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

Abstract

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.

Keywords: CXCL12; CXCR4; CXCR7; Germ cell tumors; Nanobody-drug-conjugate; Resistance; Testis cancer; Therapy.

Fig. 1

(A) Immunohistochemical evaluation of CXCR4 / CXCR7 in SEM, CC, YST and YST + EC mixed GCT. (B) Relative expression of CXCR4, CXCR7, and CXCL12 in GCT cell lines and fibroblasts (MPAF). ACTB and GAPDH were used as housekeeping genes. (C) Flow cytometry data of CXCR7-APC stained GCT cell lines (blue) compared with unstained controls (grey). (D) XTT cell viability assays of GCT cell lines and fibroblasts (MPAF) treated with CXCR4-NDC or CXCR4-NB alone for 24–96 h. (E) LD₅₀ values measured by XTT cell viability assays 72 h after treatment with cisplatin (CisPt, µM) or CXCR4-NDC (nM) and color-coded changes in cell cycle distribution (mitotic catastrophe = red; changes < 5% = grey) upon treatment with CXCR4-NDC (LD₅₀ concentrations) for 72 h as compared to the CXCR4-NB alone. (F) Lollipop graph summarizing the relative number of apoptotic cells in CXCR4⁺ GCT cells and CXCR4⁻ MPAF upon treatment with either CXCR4-NDC or CXCR4-NB alone (LD₅₀ concentrations) for 72 h

Fig. 2

(A) Densitometric evaluation of relative pixel intensities (normalized to untreated controls) of the indicated phosphorylation sites in cell lysates from GCT72, 1411H, TCam-2 and BeWo cells treated with recombinant CXCL12 (250 ng / ml) for 24 h, as measured by a human phospho-kinase array. (B) Relative migration of GCT72 and 1411H cells treated with either 100 ng / ml recombinant CXCL12, 100 nM CXCR7-NB (VUN702), or the combination of both, in comparison with the untreated control. (C) Box plot summarizing the number of proliferative GCT72 cells treated with 100 ng / ml recombinant CXCL12, 20 µM CXCR4-inhibitor AMD3100, or the combination of both for 24 h in comparison to the untreated control. (D) Box plot summarizing the number of proliferative 1411H cells treated with 100 ng / ml recombinant CXCL12, CXCR4-inhibitors WZ811 (5 µM) / LY2510924 (50 nM), or the combination of both for 32 h in comparison to the untreated control. (E) Densitometric evaluation of western blot data of phospho- and total-ERK in EC cells (2102EP, NCCIT, NT2/D1) treated daily with 100 nM ERK inhibitor SCH772984 for 96 h. (F) Relative mRNA expression of CXCR4 and CXCR7 in EC cell lines (2102EP, NCCIT, NT2/D1) treated daily with 100 nM SCH772984 for 96 h as compared to untreated controls. ACTB and GAPDH were used as housekeeping genes. (G) Model summarizing key findings related to the CXCR4 / CXCR7 / CXCL12 axis. SEM present as CXCR4⁺ / CXCR7⁻, YST as CXCR4⁺ / CXCR7⁺, CC as CXCR4⁻ / CXCR7⁺ and EC as CXCR4⁻ / CXCR7⁻. SEM (also with occult YST subpopulations), YST and CC are targetable by CXCR4- and / or CXCR7-NDC, respectively. CXCL12 stimulated CXCR4 / CXCR7 enhanced proliferation and migration in YST cells. In EC cell lines, inhibition of MAPK (ERK1 / 2) signaling allows for re-induction of CXCR7 expression (and partly CXCR4)