Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy

Abstract

The emergence of resistance to multiple unrelated chemotherapeutic drugs impedes the treatment of several cancers. Although the involvement of ATP-binding cassette transporters has long been known, there is no in situ method capable of tracking this transporter-related resistance at the single-cell level without interfering with the cell's environment or metabolism. Here, we demonstrate that scanning electrochemical microscopy (SECM) can quantitatively and noninvasively track multidrug resistance-related protein 1-dependent multidrug resistance in patterned adenocarcinoma cervical cancer cells. Nonresistant human cancer cells and their multidrug resistant variants are arranged in a side-by-side format using a stencil-based patterning scheme, allowing for precise positioning of target cells underneath the SECM sensor. SECM measurements of the patterned cells, performed with ferrocenemethanol and [Ru(NH 3 ) 6 ] 3+ serving as electrochemical indicators, are used to establish a kinetic "map" of constant-height SECM scans, free of topography contributions. The concept underlying the work described herein may help evaluate the effectiveness of treatment administration strategies targeting reduced drug efflux.ancer cells, such as lung cancer or leukemia, acquire resistance to multiple unrelated drugs in response to treatment with chemotherapeutic agents (1, 2). Resistance impedes therapeutic effectiveness, which in turn, reduces the long-term survival rate of cancer patients (2). The emergence of multidrug resistance (MDR) involves the overexpression of transmembrane proteins P-glycoprotein (P-gp) and MDR-related protein 1 (MRP1), which both belong to the family of 5′-triphosphatebinding cassette transporters (known as ABC transporters). P-gp and MRP1 act as molecular "pumps," actively removing therapeutic agents from the cancer cells, thereby preventing the drug from inducing the desired effect on the cell nucleus or cytoplasm. MDR based on P-gp is relatively well understood and involves binding of hyaluronan to the cell surface glycoprotein CD44. The resulting up-regulation of the transcriptional cofactor p300 expression and therefore the NFkappaB-specific transcriptional up-regulation lead to the production of P-gp, and with that chemoresistance in cells (3). However, the mechanism that causes MRP1-mediated MDR remains unclear.Currently, quantification of MDR relies on immunohistochemical analyses, such as real-time PCR, focusing mostly on P-gp or other members of ABC transporters (4-9). Fluorescent MRP1-specific studies were also conducted, revealing that resistant and nonresistant cancer cells have differential intracellular content of glutathione and GST, which affect their cell death mechanism during hyperthermia (10). Microvoltammetry was also used to measure the efflux of chemotherapeutic drugs from normal and MDR cancer cells on the single-cell level, with detection limits in the nanomolar range (11). Finally, flow cytometry has routinely been used to quantify and compare expression levels and activity of dif...

Electrochemical recognition and quantification of cytochrome c expression in Bacillus subtilis and aerobe/anaerobe Escherichia coli using N,N,N′,N′-tetramethyl-para-phenylene-diamine (TMPD)

“…Quantitative analyses of metabolites released from organisms have been proposed in various ways in literature, 10 – 12 and the expression of a turnover number has become a standard measure in enzymology, 13 where it describes the maximum number of substrate molecules converted by an enzyme in a given time. 14 The use of turnover numbers can also be applied to bacteria and their ability to enzymatically convert redox active substrate molecules, such as N , N , N ′, N ′-tetramethyl- para -phenylene-diamine (TMPD), per second at a given concentration during electrochemical measurements.…”

Review—Advances in Scanning Electrochemical Microscopy (SECM)

“…Several reviews have been dedicated to SECM investigations of such biological systems. [119][120][121][122][123] More recent applications have involved assessing multidrug resistance on cell co-culture patterns, 124 real-time mapping of local hydrogen peroxide production across a biofilm of oral bacteria and in observing its consumption in a polymicrobial biofilm through a mechanism involving one of the bacteria during spatial scanning, 125 in the discovery of a biofilm electrocline of oral bacteria using real-time 3D metabolite analysis, 126 and in the real-time monitoring of quorum sensing in bacterial aggregates. 127,128 Some examples are briefly discussed below.…”

Non‐Invasive Monitoring of Osteogenic Differentiation on Microtissue Arrays under Physiological Conditions Using Scanning Electrochemical Microscopy

“…In that context, scanning electrochemical microscopy (SECM) is a powerful technique since it provides access to local chemical gradients 8–10. SECM has already been applied to monitor biological samples and map the concentration of various electroactive species such as oxygen 11,12, nitric oxide 13 and redox mediators 14, 15. SECM‐based assays for evaluating the differentiation status of live single embryonic stem cells and embryoid bodies using ALP activity (undifferentiated cells have higher activity when compared to differentiated cells due to changes in ALP expression) have been reported recently 16, 17.…”