Image3_Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions.TIF

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.</p

Genetic Instability and Intratumoral Heterogeneity in Neuroblastoma with <em>MYCN</em> Amplification Plus 11q Deletion

<div><h3>Background/Aim</h3><p>Genetic analysis in neuroblastoma has identified the profound influence of <em>MYCN</em> amplification and 11q deletion in patients’ prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features.</p> <h3>Methods</h3><p>We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques.</p> <h3>Results and Conclusion</h3><p>This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.</p> </div

MLPA data.

<p>INRG Biology Committee definitions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053740#pone.0053740-Schleiermacher2" target="_blank">[11]</a>. <i>MYCN</i> amplification (MNA), up to 4-fold excess of signal numbers of the chromosomal region of interest compared with the reference signals; Gain (+), unbalanced ratio (high signal excess) between the signals of a gene and all other probes located on the same chromosome; Loss (−), unbalanced ratio (low signal excess) between the signals of the chromosomal region of interest (at least two adjacent probes) and the reference signals (at least two) of the chromosomal region of interest.</p><p>The cases have been listed according to chromosome 2 aberrations using pangenomic techniques.</p

Schematic representation of the amplicons in chromosome 2.

<p>All data are illustrated according to the NCBI build Hg19. (A) Genes included in the <i>MYCN</i> amplicon. The crossed lines in cases 1, 7, 9 and 10 indicate that the breakpoint mapped more distal in chromosome 2 than the area represented. (B) Boxes and arrows indicate position of the different amplicons in the cases with complex amplification. The cases have been listed according to chromosome 2 aberrations.</p

FISH results in the heterogeneous cases (hetMNA and het11q-del).

<p>Disomic cells (ratio 2∶2) and numeric alterations (ratios 3∶3; 4∶4…), balanced ratio between the signal numbers of chromosomal region of interest and the reference signals on the opposite arm of the chromosome; Cells with gain (ie ratios 2∶4; 3∶4…), signal numbers of the chromosomal region of interest exceed up to 4-fold the number of reference signals; Cells with imbalance (ie ratios 3∶2; 4∶3…), imbalance ratio between the signal numbers of chromosomal region of interest and the reference signals with more than 1 signals of chromosomal region of interest; Cells with deletion (ie. ratios 2∶1; 3∶1 4∶1…), unbalanced ratio between the signal numbers of the chromosomal region of interest and the reference signals with only 1 signal of the chromosomal region of interest; hetMNA, occurrence of clusters or as single cells with amplification (at least five cells per slide) surrounded by non-amplified tumor cells.</p><p>ND, not done.</p><p>The cases have been listed according to chromosome 2 aberrations using pangenomic techniques.</p

Image1_Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions.TIF

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.</p

Graphic representation of chromosomes 11, 1 and 17.

<p>Bars illustrate the deleted/gain region. The positions of the breakpoints are indicated in megabases. (A) Deletions of chromosome 11q. SRO: 1117.7 to qter (22.8 Mb). (B) Deletions of chromosome 1p. SRO: 3.5 to 8.6 Mb (5.1 Mb). (C) Gain of chromosome 17q. SRO: 41.7 to qter (37.1 Mb). The cases have been listed according to chromosome 2 aberrations.</p

Image2_Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions.TIF

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.</p

Histopathological and genetic characteristics of the tumors.

<p>uNB, undifferentiated neuroblastoma; pdNB, poorly differentiated NB; nGNB, nodular ganglioneuroblastoma; %, percentage; het, heterogeneous; ND, not done; D, diploid; BD, borderline diploid, T, triploid; TE, tetraploid; P, pentaploid.</p><p>All cases presented dmin except case number 5 (* dmin plus HSR).</p><p>The patients have been listed according to the stage of disease. ID was assigned according to genetic aberrations of chromosome 2.</p

Clinical characteristics and outcome.

<p>M, male; F, female; N, no; Y, yes; B, bone; BM, bone marrow; LN, lymph nodes; ST, soft tissue; uNB, undifferentiated neuroblastoma; pdNB, poorly differentiated NB; nGNB, nodular ganglioneuroblastoma HR-NBL1, High-Risk Neuroblastoma Study 1;INES,Infants Neuroblastoma European Study, SIOPEN protocols; CR, complete response; VGPR, very good partial response; PR, partial response; DP, disease progression; SurPR, surgical partial resection; DOD, died of disease; DOS, died of sepsis; DTC, died of treatment complication, AWD, alive with disease; ADF, alive disease-free.</p><p>The patients have been listed according to the stage of disease. ID was assigned according to genetic aberrations of chromosome 2.</p