In[. J. Cancer: 52,85-91 (1992) 8 1992 Wiley-Liss, Inc.
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DIFFERENTIATION-RELATED EXPRESSION OF ADHESION MOLECULES AND RECEPTORS ON HUMAN NEUROBLASTOMA TISSUES, CELL LINES AND VARIANTS Nicole GROSS’.Simone FAVRE,Daniel BECKand Manuela MEYER Onco-hematology Unit, Pediatric Department, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland. The expression of cellular adhesion molecules (CAM) involved in cell adhesion and immune recognition was measured on neuroblastomatissue samples, on a neuroblastoma (NB) cell line, SK-N-SH, and on 3 phenotypically different variants, SH-SY5Y. SH-EP, SH-IN, representing neuronal, Schwannianl glial or intermediate NB-cell types. lmmunohistochemical analysis of CAM expression by NB and related tumors at different stages of differentiation revealed a co-expression of several CAM (ICAM- IlCD54, LFA-3, VIA-2 and HIA-ABC) associated with low stages and more highly differentiated NB tumors and peripheral neuroepitheliomas (PN). In contrast, N-CAM was uniformly expressed on all NB tumors. Flow cytometric analysis of CAM surface expression by SK-N-SH and variant cells revealed highly variable phenotypes. Expression of ICAM- I, LFA-3, VIA-2 and HIA-ABC molecules was associated with the epithelial cell type represented by the SH-EP variant. In contrast, low expression of these molecules and high expression of N-CAM was associated with the neuronal SH-SY5Y cells. Exposure of the NB cells t o differentiation inducers (retinoic acid, 5’-bromodeoxyuridine and phorbol esters) and cytokines (7-interferon. a-tumor necrosis factor) resulted in a variable up-regulation of the expression of all CAMS, except N-CAM, regardless of the type of differentiation induced. In an attempt to establish a link between the pattern of expression of CAM on NB cells and their susceptibility to natural killer (NK) or lymphokine-activated killer (IAK) cell lysis, the analysis revealed that NB cells expressing CAM and a differentiated phenotype were less susceptiblet o NK lysis, but no difference in the sensitivity of the NB cell types t o I A K effectors was observed. Treatment of NB target cells with cytokines or PMA decreased their susceptibility t o NK and I A K lysis, while induction of differentiation with RA or BUdR resulted in no changes in the sensitivity to NK and I A K lysis. In conclusion, expression of HLA-ABC and several co-regulated CAMS was shown to be associated with a differentiated phenotype in NB, with an overall decreased sensitivity to NK/IAK effector cells. i i j 1992 Wilev-Liss,OK.
cells and in general for tumor cells with low HLA-ABC expression (de Fries and Golub. 1988; Gronberg et al., 1988; Versteeg et al., 1989). Adhesion molecules and receptors are attracting increasing interest in tumor biology since, by mediating cell-cell contacts and cell-matrix adhesion, these molecules are believed to be involved both in the interactions of tumor cells with immune effector cells and in cell migration and dissemination processes (Albelda and Buck, 1990; Springer, 1990). We have recently described the expression pattern of MHC class-I and -11 molecules in addition to adhesion molecules ICAM-1 (CD54) and N-CAM (CD56) on NB, Ewing and peripheral neuroepithelioma (PN) cell lines (Gross et al., 1991). We further reported that, whereas an overall high expression of N-CAM could be detected on all NB cell lines, ICAM-1 expression was variable and restricted to a minority of the HLA class-I (HLA-ABC)-positive NB cell lines and PN and Ewing cell lines (Gross et al., 1991). In this report we have analysed the expression and modulation on NB SK-N-SH cells and their variants of a larger panel of adhesion-related molecules and receptors, including ICAM-1, LFA-3/CD2 molecules as well as p l integrins VLA-2 and VLA-4. We have established a relationship between the pattern of CAM expression and the degree and type of differentiation by analysis of fresh tumor samples and an NB cell line and variants. Differentiation has been induced using agents such as retinoic acid (RA), 5’-bromodeoxyuridine (BUdR) and the (PMA). phorbol ester, phorbol-3’myristate-5-acetate As some of these molecules have already been shown to be involved in different mechanisms of cell-mediated anti-tumor responses (Springer, 1990; Naganuma et al., 1991: Tomita et al., 1990), we have related the pattern of expression of adhesion molecules to the sensitivity of NB cells to NK and LAK-cell lysis.
Neuroblastoma (NB), a solid childhood tumor of neuralcrest origin, is characterized by highly malignant and metastatic behavior, especially in children over l year of age (Evans, MATERIAL AND METHODS 1980). NB is also one of the rare tumors able to mature spontaneously in vivo to a less malignant form, i.e. ganglioneu- Cell lines Human NB cell lines SK-N-SH and 3 cell variants, SH-EP, roblastoma or benign ganglioneuroma, as well as to differentiate in vitro along neuronal or Schwannian lineages in re- SH-SYSY, SH-IN were obtained from Dr. J. Biedler, Sloan sponse to several differentiating stimuli (Evans, 1980; Tsokos Kettering Institute, NY. The ACN cell line was established in our laboratory from a bone-marrow specimen of a 36-monthet al., 1985; Gross et aL, 1987). Ccll lines established from several neuroblastomas have old boy with a metastatic adrenal stage-IV NB with elevated shown a diversity in cell morphology representing the in vivo catecholamines. All cell lines, including the myelocytic cell line differentiated stages with neuronal or glial components. From K562, were grown in RPMI medium supplemented with 2mM one such cell line, SK-N-SH, clonal lines comprising neuronal glutamine. 10% FCS and gentamycin. or Schwannian/glial cell types have been isolated which Monoclonal antibodies provide a useful model for analysis of NB phenotypic diversifiAntibodies to MHC class-I and -11 molecules, B9-12-1 and cation and differentiation (Ciccarone et al., 1989; Rettig et af., D1-12, have been already described (Gross et al.. 1987). 1987; Biedler et al., 1988). N-CAM molecules were detected using MAb UJ13A, kindly NB cells are likewise characterized by low but inducible provided by Dr. Kemshead, (Bristol, UK), MAb NKH-I which class-I MHC molecule expression and absence of class-I1 molecules, even after 4-days’ exposure to y-IFN (Gross et al., 1987). Poor expression of these molecules on tumor cells has ‘To whom correspondence and reprint requests should be sent. been associated with the insensitivity of NB cells to T-cell immunosurveillance (Main et af., 1985). In contrast, a higher sensitivity to NK-cell-mediated lysis has been described for NB Received: December 20,1991 and in revised form April 9. 1992
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detects an epitope on the N-CAM molecule, and an anti-CD56 MAb, kindly provided by Dr. S. Carrel (Lausanne, Switzerland). MAbs detecting the ICAM-1 molecule included P3.58, MAb anti-CD54 and Me14-Dl2 (Gross et al., 1897, 1991). Anti-LFA-3 MAb was kindly provided by Dr. K. Figdor, Amsterdam. MAb A1-43 detecting the VLA-2 molecule was a gift of Dr. E. Brocker, Munster, Germany. MAb MX-24 detecting the VLA-4 molecule and 6B-11 anti-CD2 were provided by Dr. S. Carrel.
Cytojhorometric analysis For analysis of cell-surface expression of the different antigens, the cells were recovered by 0.02% EDTAIPBS treatment of the monolayers and washed twice in EDTAcontaining RPMI medium. The cells were reacted with MAbs for 30 min on ice, then incubated for 30 min on ice with an FITC-conjugated FAb goat anti-mouse Ig antibody (Cappel, Cochranville, PA). The fluorescence was analysed on a FACScan (Becton-Dickinson, Mountain View, CA). lmmunohistochemistry Immunohistochemical staining of fresh-frozen tumor sections was performed using an indirect 3-stage immunoenzymatic assay with alkaline phosphatase as already described (Becket al., 1988). RNA extraction and Northern-blot analysis PolyA mRNA was extracted from the different cell lines by a technique already described (Gross et al., 1990). Northern blotting and hybridization were performed by standard methods (Gross et al., 1990). The following probes, already described (Gross et al., 1990, 1991) were used to detect CAM mRNA expression. HLAABC heavy-chain transcripts were detected with plasmid p44-2 containing a human HLA class-I gene. ICAM-1 transcripts were detected with plasmid p89.1 containing a 2,700-bp insert coding for ICAM-1. N-CAM transcripts were probed with the PM13 clone kindly provided by Dr. Goridis (Goridis et al., 1985). LAK cell generation and cell-mediated lysis Human peripheral mononuclear cells (PBL), isolated from the blood of healthy volunteers by Ficoll-Hypaque centrifugation, were used as source of NK effector cells. For generation of LAK effectors, cells were further washed and resuspended at 6 x lobcells/ml of LAKmedium [RPMI 1640 supplemented with 500 Ulml Interleukin-2 (IL-2) (Glaxo, Geneva, Switzerland), 10% pooled human AB serum, glutamine and gentamycin] and incubated for 3-5 days at 37°C. LAK-mediated cell lysis was measured with a standard 4-hr Tr-release assay using effector/target cell ratios (EIT) of 4O/1, lO/l and 411, as described by Rayner et al. (1985). All NK and LAK experiments were performed at least 5 times, using different blood donors. RESULTS
Expression of cell-adhesion molecules on NB and related tumor samples A total of 36 samples of NB stage I-IV and neuroectodermderived tumors, some representing differentiated forms of NB including ganglioneuromas, PN and primitive neuroectoderma1 tumors (PNET), were analysed for CAM expression. Results of the immunohistochemical analysis are summarized in Table I. Staining for CD2, LFA-3, ICAM-1, VLA-2 and VLA-4 could not be demonstrated in any of the stage-IV metastatic NB samples analysed, while HLA-ABC staining was positive in only 116 sample. In contrast, all stage-IV samples were positive for N-CAM.
TABLE I - EXPRESSION OF ADHESION MOLECULES BY NB AND RELATED TUMORS Molecules analvsed
Tumor type
NB st. IV (metastatic) NB st. 1-111 (nonmetastatic) Ganglioneuroma PN PNET EWING
HLA-ABC ICAM-1 N-CAM CD2 LFA-3 VLAZ VLA4
116
016
2115
419 15115 416 218 219 019
315
115 314 013 012
414 113 212
616 016 016 016 016
515 111 215 114 nd 414 313 313 113 112 nd 112
215 015 314 015 013 013 112 012
Fresh frozen sections of tumor samples were stained with MAhs to CAMS using a 3-stage immunoenzymatic technique. Results are given as number of positively stainedinumber of analysed samples.
Among the NB samples, stages 1-111, expression of HLAABC, ICAM-1, VLA-2, LFA-3 and CD2 was variable and more frequent than in stage-IV tumors, and usually restricted to the tissues presenting histological evidence of maturation (ganglioneuroblastoma). No VLA-4 staining was observed. In contrast, expression of HLA-ABC, ICAM-1, LFA-3 and VLA-2 was frequently positive on PN as well as ganglioneuromas representing differentiated forms of the disease, as compared with NB. Only 114 PN were positive for N-CAM. A majority of the PNET samples analysed revealed a low frequency of HLA-ABC, ICAM-1, LFA-3 and VLA2 expression, while N-CAM was expressed by 100% of samples.
Expression of cell-adhesion molecules by SK-N-SH N B cell line and subclones SK-N-SH and variant cells were tested for surface expression of HLA-ABC, LFA-3, ICAM-1, CD2, N-CAM, VLA-2 and VLA-4, by flow cytometry. Figure 1 shows that the parent cell line SK-N-SH expresses high levels of the N-CAM molecule. HLA-ABC, ICAM-1, LFA-3 and VLA-2 were expressed in variable amounts by these cells with a bimodal distribution. VLA-4 and CD2 expression was hardly detectable. The neuronal variant SH-SY5Y cell line was characterized by a similarly high expression of N-CAM, but reduced expression of LFA-3 and VLA-2 as compared to the parental cell line. A disappearance of detectable HLA-ABC and ICAM-1, together with a low CD2 expression, was observed. In contrast, a majority of cells of the SH-EP variant were strongly stained with MAbs to HLA-ABC, LFA-3, CD2 and VLA-2. ICAM-1 and VLA-4 expression was very low while N-CAM was not detected on these cells. The SH-IN variant cells demonstrated an intermediate phenotype, similar to that of the parental cell line, with no CD2 expression, low HLA-ABC, ICAM-1, LFA-3, VLA-2 and VLA-4 expression and high expression of N-CAM molecules. Northern-blot analysis of N-CAM, ICAM-1 and HLA-ABC transcript levels in SK-N-SH and subclones Northern-blot analysis of N-CAM, ICAM-1 and HLA-ABC mRNA messages on SH variants and 2 control NB cell lines revealed a pattern of expression similar to that of the surface expression, as shown in Figure 2. A major N-CAM transcript of 6.5 kb and 2 minor transcripts of 4.3 and 2.9 kb were detected by Northern blotting on the parental SK-N-SH and neuronal SH-SY5Y variant. As a control, the same level and pattern of expression were revealed on another NB cell line, IMR-32 (Fig. 2, lane E) while the ACN cell line did not express any detectable N-CAM message (Fig. 2, lane F). No N-CAM message could be detected in the SH-EP cells, while the SH-IN variants appeared to mainly express the 6.5-kb transcript. Apparently, mostly quantitative
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CELL-ADHESION MOLECULES EXPRESSION ON NB CELLS PX
HIA-ABC
ICAh-1
0
2
LFA-3
N-CAM
VIA-2
MA4
FIGURE1 - Flow cytometric analysis of the surface expression of cell adhesion molecules on NB cell line SK-N-SH and the neuronal SH-SYSY, epithelial SH-EP and intermediate SH-IN variants. Control non-specific fluorescence was evaluated using the supernatant of the PX63 myeloma clone (PX).
differences were observed in the pattern of N-CAM mRNA transcripts expressed by the different variant cell lines. ICAM-1 major 3.3 and minor 2.2-kb mRNA species were poorly expressed by all 4 variant cell lines and IMR-32 cells in the absence of any stimulation, but strongly expressed by the ACN cell line. High levels of HLA-ABC mRNA transcripts were detected in SH-EP cells and lower amounts in the parent SK-N-SH cell and SH-IN variant cell lines. A low but detectable HLA-ABC message was expressed by the neuronal SH-SY5Y variant cells, in the absence of detectable surface expression. Modulation of CAM expression by differentiating agents and cytokines To further investigate the link between CAM surface expression and lineage-specific differentiation, we treated NB variant cell lines with differentiation-inducing agents and analysed by cytofluorometry the modulation of the surface CAM expression. Figure 3 illustrates the results obtained with the SH-IN variant. Similar results were obtained with the 3 other variant cell lines (data not shown). Treatment of SH-IN cells with retinoic acid, a neuronal differentiation-inducing agent (Tsokos et a/., 1985) resulted in an increase in HLA-ABC, ICAM-1, LFA-3 and VLA-2. No modification in the expression of N-CAM or VLA-4 was observed. When the same cells were treated with BUdR for differentiation along the Schwannianiglial pathway, only HLA-ABC was slightly increased, while expression of the other molecules remained unchanged. In contrast exposure of NB cells to PMA, a known stimulator of the protein kinase C, causing NB and other cell types to differentiate along different pathways, resulted in an important increase in the expression of all molecules tested except N-CAM. Exposure of NB cells to cytokines T-IFN and a-TNF under our experimental conditions, did not affect the morphological appearance of the cells. T-IFN treatment mainly resulted in a strongly enhanced expression of HLA-ABC and ICAM-1. The increase was more moderate after exposure of the cells to a-TNF. Figure 3 shows that both cytokines likewise increased LFA-3, VLA2 and VLA4 expression.
Sensitivity of SHparental cells and clones to N K and LAK bsis To investigate the putative role of some of these molecules in MHC-unrestricted cell-mediated anti-tumor responses, we tested the sensitivity of the different clones to NK and LAK lysis in an attempt to correlate the modulation of surface phenotype with a modulation of sensitivity to NK and LAK effectors. As a high variability of NK and LAK activity occurs among different normal blood donors, results presented in Figures 4 and 5 represent one typical experiment, repeated at least 4 times. As shown in Figure 4a, a high susceptibility to NK cell lysis (29-37%) with an effector/target cell (EIT) ratio of 16011 was observed with the SK-N-SH parent cell line as well as the SH-IN and the HLA-ABC-negative variant SH-SY5Y. In contrast, only 5% of lysis was obtained with the HLA-ABCpositive SH-EP variant. As a control, 70% of the NK-sensitive K562 cells were lysed. The susceptibility of all variants to LAK cell killing was high for all 4 cell lines (80-95% specific cell lysis) and similar to that of K562 cells (Fig. 4b). A consistently slightly higher percentage of specific lysis with SK-N-SH and SH-EP clones was observed, as shown in Figure 46. Target cells were further treated with differentiation inducers or cytokines and the variation of NK and LAK activity was evaluated and related to the modulation of surface phenotypic expression of CAM. As shown in Figure 5a, no significant variation in the susceptibility to NK-cell lysis ( E / T ratio 4011) was observed when the target cells (SH-SY5Y and SH-EP) were pre-treated with differentiation inducers R A and BUdR. A moderate decrease in the percentage of specific lysis was observed after a-TNF treatment, while an important decrease in cytolytic activity was measured after 24 hr exposure of target cells to T-IFN. The same effect was measured on both SH-SYSY and SH-EP variants, although the susceptibility of untreated SH-EP cells to NK cell lysis was basically very low. Modulation of LAK cell lysis after treatment of target cells was also measured using the SH-SY5Y and SH-EP variants at an E / T ratio of 10/1 and reported in Fig. 5b. O n both variants a significant decrease of sensitivity to LAK-cell lysis was measured after T-IFN treatment. A moderate increase of lysis was observed after incubation of SH-SYSY target cells with differentiation-inducer RA, while PMA caused a decrease in sensitivity to LAK lysis. BUdR treatment did not influence the sensitivity of either of the variant cell lines to LAK-cell lysis.
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F~GURE 2 - Northern-blot analysis of the expression of N-CAM, ICAM-1 and HLA-ABC transcripts by SK-N-SH (A), SH-EP (B), SH-SYSY (C), SH-IN (D), IMR-32 (E) and ACN (F) NB cell lines. PolyA mRNA was extracted from the different cell lines and blotted on filters successively hybridized with the 3 probes. Three major N-CAM transcripts of 6.5,4.3 and 2.9-kb mRNA species are expressed by NB cells. The ICAM-1 message consists of a major 3.3-kb and a minor 2.2-kb message. A single transcript of 1.6 kb represents the HLA-ABC message. DISCUSSION
In this study we first analysed the expression of CAM on a panel of NB tissue samples representing various stages and histological differentiation. O n fresh tumor samples, expression of several molecules including HLA-ABC, ICAM-1, LFA-3 and VLA2 was observed more frequently on mature tumors such as ganglioneuromas or on low-stage NB, as compared to metastatic, stage-IV NB characterized by absence of expression of these molecules. High N-CAM expression, in contrast, was associated with all NB and PNET tumors. Peripheral neuroepitheliomas, which are closely-related neural-crest-derived tumors, were likewise characterized by a more frequent expression of this group of molecules and low expression of N-CAM as compared to NB. To investigate the potential relationship between expression of some of these molecules and lineage-associated diffcrcntiation in NB cells, we measured CAM expression and modulation using the SK-N-SH NB cells and 3 cell variants which represent distinct neuronal-crest lineages and display different and well-characterized tumorigenic and differentiation-linked biochemical and morphological properties (Biedler ef a/., 1988). Analysis of the surface expression of HLA-ABC, ICAM-1, LFA-3 and VLA-2 on SK-N-SH and variant cell lines likewise revealed a co-expression of this group of molecules. Their concomitant presence at the cell surface appeared to be mainly linked to the substrate-adherent phenotype represented by the variant SH-EP, and corresponding to the
Schwannian lineage, classically characterized by absence of neuroblastic properties, decreased growth and less tumorigenic phenotype (Ciccarone et al., 1989). The same molecules were up-regulated in parallel when NB cells were induced to differentiate by either RA, BUdR or PMA, or after exposure to different cytokines. It appears therefore that this up-regulation is related to a differentiated state regardless of the lineage in which cells are induced. In conclusion, HLA class I, LFA-3, ICAM-1 and VLA-2 seem to be co-expressed and co-regulated on cells of the Schwannian lineage or NB cells exhibiting a differentiated and less tumorigenic phenotype, and therefore could be associated with NB with a better prognosis. A possible relationship between expression of either ICAM-1, LFA-3 or VLA-2 and absence of multicopies of the MYCN proto-oncogene, a parameter also linked to better prognosis in NB, needs to be investigated on a large collection of tumor samples. Expression of ICAM-1 has recently been described exclusively on MYCNnegative NB specimens, while expression of LFA-3 and H L A class I, detected on both MYCN-positive or negative samples, was likewise shown to be linked to the degree of maturation of the cells (Favrot et al., 1991). Whether the presence of some o r a combination of these molecules on tumor cells is related and contributes to the decreased tumorigenicity of epithelial-like NB cells and mature tumors, and how, has not yet been determined. lt is accepted that tumor cells with a low or absent expression of HLA-ABC, although insensitive to MHC-restricted T-cell recognition and lysis, display a higher sensitivity to NK effector cells (Wallach, 1983). However, the nature of the recognition structure and the exact role of the HLA-ABC molecules in this occurrence are not yet clear and are somewhat controversial. A hypothetical role of some adhesion molecules in the anti-tumor response mediated by NK/LAK cells was investigated on the SK-N-SH cell line and its 3 variants. As compared to the parental cell line and the 2 other variants, a clearly decreased sensitivity to NK-cell lysis was measured with SH-EP target cells which display higher HLA-ABC and VLA2 expression but lower N-CAM expression. This suggests a putative adhesive role of N-CAM and/or a negative role of HLA-ABC or VLA2 in the NK killing. In contrast, analysis of the sensitivity of the clones to LAK cytotoxicity did not reveal important differences in their susceptibility to LAK lysis despite differences in their surface antigenic phenotypes, thus suggesting a complex role of these molecules in the LAK cell lysis mechanism. Incubation of the NB target cell lines with differentiation inducers or cytokines was performed to further investigate the possible influence of modulated expression of some adhesion molecules in the NK/LAK killing. It appeared that, whether R A or BUdR-induced differentiation generally resulted in a moderate increase or stable susceptibility to NK and LAK lysis, T-IFN as well as PMA treatments caused a decrease in susceptibility to both NK and LAK. Several molecules, including HLA-ABC, ICAM-1, LFA-3 and VLA2 are up-regulated by T-IFN and PMA treatment. The roles of HLA-ABC and of the other T-IFN or a-TNF co-regulated molecules in the NK/LAK cytolysis need to be further analysed, separately, and so far remain highly controversial (Robertson and Ritz, 1990). It is generally accepted that T-IFN treatment of target cells decreases their NK and LAK susceptibility, due to the augmentation of the expression of HLA-ABC (Wallach, 1983; de Fries and Golub, 1988; Miyatake ef a/., 1990). However, other reports indicate that T-IFN treatment increases NB-cell susceptibility to NK/LAK cytolysis (Chervenak e f al., 1988), partly as a consequence of the increase in ICAM-1 expression (Naganuma et al., 1991). Such contradictory results may be due to the use of different NB cell lines, since we also observed
89
CELL-ADHESION MOLECULES EXPRESSION ON NB CELLS
PX
HIA-AEK
ICAM-1
LFA-3
VLA-2
N-CAM
FLUORESCENCE I"SITY
VIA4
(log ,o sale, au)
I ~ ~ C U R3E- Flow cytometric analysis of the modulation of surface expression of cell adhesion molecules after exposure of the cells to RA ( S . 1 V ' M), BUdR (10 Kgiml), PMA (10 ng/ml), T-IFN (500 U/ml) or a-TNF (SO Uiml) for 2 4 4 8 hr. The grey peaks represent the
expression of different molecules after treatment as compared to the control untreated cells (white peaks).
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FIGURE4 - Effective lysis of SK-N-SH and variant clones by NK ( a ) or LAK (b) effector cells as measured by a standard 4-hr 5'Cr cytotoxicity assay using different EIT ratios. The KS62 NK-sensitive cell line was used as control. Each point represent the mean of triplicate values.
variations in the modulation of NK/LAK sensitivity using various NB lines. However, this report suggests that each of the molecules-such as N-CAM or cytokine-regulated molecules HLA-ABC, ICAM-1, LFA-3 or VLA2-probably plays an important adhesive role in the NK/LAK phenomenon, most likely in a complex synergistic interaction, which needs to be dissected and analysed for each ligand/receptor complex separately. N-CAM is a surface glycoprotein which mediates homotypic cell-cell bonds. It has at least 3 isoforms generated by differential splicing of a single gene. Additional heterogeneity can be provided by developmentally regulated glycosylation which is believed to play a crucial role in the adhesive properties of N-CAM-bearing cells (Cunningham et al., 1987). The present analysis shows that the N-CAM molecule, as detected by
several different MAbs, is uniformly and strongly expressed by most NB cells. One hundred percent of NB tumors of all stages in various degrees of differentiation, and PNET, but only 25% of PN, were N-CAM positive. When NB variant cell lines are used, the expression of N-CAM appears to be linked to a neuronal phenotype since it is hardly detectable on the epithelial variant SH-EP at either the surface or the mRNA level. As revealed by 3 different MAbs, no modulation of surface N-CAM expression occurred as a result of either neuronal, Schwannian differentiation or cytokine treatment of the 4 SH cell lines or of several other NB cell lines (Gross rt ul., 1991). Important differences in the level of N-CAM mRNA transcripts were observed between the variants, with expression of
90
GROSS E T A L . 100
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FIGURE5 - Modulation of the susceptibility of SH-SYSY and SH-EP cells to NK (a) and LAK ( b ) cell lysis. Target cells were first exposed for 24-48 hr to differentiation inducers and cytokines (same conditions as for Fig. 3), further labeled and assayed for susceptibility to NK and LAK cytotoxicity at ratios of 40/1 and lO/l respectively. Each bar represents the mean of triplicate values 2 SD.
a major transcript of 6.5 kb, and minor transcripts of 4.3 and 2.9 kb by both SH parent and SH-SY5Y neuronal variants, a pattern comparable to that of the IMR-32 NB cell line. The 6.5-kb transcript was the only detectable message in SH-IN cells, while no N-CAM message at ail could be detected in SH-EP cells. Whether these differences reflect a lineagespecific expression of certain mRNA species with specific
adhesive properties of the resulting isoform, o r a simple quantitative variation, needs to be investigated by a modulation of the pattern of m R N A transcripts in relation to lineage-specific differentiation. N-CAM, also known as Leu-19 or CD56, is expressed by all NK cells and a subpopulation of T lymphocytes (Robertson and Ritz, 1990). The functional role of N-CAM in the NK cytotoxicity and sensitivity of NB cells to NK/LAK lysis has not yet been determined. An N-CAM-mediated homotypic adhesion between target and effector cells has been proposed to participate in the sensitivity of NB cells to NK/LAK cytolysis (Nitta et al., 1989). Our finding that N-CAM-negative, HLAABC-positive variant SH-EP displayed resistance to NK cells confirms a possible role of N-CAM expression by target cells in association with HLA-ABC molecules in the NK adhesion and cytolysis mechanisms. The p l integrin VLA-4 has recently been shown to be expressed by several tumor types and to mediate cell-cell contacts with a signal transmission function (Hemler, 1990). The expression and possible function of this molecule on NB cells in relation to differentiation was investigated. No expression of this molecule was detected on fresh tumor samples and a very low or undetectable VLA-4 expression was measured on all NB tissues and SH variants. A slight but consistent induction of VLA-4 expression was observed mostly on SH-IN and SH-SY5Y variants after treatment of cells with cytokines and PMA, but did not seem to be related to differentiation as induced by RA o r BUdR. In vivo, such an induction could b e related to an inflammatory response and its potential function on tumor cells deserves further analysis. Expression of CD2, the ligand for LFA-3, originally described as a T-lymphocyte-specific molecule (Albelda and Buck, 1990; Springer, 1990), has not yet been described on NB cells. O n tumor samples, the infrequent expression of this molecule seems to be linked to low stages and could be associated with NB with a better prognosis. This molecule was detected on both neuronal and epithelial variants but not on the parental SH cells or the SH-IN variant, and therefore might be related to later stages of differentiation, either neuronal or Schwannian. The additional presence of the ligand for CD2, LFA-3, on NB cells could favor an adhesion of NB cells together o r with effector lymphoid cells bearing both LFA-3 and CD2 molecules. ACKNOWLEDGEMENTS
We are grateful to Dr. D. Rimoldi for providing several h4Abs and for helpful discussion and comments, and to Mr. P. Zaech and Mr. C. Knaberhans for analysis of samples on the FACScan. This work was supported by the Swiss National Scientific Foundation, grant 32-26340.89 and by the Muschamp Foundation.
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cule: immunoglobulin-likedomain, cell surface modulation, and alternative splicing.Science, 236,799-806 (1987). DE FRIES,R.U. and GOLUB,S.H., Characteristics and mechanisms of IFNT-induced protection of human tumor cells from lysis by lymphokine-activated killer cells.J. Immunol., 140,3686-3693 (1988). EVANS, A.E., Natural history of neuroblastoma. In: A.E. Evans (ed.), Advances in neuroblastoma research, pp. 3-12, Raven, New York (1980). FAVROT, M.C., COMBARET, V., GOILLOT.E., TABONE,E., BOUFFET,E., DOLBEAU,D., BOUVIER,R., COZE. C., MICHON,J. and PHILIP,T., Expression of leucocyte adhesion molecules on 66 clinical neuroblastoma specimens.Int. J. Cancer, 48,502-510 (1991). GORIDIS, C., HIRN,M., SANTONI, M.-J., GENNARINI, G., DEAGOSTINIBAZIN,H., JORDAN,B.R., KIEFER,M. and STEINMETZ, M., Isolation of
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mouse N-CAM-related cDNA: detection and cloning using mono- killer cells: participation of ICAM-I induction on target cells. Int. J. Cancer, 47,527-532 (1991). clonal antibodies. EMBO J., 631-635 (1985). K., Involvement of GRONBERG, A,, FERM,M.T., NG, J., REYNOLDS, C.W. and ORTALDO, NITTA, T., YAGITA,H., SATO,K. and OKUMURA, J.R., IFNT-treatment of K562 inhibits natural killer cell triggering and CD56 (NKH-1ILeu-19 antigen) as adhesion molecule in natural decreases the susceptibility to lysis by cytoplasmic granules from large killer-target cell interaction. J. exp. Med., 170, 1757-1761 (1989). granular lymphocytes.J. Immunol., 140,43974406 (1988). RAYNER, A.A., GRIMM, E.A., LOTZE,M.T., CHU,E.W. and ROSENGROSS,N.. BECK,D. and FAVRE,S., In vifro modulation and relation- BERG,S.A., Lymphokine-activated killer cell (LAK) analysis of factors ship between N-myc and HLA class 1 RNA steady-state levels in relevant to the immunotherapy of human cancer. Cancer, 55, 13271333 (1985). human neuroblastoma cells. Cancer Res., 50,7532-7536 (1990). R E T I G , W.J.. SPENGLER, B.A., CHESA,P.G., OLD,L.J. and BIEDLER, GROSS.N., BECK,D., FAVRE,S. and CARREL,S., III vifro antigenic modulation of human neuroblastoma cells induced by IFN-T, retinoic J.L., Coordinate changes in neuronal phenotype and surface expression in human neuroblastoma cell variants. Cancer Res., 47,1383-1389 acid and dibutyryl-cyclic-AMP.I n f . J. Cancer, 39,521-529 (1987). (1987). GROSS,N., CARREL,S., BECK, D. and FAVRE,S., Cell adhesion M.J. and RITZ, J., Biology and clinical relevance of molecules; expression and modulation on human neuroblastoma cells. ROBERTSON, human natural killer cells. Nature ( L o r d ) ,76, 2421-2438 (1990). Advanc. Neuroblasf.Res., 3,293-299 (1991). T.A., Adhesion receptors of the immune system. Nature HEMLER, M., VLA proteins in the integrin family: structures, functions SPRINGER, (Lond.),346,425-433 (1990). and their role on leukocytes. Ann. Rev. Immunol., 365-400 (1990). Y., NISHIYAMA, T., WATANABE, H., FUJIWARA, M. and SATO, MAIN,E.K., LAMPSON, L.A., HART,M.K., KORNBLUTH, J. and WILSON, TOMITA, D.B., Human neuroblastoma cell lines are susceptible to lysis by S., Expression of intercellular adhesion molecules-1 (ICAM-1) on natural killer cells but not by cytolytic T lymphocytes.J. Imrnunol., 135, renal cell cancer: possible significance in host immune responses. Inf.J. Cancer, 46,1001-1006 (1990). 242-249 (1985). TSOKOS, M., Ross, R.A. and TRICHE,T.J., Neuronal, Schwannian and MIYATAKE, S., KIKUSHI,H., ODA, Y., NISHIOKA, T., TAKAHASHI, J., S., MATSUMOTO, M., YAMASAKI, T., IWASAKI, K., AOKI,T., melanocytic differentiation in human neuroblastoma cells in v i m . KONDOH, S. and NAMBA,Y., Decreased susceptibility of lined Progr. din. bid. Res., 175,55-68 (1985). KASAKURA, gliosarcoma cells to lymphokine-activated killer cells cytolysis by T-IFN VERSTEEG, R., PELTENBURG, L.T.C., PLOMP,A.C. and SCHRIER, P.I., treatment. Cancer Rex, 50,596-600 (1990). High expression of the c-myc oncogene renders melanoma cells prone NAGANUMA, H., KIESSLING,R., PATTAROYO, M., HANSSON,M., to lysis by natural killer cells. J. Immunol., 143,4331-4337 (1989). HANDGRETINGER, R. and GRONBERG, A,, Increased susceptibility of WALLACH, D., Interferon-induced resistance to killing b NK cells: a IFNT-treated neuroblastoma cells to lysis by lymphokine-activated preferential effect of IFN-T. Cell. Immunol., 75,390-39s 8983).
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Publicationof the International Union Against Cancer Publicationde I Union Internationale Contre le Cancer
DIFFERENTIATION-RELATED EXPRESSION OF ADHESION MOLECULES AND RECEPTORS ON HUMAN NEUROBLASTOMA TISSUES, CELL LINES AND VARIANTS Nicole GROSS’.Simone FAVRE,Daniel BECKand Manuela MEYER Onco-hematology Unit, Pediatric Department, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland. The expression of cellular adhesion molecules (CAM) involved in cell adhesion and immune recognition was measured on neuroblastomatissue samples, on a neuroblastoma (NB) cell line, SK-N-SH, and on 3 phenotypically different variants, SH-SY5Y. SH-EP, SH-IN, representing neuronal, Schwannianl glial or intermediate NB-cell types. lmmunohistochemical analysis of CAM expression by NB and related tumors at different stages of differentiation revealed a co-expression of several CAM (ICAM- IlCD54, LFA-3, VIA-2 and HIA-ABC) associated with low stages and more highly differentiated NB tumors and peripheral neuroepitheliomas (PN). In contrast, N-CAM was uniformly expressed on all NB tumors. Flow cytometric analysis of CAM surface expression by SK-N-SH and variant cells revealed highly variable phenotypes. Expression of ICAM- I, LFA-3, VIA-2 and HIA-ABC molecules was associated with the epithelial cell type represented by the SH-EP variant. In contrast, low expression of these molecules and high expression of N-CAM was associated with the neuronal SH-SY5Y cells. Exposure of the NB cells t o differentiation inducers (retinoic acid, 5’-bromodeoxyuridine and phorbol esters) and cytokines (7-interferon. a-tumor necrosis factor) resulted in a variable up-regulation of the expression of all CAMS, except N-CAM, regardless of the type of differentiation induced. In an attempt to establish a link between the pattern of expression of CAM on NB cells and their susceptibility to natural killer (NK) or lymphokine-activated killer (IAK) cell lysis, the analysis revealed that NB cells expressing CAM and a differentiated phenotype were less susceptiblet o NK lysis, but no difference in the sensitivity of the NB cell types t o I A K effectors was observed. Treatment of NB target cells with cytokines or PMA decreased their susceptibility t o NK and I A K lysis, while induction of differentiation with RA or BUdR resulted in no changes in the sensitivity to NK and I A K lysis. In conclusion, expression of HLA-ABC and several co-regulated CAMS was shown to be associated with a differentiated phenotype in NB, with an overall decreased sensitivity to NK/IAK effector cells. i i j 1992 Wilev-Liss,OK.
cells and in general for tumor cells with low HLA-ABC expression (de Fries and Golub. 1988; Gronberg et al., 1988; Versteeg et al., 1989). Adhesion molecules and receptors are attracting increasing interest in tumor biology since, by mediating cell-cell contacts and cell-matrix adhesion, these molecules are believed to be involved both in the interactions of tumor cells with immune effector cells and in cell migration and dissemination processes (Albelda and Buck, 1990; Springer, 1990). We have recently described the expression pattern of MHC class-I and -11 molecules in addition to adhesion molecules ICAM-1 (CD54) and N-CAM (CD56) on NB, Ewing and peripheral neuroepithelioma (PN) cell lines (Gross et al., 1991). We further reported that, whereas an overall high expression of N-CAM could be detected on all NB cell lines, ICAM-1 expression was variable and restricted to a minority of the HLA class-I (HLA-ABC)-positive NB cell lines and PN and Ewing cell lines (Gross et al., 1991). In this report we have analysed the expression and modulation on NB SK-N-SH cells and their variants of a larger panel of adhesion-related molecules and receptors, including ICAM-1, LFA-3/CD2 molecules as well as p l integrins VLA-2 and VLA-4. We have established a relationship between the pattern of CAM expression and the degree and type of differentiation by analysis of fresh tumor samples and an NB cell line and variants. Differentiation has been induced using agents such as retinoic acid (RA), 5’-bromodeoxyuridine (BUdR) and the (PMA). phorbol ester, phorbol-3’myristate-5-acetate As some of these molecules have already been shown to be involved in different mechanisms of cell-mediated anti-tumor responses (Springer, 1990; Naganuma et al., 1991: Tomita et al., 1990), we have related the pattern of expression of adhesion molecules to the sensitivity of NB cells to NK and LAK-cell lysis.
Neuroblastoma (NB), a solid childhood tumor of neuralcrest origin, is characterized by highly malignant and metastatic behavior, especially in children over l year of age (Evans, MATERIAL AND METHODS 1980). NB is also one of the rare tumors able to mature spontaneously in vivo to a less malignant form, i.e. ganglioneu- Cell lines Human NB cell lines SK-N-SH and 3 cell variants, SH-EP, roblastoma or benign ganglioneuroma, as well as to differentiate in vitro along neuronal or Schwannian lineages in re- SH-SYSY, SH-IN were obtained from Dr. J. Biedler, Sloan sponse to several differentiating stimuli (Evans, 1980; Tsokos Kettering Institute, NY. The ACN cell line was established in our laboratory from a bone-marrow specimen of a 36-monthet al., 1985; Gross et aL, 1987). Ccll lines established from several neuroblastomas have old boy with a metastatic adrenal stage-IV NB with elevated shown a diversity in cell morphology representing the in vivo catecholamines. All cell lines, including the myelocytic cell line differentiated stages with neuronal or glial components. From K562, were grown in RPMI medium supplemented with 2mM one such cell line, SK-N-SH, clonal lines comprising neuronal glutamine. 10% FCS and gentamycin. or Schwannian/glial cell types have been isolated which Monoclonal antibodies provide a useful model for analysis of NB phenotypic diversifiAntibodies to MHC class-I and -11 molecules, B9-12-1 and cation and differentiation (Ciccarone et al., 1989; Rettig et af., D1-12, have been already described (Gross et al.. 1987). 1987; Biedler et al., 1988). N-CAM molecules were detected using MAb UJ13A, kindly NB cells are likewise characterized by low but inducible provided by Dr. Kemshead, (Bristol, UK), MAb NKH-I which class-I MHC molecule expression and absence of class-I1 molecules, even after 4-days’ exposure to y-IFN (Gross et al., 1987). Poor expression of these molecules on tumor cells has ‘To whom correspondence and reprint requests should be sent. been associated with the insensitivity of NB cells to T-cell immunosurveillance (Main et af., 1985). In contrast, a higher sensitivity to NK-cell-mediated lysis has been described for NB Received: December 20,1991 and in revised form April 9. 1992
86
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detects an epitope on the N-CAM molecule, and an anti-CD56 MAb, kindly provided by Dr. S. Carrel (Lausanne, Switzerland). MAbs detecting the ICAM-1 molecule included P3.58, MAb anti-CD54 and Me14-Dl2 (Gross et al., 1897, 1991). Anti-LFA-3 MAb was kindly provided by Dr. K. Figdor, Amsterdam. MAb A1-43 detecting the VLA-2 molecule was a gift of Dr. E. Brocker, Munster, Germany. MAb MX-24 detecting the VLA-4 molecule and 6B-11 anti-CD2 were provided by Dr. S. Carrel.
Cytojhorometric analysis For analysis of cell-surface expression of the different antigens, the cells were recovered by 0.02% EDTAIPBS treatment of the monolayers and washed twice in EDTAcontaining RPMI medium. The cells were reacted with MAbs for 30 min on ice, then incubated for 30 min on ice with an FITC-conjugated FAb goat anti-mouse Ig antibody (Cappel, Cochranville, PA). The fluorescence was analysed on a FACScan (Becton-Dickinson, Mountain View, CA). lmmunohistochemistry Immunohistochemical staining of fresh-frozen tumor sections was performed using an indirect 3-stage immunoenzymatic assay with alkaline phosphatase as already described (Becket al., 1988). RNA extraction and Northern-blot analysis PolyA mRNA was extracted from the different cell lines by a technique already described (Gross et al., 1990). Northern blotting and hybridization were performed by standard methods (Gross et al., 1990). The following probes, already described (Gross et al., 1990, 1991) were used to detect CAM mRNA expression. HLAABC heavy-chain transcripts were detected with plasmid p44-2 containing a human HLA class-I gene. ICAM-1 transcripts were detected with plasmid p89.1 containing a 2,700-bp insert coding for ICAM-1. N-CAM transcripts were probed with the PM13 clone kindly provided by Dr. Goridis (Goridis et al., 1985). LAK cell generation and cell-mediated lysis Human peripheral mononuclear cells (PBL), isolated from the blood of healthy volunteers by Ficoll-Hypaque centrifugation, were used as source of NK effector cells. For generation of LAK effectors, cells were further washed and resuspended at 6 x lobcells/ml of LAKmedium [RPMI 1640 supplemented with 500 Ulml Interleukin-2 (IL-2) (Glaxo, Geneva, Switzerland), 10% pooled human AB serum, glutamine and gentamycin] and incubated for 3-5 days at 37°C. LAK-mediated cell lysis was measured with a standard 4-hr Tr-release assay using effector/target cell ratios (EIT) of 4O/1, lO/l and 411, as described by Rayner et al. (1985). All NK and LAK experiments were performed at least 5 times, using different blood donors. RESULTS
Expression of cell-adhesion molecules on NB and related tumor samples A total of 36 samples of NB stage I-IV and neuroectodermderived tumors, some representing differentiated forms of NB including ganglioneuromas, PN and primitive neuroectoderma1 tumors (PNET), were analysed for CAM expression. Results of the immunohistochemical analysis are summarized in Table I. Staining for CD2, LFA-3, ICAM-1, VLA-2 and VLA-4 could not be demonstrated in any of the stage-IV metastatic NB samples analysed, while HLA-ABC staining was positive in only 116 sample. In contrast, all stage-IV samples were positive for N-CAM.
TABLE I - EXPRESSION OF ADHESION MOLECULES BY NB AND RELATED TUMORS Molecules analvsed
Tumor type
NB st. IV (metastatic) NB st. 1-111 (nonmetastatic) Ganglioneuroma PN PNET EWING
HLA-ABC ICAM-1 N-CAM CD2 LFA-3 VLAZ VLA4
116
016
2115
419 15115 416 218 219 019
315
115 314 013 012
414 113 212
616 016 016 016 016
515 111 215 114 nd 414 313 313 113 112 nd 112
215 015 314 015 013 013 112 012
Fresh frozen sections of tumor samples were stained with MAhs to CAMS using a 3-stage immunoenzymatic technique. Results are given as number of positively stainedinumber of analysed samples.
Among the NB samples, stages 1-111, expression of HLAABC, ICAM-1, VLA-2, LFA-3 and CD2 was variable and more frequent than in stage-IV tumors, and usually restricted to the tissues presenting histological evidence of maturation (ganglioneuroblastoma). No VLA-4 staining was observed. In contrast, expression of HLA-ABC, ICAM-1, LFA-3 and VLA-2 was frequently positive on PN as well as ganglioneuromas representing differentiated forms of the disease, as compared with NB. Only 114 PN were positive for N-CAM. A majority of the PNET samples analysed revealed a low frequency of HLA-ABC, ICAM-1, LFA-3 and VLA2 expression, while N-CAM was expressed by 100% of samples.
Expression of cell-adhesion molecules by SK-N-SH N B cell line and subclones SK-N-SH and variant cells were tested for surface expression of HLA-ABC, LFA-3, ICAM-1, CD2, N-CAM, VLA-2 and VLA-4, by flow cytometry. Figure 1 shows that the parent cell line SK-N-SH expresses high levels of the N-CAM molecule. HLA-ABC, ICAM-1, LFA-3 and VLA-2 were expressed in variable amounts by these cells with a bimodal distribution. VLA-4 and CD2 expression was hardly detectable. The neuronal variant SH-SY5Y cell line was characterized by a similarly high expression of N-CAM, but reduced expression of LFA-3 and VLA-2 as compared to the parental cell line. A disappearance of detectable HLA-ABC and ICAM-1, together with a low CD2 expression, was observed. In contrast, a majority of cells of the SH-EP variant were strongly stained with MAbs to HLA-ABC, LFA-3, CD2 and VLA-2. ICAM-1 and VLA-4 expression was very low while N-CAM was not detected on these cells. The SH-IN variant cells demonstrated an intermediate phenotype, similar to that of the parental cell line, with no CD2 expression, low HLA-ABC, ICAM-1, LFA-3, VLA-2 and VLA-4 expression and high expression of N-CAM molecules. Northern-blot analysis of N-CAM, ICAM-1 and HLA-ABC transcript levels in SK-N-SH and subclones Northern-blot analysis of N-CAM, ICAM-1 and HLA-ABC mRNA messages on SH variants and 2 control NB cell lines revealed a pattern of expression similar to that of the surface expression, as shown in Figure 2. A major N-CAM transcript of 6.5 kb and 2 minor transcripts of 4.3 and 2.9 kb were detected by Northern blotting on the parental SK-N-SH and neuronal SH-SY5Y variant. As a control, the same level and pattern of expression were revealed on another NB cell line, IMR-32 (Fig. 2, lane E) while the ACN cell line did not express any detectable N-CAM message (Fig. 2, lane F). No N-CAM message could be detected in the SH-EP cells, while the SH-IN variants appeared to mainly express the 6.5-kb transcript. Apparently, mostly quantitative
87
CELL-ADHESION MOLECULES EXPRESSION ON NB CELLS PX
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ICAh-1
0
2
LFA-3
N-CAM
VIA-2
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FIGURE1 - Flow cytometric analysis of the surface expression of cell adhesion molecules on NB cell line SK-N-SH and the neuronal SH-SYSY, epithelial SH-EP and intermediate SH-IN variants. Control non-specific fluorescence was evaluated using the supernatant of the PX63 myeloma clone (PX).
differences were observed in the pattern of N-CAM mRNA transcripts expressed by the different variant cell lines. ICAM-1 major 3.3 and minor 2.2-kb mRNA species were poorly expressed by all 4 variant cell lines and IMR-32 cells in the absence of any stimulation, but strongly expressed by the ACN cell line. High levels of HLA-ABC mRNA transcripts were detected in SH-EP cells and lower amounts in the parent SK-N-SH cell and SH-IN variant cell lines. A low but detectable HLA-ABC message was expressed by the neuronal SH-SY5Y variant cells, in the absence of detectable surface expression. Modulation of CAM expression by differentiating agents and cytokines To further investigate the link between CAM surface expression and lineage-specific differentiation, we treated NB variant cell lines with differentiation-inducing agents and analysed by cytofluorometry the modulation of the surface CAM expression. Figure 3 illustrates the results obtained with the SH-IN variant. Similar results were obtained with the 3 other variant cell lines (data not shown). Treatment of SH-IN cells with retinoic acid, a neuronal differentiation-inducing agent (Tsokos et a/., 1985) resulted in an increase in HLA-ABC, ICAM-1, LFA-3 and VLA-2. No modification in the expression of N-CAM or VLA-4 was observed. When the same cells were treated with BUdR for differentiation along the Schwannianiglial pathway, only HLA-ABC was slightly increased, while expression of the other molecules remained unchanged. In contrast exposure of NB cells to PMA, a known stimulator of the protein kinase C, causing NB and other cell types to differentiate along different pathways, resulted in an important increase in the expression of all molecules tested except N-CAM. Exposure of NB cells to cytokines T-IFN and a-TNF under our experimental conditions, did not affect the morphological appearance of the cells. T-IFN treatment mainly resulted in a strongly enhanced expression of HLA-ABC and ICAM-1. The increase was more moderate after exposure of the cells to a-TNF. Figure 3 shows that both cytokines likewise increased LFA-3, VLA2 and VLA4 expression.
Sensitivity of SHparental cells and clones to N K and LAK bsis To investigate the putative role of some of these molecules in MHC-unrestricted cell-mediated anti-tumor responses, we tested the sensitivity of the different clones to NK and LAK lysis in an attempt to correlate the modulation of surface phenotype with a modulation of sensitivity to NK and LAK effectors. As a high variability of NK and LAK activity occurs among different normal blood donors, results presented in Figures 4 and 5 represent one typical experiment, repeated at least 4 times. As shown in Figure 4a, a high susceptibility to NK cell lysis (29-37%) with an effector/target cell (EIT) ratio of 16011 was observed with the SK-N-SH parent cell line as well as the SH-IN and the HLA-ABC-negative variant SH-SY5Y. In contrast, only 5% of lysis was obtained with the HLA-ABCpositive SH-EP variant. As a control, 70% of the NK-sensitive K562 cells were lysed. The susceptibility of all variants to LAK cell killing was high for all 4 cell lines (80-95% specific cell lysis) and similar to that of K562 cells (Fig. 4b). A consistently slightly higher percentage of specific lysis with SK-N-SH and SH-EP clones was observed, as shown in Figure 46. Target cells were further treated with differentiation inducers or cytokines and the variation of NK and LAK activity was evaluated and related to the modulation of surface phenotypic expression of CAM. As shown in Figure 5a, no significant variation in the susceptibility to NK-cell lysis ( E / T ratio 4011) was observed when the target cells (SH-SY5Y and SH-EP) were pre-treated with differentiation inducers R A and BUdR. A moderate decrease in the percentage of specific lysis was observed after a-TNF treatment, while an important decrease in cytolytic activity was measured after 24 hr exposure of target cells to T-IFN. The same effect was measured on both SH-SYSY and SH-EP variants, although the susceptibility of untreated SH-EP cells to NK cell lysis was basically very low. Modulation of LAK cell lysis after treatment of target cells was also measured using the SH-SY5Y and SH-EP variants at an E / T ratio of 10/1 and reported in Fig. 5b. O n both variants a significant decrease of sensitivity to LAK-cell lysis was measured after T-IFN treatment. A moderate increase of lysis was observed after incubation of SH-SYSY target cells with differentiation-inducer RA, while PMA caused a decrease in sensitivity to LAK lysis. BUdR treatment did not influence the sensitivity of either of the variant cell lines to LAK-cell lysis.
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F~GURE 2 - Northern-blot analysis of the expression of N-CAM, ICAM-1 and HLA-ABC transcripts by SK-N-SH (A), SH-EP (B), SH-SYSY (C), SH-IN (D), IMR-32 (E) and ACN (F) NB cell lines. PolyA mRNA was extracted from the different cell lines and blotted on filters successively hybridized with the 3 probes. Three major N-CAM transcripts of 6.5,4.3 and 2.9-kb mRNA species are expressed by NB cells. The ICAM-1 message consists of a major 3.3-kb and a minor 2.2-kb message. A single transcript of 1.6 kb represents the HLA-ABC message. DISCUSSION
In this study we first analysed the expression of CAM on a panel of NB tissue samples representing various stages and histological differentiation. O n fresh tumor samples, expression of several molecules including HLA-ABC, ICAM-1, LFA-3 and VLA2 was observed more frequently on mature tumors such as ganglioneuromas or on low-stage NB, as compared to metastatic, stage-IV NB characterized by absence of expression of these molecules. High N-CAM expression, in contrast, was associated with all NB and PNET tumors. Peripheral neuroepitheliomas, which are closely-related neural-crest-derived tumors, were likewise characterized by a more frequent expression of this group of molecules and low expression of N-CAM as compared to NB. To investigate the potential relationship between expression of some of these molecules and lineage-associated diffcrcntiation in NB cells, we measured CAM expression and modulation using the SK-N-SH NB cells and 3 cell variants which represent distinct neuronal-crest lineages and display different and well-characterized tumorigenic and differentiation-linked biochemical and morphological properties (Biedler ef a/., 1988). Analysis of the surface expression of HLA-ABC, ICAM-1, LFA-3 and VLA-2 on SK-N-SH and variant cell lines likewise revealed a co-expression of this group of molecules. Their concomitant presence at the cell surface appeared to be mainly linked to the substrate-adherent phenotype represented by the variant SH-EP, and corresponding to the
Schwannian lineage, classically characterized by absence of neuroblastic properties, decreased growth and less tumorigenic phenotype (Ciccarone et al., 1989). The same molecules were up-regulated in parallel when NB cells were induced to differentiate by either RA, BUdR or PMA, or after exposure to different cytokines. It appears therefore that this up-regulation is related to a differentiated state regardless of the lineage in which cells are induced. In conclusion, HLA class I, LFA-3, ICAM-1 and VLA-2 seem to be co-expressed and co-regulated on cells of the Schwannian lineage or NB cells exhibiting a differentiated and less tumorigenic phenotype, and therefore could be associated with NB with a better prognosis. A possible relationship between expression of either ICAM-1, LFA-3 or VLA-2 and absence of multicopies of the MYCN proto-oncogene, a parameter also linked to better prognosis in NB, needs to be investigated on a large collection of tumor samples. Expression of ICAM-1 has recently been described exclusively on MYCNnegative NB specimens, while expression of LFA-3 and H L A class I, detected on both MYCN-positive or negative samples, was likewise shown to be linked to the degree of maturation of the cells (Favrot et al., 1991). Whether the presence of some o r a combination of these molecules on tumor cells is related and contributes to the decreased tumorigenicity of epithelial-like NB cells and mature tumors, and how, has not yet been determined. lt is accepted that tumor cells with a low or absent expression of HLA-ABC, although insensitive to MHC-restricted T-cell recognition and lysis, display a higher sensitivity to NK effector cells (Wallach, 1983). However, the nature of the recognition structure and the exact role of the HLA-ABC molecules in this occurrence are not yet clear and are somewhat controversial. A hypothetical role of some adhesion molecules in the anti-tumor response mediated by NK/LAK cells was investigated on the SK-N-SH cell line and its 3 variants. As compared to the parental cell line and the 2 other variants, a clearly decreased sensitivity to NK-cell lysis was measured with SH-EP target cells which display higher HLA-ABC and VLA2 expression but lower N-CAM expression. This suggests a putative adhesive role of N-CAM and/or a negative role of HLA-ABC or VLA2 in the NK killing. In contrast, analysis of the sensitivity of the clones to LAK cytotoxicity did not reveal important differences in their susceptibility to LAK lysis despite differences in their surface antigenic phenotypes, thus suggesting a complex role of these molecules in the LAK cell lysis mechanism. Incubation of the NB target cell lines with differentiation inducers or cytokines was performed to further investigate the possible influence of modulated expression of some adhesion molecules in the NK/LAK killing. It appeared that, whether R A or BUdR-induced differentiation generally resulted in a moderate increase or stable susceptibility to NK and LAK lysis, T-IFN as well as PMA treatments caused a decrease in susceptibility to both NK and LAK. Several molecules, including HLA-ABC, ICAM-1, LFA-3 and VLA2 are up-regulated by T-IFN and PMA treatment. The roles of HLA-ABC and of the other T-IFN or a-TNF co-regulated molecules in the NK/LAK cytolysis need to be further analysed, separately, and so far remain highly controversial (Robertson and Ritz, 1990). It is generally accepted that T-IFN treatment of target cells decreases their NK and LAK susceptibility, due to the augmentation of the expression of HLA-ABC (Wallach, 1983; de Fries and Golub, 1988; Miyatake ef a/., 1990). However, other reports indicate that T-IFN treatment increases NB-cell susceptibility to NK/LAK cytolysis (Chervenak e f al., 1988), partly as a consequence of the increase in ICAM-1 expression (Naganuma et al., 1991). Such contradictory results may be due to the use of different NB cell lines, since we also observed
89
CELL-ADHESION MOLECULES EXPRESSION ON NB CELLS
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expression of different molecules after treatment as compared to the control untreated cells (white peaks).
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EFFECTOR TO TARGET CELL RATIO
FIGURE4 - Effective lysis of SK-N-SH and variant clones by NK ( a ) or LAK (b) effector cells as measured by a standard 4-hr 5'Cr cytotoxicity assay using different EIT ratios. The KS62 NK-sensitive cell line was used as control. Each point represent the mean of triplicate values.
variations in the modulation of NK/LAK sensitivity using various NB lines. However, this report suggests that each of the molecules-such as N-CAM or cytokine-regulated molecules HLA-ABC, ICAM-1, LFA-3 or VLA2-probably plays an important adhesive role in the NK/LAK phenomenon, most likely in a complex synergistic interaction, which needs to be dissected and analysed for each ligand/receptor complex separately. N-CAM is a surface glycoprotein which mediates homotypic cell-cell bonds. It has at least 3 isoforms generated by differential splicing of a single gene. Additional heterogeneity can be provided by developmentally regulated glycosylation which is believed to play a crucial role in the adhesive properties of N-CAM-bearing cells (Cunningham et al., 1987). The present analysis shows that the N-CAM molecule, as detected by
several different MAbs, is uniformly and strongly expressed by most NB cells. One hundred percent of NB tumors of all stages in various degrees of differentiation, and PNET, but only 25% of PN, were N-CAM positive. When NB variant cell lines are used, the expression of N-CAM appears to be linked to a neuronal phenotype since it is hardly detectable on the epithelial variant SH-EP at either the surface or the mRNA level. As revealed by 3 different MAbs, no modulation of surface N-CAM expression occurred as a result of either neuronal, Schwannian differentiation or cytokine treatment of the 4 SH cell lines or of several other NB cell lines (Gross rt ul., 1991). Important differences in the level of N-CAM mRNA transcripts were observed between the variants, with expression of
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A SH-EP
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FIGURE5 - Modulation of the susceptibility of SH-SYSY and SH-EP cells to NK (a) and LAK ( b ) cell lysis. Target cells were first exposed for 24-48 hr to differentiation inducers and cytokines (same conditions as for Fig. 3), further labeled and assayed for susceptibility to NK and LAK cytotoxicity at ratios of 40/1 and lO/l respectively. Each bar represents the mean of triplicate values 2 SD.
a major transcript of 6.5 kb, and minor transcripts of 4.3 and 2.9 kb by both SH parent and SH-SY5Y neuronal variants, a pattern comparable to that of the IMR-32 NB cell line. The 6.5-kb transcript was the only detectable message in SH-IN cells, while no N-CAM message at ail could be detected in SH-EP cells. Whether these differences reflect a lineagespecific expression of certain mRNA species with specific
adhesive properties of the resulting isoform, o r a simple quantitative variation, needs to be investigated by a modulation of the pattern of m R N A transcripts in relation to lineage-specific differentiation. N-CAM, also known as Leu-19 or CD56, is expressed by all NK cells and a subpopulation of T lymphocytes (Robertson and Ritz, 1990). The functional role of N-CAM in the NK cytotoxicity and sensitivity of NB cells to NK/LAK lysis has not yet been determined. An N-CAM-mediated homotypic adhesion between target and effector cells has been proposed to participate in the sensitivity of NB cells to NK/LAK cytolysis (Nitta et al., 1989). Our finding that N-CAM-negative, HLAABC-positive variant SH-EP displayed resistance to NK cells confirms a possible role of N-CAM expression by target cells in association with HLA-ABC molecules in the NK adhesion and cytolysis mechanisms. The p l integrin VLA-4 has recently been shown to be expressed by several tumor types and to mediate cell-cell contacts with a signal transmission function (Hemler, 1990). The expression and possible function of this molecule on NB cells in relation to differentiation was investigated. No expression of this molecule was detected on fresh tumor samples and a very low or undetectable VLA-4 expression was measured on all NB tissues and SH variants. A slight but consistent induction of VLA-4 expression was observed mostly on SH-IN and SH-SY5Y variants after treatment of cells with cytokines and PMA, but did not seem to be related to differentiation as induced by RA o r BUdR. In vivo, such an induction could b e related to an inflammatory response and its potential function on tumor cells deserves further analysis. Expression of CD2, the ligand for LFA-3, originally described as a T-lymphocyte-specific molecule (Albelda and Buck, 1990; Springer, 1990), has not yet been described on NB cells. O n tumor samples, the infrequent expression of this molecule seems to be linked to low stages and could be associated with NB with a better prognosis. This molecule was detected on both neuronal and epithelial variants but not on the parental SH cells or the SH-IN variant, and therefore might be related to later stages of differentiation, either neuronal or Schwannian. The additional presence of the ligand for CD2, LFA-3, on NB cells could favor an adhesion of NB cells together o r with effector lymphoid cells bearing both LFA-3 and CD2 molecules. ACKNOWLEDGEMENTS
We are grateful to Dr. D. Rimoldi for providing several h4Abs and for helpful discussion and comments, and to Mr. P. Zaech and Mr. C. Knaberhans for analysis of samples on the FACScan. This work was supported by the Swiss National Scientific Foundation, grant 32-26340.89 and by the Muschamp Foundation.
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