Metastasis in an orthotopic murine model of melanoma is independent of RAS/RAF mutation

Abstract

Melanoma is the most lethal skin tumor in large part because of a propensity for early metastasis. Good models of this most clinically relevant feature of melanoma are lacking. Here, we report the development of an in-vivo model of metastasis that relies on orthotopic injection of green fluorescent protein-tagged lines in immunodeficient mice, serial intravital imaging of tumor progression, and quantification of distant spread by two-photon laser scanning microscopy, immunohistochemistry, and real-time PCR analysis. Using this system, we report an assessment of the in-vivo growth and metastatic properties of 11 well-characterized human melanoma cell lines. A subset of lines showed rapid in-vivo growth with invasion of host vasculature and distant seeding of viscera in this system. The ability to form metastasis in vivo did not correlate with three-dimensional collagen invasion in vitro. Surprisingly, similar lines in terms of molecular genetic events differed markedly in their propensity to metastasize to distant organs such as brain and lung. In particular, two lines harboring B-RAF mutation and high levels of phosphorylated ERK and AKT were reproducibly unable to form tumors after orthotopic injection. Similarly, two previously identified RAS/RAF wildtype 'epithelial like' lines that do not have elevated phosphorylated ERK and AKT or express TWIST1 mRNA still showed a pronounced ability for orthotopic growth and metastatic spread. All the metastatic cell lines in this model showed increased NEDD9 expression, but NEDD9 lentiviral overexpression did not convey a metastatic phenotype on nonmetastatic cells. These data suggest that melanoma metastasis is a molecularly heterogeneous process that may not require epithelial-to-mesenchymal transition or ERK activation, although both may facilitate the process.

Fig. 1. In vitro and in vivo growth

A) In vitro cell proliferation rate of the different human melanoma cell lines used in this study. B) Kaplan-Mayer curve showing tumor-free survival of SCID mice injected with 5×10⁵ GFP-tagged cell lines. UACC257, SK-MEL23, SK-MEL24 and PMWK do not form tumors even after 90 days. 3 mice were bilaterally injected per tumor cell line (6 injection sites). C) Kaplan-Mayer curve showing no difference in tumor-free survival of SCID mice injected with B-RAF mutant versus wild type cell lines.

Fig. 2. Serial imaging of ear tumors by intravital microscopy

A) Representative 2-photon laser scanning microscopy images of GFP-tagged cells 1, 7, 14 and 45 days after injection of 5×10⁵ GFP-tagged WM2664 cells i.d. in the ear of a NOD-SCID mouse, imaged at 20x (top row; scale bar: 100 μm) and 40x magnification (bottom row; scale bar: 50 μm). Single cells can be identified at all time points. B) Dextran red-labeled blood vessels showing intravasated cells 2 weeks after injection in the ear. Left: dextran red only. Note unstained area where melanoma cells are located (arrow head). Middle: GFP only. Right: merged dextran red and GFP. Scale bar: 200 μm. C) Intravasated cells can be observed by contrast in the green channel (left) or merging the red and green channels (right).

Fig. 3. Detection of metastatic cells in whole organs

Representative 2-photon images of tumors and organs of mice injected with the different human melanoma cell lines at end-point (when tumors reached 1.3 mm diameter or 90 days post-injection). SK-MEL28 cells form rapidly growing tumors, but show no signs of metastasis in any organs. WM2664 and A2058 cells form slower growing tumors that metastasize to lungs and brain. SK-MEL24 cells were not able to form overt tumors and did not give rise to metastasis in any organs, but cells were still present in the ear 90 days after injection. Note anatomical detailing of organs by autofluorescence. Scale bar: 100 μm.

Fig. 4. Immunohistochemical detection of GFP metastasis in lungs

Representative images of paraffin sections of tumors, lungs and brains of mice injected with the different human melanoma cell lines at end-point (when tumors reached 1.3 mm diameter or after 90 days). SK-MEL28 tumors stained positively for GFP (brown), but no GFP staining was observed in any organs from these mice by IHC. WM2664 and A2058 tumors and lungs were positive for GFP, confirming the 2-photon data. All other organs were GFP negative. Scale bar: 200 μm.

Fig. 5. Twist1, LKB1 and NEDD9 expression

A) Protein expression was assessed in our panel of melanoma cell lines by western blot. Note expression of NEDD9 protein by cells that are metastatic in vivo (in blue). B) Western blot confirming GFP-NEDD9 overexpression (SK-MEL24, SK-MEL28 and SK-MEL187) or shRNA downregulation of NEDD9 protein (A375, A2058 and WM2664) compared to controls.