Flow cytometric quantification of surface IGF-1R and intracellular p-AKT, p-S6, and p-ERK in Ewing sarcoma (EWS) cells

23 Background: Targeting the IGF-1R pathway has shown preclinical and clinical evidence of activity in EWS. Markers predictive of response to IGF-1R inhibition are required. The aims of the current study were to quantify surface IGF-1R expression and intracellular signaling proteins by flow cytometry. Methods: Flow cytometry for CD99 and CD45 was used to detect bone marrow micrometastatic tumor cells as CD99+CD45- events in patients with localized EWS. Surface IGF-1R was quantified on tumor cells and normal marrow cells using a commercially available antibody. RD-ES EWS cells were spiked into control PBMCs and starved for 1 hour. Cells were then treated with saline control, rapamycin 10 ng/mL, ADW742 5 uM (small molecule IGF-1R inhibitor), or alpha-IR3 0.5 ug/mL (anti-IGF-1R antibody) and levels of pS6, pAKT, and pERK quantified by phospho-flow cytometry 60 minutes later. All results were expressed as the mean fluorescent intensity (MFI) of staining using arbitrary units. Results: We detected bone marrow micrometastatic tumor cells in marrow samples from 16 patients with EWS. We observed heterogeneous levels of IGF-1R tumor cell surface expression between patients (range of IGF-1R MFI = 0 – 3355). IGF-1R levels were higher in CD99+CD45- tumor cells compared to normal marrow cells (mean MFI 840 for CD99+CD45- tumor cells vs.190 for normal marrow cells; p = 0.005). In RD-ES cells spiked into PBMCs, treatment with rapamycin resulted in 1.32 fold increase in pAKT MFI compared to saline control. Treatment with ADW742 or alpha-IR3 did not impact pAKT MFI. Treatment with rapamycin resulted in a 0.52 fold decrease and a 1.20 fold increase in pS6 and pERK, respectively. Treatment with ADW742 or alpha-IR3 each resulted in similar decreases in both pS6 (0.75-0.87 fold) and pERK (0.67-0.82 fold). In PBMCs, modest changes in pS6 in rapamycin treated cells were observed, without changes in pAKT or pERK. Neither ADW742 nor alpha-IR3 modulated pS6, pAKT, or pERK signaling in PBMCs. Conclusions: Flow cytometry detects a range of surface IGF-1R expression on bone marrow micrometastatic EWS cells. Phospho-flow cytometry detects modulation of pAKT, pS6, and pERK in EWS cells treated with IGF-1R or mTOR inhibitors.