Effect of <i>FGFR3</i> Gene Mutation on Tyrosine Kinase Inhibitor Sensitivity in Bladder Cancer

doi:10.13523/j.cb.2306001

China Biotechnology

2024, Vol. 44

Issue (2/3): 1-13 DOI: 10.13523/j.cb.2306001

Effect of FGFR3 Gene Mutation on Tyrosine Kinase Inhibitor Sensitivity in Bladder Cancer

DENG Lixinxu^1,²,CHE Wenan^1,²,XU Haibo²,DING Xue¹,SUN Yuandong^1,^**(

)

1 Hunan University of Science and Technology, Xiangtan 411201, China
2 State and Local Government Joint Engineering Laboratory of Synthetic Biology Medicine and Clinical Application of Key Technologies, Shenzhen Second People’s Hospital, Shenzhen 518036, China

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Abstract

Objective: Bladder cancer cell lines and organoid models with FGFR3 mutations were constructed at different sites first, and then their sensitivity to different tyrosine kinase inhibitors targeting FGFR3 gene mutations was determined. Methods: Cell line models with wild type FGFR3, point mutant FGFR3 (S249C, R248C and Y373C) and FGFR3-TACC3 fusion were established. Eight tyrosine kinase inhibitors were then used to test for differences in drug sensitivity in different stable cells. The phosphorylation level of FGFR3 downstream pathway protein was detected by Western blot analysis to analyze the mechanism of the difference in tyrosine kinase inhibitor (TKI) sensitivity in different stable cells. Organoid models were constructed using bladder cancer patient tissues, and the above experiment was repeated. The sensitivity of different mutant bladder cancer organoids to different TKIs was determined at the organoid level. Results: FGFR3 point mutation R248C and FGFR3-TACC3 fusion increased by 5-10 fold compared with wild type FGFR3 to half of tyrosine kinase inhibitors at the cell line level (P<0.05). Organoids derived from tumor tissue of bladder cancer patients were successfully constructed. This confirmed that the organoid has the organizational and genetic characteristics of the tumor in situ. The FGFR3 point mutations S249C and Y373C did not show significant differences in sensitivity, which can be 1-5 fold different from the wild type. The FGFR3-TACC3 fusion and the FGFR3 point mutation Y373C can significantly alter the sensitivity of cells to drugs at the organoid level. Conclusions: This study successfully constructed cell lines and organoids with different FGFR3 mutations, further demonstrating that different FGFR3 mutations have different sensitivities to tyrosine kinase inhibitors, and that organoids with the FGFR3 point mutation R248C have high sensitivity to all TKI drugs.

Key words： Bladder cancer FGFR3 Tyrosine kinase inhibitor Organoids

Received: 01 June 2023 Published: 03 April 2024

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	Lixinxu DENG
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DENG Lixinxu, CHE Wenan, XU Haibo, DING Xue, SUN Yuandong. Effect of FGFR3 Gene Mutation on Tyrosine Kinase Inhibitor Sensitivity in Bladder Cancer. China Biotechnology, 2024, 44(2/3): 1-13.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2306001 OR https://manu60.magtech.com.cn/biotech/Y2024/V44/I2/3/1

Table 1 The main reagents and their sources

Table 2 The main FGFR3 TKI

Table 3 Bladder cancer organoid media recipe

Fig.1 FGFR3 mutations are associated with survival in patients with bladder cancer

Fig.2 The expression levels of FGFR3 mRNA in sv-huc-1 FGFR3 OE cell line

Fig.3 The expression levels of FGFR3 and downstream proteins ERK and AKT in sv-huc-1 FGFR3 OE cell line A: The results of the Western blotting of the sv-huc-1 FGFR3 OE cell lines B: The FGFR3 protein expression quantification of sv-huc-1 FGFR3 OE cell lines C: The downstream protein pAKT/AKT quantification of sv-huc-1 FGFR3 OE cell lines D: The downstream protein pERK/ERK Quantification of sv-huc-1 FGFR3 OE cell lines

Fig.4 Drug sensitivity curves of FGFR3-TKIs on sv-huc-1 FGFR3 OE cell lines A: CH5183284 B: Erdafitinib C: LY2874455 D: Nintedanib E: TAS-120

Table 4 Each FGFR3 TKIs' IC₅₀

Fig.5 Western blotting of FGFR3-TKIs acting on downstream signaling pathway proteins of sv-huc-1 FGFR3 OE stable cell line

Fig.6 HE staining shows that tumor tissue is consistent with the histological characteristics of tumor-derived organoids A: Parental tumors B: Organoid. Scale bar, 50 μm

Fig.7 Immunofluorescence analysis of marker expression in parental tumors and patient-derived organoids Scale bar, 100 μm

Fig.8 Somatic mutations found in tumor tissue and organoid pairs

Fig.9 The expression levels of FGFR3 mRNA in BCO1 FGFR3 OE organoids

Fig.10 The expression levels of FGFR3 in BCO1 FGFR3 OE organoids

Fig.11 Quantification of FGFR3 protein expression in BCO1 FGFR3 OE organoids

Fig.12 Drug sensitivity curves of FGFR3-TKIs on BCO1 FGFR3 OE A. TAS-120 B. Infigratinib C. LY2874455 D. Erdafitinib E. CH5183284 F. Pemigatinib

Table 5 Each FGFR3 TKIs' IC₅₀


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