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

Abstract

SummaryThe 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 αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM05201), αvβ8 (EM13309), and pan-αv (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. 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 αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal.

Correlation between the expression of integrins in prostate cancer and clinical outcome in 1284 patients

“…Formalin-fixed and paraffin-embedded material from each tumor was stained with 6 recently established monoclonal rabbit antibodies (Table 1) directed against integrin complexes or individual chains, as previously described [23]. The biochemical specificity of the antibodies against integrins, which were used in this study, has been precisely defined [24,25]. They detect the αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM052), and αvβ8 (EM13309) heterodimeric complexes; the αv chain in all the αv heterodimeric complexes (EM01309); or the β3 chain cytoplasmic domain (EM00212).…”

A Novel Small-Molecule Integrin Antagonist Inhibits Cells Adhesion Followed By Anoikis in Endothelial Cells - A Comparative Analysis with Cilengitide

“…We show that αVβ3-expressing human endothelial cells demonstrated the highest sensitivity against AV-38/398 with IC 50 in the nanomolar range, whereas HT-29 adenocarcinoma cells, lacking αVβ3, were non-responsive to the treatment. Since HT-29 is devoid of integrin αVβ3 expression but is positive for αVβ5 [23,24; see data Figure 1], we compared AV-38/398 effects towards the αVβ3/αVβ5 integrin antagonist cilengitide. Cilengitide effectively inhibited the growth of HT-29 cells but was less efficient in endothelial cells.…”

Differential Effect on Bone Lesions of Targeting Integrins: Randomized Phase II Trial of Abituzumab in Patients with Metastatic Castration-Resistant Prostate Cancer

“…All patients also provided formalin-fixed, paraffin-embedded primary tumor material (blocks or biopsy) for assessment of integrin expression during screening. Integrin expression in formalin-fixed, paraffin-embedded tumor material was assessed by IHC using antibodies against pan-av, avb5, and avb6 as described previously (26).…”

“…175 primary prostate cancer samples were assessed for pan-av, avb5, and avb6 expression by IHC (26). A histoscore of 50 was chosen as the cut-off for the presence or absence of integrin expression.…”