFGF mRNA levels in human foreskin fibroblasts. Although growth characteristics did not correlate with the presence or absence of the bFGF response in the NSCLC cell lines, lack of epithelial differentiation features in the SK-Lu-1 lines raises the possibility of a developmental relationship to the bFGF serum response. Since the other three NSCLC cell lines were adenocarcinomas, it is equally possible that the cell type (adenocarcinoma) is the major determinant of the bFGF serum response. One possibility that cannot be excluded is that the bFGF serum response is completely independent of cell type/ differentiation, and only coincidentally occurred in the least differentiated examined in this study. Unlike the bFGF gene, the c-fos gene was serum responsive in all of the NSCLC cell lines. c-fos gene expression has previously been shown by multiple groups to be suppressed by serum starvation, and markedly increased by a re-exposure to serum."'47'4 The striking feature of this response is the rapid increase in expression, typically 5 minutes after serum exposure, followed by a rapid decline to trace/undetectable levels." The c-fos response in the NSCLC cell lines followed this general pattern. These results show that the bFGF and c-fos responses to growth factors in serum are likely caused by different mechanisms, either by different growth factors within the serum, or by different, more distal pathways activated by the same growth factor receptor(s). The preservation of the c-fos response in all of the NSCLC cell lines further shows that the serum starvation conditions did not suppress the bFGF serum response in some of the NSCLC cell lines via a nonspecific toxic effect of the experimental conditions. Previous observations of bFGF expression have shown that two dominant transcripts are produced with additional smaller transcripts in lower amounts.30'3 It has been speculated that the source of the multiple transcripts can be at least partly explained by the presence of multiple polyadenylation signals within the bFGF gene.39 The adult human lung fibroblasts (CCD-8Lu) produced bFGF transcripts of 7.1 and 3.7 kb similar to those observed in previous reports. In contrast, all four of the NSCLC cell lines had a dominant 7.1 kb transcript with trace to undetectable amounts of the 3.7 kb transcript. Since we have observed a similar pattern of bFGF expression in normal, whole, human lungs (not shown), this qualitative difference in bFGF expression is most likely tissue-related rather than malignancy-related. The bFGF

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expression in each of the NSCLC cell lines was less than that in the control human lung fibroblasts, and the relative bFGF expression among these lines did not predict serum responsiveness. The heterogeneity in serum responsiveness among the four NSCLC cell lines tested has three potential clinical implications. First, one apparent fact is that not all NSCLC responds identically to serum/growth factor stimulation. The availability of an increasing number of growth factors for clinical use will likely lead to clinical trials utilizing growth factors in individuals with NSCLC. The evaluation of recombinant growth factors as therapeutic agents in the treatment of lung cancer should address the possibility that other defined growth factor responses may be heterogeneous, as was the serum response in this study. Since results presented here suggest that different variants within the NSCLC group might have significantly different responses to growth factors, it would seem most prudent to evaluate the possibility of growthfactor-response heterogeneity in defined growth factors proposed for use in clinical trials. Second, the correlation of differentiation and/or cell type with the bFGF serum response raises the possibility that precise morphologic characterization of NSCLC might be important in future therapies using growth factors. Some growth factors may produce desired effects on a subset of NSCLC defined by cell type and/or state of differentiation. Third, the changes in bFGF expression by serum in at least one NSCLC cell line suggests that other growth factors expressed by NSCLC tissue might also be responsive to modulation. Future strategies may be developed to modify the expression of growth factors within NSCLC in a therapeutically beneficial fashion. Since so little is known about the role of bFGF in NSCLC growth, it is not clear whether the bFGF serum response differences per se are physiologically relevant. At this time, it is unknown whether bFGF has any effects on NSCLC growth, or whether NSCLC has bFGF receptors. Even in the absence of direct effects on NSCLC, bFGF could exert important effects on surrounding tissues. It has been postulated to play an important role in the growth of all solid tumors as a consequence of its angiogenesis-promoting activities.32 In addition, bFGF may promote the proliferation of other supportive stromal elements important for tumor growth. Nevertheless, the influence of bFGF on NSCLC growth will require further study.

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Cancer 1990, 40:9-26 2. World Health Organization Monograph: Histologic typing of lung tumors, 2nd ed. Geneva, WHO, 1981

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19.

20.

21.

22.

23.

24.

25. 26. 27.

28.

29.

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