CN115112892B - Methods, uses and kits for determining surgical margins of cancer - Google Patents

Abstract

The invention discloses a method, application and kit for determining a cancer surgical margin by using CREPT. The invention determines the region of CREPT positive cells through means such as immunohistochemical staining of CREPT so as to identify tissues with high risk of in-situ recurrence and guide cancer operation cutting edge. The present invention finds that elevated levels of CREPT expression are accompanied by the process of cancer cells promoting normal cellular canceration by secretion of tumor exosomes. Whereas CREPT acts as a carcinotropic protein, its elevation implies a high risk of cancerous and/or recurrent states of the cell. Therefore, the invention can realize the identification of the tissue region with high canceration risk through the detection of the CREPT level.

Description

Methods, uses and kits for determining surgical margins of cancer

Technical Field

The invention relates to the technical field of molecular biology, in particular to a method, application and kit for determining the surgical margin of a cancer tumor by using CREPT, a kit for identifying normal tissue cells and tissue cells with gene level precancerous lesions, a kit for indicating tissue cells with high risk of canceration, a kit for indicating tissue cells with inflammatory formation, a kit for indicating tissue cells with strong proliferation capability, a kit for indicating tissue cells with reduced apoptosis occurrence, and a kit for judging the recurrence risk of paracancerous tissue.

Background

Surgical resection is a radical and better prognosis approach in current tumor treatment methods, but physicians often rely on experience and rapid pathological tests in surgery, such as immunohistochemistry, in determining resection boundaries, which can only identify pathological altered paracancerous tissues, such as atypical hyperplastic tissues, but cannot effectively identify paracancerous tissues whose pathology has not been altered but whose gene expression profile has been altered before cancer, so recurrence after tumor resection is very common.

The concept of regional canceration was first proposed by Slaughter in 1953, who studied tumor specimens of 783 oral squamous carcinoma patients, found that all paracancerous regions were pathologically altered (e.g., hyperproliferative and keratinized), and that 11.2% of patients had two or more foci (Slaughter et al., 1953). The pathological change of the paracancerous tissues they find is a classical regional carcinomatous phenomenon.

As the phenomenon of regional canceration is found in various solid tumors, it is recognized that the division of the past pathological boundaries is insufficient to distinguish cells that have been changed prior to the occurrence of cancer. These precancerous cells may become cancerous during the course of tumor development, causing a situation of multiple foci, causing resistance to the tumor, and may also develop into new tumors after excision of the primary tumor, which may appear as recurrence in situ. The surgeon needs to balance the size of the resected area with the risk of tumor recurrence, such as the surgical format and scope of breast conservation for breast in situ ductal carcinoma. Therefore, there is also a need to find the molecular boundaries of tumors, and in particular to provide biomarkers that simply and effectively distinguish normal cells from those where pre-cancerous lesions have occurred at the gene level, to guide the surgeon in resecting these cells, which are highly likely to be cancerous, thereby reducing the recurrence rate of the tumor.

Current research is generally focused on exploring what changes have occurred in the tissues beside the cancer, and in cancers such as pancreatic cancer, prostate cancer and breast cancer, researchers find that the tissues beside the cancer seem to be normal through means such as sequencing and the like, and the changes are indeed related to the probability of in-situ recurrence. In particular, in breast cancer, not only is the transcriptional profile of the paracancerous tissue found to change, but also the telomere length is found to change, as is the methylation level of DNA, and these changes are correlated with distance from the in situ tumor, as well as affecting in situ recurrence of the tumor.

However, there is no study on the cause of canceration in the paracancerous normal tissue generation region. There are studies showing that tumor cells secrete soluble factors such as E-cadherein (Patil et al 2015) that promote canceration of normal epithelial cells. In addition, studies have suggested that exosomes secreted by tumor cells (a vesicle) may be involved in regional canceration of paracancerous tissue (AMIRRAD ET al, 2020), and that tumor cells may induce canceration of normal epithelial cells by secreting exosomes (bertoli et al, 2020; melo et al, 2014; wu et al, 2019; yoon et al, 2022). Multifocal and recurrent bladder cancer is considered an effect of regional carcinomatosis. Researchers have found that chronic exposure of non-malignant human urothelial cells SV-HUC to bladder cancer extracellular vesicles can trigger endoplasmic reticulum stress and promote up-regulation of IRE1 and NF-kB and down-regulation of pro-apoptotic protein CHOP, ultimately leading to cell canceration (Wu et al, 2019). Various research evidences suggest that tumor exosomes can induce regional canceration of paracancerous normal tissues as a carcinogen.

Therefore, the accurate discovery of the regional cancer causes is helpful to find reliable biomarkers so as to distinguish completely normal cells from cells with precancerous lesions on the gene level, help surgeons to accurately determine the surgical margin positions, and not only reduce postoperative recurrence of tumor resection to the greatest extent, but also protect normal tissues and functions of patients to the greatest extent. This will play an important role in the treatment of many tumors, such as breast cancer breast surgery, partial excision of stomach cancer, early stage ovarian cancer, etc. There is currently no method and tool for more precisely determining the surgical margin of cancer.

Disclosure of Invention

The invention aims to solve the technical problems of determining the surgical margin during the resection of the cancer tumor more accurately, so as to prevent the in-situ recurrence of the tumor and unavoidable multiple surgical wounds caused by the fact that the tissue with risk of canceration is not resected as much as possible while retaining normal tissues as much as possible.

In order to solve the above technical problem, the present invention provides, in a first aspect, a method of determining a surgical margin of cancer, the method comprising:

Detecting in a paracancerous tissue from a subject the expression level of a marker which is a CREPT (tumor cell cycle associated and expression enhancing protein) and a mutant having an identity of 85% or more, 90% or more, 95% or more, 98% or more or 99% or more;

And comparing the expression level of the marker with the expression level in a control sample from normal tissue of the subject or a healthy subject,

Wherein the tissue to be resected is determined to be a tissue requiring resection when the expression level of CREPT in the paracancerous tissue is increased by 10% or more, 20% or more, 50% or more, 100% or more, or 200% or more as compared to the expression level in the control sample.

In embodiments of the methods of the invention, the expression level of CREPT in the atypical proliferative tissue and in the paracancerous tissue of the surgical subject, where the gene expression profile is changed pre-cancerous but the pathology has not been altered, is increased compared to the expression level in the control sample.

In an embodiment of the method of the invention, the cancer is a solid cancer, such as cervical cancer, breast cancer, ovarian cancer, melanoma, colon cancer.

In an embodiment of the method of the invention, the expression level of the marker is detected by one or more of the following means:

immunohistochemistry, western immunoblotting, or real-time fluorescent quantitative PCR.

In a second aspect, the invention provides the use of a marker CREPT as a biomarker to be detected in the manufacture of a kit for determining the surgical margin of cancer, the kit being for performing the method of determining the surgical margin of cancer as described above,

The kit comprises:

A molecule that specifically recognizes the CREPT, and

Reagents for carrying out the detection of CREPT by said molecules specifically recognizing CREPT.

In an embodiment of the use according to the invention, the molecule specifically recognizing the marker is an antibody specifically recognizing the marker or a small molecule compound specifically binding to CREPT.

In an embodiment of the use according to the invention, the reagent for carrying out the detection of CREPT by the molecule specifically recognizing CREPT comprises: a detectable or chromogenic label conjugated or directly or indirectly specifically bound to a molecule that specifically recognizes said CREPT.

The present invention provides in a third aspect a kit for determining a surgical margin of a cancer, the kit comprising:

a molecule that specifically recognizes the CREPT,

Reagents for carrying out the detection of CREPT by said molecules specifically recognizing CREPT.

In an embodiment of the kit of the invention, the molecule that specifically recognizes the marker is an antibody that specifically recognizes the marker, a small molecule compound that specifically binds to CREPT.

In an embodiment of the kit of the invention, the reagent for carrying out the detection of CREPT by the molecule specifically recognizing CREPT comprises: a detectable or chromogenic label conjugated to a molecule specifically recognizing said CREPT as described above or specifically bound directly or indirectly afterwards.

In a fourth aspect, the invention provides the use of a reagent for detecting CREPT in the preparation of a kit for determining the surgical margin of a tumour,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In a fifth aspect, the present invention provides the use of a reagent for detecting CREPT in the preparation of a kit for identifying normal tissue cells and tissue cells having a gene level precancerous lesion,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In a sixth aspect the present invention provides the use of a reagent for detecting CREPT in the manufacture of a kit for use in the indication of high risk of cancerous tissue cells,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In a seventh aspect, the invention provides the use of a reagent for detecting CREPT in the manufacture of a kit for indicating the occurrence of inflammatory tissue cells,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In an eighth aspect, the present invention provides the use of a reagent for detecting CREPT in the preparation of a kit for indicating increased proliferation potency,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In a ninth aspect the present invention provides the use of a reagent for detecting CREPT in the preparation of a kit for use in the indication of tissue cells in which reduced occurrence of apoptosis is indicated,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

The invention provides in a tenth aspect the use of a reagent for detecting CREPT in the manufacture of a kit for determining the risk of recurrence of a paracancerous tissue,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In some embodiments of the use according to the fourth to tenth aspects of the invention, wherein the reagent for detecting CREPT comprises:

reagents for performing PCR and/or qPCR on CREPT in a sample;

An antibody that specifically recognizes CREPT; or (b)

Small molecule compounds that specifically bind to CREPT.

In some embodiments of the use according to the fourth to tenth aspects of the invention, the antibody specifically recognizing CREPT comprises: monoclonal antibodies or polyclonal antibodies.

In some embodiments of the use according to the fourth to tenth aspects of the invention, wherein, when the reagent for detecting CREPT is an antibody specifically recognizing CREPT or a small molecule compound specifically binding to CREPT, the kit further comprises a reagent for performing the detection of CREPT by the molecule specifically recognizing CREPT, comprising: a detectable or chromogenic label conjugated or directly or indirectly specifically bound to a molecule that specifically recognizes said CREPT.

In some embodiments of the use according to the fourth to tenth aspects of the invention, where the reagent for detecting CREPT is a reagent for qPCR of CREPT in a sample, it comprises the following primers:

CREPT QPCR primer

Through the method, the application and the kit, the invention can realize more accurate detection on the paracancerous tissue with the gene expression profile changed before cancer and the pathological display normal, identify normal tissue cells and tissue cells with the gene level precancerous lesions, find tissue cells with high risk of canceration, find tissue cells with inflammation, find tissue cells with strong proliferation capacity, find tissue cells with reduced apoptosis, thereby more accurately determining the surgical margin when cancer tumor is resected compared with the prior art, and reduce the probability of tumor in-situ recurrence while retaining normal tissues as much as possible.

Drawings

Embodiments of the present invention will be explained with reference to the following drawings, in which,

FIG. 1 shows the result of immunohistochemical staining of CREPT at breast cancer and paracarcinoma. In FIG. 1A, CREPT is highly expressed in ductal breast cancer (a), elevated in paracancerous epithelial tissue near the tumor (b, c), and low in normal tissue at a distance (d). In FIG. 1B, CREPT is highly expressed in breast lobular carcinoma (a), elevated in paracancerous epithelial tissue near the tumor (B, c), and low in normal tissue at a distance (d).

FIG. 2 shows immunohistochemical staining results of CREPT at the side of carcinoma of cervical cancer, colon cancer. In FIG. 2A, CREPT is highly expressed in cervical cancer (a), elevated in paracancerous epithelial tissue near the tumor (b, c), and low in normal tissue at a distance (d). In FIG. 2B, CREPT is highly expressed in colon cancer (a), elevated in paracancerous epithelial tissue near the tumor (B, c), and low in normal tissue at a distance (d).

FIG. 3 shows the results of measurement of the particle size distribution of exosomes. Nanoparticle tracking analysis of exosomes (231 EXO and 4T1 EXO) from MDA-MB-231 (231) and 4T1 cells showed that the size distribution of exosomes was between 30 and 200 nm.

FIG. 4 is a crystal violet staining result of plate clones of MCF10A (FIG. 4A) or NmuMG (FIG. 4B) cells after 2 weeks of treatment with/without 1X 10 ⁹ particles/mL 4T1EXO or 231 EXO. Statistical results were analyzed using t-test, representing p values <0.05.

FIG. 5 shows Western blot results of CREPT, p-ERK, ERK, p-AKT, AKT and Actin in 4T1EXO, 231EXO induced NMuMG, MCF10A cells. In FIG. 5A, western blot detection of CREPT, p-ERK, ERK, p-Akt, akt levels of MCF10A cells treated for 0,1, 3, 6, 9 days, actin as an internal control. In FIG. 5B, western blot detects CREPT, p-ERK, ERK, p-Akt, akt levels of NMuMG cells treated with 4T1EXO for 0,1, 3, 6, 9, 11 days, and Actin as an internal control.

FIG. 6 shows that knockout of CREPT affects proliferation of NMuMG cells after 4T1EXO treatment, nude mice become neoplastic and apoptotic, wherein,

NMuMG-WT/-KO cells after 2 weeks of treatment with/without 1×10 ⁹ particles/mL 4T1EXO were examined for 5 days of cell proliferation in CCK-8 in a of fig. 6, (cell number is represented by OD450 values normalized by OD450 on day 0, significance analysis was performed using T-test, ×representing p values <0.001, ns representing no significant difference);

in FIG. 6B, plate clones formed crystal violet staining results;

In fig. 6C, the results of the clone size statistical analysis in fig. 6B (using two-factor analysis of variance, representing p-value < 0.05);

In D of fig. 6, nude mice were tumorigenic results: these cells were injected in situ into nude mice mammary fat pads (5 per group), 1×10 ⁷ cells per point, and after 10 weeks, the neoplasia was observed;

in fig. 6E, the weight statistics of the tumors in fig. 6D (using two-factor anova, representing p-values < 0.01);

in FIG. 6F, western blot detects protein levels of Caspase3 and CLEAVED CASPASE3 of NMuMG-WT/-KO cells without/after 1X 10 ⁹ particles/mL 4T1EXO treatment, and Actin as an internal control protein.

FIG. 7 shows the knockout influence of CREPT on the clonal formation of MCF10A cells after 231EXO treatment, in which

Plate clone formation (crystal violet staining) of 10A-WT/-KO cells (left) after 2 weeks/no 2X 10 ⁸ particles/mL 231EXO treatment, right panel shows the results of statistical analysis of clone size (using two-factor analysis of variance, representing p-value < 0.05).

FIG. 8 shows CREPT knockout blocks ERK and AKT activation in 4T1EXO, 231 EXO-induced NMuMG, MCF10A cells, wherein,

In FIG. 8A, western blot detection of CREPT, p-ERK, ERK, p-Akt, akt levels of MCF10A-WT and KO cells treated for 0, 1,3, 6, 9 days, actin as an internal control;

in FIG. 8B, western blot was performed to detect CREPT, p-ERK, ERK, p-Akt, and Akt levels of NMuMG-WT and KO cells on days 0, 1, 3, 6, 9, and 11, and Actin as an internal control.

FIG. 9 shows that CREPT affects proliferation of normal epithelial cells, wherein

A, B, C of FIG. 9 is Western blot to identify CREPT over-expressed and knocked out cell lines, actin is used as an internal reference protein, wherein A of FIG. 9 is CHO cells, B of FIG. 9 is NMuMG cells, and C of FIG. 9 is MCF10A cells;

D, E, F of FIG. 9 is a proliferation assay for cell lines overexpressing and knocked out CREPT, where D of FIG. 9 is CHO cells, E of FIG. 9 is NMuMG cells, F of FIG. 9 is MCF10A cells, and cell proliferation is detected using CCK-8, and cell number is represented by OD450 values normalized by OD450 on day 0. The significance analysis uses the t-test that represents p <0.01, p <0.001, and p <0.0001.

FIG. 10 shows that overexpression of CREPT promotes the tumorigenicity of CHO cells in which

Nude mice nodulation results of CHO cells overexpressing CREPT. CHO cells (CHO-OE, lower, n=8) over-expressing CREPT and their controls (CHO-EV, upper, n=8) were injected subcutaneously under the armpits on both sides of nude mice, 1 x 10 ⁷ cells per spot, and after 5 weeks, neoplasia was observed.

FIG. 11 shows that CREPT knockdown in MCF10A cells prevented 231 EXO-induced upregulation of a portion of the genes in the TNF signaling pathway, wherein,

FIG. 11A is a heat map of the expression level of TNF signal pathway related genes in MCF10A-WT/KO cells without/with 231EXO treatment. Values are shown from green (0) to red (100) after normalization treatment for each set of maxima. FIG. 11B-F is a qPCR assay of mRNA levels of TNFRSF1B, PIK, CD, JUN, TNF, NOD2 and CSF1 in MCF10A-WT/KO cells not treated/treated with 231EXO on day 16. mRNA levels of Actin served as an internal reference.

FIG. 12 shows that CREPT knockdown in NMuMG cells prevented upregulation of a portion of the genes in the 4T1 EXO-induced TNF signaling pathway, wherein,

FIG. 12A is a thermal graph showing the expression level of genes associated with TNF signaling pathways in NMuMG-WT/KO cells untreated/treated with 4T1 EXO. Values are shown from green (0) to red (100) after normalization treatment for each set of maxima. FIG. 12B-C is a qPCR detection of mRNA levels of TNFRSF1B and PIK3CD in NMuMG-WT/KO cells on day 16 without/with 4T1EXO treatment. mRNA levels of Actin served as an internal reference.

Detailed Description

The invention provides the following non-limiting embodiments to illustrate the technical solution of the invention.

Definition:

CREPT, also known as RPRD B, is a protein which is highly expressed in tumor tissues and is lowly expressed or not expressed in normal tissues, and the high expression of CREPT can promote the proliferation of tumor cells. The protein sequence consists of 326aa (see SEQ ID NO: 1), uniProt number Q9NQG5.

Regional canceration: malignant changes in cells surrounding tumor tissue are known as regional canceration.

Atypical hyperplasia: pathological concept, meaning that an epithelial cell develops abnormal proliferation, the cell has a degree of atypical but is not sufficient to diagnose cancer. Atypical hyperplasia is classified into mild, moderate and severe atypical hyperplasia according to the degree of cellular atypical.

Tumor exosomes: exosomes are vesicles secreted by tumor cells, which serve as important tools for intercellular communication, affecting the supply of nutrients, angiogenesis, immune escape, etc.

Surgical cutting edge: cancer tumor surgery resects the edges of tissue for evaluation of whether the tumor surgery resects malignant or potentially malignant tissue or cells.

TNF: tumor necrosis factor (Tumor necrosis factor, TNF) is a pleiotropic cytokine. It is 34kDa in size and plays an important role in canceration, cancer progression and metastasis and in immunity.

Cancer: as used herein, the terms "cancer," "malignancy," "neoplasm," "tumor," and "cancer" are used interchangeably to refer to a disease, disorder, or condition in which cells exhibit or exhibit relatively abnormal, uncontrolled, and/or autonomous growth, and thus they exhibit or exhibit an abnormally elevated proliferation rate and/or abnormal growth phenotype. In some embodiments, for example, as set forth herein, the cancer may include one or more tumors. In some embodiments, for example, as set forth herein, the cancer can be or include pre-cancerous (e.g., benign), malignant, pre-metastatic, and/or non-metastatic cells. In some embodiments, for example, as set forth herein, the cancer may be or include a solid tumor. In some embodiments, for example, as set forth herein, the cancer may be or include a hematological tumor. In general, examples of different types of cancers known in the art include, for example, colorectal cancer, hematopoietic cancers including leukemia, lymphomas (hodgkin and non-hodgkin), myelomas, and myeloproliferative diseases; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the mouth, throat, larynx and lung cancers, liver cancers, genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer and endometrial cancer, renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer and nervous system cancer, benign lesions, and the like such as papilloma and the like.

Such cancers are within the scope of the present invention.

Solid tumors: as used herein, the term "solid tumor" refers to an abnormal mass of tissue including cancer cells. In various embodiments, for example, as set forth herein, a solid tumor is or includes an abnormal tissue mass that does not contain cysts or liquid regions. In some embodiments, for example, as set forth herein, a solid tumor may be benign; in some embodiments, the solid tumor may be malignant. Examples of solid tumors include carcinomas, lymphomas and sarcomas. In some embodiments, for example, as set forth herein, a solid tumor may be or include adrenal gland, bile duct, bladder, bone, brain, breast, cervix, colon, endometrium, esophagus, eye, gall bladder, gastrointestinal tract, kidney, larynx, liver, lung, nasal cavity, nasopharynx, oral cavity, ovary, penis, pituitary gland, prostate, retina, salivary gland, skin, small intestine, stomach, testis, thymus, thyroid, uterus, vagina, and/or vulva tumor.

The solid tumors described above are within the scope of the invention.

Prevention or prevention of: the terms "prevent" and "preventing" as used herein in connection with the occurrence of a disease, disorder or condition refer to reducing the risk of developing the disease, disorder or condition; delaying onset of the disease, disorder, or condition; delaying onset of one or more features or symptoms of the disease, disorder, or condition; and/or reduce the frequency and/or severity of one or more features or symptoms of a disease, disorder, or condition. Prevention may refer to prevention of a particular subject or statistical impact on a population of subjects. Prevention may be considered complete when the onset of the disease, disorder or condition is delayed by a predetermined period of time.

In some embodiments, the present invention may more effectively and more accurately achieve prevention of cancer recurrence by identifying and resecting areas of increased CREPT expression.

Treatment: as used herein, the term "treating" refers to administering a partial or complete reduction, amelioration, alleviation, inhibition, delay of onset of, reduction in severity of, and/or reduction in incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, or condition, or administration for the purpose of achieving any such result. In some embodiments, for example, as set forth herein, such treatment may be directed to a subject that does not exhibit signs of the associated disease, disorder, or condition and/or a subject that exhibits only early signs of the disease, disorder, or condition. Alternatively or additionally, such treatment may be directed to a subject exhibiting one or more determined signs of the associated disease, disorder, and/or condition. In some embodiments, for example, as set forth herein, the treatment may be directed to a subject that has been diagnosed with a related disease, disorder, and/or condition. In some embodiments, for example, as set forth herein, the treatment may be directed to a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing a related disease, disorder, or condition. In various examples, the treatment is directed to cancer.

In some embodiments, the present invention may more effectively and more accurately achieve treatment of cancer in a first surgery by identifying and resecting areas of increased CREPT expression.

Variants: as used herein, the term "variant" refers to an entity that exhibits significant structural identity to a reference entity but is structurally different from the reference entity in the presence, absence, or level of one or more chemical moieties as compared to the reference entity, e.g., the gene or protein of CREPT. In some embodiments, for example, as set forth herein, a CREPT variant is also functionally different from its reference entity, e.g., CREPT wild-type. In general, whether a particular entity is properly considered a "variant" of a reference entity depends on the degree of structural identity with the reference entity. Variants may be molecules that are comparable to, but not identical to, the reference. For example, a variant nucleic acid may differ from a reference nucleic acid at one or more differences in nucleotide sequence. In some embodiments, for example, as set forth herein, the variant nucleic acid exhibits at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity to the reference nucleic acid. In many embodiments, for example, a nucleic acid of interest is considered to be a "variant" of a reference nucleic acid if it has a sequence that is identical to the sequence of the reference but has a small sequence change at a particular position, as set forth herein. In some embodiments, for example, as set forth herein, variants have 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 substituted residues as compared to the reference. In some embodiments, for example, as set forth herein, variants have no more than 5, 4, 3,2, or 1 residue additions, substitutions, or deletions as compared to the reference. In various embodiments, for example, the number of additions, substitutions, or deletions is less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 residues, as set forth herein.

The invention may be carried out by performing the assays of the invention on a wild type of CREPT or a variant of CREPT as defined above.

In the course of the functional study of CREPT, the invention finds that the CREPT expression level of normal epithelial tissues close to tumor tissues is increased compared with that of normal epithelial tissues at the far end, and the epithelial tissues with the increased CREPT expression level are accompanied with hyperplasia or atypical hyperplasia. Atypical proliferation of paracancerous tissue predicts that growth of these cells begins to be uncontrolled, i.e., some of the tumor's characteristics are obtained. This malignant change of cells surrounding the tumor tissue is called regional canceration.

By CREPT staining in breast cancer sections with paracancerous normal breast tissue, elevated levels of CREPT expression in paracancerous tissue in close proximity to the tumor tissue were found, while CREPT expression in normal tissue at a greater distance remained low. Thus, elevated levels of CREPT expression are believed to be associated with regional canceration.

The invention proposes that the expression of the paracancel CREPT has a distance effect, namely, the expression level of the CREPT at a part which is closer to the in-situ tumor is higher, and the expression level of the CREPT at a part which is further away from the in-situ tumor is lower, which proves that the CREPT of the paracancel tissue can be influenced by a certain factor from the in-situ tumor.

In the case of elevated CREPT expression in tissues other than cancer, these tissues in which CREPT expression is elevated include tissues in which atypical hyperplasia (i.e., pathologically distinct from normal tissues) and tissues in which the gene expression profile is changed before cancer but the pathology has not been changed yet (hereinafter simply referred to as pathological tissues in which the gene expression is changed before cancer). Cancerous regions, although not necessarily morphologically altered, e.g., tissues in which gene expression is altered prior to cancer, may acquire some, but not all, of the desired phenotypic changes of the malignancy, including acceleration of proliferation, reduction of mortality, or increase of immune escape (Curtius et al, 2018).

The present invention has found that both the atypically proliferative tissue and the tissue in which the gene expression is changed prior to cancer have the potential to become cancerous, thereby causing recurrence of the cancer.

Thus, the present invention proposes that potentially malignant cells can be more accurately identified by identifying atypical proliferative tissues with elevated CREPT expression, as well as plausible normal tissues in which the expression of genes with elevated CREPT expression is altered prior to cancer, thereby achieving more accurate guidance of the surgical margin of cancer.

As described above, atypical proliferative tissues with elevated CREPT expression, as well as tissues that appear to be normal, where the expression of genes with elevated CREPT expression is altered prior to cancer, have the potential to become cancerous, thereby causing recurrence of the cancer.

The study of the mechanism of elevated levels of CREPT expression in paracancerous normal epithelial tissue may not only provide an effective marker for cells that develop regional canceration, but may also reveal the causative factors of regional canceration. Meanwhile, CREPT is taken as a potential regulatory factor for regional canceration and possibly becomes an important target for tumor in-situ recurrence, and provides a new direction for tumor treatment.

Exosomes are vesicles secreted by cells, which have received widespread attention in recent years as important tools for intercellular communication. Exosomes also play an important role in the development of tumors. The occurrence and development of tumors have been kept away from the interaction of tumor cells with their growth environment. The growth of tumors requires a suitable growth environment. Likewise, tumors secrete factors or extracellular vesicles such as exosomes to affect the environment in which they grow. Numerous studies have reported that exosomes act in tumor microenvironments, affecting stromal cells, endothelial cells, immune cells, etc. in the microenvironment, and thus affecting the supply of nutrients, angiogenesis, immune escape, etc. Then, the paracancerous normal epithelial tissue, which is also immediately adjacent to the tumor tissue, is necessarily affected by the exosomes secreted by the tumor tissue, and studies directed to the role of exosomes in the process of canceration of the normal epithelial cell region will provide new protocols for tumorigenesis, progression and prevention and treatment.

The present invention has found through research that tumors increase CREPT expression in paracancerous tissues via exosomes, and thereby induce changes in the genomic level of tumorigenesis, followed by pathological changes, such as atypical hyperplasia, and ultimately cancerous changes. Accordingly, the present invention provides the following exemplary embodiments.

[ Method of determining the surgical margin of cancer ]

The present disclosure includes, inter alia, a method of determining a surgical margin of cancer, which can be performed by: detecting the expression level of a marker in a paracancerous tissue from a subject, the marker being CREPT and variants thereof. In some embodiments of the invention, the expression level of the marker in a paracancerous tissue from a subject is compared to the expression level in a control sample. In some embodiments of the invention, the tissue in which the expression level of CREPT in the paracancerous tissue is increased by more than 10%, more than 20%, more than 50%, more than 100%, more than 200% as compared to the expression level in the control sample is determined to be in need of excision.

In some embodiments of the invention, the tissue in which the expression level of CREPT in the paracancerous tissue is increased by a factor of 2 or more, 3 or more, 4or more, 5 or more, 6 or more, 8 or more as compared to the expression level in the control sample is determined to be in need of excision.

In some embodiments of the invention, the subject is a human, preferably a human with cancer, preferably a human receiving cancer treatment, preferably a human receiving surgical removal of cancer.

In some embodiments of the invention, the CREPT variant has an identity to CREPT of 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

In some embodiments of the invention, the determination of the level of CREPT expression in the tissue may be performed by methods known to those skilled in the art, such as immunohistochemistry, western blotting, real-time fluorescent quantitative PCR, in situ hybridization (FISH) methods, and the like.

In some embodiments of the invention, the control sample is from normal tissue of a surgical subject or a healthy subject.

In some embodiments of the invention, the healthy subject is a subject judged to be free of tumors and cancers based on examination and clinical criteria.

In some embodiments of the invention, the expression level of CREPT is increased in the paracancerous tissue of the subject undergoing surgery, preferably in the paracancerous tissue whose atypical proliferative tissue and/or gene expression profile is changed pre-cancer but whose pathology has not been changed, as compared to the expression level in the control sample.

Atypical hyperplasia tissue is a state of tissue between normal tissue and cancerous tissue, i.e., a state that is pathologically different from normal tissue but not yet sufficient to be judged as cancerous, and is generally considered to have an increased likelihood of cancerous changes.

Tissues whose gene expression profile is changed before cancer but whose pathology has not been changed are those which appear the same as normal tissues in the conventional pathology test, but whose gene expression has been different from that of normal tissues. In the present invention, it is considered that a tissue whose gene expression profile is changed before cancer but whose pathology has not been changed is highly likely to be cancerous as compared with a normal tissue, but has not progressed to a stage where it is pathologically shown an atypically proliferated tissue.

In some embodiments of the invention, the cancer is a solid cancer, such as a solid tumor as defined hereinabove. In some embodiments of the invention, the solid cancer is, for example, cervical cancer, breast cancer, ovarian cancer, melanoma, colon cancer.

In some embodiments of the invention, the expression level of CREPT is detected by one or more of the following means: immunohistochemistry, western immunoblotting, real-time fluorescent quantitative PCR, in situ hybridization (FISH), and the like.

In some embodiments of the invention directed to detecting the expression level of CREPT, antibodies specifically recognizing CREPT may be employed including:

CREPT antibodies (3E 10), as described in Ren F et al (Characterization of a monoclonal antibody against CREPT,a novel protein highly expressed in tumors.Monoclon Antib Immunodiagn Immunother.2014Dec;33(6):401-8.doi:10.1089/mab.2014.0043.PMID:25545209;PMCID:PMC4278082.);

RPRD1B antibody from GeneTex, inc. (North America), cat No. gtx119969;

RPRD1B Antibody #74693 from CELL SIGNALING Technology, inc;

Anti-RPRD B polyclonal antibody (K110225P) from beijing laba technologies limited;

RPRD1B monoclonal antibody (OTI 1C 9) and RPRD B polyclonal antibody (Product #PA 5-98941) from Invitrogen.

In some embodiments of the invention employing real-time fluorescent quantitative PCR, primers obtained by designing CREPT using conventional primer design tools, such as the following primers:

CREPT QPCR primer

[ Use in the preparation of a kit for determining the surgical margin of cancer ]

In other embodiments of the present invention there is provided the use of the marker CREPT as a marker to be detected in the manufacture of a kit for determining a surgical margin of cancer for carrying out the above method of determining a surgical margin of cancer according to the present invention,

The kit comprises:

A molecule that specifically recognizes the CREPT, and

Reagents for carrying out the detection of CREPT by said molecules specifically recognizing CREPT.

In an embodiment of the use according to the invention, the molecule specifically recognizing the marker is an antibody specifically recognizing the marker, a small molecule compound specifically binding to CREPT.

The invention relates to antibodies specifically recognizing CREPT comprising:

CREPT antibodies (3E 10), as described in Ren F et al (Characterization of a monoclonal antibody against CREPT,a novel protein highly expressed in tumors.Monoclon Antib Immunodiagn Immunother.2014Dec;33(6):401-8.doi:10.1089/mab.2014.0043.PMID:25545209;PMCID:PMC4278082.);

RPRD1B antibody from GeneTex, inc. (North America), cat No. gtx119969;

RPRD1B Antibody #74693 from CELL SIGNALING Technology, inc;

Anti-RPRD B polyclonal antibody (K110225P) from beijing laba technologies limited;

RPRD1B monoclonal antibody (OTI 1C 9) and RPRD B polyclonal antibody (Product #PA 5-98941) from Invitrogen.

In an embodiment of the use according to the invention, the reagent for carrying out the detection of CREPT by the molecule specifically recognizing CREPT comprises: a detectable or chromogenic label conjugated or directly or indirectly specifically bound to a molecule that specifically recognizes said CREPT.

[ Kit for determining the surgical margin of cancer ]

In other embodiments of the invention, there is provided the use of a marker in the manufacture of a kit for determining a surgical margin of cancer. The kit comprises: a molecule that specifically recognizes CREPT, and a reagent for performing CREPT detection by the molecule that specifically recognizes CREPT.

In an embodiment of the invention, the kit refers to a component of a consumable for qualitative and/or quantitative detection of expression of CREPT in a paracancerous tissue. The core of the kit of the invention processes tissue biopsies, surgical resections, intraoperative pathological sections, thereby showing the presence or absence of CREPT expression, and/or the amount of CREPT expression.

In some embodiments of the invention, the molecule that specifically recognizes the marker, e.g., CREPT, is an antibody that specifically recognizes the marker, or a small molecule compound that specifically binds to CREPT.

In some embodiments of the invention, the reagent for performing the detection of CREPT by the molecule specifically recognizing CREPT comprises: a partner that specifically binds to a molecule that specifically recognizes the CREPT, and a label that can bind the partner and produce a detectable signal.

In addition, in some embodiments of the invention, the reagent for performing the detection of CREPT by the molecule specifically recognizing CREPT further comprises: some auxiliary agents such as buffers, protein stabilizers, e.g. polysaccharides, etc. The diagnostic kit also includes other components of the signal generating system, such as a reagent for reducing background interference, a control reagent, equipment required for completing one test, and the like, as needed. In another embodiment, the diagnostic kit comprises a conjugate of a CREPT antibody and a substance capable of producing a detectable signal.

When the molecule that specifically recognizes CREPT is an antibody that specifically recognizes the marker, the reagent used to perform the detection of CREPT by the antibody includes a reagent that promotes the binding of CREPT to the specific antibody, such as a buffer, a stabilizer, a blocking agent, a color developing agent, or the like.

For example, in one embodiment, the kit for performing a specific detection of a protein using an antibody further comprises a secondary antibody, a color developing agent coupled to the secondary antibody, and the like.

When the molecule that specifically recognizes CREPT is a small molecule compound, the reagent used to perform the detection of CREPT by the small molecule compound includes a reagent that promotes the binding of CREPT to the small molecule compound, such as a buffer, a stabilizer, a blocking agent, a color developing agent, or the like.

For example, in one embodiment, the small molecule compound may bind to the CREPT protein in a covalent or non-covalent form.

For example, in one embodiment, the small molecule compound may be a small molecule compound with a fluorescent or biotin probe.

The method for determining a surgical margin of cancer and the kit for determining a surgical margin of cancer of the present invention can reduce the risk of recurrence in situ at the time of a cancer excision operation.

In situ recurrence refers to the recurrence of a tumor at the same site or in close proximity to the primary tumor, with or without distant diffuse metastasis. The cancer which recurs in situ belongs to local tumor, and according to the illness state of a patient in clinic, the detection method and the kit can accurately identify tissues and cells with potential of recurrences in situ besides adopting local treatment of operation and radiotherapy or combined treatment of chemotherapy and the like, so that the tissues can be excised when the first cancer operation is performed, and the possibility of recurrences in situ is reduced.

In some embodiments of the invention, the subject is a human, preferably a human with cancer, preferably a human receiving cancer treatment, preferably a human receiving surgical removal of cancer.

In some embodiments of the invention, the CREPT variant has an identity to CREPT of 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

[ Application of CREPT detection reagent in preparation of kit ]

The invention also relates to a kit for preparing a kit for performing one or more of the following uses selected from: kit for determining tumor surgical margin, kit for identifying normal tissue cells and tissue cells having a gene level precancerous lesion, kit for indicating tissue cells at high risk of canceration, kit for indicating tissue cells having an inflammatory condition, kit for indicating a strong proliferation capacity, kit for indicating tissue cells having reduced apoptosis, kit for judging the risk of recurrence of paracancerous tissue,

The kit comprises:

reagents for performing the detection of CREPT in a sample from a subject.

In some embodiments of the invention directed to the above applications, wherein the reagent for detecting CREPT comprises:

reagents for performing PCR and/or qPCR on CREPT in a sample;

An antibody that specifically recognizes CREPT; or (b)

Small molecule compounds that specifically bind to CREPT.

In some embodiments of the invention directed to the above applications, the antibodies that specifically recognize CREPT comprise:

CREPT antibodies (3E 10), as described in Ren F et al (Characterization of a monoclonal antibody against CREPT,a novel protein highly expressed in tumors.Monoclon Antib Immunodiagn Immunother.2014Dec;33(6):401-8.doi:10.1089/mab.2014.0043.PMID:25545209;PMCID:PMC4278082.);

RPRD1B antibody from GeneTex, inc. (North America), cat No. gtx119969;

RPRD1B Antibody #74693 from CELL SIGNALING Technology, inc;

Anti-RPRD B polyclonal antibody (K110225P) from beijing laba technologies limited;

RPRD1B monoclonal antibody (OTI 1C 9) and RPRD B polyclonal antibody (Product #PA 5-98941) from Invitrogen.

In some embodiments of the invention directed to the above application, where the reagent for detecting CREPT is an antibody specifically recognizing CREPT or a small molecule compound specifically binding to CREPT, the kit further comprises a reagent for performing CREPT detection by the molecule specifically recognizing CREPT, comprising: a detectable or chromogenic label conjugated or directly or indirectly specifically bound to a molecule that specifically recognizes said CREPT.

In some embodiments of the present invention directed to the above application, where the reagent for detecting CREPT is a reagent for qPCR of CREPT in a sample, primers obtained by designing CREPT using a common primer design tool, for example, the following primers:

CREPT QPCR primer

All the embodiments of the invention described above can be combined with each other or the technical features of the embodiments can be combined by a person skilled in the art according to common knowledge.

Examples

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative tests in the following examples were each set up three repeated experiments, and the average value was taken as a result.

[ Experimental materials ]

1 Laboratory animal and cell

A total of 7 cell lines were used, namely mouse mammary epithelial cell NMuMG, mouse mammary cancer cell 4T1, hamster ovary epithelial cell CHO, human mammary epithelial cell MCF10A, human mammary cancer cell MDA-MB-231, human mammary cancer cell MCF7, human mammary cancer tamoxifen resistant cell line MCF7/TAM-R.

The animals used in the experiment are BALB/c nude mice, and the self-cleaning university experimental animal platform is purchased.

2 Plasmid

Myc-CREPT, CRISPR/cas9 mediated CREPT knockout plasmid PX458 (gRNA sequence shown below) and CREPT knockdown shRNA (sequence shown below) on pcDNA3.1 vector were used and constructed and stored by the present laboratory.

Knockout of CREPT: CRISPR-CAS9

Guide RNAs (gRNAs): ATCGTCTCCGTGTGGCACCG (see, SEQ ID NO.: 2); CGTGCCGTCGCTCTTCCCGC (see, SEQ ID NO.: 3)

Knock down CREPT shRNA

TRCN0000148750: CCGGCGGCAGCAGTATATTCTGAAACTCGAGTTTCAGAATATACTGCTGCCGTTTTTTG (see SEQ ID NO.: 4)

TRCN0000149087: CCGGGCACGAAGATTAGGTGCATTTCTCGAGAAATGCACCTAATCTTCGTGCTTTTTTG (see SEQ ID NO.: 5)

Knock down CREPT: siRNA (small interfering RNA)

GCAAGAACGAAGUGUUAUTT (see, SEQ ID NO.: 6)

GUCUGUUACUAGCAGAAUATT (see, SEQ ID NO.: 7)

3 Antibodies

Antibodies against p-ERK1/2 (Thr 202/Tyr204, # 4370), ERK1/2 (# 4695), p-Akt (Ser 473, # 4046), akt (# 9272) and Caspase3 (# 9662) were purchased from CELL SIGNALING Technology company. anti-Actin (a 5316) antibodies were from Sigma. CREPT antibodies (3E 10) were produced by this experiment (Ren et al, 2014). Horseradish peroxidase (HRP) -labeled goat anti-mouse (ZB-2305) and goat anti-rabbit IgG (ZB-2301) were purchased from Meter Cunninghamia bridge.

4 Reagent

DMEM (C11995500 BT), horse serum (26050088), pancreatin (Trypsin-EDTA, 25200056), blue/streptomycin (15140122), PBS powder (21600044) were all purchased from Thermo Fisher company. Fetal bovine serum (Fetal Bovine Serum, FBS) was purchased from BI company. Insulin (CC 101), cholera toxin (CC 104), epidermal Growth Factor (EGF) (CC 102), hydrocortisone (CC 103) and phenol red-free DMEM/F12 medium (CM 16405) were all purchased from Zhongke Mich (Beijing) technologies Co.

In addition, RNA extraction reagents (TRIzol, 15596018), protein molecular weight markers (26617), and chemiluminescent (ECL) substrates (34577) were purchased from Thermo Fisher, FASTKING CDNA first strand synthesis kit (KR 116) and fluorescent quantitative detection kit (FP 209) from the company diurnal biochemical technology (beijing). The high efficiency eukaryotic transfection reagent Vigofect and the dual luciferase reporter gene detection kit were all purchased from Wiggares Biotechnology (Beijing) Inc. CCK-8 kit (GB 707) was purchased from Tojindo, japan. The primers were synthesized by the biotechnology Co.Ltd of Beijing Rui Boxing.

Example 1 immunohistochemical staining shows elevated expression of CREPT in paracancerous dysplastic tissue and in partially pathologically normal tissue

Immunohistochemical staining experiments were performed using the CREPT antibody (3E 10) (Ren et al, 2014) in different types of tumor sections with paracancestral tissue to detect expression of CREPT.

Samples used in the experiments were from ductal carcinoma of the breast (a of fig. 1), lobular carcinoma of the breast (B of fig. 1), cervical carcinoma (a of fig. 2) and colon carcinoma (B of fig. 2).

The immunohistochemistry is carried out on slices by adopting slice baking, dewaxing and rehydration, slice washing, antigen restoration, sealing, primary antibody combination (primary antibody of China fir gold bridge company, CREPT antibody dilution factor of 50 times), secondary antibody combination (HRP coupled mouse/rabbit universal secondary antibody (Envision) produced by Dako company), DAB staining, nuclear dyeing, dehydration and transparentization, slice sealing, and then placing the slices on a slice frame for overnight airing.

Finally, the sections were scanned, the results of which are shown in fig. 1a and 1B and fig. 2 a and 2B, and the results of which were analyzed.

Conclusion:

An elevated level of CREPT expression was found in the paracancerous tissues of ductal breast cancer (A of FIG. 1). Looking at the sections carefully, it can be seen that CREPT of the tumor tissue appears positive (a in FIG. 1A); the increase in CREPT expression level occurs in a part of cells of a duct located closer to the tumor tissue in the paracancerous tissue, and the morphology of the duct is changed-internal parenchyma, indicating that atypical hyperplasia of the tissue occurs (b in FIG. 1A); a slightly more distant catheter, with a round of myoepithelial cells that appear positive for CREPT expression, but the tissue morphology is unchanged (c in fig. 1 a); whereas normal tissue at a far distance is completely free of CREPT expression (d in a of fig. 1). Similarly, in breast lobular carcinoma sections, we found that CREPT was highly expressed in their tumor tissues (a in B of FIG. 1). Both the paracancerous leaflet (B in fig. 1B) that is close to the tumor tissue and the somewhat distant leaflet (c in fig. 1B) present an elevated level of CREPT expression, and the portion of the leaflet that is close to the tumor tissue has hyperplasia. Whereas the expression level of CREPT is lower in the more distant leaflets (d in B of fig. 1). Furthermore, we have found that this phenomenon is also observed in cervical cancer (FIG. 2A) and colon cancer (FIG. 2B), where CREPT expression is elevated in paracancerous tissue near the tumor tissue (B, c in FIG. 2A and B, c in FIG. 2B), while normal tissue at a greater distance is negative (d in FIG. 2A and d in FIG. 2B).

EXAMPLE 2 inhibition of CREPT inhibits the canceration of normal epithelial cells by tumor exosomes

2.1. Extraction and identification of exosomes

The exosomes used were derived from mouse breast cancer cells 4T1 and human breast cancer cells MDA-MB-231 (purchased from ATCC), extracted using conventional differential ultracentrifugation, i.e. preparing supernatant, removing impurities, ultracentrifugating to extract exosomes, and then preserving: PBS resuspended exosomes can be stored briefly at 4 ℃, tested for functional verification within a week, or stored for long periods at-80 ℃.

Exosome particle size distribution and particle concentration were analyzed using NanoSight LM14 (MALVERN PANALYTICAL, UK) equipped with a nanoparticle tracking analysis (Nanoparticle TRACKING ANALYSIS, NTA) system. Data acquisition and analysis were performed using NTA analysis software version 3.1. The analysis result shows that the grain size distribution of the extracted product is between 30 and 200nm, which accords with the characteristics of the grain size distribution of exosomes (figure 3).

2.2. The process of promoting cellular canceration by tumor exosomes is accompanied by an increase in the expression level of CREPT

2.2.1 The effect of tumor exosome treatment on cell clone formation was assessed by plate clone formation experiments.

Normal mammary epithelial cells NMuMG or MCF10A were treated with PBS or 1×10 ⁹ pellet/mL 4T1 cell-derived exosomes or MDA-MB-231-derived exosomes for two weeks, followed by removal of the exosomes and cell culture using normal medium. Clone formation experiments were performed using normal mammary epithelial cells treated with control PBS or tumor exosomes, and the ability of the cells to form clones was judged according to the number of cell colony formations.

The operation steps comprise: 1. cell suspensions were prepared and 500 or 1000 cells were seeded per well in six well plates. 3 compound holes per group; culturing for 7-14 days, and changing the primary liquid every three days; after macroscopic clone formation, the medium was removed, washed once with PBS, and the clone was stained with 0.1% crystal violet (dissolved in methanol to 0.5% stock solution, diluted with distilled water before use); standing at room temperature for 15min, discarding the dyeing liquid, and washing the background with distilled water; airing; the record was scanned and analyzed using ImageJ software.

Experimental results indicate that breast tumor exosomes promote the clonal formation of normal breast epithelial cells, i.e., enhance the ability of cells to proliferate, a phenotype of regional carcinogenesis (FIG. 4).

2.2.2. 2 Tumor exosomes promote elevated levels of CREPT and p-AKT, the cancer markers, in the course of cellular canceration

Classical immunoblotting (WB) was used to detect CREPT expression levels during tumor exosome treatment and phosphorylation levels of ERK and AKT, key oncogenic signaling molecules. Results were visualized, observed and recorded using MINICHEMI610,610 imaging system (Sagecreation Service For LIFE SCIENCE).

The experimental results show that the expression level of CREPT is obviously increased in the process of inducing normal epithelial cell canceration by tumor exosomes, and the phosphorylation levels of the cancer promotion signal molecules ERK and AKT are also increased (figure 5).

This means that tumor exosomes can promote the canceration of normal epithelial cells, and this process is accompanied by an increase in the expression level of CREPT, which is involved in the process of cell canceration. Thus, elevated CREPT expression is an early molecular indicator and therapeutic target for the inflammation and canceration of paracancerous tissues.

2.3 Inhibition of CREPT inhibits the canceration of normal epithelial cells by tumor exosomes

Through cell proliferation experiments, clonogenic experiments and oncological experiments, we demonstrated that inhibition of CREPT inhibited the canceration of normal cells caused by tumor exosomes (fig. 6, 7). When the wild NMuMG cells (NMuMG-WT) are treated by a 4T1 exosome (4T 1 EXO), the proliferation capacity of the wild NMuMG cells is obviously improved; in contrast, the proliferation potency of CREPT knockout NMuMG (NMuMG-KO) was not significantly altered by 4T1EXO (FIG. 6A), i.e., the response to exosome stimulation was lost. Similarly, NMuMG-WT showed a significant increase in clonality following 4T1EXO stimulation, while NMuMG-KO showed no significant change in clonality following 4T1EXO stimulation (B, C of FIG. 6). Similarly, in MCF10A cells treated with the exosomes (231 EXO) of MDA-MB-231, the knockout of CREPT also inhibited the promotion of clonogenic effects of tumor exosomes (FIG. 8). Furthermore, the result of nude mouse tumorigenesis experiments on NMuMG-WT and NMuMG-KO cells treated by 4T1EXO shows that the 4T1EXO can promote the tumorigenesis of NMuMG-WT; the tumorigenicity of NMuMG following CREPT knockout was reduced, manifested as a decrease in tumor volume and mass, while 4T1EXO was no longer able to promote tumorigenesis of NMuMG (D, E of fig. 6). The three experiments prove that the inhibition of CREPT can inhibit the canceration of normal epithelial cells by tumor exosomes.

Furthermore, the knockout of CREPT also increased NMuMG cells in 4T1EXO treated apoptosis marker clean-Caspase 3 (F of FIG. 6), which means that CREPT also has an effect on apoptosis. When tumor exosomes treat normal epithelial cells, CLEAVED CASPASE3 is reduced, i.e., apoptosis of the cells is inhibited, which corresponds to a reduction in apoptosis of cells during regional canceration. Whereas the knockout of CREPT enhances apoptosis, i.e. also affects the canceration of cells.

At the same time, the level of ERK and AKT phosphorylation in cells following CREPT knockout was detected. As a result, CREPT knockdown was found to also suppress the increase in ERK and AKT phosphorylation levels stimulated by tumor exosomes.

The above results indicate that inhibition of CREPT can prevent tumor exosome-induced canceration of normal epithelial cells. Similarly, inhibition of CREPT can prevent tumor exosome-induced canceration of paracancerous tissue, i.e., inhibition of CREPT can inhibit in situ recurrence of tumors to some extent (FIGS. 6, 7, 8).

Example 3 cell proliferation assay-CREPT knockout affects the proliferative capacity of normal epithelial cells; CREPT overexpression promotes the tumorigenesis of CHO cells in mice.

Myc-tagged CREPT was overexpressed in CHO cells, NMuMG cells and MCF10A cells, respectively, and stable overexpressed cell lines (CHO-OE, NMuMG-OE, MCF10A-OE, control CHO-EV, NMuMG-EV, MCF10A-EV, respectively) were obtained by neomycin screening, and CREPT knockout cell lines (CHO-KO, NMuMG-KO, MCF10A-KO, control CHO-WT, NMuMG-WT, MCF10A-WT, respectively) were established using the CRISPR-Cas9 system (A, B, C of FIG. 9).

This example was performed using a protocol conventional in the art for CCK-8 (Cell Count Kit-8).

Growth curves were plotted on days by CCK-8 experiments to examine the proliferation capacity of these cells. The results indicate that overexpression of CREPT promotes cell proliferation, whereas knock-out of CREPT inhibits cell proliferation (D, E, F of FIG. 9). Surprisingly, the tumorigenicity of CHO cells was promoted when CREPT was overexpressed in hamster ovary epithelial CHO cells (fig. 10), meaning that overexpression of CREPT promoted malignant transformation of CHO cells. The above results indicate that CREPT not only affects the proliferation capacity of normal cells, but also promotes canceration of normal cells.

CREPT was found in cell proliferation experiments as a pro-oncoprotein and tumor marker, and its elevation suggests that normal cells are highly likely to undergo malignant changes (FIGS. 5, 9, 10). The unpublished data in the laboratory indicate that early stages of normal cell canceration are accompanied by elevated levels of CREPT expression during tumorigenesis. The over-expression of CREPT can promote the proliferation and the tumorigenicity of normal epithelial cells, and the phenomenon of atypical hyperplasia is associated with tissues with increased expression of CREPT beside cancer, so that the fact that the increased expression level of CREPT promotes regional canceration of normal tissues, and the regional canceration of normal cells means that the cells are at extremely high canceration risk.

EXAMPLE 4 CREPT is involved in the inflammatory response mediated by tumor exosome-induced TNF signaling pathway

The change in transcriptome of CREPT knockout cells after tumor exosome stimulation was compared to the change in transcriptome of wild type cells. As a result, 2140 (94.0%) of the 2276 upregulated genes in MCF10A cells after CREPT knockout were no longer significantly elevated after CREPT knockout, and 380 (56.8%) of the 669 upregulated genes in NMuMG cells were not upregulated after CREPT knockout, which the inventors considered were directly or indirectly regulated by CREPT.

Subsequently, KEGG analysis was performed on 2140 genes and 380 genes, respectively, to explore signaling pathways involved in the regulation of CREPT during tumor exosomes stimulated normal epithelial canceration. The results indicate that the pathways affected by both are inflammatory pathways such as Cytokine receptor interaction (Cytokine-Cytokine receptor interaction), TNF signaling pathway (TNF SIGNALING PATHWAY), AGE-RAGE signaling pathway (AGE-RAGE SIGNALING PATHWAY), and pathways associated with the development of tumorigenesis such as cancer pathway (PATHWAYS IN CANCER), MAPK signaling pathway (MAPK SIGNALING PATHWAY), PI3K-AKT signaling pathway (PI 3K-AKT SIGNALING PATHWAY).

There are 31 genes that were up-regulated in TNF signaling pathway after 231EXO stimulation of MCF10A, 28 of which were regulated by CREPT, and we mapped the expression levels of these genes to a heat map (a of fig. 11). mRNA levels of TNFRSF1B, PIK, CD, JUN, TNF, NOD and CSF1 were confirmed, and CREPT knockdown was found to indeed suppress the increase in the expression level of these genes (B-F of FIG. 11).

Similarly, NMuMG cells were stimulated with 4T1EXO and 19 genes were up-regulated in the TNF signaling pathway, 8 of which were CREPT regulated (FIG. 12A). The expression levels of TNFRSF1B and PIK3CD were affected by CREPT knockdown as verified by qPCR (B-C of FIG. 12).

TNFR2 is one of two receptors for TNF, expressed on the surface of some tumor cells and some immunosuppressive cells, to promote tumor proliferation and immune escape. TNFR2 promotes the development of tumors mainly by activating ERK, AKT, NF- κ B, MLCK, etc., and the signal paths affected by CREPT knockdown are found in the foregoing as well as MAPK and PI3K-AKT signal paths, so that ERK and AKT activation are also affected by CREPT knockdown.

By Western blot detection, it was found that the levels of ERK and AKT phosphorylation in normal epithelial cells were significantly increased with increasing days of tumor exosome treatment, while the levels of ERK and AKT phosphorylation were significantly inhibited after CREPT knockout (fig. 8). The above results indicate that CREPT is primarily involved in the activation of TNF signaling pathways during the induction of inflammatory responses by normal epithelial cells by tumor exosomes.

Thus, tumor exosomes were found to elicit an inflammatory response in normal epithelial cells and promote expression of CREPT. The high expression of CREPT further promotes the activation of signals (such as ERK and AKT) related to survival downstream of TNF signal paths, so that the viability of cells is improved, and the balance between survival and apoptosis is broken. That is, TNF signaling can promote proliferation of cells or even cancerous in the presence of CREPT.

Thus, it is proposed that by inhibiting CREPT, the inflammatory process of the paracancerous tissue can be reduced, thereby preventing cancer recurrence at an earlier stage.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

List of references:

Amirrad, f., pytak, p.a., SADEGHIANI-Pelar, n., nguyen, j.p.t., cauble, e.l., jones, a.c., and Bisoffi,M.(2020).Prostate field cancerization and exosomes:Association between CD9,early growth response 1and fatty acid synthase.Int J Oncol 56,957-968.

Bertolini,I.,Ghosh,J.C.,Kossenkov,A.V.,Mulugu,S.,Krishn,S.R.,Vaira,V.,Qin,J.,Plow,E.F.,Languino,L.R., And Altieri,D.C.(2020).Small Extracellular Vesicle Regulation of Mitochondrial Dynamics Reprograms a Hypoxic Tumor Microenvironment.Dev Cell 55,163-177e166.

Melo,S.A.,Sugimoto,H.,O'Connell,J.T.,Kato,N.,Villanueva,A.,Vidal,A.,Qiu,L.,Vitkin,E.,Perelman,L.T.,Melo,C.A., Equal .(2014).Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis.Cancer Cell 26,707-721.

Nagaishi, t., watabe, t., jose, n., tokai, a., fujii, t., matsuoka, k., nagahori, m., ohtsuka, k., and Watanabe,M.(2016).Epithelial Nuclear Factor-x03BA;B Activation in Inflammatory Bowel Diseases and Colitis-Associated Carcinogenesis.Digestion 93,40-46.

Onizawa,M.,Nagaishi,T.,Kanai,T.,Nagano,K.,Oshima,S.,Nemoto,Y.,Yoshioka,A.,Totsuka,T.,Okamoto,R.,Nakamura,T., Equal (2009).Signaling pathway via TNF-alpha/NF-kappaB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis.American journal of physiology Gastrointestinal and liver physiology 296,G850-859.

Patil,P.U.,D'Ambrosio,J.,Inge,L.J.,Mason,R.W.,and Rajasekaran,A.K.(2015).Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.J Cell Sci 128,4366-4379.

Quail, d.f., and Joyce,J.A.(2013).Microenvironmental regulation of tumor progression and metastasis.Nat Med 19,1423-1437.

Ren, f., wang, r., zhang, y., liu, c., wang, y, hu, j., zhang, l., and Chang,Z.(2014).Characterization of a monoclonal antibody against CREPT,a novel protein highly expressed in tumors.Monoclonal antibodies in immunodiagnosis and immunotherapy 33,401-408.

Slaughter, d.p., southwick, h.w., and Smejkal,W.(1953).Field cancerization in oral stratified squamous epithelium;clinical implications of multicentric origin.Cancer 6,963-968.

Suzuki,M.,Nagaishi,T.,Yamazaki,M.,Onizawa,M.,Watabe,T.,Sakamaki,Y.,Ichinose,S.,Totsuka,M.,Oshima,S.,Okamoto,R., Equal .(2014).Myosin light chain kinase expression induced via tumor necrosis factor receptor 2signaling in the epithelial cells regulates the development of colitis-associated carcinogenesis.PLoS One 9,e88369.

Wu, c.h., silvers, c.r., messaging, e.m., and Lee,Y.F.(2019).Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells.J Biol Chem 294,3207-3218.

Yoon, j.h., choi, b.j., nam, s.w., and Park,W.S.(2022).Gastric cancer exosomes contribute to the field cancerization of gastric epithelial cells surrounding gastric cancer.Gastric cancer:official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association.

Yuan,D.,Huang,S.,Berger,E.,Liu,L.,Gross,N.,Heinzmann,F.,Ringelhan,M.,Connor,T.O.,Stadler,M.,Meister,M., Equal (2017).Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.Cancer Cell 31,771-789e776.

Sequence listing

<110> University of Qinghua

<120> Methods, uses and kits for determining surgical margins for cancer

<130> GAI22CN4685

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 326

<212> PRT

<213> CREPT protein sequence

<223> Person

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Met Ser Ser Phe Ser Glu Ser Ala Leu Glu Lys Lys Leu Ser Glu Leu

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Ser Asn Ser Gln Gln Ser Val Gln Thr Leu Ser Leu Trp Leu Ile His

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His Arg Lys His Ala Gly Pro Ile Val Ser Val Trp His Arg Glu Leu

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Arg Lys Ala Lys Ser Asn Arg Lys Leu Thr Phe Leu Tyr Leu Ala Asn

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Asp Val Ile Gln Asn Ser Lys Arg Lys Gly Pro Glu Phe Thr Arg Glu

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Phe Glu Ser Val Leu Val Asp Ala Phe Ser His Val Ala Arg Glu Ala

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Asp Glu Gly Cys Lys Lys Pro Leu Glu Arg Leu Leu Asn Ile Trp Gln

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Glu Arg Ser Val Tyr Gly Gly Glu Phe Ile Gln Gln Leu Lys Leu Ser

115 120 125

Met Glu Asp Ser Lys Ser Pro Pro Pro Lys Ala Thr Glu Glu Lys Lys

130 135 140

Ser Leu Lys Arg Thr Phe Gln Gln Ile Gln Glu Glu Glu Asp Asp Asp

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Tyr Pro Gly Ser Tyr Ser Pro Gln Asp Pro Ser Ala Gly Pro Leu Leu

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Thr Glu Glu Leu Ile Lys Ala Leu Gln Asp Leu Glu Asn Ala Ala Ser

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Gly Asp Ala Thr Val Arg Gln Lys Ile Ala Ser Leu Pro Gln Glu Val

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Gln Asp Val Ser Leu Leu Glu Lys Ile Thr Asp Lys Glu Ala Ala Glu

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Arg Leu Ser Lys Thr Val Asp Glu Ala Cys Leu Leu Leu Ala Glu Tyr

225 230 235 240

Asn Gly Arg Leu Ala Ala Glu Leu Glu Asp Arg Arg Gln Leu Ala Arg

245 250 255

Met Leu Val Glu Tyr Thr Gln Asn Gln Lys Asp Val Leu Ser Glu Lys

260 265 270

Glu Lys Lys Leu Glu Glu Tyr Lys Gln Lys Leu Ala Arg Val Thr Gln

275 280 285

Val Arg Lys Glu Leu Lys Ser His Ile Gln Ser Leu Pro Asp Leu Ser

290 295 300

Leu Leu Pro Asn Val Thr Gly Gly Leu Ala Pro Leu Pro Ser Ala Gly

305 310 315 320

Asp Leu Phe Ser Thr Asp

325

<210> 2

<211> 20

<212> DNA

<213> Knockout CREPT: CRISPR-CAS9 Guide RNAs

<400> 2

atcgtctccg tgtggcaccg 20

<210> 3

<211> 20

<212> DNA

<213> Knockout CREPT: CRISPR-CAS9 Guide RNAs

<400> 3

cgtgccgtcg ctcttcccgc 20

<210> 4

<211> 59

<212> DNA

<213> CREPT-shRNA knock-down

<400> 4

ccggcggcag cagtatattc tgaaactcga gtttcagaat atactgctgc cgttttttg 59

<210> 5

<211> 59

<212> DNA

<213> CREPT-shRNA knock-down

<400> 5

ccgggcacga agattaggtg catttctcga gaaatgcacc taatcttcgt gcttttttg 59

<210> 6

<211> 20

<212> DNA

<213> Knock down CREPT: siRNA (small interfering RNA)

<400> 6

gcaagaacga aguguuautt 20

<210> 7

<211> 21

<212> DNA

<213> Knock down CREPT: siRNA (small interfering RNA)

<400> 7

gucuguuacu agcagaauat t 21

<210> 8

<211> 20

<212> DNA

<213> QPCR primer CREPT_F1

<400> 8

tgtccctttg gctcatccac 20

<210> 9

<211> 20

<212> DNA

<213> QPCR primer CREPT_R1

<400> 9

catctgcctc tctggcaaca 20

<210> 10

<211> 20

<212> DNA

<213> QPCR primer CREPT_F2

<400> 10

ctccgcaaag ccaaatcaaa 20

<210> 11

<211> 20

<212> DNA

<213> QPCR primer CREPT_R2

<400> 11

cgccatacac acttcgttct 20

<210> 12

<211> 21

<212> DNA

<213> QPCR primer CREPT_F3

<400> 12

gctccgcaaa gccaaatcaa a 21

<210> 13

<211> 20

<212> DNA

<213> QPCR primer CREPT_R3

<400> 13

gccgccatac acacttcgtt 20

<210> 14

<211> 22

<212> DNA

<213> QPCR primer CREPT_F4

<400> 14

tgaatctgtc cttgtggatg ct 22

<210> 15

<211> 19

<212> DNA

<213> QPCR primer CREPT_R4

<400> 15

tgtatgaact cgccgccat 19

<210> 16

<211> 19

<212> DNA

<213> QPCR primer CREPT_F5

<400> 16

ggacccatcg tctccgtgt 19

<210> 17

<211> 19

<212> DNA

<213> QPCR primer CREPT_R5

<400> 17

gccgccatac acacttcgt 19

Claims (4)

1. The use of reagents for detecting the marker CREPT in the preparation of a kit for determining the surgical margin of cancer indicative of the excision of atypical proliferative tissue and of paracancerous tissue of a subject whose gene expression profile has been altered prior to cancer but whose pathology has not been altered,

The kit comprises: reagents for performing detection of CREPT in a sample from a subject;

the reagent is a reagent for qPCR on CREPT in a sample, and comprises at least one group of primer pairs:

Primer pair 1: a forward sequence is shown as SEQ ID No.8, and a reverse sequence is shown as SEQ ID No. 9;

primer pair 2: a forward sequence shown as SEQ ID No.10 and a reverse sequence shown as SEQ ID No. 11;

Primer pair 3: a forward sequence shown as SEQ ID No.12 and a reverse sequence shown as SEQ ID No. 13;

Primer pair 4: a forward sequence shown as SEQ ID No.14 and a reverse sequence shown as SEQ ID No. 15;

Primer pair 5: a forward sequence shown as SEQ ID No.16 and a reverse sequence shown as SEQ ID No. 17;

A method for determining a surgical margin of cancer using the kit, the method comprising:

Detecting in a paracancerous tissue from a subject the expression level of a marker which is a CREPT (tumor cell cycle associated and expression enhancing protein) and a mutant having an identity of 85% or more, 90% or more, 95% or more, 8% or more or 99% or more;

the amino acid sequence of CREPT protein is shown as SEQ ID No. 1;

And comparing the expression level of the marker with the expression level in a control sample from normal tissue of the subject or a healthy subject,

Wherein the tissue to be resected is determined to be a tissue requiring resection when the expression level of CREPT in the paracancerous tissue is increased by 10% or more, 20% or more, 50% or more, 100% or more, or 200% or more as compared to the expression level in the control sample.

2. The use of claim 1, wherein the expression level of CREPT is elevated in the atypical proliferative tissue and in the paracancerous tissue of the subject whose gene expression profile is changed pre-cancerous but whose pathology has not been changed compared to the expression level in the control sample.

3. The use of claim 1, wherein the cancer is a solid cancer.

4. The use according to claim 3, wherein the solid cancer is selected from cervical cancer, breast cancer, ovarian cancer, melanoma, colon cancer.