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

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 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.

Keywords: Alphav; Immunohistology; Integrin; Paraffin embedded; Rabbit-monoclonal.

Fig. 1.. ELISA profile of EBNA-recombinant rabbit anti-integrin monoclonal antibodies.

Plates coated with soluble recombinant integrins (A) αvβ3; (B) αvβ5; (C) αvβ6; (D) αvβ8; or (E) native platelet gpiibβ3 (1 µg/ml) were incubated with recombinant antibodies from clones EM22703 (circles, closed); EM09902 (squares); EM05201 (triangles, up); EM13309 (diamonds); EM01309 (triangles, down); EM00212 (circles, open).

Fig. 2.. Characterization of EBNA-recombinant rabbit anti-integrin antibodies on Western blots of whole cell lysates.

Detergent lysates of tumor cell lines (10 µg protein) and purified integrins were resolved on SDS-PAGE gels. HT29 (lane 1), A549 (lane 2); M21 (lane 3); and M24 (lane 4). (A) Lanes 1–4 stained with Coomassie brilliant blue. Lanes 5–8 recombinant DTM-integrins αvβ3, αvβ5, αvβ6, and αvβ8 (750 ng). (B–F) Western blots probed with (B) EM01309; (C) EM22703; (D) EM09902; (E) EM05201; or (F) EM13309; and bound antibody detected using ECL. Molecular markers were run in parallel as indicated on the left gel margins. In (B–F), positive control integrins were loaded in lanes 6 (7.5 ng) and 7 (25 ng) vs. negative control integins (100 ng) in lane 8 as follows: (B) αvβ5 vs. gpiibβ3; (C) αvβ3 vs. αvβ6; (D) αvβ5 vs. αvβ6; (E) αvβ6 vs. αvβ3; (F) αvβ8 vs. αvβ6. Note that in each blot the integrin negative control is not stained.

Fig. 3.. Viable cell flow cytometry with the RabMabs shows strong LIBS signals from anti-αvβ3 antibody EM22703, and αvβ8 signals.

(A) Flow cytometry in the presence of physiological divalent cations (black open); 1 mM Mn²⁺ (red closed); 10 µM cilengitide in physiological cations (blue closed); 10 µM cilengitide in 1 mM Mn²⁺ (green open). Horizontal panels show staining with EM22703 (αvβ3); EM09902 (αvβ5); EM05201 (αvβ6); and EM13309 (αvβ8). 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  =  0 µM; black  =  4 nM; brown  =  40 nM; green  =  100 nM; yellow  =  400 nM; grape  =  4 µM; blue  =  100 µM.

Fig. 4.. Automated image analysis of FFPE human tumor cell line staining using anti-integrin αv RabMabs.

Image analysis of human tumor cell lines in TMAs 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. (C–E) Mammary carcinomas: MCF7; MDA-MB231; MDA-MB468. (F) Carcinoid: A431. (G–I) Colorectal carcinomas: Colo 205; HT-29; SW707. (J) Glioma: U87MG. (K–M) Lung carcinomas: A549; Calu-6; H460. (N–R) Melanomas: C8161; Lox; M21; M24-met; WM164. (S–V) Ovarian carcinomas: A2780ADR; Igrov1; OVCAR-3; SKOV3. (W) Pancreatic carcinoma: Suit7. (X–Z) Prostate carcinomas: DU145; MiaPaCa2; PC-3. (AA) SCCHN: Kyse30. AU  =  adsorption units. n.d.  =  not determined. αvβ5 histograms for Lox and M24-met are truncated for comparability from original values of 146 AU (Lox) and 178 AU (M21).

Fig. 5.. Human tumor cell lines stained in FFPE microtissue array using RabMabs.

M21 and M24-met melanoma, A549 NSCLC, HT29 CRC, MDA-MB468 mammary carcinoma, and Raji B-cell lymphoma are shown stained with EM01309 (αv), EM22703 (αvβ3), and EM09902 (αvβ5). Scale bar  =  50 µm.

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 B-cell lymphoma stained with EM05201 (αvβ6), EM13309 (αvβ8), and EM00212 (cyto-β3). Scale bar  =  50 µm.

Fig. 7.. 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 EM01309 (αv), EM22703 (αvβ3), and EM09902 (αvβ5). Scale bar  =  50 µm.

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 (αvβ6), EM13309 (αvβ8), and EM00212 (cyto-β3). Scale bar  =  50 µm.