MDA-MB-435 subcutaneous xenograft studies

All animal studies were approved by MIT’s committee on animal care (MIT protocol 0411-036-14). To generate subcutaneous grafts, 3–4 week-old female NCr nude mice (Taconic) were injected bilaterally with 5 × 10⁶ MDA- MB-435 cells per flank. Urine measurements were made prior to tumour inoculation by injecting either 0.5 μM LyP1 or non-penetrating ABNs (by protease cleavable peptide) in 200 μL PBS intravenously at most every week after inoculation. After nanoparticle injection, mice were placed in custom housing with a 96-well plate base for urine collection. After 1 hour, bladders were voided to collect between 100–200 μL of urine. For analysis, urine was diluted between 10- to 25-fold in PBS. Reporter concentration was quantified by fluorescence measurement of Cy7 using a Licor by comparing to a ladder (Odyssey). Tumour sizes were measured using digital electronic calipers (Marathon Management Co.) and volume was calculated as volume = 0.5 × length × width², where length and width are the larger and smaller dimensions, respectively. Total tumour volume was defined as the sum of the tumour volume in each flank. Mice were binned based on tumour sizes and urine signal was quantified for the bins. Receiver operating characteristic (ROC) curves were generated in GraphPad.

For organ and tumour biodistribution and quantification, mice were sacrificed at 3 hours post-NP injection and organs were removed and scanned on the LI-COR Odyssey Infrared Imaging System. Fluorescence from the nanoparticle scaffold (VT680) and the peptide (Cy7) was quantified using ImageJ software (NIH).