The progressive development of the malignant mass is depicted at four different time points, namely 6, 12, 18 and 24 days post tumor inception.

<p>Three characteristic tumor regions can be identified as the viable (red), hypoxic (blue), and necrotic (brown) tissue. Pre-existing vessels (straight brown lines) are laid out in a regular grid, maintaining normoxic conditions in the surrounding tissue. New vessels (irregular brown lines) are sprouting from the pre-existing vasculature in response to a net balance of pro-angiogenic factors released by hypoxic cells in the interior of the tumor. Field of view is 2×2 mm.</p

Blood flow rate and adhering particle fraction. The simulated blood flow rate mapped directly over the tumor and pre-existing vascular network (top).

<p>The color map is scaled by the maximum flow rate reached in the pre-existing vessels inside the tumor (10⁻⁵ m³/s). The fraction of injected 1,000 nm NPs (×10⁻³) adhering firmly at the blood vessel walls is also shown at ∼100 min after systemic injection (<b>bottom</b>). The images correspond to the tumor stages depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056876#pone-0056876-g002" target="_blank"><b>Figure 2</b></a>. Red arrows indicate the points of injection for the NPs, located upstream with respect to the tumor mass. The parameter <i>α</i> is 10¹² m⁻² in the tumor neovasculature and 10¹⁰ m⁻² in the pre-existing vessels. The parameter <i>β</i> is fixed and equals 10⁻³ m⁻² s. Note that under these conditions, the NPs accumulate mostly at the periphery of the tumor immediately downstream of the injection sites.</p

<b>Fraction of the injected NPs adhering firmly at the blood vessel walls at about 100 min after injection on day 18, upstream of the tumor mass as indicated by the red arrows in</b><b>Figure 4</b>.

<p>The colors on the right provide a measure of the particle fraction adhering mainly to the neovasculature (irregular lines). The columns are related to three different NP sizes, namely 100, 600 and 1,000 nm; whereas the three rows are related to three different values for the parameter <i>α</i> (<i>α</i> = 10¹² m⁻² top row; <i>α</i> = 10¹⁰ m⁻² middle row and <i>α</i> = 10⁸ m⁻² bottom row). For all cases, <i>β</i> = 10⁻⁴ m⁻² s while <i>α</i> for the pre-existing vessels is 100 times smaller than for the corresponding tumor-induced neovessels. Tumor colors are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056876#pone-0056876-g002" target="_blank"><b>Figure 2</b></a>.</p

Model overview showing the main components, variables, and key system interactions.

<p>Model overview showing the main components, variables, and key system interactions.</p

Assessment of TEB path tortuosity.

<p>The angle of deflection caused by bifurcation, the frequency of bifurcation, and the disparity between displacement and path tracing measurements were measured. A) Representative image of a TEB bifurcation event with the original path of the TEB noted as a solid line, the new path of each TEB as a result of the bifurcation are noted as dotted lines and the angles measured noted. B) Quantification of the angle of deflection presented as mean (whiskers denote range)(n = 62). C) Representative image of TEB’s growth path with 2 bifurcation events noted and the length of duct between noted with a solid line. D) Quantification of the total length of duct between bifurcation events is presented as mean (whiskers denote range)(n = 23). E) Total length of duct measurements were compared to corresponding displacement measurements. Displacement measurements consistently underestimated the total length by 6.1% (±0.9) (p = .0001, paired ratios t-Test, n = 23).</p

Cap cells migrate into body cell layer and undergo apoptosis.

<p>Proliferation status and apoptotic status of SMA+ cells were investigated in both the outer regions (Region 1 and 2) and the inner regions (Regions 5 and 6) of the TEB. A) Representative image of a TEB triple stained for SMA, BrdU, and CC3. B) Mean number of SMA+ cells present in each region, (whiskers denote range) n = 34 TEBs. C) SMA+ cells also positive for BrdU or pHH3 in each region presented as mean ±SEM, *p < .05, ****p < .0001, n = 34 TEBs. D) Quantification of SMA+ cells positive for CC3 in each region presented as mean, ****p<0.0001 (whiskers denote range) n = 34 TEBs.</p

Morphology characterization of the terminal end bud.

<p>Glands from 5 and 6 week old mice were embedded, sectioned and stained for epithelial markers and measurements were taken of both cells and the TEB structure. A) Regional measurements of the TEB are represented in a scaled schematic (n values: Regions 1, 2, 5, and 6 = 15 TEBs, Region 3 = 23 TEBs, Region 4 = 8 TEBs, Region 7 layer diameter = 24 TEBs, lumen = 15 TEBs, length = 29 TEBs, Region 8 = 8 ducts). B) Mean cellular dimensions of the cap and myoepithelial cells, Regions 1–4 (whiskers denote range, n values: Region 1 length = 15 TEBs/ 45 cells, Region 1 width = 10 TEBs/ 126 cells. Region 2 length = 15 TEBs/ 56 cells, Region 2 width = 10 TEBs/ 115 cells. Region 3 length = 23 TEBs/ 273 cells, Region 3 width = 9 TEBs/ 36 cells. Region 4 length = 8 TEBs/ 197 cells, Region 4 width = 8 TEBs/ 197 cells). C) Mean cellular dimensions of body and luminal cells, Regions 5–8 (whiskers denote range, n values: Region 5 width/length = 5 TEBs/ 221 cells. Region 6 width/length = 5 TEBs/ 316 cells. Region 7 length/width = 4 TEBs/ 204 cells. Region 8 length/width = 6 ducts/ 301 cells). See also Table A in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004839#pcbi.1004839.s001" target="_blank">S1 Text</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004839#pcbi.1004839.s002" target="_blank">S2 Fig</a>.</p