As soon as steady state NVP-BKM120 had been reached (typically in 60-90 seconds), three individual values were noted from the display. The median of these three values was used for further data analysis. The TBF measurement apparatus was calibrated to 250 PU in a “motility standard” reference solution (Perimed) before the measurements, and calibration was regularly confirmed. Calibration was stable over time. Harvested lungs were embedded in cryogenic embedding medium (OCT), sectioned, and visualized using an epifluorescence microscope

to determine doxorubicin signal as previously described [13]. For each lung, a series of four red green blue (RGB) images in the tumor and in the normal lung was performed using a mercury lamp coupled to a 580-nm absorbance filter. This allowed visualizing the distribution

of doxorubicin, the basic component of Liporubicin that is encapsulated in liposomes. Hereafter and throughout the text, Liporubicin quantification refers to doxorubicin signal quantification as this is the active component at the cellular level of Liporubicin. To determine the distribution of Liporubicin in the tumor, a custom-built macro for ImageJ was used as previously described [13]. Briefly, the RGB images were created, taking highly intense green images that corresponded to endothelial cell lining (red pseudocolor) and lower intense signal (green pseudocolor, Liporubicin). The dilation function was applied to the red pseudocolor image for sequential dilations. A new RGB image was Dasatinib research buy recreated, and the overlap PAK6 between

green and red channels was quantified using the RGB colocalization function in ImageJ that quantifies the overlapped green and red pixels. This signal was corrected for initial overlap and background pixel count on the nondilated image and divided by the number of vessels per image (cross-checked by conventional histology). The results represent the presence of Liporubicin pixels as a function of distance from vessels in the different treatment groups, in other words, its distribution within tumors. Liporubicin signal at increasing distances from tumor vessels was assessed using a Student’s t test in Excel (Microsoft Corporation, Redmound, WA, USA) where a bidirectional hypothesis was applied. IFP and TBF changes were compared to initial values using a paired t test and between time points using a Student’s t test where a bidirectional hypothesis was applied. Results were considered significant when P < .05.) Before L-PDT treatment, tumor IFP values were significantly higher than lung values (4 ± 1.5 mm Hg vs 0 ± 0.25 mm Hg, respectively; P < .05). To exclude hemodynamic instability caused by anesthesia, we determined continuous tumor and lung IFP values during the first 30 minutes following anesthesia induction ( Figure 1A, pre–L-PDT). IFP values remained constant throughout this time frame. IFP was then measured in a constant way during and up to 1 hour following L-PDT.