WO1990008781A1

WO1990008781A1 - Tenascin-induced cell attachment and the tenascin receptor

Info

Publication number: WO1990008781A1
Application number: PCT/US1990/000408
Authority: WO
Inventors: Erkki I. Ruoslahti; Mario A. Bourdon
Original assignee: La Jolla Cancer Research Foundation
Priority date: 1989-01-24
Filing date: 1990-01-23
Publication date: 1990-08-09
Also published as: CA2008462A1; AU5163390A

Abstract

The present invention provides a substantially pure tenascin receptor characterized in that it consists of two subunits with molecular weights of about 145,000 and 125,000 as determined by SDS-PAGE under non-reducing conditions, and specifically interacts with tenascin in an Arg-Gly-Asp dependent manner.

Description

TEASCIN-IMJϋCED CELL ATTACHMENT AND THE TENASCIN RECEPTOR

BACKGROUND OF THE INVENTION

This invention relates generally to the fields of cell and neurobiology and more specifically to cell adhesion systems.

Much of what goes on at the cell surface relates to recognition by the cell of substances around it. An important aspect of cell surface recognition is the interaction of a cell with insoluble structures that surround it. Such a structure can be the surface of another cell or the extracellular matrix.

The extracellular matrix is a complex assembly of molecules which interact with one another as well as with cells to effect a wide range of complex cellular functions seen in normal development and tumor growth and metastasis. Extracellular matrices are composed of an insoluble meshwork of protein and carbohydrate that is laid down by cells and fills most of the intercellular spaces. Matrices in different locations in the body consist of different combinations of collagens, proteoglycans, elastin, hyaluronic acid and various glycoproteins such as fibronectin and tenascin. Virtually all of the extracellular matrix glycoproteins and collagens that have been identified interact with cells.

The most readily observable result of the interaction of cells with extracellular matrix molecules is cell adhesion. The adhesive properties of the extracellular matrix proteins can be easily demonstrated jLn vitro by plating cells onto a surface coated with extracellular matrix material or with one of the purified matrix proteins. The cells will rapidly adhere to such a surface and spread on it. However, the adhesive proteins not only promote adhesion, they also stimulate cell migration. When confronted with limiting concentrations of an adhesive protein applied as a gradient on a surface, cells move toward the higher concentration. Moreover, cell proliferation and differentiation can also be controlled by the extracellular matrix interactions.

Recently, it has been determined that a number of extracellular matrix components interact with cells through specific cell surface receptors. Many of these receptors, termed integrin type receptors or integrins, recognize the tri-peptide sequence Arg-Gly-Asp (RGD) in their extracellular ligands (See Ruoslahti and Pierschbacher, Science 238:491-497, (1987)). While a number of extracellular matrix components including those mentioned above have been well characterized, new molecules are likely to be found which play specific roles in cell matrix interactions. One such novel extracellular matrix molecule is the glial-mesenchymal extracellular matrix glycoprotein tenascin described in Bourdon et al., Cancer Res. 43:2796 (1983), which is incorporated herein by reference.

Also known as glial-mesenchymal extracellular matrix (GMEM) protein, m otendinous antigen, cytotactin, and hexabrachion protein, tenascin is a component of some specialized extracellular matrices. Human tenascin is a 250 kD glycoprotein which is secreted as a high molecular weight (<10⁶ kD) disulfide-bonded oligomer. In rotary shadowing images, tenascin appears as a hexameric structure. This structurally unusual matrix molecule is further distinguished by its highly selective oncodevelopmental expression.

Developmentally, tenascin appears to be selectively expressed in the developing nervous system and in condensing mesenchyme during the initial stages of organogenesis of mammary gland, toothbud and kidney. In each of these organs, epithelial mesenchymal interactions are of key importance in normal organ development. Tenascin is largely absent in normal adult tissues, but is expressed in a variety of solid tumors. In human gliomas, it is expressed around the tumor neovasσulature and in fibrosarcomas within the stroma. This selective expression of tenascin in tumors makes it a suitable target molecule for the delivery of antibodies and antibodyrbound drugs to tumors. Moreover, the connection with tumor vasculature suggests a role in the growth of blood vessels into tumors. Promotion of tumor cell proliferation by tenascin has also been observed.

There thus exists a need to be able to promote and interfere with the ability of tumor cells to interact with tenascin. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that the interaction of cells with tenascin is mediated by an Arg-Gly-Asp directed receptor. The present invention provides a substantially pure tenascin receptor characterized in that it consists of two subunits with molecular weights of about 145,000 and 125,000 as determined by SDS-PAGE under non-reducing conditions, and specifically interacts with tenascin in an Arg-Gly-Asp dependent manner.

In one aspect of the invention, the tenascin receptor is isolated from human glioma cells. The receptor may be coated onto a substrate incorporated into lipid vesicles, or liposomes, and used to target therapeutic or other materials to tissues containing tenascin.

In another aspect of the invention, the interaction of cells with tenascin is inhibited with peptides containing the Arg-Gly-Asp sequence.

In yet another aspect of the present invention, monoclonal and polyclonal antibodies are prepared against the isolated receptor and their reactivity is assayed against the purified receptor. Antibodies that react only with the 145kd (alpha) subunit react specifically with the tenascin receptor, whereas antibodies against the smaller

(beta) subunit can react with other receptors, such as a fibronectin receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 shows a comparison of cell attachment activity to tenascin and fibronectin substrates plotted relative to maximum cell attachment to fibronectin.

FIGURE 2 shows inhibition of tenascin mediated cell attachment by anti-tenascin antibodies. Values represent relative cell attachment and standard deviation. Controls represent maximum cell attachment on each adhesion protein in the absence of added antibody.

FIGURE 3 shows effect of Gly-Arg-Gly-Asp-Ser-Pro peptide on tenascin-induced cell attachment.

DETAILED DESCRIPTION OF THE INVENTION

The cell attachment activity of tenascin is mediated through a novel integrin-type tenascin receptor which recognizes a tenascin cell-binding site functionally similar to the Arg-Gly-Asp cell attachment recognition site present in fibronectin and other adhesion molecules. Human tenascin supports the attachment of a wide range of tumor and normal cell types including cell lines derived from gliomas, sarcomas, and carcinomas as well as normal fibroblasts and endothelial cells. Titration curves comparing cell attachment to tenascin and fibronectin in a serum-free cell attachment assay indicate comparable cell attachment activities. Inhibition of cell attachment to tenascin by tenascin specific antibodies but not by anti- fibronectin antibodies indicates a specific interaction of tenascin with the cells.

The mechanism by which cells bind to tenascin appears to be similar to that of the Arg-Gly-Asp family of cell attachment proteins. Functionally, tenascin is a member of this family as demonstrated by the specific, dose dependent inhibition of cell attachment to tenascin by Arg-Gly-Asp peptides. These findings were unexpected because others have reported that tenascin does not promote cell attachment or, that when it does, the attachment is not Arg-Gly-Asp dependent.

Like other integrin-type receptors, the tenascin receptor is a detergent-soluble cell surface protein heterodimer and is composed of a 145 kd α subunit and a 125 kd β subunit. Upon reduction, the subunit co-migrates at about 140 kd with the β subunit. These changes in subunit mobilities upon reduction are a characteristic of many integrins (Ruoslahti and Pierschbacher, Supra) . The a subunits have been shown to be postranslationally processed into an heavy chain and an light chain of 20 to 25 kd. Although an light chain has not been positively identified in cell surface iodinated tenascin receptor preparations, this may well be due to a limited number of radiolabeling sites on the light chain.

The β subunit of the tenascin receptor has the same molecular weight as fibronectin receptor β subunit and reacts with a monoclonal antibody prepared against the fibronectin β subunit. These results suggest that the tenascin receptor has the same β subunit as the fibronectin receptor and a distinct α subunit. This receptor, therefore, seems to be a member of the group of receptors within the integrin superfamily that includes the mammalian fibronectin receptor, type I collagen receptor, integrins known as VIA proteins and the chicken integrin receptor complex, CSAT.

Results of Arg-Gly-Asp inhibition of cell attachment to substrates coated with tenascin, fibronectin or vitronectin indicate that the tenascin mediated cell attachment is inhibited more readily than that mediated by the other two proteins. This sensitivity of the tenascin- promoted cell attachment to the inhibition Arg-Gly-Asp peptides indicates that these peptides can be used to inhibit the tenascin attachment without substantially affecting other attachment phenomena.

A functional difference between the tenascin receptor and fibronectin or vitronectin receptors is suggested by the differences in cell morphology seen as cells attach to tenascin, fibronectin and vitronectin. As a result of attaching to tenascin, cells only partially flattened and appeared to polarize and extend pseudopodia. In contrast, cells attaching to fibronectin assumed a flattened appearance with extensive cell spreading.

As used herein, "fibronectin receptor" refers to the receptor identified and characterized in Pytela et al., Cell 40:191-198, (1985), and Argraves et al., J.Cell Biol. 105:1183-1190, (1987), which are incorporated herein by reference.

As used herein, "vitronectin receptor" refers to the receptor identified and characterized in Pytela et al., Proc. Natl. Acad. Sci. USA, 82:5766-5770 (1985), and Suzuki et al., J. Biol. Chem. 262:14080-14085 (1987), which are incorporated herein by reference. As used herein, the term "substantially purified" when used to describe to the tenascin receptor or other entity refers to the entity substantially free of proteins or other biochemical entities with which it is normally associated.

EXAMPLE I Cell attachment activity of Tenascin

Tenascin was purified from the spent culture media of U251MG human glioma cells by affinity chromatography on an 81C6 anti-tenascin monoclonal antibody (Bourdon, supra) coupled to Sepharose 4B (Sigma Chemical Co., St. Louis, MO) . The spent culture media was first concentrated by tangential flow filtration over PLMK300 filters (Millipore, Bedford, MA) . A Sepharose 4B precolumn was used to remove debris and aggregated protein prior to application of the sample to the monoclonal antibody affinity column. Non- bound proteins were washed from the antibody-Sepharose column with 0.5M NaCl, 1M urea, lOmM NaHP0₄, pH 7.4 and tenascin eluted with 0.5m NaCl, 4M urea, lOmM NaHP0₄, pH 7.4. Protein elution was monitored in a continuous flow cell UV monitor (ISCO) at 280 nm. Purity of the tenascin preparations was monitored by SDS-PAGE analysis on 7% acrylamide gels followed by Coomassie blue or silver staining and by HPLC chromatography on a TSK-400 column (7.5mm x 60mm). Fibronectin and vitronectin were purified from human plasma as described in Ruoslahti et al., Meth. Enzymol. 144:430-437, 1987, which is incorporated herein by reference. Rotary shadowing of purified tenascin was performed using standard procedures Engvall et al., J. Cell Biol. 102:703, 1986, which is incorporated herein by reference. Tenascin molecules were imaged on a Hitachi H- 60 scanning-transmission electron microscope.

The cell attachment assay was performed as follows: Cells cultured in Dulbecco's minimal essential media (DMEM) supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin were detached using 0.02% EDTA in phosphate buffered saline (PBS), pH 7.4, washed in DMEM containing 2 mg/ml bovine serum albumin (BSA) and plated at 2 x 10⁴ cells per well in 96 well flat bottom microtitration plates (Titertek, Flow Laboratories) . Wells were previously coated overnight with dilutions of cell attachment proteins in PBS and washed with a solution containing the DMEM-BSA medium, prior to their use in cell attachment assays. Assays were carried out at 37°C in a C0₂ incubator for 90 minutes. Non-adherent cells were removed by washing with PBS and adherent cells fixed with 3% paraformaldehyde and stained with 0.5% toluidine blue. Adherent cells were either counted directly or their numbers determined by lysing cells with 1% SDS and measuring dye absorbance at 600nm in a multiscan plate reader (Flow Laboratories) .

Cells adhering to tenascin included a variety of tumor cell lines of glial (U25IMG, Figure 1) , epithelial (A431) , endodermal (PFHR-9) , and mesenchymal (MG63, HT1080) origin as well as fibroblasts and human umbilical vein endothelial cells (HUVEC) . Human m21 melanoma cells, F9 mouse embryonic carcinoma cells and EL4 cells adhered poorly or not at all to tenascin.

The morphology of cells adhering to tenascin, as compared to those adhering to fibronectin or vitronectin, was distinctly different. U251MG cells adhering to tenascin generally assumed a more polar, elongated morphology with larger numbers of cellular extensions and less extensive spreading than those seen on fibronectin or on vitronectin. Despite the reduced cell spreading observed for cells adhering to tenascin, the level of cell attachment to tenascin was found to be similar to the level of cell attachment to fibronectin. As shown for the U251MG cells in Fig. 1, the cell attachment titration curves for tenascin and fibronectin closely paralleled one another with maximum cell attachment on either tenascin or fibronectin occurring at a coating concentration of 3 μg/ml protein and higher. Cell attachment to tenascin was not due to contamination with fibronectin, since no fibronectin was detected in tenascin samples by solid phase ELISA. The specificity of tenascin cell attachment was further demonstrated by anti-human tenascin antibodies which blocked cell attachment to tenascin but not to fibronectin or vitronectin (Fig. 2) . Polyclonal antisera against tenascin were prepared by immunization of rabbits with purified human tenascin and the immunoglobulin isolated on protein A-Sepharose. Antibodies were absorbed with fibronectin-Sepharose and bovine plasma protein-Sepharose. Conversely, affinity purified anti-fibronectin antibodies inhibited cell attachment to fibronectin only, Ruoslahti et al., Meth. Enzymol. 144:430-437 (1985).

EXAMPLE II Inhibition of Tenascin Attachment by RGD Peptides

The peptide Gly-Arg-Gly-Asp-Ser-Pro derived from the fibronectin cell attachment site and control peptide Gly- Arg-Gly-Gly-Ser-Pro were synthesized on an Applied Biosystems Peptide Synthesizer using solid phase chemistry according to the manufacturer's instructions. Peptides were purified by ion exchange HPLC using methods well known in the art.

Cell attachment assays were performed as described above and peptides at various concentrations were included in the incubation with cells to determine their inhibitory activities, (See Pierschbacher and Ruoslahti, Nature, 309:30-33 (1984), which is incorporated herein by reference) . Like the attachment of cells to fibronectin and vitronectin, the attachment to tenascin could be inhibited specifically by Arg-Gly-Asp-containing peptides, as shown in Fig. 3. The peptide Gly-Arg-Gly-Asp-Ser-Pro at concentrations of 300 μg/ml could completely inhibit cell attachment to tenascin while the peptide Gly-Arg-Gly-Gly- Ser-Pro, which does not inhibit Arg-Gly-Asp dependent cell attachment had no effect on cell attachment to tenascin even at high concentrations, such as 10 mg/ml.

The inhibition of cell attachment to tenascin by the Gly-Arg-Gly-Asp-Ser-Pro peptide occurred at concentrations about 30- and 150-fold lower than are needed for comparable cell attachment inhibition to either vitronectin or fibronectin respectively. The concentration of Gly-Arg- Gly-Asp-Ser-Pro resulting in a 50% inhibition of cell attachment to tenascin was 25 μg/ml, whereas 500 μg/ml and 4.5 mg/ml, respectively, were necessary to produce the same degree of inhibition to fibronectin and vitronectin under the conditions used.

EXAMPLE III Isolation of Tenascin Receptor

The receptor can be isolated from U251/MG glioma cells or MG63 fibrosarcoma (ATCC CRL 1427) or IMR90 normal fibroblasts (ATCC CCL 186) .

Cell surface labeling was performed with ¹⁵I by the lactoperoxidase labeling method as previously described (Pytela et al. , Cell 40:191 (1985), which is incorporated herein by reference) . Preparation of 50mM octylglucoside cell extracts and affinity chromatography were done as follows and previously described (Pytela, supra) . Pools of 10⁸ cells were surface labeled with ¹²⁵I and lysed in 50mM octylglucoside, ImM CaCl₂, ImM MgCl₂, 0..15M NaCl, ImM PMSF, lOmM Tris pH 7.2 for 10 minutes at room temperature. Cell extracts were clarified by centrifugation at 11,000 g for 10 minutes and the supernatant retained for receptor isolation. Cell extracts were passed over tenascin- Sepharose, Gly-Arg-Gly-Asp-Ser-Pro-Lys-Sepharose or fibronectin 120 kd frag ent-Sepharose columns, the affinity columns washed with 10 column volumes of 25 mM octylthioglucoside, 1 mM CaCl₂, 1 mM MgCl₂, 0.15 M NaCl, 1 mM PMSF, 10 μM Tris pH 7.2 and the receptors eluted with 5 column volumes of lmg/ml Gly-Arg-Gly-Asp-Ser-Pro peptide in the same buffer or alternatively with 20 mM EDTA in 25 mM octylthioglucoside, 0.15 M NaCl, 10 mM Tris pH 7.2 as previously described (Pytela supra) . Fractions were analyzed by SDS-PAGE on 7.5% acrylamide gels. Gels were dried and XAR5 x-ray film placed over the gels along with an enhancer screen, and the film was exposed at -70°C.

The receptor was isolated by tenascin-Sepharose affinity chromatography isolation from octylglucoside extracts of U251MG cells surface labeled with ¹²⁵I. SDS- PAGE analysis of material eluted from the tenascin- Sepharose column with the Gly-Arg-Gly-Asp-Ser-Pro peptide revealed a 145 kd polypeptide and a 125 kd polypeptide under non-reducing conditions. Under reducing conditions, the two polypeptides ran as a single band of about 140 kd. Direct comparison of the mobilities of the tenascin subunit with those of the mammalian integrin receptors for fibronectin and vitronectin indicated that the tenascin receptor was structurally distinct from either one of these receptors.

Cross reactivity between the tenascin receptor and other known integrins was determined by immunoblotting and immunoprecipitation. Receptors isolated as described above and in Pytela, supra and Pytela et al., Proc. Natl. Acad. Sci. USA, 82:5766-5770 (1985) from unlabeled cells were concentrated by precipitation in acetone (6 volumes, -20°C) and electrophoresed on 7.5% acrylamide gels. The separated proteins were then electroblotted onto nitrocellulose membranes. Blotted protein bands were visualized by staining the membrane with ponceau-S stain and destained in PBS. The blots were incubated in PBS containing 1% BSA for 1-2 hours to block non-specific protein binding sites and subsequently incubated with antibodies in PBS-1% BSA overnight. Following the incubation, the blots were washed and incubated with either HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG antibodies (Bio-Rad, Richmond, CA) in PBS-1% BSA for one hour. Bound antibodies were visualized by addition of diaminobenzidine tetrahydrochloride (DAB) substrate in PBS 0.01% H₂0₂ solution to washed blots.

Immunoblot and immunoprecipitation analysis showed that a monoclonal anti- fibronectin receptor β subunit antibody binds to the tenascin receptor β subunit. However, the α subunit appeared to be distinct from the fibronectin receptor subunit since it did not bind anti- peptide antibody specific for the fibronectin receptor α subunit. The tenascin receptor is, therefore, not the same as the fibronectin receptor, but may share the fibronectin β subunit. Antibodies to the α and β subunits of vitronectin did not bind to either or β subunit of the immunoblots. Polyclonal antibodies to vitronectin receptor were affinity purified on vitronectin receptor-Sepharose by the method of Suzuki et al. , J. Biol. Che . 262:14080, 1987, which is incorporated herein by reference. Monoclonal antibody 442 was specific for the β-subunit of fibronectin receptor. An antibody prepared against the COOH-terminal peptide of the fibronectin receptor α-subunit reacts with the α-subunit of the receptor, but not the β- subunit.

EXAMPLE IV Anti-Tenascin Receptor Antibodies and Their Use

Monoclonal and polyclonal antibodies against the tenascin receptor are prepared according to procedures well known in the art. Polyclonal antisera against tenascin receptor are prepared by immunization of rabbits with purified human tenascin receptor isolated as described above and the immunoglobulin isolated on protein A- Sepharose.

The polyclonal antibodies are pre-absorbed with the purified fibronectin receptor coupled to- Sepharose or immobilized on nitrocellulose or other protein binding membrane as is well known in the art. Since the β subunits of the tenascin and fibronectin receptors are similar and since the fibronectin receptor β subunit is shared by a number of other integrins Ruoslahti and Pierschbacher, supra. which is incorporated herein by reference, this treatment will eliminate antibodies directed against determinants shared by the receptors in this integrin family. It will also eliminate antibodies against contaminating proteins that would bind nonspecifically to both the tenascin and fibronectin affinity matrices. If necessary, such antibodies can also be removed by absorbing the anti-serum with "mock purified receptor." The specificity of the antibodies is examined by carrying out enzyme immunoassays, immunoprecipitation of detergent extracts from surface-iodinated and metabolically labeled U251MG cells and immunoblotting by methods well known in the art.

Monoclonal antibodies are prepared by immunizing with the isolated receptor or other material containing the receptor followed by isolation of antibody-producing hybridoma cells as is well known in the art. The appropriate hybridoma cells are selected by performing enzyme immunoassays with the purified tenascin receptor. Antibodies specific for the tenascin receptor are obtained by using immunoblotting to select those antibodies that are reactive with the α subunit of the tenascin receptor. Finally, to obtain antibodies specific for the tenascin receptor that can inhibit the tenascin-binding activity of the receptor, the α-subunit-specific monoclonal antibodies tested are inhibitors of cell attachment to tenascin.

EXAMPLE V

Reactivity of Tenascin Receptor With Anti-Inteqrin Antibodies

Polyclonal and monoclonal antibodies to " various integrin subunits were used to determine the specific reactivity of the tenascin receptor. Table I provides the particulars of the various antibodies.

TABLE I

Host Reactive Monoclonal Reference with

Subunit or or Tenascin, Antigen Polyclonal Immunogen Source Receptor?

α, rabbit α. Richard No (immuno¬ polyclonal cytoplasmic Hynes blotting) peptide (MIT)

α_c rabbit α₅ Bourdon & No (immuno¬ polyclonal cytoplasmic Ruoslahti blotting) domain J. Cell peptide Biol. 108: 1149 (1989)

α. mouse fibro- Wagner et No (immuno¬ monoclonal sarcinoma at., J. precipita¬

PIB5 HT1080 Biol. tion) 107:1881 (1988)

α_c Wagner et No (immuno- at. , Supra precipita¬ tion)

α rat mouse Sonnenberg No (immuno- monoclonal mammary et al., J. precipita- GoH3 carcinoma Biol. Chem. tion) 262:10375 (1987)

The tenascin receptor; purified as described in Example III, was tested for reactivity to the antibodies using the immunoblotting method as described in Example III, for the polyclonal antibodies and immunoprecipitation method for monoclonal antibodies. Immunoprecipitation was performed by incubating ¹²⁵I-labeled samples with antireceptor antibodies overnight at 4°C. followed by recovery of the bound label with protein A-Sepharose. Bound material was analyzed by SDS-PAGE followed by fluorography as described above. The tenascin receptor did not bind to α₃, α₅ and α₆ subunits.

Although the invention has been described with reference to the presently-preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

WE CLAIM:

1. A substantially pure receptor, comprising an α subunit with molecular weight of about 145,000 and a β subunit with molecular weight of about and 125,000 as determined by SDS-PAGE, said receptor specifically binding

5 with tenascin or cell attachment-promoting fragments of tenascin.

2. The substantially pure receptor of claim 1, further characterized in that the 145,000 subunit is not immunologically cross-reactive with the fibronectin receptor and the 125,000 subunit is cross reactive with the fibronectin receptor.

3. A composition of matter, comprising the receptor of claim 1 incorporated into lipid vesicles.

4. A composition of matter, comprising the receptor of claim 1 coated onto a substrate.

5. A composition of matter, comprising fragments of the receptor of claim 1, having tenascin binding activity.

6. A method of isolating a tenascin receptor, comprising the steps of:

a. providing a cell membrane preparation;

5 b. passing said cell membrane preparation over a column containing tenascin bound to a matrix; and

£ c. specifically eluting the material attached 10 to said tenascin matrix.

7. A receptor substantially identical to that isolated by the method of claim 6.

8. A method of inhibiting the attachment of cells to tenascin, comprising providing Arg-Gly-Asp containing peptides.

9. Antibodies reactive with the receptors of claims 1 or 7 but not with other receptors bearing a β subunit.

10. Antibodies reactive with the alpha subunit of receptor of claim 1.

11. The antibodies of claim 9, wherein said antibodies are monoclonal antibodies.

12. The antibodies of claim 9, wherein said antibodies are polyclonal antibodies.

13. A method of detecting the presence of tenascin receptor in a sample, comprising the steps of:

a. contacting said sample with antibodies reactive with the tenascin receptors, but not cross reactive with other receptors.

b. determining binding between said antibodies and components of said sample, wherein binding indicates the presence of said tenascin receptor in said sample.