CN118384178A - Application of PTEN as an immune cell activator in the preparation of drugs for treating tumors - Google Patents

Abstract

The present invention discloses the use of PTEN as an immune cell activator in the preparation of a drug for treating tumors. The present invention provides the use of PTEN in the preparation of a drug for treating tumors, wherein the drug for treating tumors is an immune cell activator. PTEN activates the JAK2-STAT1 signaling pathway by binding to the PLXDC2 protein on the surface of macrophages, thereby promoting the polarization of M2 macrophages to the M1 direction, activating T and NK cells, and then killing tumors, which can significantly reduce the size of tumor lesions. However, knocking out the PLXDC2 protein or inhibiting the JAK2-STAT1 signaling pathway cannot promote the polarization of macrophages to the M1 type, thereby failing to achieve a tumor suppressor effect. The present invention also provides the use of a reagent for detecting PLXDC2 in the preparation of a product for evaluating the effect of a PTEN-containing drug for treating tumors, and/or the use of evaluating the effect of a PTEN-containing drug for treating tumors.

Description

Application of PTEN as an immune cell activator in the preparation of drugs for treating tumors

Technical Field

The present invention relates to the field of biomedical technology, and in particular to the use of PTEN as an immune cell activator in the preparation of drugs for treating tumors.

Background technique

Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor gene with a high mutation rate, which plays a tumor suppressor role in both phosphatase function and non-phosphatase function.

In cells, PTEN, which plays a tumor suppressive role as a dual lipid and protein phosphatase, can dephosphorylate phosphatidylinositol (PtdIns), P3 (PIP3) into PIP2 by playing the role of lipid phosphatase, thereby reducing the amount of PIP3 required for the activation of intracellular serine/threonine protein kinase (AKT) and inhibiting the activity of intracellular phosphatidylinositol kinase (PI3K). As a protein phosphatase, PTEN can play a tumor suppressive role by dephosphorylating some protein substrates, such as focal adhesion kinase (FAK), tyrosine protein kinase (c-SRC), cyclic adenosine monophosphate response element binding protein (CREB), RAS-associated protein 7 (RAB7) and insulin receptor substrate 1 (IRS1). Therefore, there are many targeted drugs designed for this pathway, but unfortunately the effects are not obvious.

Tumor associated macrophages (TAMs) are an important component of the tumor microenvironment and play a role in the coordination of angiogenesis, extracellular matrix remodeling, cancer cell proliferation, metastasis, and immunosuppression, as well as resistance to chemotherapeutic drugs and checkpoint blockade immunotherapy. When properly activated, macrophages can mediate phagocytosis and cytotoxic tumor killing of cancer cells and engage in effective bidirectional interactions with components of the innate and adaptive immune systems.

Many targeted therapeutic strategies for macrophages have been designed. One of the important methods is to reprogram macrophages in the tumor microenvironment. The realization of this method depends on the regulation of macrophage surface receptors. Among the many membrane proteins of cells, plexin domain-containing protein 2 (PLXDC2) is very eye-catching and is associated with a variety of tumors.

On macrophages, the JAK2-STAT1 pathway is the main pathway for activating macrophage polarization toward M1. Nearly 60 cytokines, including many interleukins, colony stimulating factors, hormone-like cytokines, and growth factors, use the JAK-STAT pathway as the main necessary mode for initiating transcription, and the activation of this pathway is closely related to the polarization of macrophages.

Therefore, there is an urgent need to find new ways to treat cancer with PTEN in order to better exert its tumor suppressor effect.

Summary of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide the use of PTEN as an immune cell activator in the preparation of drugs for treating tumors.

The present invention discovered a new phosphatase-independent tumor suppressor function of PTEN, which activates the JAK2-STAT1 signaling pathway by binding to the PLXDC2 protein on the surface of macrophages, thereby promoting the polarization of M2 macrophages to M1 type, activating T and NK cells, and then killing tumors.

The first object of the present invention is to provide the use of PTEN in preparing drugs for treating tumors.

The second object of the present invention is to provide a reagent for detecting PLXDC2 for use in preparing a product for evaluating the effect of a PTEN-containing drug in treating a tumor, and/or for use in evaluating the effect of a PTEN-containing drug in treating a tumor.

In order to achieve the above object, the present invention is implemented by the following scheme:

The use of PTEN in preparing a drug for treating tumors is characterized in that the drug for treating tumors is an immune cell activator, and the accession number of PTEN on NCBI is Gene ID: 5728.

Furthermore, the immune cells are one or more of macrophages, NK cells and T cells.

Furthermore, the immune cell activator is an activator that promotes macrophage polarization.

Furthermore, the immune cell activator is an activator that promotes the polarization of M2 macrophages into M1 macrophages.

Furthermore, the immune cell activator is a preparation that activates the JAK-STAT signaling pathway of macrophages.

Furthermore, the immune cell activator is a preparation combined with PLXDC2, and the accession number of PLXDC2 on NCBI is Gene ID: 84898.

In a specific embodiment of the present application, experiments were conducted using B16-F10 cells as an example, and the tumor was melanoma.

The present application also provides the use of a reagent for detecting PLXDC2 in the preparation of a product for evaluating the effect of a PTEN-containing drug in treating a tumor, and/or the use of a reagent for evaluating the effect of a PTEN-containing drug in treating a tumor;

The accession number of PTEN in NCBI is Gene ID: 5728, and the accession number of PLXDC2 in NCBI is Gene ID: 84898.

Furthermore, the tumor is melanoma.

Furthermore, the drug is an immune cell activator.

Furthermore, the immune cells are one or more of macrophages, neutrophils, NK cells and T cells.

Furthermore, the immune cell activator is an activator that promotes macrophage polarization.

Furthermore, the immune cell activator is an activator that promotes the polarization of M2 macrophages into M1 macrophages.

Furthermore, the immune cell activator is a preparation that activates the JAK-STAT signaling pathway of macrophages.

Furthermore, the immune cell activator is a preparation combined with PLXDC2, and the accession number of PLXDC2 on NCBI is Gene ID: 84898.

In a specific embodiment of the present application, experiments were conducted using B16-F10 cells as an example, and the tumor was melanoma.

Compared with the prior art, the present invention has the following beneficial effects:

PTEN activates the JAK2-STAT1 signaling pathway by binding to the PLXDC2 protein on the surface of macrophages, thereby promoting the polarization of M2 macrophages to the M1 type, activating T and NK cells, and then killing tumors, which can significantly reduce the size of tumor lesions. However, knocking out the PLXDC2 protein or inhibiting the JAK2-STAT1 signaling pathway cannot promote the polarization of macrophages to the M1 type, thereby failing to achieve a tumor suppressor effect. The present invention also provides the use of a reagent for detecting PLXDC2 in the preparation of a product for evaluating the effect of a PTEN-containing drug on treating tumors, and/or the use of an agent for evaluating the effect of a PTEN-containing drug on treating tumors. It provides new ideas and tools for effective targeted treatment of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the acquisition of PTEN and its esterase-independent anti-cancer effect. A shows the purification of human PTEN recombinant protein using a prokaryotic expression system and Coomassie Brilliant Blue staining. B shows the tumor volume after intratumoral injection of PTEN or PBS into the PTEN-knockout melanoma C57BL/6J mouse model (n=5 mice/group). C shows the cell growth of B16-F10-ΔPTEN cells cultured in vitro with or without 100ng/mL PTEN treatment; NS in the figure indicates not significant.

Figure 2 shows the binding of PTEN-Flag to intratumoral macrophages (Mφ, CD45+CD11b+F4/80+), neutrophils (neu, CD45+CD11b+Ly6G+), NK cells (CD45+CD3e-NK1.1+) and T cells (CD45+CD3e+NK1.1-) in the PTEN knockout melanoma C57BL/6J mouse model after Flag-PTEN treatment (n=5). Among them, A is the MFI of each cell type calculated by comparing the relative mean fluorescence intensity (MFI) between the treatment group and the control group in flow cytometry analysis, and the dotted line indicates MFI=1. B is the proportion of IFNγ+CD8+T cells in CD8+T cells in the immune microenvironment detected by flow cytometry. C is the proportion of GZMA+NK1.1+ cells in NK1.1+ cells in the immune microenvironment detected by flow cytometry.

Figure 3 shows the binding relationship between PTEN and macrophages and PLXDC2. A shows the expression of PLXDC2 in macrophages (Mφ), neutrophils (Neu), NK cells and T cells in the tumor of the PTEN knockout melanoma C57BL/6J mouse model by flow cytometry; the relative MFI of each cell type was calculated by comparing the MFI of the anti-Flag group and the IgG group, and the dotted line indicates relative MFI = 1. B shows the extracellular segment (ECD) of mouse PLXDC2 with a C-terminal 6×His tag (PLXDC2-ECD-His) incubated with Flag or Flag-PTEN protein, followed by Flag-tag pulldown and WB analysis. C shows the binding of PTEN and PLXDC2 by immunofluorescence staining, and the cell nucleus is stained with DAPI.

Figure 4 shows the expression of related genes in M1 BMDMs and M2 BMDMs before and after PLXDC2 knockout, with or without 100 ng/mL Flag-PTEN treatment. A is a heat map of the expression of 10 M1 BMDMs-related genes and 2 M2 BMDMs-related genes in RNA-seq analysis. B is the expression of marker genes related to M1 BMDMs and M2 BMDMs in flow cytometry.

Figure 5 shows the JAK2-STAT1 pathway in M2 BMDMs with or without 100 ng/mL Flag-PTEN treatment before and after PLXDC2 knockout. A is a WB analysis of 4 JAK proteins. B is a WB analysis of 6 STAT proteins. C is a WB analysis of JAK and STAT proteins after using a JAK2 inhibitor.

Detailed ways

The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

HEK-293T cells (human embryonic kidney epithelial cells) and B16-F10 cells (mouse melanoma cells) were cultured in DMEM medium containing 10% fetal bovine serum by volume. All cell lines were cultured at 37°C and 5% CO ₂ , and no mycoplasma contamination was detected. The identity of the cell lines was confirmed by STR identification. C57BL/6J mice were provided by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. All mouse experiments followed the animal experiment ethics regulations approved by the Animal Research Ethics Committee of the School of Medicine of Shanghai Jiao Tong University. The mice used in the experiment were between 6 and 8 weeks old and were kept in the SPF (specific pathogen-free) environment of the Animal Experiment Center of the School of Medicine of Shanghai Jiao Tong University.

Example 1 Extracellular PTEN has a strong tumor suppressor function

1. Experimental Methods

1. Purification of PTEN recombinant protein

(1) Using the pET-28a prokaryotic protein expression system, the pET-28a plasmid containing the full length of PTEN was transformed into Escherichia coli Transetta (DE3, TransGen), and amplified in a shaking incubator at 37° C. to obtain recombinant Escherichia coli. The accession number of PTEN in NCBI is Gene ID: 5728.

(2) After inducing the recombinant E. coli in step (1) with 0.1 mM IPTG at 16°C for 18 h, the cells were collected by centrifugation and lysed with a lysis buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% (v/v) glycerol) containing a protease inhibitor cocktail.

(3) Ultrasonication is used to disrupt the lysed bacteria in step (2), and the bacterial fragments are removed by ultracentrifugation, while the supernatant is retained.

(4) The supernatant of step (3) was mixed with His-tagged protein agarose purification resin (YEASEN biotech), incubated at 4°C for 2h, and eluted with 300mM imidazole to obtain imidazole-eluted protein. The imidazole-eluted protein was further purified by gel filtration chromatography and replaced in PBS containing 1mM DTT (dithiothreitol) to obtain chromatographically purified protein. The chromatographically purified protein was concentrated using a concentrator column (Millipore) to obtain concentrated PTEN recombinant protein (Flag-PTEN, amino acid sequence as shown in SEQ ID NO: 1), which was stored at -80°C for future use.

2. Construction of PTEN knockout B16-F10 cells - B16-F10-ΔPTEN

(1) B16-F10 cells were transfected with LentiCRISPR v2 containing the PTEN targeting sequence (sgRNA targeting sequence: ACAAAAGGAGATATCAAGAGG).

(2) Use 2 μg/mL puromycin to screen the positive cells after transfection in step (1).

(3) Diluting the positive cells in step (2), screening the PTEN knockout B16-F10 cell monoclonal, i.e., B16-F10-ΔPTEN, and confirming the PTEN knockout in the B16-F10-ΔPTEN monoclonal cells by western blot experiment and gene locus sequencing.

(4) When culturing B16-F10-ΔPTEN cells in vitro, PTEN recombinant protein was added at a final concentration of 100 ng/mL as the treatment group, and PBS was added as the control group to observe the effect of PTEN recombinant protein on the growth and proliferation of B16-F10-ΔPTEN cells.

3. 1.0×10 ⁵ B16-F10-ΔPTEN cells were subcutaneously injected into C57BL/6J mice to construct a PTEN knockout melanoma C57BL/6J mouse model. When the subcutaneous tumor volume in the mouse model reached 50-100 mm ³ , 10 μg or 20 μg of PTEN recombinant protein was injected intratumorally into the above mouse model every day as a treatment group, and PBS was injected intratumorally as a control group. The tumor size was measured with a vernier caliper, and the volume calculation formula was: V = as (width × width × length) / 2.

2. Experimental Results

As shown in FIG1A , PTEN recombinant protein was purified in vitro.

As shown in Figure 1B, compared with the PBS control group, injection of PTEN recombinant protein significantly inhibited the subcutaneous growth of B16-F10-ΔPTEN cells in the PTEN knockout melanoma C57BL/6J mouse model.

As shown in Figure 1C , compared with the PBS control group, PTEN recombinant protein had no growth inhibitory effect on B16-F10-APTEN monoclonal cells.

From the above, it can be seen that the PTEN recombinant protein does not inhibit the growth and proliferation of tumor cells in vitro, but significantly inhibits the growth and proliferation of tumor cells in vivo.

Example 2 PTEN binds to macrophages and activates T and NK cells

1. Experimental Methods

1. Flow cytometry analysis

(1) The subcutaneous tumor tissue cells of the mice in the control group and the PTEN recombinant protein treatment group in step 3 of Example 1 were digested, stained with Zombie NIR, and live cells were sorted by flow cytometry.

(2) To prevent non-specific binding of subsequent staining, the live cells sorted in step (1) were pre-incubated with anti-CD16/32 antibody (Biolegend) on ice for 10 min to block Fc receptors and obtain Fc receptor-blocked cells. Subsequently, the Fc receptor-blocked cells were washed with flow cytometry buffer and diluted to 1-5×10 ⁵ cells/mL to obtain diluted cells.

(3) The cells diluted in step (2) were fixed and permeabilized with permeabilization buffer (eBioscience), and then stained with the corresponding antibodies. The antibodies used were as follows: Macrophage: CD45, CD11b, F4/80 antibodies; neutrophils: CD45, CD11b, Ly6G; NK cells: CD45, NK1.1, CD3e (negative selection) +); T cells: CD45, CD3e, NK1.1 (negative selection).

(4) To verify the effect of the PTEN recombinant protein of Example 1 on tumor T cells and NK cells in the treatment group and the control group in Example 1, the positive T cells sorted in step (2) were labeled with IFNγ, and the positive NK cells were labeled with GZMA antibody. The collected data were analyzed by Beckman CytoFlex S flow cytometer, and the generated data were processed by FlowJo software.

2. Experimental Results

Among the four major immune cell populations, PTEN selectively binds to macrophages and does not bind to Neu, T, and NK cells (Figure 2A). However, subcutaneous tumor cells injected with PTEN can cause T cell (Figure 2B) and NK (Figure 2C) cell activation in the tumor. This indicates that PTEN binds to macrophages, and macrophages further cause T and NK cell activation.

Example 3 PTEN binds to the macrophage membrane surface through plexin domain-containing protein 2 (PLXDC2, NCBI ID: 84898)

1. Experimental Methods

1. Anti-PLXDC2 antibody was used to detect the expression of PLXDC2 on the surface of the four immune cells (macrophages, Neu, NK cells, and T cells) isolated from the subcutaneous tumor in Example 2. The isotype control (Flag-PLXDC2, PLXDC2 with a Flag tag) was used as a negative control for the anti-PLXDC2 antibody.

2. The extracellular segment of mouse PLXDC2 with a 6×His tag at the C-terminus (PLXDC2-ECD-His) was incubated with Flag purified in vitro and Flag-PTEN protein prepared in Example 1, and then a pulldown experiment was performed using anti-Flag to determine whether PTEN directly binds to the extracellular segment of PLXDC2. PLXDC2-ECD is amino acids 1-454 of the extracellular segment of PLXDC2 (https://www.uniprot.org/uniprotkb/Q6UX71/entry#ptm_processing).

3. (1) Polarization and stimulation of bone marrow-derived macrophages (BMDMs): Bone marrow (BM) cells were collected from the femur and tibia of C57BL/6J mice. Recombinant mouse M-CSF (final concentration 50 ng/mL, Sino Biological) was added to DMEM medium (Gibco) containing 1% penicillin/streptomycin and 10% (v/v) FBS to induce differentiation of BM cells for 7 days, and non-polarized macrophages (M0 BMDMs) were obtained. IL-4 (final concentration 10 ng/mL, Biolegend) and IL-13 (final concentration 10 ng/mL, MCE) were added to DMEM medium (Gibco) containing 1% penicillin/streptomycin and 10% (v/v) FBS to induce differentiation of washed M0 BMDMs for 48 h to obtain M2 BMDMs (M2 bone marrow-derived macrophages).

(2) Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) was used in Opti-MEM ^TM I reduced serum medium to transfect M2BMDM with negative control siRNA (siNC) and PLXDC2 siRNA, respectively, according to the protocol in the reagent company's instructions, to obtain M2 BMDMs with no knockout and knockout of PLXDC2 (siNCM2 BMDMs, siPlxdc2-1M2 BMDMs, and siPlxdc2-2M2 BMDMs). Gene knockout efficiency and subsequent experiments were performed 48 to 72 hours after transfection (siPlxdc2-1 targeting sequence: GAGAAGTTGTACATCGAAT, siPlxdc2-2 targeting sequence: GAGAAGTTGTACATCGAAT, siRNA was provided by Shanghai Jima Biotechnology Co., Ltd.).

(3) The three types of M2 BMDMs prepared in step (2) were cultured with Flag and Flag-PTEN proteins at a final concentration of 100 ng/mL for 1 h each, and immunofluorescence experiments (IF) were performed using anti-Flag antibodies to obtain the cultured cells.

The immunofluorescence experiment steps are as follows: Wash the cells cultured in step (3) once with PBS, and immediately incubate with 4% paraformaldehyde (Shanghai Biotechnology) at room temperature for 15 minutes to obtain paraformaldehyde-fixed cells. Block the fixed cells with 2% (w/v) BSA diluted in PBS for 1 hour, and incubate with Flag antibody (Proteintech) at 4°C overnight to obtain antibody-incubated cells. Wash the antibody-incubated cells 3 times with PBS, and then incubate with fluorescent mouse secondary antibody (Proteintech) at room temperature for 1 hour. Use Leica TCS SP8 to collect fluorescence images.

2. Experimental Results

The binding of PTEN to PLXDC2 occurs specifically on macrophages (Figure 3A), and PTEN binds to the extracellular segment of PLXDC2 (Figure 3B). PTEN (Flag-PTEN) was observed to bind to the extracellular segment of PLXDC2 on the surface of macrophages on the surface of M2BMDM cells, but after PLXDC2 was knocked out, no binding of PTEN protein to the extracellular segment of PLXDC2 on the surface of macrophages was observed (Figure 3C).

Example 4 PTEN promotes the polarization of M2 macrophages to M1 through PLXDC2

1. Experimental Methods

1. Three types of M2 BMDMs cells (siNCM2 BMDM, siPlxdc2-1M2 BMDM and siPlxdc2-2M2 BMDM) were treated according to the method of step (3) in step 3 of Example 3, wherein the M2BMDMs were cultured with Flag and Flag-PTEN for 24 h, and the RNA of the treated cells was extracted for transcriptome sequencing to analyze the genes related to M1 and M2 macrophages.

2. M2 BMDMs (siNCM2BMDM, siPlxdc2-1M2 BMDM and siPlxdc2-2M2 BMDM) were treated according to the method of step (3) in step 3 of Example 3, wherein the M2 BMDMs were cultured with Flag and Flag-PTEN prepared in Example 1 for 24 h, and the macrophage marker proteins IL1β and CD206 were labeled, and flow cytometry analysis was performed to observe the expression of cell marker genes (IL1β and CD206).

2. Experimental Results

Analysis of RNA sequencing data showed that in siNCM2 BMDMs, genes related to M1 BMDMs were upregulated after PTEN treatment, while genes related to M2 BMDMs were downregulated. The magnitude of changes in these related genes was significantly reduced after knocking out PLXDC2 (siPlxdc2-1M2 BMDM and siPlxdc2-2M2 BMDM) (Figure 4A).

Flow cytometry was used to detect the expression of IL1β and CD206, two proteins most associated with M1 BMDMs and M2 BMDMs, indicating the polarization state of macrophages. After knocking out PLXDC2, the polarization state of macrophages was no longer affected by PTEN (Figure 4B).

When PLXDC2 was not knocked out, PTEN treated M2 BMDMs and induced M2 BMDMs to polarize toward M1 BMDMs; after PLXDC2 was knocked out, PTEN treated M2 BMDMs and could not change the polarization state of M2 BMDMs. This indicates that PTEN polarizes M2 BMDMs toward M1 BMDMs by binding to PLXDC2 on the surface of M2 BMDMs.

Example 5 PTEN activates the JAK2-STAT1 pathway through PLXDC2 to promote the polarization of macrophages towards M2.

1. Experimental Methods

1. M2 BMDMs cells (siNCM2BMDM, siPlxdc2-1M2 BMDM and siPlxdc2-2M2 BMDM) treated according to the method of step (3) in step 3 of Example 3, wherein the M2 BMDMs were cultured with Flag and Flag-PTEN prepared in Example 1 for 30 min, and the cell proteins were collected. The expression of four JAK proteins (JAK1, JAK2, JAK3, TYK2) and their phosphorylated proteins (p-JAK1, p-JAK2, J p-AK3, p-TYK2) and six STAT proteins (STAT1-6) and their phosphorylated proteins (p-STAT1-6) were detected by immunoblotting.

2. M2 BMDMs were treated with or without 100 ng/mL Flag-PTEN prepared in Example 1 for 30 min with or without the addition of JAK2 inhibitor (Z3, final concentration 100 μM) and JAK2-IN-6 (hereinafter referred to as J6, final concentration 50 μg/mL), and WB was performed to detect the expression of JAK2 protein and its phosphorylated protein (p-JAK2 ^Y1008 ) and STAT1 protein and its phosphorylated protein (p-STAT1 ^Y701 ).

2. Experimental Results

When PLXDC2 was not knocked out, JAK2 and STAT1 of M2 BMDMs were phosphorylated when PTEN was treated; after PLXDC2 was knocked out, phosphorylation of M2 BMDMs was no longer observed when PTEN was treated (Figure 5A and Figure 5B). After treatment with JAK2 inhibitors Z3 and J6, JAK2 and STAT1 were not phosphorylated regardless of PTEN treatment (Figure 5C). This indicates that PTEN activation of the JAK2-STAT1 pathway depends on the interaction between PTEN and PLXDC2.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. Application of PTEN in the preparation of a drug for treating tumors, characterized in that the drug for treating tumors is an immune cell activator, and the accession number of PTEN on NCBI is Gene ID: 5728. 2. The use according to claim 1, characterized in that the immune cells are one or more of macrophages, NK cells and T cells. 3. The use according to claim 2, characterized in that the immune cell activator is an activator that promotes macrophage polarization. 4. The use according to claim 3, characterized in that the immune cell activator is an activator that promotes the polarization of M2 macrophages into M1 macrophages. 5. The use according to claim 3, characterized in that the immune cell activator is a preparation that activates the JAK-STAT signaling pathway of macrophages. 6. The use according to claim 5, characterized in that the immune cell activator is a preparation combined with PLXDC2, and the accession number of PLXDC2 in NCBI is Gene ID: 84898. 7. The use according to any one of claims 1 to 6, characterized in that the tumor is melanoma. 8. Use of a reagent for detecting PLXDC2 in the preparation of a product for evaluating the effect of a PTEN-containing drug in treating a tumor, and/or use of a reagent for evaluating the effect of a PTEN-containing drug in treating a tumor; The accession number of PTEN in NCBI is Gene ID: 5728, and the accession number of PLXDC2 in NCBI is Gene ID: 84898. 9. The use according to claim 8, characterized in that the tumor is melanoma. 10. The use according to claim 8, characterized in that the drug is an immune cell activator.