Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients

Abstract

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

Fig. 1.

Combinatorial selection in patients. (A) Monte Carlo simulations with peptides analyzed in serial rounds of selection show nonrandom distribution of tripeptides. Thick black lines represent the Fisher’s exact test; thin blue lines represent the corresponding random permutated dataset. (B) A saturation plot (modified from ref. 17) shows the number of distinct peptides as a function of the total number of peptide sequences. All tissues reached saturation, as indicated by flattening of the slope; in contrast, unselected library showed no evidence of saturation. (C) Isolated peptides were grouped according to tissue-of-origin and subjected to Monte Carlo simulation. For every simulation, the pool of peptides was randomly distributed into groups corresponding to the number of sequences analyzed for each targeted tissue. Frequencies were calculated for each simulated organ, and Fisher’s exact test applied on the permutated dataset. A nonrandom selection of tripeptides was observed in all organs tested.

Fig. 2.

Discovery of integrin α4 subunit/ANXA4 as a shared ligand-receptor in the vasculature of multiple human tissues. (A) Binding of phage clones to the receptor ANXA4. Phage displaying the peptide CMRGFRAAC bound preferentially to its receptor, relative to negative controls. Experiments were performed three times in triplicate with similar results. Bars represent mean ± standard error of the mean (SEM). (B) Competition with the synthetic peptide shows that binding of selected phage to the purified receptor is specific. Binding of unrelated control phage, insertless phage, binding to BSA and inhibition with an unrelated peptide served as controls. (C) ELISA with preimmune and postimmune rabbit polyclonal antibodies against CMRGFRAAC and performed on recombinant integrin α4 or a control (shown is α5β1 integrin). (D) Binding of postimmune antibodies to recombinant integrin α4 is inhibited by CMRGFRAAC but not by an unrelated control peptide. (E) Binding specificity was confirmed by immunoblotting. Integrins αvβ5, α5β1, and αvβ3 were used as negative controls. Arrow points to integrin α4. (F) Direct interaction between ANXA4 and integrin α4. The binding is concentration-dependent, indicating specificity. (G) Immunostaining of sections of normal human tissues with an anti-ANXA4 polyclonal antibody. Arrows point to ANXA4-positive blood vessels. Insets show negative control staining. (Scale bar, 100 μm).

Fig. 3.

Discovery of cathepsin B/ApoE3 as a shared ligand-receptor in the vasculature of multiple human tissues. (A) Binding of CMGGHGWGC-phage to ApoE3. CMGGHGWGC- phage bound preferentially to its receptor relative to negative controls. Experiments were performed three times in triplicate with similar results. Bars represent mean ± SEM (B) Competition assay with the cognate synthetic peptide shows that binding of CMGGHGWGC-phage to the purified ApoE3 is specific. Binding of unrelated control phage, insertless phage, binding to BSA and inhibition with an unrelated peptide served as controls. (C and D) ELISA with preimmune and postimmune polyclonal antibodies against CMGGHGWGC (C) and performed on purified cathepsin B or control protein (D). (E) Binding specificity was confirmed by immunoblotting. (F) Immunostaining of human sections with an anti-ApoE3 antibody confirms that the candidate receptor ApoE3 is expressed in the normal vasculature of several human tissues (arrows). (Scale bar, 100 μm).

Fig. 4.

Discovery of ANXA2/prohibitin as a tissue-specific ligand-receptor targeting normal human tissue. (A) Immunoblotting of His₆-ANXA2 or control proteins with antiserum against CKGGRAKDC or control preimmune serum, as indicated. Arrow: His₆-ANXA2. (B) Binding of CKGGRAKDC-displaying phage is specifically inhibited by the synthetic peptide. Binding of unrelated control phage, insertless phage, binding to BSA and inhibition with an unrelated peptide served as controls. (C and D) Association of prohibitin and ANXA2 with membrane lipid rafts. Membrane proteins extracted from human WAT were subjected to immunoblotting or to fractionation enriching for noncaveolar or caveolar lipid rafts. Proteins recognized by anti-ANXA2 (C), antiprohibitin (D, upper box), and anticaveolin 1 antibodies (D, lower box) are indicated by arrows. (E) Binding of prohibitin and ANXA2 in vitro. Increasing concentrations of GST-prohibitin or GST control were captured with His₆-ANXA2 or control His₆-ANXA5. Specific binding was assessed with anti-GST antibodies. Arrow indicates GST-prohibitin (migrating as several bands). (F and G) Vascular expression of ANXA2 in human WAT. Immunohistochemistry with anti-ANXA2 and antiprohibitin antibodies on human WAT demonstrated colocalization of ANXA2 and prohibitin in the vasculature. Blood vessels identity was confirmed by staining with anti-VE-cadherin antibody (G, inset). Arrows point to blood vessels. Red insets show lower magnification of the corresponding area. (Scale bar, 100 μm).

Fig. 5.

Discovery of RAGE/PR-3 as a ligand-binding targeting human bone marrow containing cancer cells. (A) Anti-CWELGGGPC antibodies recognize human recombinant RAGE. ELISA with post- and preimmune polyclonal antibodies against CWELGGGPC was performed on immobilized CWELGGGPC, a control peptide, recombinant Fc-tagged proteins, and a control protein. (B) Anti-CWELGGGPC antibodies recognize native human RAGE. Protein extracts from human prostate cancer cell lines PC3 and DU145, or from human bone marrow mononuclear control cells, along with recombinant RAGE protein, were immunoblotted with post- and preimmune polyclonal antibodies against CWELGGGPC. Arrow points to RAGE. (C) Validation of RAGE binding to PR-3 in vitro. Either immobilized PR-3 or control protein (BSA) was subjected to RAGE, BMPRIA, BSA, and anti-PR-3 antibody. Bars represent mean ± SEM (D) RAGE binding to active PR-3 is concentration-dependent. (E) Binding of CWELGGGPC-phage is specifically inhibited by the synthetic peptide. Binding of unrelated control phage, insertless phage, binding to BSA and inhibition with an unrelated peptide served as controls. (F) Relative quantification of RAGE expression on prostate cancer patient samples. Expression of RAGE is represented as low, moderate and high expression according to a standardized pathology score. (G–I) Immunohistochemistry with RAGE-specific antibodies performed on human tissue sections derived from a panel of prostate cancer patients. (G) Organ-confined prostate cancer; (H), lymph node metastasis; and (I), bone marrow metastasis. Asterisks represent lymphoid (H) and bone (I) tissues. (Scale bar, 100 μm).