In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer - PubMed (original) (raw)
. 2009 Jun;1(5-6):382-93.
doi: 10.1039/b904890a. Epub 2009 May 11.
Affiliations
- PMID: 20023745
- PMCID: PMC2796841
- DOI: 10.1039/b904890a
In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer
Emilia S Olson et al. Integr Biol (Camb). 2009 Jun.
Abstract
Activatable cell penetrating peptides (ACPPs) are novel in vivo targeting agents comprised of a polycationic cell penetrating peptide (CPP) connected via a cleavable linker to a neutralizing polyanion (). Adsorption and uptake into cells are inhibited until the linker is proteolyzed. An ACPP cleavable by matrix metalloproteinase-2 (MMP-2) in vitro was the first one demonstrated to work in a tumor model in vivo, but only HT-1080 xenografts and resected human squamous cell carcinomas were tested. Generality to other cancer types, in vivo selectivity of ACPPs for MMPs, and spatial resolution require further characterization. We now show that ACPPs can target many xenograft tumor models from different cancer sites, as well as a thoroughly studied transgenic model of spontaneous breast cancer (mouse mammary tumor virus promoter driving polyoma middle T antigen, MMTV-PyMT). Pharmacological inhibitors and genetic knockouts indicate that current ACPPs are selective for MMP-2 and MMP-9 in the above in vivo models. In accord with the known local distribution of MMP activity, accumulation is strongest at the tumor-stromal interface in primary tumors and associated metastases, indicating better spatial resolution (<50 mum) than other currently available MMP-cleavable probes. We also find that background uptake of ACPPs into normal tissues such as cartilage can be decreased by appending inert macromolecules of 30-50 KDa to the polyanionic inhibitory domain. Our results validate an approach that should generally deliver imaging agents and chemotherapeutics to sites of invasion, tumor-promoting inflammation, and metastasis.
Figures
Figure 1. Uptake of free ACPP’s by mice harboring HT-1080 xenografts is protease dependent
(A) Images of nude mice bearing HT-1080 xenografts six hours after injection with either cleavable ACPP or an all-D-amino acid control. Skin has been removed to expose the tumor (red arrows). (B) Shows a washout curve generated from taking regions over the tumor (red or blue) and thorax (pink or light blue, dashed lines) of animals receiving 10 nmol of either cleavable suc-e8-xPLGLAG-r9-c(Cy5) (n=3) (red diamonds or pink squares) or suc-e8-xplglag-r9-k(Cy5) (n=3) (blue triangles or light blue squares) respectively. Error bars represent standard deviations. (C) Tumor uptake of peptide in animals decreased when pre-injected with pharmacological inhibitors of MMPs (SB3CT or prinomastat). Uptake values for the r9-X-k(cy5) CPP probe as well as the all D-amino acid and 2 unit PEG linker control peptides are shown for reference. r9-x-k(Cy5) n= 3, PLGLAG n= 9, SB3CT n= 3, Prinomastat n= 3, plglag n= 8, mPeg n= 3. Error bars represent standard deviation. ** represents statistically significant difference from the cleavable ACPP using a two tailed t-test. (D) Polyacrylamide gel electrophoresis of tumor homogenates differentiates cleaved versus intact peptides recovered from tumors of IV injected mice.
Figure 2. Uptake of MMP cleavable ACPP’s is significantly reduced in PyMT mice lacking the genes for MMP-2 and MMP-9
(A) Images of PyMT animals of the indicated genotype six hours after intravenous injection of the CPP r9-c(Cy5), the cleavable ACPP (suc-e8-xPLGLAG-r9-c(Cy5)), or the D-amino acid negative control (suc-e8-xplglag-r9-k(Cy5)) with the skin removed. (B) A chart showing tumor SUV’s taken six hours after injection with peptide showing a significant reduction of ACPP uptake in tumors from animals lacking the genes for MMP-2 and MMP-9. r9-c(Cy5) n=3, PLGLAG n=5, MMP-2−/− n=3, MMP-2−/− 9+/−n=3, MMP-2−/− 9−/− n=3, plglag n=5, (PEG2)2 n=3. Error bars represent standard deviations. ** denotes p<0.05 when compared to cleavable PLGLAG ACPP peptide uptake.
Figure 3. Uptake of MMP cleavable ACPP’s follows the distribution of MMP’s in adenomatous tumors and once cleaved, are delivered to the endosomes of stroma and to a lesser extent tumor cells
(A) Compares the spatial distribution of fluorescence for cleavable (suc-e8-xPLGLAG-r9-c(Cy5)) or control (suc-e8-xplglag-r9-k(Cy5)) peptide in frozen tissue slices six hours after injection of peptide to that of DQ gelatin done on the same slices post mortem. H/E stains are shown for adjacent tissue slices for identification purposes. (B) Shows confocal images of tissue from PyMT+GFP+/+ animals six hours after IV injection with cleavable (suc-e8-xPLGLAG-r9-c(Cy5)), cleavage resistant D-amino acid control or CPP (r9-c(Cy5) control peptide. Cy5 fluorescence is shown in red. Tissues were harvested and imaged five minutes post injection with rhodamine-dextran which labels tumor vasculature shown in blue, demonstrating ACPP accumulation outside of the vascular space in stroma and tumor cells. GFP-expressing tumor epithelial cells are shown in green as overlay or are absent to better visualize peptide and vascular distribution (below). Orange arrows point to areas of more obvious DCIS and red arrows point to areas of likely early invasion. (C) Enlarged window of cleavable ACPP demonstrated more clearly that the uptake into the stroma (green arrows) and tumor cell (white arrows) is reminiscent of endosomal puncta.
Figure 4. ACPP’s accumulate in spontaneous lung metastases in PyMT mice
(A) Gross images of dissected lung lobes from animals six hours after injection with either the cleavable (Suc-e8-xPLGLAG-r9-c(Cy5)) or cleavage resistant control (Suc-e8-xplglag-r9-k(Cy5)). Lungs were then frozen sectioned, imaged by fluorescence histology to visualize the microdistribution of the peptide (lower left), and serial sections were stained with H/E (lower right) to demonstrated presence of metastases. (B) Contrast of metastases as small as 100 µm (white arrow) were detected in tissue sections. (C) DQ gelatin in situ zymography was performed on the same frozen section of cleavable ACPP (imaged by Cy5 fluorescence) metastasis showing substantial colocalization of peptide uptake to enzyme activity in the metastases.
Figure 5. Free ACPP’s accumulate in cartilage, liver and kidney as well as tumor
(A) and (B) show animals six hours after injection with either the cleavable (Suc-e8-xPLGLAG-r9-c(Cy5)) (A) or control (Suc-e8-xplglag-r9-k(Cy5)) (B) peptides. Skin and pectoralis muscle have been removed, exposing the ribs and thoracic cage. Significant uptake is localized to cartilage which is specific to cleavable peptide (blue arrows). (C) and (D) show standardized uptake values for liver (C) and kidney (D) for animals injected with the r9-x-k(Cy5), the cleavable peptide at 10 and 3 nmol doses, the D-amino acid control, the (PEG2)2 control, and a PAMAM conjugated ACPP. These data show there is high uptake in liver and kidney with free ACPP peptides and that kidney uptake decreases significantly as a result of adding macromolecular weight carriers. r9-x-k(Cy5) n=3, xPLGLAG 10 nmol n=9, xPLGLAG 3 nmol n=3, (PEG2)2 n=3, PAMAM + ACPP n=2. Error bars are standard deviations.
Figure 6. Addition of a large molecular carrier to the polyglutamate results in greater uptake in tumor
(A) and (B) shows fluorescent images of live mice six and 48 hours (time of maximal contrast) after injection with cleavable peptide (suc-e8-xPLGLAG-r9-c(Cy5)) (A) and cleavable peptide attached to a generation 5 PAMAM dendrimer (PAMAM-e9-xPLGLAG-r9-k(Cy5))(B) respectively. (C) A time course of fluorescence contrast showing the difference in washout from the tumor and tissues for the two constructs. Solid lines represent tumor and dashed lines thoracic background for the free peptide (purple) and carrier attached construct (red). Data is representative of n=2 mice each and error bars are standard deviations. (D) and (E) show animals 48 hours after injection with cleavable and cleavage resistant peptides attached covalently to albumin (D) and dextran (E) respectively (albumin or dextran-e9-xPLGLAG-r9-k(Cy5)). (F) shows SUV of tumors for the cleavable and D-amino acid cleavage resistant constructs shown in (D) and (E) (n=2 each).
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