Multicellular spheroidを用いた抗癌剤の脳腫瘍細胞抑制効果の検討

EFFECTS OF ANTICANCER DRUGS ON MULTICELLULAR SPHEROID OF 9L RAT BRAIN TUMOR

　抄録　9Lラット脳腫瘍細胞よりmulticellular spheroid （以下スフェロイド）を作成し，各種抗癌剤の効果を比較検討した。スフェロイドは200-400μmのものを選別採取し，Nimustinc （ACNU），AclacinomycinA （ACR），Adriamycin （ADM），Bleomycin （BLM），Cisplatin （CDDP）は1時間，5—Fluorouracil （5—FU）は24時間作用させた後，殺細胞効果および成長抑制効果をそれぞれcolony forming assay （CFA），　growthdelay assayによって検討した。同時に単層培養細胞でもCFAを行いスフェロイドへの効果と比較した。その結果，各種抗癌剤のdose response curveは単層培養細胞，スフェロイド共にADM,BLM,5—FUはbiphasic型，ACNU,ACR,CDDPはshoulder-threshold型を示した。又ADM,ACR,5—FUは単層培迷細胞に，BLMはスフェロイドにより大きな殺細胞効果を示し，ACNU,CDDPでは同程度であった。殺細胞効果と成長抑制効果の関係はACNU,ADM,BLM,CDDPでは比例1的であるが，ACR,5—FUは殺細胞効果は少なくても成長抑制効果が大きいことが指摘された。従って抗癌剤の作用は殺細胞効果のみでなく成長抑制効渠も併せてとらえることが必要であり，スフェロイドは固形腫瘍により近い性格をもっている上，この両面から抗癌剤の効果を検討できる有用な実験系であることが示唆された。

The effects of the anticancer drugs Nimustine (ACNU), Aclacinomycin A (ACR), Adriamycin (ADM), Bleomycin (BLM), Cisplatin (CDDP), and 5-Fluorouracil (5-FU) on the multicellular spheroid of a chemically-induced 9 L rat glioma was studied. The multicellular spheroid in which cells grow in vitro as three-dimensional aggregates represents a biological model, which is intermediate between monolayer cells in vitro and solid tumors. Spheroids were initiated in bacteriological grade petri dishes seeded with 106 9L rat glioma cells, cultured for four days and thereafter transfered and further developed in a spinner flask. Spheroids of 200-400 pm diameter were sorted and exposed for 24 hours to 5-FU and one hour for other drugs. After treatment both cytotoxic effect and growth delay were analyzed. Following disaggregation using collagenase, pronase and DNAase, cytotoxic effect on multicellular spheroids was measured by coloney forming assay and were compared with those effects on 9 L monolayer culture cells in the exponential growth.

For growth delay assay, multicellular spheroids were indivisually transferred to 16 mm well con-taining 0.4 ml agarose base and 2 ml culture me-dium. Spheroid size was measured twice a week and growth curves were drawn. The growth delay was determined as the treated group vs. control differences in time required to a size four times that of the initial volume.

For cells both in the monolayer culture and the multicellular spheroid, the dose response curve for ADM, BLM and 5-FU was "biphasic" and that for ACNU, ACR and CDDP "shoulder-threshold" type. 9 L cells in monolayer culture were highly sensitive to ACNU, ADM, moderately to ACR, CDDP and resistant to BLM. A discrepancy was noted, however, between the cytotoxic effect on cells in monolayer culture vs. the cytotoxic effect on cells in multicellular spheroids. Cells in the multicellular spheroids were more resistant than cells in the monolayer cultures to ADM, ACR, 5-FU and sensitive to BLM. The cytotoxic effects of ACNU and CDDP were equivalent for both the spheroid cells and the monolayer cells.

An increased growth delay was observed along with an increased cytotoxity when multicellular spheroids were treated with ACNU, ADM, BLM and CDDP, but the growth delay observed in spheroids treated with ACR and 5-FU was greater in spite of limited cytotoxic effects. For example, although the surviving fraction of cells treated with 18ug/ml of ACR was 0.38, the growth of multicellular spheroids was completely inhibited.

The multicellular spheroid is a favorable model used to study the cytotoxic effects and the growth delay caused by anticancer drugs, which appears to mimic tumors in vivo.