CN117706091A - Application of cells with high NIK expression as therapeutic targets for controlling tumor deterioration - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to application of cells with high NIK expression as a treatment target for controlling tumor deterioration. Specifically, the invention identifies the endothelial cells and B lymphocytes with high NIK expression as malignant cells, promotes tumorigenesis, and can delay tumorigenesis and prolong the survival time by specifically treating the malignant cells.

Description

Application of cells with high NIK expression as therapeutic targets for controlling tumor deterioration

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application of cells with high NIK expression as a treatment target for controlling tumor deterioration.

Background

NF-. Kappa.B (kappa.light chain enhancement of B cells activated by nuclear factors) is a protein complex that controls DNA transcription, cytokine production and cell survival. NF-. Kappa.B is present in almost all animal cell types and is involved in the response of cells to external stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet radiation, oxidized low density lipoproteins and bacterial or viral antigens. NF- κB plays a key role in regulating the immune response to infection. Incorrect regulation of NF- κb is not associated with cancer, inflammatory and autoimmune diseases, septic shock, viral infection and immune development.

In the classical (classical) signaling pathway, NF-. Kappa.B/Rel proteins bind to and are inhibited by IκB proteins. In an alternative (or called non-classical) NF- κB pathway, the NF- κB2p100/RelB complex is inactivated in the cytosol. Signal transduction through a subset of receptors, including ltβ R, CD40 and BR3, activates the kinase NIK, which in turn activates the ikkα complex, which phosphorylates the C-terminal residue of NF- κb2p100. Phosphorylation of NF- κb2p100 leads to ubiquitination itself and proteasome processing to NF- κb2p52. Thus, NF- κBp52/RelB complex with transcription ability can be generated and transferred into nucleus to induce the target gene expression.

B lymphocytes may also be abbreviated as B cells. Multipotent stem cells derived from bone marrow. It is slightly larger in volume than T lymphocytes. Such lymphocytes, upon antigen stimulation, proliferate and differentiate into a large number of plasma cells. Plasma cells can synthesize and secrete antibodies and circulate in the blood. Mature B cells migrate out through peripheral blood, enter spleen and lymph nodes, are mainly distributed in spleen nodules, spleen chordae and lymph nodules, lymph chordae and lymph nodules under alimentary canal mucosa, differentiate and proliferate into plasma cells after being stimulated by antigen, synthesize antibodies and play a role of humoral immunity.

Disclosure of Invention

In order to accurately target malignant cells and effectively control tumor deterioration, a group of markers are screened, and the cells which express the markers are targeted for treatment can be proved to obtain good treatment effect.

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides a panel of screening markers for malignant cells comprising at least one of Nik (Nik, NF- κb-induced kinase), CD19, cxcl13, cxcr5, CD34.

Preferably, the marker is a combination of Nik and at least one of CD19, cxcl13, cxcr5, CD34.

In another aspect, the invention provides a marker for screening for malignant B cells or malignant endothelial cells, the marker comprising at least one of Nik, cxcl13, cxcr 5.

In another aspect, the invention provides the use of the above screening markers in the preparation of malignant cell screening products, cancer diagnostic products.

Preferably, the malignant cells comprise malignant B cells or malignant endothelial cells.

Preferably, malignant B cells are screened for use in combination with B cell markers, preferably cell surface markers. Exemplary B cell markers include: CD1, CD21, CD27, CD138, CD19, CD25, CD30, CXCR3, CXCR4, CXCR5, CXCR6, igA, igG, igE, CD, CD27, CD40, CD80, PDL-2, igD, CD1, CD5, CD21, CD24, TLR4, igD, CD21, CD22, CD23, igG, CD27, CD38, CD78, CD138, CD319, igM, CD19, IL-10, TGF beta, IL-6, notch2.

Preferably, the B cell surface marker comprises CD19.

Preferably, screening for malignant endothelial cells may be used in combination with endothelial cell markers, preferably cell surface markers. Exemplary endothelial cell markers include CD31, CD 45, CD34, ICAM-1/CD54, LYVE-1, tie-2/Tek, VCAM-1/CD 106, VE cadherin, VEGF-R2, von Willebrand factor (vWF)

Preferably, the endothelial cell surface marker comprises CD34.

The endothelial cells and CD19 positive B lymphocytes obtained by the screening promote tumorigenesis and are therefore called malignant cells.

More specifically, whether the screening marker of the malignant cells in the sample to be tested is positive/strong positive is detected, and if the detection result is positive, the test subject suffers from cancer.

Preferably, the assay may be one or more of a cellular activity assay, an immune-based assay, a flow cytometer assay, a colorimetric assay, a gold nanoparticle-based assay, a fluorescent assay, an ultraviolet assay;

preferably, the detection may be performed on the above screening markers, proteins of the markers, or on nucleic acids.

Preferably, the method used for protein retrieval includes hematoxylin-eosin staining (HE staining method for short), safranin O-fast green staining, western Blot (Western Blot method), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical (immunochemistry) staining method, mass spectrometry, immunoprecipitation assay, complement-binding assay, flow cytometry, and protein chip method.

Preferably, the method used for detection of nucleic acids comprises: PCR-based detection methods, southern hybridization methods, northern hybridization methods, dot hybridization methods, fluorescent in situ hybridization methods, DNA microarray methods, ASO methods, high throughput sequencing platform methods.

Preferably, the sample to be tested comprises peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.

Preferably, the sample to be tested is tissue. More preferably, the cancer tissue.

Preferably, the sample to be tested may also be a cell population suspected of containing malignant cells.

More specifically, the present invention provides methods for diagnosing cutaneous Squamous Cell Carcinoma (SCC) by detecting Nik in cancerous tissue of a subject. Also provided are methods of detecting CD19 and NIK, and thus diagnosing cancer, which are applicable to all types of cancer.

In another aspect, the invention provides the use of products targeting the above screening markers, malignant cells in the preparation of a medicament for cancer.

Preferably, the product comprises a compound, an antibody, having specific binding capacity to a screening marker, a marker.

Preferably, the antibodies include monoclonal antibodies, single chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies.

Specifically, the products targeting the above screening markers, markers may also be referred to as inhibitors of the screening markers, markers.

Preferably, the inhibitor comprises an agent that reduces NIK expression by: RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP gene recombination technology.

Preferably, the product contains CD20 antibodies.

Preferably, the antibodies include monoclonal antibodies, single chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies.

In another aspect, the invention provides the use of a CD20 antibody for the preparation of a product for reducing the increase in expression of CD19 by NIK.

Preferably, the inhibitor comprises an agent that reduces NIK expression by: RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP gene recombination technology.

Preferably, the inhibitor comprises a compound or an antibody having specific binding capacity to CD 20;

preferably, the antibodies include monoclonal antibodies, single chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies.

Preferably, the CD20 inhibitor is a CD20 antibody.

As used herein, the screening markers, refer to biochemical indicators that can label changes or changes in system, organ, tissue, cell and subcellular structure or function, with very broad utility. The method not only can discuss pathogenesis from molecular level, but also has unique advantages in accurately and sensitively evaluating early and low-level damage, can provide early warning, prognosis curative effect analysis, accurate stage parting basis of diseases and the like, and provides auxiliary diagnosis basis for clinicians to a great extent.

As used herein, "cancer" and "tumor" are interchangeable terms that refer to the growth or proliferation of any abnormal cell or tissue in an animal. As used herein, the terms "cancer" and "tumor" encompass solid cancers and blood/lymph cancers, and also encompass malignant, premalignant, and benign growths, such as dysplasia. Cancers of the invention include adenocarcinoma, squamous carcinoma, large cell carcinoma, small cell carcinoma, examples of which include, but are not limited to, cervical cancer, seminoma, lymphoma (including non-hodgkin's T-cell lymphoma, non-hodgkin's diffuse large B-cell lymphoma, non-hodgkin's B-cell lymphoma), prostate cancer, ovarian cancer, lung cancer (e.g., small cell lung cancer SCLC, non-small cell lung cancer NSCLC, lung adenocarcinoma), rectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, epithelial cancer (including urothelial cancer), leukemia (e.g., acute lymphoblastic leukemia ALL, acute myelogenous leukemia AML, chronic myelogenous leukemia CML, chronic lymphocytic leukemia CLL); brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female genital tract cancer, carcinoma in situ, neurofibromatosis, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, ductal carcinoma.

The term "treatment" as used herein includes the use of the oligonucleotides provided herein to prevent or delay the appearance of symptoms and complications of a disease, such as a tumor. Treatment may also be prophylactic. Treatment of tumors also refers to controlling tumor progression in individuals, prolonging survival of tumor patients, improving quality of life, alleviating symptoms, shrinking or even eliminating tumors, and suppressing tumor metastasis. Antitumor effects include treatment of tumors, as well as prevention of tumorigenesis, recurrence, and metastasis.

Drawings

FIG. 1 is an image of Nik-toe mice and WT mice.

Fig. 2 is a graph of the results of detecting Nik.

FIG. 3 is a graph showing the results of staining markers Ki67, SOX-9, CD34, CD31, P40 and P63.

Figure 4 is a statistical result of the survival of mice.

Figure 5 is an RNA sequencing hint that NIK induced CXCL13 and CXCR5 elevation.

Fig. 6 shows the results of CXCL13, CXCR5 and CD19 detection.

Fig. 7 shows the detection result of CD34.

FIG. 8 is a graph showing the results of NIK positive TEK cells promoting B cell proliferation and verifying CD20 versus B cell reduction.

FIG. 9 shows that TEK positive cells can differentiate into approximately 4% B cells.

Figure 10 is a statistical result of the survival of mice.

FIG. 11 is a graph of the results of detection of Nik in clinical samples.

FIGS. 12-17 are graphs showing the detection results of Nik and CD19 in clinical samples.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Reagent(s)	Manufacturer' s	Goods number
			CD20 antibody	BIOLEGEND	152104
CD 19 antibody	CST	90176
			CD19-APC antibody	Invitrogen	17-0193-80
NF-KB2 antibody	CST	4884
			Human-NIK antibody	R&D	MAB6888R
Human-NIK antibody	Invitrogen	Pa5-100732
			Cxcl13 antibody	invitrogen	PA5-47035
CXCR5 antibody	CST	72172
			Cxcl13 antibody	R&D	AF470

Example one, construction of Nik-toe mouse model

Tek-cre ^+/+ Mice and Nik-stop ^fl/+ Mice were hybridized and Tek-cre was obtained ^+/- -Nik-stop ^fl/+ Mice (hereinafter referred to as Nik-toe mice) in which Tek-cre ^+/+ Mice were purchased from Jizhikang, nik-stop ^fl/+ Reference is made to article High expression of NF-kappa B inducing kinase in the bulge region of hair follicle induces tumor. Animals were kept under controlled conditions (12 hours light/dark cycle, 60.+ -. 10% relative humidity, 22.+ -. 1 ℃ room temperature).

FIG. 1 shows that the model is successfully constructed, the HE staining result is shown in FIG. 2A, the western blot result shows that the expression level of Nik is improved (FIG. 2B), and the Ki67, SOX-9, CD34, CD31, P40 and P63 staining pairs in the Nik-toe mice and the wild-type mice are shown in FIG. 3, and the survival statistics are shown in FIG. 4.

Example two screening for Key markers

1. Mu.g of RNA was prepared for each sample to generate an RNA library. The PCR products were sequenced on the Illumina platform. Total cytokine and cytokine receptor expression were analyzed between the tissues of the Nik-toe mouse and the control group, and between the tissues of the Nik-toe mouse and the control group, to give a cytokine expression pattern shown in FIG. 5

Nik-toe-G/R three gene expression mice were established, tumor sections were washed three times with 0.3% Triton X-100/PBS and blocked with 10% horse serum for 30 minutes. The sections were then incubated with Hoechst 33342 for 1 hour at room temperature. Green GFP fluorescence was activated with 488nm laser and red Tomato fluorescence was activated at 546 nm. The results of observations under confocal fluorescence microscopy (Olympus FV 1000) are shown in fig. 6A-C. The high expression of NIK in CD19 positive cells was confirmed in Nik-toe-G/R mice by labeling CD19, CXCL13, CXCR5 and CD34, detecting the expression of CD19, CXCL13, CXCR5 and CD34, etc. The results of the hematopoietic stem/progenitor cell marker CD34 are shown in fig. 7, indicating that NIK is expressed on CD34 cells, resulting in endothelial cell and CD19 positive B lymphocyte expression and promoting tumorigenesis.

The B cells highly expressing NIK may be potential therapeutic targets for controlling tumor progression, and the B cells highly expressing NIK have CXCL 13-positive, CXCR 5-positive and CD 19-positive cell populations and can be used as markers to indicate cancer risk.

Example III detection of CD19 expression Change in CD20 treatment

3 NIK-Toe mice were given intraperitoneal injections of CD20, 25 ug/mouse, 2/week, and one week later, the expression levels of CD19 and CD3 were examined by flow cytometry. The results showed a significant decrease in CD19 in the mouse samples of the CD20 treated group. Furthermore, normal TEK-positive cells can differentiate into 4% of B lymphocytes, and NIK-highly expressed TEK cells promote B cell proliferation (fig. 8-9).

NIK-Toe mice were given 5 CD20 treatments, i.p. injections, 25 ug/2/week. The survival was prolonged by 21 days (FIG. 10, con represents non-intervening Nik-toe mice). The results show that high expression of NIK in B lymphocytes promotes tumorigenesis, and removal of B lymphocytes by CD20 antibodies delays tumorigenesis.

Example IV, verification of diagnostic function of CD19, NIK by clinical samples

Clinical samples of SCC were collected for staining and found to be present with Nik positive cells in SCC patients (fig. 10).

In addition, 197 clinical samples were collected to detect CD19 and NIK, and strong positive expression of CD19 and NIK was detected. Sample number, sampling position and disease diagnosis results are shown in table 1, and partial detection results are shown in fig. 11 to 16.

TABLE 1 clinical sample information

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims (10)

1. A panel of malignant cell screening markers comprising at least one of Nik, CD19, cxcl13, cxcr5, CD 34;

preferably, the marker is a combination of Nik and at least one of CD19, cxcl13, cxcr5, CD34.

2. A marker for screening for malignant B cells or malignant endothelial cells, the marker comprising at least one of Nik, cxcl13, cxcr 5.

3. Use of the detection reagent of the marker of claim 1 or 2 for the preparation of a malignant cell screening product and a cancer diagnosis product;

preferably, the malignant cells comprise malignant B cells or malignant endothelial cells;

preferably, the screening product for malignant B cells further comprises a B cell marker detection reagent;

preferably, the B cell marker comprises CD19;

preferably, the screening product for malignant endothelial cells further comprises an endothelial cell marker detection reagent;

preferably, the endothelial cell marker comprises CD34.

4. The use of claim 3, wherein the method of detection comprises one or more of cellular activity detection, immune-based detection, flow cytometer detection, colorimetric detection, gold nanoparticle-based detection, fluorescent detection, ultraviolet detection;

preferably, the detection protein is either detected;

preferably, the method used for protein retrieval includes hematoxylin-eosin staining, safranin O-fast green staining, western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry fluorescence assay, and protein chip method;

preferably, the method used for detection of nucleic acids comprises: PCR-based detection methods, southern hybridization methods, northern hybridization methods, dot hybridization methods, fluorescent in situ hybridization methods, DNA microarray methods, ASO methods, high throughput sequencing platform methods.

5. The use according to claim 3, wherein the sample to be tested comprises peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucous, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid;

preferably, the test sample further comprises a population of cells suspected of containing malignant cells;

preferably, the sample to be tested is tissue; more preferably, the cancer tissue.

6. The use of claim 3, wherein the cancer comprises adenocarcinoma, squamous carcinoma, large cell carcinoma, small cell carcinoma, lymphoma;

in particular, the cancer includes melanoma, fibrosarcoma, myxosarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat adenoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, neuroblastoma, craniopharyngema, ventricular omenings, pineal tumor, angioblastoma, auditory glioma, oligodendroglioma, meningioma, neuroblastoma, gastric cancer, and anterior carcinoma.

7. Use of a product targeting a marker according to claim 1 or 2, a cell expressing a marker according to claim 1 or 2, for the preparation of a medicament for cancer;

preferably, the product comprises a compound, an antibody, having the ability to specifically bind to a marker according to claim 1 or 2;

preferably, the antibodies include monoclonal antibodies, single chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies.

8. The use of claim 7, the product further comprising the inhibitor targeting the marker of claim 1 or 2;

preferably, the inhibitor comprises an agent that reduces NIK expression by: RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP gene recombination technology.

9. The use of claim 7, wherein the cancer comprises adenocarcinoma, squamous carcinoma, large cell carcinoma, small cell carcinoma, lymphoma;

in particular, the cancer includes melanoma, fibrosarcoma, myxosarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat adenoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, neuroblastoma, craniopharyngema, ventricular omenings, pineal tumor, angioblastoma, auditory glioma, oligodendroglioma, meningioma, neuroblastoma, gastric cancer, and anterior carcinoma.

Use of a CD20 inhibitor for the preparation of a product for reducing the increase in expression of CD19 by Nik;

preferably, the inhibitor comprises an agent that reduces NIK expression by: RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP gene recombination technology.

Preferably, the inhibitor comprises a compound or an antibody having specific binding capacity to CD 20;

preferably, the antibodies include monoclonal antibodies, single chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies.

Preferably, the CD20 inhibitor is a CD20 antibody.