Real-time imaging reveals the single steps of brain metastasis fo mation r
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1 Real-time imaging reveals the single steps of brain metastasis fo mation r Yvonne Kienast, Louisa von Baumgarten, Martin Fuhrmann, Wolfgang E.F. Klinkert, Roland Goldbrunner, Jochen Herms and Frank Winkler Supplementary Figures a Red fluorescence intensity (A.U.) Metastatic nodule 1 Metastatic nodule 2 Metastatic nodule b Day 14 Day 21 Day 28 5µm Supplementary Figure 1. RFP fluorescence intensity in proliferation and regression over time. (a) Increase of fluorescence intensity of three successfully proliferating metastatic nodules that could be followed from day 1 to day 51 after cancer cell injection. Mean red fluorescence intensity was determined in areas of 12x12 µm on days 1, 3, 6, 9, 14, 21, 28, 42, and 51, and given in arbitrary units (A.U.). (b) On day 21, cells apparently fragment into small apoptotic bodies with bright RFP expression. Before disappearance, regressing cancer cells sometimes show morphological features of apoptosis, but never a gradual decline in fluorescence intensity. Depth, 9-13 µm. All data was acquired by in vivo MPLSM. Nature Medicine: doi:1.138/nm.272
2 a Metastatic foci (PC14-PE6) PC14-PE6 lung carcinoma Metastatic foci (MDA-MB-435) b Tumor cell velocity (µm/s) Cell/vessel diameter ratio <1 Cell/vessel diameter ratio =1 No vessel branch Vessel branch c Tumor cell velocity (µm/s) r =.56 p = melanoma Vessel diameter (µm) d Perivascular melanoma cells Spherical cells (no cell protrusions) Longitudinal cells (cell protrusions) A258 melanoma 28 Perivascular melanoma cells Spherical cells (no cell protrusions) Longitudinal cells (cell protrusions) e Cells/growing metastasis (HTB177) Cells/growing metastases Vascular remodeling (%) Angiogenesis (%) HTB177 lung carcinoma Vascular remodeling/angiogenesis (%) Cells/growing metastasis (A258) Cells/growing metastases Vascular remodeling (%) Angiogenesis (%) A258 melanoma Vascular remodeling/angiogenesis (%) f Nodules (PC14-PE6) PC14 lung carcinoma Nodules (HTB177) HTB177 lung carcinoma Nodules (MDA-MB-435) Nodules (A258) A258 melanoma 28 g 25 Vascular density (mm/mm 3 ) PC14-PE6 lung carcinoma HTB177 lung carcinoma A258 melanoma 2 2 Vascular density (mm/mm 3 ) Vascular density (mm/mm 3 ) Growing metastases Regressing metastases Growing metastases Regressing metastases Growing metastases Regressing metastases Nature Medicine: doi:1.138/nm.272 Supplementary Figure 2 Additional quantifications.
3 . (a) Metastatic inefficiency. 17 ± PC14-PE6 lung carcinoma and 117 ± cells were detectable through the cranial window on day 1 post injection; this amount declined to 1.2 ± 1.46 (PC14- PE6, day 21, n = 6 mice) and 3.3 ± 5.63 (MDA-MB-435, day 51, n = 4 mice) metastatic nodules over time. Error bars represent standard error. (b) cell velocity in relation to the ratio of tumor cell and vessel diameter, and to the presence of vascular bifurcations (n=1435 time points analyzed, n = 3 mice; P =.1, Mann-Whitney rank sum test, error bars represent standard error). (c) Correlation between blood vessel diameter and intravascular cell velocity (n=1435 time points analyzed, n = 3 mice; r =.56, P =.1, Spearman rank order correlation). (d) Frequency of perivascular A258 (17 cells in n = 5 mice) and MDA-MB-435 (128 cells in n = 4 mice) melanoma cells displaying a spherical vs. longitudinal morphology over time. A longitudinal form is typically seen in those cells that invade and proliferate along pre-existing brain microvessels. (e) Proliferation kinetics of successfully metastasizing HTB177 lung carcinoma and A258 melanoma cells in relation to changes of the vascular bed. Vascular remodeling: vessel dilatation, capillary loop formation, and/or increasing tortuosity. Angiogenesis: formation of new blood vessels over time. HTB177 lung carcinoma: 1 macrometastasis from a total of 12 metastatic nodules in 5 mice; A258 melanoma: 5 macrometastases in 3 animals from a total of 17 metastatic nodules in 5 mice. (f) Time course of the development of micrometastases (4-5 cells) and macrometastases (>5 cells) from single extravasated PC14- PE6 (n=124 cells in 6 mice) and HTB177 (n = 12 cells in n = 5 mice) lung carcinoma cells, or MDA-MB-435 (n = 128 cells in 4 mice) and A258 (n = 17 cells in n=5 mice) melanoma cells. (g) Growth or final regression of PC14-PE6 and HTB177 lung carcinoma micrometastases is not determined by the vascular length density (mm/mm 3 ) of the site (PC14-PE6: n = 2 growing metastases in 2 mice and n = 4 regressing metastases in 4 mice; HTB177: n = 4 growing metastases in 2 mice and n = 4 regressing metastases in 4 mice). The difference of the vascular length density of growing and regressing micrometastases did not reach statistical significance in the melanoma cell line A258 (n = 4 growing metastases in 3 mice and n = 4 regressing metastases in 4 mice; P =.38). Nature Medicine: doi:1.138/nm.272
4 a b PC14-PE6, day 1 PC14-PE6, day kda VEGF-A only 21 kda Merge: VEGF-A cancer cells (RFP) blood vessels (FITC) c bevacizumab control IgG PC14-PE6 lung carcinoma Supplementary Figure 3. VEGF expression of cancer cells in vitro and in vivo, and in vitro effects of bevacizumab on cell proliferation (a) Western blot analysis of VEGF-A expression of a positive control (1, human U87 glioma cells), PC14-PE6 lung carcinoma cells (2), and cells (3), under normoxic conditions in vitro. VEGF monomers can be detected at 21 kda, dimers at 42 kda. (b) Immunofluorescence analysis of metastasizing PC14-PE6 cancer cells (RFP, red), blood vessels (FITC lectin, green), and VEGF-A protein expression (blue). While VEGF-A expression of single cells one day after injection was near the detection limit, macrometastases of more than.5 mm showed a robust VEGF-A expression. The primary antibody for these experiments was VEGF (A-2) sc-152 from Santa Cruz. Experiments were done according to the manufacturer s instructions. The images were acquired by confocal microscopy of brain sections of 16 µm thickness. (c) Bevacizumab does not inhibit the proliferation of PC14-PE6 lung carcinoma and MDA-MB- 435 melanoma cells in vitro. Cell proliferation was determined using a cell proliferation assay according to the manufacturer s instructions (CellTiter 96 AQueous non-radiactive cell proliferation Assay, Promega). Cells were incubated with different concentrations of bevacizumab or control IgG in the same volume for 48 hours. IgG vs. bevacizumab groups were compared using ANOVA on ranks; no significant differences could be determined. Each graph represents the mean +/- SEM of 4 replicates. Nature Medicine: doi:1.138/nm.272
5 Day 21 Day 28 Supplementary Figure 4. Bevacizumab treatment does not change the growth pattern of MDA- MB-435 melanoma brain metastases. Inhibition of cancer-cell derived VEGF-A by bevacizumab does not inhibit capillary loop formations (arrows) of the melanoma cell line MDA-MB-435, which grows continuously despite therapy. Images were acquired by in vivo MPLSM. Depth, 6-15 µm. 64 cells in n = 3 animals were analyzed. 5µm Day 21 Day 28 5µm Nature Medicine: doi:1.138/nm.272
6 Supplementary Methods Quantification of image parameters The following parameters were determined in the same brain regions over time and quantified for each metastatic nodule: 1) directly after intra-arterial injection: relation of cancer cell diameter to vascular diameter (µm), intravascular velocity of tumor cells (µm/sec), number of cancer cells in association with a vascular branch point; 2) from day on, for each individual cancer cell over time: a) fraction of cancer cells that were I) intravascular, II) in the process of extravasation (showing both intra- and extravascular cellular parts), III) extravasated, IV) perivascular, V) vessel-unrelated; b) cancer cell movement velocity (µ m/h, intra- vs. perivascular vs. parenchymal); c) change of cell number and metastasis diameter (µm), in relation to the anatomical localization (intra-, perivascular, parenchymal); d) blood vessel length density (mm/mm 3 ); (e) events of vascular remodeling (i.e., capillary loop formation, development of tortuous vascular structures, and/or vasodilatation), or appearance of new blood vessels (angiogenesis); (f) cell shape (longitudinal vs. spherical) and (g) cells staying dormant during the experiment, and their relation to perfused vessels. Only those cancer cells that had direct contact with a vessel, showing no gap between cancer cell signal and vascular signal at maximum magnification, were termed perivascular. Dormancy was defined as lack or arrest of cancer cell proliferation without any signs of regression during the entire length of one experiment (determined by animal survival: minimum 21, maximum 51 days). A minimum time period of 21 days without any change in cell number was required for the definition of dormancy. Vessel cooption was defined as invasion and proliferation along preexisting cerebral blood vessels, resulting in a perivascular infiltration of the brain parenchyma. were defined as multicellular cancer cell clusters of 4-5 cells (which generally means a metastasis diameter of <5 µm), and macrometastases of >5 cells (>5 µm). This size represents the mouse equivalent of the proportion of a magnetic resonance imaging-detectable brain metastasis (5 mm) to the length of a human brain. In addition, we determined blood vessel length density in regions of 15 x 15 x 3 µm of proliferating and regressing micrometastases. For image analysis we used the AxioVision Rel. 4.6 software (Zeiss) for evaluation of either single image stacks or 3-dimensional reconstructions. Nature Medicine: doi:1.138/nm.272
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