Modulation of interactions of neuroblastoma cell lines with extracellular matrix proteins affects their sensitivity to treatment with the anti-GD2 ganglioside antibody 14G2a

Abstract

Children diagnosed with high risk neuroblastoma have poor prognosis which stimulates efforts to broaden therapies of the neoplasm. GD2-ganglioside (GD2) marks neuroblastoma cells and is a target for monoclonal antibodies. We have recently shown that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 mouse monoclonal antibody 14G2a (mAb). For IMR-32 and LA-N-1 cell lines, treatment with the 14G2a mAb induced evident changes in appearance such as cell rounding, aggregation, loose contact with culture plastic, or detachment. Such findings prompted us to investigate whether modulation of attachment of neuroblastoma cells to extracellular matrix (ECM) proteins can affect their sensitivity to the 14G2a mAb treatment. First, using ultra-low attachment plates, we show that survival of the IMR-32, LA-N-1, LA-N-5, CHP-134 and Kelly cells depends on attachment. Next, we compared cellular ATP levels of the cell lines treated with the 14G2a mAb using uncoated, fibronectin-, collagen IV-coated surfaces to show that the ECM proteins slightly modulate sensitivity of the cell lines to the mAb. Then, we characterized presence of selected integrin subunits or their complexes on the cell surface. Finally, we applied small molecule inhibitors of selected integrin complexes: obtustatin (inhibiting α1β1 heterodimer), BIO 1211 (inhibiting active α4β1 heterodimer), cilengitide and SB273005 (inhibitors of αVβ3, αVβ5 heterodimers) to verify their effects on attachment of cell lines, cellular ATP levels, and in some experiments activities of apoptosis-executing caspase-3 and -7, for the compounds used alone or in combination with the 14G2a mAb. We characterized levels of total FAK (focal adhesion kinase), p-FAK (Tyr397) in IMR-32 cells treated with BIO 1211, and in LA-N-5, Kelly and SK-N-SH cells treated with SB273005. Our results extend knowledge on factors influencing cytotoxicity of 14G2a.

Hu14.18K.322A Causes Direct Cell Cytotoxicity and Synergizes with Induction Chemotherapy in High-Risk Neuroblastoma

“…Exposure to hu14 resulted in clumping and aggregate formation in our study. Similar effects were observed by Horwacik et al in NB cells treated with 14Ga, suggesting that integrins and extracellular matrix might play a role in this phenomenon [ 25 ].…”

“…The idea that antibodies themselves can cause direct cytotoxicity is interesting and may play a role in standardizing therapy. Direct cell cytotoxicity by anti-GD2 antibodies has been reported by other researchers over the years using 14G2a or 3F8, both anti-mouse monoclonal antibodies [9,25,26]. In this study, we investigated the mechanisms of direct cytotoxicity of a humanized anti-GD2 antibody, hu14.18K322A (hu14).…”

“…p53 is a tumor-suppressor protein that plays an important role in cell cycle, proliferation, and cell death [ 28 ]. Horwacik et al showed an increase in p53 levels in response to anti-GD2 antibody [ 25 ]. However, p53 decreased in SK-N-BE1 and remained unchanged in CHLA15 upon hu14 treatment in this study.…”

Anticancer Efficacy and Mechanisms of a Dual Targeting of Norepinephrine Transporter and Thyrointegrin αvβ3 Antagonist in Neuroblastoma

“…BG has been shown to target neuroendocrine tumors 32 . Horwacik and Rokita showed that targeting αvβ3 integrin can control cancer biology such as tumor angiogenesis, growth, and metastasis in neuroblastoma 33 .…”