Peptides selected for binding to clotted plasma accumulate in tumor stroma and wounds

Peptides Selected from Plasma Clot Bind to Tumor Stroma.

The phage selection scheme was designed to amplify phage that bind to plasma clots but not to anticoagulated plasma. Three rounds of selection produced a pool with 7-fold increased binding to plasma clots. Additional selection rounds did not increase the binding. Sequencing showed that the phage encoding the peptide insert CGLIIQKNEC (CLT1 peptide) and CNAGESSKNC (CLT2 peptide) were highly enriched among the clot-binding phage. Three of 24 clones sequenced encoded CLT1, and two encoded CLT2; the rest of the clones were only represented once. The CLT phage clones bound to plasma clots with 3- to 4-fold efficiency compared to control phage. They did not significantly bind to clots made out of purified fibrin, or fibronectin immobilized on plastic (results not shown).

To determine whether the CLT peptides would recognize clotted plasma proteins in vivo, we synthesized the peptides as fluorescein conjugates and imaged their accumulation in tumors. Mice bearing orthotopic MDA-MB-435 xenograft tumors were intravenously injected with fluorescein-conjugated CLT1 (n = 6), CLT2 (n = 4), or a control peptide (n = 4) with the sequence KAREC. Examination of whole tissues from these mice under blue light 3 h after the peptide injection revealed intense fluorescence in the tumors of the mice injected with CLT1 and CLT2, whereas essentially no fluorescence was detected in tumors from the control peptide-injected mice (Fig. 1). We also detected no fluorescence in healthy organs of the CLT-injected animals.

Upon histological examination, CLT peptide fluorescence in tumors appeared in a network pattern within the tumors (Fig. 1 C and G). Tumor homing of both peptides was strongly reduced after coinjection of fluorescein-conjugated CLT1 or CLT2 in combination with unlabeled CLT2 in 5 fold excess (Fig. 1 D and H). This finding indicates that the CLT1 peptide recognizes the same binding site within the tumor as CLT2. Quantification of these results showed that the intensity of fluorescence in the MDA-MB-435 tumors of mice injected with the CLT peptides was 60–130 times stronger than in mice injected with the KAREC control peptide (Fig. 1M). Also shown in Fig. 1M is that the CLT peptides specifically accumulated in all of the various tumors we tested (n = 2–8). A fibrillar pattern similar to the one shown in Fig. 1 C and G was seen in each of these other tumors (data not shown). To study the ability of the CLT peptides to recognize human clinical cancers, we developed a peptide overlay assay. Overlay with the CLT1 peptide produced a fibrillar network in mouse Lewis lung carcinoma similar to that seen in the tumors after i.v. injection of the peptide (Fig. 2A). The binding of the peptide was also inhibited by unlabeled CLT1 peptide (Fig. 2B) but not by a control peptide (Fig. 2C), showing the specificity of the binding. Sections from two clinical breast cancers (Fig. 2 D and E) and from PPC1 xenografts were positive in this overlay assay (Fig. 2F).

CLT Peptides Associate with Fibrin and Fibronectin in Tumor Stroma.

Staining of tumor sections with antibodies against fibrin(ogen) produces a fibrillar staining that is not seen in normal tissues (8). Fluorescence from intravenously injected CLT peptide (CLT1; Fig. 3A) and fibrin staining (Fig. 3B) colocalized (Fig. 3C) in tumor sections of MDA-MB-435 breast cancer xenografts. Plasma fibronectin and fibrin are deposited together into plasma clots by Factor XIII activity (13), prompting us to analyze the relationship of CLT peptide fluorescence and fibronectin staining in tumors. The results showed that CLT peptides also codistribute with fibronectin (not shown). We also studied the distribution of the CLT peptides, fibrin, and fibronectin in MMTV-PyMT transgenic breast tumors. The results (data not shown) were identical to those obtained with the MDA-MB-435 xenografts.

CLT Peptide Tumor Homing Requires Fibrin and Plasma Fibronectin.

We next used knockout mice to study the role of fibrin(ogen) and fibronectin in the homing of the CLT peptides to tumors. Fibrinogen knockout mice are viable (14) and could be used as tumor recipients. Complete absence of fibronectin is lethal, but mice that lack plasma fibronectin can be generated by postnatally deleting the fibronectin gene in the liver (15). The CLT peptides homed to B16F1 tumors grown in wild-type C57BL/6 mice (n = 10) producing a fibrillar meshwork (CLT1; Fig. 3D). In contrast, only faint, evenly distributed fluorescence was present in tumors (n = 4) grown in the fibrinogen knockout mice (which lack the ability to produce fibrin) (Fig. 3E). B16F1 tumors grown in wild-type littermates of plasma fibronectin-deficient mice also accumulated CLT peptides in a fibrillar matrix (Fig. 3F), but mice lacking plasma fibronectin (n = 6) did not (Fig. 3G). Unlike in the fibrinogen knockout mice, there was no residual peptide binding to the tumors in the plasma fibronectin-deficient mice (Fig. 3H). These results show that both fibrin and fibronectin from plasma are needed for the CLT peptides to highlight a fibrillar matrix in tumors, but that some diffuse binding of the peptide persists in the absence of fibrin.

CLT Binds Sites of Tissue Injury in Vivo.

Clotting is an important part of wound healing. Therefore, we tested CLT peptide homing to tissue injuries. Fluorescein-conjugated CLT peptides homed to deendothelialized femoral arteries (n = 5), producing strong fluorescence in the vessel wall (Fig. 4A). CLT homing was also seen in crush injuries of the muscle (n = 3; Fig. 4D) and in skin wounds resulting from incisions (n = 4; Fig. 4E). No CLT peptide homing was observed in intact arteries, muscles, skin (Fig. 4 B, F, and G), or other healthy tissues of the mice with the injuries, and a control peptide showed no homing to the injured tissues (data not shown).

Animals, Cell Lines, and Tissues.

B16F1 mouse melanoma, Lewis lung carcinoma, C8161 human melanoma, MDA-MB-435 human breast cancer, and PPC-1 human prostate cancer cells (American Type Culture Collection) were maintained in RPMI medium 1640 or DMEM supplemented with 10% FCS. Human tumor cells (1 × 10⁶) were injected into the mammary fat pad or the flank of nude BALB/c nu/nu mice to induce tumors. Mouse mammary tumor virus (MMTV) PyMT mice were provided by Robert Oshima (Burnham Institute for Medical Research). The MMTV PyMT mice develop breast cancer under the influence of a polyoma middle T antigen driven by the mouse tumor virus promoter (21). B16F1 tumors induced by injecting 1 × 10⁶ cells s.c. were grown in the flank of fibrinogen knockout mice (22), and of transgenic plasma fibronectin-deficient C57BL/6-Fn(fl/fl) Mx-Cre⁺ (15) and their wild-type littermates. Deletion of the fibronectin gene in the liver was induced by poly(I):poly(C) (15, 23). Frozen human tissues from clinical breast cancers were provided by the Western Division of the National Cancer Institute Cooperative Human Tissue Network (Vanderbilt University Medical Center, Nashville, TN).

Phage Library.

A cyclic peptide library with the general structure of CX8C (C = cysteine; X = any amino acid) was designed to express 5–15 peptide copies on a T7 10–3b phage vector (T7 Select kit; Novagen). CX8C-encoding oligonucleotides were synthesized with flanking EcoRI/HindIII adapters (n = any nucleotide, K = guanine or thymine, M = adenine or cytosine): 5′-AATTCCTGCNNKNNKNNKNNKNNKNNKNNKNNKTGCTA-3′ and 3′-GGACGNNMNNMNNMNNMNNMNNMNNMNNMACGATTCGA-5′.

The annealed oligonucleotide pairs were ligated to EcoRI/HindIII-digested phage vector according to the T7 Select protocol. The diversity of the peptide library was 5 × 10⁸ primary recombinants.

Clot Formation.

Blood was anticoagulated with 0.4% sodium citrate and centrifuged at 2,500 × g. The plasma was collected, spun again to remove remaining blood cells, and frozen at −80°C. Clotting was initiated by adding CaCl₂ to 20 mM, and the clot was repeatedly washed with PBS.

Phage Screening.

Human plasma clots were incubated with the CX8C peptide library at 22°C for 30 min and extensively washed with PBS. Plasma was then added to the clot to remove phage recognizing soluble plasma components. The phage pool that remained bound to the clot was quantified and amplified. The process was repeated until maximum clot binding was reached. The peptide-encoding DNA from 24 randomly picked phage clones in the selected pool was isolated and sequenced.

Peptide Synthesis.

Peptides were synthesized and conjugated to fluorescein isothiocyanate as described (24) and cyclized by exposure to air.

Imaging, Antibodies, and Immunohistology.

Imaging under a blue light and examination of the distribution of fluorescein-conjugated peptides after tail vein injection have been described (24, 25).

The imaging methods are briefly described as follows. A Leica fluorescence stereomicroscope, model LZ12, equipped with a 50-W mercury lamp, was used for high-magnification imaging. Selective excitation of GFP was produced through a D425y60 band-pass filter and 470 DCXR dichroic mirror. Emitted fluorescence was collected through a long-pass filter GG475 (Chroma Technology, Brattleboro, VT) on a Hamamatsu C5810 three-chip cooled color charge-coupled device camera (Hamamatsu Photonics, Bridgewater, NJ). Images were processed for contrast and brightness and analyzed with the use of image pro plus 3.1 software (Media Cybernetics, Silver Springs, MD). Images of 1,024 × 724 pixels were captured directly on an IBM PC or continuously through video output on a high-resolution Sony VCR model SLV-R1000 (Sony). Imaging at lower magnification that visualized the entire animal was carried out in a light box illuminated by blue light fiber optics (Lightools Research, Encinitas, CA) and imaged by using the thermoelectrically cooled color charge-coupled device camera as described above (26).

The mice were perfused through the heart 3 h after the peptide injection, and tissues were removed for examination. For fibrin and fibronectin immunostaining, tissues were fixed with 4% paraformaldehyde and stained with biotinylated mouse fibrin(ogen) antiserum (Nordic) or a polyclonal anti-mouse fibronectin antibody (Chemicon), followed by streptavidin Alexa Fluor 594 or anti-rabbit Alexa Fluor 594 (Molecular Probes), respectively. For peptide overlay, sections of frozen OCT-embedded tissue were incubated with 10 μg/ml fluorescein-conjugated peptide for 30 min at room temperature. Tissue fluorescence was quantified by using image pro plus software. At least two images from representative microscopic fields were analyzed from each tissue sample, and data from individual mice that received the same treatment (n = 2–8) were pooled.

Wound Assays.

The wire injury of the mouse femoral artery was induced as described (27). Thirty minutes after the wire injury, 250 μg of fluorescein-conjugated peptides were injected i.v. Muscle injuries were induced in mice by applying a crush injury to the quadriceps muscle, and 500 μg of fluorescein-conjugated peptides was injected i.v. 48 h later. Skin incisions were studied similarly. The mice were perfused through the heart 4 h after the peptide injection, and tissues were excised, fixed in 4% paraformaldehyde, and embedded in OCT.