Selective targeting of liver cancer with the endothelial marker CD146

Abstract

Hepatocellular carcinomas are well-vascularized tumors; the endothelial cells in these tumors have a specific phenotype. Our aim was to develop a new approach for tumor-specific drug delivery with monoclonal antibody targeting of endothelial ligands. CD146, a molecule expressed on the endothelial surface of hepatocellular carcinoma, was identified as a promising candidate for targeting. In the present study, endothelial cells immediately captured circulating anti-CD146 (ME-9F1) antibody, while antibody binding in tumors was significantly higher than in hepatic endothelium. Macroscopically, after intravenous injection, there were no differences in the mean accumulation of anti-CD146 antibody in tumor compared to liver tissue , due to a compensating higher blood vessel density in the liver tissue. Additional blockade of nontumoral epitopes and intra-arterial administration, improved selective antibody capture in the tumor microvasculature and largely prevented antibody distribution in the lung and liver. The potential practical use of this approach was demonstrated by imaging of radionuclide-labeled ME-9F1 antibody, which showed excellent tumor-selective uptake. Our results provide a promising principle for the use of endothelial markers for intratumoral drug delivery. Tumor endothelium-based access might offer new opportunities for the imaging and therapy of hepatocellular carcinoma and other liver malignancies.

Figure 1. Expression of CD146 on endothelium in murine and human hepatocellular carcinoma

(A, B) Representative images of immunofluorescence labeling with Alexa Fluor 488–conjugated anti-Lyve-1 (green) and PE-conjugated anti-CD146 antibodies (red). LSCM of histological slides (A) and whole-mount tissue after intravenous injection (B). Lyve-1 was strongly expressed by normal sinusoidal endothelial cells. High levels of CD146 staining were found in all tumor blood vessels, in microvessels of the periportal area, and in zone 1 of the acinus in the liver. T, tumor; L, liver. (C) Image-based analysis of CD146 staining on histological slides. We observed a high mean fluorescence intensity of CD146 on tumor blood vessels. (D–F) Comparison of CD146 expression in isolated HECs and TECs. mRNA levels (D), representative fluorescence staining with Alexa Fluor 488–ME-9F1 mAb (E), and ELISA of cell lysates (F). CD146 expression was significantly higher in TECs compared to HECs (P<0.05). (G, H) Expression of CD146 on endothelium in human tissue; immunohistochemical staining of CD146. (G): Representative images of snap-frozen tissue, L, liver tissue, C, connective tissue. (H): Sample distribution according to expression intensity, formalin-fixed samples of 41 tumors and 3 livers were included into the analysis. CD146 was overexpressed in the majority of human HCC samples.

Figure 2. Immediate binding of ME-9F1 mAb to endothelial cells

(A) Image-based immunofluorescence analysis of mAb binding to histological slides after 5 s incubation. ME-9F1 showed immediate concentration-dependent binding to tumor endothelial cells, which was significantly higher than binding to hepatic endothelial cells (P<0.05). *Indicates significant differences between tumor and liver tissue. (B) Images of PE-conjugated ME-9F1 mAb binding to tumor endothelial cells in vivo; laser scanning confocal microscopy. Intravenous injection of mAb resulted in excellent visualization of the tumor vascular system in different mouse tumor models. (C–E) Capture of ME-9F1 mAb in tumor and lung tissue after intravenous (i.v.) and intra-arterial (i.a.) injection. mAb binding in tumor (C) and tumor:liver ratio (E) after intra-arterial application was significantly higher than after intravenous injection. Higher mAb binding in the lung was found after intravenous injection (D). (F–G) Blood vessel density (F) and representative images of blood vessel staining in HCC and liver tissue using anti-CD146 or anti-CD105 mAb (G). Tumor blood vessel density in the liver was significantly higher than in HCC from AlbTag mice (P<0.05).

Figure 3. Selective access to tumor vasculature using PE-conjugated ME-9F1 mAb

(A) Representative LSCM images of selective arterial blood supply to HCC. Alexa Fluor 488 (green)- or PE (red)-labeled ME-9F1 mAb were injected during alternate clamping of the hepatic artery or portal vein. Blood vessels in the liver, as well as in small tumors, were labeled with both Alexa Fluor 488 and PE (mixed arterial and portal blood supply). Microvessels in larger tumors were labeled mainly with PE-conjugated mAb (selective arterial blood supply). (B–D) Selective enrichment of labeled ME-9F1 mAb using bioavailability blockade of nontumoral epitopes or/and intra-arterial injection. Diagrams show mAb content in the tumor, liver (C), and lung (E) and the mAb tumor:liver ratio (D). Bioavailability blockade of nontumoral epitopes by unconjugated mAb and intra-arterial injection of 10 ng/g BW mAb improved selective accumulation of labeled mAb in tumor tissue and strongly reduced mAb load in the lung. *Indicates significant differences between tumor and liver tissue. (E) LSCM images of different organs without and with blockade of nontumoral epitopes. Blockade of nontumoral epitopes resulted in visualization of solid tumors (>5mm) through selective labeling of tumor vasculature, whereas the fluorescence signal in the liver and other organs was strongly inhibited after blockade.

Figure 4. Intraarterial hepatic perfusion and static planar imaging of the whole body (top panels) and tissue pieces (bottom panels)

(A-B) The combination of a reduction in the nontumoral epitope bioavailability and administration of PE-conjugated ME-9F1 into the hepatic artery led to preferential accumulation of labeled mAb in tumor vessels and resulted in the high tumor:liver ratio. *p<0.05. (C) Combination of bioavailability blockade of nontumoral epitopes and intra-arterial perfusion with I¹²⁵-conjugated ME-9F1 mAb in a tumor-bearing mouse. Multiple small HCCs and one large tumor were macroscopically identified in the liver. Tumor tissue produced a strong signal, whereas a very weak signal was detected in the liver. T, tumor; L, liver. (D) Combination of nontumoral epitope blockade and intra-arterial perfusion with I¹²⁵-conjugated ME-9F1 mAb in a tumor-free mouse. Weak signal in the liver was detected. (E) Intraportal perfusion with I¹²⁵-conjugated ME-9F1 mAb without blockade in a tumor-free mouse. Strong signal in the liver tissue was detected.