Dados do Resumo

Título

Establishment of in vitro platform for epigenetic drug testing in neuroblastoma tumoroids

Introdução

Neuroblastoma is a pediatric tumor that originates from nerve cells of the sympathetic nervous system. Neuroblastoma substantially contributes to childhood cancer mortality, particularly in high-risk patients suffering chemo-resistant relapse, who present a poor survival rate. The study of tumor methylation profiles has been widely used for molecular classification, allowing better stratification of tumor subtypes and treatment targeting.

Objetivo

Following the molecular characterization of neuroblastoma, the aim was to establish an in vitro platform for epigenetic drug testing using tumoroids originating from patient-derived xenograft (PDX) mouse models.

Métodos

This study was approved by both CEP (CAAE 44219021.6.0000.5376) and CEUA (0017-2021). We performed whole exome sequencing and methylation analysis by Infinium MethylationEPIC BeadChip (Illumina®) in 35 neuroblastoma tumor samples from patients. Data were subjected to the DKFZ/Heidelberg CNS tumor platform (v12.5) classification. For tumoroid formation, mechanical and enzymatic dissociation protocols were implemented with Collagenase type II. Tumoroid formation was performed from 10,000 cells seeded in a 96-well plate, U-bottom, and repellent. We used DMEM-F12 medium (3:1) supplemented with glutamine, antibiotics, EGF, and FGF. After tumoroid establishment, we tested Belinostat, Romidepsin, and Panobinostat at concentrations of 25 uM, 10 uM, 1 uM, 0.1 uM, and 0.01 uM. The IC50 was established for each tumor tested, and the experiment was repeated with the IC50 dose, one dose three times higher and one dose three times lower. Viability assays were performed, as ATPGlow and Live&Dead, to verify drug response.

Resultados

Using methylation profiling, 80.0% of cases (28 out of 35) were classified into a methylation family with a high calibrated score of ≥0.90. Of these cases, 85.7% (30/35) agreed with initial diagnosis. Molecular classification stratified into 4 molecular subclasses in 88.6% of cases (31/35). Cases with initial diagnosis and molecular classification discrepancies were disregarded for further experiments. Methylation analyses also revealed a high level of methylation variation between molecular subclasses. So far, 4 cases of neuroblastomas were processed for 3D culture. The successful dissociation protocol used 6 fragments of 0.5 x 0.5mm, resulting in a range of between 3 and 4 million cells. In 100% of cases, tumoroids were formed. The 4 tumors were responsive to at least one of the epigenetic drugs tested at concentrations from 0.05 uM.

Conclusões

The molecular classification of tumors allowed stratification into subclasses in more than 80% of cases, corroborating that methylation can contribute to the diagnosis of tumors and bring insights into new therapeutic targets for such a heterogeneous group of tumors. We established an in vitro drug testing platform for neuroblastoma that successfully forms tumoroids. Drugs were chosen based on the molecular alterations and successfully killed tumor cells.

Financiador do resumo

PRONON 2500211368/2019-41

Palavras Chave

Neuroblastoma Tumoroids; Molecular Classification; Epigenetic Drug Test