Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line

Abstract

Background Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible. Methods We applied high density SNPa and NGS techniques to in vivo and in vitro models (orthotropic xenograft vitronectin knock-out mice and 3D bioprinted hydrogels with different stiffness) using two representative neuroblastoma cell lines (the MYCN-amplified SK-N-BE(2) and the ALK-mutated SH-SY5Y), to discern how tumor genomics patterns and clonal heterogeneity of the two cell lines are affected. Results We describe a remarkable subclonal selection of genomic aberrations in SK-N-BE(2) cells grown in knock-out vitronectin xenograft mice that also emerged when cultured for long times in stiff hydrogels. In particular, we detected an enlarged subclonal cell population with chromosome 9 aberrations in both models. Similar abnormalities were found in human high-risk neuroblastoma with MYCN amplification. The genomics of the SH-SY5Y cell line remained stable when cultured in both models. Conclusions Focus on heterogeneous intratumor segmental chromosome aberrations and mutations, as a mirror image of tumor microenvironment, is a vital area of future research.

Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models

“…No genomic differences were observed between hydrogels grown with SK-N-BE(2) cell line alone and co-cultured with SW10 cell line in any of the studied conditions. Genomics of SH-SY5Y cells remained stable in every studied 3D condition, identical to that of 2D cultures[22].…”

“…These data could be related to the differences observed in their genetic profile, which would indicate that stiffness has a strong effect on SK-N-BE(2). In fact, after 6 weeks of stiffness surroundings, new segmental chromosomal aberrations such as 9p (where DOCK8 and KANK1 genes are located) started to appear in SK-N-BE(2) but not in SH-SY5Y [22]. Further tests in other NB cell lines are warranted to evaluate whether the effect of stiffness on MYCNand non-MYCN-amplified cells can be generalized.…”

“…Performing digital image analysis, we have successfully defined morphological patterns associated with ECM stiffness in NB, considering different components such as blood and lymph vessels, collagen fibers, glycosaminoglycans (GAGs), and glycoproteins such as fibronectin and vitronectin (VN) [13][14][15][16]19,20]. In recent years, we have also worked with 3D cell culture in bioprinted models, showing that the biomechanical forces imposed by the scaffolds have a great impact on malignant neuroblastic cell behavior [21,22].…”

“…This makes VN a potential therapeutic target, assuming that inhibiting its interaction with tumor cell integrins and/or its ECM remodeling action could reduce tumor cell migration, which prompted us to evaluate its presence in 3D NB models. We also studied the expression dynamics of two additional genes, DOCK8 and KANK1, having detected a common atypical segmental chromosomal aberration affecting these genes located in chromosome 9p in some high-risk NB patients, as well as in orthotopic mice models and 3D NB bioprinted models [22]. DOCK8 is involved in cytoskeleton remodeling, inducing migration in macrophages, Schwann precursors cells, T cells, and dendritic cells [31][32][33][34].…”

“…In this study, we used morphometric digital analysis to identify cell characteristics and evaluate cell behavior on 3D scaffold-based NB models. Following on from our previous genetic studies [22], we have chosen replicates of the 3D cultured samples to characterize the expression pattern of the above-mentioned markers and pinpoint correlations with their genomic landscape. The specific parameters analyzed herein provide proof of concept of the wide spectrum of possibilities that these techniques offer to obtain clinically relevant in vitro models for preclinical therapeutic assessment.…”