Development of an extracellular vesicle-associated transcriptomic biomarker signature for vandetanib treatment response of anaplastic thyroid cancer cells

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Development of an extracellular vesicle-associated transcriptomic biomarker signature for vandetanib treatment response of anaplastic thyroid cancer cells

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Author(s): Christian Grätz ¹ ^, , Prashant Changoer ² , Benedikt Kirchner ¹ ^, ³ , Martin Jaeger ² , Marlene Reithmair ³ , Romana Netea-Maier ² , Michael W. Pfaffl ¹

Publication date (Electronic preprint): 25 July 2024

Journal: RExPO24 Conference

Publisher: REPO4EU

Conference name: RExPO24

Conference date: 3-5 July 2024

Keywords: anaplastic thyroid cancer, liquid biopsy, transcriptomics, extracellular vesicles, cell-free RNA, RT-qPCR, vandetanib, drug repurposing

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Abstract

Anaplastic thyroid cancer (ATC) is a relatively rare but highly aggressive form of thyroid carcinoma (TC), with a median overall survival between only 3 and 6 months due to its metastatic tendency and a lack of targeted therapy options. To increase the range of therapeutic options, ATC is one of the malignancies included in the REPO4EU drug repurposing project. In the scope of REPO4EU, we found sensitivity of the ATC cell line Cal62 to the receptor tyrosine kinase (RTK) inhibitor vandetanib, making this drug a repurposing candidate to be investigated for treatment of ATC. However, to assess response to a drug in clinical practice, imaging techniques are usually employed, meaning that it takes time before non-responders are identified, in which the tumor can grow and metastasize further. Since tumors are known to release a high amount of cell-free RNA (cfRNA), which can be found in patient blood samples, protected from degradation by association to extracellular vesicles (EVs), a liquid biopsy-based approach might provide information about the therapeutic success earlier. As a first step towards this goal, we aimed to identify an in vitro EV-associated cfRNA biomarker signature for the response of Cal62 cells to vandetanib.First, we assessed the EC50 of vandetanib for Cal62 cells in cell culture medium depleted of fetal calf serum EVs (8.8 μM). We then treated Cal62 cells with two doses of vandetanib (8.8 and 20 μM) in triplicates and isolated both the cellular and EV-associated cfRNA after 48 hours. EV concentration and size distribution was assessed by nanoparticle tracking analysis and flow cytometry. Using RT-qPCR, we observed significant transcriptional regulation of multiple transcription factors of the MAPK-pathway as well as their target genes, involved in cell-cycle control, RTK signaling feedback, and inflammation. Most notably, we measured 3.4-fold / 11.2-fold upregulation of cyclin dependent kinase inhibitor 1A (CDKN1A; p-values 0.034 and 0.007), 5.2-fold / 6.9-fold downregulation of cell division cycle 25A (CDC25A; p-values 0.018 and 0.001) and 3.1-fold / 3.6-fold downregulation of the ERBB receptor feedback inhibitor 1 (ERFFI1; p-values 0.003 and 0.003) compared to the DMSO control on cellular RNA level after treatment with 8.8 / 20 μM vandetanib treatment for 48 h, respectively. These results were confirmed in the cell-free RNA compartment.As a next step, we will assess how the transcription of these genes is regulated after different treatment times with vandetanib. Finally, we will investigate the whole transcriptomic change of Cal62 cells in the cellular and cell-free RNA by next-generation sequencing to identify an EV-associated biomarker signature that significantly predicts the response to vandetanib in vitro. These results may serve as a basis for further clinical studies, making this project an important basis towards assessing therapeutic success of vandetanib treatment for ATC patients in liquid biopsies.

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Author and article information

Conference

Title: RExPO24 Conference

Publisher: REPO4EU

Publication date (Electronic preprint): 25 July 2024

Affiliations

[1 ] Department of Animal Physiology and Immunology, School of Life Sciences, Technical University of Munich, Freising, Germany ( https://ror.org/02kkvpp62)

[2 ] Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands ( https://ror.org/05wg1m734)

[3 ] Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany ( https://ror.org/05591te55)

Author notes

[* ]Email: chris.graetz@ 123456tum.de .

Author information

Christian Grätz https://orcid.org/0000-0002-9356-7940

Prashant Changoer https://orcid.org/0009-0004-5892-8706

Benedikt Kirchner https://orcid.org/0000-0003-3878-0148

Martin Jaeger https://orcid.org/0000-0001-6994-493X

Marlene Reithmair https://orcid.org/0000-0002-9113-9643

Romana Netea-Maier https://orcid.org/0000-0002-9603-0460

Michael W. Pfaffl https://orcid.org/0000-0002-3192-1019

Article

DOI: 10.58647/REXPO.24000073.v1

SO-VID: 22a02b4d-d582-46d1-b11c-ee093f7649b5

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This work has been published open access under Creative Commons Attribution License CC BY 4.0 , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com .

Conference name: RExPO24

Conference number: 3

Conference location: Munich, Germany

Conference date: 3-5 July 2024

History

Date received : 24 July 2024

Funding

Funded by: funder-id http://dx.doi.org/10.13039/100018696, HORIZON EUROPE Health;

Award ID: 101057619

References

Cabanillas Maria E, Ryder Mabel, Jimenez Camilo. Targeted Therapy for Advanced Thyroid Cancer: Kinase Inhibitors and Beyond. Endocrine Reviews. Vol. 40(6):1573–1604. 2019. The Endocrine Society. [Cross Ref]
Mader Sonja, Pantel Klaus. Liquid Biopsy: Current Status and Future Perspectives. Oncology Research and Treatment. Vol. 40(7-8):404–408. 2017. S. Karger AG. [Cross Ref]
Théry Clotilde, Amigorena Sebastian, Raposo Graça, Clayton Aled. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology. Vol. 30(1)2006. Wiley. [Cross Ref]
Vandesompele Jo, De Preter Katleen, Pattyn Filip, Poppe Bruce, Van Roy Nadine, De Paepe Anne, Speleman Frank. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology. Vol. 3(7)2002. Springer Science and Business Media LLC. [Cross Ref]
Andersen Claus Lindbjerg, Jensen Jens Ledet, Ørntoft Torben Falck. Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Research. Vol. 64(15):5245–5250. 2004. American Association for Cancer Research (AACR). [Cross Ref]
Pfaffl Michael W., Tichopad Ales, Prgomet Christian, Neuvians Tanja P.. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters. Vol. 26(6):509–515. 2004. Springer Science and Business Media LLC. [Cross Ref]
Pfaffl M. W.. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. Vol. 29(9)2001. Oxford University Press (OUP). [Cross Ref]
Zuehlke Abbey D., Beebe Kristin, Neckers Len, Prince Thomas. Regulation and function of the human HSP90AA1 gene. Gene. Vol. 570(1):8–16. 2015. Elsevier BV. [Cross Ref]

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[1] Cabanillas Maria E, Ryder Mabel, Jimenez Camilo. Targeted Therapy for Advanced Thyroid Cancer: Kinase Inhibitors and Beyond. Endocrine Reviews. Vol. 40(6):1573–1604. 2019. The Endocrine Society. [Cross Ref]

[2] Mader Sonja, Pantel Klaus. Liquid Biopsy: Current Status and Future Perspectives. Oncology Research and Treatment. Vol. 40(7-8):404–408. 2017. S. Karger AG. [Cross Ref]

[3] Théry Clotilde, Amigorena Sebastian, Raposo Graça, Clayton Aled. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology. Vol. 30(1)2006. Wiley. [Cross Ref]

[4] Vandesompele Jo, De Preter Katleen, Pattyn Filip, Poppe Bruce, Van Roy Nadine, De Paepe Anne, Speleman Frank. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology. Vol. 3(7)2002. Springer Science and Business Media LLC. [Cross Ref]

[5] Andersen Claus Lindbjerg, Jensen Jens Ledet, Ørntoft Torben Falck. Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Research. Vol. 64(15):5245–5250. 2004. American Association for Cancer Research (AACR). [Cross Ref]

[6] Pfaffl Michael W., Tichopad Ales, Prgomet Christian, Neuvians Tanja P.. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters. Vol. 26(6):509–515. 2004. Springer Science and Business Media LLC. [Cross Ref]

[7] Pfaffl M. W.. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. Vol. 29(9)2001. Oxford University Press (OUP). [Cross Ref]

[8] Zuehlke Abbey D., Beebe Kristin, Neckers Len, Prince Thomas. Regulation and function of the human HSP90AA1 gene. Gene. Vol. 570(1):8–16. 2015. Elsevier BV. [Cross Ref]

Development of an extracellular vesicle-associated transcriptomic biomarker signature for vandetanib treatment response of anaplastic thyroid cancer cells

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