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Tumor marker, and method for collecting and detecting tumor cell in distinction from contaminant cell

A technology for tumor markers and tumor cells, which is applied in the field of recovery and detection of tumor markers and distinguishing tumor cells from intermingled cells

Pending Publication Date: 2020-10-27
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a problem that cytokeratin crosses with the mixed cells contained in blood (non-specific detection), and EpCAM has the problem that only some CTCs of epithelial origin can be recovered and detected

Method used

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  • Tumor marker, and method for collecting and detecting tumor cell in distinction from contaminant cell
  • Tumor marker, and method for collecting and detecting tumor cell in distinction from contaminant cell
  • Tumor marker, and method for collecting and detecting tumor cell in distinction from contaminant cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1 Gene expression analysis of healthy human leukocytes and various cancer cells

[0105] As cancer cell lines, human lung adenocarcinoma cells (PC9 and PC14), human breast cancer cells (MDAMB231), human breast cancer cells (SKBR3), human prostate cancer cells (22Rv1 and PC3), human liver cancer cells (HepG2 and HuH- 7) For 10 types of human pancreatic cancer cells (PANC1 and AsPC-1), the difference in gene expression between these cancer cell lines and healthy human leukocytes was analyzed by the following method using a next-generation sequencer.

[0106] (1) The cancer cell lines were respectively treated with the medium shown below in 5% CO 2 After culturing at 37°C in an ambient environment, the cells were detached from the culture medium using 0.25% trypsin / 1mM EDTA to recover cancer cells (n=4) as single cell units, and the RNA from the single cells was used by SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) was used for cDNA synthesis and ...

Embodiment 2

[0144] Example 2 Staining of cancer cells and leukocytes using anti-TM4SF1 antibody

[0145] It has been clarified in Example 1 that the TM4SF1 gene is highly expressed in cancer cells compared with healthy human leukocytes. Therefore, it was investigated whether TM4SF1 can be used for the specific detection of tumor cells contained in blood samples.

[0146] (1) A blood sample obtained by removing erythrocytes from healthy human blood by the specific gravity difference separation method was used as the blood sample used in this example, and human lung adenocarcinoma cells (PC14) or human lung adenocarcinoma cells (PC14) or human Pancreatic cancer cells (PANC1).

[0147] (2) 10 μL of an FcR blocking agent (Miltenyi Biotec) was added to 100 μL of a blood sample spiked with a cancer cell line, and a blocking treatment was performed at room temperature for 10 minutes.

[0148] (3) After blocking treatment, 10 μL of antibody solution against TM4SF1 (Human TM4SF1 Phycoerythrin MAb...

Embodiment 3

[0151] Example 3 Staining of cancer cells and leukocytes using anti-TNFRSF12A antibodies

[0152] It has been clarified in Example 1 that the TNFRSF12A gene is highly expressed in cancer cells compared with healthy human leukocytes. Therefore, it was investigated whether TNFRSF12A can be used for the specific detection of tumor cells contained in blood samples.

[0153] (1) A sample obtained by removing erythrocytes from healthy human blood by the specific gravity difference separation method was used as the blood sample used in this example, and human lung adenocarcinoma cells (PC9) or human lung adenocarcinoma cells (PC9) or human Breast cancer cells (MDAMB231).

[0154] (2) 10 μL of an FcR blocking agent (Miltenyi Biotec) was added to 100 μL of a blood sample spiked with a cancer cell line, and a blocking treatment was performed at room temperature for 10 minutes.

[0155] (3) After the blocking treatment, 2.5 μL of the antibody against TNFRSF12A (PE anti-human CD266 (Fn14...

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Abstract

Provided is a method for collecting and detecting a tumor cell contained in a sample in distinction from a contaminant cell. A tumor cell contained in a sample can be collected and detected in distinction from a contaminant cell by detecting any one polypeptide selected from (i) a polypeptide which comprises at least one amino acid sequence selected from six amino acid sequences including TM4SF1 (GenBank No. NP_055035.1) and TNFRSF12A (GenBank No. NP_057723.1), (ii) a polypeptide which comprises at least an amino acid sequence having a 70% or higher homology with the aforementioned amino acidsequence, and (iii) a polypeptide which comprises at least a spliced variant of any one of the aforementioned amino acid sequences (i.e., the aforementioned amino acid sequences (i) and (ii)) contained in the sample, or a gene encoding any one of the polypeptides.

Description

technical field [0001] The present invention relates to a method for recovering and detecting tumor markers and tumor cells contained in samples. In particular, the present invention relates to a method for distinguishing the tumor cells from foreign cells contained in the sample using proteins or genes expressed by the tumor cells contained in the sample, recovering and detecting the tumor cells. Background technique [0002] Once the tumor cells leave the primary tumor, they infiltrate into blood vessels or lymph vessels, circulate in the blood or lymph, and finally invade other organs and tissues, thus forming metastases. Here, tumor cells circulating in the blood are also called CTCs (Circulating Tumor cells, circulating tumor cells), and a large number of clinical trials and researches have been conducted. For example, the number of CTCs contained in blood collected from a patient is measured (Patent Document 1); or, by investigating protein expression, gene mutation, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C07K14/82C12N15/12C12Q1/04C12Q1/6813C12Q1/6886G01N33/53G01N33/574C12N15/115
CPCC12N15/115C07K14/4748C07K14/705C07K14/70578C07K14/70596C07K14/745C12N9/12C12Y207/10001C07K14/7055G01N33/57492G01N33/57488G01N33/56966C07K14/4725C12Q1/6886C12Q2600/158
Inventor 熊木勇一平井清华大槻诚三木大辅二见达
Owner TOSOH CORP