Fig 2: Detection of cytoplasmic and nuclear proteins in blood by single-molecule imaging.(A) Schematic diagram of intracellular cytoGFP release from a tumor cell (lime) into a blood vessel (red). (B) SMAC quantification of serum cytoGFP levels in naïve mice (gray circles; n = 8) and tumor-bearing mice 1 week after oropharyngeal (blue circles; n = 4) or subcutaneous (red circles; n = 4) injection of cytoGFP+ tumor cells (TC-1). (C to E) To induce a spontaneous cytoGFP+ tumor, mice (n = 10) were administered with DNA encoding RasG12V, p53 shRNA, cytoGFP, and luciferase. Graph depicting the relationship between tumor luciferase and serum cytoGFP concentrations assessed by SMAC at an end point of more than 2 months (C) or throughout the first 2 months (D). In (C), tumor-induced mice that displayed a grossly visible tumor were labeled “tumor” (red circles), while those that did not were labeled “pretumor” (blue circles). Using the kinetics data in (D), the time correspondence between serum cytoGFP levels and tumor burden was determined by cross-correlation analysis (E). (F) SMAC images of purified human p53 at femtomolar concentrations in aqueous buffer. Scale bar, 4 µm. (G) Comparison of the sensitivity of SMAC with shape analysis (SR counts, red circles) and ELISA [OD450nm (optical density at 450 nm), blue circles] using purified human p53. The dotted line indicates the ELISA detection limit. (H and I) To stimulate a spontaneous tumor carrying mutant human p53, mice (n = 10) were administered with DNA encoding human p53R175H, RasG12V, and luciferase. Time-course (H) and cross-correlation (I) plots depicting the relationship between tumor luciferase and serum mutant p53 levels measured by SMAC. For cross-correlation plots, each unit time lag is around 5 days. All data are expressed as means ± SD. ****P < 0.0001. P values are from a two-sided unpaired t test.