Soluble AXL: A Possible Circulating Biomarker for Neurofibromatosis Type 1 Related Tumor Burden

<div><p>Neurofibromatosis type 1 (NF1) is the most common tumor predisposition disorder affecting 1/3500 worldwide. Patients are at risk of developing benign (neurofibromas) and malignant peripheral nerve sheath tumors (MPNST). The AXL receptor tyrosine kinase has been implicated in several kinds of cancers, but so far no studies have investigated the role of AXL in NF1 related tumorigenesis. Recently, the soluble fraction from the extracellular domain of AXL (sAXL) has been found in human plasma, and its level was correlated to poor prognosis in patients with renal cancer. Compared to normal human Schwann cells, a significantly high expression level of AXL was found in three of the four MPNST cell lines and two of the three primary MPNST tissues. Similarly, the level of sAXL in conditioned media corresponded to the protein and mRNA levels of AXL in the MPNST cell lines. Furthermore, in two different human MPNST xenograft models, the human sAXL could be detected in the mouse plasma. Its level was proportionate to the size of the xenograft tumors, while no human sAXL was detect prior to the formation of the tumors. Treatment with a newly developed photodynamic therapy, prevented further tumor growth and resulted in drastically reduced the levels of sAXL compared to that of the control group. Finally, the level of sAXL was significantly increased in patients with plexiform tumors compared to patients with only dermal neurofibromas, further supporting the role of sAXL as a marker for NF1 related tumor burden.</p></div

Release of sAXL into the conditioned media of MPNST cell lines.

<p><b>A</b>, all four MPNST cell lines (S462, STS26T, ST8814 and T265) and NHSC released sAXL into the conditioned media, at levels that roughly comparable to the protein levels of full length AXL in the cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g002" target="_blank">Fig. 2A</a>). A highly-matched correlation was found between the time in culture and the sAXL concentration in all four cell lines and NHSC (symbol cross for S462 cells with a coefficient of determination, R² = 0.97; diamonds for STS26T with R² = 0.99, squares for ST8814 with R² = 0.97, triangles for T265 with R² = 0.99 and star for NHSC R² = 0.94). To further highlight that all cell lines release sAXL at a constant rate, the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g003" target="_blank">Fig. 3A</a> is presented as 5 individual graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone.0115916.s001" target="_blank">S1 Fig. <b>B–C</b></a>, Using specific lentiviral shRNA clones, the expression of AXL and TYRO3 has been knocked down in MPNST cell line T265, either alone or in combination to knockdown both genes (shDual). Successful silencing was verified with quantitative-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g003" target="_blank">Fig. 3B</a>) and Western blotting (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g003" target="_blank">Fig. 3C</a>). <b>D</b>, the levels of sAXL in the cell medium were drastically reduced in AXL-knockdown cells. In contrast, knockdown of TYRO3 did not affected the release of sAXL compared to the empty vector (GIPZ). In each case the level of sAXL in the conditioned media corresponded to the mRNA levels in the cells.</p

Mice harboring human MPNST (STS26T) xenograft tumors release human-soluble AXL into the blood.

<p>There was a trend of size dependent increasing of sAXL level until the tumors reached 2000 mm³. The coefficient of determination, R² was 0.884.</p

The plasma levels of human sAXL correlated to the tumor growth and the efficacy of Lipo-Ce6 in MPNST xenograft mice.

<p><b>A</b>, MPNST xenograft tumors (S462-TY) were allowed to grow until 100 mm³. Mice were injected with normal saline, 2 mg/kg or 2.5 mg/kg Lipo-Ce6 by tail vein injection. Two hours post injection mice were irradiated with a light dose of 100 J/cm². <b>B</b>, treatment had no adverse effect on the body weight. <b>C</b>, the tumor growth was completely suppressed in the mice receiving 2.5 mg/ml of Lipo-Ce6 followed by PDT treatment and partial suppression in the mice receiving 2 mg/kg Lipo-Ce6. <b>D</b>, the levels of sAXL corresponded to the tumor size in all three treatment groups further supporting its role as a marker for NF1 related tumor burden.</p

Demographic data, tumor burden and level of soluble AXL in patients with neurofibromatosis type 1.

<p>Dermal NFAs  =  number of skin neurofibromas; sAXL conc.  =  the concentration of soluble AXL; yrs  =  years.</p><p>Demographic data, tumor burden and level of soluble AXL in patients with neurofibromatosis type 1.</p

Increased phosphorylation activity of AXL and TYRO3 in MPNST cell lines.

<p><b>A</b>, using a phospho-RTK array, the phosphorylation levels of 42 different RTKs in four MPNST cell lines (T265, STS26T, ST8814 and S462) and NHSC were measured. Seven of the RTKs had increased phosphorylation in MPNST cells: (<b>A</b>, EGFR; <b>B</b>, MET; <b>C</b>, PDGFR-alfa; <b>D</b>, PDGFR-beta; <b>E</b>, Insulin Growth factor receptor; <b>F</b>, AXL; <b>G</b>, TYRO3). <b>B∼1D</b>, mRNA expression of AXL (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g001" target="_blank">Fig. 1B</a>), TYRO3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g001" target="_blank">Fig. 1C</a>) and GAS 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115916#pone-0115916-g001" target="_blank">Fig. 1D</a>) compared to Normal Human Schwann cells (NHSC).</p

Increased plasma levels of sAXL in patients with plexiform neurofibromas.

<p><b>A</b>, patients with neurofibromatosis had significantly higher plasma levels of sAXL. There were 72 NF1 patients (diamonds) and 46 controls (squares). The sAXL level was significantly higher in NF1 patients than that of controls (<i>p-value</i> <0.005). <b>B</b>, patients without plexiform tumors (group ‘Dermal NFA’, squares) had the same levels of sAXL compared to the healthy controls (group control, diamonds, n.s.  =  non-significant), while the levels were markedly increased in the patients with plexiform tumors (group ‘Plexiform’, triangles) compared to both the dermal NFA group (p<0.01) and the healthy control group (p<0.0001). <b>C</b>, there was not any significant difference between the male (squares) and female (diamonds) participants in any of the groups. <b>D</b>, within the dermal NFA group, males with a high number of dermal neurofibromas (>100) had significantly higher sAXL levels than that with a low NFA tumor burden (<30) (p<0.05). Nevertheless, no significant difference was found among female groups.</p