In Vitro Chemosensitivity of Two Ewing's Sarcoma Cell Lines: Implication for Autologous Bone Marrow Transplantation

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Abstract
Intensive chemotherapy requiring rescue with autologous bone marrow may be an encouraging mode of treatment for poor prognosis Ewing's sarcoma (ES). In vitro chemosensitivity test may be useful for establishing an effective purging condition. We studied the in vitro effects of a variety of chemotherapeutic agents on two established ES cell lines (ES-5838 and ES-A4573) and marrow colony-forming unit-granulocyte macrophage (CFU-GM). 4-Hydroperoxycyclophosphamide (4-HC), at 100 fiM produced complete inhibition (>5 log) of clonogenic growth of both ES cell lines and spared 6.9% of normal CFU-GM growth. Etoposide (VP-16), at 100 μM produced 3-3.5 log inhibition of ES cell lines and complete inhibition of CFU-GM growth. Adriamycin (ADR) and vincristine (VCR) were more cytotoxic to ES-5838 cells than ES-A4573 cells. ADR at 1 μM produced 99.7% inhibition of ES-5838 cells, 92.2% of ES-A4573 cells, and 86% inhibition of CFU-GM. VCR at 1 μM produced 98.6% inhibition of ES-5838 cells, only 43.7% of ES-A4573 cells, and 75% inhibition of CFU-GM growth. Addition of verapamil did not enhance VCR cytotoxicity of ES cell lines. These studies indicate that 4-HC may be a useful agent for purging metastatic ES cells from the bone marrow for autologous marrow transplantation. To develop an effective neuroblastoma (NB) purging condition, we have compared in vitro cytotoxicity of 6-hydroxydopamine (6-OHDA) and ascorbic acid, with 4-hydroperoxycyclophosphamide (4-HC) on three NB cell lines (SK-N-BE2, SMS-SAN, and LA-N-1) and also upon human hematopoietic stem cells. Our study included mixing NB cells with 20-fold excess of irradiated bone marrow buffy coat cells to simulate the borderline remission marrow. When NB cells were treated without marrow cells, all three NB cell lines were very sensitive to 6-OHDA; complete inhibition of SK-N-BE2 and SMS-SAN cells was achieved at 10 μg/ml, and >4 log inhibition of LA-N-1 was observed at 100 μg/ml of 6-OHDA. Addition of marrow cells caused marked reduction of the 6-OHDA-induced cytotoxicity of NB cells, and under similar conditions, colony-forming units-granulocyte-macrophage (CFU-GM) growth was not inhibited significantly. In the absence of normal marrow cells, 60 minutes of treatment with 100 μM of 4-HC produced complete inhibition (> 4.5 log) ofSK-N-BE2 and SMS-SAN cells, >4 log inhibition of LA-N-1 cells, and 97% of CFU-GM. Addition of marrow cells reduced the cytotoxicity of 4-HC, and 100 μM of 4-HC produced 99.8% inhibition of LA-N-1 colony growth. Shortening incubation duration to 30 minutes resulted in further reduction of 4-HC cytotoxicity; 100 μM of 4-HC caused 98.3%, 45%, and 33% inhibition of LA-N-1 cells, marrow CFU-GM, and burst-forming units-erythrocytes (BFU-E), respectively. At 200 μM, complete inhibition (>4 log) of LA-N-1 colony growth was noted, and 99% of CFU-GM and 9.3% of BFU-E growth was observed. These data favor the use of 4-HC for purging marrow of NB cells in the clinical autologous marrow transplantation.