Journal: Molecular cancer research : MCR

Article Title: Identification and characterization of cancer cells that initiate metastases to the brain and other organs

doi: 10.1158/1541-7786.MCR-20-0863

Figure Lengend Snippet: (A) Experimental strategy and procedures. Different cancer cell subpopulations are labeled in vitro (fast- vs. slow-cycling cells by PKH26, and live stemness reporters), and their characteristics is further analyzed in vitro (overlap with other labels indicating a certain population or a certain relative gene expression, cell growth), and in vivo (likelihood to master all steps of the brain metastatic cascade; overall brain metastatic efficacy). Results from different brain metastatic capacities of slow- vs. fast-cycling cancer cells are then used for identification of key molecular differences of these cell populations, finally used for knock-down studies of candidate gene(s) that can be tested regarding their influence on the brain metastatic cascade. MPLSM: multiphoton laser-scanning microscopy.(B) Representative images of a slow-cycling MDA-MB-231 breast cancer cell (remaining PKH26 staining; arrow), followed by repetitive in vivo MPLSM through all steps of the brain metastatic cascade: intravascular arrest (Days 1,3 – single cells), extravasation and colonization of the perivascular niche (Day 6 – up to 5 cells), micro- (Days 14, 21 – up to 50 cells) and macrometastasis (Day 28 - > 50 cells) formation. Green, cytoplasm of GFP-positive cancer cell(s); blue, brain microvessels labeled by TRITC angiogram; red, PKH26 staining labeling slow-cycling cancer cells. scale bars: 30μm. (C) Representative images of a fast-cycling MDA-MB-231 cell (absence of PKH26 staining) through the early steps of the brain metastatic cascade (Day 1 – single cells), until its death on day 6. scale bars: 30μm. (D) Percentage of all slow-cycling and fast-cycling MDA-MB-231 breast cancer cells, in vitro at the day of intracardial injection, and in vivo one day after. At day 1, all cancer cells were still in the state of intravascular arrest. Included cells day 0 MDA-MB-231 n=784; included cells on day 1 MDA-MB-231 n=138 (p<0.001; Chi Square test). (E) Slow-cycling JIMT1 breast cancer cell mastering all steps of the brain metastatic cascade; intravascular arrest (day 1 & day 3), extravasation and colonization of the perivascular niche (day 6), micrometastasis (day 9), macrometastasis (day 14 & day 21 & day 28). Green, cancer cell(s); blue, brain microvessels. scale bars: 30μm. (F) Fast-cycling JIMT1 breast cancer cell mastering intravascular arrest (day 1) and extravasation (day 6), but disappears afterwards until day 14. (G) Percentage of all slow-cycling and fast-cycling JIMT1 breast cancer cells, in vitro at the day of intracardial injection, and in vivo one day after. At day 1, all cancer cells were still in the state of intravascular arrest. Included cells day 0: Jimt1 n=1254; included cells on day 1: Jimt1 n=238 (p<0.001; Chi Square test). B-G: data obtained by in vivo MPLSM; scale bars: 30μm. 3 replicates per experiment.

Article Snippet: Image processing MPLSM imaging data was acquired by the Zeiss ZEN Software (Zeiss, Germany) and used to analyze the success of the single cells in performing the single steps of the brain metastatic cascade.

Techniques: Labeling, In Vitro, Gene Expression, In Vivo, Knockdown, Laser-Scanning Microscopy, Staining, Injection