Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling

Abstract

Introduction: High-risk neuroblastoma (HR-NB) presenting with hematogenous metastasis is one of the most difficult cancers to cure. Patient survival is poor. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictate tumor progression, relapse/recurrence, and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fit the CSC model.

Methods: Well-characterized clones of metastatic site-derived aggressive cells (MSDACs) from a manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit by examining epithelial-to-mesenchymal transition (EMT) (E-cadherin, N-Cadherin), survival (NFκB P65, p50, IκB and pIκB) and drug resistance (ABCG2) by immunoblotting; pluripotency maintenance (Nanog, SOX2) by immunofluorescence; and EMT and stemness related transcription of 93 genes by QPCR profiling. Plasticity of MSDACs under sequential alternation of culture conditions with serum and serum-free stem-cell conditions was assessed by clonal expansion (BrdU incorporation), tumorosphere formation (anchorage independent growth), EMT and stemness related transcriptome (QPCR profiling) and validated with MYC, SOX2, EGFR, NOTCH1 and CXCL2 immunoblotting.

Results: HR-NB MSDACs maintained in alternated culture conditions, serum-free stem cell medium to growth medium with serum and vice versa identified its flexible revocable plasticity characteristics. We observed signatures of stem cell-related molecular responses consistent with phenotypic conversions. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem cell-related signaling events, but also instigated additional events depicting aggressive adaptive plasticity.

Conclusions: Together, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fit the CSC model, and further identified the intrinsic adaptiveness of the successive phenotype switching that clarifies the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem cell-related molecular rearrangements may hold the key to the switch from favorable disease to HR-NB.

Figure 1

Morphometrics of the parental SH-SY5Y and metastatic site derived aggressive cells and the schematic representation of the experimental workflow. (A) Representative light microscope photographs of parental SH-SY5Y cells and metastatic site-derived aggressive cells (MSDACs) maintained in serum-free stem cell medium. Parental cells exhibited monolayer spreading with organized neurites (100 ×). MSDACs are more spherical, smaller in size (100 ×), and formed organized tumorospheres (10 ×). (B) Schematic representation of experimental workflow: Three well characterized individual clones of MSDACs were alternated between stabilized (three generations) microenvironment simulations with serum-free stem cell medium to growth medium with serum and reintroduced back to serum-free stem cell medium. Parental cells grown in routine medium and serum-free stem cell medium were included as controls. Cancer stem cell physiognomy, tumorosphere formation capacity, and transcriptional and translational rearrangements associated with phenotype conversions were assessed at the end of each phase and in controls.

Figure 2

Cancer stem cell characterization of MSDACs. (A) Immunoblotting analysis showing activated EMT (increased N-cadherin and decreased E-cadherin), drug resistance (increased ABCG2), and survival response (increased p65/p50 and pIκBα) in MSDACs compared with parental SH-SY5Y cells. (B) High content confocal immunofluorescence showing increased expression levels of pluripotency maintenance factors SOX2 and NANOG in three different clones of MSDACs. (C) Results of QPCR profiling analysis showing transcriptional activation of 29 stem cell-related molecules in MSDACs maintained in serum-free stem cell medium, compared with SH-SY5Y cells. EMT, epithelial-to-mesenchymal transition; MSDACs, metastatic site-derived aggressive cells.

Figure 3

Modulations in cell proliferation levels of MSDACs under alternated culture conditions. A) Representative microphotographs of random fields obtained from Operetta high-content confocal imaging showing BrdU incorporation levels in three different clones of MSDACs maintained in serum-free stem cell medium for three generations: SF-SCM-1(3G), SF-SCM-1(3G) cells transferred and maintained in growth medium with FBS for three generations (SF-SCM-1(3G) → GM-FBS(3G), or the MSDACs reintroduced in SF-SCM for three generations (SF-SCM-1(3G) → GM-FBS(3G) → SF-SCM-2(3G). Parental SH-SY5Y cells maintained in growth medium are used as controls. B) Histograms obtained from Columbus image analysis showing significant decline in BrdU positive cells in SF-SCM-1(3G) → GM-FBS(3G) MSDACs and regained and amplified cell proliferation in MSDACs reintroduced in SF-SCM (SF-SCM-1(3G) → GM-FBS(3G) → SF-SCM-2(3G). Brd-U, bromodeoxyuridine; MSDACs, metastatic site-derived aggressive cells.

Figure 4

Tumorosphere formation and anchorage independent growth capabilities of MSDACs in alternated culture conditions. (A) Representative microphotographs showing growth trends, monolayer differentiation or cell aggregation, tumorosphere formation, and phenotype conversions in three different clones of MSDACs maintained in serum-free stem cell medium for three generations: SF-SCM-1(3G), SF-SCM-1(3G) cells transferred and maintained in growth medium with FBS for three generations (SF-SCM-1(3G) → GM-FBS(3G), or the MSDACs reintroduced in SF-SCM for three generations (SF-SCM-1(3G) → GM-FBS(3G) → SF-SCM-2(3G). (B) Parental SH-SY5Y cells maintained in growth medium or SF-SCM are used as controls. (C) Representative microphotographs of soft agar colony forming assay showing anchorage-independent growth signatures of MSDACs maintained in serum-free stem cell medium for three generations, transferred and maintained in growth medium with FBS for three generations or reintroduced in SF-SCM for three generations. FBS, fetal bovine serum; MSDACs, metastatic site-derived aggressive cells; SF-SCM, serum-free stem cell medium.

Figure 5

QPCR profiling identifies regained and/or lost transcription of EMT and stem cell molecules in MSDACs cultured under alternated growth conditions. (A) Histograms of QPCR profiling analysis showing stem cell-related transcriptional responses regained with the reverted CSC phenotype when MSDACs are reintroduced into SF-SCM. Interestingly, the rescue of stem cell transcriptional responses is relatively heightened when compared with the earlier CSC phenotype stage. (B) Histograms showing the panel of stem cell-related molecules that were significantly lost in the second phase CSC phenotype. Almost all of these molecules showed decreased expression in the non-favorable niche (GM-FBS) and never regained expression when returned to a favorable environment. CSC, cancer stem cell; EMT, epithelial-to-mesenchymal transition; GM-FBS, growth medium with fetal bovine serum; MSDACs, metastatic site-derived aggressive cells; SF-SCM, serum-free stem cell medium.

Figure 6

Adaptive plasticity of stemness signaling in MSDACs cultured under alternated growth conditions. (A) Histograms of QPCR profiling analysis showing stem cell-related transcriptional responses that were exceptionally activated with the regaining of the CSC phenotype under favorable niche conditions. These molecules did not show either loss or gain under the early CSC stage or the unfavorable and differentiated stage, but demonstrated a robust and significant activation when the MSDACs regained their CSC status, suggesting an intrinsic adaptive gain in stemness. (B) Representative blots showing the phenotypic conversion associated translational modifications in MYC, SOX2, EGFR, NOTCH1, and CXCL12. The phenotype-dependent modifications of these proteins corroborated well with their transcriptional expression data and validated the regain of the CSC status. CSC, cancer stem cell; MSDACs, metastatic site-derived aggressive cells.