TAS‐115 inhibits PDGFRα/AXL/FLT‐3 signaling and suppresses lung metastasis of osteosarcoma

Osteosarcoma is the most common malignant bone tumor in adolescence and childhood. Metastatic osteosarcoma has a poor prognosis with an overall 5‐year survival rate of approximately 20%. TAS‐115 is a novel multiple receptor tyrosine kinase inhibitor that is currently undergoing clinical trials. Using the mouse highly lung‐metastatic osteosarcoma cell line, LM8, we showed that TAS‐115 suppressed the growth of subcutaneous grafted tumor and lung metastasis of osteosarcoma at least partially through the inhibition of platelet‐derived growth factor receptor alpha, AXL, and Fms‐like tyrosine kinase 3 phosphorylation. We also show that these signaling pathways are activated in various human osteosarcoma cell lines and are involved in proliferation. Our results suggest that TAS‐115 may have potential for development into a novel treatment for metastatic osteosarcoma.

Osteosarcoma is the most common malignant bone tumor in adolescence and childhood. Metastatic osteosarcoma has a poor prognosis with an overall 5-year survival rate of approximately 20%. TAS-115 is a novel multiple receptor tyrosine kinase inhibitor that is currently undergoing clinical trials. Using the mouse highly lung-metastatic osteosarcoma cell line, LM8, we showed that TAS-115 suppressed the growth of subcutaneous grafted tumor and lung metastasis of osteosarcoma at least partially through the inhibition of platelet-derived growth factor receptor alpha, AXL, and Fmslike tyrosine kinase 3 phosphorylation. We also show that these signaling pathways are activated in various human osteosarcoma cell lines and are involved in proliferation. Our results suggest that TAS-115 may have potential for development into a novel treatment for metastatic osteosarcoma.
Osteosarcoma (OS) is the most common malignant bone tumor in adolescence and childhood. Despite combined treatment with wide resection and chemotherapy, lung metastases occur in 40-50% of patients with OS [1,2]. The survival of patients with metastatic or relapsed osteosarcoma has remained virtually unchanged over the past 30 years, with an overall 5-year survival rate of 20-30% [2][3][4]. Therefore, new therapeutic options are required to improve prognoses for those patients. TAS-115 is a novel inhibitor of multiple receptor tyrosine kinases (RTKs) that has been shown to inhibit c-MET, vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), and Tyro3/AXL/ Mer (TAM) [5]. Recently, it was reported that TAS-115 has a favorable tolerability profile and exhibits antitumor activity in human gastric cancer [5,6], human lung cancer [7,8], and synovial sarcoma [9]. Specifically, TAS-115 has been reported to be effective against bone metastasis, derived from carcinomas [10,11]. However, the efficacy of this drug for the treatment of OS remains to be elucidated. Here, we aimed to investigate the therapeutic potential of TAS-115 in vitro in different OS cell lines and in vivo in a subcutaneous tumor and lung metastasis mouse model.

Cell lines
We utilized the syngeneic mouse, highly metastatic OS cell line LM8. LM8 was established from the Dunn OS cell line by eight repeated intravenous injections of the cells derived from the lung nodule [12]. We used a clone of LM8 cells that stably expresses luciferase to evaluate lung metastasis using an in vivo imaging system (IVIS), which was kindly provided by Yui. The human Yamato-SS cell line, derived from synovial sarcoma, was established from surgically resected tumors in our laboratory, as described previously [13].

Immunoblot analysis
For the lysate preparation, cells were first washed with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 1% protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentrations were determined using the bicinchoninic acid method (Thermo Scientific). The cell proteins were separated on 4-12 % Bis-Tris gels (Life Technologies, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride membranes (Nippon Genetics, Tokyo, Japan). The membranes were blocked at room temperature in Tris-buffered saline, containing 5% skim milk and Tween-20 (TBS-T). Then, the membranes were incubated overnight with primary antibodies in Can Get Signal solution 1 (Toyobo Life Science, Tokyo, Japan) at 4°C, followed by incubation with the secondary antibodies in Can Get Signal solution 2 (Toyobo Life Science) for 1 h at room temperature. After washing with TBS-T, immunoreactive bands were visualized using enhanced chemiluminescent reagents [ECL Prime (GE Healthcare Life Sciences) and ImmunoStar LD (Wako, Osaka, Japan)].

Cell proliferation and viability assay
The cell proliferation rate was measured using the premixed WST-1 cell proliferation assay system (Takara Bio, Inc., Otsu, Japan). LM8 and Yamato-SS cells were seeded in 96well plates at 2000 cells/well and treated 24 h after seeding with various concentrations of TAS-115 (0-100 lM) for 48 h. The relative cell proliferation rate was calculated by subtracting absorbance measurements obtained from a microplate reader at 690 nm from those obtained at 450 nm. To evaluate the proliferation of OS cells, 1 9 10 6 (HOS, MG63, 143B, and LM8) and 2 9 10 6 (SaOS2 and U2OS) cells were seeded in 6-well plates and allowed to attach overnight. The next day, the cells were treated with 0, 0.1, 1, and 10 lM of TAS-115. The number of cells was assessed using a Countess automated cell counter (Invitrogen, Carlsbad, CA, USA). The experiment was repeated three times for each OS cell line.

Cell cycle analysis
LM8 cells (1 9 10 6 per dish) were seeded in 10-cm culture dishes, incubated overnight, and then treated with TAS-115 or vehicle (DMSO) for 24 h. The cells were harvested and stained with propidium iodide (PI) solution (25 lgÁmL À1 PI, 0.03% NP-40, 0.02 mgÁmL À1 RNase A, and 0.1% sodium citrate) for 30 min at room temperature. For cell cycle analysis, we used the BD FACSVerse TM flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) for fluorescence-activated cell sorting (FACS) according to the manufacturer's protocol.
In vivo syngeneic mouse model Use Committee of the Osaka University Graduate School of Medicine (27-094-006, 28-015-004). In the subcutaneous inoculation model, LM8 cells (1 9 10 7 ) were inoculated subcutaneously into the flank of 5-week-old female C3H mice. One week after inoculation, TAS-115 was orally administered once daily at a dose of 50 or 200 mgÁkg À1 for 4 weeks. Lung metastasis and subcutaneous tumors were evaluated using IVIS 3 and 4 weeks after inoculation with LM8. Four weeks after inoculation, the mice were euthanized and the tumor weight was measured. Tumor volume (mm 3 ) was defined as (A 9 B 2 )/2, where A is the longest and B is the shortest diameter of the tumor. Tumor volume and mouse body weight were measured twice a week. In the intravenous injection model, LM8 cells (1 9 10 6 ) were injected into the lateral tail vein of 5-week-old female C3H mice. One week after LM8 injection, TAS-115 was orally administered once daily at a dose of 50 or 200 mgÁkg À1 for 3 weeks. Lung metastases were evaluated using IVIS at 2 and 3 weeks after injection of LM8. For immunoblot analyses of tumor tissue lysate, mice bearing tumors were orally treated with TAS-115 (200 mgÁkg À1 ) for three consecutive days. Three hours after the last drug administration, the tumors were resected and extracted in Tissue Protein Extraction Reagent (T-PER; Thermo Scientific) supplemented with 1% protease/phosphatase inhibitor cocktail. The doses of TAS-115 were selected based on previous reports in which the daily administration of TAS-115 (50-200 mgÁkg À1 ) resulted in significant growth inhibition of the tumors [5][6]9,11].

In vivo imaging
Mice received D-luciferin (Caliper Life Sciences, Waltham, MA, USA) at a dose of 100 lg per mouse by intraperitoneal injection 15 min prior to imaging. A region of interest (ROI) of the same size and shape, covering the entire thoracic cavity, was applied to every image. Total flux in the ROI was measured. Data were analyzed by using IVIS Living Image software (Caliper Life Sciences).

Immunohistochemistry
Excised tumors were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (4 lm) were deparaffinized and dehydrated. After antigen retrieval in 10 mM citrate buffer at 95°C for 30 min, endogenous peroxidase activity was blocked with methanol containing 3% H 2 O 2 for 10 min. The sections were incubated overnight with primary antibodies at 4°C, followed by 1 h of incubation with secondary antibodies. Finally, labeled sections were stained with 3,3 0 -diaminobenzidine tetrahydrochloride (DAB; Dako, Glostrup, Denmark) and counterstained with hematoxylin.

Measurement of tumor vascular density
Immunohistological staining of anti-CD31 antibody in LM8 tumors was performed for each treatment group. Pictures were taken of three fields/section of each specimen of each treatment group in the best-stained tumor area (9200). The microvessel density (MVD, number of tumor vessels per mm 2 ) was calculated using the equation below: MVD ¼ total number of vessels in three visual fields area of one visual field Â 3

Phospho-RTK arrays
A mouse phospho-RTK array kit (R&D Systems, Minneapolis, MN, USA) was used to measure the relative level of tyrosine phosphorylation of 39 distinct receptor kinases. LM8 cells were cultured with/without TAS-115 for 24 h and lysed using lysis buffer. For collecting the lysate from the subcutaneous tumor, or lung nodule in mice, a piece of the tumor sample was minced and homogenized in T-PER supplemented with 1% protease/phosphatase inhibitor cocktail on ice immediately after tumor resection. The arrays were blocked with blocking buffer and incubated with 300 lg of cell lysate overnight at 4°C. The arrays were washed, incubated with a horseradish peroxidase-conjugated phosphotyrosine detection antibody, treated with chemiluminescent, and exposed to film.

Statistical analysis
We used Student's t-tests to determine the significance between the untreated controls and the various treatments in the in vitro and in vivo experiments. Values of P < 0.05 were statistically significant.

TAS-115 suppresses the proliferation of LM8 cells in vitro
We performed the WST-1 cell proliferation assay to examine the in vitro antitumor activity of TAS-115 in LM8 cells. TAS-115 dose-dependently inhibited the proliferation of LM8 cells (Fig. 1A) and of the synovial sarcoma cell line Yamato-SS, as was previously reported [9]. Flow cytometric analyses showed that TAS-115 increased the percentage of cells in the G0/ G1 phase and decreased the percentage of cells in the S phase in the LM8 cells in a dose-dependent manner (Fig. 1B). Immunoblot analyses revealed that the levels of cleaved PARP were not elevated after 24-h treatment with TAS-115 (Fig. 1C). Furthermore, we   investigated the effect of TAS-115 on normal cell lines, MC3T3-E1 and NHDF. FACS showed that TAS-115 caused only a slight increase in G0/G1 phase in normal cells compared with LM8 (Fig. S1). These results suggest that TAS-115 suppressed cell proliferation by inducing G0/G1 cell cycle arrest without apoptotic changes in the LM8 cells, whereas the toxicity of TAS-115 in normal cells was less than that of LM8.

TAS-115 inhibits the growth of subcutaneous tumors and the incidence of lung metastasis
We investigated the growth of tumors after the subcutaneous injection of LM8. Mice bearing tumors were treated daily with an oral dose of 50 or 200 mgÁkg À1 TAS-115, or water with HPbCD as a control, 1 week after subcutaneous injection of LM8 cells. TAS-115 (50 and 200 mgÁkg À1 ) suppressed tumor growth comprising LM8 cells ( Fig. 2A,B). Marked body weight loss was not observed in TAS-115-treated mice (Fig. S2). Histologically, a broad range of necroses were observed upon TAS-115 treatment compared to the vehicle control (Fig. 2C). Immunohistochemical analyses revealed that TAS-115 dose-dependently decreased MVD (Fig. 2D), indicating that TAS-115 has an anti-angiogenesis effect. Using an IVIS 4 weeks after the inoculation of LM8 cells, TAS-115 at a dose of 200 mgÁkg À1 led to a decrease in bioluminescence intensity, not only in the subcutaneous tumor but also in the lung metastasis (Fig. 2E-G). These results suggest that TAS-115 suppressed the growth of LM8   tumors and lung metastasis. However, it remains unclear whether TAS-115 indirectly decreased the new lung metastasis formation by suppressing the growth of a subcutaneous tumor or directly suppressed the growth of a formed lung micro-metastasis.

TAS-115 attenuates the growth of lung metastasis after intravenous injection of LM8
To investigate the growth inhibitory effect of TAS-115 on a formed lung metastasis, we monitored lung metastasis after intravenous injection of LM8 cells by IVIS (Fig. 3A). One week after intravenous injection, bioluminescence signals from tumor cells were detected in the lung using IVIS (Fig. 3B) and the administration of TAS-115 commenced. Three weeks after the injection of LM8 cells, TAS-115 significantly decreased the intensities of bioluminescence in the lung compared to those of the controls (Fig. 3C). These results suggest that TAS-115 suppressed the growth of a formed lung metastasis.

Therapeutic potential of TAS-115 via PDGFRa, AXL, and FLT-3 signal inhibition for LM8
Next, we investigated the mechanism by which TAS-115 mediates growth inhibition of LM8 cells. The phospho-RTK arrays revealed that PDGFRa demonstrated the highest levels of phosphorylation in the LM8 cell lysate (Fig. 4A). However, in vivo, not only PDGFRa but also some other TKs were phosphorylated. Notably, PDGFRa and AXL were strongly phosphorylated in the lysate of LM8 subcutaneous tumors (Fig. 4B), whereas AXL and FLT-3 were in the lung metastasis lysate (Fig. 4C). These results suggest that the phosphorylation of RTKs differed between in vitro and in vivo conditions. Moreover, these phosphorylation levels of PDGFRa, AXL, and FLT-3 were decreased in lysates treated with TAS-115 in vitro and in vivo. We showed the effects of TAS-115 on the PDGFRa, AXL, and FLT-3 signaling pathways by immunoblot analyses in vitro and in vivo. PDGFRa phosphorylation was suppressed in LM8 cells after a 3-h incubation with TAS-115 at concentrations ranging from as low as 0.001 to 10 lM (Fig. 5A).
To verify the inhibitory effects of TAS-115 in vivo, we administered TAS-115 (200 mgÁkg À1 ) to mice bearing LM8 tumors. TAS-115 at a dose of 200 mgÁkg À1 inhibited the phosphorylation of PDGFRa, AXL, and FLT-3 in both LM8 subcutaneous tumors and lung metastases (Fig. 5B). These results suggest that TAS-115 inhibited LM8 tumor growth in vitro and in vivo through the inhibition of PDGFRa, AXL, and FLT-3 signaling.
PDGFRa, AXL, and FLT-3 signaling are associated with the proliferation of several human OS cell lines We investigated the phosphorylation of PDGFRa, AXL, and FLT-3 in not only the murine OS cell line but also several human OS cell lines and examined the effect of PDGFRa, AXL, and FLT-3 signaling on their proliferation using various selective inhibitors. The phosphorylation of PDGFRa, AXL, and FLT-3 was observed in almost all cell lines (Fig. 6A), and these inhibitors induced a dose-dependent growth inhibition in many cell lines (Fig. 6B-D). These results indicate that these signaling pathways were partly involved in the proliferation of OS cells. TAS-115 treatment blocked the phosphorylation of PDGFRa, AXL, and FLT-3 and suppressed the proliferation of all OS cell lines in a dose-dependent manner (Fig. 6E) (Fig. 6F). These observations indicate that TAS-115 suppressed the proliferation of OS cells by inhibiting all signaling of PDGFRa, FLT-3, and AXL.

Discussion
This study showed three main findings. First, the novel multiple RTK inhibitor TAS-115 had an antitumor effect on the highly metastatic mouse OS cell line LM8. Second, this antitumor effect of TAS-115 was partially attributed to the blocking of PDGFRa, AXL, and FLT-3 phosphorylation. Third, these signals were also associated with the proliferation of many human OS cell lines and TAS-115 inhibited the proliferation of these human OS cell lines with inhibition of these signals. These results suggested that TAS-115 could be a particularly potent agent against OS.
Our laboratory previously reported that nuclear factor-jB (NF-jB) is a potential molecular target for designing specific prophylactic interventions against distant metastasis [14][15][16]. In the past study, an NF-jB inhibitor did not suppress the growth of the pulmonary metastasis formed once. Interestingly, in this study, we showed that TAS-115 is able to directly suppress the growth of a formed lung micro-metastasis in an intravenous injection mouse model. We believe that TAS-115 may be a more useful therapeutic drug than the NF-jB inhibitor for patients who have already had pulmonary metastases.
We showed that the antitumor activity of TAS-115 is partially mediated via PDGFRa/AXL/FLT-3 signaling, thereby supporting the previous findings of Fujita et al. [5], who discovered TAS-115 and showed that it is capable of inhibiting both MET and VEGFR2 via ATP antagonism. They reported that TAS-115 inhibits MET, VEGFR2, and several other tyrosine kinases with IC 50 values of less than 1 lmolÁL À1 , including PDGFRa, AXL, and FLT-3 in mobility shift assay. We did not detect the phosphorylation of MET and VEGFR2 in the LM8 cell lysate. However, the phosphorylation of PDGFRa, AXL, and FLT-3 was detected in the LM8 lysate in vivo. Furthermore, we showed that TAS-115 inhibited the phosphorylation of PDGFRa, AXL, and FLT-3 and decreased cell proliferation in LM8 and other human osteosarcoma cell lines. Thus, our results indicated that these three signals are potential targets of TAS-115 against osteosarcomas.
We demonstrated that these signals were also activated in various human OS cell lines and that they were associated with their proliferation. Several studies have reported that these signals are associated with OS prognosis. For example, PDGFRa and PDGF-A correlate with a poor prognosis in OS [17,18], AXL is frequently overexpressed in OS [19] and promotes osteosarcoma progression by affecting p-AKT expression [20,21], and FLT-3 ligand is significantly associated with poor overall survival and event-free survival independently of metastasis [22]. However, several clinical studies of selective inhibitors, such as imatinib, demonstrated little or no activity as a single agent in OS [23,24]. On the other hand, several clinical trials of multiple TK inhibitors, such as regorafenib [25], cabozantinib [26], and apatinib [27], in OS indicated a marked effect for OS and the potential to be developed as new therapeutic agents. TAS-115 is also a novel multiple RTK inhibitor that demonstrates much less toxicity in various normal cell lines than other VEGFR-targeted kinase inhibitors. [5]. Currently, a phase 1 study investigating the effect of TAS-115 in patients with solid tumors, including metastatic and relapsed OS, is ongoing in Japan (JapicCTI-132333). TAS-115 may also be a novel treatment for these patients.
We believe that the activated signals of tumor cells are different among tumor microenvironments. Therefore, we investigated the signals of LM8 using phospho-RTK arrays in three conditions: in vitro culture, subcutaneous tumor, and pulmonary metastasis. Interestingly, PDGFRa and AXL were activated in all three conditions. However, FLT-3 was not activated in vitro but was in vivo. These observations suggested that tumor cells were affected by the tumor microenvironment. There are few detailed reports about effective multiple TK inhibitors against osteosarcoma and their underlying modes of action. Interestingly, Fioramonti et al. [28] reported that cabozantinib, an inhibitor of multiple RTKs such as VEGFR2, AXL, and c-MET, affects not only tumor cells but also osteoblasts, which induces RANKL expression, promoting tumor proliferation in the primary bone microenvironment.   However, the interaction between the tumor and the host microenvironment in lung metastasis (e.g., immune cells and cancer-associated fibroblasts) is unknown. In our study, the activated signal was different in the subcutaneous tumor compared to the one in the pulmonary metastasis. Therefore, TAS-115 may exert the same antitumor effect in several tumor microenvironments. Further studies are needed to investigate the effect against tumor and host interactions in lung metastasis.
In conclusion, our findings indicate that TAS-115 exerts antitumor effects against OS partially via the inhibition of PDGFRa, AXL, and FLT-3 signaling. This study makes a novel contribution to the literature as it is the first time that TAS-115 has been studied for its effect on osteosarcoma and metastasis derived from osteosarcoma. We suggest that TAS-115 could be a promising therapeutic option for patients with metastatic or relapsed OS.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.