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Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors.

Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, Li Y, Kong L, Kouzani AZ, Lin J, Liu K, Li L, Shigdar S, Duan W - Theranostics (2015)

Bottom Line: Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy.To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications.We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3216, Australia.

ABSTRACT
Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.

No MeSH data available.


Related in: MedlinePlus

Time-dependent penetration of PEGylated aptamer and antibody in relation to blood vessels in HT29 xenograft tumors. (a) Representative images of double staining of aptamer or antibody and blood vessels in tumor sections dissected from treated mice-bearing HT29 xenografts 3 h and 24 h after i.v. administration of aptamer or antibody at a dose of 2 nmol/mouse. Blood vessels were stained by chromogenic alkaline phosphatase (black arrow); while aptamer or antibody were stained using DAB peroxidase substrate (brown). Scale bar: 200 μm. (b-c) Quantitative determination of staining intensity against a given perpendicular distance (20-200 μm) to the blood vessels at 3 h. (b) and 24 h (c) after i.v. injection of aptamer or antibody. Data are means ± SEM (n=8).
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Figure 6: Time-dependent penetration of PEGylated aptamer and antibody in relation to blood vessels in HT29 xenograft tumors. (a) Representative images of double staining of aptamer or antibody and blood vessels in tumor sections dissected from treated mice-bearing HT29 xenografts 3 h and 24 h after i.v. administration of aptamer or antibody at a dose of 2 nmol/mouse. Blood vessels were stained by chromogenic alkaline phosphatase (black arrow); while aptamer or antibody were stained using DAB peroxidase substrate (brown). Scale bar: 200 μm. (b-c) Quantitative determination of staining intensity against a given perpendicular distance (20-200 μm) to the blood vessels at 3 h. (b) and 24 h (c) after i.v. injection of aptamer or antibody. Data are means ± SEM (n=8).

Mentions: The efficacy of drug transport inside tumors was highly restricted because of limited penetration, due to the abnormality and complication of tumor microenvironment, including poorly organized vascular system, increased interstitial fluid pressure as well as the presence of extracellular matrixes. Thus, the next generation of anticancer therapeutics must penetrate into tumor tissues efficiently and gain access to all cancer cells at a sufficient concentration to eradicate solid tumors 2, 5, 15, 47. To further determine the penetration ability of aptamer in comparison with antibody in vivo, NOD/SCID mice bearing xenograft colorectal tumous with a tumor volume of 150 mm3 received a bolus i.v. injection of 2 nmol mouse of PEGylated-aptamer or antibody. At 3 h or 24 h post injection, the mice were sacrificed and the formalin fixed paraffin embedded tumor sections were prepared for double staining of blood vessels and aptamer or antibody. To evaluate tumor penetration in vivo, the FITC-labeled aptamer or antibody were detected with an anti-FITC antibody and visualized with the aid of chromogenic substrates 3.3' diaminobenzidine (DAB); while the blood vessels were marked by anti-mouse CD31 antibody and visualized using chromogenic alkaline phosphatase solution. The distribution of aptamer or antibody in tumor sections was assessed by quantification of pixel intensity of aptamer or antibody in relation to distances from the blood vessels within the selected region of interest using Image Pro software. Shown in Fig. 6a are representative images of time-dependent distribution of PEGylated aptamers or antibodies (brown) in relation to blood vessels (red, marked by black arrows) in HT29 xenografts sections. These images show that the majority of brown staining for antibodies was localized near blood vessels, whereas regions away from vessels of the tumors show ample brown staining for aptamers. The quantification of intensity of aptamer or antibody in relation to distances from blood vessels at 3 h after intravenous injection revealed that there were significant differences in the intensity of signals between aptamers and antibodies throughout the entire 200 μm distances from the blood vessels (Fig. 6b). In fact, aptamer achieved at least 4-time better tumor penetration than that of the antibody throughout the tumor spaces studied after 3 h i.v. injection. Importantly, 24 h after administration, intratumoral antibody was hardly discernable even in the close range (20 µm) of the blood vessels. In contrast, the aptamer remained detectable even at regions as far as 200 µm away from the blood vessels (Fig. 6c). These data indicate that aptamers, even after PEGylation, can penetrate tumors much more extensively and efficiently than antibodies. Therefore, there is a great potential for aptamers to become effective modalities for targeted tumor therapeutics and imaging.


Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors.

Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, Li Y, Kong L, Kouzani AZ, Lin J, Liu K, Li L, Shigdar S, Duan W - Theranostics (2015)

Time-dependent penetration of PEGylated aptamer and antibody in relation to blood vessels in HT29 xenograft tumors. (a) Representative images of double staining of aptamer or antibody and blood vessels in tumor sections dissected from treated mice-bearing HT29 xenografts 3 h and 24 h after i.v. administration of aptamer or antibody at a dose of 2 nmol/mouse. Blood vessels were stained by chromogenic alkaline phosphatase (black arrow); while aptamer or antibody were stained using DAB peroxidase substrate (brown). Scale bar: 200 μm. (b-c) Quantitative determination of staining intensity against a given perpendicular distance (20-200 μm) to the blood vessels at 3 h. (b) and 24 h (c) after i.v. injection of aptamer or antibody. Data are means ± SEM (n=8).
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Related In: Results  -  Collection

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Figure 6: Time-dependent penetration of PEGylated aptamer and antibody in relation to blood vessels in HT29 xenograft tumors. (a) Representative images of double staining of aptamer or antibody and blood vessels in tumor sections dissected from treated mice-bearing HT29 xenografts 3 h and 24 h after i.v. administration of aptamer or antibody at a dose of 2 nmol/mouse. Blood vessels were stained by chromogenic alkaline phosphatase (black arrow); while aptamer or antibody were stained using DAB peroxidase substrate (brown). Scale bar: 200 μm. (b-c) Quantitative determination of staining intensity against a given perpendicular distance (20-200 μm) to the blood vessels at 3 h. (b) and 24 h (c) after i.v. injection of aptamer or antibody. Data are means ± SEM (n=8).
Mentions: The efficacy of drug transport inside tumors was highly restricted because of limited penetration, due to the abnormality and complication of tumor microenvironment, including poorly organized vascular system, increased interstitial fluid pressure as well as the presence of extracellular matrixes. Thus, the next generation of anticancer therapeutics must penetrate into tumor tissues efficiently and gain access to all cancer cells at a sufficient concentration to eradicate solid tumors 2, 5, 15, 47. To further determine the penetration ability of aptamer in comparison with antibody in vivo, NOD/SCID mice bearing xenograft colorectal tumous with a tumor volume of 150 mm3 received a bolus i.v. injection of 2 nmol mouse of PEGylated-aptamer or antibody. At 3 h or 24 h post injection, the mice were sacrificed and the formalin fixed paraffin embedded tumor sections were prepared for double staining of blood vessels and aptamer or antibody. To evaluate tumor penetration in vivo, the FITC-labeled aptamer or antibody were detected with an anti-FITC antibody and visualized with the aid of chromogenic substrates 3.3' diaminobenzidine (DAB); while the blood vessels were marked by anti-mouse CD31 antibody and visualized using chromogenic alkaline phosphatase solution. The distribution of aptamer or antibody in tumor sections was assessed by quantification of pixel intensity of aptamer or antibody in relation to distances from the blood vessels within the selected region of interest using Image Pro software. Shown in Fig. 6a are representative images of time-dependent distribution of PEGylated aptamers or antibodies (brown) in relation to blood vessels (red, marked by black arrows) in HT29 xenografts sections. These images show that the majority of brown staining for antibodies was localized near blood vessels, whereas regions away from vessels of the tumors show ample brown staining for aptamers. The quantification of intensity of aptamer or antibody in relation to distances from blood vessels at 3 h after intravenous injection revealed that there were significant differences in the intensity of signals between aptamers and antibodies throughout the entire 200 μm distances from the blood vessels (Fig. 6b). In fact, aptamer achieved at least 4-time better tumor penetration than that of the antibody throughout the tumor spaces studied after 3 h i.v. injection. Importantly, 24 h after administration, intratumoral antibody was hardly discernable even in the close range (20 µm) of the blood vessels. In contrast, the aptamer remained detectable even at regions as far as 200 µm away from the blood vessels (Fig. 6c). These data indicate that aptamers, even after PEGylation, can penetrate tumors much more extensively and efficiently than antibodies. Therefore, there is a great potential for aptamers to become effective modalities for targeted tumor therapeutics and imaging.

Bottom Line: Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy.To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications.We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3216, Australia.

ABSTRACT
Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.

No MeSH data available.


Related in: MedlinePlus