Characterization of new serum biomarkers in breast cancer using lipid microarrays
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- Yonekubo, Y., Wu, P., Esechie, A. et al. Tumor Biol. (2010) 31: 181. doi:10.1007/s13277-010-0027-7
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Breast cancer is the most common form of cancer among women. Compared with other serum polypeptides, autoantibodies have many appealing features as biomarkers including sensitivity, stability, and easy detection. Anti-lipid autoantibodies are routinely used in the diagnosis of autoimmune diseases, but their potential for cancer diagnosis has not been explored. Dysregulation of cellular signaling in cancer cells would be expected to lead to irregular metabolism of many lipids, which could be sensed by the immune system and cause the production of autoantibodies. Discovery of anti-lipid antibodies could be used as biomarkers for early breast cancer diagnosis. We describe here a more sensitive and accurate method for lipid microarray detection using dual fluorescent labeling, and used it to examine global anti-lipid profiles in the MMTV-Neu transgenic breast cancer model. We conclude that, at the current technology, lipid microarray is not a preferred method for anti-lipid antibody detection in breast cancer animal models. Our result will help the future application of lipid microarrays in identifying anti-lipid autoantibodies in breast cancer and other human diseases.
KeywordsBreast cancerLipidLipid microarrayAutoantibodyTransgenic mouse model
Breast cancer is the most common form of cancer among women. However, despite many advances in technology, imaging, and genomic information, few reliable biomarkers for early breast cancer diagnosis are currently available. Improved prognosis could be achieved with better early screening tests, which would lead to earlier diagnosis. The ideal cancer screening test is one that is minimally invasive, inexpensive, and highly sensitive and specific. Much effort has been expended to identify new markers for breast cancer, including gene expression profiling using oligo or cDNA microarrays  or protein expression alteration analysis using mass spectrometry . Another new and promising approach for early detection is to look for the immune response to cancer, instead of cancer itself. Multiple studies have shown that cancer patients produce detectable autoantibodies to tumor-associated antigens that are overexpressed by neoplastic cells . Compared with other serum polypeptides, autoantibodies have many appealing features as biomarkers including increased sensitivity, better stability, and easy detection. Several potential autoantibodies have already been identified in breast cancer, although the ones found thus far are not universally present [3, 4].
The use of autoantibodies as cancer diagnostic biomarkers has been limited thus far to protein antigens. In contrast, anti-lipid autoantibodies are routinely used in the diagnosis of autoimmune disease, but their potential for cancer diagnosis has not yet been explored. Metabolism of lipids immediately follows cellular stimulation, resulting in various lipid metabolites (e.g. diacylglycerol, lysophospholipid, and ceramide). In fact, several lipid metabolic/signaling pathways have been found to associate directly with cancer. For example, fatty acid alpha-methylacyl-CoA racemase, which is involved in peroxisomal β-oxidation of dietary branched-chain fatty acids, is one of the most overexpressed genes in prostate cancer and is directly associated with prostate cancer risk [5–7]. Fatty acid synthase, which synthesizes fatty acids from acetyl-CoA and malonyl-CoA, is highly expressed in aggressive localized and metastatic prostate cancer [8, 9]. Moreover, one of the genes frequently inactivated in prostate cancer is phosphatase and tensin homolog, which controls the production of phosphatidylinositol-3,4,5-triphosphate production and antagonizes the PI3K/AKT pathway . Finally, using magnetic resonance imaging, magnetic resonance spectroscopy, and mass spectrometry, specific changes in choline phospholipid metabolism has been found to associate with more aggressive cancer phenotypes [10, 11].
Dysregulation of cellular signaling in cancer cells would be expected to lead to irregular metabolism of many lipids, which would be sensed by the immune system and cause the production of novel autoantibodies. These anti-lipid antibodies may be very useful markers for cancer diagnosis and prognosis. Due to technical difficulties, there has been no systemic study on production of anti-lipid antibodies during cancer progression. The technology now exists to explore this question at a “lipidomic” level, using the newly developed approach of lipid microarrays, which exhibit great sensitivity and specificity, and have been used successfully to identify autoantibodies against lipids in multiple sclerosis (MS) patients and in an MS animal model . In the current study, we have developed a dual fluorescent labeling method for lipid array detection, and used this method to look for the potential anti-lipid antibodies in a transgenic breast cancer model.
Materials and methods
Chemicals, lipids, and antibodies
Regular chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Casein was from USB (Cleveland, OH, USA). Odyssey blocking buffer was from LI-COR Biosciences (Lincoln, NE, USA). Fatty acid-free bovine serum albumin (BSA) was from Millipore (Billerica, MA, USA). Lipids were obtained from Matreya (Pleasant Gap, PA, USA), Avanti Polar Lipids (Alabaster, AL, USA), EMD Biosciences (Gibbstown, NJ, USA), and Sigma-Aldrich. They were dissolved in the solvents suggested by the manufacturers at a working concentration of 200 μM (see Supplementary Table 1). Rabbit asialo-GM1 antibody (#1950) was from Matreya. GD3 mouse monoclonal antibody (#MAB2053) was from Millipore. Goat anti-rabbit and mouse IgG conjugated with IRDye 800 or horseradish peroxidase (HRP) were from Rockland Immunochemicals. Alexa 680–conjugated goat anti-mouse and anti-rabbit IgG were from Invitrogen (Carlsbad, CA, USA).
Preparation of lipid arrays
Mice and blood collection
All animal experiments were performed at Stony Brook University animal facility in accordance with AAALAC guidelines and with Stony Brook University Institutional Animal Care and Use Committee approval. FVB/N mice and MMTV-Neu mice on an inbred FVB/N background were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The transgenic animals were confirmed by PCR analysis of genomic DNA from tail biopsy. Blood was drawn from the submandibular area of age-matched FVB/N and MMTV-Neu mice using a goldenrod animal lancet from Medipoint (Mineola, NY, USA). After collection, the blood was allowed to clot for 30 min at room temperature in 1.1 ml Z-GEL serum collecting tubes (Sarstedt, Newton, NC, USA). After 1 min spin in a microcentrifuge at top speed, the supernatant was transferred to a new Eppendorf tube and stored in the freezer (−20°C) until use.
Probing lipid arrays
Lipid arrays were blocked overnight at 4°C with a blocking buffer in PBS (5% fatty-acid-free BSA, 1% casein or Odyssey blocking buffer). Each array was incubated with serum sample (1:200 dilution in the blocking buffer with a total volume of 1 ml), and / or the control rabbit anti Asialo-GM1 (1:5,000) or mouse GD3 mouse monoclonal antibody (1:1,000) at room temperature for 3 h. The membranes were washed twice with blocking buffer (10 min each time), and then incubated with secondary antibodies (1:5,000) diluted in the blocking buffer (Alexa 680-conjugated anti-rabbit or anti-mouse IgG, or IRDye 800-conjugated goat anti-rabbit or goat anti-mouse antibodies) for 2 h at room temperature. After two more washes (10 min each time), the arrays were scanned using an Odyssey infrared imaging system from LI-COR Biosciences. For the chemiluminescent detection, the arrays were blocked with Odyssey blocking buffer, and incubated with the rabbit anti Asialo-GM1 and HRP-conjugated goat anti-rabbit secondary antibodies. After two washes, the membranes were incubated with the SuperSignal West Pico Chemiluminescent Substrate from Thermo Fisher Scientific (Rockford, IL, USA) and detected by a charge coupled device (CCD) camera on an Alpha Innotech Gel Imaging System with the FluorChem Q application from Cell Biosciences, Inc. (Santa Clara, CA, USA). The quantification of fluorescent and chemiluminescent signals was performed using software installed on Odyssey and Alpha Innotech Gel Imaging Systems, respectively.
Results and discussion
Improvement of the currently existing lipid microarray methodology
In the lipid array protocol of identifying autoantibodies against lipids in MS patients and in an MS animal model , chemiluminescent detection was used to detect antibody reactivity to lipids and glycolipids spotted on PVDF membranes. Chemiluminescent detection relies on an enzymatic reaction to generate light, which is detected by a CCD camera or imaged on film. This enzymatic reaction is dynamic, constantly changing over time. Some samples produce bright light for a short time and others produce comparatively weak light, but for a long period of time. Therefore, images must be collected in a certain time period. This time dependence of the signal compromises quantification and accuracy. We decided to improve this technique by using a dual-labeled fluorescent detection, which can be detected by an LI-COR Odyssey infrared imaging instrument.
The first two issues we set up to test were the membrane support and blocking solution. The PVDF membrane was used to spot lipids in the original lipid arrays. However, autofluorescence was consistently high on PVDF membrane when the fluorescently labeled secondary antibodies were used (result not shown). Although the infrared dyes used in the LI-COR Odyssey imaging system is supposed to have low background, the mouse serum generated a very high autofluorescent background. We then decided to test if other membranes could lower the autofluorescent background. Nitrocellulose membrane has an intrinsic low background and is recommended for Western blotting using fluorescently labeled antibodies. However, this type of membrane reacted to some solvents used to dissolve lipids, which generated high background (data not shown). We then chose to use a new type of PVDF from Amersham, Hybond-LFP, which the manufacturer claimed to have low fluorescent background for the Western blotting application. We compared the performance of regular PVDF and Hybond-LFP in three different blocking buffers. Different amounts of asialo-GM1 (200, 40, 8, 3.2, and 0.64 pmol) were spotted on either Hybond-LFP or regular PVDF (BioRad). The membranes were blocked in BSA, casein or Odyssey blocking buffers, detected by a polyclonal rabbit asialo-GM1 antibody, followed by IRDye 800 goat anti-rabbit (green channel) secondary antibody. Although no secondary antibodies were used for channel 700, the background in this channel is extremely high on the regular PVDF membranes in all three blocking conditions (Fig. 1a, right panel). The background in channel 700, however, was minimal on Hybond-LFP (Fig. 1a, left panel). The background in channel 800 was low and was very similar in all blocking solutions on both regular PVDF and Hybond-LFP. Among all blocking solutions, Odyssey blocking buffer was able to detect as little as 0.64 pmol of Asialo-GM1, thus gave the best sensitivity (Fig. 1a, bottom, left panel). We, therefore, decided to use the Hybond-LFP PVDF membrane and Odyssey blocking buffer for our subsequent experiments.
In the previous chemiluminescent detection, 10-100 pmol of lipids were used to prepare the lipid arrays  suggesting our fluorescent detection method is more sensitive. To avoid the variations caused by different reagents and blocking solution, we performed experiments to directly compare the chemiluminescent detection using a CCD camera and our fluorescent detection using the Odyssey imaging system. The chemiluminescent signal was detected by one of the most advanced chemiluminescent detection system, Alpha Innotech Gel Imaging System, which can approach the sensitivity of film, while far exceeding film with respect to linear dynamic range and the ability to obtain quantitative information from the image (Based on Alpha Innotech Applicatin Technique Note 122). Different amounts of asialo-GM1 were spotted in duplicate on Hybond-LFP as described above. After blocking with Odyssey blocking buffer and incubating with the rabbit asialo-GM1 antibody, the membranes were then incubated with goat anti-rabbit labeled with HRP (for chemiluminescent detection), or with Alexa-680 (for LI-COR Odyssey detection), respectively. To compare two different imaging systems, we generated the relative intensity values based on that of 8 pmol asialo-GM1 (Fig. 1b). The signals detected by all antibody-based methods are not linear to the level of their targets. However, both methods were able to reflect the differences of the relative amounts of lipid. Compared to the chemiluminescent detection, one advantage of the fluorescent detection method is the sensitivity. While the chemiluminescent detection barely detected the lowest amount of Aisalo-GM1 tested in our experiment (0.64 pmol), the fluorescent detection method generated a stronger or more visible signal from the same amount of lipid. Another advantage of the fluorescent detection is the better dynamic range. In the tested dilution range, the relative intensity value is from 0 (background) to 3.6 for the fluorescent detection, and from 0 (background) to 2.1 for the chemiluminescent detection. In summary, our results suggest that the fluorescent dye-based detection improved the sensitivity of detection as well as provided the better assessment of relative lipid levels.
Profiling lipid-specific antibody responses in sera at different stages of breast cancers
Several possibilities may account for the lack of the serum reactivity in the transgenic breast cancer mice in the current study. First, the number of lipids tested in the current studies is far less than the number of lipids that exists in cells. Lipids are structurally highly diverse owing to the many possible variations of the lipid building blocks and how these blocks are linked. A conserved estimation of the theoretical number of lipids covering major lipid classes is close to 200,000 . Second, not all known cellular lipids have been chemically synthesized or purified from cells or tissues, thus they are not commercially available for preparing lipid arrays. Third, the lipids spotted on the PVDF membranes may be misoriented and have different conformations comparing to those in cell membranes. The anti-lipid antibodies in the transgenic mice, if there is any, recognize only the lipids in cell membranes, and cannot recognize the “denatured” cognate lipids. Finally, our current detection method has not reached the level of sensitivity to detect the weak immune response that may occur. Based on our current pilot study, we conclude that at present, the lipid microarray technology is not a preferred method for anti-lipid antibody detection in breast cancer diagnosis and prognosis.
Nevertheless, as the major components of cell structure and signaling molecules, deregulation of lipid levels is expected to play important roles in tumorigenesis and trigger immune responses. Indeed, recent reports describe anti-lipid antibody production in cancer patients . In one study, a patient with breast cancer and another with colorectal carcinoma experienced dramatic exacerbation of their pre-existing anti-phospholipid syndrome after surgery . In another study, anti-GD1 IgM was found to be specifically augmented in patients with organ-confined prostate cancer (stage T1/T2), but not in patients with unconfined prostate cancer (T3/T4), benign prostate hyperplasia or healthy people . In principle, lipid microarray is still the most powerful method to explore potential anti-lipid autoantibodies in breast cancer patients at a “lipodomic” level. Compared to DNA and protein microarrays, the production of lipid arrays, especially the handling of the solvents used to dissolve lipids, is relatively challenging. Development of better lipid array techniques in the future will definitely help to delineate the functions of lipids in cancer and lead to discover some useful lipid or anti-lipid biomarkers. Some recent progress on this topic has shed light on increasing the accessibility of lipid microarray to the research community. Among those, a new method has recently been developed to create micropatterned lipid bilayer arrays using a 3D microfluidic system . The dual fluorescent labeling detection based on the LI-COR infrared imaging system and preliminary results described in our current study would certainly provide some useful information for identifying new anti-lipid autoantibodies in the future.
The authors thank Ms. Yue Zeng and Dr. Wenjuan Su for their help with mouse maintenance and blood collection. This work was supported by a concept award from the Department of Defense (DOD) Breast Cancer Research Program (W81XWH-06-1-0690) and a research grant from National Institutes of Health (GM071475).