Three-Dimensional Cultivation of Germ Cell Cancer Cell Lines as Hanging Drops

Skowron, Margaretha A.; Watolla, Meike M; Nettersheim, Daniel

doi:10.1007/978-1-0716-0860-9_6

Cited by 4 publications

(4 citation statements)

References 20 publications

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“…A detailed description has been published previously [25]. Briefly, a total amount of 3 × 10 3 cells per 40‐μL drop have been plated onto an inverted lid of a 15‐cm cell culture plate.…”

Section: Methodsmentioning

confidence: 99%

“…Cluster of differentiation 24 (CD24) is a small, mucin-like glycosylphosphatidylinositol (GPI) anchored membrane molecule that is discussed to function in both, signal Three-dimensional cell co-cultivation and cell sorting A detailed description has been published previously [25]. Briefly, a total amount of 3 x 10 3 cells per 40 μl drop have been plated onto an inverted lid of a 15 cm cell culture plate.…”

Section: Introductionmentioning

confidence: 99%

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The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas

et al. 2021

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demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, i.e. histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3-ubiquitin ligases or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9-deficiency model, we demonstrate that CD24 fulfils a bivalent role in differentiation via regulation of homeobox, phospho-and glycoproteins, i.e. it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity towards cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas

et al. 2021

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“…Confirmation of staining has been shown previously [26]. The three‐dimensional co‐cultivation of GCT and TM cells (3 × 10 3 cells per 40‐μL drop) has been performed as described previously [26,32].…”

Section: Methodsmentioning

confidence: 70%

Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level

et al. 2022

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The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T‐lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high‐throughput approaches of three‐dimensional (3D) co‐cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin‐based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass‐spectrometry‐based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin‐like growth factor (IGF) transport. To evaluate direct cell–cell contacts, green fluorescent protein (GFP)‐expressing GCT cells and mCherry‐expressing TM cells were co‐cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re‐analyses of secretome data with regard to lysine‐ and proline‐hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV‐ or fibronectin‐coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro‐tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin‐based chemotherapy for GCT patients.

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“…Establishment of CD24-deficient EC cell lines (DCD24) via CRISPR/Cas9 technology was performed as reported previously [4,17,18]. For 3D cultivation in hanging drops, similar to our previous studies [19,20], 3 9 10 3 tumour cells per drop (40 lL) were seeded onto an inverted lid of a 15-cm cell culture dish before being inverted back onto the bottom chamber filled with phosphate-buffered saline for humidity. The next day, NK-92 or NK-92-CD24-CAR cells were added in the same manner at a ratio of 1 : 3 (9 x 10 3 ) in a volume of 10 lL per drop, supplemented with recombinant human interleukin-2 (Peprotech via Biozol, Eching, Germany), with a final concentration per drop of 5 ngÁmL À1 .…”

Section: Cell Culturementioning

confidence: 99%

CD24 targeting with NK‐CAR immunotherapy in testis, prostate, renal and (luminal‐type) bladder cancer and identification of direct CD24 interaction partners

et al. 2023

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Alternative therapeutic options targeting urologic malignancies, such as germ cell tumours, as well as urothelial, renal and prostate carcinomas, are still urgently needed. The membrane protein CD24 represents a promising immunotherapeutical approach. The present study aimed to decipher the molecular function of CD24 in vitro and evaluate the cytotoxic capacity of a third‐generation natural killer (NK) cell chimeric antigen receptor (CAR) against CD24 in urologic tumour cell lines. Up to 20 urologic tumour cell lines and several non‐malignant control cells were included. XTT viability assays and annexin V/propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co‐immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of CD24 expression by SOX2. N‐ and O‐glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. The study demonstrates that SOX2 transactivates CD24 expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding and post‐translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumour cells with NK‐CD24‐CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24+ tumour cells. Limitations of the study include the in vitro setting, which still has to be confirmed in vivo. In conclusion, we show that CD24 is a promising novel target for immune therapeutic approaches targeting urologic malignancies.

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Three-Dimensional Cultivation of Germ Cell Cancer Cell Lines as Hanging Drops

Cited by 4 publications

References 20 publications

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas

Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level

CD24 targeting with NK‐CAR immunotherapy in testis, prostate, renal and (luminal‐type) bladder cancer and identification of direct CD24 interaction partners

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