Purpose: Platelet-derived growth factor receptor α (PDGFRα) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC-3G3) is a human mAb that exclusively binds to PDGFRα and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors.

Experimental Design: PDGFRα expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor.

Results:In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin.

Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847–57. ©2017 AACR.

Translational Relevance

Olaratumab (LY3012207/IMC-3G3) is a human mAb against the platelet-derived growth factor receptor α (PDGFRα), which recently received accelerated approval from the FDA and conditional EMA approval for treatment of adult advanced soft tissue sarcoma patients in combination with doxorubicin. Pediatric patients with bone and soft tissue tumors are treated with intensive, multimodal therapeutic regimens that often result in debilitating long-term side effects. PDGFRα has been implicated in these pediatric malignancies. In this study, we demonstrate the antitumor activity of olaratumab in combination with standard chemotherapy in preclinical models of pediatric bone and soft tissue tumors. These data support clinical evaluation of olaratumab in combination with chemotherapy in pediatric patients with solid tumors (NCT02677116).

The platelet-derived growth factor (PDGF) pathway is composed of two receptors (PDGFRα and PDGFRβ), four homodimeric ligands (PDGF-AA, -BB, -CC, and -DD), and one heterodimeric ligand (PDGF-AB). After ligand binding, PDGFRα and/or PDGFRβ form complexes as homo- or heterodimers, resulting in receptor transphosphorylation and activation of downstream signaling pathways, which in turn regulate normal cellular processes, such as proliferation, migration, and survival (1). Aberrant activation of the PDGF pathway through overexpression of key nodes or receptor mutation often facilitates tumorigenesis and disease progression across several cancer subtypes (2, 3). In addition, PDGF signaling in tumor-associated stromal cells promotes fibroblast activation and angiogenesis (4–7).

The PDGF pathway has been implicated in bone and soft tissue sarcoma, a collection of mesenchymal malignancies comprised of nearly 80 distinct histologies. Olaratumab (LY3012207/IMC-3G3) is a fully human mAb that specifically binds to and inhibits PDGFRα (8). In a phase II clinical study, olaratumab in combination with doxorubicin improved advanced adult sarcoma patient outcome with a median overall survival benefit of 11.8 months when compared with doxorubicin alone (9). On the basis of these data, olaratumab received accelerated approval from the FDA and conditional approval from the European Medicines Agency for treatment of advanced soft tissue sarcoma in combination with doxorubicin in adult patients.

Sarcoma subtypes occurring primarily in the pediatric population, including osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma, account for approximately 15% of childhood cancers (10). Despite intensive multimodal therapy, which typically includes a combination of chemotherapies, surgery, and/or radiotherapy, the current 5-year overall survival rate for pediatric sarcoma patients is approximately 60%; for those who experience a relapse or have metastatic disease, survival drops to only 20% to 30% (11). Malignant rhabdoid tumor (MRT) is a highly aggressive pediatric cancer typically occurring in the kidney and soft tissues or the central nervous system [where it is referred to as atypical teratoid/rhabdoid tumor (AT/RT)] and is characterized by a loss of SMARCB1 (12, 13). Currently, no standard of care exists for this patient population, and overall survival remains poor (14–16). For pediatric cancer survivors, the possibility of debilitating long-term side effects, chronic health conditions, and even secondary cancers resulting from demanding therapeutic regimens remains (17, 18). Therefore, it is of the utmost importance to identify and evaluate targeted agents in the pediatric setting to improve patient outcome.

Although the prevalence of PDGFRA genetic aberrations in pediatric cancer is reported to be only about 2% (19), members of the PDGF pathway are highly expressed in several subtypes of pediatric bone and soft tissue tumors, including rhabdomyosarcoma (20, 21), synovial sarcoma (22), osteogenic sarcoma (23), Ewing sarcoma (24), and MRT (25). In addition, PDGFRα expression is linked to adverse outcomes in pediatric patients with rhabdomyosarcoma (26). In this study, we investigated olaratumab alone and in combination with standard-of-care (SOC) chemotherapy in preclinical models of pediatric bone and soft tissue tumors.

Test compounds

Olaratumab (LY3012207/IMC-3G3, Eli Lilly and Company) was prepared in PBS for both in vitro and in vivo use. The mouse anti-PDGFRα antibody, 1E10, was also prepared in PBS for in vivo experiments.

Cell culture

The HuO9 osteosarcoma cell line was purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Rh18, Rh28, Rh36, and Rh41 rhabdomyosarcoma cell lines were obtained from St. Jude Children's Research Hospital (Memphis, TN). Other pediatric cancer cell lines and WS-1 normal human fibroblasts were ordered from ATCC. Cell lines were Mycoplasma negative prior to freezing working stocks. The frozen working stocks of each cell line were authenticated by STR-based DNA profiling and multiplex PCR. The genetic profiles for the samples were identical to the genetic profiles reported for these cell lines. BT-12 and BT-16 AT/RT cell lines were a gift from Dr. Peter Houghton (Greehey Children's Cancer Research Institute, San Antonio, TX). Cell line details are listed in Supplementary Table S1. All cells were maintained at 37°C and 5% CO2 in tissue culture–treated flasks. Cells were used for experiments within 10 to 20 passages and then discarded.

Reagents and antibodies

Specific details regarding recombinant human PDGFs (AA, BB, CC, DD, and AB) are displayed in Supplementary Table S2. Antibodies against PDGFRα, PDGFRα Y754, PDGFRβ, PDGFRβ 751, ERK1/2, ERK1/2 T202/Y204, AKT, and AKT S473 were purchased from Cell Signaling Technology or Santa Cruz Biotechnology; details are included in Supplementary Table S2.

Western blot analysis

To determine receptor status and baseline pathway signaling in a panel of pediatric bone and soft tissue tumor lines, cells were serum starved overnight and harvested into 1% SDS lysis buffer containing 1× HALT protease and phosphatase inhibitor (Thermo Fisher Scientific, cat #78441). To determine effects of olaratumab treatment, cell lines were treated with either an IgG control antibody or olaratumab for 15 minutes and then stimulated with individual PDGFs (final concentration, 5 nmol/L) for an additional 15 minutes. Cells were immediately washed with PBS and harvested into 1% SDS lysis buffer containing 1× HALT protease and phosphatase inhibitor.

Protein expression and phosphorylation status was assessed by SDS-PAGE and immunoblotting as described previously (27). Briefly, whole-cell lysates (30–50 μg of protein/sample) were separated on gradient Tris-Glycine protein gels (Novex, Thermo Fisher Scientific) and transferred to nitrocellulose via TransBlot Turbo (Bio-Rad, cat #170–4159). After blocking with 5% milk in TBST, membranes were probed with primary antibody overnight at 4°C, washed 3 times with TBST, and incubated with secondary antibodies for 1 hour at room temperature. Following 3 washes with TBST, membranes were developed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, cat #34095) and imaged with a Bio-Rad ChemiDoc XRS.

Cell proliferation assay

To determine antiproliferation EC50 values for olaratumab, pediatric cancer cells were seeded in 96-well plates. The next day, cells were incubated with increasing concentrations of olaratumab or the IgG antibody control in serum-free media for 2 hours at 37°C and then stimulated with individual PDGFs. Cell proliferation was assayed by CellTiter Glo Luminescent Cell Viability Assay (Promega, cat #G7571) 72 hours after stimulation. Data were normalized to the unstimulated IgG-treated control. Relative EC50 values were calculated using GraphPad Prism software. Experiments were repeated in triplicate and data from a representative experiment are displayed in Table 1.

Table 1.

Receptor status and relative EC50 values for proliferation inhibition in a panel of pediatric bone and soft tissue tumor cell lines

Relative EC50 (nmol/L) - antiproliferation
Cell lineDiseasePDGFRαaPDGFRβa+AA+BB+CC+DD+AB
A-204 MRT +  45.74 170.7 71.42 NDb 123 
BT-12 AT/RT  + Not evaluated 
BT-16 AT/RT   Not evaluated 
G-401 MRT  + Not evaluated 
G-402 MRT + 167.3 498.2 70.95 ND ND 
RD Embryonal RMS   Not evaluated 
RD-ES Ewing sarcoma   Not evaluated 
Rh18 Alveolar RMS  + Not evaluated 
Rh28 Alveolar RMS   Not evaluated 
Rh36 Embryonal RMS   Not evaluated 
Rh41 Alveolar RMS +  Not evaluated 
SJCRH30 Alveolar RMS   Not evaluated 
HuO9 Osteosarcoma +  204.7 311.3 35.11 20.1 164 
KHOS/NP Osteosarcoma + + ND 
MG-63 Osteosarcoma + + 52.37 186 38.03 ND 101.1 
MNNG/Hos Osteosarcoma + + ND 
Saos-2 Osteosarcoma +  Not evaluated 
SJSA-1 Osteosarcoma + + ND 
WS-1 Normal fibroblast + + Not evaluated 
Relative EC50 (nmol/L) - antiproliferation
Cell lineDiseasePDGFRαaPDGFRβa+AA+BB+CC+DD+AB
A-204 MRT +  45.74 170.7 71.42 NDb 123 
BT-12 AT/RT  + Not evaluated 
BT-16 AT/RT   Not evaluated 
G-401 MRT  + Not evaluated 
G-402 MRT + 167.3 498.2 70.95 ND ND 
RD Embryonal RMS   Not evaluated 
RD-ES Ewing sarcoma   Not evaluated 
Rh18 Alveolar RMS  + Not evaluated 
Rh28 Alveolar RMS   Not evaluated 
Rh36 Embryonal RMS   Not evaluated 
Rh41 Alveolar RMS +  Not evaluated 
SJCRH30 Alveolar RMS   Not evaluated 
HuO9 Osteosarcoma +  204.7 311.3 35.11 20.1 164 
KHOS/NP Osteosarcoma + + ND 
MG-63 Osteosarcoma + + 52.37 186 38.03 ND 101.1 
MNNG/Hos Osteosarcoma + + ND 
Saos-2 Osteosarcoma +  Not evaluated 
SJSA-1 Osteosarcoma + + ND 
WS-1 Normal fibroblast + + Not evaluated 

Abbreviations: ND, not detected; RMS, rhabdomyosarcoma.

aExpression determined by Western blot analysis; boldface indicates strong signal observed.

bND, not determined due to ambiguous or nonconverged curves.

To evaluate the effects of combination treatment on pediatric cancer cell proliferation, cells were plated in 96-well microtiter plates in normal media containing 10% FBS. The next morning, media were changed to media containing 0.5% FBS. After 4 hours, cells were costimulated with 5 nmol/L PDGF-AA and 5 nmol/L PDGF-CC and treated with 1 μmol/L olaratumab, 0.5 μmol/L doxorubicin, or 2 μmol/L cisplatin or combination of olaratumab and chemotherapy. Cell proliferation was assayed after 72 hours by CellTiter Glo. Luminescence was normalized to the average of the untreated control for each cell line. Results are presented as the mean of duplicate or triplicate experiments (indicated in figure legends) ± SEM. Statistical significance was determined by the Student t test.

Evaluation of cell invasion

Invasion assays were performed using the CultreCoat Low BME Cell Invasion Assay (Trevigen, cat #3481–096-K) per the manufacturer's instructions. Briefly, 2.5 × 104 pediatric cancer cells were seeded in the upper chamber of the well with 1 μmol/L IgG control antibody or olaratumab. Serum-free media was added to the lower chamber, and individual PDGFs were used as a chemoattractant. Media with 10% FBS as a chemoattractant served as a positive control. Final concentration of ligand was 5 nmol/L for each, with the exception of PDGF-DD, which was 20 nmol/L. After 48 hours, cells able to invade through the membrane to the lower chamber were dissociated, and plates were read at 485 nm excitation, 520 nm emission. Results were normalized to the serum-free media, IgG control for each cell line. Results are presented as the average of duplicate or triplicate experiments (indicated in figure legends) ± SEM. Significance was determined by the Student t test.

Expression profiling of patient-derived xenograft mouse models

Samples from pediatric sarcoma patient-derived xenografts (PDX) were obtained from Champions Oncology, START Discovery, and Oncotest (Charles River Laboratories). Frozen tumors were manually dissociated using a TissueLyser (Qiagen). RNA extraction was performed using the MagMAX-96 total RNA Isolation Kit (Thermo Fisher Scientific, cat#AM1830) per the manufacturer's instructions. The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, cat #4374967) was used to generate cDNA. TaqMan Gene Expression Master Mix (cat#4369016) and TaqMan probes for human PDGFRA, PDGFRB, PDGFA, PDGFC, and 18S were purchased from Thermo Fisher Scientific (cat #4331182). Probe IDs are as follows: PDGFRA – HS00998018_m1; PDGFRB – HS01019589_m1; PDGFA – HS00964426_m1; PDGFC – HS01044219_m1; and 18S – HS03928985_g1. A standard curve was generated for each gene using a plasmid DNA template, and data were extracted using this curve. Values are expressed as copies of mRNA/ng of cDNA.

In vivo evaluation of olaratumab

A colorimetric ELISA was used to quantify the concentration of olaratumab in mouse serum and mouse plasma. Briefly, the PDGFRα extracellular domain was immobilized in coating buffer in 96-well microtiter plate. After washing the plates, wells were blocked with block/diluent buffer between 1 and 2 hours. Plates were then washed and analytes (diluted 1:500 in block/diluent buffer) were added to each well, incubated for approximately 1 hour, and washed. Goat α-human IgG F(ab)2 was added to each well and incubated for approximately 1 hour. The plates were washed, followed by the addition of tetramethylbenzidine to each well and incubation for 15 minutes. The reaction was terminated with the addition of a Stop Solution. Plates were read at 450 nm for detection of olaratumab and at 620 nm for background signal. The concentration of olaratumab was determined on a standard curve.

In vivo studies were performed in accordance with American Association for Laboratory Animal Care institutional guidelines. A-204 and HuO9 in vivo experiments were approved by the Eli Lilly and Company Animal Care and Use Committee. To evaluate the effects of olaratumab in HuO9 and A-204 cell line–derived xenografts, cells were harvested during log-phase growth and resuspended in Hank balanced salt solution. Cell suspensions containing 10 × 106 (HuO9) or 5 × 106 (A-204) cells (0.2 mL total volume) were subcutaneously injected into the right flank of female athymic nude mice and tumor growth monitoring started 1 week postinjection. When tumor volumes averaged 200 mm3, mice were randomized into treatment groups (n = 6/group) based on tumor volume and body weight. In vivo experiments utilizing the osteosarcoma PDX model CTG-1095 were conducted at Champions Oncology. The CCSARC005 osteosarcoma PDX model was generated and evaluated at Covance, Inc.

Mice with A-204 xenografts were treated with 40 mg/kg IgG or olaratumab three times a week for 4 weeks. Animals bearing HuO9 or CTG-1095 tumors were treated with control (80 mg/kg IgG, i.p. + vehicle), cisplatin (5 mg/kg once weekly, i.p.), doxorubicin (5 mg/kg once weekly, i.v.), olaratumab + 1E10 (60 and 20 mg/kg, respectively, twice weekly, i.p.), or combination of olaratumab + 1E10 and a SOC for 5 weeks. Mice with CCSARC005 xenograft tumors were treated with control (60 mg/kg IgG, i.p. + vehicle), olaratumab (60 mg/kg, twice weekly, i.p.), cisplatin (4 mg/kg once weekly, i.p.), or a combination of the two. Tumor volume was monitored twice weekly. For all studies, experiments were terminated within 2 weeks following the end of the treatment period.

Expression of PDGF pathway components is detected in several pediatric sarcoma cell lines

Gene expression of PDGFRA and PDGFRB and of PDGFRα-activating ligands PDGFA and PDGFC was evaluated in 7 pediatric bone and soft tissue tumor cell lines (Fig. 1). The osteosarcoma cell line HuO9 and the MRT cell line A-204 exclusively expressed PDGFRA, while the transcripts of both receptors were detected in MG-63, KHOS/NP, and MNNG/Hos osteosarcoma cell lines (Fig. 1A). A-204, HuO9, and MG-63 also expressed PDGFA and/or PDGFC, indicating the potential for autocrine activation of PDGFRα (Fig. 1B). Gene expression correlated with PDGFRα and PDGFRβ protein expression (Supplementary Fig. S1). Furthermore, PDGFRα expression was detected in G-402 MRT cells but not in G-401 MRT cells nor in BT-12 or BT-16 AT/RT cells (Supplementary Fig. S1A). Expression of PDGFRα and β was also not observed in the embryonal rhabdomyosarcoma cell line RD or the Ewing's sarcoma cell line RD-ES (Supplemental Fig. S1B). Surprisingly, although previous studies reported robust PDGFRα expression in human and murine rhabdomyosarcoma (20, 28), low levels of PDGFRA and PDGFRB transcript were detected in the alveolar rhabdomyosarcoma cell line SJCRH30 (Fig. 1A). Similarly, PDGFRα was not detected at the protein level in 3 of 4 rhabdomyosarcoma cell lines (Supplementary Fig. S1C). As olaratumab is a selective anti-PDGFRα antibody, this study focused on receptor-expressing pediatric models (Table 1; Fig. 1; and Supplementary Fig. S1). Receptor status for all evaluated cell lines is summarized in Table 1.

Figure 1.

Pediatric bone and soft tissue tumor cell lines express platelet-derived growth factors and their receptors. A panel of pediatric bone and soft tissue tumor cell lines were evaluated for receptor and ligand expression by qPCR. A, Results are plotted as expression of PDGFRA by PDGFRB on a log10 scale. Note: expression of PDGFRB did not exceed 1,000 copies/ng cDNA. B, Expression of PDGFA and PDGFC are presented on a linear scale.

Figure 1.

Pediatric bone and soft tissue tumor cell lines express platelet-derived growth factors and their receptors. A panel of pediatric bone and soft tissue tumor cell lines were evaluated for receptor and ligand expression by qPCR. A, Results are plotted as expression of PDGFRA by PDGFRB on a log10 scale. Note: expression of PDGFRB did not exceed 1,000 copies/ng cDNA. B, Expression of PDGFA and PDGFC are presented on a linear scale.

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PDGFRα phosphorylation is blocked by olaratumab in pediatric osteosarcoma cell lines

Olaratumab specifically binds PDGFRα and prevents ligand binding to the receptor and stimulating transphosphorylation (8). The influence of PDGFRβ expression and potential PDGFRα:PDGFRβ heterodimers on olaratumab activity is currently not well understood; therefore, in vitro evaluation of olaratumab was conducted using cell lines with varying expression levels of PDGFRα and/or PDGFRβ. Furthermore, BT-16, an AT/RT cell line that does not express either receptor, was investigated alongside PDGFR+ cell lines as a negative control. The WS-1 normal fibroblast cell line was used as a nontransformed cell control.

Phosphorylation status of the PDGF receptors and their downstream effectors was assessed following stimulation by individual PDGFs (Fig. 2; Supplementary Figs. S2 and S3A). Phosphorylation of PDGFRα at tyrosine 754 (Y754) was detected in PDGFRα-positive cell lines, albeit to varying degrees, and indicated receptor activation. Stimulation with PDGF-BB or -DD elicited phosphorylation of PDGFRβ at tyrosine 771 (Y771) in PDGFRβ-expressing cell lines (Fig. 2B and C; Supplementary Fig. S2); however, phospho-PDGFRβ was not detected in BT-12 AT/RT cells (Supplementary Fig. S3A). Stimulation of HuO9 and MG-63 OS cells with PDGFs resulted in activation of downstream PI3K and MAPK pathway signaling [as measured by phosphorylation of AKT at serine 473 (S473) and ERK1/2 threonine 202/tyrosine 204 (T202/Y204); Fig. 2A and B]. Baseline AKT and ERK1/2 phosphorylation was observed in all MRT and AT/RT cell lines evaluated regardless of PDGF receptor status (Fig. 2; Supplementary Fig. S3A). Stimulation with any PDGF (in the case of A-204 and G-402; Fig. 2A and B) or PDGFRβ-activating ligands (in G-401 and BT-12 cells; Fig. 2C; Supplementary Fig. S3A) resulted in a detectable increase in pAKT and pERK1/2. Surprisingly, PDGF-CC stimulation elicited a marked increase in AKT and ERK1/2 phosphorylation in BT-16 cells, suggesting that PDGFRα may be expressed below the level that can be detected by Western blot analysis (Supplementary Fig. S3A).

Figure 2.

Olaratumab prevents ligand-induced phosphorylation of PDGFRα. A–C, A-204 and HuO9 cells (A), which only express PDGFRα, G-402 and MG-63 cells (B), which express both PDGF receptors, or G-401 cells (C), which are PDGFRβ positive, were treated with 1 μmol/L IgG or olaratumab for 15 minutes and then stimulated with individual PDGFs (5 nmol/L). Whole-cell lysates were probed for phosphorylation of PDGF receptors and downstream effectors AKT and ERK1/2.

Figure 2.

Olaratumab prevents ligand-induced phosphorylation of PDGFRα. A–C, A-204 and HuO9 cells (A), which only express PDGFRα, G-402 and MG-63 cells (B), which express both PDGF receptors, or G-401 cells (C), which are PDGFRβ positive, were treated with 1 μmol/L IgG or olaratumab for 15 minutes and then stimulated with individual PDGFs (5 nmol/L). Whole-cell lysates were probed for phosphorylation of PDGF receptors and downstream effectors AKT and ERK1/2.

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As expected, incubation of cells with olaratumab prior to ligand stimulation blocked PDGFRα Y754 phosphorylation in PDGFRα-positive cell lines (Fig. 2; Supplementary Fig. S2). Interestingly, olaratumab blocked activation of AKT and ERK1/2 resulting from stimulation with any ligand in HuO9 cells (Fig. 2A). Similarly, phosphorylation of AKT was strongly inhibited with olaratumab treatment in A-204 cells, whereas ERK1/2 phosphorylation was reduced to baseline signal with treatment (Fig. 2A). PDGFRβ phosphorylation at Y771 was sustained following olaratumab treatment in PDGFRβ-expressing MG-63 and G-401 as well as WS-1 fibroblasts (Fig. 2B and C; Supplementary Fig. S2). However, PDGF-BB and -AB-stimulated PDGFRβ phosphorylation was noticeably reduced with olaratumab treatment in G-402 MRT cells (Fig. 2B). Reduced phosphorylation of AKT and ERK1/2 in PDGFRα/PDGFRβ coexpressing cells (Fig. 2B; Supplementary Fig. S2) was observed only when olaratumab prevented PDGFRα stimulation by PDGF-AA, -AB, or (with the exception of MG-63) PDGF-CC; furthermore, AKT and ERK1/2 phosphorylation in G-401 cells did not change with olaratumab treatment (Supplementary Fig. S2A), indicating that PDGFRβ homodimeric signaling is not affected by olaratumab. Interestingly, olaratumab treatment slightly reduced pAKT and pERK1/2 in BT-16 cells stimulated with PDGF-AA or -CC (Supplementary Fig. S3A).

PDGF-stimulated cell proliferation is inhibited by olaratumab alone and in combination with chemotherapeutic agents

As the PDGF pathway supports tumor cell growth (1), the potential antiproliferative effect of olaratumab was assessed in a panel of pediatric bone and soft tissue tumor cell lines (summarized in Table 1). In addition, olaratumab was evaluated in combination with either doxorubicin or cisplatin in osteosarcoma and rhabdoid tumor cell lines (Supplementary Figs. S3B, S3C, and S4; Supplementary Table S3). Stimulation with PDGF-AA and PDGF-CC increased cell proliferation in PDGFRα-expressing cell lines when compared with unstimulated control cells (Supplementary Fig. S4A–S4D; P values reported in Supplementary Table S3). The increase in A-204 and HuO9 proliferation in response to ligand stimulation was modest when compared with the unstimulated control; however, these cell lines also express PDGFRα-activating ligands and likely activate the receptor in an autocrine manner (Supplementary Fig. S4A and S4B). As expected, PDGFRα-null rhabdoid cell lines G-401, BT-12, and BT-16 did not respond to PDGF-AA/-CC treatment with increased proliferation (Supplementary Figs. S3B, S3C and S4E).

Doxorubicin significantly reduced tumor cell proliferation regardless of receptor status or stimulation with exogenous ligand (Supplementary Figs. S3B, S3C, and S4). Combination of doxorubicin with olaratumab in A-204 and HuO9 modestly enhanced this reduction by an additional 10% (Supplementary Fig. S4A and S4B). Cisplatin plus olaratumab resulted in a 20% to 30% further reduction in proliferation in PDGFRα-positive osteosarcoma cell lines when compared with single-agent treatment (Supplementary Fig. S4A–S4D). As expected, olaratumab treatment did not affect proliferation in cells that do not express the receptor (Supplementary Figs. S3B, S3C, and S4E).

Olaratumab has varying effects on PDGF-driven tumor cell invasion

The ability of olaratumab to modulate cell invasion through PDGFRα inhibition was also evaluated in osteosarcoma and rhabdoid tumor cell lines (Fig. 3; Supplementary Fig. S3D and S3E). Serum elicited tumor cell invasion when used as a chemoattractant regardless of PDGF receptor status. Interestingly, serum-stimulated A-204, HuO9, and G-402 cell invasion was reduced with olaratumab treatment by approximately 50% when compared with the IgG control treatment group (Fig. 3A–C). Unexpectedly, A-204 and HuO9 cell invasion did not increase substantially when any of the individual PDGFs were used as the chemoattractant, which again suggests that PDGFRα is already activated via an autocrine loop (Fig. 3A and B). In contrast, all PDGFs (with the exception of PDGF-AB in the case of MG-63) promoted invasion of cells that coexpressed PDGFRα and PDGFRβ (Fig. 3C and D). MG-63 cells were particularly responsive to the ligands when compared with the 10% FBS control chemoattractant (Fig. 3D). Olaratumab treatment significantly blocked HuO9, G-402, and MG-63 cell invasion stimulated by the different ligands; however, although olaratumab treatment reduced HuO9 invasion, these changes were less than 20% of the untreated control. Furthermore, olaratumab did not inhibit the invasive potential of tumor cells that lacked PDGFRα expression (G-401, BT-12, and B-16; Fig. 3E; Supplementary Fig. S3D and S3E).

Figure 3.

Olaratumab reduces osteosarcoma and MRT cell invasion in vitro. A–E, A-204 (A), HuO9 (B), G-402 (C), MG-63 (D), or G-401 cells (E) were evaluated for cell invasion using a simplified Boyden chamber assay. Cells were incubated with IgG or olaratumab; individual PDGFs (5 nmol/L each, with the exception of 25 nmol/L PDGF-DD) or FBS served as chemoattractants. After 48 hours, the number of invasive cells was quantified using a fluorescence-based method and normalized to the serum-free media (SFM) IgG control. Results are presented as the summary of duplicate (G-402) or triplicate experiments ± SEM. P values were calculated using the Student t test. Significance indicators: *, P < 0.05; ***, P < 0.001.

Figure 3.

Olaratumab reduces osteosarcoma and MRT cell invasion in vitro. A–E, A-204 (A), HuO9 (B), G-402 (C), MG-63 (D), or G-401 cells (E) were evaluated for cell invasion using a simplified Boyden chamber assay. Cells were incubated with IgG or olaratumab; individual PDGFs (5 nmol/L each, with the exception of 25 nmol/L PDGF-DD) or FBS served as chemoattractants. After 48 hours, the number of invasive cells was quantified using a fluorescence-based method and normalized to the serum-free media (SFM) IgG control. Results are presented as the summary of duplicate (G-402) or triplicate experiments ± SEM. P values were calculated using the Student t test. Significance indicators: *, P < 0.05; ***, P < 0.001.

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Olaratumab, alone or in combination with chemotherapy, delays xenograft growth

Single-agent olaratumab was first investigated in mice bearing A-204 MRT xenografts, as the A-204 cell line expresses both PDGFRα and its activating ligand PDGFC. Tumor-bearing animals were given 40 mg/kg olaratumab or control antibody three times weekly for 4 weeks. Using this dosing schedule, olaratumab treatment significantly delayed A-204 xenograft growth in all treated animals, with stable disease observed in 55% of the treated arm (Fig. 4A and B; Supplementary Table S4).

Figure 4.

Olaratumab inhibits tumor growth in A-204 and HuO9 cell line–derived xenograft mouse models. A and B, Mice with A-204 subcutaneous xenografts were treated with either 40 mg/kg IgG control or 40 mg/kg olaratumab three times weekly for 4 weeks. A, Average tumor volume ± SEM (n = 11/group) during the treatment period is shown. B, Waterfall plot of %Δtumor/control or %regression for individual animals at day 30. One animal in the treatment arm was found dead on day 17 and was not included in downstream analysis. C and D, Mice bearing HuO9 subcutaneous xenografts were treated with olaratumab (60 mg/kg)/1E10 (20 mg/kg) twice weekly and/or cisplatin or doxorubicin (5 mg/kg) once weekly. Treatment began at day 44 (dotted line) and continued for 5 weeks. C, A single HuO9 in vivo experiment is displayed in two separate graphs with the same control and olaratumab/1E10 arms but different chemotherapy-containing arms containing chemotherapy (left, cisplatin; right, doxorubicin). The graph displays average tumor volume ± SEM (n = 6/group). D, Waterfall plot of individual %Δtumor/control or %regression at day 60. P values for tumor growth curves are displayed in Supplementary Table S4. PD, progressive disease (>10% growth); SD, stable disease (−50%–10% growth); PR, partial regression (≤−50% and >14 mm3).

Figure 4.

Olaratumab inhibits tumor growth in A-204 and HuO9 cell line–derived xenograft mouse models. A and B, Mice with A-204 subcutaneous xenografts were treated with either 40 mg/kg IgG control or 40 mg/kg olaratumab three times weekly for 4 weeks. A, Average tumor volume ± SEM (n = 11/group) during the treatment period is shown. B, Waterfall plot of %Δtumor/control or %regression for individual animals at day 30. One animal in the treatment arm was found dead on day 17 and was not included in downstream analysis. C and D, Mice bearing HuO9 subcutaneous xenografts were treated with olaratumab (60 mg/kg)/1E10 (20 mg/kg) twice weekly and/or cisplatin or doxorubicin (5 mg/kg) once weekly. Treatment began at day 44 (dotted line) and continued for 5 weeks. C, A single HuO9 in vivo experiment is displayed in two separate graphs with the same control and olaratumab/1E10 arms but different chemotherapy-containing arms containing chemotherapy (left, cisplatin; right, doxorubicin). The graph displays average tumor volume ± SEM (n = 6/group). D, Waterfall plot of individual %Δtumor/control or %regression at day 60. P values for tumor growth curves are displayed in Supplementary Table S4. PD, progressive disease (>10% growth); SD, stable disease (−50%–10% growth); PR, partial regression (≤−50% and >14 mm3).

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A dose-dependent increase in mean trough serum concentration was measured following olaratumab dose escalation in two cell line–derived xenograft mouse models (Supplementary Fig. S5A). On the basis of these results, 60 mg/kg olaratumab twice weekly was selected as the dosing schedule used in future in vivo experiments. Trough mean serum concentrations of olaratumab were determined in several cell line–derived and PDX mouse models and were found to be relatively consistent across the evaluated models (Supplementary Fig. S5B).

Olaratumab was further investigated in mice bearing HuO9 osteosarcoma xenografts (Fig. 4C and D; Supplementary Table S4). To interrogate and potentially disrupt the relationship between the human tumor and mouse stroma, the mouse anti-PDGFRα antibody 1E10 was given in conjunction with olaratumab. In addition to single-agent olaratumab/1E10 treatment, combination with doxorubicin or cisplatin (two SOC agents used in the treatment of osteosarcoma patients) was also evaluated. Administration of olaratumab/1E10, cisplatin, or doxorubicin alone delayed tumor growth when compared with the control arm (Fig. 4C). Furthermore, stable disease was achieved in 1 or 2 animals in each group (Fig. 4D). The combination of olaratumab/1E10 plus cisplatin (Fig. 4C, left) resulted in stable disease in 4 of 6 mice (Fig. 4D), whereas combination with doxorubicin (Fig. 4C, right) elicited a partial regression in one animal in addition to 3 of 6 animals with stable disease.

PDX models of pediatric bone and soft tissue sarcoma were profiled for expression of PDGFRA, PDGFRB, PDGFA, and PDGFC (Supplementary Table S5). Expression of each gene was variable, both within and between sarcoma subtypes. PDGFRA transcript was detectable in 50% (11/22) of models assayed with a cutoff of at least 500 copies mRNA/ng cDNA. Osteosarcoma PDXs made up more than half (6/11) of the PDGFRA-expressing models. Activity of olaratumab was evaluated in the CTG-1095 model, an osteosarcoma PDX that expressed moderate levels of PDGFRA, PDGFRB, and PDGFC (Supplementary Table S5). Olaratumab/1E10 treatment impaired tumor growth, as evidenced by a reduction in tumor volume when compared with the control arm (Fig. 5A and B; Supplementary Table S4). Although doxorubicin did not demonstrate any activity in the xenograft tumors, single-agent cisplatin significantly delayed tumor growth. In addition, stable disease was achieved in 2 animals receiving olaratumab/1E10 in combination with cisplatin (Fig. 5B).

Figure 5.

Olaratumab, alone or in combination with cisplatin, reduces tumor volume in two osteosarcoma PDX mouse models. A and B, Mice bearing CTG-1095 osteosarcoma PDX tumors were treated with olaratumab/1E10 (60 mg/kg olaratumab/20 mg/kg 1E10) twice weekly and/or SOC [cisplatin or doxorubicin (5 mg/kg)] once weekly (n = 5/group). One animal in the olaratumab/1E10 group was sacrificed prior to experiment termination due to circumstances unrelated to treatment. A, Dosing began on day 0, and average tumor volume ± SEM over the 5-week dosing period is shown. B, Waterfall plot of %Δtumor/control or %regression for individual animals at day 21. C and D, Animals with CCSARC005 osteosarcoma PDX tumors were treated with olaratumab (60 mg/kg) twice weekly and/or 4 mg/kg cisplatin once weekly (n = 5/group). C, Dosing period is indicated by the dotted vertical lines. Average tumor volume ± SEM is shown. D, Waterfall plot of %Δtumor/control or %regression for individual mice at day 62. P values for tumor growth curves are displayed in Supplementary Table S4. PD, progressive disease (>10% growth); SD, stable disease (−50%–10% growth); PR, partial regression (≤−50% and >14 mm3).

Figure 5.

Olaratumab, alone or in combination with cisplatin, reduces tumor volume in two osteosarcoma PDX mouse models. A and B, Mice bearing CTG-1095 osteosarcoma PDX tumors were treated with olaratumab/1E10 (60 mg/kg olaratumab/20 mg/kg 1E10) twice weekly and/or SOC [cisplatin or doxorubicin (5 mg/kg)] once weekly (n = 5/group). One animal in the olaratumab/1E10 group was sacrificed prior to experiment termination due to circumstances unrelated to treatment. A, Dosing began on day 0, and average tumor volume ± SEM over the 5-week dosing period is shown. B, Waterfall plot of %Δtumor/control or %regression for individual animals at day 21. C and D, Animals with CCSARC005 osteosarcoma PDX tumors were treated with olaratumab (60 mg/kg) twice weekly and/or 4 mg/kg cisplatin once weekly (n = 5/group). C, Dosing period is indicated by the dotted vertical lines. Average tumor volume ± SEM is shown. D, Waterfall plot of %Δtumor/control or %regression for individual mice at day 62. P values for tumor growth curves are displayed in Supplementary Table S4. PD, progressive disease (>10% growth); SD, stable disease (−50%–10% growth); PR, partial regression (≤−50% and >14 mm3).

Close modal

Olaratumab treatment, alone or in combination with cisplatin, was also investigated in the CCSARC005 osteosarcoma PDX model. IHC analysis of xenograft tumors revealed that a small number of tumor cells express PDGFRα; however, PDGFRβ was readily detectable by Western blot analysis (Supplementary Fig. S6). Treatment with olaratumab or cisplatin alone had no effect on tumor growth when compared with tumors from vehicle-treated animals (Fig. 5C). The combination of olaratumab and cisplatin significantly inhibited xenograft tumor growth with tumors in 2 of 5 mice meeting the criteria for stable disease (Fig. 5C and D) and the remaining mice within the cohort responding with strong tumor growth delay. Tumors from the combination arm were significantly smaller when compared with vehicle- or single agent–treated tumors at study termination (Fig. 5C; Supplementary Table S4).

Aberrant expression and/or activation of PDGFRα is implicated in several subtypes of adult and pediatric sarcoma and supports protumorigenic processes in both the tumor cells and associated stroma (1). Therefore, anti-PDGFRα therapies are of high interest; several molecules targeting PDGFRα have been developed and evaluated both preclinically and clinically in adult and pediatric malignancies (29–31). Olaratumab (LY3012207/IMC-3G3) is a fully human IgG1 mAb, which specifically binds to PDGFRα, thus inhibiting ligand-binding and receptor activation (8). Following a pivotal randomized phase II trial, olaratumab received accelerated FDA approval and conditional EMA approval for treatment of advanced soft tissue sarcoma in adult patients in combination with doxorubicin (9). In this study, we demonstrate olaratumab activity in preclinical pediatric bone and soft tissue tumor models. Olaratumab specifically blocked phosphorylation of PDGFRα and attenuated associated downstream MAPK and PI3K pathway signaling in PDGFRα-positive tumor cell lines. Furthermore, as a single agent, olaratumab significantly delayed osteosarcoma and rhabdoid xenograft growth; combination with doxorubicin or cisplatin resulted in stable disease in three mouse models of human osteosarcoma.

Therapeutic targeting of PDGFRα with small-molecule inhibitors, such as sunitinib and imatinib, has been investigated preclinically and clinically in multiple pediatric cancer types, including osteosarcoma and MRT (25, 32–35). As many of these drugs are designed to bind the ATP site within the kinase domain, target specificity is often lacking, resulting in inhibition of multiple receptor tyrosine kinases and associated toxicity (34, 36). This target promiscuity makes it difficult to assign the direct contribution of PDGFRα to tumor growth and cancer cell survival in preclinical studies using small-molecule inhibitors. In contrast, mAbs are engineered toward a specific target, and cross-reactivity has not been reported (36). Olaratumab blocks ligand binding and receptor dimerization by specifically binding to PDGFRα and as such is able to suppress potential kinase domain-independent signaling (37). Furthermore, olaratumab-bound PDGFRα is internalized and degraded, which attenuates downstream pathway activation and likely further contributes to drug activity (8, 37).

Although PDGFRA is not frequently altered in childhood cancers (19, 38, 39), receptor expression is associated with progressive disease and poor prognosis in some pediatric malignancies of bone and soft tissue (11, 26, 40). Expression of the receptor was readily detected in several osteosarcoma cell lines and PDX models, which was expected, as 50% of osteosarcoma primary samples were previously found to be PDGFRα positive (23). In addition, PDGFRα-activating ligands (namely, PDGFA and/or PDGFC) were also expressed in these cells lines. Conversely, PDGFRα was not expressed at the gene or protein level in the majority of rhabdomyosarcoma cell lines and PDX models in our study nor in a previous study that examined PDGFRα in SJCRH30 and other rhabdomyosarcoma cell lines (41). These findings were particularly surprising, as PDGFRα is readily detectable in rhabdomyosarcoma clinical specimens by qPCR or IHC and PDGFRA is a transcriptional target of the fusion protein driving alveolar rhabdomyosarcoma (PAX3-FOXO; refs. 20, 22, 26). Similarly, the Ewing sarcoma PDX models evaluated in this study were negative for PDGFC expression, despite previous work identifying PDGFC as a target gene of the EWS-FLI1 fusion transcription factor (42). These data are suggestive of potential alterations in the tumor transcriptome when cultured or propagated outside of its native environment and are reminiscent of a previous report describing changes in the epigenome and transcriptome of mammalian cells isolated from primary tissues upon exposure to cell culture (43). Furthermore, a recent study demonstrated that an anti-PDGFRα polyclonal antibody frequently used in previous IHC analyses cross-reacts with PDGFRβ, further confounding previous reports regarding the prevalence of PDGFRα expression in pediatric bone and soft tissue tumor patient samples (44). In light of this discordance in receptor expression between available preclinical bioresources and clinical specimens, it is difficult to investigate the full range of olaratumab activity in pediatric sarcoma in a preclinical setting and to identify potential predictive biomarkers. Furthermore, although PDGFRα expression in cell lines and xenograft models was necessary for olaratumab to demonstrate some antitumor activity in preclinical models, expression alone was insufficient to predict sensitivity, echoing the findings of the pivotal phase II study in adult STS patients (9). Further development of preclinical models that better recapitulate human disease is necessary to predict the clinical activity of novel agents and to understand additional factors that may influence sensitivity.

PDGFC has been reported to be highly expressed in A-204 cells, suggesting the potential for an autocrine manner of PDGFRα activation (41); indeed, shRNA-mediated silencing of PDGFC significantly reduced A-204 proliferation, comparable with the knockdown of PDGFRA. We confirmed that PDGFC was expressed in A-204 cells and found that HuO9 expressed both PDGF-AA and -CC, indicating another potential autocrine loop. Furthermore, we observed that exogenous PDGFs were not chemoattractive and could not promote invasion of serum-starved HuO9 or A-204 cells. Insensitivity of HuO9 and A-204 to exogenously supplied PDGFs may be explained by saturation of PDGFRα via endogenously produced ligands. Interestingly, the addition of serum elicited an increase in A-204 and HuO9 osteosarcoma cell invasion well above that observed when PDGFs were used as chemoattractants. Olaratumab subsequently blocked this serum-induced invasion, suggesting that PDGFRα may accommodate signaling initiated by non-PDGF ligands and/or through noncanonical receptor dimerization partners.

As expected, olaratumab blocked PDGF-stimulated PDGFRα phosphorylation in alpha-positive normal and pediatric tumor cell lines. PDGFRβ phosphorylation and PDGFRβ-activated downstream pathways were not inhibited by treatment, further illustrating the specificity of olaratumab for the alpha receptor. Interestingly, varied effects on downstream MAPK and PI3K pathway signaling were observed following ligand stimulation and olaratumab treatment in MG-63 and WS-1 cells, potentially influenced by coexpression of PDGFRα and PDGFRβ. Dimerization and subsequent activation of PDGFRα is primarily driven by binding PDGF-AA and PDGF-CC, whereas PDGFRβ has a higher affinity for PDGF-BB and PDGF-DD (1, 45). Heterodimerization can occur after binding any ligand except for PDGF-AA. The expression of PDGFRα alone in HuO9 OS cells and A-204 MRT cells seemingly heightens the affinity of the receptor for individual PDGFs, and olaratumab is therefore highly effective at blocking MAPK and PI3K pathway activation caused by PDGFRα signaling. In contrast, MG-63 and WS-1 cells express both PDGFRα and PDGFRβ and downstream signaling persisted after PDGF-BB, -DD, and -AB stimulation, even in the presence of olaratumab. Furthermore, PDGFRβ phosphorylation and downstream pathway signaling was unaffected by olaratumab in G-401 MRT cells, which only express the beta receptor; however, olaratumab reduced PDGF-BB or -AB-stimulated PDGFRβ phosphorylation in G-402 MRT cells. The differences in signal propagation initiation and maintenance are most likely influenced by the patterns of homo- and heterodimerization. Furthermore, although it is generally accepted that specific PDGFs activate PDGFRα or PDGFRβ, our data suggest that receptor affinity for ligand may be flexible and dependent on relative expression levels of PDGFRα and PDGFRβ protein.

To our knowledge, this study is the first to demonstrate that the antitumor activity of olaratumab observed in adult sarcomas can extend to preclinical models of pediatric bone and soft tissue malignancies. In the phase II clinical trial combining olaratumab with doxorubicin, progression-free survival was improved by 2.5 months, and overall survival was extended by a median gain of 11.8 months with combination treatment when compared with single-agent doxorubicin (9). In this study, single-agent olaratumab delayed tumor growth in preclinical mouse models of osteosarcoma and MRT, indicating that PDGFRα signaling is necessary to drive some aspect of xenograft growth; however, a recent report demonstrated that inhibition of PDGFRα alone is insufficient for durable control of MRT growth (25), and combination with other targeted agents or chemotherapy is most likely necessary. Indeed, we observed enhanced antitumor activity with coadministration of olaratumab and either doxorubicin or cisplatin in osteosarcoma mouse models. These data indicate that the improvement in activity observed upon combining SOC with olaratumab is not limited to doxorubicin. Therefore, other chemotherapies commonly used in pediatric tumors, such as ifosfamide, cyclophosphamide, or vincristine (46, 47), could potentially combine with olaratumab to enhance antitumor activity in preclinical models of pediatric bone and soft tissue tumors.

B.P. Rubin reports receiving speakers bureau honoraria from and is a consultant/advisory board member for Eli Lilly. L.F. Stancato has ownership interests in Eli Lilly. No potential conflicts of interest were disclosed by the other authors.

Conception and design: C.D. Lowery, W. Blosser, M. Dowless, J. Stephens, H. Li, N. Loizos, G.J. Oakley III, L. Stancato

Development of methodology: W. Blosser, M. Dowless, J. Stephens, H. Li, G.J. Oakley III, Q. Guo, S. Iyer

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Blosser, M. Dowless, S. Knoche, J. Stephens, H. Li, D. Surguladze, N. Loizos, D. Luffer-Atlas, G.J. Oakley III, Q. Guo, B.P. Rubin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.D. Lowery, W. Blosser, S. Knoche, J. Stephens, H. Li, D. Surguladze, N. Loizos, D. Luffer-Atlas, G.J. Oakley III, S. Iyer, L. Stancato

Writing, review, and/or revision of the manuscript: C.D. Lowery, D. Surguladze, N. Loizos, D. Luffer-Atlas, G.J. Oakley III, L. Stancato

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.D. Lowery, W. Blosser, S. Iyer

Study supervision: N. Loizos, S. Iyer

The authors would like to thank Dr. Peter Houghton for his gift of the atypical teratoid/rhabdoid tumor cell lines BT-12 and BT-16. This study was funded by Eli Lilly and Company, Lilly Corporate Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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