Metabolic Study of Cancer Cells Using a pH Sensitive Hydrogel

Dec 29, 2016 - changes in pH of the media as cancer cells consume glucose and release ... indicated the importance of monitoring metabolic alterations...
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Metabolic Study of Cancer Cells Using a pH Sensitive Hydrogel Nanofiber Light Addressable Potentiometric Sensor Parmiss Mojir Shaibani,‡ Hashem Etayash,†,‡ Selvaraj Naicker,‡ Kamaljit Kaur,†,§ and Thomas Thundat*,‡ †

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2V4, Canada § Chapman University School of Pharmacy (CUSP), Harry and Diane Rinker Health Science Campus, Chapman University, Irvine, California 92618-1908, United States ‡

ABSTRACT: We report a simple, fast, and cost-effective approach that measures cancer cell metabolism and their response to anticancer drugs in real time. Using a Light Addressable Potentiometric Sensor integrated with pH sensitive hydrogel nanofibers (NF-LAPS), we detect localized changes in pH of the media as cancer cells consume glucose and release lactate. NF-LAPS shows a sensitivity response of 74 mV/pH for cancer cells. Cancer cells (MDA MB231) showed a response of ∼0.4 unit change in pH compared to virtually no change observed for normal cells (MCF10A). We also observed a drop in pH for the multidrug-resistant cancer cells (MDA-MB-435MDR) in the presence of doxorubicin. However, inhibition of the metabolic enzymes such as hexokinase and lactate dehydrogenase-A suggested an improvement in the efficacy of doxorubicin by decreasing the level of acidification. This approach, based on extracellular acidification, enhances our understanding of cancer cell metabolic modes and their response to chemotherapies, which will help in the development of better treatments, including choice of drugs and dosages. KEYWORDS: cancer cells, metabolism, nanofibers, potentiometric sensor, Warburg effect, extracellular acidification

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that an altered energy mechanism is what distinctly separates cancer cells from normal cells.8 The Light Addressable Potentiometric Sensor (LAPS) has been widely used in pH sensing for chemical and biological measurements.9−11 The LAPS is a photoelectrochemical sensor that combines the simplicity of electrochemical sensors with the advanced features of semiconductor-based sensors. LAPS is a simple technique compared to other conventional detection techniques such as the cell plating and the ELISA assay and has comparable sensitivity to other rapid noninvasive tools of detection such as the impedance spectroscopy,12 surface plasmon resonance (SPR),13 microfluidic resonance,14,15 and microcantilevers.16,17 LAPS is less expensive, simple to use, requires a short operation time, and is highly sensitive since only parts of the sensor which are illuminated will generate a photocurrent.10,18 Therefore, a focused light beam can be used to create a spatial map by monitoring changes at various regions on the surface of the sensor. Moreover, LAPS can be easily configured as a portable device for field applications. In our

bserved metabolic properties of cancer cells differ significantly from those of normal cells.1,2 Emerging evidence shows that tumor cells utilize glucose and glutamine metabolic processes to meet the increased energy demands needed for proliferation, invasion, angiogenesis, and metastasis.3 The production of energy in cancer cells relies abnormally on aerobic glycolysis, a phenomenon known as the Warburg effect.3 The Warburg effect is defined by an increased level of glucose utilization via glycolysis. In addition, such highly proliferating cells depend on other atypical metabolic properties like increased fatty acid synthesis and elevated rates of glutamine breakdown.3 Previous studies have shown that many characteristics of cancer cells, such as dysregulated Warburg-like glucose metabolism, fatty acid synthesis, and glutaminolysis, are linked to therapeutic resistance in cancer treatment.4−6 Furthermore, a number of recent reports have shown that targeting cellular metabolism may improve the response to cancer therapeutics;7 thus, combining chemotherapeutics with metabolic enzyme inhibitors may represent a promising strategy to overcome drug resistance in cancer therapy. Del Ben et al. also recently indicated the importance of monitoring metabolic alterations in cancer cells to detect circulating tumor cells (CTCs), stating © XXXX American Chemical Society

Received: October 11, 2016 Accepted: December 29, 2016 Published: December 29, 2016 A

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Figure 1. pH sensitive hydrogel nanofiber integrated light addressable potentiometric sensor (NF-LAPS) setup. The diagram on the right shows the sensor setup combing three different electrodes, a semiconductor working electrode (WE), a reference electrode (RE), and a counter electrode (CE). The pH sensitive hydrogel was fabricated on top of p-type SiO2 substrate to work as a pH sensitive adlayer (scanning electron microscopy shows the nanofiber layer). As the light sparkles, it generates electron−hole pairs in the semiconductor; a photocurrent is then produced while the electrode is in a state of depletion. The generated photocurrent curve typically shifts to higher potential values when pH increases, and the reverse is true. Sensitive Layer Characterization. The nanofiber (NF) layer was characterized according to the methods described previously using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). The swelling behavior of the NFs was also studied as described earlier using weighing.9 Cell Culture. The human breast cancer cells line MDA-MB-231 (American Type Culture Collection, Manassas, VA) was used in this study. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 IU mL−1 penicillin, and 100 IU mL−1 streptomycin. The human mammary epithelial cell line MCF10A was also used as a representative of normal breast cells. MCF10A was cultured in a minimal essential growth medium (MEGM, Lonza, Cedarlane) supplemented with the same additives as above. Resistant type cancer cell line (MDA-MB-435-MDR) was used as a resistive breast cancer cells in order to investigate the chemoresistance and response to antibiotics in the presence of metabolic enzyme inhibitors. All cells were cultivated at 37 °C in a 5% CO2−95% O2 incubator, and the growth media were replaced every 48 h. NF-LAPS Experimental Measurements. As indicated earlier, we base our study on measurements of the extracellular pH fluctuation (acidification) initiated by cancer cells in the surrounding media. Due to the high sensitivity of the hydrogel NFs, very small changes (down to ∼0.02 pH) can be easily detected. In the first set of the experiments, we calibrated the physiochemical properties of the NF-LAPS with DMEM medium at pH 7.3 using a linear sweep voltammetry (LSV) method. In 400 μL culture medium and at a concentration of 1 × 105 cells mL−1, harvested cancer cell lines MDA-MB231 or the noncancerous cell line MCF10A were transferred to the sensor chamber and kept under standard culture conditions for growth. The NF-LAPS readings were recorded directly after addition of the culture and continued for ∼90 min. Control samples included a blank medium free of cell lines and a culture medium containing noncancerous cell lines were used as a reference for comparison. The rate of acidification was simply calculated from the photocurrent measurements. The acidification of cells was also recorded in the absence of glucose and in the presence of different glucose levels (2 mM to 10 mM). In addition, to measure sensitivity of the sensor, we recorded the acidification in the presence of different concentrations of cancer cells (103−106 cells mL−1). For monitoring drug resistance, we used the multidrugresistant cell line (MDA-MB-435-MDR) and monitored the acidification as above, for approximately 90 min in the presence of doxorubicin (solely at 1 μM) or doxorubicin with metabolic enzyme inhibitors, 2-deoxyglucose (2-DG), or Oxamate at (1 μM). Statistically, experiments were performed using multiple chips and run under same conditions to ensure the reproducibility of the experiments. All

approach, enhanced sensitivity is obtained by immobilizing pH sensitive nanofibers (NFs) on the semiconductor surface, which also increases the surface area for interaction with targeted analytes.9,19 The pH responsive hydrogel NFs have also enhanced properties compared to their bulk form when swelling, since nanometer size affects the response time of the hydrogel. The physical swelling of the NFs reflects in the overall change in the photocurrent signal and thereby the pH response.9 In this study, we report on measuring cancer cell acidification and monitor their response to therapeutics over a period of 2 h using NF-LAPS in an effort to contribute to the current understanding of cancer metabolism (Figure 1). We also explored the implications of the metabolic enzyme inhibitors in extracellular acidification, tumor proliferation, and potential shifts of tumor metabolism in combination with cancer chemotherapy.

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EXPERIMENTAL SECTION

NF-LAPS Design and Fabrication. Fabrication of the sensor chips with pH sensitive poly(vinyl alcohol)/poly(acrylic acid) (PVA/ PAA) hydrogel was carried out using a homemade electrospinning setup as described previously in our report.9 Briefly, cleaned p-type Si substrates with a thickness of 525 ± 25 μm with a natural SiO2 layer were chosen as substrates. The Si chips were used as a collecting target on a grounded conducting plate inside the electrospinning unit. The PVA/PAA blend with a 1/5 wt % ratio was electrospun for 2 h under 20 kV with a collector distance of 15 cm. The flow rate was kept constant at 0.3 mL/h. Following the electrospinning steps, the Si chips were annealed at 145 °C for 30 min in a conventional vacuum oven. After the annealing process was complete, the sensor chips were prepared for the LAPS measurements by making an ohmic contact using eutectic Ga:In. The LAPS measurement setup was established using a threeelectrode electrochemical arrangement. The silicon chip acted as a working electrode (WE). Ag/AgCl and Pt wires were used as a pseudo reference electrode (RE) and counter electrode, respectively. The illumination source of the LAPS experiments was a DLP Lightcrafter evaluation module (EVM). Red light was used with a 12 kHz frequency modulation. Measurements were carried out on a Gamry Reference 3000 potentiostat. All measurements were performed using a linear sweep voltammetry (LSV) method with 2 mV increments in voltage steps and a scanning rate of 40 mV/s. B

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Figure 2. Metabolic monitoring of cancer cells by detecting the extracellular acidification on microenvironment. (a) Standard pH fluctuation monitored in a culture medium (DMEM pH 7.3) to show stability of the NF-LAPS sensor performance. (b) Detection of extracellular acidification (pH change) of breast cancer cells (MDA MB231) in microenvironment (400 μL sample) at low and high levels of glucose moieties. As indicated the level of acidification was compared to normal cell lines (MCF10A) in the same environment with low and high levels of glucose. (c) ΔpH max where statistics of different pH changes were recorded and averaged values (mean ± SD) were presented from three replicates carried out in the same environment and over different time periods.

Figure 3. NF-LAPS sensitivity and concentration dependence measurements. (a) pH shift as a function of time at different glucose levels and fixed concentration of cancer cells (106 cells per mL). (b) Correlation between pH and the different concentrations of cancer cells after stabilization in the sensor chamber. The results suggest that the extracellular pH decreases with time at higher number of cancer cells; i.e., the values of pH scale inversely with the number of cancer cells in the sample. The fitted curves indicate the exponential decay in pH change. (c) pH shift in the NF-LAPS after exposure to serial concentrations of cancer cells shows the sensitivity of the sensor. The corresponding fit is a linear function and error bars represent standard deviations (n = 3). All data represent an average of 3 replicates and error bars correspond to standard deviations. measurements were carried out under STP settings. All measurements were averaged and each experiment was performed at least three times. Data are presented as mean ± SD throughout the manuscript. The statistical difference was tested either using the unpaired t test or the one-way ANOVA test. In all statistical analysis the significance level (P value) was set at 0.05.

We initially tested the performance of the sensor using the culture medium (DMEM at pH 7.3). This was also used as a standard reference for our next experiments (Figure 2a). From the statistics, the fluctuation in the potential of the NF-LAPS was found to be very stable. For subsequent stability testing, we monitored the extracellular acidification (the pH sensitivity of the NF-LAPS) by scanning the photocurrent−voltage characteristic curves under series of pH conditions. The fluctuation in the pH can be easily generated from the photocurrent change by the shift in the inflection point of photocurrent voltage curves and the pH sensitivity of the calibrated curve. First, results obtained with low glucose level (DMEM low glucose) media spiked with breast cancer cell lines (MDA MB231), or normal breast cell line (MCF10A) at a concentration of 105 cells mL−1, showed no significant changes in the pH over 80 min of monitoring (Figure 2b and c). However, when a glucose supplement (10 mM) was added to the media, a decrease in the pH in the cancer cell spiked medium compared to the normal cell containing medium, as well as to the reference control (medium free of cells), was observed (P value ≤0.05).

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RESULTS AND DISCUSSION The hydrogel NFs were fabricated on p-type Si substrates with a thickness of 525 ± 25 μm. As-spun and annealed hydrogel NFs were characterized before experiments in order to verify their nature and surface morphology. Figure 1 shows a scanning electron microscopy (SEM) image of annealed PAA/PVA hydrogel NFs on surface of S substrate with an average diameter of 330 ± 50 nm. The images indicate that cylindrical hydrogel NFs are formed with uniform topography. In order to maintain consistency of the experiments, we characterized all hydrogel NFs’ chips before experiments and used the ones which have more than 90% similarity. Device setup, instrumentation for data acquisition, and software for analysis are detailed in our previous work.9 C

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Figure 4. Experiments display the response of the NF-LAPS system to the resistant cancer cell line (MDA MB435-MDR) in the presence of Doxorubicin (DOX) alone/or in combination with metabolic enzyme inhibitors, 2-DG or Oxamate. (a) Detection of extracellular acidification (pH change) due to the resistant cancer cells (MDA MB435-MDR), alone, in the presence of Dox, or in the presence of DOX with metabolic enzyme inhibitors, as specified. As results indicated, a significant drop in pH was observed when cancer cells were incubated alone or with DOX; however, lower acidification rate (no change in pH) was observed when DOX was coadministered with the enzyme inhibitors, 2-DG or the Oxamate. Results suggest a synergistic effect of the combined therapy on cancer metabolism. An exponential decay trend is observed for the changes in pH in the absence of inhibitors, seen as the fitted curves. (b) Statistical data derived from three replicate studies conducted in the same environment. Averaged values are presented with error bars indicating standard deviations.

Furthermore, experiments where different concentrations of glucose were added showed different degree of shift toward acidic pH. Higher acidity (lower pH) was observed when a higher concentration of glucose was injected (∼0.4 pH change, which is equivalent to ∼29 mV, Figure 3a). The noise of NFLAPS was less than 5 nA, and the sensitivity of detection was ∼74 mV/pH. The sensitivity for different concentrations of the cancer cells (with glucose added) shows a slope of 0.092 cell mL−1 and pH resolution reaches 0.02 pH. It should be noted that the pH values are calculated based on the LAPS photocurrent curves. The values can be determined down to 0.02 pH units. We can determine the accuracy of the measured pH down to a level of 0.1 pH confidence. The changes in pH in the control experiments also confirm that margin of error. This was very consistent with previous results of sensor performance tests. We strongly believe that pH begins to decrease after the addition of glucose as a result of the accelerated glucose fermentation process by cancer cells. In contrast, there was no evidence of fermentation in the media spiked with normal cell lines, as the pH value remained virtually the same with respect to the tested time period. These results are also in agreement with previous reports and confirm further that the energy production in cancer cells is abnormally reliant on aerobic glycolysis.20,21 To identify sensitivity of the NF-LAPS sensor in detecting extracellular acidification, serious experiments where glucoserich media having various concentrations of cancer cells (MDA MB231) were tested for pH change over a 90 min period. Figure 3b shows the pH change over time for different concentrations of cancer cells after stabilization in the sensor chamber. The fitted lines represent exponential decay of the pH change with time. While in Figure 3c, we observe the sensitivity line for the NF-LAPS with respect to concentration of the cancer cells in the presence of glucose. It is clear that the pH scales inversely with the number of cancer cells in the sample. The detection limit, the minimum number of cells which can show detectable change in the pH, was found to be 103 mL−1

for a signal-to-noise ratio of 3. The rate of acidification of cancer cells, based on the serial concentrations of cancer cells used, was found to vary with the NF-LAPS sensor. Although acidification rate per cell should be identical for all the concentrations, the observed values vary, most probably due to the increase in the number of cells while the amount of nutrients remains the same. Therefore, at lower concentrations, the cancer cells may have more glucose to ferment leading to higher rates of acidification. On the contrary, at higher concentration of cancer cells, cells may get less glucose to ferment causing a lower level of acidification. This observation is based on the k values of the fitted curves in Figure 3b for the various concentrations of the cells. The k values are proportional to the rate of acidification as they represent the rate constant of the fitted curves. For the experiments with 103 and 106 cells mL−1, k values are similar at 0.007 and 0.005, respectively. However, they vary from k calculated for 104 and 105 cells mL−1 with 0.023 and 0.028, respectively. This means that the acidification rates, as well as the pH change, are not identical for different concentrations of MDA MB231. Next, we determined the extracellular acidification of the multidrug-resistant cancer cell line (MDA-MB-435-MDR), alone and in the presence of doxorubicin in glucose-rich media. Monitoring the extracellular acidification over a period of ∼90 min showed a dramatic drop in the pH due to glucose fermentation by cancer cells (Figure 4a,b). This is in contrast with what is observed with the nonresistant cancer cells (MDA MB231) when they were treated with the same anticancer drug (Figure 4b). The results suggest that resistant cancer cells are not provoked by the introduction of doxorubicin. Instead, their metabolic processes remained active by generating lactic acid metabolite. Further, to estimate the effect of metabolic enzyme inhibitors on cancer cells, culture media with or without doxorubicin was treated with metabolic enzyme inhibitors (2deoxyglucose (2-DG), or Oxamate) at 1 μM. The results are illustrated in Figure 4a,b, which show the effect of metabolic enzyme inhibitors on cancer cell acidification over a 90 min D

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period. While in the presence of enzyme inhibitors alone, the pH has dropped to acidic, a notable increase in the pH (lower acidification) was observed when doxorubicin was coadministered with the enzyme inhibitors. It appears that both enzyme inhibitors have generated a synergistic mechanism by which the anticancer activity of doxorubicin was improved. The 2-DG is a glucose analog metabolic inhibitor that acts with other chemoand radiotherapy to enhance the anticancer activity. The analog is phosphorylated by hexokinase enzyme to generate a stable 2DG-phosphate. This stable product accumulates in the cells, inhibits further metabolic process, decreases glycolysis, and causes ATP depletion.7,22 The Oxamate, on the other hand, is a pyruvate analog that blocks glycolysis by blocking the conversion of pyruvate to lactate.7,23 Results illustrate that combination of doxorubicin with 2-DG or Oxamate generates a glycolysis inhibitory effect on doxorubicin resistant cells, which may have led to a synergistic anticancer effect on cancer. Although a number of key studies remain to be explored, we anticipate these findings will contribute well to the current understanding of cancer metabolism and its related mechanism of drug resistance. The device will be very useful for a variety of applications including drug discovery and testing pharmaceutical ingredients.

REFERENCES

(1) Vander Heiden, M. G.; Cantley, L. C.; Thompson, C. B. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science 2009, 324 (5930), 1029−1033. (2) Kroemer, G.; Pouyssegur, J. Tumor Cell Metabolism: Cancer’s Achilles’ Heel. Cancer Cell 2008, 13 (6), 472−482. (3) Hsu, P. P.; Sabatini, D. M. Cancer Cell Metabolism: Warburg and Beyond. Cell 2008, 134 (5), 703−707. (4) Pandey, P. R.; Liu, W.; Xing, F.; Fukuda, K.; Watabe, K. AntiCancer Drugs Targeting Fatty Acid Synthase (FAS). Recent Pat. AntiCancer Drug Discovery 2012, 7 (2), 185−197. (5) Wise, D. R.; Thompson, C. B. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem. Sci. 2010, 35 (8), 427− 433. (6) Birsoy, K.; Sabatini, D. M.; Possemato, R. Untuning the tumor metabolic machine: Targeting cancer metabolism: a bedside lesson. Nat. Med. 2012, 18 (7), 1022−1023. (7) Zhao, Y.; Butler, E. B.; Tan, M. Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis. 2013, 4, e532. (8) Del Ben, F.; Turetta, M.; Celetti, G.; Piruska, A.; Bulfoni, M.; Cesselli, D.; Huck, W. T. S.; Scoles, G. A Method for Detecting Circulating Tumor Cells Based on the Measurement of Single-Cell Metabolism in Droplet-Based Microfluidics. Angew. Chem., Int. Ed. 2016, 55 (30), 8581−8584. (9) Shaibani, P. M.; Jiang, K.; Haghighat, G.; Hassanpourfard, M.; Etayash, H.; Naicker, S.; Thundat, T. The detection of Escherichia coli (E. coli) with the pH sensitive hydrogel nanofiber-light addressable potentiometric sensor (NF-LAPS). Sens. Actuators, B 2016, 226, 176− 183. (10) Hu, N.; Ha, D.; Wu, C.; Zhou, J.; Kirsanov, D.; Legin, A.; Wang, P. A LAPS array with low cross-talk for non-invasive measurement of cellular metabolism. Sens. Actuators, A 2012, 187, 50−56. (11) Hu, N.; Wu, C.; Ha, D.; Wang, T.; Liu, Q.; Wang, P. A novel microphysiometer based on high sensitivity LAPS and microfluidic system for cellular metabolism study and rapid drug screening. Biosens. Bioelectron. 2013, 40 (1), 167−173. (12) Etayash, H.; Jiang, K.; Thundat, T.; Kaur, K. Impedimetric Detection of Pathogenic Gram-Positive Bacteria Using an Antimicrobial Peptide from Class IIa Bacteriocins. Anal. Chem. 2014, 86 (3), 1693−1700. (13) Bellassai, N.; Spoto, G. Biosensors for liquid biopsy: circulating nucleic acids to diagnose and treat cancer. Anal. Bioanal. Chem. 2016, 408, 7255. (14) Burg, T. P.; Godin, M.; Knudsen, S. M.; Shen, W.; Carlson, G.; Foster, J. S.; Babcock, K.; Manalis, S. R. Weighing of biomolecules, single cells and single nanoparticles in fluid. Nature 2007, 446 (7139), 1066−1069. (15) Etayash, H.; Khan, M. F.; Kaur, K.; Thundat, T. Microfluidic cantilever detects bacteria and measures their susceptibility to antibiotics in small confined volumes. Nat. Commun. 2016, 7, 12947. (16) Etayash, H.; McGee, A. R.; Kaur, K.; Thundat, T. Nanomechanical sandwich assay for multiple cancer biomarkers in breast cancer cell-derived exosomes. Nanoscale 2016, 8 (33), 15137−15141. (17) Etayash, H.; Jiang, K.; Azmi, S.; Thundat, T.; Kaur, K. Real-time Detection of Breast Cancer Cells Using Peptide-functionalized Microcantilever Arrays. Sci. Rep. 2015, 5, 13967. (18) Yun, J.; Jin, D.; Lee, Y.-S.; Kim, H.-I. Photocatalytic treatment of acidic waste water by electrospun composite nanofibers of pHsensitive hydrogel and TiO2. Mater. Lett. 2010, 64 (22), 2431−2434. (19) Yun, J.; Im, J. S.; Oh, A.; Jin, D.-H.; Bae, T.-S.; Lee, Y.-S.; Kim, H.-I. pH-sensitive photocatalytic activities of TiO2/poly(vinyl alcohol)/poly(acrylic acid) composite hydrogels. Mater. Sci. Eng., B 2011, 176 (3), 276−281. (20) Tennant, D. A.; Duran, R. V.; Gottlieb, E. Targeting metabolic transformation for cancer therapy. Nat. Rev. Cancer 2010, 10 (4), 267−277. (21) Vander Heiden, M. G. Targeting cancer metabolism: a therapeutic window opens. Nat. Rev. Drug Discovery 2011, 10 (9), 671−684.

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CONCLUSION A LAPS device modified with very sensitive, biocompatible PAA/PVA hydrogel NFs was used for investigating metabolic activities of cancer cells in a noninvasive fashion. Results show the ability of the NF-LAPS to monitor the extracellular acidity of cancer cells in real time. It was used to monitor chemoresistance of cancer cells and shows the effects of metabolic enzyme inhibitors on anticancer activity when they are combined with chemotherapeutics. Reducing drug resistance would have a significant benefit for cancer patients. Designing simple tools to detect drug resistance and measuring drug efficacy would have significant impact on the current use of drugs, efficacy monitoring, and in future for new drug discovery.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 780.492.2068. Fax: 780.492.2881. ORCID

Hashem Etayash: 0000-0001-6386-2407 Author Contributions

H.E. conceived the project, designed, and ran experiments. P.M.S. designed and fabricated the NF-LAP sensor. P.M.S. and S.N. analyzed the experimental data. S.N. and P.M.S. performed calculations and statistical analysis. T.T. and K.K. supervised the project and contributed to the article composition. H.E. wrote the manuscript. Authors have read and proof-edited the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Excellence Research Chair (CERC) Program for support. H. Etayash and P.M. Shaibani are the recipients of an Alberta Innovates − Technology Futures Scholarship. E

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ACS Sensors (22) Pelicano, H.; Martin, D. S.; Xu, R. H.; Huang, P. Glycolysis inhibition for anticancer treatment. Oncogene 2006, 25 (34), 4633− 4646. (23) Zhou, M.; Zhao, Y.; Ding, Y.; Liu, H.; Liu, Z.; Fodstad, O.; Riker, A. I.; Kamarajugadda, S.; Lu, J.; Owen, L. B.; Ledoux, S. P.; Tan, M. Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol. Mol. Cancer 2010, 9, 33.

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