WO2024230090A1 - Use of sialyltransferase inhibitor in preparing medicament for neutralizing acidic tumor microenvironment - Google Patents

Abstract

The present application relates to the field of tumor treatment technology, and in particular, to use of a sialyltransferase inhibitor in preparing a medicament for neutralizing an acidic tumor microenvironment. By providing the use of the sialyltransferase inhibitor in preparing the medicament for neutralizing the acidic tumor microenvironment, the sialyltransferase inhibitor can neutralize the acidic tumor microenvironment and establish an ecological barrier against tumor metastasis, thus providing a new way of preventing and treating tumors.

Description

Application of sialyltransferase inhibitors in the preparation of drugs for neutralizing acidic tumor microenvironment

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 2023105179746 filed with the State Intellectual Property Office of China on May 9, 2023, entitled “Application of sialyltransferase inhibitors in the preparation of drugs for neutralizing acidic tumor microenvironment”, the entire contents of which are incorporated by reference into this application.

Technical Field

The present disclosure relates to the technical field of tumor treatment, and in particular, to the use of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment.

Background Art

Cancer metastasis is an extremely complex process that is affected by many factors, including acidity, which is generally believed to be mainly caused by lactate/H ⁺ produced by glycolysis. Acidity is one of the main characteristics of many cancers. It is currently believed that acidity is caused by the accumulation of metabolic waste caused by cancer cells with high metabolic activity and insufficient blood perfusion. During tumor growth and metastasis, the acidic tumor microenvironment (ATME) is believed to be caused by the removal of protons and H ⁺ produced by lactate/H ⁺ , bicarbonate and ^CO2 hydrogenation from the cytosol to the interstitial space. The pH value of the acidic tumor microenvironment (ATME) is generally 5.6-7.0. The acidic microenvironment not only helps cancer cells migrate through the extracellular matrix (ECM) to blood vessels with a relatively high pH (7.35-7.45), but also hinders the infiltration of active immune cells into tumor tissues by reducing the activity of T cells and NK cells, thereby achieving tumor metastasis. To date, no other mechanism that can cause the generation of an acidic tumor microenvironment (ATPME) has been found.

According to surveys, breast cancer has the highest incidence rate among all cancers, with 2.26 million new cases of breast cancer and approximately 685,000 cancer deaths in 2020. Almost all breast cancers are carcinomas that originate from mammary epithelial cells, which are arranged in a double-layer structure between the lobules and the terminal ducts. The inner layer contains secretory luminal epithelial cells, and the outer layer is composed of contractile myoepithelial cells (or basal cells) surrounded by a basement membrane (BM). The integrity of the epithelial bilayer is important for maintaining the normal development of the mammary gland and preventing the malignant transformation of epithelial cells. In addition, the spread of malignant cells to secondary organs depends on their ability to destroy the basement membrane and basal layer, which mainly contain laminin and collagen IV, their ability to invade the extracellular matrix, their ability to infiltrate the circulation, extravasate from blood vessels, and grow in distant organs. Therefore, the study of tumor growth and metastasis plays a key role in the treatment and prevention of cancer.

In view of this, the present disclosure is proposed.

Summary of the invention

The present disclosure provides a use of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment.

Optionally, the sialyltransferase inhibitor neutralizes the acidic tumor microenvironment by downregulating the expression of Vegfa and/or VegfIl6.

Optionally, the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.

Optionally, the sialyltransferase inhibitor inhibits the expression of polysialyltransferase and/or polysialyltransferase receptor.

Optionally, the polysialyltransferase comprises St8sia4.

Optionally, the polysialyltransferase receptor comprises at least one of soluble E-selectin and L-selectin.

Optionally, the drug for neutralizing the acidic tumor microenvironment also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.

The present disclosure provides an application of a sialyltransferase inhibitor in preparing a drug for treating Brca1 defect-related tumors.

Optionally, the drug for treating Brca1 deficiency-related tumors also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.

Optionally, the Brca1 deficiency-associated tumor is Brca1 deficiency-associated breast cancer.

The present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for preventing or treating cancer. The drug for preventing or treating cancer includes drugs for preventing or treating any one of the occurrence, metastasis or growth of cancer.

Optionally, the drug for preventing or treating cancer also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.

Optionally, the cancer includes any one of breast cancer, liver cancer, and spleen cancer.

The present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for treating the destruction of the double-layer structure of mammary epithelium.

Optionally, the drug for treating destruction of the double-layer structure of mammary epithelium also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.

The present disclosure provides a pharmaceutical composition, comprising the aforementioned sialyltransferase inhibitor and a pharmaceutically acceptable excipient.

Optionally, the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.

Optionally, the dosage form of the drug includes any one of injection, injection, tablet, granule, granule or capsule.

Optionally, the drug is in the form of nanoparticles.

The present disclosure provides a method of neutralizing an acidic tumor microenvironment, the method comprising administering to a subject in need thereof an effective amount of a sialyltransferase inhibitor.

The present disclosure provides a method for treating a tumor associated with Brca1 deficiency, the method comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof;

Optionally, the Brca1 deficiency-associated tumor is Brca1 deficiency-associated breast cancer.

The present disclosure provides a method for preventing or treating cancer, comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof; wherein the drug for preventing or treating cancer includes drugs for preventing or treating any one of the occurrence, metastasis or growth of cancer.

Optionally, the cancer includes any one of breast cancer, liver cancer, and spleen cancer.

The present disclosure provides a method for treating disruption of the bilayer structure of mammary epithelium, the method comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof.

Optionally, the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.

Optionally, the above method comprises the combined use of a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.

The present disclosure provides a sialyltransferase inhibitor for the following purposes:

i. Neutralize the acidic tumor microenvironment;

ii. Treating Brca1 deficiency-related tumors; optionally, the Brca1 deficiency-related tumor is Brca1 deficiency-related breast cancer;

iii. Preventing or treating cancer; wherein preventing or treating cancer includes preventing or treating any of the occurrence, metastasis or growth of cancer; optionally, the cancer includes any of breast cancer, liver cancer, spleen cancer;

iv. Treat the destruction of the double-layer structure of mammary epithelium.

Optionally, the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.

Optionally, the use includes a sialyltransferase inhibitor combined with a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

FIG1 is a result of the destruction of the double-layer structure of the mammary gland associated with the increase of sialyltransferase provided by the embodiments of the present disclosure;

FIG2 is a result diagram of the increase of sialyltransferase in mouse mammary tissue provided by the embodiments of the present disclosure;

FIG3 is a graph showing the increase in polysialic acid content in mouse Brca1-deficient cells and human Brca1-deficient breast cancer samples provided by the embodiments of the present disclosure;

FIG4 is a graph showing changes in sialyltransferases in Brca1-deficient mice and non-Brca1-deficient mice provided in an embodiment of the present disclosure;

FIG5 is a diagram showing the effect of sialyltransferase on breast cancer metastasis provided by an embodiment of the present disclosure;

FIG6 is a diagram showing the effect of sialyltransferase expression on tumor growth and metastasis provided by an embodiment of the present disclosure;

FIG. 7 is a diagram showing the results of detecting the expression of sialyltransferase in mammary epithelial cells provided by an embodiment of the present disclosure;

FIG8 is a diagram showing that the Vegfa/Il6 signaling pathway upregulates TGF-β signals in Brca1-deficient mammary epithelial cells and macrophages according to an embodiment of the present disclosure;

FIG9 is a result of the tumor immunosuppressive microenvironment induced by high sialylation in breast tissues of mice and human breast cancer patients provided by the embodiments of the present disclosure;

FIG10 is a graph showing the relationship between the increase in the number of MDSCs in Brca1-deficient mice and the effect of Vegfa/Il6 provided in an embodiment of the present disclosure;

FIG. 11 shows the results of inhibiting breast tumor growth and metastasis by using a sialyltransferase inhibitor and a combination of the sialyltransferase inhibitor and a drug provided in an embodiment of the present disclosure;

FIG. 12 shows the results of inhibiting breast tumor growth and metastasis after intraperitoneal injection of sialyltransferase inhibitors and drugs used in combination therewith provided in the embodiments of the present disclosure;

FIG. 13 is a diagram showing the results of treating breast tumors with high sialyltransferase expression using a sialyltransferase inhibitor in combination with αPD-1 according to an embodiment of the present disclosure;

FIG. 14 shows the effects of the sialyltransferase inhibitor provided in the embodiments of the present disclosure in combination with αPD-1 on different cells during breast tumor growth and metastasis.

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

The term “Brca1” stands for breast cancer associated gene 1, a tumor suppressor gene whose germline mutations cause familial breast cancer.

The term "EMT" stands for epithelial-mesenchymal transition, which refers to the transformation of epithelial to mesenchymal cells, which endows cells with the ability to metastasize and invade, including stem cell characteristics, reduces apoptosis and senescence, and promotes immunosuppression. It not only plays a key role in the development process, but also participates in processes such as tissue healing, organ fibrosis, and cancer occurrence.

The term "MDSCs" refers to myeloid-derived suppressor cells, which are precursors of dendritic cells, macrophages and/or granulocytes and have the ability to significantly suppress immune cell responses.

The term "ATPME" stands for acidic tumor microenvironment.

The terms "Vegfa" and "VegfIL6" are both transcriptional regulators involved in angiogenesis and tumor cell invasion, where Vegfa is vascular endothelial growth factor A (VEGF-A).

VegfIL6 is vascular endothelial growth factor (VEGF) and IL-6, IL-6 is interleukin-6, a cytokine belonging to the chemokine family.

The sialyltransferase genes St8sia4 and St3gal1 are responsible for the production of polysialic acid (PSA) and Sialyl-Lewis A (sLeA), respectively.

3Fax-Peracetyl Neu5Ac, molecular formula: C ₂₂ H ₃₀ FNO ₁₄ , structural formula is as follows:

As a cell permeable silyl analog, 3Fax-Peracetyl Neu5Ac can be converted to CMP-Neu5Ac and inhibit silyl transferase. It is used as a sialyltransferase inhibitor in this application.

Stattic is a STAT3 inhibitor, with a molecular formula of C ₈ H ₅ NO ₄ S and a structural formula as shown below:

The present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment. Through research, it is found that the sialyltransferase inhibitor can neutralize the acidic tumor microenvironment and establish an ecological barrier to tumor metastasis, providing a new approach for the prevention and treatment of tumors.

The sources of human tissue microarrays and human breast cancer samples in the following experiments are as follows: 95 tissue microarray human breast cancer samples were kindly provided by the Third Affiliated Hospital of Sun Yat-sen University. 25 human breast cancer samples for immunofluorescence staining were kindly provided by the First Affiliated Hospital of Sun Yat-sen University, and BRCA mutations were sequenced and identified. Among these 25 primary tumor samples, 4 were BRCA1 mutation carriers and 21 were non-BRCA1 mutation carriers. Human tissues were fixed in formalin, embedded in paraffin, and sectioned for experiments.

The sources of mice and experimental cell lines in the following experiments are as follows: All mouse experiments performed in this study were approved by the Animal Ethics Committee of the University of Macau.

The Brca1 conditional knockout mouse model (Brca1co/co; MMTV-Cre) has been established in the inventors' laboratory.

To establish tumor allograft models, constructed Brca1WT (B477) mammary epithelial cells (2×10 ⁵ ) and Brca1-MT (G600) mammary epithelial cells (2×10 ⁵ ) were orthotopically implanted into the fourth mammary fat pad on the right side of 5-6 week-old nude mice. To establish tumor metastasis models, constructed 545 (Brca1 mutant low metastasis), 628W (Brca1 wild type high metastasis) and EMT6 cells (5×10 ⁵ ) were orthotopically injected into the fourth mammary fat pad on the right side of nude mice and Balb/c mice or directly injected through the tail vein (5×10 ⁴ cells). The tumor volume was measured and calculated using the formula: V=0.5×L×W ² , where L represents the major diameter and W represents the minor diameter. For drug treatment experiments, when the tumor volume reached about 50 ^mm3 , different drugs were given, including intraperitoneal injection of sialyltransferase inhibitor 3Fax-P-Neu5Ac (20 mg/kg) for 7 consecutive days and intraperitoneal injection of Stattic (10 mg/kg) 3 times a week. For nanoparticle delivery, Stattic (10 mg/kg) and 3Fax-P-Neu5Ac (20 mg/kg) were injected intravenously once every three days for a total of 3 times.

The ratiometric fluorescent probe was kindly provided by Professor Zhang Xuanjun of the University of Macau, and the Stattic and 3Fax-P-Neu5Ac nanoparticles were kindly provided by Professor Dai Yunlu of the University of Macau. The PD1 antibody, i.e., αPD-1 used in the examples of this application, was purchased from Bio X Cell InVivoMab, catalog number: BE0273-5mg.

The sources of RNA-seq data and human datasets in the following experiments are as follows: Bone marrow, blood, spleen, peritoneal cells, and mammary tissues from 10-month-old mice and breast tumors collected from Brca1co/co tumor mice were isolated in Trizol, and total RNA was subjected to mRNA isolation and library construction, followed by RNA sequencing (Hiseq, paired-end, 6GB raw data per sample). The data were analyzed using HISAT, StringTie, and Ballgown to obtain differentially expressed genes. FPKM values were extracted for each relevant gene to generate a matrix, and heat maps were drawn for different important genes in different organs by RStudio. The immune cell composition in breast and breast tumors was calculated by ImmuCC and plotted by heat maps. To predict the correlation of immune cell abundance with St8sia4 expression, the human dataset of BRCA samples was obtained from TISIDB (http://cis.hku.hk/TISIDB/index.php). The data on the transcription or protein levels of St8sia4 and St3gal1 genes were obtained from breast cancer patients in the TCGA database, and the graphs were obtained from Linkedomics (http://www.linkedomics.org/login.php). The Kaplan-Meier plotter (https://kmplot.com/analysis/) was used to obtain the survival curves of breast cancer patients with high or low gene expression.

The sources of immunofluorescence staining of tissue sections in the following experiments are as follows: All human and mouse paraffin slides were dewaxed and rehydrated according to standard protocols. The slides were washed with PBS and then heated in R-Buffer-A (10 mL in 90 mL water). After the treatment was completed (or overnight incubation), the slides were washed with PBS and then treated with 0.5% TritonX-100 and 0.5 mg/ml sodium borohydride (in PBS) for 10 minutes at room temperature, respectively. The slides were then incubated with blocking solution (50% 3% BSA and 50% Animal-FreeBlocker) at room temperature overnight or at least 1 hour, and incubated with primary antibodies from different sources overnight at 4°C. Secondary antibodies and DAPI were incubated for 1 hour at room temperature, anti-fading reagents were covered on the tissues, and coverslips were placed on each slide, and images were scanned by CarlZeissLSM880 super-resolution microscope.

The source of cell immunofluorescence staining in the following experiments is as follows: cells were seeded in a 4-well chamber, washed twice with PBS, and fixed with 4% formaldehyde for 15 minutes. Then, cells were thoroughly washed with PBS and treated with 0.5% TritonX-100 for 10 minutes, and then incubated with blocking solution at room temperature for at least 1 hour. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature using a Carl Zeiss LSM880 ultrasonoscope. High-resolution microscope scan image.

The sources of the cytokine antibody array in the following experiments are as follows: serum from 6-month-old mice and supernatants from different cell lines were diluted with 1× blocking buffer. The diluted samples were added to the membrane in a 4-well tray and pre-incubated with 1× blocking buffer for 30 minutes at room temperature and incubated overnight at 4°C. The membranes were placed in a clean container and washed with 20-30mL of 1× wash buffer I for 30-45 minutes per membrane. They were then returned to the 4-well tray and washed twice with 2ml wash buffer II at room temperature. 2ml 1× Biotin-Conjugated Anti-Cytokines was pipetted into each well and the membrane was incubated overnight at 4°C. 2mL of 1× HRP-conjugated streptavidin was added to each well and the membrane was incubated overnight at 4°C. The membranes were washed and processed the next day as described previously. Detection buffer C and detection buffer D were mixed in equal volumes (1:1) and covered with the membranes in the tray. After incubation for 2 min at room temperature, the membrane was immediately exposed and images were captured using an immunoblot imaging system.

The source of tissue pH detection in the following experiments is as follows: Different cell lines were seeded into 24-well plates at the same initial concentration, and after incubation for 48 hours or 72 hours, the pH of the supernatant from different wells was measured using a cell culture biochemical analyzer. To determine the intracellular pH, the ratiometric fluorescent probe was diluted in culture medium (10 μM) and then added to different cultured cells, followed by incubation at 37°C for 1 hour. The cells were washed with PBS in a 4-well confocal chamber, and green and red fluorescence was captured under a Carl Zeiss LSM880 super-resolution microscope. To determine the pH of the tissue, fresh tumor samples were cut into 250-μm sections and stained with the ratiometric fluorescent probe (40 μM) at 37°C for 1 hour, followed by washing with PBS. Images were acquired using a Leica M165FC stereomicroscope. The pH value was determined according to the formula: (y = 31.8403-4.4898x, R = 0.9928, where y represents the pH value and x represents the ratio of red and green fluorescence intensities).

The sources of protein extraction and immunoblotting in the following experiments are as follows: RIPA buffer (150mM NaCl, 50mMTris-HCl, pH7.4, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1mM EDTA) supplemented with protease inhibitors and phosphatase inhibitor cocktails. The membranes with different target proteins were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hour at RT. Images were acquired by a dual-color infrared laser imaging system ODYSSEYCLx.

The methods for RNA extraction and real-time qPCR analysis in the following experiments are as follows: RNA was isolated from fresh breast, tumor and spleen tissues using Trizol and reverse transcribed using PrimeScriptTMRT kit and gDNAEraser (Takara #RR047Q). Real-time qPCR was performed using a fluorescent quantitative PCR kit (Roche, 24759100), and data were acquired using a real-time fluorescent quantitative PCR system (ThermoFisher).

The mass spectrometry analysis method in the following experiments was as follows: Single cells from mouse tumor-adjacent mammary tissue and tumor tissue were collected and digested for 2 hours at 37°C with digestion medium containing 5% fetal bovine serum, 5 μg/ml insulin (I-1882; Sigma-Aldrich), 500 ng/ml hydrocortisone (H0888; Sigma-Aldrich), 300 U/ml collagenase III (S4M7602S, Worthington, Lakewood, NJ) and 100 U/ml hyaluronidase (H3506; Sigma-Aldrich), and then washed with HPSS (14170-112; Life Technologies). Lysed in 1xRBC lysis buffer for 3 minutes at room temperature (RT) to deplete red blood cells and quenched by HBSS. The suspension was then filtered through a 40 μm cell strainer and washed with PBS. Resuspend the cells to a density of 2x107 cells/ml in pre-warmed serum-free medium, and add an equal volume of 10μM cisplatin compound solution to the cell suspension (final concentration of cisplatin compound: 5mM). Resuspend the cells and incubate at room temperature for 5 minutes. Then, add 5 volumes of Maxpar Cell Staining Buffer to quench cisplatin staining, and centrifuge the cells at 300x g for 5 minutes. Then wash the cells once with 1ml Maxpar Cell Staining Buffer, and suspend 3 million cells in 80μl Maxpar Cell Staining Buffer. Add the suspended cells to 20μl of Fc blocking solution. Incubate the cells at 4°C for 10 minutes and then resuspend in 50μl Maxpar Cell Staining Buffer. Add 50μl of antibody mixture to each test tube, and incubate the cells at room temperature for 30 minutes after gentle vortexing. The cells were then washed twice with 1 ml Maxpar Cell Staining Buffer and 1 ml of cell embedding solution (Cell-ID Intercalator-Ir diluted with Maxpar Fix and Perm Buffer) was added. The samples were mixed thoroughly and placed overnight at 4°C. The next day, the cells were washed twice with 1 ml Maxpar Cell Staining Buffer and 1 ml Maxpar water. The cell concentration was adjusted to 2.5-5 x 10 ⁵ / ml with a positive control bead buffer (EQTM four-element calibration, Cat. # 201078), and then data was collected on a mass spectrometer (Fluidigm).

The flow cytometry analysis method in the following experiments was as follows: Single cells were collected from nude mouse breast tumors, bone marrow, blood, spleen, and peritoneum, then treated with RBC lysis buffer and washed with PBS. The cells were resuspended in 100 μL PBS and incubated with the conjugated antibody on ice for 30 minutes. The cells were then washed twice and resuspended in PBS, and the data were acquired using a flow cytometer and analyzed using flow data processing software.

The preparation and treatment methods of nanoparticles in the following experiments are as follows: The materials used to prepare the nanoparticle drugs were provided by Professor Dai Yunlu's laboratory. Polyethylene glycol and drugs were dissolved in methanol, and the mixture was added to a solution of deionized water/tetrahydrofuran and sonicated for 5 minutes. After the organic phase was removed by vacuum suction, the unencapsulated components were filtered using a 220 nm filter membrane, and the remaining solution was concentrated and stored in a dark bottle at 4 °C. The morphology of the nanoparticles was studied by transmission electron microscopy, and the UV-visible absorption spectrum was measured by a spectrophotometer. 200 μL of RFP-labeled control group polyethylene glycol was injected into tumor-bearing nude mice through the tail vein, and the enrichment of nanoparticles in the tumor site was observed using the IVIS Illumina in vivo system. When the average tumor size reached about 50 mm ³ , PEG containing the drug was injected three times through the tail vein, and the mice were killed on the 26th day.

The statistical analysis methods of the data obtained in the following experiments are as follows: Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, USA). Unpaired Student's t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. Correlation analysis was performed by Pearson's correlation coefficient test. Data are expressed as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered statistically significant compared with the control group.

Most breast cancers are sporadic and have no family history, but approximately 5-10% of breast cancers are heritable. Breast cancer associated gene 1 (Brca1) is a tumor suppressor gene whose germline mutations can lead to familial breast cancer. Brca1 has many important functions, including transcriptional regulation, DNA damage repair, centrosome duplication, cell cycle checkpoints, chromatin remodeling, and protein ubiquitination. Current studies have found that approximately 25% of mice with Brca1 deficiency develop breast tumors at 1.5 years of age, and approximately 20% of these mice have tumor metastases to distant organs. Further studies have shown that Brca1 deficiency leads to many abnormalities in mammary gland development, such as blunted ductal morphogenesis, dysregulated genome-wide gene expression, enhanced discharge of DNA replication forks, and increased apoptosis; this can be partially supported by the loss of p53, Atm, Chk2, and 53PB1 function or the activation of certain oncogenes. Breast cancer patients with Brca1 mutations also experience a high frequency of multi-organ metastasis. Therefore, the inventors believe that relevant research on Brca1 mutations is of great significance for the prevention and treatment of breast cancer and multi-organ metastasis of tumors.

The inventors first used immunofluorescence staining to label luminal epithelial cells (CK18), basal or myoepithelial cells (CK14) and basement membrane (collagen IV) in human breast cancer patients with or without Brca1 mutation and Brca1-MSK mouse model to detect the mammary double-layer structure composed of luminal and basal layers and surrounded by basement membrane, and obtained the results shown in Figure 1. The marks in the figure are: WT indicates non-BRCA1 mutation, and MT indicates BRCA1 mutation.

Among them, Figure 1 A is a representative image of the tumor-adjacent breast of a breast cancer patient; Figure 1 C is a representative image of the tumor tissue of a breast cancer patient; the experiment used CK18/CK14 co-staining, and calculated the percentage of the affected basal layer in the patient's tumor-adjacent breast and tumor tissue, and obtained the results shown in Figure 1 B and Figure 1 D, respectively. As shown in Figure 1 A-D, compared with non-BRCA1 mutations, the basal layer in the tumor-adjacent breast tissue and tumor tissue of breast cancer patients with BRCA1 mutations is severely missing.

Figure 1E is a representative image of the tumor-adjacent mammary gland of Brca1-MSK mice; Figure 1G is a representative image of the tumor tissue of Brca1-MSK mice; the experiment used CK18/Collagen IV co-staining, and calculated the percentage of the affected basal layer in the tumor-adjacent mammary gland and tumor tissue of the mice, and obtained the results shown in Figure 1F and Figure 1H, respectively. It can also be found that compared with non-BRCA1 mutations, the basal layer in the tumor-adjacent mammary gland tissue and tumor tissue of Brca1-MSK mice with BRCA1 mutation is severely missing. This shows that BRCA1 defect-related tumor occurrence has a greater impact on the structural integrity of the mammary gland than sporadic tumor occurrence.

Further, the inventors used immunofluorescence staining to study the structural integrity of the mammary glands of 8-month-old and 10-month-old non-mutated and mutated Brca1-MSK mice, all of which were in the stage before or after tumor formation. As shown in I-K of Figure 1, the mammary gland structure of non-BRCA1 mutant mice is more complete, while the mammary gland structure of mice after BRCA1 mutation is abnormal. The 10-month-old mice were further experimented with. Representative images of tumor-adjacent mammary tissues of 10-month-old non-mutated mice and control mice (shown in L of Figure 1) were co-stained with CK18 and CK14, and the percentage of the destroyed basal layer was calculated (shown in M of Figure 1). Representative images of tumor tissues of 10-month-old non-mutated mice and control mice (shown in N of Figure 1) were co-stained with CK18 and CK14, and the percentage of the destroyed basal layer was calculated (shown in O of Figure 1). As shown in L-O of Figure 1, the basal layer of 10-month-old mutant mice was severely missing and the mammary ducts were destroyed. These results suggest that mammary epithelial cells and Brca1 deficiency in mice display structural abnormalities before and during tumorigenesis.

Cancer metastasis is a very complex process, and epithelial-mesenchymal transition (EMT) is acquired by malignant cells before metastasis. Examination of tumor-adjacent breast tissues of breast cancer patients with vimentin antibodies showed that higher levels of vimentin were detected in BRCA1, and more multi-organ metastases were observed in mutation carriers than in non-mutation carriers (Figure 2, H) in the Brca1-MSK mouse model (Figure 2, D-G).

By comparing the gene expression profiles of RNA sequence analysis of age-matched Brca1 wild-type breast tissue (WTMG), Brca1 mutant breast tissue (MTMG) and Brca1 mutant primary tumor (MTPT), the results shown in Figure 2 were obtained. As shown in Figure 2I, compared with the process from WTMG to breast tumor tissue, the regulatory factors of EMT markers, such as Ddr2 and Itga5, and EMT markers, such as vimentin, Fn1, MMP2, MMP3, MMP14, Tcf3, Twist and Zeb2, were expressed in the process from MTMG to breast tumor tissue. The expression of Krt19, Krt18, Krt5, and Krt14 in mammary epithelial tumor tissues of Brca1-MSK mice continued to increase during the process of differentiation, while markers of mammary epithelial differentiation, including Krt19, Krt18, Krt5, and Krt14, were significantly reduced in mammary tumor tissues of Brca1-MSK mice. These findings suggest that mammary epithelial cells in Brca1-MSK mice undergo a different process than currently recognized.

Furthermore, the inventors performed a cytokine array screening experiment using the sera of 6-month-old non-Brca1 mutant (WT) and Brca1 mutant (Brca1-MSK) mice, and obtained the results shown in FIG. 1 and FIG. 2 .

As shown in P in Figure 1 and J in Figure 2, 34 differentially expressed cytokines were detected in the blood of Brca1-MSK mice compared with WT mice, including 20 increased cytokines and 14 decreased cytokines. Among them, the inventors found that sialic acid binding receptors, such as soluble E-selectin (Sele), L-selectin (Sell), Vcam1 and Madcam1, were significantly increased in the blood of Brca1 mutant mice. This indicates that the interaction between sialic acid ligands on tumor cells of Brca1-MSK mice and receptors on endothelial cells and immune cells is increased.

Furthermore, the real-time fluorescence quantitative PCR method was used to detect the mammary epithelial cell lines of G600 (Brca1-MT) and B477 (Brca1-WT), respectively. As shown in Q of Figure 1, compared with B477 cells, the mRNA levels of 8 of them in G600 cells were significantly increased; as shown in K to M of Figure 2, compared with Brca1-WT mice, the mRNA levels of Sele, Sell, St8sia4 and St3gal1 were found to be increased in the mammary tissue of Brca1-MSK mice; compared with the B477 cell line, the mRNA levels of St8sia4 and St3gal1 in G600 mammary epithelial cells of Brca1-MT were increased. In addition, see R of Figure 1, which shows the protein level of St8sia4 in the B477 control cell line and the Brca1-MT cell line. As shown in Q-U of Figure 1, the inventors also proved that the protein levels of the above genes were increased by Western blotting analysis. The above experiments suggest that elevated sialyltransferases St8sia4 and St3gal1 and the receptors for sialic acid ligands Sele and Sell may lead to hypersialylation and relieve malignant cells from their original site.

The expression of the two genes St8sia4 and St3gal1 in B477 cells expressing the Brca1 knockdown plasmid (shBrca1) at different concentrations was detected by real-time fluorescence quantitative PCR, and the results shown in Figure 3 were obtained. As shown in Figure 3A, both St8sia4 and St3gal1 are negatively regulated by Brca1. However, it is not clear whether acidic conditions exist in precancerous breast tissue and breast cancer tissue of patients carrying BRCA1 mutations. It is also unknown whether acidity, if present, will affect the breast integrity and cancer metastasis of these patients. Based on this, the inventors continue to propose the following solution.

The two genes St8sia4 and St3gal1 are responsible for the production of polysialic acid (PSA) and Sialyl-Lewis A (sLeA), respectively. Polysialic acid (PSA) is a highly negatively charged polymer molecule that can lower the local pH by charge attraction in the presence of polyanions on the cell membrane. The inventors used PSA antibodies to detect the level of polysialic acid (PSA) in mammary epithelial cells of mice and humans with or without BRCA1 defects, and obtained the results shown in Figure 3. As can be seen from Figure 3 B-E, Figure 3 B-C is the detection result of mice, specifically, by IF staining, PSA was used to quantify the PSA distribution pattern (Figure 3 B-a), tumor adjacent mammary tissue (Figure 3 B-b) and tumor epithelial cells (Figure 3 B-c) of 10-month-old Brca1-MSK mice. Figure 3 D-E shows the detection results of human mammary epithelial cells, specifically, the PSA distribution pattern (Figure 3 D) and quantity (Figure 3 E) in the epithelial cells of breast cancer patients (n=4 BRCA1 mutation carriers and n=21 non-BRCA1 mutation carriers) were quantified by PSA staining. Among them, the PSA levels of mammary epithelial cells of BRCA1-deficient mice and humans were both elevated.

Furthermore, the inventors used a ratiometric fluorescent probe method to detect the pH values of mammary tissues of 8-month-old Brca1-MSK mice and WT mice of the same age, as well as tumors from Brca1-MSK and WT mice, and obtained the color intensity ratios as shown in Figure 3 F. As shown in Figures 3 F-G, the average pH values of mammary tissues and tumors of Brca1-MSK mice were 6.6 and 6.25, respectively, while the pH value of control mice was 7.4.

Furthermore, the inventors conducted tissue microarray analysis on 95 human breast cancer samples and obtained the results shown in Figure 3. Among them, conventional IHC or IF staining was performed on breast cancer tissue using BRCA1 antibody and PSA (as shown in H of Figure 3), and PSA in breast cancer patients with high expression of BRCA1, medium expression of BRCA1 and low expression of BRCA1 was quantified (as shown in I of Figure 3). As shown in H-I of Figure 3, the expression of Brca1 is negatively correlated with the expression of PSA. In addition, as shown in D-F of Figure 4, no such correlation was detected between Brca1 and LDHA (Figure 4D-E) and PSA and LDHA (Figure 4F), indicating that the acidic tumor microenvironment induced by the lack of or low expression of BRCA1 is not caused by excessive production of lactic acid.

Therefore, the inventors proposed a new method for neutralizing the acidic tumor microenvironment, which includes using a sialyltransferase inhibitor to neutralize the acidic tumor microenvironment.

Mammary fat blocks were implanted with B477 cells carrying OE-St8sia4 or G600 cells carrying sgSt8sia4, and empty and The white control was used to examine the changes in pH values, and the results were shown in Figure 3. The tumor images in B477 cells without or with OE-St8sia4 are shown in Figure 3 J, the tumor slice images using the ratio fluorescence probe are shown in Figure 3 K, and the interstitial pH is shown in Figure 3 L. As shown in Figure 3 JL, compared with the parental tumor tissue, the interstitial tissue in the breast tumor tissue with OE-St8sia4 has a larger tumor volume and a lower pH value; conversely, the tumor images in G600 cells without or with sgSt8sia4 are shown in Figure 3 M, the tumor slice images using the ratio fluorescence probe are shown in Figure 3 N, and the interstitial pH is shown in Figure 3 O. As shown in Figure 3 M, the tumor volume is smaller. At the same time, as shown in Figure 3 NO, the expression of sgSt8sia4 in G600 cells increased the pH value of the interstitial breast tissue from pH 6.5 to pH 7.0, indicating that the increase in sialyltransferase in the breast tissue observed earlier may lead to acidosis. Further, as shown in FIG4A, St8sia4 mRNA expression in G600 cells without or with Brca1 for 72 hours was detected by qPCR. As shown in FIG4B, representative images of mammary tissues of MMTV-cNeu, FGFR2-S252-W and FGFR2-S252-W/Brca1-MSK mice co-stained with PSA (green) and E-cadherin (red) and imaged in an LSM880 high-resolution microscope (n=3 mice/group). As shown in FIG4C, higher St8sia4 expression in human breast cancer patients is associated with poorer survival outcomes in breast cancer patients.

The above experiments indicate that, in addition to the currently known production of acidic conditions in tumors mainly through cancer cell metabolic activities, such as lactate accumulation, the hypersialylation caused by the lack or insufficiency of BRCA1 and associated with high levels of sialyltransferase in breast tissue can establish an acidic tumor microenvironment (ATPME) in precancerous and breast tumor tissues.

As shown in Figure 5A, St8sia4 was overexpressed in the Brca1-MT545 cell line with low metastatic ability and the EMT6 cell line with moderate metastatic ability, and the integrity of the adjacent mammary glands was examined on days 7, 14, and 26 of tumor growth. By co-staining with CK14 and CK18 antibodies, in the EMT6 mouse model, destroyed adjacent mammary gland structures were observed on day 14, and more basal and luminal layer damage could be detected on day 26. When the sialyltransferase inhibitor 3Fax-P-Neu5Ac (STi) was used, the damaged mammary gland structure could be restored, as shown in Figure 5B. Similarly, representative images of tumor-adjacent mammary tissues of nude mice implanted with 545 and OE-St8sia4-545 cells in the fat pad by IF staining of CK18 and CK14 (Figure 5C), PSA/E-cadherin and CK18/CollagenIV (Figure 5D) of tumor tissues of OE-St8sia4-545 cells, and representative images of CK18/vimentin (Figure 5E), scale bar is 10μm. As shown in Figure 5C-E, the same mammary structural damage phenotype and EMT state observed in the EMT6 cell line were also observed in the 545 mouse model. This indicates that high sialylation induced by elevated St8sia4 may lead to the destruction of mammary gland structure and enhance the effect of mammary epithelial cells on EMT.

Further, the inventors used two cell lines, 628W and 545, for experiments, wherein the 628W cell line has high metastatic ability and expresses higher levels of St8ia4 and St3gal1; the 545 cell line has low metastasis and has low levels of St8ia4 and St3gal1 expression. As shown in A of FIG5 and B of FIG6, St8sia4 and St3gal1 were first overexpressed in 545 cells, and St8sia4 and St3gal1 in 628W cells were knocked out by expression of sgSt8sia4 and St3gal1. As shown in F of FIG5 and C-D of FIG6, the migration state of these cells was studied in vitro, and the research methods were fat pad implantation and tail vein injection in vivo. As shown in E-F of FIG6, compared with 545 parental cells, the transwell migration assay detected much more migration from 545 cells with OE-St8sia4 and St3gal1, indicating that when St8sia4 and St3gal1 are overexpressed, cell morphology may change.

As shown in Figure 6G-J, compared with the parental cells in 2D culture, EMT6 with OE-St8sia4 acquired EMT characteristics, while EMT6 and B477 cells with OE-St8sia4 acquired invasive characteristics in 3D culture.

In the mammary fat pad implantation experiment, as shown in Figure 5G, overexpression of St8sia4 or St3gal1 in 545 cells promoted tumor growth, and as shown in Figure 5H-I, stronger GFP metastasis signals were detected in the lungs of cells with OE-St8sia4, but not with OE-St3gal1. In contrast, as shown in Figure 5J-K, by tail vein injection experiments, stronger GFP metastasis signals were detected in the lungs after injection of 545 cells with OE-St3gal1 than with OE-St8sia4. These observations suggest that hypersialylation induced by St8sia4 and St3gal1 plays different roles in different steps of tumor metastasis. Similar obvious effects on cancer metastasis were observed in 628W cells carrying sgSt8sia4 or sgSt3gal1, depending on their injection sites. Although sgSt8sia4 could only block tumor metastasis when 628W-sgSt8sia4 cells were injected into the fat pad but not into the tail vein, knocking out St3gal1 in 628W cells blocked tumor metastasis when cells were injected at both sites, as shown in Figure 5, L-O and Figure 6, K-L. The above data suggest that polysialic acid (PSA) accumulation induced by elevated St8sia4 primarily establishes acidic pre-metastatic niches at the primary tumor site, allowing malignant cells to ease access to blood vessels from damaged mammary gland structures, while elevated St3gal1 enhances tumor cell entry into remote organs of cancer metastasis in the circulation and extravasation steps, see Figure 5, P.

Furthermore, histological analysis of tumor tissues derived from the allogeneic transplant mouse model initiated with Brca1-MT cells showed extensive angiogenesis in the tumor tissues by staining with antibodies against CD31 and endothelial mucin (Endomucin) in FIG8A . This observation was further supported by increased mRNA (Fig. 8, B) and protein levels (Fig. 8, C) of Vegfa and Il6 in Cd24- and Cd29-sorted luminal subsets of GFP-positive cells in Brca1-MSK mice, suggesting that Brca1 deficiency may trigger increased expression of these oncogenes in mutant cells prior to tumor formation.

In addition, by monitoring the expression of Vegfa and Il6 in shBrca1-B477 cells and OE-Brca1-G600 cells, it was found that the mRNA levels of Vegfa and Il6 increased with the transfection of shBrca1 into B477WT cells (Figure 7A), and decreased in Brca1-MTG600 cells with OE-Brca1 cDNA (Figure 7B). As shown in Figure 7C-D, higher promoter activity of Vegfa and Il6 was observed in G600 cells compared with B477 cells, and their activities were inhibited when mBrca1 was expressed in both types of cells, indicating that Brca1 negatively regulates the expression of Vegfa and Il6.

By detecting the protein levels of St8sia4 and St3gal1 in B477 cells expressing shBrca1, shBrca1/sgVegfa and shBrca1/sgIl6 and G600 cells with OE-mBrca1, OE-Brca1/OE-Vegfa and OE-Brca1/OEIl6, the results shown in Figures 7 and 8 were obtained. The data showed that in B477 cells expressing shBrca1 alone, the protein levels of St8sia4 and St3gal1 increased, but this increase was reversed by co-expression of sgVegfa or sgIl6 (as shown in Figure 7E). On the other hand, the inhibition of St8sia4 by OE-Brca1 in G600 cells was partially released by the expression of OE-Vegfa or OE-Il6 (as shown in Figure 7F). These data indicate that Brca1 negatively regulates St8sia4 and St3gal1 through Vegfa/Il6.

Vegfa and VegfIL6 are both transcriptional regulators involved in angiogenesis and tumor cell invasion, and Vegfa signaling can stimulate the production of TGF-β in A549 cells. Therefore, the inventors used protein immunoblotting to conduct experiments and found that the protein levels of Vegfa and VegfIl6 increased compared with the control group, and the levels of TGF-β1 and its downstream proteins pSmad3/total-Smad3 and pStat3/totalStat3 in the precancerous breast (G in FIG. 7 ) and tumor tissue (H in FIG. 7 ) of Brca1-MSK mice were increased. In addition, the inventors also found that there is a positive mutual regulation cycle between Vegfa and VegfIl6, that is, overexpression (OE) or knockout (KO) of any one of them may affect the other (as shown in FIG. 7 I). Thus, OE-Vegfa or OE-Il6 in G477 cells increased their own expression together with TGF-β, St8sia4 and St3gal1, whereas KO of Vegfa or Il6 reduced the expression levels of all these cells (as shown in Figure 7 I). Similar results were obtained in EMT6 and MDA-MB-231 cells (as shown in Figure 8 D-E).

Furthermore, the mRNA expression and the displacement of polysialic acid (PSA) and S-selectin (Sele) on the cell membrane in epithelial cells, RAW264.7 cells and SVEC4-10 cells were detected after treatment with TGF-β and its inhibitor in G600. The data showed that TGF-β could induce the expression of St8sia4 and St3gal1 and other sialyltransferases in WT and Brca1-MT cells (as shown in J of FIG7 and F-G of FIG8), and the protein levels of St8sia4 and St3gal1 were reduced after treatment with TGF-β inhibitor (as shown in K of FIG7). After treatment with TGF-β inhibitor or introduction of mBrca1 cDNA into G600 cells (as shown in L-M of FIG7) and SVEC4010 cells (as shown in H-I of FIG8), the displacement of PSA and Sele on the cell membrane was significantly increased and decreased. Consistent with the above analysis, knockout of Vegfa or VegfIl6 in 628W and G600 cells decreased the protein levels of St8sia4 and St3gal1 (as shown in FIG. 8 J) and the production of polysialic acid PSA (as shown in FIG. 8 L-M). In contrast, overexpression of Vegfa or VegfIl6 in B477 cells increased the content of St8sia4 and St3gal1 (as shown in FIG. 8 K). In nude mice implanted with G600 cells expressing sgVegfa or sgVegfIl6, the tumor volume was reduced (as shown in FIG. 7 N-O) and the transfer signal of green fluorescent protein GFP was inhibited (as shown in FIG. 8 P-R). There was also a positive correlation between TGF-β and St8sia4, VegfIL6 and St3gal1, and St3gal1 and Vegfa.

The spread of cancer cells requires the joint support of the tumor immunosuppressive microenvironment (TIME) from the original lesion and distant metastatic organs. The inventors used antibodies to CD45, CD11b, Ly6G and Ly6C, CD3, CD4 and CD8 to various immune cell populations in breast tumor tissues from Brca1-MSK mice, as well as EMT6 parental cells, OE-St8sia4 cells, and OE-St8sia4/sgSt8sia4 cells. Mass spectrometry flow analysis was performed on the 14th and 26th days after implantation, and the results of A-B in Figure 9 were obtained. The data showed that compared with normal breast tissue, the parental tumor accumulated more polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and mononuclear myeloid-derived suppressor cells (M-MDSC), and reduced CD3+ and CD4+ cells. OE-St8sia4 in primary tumors significantly enhanced this phenotype, whereas sgSt8sia4 reversed it when analyzed at days 14 and 26.

Immunohistochemistry and IF staining were performed in the spleen and breast tumor tissues of the OE-St8sia4-EMT6 and OE-St8sia4-545 allogeneic transplant mouse models, where the MDSCs antibody was Gr1 (Ly6G and Ly6C), the M1-like macrophage antibody was F4/80 and CD86, and the M2-like macrophage antibody was F4/80 and CD206. As shown in Figures 10A-C, MDSCs in the spleen of Brca1-MSK mice increased. In the tumor tissues of the OE-St8sia4-EMT6 and OE-St8sia4-545 mouse models, M1-like macrophages decreased and M2-like macrophages increased (CE in Figure 9 and DF in Figure 10). Compared with control mice, the GI in Figure 10 was obtained by FACS flow cytometry analysis. The above phenotypes were also observed in different ages and organs of Brca1-MSK mice, which is because Brca1 negatively regulates the expression of St8sia4. Da.

By detecting the MDSC population, immunohistochemistry using S100a9 antibody, and flow cytometry detection of Cd11b and Ly6G in spleen and tumor tissues expressing sgVegfa or sgVegfIl6, it was found that MDSCs were reduced in tumor tissues (as shown in Figure 9 F-G), increased in the spleen of OE-St8sia4-545 tumor-bearing mice, and reduced in the spleen of 628W tumor-bearing mice expressing sgSt8sia4 (Figure 9 H-I). This view was further supported by the reduction of MDSCs in spleen and breast tumor tissues expressing sgVegfa or sgVegfIl6 by flow cytometry analysis, as shown in Figure 10 J-L.

The inventors further analyzed bulk RNA-seq from bones, blood, spleen, and peritoneum of WT mice, Brca1-MSK mice without tumors, and Brca1-MSK mice carrying primary breast tumors, and then compared the expression patterns obtained in the TCGA database. In the Brca1-MSK mouse model, consistent with the above analysis, B cells, T cells, and dendritic cells (DCs) began to decrease, while immune cells from the myeloid cell lineage began to increase in the blood and spleen of Brca1 mutant mice without tumors, and further increased in Brca1 mutant tumor-bearing mice (M in Figure 10). Gene expression of immune cells further illustrates the increase in macrophages and monocytes in bones, blood, and spleen, especially in the peritoneum of Brca1-MSK compared with WT mice (N in Figure 10). The characteristic gene expression pattern in immune cells showed highly expressed immunosuppressive genes, including TGF-β1, Smad3, Mmp9, Arg1, Arg2, Il10, Il1β and PD-L1 in blood, spleen and peritoneum (O in Figure 10), indicating that Brca1 deficiency leads to a stronger immunosuppressive environment in those organs. The above gene expression analysis was further supported by the increased levels of TGF-β signaling proteins by Western blotting (P in Figure 10). Correlation analysis of st8sia4 and St3gal1 expression with immune cell populations in the TCGA database showed that St8sia4 expression was positively correlated with MDSC, macrophages and Treg cells (J-N in Figure 9), but not with St3gal1. These data suggest that elevated St8sia4, Vegfa/Il6 and Brca1 deficiency reshape the tumor immunosuppressive microenvironment that may be conducive to tumor cell growth and metastasis.

The acidic tumor microenvironment (ATPME) induced by high sialylation may damage the structure of the mammary bilayer, thereby promoting the entry of malignant cells into blood vessels through the extracellular matrix (ECM). The inventors next studied whether the neutralization of ATPME could inhibit tumor metastasis. 3Fax-P-Neu5Ac (STi) is a naturally occurring hyperacetylated analog of Neu5Ac that inhibits sialyltransferase function. Another drug selected was Stattic, an inhibitor that can disrupt TGF-β signaling induced by pStat3 and pSmad3 activation, because the levels of proteins pStat3 and pSmad3 are both elevated in cells with low Brca1 expression or high Vegfa/Il6 expression. First, in allogeneic transplanted tumors generated by 628W cells, these two drugs were used for monotherapy and combination therapy by intraperitoneal delivery (as shown in A of Figure 12). The data showed that intraperitoneal injection of STi (20 mg/kg) or Stattic (10 mg/kg) for single treatment can inhibit primary tumor growth (as shown in Figure 12 B-C) and GFP metastasis signals in the lungs (as shown in Figure 12 D-E) to a certain extent, while reducing lung weight (as shown in Figure 12 F). The combination of these two drugs further reduced tumor growth (as shown in Figure 12 B-C) and reduced metastasis signals in the lungs (as shown in Figure 12 D-E), indicating that neutralizing the acidic environment and inhibiting TGF-β signals can synergistically delay the growth of primary tumors and block the metastasis that reverses the splenomegaly phenotype. Moreover, no obvious cytotoxic effects were observed in the lungs, kidneys, liver, or spleen using H&E staining (as shown in Figure 12 F).

Currently, although an effective dose (25 mg/kg) of 3Fax-P-Neu5Ac can inhibit the growth of myeloma tumors, it can also cause edema in the peritoneal cavity of mice and toxicity to the liver and kidneys. Due to the potential side effects of these anticancer drugs, the inventors packaged the drugs into nanoparticles with amphiphilic polymers and administered them three times by intravenous injection every three days when the tumor size reached about 50 mm3 after implantation (Figure 11 A). The data showed that nanoparticles can be enriched in breast tissue 48 hours after tail vein injection (Figure 11 B). Nanoparticles containing Stattic or STi significantly reduced primary tumor growth and GFP metastasis signals (Figure 11 C-F).

Furthermore, the pH of tumor tissue was measured in the same group of mice by using a ratiometric fluorescent probe. The ratio of green to red was much larger in the control group (PBS and PEG treatment groups) compared with the drug treatment group (Figure 11G-H) and after treatment with Stattic or STi, the pH of breast tissue recovered from approximately 6.5 to above 6.8 to 7.0 (Figure 10H).

Further, the inventors used STi, Stattic or double treatment with STi and Stattic, and co-stained tumors and tumor-adjacent breasts with CK18/CollagenIV or CK18/CK14 antibodies. The H-J data of Figure 11 show that when treated with a single or combined drug, more collagen bundles are formed in the tumor tissue detected by CK18/Collagen IV staining in the mammary basement membrane compared with the parental control tumor tissue (D of Figure 11), which can prevent tumor cells from escaping from the original position; by double staining CK14 and CK18 antibodies (J of Figure 11), more mammary ducts with complete basal cell layers are observed after treatment with a single drug or two drugs together (I-J of Figure 11), indicating that the acidic conditions in the mammary tissue caused by high sialylation can be specifically neutralized by nanoparticles containing pan-inhibitors of sialyltransferases and inhibitors of TGF-β signaling molecules.

In the treatment of cancer patients, immunotherapy resistance is a very challenging problem. Increased expression reduces effector T cells and accumulates MDSCs in mammary tissue. The inventors first examined the expression levels of PD1, CD3, PD-L1 and CK18 in the mammary tissue of Brca1-MSK mice implanted with parental EMT6 cells and EMT6/OE-St8sia4 cells. Very weak and abnormal expression of PD-1 and CD3 was detected in tumors caused by EMT6 parental cells. CD3-positive cells were significantly reduced, accompanied by OE-St8sia4 in breast tumor tissues with clear PD-1 positive immune cells for 7 days (Figure 13 A), while very strong PD-L1-St8sia4 was detected in the parental tumors and OE tumors of the control for 7 days (Figure 13 B), indicating that ATPME induced by OE-St8sia4 triggered T cell death.

Because polysialic acid (PSA) is a highly negatively charged polymer molecule, tumor-bearing mice overexpressing St8sia4 were treated with αPD-1, STiNPs, and STiNPs together with αPD-1 (Figure 14A). The data showed that OE-St8sia4 promoted tumor growth, treatment with αPD1 alone did not change tumor growth at all, and treatment with sialyltransferase inhibitor nanoparticles (STiNPs) alone significantly reduced tumor growth, however, combined treatment with STi and αPD-1 further inhibited tumor growth compared to parental tumors, tumors with OE-St8sia4, and tumors treated with αPD-1 alone (Figure 137C-D). The splenomegaly phenotype was also reversed in the combined treatment groups of STi and αPD-1 (Figure 14B and Figure 13E), respectively, while there was no significant change in liver weight (Figure 14C-D).

To further examine the changes in MDSC, Cd4+ and Cd8+ cell populations in tumor tissues before and after OE-St8sia4 treatment, mass spectrometry flow cytometry analysis was performed to support the above viewpoints. The data showed that 1) OE-St8sia4 reduced Cd4+ and Cd8+ cell populations and increased M-MDSCs and PMN-MDSCs; 2) there was no significant change in the immune population after αPD-1 monotherapy compared with the control group; 3) 3Fax-P-Neu5Ac (STi) monotherapy restored the Cd4+ and Cd8+ populations and reduced the M-MDSCs and PMN-MDSCs populations; 4) in the αPD-1 and 3Fax-P-Neu5Ac combined treatment group, the Cd4+ and Cd8+ populations continued to increase, and the M-MDSCs and PMN-MDSCs populations were much less than those in the αPD-1 or 3Fax-P-Neu5Ac single treatment group, as shown in Figure 13F and Figure 14E), indicating that MDSCs were reduced and T cells were increased after STi-αPD-1 combined treatment.

Consistent with mass spectrometry analysis, the levels of CD3-positive cells and Caspase3 protein increased and the levels of polysialic acid decreased in the 3Fax-P-Neu5Ac and 3Fax-P-Neu5Ac+αPD-1 treatment groups (Figure 13G-H). H&E staining also showed that the lung morphology of metastatic lungs was also restored in the 3Fax-P-Neu5Ac and 3Fax-P-Neu5Ac+αPD-1 treatment groups, and there was no obvious toxic effect on other organs including the liver and spleen before and after treatment, as shown in Figure 13H and Figure 14D. In summary, these data indicate that ATPME caused by OE-St8sia4 is associated with resistance to immunotherapy, and the combination of 3Fax-P-Neu5Ac and αPD-1 sensitizes breast tumors with ATPME to αPD-1 treatment.

In summary, the inventors have determined that breast cancer caused by Brca1 deficiency has a sialyltransferase-mediated mechanism that can induce an acidic tumor microenvironment (ATPME) in Brca1 mutations and most Brca1 low-expressing breast cancers, and that high sialylation caused by polysialic acid (PSA) damages the bilayer structure of the mammary epithelium and establishes an acidic tumor microenvironment (ATPME) and tumor immunosuppressive microenvironment (TIME) that contribute to cancer metastasis. In addition, the carcinogenic effect of Vegfa/Il6 induced by Brca1 deficiency or insufficiency activates TGFβ-St8sia4 signaling, increases the accumulation of polysialic acid on the mammary epithelial membrane, and forms a malignant niche that promotes cell escape from immune surveillance, ultimately leading to tumor metastasis and resistance to αPD-1 treatment. Therefore, the inventors proposed to use sialyltransferase inhibitors to prevent or treat breast cancer caused by Brca1 deficiency. By using sialyltransferase inhibitors, the expression of sialyltransferase can be reduced, and the acidic tumor microenvironment formed can be neutralized, thereby achieving the effect of treating or even reversing the basement membrane defects of breast epithelial cells. In addition, by combining sialyltransferase inhibitors with drugs PD-1 or Stattic, ATPME can be further neutralized and tumor growth and metastasis can be blocked, greatly reducing the chance of tumor metastasis.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Industrial Applicability

The present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment, and relates to the technical field of tumor treatment. The present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment. The sialyltransferase inhibitor can neutralize the acidic tumor microenvironment and establish an ecological barrier for tumor metastasis, providing a new approach for the prevention and treatment of tumors, and having excellent industrial applicability.

Claims (18)

A use of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment. The use according to claim 1 is characterized in that the sialyltransferase inhibitor neutralizes the acidic tumor microenvironment by downregulating the expression of Vegfa and/or VegfIl6. The use according to claim 1 is characterized in that the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic. The use according to claim 1, characterized in that the sialyltransferase inhibitor inhibits the expression of polysialyltransferase and/or polysialyltransferase receptor. The use according to claim 4, characterized in that the polysialyltransferase comprises St8sia4. The use according to claim 4, characterized in that the polysialyltransferase receptor comprises at least one of soluble E-selectin and L-selectin. The use according to any one of claims 2 to 6 is characterized in that the drug for neutralizing the acidic tumor microenvironment also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody. Use of a sialyltransferase inhibitor in the preparation of a drug for treating Brca1 defect-related tumors; Preferably, the drug for treating Brca1 deficiency-related tumors also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody; Optionally, the Brca1 deficiency-associated tumor is Brca1 deficiency-associated breast cancer. A use of a sialyltransferase inhibitor in the preparation of a drug for preventing or treating cancer, wherein the drug for preventing or treating cancer includes any one of preventing or treating the occurrence, metastasis or growth of cancer; Optionally, the drug for preventing or treating cancer further includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody; Optionally, the cancer includes any one of breast cancer, liver cancer, and spleen cancer. Use of a sialyltransferase inhibitor in the preparation of a drug for treating the destruction of the double-layer structure of mammary epithelium; Optionally, the drug for treating destruction of the double-layer structure of mammary epithelium also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody. A pharmaceutical composition comprising a sialyltransferase inhibitor and a pharmaceutically acceptable carrier; Optionally, the sialyltransferase inhibitor comprises at least one of 3Fax-Peracetyl Neu5Ac or Stattic; Optionally, the dosage form of the drug includes any one of injection, injection, tablet, granule, granule or capsule; Optionally, the drug is in the form of nanoparticles. A method of neutralizing an acidic tumor microenvironment, the method comprising administering to a subject in need thereof an effective amount of a sialyltransferase inhibitor. A method for treating a tumor associated with Brca1 deficiency, the method comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof; Optionally, the Brca1 deficiency-associated tumor is Brca1 deficiency-associated breast cancer. A method for preventing or treating cancer, comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof; wherein the drug for preventing or treating cancer includes any one of preventing or treating the occurrence, metastasis or growth of cancer; Optionally, the cancer includes any one of breast cancer, liver cancer, and spleen cancer. A method for treating disruption of the bilayer structure of mammary epithelium, the method comprising administering to a subject in need thereof an effective amount of a sialyltransferase inhibitor. The method according to any one of claims 12 to 15, wherein the sialyltransferase inhibitor comprises at least one of 3Fax-Peracetyl Neu5Ac or Stattic; Optionally, the method comprises the combined use of a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody. A sialyltransferase inhibitor for use in: i. Neutralize the acidic tumor microenvironment; ii. Treating Brca1 deficiency-related tumors; optionally, the Brca1 deficiency-related tumor is Brca1 deficiency-related breast cancer; iii. Preventing or treating cancer; wherein preventing or treating cancer includes preventing or treating any of the occurrence, metastasis or growth of cancer; optionally, the cancer includes any of breast cancer, liver cancer, spleen cancer; iv. Treat the destruction of the double-layer structure of mammary epithelium. The sialyltransferase inhibitor according to claim 17, wherein the sialyltransferase inhibitor comprises at least one of 3Fax-Peracetyl Neu5Ac or Stattic; Optionally, the use includes a sialyltransferase inhibitor combined with a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.