Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

Biology Open, Apr 2012

Simon L. Goodman, Hans Juergen Grote, Claudia Wilm

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Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

Simon L. Goodman () 1 Hans Juergen Grote 0 Claudia Wilm 0 0 Department of Target Research, Therapeutic Area Oncology , Merck KGaA, Darmstadt, 64217, Germany 1 Department of Cellular Pharmacology Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins avb6 and avb8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of av integrins that react with both native integrins and n formalin fixed, paraffin embedded (FFPE) human tissues. e p These RabMabs, against avb3 (EM22703), avb5 (EM09902), O avb6 (EM05201), avb8 (EM13309), and pan-av (EM01309), y recognize individual integrin chains in Western blots and in g lo flow cytometry. EM22703 detected a ligand-induced binding io site (LIBS), reporting an epitope enhanced by the binding of B an RGD-peptide to avb3. avb8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed avb8, as did some melanoma cells, whereas U87MG glioma lacked avb8 expression. We observed an - Introduction Five integrins share the av chain: avb3, avb5, avb6, avb8, and avb1. avb3 and avb5 are well characterized, avb6 less so, and avb8 and avb1 the least, which may reflect the availability of specific antibodies. For example, in the case of murine monoclonal antibodies (MuMabs), LM609 binds to and inhibits avb3 (Cheresh and Spiro, 1987; Lin et al., 1998), PIF6 binds to avb5 (Weinacker et al., 1994), and 5C4 binds to b6 (Sipos et al., 2004). However, specific antibodies to b5, b6, and b8 chains are rare. This is unfortunate as the av biologies are complex and interesting. For example, avb3 and avb5 are involved in tumor growth and angiogenesis (Desgrosellier and Cheresh, 2010), whereas avb6 and avb8 enhance the activation of latent TGFbeta (Lacy-Hulbert et al., 2007; Sheppard, 2005), and avb8 expression has been connected to the inhibition of tumor cell growth (Cambier et al., 2000; Fang et al., 2011). However, limitations in detecting antibodies make it hard to identify integrin distributions or activation states that could be relevant to human pathologies. Integrins are dimeric cell surface proteins which control cell attachment. 14 alpha chains associate with 8 beta chains to form 24 receptors, each an obligate heterodimer. Integrins are unexpected strong expression of avb6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. avb3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express avb3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing av integrin biology, and suggest that especially avb6 and avb8 biologies still have much to reveal. 2012. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License ( differentially glycosylated, spliced, and activated in response to their cellular and extracellular environments (Bellis, 2004; Campbell and Humphries, 2011; Fornaro and Languino, 1997). This subtle regulation of cell attachment to extracellular matrix and to other cells, coordinates intracellular signaling responses to growth factors, and drives diverse cell behaviors (Hynes, 2002; Schwartz and Ginsberg, 2002). Occasionally, valuable antibodies have been described which detect those LIBS that alter following ligation or activation of integrins (Honda et al., 1995; Mould et al., 1995). The structural basis of such LIBS is unknown, with few exceptions (Honda et al., 1995). av integrins are being targeted in the clinics, notably in cancer therapies, so the characterization of their distribution in human tissue is important (Cox et al., 2010). av expression in tumors may reveal significant information for diagnosis, prognosis, and therapeutic outcome. Yet despite 25 years of research, and hundreds of specific antibodies that can stain fresh frozen tissues, monoclonal antibodies that specifically stain integrins in FFPE material remain rare. Sampling, storage, and logistics for frozen tissue are challenging, involving strict maintenance of coolchains. Furthermore, histomorphology is not optimally maintained in cryostat sections, compared with FFPE biopsies, so information on target distribution may be lost. In addition, some tissues are difficult to obtain, except as FFPE biopsies. The FFPE process involves cross-linking, dehydration, hydrophobic environments, and heat, all of which can destroy or conceal epitopes. Integrins are large, conformationally active transmembrane proteins and have many epitopes that might be lost. Nevertheless, some antibodies do bind to av integrins in FFPE material; often these are rabbit polyclonal antibodies, targeting the short conserved integrin cytoplasmic domain. However, polyclonal antibodies have numerous disadvantages, not least mortality of their hosts. In addition, antibodies against integrins cytoplasmic domains may complicate the interpretation of the results as staining can report ambiguous distribution, while the location of functional integrin heterodimers is unambiguously at the cell surface. Optimal antibodies should recognize the active heterodimeric extracellular domains. Monoclonal antibodies are the reagents of choice, being uniform and available in essentially unlimited amounts. Thus, there are gaps in our knowledge of integrin pathology due to the lack of monoclonal antibodies that can identify integrins in FFPE material. As rabbit polyclonal antibodies can bind epitopes in FFPE material (Pytela et al., 2008), we thought that RabMabs might also recognize integrin extracellular domains there. Lagomorphs have longer complementarity-determining regions (CDRs) than rodents, and thus potentially have high binding affinity; also, as n they are out-bred, their MHC loci are more diversified than the e p in-bred rodent populations often used for monoclonal production. O Here we describe RabMabs against extracellular domains of av yg integrins, one of which detects a LIBS on avb3. These RabMabs lo have helped to better understand the relationships between io expression of native heterodimers and cell proliferation on viable B cells, in archival FFPE material, and with biochemistry. Results Rabbit monoclonal antibody generation Primary bleeds of approximately one third of the immunized animals reacted with the DTM-integrins by ELISA. IHC using primary bleeds gave strong membrane staining of cells known to express the target integrins (Table 1) being stained on FFPE cellline microarray (CMA). Twenty to thirty percent of the resulting fused spleenocytes multiclones had the desired specificity by ELISA and 12% were reactive in IHC. Multiclones showing the lowest background staining on Raji and Sf9, and the strongest specific membrane staining were recloned to monoclonality and then banked. cDNA from the heavy and light chains was cloned, sequenced, and expressed in a 293-EBNA cell line, and the recombinant RabMabs were purified and studied further. Integrin expression profile on human tumor cells To generate integrin target profiles, we used MuMabs in flow cytometry of a panel of human cells (Table 1). In general, our results are in agreement with what has been reported in literature regarding integrin expression on these cells. For example, the M21 cell series M21 (avb3 +; avb5 +; avb6 2) and M21-L (avb3 +/2; avb5 2; avb6 2), the colon carcinoma line HT29 (avb32; avb5+; avb6+), the NSCLC line A549 (avb32; avb5+; avb62), and HUVECs (avb3+; avb5+; avb62) had the integrin expression profiles reported in the literature (Table 1). Some integrin expression patterns are controversial. Expression of avb3 integrin on the prostate cell line PC-3 has been reported as being high (Zheng et al., 2000), low (Witkowski et al., 1993), or not occurring (Haywood-Reid et al., 1997), and similar results have been found on MDA-MB231 breast carcinoma. We confirmed that the PC-3 cell line does not express avb3 and that low levels of this integrin are detected on MDA-MB-231. Expression of avb6 is not extensively documented. We confirmed avb6 expression on HT29 cells (Kemperman et al., 1997), and found high levels of this integrin on the SCCHN line Kyse30, and low levels on MDA-MB231 and -468 breast carcinoma lines. avb6 expression on clinical SCCHN has been associated with invasive tumor behavior (Janes and Watt, 2004; Sipos et al., 2004; Xue et al., 2001). avb8 integrin expression has not been reported for the tumor cell lines we examined in this study, and no avb8-specific monoclonal reagent for flow cytometry appears to be commercially available. As our results otherwise agreed with the literature descriptions, we arrayed the cell lines as paraffin CMAs for the primary FFPE screening of the RabMabs. The RabMabs bind purified intact integrins via individual chains of the complex The binding specificities of the RabMabs were characterized on recombinant DTM-integrins (i.e. the immunogens) and native gpiibb3 using ELISA in physiological divalent cations (Fig. 1). EM22703 bound to avb3 and gpiibb3 (EC50 2 ng ml21) (Fig. 1A,E). EM09902 bound to avb5 (EC50 5 ng ml21) and also weakly bound to avb3 and gpiibb3 (EC50 ,7 mg ml21) (Fig. 1A,B,E). EM05201 bound to avb6 (EC50 ,5 ng ml-1), EM13309 bound to avb8 (EC50 ,10 ng ml21), and EM01309 bound to all av integrins (EC50 ,10 ng ml21) but not gpiibb3. These data suggested that the RabMabs bound to the extracellular domains of native integrin complexes. Unexpectedly, EM00212 did not bind even to native gpiibb3 (EC50..10 mg ml21). EM22703 did not bind to the b3 cytoplasmic domain (it binds to DTM-avb3 that lacks this domain), but it did react with gpiibb3. The EC50s suggest the antibodies have picomolecular binding affinities. We next studied the antibodies on Western blots of whole cell lysates. Preliminary experiments showed that the RabMabs blotted the DTM-integrins, and that the signal from the nonreduced proteins was stronger than from the reduced proteins, suggesting some conformational specificity; detection limits were below 5 ng integrin per lane. We calculated that this sensitivity might detect integrins in cell lysates (Fig. 2). In octylglucoside lysates of M21, HT29, M24, and A549 cells (Fig. 2A, lanes 14), the pan-av antibody EM01309 bound to a band of ,150 kDa (Fig. 2B, lanes 14), and to the a-chain of recombinant avb3 (Fig. 2B, lanes 6, 7) but not to gpiibb3 (Fig. 2B, lane 8). There was weak reactivity with a doublet component and with a protein of approximately 95 kDa. Faster migrating proteins were also weakly detected in the positive control lanes, and may be partially degraded av. Both av and b chains of DTM-integrins are transmembrane truncated and so migrate faster than the major staining bands in the cell lysate that, therefore, likely identify the cellular integrin av chain (Kraft et al., 1999; Mehta et al., 1998). The avb3-antibody (EM22703) bound to the b3 chain in Western blots (Fig. 2C), strongly stained a protein in M21 and M24 melanoma cells running at ,85 kDa (lanes 3, 4), that can be seen only very faintly in HT29 and A549 cells (lanes 1, 2). In DTM-avb3 (Fig. 2C, lanes 6, 7), a band running at the same position as the recombinant DTM-b3 chain was observed. Melanoma cells (e.g. M21) expressed avb3, whereas HT29 and Murine Mabs flow cytometry av Integrin expression citation A549 did not. Similar results have been previously reported for both melanoma cells (Clark et al., 1994; Felding-Habermann et al., 1992; Kieffer et al., 1991) and HT29 and A549 lines (Bauer et al., 2007; Kemperman et al., 1997). The avb5-specific antibody EM09902 bound to a diffuse band of 8095 kDa in all tumor cells examined (Fig. 2D), and to a protein with the mobility of recombinant b5 on DTM-avb5 (Fig. 2D, lanes 6, 7), but no binding to DTM-avb6 controls was observed (lane 8). Flow cytometry showed that HT29 and A549 expressed avb5 strongly, whereas M21 expressed this integrin weakly and expression in the M24 line was moderate. These cell lines have previously been reported to express avb5 (Burvenich et al., 2008; Felding-Habermann et al., 1992; Kemperman et al., 1997). The avb6-specific antibody EM05201 bound to a compact protein band at ,90 kDa in HT29 cells (Fig. 2E, lane 1). It also stained a protein migrating in the same position as the DTM-b6 chain (Fig. 2E, lanes 6, 7), but did not stain DTM-avb3 (Fig. 2E, lane 8). To our knowledge, the avb6 expression pattern for M21, M24, or A549 cells has not been reported. However, HT29 cells have been shown to express avb6 (Kemperman et al., 1997), an epithelial integrin, not yet reported on melanoma (Sheppard, 1996). The avb8-specific antibody EM13309 bound weakly to a protein band migrating at ,90 kDa in HT29 cells and M21 cells; binding was moderate in M24 cells (Fig. 2F, lanes 14). EM13309 also stained a protein migrating in the same position as the recombinant DTM-b8 chain, but did not stain DTM-avb6 (Fig. 2F, lanes 68). We were unable to find an avb8 expression profile for HT29, M21, M24, or A549 cells in the literature; however, avb8 is expressed in cells that, like melanocytes, are in the neural crest lineage (Nishimura et al., 1998). The RabMabs recognize the integrin heterodimers by flow cytometry on viable cells We next investigated whether the RabMabs could bind their targets on viable cells. The antibodies bound strongly and specifically to a similar set of cells stained in FFPE material (Table 1; Fig. 3A), but with interesting variations. The patterns Fig. 1. ELISA profile of EBNA-recombinant rabbit anti-integrin n monoclonal antibodies. Plates coated with soluble recombinant integrins e (A) avb3; (B) avb5; (C) avb6; (D) avb8; or (E) native platelet gpiibb3 p (1 mg/ml) were incubated with recombinant antibodies from clones EM22703 O (circles, closed); EM09902 (squares); EM05201 (triangles, up); EM13309 yg (diamonds); EM01309 (triangles, down); EM00212 (circles, open). o l o i B of expression in flow cytometry with EM22703 (anti-avb3) and EM09902 (anti-avb5) matched literature and our in-house data obtained using MuMabs LM609 (anti-avb3) and P1F6 (antiavb5). M21 cells and HUVECs expressed avb3, HT29 and A549 cells displayed expression levels approaching background, and M21-L cells did not express any of the integrins. However, EM22703, which in ELISA also recognized gpiibb3, bound to M21-gpiib but not to M21-L cells. Thus, EM22703 binds to an epitope on the b3 chain, in a complex with either av or gpiib. EM09902 (anti-avb5) stained most cells with the exception of M21-L and M21-gpiib, which do not express av, and so lack cell surface avb5. M21, HUVEC, A549, and HT29 cells express avb5 (Table 1). EM05201 (anti-avb6) detected a strong signal on HT29 and on no other cell line, a pattern similar to the avb6 specific murine antibody 10C5 (Table 1). EM13309 (anti-avb8) detected a signal on HT29 cells and on two melanoma lines (M21 and M24-met), but not on A549, HUVECs, M21-L, nor M21gpiib lines. The EM01309 antibody unambiguously detected av chains in ELISA of the intact recombinant proteins (Fig. 1) and on Western blots (Fig. 2). However, in viable cell flow cytometry, it recognized the av chain only on HUVECs and weakly stained M24-met cells. Nevertheless, M21, A549, and HT-29 cells express av (Table 1; Fig. 4). Control murine anti-av Mab, 17E6 (Mitjans et al., 1995), bound strongly in flow cytometry to all cells with the exception of M21-L and M21gpiib. EM00212 (anti- b3A-cytoplasmic domain) gave the same signal as the second layer antibody, and it acted as a control for cellular integrity. Thus the RabMabs recognized the dissociated preferably nonreduced integrin chains in blots. They also bound to them specifically in the context of intact, native integrin heterodimers on the cell surface, and in the functionally active heterodimeric DTM-immunogens. EM22703 recognizes a LIBS epitope As integrins undergo conformational changes on binding ligands (to produce LIBS) whose gain or loss can be reported by antibodies (Du et al., 1993; Luo et al., 2005), we investigated whether the RabMabs reported such LIBS. av-integrins bind the Arg-Gly-Asp sequence in their ligands (Xiong et al., 2002). The RabMabs showed little change in binding to cells co-incubated with an RGD peptide, cilengitide, during antibody staining. The only exception was EM22703, in which the signal increased by 310 fold following incubation with RGD-peptide on M21, M24Met and HUVEC cells. In M24-Met line, only a sub-population of ,15% of the cells developed the LIBS epitope on exposure to cilengitide, whereas for both M21 and HUVECs the entire detected population expressed the novel epitope (Fig. 3A). The signal increase was concentration dependent (Fig. 3B), with an EC50 of ,100 nM on M21 cells (Fig. 3C). The change in LIBS expression was not a result of activation, as high concentrations of Mn2+, a known activator and conformational modulator of av integrins, had no effect on the antibody signals. Also, the signal that developed in physiological divalent cations with cilengitide was not affected by Mn2+ (Fig. 3A). The RabMabs recognize specific groups of human cell lines in FFPE preparations To develop robust staining protocols, the RabMabs were screened using a widely used automated clinical processing machine. The RabMabs were screened on sectioned FFPE CMAs and tumor-xenografts, where they stained strongly and specifically. Mild protease treatment was optimal for the antibodies against avb3, avb5, avb6, and avb8. Tris-EDTA pre-treatment was optimal for the antibodies against b3 cytoplasmic domain and av. The antibodies strongly stained cell plasma membranes, with some punctuate intracellular staining. For avb8 pronounced staining was also frequently observed in cytoplasm. Quantitative image analysis of the CMA stainings is shown in Fig. 4. EM01309: This pan-av-specific antibody stained all adherent human cell lines, but did not stain Raji B-cell lymphoma or Sf9 insect cells. It labeled membranes, with some punctuate intracellular staining (Figs 4, 5). It also stained cell lines when these were cultivated as subcutaneous xenografts in immunesuppressed mice, with pronounced membrane staining. EM22703: This avb3-specific antibody stained a subset of cell lines, including melanomas (3/4) (WM164; M24met; M21; but not Lox), glioma (U87MG), and most ovarian carcinomas (Igrov1; SKOV3 weakly Ovcar3; but not A2780ADR), whereas CRC (Colo205; HT29; SW707), NSCLC (Calu6; NHI-H460; A549), SCCHN (Kyse30), mammary (MCF7; MDA-MB468; MDA-MB231), and prostate lines (PC3; DU145; MiaPaCa2) were unstained (Figs 4, 5). EM22703 strongly labeled plasma membranes. EM09902: This avb5-specific antibody stained all adherent tumor cells on the CMA (Figs 4, 5). Some stainings were intense (e.g. M21, M24met, HT29, and A549). EM09902 labeled plasma membranes. It also stained sectioned subcutaneous HT29 tumor xenografts from mice. Although EM09902 staining was intense, it was specific: Raji lymphoma and Sf9 insect cells were unstained. EM05201: This avb6-specific antibody stained plasma membranes of a subset of cell lines, including CRC (3/3) (HT29; Colo205; SW707), SCCHN (Kyse30), mammary (1/3) (MDA-MB468), and carcinoid (A431) lines, but not melanomas (M21) glioma (U87MG), or ovarian carcinomas (3/4) (Igrov1; A2780ADR; Ovcar3). Prostate lines (DU145; MiaPaCa2) were unstained or faintly positive (PC3) (Figs 4, 6). EM13309: This avb8-specific antibody stained a subset of tumor cell lines, including ovarian carcinomas (3/3) (Ovcar3; Igrov1; SKOV3), some melanomas (2/4) (Lox; M24-met; and M21, but only weakly), and a breast carcinoma (1/3) (MDA-MB468), and weakly stained a CRC line (1/4: HT29). A prostate line (1/2) (PC3) stained weakly. It stained no lung (0/4) or glioma lines (Figs 4, 6). Staining defined the plasma membrane; but, in contrast to the other antibodies, EM13309 also often stained the cytoplasm that sometimes dominated the membrane staining. In flow cytometry, viable cells showed a clear staining. It was notable that the cell lines that stained well for avb8 were strongly proliferative ovarian carcinomas, and did not include the glioblastoma derived line U87MG (Fang et al., 2011). EM00212: As the staining patterns with the avb6 and avb8 antibodies were unexpected, we studied the antibody EM00212, an anti-b3 cytoplasmic domain reagent, to verify specificity. The low staining intensity of Sf9 and Raji, known to not express avb3, was considered as background. EM00212 stained the same cell lines as EM22703, with some variations in intensity of staining; for example, the faint signal on Lox with EM22703 was not seen. EM00212 strongly marked plasma membranes (Figs 4, 6). Thus the staining from an anti-cytoplasmic b3 RabMab closely matched the results from the extracellular domainspecific reagent EM22703, and was distinct from the staining with EM13309 and EM05201. Cells that stained for avb3 tended not to stain for avb6, with the exception of the SKOV3 and OVCAR3 ovarian carcinoma lines. The RabMabs recognize their targets in FFPE archival human tumor samples Human TMAs and normal archival paraffin tissue specimens were stained with the RabMabs. Strong staining without unspecific background was observed in a subset of samples. Six to nine samples of each tumor were examined, and representative galleries of data are shown (Figs 7, 8). Archival FFPE samples of solid tumors, shown for melanoma, NSCLC, CRC, mammary, and prostate carcinomas, strongly expressed membranous av integrins. Melanomas stained for avb3, as previously reported (Albelda et al., 1990; Hart et al., 1991), as well as for avb5, particularly in the connective tissues and vasculature (Fig. 7). avb6 expression was absent, and avb8 was weakly expressed (Fig. 8). In NSCLC, avb3 strongly labeled complexes of tumor cells at the invasion front, and Fig. 3. Viable cell flow cytometry with the RabMabs shows strong LIBS signals from anti-avb3 antibody EM22703, and avb8 signals. (A) Flow cytometry in the presence of physiological divalent cations (black open); 1 mM Mn2+ (red closed); 10 mM cilengitide in physiological cations (blue closed); 10 mM cilengitide in 1 mM Mn2+ (green open). Horizontal panels show staining with EM22703 (avb3); EM09902 (avb5); EM05201 (avb6); and EM13309 (avb8). Vertical panels show staining on HUVECs, M21, A549, HT29, and M24Met. Gray shading shows binding of EM00212 and the second layer controls, which superimpose. (B,C) Variation of EM22703 flow cytometry signal on M21 cells with cilengitide concentration. (B) red 5 0 mM; black 5 4 nM; brown 5 40 nM; green 5 100 nM; yellow 5 400 nM; grape 5 4 mM; blue 5 100 mM. Fig. 4. Automated image analysis of FFPE human tumor cell line staining using anti-integrin av RabMabs. Image analysis of human tumor cell lines in TMAs n stained with RabMabs. The cells are grouped by tumor-of-origin: (A) T-lymphoma: Raji: and (B) insect production cell line: Sf9, serve as negative controls. pe (CE) Mammary carcinomas: MCF7; MDA-MB231; MDA-MB468. (F) Carcinoid: A431. (GI) Colorectal carcinomas: Colo 205; HT-29; SW707. (J) Glioma: O U87MG. (KM) Lung carcinomas: A549; Calu-6; H460. (NR) Melanomas: C8161; Lox; M21; M24-met; WM164. (SV) Ovarian carcinomas: A2780ADR; Igrov1; yg nO.Vd.C5ARn-o3t;dSeKteOrmVi3n.ed(W.a)vPba5nhcirsetaotgicracmarscfinoormLao:xSaunidt7M.(2X4-Zm)etParorestatrtuenccaartceidnofomracso:mDpUa1r4ab5i;liMtyiafProamCao2r;igPiCna-3l.v(aAluAes) SofC1C4H6NA: UKy(Lseo3x0). aAnUd 1578adAsUorp(Mtio2n1)u.nits. o l o i B tumor vasculature (Fig. 7). avb5 was prevalent in cells within the tumor stroma and occasionally on tumor cells. avb6 was intensely expressed on tumor cells, but absent from interstices and vasculature (Fig. 8). A weak membrane staining for avb8 was frequently observed in NSCLC (Fig. 8). In CRC, only vasculature stained for avb3, whereas avb5 was heavily expressed throughout the tumor and its interstices (Fig. 7). avb6 expression was confined to apparently invasive cohorts, and avb8 expression was absent (Fig. 8). A different pattern was seen in mammary carcinomas, where avb3 expression was absent except on vasculature, avb5 was confined to interstices and vasculature (Fig. 7), and avb6 was strongly expressed on the tumor cells (Fig. 8). There was no avb8 expression in mammary carcinomas (Fig. 8). Prostate cancer samples expressed essentially only avb5 in the tumor environment, with vasculature expressing some avb3 (Figs 7, 8). Discussion Effective and specific antibodies have provided foundations for progress and understanding in the field of integrin research. Here we describe a matched set of RabMabs that can stain integrins in archived FFPE tissue. The RabMabs also function in flow cytometry of viable cells, where EM22703 acts as a LIBS reagent, reporting binding of nanomolar concentrations of an antagonistic avb3 inhibitor cilengitide, and in Western blotting, on DTM-integrins. The antibodies revealed unexpected distributions of the av integrins in human archival paraffin embedded materials, notably avb3 in some invasive NSCLC, and avb6 in mammary carcinoma. Integrins are large complex molecules, where ligand binding in the extracellular head domains depends on divalent cations which can act to stimulate (usually Mg2+) or inhibit (usually Ca2+) ligand binding. Mn2+ is often a strong-activator. EM22703 is a LIBS antibody. It binds to unligated avb3 on viable cells and in FFPE tissues. However, in flow cytometry, the EM22703 signal is increased 310 fold in the presence of nanomolar amounts (>50 nM) of cilengitide (Dechantsreiter et al., 1999), and is not inhibited by micromolar levels of this RGD peptide, nor it is affected by Mn2+. Thus it is not a ligand-mimetic antibody like WOW-1 and PAC-1 (Pampori et al., 1999) or an ion-sensitive reporter. Cilengitide induces large conformational changes in avb3 upon binding (Arnaout et al., 2007; Xiong et al., 2002). The precise molecular basis for the EM22703 LIBS signal is not clear, but it is not induced by manganese ions, which both activate and cause conformational changes (Hynes, 2002). LIBS antibodies that report b3 activation (Honda et al., 1995) may be sensitive to divalent cations or to the presence of ligands, or may modulate ligand binding (Frelinger et al., 1990; Frelinger et al., 1991). However, EM22703 does not report this type of epitope. It still needs to be determined whether the EM22703 LIBS signal might be detectable in clinical specimens during RGD-based therapy. Currently, standard antibodies recognizing av integrin (e.g. LM609; P1F6; 10C5; 17E6) can only detect their epitopes in cryopreserved tissues and, with few exceptions, do not show specific staining in paraffin tissue blocks. This has locked integrin researchers out of the FFPE archives that bank many important and rare clinical samples. We used commercial methodology to generate FFPE-competent anti-integrin RabMabs, five of which we describe here: it seems the techniques may be generally applicable for producing FFPE-reactive antibodies against integrin extracellular domains. In summary, the antibodies stains in FFPE material support published integrin distributions seen in cryostat materials. However, this now enables the greater spatial resolution of FFPE immunohistology. We also show new characterizations of avb8 for which no FFPE-capable monoclonal antibodies have been described, and find that ovarian carcinoma cell lines express avb8. Western blotting showed the epitopes of the RabMabs localized to individual integrin chains, though they bind the intact heterodimers on viable cells. The signals in Western blots were decreased under reducing conditions, implying a conformational aspect to the epitopes. The immunogens were made in insect cells with divergent glycosylation from mammalian forms. The above mentioned cross reactivity and conformational aspects suggest that sugars are not involved in the RabMab epitopes. Integrins are obligate heterodimers, so antibodies binding only one chain may define the complex in IHC, providing (a) they recognize external domain of the intact complex, (b) they recognize a chain with a limited number of partners, and, not least, (c) they function in IHC. Three integrin beta chains are believed monogamous for av: b5, b6 and b8. b3 Fig. 6. Human tumor cell lines stained on FFPE microtissue array using RabMabs. EM05201, EM13309, and EM00212, M21 and M24-met melanoma, A549 NSCLC, HT29 CRC, MDA-MB468 mammary carcinoma, and Raji Bcell lymphoma stained with EM05201 (avb6), EM13309 (avb8), and EM00212 (cyto-b3). Scale bar 5 50 mm. also binds gpiib, which is expressed only in the megakaryocyte lineage, so IHC staining for b3 can often be unequivocally assigned to avb3 (Fig. 7: NSCLC). As such, binding of the RabMabs to external domains of integrin chains may be used for the tissue localization of the avbx complexes. Furthermore, it is known that the epitope of antibody LM609 maps to the b3 chain but it detects only avb3, not gpiibb3 (Cheresh and Spiro, 1987; Lin et al., 1998). Consequently, experimental context may affect whether a single chain or a complex-dependent epitope is detected. The RabMabs described here recognize conformational epitopes on individual chains, but clearly they do so in the context of the intact integrin heterodimers on vital cells. In this study, this was highlighted by the EM22703 LIBS antibody against avb3, and by EM01309 against av. For the RabMab EM01309, biochemistry and FFPE techniques reported an identical pan-av profile, which was consistent with results reported with 17E6 (Mitjans et al., 1995) and LM142 (Lawler and Hynes, 1989). Interestingly, however, these profiles did not match the flow-cytometry patterns of EM01309. It strongly bound to HUVECs, moderately bound M24 cells, but did not bind to M21. The molecular basis for this unusual recognition profile is under investigation, but it does not seem to be modulated by Mn2+ or RGD peptides. In summary, data found by FFPE IHC supported the distributions of avb3, avb5, and avb6 found using biochemistry and cell biological tools. For example, flow cytometry profiles with LM609 and EM22703 (binding avb3) are similar. However, unlike LM609, EM22703 also recognizes b3 on Western blots, on FFPE material, and is a LIBS antibody. P1F6 and EM09902 (antiavb5) stained all adherent tumor cells, but not M21-L or Raji lymphoma, both known to not express avb5 (Nagel et al., 2003). For EM05201 (anti-avb6), cells with an SCC origin and breast carcinoma, NSCLC, and CRC tissues were strongly stained. For EM13309 (anti-avb8), we had no appropriate comparator antibody available, but the biochemical, cytometric, and IHC patterns were unequivocal. These surprisingly revealed avb8 expression in ovarian tumor cell lines. The distribution predicted by staining of tumor cell lines is largely reflected in clinical archival material. The integrin distributions we observed on FFPE material were unexpected. avb8 is implicated in activation of TGFb (Lacy-Hulbert et al., 2007) and, as confirmed here, is present on astrocytes (Milner et al., 1999; Nishimura et al., 1998). avb8 inhibits angiogenesis and growth of transformed epithelial and glioblastoma multiform (GBM) (Tchaicha et al., 2011), and promotes invasion in GBM cell lines (Cambier et al., 2000; Fang et al., 2011). A microRNA miR-93 that targets b8 in U87MG cells promotes cell growth, but here we found that U87MG expresses little avb8, confirming recent reports by others Fig. 8. IHC of archival human tumors. Malignant melanoma (MaMe) and non-small-cell lung carcinoma (NSCLC), colorectal carcinoma (CRC), invasive ductal breast carcinoma (BrCa), and prostate carcinoma (PrCa) are shown stained with EM05201 (avb6), EM13309 (avb8), and EM00212 (cyto-b3). Scale bar 5 50 mm. (Tchaicha et al., 2011). This difference may reflect the antibodies used. avb8 was also unexpectedly expressed in several ovarian carcinoma cell lines, in some melanoma lines (e.g. M24-met), and in a breast carcinoma MDA-MB468 line. Nishimura and colleagues (Nishimura et al.,1994) reported b8 mRNA expression in a normal ovary, but we could find no report of its expression in ovarian or mammary carcinomas. avb8 was not strongly expressed in the restricted number of breast carcinoma or melanoma tissue samples that were examined in this study. We note that M24-met and MDA-MB468 are derived from metastases: if avb8-dependent TGFb activation mediated immune suppression at metastatic sites, one could speculate that this would be an appropriate expression pattern. However, this speculation clearly awaits detailed study. avb6 is also implicated in TGFb activation (Margadant and Sonnenberg, 2010; Sheppard, 2005), and is associated with SCCHN (Xue et al., 2001), with a poor prognosis in CRC (Bates et al., 2005) and with inflammation of the lung (Horan et al., 2008). We confirmed avb6 expression in NSCLC and CRC, and found it strongly expressed in human mammary carcinomas and on the mammary line MDA-MB468. avb6 expression in mammary carcinoma has been previously noted (Arihiro et al., 2000). In the present study, we confirmed and extended previous results, confirming observations on mRNA that avb6 was expressed in the basal layers of normal colonic epithelium and in a set of kidney tubuli. As with avb8, expression of avb6 in invasive tumors of the breast and lung supports the concept of an immunosuppressive role for avb6 via local activation of latent TGFb at tumor margins (Thomas et al., 2006; Xue et al., 2001). We found that avb5 was ubiquitous on attached tumor cells and in tumors. The expression of avb5 and avb6 was often complementary: where both were expressed, avb5 was frequently in stromal compartments, with avb6 in the tumor, for example in the breast and, to a lesser extent, in the lung and colon. In prostate carcinoma (which generally lacked avb6 and avb8 and, except the vasculature, avb3), avb5 was expressed both in the tumor cells and in tumor stroma. A switch between avb5 and avb6 which supports survival of squamous cell carcinomas has been noted (Janes and Watt, 2004), while stratified carcinomas maintain avb5 expression. avb3 is well characterized, and we confirmed its expression in melanoma cell lines, in malignant melanoma and its vasculature, and in ovarian cancer cell lines. We also confirmed avb3 expression on glioma, but found it was not expressed on PC-3 prostate carcinoma cells. Unexpectedly, in clinical specimens, avb3 was occasionally expressed on invasive cohorts in NSCLC. Why do RabMabs function so well in FFPE material, where murine antibodies largely fail to recognize FFPE-embedded integrins? The immunogen integrin was made in insect cells, where glycoprotein sugar processing differs from mammalian forms, and this can enhance immunogenicity (Gu et al., 2009; Tomiya et al., 2004; Wilson et al., 1998). Rabbits are out-bred, so y g the diversity of type I MHC loci and antibody diversity is greater o lo than the in-bred rodent populations often used for monoclonal i B antibody production. Finally, lapidomorphs express CDR domains that are longer than those in rodentia (Liu and Wolf, 1998), which permits more precise spatial imaging of structures. The resulting antibody response detects integrin epitopes in FFPE material that are apparently invisible to rodent immune systems. It is very fitting that Dr. Robert Pytela, a discoverer of the integrins, invented rabbit monoclonal antibody technologies. We have struggled to identify integrin antibodies that react reproducibly in FFPE material with integrin extracellular domains, though occasional monoclonals may do so (e.g. GoH3). FFPE tissue banks are a resource of great importance, from which integrin researchers have been essentially locked out. Of course, some polyclonal antibodies directed against the cytoplasmic domains do recognize integrins in FFPE material, but such antibodies cannot be used in viable cell flow cytometry. In addition, polyclonal antibodies are mortal producer animals die. When discussing the use of antibodies in clinical and especially in diagnostic applications, monoclonals are the gold standard. The RabMabs show that, although hard, the situation is not hopeless. In conclusion, we have routinely generated rabbit monoclonal antibodies against extracellular domains of human av integrins. They function in biochemistry, cell biology, and FFPE immunohistology, and can report receptor RGD-occupancy of avb3 at nanomolar concentrations in viable cells. These RabMabs reveal unexpected integrin expression patterns, notably of avb8 in ovarian and mammary carcinomas, and avb6 in mammary carcinomas, and are proving to be invaluable tools for investigating the links between cell biology and pathologies driven by integrins. Materials and Methods Human recombinant transmembrane-truncated (DTM) extracellular domains of integrins avb3, avb5, avb6, and avb8 were generated using the baculovirus system, and purified from High Five insect cell lines (Kraft et al., 1999; Mehta et al., 1998; Xiong et al., 2007). Integrin chains were truncated at the juxtamembrane residues C-termini (av chain: IQP987; b3: GPD718; b5: TPN719; b6: PPN706; and b8: YLR684) (Mehta et al., 1998; Ulmer, 2010). A b3A cytoplasmic domain (H722DRRGT762) was produced as C-terminal fusion on glutathione Stransferase. Cells and mouse monoclonal antibodies MuMabs against integrins avb3 (LM609), avb5 (P1F6), avb6 (10C5), av (17E6), and b1 (P4C10) were obtained from Millipore (Schwalbach, Germany), antimouse-FITC was obtained from Becton Dickinson (Heidelberg, Germany), and anti-rabbit-Alexa-488 was obtained from Invitrogen (Darmstadt, Germany). Cells from ATCC (Wessel, Germany) were cultured as recommended, in DMEM (A431; A549; Calu-6; Colo205; HT29; M21 series; M24met; MCF7; MDA-MB-231; MiaPaCa2; PC3; SKOV3; SW707; Suit-S2; U87MG; WM164), RPMI (A2780 ADR; Igrov1; Lox; MDA-MB 468; NCI-H460; Ovcar3; Raji), RPMI/Hams F12 (1:1) (Kyse30), DMEM/Hams F12 (C8161), or MEM alpha+ (DU145) supplemented with 10% FCS in a 5% CO295% air atmosphere. Human umbilical vein endothelial cells (HUVECs) were produced in-house and maintained as previously described in complete EGM MV medium (EBM: Promo cell, Heidelberg, Germany): 2% (v/v) endothelial cell growth supplement, 10 ng/ml EGF, 1 mg/ml hydrocortisone (Mikkelsen et al., 2008). Cells with previously well characterized integrin profiles were used to investigate RabMab staining and LIBS activities. M21 cells express avb3 and avb5 (Wayner et al., 1991), and M21-L is a sub-clone with low av expression (Cheresh and Spiro, 1987; Mitjans et al., 1995). M21-gpiib is M21-L transfected with the integrin gpIIb and expresses gpiibb3 (Kieffer et al., 1991; Mitjans et al., 1995). HT29 cells express avb5 and avb6 (Kemperman et al., 1997). A549 cells express avb5, but no avb3 or avb6 (Burvenich et al., 2008; Falcioni et al., 1994). Immunization and primary screening RabMabs against DTM-avb3, DTM-avb5, DTM-avb6, DTM-avb8, and cytoplasmic domain of b3 were generated under proprietary protocols by Epitomics (Burlingame, CA, USA) (Epitomics inc, 2010; Pytela et al., 2008; Spieker-Polet et al., 1995), and screened by ELISA to identify antibodies specifically binding DTM-integrins (Mehta et al., 1998). Reactive hybridomas were subsequently screened by immunohistology on FFPE-sectioned human tumor cells pre-screened by flow cytometry (Table 1). Hybridomas were recloned by serial dilution and expanded. The monoclonal RabMab cells were harvested and banked. Immunoglobulin genes were amplified by PCR from the monoclonals and the heavy and light chain cDNA cloned into an EBNA expression system for recombinant antibody production. The IgGs were purified and stored under aseptic conditions at 4C, or for long storage at 280C. The binding characteristics of the final EBNA recombinant antibodies described here were identical with the hybridoma-derived antibodies. Cultured human tumor-derived cell lines were harvested, fixed in buffered formaldehyde (4%; pH 7.0; 1624 h, 20C), embedded in paraffin, and set en bloc for cell-line microarrays (CMAs). Sections 3 mm thick were mounted on positively charged slides (SuperFrost Plus; Menzel-Glaser, Braunschweig, Germany) and stored at 280C under desiccant. An automated tissue immunohistology processing machine (Discovery XT; Ventana Medical Systems Inc., Tuscon, AZ, USA [VMSI]) was used for tissue staining. Optimal procedures involved heating the de-paraffinized sections in Tris-EDTA buffer pH 8 or incubating them with protease 1 (0.5 U/ml, 8 min; 37C: VMSI) or protease 2 (0.1 U/ml, 12 min; 37C: VMSI). Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide. Sections were incubated with RabMabs diluted in PBS, followed by incubation with HRP conjugated secondary anti-rabbit antibody (16 min; 37C: Ventana OmniMap or UltraMap Kits), and development with 3,39diaminobenzidine tetrahydrochloride (DAB)/H2O2. Counterstaining was done with haematoxylin and samples were washed, dehydrated, and mounted under EntellanH Neu (VWR, Darmstadt, Germany). The milder protease treatment (0.1 U/ml) was optimal for antibodies against avb3, avb5, avb6, and avb8. The Tris-EDTA treatment was optimal for the antibodies against b3 cytoplasmic domain and av. Commercial FFPE human normal tissue (provitro GmbH, Berlin, Germany) and human tumor tissue (Asterand plc, Detroit, MI, USA) microarrays (TMAs) were processed in parallel to the cell line microarrays as described above. The stained CMAs were digitized with the MiraxScan device (Zeiss, Oberkochen, Germany) to a resolution of 1 pixel 5 0.2360.23 mm2. The MiraxScan calibrated brightness for each slide prior to scanning. The scans were analyzed with the Visiopharm Integrator System image analysis software (Visiopharm A/S, Hoersholm, Denmark). Cells were detected by their darker blue nuclear and cytoplasmic staining. The positive (brown) stained area was calculated as percent area of the viable tissue area. Antibody staining (arbitrary unit) was calculated as Antibody staining 5 Area fraction * (255-Intensity) of the brown color Each RabMab was cloned into an identical IgG backbone, so their relative staining intensities reported by a labeled second layer could be directly compared. Western blotting Representative cell lines, shown by IHC or flow cytometry to express the target integrins, were grown to semiconfluency, harvested, and processed for Western blotting (Mitjans et al., 1995; Sipos et al., 2004). The solubilized detergent extracts were resolved by PAGE under reducing and non-reducing conditions (412% SDS-PAGE Bis-Tris gels; MOPS buffer system) (Invitrogen; NuPAGE-MOPS system). Molecular weight standards for enhanced chemoluminescence (ECL; Magic Marker XP, Invitrogen, Karlsruhe, Germany), Coomassie blue staining (SeeBlue2; Invitrogen, Karlsruhe, Germany), and DTM-recombinant integrins were run in parallel as mass and blotting controls. The gels were blotted onto nitrocellulose papers in a semi-dry apparatus (Trans-Blot: Biorad, Munich, Germany), blocked (PBS; 5% w/v BSA; 0.1% Tween-20), and the transferred proteins probed with the RabMabs (0.0210 mg/ml) diluted in T-PBS (PBS; 0.1% Tween-20). After washing in T-PBS, bound RabMabs were detected using HRPconjugated goat-anti-rabbit antibodies (1:100,000 in T-PBS: Biorad, Munich, Germany), and visualized using enhanced chemoluminescence (Lumi-Light plus; Roche, Mannheim, Germany). The images were captured in digital format (Versadoc; Biorad, Munich, Germany). Viable cell flow cytometry Viable cell flow cytometry on human tumor cell lines was performed essentially as detailed elsewhere (Mitjans et al., 1995). Cells were harvested from culture using trypsin (0.5 mg/ml)/EDTA (0.2 mg/ml), which did not affect expression of the integrins, and washed in FACs saline buffer (PBS; 0.9 mM CaCl2; 0.5 mM MgCl2; 0.5% w/v BSA). They were then incubated with antibody diluted in FACs buffer n (1 mg/ml; 60 min; 4C), washed and stained using Alexa488 labeled goat-antie rabbit IgG (Invitrogen, Karlsruhe, Germany) (30 min; 4C). Finally, cells were rep washed and subjected to flow-cytometry collecting 20000 events. Murine antiO integrin antibodies were used under identical conditions (but at 10 mg/ml). The y EM00212 antibody against the b3 cytoplasmic domain does not recognize its g epitope on viable cells, and was used as the isotype matched RabMab control. The lo mean intensity of fluorescence (MIF) was expressed as the ratio to the MIF of the io negative control (cells stained with PI, with an isotype matched control, and B secondary labeled antibody). For detection of LIBS epitopes, cells were washed and suspended in FACS saline buffer and then incubated for 15 min with various concentrations of cyclic RGD peptide, reactive with integrins avb3 and avb5 (Goodman et al., 2002). Cells were then incubated with RabMabs in the presence of the peptide; the washing, staining, and flow-cytometry procedure was done as described above. Acknowledgements Immunization, fusion, ELISA primary screening, antibody molecular cloning procedures and recombinant antibody production were performed under a service agreement with Epitomics. We thank the Epitomics experimental teams in Burlingame, CA, USA under Dr. Li Li and the team in Vienna, under Dr. Franz Leichfried and Lis Knogler, for their expert work. All screening protocols, IHC, biochemistry, and cell biology experimental studies were devised and performed in the authors laboratories. We thank the molecular biology and protein technology teams at Merck KGaA under Dr. Detlev G ussow and Dirk Mueller-Pompalla for the cDNA cloning and production of the immunogens, and Kerstin Leidinger, Ina Seibel, Catherine Eichhorn, and Jutta Welge for expert technical support, and Dr. Francesc Mitjans who supported the early stages of this project. Dr. Sandra Mendes kindly commented on the manuscript. References Albelda, S. M., Mette, S. A., Elder, D. E., Stewart, R., Damjanovich, L., Herlyn, M. and Buck, C. A. (1990). Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression. Cancer Res. 50, 6757-6764. Albert, J. M., Cao, C., Geng, L., Leavitt, L., Hallahan, D. E. and Lu, B. (2006). Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in

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Simon L. Goodman, Hans Juergen Grote, Claudia Wilm. Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors, Biology Open, 2012, 329-340, DOI: 10.1242/bio.2012364