Ligand-directed targeting of lymphatic vessels uncovers mechanistic insights in melanoma metastasis

Abstract

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

Keywords: cell surface peptide; cell–cell interaction; lymphatic targeting; phage display.

Fig. 1.

Two-step screening methodology based on molecular mimicry. (A) Flowchart depicting the separate screenings based on an LyV culture and consecutive LEC screening rounds with the Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) method. (B) Identification of the native protein-mimicking ligands with high-density protein microarrays. Antipeptide serum and control serum reactivity to proteins on a nitrocellulose-dotted human microarray was evaluated. (C) Sequences of peptides enriched during rounds 2–4. The enrichment was calculated based on peptide abundance from the total number of peptides sequenced. (D) Melanoma cell immunoreactivity against the corresponding antipeptide sera by immunofluorescence. Anti-GLTFKSL serum recognizes a protein on the cell surface of C8161 melanoma cells compared with anti-AEKSSYV, anti-FSGWSTV, anti-VSQRNEL, and negative control serum. (E) Specific peptide cell surface binding to LECs and C8161 melanoma cells. Binding of GLTFKSL displayed on phage was evaluated using the BRASIL method. GLTFKSL phage specifically bound to LECs and C8161 melanoma cells compared with insertless control phage (Fd phage) and human umbilical vein endothelial cells (HUVECs). Relative transducing units (T.U.) are displayed as means ± SEMs. (F) Nonpermeabilized and permeabilized LECs and melanoma cells were immunoreactive against GLTFKSL Abs.

Fig. 2.

GLTFKSL peptide is the molecular mimic of PPP2R1A. (A, Left) Expression of the truncated PPP2R1A 457–589 and a control protein from the microarray were verified by Coomassie blue staining. (A, Right) Anti-GLTFKSL Ab recognizes the truncated recombinant PPP2R1A protein by Western blot. (B) Binding specificity of GLTFKSL Ab to truncated recombinant PPP2R1A. The Ab reactivity against the recombinant protein was inhibited by addition of GLTFKSL peptide. (C) Anti-GLTFKSL recognizes recombinant human PPP2R1A by Western blot. (D) Reciprocal immunoprecipitation. Both anti-PPP2R1A and anti-GLTFKSL immunoprecipitate PPP2R1A from C8161 melanoma cell extracts. (E) Transmission EM of anti-GLTFKSL and anti-PPP2R1A binding to C8161 cells. Secondary Abs labeled with 18-nm gold particles were used for detection. (F) Silencing of PPP2R1A by shRNA in C8161 melanoma cells. Silencing was confirmed by immunoblotting with the anti-PPP2R1A and anti-GLTFKSL Abs. NT shRNA was used as control vector. (G) Cell surface binding of anti-PPP2R1A to a panel of human melanoma cell lines. The percentage of cell surface binding as analyzed by FACS is depicted as mean ± SD. Rabbit IgG was used as a negative control. IP, immunoprecipitation; WB, Western blot.

Fig. 3.

PPP2R1A mediates the cell–cell interaction between LECs and melanoma. (A) GLTFKSL phage bound specifically to the GLTFKSL peptide compared with control peptides, BSA, and insertless phage. (B) PPP2R1A was immunocaptured from C8161 cell extracts with the anti-PPP2R1A and anti-GLTFKSL Abs. GLTFKSL phage bound to both immunocaptured proteins compared with control. All relative transducing units (T.U.) were expressed as means ± SEMs. (C) The Biopanning and Rapid Analysis of Selective Interactive Ligands method was used to evaluate binding of GLTFKSL phage to C8161 cells stably transduced with PPP2R1A shRNA or NT shRNA. Binding of GLTFKSL phage to PPP2R1A-silenced cells was reduced by about 50%. Insertless control phage (Fd phage) served as a negative control. (D) Ab against PPP2R1A inhibits cell–cell interaction between melanoma and lymphatic cells. (E) Lymphatic and melanoma cell adhesion to the ECM was evaluated in vitro with or without addition of anti-PPP2R1A Ab. (F, Left) Silencing of PPP2R1A in C8161 decreases melanoma cell attachment to LECs. (F, Right) Cell adhesion to the ECM was not compromised after PPP2R1A knockdown. (G) Melanoma–lymphatic cell interaction inhibited by the GLTFKSL peptide. Cell migration is mediated by surface expression of PPP2R1A. (H) Anti-PPP2R1A Ab. (I) GLTFKSL peptide. CTL, control. *P < 0.05; **P < 0.01.

Fig. 4.

PPP2R1A is expressed on LyVs in vivo. (A) Confocal microscopy was performed on C8161 tumor frozen sections stained with PPP2R1A (red), Podoplanin (green), and CD34 (blue). Colocalization (yellow) of PPP2R1A and Podoplanin is depicted in the merged image. Neg. CTRL, negative control. (B) Immunohistochemical analysis of GLTFKSL phage homing. Positive staining (red arrows) is shown in the tumor of GLTFKSL-injected mice, whereas insertless phage staining in the tumor is negative. Muscle was the negative control organ. Liver is part of the reticuloendothelial system, and therefore, phage is present. Representative pictures are shown (magnification: 200×). (C) PPP2R1A expression in C8161 tumors. PPP2R1A is highly expressed in C8161 tumors as shown by immunohistochemistry. CTRL, control. (Magnification: 200×.)

Fig. 5.

PPP2R1A expression on melanoma patient samples. (A) PPP2R1A is expressed on the cell surface of human metastatic melanoma samples (grade IV). Flow cytometry analysis of melanoma samples is graphically represented as a waterfall blot. A positive correlation is observed between expression of PPP2R1A on the surface of LECs and melanoma cells as calculated by the Pearson correlation method (r = 4, P = 0.017). NHEM, normal human melanocyte. (B) Histological analysis of human melanoma samples. Primary melanoma (Prim. Tumor), in-transit metastasis (In-transit), and LN metastasis (LN Met) samples stain positive for PPP2R1A. Neg. CTRL, negative control. (Magnification: 200×.)