CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling

CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.


Immunoistochemistry staining
Sections (5 μm) from formalin-fixed, paraffinembedded tumor xenografts were placed on poly-L-lysine coated slides (Sigma). Avidin-biotin-peroxidase procedure was used for immunostaining. Briefly, sections were treated sequentially with xylene and ethanol to remove paraffin. For immunohistochemical detection of IGF-IR, sections were pretreated with a citrate buffer solution [0.01 mol/L citric acid and 0.01 mol/L sodium citrate (pH 6.0)] in a microwave oven at 750 W for three cycles of 5 min each. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 30 min at room temperature. A blocking step with normal goat serum (Vector, Burlinga me, CA) was used. This treatment ensured antigen retrieval from samples. The following primary antibodies (anti-IGF-1R β-subunit, clone C-20, diluted 1:50; anti-RAS, clone FL-189, diluted 1: 100 and anti-Rac1, clone C14, diluted 1: 100, Santa Cruz Biotechnologies) were applied overnight in a moist chamber at 4°C. The following day, tissue sections were incubated with a secondary biotinylated anti-rabbit antibody and with an avidin-biotin-peroxidase complex (Vector).
The final reaction product was revealed by exposure to 0.03% diaminobenzidine (Sigma), and nuclei were counterstained with Mayer's hematoxylin.

Acridine orange (AO) immunofluorescence
6647 and LAP-35 cells were seeded on fibronectin coated-dishes and after 48h stimulated with anti-CD99 0662mAb (3μg/ml) for 3h. After treatment, cells were washed with PBS and then stained with 1 μg/ml AO in PBS for 15 min at 37°C and nuclei were counterstained with vital Hoechst 33342 (Sigma). Apoptosis was induced in 6647 or LAP-35 dominant negative RAS cells (hRAS N17) by treating them with 3μg/ml 0662mAb for 3h at 37°C. Control or treated cells were stained with AO 15 min at 37°C and nuclei were counterstained with vital Hoechst 33342 (Sigma).

CD99 internalization
ELISA assay was performed on 70,000 cells/timepoint seeded in multi 24 plates and starved for 3h, then mAb; subsequently cells were washed in TBS 1X, fixed in 3.7% TBS-paraformaldehyde and blocked in 1% TBS-BSA. After the incubation with the anti-mouse secondary antibody, the relative substrate (BIO-RAD Cat# 172-1063) was added to the cells. The reaction was stopped by adding NaOH 0.4 N, then absorbance was read at 405 nm.
CD99 exposure was evaluated by indirect immunofluorescence: after 0662mAb exposure, a pellet of 500 000 cells has incubated with goat anti-mouse IgG (H+L) FITC (Thermo Fisher Scientific Inc) for 30 min, the reaction was stopped by adding serum-free medium. Cells resuspended in 1μg/ml ethidium bromide-PBS were processed for FACS analysis.

Treatment with inhibitor or siRNA of Rac1
The Rac1 inhibitor, EHT 1864 (Tocris), was added at 25 μM in culture medium 24h after cell seeding and renewed every day. After 48h control or treated cells were exposed to 0662mAb and subsequently analyzed. Transient Rac1 depletion was achieved by siRNA approaches using anti-Rac1 oligos commercially available. Scrambled (ON-TARGETplus siCONTROL, Thermo Scientific) was used as control. All were administered at 100 picomoles. Cells were seeded in 60-mm plates in standard medium (3 × 10 5 cells per plate) and transfected after 24h (Lipofectamine 2000, Life Technologies). 48h later, cells were treated with 3 μg/ml of 0662mAb for 2h and harvested for vital count.

Network analyses of gene expression array
Biological pathways were defined according to "GeneGO Pathway Maps" manually curated database, instead ontologies according to Gene Ontology database. Enrichment was tested for significance by modified Fisher's Exact Test corrected by FDR and considered significant if p-value ≤ 0.1. Network analysis of biologically related terms was performed with the direct interaction method in GeneGO, were an edge connecting two genes indicates their direct biological relation according to GeneGO database.

In vivo treatments with anti-CD99 antibody
Female athymic 4-5-week-old Crl:CD-1nu/nuBR mice (Charles River Italia, Italy) were subcutaneously injected with 5 × 10 6 EWS cells. The animals were randomised into two groups. In the group treated with anti-CD99 antibody alone, each mouse received s.c. injections of antibody (1mg/injection) in the proximity of the tumor for each day for two subsequent cycles of five days. Control mice received s.c. injections of PBS (same volume/injection; 10 injections). Tumor growth and body weight was assessed once a week by measuring tumor volume, calculated as π/2 [ √(a b)]3/6 where a and b are the two maximum diameters. For ethical reasons, mice with local tumors were killed when they achieved a tumor volume of 3 ml and necropsied. All animal experiments were performed according to Italian law 116/92 and European directive 2010/63/UE. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee ("Comitato Etico Scientifico per la Sperimentazione Animale") of the University of Bologna, and forwarded to the Italian Ministry of Health with letter 4783-X/10 (Responsible Researcher Prof. C. De Giovanni). Figure S1: Vesicles are induced upon 0662 mAb exposure, but cell death does not occur through necroptosis and it is independent from autophagy. A. Treatment with 0662 mAb (30 min) with or without Nec-1 pretreatment (30μM for 30 min or 50μM overnight) (Annexin V/PI assay). B. Transmission electron microscopy of 0662 mAb treated LAP-35 cells with or without 3-MA treatment at low (left, scale bar 2μm) and high magnification (right, scale bar 0.5μm). C. Upper panel: percentage of live (dark grey) or dead (propidium iodide-positive; light grey) EWS cells transfected with an inducible system for ATG7 silencing before (-) and after (+) treatment with 0662 mAb. Lower panel: western blotting of ATG7 protein levels before and after treatment with tetracycline (TET) 2.5μg/ml and/or with 0662 3μg/ml in 6647 cells. Equal loading was monitored by GAPDH. D. Western blotting of LC3IIB on cell extracts from control (-) or treated LAP-35 cells. Equal loading was monitored by GAPDH. E. Immunofluorescence analysis of LC3 in untreated or 0662 mAb treated (3h) LAP-35 cells (scale bar 20μm).