Marker ibsp for lung cancer bone metastasis and application thereof

Abstract

The present application relates to a marker IBSP of lung cancer bone metastasis and application thereof. Specifically, the present inventors found that IBSP is highly expressed in both plasma and bone metastasis foci of lung cancer patients, and positively correlates with bone metastasis, thus can be used as a biomarker for detecting and diagnosing bone metastasis. Further studies have proved that the differentiation of macrophages to osteoclasts induced by IBSP is a necessary and sufficient condition for promoting lung cancer bone metastasis, thus IBSP inhibitors can inhibit bone metastasis. In addition, the present inventors first found that IBSP can directly induce the positive differentiation of macrophages to osteoclasts by regulating the Rac1-NFAT signaling pathway, thus inhibiting Rac1 can block the lung cancer bone metastasis induced by IBSP. The present application provides an effective target for diagnosing and treating lung cancer bone metastasis, and provides a new method for the prevention, diagnosis and treatment of bone metastasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a marker IBSP for lung cancer bone metastasis and application thereof. BACKGROUND

[0002] Lung cancer is the highest mortality rate of malignant tumor in the world, with more than 1.5 million deaths per year. More than 85% of lung cancer pathological types are non-small cell lung cancer (NSCLC), of which lung adenocarcinoma (LUAD) accounts for more than 40%. Bone metastasis (BM) is a common phenomenon in the progression of advanced NSCLC, with a prevalence of 40%-50%, and is one of the most common metastatic sites. NSCLC patients with bone metastasis have a very poor prognosis, with a median survival of only 6 months. In addition, nearly half of NSCLC patients with bone metastasis will suffer from skeletal-related events (SRE), which greatly reduces their quality of life and further shortens their life span. In addition to imaging diagnosis, there is currently a lack of effective biomarkers to predict the occurrence of bone metastasis in lung adenocarcinoma patients. In addition, the current treatment methods for lung adenocarcinoma bone metastasis are still limited.

[0003] Clinical data shows that patients with bone metastasis are accompanied by osteoclast lesions, in which osteoclasts play a key role. As an important component of the tumor microenvironment, monocyte macrophages have the ability to differentiate into osteoclasts and can be recruited in certain areas of the bone to mediate the process of tumor bone metastasis. RANKL / M-CSF plays a crucial role in the differentiation of macrophages into osteoclasts. Anti-RANKL antibody Denosumab is a standard drug for directly targeting osteoclasts to treat osteoporosis. Although RANKL inhibitors are effective in addressing osteolysis, some limitations of anti-RANKL therapy for lung cancer bone metastasis cannot be ignored. Not all lung cancer patients with bone metastasis can benefit from anti-RANKL therapy, and RANKL is a membrane protein that cannot be quantified as an important standard biomarker to guide treatment.

[0004] Therefore, there is an urgent need in the art to study more effective diagnostic and therapeutic targets to achieve dynamic monitoring and intervention of lung cancer bone metastasis. SUMMARY

[0005] The purpose of the present application is to provide a target IBSP for lung cancer bone metastasis diagnosis and treatment and application thereof.

[0006] In a first aspect of the present application, there is provided the use of an integrin-binding sialoprotein (IBSP) or a detection reagent thereof for the manufacture of a reagent or kit for (a) assessing the risk of bone metastasis of a tumor or cancer; and / or (b) assessing the prognosis (such as survival) of a cancer patient.

[0007] In another preferred embodiment, the detecting comprises detecting a sample of a primary tumor, a metastatic tumor, and / or blood.

[0008] In another preferred embodiment, the detecting is detecting a blood sample, a plasma sample, or a serum sample.

[0009] In another preferred embodiment, the IBSP detection reagent comprises a reagent for detecting IBSP gene, mRNA, cDNA, or protein.

[0010] In another preferred embodiment, the IBSP detection reagent comprises a reagent for detecting the expression level of IBSP.

[0011] In another preferred embodiment, the expression level comprises protein expression level or mRNA expression level.

[0012] In another preferred embodiment, the detection reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (such as a DNA chip), or a protein chip.

[0013] In another preferred embodiment, the detection reagent is a nucleic acid reagent for detecting IBSP mRNA or cDNA.

[0014] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0015] In another preferred embodiment, the diagnostic reagent or kit comprises one or more detection reagents selected from the group consisting of:

[0016] (a) a specific antibody of IBSP, a specific binding molecule of IBSP; and / or

[0017] (b) a primer or primer pair, a probe, or a chip for specifically amplifying IBSP mRNA or IBSP cDNA.

[0018] In another preferred embodiment, the detection reagent is coupled with or carries a detectable label.

[0019] In another preferred embodiment, the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

[0020] In another preferred embodiment, the specific antibody of IBSP is a monoclonal antibody or a polyclonal antibody.

[0021] In another preferred embodiment, the expression level of IBSP protein in the sample is detected by RT-PCR or immunohistochemistry.

[0022] In another preferred embodiment, the tumor or cancer is lung cancer.

[0023] In another preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0024] In another preferred embodiment, the lung cancer is lung adenocarcinoma.

[0025] In another preferred embodiment, the IBSP gene, mRNA, cDNA, or protein is derived from a mammal, preferably from a rodent (e.g., mouse, rat), a primate, and more preferably from a patient diagnosed with a tumor.

[0026] In another preferred embodiment, the IBSP gene, mRNA, cDNA, or protein is derived from a patient with a tumor.

[0027] In another preferred embodiment, the detection kit further comprises one or more detection reagents selected from the group consisting of:

[0028] (Y1) a reagent for detecting a Rac1 gene, mRNA, cDNA, or protein;

[0029] (Y2) a reagent for detecting a Nfatcl gene, mRNA, cDNA, or protein;

[0030] (Y3) a reagent for detecting a Nfatc2 gene, mRNA, cDNA, or protein;

[0031] (Y4) a reagent for detecting an ENPP1 gene, mRNA, cDNA, or protein;

[0032] (Y5) a reagent for detecting a TNFRSF11B gene, mRNA, cDNA, or protein;

[0033] (Y6) a reagent for detecting a SRI gene, mRNA, cDNA, or protein;

[0034] (Y7) a reagent for detecting a PRKCD gene, mRNA, cDNA, or protein;

[0035] (Y8) a reagent for detecting an ATP2A3 gene, mRNA, cDNA, or protein;

[0036] (Y9) a reagent for detecting a PSMB10 gene, mRNA, cDNA, or protein;

[0037] (Y10) a reagent for detecting a macrophage marker gene, mRNA, cDNA, or protein, wherein the macrophage marker gene is selected from the group consisting of CD86, CD115, and CD206.

[0038] In another preferred embodiment, the detection reagent is used to detect whether the expression of one or more genes selected from Y1 to Y10 is significantly up-regulated.

[0039] In another preferred embodiment, the detection reagent is for detecting a blood sample or a serum sample.

[0040] In another preferred embodiment, when the sample is a serum sample, if the serum concentration of IBSP is significantly up-regulated, it indicates that the subject has a significantly higher risk of developing lung cancer bone metastasis than a general lung cancer patient.

[0041] In another preferred embodiment, when the sample is a serum sample, if the serum concentration of IBSP is ≥1056±100 ng / L, it indicates that the subject has a significantly higher risk of developing lung cancer bone metastasis than a general lung cancer patient.

[0042] In another preferred embodiment, the kit further comprises a reagent for detecting a differentiation-related signature gene of macrophages selected from the group consisting of CD11b, CD24, CD44, CD172a, CD66a, CD62L, CD14, MERTK, CD68, CD64, CD172a, CD38, or a combination thereof.

[0043] In another preferred embodiment, if the expression of the differentiation-related signature gene of macrophages is significantly up-regulated, the differentiation-related signature gene of macrophages (or mRNA, protein thereof) can be a biomarker indicating that the patient has tumor bone metastasis.

[0044] In another preferred embodiment, the prognosis is a prognosis for a lung cancer patient.

[0045] In another preferred embodiment, the prognosis is a prognosis of the survival period of the patient.

[0046] In another preferred embodiment, the prognosis is a prognosis of the survival period of the patient according to the following criteria: if the tumor tissue of the patient is positive for IBSP by immunohistochemical staining and / or the serum sample of the patient is positive for IBSP, it indicates that the patient has a shorter survival period; if the tumor tissue of the patient is negative for IBSP by immunohistochemical staining and / or the serum sample of the patient is positive for IBSP, it indicates that the patient has a longer survival period.

[0047] In another preferred embodiment, the shorter or longer survival period is compared with the average survival period of the same cancer patients without distinguishing between IBSP positive and negative cases.

[0048] In another preferred embodiment, when the sample is a serum sample, when the serum concentration of IBSP is ≥ 1000 ng / L, and the subject is a lung cancer patient (or a lung cancer patient with bone metastasis), it is suggested that the subject (or patient) has a shorter survival period; while the serum concentration of IBSP is ≤ 1000 ng / L, and the subject is a lung cancer patient (or a lung cancer patient with bone metastasis), it is suggested that the subject (or patient) has a longer survival period.

[0049] In a second aspect of the present application, there is provided a use of a reagent combination for the preparation of a kit for diagnosing bone metastasis of a tumor or cancer;

[0050] In the reagent combination, the reagent combination comprises: (Y0) a reagent for detecting IBSP gene, mRNA, cDNA, or protein;

[0051] and one or more detection reagents selected from the group consisting of:

[0052] (Y1) a reagent for detecting Rac1 gene, mRNA, cDNA, or protein;

[0053] (Y2) a reagent for detecting Nfatc1 gene, mRNA, cDNA, or protein;

[0054] (Y3) a reagent for detecting Nfatc2 gene, mRNA, cDNA, or protein;

[0055] (Y4) a reagent for detecting ENPP1 gene, mRNA, cDNA, or protein;

[0056] (Y5) a reagent for detecting TNFRSF11B gene, mRNA, cDNA, or protein.

[0057] In a third aspect of the present application, there is provided a use of an IBSP inhibitor for the preparation of a composition or preparation for (a) preventing and / or blocking bone metastasis of a tumor or cancer; and / or (b) inhibiting differentiation of macrophages into osteoclasts.

[0058] In another preferred embodiment, the tumor or cancer is lung cancer.

[0059] In another preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0060] In another preferred embodiment, the tumor or cancer is lung adenocarcinoma.

[0061] In another preferred embodiment, the IBSP inhibitor is an inhibitor of IBSP gene, RNA, or protein encoded thereby.

[0062] In another preferred embodiment, the IBSP inhibitor is selected from the group consisting of an antibody, a small molecule compound, a microRNA, an siRNA, an shRNA, an antisense oligonucleotide (ASO), an aptamer, a gene editor, or a combination thereof.

[0063] In another preferred embodiment, the IBSP inhibitor is an antibody.

[0064] In another preferred embodiment, the IBSP inhibitor is further used to impede the pre-fusion differentiation of macrophages into osteoclasts.

[0065] In another preferred embodiment, the composition is a pharmaceutical composition.

[0066] In another preferred embodiment, the pharmaceutical composition is administered to a subject having one or more of the following phenotypes:

[0067] (Z1) lung cancer positive for IBSP;

[0068] (Z2) tumor susceptible to bone metastasis;

[0069] (Z3) tumor with up-regulated expression of one or more of the following co- markers: Rac1, Nfatc1, Nfatc2, ENPP1, TNFRSF11B.

[0070] In another preferred embodiment, the tumor susceptible to bone metastasis refers to a tumor with high risk of bone metastasis (e.g., >10%).

[0071] In another preferred embodiment, the tumor susceptible to bone metastasis is selected from the group consisting of lung cancer, prostate cancer, thyroid cancer, renal cancer, or a combination thereof.

[0072] In a fourth aspect of the present application, there is provided a use of a Rac1 inhibitor for the manufacture of a composition or a medicament for one or more of the following uses:

[0073] (1) preventing and / or blocking bone metastasis of a tumor or cancer;

[0074] (2) inhibiting differentiation of macrophages into osteoclasts.

[0075] In another preferred embodiment, the inhibition of differentiation of macrophages into osteoclasts comprises impeding the pre-fusion differentiation of macrophages into osteoclasts.

[0076] In another preferred embodiment, the Rac1 inhibitor is selected from the group consisting of an antibody, a small molecule compound, a microRNA, an siRNA, an shRNA, an antisense oligonucleotide, an aptamer, a gene editor, or a combination thereof.

[0077] In another preferred embodiment, the Rac 1 inhibitor is an siRNA.

[0078] In another preferred embodiment, the Rac 1 inhibitor is a small molecule compound selected from the group consisting of EHT-1864, azathioprine, or a combination thereof.

[0079] In another preferred embodiment, the tumor or cancer is lung cancer.

[0080] In another preferred embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0081] In another preferred embodiment, the tumor or cancer is lung adenocarcinoma.

[0082] In a fifth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0083] (a) an IBSP inhibitor as a first active ingredient;

[0084] (b) a Rac 1 inhibitor as a second active ingredient; and

[0085] (c) a pharmaceutically acceptable carrier.

[0086] In a sixth aspect of the present application, there is provided a use of an active ingredient combination for the manufacture of a pharmaceutical composition for preventing and / or treating lung cancer bone metastasis, the active ingredient combination comprising: (a) an IBSP inhibitor as a first active ingredient; and (b) a Rac 1 inhibitor as a second active ingredient.

[0087] In a seventh aspect of the present application, there is provided a kit-of-parts comprising:

[0088] (a) a first pharmaceutical composition comprising (al) an IBSP inhibitor as a first active ingredient; and (a2) a pharmaceutically acceptable carrier; and

[0089] (b) a second pharmaceutical composition comprising (bl) a Rac 1 inhibitor as a second active ingredient; and (a2) a pharmaceutically acceptable carrier.

[0090] In another preferred embodiment, the first and second pharmaceutical compositions are each independent.

[0091] In an eighth aspect of the present application, there is provided a method for inducing differentiation of macrophages into osteoclasts in vitro, the method comprising the step of: (a) culturing macrophages in the presence of IBSP, thereby promoting differentiation of the macrophages into osteoclasts.

[0092] In another preferred embodiment, the method further comprises detecting the expression level of a differentiation-related signature gene of the macrophage in step (a).

[0093] In another preferred embodiment, the differentiation-related signature gene is a significantly up-regulated signature gene.

[0094] In another preferred embodiment, the signature gene is selected from the group consisting of Cd11b, Ly6C, Cd24, Cd44, F4_80, Cd172a, Cd66a, Cd62L, Cd14, Mertk, Cd68, Cd64, Cd172a, Cd38, or a combination thereof.

[0095] In a ninth aspect of the present application, there is provided a method of inhibiting differentiation of macrophages into osteoclasts in vitro, comprising the step of: culturing macrophages in the presence of an IBSP inhibitor, thereby inhibiting differentiation of macrophages into osteoclasts.

[0096] In another preferred embodiment, the inhibiting differentiation of macrophages into osteoclasts comprises hindering the pre-osteoclast differentiation of macrophages.

[0097] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0098] In a tenth aspect of the present application, there is provided a method of screening candidate compounds for preventing and / or treating bone metastasis, the method comprising the steps of:

[0099] (a) testing the expression level (El) and / or activity (Al) of IBSP in cells of a test group, wherein the test compound is added to the culture system of the cells, and observing the expression level (E0) and / or activity (A0) of IBSP in cells of a control group, wherein the test compound is not added to the culture system of the cells;

[0100] wherein if the expression level (El) and / or activity (Al) of IBSP in the cells of the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate compound for preventing and / or treating bone metastasis by inhibiting the expression and / or activity of IBSP.

[0101] In another preferred embodiment, the expression level of IBSP is obtained by qPCR.

[0102] In another preferred embodiment, the method further comprises the step of:

[0103] (b) further testing the candidate compound obtained in step (a) for its inhibitory effect on the pre-osteoclast differentiation of macrophages; and / or further testing whether it has a down-regulating effect on the IBSP gene.

[0104] In another preferred embodiment, the method comprises step (c) administering the candidate compound identified in step (a) to a mammalian model and determining its effect on the mammal.

[0105] In another preferred embodiment, the mammal is a mammal having cancer bone metastasis.

[0106] In another preferred embodiment, "significantly lower" means E1 / E0≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 4.

[0107] In another preferred embodiment, "significantly lower" means A1 / A0≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 4.

[0108] In another preferred embodiment, the cell comprises lung adenocarcinoma cell.

[0109] In another preferred embodiment, the cell is a cell cultured in vitro.

[0110] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0111] In an eleventh aspect of the present application, there is provided a method for preventing and / or treating tumor bone metastasis, comprising the step of administering an IBSP inhibitor to a subject in need thereof.

[0112] In another preferred embodiment, the subject comprises a human or a non-human mammal.

[0113] In another preferred embodiment, the subject is a lung cancer patient.

[0114] In a twelfth aspect of the present application, there is provided a method for inhibiting the differentiation of macrophages into osteoclasts, comprising the step of administering an IBSP inhibitor to a subject in need thereof, thereby inhibiting the differentiation of macrophages into osteoclasts.

[0115] In another preferred embodiment, the subject comprises a human or a non-human mammal.

[0116] In another preferred embodiment, the subject is a lung cancer patient.

[0117] In a thirteenth aspect of the present application, there is provided a method for inducing the differentiation of myeloid cells / macrophages in vivo, the method comprising the step of administering IBSP to a subject in need thereof, thereby inducing the differentiation of myeloid cells / macrophages into osteoclasts.

[0118] In another preferred embodiment, the subject includes a human and a non-human mammal.

[0119] In another preferred embodiment, the subject includes a rodent (e.g., a mouse).

[0120] In another preferred embodiment, the administration includes intravenous, intraperitoneal, intramuscular administration, or a combination thereof.

[0121] In another preferred embodiment, the method includes administering IBSP to a mouse by intraperitoneal injection, 5-10 times, with 1-3 days interval.

[0122] In another preferred embodiment, the method includes administering IBSP to a mouse by intraperitoneal injection, once every 2 days, and after 2 weeks, detecting the number or proportion of myeloid cells in bone, and the expression level of a differentiation-related signature gene of macrophages.

[0123] In another preferred embodiment, in a subject administered with IBSP, the proportion of myeloid cells in bone is significantly up-regulated, and the differentiation-related signature gene is significantly up-regulated.

[0124] In another preferred embodiment, the differentiation-related signature gene is selected from the group consisting of Cdllb, Ly6C, Cd24, Cd44, F4_80, Cd172a, Cd66a, Cd62L, Cd14, Mertk, Cd68, Cd64, Cd172a, Cd38, or a combination thereof.

[0125] In a fourteenth aspect of the present application, a method for detecting a biomarker of tumor bone metastasis is provided, comprising the steps of:

[0126] The detection includes (i) detecting the proportion of myeloid cells, and (ii) detecting the expression level of a differentiation-related signature gene of macrophages selected from the group consisting of CDllb, CD24, CD44, CD172a, CD66a, CD62L, CD14, MERTK, CD68, CD64, CD172a, CD38, or a combination thereof.

[0127] In another preferred embodiment, if the proportion of myeloid cells is significantly up-regulated, and the expression of the differentiation-related signature gene of macrophages is significantly up-regulated, it indicates that the patient has a high risk of tumor bone metastasis or has bone metastasis.

[0128] In a fifteenth aspect of the present application, a method for establishing a rodent model of lung cancer bone metastasis is provided, comprising the step of: injecting genetically engineered lung cancer cells overexpressing IBSP into a rodent, thereby establishing a rodent model of lung cancer bone metastasis.

[0129] In another preferred embodiment, the lung cancer cells are stable cell lines.

[0130] In another preferred embodiment, the lung cancer cells stably express exogenous IBSP.

[0131] In another preferred embodiment, the lung cancer cells are NSCLC.

[0132] In another preferred embodiment, the cells are LLC cells.

[0133] In another preferred embodiment, the injecting comprises intracardiac injection.

[0134] In another preferred embodiment, the rodent model of lung cancer bone metastasis with tumor bone metastasis foci is formed about 2 weeks after the injecting.

[0135] In another preferred embodiment, the rodent comprises a mouse.

[0136] In a sixteenth aspect of the present application, a method for detecting a tumor bone metastasis biomarker is provided, comprising the step of: detecting a sample from a primary tumor site, a metastatic site, and / or blood of a tumor patient, the detecting comprising detecting whether the expression level of a macrophage signature gene selected from the group consisting of CD86, CD115, CD206, or a combination thereof is significantly up-regulated;

[0137] wherein if the expression level of the macrophage signature gene is significantly up-regulated, it indicates that the patient has a high risk of developing tumor bone metastasis or has developed bone metastasis.

[0138] In a seventeenth aspect of the present application, a method for detecting a tumor bone metastasis biomarker is provided, the method comprising the step of: detecting a sample from a primary tumor site, a metastatic site, and / or blood of a tumor patient, the detecting comprising detecting whether the expression level of a gene selected from the group consisting of SRI, NFATC2, PRKCD, ATP2A3, PSMB10, or a combination thereof is significantly up-regulated;

[0139] wherein if the expression level of the gene is significantly up-regulated, it indicates that the patient has a high risk of developing tumor bone metastasis or has developed bone metastasis.

[0140] In another preferred embodiment, the significant up-regulation refers to an expression level or protein content of 1.5-fold or more, preferably 2.0-fold or more, more preferably 3-fold or more, of a reference value.

[0141] In another preferred embodiment, the reference value is the expression level or protein content of healthy controls; or the expression level or protein content of the same disease patients who are IBSP negative.

[0142] In another preferred embodiment, the same disease patients are lung cancer patients who are IBSP negative and do not have bone metastasis.

[0143] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described below (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0144] Figure 1 IBSP is highly expressed in advanced lung adenocarcinoma and is associated with bone metastasis.

[0145] Figure 2 IBSP promotes macrophage differentiation into osteoclasts independent of RANKL / M-CSF.

[0146] Figure 3 IBSP-induced macrophage differentiation into osteoclasts is required for lung cancer bone metastasis.

[0147] Figure 4 IBSP induces macrophage pre-osteoclast differentiation, leading to early osteolysis and bone metastasis.

[0148] Figure 5 IBSP mediates macrophage pre-osteoclast differentiation by regulating the NFAT signaling pathway.

[0149] Figure 6 Rac1 is a target gene of IBSP-induced NFAT signaling leading to bone metastasis, which can be restored by Rac1 inhibition.

[0150] Figure 7 Single-cell sequencing analysis reveals the differentiation trajectory of macrophages into osteoclasts in lung adenocarcinoma bone metastasis.

[0151] Figure 8 IBSP promotes macrophage differentiation into osteoclasts.

[0152] Figure 9 Rac1 is a target gene in IBSP-induced NFAT signaling. DETAILED DESCRIPTION

[0153] Through extensive and in-depth research, the present inventors found that IBSP is highly expressed in the plasma of lung adenocarcinoma patients and bone metastases. Further research proved that the IBSP secreted by lung cancer cells is a key regulator of niche remodeling before tumor cell metastasis and bone metastasis, and the differentiation of macrophages into osteoclasts induced by IBSP is a necessary and sufficient condition for promoting lung cancer bone metastasis, so inhibiting IBSP can block bone metastasis. In addition, the present inventors first found that IBSP can directly induce the positive differentiation of macrophages into osteoclasts by regulating the Rac1-NFAT signaling pathway, leading to early bone resorption, so inhibiting Rac1 can block the IBSP-induced lung cancer bone metastasis. Based on the above findings, IBSP can be used as a biomarker for diagnosing and dynamically monitoring lung adenocarcinoma bone metastasis, and inhibiting the expression and / or function of IBSP or Rac1 can effectively block bone metastasis of cancer cells. On this basis, the present application is completed.

[0154] IBSP gene and protein

[0155] IBSP is a secreted protein belonging to the Small Integrin-Binding Ligand N-linked Glycoproteins (SIBLINGs) family. IBSP is a non-collagenous component protein of the extracellular matrix of mineralized tissues such as bone, calcified cartilage, and dentin, and has been considered to play a role only in bone polymorphic formation. Its normal expression is widely present in bone microenvironment cells, mainly in osteoblasts, osteoclasts, and bone cells. Under physiological conditions, the expression of IBSP is precisely regulated by the bone microenvironment homeostasis.

[0156] Rac1 gene and protein

[0157] Rac1 gene is a gene encoding a protein named Ras-related C3 amino-terminal peptide acyltransferase 1 (Rac1). Rac1 belongs to the Rho family of small GTPases and plays an important regulatory role in cells. Rac1 protein is a key molecule in cell signaling and dynamic regulation of cytoskeleton, and is involved in various biological processes, including cell migration, proliferation, adhesion, differentiation, and morphogenesis. Rac1 regulates the dynamic changes of cytoskeleton, affecting cell motility, shape, and adhesion properties. The activity regulation of Rac1 is mainly achieved through the conversion of its GDP / GTP binding state. When in the activated state, Rac1 can interact with effector proteins, thereby promoting the assembly and remodeling of cytoskeleton and regulating cell shape and movement.

[0158] IBSP inhibitor

[0159] Through various conventional screening methods, substances that interact with IBSP gene or protein, especially inhibitors, etc. can be screened.

[0160] In the present application, the IBSP inhibitor can be used to prepare a composition or formulation for (a) preventing and / or blocking bone metastasis of a tumor or cancer; and / or (b) inhibiting differentiation of macrophages into osteoclasts.

[0161] The IBSP inhibitor (or antagonist) useful in the present application includes any substance that can inhibit the expression and / or activity of IBSP gene or its encoded protein.

[0162] For example, the IBSP inhibitor includes an antibody of IBSP, an antisense RNA, siRNA, shRNA, miRNA, gene editor of IBSP nucleic acid, or an inhibitor of IBSP activity. A preferred IBSP inhibitor refers to an antibody that can inhibit the expression of IBSP.

[0163] In a preferred embodiment, the method and procedure of inhibiting IBSP include neutralizing its protein with an antibody of IBSP, silencing IBSP gene with shRNA or siRNA or gene editor carried by a virus (such as adeno-associated virus).

[0164] The inhibition rate of IBSP is generally at least 50% or more, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be controlled and detected based on conventional techniques, such as flow cytometry, fluorescent quantitative PCR or Western blotting, etc.

[0165] Rac1 inhibitor

[0166] In the present application, the Rac1 inhibitor can be used to prepare a composition or formulation for (1) preventing and / or blocking bone metastasis of a tumor or cancer; and / or (2) inhibiting differentiation of macrophages into osteoclasts.

[0167] The Rac1 inhibitor useful in the present application includes any substance that can inhibit the expression and / or activity of Rac1 gene or its encoded protein.

[0168] For example, the Rac1 inhibitor includes an antibody of Rac1, an antisense RNA, siRNA, shRNA, miRNA, gene editor of Rac1 nucleic acid, or a small molecule compound that inhibits the activity of Rac1. A preferred Rac1 inhibitor refers to a small molecule compound that can inhibit the activity of Rac1.

[0169] In a preferred embodiment, the method and procedure of inhibiting Rac1 include silencing Rac1 gene with shRNA or siRNA or gene editor carried by a virus (such as adeno-associated virus).

[0170] The inhibition rate of Rac1 is generally at least 50% inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be controlled and detected based on conventional techniques.

[0171] Pharmaceutical composition

[0172] The IBSP or Rac1 inhibitors (including antibodies, antisense nucleic acids, gene editors, and other inhibitors) of the present application, when administered (given) therapeutically, can inhibit the expression and / or activity of IBSP protein or Rac1 protein, thereby inhibiting the differentiation of macrophages into osteoclasts, thereby preventing and / or blocking bone metastasis of tumors or cancers. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance being formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to: topical, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction culture and reinfusion, etc.

[0173] The present application also provides a pharmaceutical composition containing a safe and effective amount of the inhibitors (such as antibodies, gene editors, antisense sequences (such as siRNA), or inhibitors) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. The pharmaceutical composition such as needle, solution, tablet and capsule should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram-10 milligrams per kilogram of body weight per day.

[0174] The main advantages of the present application include:

[0175] (1) The present application discloses for the first time that the expression level of IBSP is positively correlated with lung adenocarcinoma bone metastasis, providing the application of IBSP in predicting, diagnosing and dynamically monitoring lung adenocarcinoma bone metastasis.

[0176] (2) The present application provides for the first time the application of IBSP inhibitors in inhibiting and treating lung cancer bone metastasis.

[0177] (3) The present application first discloses that IBSP mediates the positive differentiation of macrophages to osteoclasts through regulating the NFAT signal pathway. In addition, the Rac1 inhibitor can also be used for inhibiting and treating lung cancer bone metastasis.

[0178] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0179] Experimental methods

[0180] 1. siRNA transfection and Rac1 inhibitor treatment

[0181] The siRNAs targeting Rac1, NFATC1 and NFATC2 and the negative control siRNA are commercially available (RiboBio Biotechnology Co., Ltd.). The siRNAs are transfected with Jetprime reagent (Ployplus 101000046, China). The mouse macrophage cell line Raw264.7 cell strain is cultured with 10 or 50 uM EHT-1864 for 48 h, and EHT-1864 is dissolved or diluted in DMSO. Then the cells are collected, and total RNA and protein are extracted.

[0182] 2. Reverse transcription PCR and real-time quantitative PCR

[0183] The total RNA is extracted from clinical tissue specimens and cell lines using an RNA extraction kit (Fastagen, China). The CDNA synthesis is performed using M-MLV (H-) reverse transcriptase (Vazyme, China). The quantitative real-time PCR is performed using Hieff gPCR SYBR Green Master Mix (YEASEN, China) and Light Cycler 480 II real-time PCR system (Roche, USA). The expression of the indicated genes is normalized to the endogenous reference control GAPDH using the 2-AACt method.

[0184] 3. Western blot analysis

[0185] Cell lysates were prepared using SDS lysis buffer containing protease inhibitor cocktail (Beyotime, China). Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo, USA). Western blot analysis of cell lysates was performed using primary antibodies (Rac1, NFATC1, B-actin) and horseradish peroxidase (HRP)-conjugated secondary antibodies (BBI D110087). Western blot analysis was performed using a chemiluminescence system (Tanon, China).

[0186] 4. Cancer cell characterization analysis

[0187] The effect of IBSP on LLC cell invasion and proliferation was detected by Transwell assay, scratch assay and colony formation assay.

[0188] 5. Protein preparation and mass spectrometry analysis

[0189] Proteome variations were analyzed by mass spectrometry (MS). The collected cell samples were processed using Pierce Mass Spectrometry Sample Preparation Kit and EasyPep MS Sample Preparation Kit (Thermo Scientific, Cat No. 84840 and A40006). All samples were analyzed using a hybrid trapped ion mobility spectrometry quadrupole Krow time mass spectrometer equipped with a nano-electrospray ion source.

[0190] 6. Bulk RNA sequencing

[0191] Lung adenocarcinoma samples were collected and stored at -80°C. Total RNA extraction was performed using RNeasy Mini Kit (Qiagen, 74104, Germany). Specific libraries were prepared using VAHTS Universal V6 RNA-seo Library Prep Kit for Illumina (Vazyme, NR604-02, China) according to the manufacturer's instructions. Purified libraries were quantified using Qubit 2.0 Fluorometer (Life, Q32866, USA) and verified using Agilent 2100 Bioanalyzer (Agilent, 2100 Bioanalyzer, USA) to confirm insert size and calculate molarity. Libraries diluted to 10 pm were used to generate clusters by cBot and sequenced on NovaSeg 6000 sequencing system (Illumina, NovaSeq 6000, USA).

[0192] 7. Single-nuclei RNA sequencing

[0193] Bone metastasis samples were collected prospectively and stored in liquid nitrogen. Cell nuclei were isolated using a nuclei isolation kit (SHBIO, CA INo.52009-10), counted using a Countess II Cell Counter (Thermo, USA), and single nuclei RNA sequencing was performed using the Chromium Single Cell 3’ Library and Gel Bead Kit (10X Genomics, USA). Nuclei were loaded onto a Chromium Single Cell Processor (10X Genomics, USA) for barcoding of RNA from each nucleus. Sequencing libraries were constructed following the manufacturer’s instructions (10X Genomics, 1000121, USA) and sequenced on a NovaSeq 6000 sequencing system (Illumina, NovaSeq 6000, USA).

[0194] 8. Monocyte / macrophage differentiation induction and identification

[0195] Monocyte / macrophage differentiation to osteoclasts was performed using human IBSP or mouse IBSP. SRANKL+M-CSF was used as a positive control. TRAP / ALP staining and Giemsa beam color were used to identify differentiated osteoclasts, with more than 3 nuclei as the main feature of identification. In vitro bone resorption assay was used to verify the osteolytic function of induced osteoclasts. Cells were lysed in 6-well plates using 500 μΐ TRIzol (Invitrogen, USA), and RNA was extracted and quantified. DNA was synthesized by reverse transcription, and real-time PCR was used to detect osteoclast-related genes.

[0196] 9. Cell staining

[0197] Cells were collected and fixed on 24-well plates with pre-cooled methanol, and staining was performed according to the procedure provided by the TRAP / ALP staining kit (Wako, Cat No. 294-67001). Giemsa staining solution was diluted 1:10 to obtain the working solution. Subsequently, the working solution was added to the fixed cells in the 24-well plates, and incubated for 1 min, followed by the addition of 100 μΐ PBS to the wells and incubation for 25 min. The culture plates were then washed 3 times.

[0198] 10. Bone resorption assay

[0199] In vitro bone resorption assay was performed using a bone resorption detection kit (Cosmo, CSR-BRA-48X2, No. 648652670). Before use, a solution of fluorescein-amine-labeled chondroitin sulfate (Facs) was filled into the culture plates to prepare the culture plates. Cells were counted and seeded at a density of 5 x 10 3 phenol red-free cell culture medium was used throughout the experiment. Raw264.7+M-CSF and RANKL were used as positive controls, and IBSP+Raw264.7 and control Raw264.7 were used as experimental and negative controls, respectively. After 7 days of culture, the culture medium was extracted to determine the fluorescence and determine the bone resorption.

[0200] 11. Multicolor flow cytometry

[0201] Mice were euthanized after anesthesia, and tumor tissue was harvested from the tibia. Tumor samples were processed using the Miltenyi Tumor Dissociation Kit (130-096-730) and the Gentmacs Octo Dissociation Platform. Cells were labeled and loaded onto a Beckman CytOFLEXLX cytometer (Beckman, Coulter, USA).

[0202] 12. Cell Count

[0203] To verify the effect of IBSP on macrophages in the bone immune environment of mice, mice were injected with the same pathological dose of mouse IBSP. Intraperitoneal injections were administered every 2 days for a total of 7 injections, with the injection volume depending on the mouse's body weight. Cell suspensions were obtained from the tibia and femur of mice after digestion. Cell samples were prepared and labeled with antibodies, followed by cell counting analysis (Helios, Fluidigm).

[0204] 13. Live imaging

[0205] In vivo fluorescein imaging was used in DTC models and pre-metastatic niche models for multiple organ metastases to locate and assess tumor implantation. In vivo SPECT-CT imaging was used to simulate bone metastasis models to validate early osteolysis and prograde osteoclastogenesis. Bone structure was assessed by CT.

[0206] 14. Bone sections and staining

[0207] H&E staining was performed on bone tissue from a tumor-implanted model simulating the bone metastasis microenvironment to observe its pathological structure.

[0208] 15. Statistical Analysis

[0209] In this invention, unless otherwise stated, at least three replicates are used for each observation dimension. In animal experiments, at least six replicates are used for each observation dimension. Both cell and animal experiments are statistically analyzed using the Wilcoxon test. A p-value <0.05 is considered significant, *P<0.05, **P<0.01, ***P<0.001.

[0210] Example 1: IBSP levels were significantly elevated in patients with lung adenocarcinoma bone metastases and were positively correlated with lung adenocarcinoma bone metastases.

[0211] To determine the key regulatory genes that mediate lung cancer bone metastasis, the inventors collected lung lesion samples from lung adenocarcinoma bone metastasis (BM) or intrapulmonary metastasis (IM) patients, and analyzed them by RNA sequencing. In addition, bone lesion samples from lung adenocarcinoma bone metastasis patients were compared with normal bone tissue to further characterize the highly expressed genes that promote bone metastasis. The results are shown in Figure 1 a, Figure 1 b and Figure 1 c. The highly expressed genes were cross-referenced with the gene sets of the Human Cancer Metastasis Database (HCMDB) to determine bone metastasis-related genes. A total of 4 genes were obtained, of which IBSP was the most prominent Figure 1 d).

[0212] IBSP is a secreted protein, so ELISA was used to detect the expression of IBSP in the serum of lung adenocarcinoma patients. The results proved the high expression of IBSP in bone metastasis patients Figure 1 a). In addition, it was also confirmed by immunohistochemistry that IBSP was more highly expressed in BM patient lesions Figure 1 e).

[0213] To study whether IBSP can be a key gene that regulates lung adenocarcinoma bone metastasis, single-cell transcriptome sequencing was used to analyze the differences between lung adenocarcinoma primary lung lesion tissue and bone metastasis tissue. The data sets of lung lesions with bone metastasis and bone metastasis lesions were clustered into 18 cell populations. Among them, the C5 and C1 subpopulations highly expressed metastasis marker genes, as shown in Figure 1 f(1). Further analysis found that the expression of IBSP in the C5 and C1 subpopulations was significantly higher than that in other cancer cell populations, as shown in Figure 1 f(2). In addition, the proportion of IBSP-positive cells in bone metastasis lesions was the highest in the C5 and C1 subpopulations, as shown in Figure 1 f(3).

[0214] Since IBSP can be secreted by metastatic lung cancer cells as a regulator of remodeling bone metastasis niches, it can be considered as an important diagnostic biomarker for lung adenocarcinoma with bone metastasis potential. Serological analysis showed that the expression of IBSP in the serum of patients with simple bone metastasis (BM) and multiple metastasis (including BM and IB) was significantly higher than that in patients with intrapulmonary metastasis (IM), while there was no significant difference between the simple BM group and the multiple metastasis group Figure 1 g). Serological analysis showed that the sensitivity of detecting BM was 0.827, and the specificity was 0.613 Figure 1 h).

[0215] In addition, the results of the external data set showed that the overall survival of the cohort of lung adenocarcinoma patients with high expression of IBSP was significantly shorter than that of the cohort with low expression of IBSP, with a hazard ratio of 1.65Figure 7 i).

[0216] The above results indicate that IBSP is highly expressed in cancer cells of patients with bone metastasis of lung adenocarcinoma and is positively correlated with bone metastasis. Therefore, IBSP can be used as a diagnostic biomarker for bone metastasis of lung adenocarcinoma.

[0217] Example 2: Single-cell sequencing analysis reveals the macrophage-osteoclast differentiation trajectory in lung adenocarcinoma bone metastases.

[0218] To further investigate the potential mechanisms promoting lung cancer bone metastasis, single-cell sequencing data from lung adenocarcinoma bone metastases and normal bone tissue were analyzed. Figure 7 a, Figure 7 b) A significant change in a large number of immune cells was found in lung adenocarcinoma bone metastases. Figure 7 c).

[0219] Clinical studies have shown that patients with adenocarcinoma bone metastases exhibit osteoclastosis, in which osteoclasts play a crucial role. UMAP dimensionality reduction analysis revealed that cell clusters 5 (C5) and 21 (C21) were annotated as macrophage and osteoclast clusters, respectively, exhibiting continuous cell trajectory characteristics. Figure 7 d(1)). Through pseudo-time analysis, a trend of macrophage differentiation into osteoclasts was found ( Figure 7 d(2)-(4)). Further analysis revealed that macrophage-related genes were highly expressed in the C5 subset, while osteoclast-related genes were highly expressed in both the C5 and C21 subsets. Figure 7 e). By screening for family-conserved expressed genes, characteristic genes from the C5 and C21 subsets were found to be involved in regulating osteoclast differentiation. Figure 7 f).

[0220] Pseudo-time cell clustering was used to define key regulatory genes mediating macrophage differentiation into osteoclasts. Among them, osteoclast-specific tag genes NFATC1, CTSK, ACP5, and TNFRSF11A (RANK) were highly enriched in the late stages of the pseudo-timeline of osteoclast differentiation, while osteoclast differentiation regulatory genes FCGR1A, FCGR1B, FCGR3A, FCGR3B, STIM1, LRP1, and CD74 were highly enriched in the early and mid stages of the pseudo-timeline. Figure 2 g).

[0221] The results indicate the trajectory of macrophage differentiation into osteoclasts in lung adenocarcinoma bone metastases, suggesting that this differentiation process may mediate bone metastases in lung cancer patients.

[0222] Example 3: IBSP promotes macrophage differentiation into osteoclasts without relying on RANKL / IMI-CSF

[0223] The present inventors confirmed that IBSP is a key regulatory gene highly expressed in bone metastatic lung cancer cells in Example 1, and further confirmed that the differentiation trajectory of macrophages into osteoclasts plays a regulatory role in bone metastasis of lung adenocarcinoma in Example 2. Therefore, the present inventors speculated that IBSP promotes bone metastasis of lung adenocarcinoma by inducing differentiation of macrophages into osteoclasts, and proved it with the following methods:

[0224] IBSP was injected into wild-type mice, and the differentiation effect of IBSP was studied using a cell counting technique (CyTOF) Figure 2 a), and the cell clusters were annotated according to cell-specific expressed proteins. The results showed that the bone marrow-related cells in the IBSP-treated group were significantly increased compared with the control group Figure 2 b、 Figure 2 c). In the C20 and C36 subgroups, macrophage-specific biomarkers and differentiation-related markers were highly expressed in the IBSP-induced group Figure 2 d、 Figure 2 e), indicating that IBSP induced differentiation of macrophages in vivo.

[0225] To study the important role of IBSP in regulating macrophage differentiation, human monocyte cell line THP-1 and mouse macrophage cell line Raw264.7 were treated with IBSP, respectively. The results showed that IBSP induced differentiation of macrophages into osteoclast-like cells with multinuclei characteristics Figure 2 f).

[0226] The present inventors further constructed an IBSP-high-expressing mouse macrophage cell line Raw264.7 cell line to confirm the important role of IBSP in macrophage differentiation. TRAP / ALP staining showed that the IBSP-high-expressing mouse macrophage cell line Raw264.7 could directly differentiate into osteoclast-like cells Figure 8 g).

[0227] To explore the mechanism of macrophage differentiation, proteomics analysis was performed. In Raw264.7 cells, it was found that high expression of IBSP could up-regulate the biological processes of osteoclast differentiation, bone marrow differentiation, cell adhesion and bone destruction Figure 8 e、 Figure 8 f、 Figure 8 g). IBSP could up-regulate the NFKB / TNF signaling pathway in THP-1 cells and the biological process of actin bundle assembly related to osteoclast differentiation Figure 8 h、 Figure 8 i、 Figure 2 j).

[0228] It is known that RANKL / M-CSF is considered as a major regulatory factor for inducing osteoclast differentiation. The present inventors further found that IBSP can increase osteoclast differentiation-related genes (e.g., DCSTAM, DCBLD2, and CTSK) independently of RANKL / M-CSF (h). Figure 2 h) In addition, in vitro bone resorption experiments showed that the IBSP high expression group had stronger bone destruction ability compared with the control group, and its osteolytic effect was similar to that of the RANKL / M-CSF stimulation group (i). Figure 3 i).

[0229] The above results show that IBSP can directly promote the differentiation of macrophages into osteoclasts without RANKL / M-CSF.

[0230] Example 3: IBSP-induced differentiation of macrophages into osteoclasts is a necessary condition for lung cancer bone metastasis

[0231] To further investigate the role of IBSP in lung cancer bone metastasis, the present inventors constructed an IBSP high expression cell line using the Lewis lung cancer (LLC) cell line and a disseminated tumor cell mouse model (DTC model) by intracardiac injection of cancer cells. Figure 3 a) After 14 days of intracardiac injection of cancer cells, in vivo fluorescence imaging analysis showed that the proportion of IBSP high expression LLC bone metastasis (9 / 10) was significantly higher than that of the control group (1 / 10) Figure 3 b). H&E staining detected bone metastasis lesions, and the results showed that metastatic tumor cells formed lesions under the cortical bone and induced osteolysis of the surrounding bone tissue Figure 3 c). These DTC model data show that IBSP can promote lung cancer cell bone metastasis.

[0232] To verify the important role of macrophages in IBSP promoting lung cancer bone metastasis, the present inventors intracardially injected LLC and in vivo depleted macrophages with Clodronate liposomes (CH). After 14 days, bone metastasis lesions were evaluated Figure 3 d(1)). In vivo fluorescence imaging showed that the number of IBSP-promoted bone metastasis was significantly reduced after macrophage depletion Figure 3 d(2)-(4)). These results show that macrophages are essential for IBSP-promoted bone metastasis.

[0233] Finally, to verify the effect of IBSP-induced macrophages on bone metastasis, the present inventors constructed a pre-metastatic niche model in multiple organs, see Figure 3e(l). In vivo fluorescence imaging found that LLC control group could not form detectable tumor lesions within 14 days. While IBSP high expression group could form a small local lesion near the tibial site within 14 days. Interestingly, IBSP-induced macrophages co-injected with control LLC group could form lesions in the bone and abdominal region, see Figure 4 e(3). Therefore, these multi-organ metastasis pre-niche model data suggest that IBSP-induced macrophages are sufficient for bone colonization in lung cancer cells.

[0234] In summary, IBSP-induced macrophage differentiation into osteoclasts is a necessary and sufficient condition for promoting lung cancer bone metastasis.

[0235] Example 4: IBSP-induced macrophage pre-migration differentiation leads to early osteolysis and bone metastasis

[0236] The pre-migration of macrophage differentiation into osteoclasts can mediate early osteolysis, leading to skeletal-related events (SRE), which are clinical complications of bone metastasis. To investigate whether IBSP plays a promoting role in it, the inventors verified the following methods:

[0237] A simulated bone metastasis niche model was established by injecting LLC into the bone at day 2, day 5 and day 7, respectively. CH was administered at the 3 time points to deplete macrophages Figure 4 a). CT was used to evaluate the state of trabecular bone and cortical bone integrity to determine the state of bone erosion. Compared with the control group, the inventors found that the IBSP high expression group had obvious bone erosion at day 5 and day 7. However, the CH treatment group showed a significant recovery of osteolysis Figure 4 b). Further results showed that the IBSP high expression group had significantly lower bone trabecular density and morphological changes such as rod-like trabeculae at day 5 and day 7 than the control group. However, the CH treatment group showed a significant improvement Figure 4 c).

[0238] To clarify the early osteolysis caused by IBSP-induced macrophage differentiation into osteoclasts, the inventors also performed mTc-MDE SPECT-CT imaging on the simulated bone metastasis niche model implanted with cancer cells and CH at day 2, day 5 and day 7.

[0239] The results showed that the osteoclasts in the IBSP high expression group were activated at day 5, with a lower radioactivity concentration in the tibia than in the contralateral limb. In contrast, the radioactivity concentration of the tibia in the CH treatment group and the control group was symmetrical, indicating a balance of osteoclasts Figure 4 d). Bone lesion H&E staining results showed that the IBSP high expression group had cortical bone erosion and tumor cell infiltration at day 5. However, in the CH treatment group, the cortical bone remained relatively intact, and tumor growth was limited outside the bone tissue without cortical bone infiltrationFigure 4 e) It is generally accepted that M-CSF / RANKL is required to stimulate 7 days in vitro to induce osteoclast precursor differentiation into osteoclasts. However, the present inventors found that IBSP stimulation alone can induce osteoclast formation in vivo within 5 days.

[0240] The above results show that IBSP-induced macrophages with pre-mature differentiation play an important role in bone erosion of lung cancer bone metastasis.

[0241] In addition, the present inventors performed flow cytometry on cells in bone lesions and selected Cd45+ Cd326- myeloid cells for subsequent analysis. Compared with the control group, the IBSP high expression group with independent cell subpopulation ratio was distributed according to time characteristics, and according to the pseudo-time sequence, three cell subpopulations showed obvious peaks ( Figure 4 f) By establishing a pseudo-time cell evolution trajectory model, a cell cluster tree showing the proportion of cell subpopulations was obtained Figure 4 g) Cells with more than 50% cell subpopulations and expressing Cd11b at day 2, day 5 and day 7 were defined as early, intermediate and late myeloid cell clusters, respectively. Similarly, the present inventors defined the cell subpopulation expressing Cd116 as a cell subpopulation not in the same trajectory tree branch as other myeloid cells, and defined the cells not expressing Cd116 as non-myeloid cells. Interestingly, in order to further determine the characteristics of all subpopulations, the present inventors analyzed the flow cytometry and cluster-specific genes, and found that the macrophage-specific biomarkers F480, Cd86, Cd115 and Cd206 were enriched in the intermediate stage and increased in the late stage. While Ly6c was expressed the highest in the intermediate stage and decreased in the late stage Figure 5 h) In combination with existing research, the present inventors speculate that macrophages may also retain the biomarkers of their precursors during the fusion and differentiation process of bone metastasis.

[0242] In summary, the above results show that IBSP regulates the pre-mature differentiation of macrophages into osteoclasts in vivo, promotes bone erosion and mediates bone metastasis of lung cancer.

[0243] Example 5: IBSP mediates the forward shift of macrophages to osteoclasts by regulating the NFAT signaling pathway

[0244] In order to study the mechanism of IBSP promoting the pre-mature differentiation of macrophages to osteoclasts, an in vitro cell model of IBSP-induced differentiation of THP-1 and Raw264.7 to osteoclasts was constructed. Proteomics-mass spectrometry was used to find the mechanism of time differentiation, and cell samples were collected at day 2, day 5 and day 7 Figure 5 a(1)) Using unsupervised time clustering algorithm, the present inventors located proteins with the same trend of change in 3 different times. Among the 4 change trends of these proteins, there are up, down, peak and valley, respectivelyFigure 5 a(2)). Taking the intersection of Raw264.7 and THP-1, which have the same variation trend, some proteins related to osteoclast differentiation were located. Figure 5 b).

[0245] Natc1 / 2 is crucial for osteoclast differentiation. Given that Natc2 is a protein that maintains a sustained upward trend, this suggests that NFAT may play an important role in IBSP-induced macrophage-to-osteoclast differentiation. The inventors screened differentially expressed proteins across three sampling time points and found that IBSP-induced osteoclast-like differentiation occurred in Raw264.7 cells on day 5. Figure 5 c. Figure 5 d).

[0246] To further confirm the changes that occurred 5 days after IBSP stimulation, the genes specifically upregulated on day 5 were clustered and found to be mainly involved in myelocyte differentiation and osteoclast differentiation. Figure 5 e). The results showed that no enrichment of genes associated with ascending signaling pathways related to osteoclast differentiation was found. These results indicate that IBSP-induced macrophage differentiation into osteoclasts has a significant precession characteristic, with the average differentiation time reduced from the classically accepted 7 days to 5 days.

[0247] It is worth noting that the inventors also found that Natc1 and Nfatc2 were significantly elevated in macrophages with high IBSP expression. Figure 5 f). Interestingly, knocking out Nfatc1 also downregulated the expression of Nfatc2; knocking out Nfatc2 also downregulated the expression of Nfatc1. Figure 5 These results indicate that IBSP-induced upregulation of Nfatc1 and Nfatc2 is mutually influential. Finally, the inventors found that downregulating Nfatc1 or Nfatc2, respectively, significantly alleviated IBSP-induced osteoclast differentiation (g). Figure 9 h、 Figure 9 i).

[0248] In summary, IBSP can activate the NEAT signaling pathway and promote the positive differentiation of macrophages into osteoclasts.

[0249] Example 6: Inhibition of Rac1 can block IBSP-induced bone metastasis

[0250] To explore key target genes of the IBSP-upregulated NEAT signaling pathway, the inventors screened candidate target genes that may be involved in the regulation of the NFAT signaling pathway through IBSP upregulation, and obtained Rac1, Fyn, and Epha2. Figure 9a) The inventors found that the expression levels of Nfatcl and Nfatc2 did not change consistently in Raw264.7 cells excluding Fyn or Epha2 Figure 6 b) or no significant difference Figure 6 c) However, in IBSP+Raw264.7 cells, the expression levels of Nfatcl and Nfatc2 were significantly down-regulated after knocking down Rac1 Figure 6 a, Figure 9 b) Interestingly, the inventors also found that knocking down Nfatcl or Nfatc2 could lead to down-regulation of Rac1 expression Figure 9 c), which suggests that there may be positive feedback regulation of Rac1 and NEAT signaling pathways up-regulated by IBSP.

[0251] Further examination found that the differentiation of macrophages into osteoclast-like cells induced by IBSP depends on Rac1 Figure 6 d, Figure 6 e) In vitro bone resorption experiments showed that the high bone destruction ability of the IBSP high expression group was significantly reduced after knocking out Rac1 Figure 6 d) In addition, in the C21 group of osteoclast-related cells in the bone metastasis tissue of lung adenocarcinoma patients, the expression level of Rac1 was positively correlated with the genes related to osteoclast differentiation signaling pathway Figure 6 e).

[0252] To further evaluate the effect of interfering with Rac1 on IBSP promoting lung cancer bone metastasis, the inventors used a specific inhibitor of Rac1, EHT-1864. The inventors found that EHT-1864 can significantly down-regulate the expression of Rac1, and the expression of Nfatcl and Nfatc2 is also down-regulated in Raw264.7 cells Figure 6 f, Figure 6 g) In addition, after EHT-1864 treatment, the genes related to osteoclast differentiation signaling pathway were also down-regulated Figure 6 h) In vitro bone resorption experiments showed that the enhanced bone destruction of IBSP was significantly reduced after inhibiting Rac1 Figure 6 i) These results show that EHT-1864 can inhibit Rac1, thereby down-regulating the NEAT signaling pathway and blocking osteoclast differentiation.

[0253] To further explore the role of Rac1 in IBSP promoting lung cancer bone metastasis, the inventors used IBSP high expression cell lines, used LLC and KP cell lines, and used DTC model of intracardiac injection of cancer cells, and treated with EHT-1864 or clinical drug azathioprine that can inhibit Rac1 activity Figure 6 j(1)) The results showed that the addition of EHT-1864 or azathioprine significantly reduced the promotion of lung cancer bone metastasis by IBSPFigure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 j(2)-(4))).

[0254] The above results demonstrate that the inhibition of Rac1 by EHT-1864 or azathioprine can block the IBSP promotion of bone metastasis.

[0255] Discussion

[0256] In the present application, the present inventors found that the expression level of IBSP in lung adenocarcinoma bone metastasis is elevated, and the prognosis of lung adenocarcinoma patients with high expression of IBSP is poorer than that of patients with low expression of IBSP. In addition, serological tests also show that the serum IBSP of bone metastasis patients is significantly elevated, indicating that IBSP is a valuable serological diagnostic biomarker for lung adenocarcinoma bone metastasis.

[0257] On the other hand, the experiments of the present inventors show that IBSP promotes macrophage-dependent bone metastasis by regulating the Rac1-NFAT signaling pathway and directly inducing the forward movement of macrophages to osteoclasts.

[0258] In summary, the research of the present inventors shows that IBSP can be a potential serological diagnostic marker, and IBSP is a strong therapeutic target for lung adenocarcinoma bone metastasis.

[0259] Previously, it was generally believed that at least 7 days were needed to induce the differentiation of macrophage precursors into osteoclast-like cells in vitro, and RANKL / M-CSF was needed to induce the maturation of osteoclasts. The present inventors screened differentially expressed proteins at 2, 5, and 7 days after IBSP induction, and found that the expression of multiple osteoclast differentiation-related genes reached a peak at 5 days, indicating that IBSP induced the differentiation of macrophages into osteoclasts within 5 days. The research results of the present inventors also found that IBSP can induce the differentiation of macrophages into RANKL / M-CSF-independent osteoclasts.

[0260] In the present invention, the inventors demonstrated that IBSP can upregulate the Rac1-NFAT signaling pathway, leading to early osteolysis. The inventors determined that IBSP can upregulate the expression level of Rac1, and downregulation of Rac1 by siRNA or its inhibitor EHT-1864 can significantly block IBSP-induced expression of NFATC1 and NFATC2. Notably, knockdown of NFATC1 or NFATC2 also partially reduced IBSP-induced Rac1 expression. This suggests that Rac1 and NFAT signaling pathways form a positive feedback loop, mediating IBSP-induced differentiation of macrophages into osteoclasts. TRAF can activate Rac1 to promote osteoclast differentiation. The results show that IBSP can activate TRAF in Raw264.7 and THP1 cells. Therefore, the inventors speculate that IBSP can promote the forward differentiation of macrophages into osteoclasts by activating TRAF, upregulating the Rac1-NFAT signaling pathway, and further enhancing the osteolytic bone metastasis of lung cancer.

[0261] Since IBSP can directly induce the forward differentiation of macrophages into osteoclasts by regulating the Rac1-NFAT signaling pathway, the inventors investigated the therapeutic effect of using Rac1 inhibitors (EHT-1864 and azathioprine) and found that Rac1 inhibitors can significantly block IBSP+LLC bone metastasis. Therefore, Rac1 is a strong therapeutic target for lung cancer bone metastasis.

[0262] To further clarify the therapeutic effect of EHT-1864 and azathioprine, the inventors established another highly metastatic lung cancer cell line, the IBSP-high-expressing KP cell line (IBSP+KP). The results were consistent with the IBSP+LLC bone metastasis mouse model, and the administration of EHT-1864 or azathioprine can significantly reduce the bone metastasis of IBSP+KP cells. Notably, the inventors also found that some brain metastasis also occurred in the IBSP+KP cell metastasis mouse model, and EHT-1864 or azathioprine can partially alleviate this metastasis. These results show that EHT-1864 and azathioprine are effective blockers of lung cancer bone metastasis and potential therapeutic options for lung cancer brain metastasis. Therefore, the inventors found that inhibition of Rac1 by EHT-1864 or azathioprine can block IBSP-induced lung cancer bone metastasis in vivo.

[0263] All documents mentioned in the present invention are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various modifications and changes can be made to the present invention by those skilled in the art which do not part from the spirit and scope of the present invention.

Claims

1. The use of an integrin-binding salivary protein (IBSP) detection reagent, characterized in that, This kit is used to prepare a reagent for assessing the risk of bone metastasis in a tumor or cancer, wherein the tumor or cancer is lung adenocarcinoma; The test reagent is used to detect serum samples; when the serum concentration of IBSP is ≥1056±100ng / L, it indicates that the risk of bone metastasis of lung adenocarcinoma in this subject is significantly higher than that of general lung adenocarcinoma patients. The IBSP detection reagent is a reagent used to detect IBSP mRNA, cDNA, or protein; The kit also includes reagents for detecting Rac1 mRNA, cDNA, or protein.

2. The use of a reagent combination, characterized in that, This kit is used to prepare a reagent for diagnosing bone metastases of a tumor or cancer, specifically lung adenocarcinoma. The reagent combination includes: (Y0) A reagent for detecting IBSP mRNA, cDNA, or protein; and (Y1) A reagent used to detect Rac1 mRNA, cDNA, or protein.

3. The use as described in claim 2, characterized in that, The aforementioned test reagent is used to test serum samples.

4. The use of a Rac1 inhibitor, characterized in that, Used to prepare a composition or formulation for preventing and / or blocking bone metastasis of tumors or cancer; The tumor or cancer mentioned is lung adenocarcinoma; The Rac1 inhibitor is EHT-1864 or azathioprine.

5. The use as described in claim 4, characterized in that, The lung adenocarcinoma is a tumor with upregulated Rac1 expression.

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