Characterization of undifferentiated neuroblastoma cells and their contribution to aggressiveness

Abstract

Neuroblastoma (NB) represents the most prevalent extracranial solid tumor among children, originating from cells within the sympathoadrenal lineage of the neural crest. This disease is characterized by its high clinical heterogeneity, ranging from spontaneous regression to aggressive metastasis. A key aspect of NB is its intratumoral heterogeneity, which underpins the aggressive nature of the disease. Recent transcriptomic studies have revealed that low-risk NB primarily consists of adrenergic differentiated cells, whereas high-risk NB is characterized by a higher proportion of undifferentiated tumor cells, which correlate with progenitor, migratory, and metastatic signatures. These neural crest stem-like cells exhibit cancer stem cell (CSC) properties and are associated with tumor progression, relapse/recurrence, metastasis, and poor clinical outcomes. The development of aggressive metastases involves a complex process requiring cellular properties such as adhesion, migration, and invasion, all of them typical features of stem cells derived from the neural crest. To investigate these neural crest stem-like cells, we employed the tumorsphere (TS) formation culture method, which isolates undifferentiated cells (UC) with self-renewal and proliferative capabilities. We then examined their phenotypic attributes, particularly focusing on their adhesive, migratory, and invasive capacities that set them apart from other tumor cell populations. Our findings indicate that TS cultures enrich for a population of multipotent progenitor cells, resembling progenitors from early developmental stages. These UC demonstrate distinct adhesive properties and a unique cytoskeletal structure that endows them with cytoskeletal plasticity. They also display specific actin-rich protrusions and a unique mode of motility characterized by slow but directional movement, enhancing their response to chemotactic stimuli. Furthermore, these undifferentiated cells exhibit a robust capacity for degrading and invading the extracellular matrix (ECM). Additionally, transcriptomic analysis revealed that this progenitor population expresses genes and pathways directly linked to aggressiveness and metastasis. UC cells also possess the ability to form tumors in vivo, and to develop aggressive metastases in distant organs. We argue that these insights open promising avenues for developing targeted therapeutic strategies aimed at eliminating these highly aggressive cancer cells.