Roth J A, Thomsen S, Fry K D, Scannon P, Chang C
Department of Thoracic Surgery, University of Texas M D. Anderson Cancer Center, Houston, Texas 77030.
Cancer Res. 1989 Oct 15;49(20):5708-12.
Ricin A chain immunotoxin (IT) 45-2D9-RTA mediates regression of spontaneous pulmonary metastases and lung colonies from K-ras transformed rat fibroblasts (TRF cells). However, residual metastases are frequently noted after IT therapy, and therefore, possible mechanisms mediating tumor cell escape were investigated. Individual lung colonies were dissected from lungs of BALB/c mice, and single-cell suspensions of fresh cells from short-term cultures (eight passages) were tested. Immunoperoxidase staining with 45-2D9 monoclonal antibody showed that stable loss of surface antigen by cells cultured from IT-treated mice did not occur after four injections of specific IT. Sensitivity to specific IT in vitro was equal for metastatic tumor cells from mice treated with either two or four doses of specific IT compared to cells from nonspecific IT-treated mice and to parental cells. Clones derived from metastases of IT-treated mice were not resistant to IT. Clones derived from metastases of specific IT-treated mice internalized bound antibody or IT at the same rate as untreated cells. Freshly disaggregated cells from specific IT-treated mice were as sensitive to specific IT as were cells from nonspecific IT-treated or untreated mice. Specific IT successfully mediated reduction of lung colonies derived from fresh suspensions of lung colony TRF cells from IT-treated mice. This reduction was equivalent to that seen for cells not previously exposed to specific IT. Immunoperoxidase stains of lung sections with 45-2D9 showed that colonies consisting entirely of unstained TRF cells were present in both specific IT and phosphate buffered saline-treated mice. There was a trend toward a higher percentage of antigen-negative colonies in mice treated with IT, although 9 days following specific IT therapy, greater than 80% of lung colonies expressed gp74 antigen. When TRF cells were grown on agar plugs, which promoted three-dimensional growth, groups of cells showing absence of immunoperoxidase staining with antibody to gp74 were identified during 2 weeks of growth. Thus, stability of antigen-negative variants is favored by three-dimensional growth conditions and the selective pressure of IT administration. Our results also suggest that impaired trafficking of IT to antigen-positive cells may also contribute to escape from IT therapy.
蓖麻毒素A链免疫毒素（IT）45-2D9-RTA可介导K-ras转化的大鼠成纤维细胞（TRF细胞）自发肺转移灶和肺集落的消退。然而，IT治疗后经常会发现残留转移灶，因此，对介导肿瘤细胞逃逸的可能机制进行了研究。从BALB/c小鼠的肺中解剖出单个肺集落，并对短期培养（八代）的新鲜细胞的单细胞悬液进行检测。用45-2D9单克隆抗体进行免疫过氧化物酶染色显示，在注射四次特异性IT后，从IT治疗小鼠培养的细胞未出现表面抗原的稳定丢失。与未用特异性IT治疗的小鼠细胞和亲本细胞相比，用两剂或四剂特异性IT治疗的小鼠的转移瘤细胞在体外对特异性IT的敏感性相同。源自IT治疗小鼠转移灶的克隆对IT不耐药。源自特异性IT治疗小鼠转移灶的克隆内化结合抗体或IT的速率与未处理细胞相同。来自特异性IT治疗小鼠的新鲜解离细胞对特异性IT的敏感性与未用特异性IT治疗或未处理小鼠的细胞相同。特异性IT成功介导了来自IT治疗小鼠肺集落TRF细胞新鲜悬液的肺集落减少。这种减少与未预先暴露于特异性IT的细胞所见的减少相当。用45-2D9对肺切片进行免疫过氧化物酶染色显示，在特异性IT和磷酸盐缓冲盐水处理的小鼠中均存在完全由未染色的TRF细胞组成的集落。尽管在特异性IT治疗9天后，超过80%的肺集落表达gp74抗原，但在IT治疗的小鼠中，抗原阴性集落的百分比有升高趋势。当TRF细胞在促进三维生长的琼脂塞上生长时，在生长的2周内鉴定出了用gp74抗体免疫过氧化物酶染色阴性的细胞群。因此，三维生长条件和IT给药的选择压力有利于抗原阴性变体的稳定性。我们的结果还表明，IT向抗原阳性细胞的转运受损也可能导致逃避IT治疗。
