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

Abstract

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.

Keywords: breast cancer; invasion; migration; multicellular streaming; multiphoton imaging.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1

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.

Figure 2

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. Data for TN1 were performed in this study for the first time. Error Bars: SEM, n = 3 mice per group.

Figure 3

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.

Figure 4

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).

Figure 5

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.

Figure 6

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.