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. 2014 Oct 14:4:6609.
doi: 10.1038/srep06609.

In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance

Wei Wang  1 , Linliang Yin  2 , Laura Gonzalez-Malerva  3 , Shaopeng Wang  4 , Xiaobo Yu  3 , Seron Eaton  3 , Shengtao Zhang  5 , Hong-Yuan Chen  1 , Joshua LaBaer  3 , Nongjian Tao  6
Affiliations

In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance

Wei Wang et al. Sci Rep. .

Abstract

Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

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Figures

Figure 1
Figure 1
Top: Under “bypass hypothesis”, in situ binding kinetics of Herceptin with resistant cell was not affected. Bottom: “Alternation hypothesis” suggests an altered binding kinetics due to the influences of relevant proteins surrounding Her2.
Figure 2
Figure 2
A) Experimental setup. A p-polarized LED light is directed onto a gold-coated glass slide mounted on a SF-11 prim to create SPR on the gold surface, which is captured with a CCD camera. In situ binding kinetics can be extracted from the time lapse SPR image when drug flown over a cell adhered on the gold chip. (B) Short-term noise level when using a superluminescent (SLD, red curves) and a light emitting diode (LED, black curves) as light source. (C) Long-term noise level for a LED light source with and without temperature control. D) Typical SPR image of a few tens of SK-BR3 cells adhered on a gold-coated coverslip. E) The SPR sensorgrams of each individual cells (black: average SPR sensorgram, red: fitted curve, grey background: cell-to-cell variation) and the surrounding regions without cell coverage (blue curve). F) The concentration-dependent SPR sensorgrams and global fitting (red curves). The Herceptin concentration was 21, 8.4, 4.2, 2.1, 1.05 and 0.42 μg/mL from top to bottom curve, respectively. Scale bar, 100 μm.
Figure 3
Figure 3. SPR sensorgrams when introducing 2.1 μg/mL Herceptin over A) BT474, B) MCF7-Her2, C) HeLa and D) MCF7-WT, respectively.
Figure 4
Figure 4. Herceptin interactions with SK-BR3 Herceptin-resistant (H6 and C5) and SK-BR3 Herceptin-sensitive (E8 and C11) clones.
A) The average SPR sensorgram of each clone and the corresponding two-constant (H6 and C5) or one-constant (E8 and C11) fitting. B) The resistance index (RI) map of each clone, demonstrating the potency of individual cell being resistant to Herceptin treatment. C) SPR sensorgrams of two individual cells are shown to demonstrate the cell-to-cell variation when interacts with Herceptin. Scale bars, 100 μm.
Figure 5
Figure 5. Molecular-scale mechanism of ineffective Her2 population.
A) Resistant clones (H6 and C5) overexpress MUC4 while sensitive clones (E8 and C11) do not. B) Dual-staining of Her2 and MUC4 in one single cell showing negative co-localization for Her2 and MUC4. Similar features were observed in multiple cells, but only a representative one was shown here. Scale bars, 50 μm.
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