van Leeuwen-Stok A E, Schuurhuis G J, Dräger A m, Visser-Platier A W, van Dieren E B, Teule G J, Huijgens P C
Department of Hematology, Free University Hospital, Amsterdam, The Netherlands.
Int J Radiat Oncol Biol Phys. 1996 Jun 1;35(3):507-17. doi: 10.1016/s0360-3016(96)80013-7.
The purpose of the present study was to investigate if there were differences between U715 spheroids and single cells in the radiotoxic effect of 67Ga on cell growth and clonogenic capacity in vitro and to generate dosimetric approaches for the multicellular tumor model.
Human lymphoma U715 cells were cultured in vitro as single cells and multicellular spheroids, grown with the use of a combination of fibrin clot technique, spinner flasks, and liquid-overlay culture. Cells were incubated with 2.96-8.88 MBq/ml 67Gallium for 4 days. Spheroids were dispersed to single cells by treatment with plasmin. Residual proliferative and clonogenic capacity after 67Ga incubation were assayed using the MTT-test and clonogenic test, respectively. Autoradiography was performed with 1 microm sections and Ilford L4 liquid photographic emulsion. Dosimetric approaches were made, based on the MIRD-approach.
During 67Ga incubation proliferation was inhibited. The residual proliferative or clonogenic capacity was inhibited by 8.88 MBq/ml for 39 and 88%, respectively. For single cells with 6.66 MBq/ml these inhibitions were 64 and 96%, respectively. Autoradiography showed an homogeneous distribution of 67Ga in spheroids and single cells. In single cells a 2.1-3.5 times higher 67Ga uptake/cell than in spheroids produced an equitoxic effect. The uptake parameters were implemented in new dosimetric approaches and showed that the efficacy of intracellular 67Ga was two times higher in spheroid clusters than in single cells due to energy deposition of internal conversion electrons within the cell clusters with a mean diameter size of nine cells. Both for proliferative and clonogenic capacity the exponential survival curves were superimposed.
With the new approaches made in our dosimetric model the discrepancy found between 67Ga accumulation and radiotoxic effect in spheroids as compared to single cells can be explained by additional effects of the crossfire of internal conversion electrons within clusters of about nine cells in diameter in spheroids. Only twice as much 67Ga was needed to reach equitoxic absorbed doses in spheroids than was needed in single cells. Such might be important for the use of 67Ga treatment of small metastasis of malignant lymphoma.
本研究旨在探讨U715球体与单细胞在体外67Ga对细胞生长和克隆形成能力的放射毒性作用方面是否存在差异，并为多细胞肿瘤模型建立剂量学方法。
人淋巴瘤U715细胞在体外培养为单细胞和多细胞球体，采用纤维蛋白凝块技术、转瓶和平板覆盖培养相结合的方法进行培养。细胞与2.96 - 8.88 MBq/ml的67镓孵育4天。用纤溶酶处理将球体分散为单细胞。67Ga孵育后剩余的增殖和克隆形成能力分别用MTT试验和克隆形成试验进行测定。用1微米切片和伊尔福L4液体照相乳剂进行放射自显影。基于MIRD方法建立剂量学方法。
在67Ga孵育期间，增殖受到抑制。8.88 MBq/ml时，剩余增殖或克隆形成能力分别被抑制39%和88%。对于6.66 MBq/ml的单细胞，这些抑制分别为64%和96%。放射自显影显示67Ga在球体和单细胞中分布均匀。在单细胞中，67Ga摄取/细胞比在球体中高2.1 - 3.5倍时产生同等毒性效应。摄取参数被应用于新的剂量学方法中，结果表明，由于平均直径为9个细胞的细胞簇内内转换电子的能量沉积，细胞内67Ga在球体簇中的效能比在单细胞中高两倍。对于增殖和克隆形成能力，指数存活曲线相互叠加。
通过我们剂量学模型中的新方法，与单细胞相比，在球体中发现的67Ga积累与放射毒性效应之间的差异可以通过直径约为9个细胞的簇内内转换电子的交叉火力的额外效应来解释。在球体中达到同等毒性吸收剂量所需的67Ga仅为单细胞的两倍。这对于使用67Ga治疗恶性淋巴瘤的小转移灶可能很重要。
