Axon-like protrusions promote small cell lung cancer migration and metastasis
- Cancer Biology Program, Stanford University School of Medicine, United States
- Stanford University School of Medicine, United States
- University Hospital Essen, Germany
- German Cancer Consortium (DKTK) partner site Essen, Germany
- Tufts University, United States
Figures
Figure 1 with 1 supplement
SCLC cells grow protrusions in culture and in vivo.
(A) Representative bright field images of three murine SCLC (mSCLC) cell lines (KP22, N2N1G, and 16T). Cells extend protrusions into a cell-free scratch generated in monolayer cultures. Protrusions …
Figure 1—figure supplement 1
SCLC cells grow protrusions in culture and in vivo.
(A) Representative images of mSCLC N2N1G and 6PFG cells growing in dense culture from N = 3 independent experiments. At the time of plating, 3–5% cells expressing membrane-GFP (mGFP, green …
Figure 2 with 2 supplements
SCLC cells with protrusions migrate in a saltatory fashion similar to neuroblasts.
(A) Representative immunofluorescence images of N2N1G mSCLC cells expressing membrane-GFP (GFP, green) and stained (red) for expression of the neuronal marker Tuj1, the axonal marker TAU, or the …
Figure 2—figure supplement 1
SCLC protrusions resemble axons and enable rapid cell movement.
(A) Representative fluorescence images of a mouse brain section stained with Tuj1, TAU, and MAP2 antibodies (positive controls, red). DAPI marks the nuclei of cells in blue. Scale bars, 50 μm. (B–C) …
Figure 2—figure supplement 2
Mouse and human SCLC cells express axonal markers in vivo.
(A) Representative immunofluorescence staining of SCLC cells in the liver of a TKO;mTmG mouse (in which SCLC cancer cells express membrane GFP (GFP)). These cancer cells have protrusions positive …
Figure 3 with 3 supplements
The axonal-like protrusions contribute to the migratory ability of SCLC cells in culture.
(A) Quantification of the number of cells with protrusions when mGFP-labeled N2N1G mSCLC cells were allowed to grow into a cell-free scratch generated in monolayer cultures under Matrigel. N = 3 …
Figure 3—figure supplement 1
The expression of the 13 genes selected for their possible role in the formation of protrusions is in part regulated by NFIB.
(A) mRNA levels of candidate genes in human primary SCLC tumors (RNA-seq from George et al., 2015). (B) Network representation of the 13 candidates. Edges in the STRING analysis represent …
Figure 3—figure supplement 2
The 13 genes selected for their possible role in the formation of protrusions are expressed in human SCLC but do not play a key role in the expansion of SCLC cell populations.
(A) DepMap analysis (depmap.org) of the requirement for the 13 candidate genes in 25 human SCLC cell lines (RNAi combined analysis). Note that in a number of cell lines, the knock-down of candidate …
Figure 3—figure supplement 3
Knock-down of GAP43 and FEZ1 disrupts the formation of protrusions and cell migration in mouse SCLC cell lines in culture.
(A) Representative images of the data quantified in Figure 3A and C with knock-down of Fez1. Scale bars, 100 μm. (B–C) Immunoblot analysis of GAP43 (B) or FEZ1 (C) levels in control and knock-down …
Figure 4 with 2 supplements
Genes involved in the generation of protrusions also control the formation of metastases.
(A) Diagram of the approach to investigate the formation of liver metastases (met.) after intravenous injection of SCLC cells. (B–C) Quantification of the number of metastases 4 and 5 weeks after …
Figure 4—figure supplement 1
Increased expression of axonal markers in metastatic SCLC.
(A) Representative images of immunohistochemistry experiments on lung sections from TKO mice 3 months and 6 months after SCLC initiation with Ad-CMV-Cre. None of the mice had metastases at the 3 …
Figure 4—figure supplement 2
Reduced formation of metastasis upon knock-down of GAP43 and FEZ1 in SCLC cells.
(A) Representative live and epifluorescence images (GFP, green) of liver section of mice 4 weeks after intravenous injection of GFP-positive N2N1G mSCLC cells, with control knock-down or knock-down …
Videos
Video 1
Time-lapse video of 16T mouse SCLC cells (images collected every 15 min).
Video 2
Time-lapse video of KP22 mouse SCLC cells (images collected every 15 min).
Video 3
Time-lapse video of 16T mouse SCLC cells (images collected every 15 min).
Video 4
Time-lapse video of N2N1G mouse SCLC cells (images collected every 15 min).
Additional files
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Supplementary file 1
Key Resources table.
- https://cdn.elifesciences.org/articles/50616/elife-50616-supp1-v2.docx
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Supplementary file 2
This Excel file contains all the Tables associate with the manuscript.
Table 1: A Summary of all the cell line that are used and tested for protrusion formation. Table 2: Expression of genes involved in axonogenesis and axon guidance in primary human SCLC tumors by RNA-seq Table3: Expression of genes involved in axonogenesis and axon guidance in mouse SCLC tumors and metastases by RNA-seq Table 4: Metascape analysis of the top 20 clusters with their representative enriched terms (one per cluster) for the 69 candidates. Table 5: Biological process GO term analysis for the 13 selected proteins Table 6: 13 candidate genes that may control the growth of axonal-like protrusions on SCLC cells Table 7: Expression levels and dependency scores for the 13 candidates in human SCLC cells Table 8: Knock-down of the 13 candidate genes in mouse SCLC cells Table 9: RNA-seq analysis of N2N1G and KP22 mouse SCLC cells
- https://cdn.elifesciences.org/articles/50616/elife-50616-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50616/elife-50616-transrepform-v2.docx
