Article Figures & Data
Figures
- Fig. 1.
Staining for bound trastuzumab and HER2. Tumor cryosections are shown from MDA-435-LCC6HER2-overexpressing xenografts treated with 20 mg/kg trastuzumab for 3 h or left untreated. Overlaid images of a treated tumor show bound trastuzumab (black) relative to blood vessels (CD31; dark blue) and the perfusion marker DioC7(3) (cyan; A); additional staining of the same section for HER2 (red) shows that areas with no bound trastuzumab are overexpressing HER2 (B). An untreated MDA-435-LCC6HER2 tumor shows no bound trastuzumab (C) but relatively homogeneous HER2 expression (D). Similarly stained MDA-435-LCC6vector tumors display no bound trastuzumab or unbound HER2 in treated or untreated tumors (not shown).
- Fig. 2.
Distribution of trastuzumab over time. The relative amount of bound trastuzumab in cryosections obtained 2 to 2.5 mm from the tumor edge of MDA-435-LCC6HER2 tumors is assessed using quantitative analysis of immunohistochemical staining. Representative portions of whole-tumor sections are shown with trastuzumab (red), CD31 (dark blue), and perfusion dye, DioC7(3) (cyan): an untreated control tumor (A), a single dose of 4 mg/kg trastuzumab at 8 h (B), 24 h (C), 72 h (D), and 96 h (E). The average fraction of tissue stained positively for bound trastuzumab shows detectable levels as early as 3 h, peak levels at 24 h, with significant decreases at 72 and 120 h (F).
- Fig. 3.
Heterogeneous distribution of trastuzumab through whole tumors. Tumors grown to 8 to 10 mm in diameter were dosed with 20 mg/kg trastuzumab and harvested at 3 h (A) or 26 h (B); 10 μm thick transverse sections were obtained at 1mm intervals from the tumor edge, were imaged for perfusion marker DioC7(3) (cyan), and stained for bound trastuzumab (red) and CD31 (dark blue). Examples of vessels with very little or no extravasating trastuzumab are illustrated (green arrows). Those sections obtained closer to the gross tumor edge of both the 3 h (A, left) and 26 h (B, left) show a greater proportion of tissue stained for trastuzumab than the more heterogeneous central sections (A and B, right). Quantitative analysis shows percentage of pixels positive for trastuzumab in sections at increasing distances from the tumor edge (C, left). The microregional distribution of trastuzumab as a function of distance from vasculature was analyzed on sections obtained 1 mm from tumor edge and compared with those at 4 mm; data is shown for tumors harvested at 3 h (C, middle) and 26 h (C, right) after treatment.
- Fig. 4.
Heterogeneous microregional distribution of trastuzumab. Tumors grown to 8 to 10 mm in diameter were dosed with 20 mg/kg trastuzumab and harvested at 3 h (A) or 26 h (B); 10 μm cryosections were imaged for perfusion marker DioC7(3) (cyan) and stained for bound trastuzumab (red) and CD31 (dark blue). Trastuzumab is seen to extravasate in a heterogeneous pattern in all regions of the tumor sections at 3 h (A, right); inset (A, left) illustrates presence of many perfused vessels not associated with significant amounts of trastuzumab (green arrows). At 26 h, tissue free of bound trastuzumab can still be found on cryosections (B, middle) in areas demonstrating both a high density of perfused vessels (B, left) and low density (B, right). All scale bars, 200 μm.
- Fig. 5.
Quantitative analysis of tumor microenvironment. Analyses of vascular architectural features were done on MDA-435-LCC6HER2 tumors treated with 20 mg/kg trastuzumab for either 3 or 26 h (A); transverse sections were obtained at 1 mm intervals from the tumor edge. No differences are seen in the density of CD31-stained microvessels (Ai), or the fraction of vessels staining dual positive for perfusion marker DioC7(3) (Aii), or collagen type IV (Aiii); however, dual staining of CD31 with pericyte marker desmin D33 shows a decreasing trend at distances farther from the tumor edge (Aiv). Tumor microenvironmental features were also assessed at longer time points after a single 4 mg/kg dose of trastuzumab (B), with no significant changes in the fraction of perfused vessels (Bi), S-phase cells detected using BrdUrd incorporation (Bii), or the relative fraction of necrotic tumor tissue at time points up to 96 h after administration (Biii); however, the fraction of necrotic tissue that stains positive for bound trastuzumab does increase with time after treatment (Biv).
Volume 14, Issue 7
