Mice bearing triple-negative breast cancer tumours were administered five different barcoded nanoparticles, simultaneously. The nanoparticles contained either one of the anticancer agents: doxorubicin, gemcitabine or cisplatin, or were loaded with caffeine, or empty (barcode alone). The barcoded nanoparticle cocktail was injected intravenously and accumulated in the tumour cells over a period of 48 h. After 48 h, the tumour tissue was biopsied and dissociated into single cells. The cells were sorted according to their viability (live/dead) and the barcodes in each of these populations were extracted, analysed and quantified using RT–PCR and sequencing. The activity of each of the agents was measured at the single-cell level; data from 80 representative cells are shown (
a, b). A noise level below 20% was set as the threshold for determining the activity of a single agent at the single-cell level. In addition, ( c) the overall activity of each of the agents in the tumour was determined by analysing the barcode abundance in groups of at least three million live or dead cells. ( d) To compare between the potency of the different drugs, a potency scale was plotted. The comparative potency is based on the ratio of barcodes found in the dead cells to those found in live cells. On the basis of this comparative diagnostic scale, a treatment protocol was devised. To ensure statistical significance, each screen was based on at least three million cells extracted from the tumour. The data were calculated as the mean±s.e.m. of n=6, in two independent experimental replicates.