Dados do Resumo

Título

Establishing an in vitro model for drug screening for pediatric cancers

Introdução

New treatment strategies are necessary for children with cancer, especially for those which have achieved the limits for the current drug regimens and they are still at risk of either long term effects or complete therapy failure. Pediatric precision oncology studies identified potentially targetable molecular findings in most high-risk cancers but the targeted therapy has facing challenges due to uncertainty regarding the efficacy and benefit–risk balance of precision-guided treatment.

Objetivo

To establish a platform of in vitro models of pediatric cancers to further enable drug screening based on molecular alterations.

Métodos

This study was approved by both CEP (CAAE 44219021.6.0000.5376) and CEUA (0017-2021). We used fresh and frozen tumor tissues from pediatric patients diagnosed with several cancer types. Following the organization of flowchart that included tests of dissociation types, cell plates, media and scaffold combinations, cell number seeding, several were tested across carcinomas, embryonal tumors, lymphomas, sarcomas and central nervous system tumors. For dissociation, the conditions included mechanical dissociation, three enzymes as well as combinations. If cell viability was lower than 75%, dead cell and debris cleaning protocols were applied. Protocols with combined media and supplements, and polyvinyl alcohol (PVA) 0,4% and magnetic beads or no scaffold were tested. Between 1.000 to 20.000 cells were seeded in 96 (round or flat) well-plates. Tumoroid formation was daily monitored.

Resultados

In total, 87 samples (19 fresh tissue and 68 frozen tissue) comprising of 5 tumor types (carcinomas, embryonal tumors, lymphomas, sarcomas and central nervous system tumors). One to 12 fragments of 1 to 7 millimeters were tested for 8 dissociation protocols. Following dissociation, cell viability ranged from 2% to 100%. For 44 samples with cell viability lower than 75% and more than 1 million cells, Ficoll, Percoll or BSA cleaning protocols were applied resulting in recovered cell viability from 2.8% to 98.6%. The use of collagenase and a combination of collagenase and trypsin were the best enzymes for dissociation of soft sarcomas and embryonal tumors, and osteosarcomas, respectively. Cells were plated without the using scaffolds, with 0.4% PVA and/or magnetic beads. Tumoroids formed in 63 of 88 (72% of success) samples. The most successful protocol relied on the use of PVA or the absence of scaffolds. Enrichment for viable cells also improved the time for tumoroids formation.

Conclusões

The best protocol for tumoroid formation relied to a large extent on the tumor type, with some samples not forming regardless of the protocol used. We implemented a platform of three‐dimensional cellular models that will enable drug screening in a precision medicine context for children with cancer, that will have an important impact for children with high-risk tumors that typically have few established treatment options, in spite of advancements in standard therapy.

Financiador do resumo

PRONON 2500211368/2019-41

Palavras Chave

Tumoroids; precision medicine; Pediatric Oncology