Cy5-labeled ACPPD cause localized uptake in tumors lasting at least 48 h. (A) ACPPD schematic. Each ACPPD consists of a dendrimer (gray circle) covalently attached to the polycationic segments (blue) of several ACPPs (typically about six). In tumors, proteases such as MMP-2 and -9 cleave the linkers (green), releasing polyanions (red) and leaving a highly cationic molecule to stick to and enter cells. Yellow ovals indicate payloads, which can be Cy5 (ACPPD-Cy5) or Gd-DOTA (ACPPD-Gd) or both (dual ACPPD). (B–D) Representative fluorescence images (skin removed) 48 h after injection with ACPPD-Cy5 or ACPP-Cy5, each containing 10 nmol of Cy5. Yellow arrows point to tumors. D has been brightened fivefold relative to B and C to make the dim signal from free ACPP-Cy5 visible. For corresponding images before skin removal, see Fig. S2. (E) Time courses of tumor fluorescence, viewed through intact skin, from animals injected with cleavable (3 nmol Cy5, n = 2, red diamonds) and all-D-amino acid (3 nmol Cy5, n = 2, blue squares) ACPPD-Cy5 and an animal injected with cleavable ACPP-Cy5 (10 nmol Cy5, gray diamonds). (F) Standardized uptake values in solubilized samples of tumor, liver, kidney, and muscle, 48 h after ACPPD-Cy5 injection (n = 8 for cleavable, n = 5 for D-amino acid control) and 6 h after ACPP-Cy5 injection (n = 5). Pairwise P values are shown for each organ type.

ACPPD-Cy5 gives equal or better tumor to background contrast than existing dequenching probes. (A) Fluorescence images through intact skin of MMTV-PyMT transgenic and HT-1080 xenografted mice 48 h after injection with 1 nmol cleavable ACPPD-Cy5 or 24 h after injection with 2 nmol ProSense or MMPSense. Yellow arrows point to tumor and red arrows indicate liver. Dim images have been brightened by the factors indicated, to enable comparisons. (B) Tumor to background ratios for ACPPD-Cy5 and ProSense in the MMTV-PyMT model. Black bars show ratios with skin attached, and gray bars show ratios after skin removal. P values for MMPSense and ProSense relative to ACPPD-Cy5s are shown.

Regions of especially bright Gd and Cy5 labeled dual-ACPPD uptake on MRI correspond to regions of active MMP’s, infiltrative tumor and associated inflammation on histology. (A) Axial MR and (B) fluorescence (skin off) images of a transgenic PyMT mouse with high tumor burden (white arrows) (C) Axial MR and (D) fluorescence images of the same mouse after surgical removal of tumor under white light. Red arrows point to regions of residual hyperintensity on MR and fluorescence imaging. Gamma has been adjusted for image clarity. (E) Regions of hyperintensity on MRI were resected using fluorescence imaging and stained with hematoxylin/eosin to verify the presence of tumor. (F-H) Residual tumor collected under similar conditions from a nude mouse bearing an HT-1080 xenograft (F). The red arrow points to an area of infiltrative tumor that was largely missed at the time of surgery. The residual tissue was removed and analyzed further. (G) Fluorescence image of a section showing intense dual ACPPD uptake in inflammation surrounding the infiltrative edge of tumor (H). (I–L) Fluorescence histology confirming that the hyperintense region of ACPPD uptake corresponds to the presence of MMP activity and infiltrative tumor. (I) Fluorescence imaging of dual-ACPPD uptake and (J) DQ-gelatin on the same slice verified the presence of scattered protease producing cells. (K) Boxed region from I and J, overlaid to show rough colocalization of Cy5 (red) and DQ-positive regions (green). (L) Same section restained with hematoxylin/eosin to verify tissue identity. Purple hematoxylin has been image-enhanced for clarity.

ACPPD-Cy5 highlights primary and residual tumors in MMTV-PyMT mice. (A and B) Fluorescence of PyMT mouse 48 h after injection of cleavable ACPPD-Cy5, before (A) and after (B) skin removal. Tumors are present in all of the mammary fat pads as indicated by blue arrows. (C) Image of the same mouse after gross removal of tumor. A strip of protease activity corresponding to tumor (yellow arrows in B and C) remains attached to the pectoralis muscle (red arrows). (D and E) Fluorescence (D) and hematoxylin/eosin-stained (E) frozen section histology confirm the identity of the residual tumor, in this case no larger than 400 μm across (F) Gross image of removed lung containing metastases. (G and H) Fluorescence (G) and hematoxylin/eosin (H) images showing ACPPD-Cy5 uptake at the edges of a single lung metastasis. (I) Fluorescence image of a tumor-draining lymph node. Each fluorescence image is individually scaled to max intensity.

Gd-labeled ACPPD’s allow visualization of protease activity by T₁-weighted MRI. (A) MR axial slices of animals bearing HT-1080 xenografts before injection or 48 h after injection of cleavable ACPPD-Gd or an all-D-amino acid control as indicated. Yellow arrows point to tumors. Each image includes phantoms containing 250, 50, 10, and 0 μM Gd-DTPA from left to right. (B) Tumor intensities (black) and R₁ (gray) for cleavable and D-amino acid control ACPPD-Gd relative to those of uninjected mice. Tumor intensities have been normalized to water phantom intensities on the same image to account for variability between scans. P values are given for R₁ and normalized intensity relative to uninjected control animals. (C) Standardized uptake values at 48 h for Gd (quantitated using ICPMS) in tissues from animals injected with either the cleavable ACPPD-Gd or D-amino acid control. Tu = tumor, M = muscle, Bl = blood.