CN114280303A - Immunohistochemical staining kit for detecting tumor metastasis seed cells and application thereof - Google Patents
Immunohistochemical staining kit for detecting tumor metastasis seed cells and application thereof Download PDFInfo
- Publication number
- CN114280303A CN114280303A CN202011237078.7A CN202011237078A CN114280303A CN 114280303 A CN114280303 A CN 114280303A CN 202011237078 A CN202011237078 A CN 202011237078A CN 114280303 A CN114280303 A CN 114280303A
- Authority
- CN
- China
- Prior art keywords
- tumor
- igsf9
- prdm16
- aldh2
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 78
- 206010027476 Metastases Diseases 0.000 title claims abstract description 44
- 230000009401 metastasis Effects 0.000 title claims abstract description 38
- 238000011532 immunohistochemical staining Methods 0.000 title claims abstract description 17
- 101000642195 Homo sapiens Protein turtle homolog A Proteins 0.000 claims abstract description 52
- 102100033219 Protein turtle homolog A Human genes 0.000 claims abstract description 51
- 230000014509 gene expression Effects 0.000 claims abstract description 51
- 101000686942 Homo sapiens Histone-lysine N-methyltransferase PRDM16 Proteins 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 45
- 102000009645 Mitochondrial Aldehyde Dehydrogenase Human genes 0.000 claims abstract description 44
- 102100024594 Histone-lysine N-methyltransferase PRDM16 Human genes 0.000 claims abstract description 43
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 claims abstract description 42
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 39
- 201000011510 cancer Diseases 0.000 claims abstract description 18
- 230000002055 immunohistochemical effect Effects 0.000 claims abstract description 16
- 238000004393 prognosis Methods 0.000 claims abstract description 9
- 238000011282 treatment Methods 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 49
- 210000004881 tumor cell Anatomy 0.000 claims description 31
- 206010061289 metastatic neoplasm Diseases 0.000 claims description 25
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 claims description 9
- 238000003745 diagnosis Methods 0.000 claims description 6
- 102000054485 human PRDM16 Human genes 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 101000779444 Homo sapiens Aldehyde dehydrogenase, mitochondrial Proteins 0.000 claims description 2
- 102000048738 human IGSF9 Human genes 0.000 claims 1
- 230000000984 immunochemical effect Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000009472 formulation Methods 0.000 abstract 1
- 239000000203 mixture Substances 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 29
- 238000010186 staining Methods 0.000 description 18
- 230000001394 metastastic effect Effects 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 7
- 238000013115 immunohistochemical detection Methods 0.000 description 7
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 229940122255 Microtubule inhibitor Drugs 0.000 description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- 102000038627 Zinc finger transcription factors Human genes 0.000 description 3
- 108091007916 Zinc finger transcription factors Proteins 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 231100000782 microtubule inhibitor Toxicity 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 206010055113 Breast cancer metastatic Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 206010014733 Endometrial cancer Diseases 0.000 description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 201000006107 Familial adenomatous polyposis Diseases 0.000 description 1
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 241000270666 Testudines Species 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000003934 aromatic aldehydes Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000001593 brown adipocyte Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 208000015606 cardiovascular system disease Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 208000029664 classic familial adenomatous polyposis Diseases 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004149 ethanol metabolism Effects 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 201000010175 gallbladder cancer Diseases 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000004683 skeletal myoblast Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 210000004003 subcutaneous fat Anatomy 0.000 description 1
- 230000027912 synapse maturation Effects 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 230000035924 thermogenesis Effects 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an immunohistochemical staining kit for detecting tumor metastasis seed cells and application thereof, belonging to preparation of immunochemical experimental substances for cancers. The kit mainly comprises three protein immunohistochemical antibodies of cancer suppressor molecules PRDM16, IGSF9 and tumor sternness molecules ALDH2, and is used for detecting the protein expression levels of the three molecules in an isolated tumor tissue of a subject. The invention is used for determining the tumor metastasis seed cells, namely the tumor metastasis seed cells with low/no expression of PRDM16 and IGSF9 proteins and high expression of ALDH2 proteins, according to the expression levels of the corresponding proteins of the three molecules, provides a basis for the formulation of a clinical accurate treatment scheme and the prognosis prediction of a patient, and has wide application prospect.
Description
Technical Field
The invention relates to preparation of immunochemical experimental substances for cancer, in particular to an immunohistochemical staining kit for detecting tumor metastasis seed cells and application thereof.
Background
Malignant tumors have become one of the most serious diseases endangering human health. The mortality rate is second to the cardiovascular system diseases, and the disease rate is on the rise year by year, which consumes a great deal of medical resources. The metastasis of tumor is a direct cause of death of patients, but how to diagnose tumor cells with metastatic capacity (namely tumor metastasis seed cells) and guide accurate treatment is still blank at present, and research and solution are urgently needed.
The PR domain gene family member 16 (PRDM 16) gene was first discovered in 2003 and is a member of the PR domain gene family, and the encoded protein is a zinc finger transcription factor comprising an N-terminal PR domain. PRDM16 is involved in the switch between skeletal myoblasts and brown adipocytes, thereby promoting thermogenesis and mitochondrial function. PRDM16 has different expression levels and different functions in different types of tumors, and may be used as cancer suppressor gene in lung cancer and oncogene in acute myelogenous leukemia, glioma, gastric cancer and rectal cancer. The expression level of PRDM16 is highly related to the methylation state of the promoter of the gene, PRDM16 is highly expressed when the promoter is hypomethylated, and the expression level is reduced when the promoter is hypermethylated.
Immunoglobulin superfamily member 9(Immunoglobulin superfamily member 9, IGSF9) is located at 1q23.2, is homologous to the drosophila gene turtle, and its presence was reported in humans in 2002. IGSF9 is a member of the immunoglobulin superfamily that mediates cell-to-cell adhesion between similar and heterogeneous species. IGSF9 is involved in dendritic formation, synaptic maturation and synaptic transmission in the murine nervous system. IGSF9 has different reported relationships with tumors: IGSF9 is up-regulated in endometrial cancer, and the endometrial cancer patient with IGSF9 over-expression has strong tumor invasion capacity, so that the prognosis of the patient is poor. High grade serous ovarian cancer IGSF9 is up-regulated by about 5-9 fold compared to normal oviduct tissue. IGSF9 is one of the most strongly expressed genes in gallbladder cancer (about 18-fold) compared to normal gallbladder tissue. And down-regulated in melanoma, familial adenomatous polyposis and adenomatosis.
The Aldehyde dehydrogenase (ALDH) superfamily is an nad (p) + dependent enzyme and is a class of proteins that exert a multifunctional role. At least 19 ALDH genes have been found in the human genome to date. Mainly in mitochondria, cytosol and cytoplasmic microsomes. The Aldehyde dehydrogenase family member 2 (ALDH 2) is a member of ALDH family, is positioned in a mitochondrial matrix, is responsible for catalyzing the reaction of oxidizing Aldehyde into acetic acid, and plays a key role in the in vivo ethanol metabolism process. ALDH2 is also effective in promoting the oxidation of short chain aliphatic and aromatic aldehydes in addition to acetaldehyde. It is expressed in multiple tissues of human body, most commonly found in liver and subcutaneous adipose tissue, and is closely related to the occurrence of alcoholic liver disease. ALDH2 plays a key role in maintaining cancer cell sternness and mediating microtubule inhibitor resistance. Overexpression of ALDH2 in leukemia cells and lung cancer cells promotes cell proliferation and clonogenic capacity, and increases resistance to doxorubicin.
At present, no report is reported about the functions of PRDM16, IGSF9 and ALDH2 in the invasion and metastasis mechanism of malignant tumors such as breast cancer. Moreover, there is no kit for identifying (screening) tumor metastasis seed cells.
Disclosure of Invention
The invention aims to solve the diagnosis problem of tumor metastasis seed cells, and provides an immunohistochemical staining kit for detecting the tumor metastasis seed cells and application thereof.
Since the protein encoded by PRDM16 is a zinc finger transcription factor, this protein family is involved in signal transduction, controlling cell proliferation, differentiation and apoptosis. It is not expressed or is low expressed in tumor cells with high metastasis. IGSF9 is a member of the immunoglobulin superfamily that mediates cell-to-cell adhesion between similar and dissimilar cells. It is not expressed or is low expressed in tumor cells with high metastasis. ALDH2 is located in the mitochondrial matrix and plays a key role in maintaining the sternness of cancer cells and mediating microtubule inhibitor resistance, and ALDH2 is highly expressed in highly metastatic tumor cells.
Therefore, the detection of the protein expression of PRDM16, IGSF9 and ALDH2 plays an important role in the diagnosis, treatment, prognosis prediction and the like of high-metastasis tumor cells.
The invention is realized according to the following technical scheme:
an immunohistochemical staining kit for detecting tumor metastasis seed cells mainly comprises three protein immunohistochemical staining detection antibodies of cancer suppressor molecules PRDM16, IGSF9 and tumor desiccation molecules ALDH 2.
The immunohistochemical staining kit for detecting the tumor metastasis seed cells is characterized in that three protein immunohistochemical antibodies of the kit are human PRDM16, IGSF9 and ALDH2 monoclonal antibodies for detecting the expression degree of PRDM16, IGSF9 and ALDH2 proteins in isolated malignant tumor tissues.
The application of three protein immunohistochemical antibodies PRDM16, IGSF9 and ALDH2 in the preparation of an immunohistochemical staining kit for detecting tumor metastasis seed cells, wherein PRDM16 and IGSF9 with cancer inhibition function have low protein expression or expression loss in high-metastasis tumor cells, and tumor stem molecules ALDH2 have high protein expression in high-metastasis tumor cells, so that the tumor metastasis seed cells are screened.
The application of three protein immunohistochemical antibodies PRDM16, IGSF9 and ALDH2 in the preparation of an immunohistochemical staining kit for detecting tumor metastasis seed cells, and the protein expression level of the combination of protein molecules PRDM16, IGSF9 and ALDH2 provides an extremely important basis for the diagnosis, treatment and prognosis prediction of high metastasis tumor cells.
According to the invention designed in the way, the protein expression levels of three molecules of PRDM16, IGSF9 and ALDH2 in the isolated tumor tissue from the subject are jointly detected, the sequencing analysis is carried out on the tumor single cell clusters in the primary focus and the metastatic focus of the high metastatic breast cancer, the tumor metastatic seed cells with the metastatic capacity are screened out, and the important basis is provided for the accurate diagnosis and treatment and prognosis prediction of the tumor.
Drawings
FIG. 1 is a sequence chart of single cell mass in highly metastatic tumor tissue;
FIG. 2 is a graph comparing the similarity of genomic variations of tumor cells;
FIG. 3 is a schematic diagram of molecular modification of metastatic seed cells of a highly metastatic tumor;
FIG. 4 is an immunohistochemical detection map of IGSF9 of tumor cells;
FIG. 5 is an immunohistochemical map of PRDM16 of tumor cells;
FIG. 6 is an immunohistochemical assay of ALDH2 of tumor cells;
FIG. 7 is a graph of protein expression levels of three molecules as a function of patient survival time.
Detailed Description
The invention is further illustrated by the specific example of immunohistochemical detection of tumor tissue samples from patients with highly metastatic tumors.
1) Tumor tissues to be detected:
the tumor tissue excised by the operation of a patient is fixed, taken, dehydrated, embedded and HE stained according to the conventional method for pathological diagnosis, and 10 pieces of serial sections (4-6 microns thick/piece) are used for immunohistochemical detection of tumor metastasis seed cell identification. Referring to FIG. 1, the sequencing results of 50 single cell clusters in tumor tissue (accession number P18) of a patient with highly metastatic breast cancer are shown. Comprises 17 tumor single cell clusters obtained by laser capture micro-excision from tumor tissues of isolated tumor primary focuses and 11, 8 and 14 tumor single cell clusters obtained by respective excision from 3 metastatic lymph nodes (L01, L02 and L03). The genomic variation evolutionary tree revealed that the cell mass of the metastatic focus L2 evolved from a single cell mass in the primary focus and then evolved continuously in the metastatic focus, with the evolution process being T-L2-L3-L1. Accordingly, highly metastatic tumor seed cells were identified.
FIG. 2 shows the results of comparison of the genomic variation similarity between primary and metastatic tumor cells in 6 patients with highly metastatic tumors (P14, P16, P18, P19, P22, P23). Metastatic seed cells were found in each highly metastatic tumor tissue (T3, T1, T2, T3, T1 and T3).
Figure 3 shows that deletion of IGSF9 and PRDM16, ALDH2 expansion is a molecular alteration common to tumor metastasis seed cells.
2) Immunohistochemical detection kit for tumor metastasis seed cells
The protein based on PRDM16 is a zinc finger transcription factor, and the protein family is involved in signal transduction, controls cell proliferation, differentiation and apoptosis, and has cancer inhibiting effect. It is not expressed or is low expressed in tumor cells with high metastasis. IGSF9 is a member of the immunoglobulin superfamily that mediates cell-to-cell adhesion between similar and dissimilar cells. The cancer inhibiting effect is not expressed or is low expressed in high metastatic tumor cells. ALDH2 is located in the mitochondrial matrix and plays a key role in maintaining the sternness of cancer cells and mediating microtubule inhibitor resistance, and ALDH2 is highly expressed in highly metastatic tumor cells.
An immunohistochemical staining kit for detecting tumor metastasis seed cells mainly comprises three protein immunohistochemical antibodies of cancer suppressor molecules PRDM16, IGSF9 and tumor desiccating molecules ALDH 2.
The kit comprises: human PRDM16, IGSF9, ALDH2 monoclonal antibodies and immunohistochemical detection reagents for detecting the expression degree of PRDM16, IGSF9 and ALDH2 proteins in isolated malignant tumor tissues. Wherein, the ordering companies and the goods numbers of the three protein immunohistochemical antibodies are respectively: PRDM16(Affinity, DF13303), IGSF9(Sigma, HPA037753) and ALDH2(Abcam, ab 108306).
The immunohistochemical detection reagent in the kit comprises: xylene, ethanol, 3% H2O2Normal goat serum working solution for sealing, antibody diluent and biotin labelA secondary antibody working solution, a DAB chromogenic reagent, a horseradish peroxidase labeled chain avidin working solution, hematoxylin, PBS and EDTA antigen repairing solution.
The immunohistochemical detection steps of the kit are as follows:
(a) the prepared paraffin slides were baked (60 ℃, 2 hours), and then placed in a 60 ℃ incubator overnight.
(b) Dewaxing by xylene, soaking once for 20 minutes for about 60 minutes for three times, and hydrating by (100%, 95%, 80%, 75%) gradient alcohol;
(c) washing the slide with distilled water for 3 times, and washing with PBS buffer solution for one time;
(d) heating an autoclave filled with a proper amount of EDTA antigen repairing liquid to boiling, putting the prepared tumor tissue slices (white slices) for staining into the autoclave, and ensuring that the liquid submerges tissues to be stained on the slices; tightly covering the cooker cover, timing when the pressure cooker starts to jet air, stopping heating after 2 minutes and 30 seconds, opening the cooker cover, standing at room temperature, and naturally cooling the slices to be sliced;
(e) washing the slices with distilled water for 3 times, and then washing with PBS for 1 time;
(f) soaking the glass slide in 3% hydrogen peroxide for 10min to block the activity of endogenous peroxidase, washing with clear water, and washing with PBS for 1 time;
(g) taking out the section and slightly wiping off water around the tissue, dropwise adding goat serum on the tissue, placing at room temperature, sealing for 30 minutes, and covering the tissue with liquid to avoid non-specific staining;
(h) discarding serum, without washing, directly dripping diluted primary antibody on the tissue by using PBS as a negative control, placing the tissue in a wet box, and placing the wet box in a refrigerator at 4 ℃ for incubation overnight;
(i) taking out the wet box, and then putting the wet box at room temperature for rewarming for about 60 minutes;
(j) PBS washes 3 times, 5 minutes each;
(k) slightly wiping off water around the tissue by using absorbent paper, dripping secondary antibody on the tissue, and then placing the tissue in a humid box to incubate for 30 minutes at 37 ℃;
(l) PBS washes 3 times, 5 minutes each;
(m) wiping off water around the tissue by using absorbent paper, dripping horse radish peroxidase on the tissue, and then placing the tissue in a wet box to incubate for 30 minutes at 37 ℃;
(n) PBS wash 3 times, 5 minutes each;
(o) wiping water around the tissue by using absorbent paper, dropwise adding DAB (1: 20) which is freshly prepared and stored in a dark place on the tissue, standing at room temperature for developing for 1-3 minutes, observing a developing result under a microscope, and once positive control is sufficiently developed, washing the slide for multiple times by using running water to stop developing;
(p) counterstaining hematoxylin for 3 minutes, cleaning redundant hematoxylin by clear water, placing the slide in a hydrochloric acid water solution for several seconds for differentiation, washing by tap water, then placing the slide in ammonia water for several seconds for returning blue, and washing by tap water;
(q) dehydration was performed with a gradient of 80%, 95%, 100% alcohol concentration, and then placed in xylene for 10 minutes each time for 2 permeabilizations. Followed by mounting with neutral gum.
3) Joint detection and evaluation of expression of three proteins PRDM16, IGSF9 and ALDH2
Attached watch
The attached table shows that the PRDM16 and IGSF9 protein low expression/deletion and ALDH2 protein high expression of the tumor cells are positively correlated with the lymph node metastasis number of the tumor cells.
Evaluation criteria of immunohistochemical results of PRDM16 protein: the tumor cell nucleus is brown and is the expression positive tumor cell. Staining intensity and scores were dark brown (3 points ═ strong staining), dark brown (2 points ═ medium intensity staining), light brown (1 points ═ weak staining), no staining (0 points ═ no staining); the percentage of positive cells of the tumor is more than 75% (4 min), 51-75% (3 min), 26-50% (2 min), 1% -25% (1 min) and 0% (0 min). Performing statistical analysis according to the percentage of the intensity x, wherein the product of the two scores is more than or equal to 6, and the PRDM16 protein expression is defined as high expression; PRDM16 protein expression was defined as low expression if the product of the two scores was <6, see fig. 5, showing low expression (foci) and absence of PRDM16 in highly metastatic tumor cells.
Evaluation criteria for immunohistochemical results of IGSF9 protein expression: the tumor cytoplasm is expressed as positive tumor cells. The staining intensity of the positive cells of the tumor is classified into dark brown (3 points are strong staining), dark brown (2 points are medium staining), light brown (1 point is weak staining), and no staining (0 point is colorless); the percentage of positive cells in the tumor was > 50% (3 min), 25-50% (2 min), 1-25% (1 min), 0% (0 min). Statistical analysis was performed as intensity x percent, with the product of the two scores >4, and IGSF9 protein expression was defined as high expression group; if the product of the two scores is less than or equal to 4, the expression of IGSF9 protein is defined as a low expression group, see FIG. 4, which shows that IGSF9 has no expression in highly metastatic tumor cells.
Evaluation criteria for immunohistochemical results of ALDH2 protein expression: the tumor cytoplasm is expressed as positive tumor cells. The staining intensity of the positive cells of the tumor is classified into dark brown (3 points are strong staining), dark brown (2 points are medium staining), light brown (1 point is weak staining), and no staining (0 point is colorless); the percentage of positive cells in the tumor was > 50% (3 min), 25-50% (2 min), 1-25% (1 min), 0% (0 min). Performing statistical analysis according to the intensity x percentage, wherein the product of two scores is greater than 4, and the ALDH2 protein expression is defined as a high expression group; when the product of the two scores is less than or equal to 4, the ALDH2 protein expression is defined as a low expression group, see FIG. 6, which shows that ALDH2 is highly expressed in highly metastatic tumor cells.
4) Screening of tumor metastasis seed cells
The application of antibodies of three proteins of PRDM16, IGSF9 and ALDH2 in immunohistochemical staining kit for preparing tumor metastasis seed cells, PRDM16 and IGSF9 with cancer inhibition function have low protein expression or expression loss in high-metastasis tumor cells, and tumor stem molecules ALDH2 have high protein expression in high-metastasis tumor cells, so that the tumor metastasis seed cells are screened.
And (3) screening out the tumor metastasis seed cells with low/no expression of IGSF9 and PRDM16 and high expression of ALDH2 according to the evaluation standard of the immunohistochemical results expressed by the three proteins. The tumor tissue in which the seed cell for tumor metastasis is detected has a strong metastatic ability, and the prognosis of the patient is poor. Referring to FIG. 7, the relationship between the protein expression levels of three molecules (PRDM16, IGSF9, ALDH2) of tumor metastasis seeds and the survival time of tumor patients is shown. The lower the expression of IGSF9, PRDM16 protein and the higher the expression of ALDH2 protein, the shorter the survival time of the patient.
The application of antibodies of three protein immunohistochemistry of PRDM16, IGSF9 and ALDH2 in the preparation of an immunohistochemical staining kit for detecting tumor metastasis seed cells, and the protein expression level of the combination of protein molecules PRDM16, IGSF9 and ALDH2 provides an extremely important basis for diagnosis, treatment and prognosis prediction of high metastasis tumor cells.
According to the experimental results, the conclusion can be drawn that the kit for jointly detecting the protein expression of PRDM16, IGSF9 and ALDH2 by using the immunohistochemical method disclosed by the invention is applied to the identification of tumor metastasis seed cells, namely the low/loss expression of IGSF9 and PRDM16 and the high expression characteristic of ALDH2 can provide direct basis for clinically formulating an accurate treatment scheme and predicting the prognosis of patients.
Claims (4)
1. An immunohistochemical staining kit for detecting tumor metastasis seed cells, which is characterized in that: the kit mainly comprises three protein immunohistochemical antibodies of cancer suppressor molecules PRDM16, IGSF9 and tumor sternness molecules ALDH 2.
2. The immunohistochemical staining kit for detecting tumor metastasis seed cells according to claim 1, characterized in that: the three protein immunohistochemical antibodies are human PRDM16, IGSF9 and ALDH2 monoclonal antibodies for detecting the expression degree of PRDM16, IGSF9 and ALDH2 proteins in isolated malignant tumor tissues.
The application of three protein immunohistochemical antibodies PRDM16, IGSF9 and ALDH2 in the preparation of an immunohistochemical staining kit for detecting tumor metastasis seed cells is characterized in that: PRDM16 and IGSF9 with cancer inhibiting effect are low expressed or lack of expression in the protein of high metastatic tumor cells, and the tumor stem molecule ALDH2 is high expressed in the protein of high metastatic tumor cells.
The application of three protein immunohistochemical antibodies PRDM16, IGSF9 and ALDH2 in the preparation of an immunohistochemical staining kit for detecting tumor metastasis seed cells is characterized in that: the protein expression level of the combination of the protein molecules PRDM16, IGSF9 and ALDH2 provides an extremely important basis for diagnosis, treatment and prognosis prediction of high-metastasis tumor cells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011044088 | 2020-09-28 | ||
| CN2020110440889 | 2020-09-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114280303A true CN114280303A (en) | 2022-04-05 |
Family
ID=80868096
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011237078.7A Pending CN114280303A (en) | 2020-09-28 | 2020-11-09 | Immunohistochemical staining kit for detecting tumor metastasis seed cells and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114280303A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251013A (en) * | 2011-02-22 | 2011-11-23 | 北京市肿瘤防治研究所 | Antibody and antigen for recognizing tumor initiator cell and application thereof |
| CN102329871A (en) * | 2011-09-29 | 2012-01-25 | 中南大学 | Application method of low-methylation gene PRDM 16 (PR domain containing 16) |
| US20130331281A1 (en) * | 2012-03-29 | 2013-12-12 | National Health Research Institutes | Molecular markers for prognostically predicting prostate cancer, method and kit thereof |
| US20160061842A1 (en) * | 2013-04-15 | 2016-03-03 | Cedars-Sinai Medical Center | Methods for detecting and treating cancer metastasis |
| CN110563845A (en) * | 2019-09-12 | 2019-12-13 | 滨州医学院 | anti-IGSF 9 antibody, pharmaceutical composition and application thereof |
-
2020
- 2020-11-09 CN CN202011237078.7A patent/CN114280303A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251013A (en) * | 2011-02-22 | 2011-11-23 | 北京市肿瘤防治研究所 | Antibody and antigen for recognizing tumor initiator cell and application thereof |
| CN102329871A (en) * | 2011-09-29 | 2012-01-25 | 中南大学 | Application method of low-methylation gene PRDM 16 (PR domain containing 16) |
| US20130331281A1 (en) * | 2012-03-29 | 2013-12-12 | National Health Research Institutes | Molecular markers for prognostically predicting prostate cancer, method and kit thereof |
| US20160061842A1 (en) * | 2013-04-15 | 2016-03-03 | Cedars-Sinai Medical Center | Methods for detecting and treating cancer metastasis |
| CN110563845A (en) * | 2019-09-12 | 2019-12-13 | 滨州医学院 | anti-IGSF 9 antibody, pharmaceutical composition and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| HUAN-MING HSU等: "six novel immunoglobulin genes as biomarkers for better prognosis in triple-negative breast cancer by gene co-expression network analysis", 《SCIENTIFIC REPORTS》, vol. 9, 14 March 2019 (2019-03-14), pages 3 - 4 * |
| PAOLA MARCATO等: "Aldehyde Dehydrogenase Activity of Breast Cancer Stem Cells is Primarily Due to Isoform ALDH1A3 and Its Expression is Predictive of Metastasis", 《STEM CELLS》, 31 December 2011 (2011-12-31), pages 42 * |
| 费良儒: "PRDM16抑制肺腺癌转移的机制研究", 《中国优秀博士学位论文全文数据库》, vol. 2020, no. 01, 15 January 2020 (2020-01-15) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2619321B1 (en) | Biomarkers for differentiating melanoma from benign nevus in the skin | |
| JP5683108B2 (en) | Cancer biomarkers | |
| Savci-Heijink et al. | The role of desmoglein-3 in the diagnosis of squamous cell carcinoma of the lung | |
| CN107085106B (en) | Detection reagent and kit for prostate cancer progression prognosis | |
| US20110306513A1 (en) | Novel biomarker for liver cancer and applications for same | |
| Matsumoto et al. | A novel tumor marker, Niban, is expressed in subsets of thyroid tumors and Hashimoto's thyroiditis | |
| CN110187110B (en) | Cardiac cancer prognosis prediction marker and application thereof | |
| JP5747419B2 (en) | Methods and compositions for detecting lethal cells and uses thereof | |
| US20230136619A1 (en) | Dcis recurrence and invasive breast cancer | |
| Peiro et al. | Analysis of HER-2/neu amplification in endometrial carcinoma by chromogenic in situ hybridization. Correlation with fluorescence in situ hybridization, HER-2/neu, p53 and Ki-67 protein expression, and outcome | |
| Kinugasa et al. | Overexpression of transforming growth factor-β1 in scirrhous carcinoma of the stomach correlates with decreased survival | |
| Wells et al. | Expression of “Spot 14”(THRSP) predicts disease free survival in invasive breast cancer: immunohistochemical analysis of a new molecular marker | |
| US11821900B2 (en) | Method of selection for treatment of subjects at risk of invasive breast cancer | |
| US20200232039A1 (en) | Adm2 gene marker for diagnosis or prognosis prediction of thyroid cancer and uses thereof | |
| SU et al. | Decreased expression of KAI1 metastasis suppressor gene is a recurrence predictor in primary pTa and pT1 urothelial bladder carcinoma | |
| CN114280303A (en) | Immunohistochemical staining kit for detecting tumor metastasis seed cells and application thereof | |
| US7267951B2 (en) | Method for evaluating a tissue or biopsy sample to determine if the sample is early-stage melanoma | |
| US20140248637A1 (en) | Composition for diagnosis of lung cancer and diagnosis kit of lung cancer | |
| CN111948395A (en) | Quadruple marker for diagnosing immune regulation subtype of triple negative breast cancer and application thereof | |
| CN114705863B (en) | Application of SSH3 protein in cardiac adenocarcinoma prognosis prediction | |
| Terada et al. | N-myc downstream regulated gene-1 may play an important role in the prognosis of ovarian cancer, in its association with beta-catenin | |
| KR20120004736A (en) | A method for diagnosing the risk of lymph node metastasis in papillary thyroid carcinoma | |
| US20140356868A1 (en) | Macc1 as a prognostic biomarker for hepatobiliary tumors | |
| CN117092342A (en) | Application of CHD4 protein in prognosis prediction of esophageal squamous carcinoma | |
| CN113671187A (en) | IHC detection kit for detecting MYCN protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |