TWI727132B - Biomarkers for lung cancer stem cells - Google Patents

Abstract

Provided herein are methods and kits for analyzing a sample such as a biological sample obtained from a subject having, suspected of having, or being at risk for a cancer to assess presence of cancer stem cells in the sample, which is indicative of poor cancer prognosis.

Description

Biomarkers of lung cancer stem cells

The present invention relates to a biomarker of cancer, and particularly relates to a biomarker of lung cancer stem cells.

Lung cancer is the most common fatal malignant tumor in the world, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Due to the success of driver gene judgment and specific targeted therapy, many patients with lung cancer have shown a good initial response to treatment. However, most patients eventually develop drug resistance and cancer recurrence within a year.

The traditional anti-cancer treatment strategy is to target non-specific types of cancer cells. However, for example, solid tumors, such as lung cancer, are usually organized and contain heterogeneous cell populations. Complex cell-cell interactions that form a tumor microenvironment (or niche) are involved in small cell populations called cancer stem cells (CSCs), which may cause most malignant tumors. Both CSCs and tumor niches play important roles in cancer recurrence, metastasis, and drug resistance.

Therefore, for individuals with cancer (such as lung cancer), especially cancers related to poor prognosis, it is very useful to confirm the biomarker of CSC to develop a reliable and evaluable prognostic method. significance. These biomarkers will also facilitate the study of lung cancer mechanisms and help develop new effective treatment methods for lung cancer.

The present invention discloses the identification of novel biomarkers based at least in part on cancer stem cells (such as lung cancer stem cells), for example, CD14, or a combination of CD14 and CD44. Through the analysis of cells via transcriptomic and proteosome, compared with differentiated cancer cells, the present invention has found that CD14 and CD44 are differentially present in lung cancer stem cells co-cultured with cancer-associated fibroblasts (CAF). Furthermore, the present invention found that CD14 and CD44 are associated with poor prognosis of lung cancer patients. In addition, CD14 is present in various types of cancer cells, including liver cancer, colon cancer, and pancreatic cancer.

Therefore, an aspect disclosed in the present invention provides a method of analyzing a sample, the method comprising: (i) providing a sample suspected of containing cancer stem cells, and (ii) measuring the content of CD14 in the sample, and also selectively measuring The content of CD44.

In some embodiments, the method comprises measuring the content of CD14 protein, the content of CD44 protein, or both. In some examples, the content of membrane-bound CD14 protein and/or the content of membrane-bound CD44 protein in the sample is measured. In other embodiments, the content of circulating (soluble) CD14 and/or the content of circulating (soluble) CD44 in the sample is measured. The content of CD14 and/or CD44 protein can be measured by immunohistochemistry analysis, immunoblotting analysis, ELISA, or flow cytometry analysis.

In some embodiments, the method comprises measuring the content of nucleic acid encoding CD14, the content of nucleic acid encoding CD44, or both. In some embodiments, the nucleic acid encoding CD14 is measured The content of CD44, the content of the nucleic acid encoding CD44, or the content of both are determined by real-time reverse transcription-PCR (RT-PCR) analysis or nucleic acid wafer analysis.

The sample in any of the analysis methods described herein may be a biological sample of a human patient suffering or suspected of having cancer, such as lung cancer (for example, non-small cell lung cancer or NSCLC), liver cancer, colon cancer, or pancreatic cancer. In some embodiments, the biological sample is a tissue sample that can be obtained from a human patient as described herein, for example, from a tumor location or a suspected tumor location. In other embodiments, the biological sample is a liquid biopsy from a human patient as described herein.

Any analysis method described herein may further comprise based on the content of CD14, the content of CD44, or the combination of CD14 and CD44, to determine the cancer stem cells (for example: lung cancer stem cells, liver cancer stem cells, colon stem cells or pancreatic stem cells) in the sample. exist. An increase in CD14 content, an increase in CD44 content, or an increase in CD14 and CD44 content means that cancer stem cells are present in the sample.

Any analysis method described herein, or in addition, may further include determining the survival rate of the human patient from the obtained biological sample. An increase in the content of CD14, an increase in the content of CD44, or an increase in the content of CD14 and CD44 indicates poor survival. In some embodiments, the method further comprises administering a cancer treatment to the human patient, for example, lung cancer (eg, NSCLC) treatment, liver cancer treatment, colon cancer treatment, or pancreatic cancer treatment.

In another aspect, a method for detecting poor prognosis related to cancer is provided, comprising: (i) providing a sample of an individual with cancer, (ii) measuring the content of CD14 in the sample, and optionally the content of CD44, And (iii) based on the content of CD14 in the sample, or the content of both CD14 and CD44, determine whether the individual has a poor prognosis for cancer. If the content of CD14 in this sample If the amount, or the content of both CD14 and CD44 is higher than a predetermined content, it is judged that the individual has a poor prognosis of cancer. In some examples, the cancer can be lung cancer (eg, NSCLC), liver cancer, colon cancer, or pancreatic cancer.

In another aspect, a method for increasing cancer stem cells (as described herein) is provided, the method comprising: (i) providing a sample suspected of containing cancer stem cells, and (ii) isolating CD14 ⁺ cells from the sample and selecting Sexually have CD14 ⁺ /CD44 ^Hi cells.

This document also provides one or more detection reagents for measuring the content of CD14 and selectively using the content of CD44 for the prognosis of an individual's cancer, wherein the prognosis of the cancer is performed by the analysis method described herein.

The details of one or more embodiments of the present invention will be described later. Through the drawings of the present invention, the implementation manners containing several embodiments, and the scope of the appended patent applications, other features or advantages of the present invention will be clear and easy to see.

The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure. By combining the detailed description of the specific embodiments presented herein, a better understanding can be obtained by referring to one or more of these drawings. .

Figure 1 shows that CD14 and CD44 are involved in maintaining the stemness of lung cancer stem cells (LCSCs). A: Through image-based high-throughput analysis of Nanog-positive stem cells in each cell population. CLS1 cells were co-cultured with CAFs or not with CAFs (N=4). The scale bar is 50μm. Top image: photo image; bottom image: quantitative image. B: Differentiation of adipocytes, and positive staining of CLS1/CAFs cells with Oil Red O dye staining oil droplets (above). RT Q-PCR analysis of adipocyte PPARγ and LPL markers in CLS1/CAFs cells was performed at different time points (bottom figure). C: The differentiation of osteoblasts was performed, and the calcium-deposited Alizarin Red S was used for positive staining of CLS1/CAFs cells (above). RT Q-PCR analysis of osteoblast marker alkaline phosphatase and osteocalcin in CLS1/CAFs cells was performed at different time points (bottom figure). D: Determine the different cell numbers (1×10 ⁴ , 1×10 ³ , 1×10 ² and 10 cell numbers) of differentiated CLS1 cells (N≧3 mice) after subcutaneous injection into NSG mice, from CLS1 The incidence of xenograft tumors in mice co-cultured with CAF (N≧3 mice). L-calc limiting dilution analysis software was used to calculate the initial tumor frequency (TIF) of CSCs. CI: Confidence interval. E: Schematic diagram of cancer stem cell marker screening strategy. CSCs/CAFs and differentiated cancer cells (CLS1 p.6) were subjected to Affymetrix chip and membrane proteomic analysis to determine the high expression surface proteins of CSCs/CAF. Through the published cohort, the candidate marker is related to the survival rate of the patient. F: Patients with CD44 and CD14 high or low performance (cutoff value = median risk score). The results showed a significant difference in the Kaplan-Meier test estimated non-relapse-survival rate between high-performance or low-performance groups. The P value was obtained through two-sided log-rank tests. The patient was divided into three groups: high CD44/high CD14 group, low CD44/low CD14 group and other groups. This result shows a significant difference in the overall and non-relapse survival rates estimated by Kaplan-Meier test. The P value obtained from a two-sided logarithmic level test.

Figure 2 shows the clinical significance of CD44 and CD14 in cancer cells of NSCLC patients in the first stage. A: IHC staining of CD44 and CD14 of cancer cells from 80 patients with primary tumor samples collected from clinical cohort with first-stage NSCLC surgical resection. The images were obtained from different patients with low CD44 (score<median risk score) and high (score≧median risk score) in cancer cells (original magnification, 100x). Or, the image is the negative group (score=0) and the positive group (score>0) of CD14 in cancer cells (original magnification, 100x) obtained from different patients. B and C: Patients with CD44 high or low performance (cutoff value = median risk Score); negative and positive CD14 performance. This result shows a significant difference in the overall (B) and non-recurrence survival rate (C) estimated by the Kaplan-Meier test. The P value obtained from a two-sided logarithmic level test. By combining the expression levels of CD44 and CD14 in cancer cells, the patients were divided into three groups: high CD44/positive CD14 group, low CD44/negative CD14 group, and other groups. This result shows a significant difference in the overall and non-relapse survival rates estimated by the Kaplan-Meier test. The P value obtained from a two-sided logarithmic level test.

Figure 3 shows the enrichment of CD44 and CD14 on the surface of cancer cells co-cultured with CAFs feeder cells. A: RT Q-PCR verification of CD44 and CD14. CLS1 cells are derived from CLS1 spheres, broken down into individual cells and then subcultured with different passage times (p3, p6 and p14) without CAF. CAF serves as a feeder cell control group. The data represents the mean ± SEM (N=3). B: Analysis of CD44, CD14, and Nanog in cancer cells (EKVX) by RT Q-PCR. The cancer cells were co-cultured with CAFs (N=4) obtained from patients or without CAFs. The results show that there are significant differences in the cancer cells co-cultured with NFs or CAFs (L2, L5 and L8) of different patients in the MANOVA test. C: The protein expression of CD44 and CD14 in CLS1 cells cultured with (CLS1/CAF) or without CAFs (CLS1p.6) was evaluated by immunofluorescence staining (left picture) and Western blot analysis (right picture). D: Perform RT Q-PCR analysis to evaluate the dryness markers Nanog and Oct3 ^{of the CD14 +} CD44 ^Hi , CD14 ^- CD44 ^Hi, and CD14 ^- CD44 ^Low populations sorted from primary lung cancer cells (CL152 cells) (N=3) /4 performance. ^{E: CD14 +} CD44 ^Hi , CD14 ^- CD44 ^Hi and CD14 ^- sorted from primary lung cancer cells (CL152 cells) in MCDB201 medium containing EGF (20ng/ml) and bFGF (20ng/ml) for 21 days The ball formation ability of the CD44 ^Low group (below) and style (above). The scale bar is 100 μm. F: Perform subcutaneous injection of different cell numbers (1×10 ² and 10 cell numbers) into NSG mice to detect CD14 ⁺ CD44 ^Hi , CD14 ^- CD44 ^Hi and CD14 ^- CD44 sorted from CIS1 lung cancer cells (N≧3) The incidence of tumors in xenograft mice in the ^{Low population.} L-calclimiting dilution analysis was used to calculate the initial tumor frequency (TIF) of CSCs. CI, confidence interval.

Figure 4 shows that the CD14 ⁺ CD44 ^Hi group sorted from primary lung cancer cells has a higher tumor initial capacity. Above: Flow cytometric analysis of double staining for CD44 and CD14 in different primary lung cancer cells: CL100 (A), CL141 (B), and CL152 (C). Different primary lung cancer cells showed a variable double-positive population (5.8% for CL100, 0.2% for CL141, and 3.5% for CL152). ^{Bottom: The incidence of tumors in xenograft mice in the CD14 +} CD44 ^Hi , CD44 ^Hi CD14 ^- and CD14 ^- CD44 ^Low groups sorted from primary lung cancer cells: subcutaneous injection of different cell numbers (1×10 ⁴ , 1×10 ³ and 1×10 ² cells) to SCID mice (N=6), and judge CL100 (D), CL141 (E), and CL152 (F). L-calclimiting dilution analysis was used to calculate the initial tumor frequency (TIF) of CSCs. CI, confidence interval.

Figure 5 shows the enrichment of CD44, CD14 and Nanog on the surface of cancer cells co-cultured with CAFs feeder cells. A: Cancer cells co-cultured with or without CAFs obtained from patients were analyzed for CD44, CD14 and Nanog performance by laser extraction of colony cancer cells (A549) (N=3 patients). B: CD44 and CD14 in CLS1 cells cultured with or without CAFs feeding cells were analyzed by flow cytometry.

Figure 6 shows the flow cytometric analysis of CD44 and CD14 on the surface of cancer cells by flow cytometry. The CD44 and CD14 double staining positive groups were analyzed in different lung cancer cell lines (A549, EKVX, PC9 and HCC827) and primary lung cancer cells (CL25, CL83, CL97, CL100, CL141 and CL152).

Cancer stem cells (CSC) represent a subset of tumor stem cell-like and multifunctional cells in tumors. CSCs have the ability to self-renew, differentiate into specific cell types, and/or develop into cancer. The phenotype of "cancer stemness" may be the driving force of cancer. CSCs are thought to be related to resistance and/or metastasis to chemotherapy or radiotherapy. More and more evidences show that CSCs exist in leukemia and in various solid tumors, including lung cancer. In order to maintain "stemness", most stem cells rely on direct contact in the microenvironment or an interaction mode with, for example, fibroblasts as "feeding cells". In the tumor microenvironment, CSCs can rely on direct contact with cancer-associated fibroblasts (CAF) to maintain dryness. This interaction can create a niche that is conducive to tumor growth and/or metastasis.

The present invention discloses identification based at least in part on markers, through analysis of transcripts (cells via transcriptomic) and proteosome, including CD14 and CD44, which are highly expressed in CAF co-cultured CSC cells. For example, CD14 is defined as a novel biomarker for CSCs (such as lung cancer, liver cancer, colon cancer, or pancreatic cancer CSCs). It was judged that CSCs showed higher levels of cell surface protein biomarkers (such as CD14 and CD44) than differentiated cancer cells. Therefore, the content of the protein biomarkers (such as CD14 and CD44) is related to the presence and/or content of CSCs, and therefore the prognosis of cancer patients from which the tumor sample is derived is poor.

Therefore, some aspects disclosed in the present invention provide methods for analyzing samples based on the content of CD14 and also selective CD44 content (membrane-bound or circulating molecules (soluble CD14 or soluble CD44)). The sample is, for example, a suspected A sample of cancer stem cells. The analysis method can be used for clinical purposes, such as: determining the presence of cancer stem cells in a sample, which can indicate poor prognosis of cancer, selecting candidate markers for treatment based on the presence/content of cancer stem cells, monitoring cancer progress, and evaluating anti-cancer treatments Effect, confirm the treatment process, evaluate whether the individual is in cancer The risk of recurrence. The analytical methods described herein can also be used in non-clinical applications, for example, for research purposes, including, for example, studying the mechanism of cancer development and metastasis and/or biological pathways/processes related to cancer, and developing new cancer treatments based on the research.

Analytical methods for determining cancer stem cell (CSC) markers

This article provides methods for analyzing samples to confirm the presence and/or content of biomarkers related to cancer stem cells.

(i) CSC biomarker

The term "biomarker" or "biomarker group" as used herein refers to a specific cell population (such as CSC s) refers to the biomolecule (such as: protein) or the content of the biomolecule group in a specific cell population, or It is derived from the content of the same molecule in different cell groups. For example, a biomarker representing CSCs may have a reduced content or an increased content in CSCs relative to the content of the same marker in differentiated cancer cells of the same type or non-cancer cells. This article describes the content of CSCs biomarkers that can be contained in CSCs, and the content of the same marker (up or down) in differentiated cancer cells derived from the same type or non-cancer cells is at least 20% (eg: 30%, 30%, 50%, 80%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times or more). This biomarker/biomarker panel can be used for diagnostic/prognostic applications as well as non-clinical applications (e.g., for research purposes).

In some examples, the biomarker includes CD14. CD14 is a glycoprotein that is a co-receptor of bacterial lipopolysaccharide (LPS) and binds to LPS in the presence of lipopolysaccharide binding protein (LBP). There are two types of CD14, the membrane-bound type (mCD14) and the soluble type (sCD14). Both types can be used as biomarkers of cancer stem cells. Human mCD14 and sCD14 The peptide sequence is provided by GenBank accession number NP_001167575.0, PDB: 4GLP_A, UniGene: Hs.163867, and GeneCards GCID: GC05M14059.

In addition, the biomarker may include CD44. CD44 is a cell-surface glycoprotein system involved in cell-cell interaction, cell adhesion and migration. CD44 is a receptor for hyaluronic acid and can also interact with other ligands such as osteopontin, collagen, and matrix metalloproteins (MMPs). The amino acid sequence of the human CD44 is provided by GenBank accession number ACI46596.1.

In addition to CD14 and CD44, CP and ABCA8 in cancer stem cells were found to be different from CAFs, so they are also used as markers in the analysis methods described herein.

Exemplary biomarkers representing cancer stem cells (such as lung cancer stem cells, liver cancer stem cells, colon cancer stem cells, or pancreatic cancer stem cells) are provided in Table 1.

Any of the CSC biomarkers described herein, whether alone or in combination (such as CD14, or a combination of CD14 and CD44), can be used in the analysis method of samples suspected of containing lung cancer stem cells as described herein. The results obtained from this analysis method can be used for both clinical and non-clinical applications, including but not limited to those described herein.

(ii) Analysis of biological samples

Any sample containing cancer stem cells (for example, lung cancer stem cells, liver cancer stem cells, colon cancer stem cells, or pancreatic cancer stem cells) can be analyzed by analysis methods known in the art and/or herein. The method described herein involves a method of providing a sample suspected of containing lung cancer stem cells. In some examples, the sample can be a sample analyzed in vitro , for example, to study the behavior and/or mechanism of CSC cell culture in vitro. In some examples, the sample through the analysis method described herein may be a biological sample. As used herein, "biological sample" refers to a composition containing tissue, such as blood, plasma or protein from an individual. The biological sample can be an initial unprocessed sample from an individual or a subsequent processed sample, such as a partially purified or preserved type. In some embodiments, the biological sample may be a body fluid sample, for example, serum, plasma, tears, urine, or saliva nourishment. Alternatively, the biological sample may be a tissue sample, for example, a tissue sample obtained from a tumor location or a suspected tumor location (a tissue location suspected of containing cancer cells). In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or more) biological samples may be collected from an individual over time or at specific intervals, for example to evaluate disease progression or evaluate the effect of treatment.

A biological sample refers to that it can be obtained from an individual using any method known in the art. For example, a sample (such as a tumor tissue sample) obtained from an individual through removal, such as a biological sample obtained through a surgical operation (such as a thoracotomy), a needle aspiration, or a biopsy procedure through thoracentesis (Including, blood and pleural or abdominal effusion).

The terms "patient," "individual," or "individual" can be used interchangeably and refer to individuals who need to perform the analysis described in this article. In some embodiments, the individual is a human or non-human mammal. In some embodiments, human individuals who are suspected of having or are at high risk of cancer, such as lung cancer, colon cancer, liver cancer, or pancreatic cancer. The individual may exhibit one or more cancer-related symptom. In addition, the individual may have one or more cancer risk factors, for example, environmental factors or genetic factors related to cancer (such as exposure to pollution).

Alternatively, the individual in need of the analysis described herein may be a patient suffering from cancer, such as lung cancer, colon cancer, liver cancer, or pancreatic cancer. The individual may currently relapse, or may have had a disease in the past (e.g., does not currently relapse). In some examples, the individual may be a human patient for cancer treatment, for example, treatment involving surgery, chemotherapy, immunotherapy, or radiation therapy. In some cases, the human patient may not be treated.

In some examples, the cancer is lung cancer. Examples of lung cancer include, but are not limited to, non-small cell lung cancer (NSCLC), lung adenocarcinoma, adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), squamous cell carcinoma, large cell carcinoma, and large cell carcinoma. Cellular neuroendocrine tumors, small cell lung cancer (SCLC), mesothelioma, and carcinoid tumors.

Analyzing any of the samples described herein using the analytical methods described herein involves measuring the content of one or more CSC biomarkers described herein. In some examples, the biomarker is CD14 (membrane-bound or soluble), or CD44 (membrane-bound or soluble). In other examples, the biomarker is a combination of CD14 and CD44. The content (eg, amount) of the biomarker disclosed herein, or the change in the content of the biomarker, can be assessed using general analysis or the methods described herein.

The term "measurement" or "measurement", or "detection" or "detection" is used in this article to indicate the presence, absence, number, or amount of a substance in a sample (which can be an effective amount), including the qualitative or quantitative nature of the substance The derivation of the content of the concentration, or other methods to evaluate the value or classification of a body.

In some embodiments, the content of the biomarker can be assessed or measured by directly detecting the protein in a sample, such as a biological sample. Or in addition, through biological samples Then assess or measure the protein content, for example, by measuring the protein activity content (such as enzyme analysis).

The protein content (CD14 and/or CD44) can be measured using immunoassay. Examples of immunoassays include, but are not limited to, immunoblotting analysis (such as Western blotting), immunohistochemical analysis, flow cytometry analysis, immunofluorescence analysis (IF), enzyme-linked immunosorbent analysis assays, ELSIAs) (such as sandwich ELSAs), radioimmunoassays, electrochemiluminescence-based detection analysis, magnetic immunoassays, lateral flow assays, and related technologies. For those skilled in the art, other suitable immunoassays for detecting the provided biomarkers are obvious.

The immunoassay involves the use of reagents (such as antibodies) that are specific to target biomarkers, such as CD14 or CD44. For example, detection reagents for antibodies that "specifically bind" to target biomarkers are terms understood in the technical field, and methods for such specific binding are also known in the technical field. If an antibody interacts with a specific target biomarker more frequently, more rapidly, with a longer duration, and/or with greater affinity than other biomarkers, it is said to exhibit "specific binding". It can also be understood by reading this definition that, for example, an antibody that specifically binds to a first target peptide may or may not specifically or preferentially bind to a second target peptide. Therefore, "specific integration" or "priority integration" does not necessarily require (although it can include) specific integration. Generally speaking, but not necessarily, the reference to combination means preferential combination. In some instances, an antibody that "specifically binds" to a target peptide or epitope thereof cannot bind to other peptides or other epitopes in the same antigen.

The term "antibody" as used herein refers to a protein comprising at least one immunoglobulin variable region or immunoglobulin variable region sequence. For example, the antibody may comprise a heavy (H) chain variable region (abbreviated herein as V _H), and light (L) chain variable region (abbreviated herein as V _L). In other examples, the antibody contains two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" includes antigen-binding fragments of antibodies (such as single-chain antibodies, Fab and sFab fragments, F(ab') ₂ , Fd fragments, Fv fragments, scFv, and regional antibody (dAb) fragments (de Wildt et al. al., Eur J Immunol. 1996; 26 (3):629-39.)) and complete antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes). Antibodies can be from any source, but primates (human and non-human primates) and primatization are preferred.

In some embodiments, the antibodies described herein can bind to a detectable label, and the binding of the detection reagent to the peptide of interest can be determined based on the intensity of the signal released from the detectable label, or a second specific detection reagent is used. Antibody. One or more antibodies can be conjugated to a detectable label. Any suitable label known in the art of the present invention can be used in the assay methods described herein. In some embodiments, the detectable label comprises a fluorophore. As used herein, the term "fluorescent group" (also "fluorescent label" or "fluorescent dye") refers to a functional group that absorbs light energy at a specific excitation wavelength and emits light energy at different wavelengths. In some embodiments, the functional group to be detected and/or comprises an enzyme. In some embodiments, the enzyme produces a colored product from a colorless substrate (eg, β-galactosidase).

In some examples, the analysis methods described herein are applied to measure the content of cell surface biomarkers, for example, the sample contains membrane-bound CD14 and/or CD44. The cells can be collected by routine operations and the content of cell surface biomarkers can be measured by routine methods, such as FACS.

In other examples, the analysis method described herein is applied to measure the content of circulating biomarkers (soluble biomarkers), such as CD14 in a sample, which can be a blood sample or Plasma sample. Any analysis known in the art, such as immunoassay, can be used to measure the content of the biomarker.

It will be understood in the art that the present disclosure is not limited to immunoassays. Detection and analysis are not based on antibodies, such as mass spectrometry, which is also used for the detection and/or quantification of lung CSC biomarkers provided herein. Chromogenic substrate analysis can also be used for the detection and/or quantification of lung CSC biomarkers.

On the other hand, the amount of nucleic acid that can encode CSC biomarkers in a sample can be measured by routine methods. In some embodiments, measuring the expression level of a nucleic acid encoding a CSC biomarker comprises measuring mRNA. In some embodiments, the expression level of mRNA encoding CSC biomarkers can be measured by real-time reverse transcription (RT) Q-PCR analysis or nucleic acid wafer measurement. Methods for detecting nucleic acid sequences of biomarkers include, but are not limited to, polymerase chain reaction (PCR), reverse green-PCR (RT-PCR), in situ PCR, quantitative PCR (Q-PCR), real-time quantitative PCR (RT QPCR) ), in situ hybridization, southern blotting method, northern blotting method, sequence analysis, chip analysis, detection of reporter genes, or other DNA/RNA hybridization platforms.

Any binding reagent that specifically binds to the desired biomarker can use the methods and kits described herein to measure the content of the biomarker in the sample. In some embodiments, the binding reagent is an antibody or aptamer that specifically binds to the desired protein biomarker. In other embodiments, the binding reagent may be one or more oligonucleotides complementary to the encoding nucleic acid or its protein. In some embodiments, the sample can be contacted with more than one binding reagent that binds to different protein markers simultaneously or sequentially (eg, multiplex analysis).

To measure the content of the target biomarker, the sample can be contacted with the binding reagent (detection reagent) under suitable conditions. Generally, the term "contact" refers to the exposure of the sample or cells collected from it to the nodule Combine the reagent for an appropriate and sufficient time to form a complex between the binding reagent and the target biomarker in the sample. In some embodiments, the contact is via capillary action, and the sample moves across the surface of the supporting membrane.

In some embodiments, the analysis can be performed on a low-throughput platform, including a single analysis format. For example, a low-throughput platform can be used to measure the presence and amount of protein in biological samples (such as biological tissues, tissue extracts) for use in diagnostic methods, monitoring disease and/or treatment progress, and/or predicting disease or Illnesses can benefit from specific treatments.

In some embodiments, it may be necessary to fix the bonding agent to the support member. Considerations for the method of immobilizing the binding reagent, such as the nature of the binding reagent and the material of the supporting member, may require a specific buffer. This method is obvious to those skilled in the art. For example, the set of biomarkers in a biological sample as described herein can be measured using any set and/or detection device as described herein.

The type of detection used for the detection and/or quantification of cancer stem cell biomarkers described herein will depend on specific conditions (such as clinical or research applications), and will depend on the type and quantity of biomarkers to be tested, and/or depending on the use It depends on the type and quantity of patient samples.

The analysis method described herein can be used for both clinical and non-clinical purposes. This article provides some examples.

Diagnosis and/or prognostic application

Measuring the content of one or more CSC biomarkers in a biological sample from an individual via the analysis method described herein can be used for various clinical purposes, for example, to detect cancer cells, especially from individuals (such as human patients). The lung cancer stem cells in the sample are judged as individuals with poor prognosis of cancer based on disease development, prognostic results and/or the effects of current treatments. Monitor the progress of the individual's cancer development, evaluate the effect of the individual's cancer treatment, determine the specific treatment suitable for the patient, anticipate the individual's cancer recurrence, and/or adjust the cancer treatment. Therefore, based on one or more of the CSC biomarkers described herein, the cancer treatment and prognosis methods described herein, for example, cancers related to poor prognosis, such as CD14 or a combination of CD14 and CD44. Exemplary cancer types include lung cancer (eg, NSCLC as described herein), liver cancer, colon cancer, and pancreatic cancer.

When necessary, the content of the biomarker in the sample confirmed by the analysis method described herein can be normalized in the internal control group or standard sample (with the preset amount of the biomarker) in the same sample to obtain a standardized value. Both the original value or the standardized value of the biomarker can then be compared to the value of the reference sample or the control sample. The increase in the value of biomarkers in samples obtained from individuals indicates the presence of CSCs in the samples. Individuals with CSCs indicate that the individual has the target cancer described herein, for example, it is related to the poor prognosis of the cancer or the risk of cancer discovery.

In some embodiments, the content of the biomarker in the sample obtained from the individual is compared with the preset threshold value of the biomarker. An increase from this value may indicate that the individual has CSCs and therefore may have the target cancer, such as: The target cancer has a poor prognosis or risk related to cancer development and/or metastasis.

The control sample or reference sample may be a biological sample obtained from a healthy individual, which may be the same race, age, and/or gender as the sample obtained for analyzing the individual. Alternatively, the control sample or reference sample contains known amounts of biomarkers to be evaluated. In some embodiments, the control sample or reference sample is a biological sample obtained from a control individual.

As used herein, control individuals are healthy individuals, such as individuals who clearly do not contain target cancers (such as lung cancer, liver cancer, colon cancer, or pancreatic cancer, such as NSCLC) or have no history of disease when measuring protein content . The control group individuals can also represent a group of healthy individuals, which preferably have characteristics (such as age, gender, race) that are consistent with the analysis of the individuals through the analysis methods described herein.

The content of the control group can be a preset content or threshold. The preset content may represent the protein content in a group of individuals who do not suffer from the target disease or are not at risk of the target disease (for example, the average content of a healthy individual group). It can also mean the amount of protein in a population of individuals suffering from the target disease.

The preset content can take various forms. For example, a single cut-off value, such as the median or average. In some embodiments, the preset content may be established based on comparison groups, for example, one of the determined groups is known to have the target cancer and the other determined group is not known to have the target cancer. Alternatively, the preset content can be a range, for example, representing the range of protein content in the control group.

In some examples, the predetermined content may be a median risk score related to the target cancer described herein. The median risk score is a reference point that is known to represent one or more biomarkers and is used to distinguish high-risk groups from low-risk groups. It also indicates that the high-risk of a particular disease versus the low-risk (such as cancer occurrence, cancer Prognosis, or treatment effect). See, for example, Chen et al., N. Engl. J. Med. 356(1): 11-20 (2007). It is used as the cut-off value in the diagnosis/prognosis methods described herein. In some examples, the median risk score is identified below. The content of the biomarker can be confirmed in healthy patients (patients without the target disease) and patients with the target disease (such as cancer as described herein). The frequency distribution of biomarker content in test patients can be plotted to confirm each patient's risk score, which represents the risk profile of the test patient related to the target disease. The risk score is then used to classify patients into high-risk or low-risk groups. To avoid the influence of extreme values in the training data set and equal in the two groups (high to low risk group) setting the number of patients in the 50 ^th percentile (median risk score) may be used as the cutoff value, which can be It is the default value of the method used in this article.

The content of the control group described herein was confirmed by routine techniques. In some examples, in the control sample described herein, the content of the control group is obtained by a general method (eg, the same analysis for obtaining the protein content of the test sample as described herein). In other examples, the protein content can be obtained from the members of the control group, and the results of the calculation program are analyzed to obtain the control group content (preset content) representing the protein content in the control group.

Comparing the content of the biomarker in the sample obtained from the candidate individual with the reference value described herein can confirm whether the candidate individual has a target cancer risk or is related to a poor prognosis. For example, if the content of the biomarker in the sample of the candidate individual is increased compared to the reference value, the candidate individual can be determined to have a target cancer or risk related to a poor prognosis. When the value range of the biomarker content in the individual population represented by the reference value is that it has cancer stem cells and/or has a poor prognosis of cancer, the value of the biomarker in the candidate's sample falls within this range, which indicates that the candidate has and the prognosis Undesirable related target cancer or risk.

The use of "increase in content" or "content above the reference value" in this article means that the content of the biomarker is higher than the reference value, such as a predetermined threshold of the content of the biomarker in the control sample. The content of the control group described herein. The increased biomarker content includes biomarker content, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150 %, 200%, 300%, 400%, 500% or higher than the reference value. In some embodiments, the biomarker content in the test sample is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200, 300, 400, 500, 1000, 10000-fold or higher than the biomarker content in the reference sample.

In some examples, the biomarker includes CD14. Compared with samples of the same type of control individuals, samples obtained in candidate individuals such as human patients have the presence of CD14 (membrane-bound and/or soluble CD14) or increase the content of CD14 in the sample, indicating that the candidate The presence of cancer stem cells in an individual also indicates a poor prognosis for cancer.

In some examples, the biomarker may further include CD44. The sample obtained from the candidate individual has a high expression level of CD14, and optionally combined with the increase in the content of CD14 as described herein, indicates the presence of cancer stem cells in the candidate individual, which also indicates that the prognosis of the cancer is poor. In some examples, the CD44 high expression volume refers to the expression volume higher than the preset content, for example, not less than the median risk score corresponding to the target cancer.

In some embodiments, the candidate individual is a patient suffering from symptoms of a target cancer (eg, lung cancer, liver cancer, colon cancer, or pancreatic cancer, such as NSCLC). For example, suffering from fatigue, cough, shortness of breath, chest pain, loss of appetite, weight loss, hoarseness, hemoptysis, chronic bronchitis, chronic pneumonia, wheezing, or a combination thereof. In other embodiments, the individual did not have the target cancer symptoms, did not have a history of the target cancer symptoms, or did not have a history of the target cancer when the sample was collected. In other embodiments, the individual is resistant to chemotherapy, radiation therapy, immunotherapy, or a combination thereof.

The individual judged in the method described herein may be given appropriate treatment, such as the chemotherapy described herein, because of cancer stem cells of the target cancer or suffering from the target cancer (such as: lung cancer related to poor prognosis).

The analysis methods and kits described herein can be applied to evaluate, for example, the therapeutic effect of the target cancer described herein, considering the correlation between the content of biomarkers and the cancer. For example, various biological samples (such as tissues) can be collected from individuals before and after treatment or during treatment. Samples or body fluid samples). The content of the biomarker is measured via any of the analysis methods described herein, and thus the value (e.g., amount) of the biomarker is detected. For example, if the increased biomarker content represents an individual with lung cancer, and the biomarker content decreases after or during the treatment (the biomarker content in the sample collected later is compared to the biomarker content in the sample collected earlier), It represents the effect of treatment. In some examples, the treatment involves an effective amount of a therapeutic agent, such as a chemotherapeutic agent. Examples of the chemotherapeutic agent include, but are not limited to, carboplatin (Paraplatin) or cisplatin (Platinol), docetaxel (Docefrez, Taxotere), gemcitabine (Gemzar), albumin-bound paclitaxel (Abraxane), Paclitaxel (Taxol), Pemetrexed (Alimta), and Vinorelbine (Navelbine).

If the individual judges that there is no response to the treatment, a higher dose and/or frequency dose of the therapeutic agent is given to the judged individual. In some embodiments, the dosage or frequency dosage of the therapeutic agent is maintained, reduced, or stopped. Alternatively, different treatments can be applied to individuals who are found not to respond to the first treatment.

In other embodiments, the value of the biomarker or biomarker group may also depend on the target cancers described herein that are judged to be treatable, such as chemotherapeutic agents. In order to carry out this method, the biomarker content is measured in samples collected from individuals with target cancers (eg tissue samples or body fluid samples) via appropriate methods, such as those described herein, such as Western blots or RT Q-PCR analysis. If the biomarker content is higher than the reference value, it indicates that the chemotherapeutic agent may be effective in treating the disease. If the disease is judged to be sensitive to (treatable) chemotherapeutic agents, the method may further comprise administering to the individual suffering from the disease an effective dose of chemotherapeutic agents, such as carboplatin (Paraplatin) or cisplatin (Platinol), Sitaxel (Docefrez, Taxotere), gemcitabine (Gemzar), albumin-bound paclitaxel (Abraxane), paclitaxel (Taxol), pemetrexed (Alimta), and vinorelbine (Navelbine).

Likewise, methods for assessing the severity of target cancers as described herein are within the scope disclosed by the present invention. For example, as described herein, the target cancer can be in a quiescent state (remission), during which the individual will not experience symptoms of the disease. For example, recurrence of lung cancer is usually a recurrent episode, where the individual may experience lung cancer symptoms including, but not limited to, fatigue, cough, shortness of breath, chest pain, loss of appetite, weight loss, hoarseness, hemoptysis, chronic bronchitis, chronic pneumonia, Wheezing, or a combination thereof. In other embodiments, the content of one or more biomarkers indicates whether the individual will experience, or will soon experience, the recurrence of the target cancer (eg, lung cancer recurrence). In some embodiments, the method involves comparing the biomarker content in a sample from the same individual with the biomarker content in a sample obtained from an individual with the target cancer, such as a sample obtained from the same individual in remission or during relapse A sample obtained from the same individual at the time.

Non-clinical applications

Furthermore, the content of any CSC biomarker described herein can be applied to non-clinical purposes, for example for research purposes. In some embodiments, the methods described herein can be applied to the behavior and/or mechanism of cancer stem cells (eg, discovery of novel biological pathways or processes involving cancer stem cells that are involved in cancer development and/or metastasis).

In some embodiments, the content of the biomarker panel as described herein can be used for the development of new treatments for lung cancer. For example, measuring the biomarker content of samples obtained from individuals given new treatments (eg, clinical trials). In some embodiments, the content of the biomarker group may indicate the effect of the new treatment or development of the individual's cancer before, during, or after the new treatment.

Furthermore, one or more CSC biomarkers described herein can be used to increase cancer stem cells, which can be used for various purposes, including cancer biology research and the development of new anti-cancer agents specific to target cancer stem cells. In this article, the term "increasing lung cancer stem cells" refers to lung cancer stem cells isolated or separated from a biological sample, so that the lung cancer stem cells in the biological sample are completely free of other substances. In some embodiments, increasing cancer stem cells comprises separating lung cancer stem cells from non-stem cells.

Set and detection device for measuring protein biomarkers

Also provided herein are kits and detection devices for measuring the content of one or more of the biomarkers described herein. The kit or detection device may include one or more binding reagents that specifically bind to the target biomarker, as listed in Table 1. For example, the kit or detection device may include at least one binding reagent selected from Table 1 that is specific to a protein biomarker. In some examples, the kit or detection device includes binding reagents that are specific to two or more protein biomarker members described herein.

In some embodiments, one or more binding reagents (such as detection reagents) may be antibodies that specifically bind to the protein biomarker panel. In some embodiments, one or more binding reagents are aptamers, such as peptide aptamers or oligonucleotide aptamers of a specific protein binding biomarker set.

In some embodiments, the kit further includes a detection reagent (eg, an antibody that binds to the binding reagent) for detecting the reagent that binds to the protein biomarker set. The detection reagent can be bound to the label. In some embodiments, the detection reagent is an antibody that specifically binds to at least one binding reagent. In some embodiments, the binding reagent includes a tag that is judged to be a tag, and is directly or indirectly bound by the detection reagent.

In the kit or detection device, one or more binding reagents can be fixed on a supporting member, such as a membrane, a bead, a glass slide, or a porous disk. Choosing an appropriate support member for analysis will take into consideration various factors such as the number of samples and the detection method of the release signal of the label attached to the reagent.

In some embodiments, the support member is a membrane, such as a nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane, or a cellulose acetate membrane. In some examples, the analysis can be a Western blot analysis format or a nucleic acid chip analysis format.

In some embodiments, the support member is a porous disk, such as an ELISA disk. In some embodiments, the immunoassay described herein can be performed on a high-throughput platform. In some embodiments, multi-well discs for high-throughput immunoassays, such as 24-, 48-, 96-, 384- or more multi-well discs can be used. Each immunoassay can be performed in parallel in each hole. Therefore, it is often necessary to use a disc reader to measure the porosity that increases the analytical throughput in parallel. In some embodiments, the disc reader can be used for parallel multi-hole (eg, 4, 16, 24, 48, 96, 384, or more multi-hole) imaging of the platform. For example, commercially available disc readers (e.g. disc vision systems are available from Perkin Elmer, Waltham, MA). The disc reader can be powered-based fluorescent analysis. The disc vision system has high optical collection efficiency and special optics for parallel 96-well analysis. Other suitable parallel disk readers include but are not limited to SAFIRE (Tecan, San Jose, CA), FLIPRTETRA ^® (Molecular Devices, Union City, CA), FDSS7000 (Hamamatsu, Bridgewater, NJ) and CellLux (Perkin Elmer, Waltham) , MA).

The kit may also include one or more buffers as described herein but not limited to coating buffer, blocking buffer, washing buffer, and/or stop buffer.

In some embodiments, the kit may include an operating manual according to any method described herein. The included operating manual may contain instructions on how to use the components in the kit to measure the biomarker set (such as protein or nucleic acid) in an individual biological sample, such as a human patient.

Operating manuals on how the kits are used usually contain information on the amount of each component and the appropriate conditions for performing the analytical methods described herein. The components in the kit can be unit doses, bulk packages (eg, multi-dose packages), or sub-unit doses. The operating manuals provided in the kits disclosed in this article are usually written operating manuals in the labeling or packaging instructions (such as the paper included in the set), but the machine operating manuals that can be read (such as on magnetic or optical storage CDs) It is also acceptable.

The label or packaging instructions of the kit indicate that it is used to assess the content of the biomarker kit, and an operation manual can be provided to perform any of the methods described herein.

The kit of the present invention has suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (such as sealed Mylar or plastic bags) and the like. At the same time, a package that can be combined with a specific device such as a PCR machine, nucleic acid analysis, or flow cytometry system is also considered.

Sets usually provide other components such as explanatory information, controls, and/or standards or reference samples. Usually, the set includes the container and the marking or packaging instructions on or related to the container. In some embodiments, the present invention provides documents containing the contents of the kits described herein.

Cancer treatment

Judging by the methods described herein, individuals who are at high risk or suffering from lung cancer described herein (eg, lung cancer, liver cancer, colon cancer, or pancreatic cancer related to poor prognosis) can be treated with any suitable therapeutic agent. In some embodiments, the provided method includes the treatment selected by the individual based on the results produced by the method, such as measuring the content of the biomarker panel.

In some embodiments, the method includes selecting one or two therapeutic agents, such as chemotherapy, radiotherapy, surgical treatment, and/or immunotherapy, based on analysis results such as biomarker detection.

In some embodiments, the individual is administered one or more therapeutic agents. The therapeutic agent can be administered with other treatments as part of a combination therapy for the treatment of lung cancer, such as chemotherapy, radiation therapy, surgical treatment and/or immunotherapy. Combination therapy such as chemotherapy and radiation therapy can be provided in many different ways. The first treatment can be given before or after other treatments are given. In some cases, the first treatment or other treatments (such as therapeutic agents) are administered at the same time or close in time (such as short time intervals between treatments, such as during the same treatment course). The first agent and other treatments can also be given at longer intervals.

In some embodiments, chemotherapy is administered to the individual. For example, examples of chemotherapeutic agents include, but are not limited to, carboplatin or cisplatin, docetaxel, gemcitabine, albumin-bound paclitaxel, paclitaxel, pemetrexed, and vinorelbine.

In some embodiments, radiation therapy is administered to the individual. Radiation therapy includes, but is not limited to, ionizing radiation, gamma radiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, whole body radioactive isotopes, and radiosensitizers.

In some embodiments, surgical treatment is given to the individual. Surgical treatments include, but are not limited to, lobectomy, wedge resection, segmentectomy, and pneumonectomy.

In some embodiments, an immunotherapeutic agent is administered to the individual. In some embodiments, the immunotherapeutic agent is a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the immunotherapeutic agent is Nivolumab. In some embodiments, the immunotherapeutic agent is Pembrolizumab.

Other examples of chemotherapy include, but are not limited to, molybdates such as Carboplatin, Oxaliplatin, Cisplatin, Nedaplatin, Satraplatin, Lopi Platinum (Lobaplatin), adenosine triphosphate (Triplatin), tetranitrate (Tetranitrate), picoplatin (Picoplatin), Prolindac, Aroplatin and other derivatives; Topoisomerase I inhibitor (Topoisomerase I) inhibitors), such as camptothecin, topotecan, irinotecan/SN38, rubitecan, Belotecan and other derivatives; topoisomerase Topoisomerase II inhibitors, such as: Etoposide (VP-16), Daunorubicin, doxorubicin reagents (such as: doxorubicin in liposomes, Doxorubicin HCl, doxorubicin analogs, or doxorubicin and its salts or analogs in), Mitoxantrone (Mitoxantrone), Aclarubicin (Aclarubicin), Epirubicin (Epirubicin), Idarubicin, Amrubicin, Amsacrine, Pirarubicin, Valrubicin, Zorubicin, Teniposide (Teniposide) and other derivatives; Antimetabolites, such as Folic family (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin) (Aminopterin and related substances); Purine antagonists (Thioguanine, Fludarabine, Cladribine, 6-Mercaptopurine, Penstostatin ), clofarabine and related substances) and pyrimidine antagonists (Cytarabine, Floxuridine, Azacitidine, Tegafur, Carmofur) (Carmofur), kanamyce (Capacitabine), gemcitabine (Gemcitabine), hydroxyurea (hydroxyurea), 5-fluorouracil (5FU) and related substances); alkylating reagents, such as nitrogen mustard substances (such as cyclophosphamide (Cyclophosphamide), US Melphalan, Chlorambucil, Mechlorethamine, Ifosfamide, Mechlorethamine, Trofosfamide, Prednimustine, Bendamustine (Bendamustine, Uramustine, Estramustine and related substances); Triazenes (such as Dacarbazine, Altretamine, Temozolomide) ) And related substances); Alkyl sulphonates (for example, Busulfan, Mannosulfan, Treosulfan and related substances); Procarbazine; (Mitobronitol) and Aziridines (e.g. Carboquone, Triaziquone, ThioTEPA, triethylenemalamine and related substances); antibiotics, e.g. Hydroxyurea, Anthracyclines (e.g. doxorubicin) , Daunorubicin, epirubicin and other derivatives); Anthracenediones (for example, Mitoxantrone and related products); Streptomyces family (for example, Bleomycin) , Mitomycin C (Mitomycin C), Actinomycin (Actinomycin), Plicamycin (Plicamycin); and ultraviolet light.

Without further explanation, a person with ordinary knowledge in the field of the present invention can implement the present invention to the greatest extent based on the above description. The following are specific embodiments, so they are only examples, and cannot limit the rest of the present invention in any way. All published documents cited in this manual are incorporated into this manual for reference purposes or as reference objects in this manual.

Example Example 1: Confirmation of the differential existence of lung cancer stem cells (CSCs) protein compared to primary lung cancer cells Materials and Methods Lung Cancer Cell Beads

Human non-small cell lung cancer cell lines NCI-A549, EKVX, PC9 and HCC827, obtained from the National Cancer Institute (National Institutes of Health, Bethesda, MD, USA) or the American Culture Collection (ATCC, Manassas, VA, USA) ). Human lung cancer cell lines (CLS1, CL100, CL141 and CL152) were established from primary cultures of lung cancer patients with adenocarcinoma. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, and cultured in a humidified environment at 37°C, maintaining 20% O ₂ and 5% CO ₂ .

Cultivation of CSCs and CAFs co-cultivation system

Human lung CSCs and CAFs were established from freshly resected lung tumor tissues from lung cancer patients. The tumor and matched normal tissues were harvested within 30 minutes after resection to isolate primary lung CSC, CAF, and NF cultures using a modification procedure. Lung CSCs are separated from the cancer-related areas of the resected tissues of NSCLC patients and are cultured and maintained with feeder cells, namely: stromal fibroblasts. The samples are purchased and implemented in accordance with the approved IRB agreement for human subject research. All patients obtained written informed consent. The non-cancer-related interstitium was sampled by a pathologist, within 30 minutes after pathological resection, at least 5 cm away from the tumorous lesion (under aseptic conditions), and visually inspected during surgical resection and subsequent tissue analysis.

The organization operates with the parameters described in previous studies with some modifications. Briefly, the tissue was minced and cultured in the presence of deoxyribonuclease 1 (deoxyribonuclease 1; 1 mg/ml; Bioshop) and protease (1 mg/ml; Sigma) in S-MEM medium (GIBCO) at 4°C 6~12 hours. After digestion, the cell mass is sieved through a 40-μm cell strainer (Falcon) to obtain a single cell suspension. The collected cells were cultured in 24-well culture plates at different cell densities (5×10 ⁵ ). The medium was RPMI1640 supplemented with 10% fetal bovine serum under modified culture conditions, and cultured at 37°C with 20% O ₂ and 5 % CO ₂ in humidified air. After 30 days of culture, spherical-like colonies can be identified around the mesenchymal cells. These spherical-like cells were continuously cultured in the modified manner described above. After 7-10 days, the spheroids were collected by gentle centrifugation (58g), and enzymatically decomposed (0.05% trypsin, 0.53mM EDTA; Invitrogen for 10 minutes), and then mechanically destroyed. The lung CSCs that passed through the 100-μm mesh were collected, and the status of a single cell was microscopically analyzed. The single cell, with a density of 5,000 viable cells/ml, was placed in a culture dish (5×10 ⁵ cells/well) pre-implanted with CAF as a feeder. For single cell/well multiplication experiments (clone experiments), these cells were cultured in a 96-well plate using a cell sorter (FACS Ariel), and feeder cells (2,000 cells/well) were pre-implanted in the wells. ). The continued cultivation of lung cancer stem cells is carried out in a slightly modified manner as described in the previous study. Briefly, the spheroids were collected via gentle centrifugation (800 rpm), enzymatic digestion (10 minutes of 0.25% trypsin, 1 mM EDTA; Invitrogen), and mechanical destruction. The lung cancer stem cells were separated from the 100-μm filter, and the sieved cells were analyzed under a microscope. These single cells were cultured at a density of 5,000 viable cells/ml in a 10-cm culture dish with pre-implanted cancer-related fibroblast feeding cells (5×10 ⁵ cells/well).

Image-based high-throughput analysis

Pulmonary CSC or cancer cells (200 cells/well) were added to a 96-well dish pre-implanted with CAFs (2,000 cells/well) and allowed to attach to the dish overnight. After different treatments, the primary antibody against Nanog (ReproCELL; 1:300) (as a cancer stem cell marker) and the mouse anti-human CD90 FITC-binding (5E10; BD Pharmingen; 1:100) antibody (as a CAF marker) in 4 ℃ overnight. Then, the primary antibody and TRITC-conjugated secondary antibody [goat anti-rabbit IgG (H+L) conjugate; Invitrogen] were incubated at room temperature for 2 hours. Use Hoechst 33342 dye for nuclear counterstaining. To determine the degree of background fluorescence of the secondary antibody, each plate contains a control hole containing only the secondary antibody (stained with Hoechst 33342 dye). Use an automated fluorescence microscope platform to obtain images of stained cells.

Image acquisition and analysis

The stained cells are captured using a high-throughput analysis platform equipped with a 4x objective lens. Each hole has 12 fields of view, and images of each wavelength are captured and montaged for further image analysis. The images were analyzed using MetaXpress® software (Molecular Devices). First, a multi-wavelength cell sorter was used to determine cancer cell nuclei (cells without FITC staining, CD90-). The part of the cancer cell nucleus in the image is dilated and smoothed using Morphology Filters to create a cell cluster mask. The positive cells stained with TexRed were determined to be Nanog-positive cells.

Real-time reverse transcription (RT) Q-PCR

The degree of expression of genes related to stemness and the verification of Affymetrix chip data of CAF, CLS1/CAF and CLS1 were performed by RT Q-PCR using ABI Prism 7900 sequencer (Applied Biosystems). The primers designed by Primer Express 3.0 (Applied Biosystems) were used (Table 2). Use β-actin as an internal control. The expression level was standardized by β-actin and defined as -△CT=-[CTtarget-CTβ-actin]. Calculate the relative performance ratio as the change multiple relative to the internal control (2-△△CT). The experiment was repeated in three.

Adipocyte differentiation

In order to detect whether cancer stem cells can differentiate into adipocyte lineage, CLS1 cells are co-cultured with CAFs, and mesenchymal stem cells are used as a positive control group. The procedure of adipogenesis analysis (Millipore, Cat.#SCR020) is used for inspection. ^{In short, a 24-well plate was inoculated with 6×10 4} cells/well cell density in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. After the cells are dense, the fat is used for adipogenic cell culture in a fat-generating basic growth medium supplemented with 1 μM dexamethasone, 10 μg/ml insulin, and 0.5 mM isobutylmethylxanthine (IBMX). Induction medium (Millipore, Cat. #SCR020). The medium is changed every 3 days. After 21 days of culture, the cells were fixed with 4% PFA to assess the formation of adipocytes and stained with Oil Red O dye for 50 minutes at room temperature. The pictures were taken with an Axiovert 200 microscope. Analyze gene expression during differentiation through adipocytes, and collect total mRNA from adipocytes. The samples as described herein were subjected to real-time RT-PCR against peroxisome proliferator-activated receptor-γ (PPAR-γ) and lipoprotein lipase (LPL).

Differentiation of osteoblasts

To test whether cancer stem cells can differentiate into osteogenic lines, CLS1 cells were co-cultured with CAFs, and mesenchymal stem cells were used as a positive control group, with 0.1μM dexamethasone, 0.2mM ascorbic acid 2-phosphate and 10mM glycerol 2- The basic growth medium of phosphoric acid was cultured for 14 days and examined by the procedure of osteoblast analysis (Millipore, Cat.#SCR028). ^{In short, inoculate 6×10 4} cells/well in RPMI 1640 medium supplemented with 10% FBS, 100U/ml penicillin and 100μg/ml streptomycin in a 24-well plate coated with vitronectin/collagen Cell density adipocytes. Change the medium every 3 days. After 14 days, the cells were washed twice with PBS and fixed with ice-cold 70% ethanol for 1 hour at room temperature. Analysis was performed by Alizarin Red S (Mippipore, Cat. #2003999) staining for 30 minutes at room temperature. For gene expression during osteoblast analysis, the total mRNA obtained from cells grown in osteoblast differentiation medium undergoes 0, 3, or 7 days of differentiation. These samples were subjected to real-time RT-PCR for AP and osteocalcin (OC).

Membrane proteome analysis

Membrane protein samples are extracted from CLS1 cells with or without co-cultivation with CAFs, and the samples are given internal standards, gel-assisted decomposition, and LC-MS/MS triple analysis. The IDEAL-Q software developed by the laboratory of Dr. Chen Yuru, Institute of Chemistry, Academia Sinica, performs triple-repeat LC-MS/MS label-free quantification of cell lines. The ratio of peptides is determined by the weighted average of normalized peptide ratios.

Gene expression profile

The gene expression profiles of CLS1/CAF and CLS1 were obtained from the Affymetrix GeneChip system (Affymerix, Inc., Santa Clara, Ca, USA) according to the manufacturer's procedures. Affymetrix GeneChip system (Affymerix, Inc., Santa Clara, Ca, USA) was used for gene expression mapping according to the manufacturer's procedure.

The chip data is processed by the National Taiwan University Microarray Core Facility for Genomic Medicine. In short, total RNA isolated from CAFs, lung CSCs, and cancer cells was used to _{generate cDNA using T7-(dT) 24} primer (SuperscriptChoice System, Gibco BRL Life Technologies). The BioArray high-yield RNA transcription labeling kit (EnzoDiagnostic, Inc.) was used to synthesize biotin-labeled ribonucleotides and hybridized to the human genome U133 Plus 2.0 chip (Affymetrix).

result Lung CSCs maintain cancer stemness and high tumorigenicity when co-cultured with CAF feeder cells

Compared with primary lung cancer cells, proteins are judged to be differentially present in lung cancer stem cells (CSCs). CAF-co-cultured CLS1 cells (CLS1/CAF) maintain ^{a high population of Nanog+} cells (Figure 1(A)) and have the ability to differentiate into adipocytes and osteoblasts (Figure 1(B) and (C)), and When injected with a lower cell number (<100 cell number), it has the ability to produce tumors in xenograft mice (Figure 1(D)). However, when CAFs were removed during the passage, the stemness characteristics of the cells were lost, and the Nanog positive population was significantly reduced (Figure 1(A)) and the initial frequency of tumors was reduced from 1/11 to 1/1774 (Figure 1 (D)). Therefore, the CSCs/CAFs co-culture module provides a platform for culturing cancer stem cells and maintaining the stemness of cancer cells.

Compared with primary lung cancer cells, there are higher levels of CD14 and CD44 in CSCs

In order to confirm the role of CSCs cell surface proteins and surrounding CAFs, we performed CSCs/CAFs and differentiated cancer cell transcriptomic and membrane proteomic maps through the CSC marker screening strategy (Figure 1(E)). This analysis showed that CD14 and CD44 cell surface proteins were up-regulated in CSCs/CAFs. Subsequent analysis focused on the CD14 and CD44 proteins with the most relevant profile data in CSC s/CAF (Table 1 above).

In order to study candidate CSC markers, the performance of CD14 and CD44 is related to the clinical risk ratio of 152 first-stage lung cancer patients from the published Japan cohort. The results showed that compared with patients with low CD14 expression, patients with high CD14 expression in tumor cells showed a significantly poorer survival rate without recurrence (P<0.05, Kaplan-Meier test; Figure 1(F)). The CD14 and CD44 analysis of patient prognosis showed that compared with patients with low expression of CD14 and CD44 in tumor cells, patients with high expression of CD14 and CD44 in tumor cells showed poor non-recurrence survival rate (CD14+CD44, P< 0.05, Kaplan-Meier test; Figure 1(F)). Example 2: The correlation between CD14 and CD44 with poor prognosis Materials and Methods Patient and tumor samples

Lung tumor tissue samples were obtained from patients with NSCLC who had undergone complete surgical resection at the National Taiwan University Hospital (Taipei, Taiwan) between December 28, 1995 and December 26, 2005, confirmed by histology (N=80). This research was approved by the Institutional Review Board of National Taiwan University (201103028RC). Participating patients are classified as stage I, and they have not previously been treated with neoadjuvant chemotherapy or radiation. All patients provided informed consent. All samples were fixed with formalin, sectioned, stained with H&E, and examined under a microscope. The postoperative pathological staging is based on the international staging system of lung cancer.

Immunochemical analysis of tumor samples from lung cancer patients

Perform immunochemical analysis of tumor samples according to standard procedures and modification procedures described herein. Double immunochemical staining of CD14 (clone EPR3653; Epitomics; dilution 1:400) and CD44 (clone DF1485; BioGenex; dilution 1:200). After deparaffinization and dehydration, 1 mM Tris-EDTA buffer was used to perform heat-mediated antigen retrieval of sections with a thickness of 5 μm, each section for 10 minutes. After blocking with hydrogen peroxide and Ultra V Block, the sample was incubated with the primary antibody for 2 hours at room temperature. The washing step was performed with TBST buffer (Trist buffer containing 0.1% Tween 20 [TBS: 50 mM, pH 7.6]). Through the use of rabbit anti-human antibody (CD14) HRP-binding polymer and mouse anti-human antibody (CD44) alkaline phosphatase-binding polymer using a multivision polymer detection system (Thermo scientific, TL-012-MARH) for double staining. The slices used for IHC analysis of Nanog protein expression are first sterilized by autoclaving at 121°C for 10 minutes in antigen retrieval AR-10 solution (Biogenex) or antigen retrieval Citra solution (Biogenex). The sample was then _{treated with 3% H 2} O ₂ -methanol and then incubated with Ultra V Block (Lab Vision Corporation) for 10 minutes and incubated with rabbit monoclonal anti-Nanog (D73G4, Cell signaling; 1:300) at room temperature 2 hour. Use the ultra-sensitive non-biotin polymer HRP detection system (BioGenex) to detect immunostaining, according to the original factory operation manual.

result

To confirm the clinical significance and importance of CD14 and CD44 in the early stages of tumorigenesis, tumor samples were collected from 80 patients with stage I NSCLC, and each sample was sliced and stained with anti-CD14 and CD44 antibodies by IHC. A representative tissue section is shown in Figure 2(A). The clinical characteristics of these patients are summarized in Table 3 and Table 4 below.

Score the expression of CD14 in tumor cells, and divide it into two categories: positive and negative expression of CD14 protein. Score the performance of CD44, and divide it into high (score ≥ average risk score) or low (score <average risk score) CD44 protein performance classification. Cox proportional hazards regression analysis evaluated independent prognostic factors related to patient survival (Table 5).

The results showed that independent prognostic factors included CD14 performance (hazard ratio (HR)=2.79, 95%CI=1.29 to 6.03; P=0.009, Cox proportional hazard regression analysis) and CD44 performance (hazard ratio (HR)=9.25, 95% CI =3.78-22.64; P<0.0001, Cox proportional hazard regression analysis). The independent prognostic factors associated with metastasis are CD14 performance (hazard ratio (HR)=2.63, 95% CI=1.09 to 6.35; P=0.032, Cox proportional hazard regression analysis) and CD44 performance (hazard ratio (HR)=5.89, 95 % CI=2.17 to 16.01; P<0.0001, Cox proportional hazard regression analysis, Table 6).

The prognosis of patients with CD44 and CD14 expression levels combined and analysis of individual effects showed that compared with patients with low expression levels of CD44 and CD14 in tumor cells, patients with high expression levels of CD44 and CD14 in tumor cells showed the worst overall (CD44+CD14, P<0.0001, Kaplan-Meier analysis; Figure 2(B); HR=3.33, 95% CI=2.12 to 5.24; P<0.0001, Cox proportional hazard regression analysis; Table 5) and survival rate without recurrence (CD14+CD44, P<0.05, Kaplan-Meier analysis; Figure 2(C); HR=2.88, 95% CI=1.68 to 4.94; P=0.0001, Cox proportional hazard regression analysis; Table 6). These results further show that CD44 and/or CD14 provide a novel prognostic index (P=0.0001, Cox proportional hazard regression analysis) for predicting metastasis in early-stage NSCLC patients, and overall survival rate (P<0.0001, Cox proportional hazard regression analysis), See Table 5 and Table 6.

These results show that compared with patients with low CD14 and/or CD44 expression, patients with high CD14 and/or CD44 expression in tumor tissues significantly indicate poorer overall survival rates and non-recurrence survival rates.

Example 3: Co-cultivation with CAFs resulted in lung cancer stem cells CD44 ⁺ CD14 ⁺ Enrichment Materials and methods Immunofluorescence microscope

The cells were fixed with 4% paraformaldehyde phosphate buffered solution (PBS) at room temperature. Follow the standard immunofluorescence procedure. Interference and hybridization were performed in 3% (wt/vol) bovine serum albumin (BSA) in PBS. Use monoclonal antibodies (mAbs) against Nanog (ReproCELL; 1:300), CD90 FITC-binding (5E10; BD Pharmingen; 1:100), CD44 FITC-binding (G44-26; BD Pharmingen; 1:10); And CD14 PE-binding (HCD14; Biolegend; 1:20) antibodies are used. Axiovert 200 microscope (Carl Zeiss, Göttingen, Germany), a confocal laser scanning microscope (Clsi, Nikon, Japan) were used with MetaXpress® (Molecular Devices) to inspect the stained cells.

Analysis of Western Ink Spot Method

The detailed method is carried out according to standard procedures. Nanog (D73G4; 1:1000) primary antibody was purchased from Cell Signaling Technology, CD44 (MAB4073; 1:1000) primary antibody was purchased from Cell marque, and CD14 (EPR3653; 1:1000) primary antibody was purchased from Cell marque. Monoclonal murine anti-β-actin (Chemicon, Millipore; 1:5000) was used as load control. The membrane was washed three times with TBST, and then a secondary antibody (1:5,000) combined with horseradish peroxidase (HRP) was cultured in TBST containing 2% skimmed milk. The bound antibody was detected by the Enhanced Chemiluminescence System (Santa Cruz, CA). A Fujifilm LAS 3000 system (Fujifilm, Tokyo, Japan) was used to capture chemiluminescence signals. All experiments were carried out with at least three repeated experiments.

Ultra-low sphere-forming analysis

The ultra-low ball formation analysis was performed according to the standard procedure and the modification procedure described in this article. Cultured in a single cell suspension of MCDB201 serum-free medium (Invitrogen) lung CSCs supplemented with 20ng/ml EGF (Sigma) and 20ng/ml bFGF (Invitrogen), implanted with ultra-low attachment In a 24-well culture dish (Corning, Corning, NY, USA; 200 viable cells/well). Add fresh growth factors to the culture medium twice a week. After three weeks, the formed spheres were observed with an Axiovert 200 microscope.

result

In order to verify the increased CD14 and CD44 performance in the CSCs/CAD co-culture membrane group relative to differentiated cancer cells, compare the CLS1/CAF gene expression profiles of CLS1-differentiated cancer cells cultured with different subcultures and without feeder cells Atlas of gene expression. This comparison shows that the co-culture of CAFs with CLS1 cells induces CD14 and CD44 expressions compared to subculture without CAFs (Figure 3(A)).

To further evaluate whether CD14 and CD44 will increase in lung cancer cells co-cultured with CAFs from various tumors. CAFs are isolated from different lung cancer patients. The laser-extracted colony cells from different cancer cell lines (A549 and EKVX cells) showed higher expression levels of CD14 and CD44 (Figure 3(B) and Figure 5(A)). The characteristics of 12 CAF samples isolated from patients are shown in Table 7.

Immunofluorescence staining and Western blotting method confirmed that CD14 and CD44 were mainly expressed in lung CSCs (CLS1/CAFs), rather than differentiated CLS1 cells (Figure 3(C)). Flow cytometry analysis showed that CD14 and CD44 were highly expressed in lung CSCs (CLS1/CAFs) (CD14: 55%; CD44: 98.1%) and the expression of CD44 and CD14 decreased after differentiation (CD14: 11%; CD44: 30.3%) (Figure 5(B)). Confirm the percentage of CD44 positive groups and CD14/CD44 double positive groups in different lung cancer cell lines (A549, EKVX, PC9 and HCC827) and primary lung cancer cell lines (CL25, CL83, CL97, CL100, CL141 and CL152), the results It shows that the CD44 positive group is abundant in different lung cancer cells (79.6±29.7%). Figure 6. The percentage of the CD14/CD44 double positive group was significantly reduced in these cancer cells (19.0±23.9%). The analysis showed that CD44 and CD14 significantly represent cancer stem cell markers in the tumor niche.

Example 4: CD44 ^Hi CD14 ⁺ Cancer cells have a higher initial frequency of tumors Materials and methods Flow cytometry

Analyze and screen the population of cancer stem cell markers by flow cytometry. Antibodies for human antibodies CD44 FITC-binding (G44-26; BD Pharmingen; 1:10) and CD14 PE-binding (HCD14; Biolegend; 1:20) are commercially available. Lung cancer cell lines and primary lung cancer cells were double stained with CD14 and CD44 in PBS at room temperature for 30 minutes. After 30 minutes, the stained cells were washed with excess unbound antibody and resuspended in screening buffer (1 mM EDTA and 2% FBS in PBS). Flow sorting was performed using BD FACS AriaIII cell sorter (BectonDickinson), and analysis was performed in FACSC (BectonDickinson). To screen sample cells to confirm single cell screening, evaluate the forward scattering height versus forward scattering width (FSC-H versus FSC-W) and lateral dispersion area versus lateral dispersion width (SSC-A versus SSC-W) Cell mass. Eliminate dead cells through propidium iodide (PI, dead cell staining, Molecular Probes) cells, which increases the effect of large-scale screening and active cells for single-cell experiments.

result

^{Measure the tumor-initial ability of CD44 Hi} and CD14 ⁺ in lung cancer cell lines and primary lung cancer cells, which represents the definition of cancer stem cell operation. The lung cancer cells selected at limited dilutions (1 ^{×10 4} , 1 ^{×10 3} , and 1 ^{×10 2} cells/mouse) were injected subcutaneously into NOD/SCID/IL2Rγ_(NSG) mice. Compared with the CD44 ^Hi CD14 ^- group (1/278), the CD44 ^Hi CD14 ⁺ group screened from lung cancer cell lines will show a higher tumor-initial frequency (1/10). CD44 ^low CD14 ⁺ group has not been confirmed.

The initial tumor frequency ^{of CD44 Hi} and CD14 ⁺ groups screened in lung cancer cell lines (CLS1 and A549 cells) and primary lung cancer cells (CL100, CL141 and CL152) were measured. A limited dilution (1 ^{×10 4} , 1 ^{×10 3} , and 1 ^{×10 2} cells/mouse) cell group was injected subcutaneously into SCID mice. Compared with cancer cells in the CD44 ^Hi CD14 ^- and CD44 ^Low CD14 ^- groups, the CD44 ^Hi CD14 ⁺ group in xenograft cells showed a higher tumor-initial frequency (Table 8 and Figure 4(A) to (F) )). These results suggest that the CD44 ^Hi CD14 ⁺ group of lung cancer cells can be tumor stem cells with tumorigenesis.

The analysis of transcriptomic and proteosome described herein judged that CD14 and/or CD44 were present at a higher level in CSC/CAF co-culture than in CAF culture. Any protein judged herein can be used as a biomarker (individually or in combination) for lung cancer stem cells, such as detecting the presence of lung cancer stem cells in a sample, judging patients with lung cancer related to poor prognosis, and selecting candidates for treatment Marking, monitoring the progress of lung cancer, evaluating the effect of anti-cancer treatment, confirming the treatment process, evaluating whether the individual is at risk of cancer recurrence, and/or for research purposes, including, for example, studying the mechanism of lung cancer based on the development of new cancer treatments and/or Biological pathways/processes related to lung cancer.

Example 5: CD14 manifestations of various types of cancer stem cells

According to the manufacturer’s manual, cell lines of various cancer types including liver cancer cells, colon cancer cells, and pancreatic cancer cells were stained with PE-conjugated anti-CD14 antibody (HCD14; Biolegend; 1:20 in PBS) at room temperature 30 minutes. An isotope antibody was used as a negative control group for non-specific binding. The cells were then washed with PBS to remove unbound antibodies, and resuspended in screening buffer PBS (1 mM EDTA and 2% FBS in PBS). Fluorescence activated cell sorting (FACS) system was used ^{for flow sorting using BD FACSAsia TM} Fusion cell sorter (Becton Dickinson).

As shown in Table 9, the expression of CD14 was observed in various types of cancer cells.

The results of this study indicate that CD14 can be used as a biomarker for various types of cancer cells, such as cancer stem cells.

Other embodiments

All the features disclosed in this specification can be combined in any way. Each feature disclosed in this specification can be replaced with another feature of the same, equal or similar use. Therefore, unless explicitly stated, each feature disclosed is only one example in a broad series of equivalent or similar features. From the foregoing, those with ordinary knowledge in the field of the present invention can easily determine the necessary technical features of the present invention, and without exceeding the spirit and scope of the present invention, can change and modify the present invention in various ways for different usages and conditions. . Therefore, other embodiments are also included in the scope of the patent application.

Equivalents and scope

Those with ordinary knowledge in the field of the present invention will understand, or only use routine experiments, to confirm many equivalents of the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not limited by the above disclosure, but as defined by the scope of the appended application.

Terms such as "一 (a, an)" and "the (the)" in the scope of the patent application can mean one or more than one, unless the context indicates the opposite or otherwise indicates. If one, more than one, or all of the components are present in, used in, or otherwise related to the substance or method of administration, the “or” between the one or more components or the disclosure shall be deemed to be Where it is appropriate to express the situation, unless the context indicates the opposite or indicates otherwise. The present invention discloses related embodiments including a substance or method that a component of the group is present in, used in, or otherwise and administered. The present disclosure includes embodiments in which more than one or all of the components are present in, used in, or otherwise administered with objects or methods.

In addition, it should be understood that unless otherwise stated or unless contradictions or inconsistencies are obvious to those skilled in the art, the disclosure of the present invention includes one or more of the scope of the listed patent application (one or more limitations, elements, clauses). , Descriptive terms, etc. introduce all the changes, combinations and permutations in another technical solution that is attached to the same basic application patent scope (or any other related application patent scope). If it is in the form of a list (for example, in the Markusi group (Markush group or similar format), it should be understood that the present invention also discloses a subgroup of each element and any element can be removed from the group. It should be understood that, generally, if you call the present disclosure or the present disclosure Aspects include specific elements, features, etc., then certain embodiments of the present invention or aspects of the present invention consist of or mainly consist of these elements, features, etc. For the sake of brevity, the text is not specific in too many languages. State each case of these embodiments. It should be noted that the terms "including" and "including" are intended to open up and allow other elements or step inclusions. When a range is given, endpoints are included. In addition, unless the context is Indicating the opposite situation or otherwise explaining and those with ordinary knowledge in the field of the present invention will understand that the numerical value expressed as a range can adopt the range described in the different embodiments of the present disclosure. Any specific value or sub-range within the range, unless the context indicates otherwise, is one-tenth of the lower limit unit of the range.

This application involves various published patents, published patent applications, journal articles and other publications, all of which are cited in this article. If there is a conflict between any merged reference and this specification, the specification shall be defined. In addition, any specific embodiment of the present disclosure that belongs to the prior art may be explicitly excluded from the scope of any one or more patent applications. Because such embodiments are considered to be known to those having ordinary knowledge in the field of the present invention, they can be excluded even if the exclusion is not explicitly stated herein. Regardless of whether it involves the existence of prior art, any specific embodiment of the present disclosure can be excluded from the scope of any patent application.

Those of ordinary knowledge in the field of the present invention will recognize or be able to determine many equivalents to the specific embodiments described herein by merely using routine experimentation. The foregoing embodiments are intended to describe and illustrate the technical features and specific implementations of the present invention, so that those who are familiar with the field of the present invention can implement them with ordinary knowledge, and are not intended to limit the protection scope of the present invention. Although those familiar with the technical field of the present invention can easily make some changes or modifications to the present invention accordingly, these changes will not deviate from the scope defined by the scope of the patent application of the present invention. Accordingly, the scope of protection requested by the present invention is defined by the scope of the patent application, rather than by the disclosure of the implementation description and the examples.

<110> National Taiwan University

<120> Biomarkers of lung cancer stem cells

<130> S1926.70003WO00

<150> US 62/442,077

<151> 2017-01-04

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 1

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 2

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 3

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 4

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 5

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 6

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 7

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 8

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 9

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotides

<400> 10

Claims (9)

A method for analyzing a sample, the method comprising: (i) providing a sample suspected of containing cancer stem cells, (ii) measuring the content of CD14 in the sample; and (iii) measuring the content of CD44 in the sample; wherein the sample A biological sample of a human patient with or suspected of having cancer. The biological sample is a body fluid sample or a tissue sample obtained from a tumor location or a suspected tumor location; judgment based on the content of CD14 and CD44 The presence of cancer stem cells in the sample, where the increase in the content of CD14 and CD44 means that the cancer stem cells are present in the sample; the cancer is lung cancer; step (ii) and step (iii) include measuring soluble or performance The content of CD14 and CD44 on the cell surface. The method described in item 1 of the scope of the patent application, wherein the step (ii) comprises measuring the content of CD14 protein. The method according to item 1 or item 2 of the scope of the patent application, wherein the content of the CD14 protein is measured by immunohistochemistry analysis, immunoblotting analysis, or flow cytometry analysis. The method described in item 1 of the scope of the patent application, wherein the step (ii) comprises measuring the content of the nucleic acid encoding CD14. According to the method described in item 4 of the patent application, the content of the nucleic acid encoding CD14 can be measured by a real-time reverse transcription-PCR (RT-PCR) analysis or a nucleic acid wafer analysis. The method described in item 1 of the scope of patent application, wherein the human patient has non-small-cell-lung-cancer (NSCLC). The method described in item 6 of the scope of the patent application further includes judging the survival rate of the human patient, wherein an increase in the content of CD14, or an increase in the content of CD14 and CD44 indicates a poor survival rate. A method for detecting poor prognosis of cancer, comprising: (i) providing a sample of an individual with cancer, (ii) measuring the amount of CD14 in the sample, (iii) measuring the amount of CD44 in the sample, and (iv) Determine whether the individual has a poor prognosis of cancer. If the content of CD14 and the content of CD44 in the sample are both higher than a predetermined level, then the individual is judged to have a poor prognosis of cancer; the individual is a patient with lung cancer Human patients; this step (ii) and this step (iii) include measuring the content of CD14 and CD44 that are soluble or expressed on the cell surface. The method according to item 8 of the scope of patent application, wherein the human patient has non-small cell lung cancer.

Images