Nanoimmunoassay to Detect Responses in Head and Neck Cancer: Feasibility in a Mouse Model

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Abstract

Objective

To demonstrate the feasibility of detecting and quantifying extracellular signal-related kinase (ERK) phosphorylation status using nanoimmunoassay (NIA).

Study Design

Analyses using Cal27, SCC25, and OSC19 head and neck squamous carcinoma cell lines in vitro and in a murine xenograft model.

Subjects and Methods

NIA and immunoblot were performed on whole-cell lysates, tumor lysates, and fine-needle aspirate biopsies to detect ERK phosphorylation states.

Results

Using NIA, all 6 isoforms of ERK1/2, including nonphosphorylated, monophosphorylated, and diphosphorylated species, could be reliably detected, distinguished, and quantified in a single assay using a single antibody. In vitro treatment of Cal27 cells with the epidermal growth factor receptor inhibitor gefitinib abolished phospho-ERK detection by immunoblot but resulted in residual detectable species by NIA. Residual phospho-ERK in gefitinib-treated cells could be further reduced by the addition of the insulin-like growth factor 1 receptor inhibitor OSI-906; this correlated with an additional decrease in proliferation over gefitinib alone. In a pilot study of 4 murine xenograft tumors, NIA performed on tumor lysates and fine-needle aspirate biopsies demonstrated altered ERK profiles after 2 days of gefitinib treatment compared with untreated mice.

Conclusion

NIA offers a novel approach to quantitating the activation state of signaling molecules such as ERK in nanoscale in vitro and in vivo samples across a wide dynamic range. As such, it has potential to provide molecular diagnostic information before, during, and after treatment using a minimally invasive technique. Further study is warranted to determine its utility in assessing signaling proteins as biomolecular outcome predictors in clinical trials.

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