PMC

A) In vivo images showed fluorescent rims around different sizes of peritoneal tumors/ metastasis (white arrow) which are validated using bioluminescence. Li= Liver, B= Bladder, T=Tumors. B) Ex vivo images of the peritoneal tumors/metastases confirmed the rim shaped signals around the tumors. T= Tumors. Arrows indicate very small metastases (≤1-2mm) which were fluorescently delineated. Red dotted circles = Regions of interest (with B as Background).

A) Flow cytometer analyses show high uPAR expression on HT-29 cells while no expression is detectable on the Caco-2 cell line. B) Graph shows the serum stability of hybrid ATN-658. An increase in aggregates and albumin bound agents is seen over time, with 60% of the agent still free after 48 h. C) Cell based plate assay analyses show the specific binding of hybrid ATN-658 on uPAR expressing HT-29 cells. Hybrid ATN-658 signal intensities differed significantly from the control hybrid MOPC-21 at all dose groups except 0 nM. D) No specific binding on the control cell line Caco-2 and there were no significant differences between both tracers at all dose groups. A.U.= arbitrary units.

A) UPPER ROW: Representative images of a mouse injected with 75 μg/0.5 nmol of hybrid ATN-658. The tumor on the cecum is exposed using a laparotomy, and measured after 72 h. Fluorescent signals in the colon surrounding the tumor are due to ingested agents as the signals is disappeared when the colon is emptied as seen on the ex vivo images. White arrow indicate the tumor. LOWER ROW: Mouse injected with 75 μg/0.5 nmol hybrid MOPC-21 which showed no specific NIR fluorescent signals. White arrow indicate the tumor. Red dotted circles = Regions of interests (with B as Background). B) Merged images (NIR fluorescent microscopy and histology) show co-localization of the uPAR specific multimodal agent, especially at the tumor border (border surrounded with dashed line). Apart from minor fluorescent signals in necrotic areas (black arrow), no tumor-specific signals are seen with the control agent. (magnification 40x) C) Graph shows the mean TBR and SD (n=3 for each group) of mice with orthotopic colorectal tumors after 72 h (P < 0.05).

A) Biodistribution data of hybrid ATN-658 in the HT-29 subcutaneous colorectal model in mice. Graph shows increased tumor uptake over time and decreasing signals in the urine, blood, heart and lungs, as determined by a gamma counter and represented as %ID/weight. B) Mean tumor-to-cecum and tumor-to-muscle ratios over time up to 72 h post injection are shown including standard deviations. Images show SPECT scans from C) 24 h (mouse with 3 tumors) and D) 72 h (mouse with 4 tumors) post injection, revealing the broad imaging window. Activity was seen in the tumors and the metabolizing organs (kidneys and liver). Inserted are representative NIR fluorescent images taken subsequent to SPECT imaging with the pre-clinical PEARL system at 24h and 72h post injection, showing the usability of the multimodal agent. Red dotted circles = Regions of interest (with B as Background), T= Tumor, Li= Liver, K= Kidney, B= Bladder, H=Heart.

A) UPPER ROW: The images show representative fluorescent signals in a mouse injected with 150 μg/1 nmol of hybrid ATN-658 and measured at 24 h post injection with a mean TBR of 4.1. White arrows indicate tumors. LOWER ROW: Mouse injected with 150 μg/1 nmol of hybrid MOPC-21 with a mean TBR of 1.6. White arrows indicate tumors. Red dotted circles = Regions of interest (with B as Background). B) TBRs over time up-to 120h post injection of mice receiving 1 nmol of the different agents. At each time point, at least 3 mice for each group were measured. Hybrid ATN-658 TBRs were significantly different from all three controls at all time-points. C) TBRs of various doses of hybrid ATN-658. Overall there were no significant differences between the TBRs of all three dose groups except at 24 h between 50 ug and 150 ug (p=0.05). Furthermore, a decreasing trend in TBR was seen in the lower dose groups. D) Absolute signals from the different dose groups and agents in the tumors. Significant differences were seen between the lowest dose group (50 μg/0.34 nmol), the control agent (15 0μg/1 nmol) and the two highest uPAR specific dose groups (100 μg/0.67 nmol and 150 μg/1 nmol) at all time points. Between the 150 μg hybrid ATN-658 and 100 μg hybrid ATN-658 were no significant differences found. A.U.= arbitrary units