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.

Search results

Nanoimmunoassay to Detect Responses in Head and Neck Cancer

Corrigendum