Supplementary Figures (S1-S18) from Syndecan-Mediated Ligation of ECM Proteins Triggers Proliferative Arrest of Disseminated Tumor Cells

Supplementary Figure S1. Involvement of integrin:FAK-signaling in driving ERK activation and proliferation in vivo, related to Figure 1; Supplementary Figure S2. In vitro and in vivo behaviors of various mammary carcinoma cell types, related to Figure 1; Supplementary Figure S3. MoT culture as an in vitro model system to study signaling in DTCs, related to Figures 1 and 2; Supplementary Figure S4. ECM proteins in the lungs that surround recently-extravasated solitary DTCs, related to Figures 1 and 2; Supplementary Figure S5. KSR phosphorylation in nonaggressive/aggressive cancer cell types, related to Figure 2; Supplementary Figure S6. Restoration of ERK activation and proliferation by genetic depletion of Par-1, related to Figure 3; Supplementary Figure S7. Par-1:KSR:Ras/ERK signaling in various cell line models, related to Figure 3; Supplementary Figure S8. Par-1b phosphorylation and its effect on KSR:Ras/ERK signaling, related to Figure 3; Supplementary Figure S9. Function and expression of polarity-regulating proteins, related to Figure 4; Supplementary Figure S10. Distribution of polarity-regulating proteins in 3D conditions, related to Figure 4; Supplementary Figure S11. Restoration of ERK activation and proliferation by genetic depletion of Par-3, related to Figure 5; Supplementary Figure S12. Syndecans as crucial cell-surface sensors for the 3D ECM configuration, related to Figure 6; Supplementary Figure S13. Functional role of syndecans in regulating D2.1 cell behaviors in 3D conditions, related to Figure 6; Supplementary Fig. S14. Genetic inactivation of syndecans and its effect on aggressive cell behaviors, related to Figure 7; Supplementary Fig. S15. Concomitant operations of integrin:FAK-mediated pro-proliferative signaling and syndecan-mediated anti-proliferative signaling in the aggressive D2A1 cells, extended experimental results; Supplementary Fig. S16. Restoration of Par-3 and syndecan-1 expression in the B16F10 melanoma cells, extended experimental results; Supplementary Fig. S17. Evidence for genetic alterations to inactivate syndecan-mediated anti-proliferative signaling machinery in human cancers, extended analyses; Supplementary Fig. S18. Syndecan-mediated anti-proliferative signaling machinery that prevents the outgrowth of solitary DTCs, a summary illustration</p

Supplementary Methods from Syndecan-Mediated Ligation of ECM Proteins Triggers Proliferative Arrest of Disseminated Tumor Cells

Extended descriptions on experimental strategy</p

Supplementary Movie 3 from The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions

MOV file - 3.4MB, Direct conversion of FLP-associated integrin clumps into the ones constituting adhesion plaques</p

Supplementary Movie 2 from The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions

MOV file - 3.4MB, Kinetics of the assembly and disassembly of FLPs in cells cultured under MoT conditions</p

Supplementary Figures 1-17 from The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions

PDF file - 1.8MB</p

Supplementary Methods from The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions

PDF file - 216K</p

Supplementary Movie 1 from The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions

MOV file - 3.4MB, Kinetics of the assembly and disassembly of filopodia in cells cultured under monolayer conditions</p

Supplementary Table 1- Incidence of lung metastases in D2A1-d & parental D2A1 cells from Inflammation Triggers Zeb1-Dependent Escape from Tumor Latency

Summary of incidence of metastasis formation at limiting dilutions in D2A1-d and parental D2A1 cells</p

2PEM Imaging of D2.0R Cells Xenotransplanted in Mouse Ear Skin.

<p>A. Two weeks after injection with LifeAct-Ypet expressing D2.0R cells, a tumor is formed in the mouse ear skin (top left panel, outlined in green). The green fluorescent signal of the D2.0R cells can be observed in the tumor region (top right panel). A histological cross-section through the tumor region was obtained from the second ear injected at the same time with the same cell type. The Trichrome Masson staining colors the collagen in blue (bottom panel). B. Maximum projection of a z-stack obtained from an area in the periphery of the invasive tumor (top left panel A, white square). The D2.0R cells are shown in green, and the tumor is outlined in yellow. A background signal from the Evans Blue (red) can be observed in the vicinity of the blood vessels, potentially caused by the ‘leaky’ nature of the tumor vessels, its uptake by residing immune cells and the repeated injections. Scale bar: 50 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>. C. Higher magnification z-projection of the cell of interest boxed in B. Scale bar: 10 µm. D. Time-lapse imaging of a D2.0R tumor cell in vivo. The different panels depict, in black against a white background, the fluorescent signal of the actin in the cell at the indicated time-points. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>. E. Color-coded map of the structural changes of the cell of interest (boxed in B and C) over time. Scale bar: 10 µm.</p

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

<div><p>Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior <i>in vivo</i> with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.</p></div