Determination of Cadherin-17 in Tumor Tissues of Different Metastatic

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Determination of cadherin-17 in tumor tissues of different metastatic grade using a single incubation-step amperometric immunosensor Alejandro Valverde, Eloy Povedano, Víctor Ruiz-Valdepeñas Montiel, Paloma Yanez-Sedeno, María Garranzo-Asensio, Nuria Rodríguez, Gemma Domínguez, Rodrigo Barderas, Susana Campuzano, and José M. Pingarrón Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03506 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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Analytical Chemistry

Determination of cadherin-17 in tumor tissues of different metastatic grade using a single incubation-step amperometric immunosensor Alejandro Valverde,† Eloy Povedano,† Víctor Ruiz-Valdepeñas Montiel,† Paloma Yáñez-Sedeño,† María Garranzo-Asensio,‡ Nuria Rodríguez,ǁ Gemma Domínguez, ⊥ Rodrigo Barderas, ‡,* Susana Campuzano, †,* José M. Pingarrón. †,* †

Departamento de Química Analítica, Universidad Complutense de Madrid, E-28040 Madrid, Spain. UFIEC, CROSADIS, National Institute of Health Carlos III, Majadahonda, E-28222, Madrid, Spain. ǁ Medical Oncology Department, Hospital Universitario La Paz, E-28046 Madrid, Spain. ‡

⊥

Departamento de Medicina, Facultad de Medicina, Instituto de Investigaciones Biomédicas “Alberto Sols”, CSIC-UAM, E28029, Madrid, Spain. ABSTRACT: This paper reports the development of an amperometric immunosensing platform for the determination of cadherin17 (CDH-17), an atypical adhesion protein involved in the progression, metastatic potential and survival of high prevalence gastric, hepatocellular, and colorectal tumors. The methodology developed relies on the efficient capture and enzymatic labeling of the target protein on the magnetic microparticles (MBs) surface using commercial antibodies and amperometric transduction at screenprinted carbon electrodes (SCPEs) through the HRP/H2O2/HQ system. The developed immunosensing platform allows the selective determination of the target protein at low ng mL-1 level (LOD of 1.43 ng mL-1) in 45 min and using a single incubation step. The electrochemical immunosensor was successfully used for the accurate determination of the target protein in a small amount (0.5 µg) of raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade.

INTRODUCTION Cadherins are cellular adhesion molecules crucial for intracellular adhesion and many of them are associated with cancer development, progression and metastasis.1 Transition from the epithelial to mesenchymal stage, often found during tumor proliferation and cell invasion, involves dysregulation of cadherin-mediated cell adhesion.2,3 In fact, expression of cadherin-17 (CDH-17) in human tissues has been considered as an important prognosis marker candidate for gastric, ovarian, hepatocellular, and colorectal tumors.4-6 CDH-17, also called liver–intestine cadherin (LI‑cadherin), is a calcium-dependent transmembrane glycoprotein that belongs to the seven domain (7D)-cadherin family and functions as a peptide transporter and a cell adhesion molecule to maintain tissue integrity in epithelia.7 Structurally, CDH‑17 protein has seven extracellular ectodomains, one transmembrane region, and one intracellular cytoplasmic domain which has only about 20 amino acids unlike classical cadherins which contain a highly conserved cytoplasmic domain of 150–160 amino acids forming complexes with catenins.6,8-11 CDH-17 functions in cell adhesion without dependence on cytoplasmic components, such as catenins or the actin cytoskeleton. In humans, expression of CDH‑17 is limited to intestinal epithelial cells and pancreatic ductal epithelial cells. However, recent findings have shown aberrant expression of CDH-17 in gastrointestinal malignancies including hepatocellular carcinoma,12,13 gastric,14, colorectal5,15,16 and pancreatic17 cancers,

and has been clinically associated with tumor metastasis and advanced tumor stages.6,10,11,18 New function for CDH-17 to regulate α2β1 integrin signaling in cell adhesion and proliferation in colon cancer cells for liver metastasis, the major cause of colorectal cancer-associated death, was also reported.18 The determination of this relevant biomarker is performed by immunochemistry (IHC), immunoblotting, flow cytometry, tissue microarrays and, more commonly, by enzyme-linked immunosorbent assays (ELISAs). However, these methodologies do not comply the requirements of ease use and portability that clinical oncology is demanding more and more for routine and decentralized screening of cancer onset, metastasis and relapse. In this sense, electrochemical immunosensors exhibit very attractive characteristics and have demonstrated successful applicability for the reliable determination of biomarkers related to diagnosis and evolution of carcinogenic processes in complex biological samples (serum, saliva and lysate and whole cells) after minimal treatments and using simple test protocols.19-21 Currently, an attractive strategy for the development of electrochemical immunosensors involves the combined use of magnetic microparticles (MBs) and screen-printed electrodes (SPEs). The use of MBs avoids the need for applying and optimizing laborious protocols for modification of electrode substrates surface, and improves the analytical performance of the resulting immunosensors in terms of sensitivity, test time and matrix effect.22-26 Moreover, SPEs are particularly attractive for clinical applications allowing the use of small sample volumes. In addition, SPEs can be mass and inexpensively produced from a variety of materials,

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in different geometries and in miniaturized and multiplexed formats. Their planar shape facilitates the incorporation of the magnetic bioconjugates on their surface in a stable and reproducible way by simple magnetic attraction. Additional features of electrochemical immunosensors include the requirement of easily automated, portable and cost-effective instrumentation, which make them attractive and user-friendly tools to monitor routinely biomarkers at different settings. However, we are not aware that to date this type of electrochemical immunosensing platforms have been applied for direct determination in tissue samples extracts. This paper reports the first biosensor for the determination of CDH-17 as well as the pioneering application of an electrochemical biosensor for the determination of a target protein directly in tissues extracts. The strategy involved in the developed methodology is based on a sandwich immunoassay carried out onto the MBs and amperometric detection at SPCEs. The immunosensors allow fast quantification of CDH-17 in raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade.

EXPERIMENTAL SECTION

Apparatus and electrodes. Amperometric measurements were made with a CHI812B potentiostat (CH Instruments) controlled by software CHI812B. Screen-printed carbon electrodes (SPCEs, DRP-110), with a 4-mm carbon working electrode, a carbon counter electrode and a Ag pseudoreference electrode, and the specific cable connector (DRPCAC), used as electrochemical transducers and interface between the SPCEs and the potentiostat, respectively, were purchased from DropSens, S.L. A Bunsen AGT-9 Vortex, a Thermomixer MT100 constant temperature incubator shaker (Universal Labortechnik), a magnetic separator DynaMag-2 Magnet (ThermoFisher Scientific) and a homemade Teflon casing with a neodymium magnet (AIMAN GZ) embedded were also employed. Reagents and solutions. All used reagents were of the highest available analytical grade. 2.7-µm Ø carboxylic acidmodified magnetic beads (Ref: 14305D from Thermo Fisher Scientific), 2-(N-morpholino)ethanesulfonic acid (MES), sodium chloride, potassium chloride, sodium di-hydrogen phosphate, di-sodium hydrogen phosphate and Trishydroxymethyl-aminomethane-HCl (Tris-HCl) were purchased from Scharlab. N-(3-dimethyl-aminopropyl)-N’ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (SulfoNHS), ethanolamine, hydroquinone (HQ) and hydrogen peroxide (H2O2) (30 %, w/v) were purchased from Sigma-Aldrich. A ready-to-use PBS solution of 1% w/v purified casein (BB, Ref; 37528 from Thermo Scientific) was used as blocker solution. Mouse monoclonal IgG1 Kappa Anti-CDH17 antibody (WH0001015M1, from Sigma-Aldrich) was used as capture antibody (CAb). Purified recombinant protein of Human cadherin 17, LI cadherin (liver-intestine) (CDH-17), transcript variant was from Origene (Ref: TP720740). Anti-CDH17 antibody produced in rabbit (SAB4500044 from SigmaAldrich) was employed as detector antibody (DAb) further conjugated with goat anti-rabbit IgG (H+L)-HRP conjugate (Cat #170-6515, from BioRad) (HRP-Ab).

Recombinant full length human p53 protein (Catalog#14865) from EMD Millipore Corporation, bovine serum albumin (BSA Type VH) from Gerbu Biotechnik, GmbH, human hemoglobin (H7379), recombinant human TNF protein (Ref. IM1121) from Immunotech and N-terminal GST-tagged, IgG from human serum (I2511) from Sigma-Aldrich (USA), human CD105 (endoglin) from Sino Biological Inc., recombinant human fibroblast growth factor receptor 4 (FGFR4, a component of the Human Total FGFR4 DuoSet® IC ELISA kit from R&D Systems Inc., Catalog Number DYC685-2), Human Total E-Cadherin Standard (E-Cadherin, a component of the Human Total E-Cadherin DuoSet® IC ELISA kit from R&D Systems Inc., Catalog Number DYC4225-2) and recombinant human interleukin 13 soluble receptor alpha 2 (IL13sRα2, a components of the Human IL-13sRα2 DuoSet® IC ELISA kit from R&D Systems, Inc., Catalog Number: DY614), were tested as potential interfering compounds. All buffer solutions were prepared with water from Millipore Milli-Q purification system (18.2 MΩ cm): 0.1 M phosphate buffer solution, pH 8.0; 0.05 M phosphate buffer, pH 6.0; 0.1 M phosphate buffer, pH 8.0; phosphate-buffered saline (PBS) consisting of 0.01 M phosphate buffer solution containing 0.137 M NaCl and 0.0027 M KCl, pH 7.5; 0.025 M MES buffer, pH 5.0; 0.1 M Tris-HCl buffer, pH 7.2 and PBS:BB (1:1) buffer solution. An EDC/sulfo-NHS mixture solution (50 mg mL-1 each) in MES buffer, pH 5.0 and a 1.0 M ethanolamine solution (prepared in 0.1 M phosphate buffer solution, pH 8.0) were used for activation and blocking of the HOOC-MBs, respectively.

MBs modification with the sandwich immunocomplexes. 3 µL of the HOOC-MBs commercial suspension were placed in 1.5 mL microcentrifuge tube. Two washings were made with 50 µL of MES buffer (0.025 M, pH 5.0) with incubation for 10 min each (25ºC, 950 rpm). The carboxylic groups were then activated by incubating with 25 µL of the EDC/Sulfo-NHS 50 mg mL-1 solution (prepared in MES buffer 0.025 M, pH 5.0) for 35 min (25ºC, 950 rpm). Two washings were then carried out with 50 µL of MES buffer. Activated HOOC-MBs were incubated with 25 µL of capture antibody solution (25 µg mL-1 prepared in MES buffer) for 45 min (25ºC, 950 rpm). Thereafter, the CAb-MBs were washed twice with 50 µL of MES buffer and incubated with 25 µL of 1 M ethanolamine blocking solution (prepared in 0.1 M phosphate buffer, pH 8.0) for 60 min (25ºC, 950 rpm). Three washings were then made. The first one with 50 µL of Tris-HCl (0.1 M, pH 7.2) and the other two with 50 µL of a PBS solution (10 mM, pH 7.5). The CAb-MBs were then incubated with 25 µL of a mixture solution containing CDH-17 and the detection antibody at a concentration of 0.5 µg mL-1 and secondary antibody 1/500 diluted, prepared in a mixture of PBS:BB (1:1), for 45 min (25ºC, 950 rpm). The as-modified magnetic micro-carriers were washed twice with PBS:BB (1:1) solution, and re-suspended in 50 µL of 0.05 M phosphate buffer, pH 6.0, to perform the amperometric measurements.

Amperometric measurements. The SPCE was placed in the neodymium magnet-encapsulated Teflon casing and the 50 µL-aliquot of modified MBs suspension was pipetted onto the surface of the working electrode. Then, the ensemble SPCE/magnet holding block was immersed into an electrochemical cell containing 10.0 mL of 0.05 M phosphate buffer, pH 6.0, and 1.0 mM HQ (prepared just before the


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electrochemical measurement). Amperometric readouts were recorded in stirred solutions by applying a constant detection potential of −0.20 V (vs. the Ag pseudo-reference electrode) upon addition of 50 µL of a 0.1 M H2O2 solution and waiting until the steady-state was reached (approx. 100 s). The amperometric signals given through the manuscript corresponded to the difference between the steady-state and the background currents.

Determination of CDH-17 in cell lysates and tumor tissues extracts. SW480 and SW620 (from the

Figure 1. Schematic display of the immunosensor developed for the determination of CDH-17 involving the formation of sandwich immunocomplexes on the MBs and amperometric transduction at SPCEs using the H2O2/HQ system after capturing the magnetic bioconjugates on their surface.

Optimization of the experimental variables. The working protocol used for the amperometric determination of CDH-17 was optimized taking as the selection criterion the relationship between the amperometric responses obtained for 500 (S) and 0 (N) ng mL-1 CDH-17 standard solutions (S/N ratio). The variables tested are summarized in Table 1 and the results shown in Figure 2. a)

10

400

5

0

0

5

10

25

[CAb], µg mL-1

1200

50

Figure 1 schematically displays the fundamentals of the electrochemical immunosensor prepared for the determination of CDH-17. Sandwich-type immunocomplexes were formed on the surface of commercial MBs and amperometric transduction was accomplished after capturing of the resulting magnetic bioconjugates on the surface of the SPCE using the HRP/H2O2/HQ system.28

15

30

45

60

90

S/N

800

0

Inc. timeCAb, min

c)

15

d)

10

400

5

0

1

2A

2B

3

0.05 0.1 0.25 0.5

1

Number of Steps

[DAb], µg mL-1

e)

f)

1200

2.5

S/N

800

0

15

800

10

400

5

0

1/250

1/500 1/1,000 1/5,000

HRP-Ab dilution

RESULTS AND DISCUSSION

15

b)

15

30

45

60

90

S/N

-i, nA

1200

-i, nA

American Type Culture Collection (ATCC) cell repository), and KM12C and KM12SM (from I. Fidler's laboratory, MD Anderson Cancer Center, Houston, TX) cell lines were grown according to established protocols in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, penicillin and streptomycin, and 2.5 mM Lglutamine (GIBCO-Invitrogen, Carlsbad, CA, USA). Cell lysates were prepared following the protocol described previously and stored at −80 ºC.21 Total protein concentration of the resultant cell lysates were estimated using a BCA protein assay kit (Pierce, Rockford, IL). Paraffin embedded paired tumor and normal tissues were obtained from Hospital La Paz (Madrid, Spain) assessed by a pathologist. Written informed consent were obtained and approved by the Ethical Committee of the Hospital La Paz. Tumor (T) or normal (N) tissue extracts were obtained as previously described using three 6-µm sections of paraffin embedded tissues, its total protein concentration calculated as above, placed in vials and stored at -80ºC until use.27 Semiquantitative analysis of CDH-17 in both cell lysates and paraffin embedded tissue extracts was carried out using dot-blot analysis. Briefly, 5 µg of protein extracts from N and T tissue samples and 10 µg of KM12C, KM12SM, SW480, and SW620 cell lysates were blotted to nitrocellulose membranes. After blocking with PBS containing Tween 20 0.1% and 3% skimmed milk, membranes were incubated with the 1:1,000diluted DAb overnight at 4°C followed by 1 h incubation with a 1:1,000-diluted HRP-anti-rabbit IgG (Sigma) solution. Specific reactivity was visualized with SuperSignal West Pico Maximum Sensitivity Substrate (Pierce). Protein bands were semi-quantified by densitometry using Quantity One 1-D analysis software (BioRad). All quantifications were normalized using RhoGDi as loading control, and the ratio of CDH17 expression calculated. Considering the low content of the target protein in the analyzed samples, standard additions calibration plots for CDH17 were constructed between 0 and 500 ng mL-1 in 0.5 g of cell lysates or tumor tissue extracts. Then, the endogenous concentration of CDH-17 was quantified by simple extrapolation of the measured current into the corresponding standard additions calibration plot.

-i, nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

Inc. timeCDH17+DAb+HRP-Ab, min

Figure 2. Dependence of the amperometric signals obtained for 500 (S, grey bars) and 0.0 (N, white bars) ng mL-1 CDH-17 standards and the corresponding S/N ratio, with immunosensors prepared by varying the: CAb concentration (a), incubation time of the activated HOOC-MBs in the CAb solution (b), number of incubation steps (c), DAb concentration (d), HRP-Ab dilution (e) and incubation time of the CAb-MBs in the CDH-17, DAb and



Figure 2a shows that some discrimination was observed in the presence and absence of the target protein on MBs unmodified with the capture antibody. This undesired interaction may be attributed to the direct covalent immobilization of the protein on some of the remaining activated groups after blocking of the MBs with ethanolamine. The S/N current ratio increased notably with the immobilized CAb concentration indicating the major role of the sandwich-immunocomplexes formation on the MBs surface on the specific response. As a compromise between sensitivity and cost per assay, a CAb concentration of 25 µg mL-1 was selected for further work. An incubation time of 45 min in the CAb solution provided a larger S/N ratio (Figure 2b). The decrease in the S/N ratio observed for longer incubation times can be attributed to lower efficiency in the immunorecognition process for large concentrations of immobilized CAb.26 The number of incubation steps (30 min each) involved in the modification of the MBs was also optimized. The compared protocols were: (a) 1 single incubation step in a mixture solution containing CDH-17, DAb and HRP-Ab (bars 1); (b) 2 steps, an incubation in a mixture of CDH-17 and DAb and a further one in the HRP-Ab solution (bars 2A) or an incubation in the CDH-17 solution and a second incubation in a mixture of DAb and HRP-Ab (bars 2B); (c) 3 successive incubation steps in CDH-17, DAb and HRP-Ab solutions, respectively. Figure 2c shows that only when the target protein, previously captured on the MBs, was recognized with the DAb labeled with the HRP-Ab the S/N current ratio was significantly lower. Accordingly, with the aim of developing an assay as short and simple as possible, the protocol involving only one incubation step was selected to construct the immunosensor. Figures 2d-f show better S/N ratios were obtained using a Dab concentration of 0.5 µg mL-1, a HRP-Ab dilution of 1/500 and a 45 min incubation time of the CAb-MBs in the CDH-17, DAb and HRP-Ab mixture solution. Table 1. Optimization of the different experimental variables involved in the development of the amperometric immunosensor for the determination of CDH-17. Variable

Checked range

Selected value

[CAb], µg mL-1

0-50

25

Incubation timeCAb, min

15-90

45

Number of incubation steps

1-3

1

[DAb], µg mL-1

0.05-2.5

0.5

HRP-Ab dilution

1/250-1/5,000

1/500

15-90

45

Incubation 17+DAb+HRP-Ab,

timeCDHmin

Other experimental variables used in the development and functioning of the immunosensor including the amount of HOOC-MBs, the applied potential (-0.20 vs Ag pseudo-

reference electrode) and the concentration of H2O2 and HQ were taken from previous works.25,29-31

Analytical characteristics. The calibration plot for CDH-17 (Figure 3) exhibited a linear dependence of the measured cathodic current with the protein concentration between 4.76 and 1,000 ng mL-1 (r = 0.999), with slope and intercept values of (1.253 ± 0.006) nA mL ng-1 and (95 ± 3) nA, respectively. The detection and determination limits were estimated according to the 3sb/m and 10sb/m criteria (sb is the standard deviation of 10 amperometric measurements obtained for 0.0 ng mL-1 CDH-17 and m the slope of the calibration plot). The calculated values were 1.43 and 4.76 ng mL-1, respectively. a)

b)

1600

[CDH-17] ng mL-1 0 100

1200

250

800 500

400 0

0

200 400 600 800 1000

[CDH-17], ng mL-1

750

300 200 nA nA

HRP-Ab mixture solution (f). Error bars are three times the standard deviation of three replicates.

-i, nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1000

55 ss

Figure 3. Calibration plot for the amperometric determination of CDH-17 constructed with the developed immunosensor. Error bars are three times the standard deviation of three replicates.

The reproducibility between the amperometric responses obtained with different immunosensors and the storage stability of the CAb-MBs were tested. A relative standard deviation (RSD) value of 4.5% was calculated from the amperometric responses recorded for 500 ng mL-1 CDH-17 with 8 different immunosensors prepared in the same way, thus indicating a good reproducibility of the whole experimental procedure this involving both the sensor fabrication (formation of the sandwich immunocomplexes onto the MBs and their magnetic capture on the SPCE surface) and the amperometric transduction. The storage stability of the CAb-MBs (stored at 4 °C in Eppendorf tubes containing 50 µL of filtered PBS, pH 7.5) was excellent (data not shown). No significant differences between the S/N ratios calculated for 500 and 0 ng mL-1 CDH-17, obtained with the immunosensors prepared from the stored CAb-MBs, were observed for at least 40 days (no longer times were tested). Since the immunosensor described in this work is the first one reported so far for this biomarker, its analytical characteristics can be only compared with those of methodologies based on commercial spectrophotometric sandwich ELISA assays. These kits, commercialized by different companies, provide LODs (10-100 pg mL-1) lower than that achieved with the immunosensor. However, it is important to point out that ELISAs require 3 different incubation steps with assay times between 4 and 4.5 h (once prepared and blocked the CAbplate) conversely to the single incubation step and the 45 min assay time (once prepared and blocked the CAb-MBs) needed with the immunosensor. In addition, as it is demonstrated below, the sensitivity achieved with the immunosensor is sufficient to determine the concentration of the target protein even in paired healthy tissues using only 0.5 µg of extract. These characteristics together with the portable and cost-


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Analytical Chemistry effective electrochemical instrumentation required, make the developed immunosensor an attractive and user-friendly tool to perform routine determinations at different settings.

Immunosensor selectivity. Taking into account that the determination of CDH-17 is of interest in a wide variety of clinical samples including serum, plasma, cell lysates and tissue extracts, the selectivity of the immunosensor was checked against different proteins that may be present with the target protein in these samples. The amperometric responses obtained with the immunosensor for 0 and 500 ng mL-1 CDH17 were compared in the absence and the presence of each of the potentially interfering proteins at the concentration levels at which they are usually found in the clinical samples. The results are shown in Figure 4. Smaller signals were observed for 0 and 500 ng mL-1 CDH-17 in the presence of most of the non-target proteins assayed which may be attributed to some kind of matrix effect. However, it is important to mention that no significant differences in the S/N ratios were apparent for any of the tested proteins at the concentration level indicated. This is particularly relevant for E-cadherin, expressed together with CDH-17 in basolateral plasma membrane of enterocytes and goblet cells to keep tissue integrity18 and for IL-13Rα2, an emerging extracellular receptor recently found to be hyperexpressed in highly metastatic colon cancer.21,32

Figure 4. Comparison of the amperometric signals measured for 0 (white bars) and 500 (grey bars) ng mL-1 CDH-17 solutions prepared in the absence (1) and in the presence of 200 ng mL-1human p53 (2); 5 mg mL-1 BSA (3); 10 ng mL-1 hemoglobin (4); 10 ng mL-1TNF (5); 0.1 mg mL-1 human IgG (6); 10 ng mL-1 endoglin (7), 1 ng mL-1 FGFR4 (8), 500 ng mL-1 E-cadherin (9) and 10 ng mL-1 IL-13Rα2 (10). Eapp = −0.20 V vs Ag pseudo-reference electrode. Other experimental conditions are that summarized in Table 1. Error bars are three times the standard deviation of three replicates.

In vitro analysis of CDH-17 in cells lysates and tumor extracts. The developed immunosensor was applied to the determination of the target protein in raw lysates of colon cancer cells with different metastatic potential and in extracts of tumor (T) and their paired healthy (N) tissues from patients diagnosed with colorectal cancer at different stages. In addition, cell lysates and representative paired N/T tissue protein extracts were analyzed by dot-blot. As it can be seen in Figure 5a) and data summarized in Table 2, overexpression of CDH-17 was found in metastatic colon cancer cell lines SW620 and KM12SM in comparison to their respective isogenic non-metastatic colon cancer SW480 and KM12C cells, respectively. Quantification of CDH-17 protein in cell lysates and tumor extracts was accomplished with the developed immunosensor. Given the limited availability and the intrinsic heterogeneity of tissue samples and the reduced expression level of the target protein in both types of samples (around a

few ng per µg and similar to the LOD of the immunosensor), their analysis was carried out using the standard additions method by adding increasing concentrations of CDH-17 between 0 and 1,000 ng mL-1 in the presence of 0.5 g of each cell lysate or tumor extract analyzed.

Figure 5. (a) Assessment of CDH-17 status in SW480, SW620, KM12C and KM12SM cells and representative N and T tissue protein extracts by dot-blot. 10.0 µg of cell lysates or 5.0 µg of paired N/T tissue extracts were blotted to nitrocellulose membranes, and probed with the DAb and the RhoGDi (as loading control) antibodies. Protein spots were quantified by densitometry, and normalized in comparison to its corresponding loading control prior to ratio calculation. (b) Illustrative example of amperometric measurements provided by the immunosensor for 0.5 µg of protein extracts obtained from a tumor and its paired healthy tissue (Tissue 2) as well as the CDH-17 concentrations calculated in the analyzed tissue extracts. Error bars are three times the standard deviation of three replicates.

Table 2. Determination of CDH-17 (in ng µg-1) in 0.5 µg of cell lysates and tissue extracts.

Sample

Cell lysate*

Metastatic potential /Colorectal tissues (Stage in T)

Cell or Tissue reference

[CDH-17], ng µg-1

Nonmetastatic

SW480

1.7 ± 0.3

Metastatic

SW620

3.9 ± 0.6

Nonmetastatic

KM12C

1.6 ± 0.4

Metastatic

KM12SM

2.9 ± 0.5

N T (IIIC) N Tissue extracts**

T (IIIB) N T (IIIB) N T (IV) N


1 2 3 4 5

3.06 4.09 1.58 2.48 5.91 7.03 1.93 2.06 3.44

Metastatic/Nonmetastatic or T/N ratio

2.3

1.8

1.3 1.6 1.2 1.1 1.5


T (IIIA)

5.20

* Mean values ± ts/√n; n=3; α = 0.05. ** The limited availability of these samples avoided performing replicates for the determination in tissues extracts.

The results obtained show approximately twice CDH-17 expression in metastatic cells compared to non-metastatic cells, in agreement with the results observed by dot-blot analysis. In tissues extracts, the discrimination is poorer but larger expression of the target protein was also found in tumor tissues compared to paired non-tumor ones. It is important to note that the T/N ratio of CDH-17 expression obtained in tissue extracts is also in good agreement with those found by densitometry in dot blot (see results with tissue extracts 2 and 5 in Figure 5a). However, that ratio is rather variable which can be attributed to the different clinic-pathological grade of the analyzed tissues and to the individual variability of colorectal cancer patients. Results obtained in cell lysates are in agreement with data reported by other authors using proteomic analysis and tissue microarrays demonstrating overexpression of CDH-17 in highly metastatic cells (SW620, KM12SM) and lower expression in their paired cell lines with poorer metastatic capacity (SW480 and KM12C).18,33,34 Regarding tumor tissues, the obtained results seem to confirm overexpression of CDH-17 in tumor tissues. However, limited conclusions can be drawn at this point because of the small number of samples available. The results obtained for T tissue 4 (the only metastatic tissue analyzed) are in good agreement with the reported reduced expression of the target protein associated with lymph node metastasis of human colorectal carcinoma.5,35-37 It is worth to remark that the developed electrochemical immunosensor is able to provide quantitative data for CDH-17 expression in human tissues using a small amount of raw extract and a simple working protocol. This ability makes the immunosensor especially relevant to ending the discrepancy found in literature regarding CDH17 expression in colorectal cancer which might be attributed to technical reasons due to different treatment conditions such as, for instance, sample fixation time, long-term archival storage, quality of antibodies, and so on.11,18 However, it should be noted that the results reported in this work, although very promising, must be thoroughly validated with a much larger and more varied cohort of patients and further to be included in an appropriate retrospective or prospective trial in order to demonstrate that this promising candidate biomarker is truly useful.38 It worth to remark also that the determinations with the immunosensor required 10-20 times lower amounts of both types of samples (0.5 vs 5.0 and 10.0 µg in tissue extracts and cell lysates, respectively), which is particularly important in the analysis of samples with limited availability such as tissues.

CONCLUSIONS This paper describes the first immunosensor described to date for the determination of CDH-17, a biomarker of relevance in hepatic and gastrointestinal malignancies associated to metastasis. The approach is based on amperometric transduction using the HRP/H2O2/HQ system on SPCEs after magnetic capture on their surface of MBs modified with sandwich immunocomplexes. The electrochemical immunosensing platform is sufficiently sensitive (LOD of 1.43 ng mL-1) to allow the rapid determination of the target protein (just 45 minutes and using a single incubation step) in a small amount

(0.5 µg) of raw cellular lysates and, for the first time using an electrochemical immunosensor, in tissue extracts. The achieved results in the analysis of colorectal cancer cells and paired tumor/healthy tissue samples are in good agreement with the semi-quantitative results provided by the conventional dot-blot analysis but using 10-20 times lower amounts of biological samples. However, it should be remarked that a thorough validation of the reported approach with a much larger and varied cohort of patients as well as in carefully designed inter-laboratory trials is still required to fully demonstrate the reliability of the methodology and the truly clinical usefulness of CDH-17 biomarker for patient management and personalized treatment (i.e. by passive immunotherapy). Since different methodologies seem to interrogate and display the expression levels differently, quantitative data need to be agreed on to define clinically relevant thresholds of CDH-17 expression for such purposes. Once this promising biomarker and the new immunosensor are independently validated they can be included in an appropriate retrospective or prospective trial and, only after demonstrating their usefulness in these studies, they can reach wider application in a clinical setting. Indeed, this lack of standardization, which allows accurate correlation with clinical outcomes, is currently the largest hurdle to overcome the process of implementing emerging biomarkers and new quantification strategies into molecular diagnostic laboratories.38 The developed methodology should be further re-optimized, if necessary, to meet the strict demands of hospital laboratories and prove its practical superiority against other available methodologies, in which clinicians already trust on. A new breed of molecular pathologist willing to include and interpret this new information in their routine will be also required. Moreover, additional studies able to balance the importance, reliability and cost of the new information are strongly required. Although electrochemical biosensors could potentially be used as point-of-care (POC) clinical tools, their integration into fully automated diagnostics mass-produced devices, able to work with no human intervention, also demands collaborative research between basic and clinical groups enabling the pooling of the required multidisciplinary knowledge in electronics, microfluidics, chemistry, biology, medicine and material science. Despite these major challenges to be addressed, the preliminary but very important findings shown in this paper are of special relevance to take a privileged place for continue investigating the clinical relevance/role of this atypical cadherin in the proliferation, metastatic capability acquisition and overall survival of poor prognostic tumors. In addition, it should be mentioned the easy translation of the developed methodology to determine other emerging candidate biomarkers as well as the facile integration into multiplexed and/or microdevice bio-platforms. The characteristics of the developed methodology in terms of simplicity, speed, affordability, portability and the possibility of providing quantitative data using small amounts of challenging samples with minimal treatments and manipulations makes it more compatible with the clinical demands than other more complex strategies. Therefore, the methodology can open the way for bringing the determination of clinically validated biomarkers from the laboratory into real clinical or home diagnostics “sample-to-result” devices to perform routine screening of onset and relapse of high prevalence cancers, offering the opportunity of improving the patient management and getting an efficient therapeutic intervention and therefore a better disease outcome.


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ASSOCIATED CONTENT Supporting Information. No Supporting Information.

AUTHOR INFORMATION *Corresponding authors *

Tel.: +34 918223231. E-mail: [email protected] (Rodrigo Barderas). *Tel.: +34 913944368. Fax: +34 913944329. E-mail: [email protected] (Susana Campuzano). * Tel.: +34 913944315. Fax: +34 913944329. E-mail. [email protected] (José M. Pingarrón).

Author Contributions AV, EP, VRVP and MGA performed research. PYS, RB, SC and JMP designed the experiments. NR and GD collected the clinical samples. PYS, RB, SC and JMP wrote the manuscript. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The financial support of the Spanish Ministerio de Economía y Competitividad, CTQ2015-70023-R and CTQ2015-64402-C2-1-R and the NANOAVANSENS Program from the Comunidad de Madrid (S2013/MT−3029) and predoctoral contract from the Spanish Ministerio de Economía y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.-V.) are gratefully acknowledged. R.B. acknowledges the financial support of the PI17CIII/00045 grant from the AES-ISCIII program. M.G.A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.

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First electrochemical immunosensor for rapid and single-step determination of CDH-17. Application to the direct determination in tumor tissues extracts of different metastatic grade.


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Determination of Cadherin-17 in Tumor Tissues of Different Metastatic

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