Quantification of the binding potential of cell-surface receptors in fresh excised specimens via dual-probe modeling of SERS nanoparticles

Abstract

The complete removal of cancerous tissue is a central aim of surgical oncology, but is difficult to achieve in certain cases, especially when the removal of surrounding normal tissues must be minimized. Therefore, when post-operative pathology identifies residual tumor at the surgical margins, re-excision surgeries are often necessary. An intraoperative approach for tumor-margin assessment, insensitive to nonspecific sources of molecular probe accumulation and contrast, is presented employing kinetic-modeling analysis of dual-probe staining using surface-enhanced Raman scattering nanoparticles (SERS NPs). Human glioma (U251) and epidermoid (A431) tumors were implanted subcutaneously in six athymic mice. Fresh resected tissues were stained with an equimolar mixture of epidermal growth factor receptor (EGFR)-targeted and untargeted SERS NPs. The binding potential (BP; proportional to receptor concentration) of EGFR – a cell-surface receptor associated with cancer – was estimated from kinetic modeling of targeted and untargeted NP concentrations in response to serial rinsing. EGFR BPs in healthy, U251, and A431 tissues were 0.06 ± 0.14, 1.13 ± 0.40, and 2.23 ± 0.86, respectively, which agree with flow-cytometry measurements and published reports. The ability of this approach to quantify the BP of cell-surface biomarkers in fresh tissues opens up an accurate new approach to analyze tumor margins intraoperatively.

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

“…Based on flow cytometry validation data, the relative ratio of EGFR expression between A431 cell and U251 cell was used as a method to validate the accuracy of receptor concentration estimations. The expected ratio for EGFR expression was 3.05 ± 0.88 from an in vitro study (Sinha et al , 2015). Based on the experimental result, the estimated EGFR expression ratio (A431 vs. U251) was 1.70 ± 1.93, 4.20 ± 4.56, 2.09 ± 2.47, and 2.31 ± 1.81 for fluorescence, ratiometric, DPM-NS, and RPAM estimations, respectively.…”

“…To evaluate the accuracy of receptor concentration estimation on ex vivo tissue samples, data from this study were compared with data collected in a previous study (Sinha et al , 2015). Briefly, in the previous study, excised A431 and U251 tumor xenografts were developed subcutaneously in male nude mice ( n = 8 for U251; n = 9 for A431).…”

“…Recent work has established the potential for paired-agent methods to overcome the limitations of direct topical staining of thick tissues (Davis et al , 2013; Liu et al , 2009; Sinha et al , 2015; Wang et al , 2014a; Wang et al , 2016; Wang et al , 2015). Such methods utilize the signal from a control (untargeted) imaging agent that is co-administered with one or more targeted agent(s) to account for background staining and variable optical properties (Tichauer et al , 2015).…”

“…, dividing the targeted agent signal by the control agent signal (ratiometric). These methods have been shown to correlate with receptor concentration under certain conditions (Sinha et al , 2015); however, the linearity of the correlation is highly dependent on the sample type and experimental protocol.…”

Surface-enhanced Raman spectroscopy for in vivo biosensing

“…Towards this, a dual-probe approach has been used whereby tissues from mouse tumours were incubated with two types of SERS-active NP, one featuring a EGFR-specific antibody and one with no targeting moiety. EGFR concentrations in fresh tissue samples can thus be measured by subtracting the background signals obtained 146 . A single excitation wavelength was used to obtain distinct spectra from the two different probes, and it seems that the method will be extendable to include additional probes targeting different cancer markers, enabling significantly more accurate tumour detection.…”