Aminolevulinic acid (ALA) has been approved as an intraoperative molecular imaging probe for protoporphyrin IX (PpIX) fluorescence-guided resection of glioma. Here we explored its potential application for renal cell carcinoma (RCC) that is showing increased incidence in recent years. ALA-mediated PpIX in cell lysates (intracellular) and culture medium was measured in five human RCC cell lines (786-O, 769-P, A-704, Caki-1, Caki-2) and a non-tumor human kidney epithelial cell line HK-2 by spectrofluorometry and flow cytometry. The activity of PpIX bioconversion enzyme ferrochelatase (FECH) and PpIX efflux transporter ABCG2 was determined to correlate with the PpIX level. We found that ALA-PpIX fluorescence was highly variable among RCC cell lines and A-704 was the only RCC cell line exhibiting significantly higher intracellular PpIX than HK-2 cells. Neither the intracellular PpIX level nor the total amount of PpIX (including PpIX in cell lysates and the medium) had significant correlation with the activity of FECH or ABCG2. To enhance the intracellular PpIX, cells were treated with Ko143, a pharmacological inhibitor of ABCG2. Ko143 significantly increased the intracellular PpIX in cell lines with ABCG2 activity, but not in cell lines with little ABCG2 activity. In fact, there was a positive correlation between the ABCG2 activity and Ko143-induced PpIX enhancement across kidney cell lines. To identify clinically relevant ABCG2 inhibitors, small molecule inhibitors targeting various cell signaling pathways, some of which are known to inhibit ABCG2, were evaluated for the enhancement of ALA-PpIX in Caki-2 cells that had the highest ABCG2 activity in the RCC cell panel. Our screening led to the identification of several clinically available inhibitors that significantly increased the intracellular PpIX. Particularly, kinase inhibitor lapatinib exhibited the strongest enhancement effect. These clinical inhibitors can be used for the enhancement of ALA-PpIX fluorescence in tumors with elevated ABCG2 activity.

氨基乙酰丙酸 (ALA) 已被批准用作术中分子成像探针，用于原卟啉 IX (PpIX) 荧光引导的胶质瘤切除术。在这里，我们探讨了其在近年来发病率增加的肾细胞癌 (RCC) 中的潜在应用。在五种人 RCC 细胞系（786-O、769-P、A-704、Caki-1、Caki-2）和非肿瘤人肾上皮细胞中测量细胞裂解物（细胞内）和培养基中 ALA 介导的 PpIX通过荧光分光光度法和流式细胞术检测细胞系 HK-2。PpIX 生物转化酶亚铁螯合酶 (FECH) 和 PpIX 外排转运蛋白 ABCG2 的活性被确定为与 PpIX 水平相关。我们发现 ALA-PpIX 荧光在 RCC 细胞系中变化很大，A-704 是唯一表现出比 HK-2 细胞更高的细胞内 PpIX 的 RCC 细胞系。细胞内 PpIX 水平和 PpIX 总量（包括细胞裂解物和培养基中的 PpIX）均与 FECH 或 ABCG2 的活性没有显着相关性。为了增强细胞内 PpIX，用 ABCG2 的药理学抑制剂 Ko143 处理细胞。Ko143 在具有 ABCG2 活性的细胞系中显着增加细胞内 PpIX，但在具有很少 ABCG2 活性的细胞系中没有显着增加。事实上，ABCG2 活性与 Ko143 诱导的肾细胞系 PpIX 增强之间存在正相关。为了鉴定临床相关的 ABCG2 抑制剂，评估了针对各种细胞信号通路的小分子抑制剂，其中一些已知抑制 ABCG2，评估了在 RCC 细胞组中具有最高 ABCG2 活性的 Caki-2 细胞中 ALA-PpIX 的增强. 我们的筛选导致鉴定出几种临床上可用的抑制剂，这些抑制剂显着增加了细胞内 PpIX。特别是激酶抑制剂拉帕替尼表现出最强的增强作用。这些临床抑制剂可用于增强 ABCG2 活性升高的肿瘤中的 ALA-PpIX 荧光。