Lustenberger Severin K, Castro Jaramillo Claudia A, Bärtschi Lena A, Williams Rich, Schibli Roger, Mu Linjing, Krämer Stefanie D
Radiopharmaceutical Sciences, Institute of Pharmaceutical Sciences, ETH Zürich, 8092 Zürich, Switzerland.
Queens University Belfast, BT7 1NN Belfast, United Kingdom of Great Britain and Northern Ireland.
Nucl Med Biol. 2024 Nov-Dec;138-139:108951. doi: 10.1016/j.nucmedbio.2024.108951. Epub 2024 Sep 13.
M2-type tumor-associated macrophages (TAM) residing in the tumor microenvironment (TME) have been linked to tumor invasiveness, metastasis and poor prognosis. M2 TAMs suppress T cell activation, silencing the recognition of the cancer by the immune system. Targeting TAMs in anti-cancer therapy may support the immune system and immune-checkpoint inhibitor therapies to fight the cancer cells. We aimed to develop a PET tracer for the imaging of M2 TAM infiltration of cancer, using activated legumain as the imaging target.
Two P1-mimicking inhibitors with a cyano-warhead were labeled with carbon-11 and evaluated in vitro and in vivo with a CT26 tumor mouse model. Target expression and activity were quantified from RT-qPCR and in vitro substrate conversion, respectively. The co-localization of legumain and TAMs was assessed by fluorescence microscopy. The two tracers were evaluated by PET with subsequent biodistribution analysis with the dissected tissues. Parent-to-total radioactivity in plasma was determined at several time points after i.v. tracer injection, using reverse phase radio-UPLC.
Legumain displayed a target density of 40.7 ± 19.1 pmol per mg total protein in tumor lysate (n = 4) with high substrate conversion and colocalization with M2 macrophages in the tumor periphery. [C]1 and [C]2 were synthesized with >95 % radiochemical purity and 12.9-382.2 GBq/μmol molar activity at the end of synthesis. We observed heterogeneous tumor accumulation in in vitro autoradiography and PET for both tracers. However, excess unlabeled 1 or 2 did not compete with tracer accumulation. Both [C]1 and [C]2 were rapidly metabolized to a polar radiometabolite in vivo.
The legumain tracers [C]1 and [C]2, synthesized with high radiochemical purity and molar activity, accumulate in the legumain-positive CT26 tumor in vivo. However, the lack of competition by excess compound questions their specificity. Both tracers are rapidly metabolized in vivo, requiring structural modifications towards more stable tracers for further investigations.
存在于肿瘤微环境（TME）中的M2型肿瘤相关巨噬细胞（TAM）与肿瘤侵袭、转移及不良预后相关。M2型TAM抑制T细胞活化，使免疫系统对癌症的识别沉默。在抗癌治疗中靶向TAM可能会增强免疫系统及免疫检查点抑制剂疗法来对抗癌细胞。我们旨在开发一种用于成像癌症中M2型TAM浸润情况的正电子发射断层显像（PET）示踪剂，以活化的天冬酰胺酶作为成像靶点。
用碳-11标记两种带有氰基弹头的P1模拟抑制剂，并在CT26肿瘤小鼠模型中进行体外和体内评估。分别通过逆转录定量聚合酶链反应（RT-qPCR）和体外底物转化来定量靶点表达和活性。通过荧光显微镜评估天冬酰胺酶与TAM的共定位。通过PET对这两种示踪剂进行评估，随后对解剖后的组织进行生物分布分析。在静脉注射示踪剂后的几个时间点，使用反相放射性超高效液相色谱法测定血浆中母体与总放射性。
天冬酰胺酶在肿瘤裂解物中的靶点密度为每毫克总蛋白40.7±19.1皮摩尔（n = 4），具有高底物转化率且与肿瘤周边的M2巨噬细胞共定位。[C]1和[C]2合成时的放射化学纯度>95%，合成结束时的摩尔活度为12.9 - 382.2吉贝可/微摩尔。在体外放射自显影和PET中，我们观察到两种示踪剂在肿瘤中的摄取均不均匀。然而，过量的未标记的1或2并不与示踪剂的摄取竞争。[C]1和[C]2在体内均迅速代谢为一种极性放射性代谢物。
以高放射化学纯度和摩尔活度合成的天冬酰胺酶示踪剂[C]1和[C]2在体内会在天冬酰胺酶阳性的CT26肿瘤中蓄积。然而，过量化合物缺乏竞争性这一点对其特异性提出了质疑。两种示踪剂在体内均迅速代谢，需要对结构进行修饰以获得更稳定的示踪剂用于进一步研究。
