Fig 1.
Quantification of histological and MRI measures of a tumor in the control group.
(A) DAPI staining. (B) The staining density map of DAPI staining shown in (A). Also shown are the corresponding ADC (C) and Cmax (D) maps at the center of the same tumor. Two blue lines indicate a 0.2 mm-wide band immediately inside of the boundary of the tumor for the rim ROI, while the red line delineates the core ROI.
Fig 2.
Quantification of immature vessels that lack COLIV staining for basement membrane.
(A) A representative patch (500 x 500 pixels; 0.2 x 0.2 mm2) with vessels stained positive mostly for both CD31 and COLIV, which has a high covariance between the two staining density maps. (B) A representative patch with vessels that has strong staining for CD31, but weak staining for COLIV. This case has a lower covariance between the two staining density maps. (C) The whole slice CD31 and COLIV stained images for a case in the control group. (D) The CD31 and COLIV density maps estimated from the corresponding IHC images shown in (C). The immature vessel density map shown in the last column was generated using the stain covariance map between CD31 and COLIV as a mask to show CD31 densities only for the patches with low covariances.
Fig 3.
Estimated tumor volume sizes compared between control (blue line) and metronomic chemotherapy-treated mice (red) at day 7 and day 17.
A significant decrease in volume size was noted in the MCT mice (p < 0.005).
Fig 4.
Pre-contrast and post-contrast DCE images and corresponding parametric ADC mappings of the central slice of 4T1 tumors for control and MCT mouse.
An increase in ADC is seen with MCT tumors and confirms that there is a more geometrically favorable water environment with treatment. The yellow lines delineate tumor boundaries.
Fig 5.
Average DCE enhancement curves of control (A) and MCT (B) mice in each group pre- and post-chemo time points.
There is a decrease in enhancement with the control group, while the MCT mice remained fairly level after the scheduled chemotherapy regimen.
Fig 6.
DCE empirical mathematical model parameters A (A), alpha (B), q (C), β (D) and Cmax (E) in treated and untreated mice.
Significance between the two groups and p values shown accordingly.
Fig 7.
(A) Log of normalized MR signal from DWI data versus b value for MCT and control groups before and after treatment. MCT ADC values were significantly larger than the controls at the post-scan time point, which translates to the MCT tumor cellular membranes allowing for more water movement across the semi-permeable phospholipid bilayer. (B) Estimated Apparent Diffusion Coefficient (ADC) between pre and post-MRI shows an increase in diffusion with the MCT group.
Fig 8.
Representative whole slide histology images for control and MCT mice and the corresponding staining density maps.
Immature vessles were identified by determining which vessels with CD31 expression did not have a basement membrane marker COLIV.
Fig 9.
Correlation between water Apparent Diffusion Coefficient (ADC) and cell density (DAPI).
Control tumors had a constant diffusion over the course of tumor progression and cell growth, while MCT mice had greater diffusion with cell death (lower cell densities in both core and rim). Ki67, a cell proliferative marker, densities are corresponding with control (blue) and MCT (red) mice in the core and periphery of 4T1 tumors. Tumor cores did not vary between the treated and untreated groups. However, the rim of the MCT mice had significantly more Ki67.
Fig 10.
Correlation between maximum CA concentration (Cmax) and vasculature marker CD31 (A) and Cmax and immature vasculature (B).
Vascular density was greater in the periphery of the tumors regardless of treatment and was statistically similar between the groups; however, the MCT mice had more enhancement than the control group (A). (B) The rim of all tumors was composed of more immature vessels than the core for both the control and MCT mice. Immature vasculature was more profound in the MCT group than the control group.