tn1 cells  (ATCC)

Journal: Intravital

Article Title: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

doi: 10.4161/intv.25294

Figure Lengend Snippet: General characterization of in vivo migration in MDA-MB-231 and TN1 human breast tumors by intravital multiphoton microscopy. MDA-MB-231 and TN1 primary tumors were imaged by multiphoton intravital microscopy as described in Methods. (A) Quantification of general motility as average percentage of randomly imaged fields per mouse that contained either any type of motility (single cell or streaming), or streaming over total number of fields imaged. Error bars: SEM. (B) Representative images of cells migrating in either single or multicellular streaming patterns in MDA-MB-231 tumors. Still images shown here were derived from Videos S1 and S2. (C) Representative images of cells migrating in either single or multicellular streaming patterns in TN1 tumors. Still images shown here were derived from Videos S3 and S4. n = 4–6 fields per mouse imaged, 9 different mice for MDA-MB-231 and 8 different mice for TN1 tumors. In the images: Migrating tumor cells (green) are outlined in white and numbered. Stromal cells (black) co-migrating with the tumor cells in the streams are outlined in red and numbered. Collagen fibers (blue) are imaged by second harmonic generation. Yellow arrows in the leftmost panels denote direction of migration. Rightmost panels show a still image at t = 30 min with the cumulative centroid tracks of the motile cells overlaid. Scale bar: 20 µm.

Article Snippet: TN1 cells are a primary sample and therefore cannot be passaged in culture; instead TN1 cells were only passaged in vivo in NOD.SCID mice (Jackson Laboratories) by orthotopic injection of 10 6 cells mixed with 50% matrigel (BD Biosciences, cat # 354234) directly into the lower left mammary fat pad.

Techniques: In Vivo, Migration, Microscopy, Intravital Microscopy, Derivative Assay

Journal: Intravital

Article Title: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

doi: 10.4161/intv.25294

Figure Lengend Snippet: Quantification of in vivo migration patterns in MDA-MB-231 and TN1 human breast tumors. (A) Quantification of average cell velocity, net path length, directionality, average turning frequency, average cell area and minimum cell area for single or streaming moving cells in both MDA-MB-231 and TN1 tumors. Error Bars: SEM, n ≥ 20 cells per group from at least 4 different mice, *p < 0.05, **p < 0.01 (by Student’s t-test). (B) Quantification of migration patterns as average percentage of total motile cells per mouse that were involved in either single cell migration or multicellular streaming migration. Error Bars: SEM, n = 4–6 fields per mouse imaged, 9 different mice for MDA-MB-231 and 8 different mice for TN1 tumors. (C) Immunostaining with cell type-specific antibodies (anti-pan-cytokeratin for tumor cells and anti-F4/8 for macrophages) was performed in migratory cells as collected from MDA-MB-231 and TN1 tumors with the in vivo invasion assay. Results are reported as average percentage for each cell type over the total number of cells collected per mouse. Total cells were counted by nuclear counterstain using DAPI. Data shown for MDA-MB-231 were re-analyzed from previously reported experiments.25 Data for TN1 were performed in this study for the first time. Error Bars: SEM, n = 3 mice per group.

Article Snippet: TN1 cells are a primary sample and therefore cannot be passaged in culture; instead TN1 cells were only passaged in vivo in NOD.SCID mice (Jackson Laboratories) by orthotopic injection of 10 6 cells mixed with 50% matrigel (BD Biosciences, cat # 354234) directly into the lower left mammary fat pad.

Techniques: In Vivo, Migration, Immunostaining, Invasion Assay

Journal: Intravital

Article Title: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

doi: 10.4161/intv.25294

Figure Lengend Snippet: Multicellular streaming migration is significantly associated with proximity to blood vessels. (A) Migration was quantified in vivo in MDA-MB-231 and TN1 tumors by intravital multiphoton microscopy, in tumor-bearing mice where blood vessels were labeled by intravenous injection of fluorescent dextrans. A representative image of a field with multicellular streaming directed close to a blood vessel is shown (still image was derived from Vid. S5). In this image, tumor cells are green, blood vessels are red, and collagen I fibers are blue. Migrating cells are outlined in white and numbered for ease of reference. The yellow arrow indicates the direction of migration. Quantification is shown as average percentage of total migrating cells per mouse either close to blood vessels (vascularized microenvironment) or away from blood vessels (avascular microenvironment). This quantification was repeated separately for the singly migrating cells and the cells migrating in multicellular streams. Error Bars: SEM, n = 4–6 fields per mouse imaged, 4 different mice for each MDA-MB-231 and TN1 tumors, *p < 0.05, ***p < 0.001. Scale bar: 20 m. (B) Immunohistochemistry of fixed tissue sections from MDA-MB-231 tumors was performed against pan-cytokeratin for all tumor cells (pink), endomucin for blood vessels (blue), and Iba1 for macrophages (brown), in order to identify the spatial correlation between these three cell populations in tumors in situ. A representative image is shown at 20× magnification at the left panel. The right panel is a magnification of the inset, where a potential multicellular stream involving both tumor cells and macrophages can be seen in close proximity to a blood vessel. BV, blood vessel (blue arrow); TC, tumor cell (pink arrows); M, macrophage (brown arrows). Scale bar: 50 m.

Article Snippet: TN1 cells are a primary sample and therefore cannot be passaged in culture; instead TN1 cells were only passaged in vivo in NOD.SCID mice (Jackson Laboratories) by orthotopic injection of 10 6 cells mixed with 50% matrigel (BD Biosciences, cat # 354234) directly into the lower left mammary fat pad.

Techniques: Migration, In Vivo, Microscopy, Labeling, Injection, Derivative Assay, Immunohistochemistry, In Situ

Journal: Intravital

Article Title: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

doi: 10.4161/intv.25294

Figure Lengend Snippet: Macrophage function is required for multicellular streaming migration and intravasation in vivo. MDA-MB-231 and TN1 tumor-bearing mice were treated with either control (PBS) liposomes or clodronate liposomes, in order to functionally impair macrophages. (A) Quantification of total migrating cells in the treated tumors that follow either a single cell or multicellular streaming pattern. Shown in the graph is the average percentage of cells migrating in either pattern over the total number of migrating cells per mouse. Error Bars: SEM, n = 4–6 fields per mouse imaged, 5 different mice for each MDA-MB-231 and TN1 tumors, **p < 0.01, ***p < 0.001 (by Student’s t-test). (B) Intravasation was measured in treated MDA-MB-231 tumor-bearing mice as count of circulating tumor cells in the blood of the mice. Shown is the average number of circulating tumor cells per ml of blood per mouse. Error Bars: SEM, n = 5 mice per condition, **p < 0.01 (by Student’s t-test).

Article Snippet: TN1 cells are a primary sample and therefore cannot be passaged in culture; instead TN1 cells were only passaged in vivo in NOD.SCID mice (Jackson Laboratories) by orthotopic injection of 10 6 cells mixed with 50% matrigel (BD Biosciences, cat # 354234) directly into the lower left mammary fat pad.

Techniques: Migration, In Vivo, Control, Liposomes

Journal: Intravital

Article Title: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

doi: 10.4161/intv.25294

Figure Lengend Snippet: Coordinated gene expression changes in the migratory cells from MDA-MB-231 and TN1 primary tumors fall into path-ways that initiate protrusive force and chemotaxis. (A) mRNA expression for genes in known motility pathways was quantified in the migratory tumor cells from MDA-MB-231 and TN1 tumors, as isolated with the in vivo invasion assay. Results are shown here as relative mRNA expression compared with the bulk primary tumor cells, isolated from the same primary tumors (shown in a log2 scale for ease of presentation). Error bars: SEM, n = 4 different mice per group, all results shown in this graph are significant with p < 0.05. (B) Gene expression changes from the real-time PCR results of panel A were superimposed in motility pathway protein maps, for ease of comparison. All genes that are present on the map were assayed by real-time PCR, and if change was not significant the gene is denoted in plain black font. Upregulated genes are in bold red font, downregulated genes are in bold green font, and genes that coordinately regulated in both the MDA-MB-231 and TN1 tumors are denoted by an asterisk next to the gene name. The fold change in expression is shown next to each gene.

Article Snippet: TN1 cells are a primary sample and therefore cannot be passaged in culture; instead TN1 cells were only passaged in vivo in NOD.SCID mice (Jackson Laboratories) by orthotopic injection of 10 6 cells mixed with 50% matrigel (BD Biosciences, cat # 354234) directly into the lower left mammary fat pad.

Techniques: Gene Expression, Chemotaxis Assay, Expressing, Isolation, In Vivo, Invasion Assay, Real-time Polymerase Chain Reaction, Comparison

Journal: Intravital

Article Title: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

doi: 10.4161/intv.25294

Figure Lengend Snippet: In vivo migratory cells from human breast tumors show increased barbed end activity. The EGF-induced barbed ends at the leading edge were measured in the migratory tumor cells (isolated with the in vivo invasion assay) and the bulk primary tumor cells population (isolated from the same tumors by sorting all GFP-positive tumor cells), in both the (A) MDA-MB-231 tumors and (B) TN1 tumors. Shown are representative images of the immunofluorescence staining of the cells for the actin barbed ends, as well as Arp2 protein for the visualization of the leading edge (traced by dotted white lines). Graphs show quantification of three independent experiments per tumor, as average intensity of barbed ends in the cell leading edge. Error bars: SEM, n ≥ 20 cells from 3 different mice for each tumor, *p < 0.05, ***p < 0.001 (by Student’s t-test). Scale bar: 10 µm.

Article Snippet: TN1 cells are a primary sample and therefore cannot be passaged in culture; instead TN1 cells were only passaged in vivo in NOD.SCID mice (Jackson Laboratories) by orthotopic injection of 10 6 cells mixed with 50% matrigel (BD Biosciences, cat # 354234) directly into the lower left mammary fat pad.

Techniques: In Vivo, Activity Assay, Isolation, Invasion Assay, Immunofluorescence, Staining