Molecular characterization of the CXCR4 / CXCR7 axis in germ cell tumors and its targetability using nanobody-drug-conjugates

Being stimulated by the chemokine CXCL12, the CXCR4 / CXCR7 cascade is involved in tumor proliferation, migration, and metastasis. The interaction between CXCL12, secreted by cells from the microenvironment, and its receptors is complex and has been ascribed to promote chemotherapy resistance. However, the role of this signaling axis and its targetability in germ cell tumors (GCT) is not fully understood. Thus, this study investigated the therapeutic efficacy of a nanobody-drug-conjugate targeting CXCR4 (CXCR4-NDC) and functionally characterized this signaling pathway in GCT using small molecule inhibitors and nanobodies. As shown by diminished cell viability, enhanced apoptosis induction, and detection of mitotic catastrophes, we confirmed the cytotoxic efficacy of the CXCR4-NDC in CXCR4+ GCT cells (i.e. seminoma and yolk-sac tumor), while non-malignant CXCR4−-fibroblasts, remained largely unaffected. Stimulation of CXCR4+ / CXCR7+ GCT cells with CXCL12 resulted in an enhanced proliferative and migratory capacity, while this effect could be reverted using CXCR4 inhibitors or a CXCR7-nanobody. Molecularly, the CXCR4 / CXCR7-signaling cascade could be activated independently of MAPK (ERK1 / 2)-phosphorylation. Although, in CXCR4− / CXCR7− embryonal carcinoma cells, CXCR7-expression was re-induced upon inhibition of ERK1 / 2-signaling. This study identified a nanobody-drug-conjugate targeting CXCR4 as a putative therapeutic option for GCT, i.e. seminoma and yolk-sac tumors. Furthermore, this study shed light on the functional role of the CXCR4 / CXCR7 / CXCL12-signaling cascade in GCT, demonstrating an important influence on proliferation and migration.

在趋化因子CXCL12（chemokine CXCL12）的刺激下，CXCR4/CXCR7信号级联参与肿瘤的增殖、迁移与转移过程。由肿瘤微环境细胞分泌的CXCL12与其受体之间的相互作用机制复杂，且被证实可促进肿瘤化疗耐药。然而，该信号轴及其靶向性在生殖细胞肿瘤（germ cell tumors, GCT）中的作用尚未完全阐明。为此，本研究评估了靶向CXCR4的纳米抗体药物偶联物（CXCR4-NDC）的治疗效果，并通过小分子抑制剂与纳米抗体，对生殖细胞肿瘤中该信号通路的功能进行了表征。实验结果显示，CXCR4阳性的生殖细胞肿瘤细胞（包括精原细胞瘤与卵黄囊瘤细胞）经CXCR4-NDC处理后，细胞活力显著降低、凋亡诱导增强且可观察到有丝分裂灾难现象，由此证实了该偶联物对这类细胞的细胞毒性效果；而CXCR4阴性的非恶性成纤维细胞则基本未受影响。用CXCL12刺激CXCR4+/CXCR7+的生殖细胞肿瘤细胞，可增强其增殖与迁移能力，而该效应可通过CXCR4抑制剂或CXCR7纳米抗体予以逆转。分子层面的实验表明，CXCR4/CXCR7信号级联的激活可不依赖于丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）ERK1/2的磷酸化。不过，在CXCR4阴性/CXCR7阴性的胚胎癌细胞中，抑制ERK1/2信号通路可重新诱导CXCR7的表达。本研究证实，靶向CXCR4的纳米抗体药物偶联物可作为生殖细胞肿瘤（精原细胞瘤与卵黄囊瘤）的潜在治疗手段。此外，本研究还阐明了CXCR4/CXCR7/CXCL12信号级联在生殖细胞肿瘤中的功能作用，证实其对肿瘤增殖与迁移具有重要调控作用。