Method for detecting content of serum aldehyde dehydrogenase in early non-small cell lung cancer

Abstract

The technical problem to be solved by the invention is to provide the serum aldehyde dehydrogenase content for detecting the non-small cell lung cancer and the detection method, the detection method is more sensitive and higher in specificity, the early diagnosis rate of the non-small cell lung cancer is favorably improved, the five-year survival rate of the small cell lung cancer is further improved, and the prognosis is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting the content of aldehyde dehydrogenase in lung cancer serum. The detection method is more sensitive and has higher specificity. Background Art

[0002] Lung cancer is the most common malignant tumor in the world. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) according to different tissue types. NSCLC accounts for more than 80% of all lung cancers, including adenocarcinoma, squamous cell carcinoma and large cell carcinoma, among which adenocarcinoma is the most common. The clinical manifestations of lung cancer in the early stage are not obvious. Improving the early diagnosis rate of lung cancer is the key to improving the 5-year survival rate. The detection of serum markers for lung cancer has received widespread attention because it is simple, non-invasive and low-cost.

[0003] Although treatment options for lung cancer include surgery, radiotherapy, and chemotherapy, lung cancer remains the most common cause of cancer death worldwide, with a 5-year survival rate of approximately 15%. Non-small cell lung cancer (NSCLC) is the most common histological type, accounting for 80-85% of newly diagnosed cases. For functionally viable early and resectable stage III ANSCLC, surgery is the standard treatment with potential for cure. However, only 20% of NSCLC is diagnosed and resectable at an early stage, so early detection strategies for lung cancer remain an unresolved clinical challenge.

[0004] To date, many studies have mainly explored the potential impact of chest X-ray and low-dose spiral CT imaging and sputum cytology on the detection of lung cancer. Although these studies have shown encouraging results in stage distribution, favoring early-stage disease, better surgical resection of tumors, and improved survival, improvements in overall mortality cannot be determined. Serum markers such as carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and Cyfra21-1 are serum markers used to monitor treatment response in lung cancer patients, but their application as screening biomarkers remains a challenging issue.

[0005] Tarro et al. isolated a tumor-associated antigen from non-small cell lung cancer and named it tumor-released protein (TLP). By immunohistochemical methods, TLP was localized in small and large granules in the cytoplasm. Given that TLP was also detected in the lumen of atypical glands and in the bronchial secretions of some specimens, TLP can be considered a secretory product of cancer cells. Studies have shown that when TLP is isolated and purified from a patient's tumor and reintroduced into the body, it enhances the host's immune response. Based on partial sequence analysis of this protein, the corresponding antigenic peptide fragments have been synthesized and used to generate rabbit antiserum. Among the four TLP-derived peptides identified by Tarro et al., anti-RTNKEASI rabbit serum reacted specifically with NSCLC tumor extracts and lung cancer patient sera. TLP is detected in the sera of patients with non-small cell lung cancer mainly at early disease stages.

[0006] It has been shown that TLP (or a component of this complex) corresponds to aldehyde dehydrogenase (ALDH) isoform 1A1 (ALDH1A1). ALDH is a class of enzymes that are widely present in cells and participate in cellular detoxification, differentiation and drug resistance processes by converting exogenous and endogenous aldehydes into carboxylic acids. A large number of studies have shown that ALDH plays a biological role in cancer, including breast cancer, colon cancer, head and neck cancer, papillary thyroid cancer, and mainly lung cancer, providing supportive evidence for the correlation between ALDH activity and lung cancer stem cells. In addition, Cao et al. found that ALDH1A1 levels were increased in the serum of NSCLC patients. Studies have shown that ALDH isoforms other than 1A1 may be released into the blood of NSCLC patients. Therefore, screening sensitivity may be further improved by adopting isoform-nonspecific overall ALDH detection without significantly reducing specificity.

[0007] Therefore, detecting the serum aldehyde dehydrogenase level of lung cancer can help improve the early diagnosis rate of non-small cell lung cancer, thereby increasing the 5-year survival rate of small cell lung cancer and improving the prognosis. Summary of the invention

[0008] The present invention provides a method for detecting the content of aldehyde dehydrogenase in lung cancer serum. The method can detect the content of aldehyde dehydrogenase in lung cancer serum. The detection method is more accurate and sensitive, and is of great significance for the early screening and prognosis of lung cancer patients.

[0009] The technical solution of the present invention:

[0010] 1. Preparation of rabbit anti-RTNKEASI serum: RTNKEASI peptide fragments were obtained from the tumor-released protein (TLP) complex, and then the peptide fragments were used for rabbit immunization, and finally rabbit anti-RTNKEASI serum was collected.

[0011] 2. Immunoblotting: Rabbit anti-RTNKEASI serum was used to perform immunoblotting on lung cancer cell line A549 to evaluate the response of the serum to A549 cells. The effectiveness of the immune response was determined by observing whether a signal corresponding to the target protein band appeared.

[0012] 3. Mass spectrometry analysis: The target protein band detected by immunoblotting and reacting with rabbit anti-RTNKEASI serum was subjected to mass spectrometry analysis to determine its molecular mass and identify the protein identity.

[0013] 4. ELISA: Serum samples from NSCLC patients and healthy donors were analyzed using ELISA technology. ALDH1A1 and comprehensive ALDH levels were detected to evaluate the presence of these proteins in NSCLC patients and healthy donors. DETAILED DESCRIPTION

[0014] The technical solution of the present invention will be further described below, but the present invention is not limited thereto.

[0015] 1. Patient sample collection

[0016] Blood samples were collected from 20 patients with newly diagnosed early-stage non-small cell lung cancer during routine diagnostic procedures, as well as from 20 healthy donors.

[0017] 2. Antigen synthesis and antibody production

[0018] Two different rabbit polyclonal anti-RTNKEASI immune sera and TLP-derived peptide RTNKEASI were produced by Rockland and Berlin, Germany. The anti-RTNKEASI immune sera produced by the Rockland strain were further purified using a chromatography method targeting the RTNKEASI peptide fragment.

[0019] 3. Cell culture

[0020] Human cell lines MRC-5, A549, Hela, CA46, HL60, PC3, and MCF-7 were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2 in air. Specific culture conditions: CA46 cell line was cultured in Glutamax RPMI 1640 medium; PC3 cell line was cultured in Glutamax HAM F-12 medium; MRC-5, HL60, MCF-7, A549, and Hela cell lines used Glutamax modified Dulbecco's Eagle medium. All culture media (Gibco, Life Technologies) were supplemented with 10% fetal bovine serum (Berlin, Germany).

[0021] 4. Western Blot

[0022] Cell pellets and lysates were prepared from MCF-7, CA46, HL60, PC3MRC-5, A549, and Hela cell lines as described previously. Cell culture supernatants of A549 cells were frozen with 10% trichloroacetic acid (TCA) and then incubated with 90% acetic acid at -20°C. Subsequently, the samples were centrifuged and the pellets were dried at 65°C for 30 minutes.

[0023] The separated proteins were mixed in Laemmli sample buffer (20% glycerol, 4% sodium dodecyl sulfate (SDS), 100mM Tris-HCl, 200mM dithiothreitol (DTT) and 0.01% bromophenol blue) and loaded into SDS PAGE gel after heating. Finally, the protein was blotted onto the membrane and blocked in skim dry milk 8% + PBS + 0.1% Tween-20, and then hybridized overnight at 4°C with anti-RTNKEASI serum diluted to 1:1000 or β-actin (Biolegend, San Diego, CA) at 1:20000. After the primary antibody, the membrane was probed with a secondary antibody (1:30000 in PBS + 0.1% Tween-20 + BSA 5% buffer), and then the protein was visualized by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).

[0024] 5. Principal Component Analysis

[0025] According to the Rockland protocol, anti-RTNKEASI serum (1:1000) was pre-incubated with RTNKEASI peptide (500-fold molar excess) or KDSGNEQTFLPP as a control peptide and then hybridized with the blotted A549 samples for 2 hours. The membrane was treated with secondary antibodies, and immunoreactive bands were detected by enhanced chemiluminescence.

[0026] 6. Two-dimensional polyacrylamide gel electrophoresis. Protein separation of cell lines was performed as previously described.

[0027] For the first dimension isoelectric focusing, a 7 cm fixed pH gradient (IPG) dry gel strip and a linear pH 4-7 gradient (GE Healthcare) were used. The dissolved protein (50 μg) was placed on the strip, rehydrated, and incubated overnight. The protein was resolved by the PROTEAN IEF system (Bio-Rad) (at 20 ° C, the voltage gradient lower limit was 50 μA) under the following conditions: 4 h at 250 V, linear gradient 8000 V to 15000 Vh, fast 8000 V to 75000 Vh, and a total voltage of 90 kV h. After equilibration in buffer 1 (130 mM DTT, 6 M urea, 20% glycerol, 0.05 M TrisHCl, 2% SDS) and buffer 2 containing iodoacetamide instead of DTT, the first dimension band was positioned on the top of an 8% acrylamide gel (Applichem GmbH, Darmstadt, Germany) for second dimension electrophoresis. The separated proteins were silver stained or blotted and detected with pre-sera or anti-RTNKEASI serum.

[0028] 7. Protein identification by LC-MS / MS

[0029] Gel spots were treated with DTT (10 mM, 56 °C, 30 min) and iodoacetamide (55 mM, room temperature, 20 min, protected from light). Subsequently, proteins were digested with 13 ng / μl trypsin (sequencing grade modified trypsin, Promega) in 50 mM ammonium bicarbonate at 37 °C for 16 h. Tryptic peptides were extracted in 65% ethyl acetate (ACN) / 5% formic acid (FA), dried under vacuum, and dissolved in 15 μL 0.1% FA. Tryptic peptides were separated on an UltiMate 3000 Rapid Separation Liquid Chromatography (RSLC) system (Dionex, Thermo Scientific, Bremen, Germany) by online electrospray ionization (ESI) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany). Samples were loaded onto the trapping column at a flow rate of 5 μl / min. (Acclaim PepMap μ-precolumn, C18, 300 μm × 5 mm, 5 μm, Thermo Scientific, Bremen, Germany; nanoACQUITY-UPLC Symmetry C18 trapping column, 180 μm × 20 mm, 5 μm, Buffer A: 0.1% FA in HPLC-H2O; Buffer B: 0.1% FA in acetonitrile) containing 2% buffer B. The loaded sample on the capture column was first washed with 2% buffer B (5 μl / min) for 5 minutes, and then the peptides were eluted (200 nl / min) onto the separation column. The trypsin-digested peptides were separated on a reverse phase C18 column (Acclaim PepMap100, 75 μm × 25 mm, 2 μm, Thermo Scientific, Bremen, Germany; nanoACQUITY-UPLC column, BEH 130C18, Waters; 75 μm × 250 mm, 1.7 μm, ), flow rate 200nl / μm, using solvent A (0.1% FA, HPLC-H2O) and solvent B (0.1% FA, acetonitrile) to form a binary buffer system. The peptides were eluted with a gradient of 2-30% solvent B for 30min. MS analysis was performed in positive ion mode and programmed acquisition in data-dependent mode (DDA). Full scans were acquired on a Fusion Orbitrap mass analyzer with a resolution of 120,000 FWHM, m / z 200, MS level, m / z range from 400 to 1500 (maximum injection time: 50ms, automatic gain control target: 4e 5 ). At the intensity threshold of 1e 4 Fragmentation was performed without ion fragmentation, and the fragmented ions were accumulated in the linear ion trap in fast mode. Only the +2 to +5 charge states and the most intense precursor ions were selected for fragmentation. The most intense ions were isolated to a target value of 1e4 with a maximum injection time of 50-150ms. Raw data were processed using Proteome Discoverer v1.4.1.14 (Thermo Scientific). Protein identification was performed using the UniProtFASTA database.

[0030] 8. ALDH and ALDH1A1 ELISA

[0031] Serum specimens collected from patients with early-stage non-small cell lung cancer (20 cases) and healthy blood donors (20 cases) were stored at -80°C, and then the serum was tested using a specific monoclonal antibody (Bluegene, Biotech, Shanghai, China) according to the instructions of the Global ALDH ELISA Kit (Bluegene, Biotech, Shanghai, China), and the 1A1 subtype was tested using an ALDH1A1 ELISA kit using a specific monoclonal antibody (Cloud-Clone Corp, Houston, Texas, USA). The data are presented as the mean ± standard deviation of three independent experiments.

[0032] 9. Statistics

[0033] All data are expressed as mean ± standard deviation (SD). Differences in serum levels of ALDH and ALDH1A1 between early-stage NSCLC patients and healthy donors were analyzed. Correlation coefficients were calculated using the Pearson method. Version 22 or GraphPad Prism TM The analysis was performed using the software version 5. A p value < 0.05 was considered statistically significant.

Claims

1. A method for detecting serum aldehyde dehydrogenase content in early non-small cell lung cancer, wherein: The detection method not only includes detecting the serum aldehyde dehydrogenase 1A1 subtype, but also can detect the whole type of aldehyde dehydrogenase, and is characterized in that it includes the following steps: Step 1: During routine diagnosis, blood samples were collected from 20 newly diagnosed patients with early-stage non-small cell lung cancer and 20 healthy donors to process the obtained samples; Step 2, obtain the RTNKEASI peptide from the tumor-released protein (TLP) complex, then use the peptide to immunize rabbits, and finally collect rabbit anti-RTNKEASI serum. Immunoblotting was performed on the lung cancer cell line A549 with rabbit anti-RTNKEASI serum to evaluate the response of the serum to A549 cells. The effectiveness of the immune response was determined by observing whether a signal corresponding to the target protein band appeared, and the target protein band detected by immunoblotting and reacting with rabbit anti-RTNKEASI serum was subjected to mass spectrometry to determine its molecular mass and identify the protein identity. Serum samples from patients with non-small cell lung cancer (NSCLC) and healthy donors were analyzed using ELISA technology. The presence of these proteins in early non-small cell lung cancer (NSCLC) patients and healthy donors was evaluated by detecting ALDH1A1 and comprehensive ALDH levels.

2. The method according to claim 1, characterized in that The non-small cell lung cancer patients only include stage I and stage II patients.

3. The method according to claim 1, characterized in that The experimental samples in step 1 include, but are not limited to, ADC and SCC patient samples.

4. The detection method according to claim 2, characterized in that: The following steps are involved: Step 1: Antigen synthesis and antibody production Two different rabbit polyclonal anti-RTNKEASI immune sera and TLP-derived peptide RTNKEASI were produced by Rockland and Berlin, Germany. The anti-RTNKEASI immune sera produced by the Rockland strain were further purified using a chromatography method targeting the RTNKEASI peptide fragment. Step 2: Cell culture Human cell lines MRC-5, A549, Hela, CA46, HL60, PC3, and MCF-7 were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2 in air. Specific culture conditions: CA46 cell line was cultured in Glutamax RPMI 1640 medium; PC3 cell line was cultured in Glutamax HAM F-12 medium; MRC-5, HL60, MCF-7, A549, and Hela cell lines used Glutamax modified Dulbecco's Eagle medium. All culture media (Gibco, Life Technologies) were supplemented with 10% fetal bovine serum (Berlin, Germany). Step 3: Western Blot Cell pellets and lysates were prepared from MCF-7, CA46, HL60, PC3 MRC-5, A549, and Hela cell lines as described previously. The cell culture supernatant of A549 cells was frozen with 10% trichloroacetic acid (TCA) and then incubated with 90% acetic acid at -20° C. Subsequently, the sample was centrifuged and the precipitate was dried at 65° C. for 30 minutes. The separated proteins were mixed in Laemmli sample buffer (20% glycerol, 4% sodium dodecyl sulfate (SDS), 100mMTris-HCl, 200mM dithiothreitol (DTT) and 0.01% bromophenol blue) and loaded into SDS PAGE gel after heating. Finally, the protein was blotted onto the membrane and blocked in skim dry milk 8% + PBS + 0.1% Tween-20, and then hybridized overnight at 4°C with anti-RTNKEASI serum diluted to 1:1000 or 1:20000 β-actin (Biolegend, San Diego, CA). After the primary antibody, the membrane was probed with a secondary antibody (1:30000 in PBS + 0.1% Tween-20 + BSA 5% buffer), and then the protein was visualized by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK). Step 4: Principal Component Analysis According to the Rockland protocol, anti-RTNKEASI serum (1:1000) was pre-incubated with RTNKEASI peptide (500-fold molar excess) or KDSGNEQTFLPP as a control peptide and then hybridized with the blotted A549 samples for 2 h. The membrane was treated with secondary antibodies and immunoreactive bands were detected by enhanced chemiluminescence. Step 5: Two-dimensional polyacrylamide gel electrophoresis Protein separation of cell lines was performed as described previously. For the first dimension isoelectric focusing, a 7 cm fixed pH gradient (IPG) dry gel strip and a linear pH 4-7 gradient (GE Healthcare) were used. The dissolved protein (50 μg) was placed on the strip, rehydrated, and incubated overnight. The protein was resolved by the PROTEAN IEF system (Bio-Rad) (at 20 ° C, the voltage gradient lower limit was 50 μA) under the following conditions: 4 h at 250 V, linear gradient 8000 V to 15000 V h, fast 8000 V to 75000 V h, and a total voltage of 90 kV h. After balancing in buffer 1 (130 mM DTT, 6 M urea, 20% glycerol, 0.05 M TrisHCl, 2% SDS) and buffer 2 containing iodoacetamide instead of DTT, the first dimension band was positioned on the top of an 8% acrylamide gel (Applichem GmbH, Darmstadt, Germany) for second dimension electrophoresis. The separated proteins were silver stained or blotted and detected with pre-sera or anti-RTNKEASI serum. Step 6: Protein identification by LC-MS / MS Gel spots were treated with DTT (10 mM, 56 °C, 30 min) and iodoacetamide (55 mM, room temperature, 20 min, protected from light). Subsequently, proteins were digested with 13 ng / μl trypsin (sequencing grade modified trypsin, Promega) in 50 mM ammonium bicarbonate at 37 °C for 16 h. Tryptic peptides were extracted in 65% ethyl acetate (ACN) / 5% formic acid (FA), dried under vacuum, and dissolved in 15 μL 0.1% FA. Tryptic peptides were separated on an UltiMate 3000 Rapid Separation Liquid Chromatography (RSLC) system (Dionex, Thermo Scientific, Bremen, Germany) by online electrospray ionization (ESI) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany). Samples were loaded onto the trapping column at a flow rate of 5 μl / min. (Acclaim PepMap μ-precolumn, C18, 300 μm × 5 mm, 5 μm, Thermo Scientific, Bremen, Germany; nanoACQUITY-UPLC Symmetry C18 trapping column, 180 μm × 20 mm, 5 μm, Buffer A: 0.1% FA in HPLC-H2O; Buffer B: 0.1% FA in acetonitrile) containing 2% buffer B. The loaded sample on the capture column was first washed with 2% buffer B (5 μl / min) for 5 min, and then the peptides were eluted (200 nl / min) onto the separation column. The trypsin-digested peptides were separated on a reverse phase C18 column (Acclaim PepMap 100, 75 μm × 25 mm, 2 μm, Thermo Scientific, Bremen, Germany; nanoACQUITY-UPLC column, BEH 130C18, Waters; 75 μm × 250 mm, 1.7 μm, ), flow rate 200 nl / μm, using a binary buffer system consisting of solvent A (0.1% FA, HPLC-H2O) and solvent B (0.1% FA, acetonitrile). Peptides were eluted with a gradient of 2-30% solvent B for 30 min. MS analysis was performed in positive ion mode and programmed acquisition in data-dependent mode (DDA). Full scans were acquired on a Fusion Orbitrap mass analyzer with a resolution of 120,000 FWHM, m / z 200, MS level, and an m / z range of 400 to 1500 (maximum injection time: 50 ms, automatic gain control target: 4e5). Fragmentation was performed with an intensity threshold of 1e4, and fragmented ions were accumulated in a linear ion trap in fast mode. Only +2 to +5 charge states and the most intense precursor ions were selected for fragmentation. The most intense ions were isolated to a target value of 1e4 with a maximum injection time of 50-150 ms. The raw data were processed using Proteome Discoverer v1.4.1.14 (Thermo Scientific), and protein identification was performed using the UniProt FASTA database. Step 7: ALDH and ALDH1A1 ELISA Serum specimens collected from patients with early-stage non-small cell lung cancer (20 cases) and healthy blood donors (20 cases) were stored at -80°C, and then the serum was tested using a specific monoclonal antibody (Bluegene, Biotech, Shanghai, China) according to the instructions of the Global ALDH ELISA Kit (Bluegene, Biotech, Shanghai, China), and the 1A1 subtype was tested using an ALDH1A1 ELISA kit using a specific monoclonal antibody (Cloud-Clone Corp, Houston, Texas, USA). The data are presented as the mean ± standard deviation of three independent experiments. Step 8: Statistics All data are expressed as mean ± standard deviation (SD). The differences in serum levels of ALDH and ALDH1A1 between early-stage NSCLC patients and healthy donors were analyzed. Correlation coefficients were calculated using the Pearson method. Version 22 or GraphPad Prism TM The analysis was performed using the software version 5. A p value < 0.05 was considered statistically significant.

5. The detection method according to claim 2 is characterized in that not only the serum aldehyde dehydrogenase 1A1 subtype can be detected, but also the whole type of aldehyde dehydrogenase can be detected. The detection method is more sensitive and specific, which helps to improve the early diagnosis rate of non-small cell lung cancer, thereby improving the 5-year survival rate of small cell lung cancer and improving the prognosis.