Abstract B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric malignance, but 35% of patients still relapse after chemotherapy treatment, mainly in the central nervous system (CNS). In order to search for new biomarkers that could predict CNS infiltration and relapse, many studies are trying to find gene products aberrantly expressed in these patients, including osteopontin (OPN). The OPN primary transcript undergoes alternative splicing, generating at least five OPN splicing isoforms (OPN-SI), named OPNa, OPNb, OPNc, OPN4 and OPN5, that have tumor specific roles and have been reported as biomarkers in solid tumors. However, knowledge regarding OPN-SI in B-ALL is scarce. This study aimed to investigate the expression patterns and roles of OPN-SI in CNS infiltration in B-ALL. OPN-SI transcript levels were analyzed by quantitative real time PCR (qRT-PCR) assays using RNA extracted from 54 B-ALL pediatric patient blood samples or from RS4,11 cell line. OPNc transcript knock-down in RS4;11 cells (a B-ALL cell line containing the MLL gene fusion) was performed using anti-OPNc or control scrambled antisense DNA oligomers (anti-OPNc-ASOs and Scbl-ASO, respectively). These oligomers were transfected in RS4;11 cells and the in vitro effects of OPNc knock-down were evaluated by several functional assays, such as cell viability by MTT analysis, cell adhesion, and transmigration and invasion assays using a boyden chamber device. Among tested OPN-SI, OPNc is the most highly expressed OPN-SI in B-ALL patient samples containing the MLL fusion gene, when compared to those samples harboring the ETV6-RUNX1 fusion gene (p<0,005). High OPNc expression levels were significantly associated with poor prognostic features, such as high-risk classification (p<0,005), CNS infiltration and high white blood cell counting (p<0,05). The RS4;11 cells in which OPNc was knocked-down decreased their OPNc expression levels in 85% in relation to control Scbl-ASO transfected cells (p<0,005). These cells decreased their viability in 41% (p<0,05), besides displaying a 55% decrease in RS4;11 cell adhesion to matrigel. In addition, these cells decreased their capacity to transmigrate (54%) and invade the matrigel (51%) in comparison to control cells. In summary, these results suggest that OPNc transcript levels are associated with B-ALL worse prognostic features and that it may mediate some key steps involved on CNS infiltration mechanistic. Hence, our data provide important basis for the potential use of OPNc as a biomarker for B-ALL CNS relapse and for the involvement of this isoform on some steps that can mediate CNS invasion, opening new opportunities to improve B-ALL pediatric patients’ survival rates and quality of life. Citation Format: Ana Clara Ana Clara Santos da Fonseca Bastos, Luciana Bueno Ferreira, Mariana Emerenciano, Etel Rodrigues Pereira Gimba. Osteopontin-c is worse prognostic marker and mediate key steps associated with central nervous system infiltration in B-cell acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6719.