P-selectin is a nanotherapeutic delivery target in the tumor microenvironment

Abstract

Disseminated tumors are poorly accessible to nanoscale drug delivery systems because of the vascular barrier, which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases by arresting nanomedicines at the tumor endothelium. We found that P-selectin is expressed on cancer cells in many human tumors. To develop a targeted drug delivery platform, we used a fucosylated polysaccharide with nanomolar affinity to P-selectin. The nanoparticles targeted the tumor microenvironment to localize chemotherapeutics and a targeted MEK (mitogen-activated protein kinase kinase) inhibitor at tumor sites in both primary and metastatic models, resulting in superior antitumor efficacy. In tumors devoid of P-selectin, we found that ionizing radiation guided the nanoparticles to the disease site by inducing P-selectin expression. Radiation concomitantly produced an abscopal-like phenomenon wherein P-selectin appeared in unirradiated tumor vasculature, suggesting a potential strategy to target disparate drug classes to almost any tumor.

Fig. 1. P-selectin expression in human cancers, preparation of targeted nanoparticles, and binding studies to reconstituted protein

(A) Human cancer tissues stained with P-selectin antibody. Lymphoma stroma: non-Hodgkin’s B cell lymphoma (lymph node); cancer cells: brain metastases of non-Hodgkin’s B cell lymphoma. Ovarian stroma: lymph node metastases of serous papillary adenocarcinoma; cancer cells: endometrioid carcinoma. Breast cancer ECs: infiltrating ductal carcinoma; cancer cells: advanced infiltrating ductal carcinoma. Lung cancer stroma: lung squamous cell carcinoma; cancer cells: small cell undifferentiated carcinoma. (B) Percentage of positively stained samples from tumor microarrays. (C) SELP RNA expression (RNASeq Version 2) in patients from TCGA. A threshold for high expression was set at the highest expression of the lowest expressing cancer. ALL, acute lymphoblastic leukemia; SCC, squamous cell carcinoma. (D) Synthesis schemes of P-selectin–targeted nanoparticles. Top: Preparation of fucoidan-encapsulated paclitaxel and MEK162 nanoparticles (FiPAX and FiMEK) and dextran sulfate–encapsulated controls by nanoprecipitation. Bottom: Preparation of fucoidan-encapsulated doxorubicin nanoparticles (FiDOX) and dextran sulfate–encapsulated control (DexDOX) via layer-by-layer assembly. Right: Scanning electron microscopy (SEM) images of FiPAX and FiDOX nanoparticles. Scale bars, 100 nm. (E) Binding assay of FiPAX to immobilized recombinant proteins. Error bars are ± SD of the mean (n = 4); from left to right, P = 0.0062, 0.0028, 0.0022. *P < 0.05, **P < 0.01. a.u., arbitrary units.

Fig. 2. In vitro studies of nanoparticle penetration of endothelium and tumor

(A) Liver tumor spheroid model, SK-136, which constitutively expresses P-selectin at the surface. Top: Bright-field microscopy and SEM. Bottom: Hematoxylin and eosin (H&E) and IHC staining with a P-selectin antibody. Scale bars, 10 µm. (B) Fluorescence microscopy of NIR dye emission from nanoparticles in the tumor spheroids. Red, NIR fluorescence; blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar, 20 µm. (C) Quantification of nanoparticle emission in tumor spheres. Bars show means ± SD of n = 6 spheres; P = 0.0042. (D) Fluorescence images of human endothelial monolayer (EA.hy926) treated with TNFα and ionizing radiation (6 Gy) to induce P-selectin. Red, NIR dye in FiPAX or control DexPAX nanoparticles; green, CellMask membrane stain; blue, DAPI nuclear stain. Scale bar, 5 µm. (E) Nanopartide-mediated cytotoxicity of bEnd.3 cells activated by TNFα or 6 Gy, as measured by MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. (F) Diagram of assay to test penetration of nanoparticles into an activated endothelial monolayer barrier and infiltration into non–P-selectin–expressing tumor spheroids, LX33, composed of primary human small cell lung cancer (SCLC) cells. (G) Targeted (FiPAX) and control (DexPAX) nanoparticle emission in the upper and lower chambers of a Transwell system. Plots show means ± SD (n = 4).

Fig. 3. Nanoparticle treatment of P-selectin–expressing and nonexpressing tumors in vivo

(A) Top: Tissue section of SCLC PDX stained with an antibody against P-selectin. Scale bar, 50 µm. Bottom: Targeted (FiPAX) and control (DexPAX) nanoparticle fluorescence in the PDX tumors, imaged in vivo 24 and 72 hours after injection. Bars show means ± SD of total fluorescence intensity from tumor region of interest (ROI); n = 4 per group; P = 0.0091. TFE, total fluorescence efficiency. (B) Tumor growth inhibition of PDX model after administration of a single dose of indicated treatments on day 12. Plot shows means ± SD (n = 10 per group). (C) Immunofluorescence measurements of 3LL tumors extracted from mice after radiation treatment. Green, P-selectin; blue, DAPI nuclear stain. Scale bar, 50 µm. (D) Percentage of blood vessels stained positive for P-selectin in the irradiated tissue at 4, 24, and 48 hours (respective P values are 0.058, 0.0041, and 0.0076). Blood vessels were stained with a CD31 antibody. (E) Targeted (FiPAX) and control (DexPAX) nanoparticle fluorescence from the right (irradiated) flank of 3LL-inoculated mice. Plot shows means ± SD; n = 5; P = 0.0096. (F) Tumor growth inhibition after irradiation and single-dose administration of drug treatments. All treatments were given on day 10 after tumor inoculation. The color key lists nanoparticle concentrations in terms of the amount of encapsulated drug. FiPAX (20 mg/kg) + 6 Gy refers to the tumors on the irradiated (right) flanks of the mice, and (FiPAX, 20 mg/kg) + 0 Gy refers to the tumors on the unirradiated (left) flanks of the same irradiated mice. The other groups refer to the tumors on separate animals receiving no treatment (NT), nanoparticles only (FiPAX, 20 mg/kg), or radiation only (6 Gy). P =0.0016; n = 5 mice per group. Plots show means ± SD.

Fig. 4. P-selectin–targeted nanoparticle treatment of metastatic cancer models

(A) Representative images of P-selectin and vasculature (CD31) staining in a B16F10 melanoma experimental lung metastasis model 14 days after inoculation. Scale bar, 50 µm. (B) Representative images of P-selectin and CD31 staining in an MDA-MB-231 breast cancer experimental lung metastasis model 18 days after inoculation. Scale bar, 50 µm. (C and D) Survival data from two experiments using the B16F10 model treated 7 days after tumor inoculation with a single intravenous administration of the indicated treatments. (E) In vivo luciferase bioluminescence of the B16F10 model acquired 7 days after a single administration of the indicated treatments. (F) Median bioluminescence photon count from experiment shown in (D). (G) In vivo bioluminescence images acquired 21 days after a single administration of the indicated treatments to the luciferase-expressing MDA-MB-231 lung metastasis model. (H) Survival data from the MDA-MB-231 model treated on day 7 after inoculation with a single intravenous injection of the indicated treatments. The color key in each figure lists concentrations of nanoparticles and drug-polymer conjugates in terms of the amount of drug. Arrows denote day of administration for all treatments. n = 5 per group for all survival experiments.

Fig. 5. P-selectin–targeted delivery of an inhibitor of the MEK/ERK pathway

(A) Top: Proliferation of cell lines measured after 4 days of treatment with MEK162 or FiMEK as indicated. Bottom: Biochemical analysis of A375 and A549 cell lines treated for 4 hours with MEK162 or FiMEK. (B) Growth of tumor xenografts after a single dose of vehicle, MEK162, and FiMEK or a daily dose of MEK162. x axis represents days after first treatment; n = 6 per group. P^(A375) = 0.0048, P^{(SW520,FiMEK)} = 0.0071, P^(SW520,MEK) = 0.0055. (C) Biochemical quantification (Western blot) of pERK and PARP cleavage in xenografts of A375 tumors treated for 2 or 16 hours with MEK162 or FiMEK. P = 0.0089 and 0.0053, respectively. (D) IHC of cleaved caspase 3 in HCT116 xenografts treated with MEK162 or FiMEK. Scale bar, 50 µm. (E) IHC of pERK in skin and tumors of HCT116 xenografts treated with MEK162 (30 and 300 mg/kg) or FiMEK (30 mg/kg) after 24 hours. Scale bar, 100 µm. Nanoparticle concentrations are listed in terms of the equivalent amount of encapsulated drug. Plots show means ± SD.