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METHODS FOR CANCER DETECTION
This application claims the benefit of Provisional Patent Application Ser. No. 60/625,875 filed on Nov. 8, 2004, the disclosure of which is incorporated herein in its entirety by 5 reference.
The present invention relates to methods for early detection i o of cancer, and for monitoring progression, metastasis, and/or treatment efficacy of cancer. In particular, the invention provides a method for detection of cancer comprising detection of analytes indicative of cancer or a metastatic disease using a biosensor. 15
BACKGROUND OF THE INVENTION
It is known that early detection of cancer is highly correlated with patient survival. Accordingly, research has focused 2o on the discovery and recognition of new diagnostic biomarkers that relate to the presence of cancer, its severity, and its subsequent progression. Certain biomarkers are specific to certain cancer types, i.e., prostate specific antigen as a diagnostic marker for prostate cancer. However, other, non-spe- 25 cific biomarkers have value in early detection of cancer, determination of prognosis, monitoring of effectiveness of cancer treatment, detection of recurrence post-treatment, and the like. In this respect, it is known that various cytokines, growth factors, toxins, chemokines, and hormones are found in 30 elevated concentrations at the onset of cancer and during metastasis of cancer.
One such cytokine is vascular endothelial cell growth factor (VEGF), also called vascular permeability factor (VPF). Vascular endothelial cell growth factor is an endothelial cell 35 mitogen, and is known to be present in very small quantities in normal human blood in order to facilitate activities such as wound healing. In contrast, soluble VEGF is released in large quantities by tumorigenic cells (e.g. carcinomas of the breast, large and small intestine, pancreas, kidney, and the like) due 40 to its critical role in angiogenesis. During angiogenesis, new blood vessels form from existing vessels to supply nutrients to the developing tumor cells. Due to VEGF's mitogenic nature, it facilitates the formation of the new blood vessels by selective action on endothelial cells. 45
Quantities of VEGF in serum and plasma of patients with tumors from a multitude of cancers (small-cell lung cancer, primary breast cancer, non-Hodgkin's lymphoma, prostate cancer, and the like) have been found to be significantly elevated. The concentration ofVEGF has also been correlated 50 with the presence of metastatic disease, disease stage in colorectal cancer, and poor prognosis in patients with small cell lung cancer.
Conventional assays for cytokines such as VEGF for use in detecting, diagnosing, or monitoring the progress or progno- 55 sis of cancer, or for monitoring treatment efficacy, generally rely on specific immunological assays for the protein. However, there is a need in the art for a more rapid screening method for early detection and monitoring of cancer. The screening method should be simple, reliable, and allow detec- 60 tion of indices of cancer such as VEGF and other analytes known to be produced in elevated quantities during onset, progression, and metastasis of various cancers.
The present invention satisfies this need in the art by providing methods and systems for detecting and monitoring 65 cancer and its progression via detection and quantitation of a cytokine using a whole-cell biosensor. The invention relies on
induction of permeability in the cell portion of the biosensor to detect and quantitate the cytokine.
SUMMARY OF THE INVENTION
In accordance with the purposes of the present invention as described herein, in one aspect of the invention a method is provided for detecting an analyte indicative of a cancer or a metastatic disease condition, comprising providing a biosensor comprising a barrier which is substantially impermeable to an ion, a permeable membrane which is selective for the ion, and a detector capable of detecting the ion. The biosensor is contacted with a sample including at least one of the ion and an analyte indicative of a cancer or a metastatic disease condition, wherein the analyte causes at least a portion of the ion to pass through the barrier and the membrane to allow detection of the presence or absence of the ion and thereby indirect detection of the presence or absence of the analyte.
The barrier is a live cell layer known to be responsive to the analyte being detected. In one embodiment of the invention, the barrier is a transformed human endothelial cell line, and the analyte is VEGF. The cell barrier is typically provided as a substantially confluent cell monolayer which is contiguous with the permeable membrane. In use, the cell monolayer is substantially impermeable to the ion, but becomes at least partially permeable to the ion upon contact with the analyte. The detector may be coupled to a transducer to provide a measurable signal upon detection of the ion.
The analyte being detected will typically be selected such that the amount of the analyte in the sample is indicative of the presence, progression, or severity of a cancer or a metastatic disease condition, or of the efficacy of a treatment therefor. The analyte may be selected from the group of cancer-related analytes consisting of cytokines, growth factors, hormones, chemokines, and toxins, including but not limited to tumor necrosis factor, basic fibroblast growth factor, interleukin 6, interleukin 8, hepatocyte growth factor, or any permeability modifying agent released by cancer, and combinations thereof. In one embodiment, the analyte detected is VEGF.
The membrane of the biosensor may be selected to be natively selective for the ion of choice, or may be chemically modified for selectivity for the ion. The permeable membrane may be chemically modifyied using an ionophore to induce selectivity for the ion. In one embodiment, a permeable cellulose triacetate membrane is modified with valinomycin to induce selectivity for potassium.
In another aspect of the present invention, a method is provided for monitoring the presence, progression or severity of a cancer or a metastatic disease, or the efficacy of a treatment therefor, in a patient during a predetermined time interval, comprising the steps of (1) obtaining a biological sample from a patient having or at risk of having a cancer or a metastatic disease, (2) combining the biological sample with a predetermined amount of an ion to form a mixture, (3) contacting the mixture with a biosensor as described above, (4) detecting the presence or absence of an analyte which is indicative of a cancer or a metastatic disease condition in the mixture, wherein the analyte causes the live cell barrier to become at least partially permeable to the ion whereby the ion is detected by the detector, (5) correlating the amount of the ion detected with the quantity of the analyte in the biological sample, and (6) repeating steps 1 through 5 over time to measure the progress of the cancer or the treatment. It will be appreciated that if the sample naturally contains a known, fixed concentration of the ion, inclusion of additional amounts of the ion is unnecessary.
The biological sample may be selected from the group consisting of serum, whole blood, plasma, saliva, tears, milk, urine, other biological/physiological fluids, a tissue biopsy, an organ biopsy, lymphatic fluid, and any combination thereof. The quantity of the analyte detected in the biological 5 sample may be then correlated with the presence, progress, or severity of the cancer or metastatic disease, or with the efficacy of the treatment. The biosensor used in this method is substantially as described above. In one embodiment, the present method comprises use of a biosensor as described 10 above, comprising a human umbilical vascular endothelial cell line grown on a cellulose triacetate membrane chemically modified with valinomycin to induce potassium selectivity. The biosensor is used to detect and quantify VEGF in the biological samples. 15
In yet another aspect of the present invention, a system for detecting the presence of an analyte indicative of a cancer or a metastatic disease condition, comprising a cell-based biosensor, an ion, and at least one standard sample containing a predetermined quantity of an analyte indicative of a cancer or 20 a metastatic disease condition. In one possible embodiment, the cell-based biosensor comprises a monolayer of human umbilical endothelial cells on a cellulose triacetate membrane modified for potassium selectivity. The ion is potassium, and the analyte is VEGF. The biosensor includes an electrode 25 supporting the membrane, with the electrode coupled to a transducer which provides a signal which can be detected upon detection of the ion by the electrode.
It should be appreciated that the embodiments shown and described herein are illustrations of some of the modes best 30 suited to carry out the invention. It will be realized that the invention is capable of other different embodiments and its several details are capable of modification in various, obvious aspects all without departing from the invention. Accordingly, the drawings and descriptions will be regarded as illus- 35 trative in nature, and not as restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings incorporated in and forming 40 a part of the specification, illustrate several aspects of the present invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 schematically depicts an embodiment of the biosensor of the present invention; 45
FIG. 2 shows the preparation of a modified ion-selective cellulose triacetate membrane (selective for potassium) and attachment of a HUVEC monolayer;
FIG. 3 shows the biosensor response under various conditions: FIG. 3a shows the ratio of the electrode response 50 obtained at 0.1 M KC1 following treatment to the response for the membrane without cells or VEGF at 0.1 M KC1. Experimental conditions were (A) 1 (xg/ml VEGF only, (B) cells only, (C) 1 ng/ml VEGF for 2 hr, (D) 1 ng/ml VEGF for 4 hr, (E) 1 (xg/ml VEGF for 5 hr, (F) 1 (xg/ml VEGF for 6 hr (error 55 bars represent SEM); FIG. 3b shows calibration curves for the biosensor under conditions A-F above, with the top line representing the average biosensor response without cells or VEGF;
FIG. 4 shows the ratio of the biosensor response following 60 exposure to 100 ng/ml VEGF to the response for the biosensor without cells or VEGF. Experimental conditions were (A) cells only, (B) after 6 hr exposure, and (C) after 8 hr exposure (error bars represent SEM);
FIG. 5 shows the ratio of the biosensor response obtained at 65 0.1 M KC1 following exposure to 1 ng/ml VEGF to the response for the biosensor without cells or VEGF. Experi
mental conditions were (A) cells only, (B) after 8 hr exposure, and (C) after 10 hr exposure (error bars represent SEM); and
FIG. 6 shows the ratio of the biosensor response obtained at 0.1 M KC1 following 10 hr exposure to VEGF concentrations ranging from 0 to 1,000 pg/ml, with the data fitted using TableCurve 2D v5.01; also shown are the biosensor responses to media aspirated from metastatic melanoma cell cultures (1205LU) at seeding densities of lxlO6, 5xl05, and 1x10s cells/well; all results are plotted against the average concentration of VEGF as measured by ELIS A for the same samples.
Reference will now be made in detail to the present preferred embodiment of the invention, an example of which is illustrated in the accompanying drawings.
DETAILED DESCRIPTION OF THE INVENTION
Conventional methods of cancer detection and monitoring often rely on detection of specific biomarkers which may be diagnostic of specific types of cancer. However, it is known that blood concentrations of various cytokines and growth factors, while not necessarily diagnostic of specific cancers, can be correlated to early stages of cancer, to likelihood and/or aggressiveness of metastasis, and to efficacy of cancer treatment.
It was considered that a method of rapid screening for such cancer-related analytes would be useful as a general method of monitoring presence of cancer, effectiveness of cancer therapy, determination of prognosis, and/or likelihood of recurrence. Accordingly, a biosensor-mediated method for early detection of cancer, or for monitoring the progress and/ or treatment efficacy of a cancer or a metastatic disease, was developed. As an example, VEGF, a multiple-isoform glycoprotein which is a known endothelial cell mitogen, is present even during the early stages of cancer and is released in soluble form by tumorigenic cells due to its vital role in angiogenesis [Ferrara (2004) Endocrine Reviews 25, 581611; incorporated herein by reference]. Therefore, in one embodiment of the invention, the biosensor utilizes the ability of VEGF to induce permeability of a human endothelial cell line monolayer, passage of an ion therethrough, and detection of the ion, as an indirect detection method for the analyte.
In one embodiment, depicted in FIG. 1, the biosensor 10 utilized in the method of the present invention comprises a barrier 12 which is substantially impermeable to an ion, a permeable membrane 14 which is selective for the ion, and a detector 16 capable of detecting the ion. In the depicted embodiment, the barrier 12 is a human umbilical vein endothelial cell monolayer coupled to an ion-selective cellulose triacetate membrane 14 by a linker molecule 18. The barrier 12/membrane 14 complex is carried by the detector 16 to provide an ion-selective electrode 20 as will be described in greater detail below. A reference electrode 22 may also be provided as part of the detector 16 as is known in this art to allow detection of a potentiometric difference in response to detection of the ion.
A sample 24, contained in a receptacle 26, may be contacted with the biosensor 10, wherein the sample 24 includes at least one of the ion and an analyte of interest. It will be appreciated that the analyte will typically be indicative of a cancer and/or a metastatic disease condition, and further causes at least a portion of the ion to pass through the barrier 12 and the permeable membrane 14 to be detected by the detector 16.
The following examples are presented in support of and to further illustrate the invention as described herein. However, the invention is not to be considered as limited thereto.
Referring to FIG. 2, a cellulose triacetate (CTA; Eastman Kodak, Rochester, N.Y.) membrane was prepared as an ionselective support for a cell layer. The base layer of the mem- 5 brane was prepared by dissolving 74 mg of CTA pellets in 1.1 mL of methylene chloride (Aldrich, Milwaukee, Wis.), 0.4 mL of chloroform (Aldrich), and 0.40 mL of 1,1,2,2-tetrachloroethane (Aldrich). The mixed solution was then cast in a 31 mm i.d. glass ring placed on a TEFLON plate. After 10 solvent evaporation, the membrane was removed and floated on 1.0 M sodium hydroxide for 4.5 hr. Raised edges of the membrane ensured that only the lower surface of the membrane was hydrolyzed to provide free OH groups. The membrane was then rinsed with deionized water. 15
A second membrane layer was cast using an ionophore cocktail to induce selectivity for potassium. The ionophore cocktail consisted of 1 mg of valinomycin (Calbiochem, San Diego, Calif), a plasticizer (100 uL of o-nitrophenyl octyl ether; Fluka, Ronkonkoma, N.Y.), and a lipophilic salt (0.42 20 mg potassium tetrakis(chlorophenyl)borate; Fluka) with 35 mg of CTA. The mixture was dissolved in a solvent mixture composed of 0.80 mL of methylene chloride and 0.80 mL of chloroform. The solvent was allowed to evaporate for 2 days as the two membrane layers fused into a single asymmetric 25 membrane.
The bottom side of the basic membrane (having the free OH groups) was immersed in cold deionized water. Next, 324 mg of carbonyldiimidazole (CDI; Sigma, St. Louis, Mo.) was added in increments over a 15 min period, giving a final 30 concentration of 0.10 M CDI to activate the surface for peptide coupling. The membrane was then immediately incubated overnight in a 0.1 M sodium carbonate solution (pH 9.5) containing 400 ug of a synthetic RGD peptide sequence (Bachem, King of Prussia, Pa.) to promote cell binding to the 35 membranes. The peptide sequence used was Gly-Arg-GlyAsp-Ser (GRGDS) to promote covalent peptide attachment from the N-terminal amine. This is because the arg-gly-asp (RGD) segment of fibronectin is one of the most widely recognized protein sequences to which human umbilical vein 40 endothelial cells (HUVECs) bind. The membranes were then rinsed sequentially with 0.10 M NaHC03, deionized water, acetate buffer (pH 4.0), and deionized water. Protein immobilization was confirmed using a micro-BCA protein assay (Pierce Biotechnology, Rockford, 111.). 45
Six mm i.d. disks cut from the membranes prepared as described above were mounted onto Philips IS-561 electrode bodies (Glasblaserei Moller, Zurich) with the RGD-modified surface of the membrane facing the sample solution. Human umbilical vein endothelial cells (Cambrex Bioscience, East 50 Rutherford, N.J.) were cultured in an EGM-2 media (Cambrex Bioscience) supplemented with fetal bovine serum, hydrocortisone, insulin-like growth factor, basic fibroblast growth factor, VEGF, human epidermal growth factor, ascorbic acid, human fibroblast growth factor, gentamicin sulfate, 55 amphotericin-B, and heparin. The HUVECs were used experimentally up to passage 5. The HUVECs were seeded onto the bottom surface of the electrode-mounted membrane disks at a density of lxl 15 cells/mL. The cells were allowed to spread and form a confluent monolayer over the membrane 60 surface for 24 h at 37° C. in a humidified incubator with 5%
The internal filling solution for the electrode was 0.01 M KC1, and the internal reference electrode was Ag/AgCl. The external reference electrode was a double-junction Ag/AgCl 65 electrode (Orion Model 90-02-00) with an Orion (90-02-02) internal filling solution and 0.1 M Tris buffer (pH 7.5) in the
outer compartment. Potentiometric responses were measured with a four-channel high impedance amplifier interface (World Precision Instruments) connected to a Model 100 Instrunet A/D converter. Data were analyzed using Instrunet software on a Macintosh Power PC. Prior to initial use, the electrodes were conditioned in sterile 0.1 M KC1 solution. Calibration plots were constructed by plotting the measured potential (mV) versus the logarithm of the concentration of potassium ions present in the bulk solution.
The response of the biosensor as described in Example 1 was first evaluated after protein immobilization and as a function of cell confluency. After confirmation of the inhibited response following 24 h of cell growth (i.e., diminished transport of potassium through the confluent monolayer), the cellbased membranes were exposed to concentrations of VEGF ranging from 100 pg/mL to 1 ug/mL (pH 7.0 in 0.1% BSA in PBS) for times ranging from 2 to 10 hr. The biosensor response was measured for the following conditions at a final concentration of 0.1 M. KC1: (1) membrane without cells or VEGF exposure to confirm that the membrane response was not affected by RGD; (2) membrane without cells and with VEGF (1 ug/mL) to confirm that the highest concentration of VEGF did not affect the response in the absence of HUVECs (FIGS. 3a, 4, and 5); (3) membrane with cells and without VEGF to confirm the inhibited sensor response after 24 h of cell growth (FIGS. 3a, 4, and 5); and (4) membrane with cells and with varying concentrations of VEGF at different exposure times. Data were reported as a ratio of the potential response for ion-selective electrodes (ISEs) with HUVECs and/or VEGF to the potential response of the ISEs without HUVECs or VEGF for a final concentration of 0.1 M KC1 to account for slight variations between fabricated membranes. Three to five replicates were performed for each VEGF concentration.
Response of the biosensor to a high concentration ofVEGF (1 ug/mL) was evaluated. Referring to FIG. 3, no significant response from the sensor was obtained at 2 and 4 hr of exposure. Increasing the exposure time to 5 hr significantly increased the response, and at 6 hr of exposure the response was nearly double that of the 5 hr response period. Calibration curves for each exposure time supported these findings (FIG. 3b).
It was desired also to evaluate the lower detection limit of the sensor. The biosensor of Example 1 was exposed to a reduced concentration (100 ng/ml) of VEGF. Experimental conditions were substantially as described previously. The response ratio was very low after exposing the biosensor to 100 ng/ml of VEGF for 6 hr, but approximately tripled after 8 hr (FIG. 4). Reducing the VEGF concentration to 1 ng/ml with an 8 hr exposure period resulted in a response ratio nearly equal to the control response (monolayer without VEGF). However, increasing the response time to 10 hr significantly increased the response ratio (FIG. 5).
The response ratio from the biosensor was evaluated as a function of the concentration of VEGF. The sensor was exposed to increasing concentrations (100, 300, and 600 pg/ml) of VEGF, over a 10 hr exposure period based on the results described in Example 2. Referring to FIG. 6 (see the plot of "Obtained sensor values"), as the concentration of VEGF increased, the response ratio also increased. The curve shape was characteristic of binding events showing a rapid