Abstract A24: Brain tumor induction after somatic inactivation of rb1 in zebrafish

Abstract

Abstract Purpose: We found that targeted somatic inactivation of rb1 leads to solid tumor formation in the central nervous system of adult zebrafish. The purpose of our study was to characterize the histological and genetic features of the tumors. Methods: Zebrafish rb1 tumor suppressor was inactivated in somatic tissue by injection of transcription activator-like effector nucleases (TALENs) targeting exon 2 and exon 3 at the one-cell stage. Histopathology and immunohistochemistry with s100 and pH3 was performed on tumor tissue. To confirm TALEN-mediated editing of the targeted rb1 loci and characterize the frequency and type of indel alleles in tumor and somatic tissues we sequenced cloned amplicons from each locus. Results: Zebrafish injected with TALENs targeting exon 2 or exon 3 of rb1 presented with cranial tumors beginning at six months of age. By one year the frequency of adults with tumors was 33-50% and 11%, respectively. Histopathological analyses of tumors from eight exon 2-targeted and six exon 3-targeted fish revealed a range of undifferentiated and differentiated tumor phenotypes. Seven tumors exhibited features suggestive of primitive neuroectodermal tumors (PNET). The remainder exhibited more differentiated tumor phenotypes resembling glioma. S100 was detected in a subset of the tumors. pH3 labeled regions of high mitotic activity in each of the tumors. In 4 out of 4 tumors the frequency of rb1 mutant alleles (61%-87% of cloned amplicons) was consistently higher than wild type. However, only one or two specific rb1 alleles were present in 3 out of 4 tumors. In contrast, the mutation frequency in retinal, muscle, and germline tissues was highly variable (3%-100%), and represented by up to seven different mutant rb1 alleles. Conclusion: Somatic inactivation of rb1 resulted in brain tumors showing varying degrees of differentiation suggesting the possibility that transformed neural progenitors give rise to tumors. rb1 allele frequency is consistent with clonal expansion contributing to tumor growth. Predisposing zebrafish to brain tumors by somatic inactivation of rb1 with site-specific nucleases suggests a simple method for investigating pathways cooperating in tumor progression. Citation Format: Staci L. Solin, Heather R. Shive, Jeffrey J. Essner, Maura McGrail. Brain tumor induction after somatic inactivation of rb1 in zebrafish. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A24.