Stackpole C W, Groszek L, Kalbag S S
Department of Experimental Pathology, New York Medical College, Valhalla 10595.
J Natl Cancer Inst. 1994 Mar 2;86(5):361-7. doi: 10.1093/jnci/86.5.361.
Benign tumors apparently become malignant by generating a succession of variants with ever-greater growth potential and autonomy. Such stepwise progression has not been achieved in vitro under conditions likely to occur in developing tumors.
Tumors initiated by clone G3.5 of the mouse B16 melanoma regularly generate stable variants that are more malignant. We investigated the possibility that hypoxia might promote stepwise progression along a benign-to-malignant pathway in monolayer cultures of G3.5 cells.
Confluent monolayers of metastatic clone G3.5 and the nonmetastatic clone G3.15 were subjected to severe hypoxia (< 50 ppm O2) for up to 72 hours, or to moderate hypoxia (300-1200 ppm O2) for up to 12 days, and were then maintained subconfluent or at confluence for several weeks to permit emergence of progression variants. The relative malignancy of variants was assayed in vivo after subcutaneous injection into mice, by measuring tumor growth rate and counting lung metastases, and after intravenous injection, by counting lung colonies. In vitro assessment of the variants involved growth as monolayers with or without serum, growth in soft agar, and measurement of invasiveness.
G3.5 cells were converted to a more malignant variant (G3.5*) by 12-48 hours of severe hypoxia, or longer periods of moderate hypoxia, when followed by maintenance at confluence for 3-5 weeks. Conversions occurred in discrete foci of morphologically-discernible cells (optimum focus formation about one in 1-2 x 10(5) cells) that rapidly expanded to dominate the cultures. The G3.5* phenotype was comparable to the conversion phenotype generated in tumors and included acquisition of growth autonomy in serum-free medium. G3.15 cells were converted to a G3.5-like phenotype by one round of exposure to hypoxia and confluence, and then to the G3.5* phenotype during a second round, at a low frequency (one focus in 5 x 10(6) cells). This behavior was consistent with a failure of all but the largest G3.15 tumors to generate G3.5* conversion cells.
Progression from a relatively benign phenotype, G3.15, to a highly malignant phenotype, G3.5*, can be produced in monolayer culture in two stable stages by sequential rounds of exposure to hypoxia and confluence. The resulting conversions corresponded to phenotypes generated within tumors. Both conversions resulted in populations with enhanced growth capabilities, which could establish dominance within tumors.
The stepwise conversion of B16 melanoma clones provides a unique model for the in vitro investigation of mechanisms underlying acquisition of malignancy during tumor development.
良性肿瘤显然是通过产生一系列具有越来越大生长潜力和自主性的变体而转变为恶性肿瘤的。在发育中的肿瘤可能出现的条件下，尚未在体外实现这种逐步进展。
由小鼠B16黑色素瘤的G3.5克隆引发的肿瘤经常产生更具恶性的稳定变体。我们研究了缺氧是否可能促进G3.5细胞单层培养物中沿着良性到恶性途径的逐步进展。
将转移性克隆G3.5和非转移性克隆G3.15的汇合单层细胞置于严重缺氧（<50 ppm O2）长达72小时，或中度缺氧（300 - 1200 ppm O2）长达12天，然后保持亚汇合或汇合状态数周，以允许进展变体出现。将变体皮下注射到小鼠体内后，通过测量肿瘤生长速率和计数肺转移灶来测定其相对恶性程度，静脉注射后，通过计数肺集落来测定。对变体的体外评估包括在有或无血清的情况下作为单层生长、在软琼脂中生长以及侵袭性测量。
当G3.5细胞在严重缺氧12 - 48小时或更长时间的中度缺氧后，接着在汇合状态下维持3 - 5周时，会转变为更具恶性的变体（G3.5*）。转变发生在形态上可辨别的细胞离散灶中（最佳灶形成约为1 - 2×10⁵个细胞中的1个），这些灶迅速扩展以主导培养物。G3.5表型与肿瘤中产生的转变表型相当，包括在无血清培养基中获得生长自主性。G3.15细胞通过一轮缺氧和汇合暴露转变为G3.5样表型，然后在第二轮中以低频率（5×10⁶个细胞中的1个灶）转变为G3.5表型。这种行为与除最大的G3.15肿瘤外所有肿瘤未能产生G3.5*转变细胞一致。
通过连续几轮暴露于缺氧和汇合，在单层培养中可以分两个稳定阶段从相对良性的表型G3.15进展到高度恶性的表型G3.5*。产生的转变与肿瘤内产生的表型相对应。两种转变都导致生长能力增强的群体，它们可以在肿瘤内建立优势。
B16黑色素瘤克隆的逐步转变为肿瘤发生过程中恶性获得机制的体外研究提供了一个独特的模型。
