Isolation of circulating tumor cells in a preclinical model of osteosarcoma: Effect of chemotherapy

Abstract

Osteosarcoma is a rare primary bone tumor, which mainly affects children and adolescents and has a poor prognosis, especially for patients with metastatic disease. A poor therapeutic response to the conventional chemotherapy is observed with the development of lung metastases, highlighting the need for improving the current regimens and the identification of early markers of the recurrent and metastatic disease. Circulating Tumour Cells (CTCs) play a key role in the metastatic process and could be powerful biomarkers of the progressive disease. The present study aimed to isolate CTCs by using a pre-clinical model of human osteosarcoma and to monitor their kinetic of release and their modulation by ifosfamide. CTCs were detectable into the bloodstream before any palpable primary tumors. Ifosfamide increased CTCs count and in contrast decreased the number of lung tumor nodules. On established tumors, ifosfamide slowed down the tumour growth and did not modulate CTC count that could be explained by a release of cancer cells from the primary tumour with reduced properties for inducing lung metastases. This report highlights the biological interest of CTCs in osteosarcoma.

Keywords: Biomarker; Circulating tumor cells; Metastatic process; Osteosarcoma; Recurrent disease.

Fig. 1

CTC capture and characterization workflow. CTCs were isolate from a xenograft murine model of human osteosarcoma cells. Human GFP-MMNG/HOS osteosarcoma cells were inoculated in close contact to the tibia of immunodeficient mice. The kinetic study of CTCs detection into the bloodstream was then carried out. A first pre-enrichment step was performed including successively: i) a reduction of blood volume by centrifugation; ii) two lyses of erythrocytes; iii) an immunomagnetic depletion of leukocytes and residual thrombocytes. In a second step, enriched samples were processed for isolating CTCs by flow cytometry or dielectrophoresis technology (DEPArray™). Isolated cells were enumerated, cultured and singles cells were analysed by RTqPCR.

Fig. 2

Monitoring of CTC release into the bloodstream. (A) Summary of the experimental protocole. Human GFP-MMNG/HOS osteosarcoma cells were inoculated in paratibial site of nude mice. Tumor growth monitored (B) twice a week and the CTC dynamic (C,D) in blood were monitored at day 10, 30 and 45 after tumor cell inoculation. CTCs were enumerated by flow cytometry (C) or by DEPArray™ (D). CTCs are precociously detectable in mice before any palpable tumor mass and can be considered an early event associated to the tumor development. D10: day 10; * p < 0.05; ** p < 0.01. n = 5/condition.

Fig. 3

Opposite effect of one cycle of ifosfamide on the number of lung metastases and CTCs at early stage of osteosarcoma development. (A) Summary of the experimental protocole. Human GFP-MMNG/HOS osteosarcoma cells were inoculated in paratibial site of nude mice. Three days post-inoculation of osteosarcoma cells, mice were treated with ifosfamide at doses of 15 mg/kg or 30 mg/kg i.p. per day, 3 days consecutively. Control mice were injected in the same conditions with an equivalent volume of PBS. Tumor volume (B), number of lung metastases/mouse (lung Met.) (C) and CTCs (C) were enumerated 15 and 30 days after inoculation of cancer cells.

Fig. 4

At late stage of osteosarcoma development, one cycle or two cycles of ifosfamide slow down the tumor volume without any impact on CTC release into the bloodstream. (A) Summary of the experimental protocole. Human GFP-MMNG/HOS osteosarcoma cells were inoculated in paratibial site of nude mice. Three weeks post-inoculation of osteosarcoma cells, mice were treated with one (B) or two cycles (C) of 30 mg/kg/day i.p ifosfamide administered 3 days consecutively. Control mice were injected in the same conditions with an equivalent volume of PBS. Tumor volume and CTCs were measured at the end-point. D0: day 0. ** p < 0.001; *** p < 0.0001. n = 8/group.

Fig. 5

Gene expression of CTCs compared to the parental cell line. Isolated CTCs were cultured and used for developing new cell lines. Gene expression profile for CD99, LTK, ENDRA and ADAM8 was analysed by RT-qPCR compared to Hprt1 used as house keeping gene. Relative expression of different genes was obtained with DDCt method.