Preparation method and application of high heat treatment cell drug delivery system

Abstract

The invention is suitable for the technical field of biological medicine, and provides a preparation method and application of a high heat treatment cell drug delivery system. Mitoxantrone (MTX) is loaded in human umbilical cord mesenchymal stem cells (PD-MSCs) derived on the basis of high heat treatment to prepare the drug delivery system PD-MTX, drugs can be accurately delivered to tumor sites, release is controlled through a pH sensitive mechanism, the treatment effect is improved, and the side effects of chemotherapy are reduced. The PD-MSCs are inactivated through high heat treatment, so that the safety is ensured. PD-MTX and alphaPD-L1 are combined for use, so that a synergistic effect of chemotherapy and immunization is formed, and accurate and safe treatment is realized. The method for high heat treatment of cells and drug loading is not limited to human umbilical cord mesenchymal stem cells and mitoxantrone, and can be popularized to other cells and drugs. The composition has a remarkable effect in treatment of prostatic cancer and prostatic cancer osseous metastasis, can be popularized to treatment of other diseases, and has wide application potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a preparation method and application of a heat-treated cell drug delivery system. Background Art

[0002] Prostate cancer is one of the most common malignancies in men worldwide. Especially in the advanced stage, about 90% of patients will develop bone metastasis, leading to complications such as severe bone pain, fractures, spinal cord compression, and hypercalcemia, which seriously affect the quality of life of patients. Currently, the commonly used treatment methods include surgical resection, radiotherapy, chemotherapy, endocrine therapy, and targeted therapy. However, these treatment methods have obvious limitations: 1) Chemotherapy and radiotherapy: Although they can extend the survival period of patients, drugs or radiation often cause systemic toxic side effects due to non-specific effects, and the concentration of drugs at the tumor site is often insufficient, resulting in limited treatment effects. 2) Endocrine and targeted therapy: Long-term use is likely to lead to drug resistance and side effects (such as osteoporosis and cardiovascular risks), and most patients will eventually develop castration-resistant prostate cancer (CRPC).

[0003] In order to increase the drug concentration at the tumor site and reduce systemic toxicity, various drug delivery systems (such as nanoparticles, liposomes, polymer micelles, etc.) have been proposed in recent years. However, the existing drug delivery systems generally have the following defects: 1) Structural and functional limitations: Their morphologies are mostly solid or colloidal microparticles. Although their structural designs are helpful for drug loading, it is difficult to achieve efficient targeted enrichment and sustained slow release. 2) Release control problems: They lack sensitivity to the tumor microenvironment (especially the acidic environment), resulting in a lack of precise control over drug release and uneven drug efficacy. 3) Safety hazards: Some carrier materials have problems such as poor biocompatibility, long-term toxicity, or immunogenicity, which are not conducive to clinical applications.

[0004] In recent years, the use of mesenchymal stem cells (MSCs) as a drug delivery platform has received much attention. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are considered ideal carriers due to their natural tumor tropism, low immunogenicity, and high cell activity. They are spindle-shaped in morphology and have a complete cell membrane structure, and can migrate to the tumor area through natural chemokines. However, active MSCs have the following problems in practical applications: 1) Proliferation and tumor promotion risks: Active cells have the ability of self-proliferation, which may exacerbate the development of the original tumor in vivo. 2) Ethics and immune responses: The source and operation of active stem cells may cause immune responses. 3) Limited drug loading capacity: Traditional active cells have limitations in drug loading and release control, and it is difficult to meet the requirements of efficient anti-cancer treatment. Therefore, the present invention provides a preparation method and application of a heat-treated cell drug delivery system. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method and application of a high-temperature treated cell drug delivery system, aiming to solve the problems presented in the above-mentioned background technology.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] A preparation method of high-temperature treated cells, wherein the high-temperature treated cells are PD-MSCs, and the preparation method is as follows:

[0008] Pretreatment of hUC-MSCs: After trypsin digestion of hUC-MSCs, they are collected into a 15 mL centrifuge tube, centrifuged at 500 RCF for 5 min, and the supernatant is discarded;

[0009] Preparation of PD-MSCs: The pretreated hUC-MSCs are heat-treated at 45 °C for 1 h to be transformed into high-temperature treated derived mesenchymal stem cells, namely PD-MSCs.

[0010] A high-temperature treated cell prepared according to the above-mentioned preparation method.

[0011] An application of the high-temperature treated cell according to the above-mentioned in loading drugs.

[0012] A preparation method of a high-temperature treated cell drug delivery system, wherein the high-temperature treated cell drug delivery system is PD-MTX, and the preparation method is as follows:

[0013] The above-mentioned high-temperature treated cells are co-incubated with 1 mL of mitoxantrone at a concentration of 500 μg / mL at 4 °C for 24 h, and the supernatant is discarded to obtain high-temperature treated derived stem cells loaded with mitoxantrone, namely PD-MTX;

[0014] PBS is added to PD-MTX and stored in a 4 °C environment.

[0015] A high-temperature treated cell drug delivery system prepared according to the above-mentioned preparation method.

[0016] An application of the high-temperature treated cell drug delivery system according to the above-mentioned in disease treatment.

[0017] An application of the high-temperature treated cell drug delivery system according to the above-mentioned combined with αPD-L1 in disease treatment.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] In the present invention, mitoxantrone (MTX) is loaded into human umbilical cord mesenchymal stem cells (PD-MSCs) derived from high-temperature treatment to prepare a drug delivery system PD-MTX, which realizes the precise delivery of the drug to the tumor site and achieves controlled release in the tumor microenvironment through a pH-sensitive release mechanism, thereby significantly improving the therapeutic effect and greatly reducing the side effects of traditional chemotherapy. High-temperature treatment renders PD-MSCs unable to proliferate, eliminating the risk of tumor promotion and ensuring the safety of treatment. In addition, PD-MTX not only directly inhibits tumors but also promotes the body's anti-tumor immune defense by regulating the immune response in the tumor microenvironment. The good drug-loading capacity and release stability of PD-MSCs further improve the effectiveness and stability of drug delivery. The present invention also combines PD-MTX with an immune checkpoint inhibitor (αPD-L1), which releases tumor antigens by inducing immunogenic cell death, activates and remodels immune cells in the tumor microenvironment, and further improves the overall anti-tumor effect. The method of high-temperature treating cells and loading drugs in the present invention is not only applicable to human umbilical cord mesenchymal stem cells and mitoxantrone but can be extended to other cells and drugs. In addition, loading drugs after high-temperature treating cells shows excellent effects in the treatment of prostate cancer and has broad application potential, which can be extended to the treatment of other types of diseases, demonstrating significant application value. Description of the Drawings

[0020] Figure 1 Characterization of the morphology and biological properties of PD-MSCs; wherein, a are bright field images of optical microscopy and confocal microscopy of hUC-MSCs and PD-MSCs, b is the diameter analysis of hUC-MSCs and PD-MSCs, c are trypan blue staining images (upper) and confocal microscopy images of Calcein-AM / PI double staining (lower), d is the changing trend of the proliferation ability of hUC-MSCs and PD-MSCs determined by CCK-8 assay, e are flow cytometry images of apoptosis of hUC-MSCs and PD-MSCs, f is Figure 1 the quantitative results of e in, g are scanning electron microscopy (SEM) images (upper) and transmission electron microscopy (TEM) images (lower) of hUC-MSCs and PD-MSCs.

[0021] Figure 2Drug loading capacity and release characteristics of MTX-loaded PD-MSCs; where, a is the schematic diagram of the preparation of PD-MSCs and PD-MTX, b is the confocal microscopy image of MTX-loaded PD-MSCs, c is the frequency distribution diagram of MTX in different treatment groups, d is the fluorescence percentage of NC and PD-MTX, e is the Zeta potential measurement of PD-MSCs and PD-MTX, f is the standard curve of the MTX loading experiment, g is the HPLC analysis of MTX loading into PD-MTX under in vitro conditions, h is the change of the drug loading amount (left) and drug loading rate (right) of PD-MSCs with the increase of MTX concentration, i is the standard curve of the MTX release experiment, j is the HPLC analysis of MTX release from PD-MTX under in vitro conditions, k is the drug release curve of PD-MTX under different pH conditions.

[0022] Figure 3 In vitro inhibitory effect of PD-MTX on tumor cells; where, a is the scanning electron microscopy (SEM) images of each group, b is the high-content imaging at different times, c is the flow cytometry analysis of the average fluorescence intensity distribution of MTX, d is Figure 3 The quantitative analysis of c in Figure 3 e is the flow cytometry analysis of the intensity distribution of tumor cell killing in each group, f is Figure 3 The quantitative analysis of e in Figure 3 g is the curve of cell survival rate changing with time, h is the invasion experiment, i is the comparison chart of cell migration rate in different treatment groups, j is the flow cytometry analysis of the average fluorescence intensity distribution of CD86, k is

[0023] Figure 4 The tumor-targeting ability of PD-MTX in a tumor-bearing mouse model; where, a is the schematic diagram of the experiment for determining the anti-tumor efficacy of PD-MTX in a tumor-bearing mouse model, b is the in vivo fluorescence imaging of mice in the MTX-Cy5.5 and PD-MTX-Cy5.5 treatment groups at different time points, c is the average fluorescence intensity change curve of MTX-Cy5.5 and PD-MTX-Cy5.5 in the tumor-bearing mice within 24 h, d is the change diagram of the fluorescence intensity of each organ with time (i - tumor, ii - kidney, iii - liver, iv - heart, v - lung, vi - spleen), e is the fluorescence intensity time curve of each organ (i - tumor, ii - kidney, iii - liver, iv - heart, v - lung, vi - spleen).

[0024] Figure 5 Therapeutic effect of PD-MTX in a subcutaneous tumor model of prostate cancer; among them, a is a schematic diagram of the experiment for detecting the therapeutic effect of the PD-MTX subcutaneous tumor model, b is a picture of the mouse tumor tissue, c is a curve of tumor tissue volume changing with time, d is a curve of mouse body weight changing with time, e is HE staining analysis of mouse tumor tissue, f is quantification of the proportion of pathological mitotic figures in tumor tissue, g is survival analysis of different treatment groups, h is immunofluorescence staining results of immunogenic death (ICD) markers CRT and HMGB1, i is analysis of the staining results of CRT and HMGB1, j is immunofluorescence staining results of ICD markers CD8 and Granzyme B, k is analysis of the staining results of CD8 and Granzyme B, l is the level changes of inflammatory factors IFN-γ, TNF-α, IL-6 and IL-8 in tumor tissue.

[0025] Figure 6 Therapeutic effect of PD-MTX + αPD-L1 combination therapy in a bone metastasis tumor model of prostate cancer; among them, a is a schematic diagram of the experiment for detecting the therapeutic effect of the PD-MTX + αPD-L1 combination therapy bone metastasis tumor model, b is a picture of the mouse tumor tissue, c is a curve of tumor tissue volume changing with time, d is a curve of mouse body weight changing with time, e is survival analysis of different treatment groups, f is the level changes of inflammatory factors TNF-α, IL-10 and IL-12, g is the concentration changes of VEGF, PSA and RANKL, h is immunofluorescence staining results of the ICD marker PD-L1, i is immunofluorescence staining results of the ICD marker Runx2, j is staining quantification analysis of PD-L1 and Runx2, k is coronal plane reconstructed CT images of the bones of mice in different treatment groups, l is detection of osteoblastogenesis by measuring bone mineral density (BMD) and bone volume / total volume ratio (BV / TV), m is detection of osteoclastogenesis by measuring bone resorption surface / bone surface (BS / BV) and trabecular thickness (Tb.Th).

[0026] Figure 7 Effect of PD-MTX + αPD-L1 combination therapy on immune-related markers in the tumor microenvironment; among them, a is a schematic diagram of the experiment for detecting the effect of PD-MTX on immune-related markers in the tumor microenvironment, b is flow cytometry analysis of the expression of CD4 and CD8, c is the proportion change of CD8+ / CD4+ T cell subsets, d is flow cytometry analysis of the mean fluorescence intensity distribution of FOXP3, e is Figure 7Quantitative results of d, f is the immunofluorescence staining results of immunogenic cell death (ICD) markers CD11c and CD86, g is the flow cytometry analysis of the expression of MHC II-positive cells, h is the graph of detecting the activation of DCs by CD86 expression and the percentage of MHCII-positive cells, i is the immunofluorescence staining results of ICD markers F480 and iNOS, j is the flow cytometry analysis of the expression of CD86, k is the expression of M1 macrophage polarization markers iNOS and CD86, l is the flow cytometry analysis of the expression of CD69 to detect the activation of cytotoxic T cells, m is Figure 7 Quantitative results of l, n is the flow cytometry analysis of the expression of NK1.1 in NK cells, o is Figure 7 Quantitative results of n, p is the immunofluorescence staining results of ICD markers CD8 and Granzyme B, q is Figure 7 Quantitative results of p, r is the immunofluorescence staining results of Perforin protein expression, s is Figure 7 Quantitative results of r. Detailed implementation manners

[0027] For a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0028] The present invention provides a heat-treated cell drug delivery system, and the heat-treated cell drug delivery system is PD-MTX, and its preparation method is as follows:

[0029] Pretreatment of hUC-MSCs: Human umbilical cord mesenchymal stem cells (hUC-MSCs) are digested with trypsin, collected in a 15 mL centrifuge tube, centrifuged at 500 RCF for 5 min, and the supernatant is discarded.

[0030] Preparation of PD-MSCs: The pretreated hUC-MSCs are heat-treated at 45 °C for 1 h to be transformed into pyro-derived mesenchymal stem cells (PD-MSCs).

[0031] Storage of PD-MSCs: PBS is added to PD-MSCs and stored in a 4 °C environment.

[0032] Preparation of PD-MTX: PD-MSCs and 1 mL of mitoxantrone (MTX) at a concentration of 500 μg / mL were co-incubated at 4°C for 24 h. The supernatant was discarded to obtain pyro-derived hUC-MSCs loading mitoxantrone (PD-MTX).

[0033] Storage of PD-MTX: After adding PBS to PD-MTX, it was stored in an environment at 4°C.

[0034] In the present invention, pyro-derived human umbilical cord mesenchymal stem cells (PD-MSCs) were used as a drug carrier, and a drug delivery system PD-MTX was prepared by loading MTX into PD-MSCs to achieve precise delivery of the drug to the tumor site. The present invention also combined PD-MTX with an immune checkpoint inhibitor (αPD-L1) to further improve the overall anti-tumor effect. The method of heat-treating cells and loading drugs in the present invention is not only applicable to human umbilical cord mesenchymal stem cells and mitoxantrone, but can be extended to other cells and drugs. In addition, loading drugs after heat-treating cells not only shows excellent effects in the treatment of prostate cancer, but also has broad application potential and can be extended to the treatment of other types of diseases.

[0035] The following studies were conducted in the present invention:

[0036] To systematically evaluate the effects of heat treatment on human umbilical cord mesenchymal stem cells (hUC-MSCs), Figure 1 The differences between hUC-MSCs and PD-MSCs obtained by heat treatment at 45°C for 1 h were compared in terms of morphology, particle size distribution, cell viability, proliferation ability, apoptosis level, and ultrastructure. It was specifically divided into seven parts (a) to (g) as follows:

[0037] (a) Bright-field imaging with an optical microscope and a confocal microscope;

[0038] Normal cultured hUC-MSCs and PD-MSCs were respectively inoculated into culture dishes, and the cell morphology was observed using an optical microscope and a confocal microscope at the same magnification. The results showed that hUC-MSCs had a tendency to adhere and grow, with clear cell edges and good refraction; PD-MSCs still maintained a complete cell contour, but the cells were in a suspended state, and some cells showed irregular or shrunk contours. This indicates that heat treatment did not completely destroy the basic spindle shape of hUC-MSCs, but significantly reduced the cell growth state, suggesting that heat treatment would cause a certain degree of change in cell activity.

[0039] (b) Particle size analysis;

[0040] The average particle size and distribution range of hUC-MSCs and PD-MSCs were measured using a laser particle size analyzer. The results showed that the average particle size and distribution range of hUC-MSCs were slightly larger than those of PD-MSCs, and the curve showed that the peak of the particle size distribution of PD-MSCs shifted towards the direction of smaller particle size. This indicates that heat treatment may cause changes in cell membrane permeability and cell volume, manifested as a reduction in particle size distribution, providing potential advantages for subsequent drug loading.

[0041] (c) Trypan blue staining (upper) and Calcein-AM / PI double staining (lower);

[0042] Experimental design:

[0043] 1. Trypan blue staining: hUC-MSCs and PD-MSCs were respectively stained with trypan blue, and the ratio of live cells (unstained) to dead cells (stained) was observed under a microscope.

[0044] 2. Calcein-AM / PI double staining: Calcein-AM and PI were simultaneously added to the cell culture system, and the distribution of live cells (green fluorescence) and dead / apoptotic cells (red fluorescence) was observed through a confocal microscope.

[0045] The results of trypan blue staining showed that hUC-MSCs were almost unstained, indicating a high cell viability; PD-MSCs were all stained, suggesting cell non-viability. The results of Calcein-AM / PI double staining showed that hUC-MSCs mainly presented green fluorescence, and a certain number of red fluorescent cells could be observed in PD-MSCs. This indicates that high heat treatment can cause cell death, but PD-MSCs still retain certain biological activities and have the potential to be used as drug carriers.

[0046] (d) CCK-8 proliferation viability detection;

[0047] Equal amounts of hUC-MSCs and PD-MSCs were respectively inoculated into 96-well plates, and the cell proliferation viability was detected using CCK-8 reagent at 24 h, 48 h, and 72 h. The results showed that the OD value of hUC-MSCs increased significantly with the culture time, indicating a strong proliferation ability; while the OD value of PD-MSCs changed insignificantly at each time point, and the proliferation viability was significantly reduced. This indicates that after high heat treatment, the cells basically lose their proliferation ability, reducing the risk of tumor promotion, which is an important reason why PD-MSCs are superior to active stem cells in terms of safety.

[0048] (e) Flow cytometry for detecting cell apoptosis;

[0049] The apoptosis rates of hUC-MSCs and PD-MSCs, including the proportions of early apoptosis and late apoptosis, were detected by flow cytometry using Annexin V-FITC / PI double staining. The flow cytometry results showed that hUC-MSCs were mainly distributed in the viable region with a low apoptosis rate; the proportions of early and late apoptotic cells in PD-MSCs were significantly increased. This indicates that heat treatment significantly induced the apoptosis process of hUC-MSCs, further confirming that PD-MSCs are significantly different from active stem cells in biological characteristics and reducing the potential risks of proliferation and tumorigenesis.

[0050] (f) Figure 1 Quantitative analysis of (e);

[0051] Statistical processing of the flow cytometry data in (e) was performed to compare the survival rates and apoptosis rates of the two types of cells. The quantitative analysis results showed that the proportion of viable cells in PD-MSCs decreased significantly, while the apoptosis proportion increased significantly. Consistent with the results in (e), high heat treatment caused apoptosis of some cells in PD-MSCs and loss of their proliferative ability, which has a positive significance for subsequent safety.

[0052] (g) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging;

[0053] hUC-MSCs and PD-MSCs were respectively fixed, dehydrated, sputter-coated with gold (SEM) and embedded, ultrathin-sectioned (TEM) to observe the cell surface morphology and internal ultrastructure. The SEM images showed that the cell surface of hUC-MSCs was relatively smooth and the cell morphology was intact; the cell surface of PD-MSCs showed slight shrinkage or depression, and the cell surface structures such as microvilli were reduced. The TEM images showed that the internal organelles of hUC-MSCs were clearly distributed and the mitochondrial morphology was intact; the organelle structure of PD-MSCs was slightly damaged, and the mitochondria showed vacuolization or reduced density. This indicates that high heat treatment caused certain damage to the cell surface and internal structures, but PD-MSCs could still retain the basic morphology, laying a foundation for subsequent drug loading and delivery.

[0054] Summary: Figure 1 The experimental results showed that high heat treatment did not completely destroy the basic structure and biological activity of hUC-MSCs, but effectively reduced their proliferative ability and induced cell death. This enabled PD-MSCs to significantly reduce the tumorigenesis risk while retaining a certain degree of tumor targeting, providing strong support for its safe application in drug delivery systems.

[0055] Figure 2Demonstrated the loading and release characteristics of heat-treated derived mesenchymal stem cells (PD-MSCs) on the chemotherapeutic drug mitoxantrone (MTX). Specifically divided into eleven parts from (a) to (k) as follows:

[0056] (a) Schematic diagram of the preparation of PD-MSCs and PD-MTX;

[0057] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were incubated at 45 °C for 1 h to obtain heat-treated derived mesenchymal stem cells (PD-MSCs). PD-MSCs and mitoxantrone (MTX) were co-incubated at 4 °C for 24 h, and the supernatant was removed by centrifugation to obtain PD-MTX. PD-MTX was stored at 4 °C for subsequent characterization and in vitro / in vivo experiments. The schematic diagram visually demonstrated the preparation process from hUC-MSCs to PD-MSCs and then to PD-MTX, highlighting the two key steps of "heat treatment" and "drug incubation". Through heat treatment, the stem cells lost their proliferative ability, and their retained tumor targeting ability was used to load the chemotherapeutic drug MTX, successfully constructing a safe and efficient drug delivery system.

[0058] (b) Confocal microscopy images of PD-MSCs loaded with MTX;

[0059] The incubated PD-MTX cells were fixed and stained (the spontaneous fluorescence of MTX itself could be used for labeling), and the distribution of MTX in the cells was observed by confocal microscopy to evaluate the loading effect of PD-MSCs on MTX. The results showed that obvious red fluorescence signals (characteristic fluorescence of MTX) were visible in the cells, and the fluorescence signals were concentrated in the cytoplasmic region. This indicated that PD-MSCs successfully took up and loaded MTX, suggesting that the cells still retained intact cell membranes and intracellular structures after heat treatment and could be used for drug loading.

[0060] (c) Frequency distribution diagrams of MTX in different treatment groups;

[0061] The control group (NC) and PD-MTX were respectively subjected to fluorescence or absorption detection to determine the fluorescence / absorption intensity of MTX. The distribution frequency curves of MTX in different samples were plotted to compare the differences between drug-loaded and non-drug-loaded cells. The results showed that the intensity distribution curve of MTX in the PD-MTX sample shifted towards the high-value end as a whole, indicating that the cells contained a higher concentration of the drug; the values of the control group were lower or had almost no obvious peak. This indicated that PD-MTX successfully loaded MTX, further confirming the ability of PD-MSCs in drug enrichment.

[0062] (d) Fluorescence percentages of NC and PD-MTX;

[0063] Flow cytometry or image analysis software was used to perform fluorescence quantitative analysis on the NC group and the PD-MTX group. The percentage of fluorescent positive cells in the two groups was compared to evaluate the drug uptake by cells. The results showed that the proportion of fluorescent positive cells in the PD-MTX group was significantly higher than that in the NC group (drug-free or untreated group). This indicated that most of the PD-MSCs were successfully loaded with MTX, confirming that the cells still had good drug uptake and loading ability after high-temperature treatment.

[0064] (e) Zeta potential measurement of PD-MSCs and PD-MTX;

[0065] PD-MSCs and PD-MTX suspensions were taken separately, and their surface charges were measured using a Zeta potential analyzer. The Zeta potential differences between the two groups were compared to evaluate the effect of drug loading on the cell surface charge. The results showed that the Zeta potential of PD-MTX changed significantly compared with that of PD-MSCs (generally manifested as an increase or decrease in the absolute value of the potential), suggesting that the binding or entry of drug molecules into the cells changed the cell surface charge environment. This indicated that MTX loading would affect the surface physicochemical properties of PD-MSCs, and the change in Zeta potential further proved that the drug was successfully bound or entered the cells.

[0066] (f) Standard curve for the MTX loading experiment;

[0067] MTX solutions with different concentrations were prepared, and their absorbances / peak areas were detected using a UV-visible spectrophotometer or HPLC. With the concentration as the abscissa and the absorbance or peak area as the ordinate, a standard curve was plotted and the correlation coefficient was calculated. The results showed that the standard curve had a good linear relationship (R 2 close to 1), providing a basis for subsequent quantitative analysis of the MTX content in PD-MTX. This indicated that the established MTX standard curve had high accuracy and precision and could be used for quantitative calculation of the drug loading and release amounts of cells.

[0068] (g) HPLC analysis of MTX loading in PD-MTX under in vitro conditions;

[0069] The PD-MTX cells were lysed, and the MTX content was detected using high-performance liquid chromatography (HPLC). According to the retention time and peak area, it was judged whether the cells were successfully loaded with MTX, and the drug loading amount was calculated. The results showed that an obvious chromatographic peak appeared in the PD-MTX group at the characteristic retention time of MTX. The HPLC detection results were consistent with the aforementioned fluorescence detection results, further confirming that PD-MSCs had good loading effect and drug loading amount for MTX.

[0070] (h) Changes in the drug loading amount (left) and drug loading rate (right) of PD-MSCs with the increase in MTX concentration;

[0071] A series of MTX solutions with different initial concentrations were set up and incubated with the same number of PD-MSCs. The MTX content in the incubated PD-MTX was detected using HPLC or ultraviolet-visible spectrophotometer, and the drug loading amount (μg MTX / cell number) and drug loading rate (amount of MTX loaded / total initial MTX amount × 100%) were calculated. The results showed that as the initial concentration of MTX increased, the drug loading amount of PD-MSCs continuously increased; the drug loading rate might show a plateau or slightly decrease after reaching the peak value. This indicates that PD-MSCs have a certain absorption and saturation value for MTX, and the saturated drug loading capacity will be reached or approached at high concentrations, providing a basis for optimizing the drug concentration.

[0072] (i) Standard curve for the MTX release experiment;

[0073] Referring to the steps in (f), a series of MTX solutions with known concentrations were prepared, and the peak area / absorbance at a specific wavelength or retention time was detected. A linear relationship graph of concentration-absorbance / peak area was plotted to obtain the standard curve for quantitative determination of the release amount. The results were similar to those of the loading experiment, and this standard curve showed a good linear relationship (R 2 close to 1), which was suitable for quantitative analysis of the MTX release process. This indicates that when detecting the MTX content in the release solution under different pH or time conditions in the follow-up, the actual release amount can be quickly calculated through this standard curve.

[0074] (j) HPLC analysis of the release of MTX from PD-MTX under in vitro conditions;

[0075] The PD-MTX suspension was placed in simulated body fluid or other buffer solutions, and samples were taken at regular intervals. The peak area of MTX in the release solution was detected using HPLC, and then the cumulative release amount of MTX at each time point was calculated. The results showed that an MTX peak was detected in the buffer solution, indicating drug release. This shows that PD-MSCs can stably release MTX in the in vitro environment, providing a basis for subsequent in vivo release kinetics studies.

[0076] (k) Drug release curves of PD-MTX under different pH conditions;

[0077] PD-MTX was placed in buffer solutions with different pH values such as pH 7.4 (simulating the normal tissue environment) and pH 5.5 or below (simulating the tumor microacidic environment). Samples were taken at regular intervals and the MTX release amount at each time point was detected using HPLC, and the release curve was plotted. The results showed that in the pH 5.5 or more acidic environment, both the release rate and cumulative release amount of MTX were significantly higher than those under the pH 7.4 condition. This indicates that PD-MSCs have a certain responsiveness to the acidic microenvironment and can accelerate drug release in tumor tissues (with a slightly acidic pH), improving the anti-tumor efficiency and reducing the toxic and side effects on normal tissues.

[0078] Summary: Figure 2 The experimental results of Figure 2 show that PD-MSCs can efficiently load MTX and accelerate drug release in an acidic environment. Specifically, it is manifested as follows:

[0079] 1. In terms of loading: PD-MSCs have a high drug uptake capacity, can effectively load MTX and maintain good stability.

[0080] 2. In terms of release: In an acidic environment (such as the tumor microenvironment), PD-MTX can accelerate the release of MTX, thereby increasing the drug concentration at the tumor site and reducing the toxicity to normal tissues.

[0081] These characteristics prove that PD-MSCs can be used as a safe, efficient and pH-sensitive drug delivery platform, providing new ideas and technical support for the precise treatment of prostate cancer and its bone metastases.

[0082] Figure 3 The in vitro inhibitory effect of PD-MTX on tumor cells is shown, which is specifically divided into sixteen parts from (a) to (p) as follows:

[0083] (a) Scanning electron microscopy (SEM) images of each group;

[0084] The co-culture time of the control group (NC) or different treatment groups (such as PD-MSCs, MTX, PD-MTX, etc.) with tumor cells. After the treated tumor cells were fixed, dehydrated and sputter-coated with gold, the surface morphological changes of the cells were observed by scanning electron microscopy (SEM). The results showed that compared with the NC group, obvious shrinkage, microvilli collapse or membrane vesiculation and other damage characteristics appeared on the surface of tumor cells in the PD-MTX group; while the cell morphology of the cells treated without drugs or only with PD-MSCs was relatively intact. This indicates that PD-MTX can significantly damage the surface structure of tumor cells, suggesting that it has a stronger damaging or killing effect on tumor cells.

[0085] (b) High-content imaging at different times;

[0086] The tumor cells were seeded in 96-well plates and co-cultured with the PD-MTX group. At different time points of 0 h, 2 h, 4 h, and 6 h, the targeting of PD-MTX to RM1 tumor cells and the cell morphology, number of live cells and apoptosis of RM1 were detected in real time using a high-content screening (HCS) system. The results showed that as time extended, PD-MTX targeted to the periphery of tumor cells and released drugs, the proliferation of RM1 tumor cells was significantly inhibited, some cells showed shrinkage or apoptotic phenotypes, and the number of live cells decreased significantly. This indicates that PD-MTX can effectively inhibit the growth of tumor cells at different time periods and shows a trend of increasing in a time-dependent manner.

[0087] (c) Flow cytometry analysis of the mean fluorescence intensity distribution of MTX;

[0088] Since MTX usually has fluorescence, the fluorescence signal intensity of MTX in tumor cells was detected using a flow cytometer. The mean fluorescence intensity of PD-MTX and other control groups (such as untreated or MTX alone group) in tumor cells was compared. The results showed that the MTX fluorescence peak in the PD-MTX group in tumor cells was significantly higher than that in the control group, and the peak shifted to the right, indicating that tumor cells took up or were exposed to a higher concentration of MTX. This shows that through the drug delivery of PD-MSCs, higher MTX accumulation can be achieved in tumor cells, laying the foundation for subsequent killing effects.

[0089] (d) Figure 3 Quantitative analysis of (c);

[0090] The fluorescence peak area or geometric mean fluorescence intensity (Geo Mean) obtained by flow cytometry in (c) was statistically analyzed. The results showed that the mean fluorescence intensity of the PD-MTX group was significantly higher than that of other groups, and the difference was statistically significant. The quantitative data was consistent with the flow peak shape distribution, further confirming that PD-MTX can significantly increase the accumulation of MTX in tumor cells.

[0091] (e) Flow cytometry analysis of the intensity distribution of tumor cell killing in each group;

[0092] Tumor cells were co-cultured with the NC group, PD-MSCs group, MTX group, PD-MTX group, etc. respectively. An Annexin V / PI or other killing detection kit was used, and a flow cytometer was used to evaluate the apoptosis and mortality of tumor cells. The results showed that the proportion of apoptotic and dead cells in the PD-MTX group was significantly higher than that in other groups, indicating a stronger killing effect. This shows that while PD-MTX increases the drug loading and accumulation of tumor cells, it significantly enhances the cytotoxic effect on tumor cells.

[0093] (f) Figure 3 Quantitative analysis of (e);

[0094] Statistical processing was performed on the proportion of apoptotic / dead cells in each group in (e). The results showed that the apoptosis rate or mortality rate of the PD-MTX group was the highest, with a significant difference compared with the NC group or the MTX alone group. This shows that PD-MTX can more effectively kill tumor cells in vitro, providing strong data support for its application in tumor treatment.

[0095] (g) Curve graph of cell survival rate changing with time;

[0096] Tumor cells were co-cultured with different treatment groups respectively, and multiple time points were set (such as 12 h, 24 h, 48 h, 72 h, etc.). The CCK-8 cell viability detection method was used to calculate the cell survival rate and draw the time-survival rate curve. The results showed that the cell survival rate of the PD-MTX group decreased significantly with the culture time; while the decrease in the NC group or the PD-MSCs group was relatively small.

[0097] This indicates that the inhibitory effect of PD-MTX on tumor cells accumulates over time, showing a persistent and obvious killing effect.

[0098] (h) Invasion experiment;

[0099] A scratch experiment was performed using a 6-well plate to evaluate the effect of different treatment groups on the proliferation ability of tumor cells. The NC group, PD-MSCs group, MTX group, and PD-MTX group were set, and observations were made after 0 h and 24 h of culture. The results showed that after treatment with PD-MTX, the cell proliferation ability decreased significantly, indicating that the proliferation ability of tumor cells was significantly inhibited. This shows that in addition to directly killing tumor cells, PD-MTX can also effectively inhibit their proliferation ability, suggesting its potential role in blocking tumor metastasis.

[0100] (i) Comparison chart of cell migration rates of different treatment groups;

[0101] The effect of each group's treatment on the migration rate of tumor cells was compared through a conventional scratch experiment (Wound Healing). The scratch healing area or the number of cells that penetrated the membrane was counted after a certain period of time. The results showed that compared with the NC group, the scratch healing degree of the PD-MTX group was lower or the number of cells that penetrated the membrane was significantly reduced, indicating that the migration of tumor cells was inhibited. This shows that PD-MTX not only has a killing effect on tumor cells, but also can reduce their proliferation potential, providing more possibilities for comprehensive anti-cancer strategies.

[0102] (j) Flow cytometry analysis of the mean fluorescence intensity distribution of CD86;

[0103] Tumor cells after different treatments were co-cultured with macrophages, and the expression of CD86 on the surface of M1 macrophages was detected. The mean fluorescence intensity (MFI) of CD86 was analyzed using a flow cytometer to evaluate the level of immune activation. The results showed that the MFI of CD86 in the PD-MTX group was higher than that in the NC group, suggesting that it has a certain degree of activation effect on macrophages and others. This shows that PD-MTX can not only directly kill tumor cells, but may also promote the activation of macrophage phenotypes, thereby enhancing the immune response.

[0104] (k) Figure 3 Quantitative analysis in (j);

[0105] Statistical analysis was performed on the CD86 fluorescence intensity data in (j) to compare the differences between the PD-MTX group and other groups. The results showed that the expression level of CD86 in the PD-MTX group was significantly increased, and the difference was statistically significant. The quantitative results supported the positive effect of PD-MTX on immune cell activation, laying a foundation for its possible immunotherapy combination.

[0106] (l) Confocal microscopy images of the detection of immunogenic death representative proteins HMGB1 and CRT;

[0107] Tumor cells were co-incubated with different treatment groups respectively, and then their HMGB1 and CRT proteins were detected. The distribution of CRT protein (green) and HMGB1 protein (red) was observed by confocal microscopy. The results showed that the proportion of HMGB1 protein in the PD-MTX group decreased significantly, and the proportion of CRT protein increased significantly, indicating that PD-MTX has a significant ability to induce tumor immunogenic death.

[0108] (m) Distribution diagrams of the mean fluorescence intensity of HMGB1 and CRT proteins;

[0109] HMGB1 and CRT are important molecules related to immunogenic cell death (ICD). The expression levels of HMGB1 and CRT in each group of cells were detected respectively, and fluorescence quantitative analysis was performed. The results showed that the translocation of HMGB1 and the externalization of CRT membrane in the PD-MTX group were more obvious, indicating that immunogenic death occurred in the cells. This indicates that PD-MTX causes changes in ICD-related molecules in tumor cells, suggesting that it may enhance the anti-tumor immune effect by inducing immunogenic cell death.

[0110] (n) Flow cytometry analysis of the expression of CD86 and CD11c;

[0111] After dendritic cells (DCs) were co-incubated with PD-MTX-treated tumor cells, the expression of CD86 and CD11c on the surface of immune cells was detected. Flow cytometry was used to determine the degree of maturation or activation of immune cells. The results showed that after PD-MTX treatment, the expression levels of CD86 and CD11c on DCs were significantly increased, suggesting that they were activated or matured. This shows that after PD-MTX kills tumor cells, the dead tumor cells can also release immune activation signals, promoting the maturation of antigen-presenting cells and enhancing the immune response.

[0112] (o) Figure 3 Quantitative analysis in (n);

[0113] Statistically compare the flow cytometry results in (n), and quantify the positive rates or mean fluorescence intensities of CD86 and CD11c. The results showed that the indicators of immune cell maturation or activation in the PD-MTX group were significantly higher than those in the NC group, with statistical significance. The quantitative results further supported the role of PD-MTX in immune activation, providing data support for its possible combined immunotherapy applications.

[0114] (p) Schematic diagram of the experiment of co-culturing DCs or macrophages with PD-MTX;

[0115] Co-culture DCs or macrophages with the tumor cells treated with each group. Evaluate their immune activation effects by detecting immune cell maturation markers.

[0116] Summary: Figure 3 The experimental results of indicate that PD-MTX has a significant killing effect on tumor cells and can enhance the anti-tumor effect through the following mechanisms:

[0117] 1. Direct killing: PD-MSCs significantly inhibit tumor cell proliferation and induce apoptosis by efficiently loading MTX.

[0118] 2. Inhibiting migration and invasion: PD-MTX can reduce the migration ability of tumor cells and block their metastatic potential.

[0119] 3. Immune activation: PD-MTX induces immunogenic death of tumor cells, promotes the maturation of antigen-presenting cells, and enhances the immune response.

[0120] Figure 4 Demonstrates the tumor-targeting ability of PD-MTX in a tumor-bearing mouse model, which is specifically divided into five parts (a) to (e) as follows:

[0121] (a) Schematic diagram of the experiment for determining the anti-tumor efficacy of PD-MTX in a tumor-bearing mouse model;

[0122] Inoculate prostate cancer cells subcutaneously in mice, and conduct the experiment after the tumors grow to a certain volume. Inject MTX labeled with Cy5.5 fluorescence (MTX-Cy5.5) and PD-MTX (PD-MTX-Cy5.5) intravenously (i.v.) via the tail vein. Image the mice at different time points after administration using an in vivo fluorescence imaging system (IVIS) or a near-infrared fluorescence imager to observe the distribution of the drug in the body and its enrichment in the tumor site.

[0123] (b) In vivo fluorescence imaging of mice in the MTX-Cy5.5 and PD-MTX-Cy5.5 treatment groups at different time points;

[0124] The mice were divided into the MTX-Cy5.5 group and the PD-MTX-Cy5.5 group. In vivo fluorescence imaging was performed on the mice at multiple time points such as 2 h, 4 h, 8 h, 12 h, and 24 h after drug administration. The focus was on the change in the fluorescence signal intensity at the tumor site, while also paying attention to the whole-body distribution. The results showed that the fluorescence signal at the tumor in the MTX-Cy5.5 group was somewhat enriched in the early stage (2 - 4 h), but the fluorescence signal decayed relatively quickly with the extension of time, and obvious distribution was also observed in organs such as the liver and kidney. The fluorescence signal at the tumor site in the PD-MTX-Cy5.5 group was significantly higher than that in the MTX-Cy5.5 group at each time point, and the decay rate was slower. This indicates that the cell-loaded drug (PD-MTX-Cy5.5) can better deliver MTX to the tumor region and retain it for a long time, showing stronger tumor targeting ability compared to free MTX.

[0125] (c) Curves of the average fluorescence intensity changes of MTX-Cy5.5 and PD-MTX-Cy5.5 in tumor-bearing mice within 24 h; the in vivo imaging data in (b) were quantified, and the region of interest (ROI) in the tumor area was selected to measure the fluorescence intensity. Curves of the average fluorescence intensity (Mean Radiance or Fluorescence Intensity) changes at different time points after drug administration (such as 2 h, 4 h, 8 h, 12 h, 24 h) were plotted. The results showed that the fluorescence intensity within the tumor ROI in the MTX-Cy5.5 group was relatively low and decreased significantly with time. The fluorescence intensity within the tumor ROI in the PD-MTX-Cy5.5 group was initially higher and remained at a high level for a longer time, with a relatively slow decay. The quantitative curves confirmed that PD-MTX had a higher peak fluorescence intensity and a longer retention time at the tumor site, achieving efficient accumulation and continuous release in the tumor.

[0126] (d) Graph of the fluorescence intensity changes of each organ over time (i - tumor, ii - kidney, iii - liver, iv - heart, v - lung, vi - spleen);

[0127] The mice were sacrificed at the time points of 1 h, 2 h, 4 h, 12 h, and 24 h, and the main organs (tumor, liver, kidney, heart, lung, spleen) were removed for ex vivo fluorescence imaging. The dynamic changes in the fluorescence distribution of different organs over time were analyzed. The results showed:

[0128] Tumor (i): The fluorescence signal in the PD-MTX-Cy5.5 group was stronger and lasted longer; the fluorescence in the MTX-Cy5.5 group was relatively weaker.

[0129] Liver and kidney (ii, iii): These two detoxification and excretion organs usually have a relatively high uptake / metabolism of exogenous substances. The fluorescence signal in the MTX-Cy5.5 group was more concentrated in the liver and kidney; the PD-MTX-Cy5.5 group was also distributed in the liver and kidney, but was more inclined to accumulate in the tumor.

[0130] Heart, lung, spleen (iv, v, vi): There was a certain degree of distribution in both groups, but the fluorescence signal of the PD-MTX-Cy5.5 group in these organs might be lower, indicating better safety and targeting.

[0131] This indicates that PD-MTX is more significantly enriched in the tumor site, while the non-specific distribution in organs such as the liver and kidneys is reduced, helping to reduce toxic side effects.

[0132] (e) Fluorescence intensity time curves of each organ (i - tumor, ii - kidney, iii - liver, iv - heart, v - lung, vi - spleen);

[0133] Quantitatively analyze the ex vivo imaging data in (d), and plot the fluorescence intensity curves of each organ at different time points. Compare the differences in the accumulation and clearance kinetics of PD-MTX-Cy5.5 and MTX-Cy5.5 in each organ.

[0134] The results showed:

[0135] Tumor curve (i): The peak value of the curve in the PD-MTX-Cy5.5 group was significantly higher than that in the MTX-Cy5.5 group, and the decline rate was slower.

[0136] Liver and kidney curves (ii, iii): The fluorescence intensity of the MTX-Cy5.5 group in the liver and kidneys was higher and lasted longer, indicating that more free drugs were taken up by the liver or excreted through the kidneys; that of PD-MTX-Cy5.5 was relatively reduced.

[0137] Heart, lung, spleen curves (iv, v, vi): Both groups showed a certain degree of physiological distribution, but the peak value and total exposure in these organs of the PD-MTX-Cy5.5 group were significantly reduced.

[0138] This further verified the efficient enrichment of PD-MTX in the tumor site from the perspective of the time-intensity curve quantification, while reducing the distribution and potential toxicity to normal organs, providing important data support for its safe and efficient drug delivery in vivo.

[0139] Comprehensive summary: Figure 4 The experimental results verified the excellent tumor targeting characteristics of PD-MTX in vivo from the whole body to the organ level: In vivo imaging showed that PD-MTX-Cy5.5 had a higher and more persistent fluorescence signal in the tumor site, indicating that it could efficiently reach and retain in the tumor area. Ex vivo organ imaging and quantitative analysis showed that PD-MTX reduced the non-specific distribution in major metabolic organs such as the liver and kidneys, and was expected to reduce systemic toxicity. Overall, PD-MTX demonstrated good tumor enrichment ability and relative safety in the tumor-bearing mouse model, laying a solid foundation for subsequent in vivo treatment experiments and clinical translation.

[0140] Figure 5 Shows the therapeutic effect of PD-MTX in a subcutaneous tumor model of prostate cancer. It is specifically divided into twelve parts from (a) to (l) as follows:

[0141] (a) Schematic diagram of the experiment to detect the therapeutic effect of the PD-MTX subcutaneous tumor model;

[0142] RM-1 prostate cancer cells were subcutaneously inoculated into the right groin of immunocompetent C57BL / 6 mice. After the tumor volume reached a certain size, the mice were randomly divided into a control group (NC), a PD-MSCs group, an MTX group, and a PD-MTX group, and drugs were administered by tail vein injection or peritumoral injection. After drug administration, the tumor volume was measured regularly, the body weight of the mice was recorded, the survival time was observed, and samples were taken for detection at the end of the experiment (including histopathology, immunofluorescence staining, and immune factor detection, etc.). The schematic diagram shows the establishment of subcutaneous tumors, the drug administration strategy, and the multi-dimensional evaluation process, intuitively reflecting the overall idea of "cell drug loading + immune regulation" in anti-tumor in vivo. Through this model, the in vivo anti-tumor effect of PD-MTX in prostate cancer, its safety impact on the body, and the possible immune enhancement effect can be systematically investigated.

[0143] (b) Pictures of mouse tumor tissues;

[0144] At the end of the treatment cycle or when the tumor reached the ethical requirements, the mice were sacrificed and the subcutaneous tumor tissues were dissected. The tumors of each group were photographed side by side to compare their sizes and appearances. The results showed that the tumors in the NC group were larger, dark red in color, and had rich blood supply. Compared with the NC group, the tumor volume in the PD-MSCs group showed little difference. Compared with the NC group, the tumors in the MTX group were slightly reduced, but the inhibitory effect was limited. The tumor volume in the PD-MTX group was significantly reduced, with a harder texture or reduced blood supply, indicating a strong inhibitory effect. This shows that the PD-MTX group significantly inhibits tumor growth, providing support for subsequent quantitative analysis and histological detection.

[0145] (c) Curve of tumor tissue volume changing with time;

[0146] During drug administration, the long and short diameters of subcutaneous tumors were measured every 2 - 3 days using calipers, and the tumor volume was calculated (common formula: V = 0.5 × long diameter × (short diameter) 2) And draw the change curves to compare the tumor volume growth rates of each group. The results showed that the tumor volume in the NC group increased rapidly over time. The tumor volume in the PD-MSCs group increased over time. In the MTX group, a certain degree of growth inhibition was observed, but tumor development was not completely blocked. The tumor growth rate in the PD-MTX group slowed down significantly, and even showed a stable or slightly shrinking trend in the later stage, and the difference was statistically significant compared with other groups. This indicates that PD-MTX can effectively inhibit the growth of subcutaneous prostate cancer tumors in vivo and exhibits anti-tumor potential superior to that of single chemotherapy drugs.

[0147] (d) Curve of mouse body weight change over time;

[0148] The body weights of the mice were regularly measured during the experimental period to evaluate the impact of the treatment on the overall health and toxicity of the body. The body weight data were plotted into curves corresponding to the tumor volume curves. The results showed that the body weights of the mice in the NC group fluctuated slightly over time. The body weights of the mice in the PD-MSCs group increased slowly. In the MTX group, the body weights of some mice decreased significantly, indicating the systemic toxicity caused by the chemotherapy drug. The body weight curve of the mice in the PD-MTX group was relatively stable and there was no significant decrease compared with the NC group, indicating that while PD-MTX exerted its anti-tumor effect, it did not cause obvious toxic side effects. This shows that compared with traditional chemotherapy, PD-MTX has a milder impact on the body, indicating that the cell drug-loading strategy can reduce drug toxicity and improve treatment safety.

[0149] (e) HE staining analysis of mouse tumor tissues;

[0150] The excised tumor tissues were fixed, paraffin-embedded, and sectioned, and then hematoxylin-eosin (HE) staining was performed. The histological features such as the morphology of tumor cells, necrosis areas, and inflammatory infiltration were observed under the microscope. The results showed that the tumor cells in the NC group were densely arranged, the mitotic figures increased, and the blood vessels were abundant. There was little difference between the PD-MSCs group and the NC group. In the PD-MTX group, a larger area of necrosis or apoptosis was visible, the density of tumor cells decreased, and inflammatory cell infiltration might occur in the stroma. This indicates that PD-MTX can effectively induce tumor tissue necrosis or apoptosis and block tumor cell proliferation.

[0151] (f) Quantification of the proportion of pathological mitotic figures in tumor tissues;

[0152] Multiple fields of view were randomly selected under the HE sections, and the number of mitotic figures in tumor cell nuclei was counted. The average number of mitotic figures per field of view was used as an index to measure the activity of cell proliferation. The results showed that the proportion of mitotic figures in the NC group was the highest and the tumor proliferation was active. The number of mitotic figures in the PD-MTX group decreased significantly, indicating that cell proliferation was significantly inhibited. This shows that this index proves at the pathological level that PD-MTX effectively blocks the mitotic process of tumor cells and inhibits tumor growth.

[0153] (g) Survival analysis of different treatment groups;

[0154] Record the time from the start of treatment to natural death of the mice, and calculate the survival rate. Use the Kaplan-Meier curve to plot and perform statistical analysis. The results show that the survival rate of the NC group decreased rapidly. There was little difference between the PD-MSCs group and the NC group. The survival time of the MTX group was relatively prolonged, but the improvement was limited. The survival rate of the PD-MTX group was significantly increased, and the overall survival period was significantly prolonged. This indicates that PD-MTX has obvious advantages in prolonging the survival of tumor-bearing mice, and once again confirms its strong and safe anti-tumor effect.

[0155] (h) Immunofluorescence staining results of immunogenic cell death (ICD) markers CRT and HMGB1;

[0156] Perform double immunofluorescence staining on tumor tissue sections to detect the extracellularly flipped calreticulin (CRT) and extracellularly translocated HMGB1 respectively. Observe the staining location and intensity under a fluorescence microscope. The results show that CRT and HMGB1 in the NC group were mainly distributed intracellularly with low expression levels. In the PD-MTX group, a large amount of CRT was found to be flipped on the cell membrane, and HMGB1 was translocated to the extracellular space or intercellular space, with significantly enhanced fluorescence signals. This indicates that PD-MTX can induce immunogenic cell death (ICD), promote the exposure of tumor antigens and immune recognition through the flipping / translocation of CRT and HMGB1, and provide signals for the immune system to eliminate tumor cells.

[0157] (i) Analysis of the staining results of CRT and HMGB1;

[0158] Quantify the fluorescence intensity in (h) using image analysis software, and compare the staining intensities of each group. Display the results in the form of a bar chart to evaluate the expression levels of CRT / HMGB1 in the cell membrane or extracellular region. The results show that the average fluorescence intensities of CRT and HMGB1 in the PD-MTX group were significantly higher than those of other groups, and the difference was statistically significant. The quantitative analysis was consistent with the microscopic observation results, further confirming that PD-MTX can significantly enhance the ICD level of tumor cells and has a potential immune activation effect.

[0159] (j) Immunofluorescence staining results of ICD markers CD8 and Granzyme B;

[0160] Perform immunofluorescence staining on tumor tissue to detect the infiltration of CD8 + T cell infiltration and the expression level of the cytotoxic factor Granzyme B. Observe the distribution and co-localization of CD8 and Granzyme B through a fluorescence microscope. The results show that CD8 in the tumor stroma of the PD-MTX group +The number of T cells increased significantly, and the staining intensity of Granzyme B enhanced, indicating a large infiltration of activated cytotoxic T lymphocytes (CTLs) into the tumor. This suggests that PD-MTX treatment is beneficial for recruiting and activating more CD8 + T cells into the tumor site, enhancing the cytotoxic immune response, and further promoting tumor cell clearance.

[0161] (k) Analysis of the staining results of CD8 and Granzyme B;

[0162] Statistical analysis was performed on the CD8 and Granzyme B positive cells or fluorescence intensity of the immunofluorescence sections in (j). Bar charts or scatter plots were drawn to compare the differences between groups. The results showed that the number of infiltrating CD8 + T cells and the expression level of Granzyme B in the PD-MTX group were significantly higher than those in other groups. The quantitative results were consistent with the microscopic observation results, further corroborating that PD-MTX can significantly enhance the cytotoxic immune response at the tumor site.

[0163] (l) Changes in the levels of inflammatory factors IFN-γ, TNF-α, IL-6, and IL-8 in tumor tissues;

[0164] Serum was collected, and the concentrations of inflammatory factors such as IFN-γ, TNF-α, IL-6, and IL-8 were measured by ELISA. The differences in inflammatory factors after treatment in each group were compared. The results showed that the levels of anti-tumor immune factors such as IFN-γ and TNF-α in the PD-MTX group were significantly increased, indicating a stronger Th1-type immune response; some pro-inflammatory factors (such as IL-6 and IL-8) may also show a certain degree of upregulation or complex changes, reflecting the remodeling process of the tumor microenvironment. This indicates that PD-MTX triggers a stronger immune-inflammatory response in the local tumor microenvironment, especially the upregulation of cytokines related to anti-tumor, providing more evidence for the immune-enhancing effect of PD-MTX.

[0165] Summary: Figure 5 The experimental results fully demonstrate the multiple therapeutic advantages of PD-MTX in the subcutaneous tumor model of prostate cancer:

[0166] 1. Outstanding tumor suppression effect: Compared with the NC group and the simple MTX group, PD-MTX can significantly inhibit tumor volume growth and extend the survival time of mice.

[0167] 2. Reduced toxic and side effects: The curve of mouse body weight change showed that there was no obvious weight loss in the PD-MTX group, indicating good safety.

[0168] 3. Immune enhancement potential: PD-MTX induces immunogenic cell death of tumor cells (upregulation of CRT and HMGB1), and promotes CD8 +T cell and Granzyme B infiltration enhance the secretion of anti-tumor factors such as IFN-γ and TNF-α, thus forming a stronger immune response.

[0169] Figure 6 The therapeutic effect of PD-MTX + αPD-L1 combination therapy in a prostate cancer bone metastasis tumor model is demonstrated. It is specifically divided into thirteen parts from (a) to (m) as follows:

[0170] (a) Schematic diagram of the experiment to detect the therapeutic effect of PD-MTX + αPD-L1 combination therapy on the bone metastasis tumor model;

[0171] C57BL / 6 immunocompetent mice were selected, and prostate cancer cells were inoculated into the mouse bone marrow cavity to simulate the formation of bone metastasis. Treatment was started after the tumor had grown stably in the bone. The mice were randomly divided into a control group (NC), an αPD-L1 group, a PD-MSCs group, a PD-MSCs + αPD-L1 group, a PD-MTX group, and a combination therapy group (PD-MTX + αPD-L1). Administration was carried out by intravenous (i.v.) injection, etc. Body weight was measured regularly and behavior was observed during the administration period; multiple endpoint detections (tumor volume, degree of bone destruction, immune indexes, etc.) were set, and finally imaging and pathological analyses of bone tissue were performed. This model can systematically study the anti-tumor effect of PD-MTX + αPD-L1 in the bone metastasis environment and its protective or remodeling effect on the bone microstructure, providing an important basis for subsequent clinical translation.

[0172] (b) Pictures of mouse tumor tissues;

[0173] After the end of the treatment cycle or reaching the experimental endpoint, the mice were dissected and the femurs, tibias, and surrounding soft tissues were removed; macroscopic photos of the tumor tissues of each group were taken for visual comparison of tumor size and bone appearance. The results showed that an obvious extra-bone tumor mass was visible in the NC group, and erosion and depression appeared on the bone surface, indicating severe bone destruction. The tumor volume in the PD-MTX + αPD-L1 group was significantly reduced, the degree of bone surface damage was alleviated, and the local bone structure was relatively intact. This indicates that PD-MTX + αPD-L1 combination therapy effectively inhibits the growth of bone metastasis tumors and protects the bone structure to a certain extent.

[0174] (c) Curve of tumor tissue volume changing with time;

[0175] The tumor volume was regularly evaluated by measuring soft tissue masses. The measurement results were plotted as a line graph to compare the tumor growth trends of each group over time. The results showed that the tumor volume in the NC group increased rapidly, indicating an accelerated bone metastasis process. The tumor growth in the PD-MTX + αPD-L1 group was significantly delayed, with the lowest curve growth slope, suggesting stronger anti-bone metastasis ability. The quantitative results were consistent with the macroscopic observations, demonstrating that PD-MTX + αPD-L1 was superior to other groups in inhibiting bone metastases.

[0176] (d) Curve of mouse body weight over time;

[0177] The body weights of the mice were regularly measured during the administration period to evaluate the systemic toxicity and overall health effects of the treatment on the body. The changes in body weight over time were plotted as a curve corresponding to the changes in tumor volume. The results showed that the body weight of the mice in the NC group decreased slightly due to factors such as increased tumor burden or bone pain. The body weight in the PD-MTX + αPD-L1 group was relatively stable or slightly fluctuated, without significant decrease, indicating relatively low toxic side effects. This shows that PD-MTX + αPD-L1 has relatively low toxic side effects and good safety while inhibiting tumors.

[0178] (e) Survival analysis of different treatment groups;

[0179] The time from the start of treatment to natural death or reaching the humane endpoint of the mice in each group was recorded, and the survival rate was calculated. The Kaplan-Meier survival curve was plotted and a statistical test (such as the Log-rank test) was performed. The results showed that the survival curve of the NC group dropped rapidly, indicating an increased mortality rate due to aggravated bone metastasis. The PD-MTX + αPD-L1 group significantly prolonged the survival period of the mice, with the highest survival rate. This shows that compared with other groups, the PD-MTX + αPD-L1 group significantly improved the survival status in the bone metastasis mouse model.

[0180] (f) Changes in the levels of inflammatory factors TNF-α, IL-10, and IL-12;

[0181] Serum was collected, and the concentrations of TNF-α, IL-10, and IL-12 were detected by ELISA. The regulation of inflammatory factors in the bone metastasis environment of each group was compared.

[0182] The results showed:

[0183] TNF-α, IL-12 (anti-tumor immune factors): The levels were significantly increased in the PD-MTX + αPD-L1 group, suggesting that stronger immune activation or pro-inflammatory responses are beneficial for tumor clearance.

[0184] IL-10 (immune inhibitory factor): The level in the PD-MTX + αPD-L1 group decreased relatively or did not increase significantly, indicating that the inhibitory microenvironment may be alleviated.

[0185] This indicates that PD-MTX + αPD-L1 can regulate the secretion of inflammatory factors in the bone metastasis environment, tending to promote anti-tumor immune responses and inhibit the immunosuppressive pathway.

[0186] (g) Changes in the concentrations of VEGF, PSA, and RANKL;

[0187] Using a sampling method similar to that in (f), detect the levels of VEGF (an angiogenic factor), PSA (prostate-specific antigen), and RANKL (an important factor related to bone destruction) in the serum. Compare the differences among groups in terms of angiogenesis, prostate cancer specificity, and bone destruction signals.

[0188] The results showed that:

[0189] VEGF: The content of VEGF in the PD-MTX + αPD-L1 group decreased significantly, indicating that angiogenesis was inhibited.

[0190] PSA: As a prostate cancer-specific marker, the PSA level increased in the PD-MTX group, suggesting a reduction in tumor burden.

[0191] RANKL: The RANKL level in the PD-MTX + αPD-L1 group also decreased, indicating a reduction in osteoclast activation and inhibition of the bone destruction process.

[0192] This indicates that PD-MTX + αPD-L1 can effectively inhibit angiogenesis and osteoclasts during the bone metastasis process of prostate cancer, and has multiple intervention effects on bone metastatic tumors.

[0193] (h) Immunofluorescence staining results of PD-L1;

[0194] Immunofluorescently label bone metastasis tumor tissue sections to detect the expression and distribution of PD-L1 (programmed death ligand 1). Observe the positive signals of PD-L1 under a fluorescence microscope. The results showed that PD-L1 was moderately or highly expressed in the NC group, suggesting the existence of an immune escape mechanism in the tumor microenvironment. The expression level of PD-L1 decreased in the PD-MTX + αPD-L1 group. This indicates that during the treatment with PD-MTX + αPD-L1, PD-L1 is a key immune checkpoint molecule, and its expression change indicates the interaction between tumor cells and immune cells, and also provides ideas for the combination of immune checkpoint inhibitors.

[0195] (i) Immunofluorescence staining results of Runx2;

[0196] Runx2 is a key transcription factor for osteoblast differentiation and also plays a certain role in bone metastasis of prostate cancer. Immunofluorescence staining was performed on bone metastatic tumor cells to observe the localization and expression level of Runx2. The results showed that Runx2 was highly expressed at the tumor-bone interface or within tumor cells in the NC group, promoting abnormal osteogenesis or carcinogenesis. The staining intensity of Runx2 in the PD-MTX + αPD-L1 group was weakened, indicating that the intervention in the osteogenesis-tumor interaction blocked the imbalance of tumor-induced bone remodeling. This indicates that while inhibiting tumor growth, PD-MTX + αPD-L1 can also regulate the expression of the key transcription factor Runx2 in the bone microenvironment, thereby inhibiting abnormal bone formation or the vicious cycle of tumor-bone mutual promotion.

[0197] (j) Quantitative analysis of the staining of PD-L1 and Runx2;

[0198] The fluorescence images obtained in (h) and (i) were quantified to measure the mean fluorescence intensity or the proportion of positive cells of PD-L1 and Runx2. The results were plotted as histograms to compare the differences between different groups. The results showed that the positive signals of PD-L1 and Runx2 in the PD-MTX + αPD-L1 group showed a certain degree of decrease or local changes compared with the NC group or other groups, and the differences were statistically significant. The quantitative results corroborated the microscopic observations, indicating the dual regulatory effects of PD-MTX + αPD-L1 on immune escape molecules and key factors in bone metastasis.

[0199] (k) Coronal reconstruction CT images of the bones of mice in different treatment groups;

[0200] Micro-CT was used to scan the bones of mice to obtain high-resolution bone structure data. Coronal or three-dimensional reconstruction images were selected to visually compare the destruction or repair of the bone cortex and trabeculae in each group. The results showed that there were bone defects, sparse or broken trabeculae in the NC group, indicating obvious signs of bone destruction. The continuity of the bone cortex in the PD-MTX + αPD-L1 group was more complete, and the trabecular structure was relatively dense, suggesting a protective or reparative effect on bone mass. This indicates that PD-MTX + αPD-L1 can reduce or reverse the bone destruction caused by bone metastasis of prostate cancer and improve the integrity of bone structure.

[0201] (l) Detection of bone density by measuring bone mineral density (BMD) and bone volume / total volume ratio (BV / TV);

[0202] Based on Micro-CT data, a specialized software was used to analyze bone mineral density (BMD) and bone volume / total volume (BV / TV). Higher BMD and BV / TV generally indicate denser bone mass and higher bone quantity. The results showed that both BMD and BV / TV were lower in the NC group, indicating severe bone mass loss. In the PD-MTX + αPD-L1 group, BMD and BV / TV increased significantly, and bone mass increase or destruction was alleviated. This indicates that PD-MTX + αPD-L1 can prevent bone loss, increase bone density, and improve the bone volume fraction in the context of prostate cancer bone metastasis.

[0203] (m) The bone structure was detected by measuring the bone resorption surface / bone surface (BS / BV) and trabecular thickness (Tb.th);

[0204] Using the Micro-CT data, the ratio of bone resorption surface (BS) to bone surface (BV), and trabecular thickness (Tb.th) were also analyzed. Lower BS / BV and lower Tb.th generally represent stable bone structure. The results showed that compared with the Normal group, the BS / BV ratio and Tb.th increased in the NC group, indicating poor bone structure stability. Compared with the NC group, the BS / BV decreased and Tb.th decreased significantly in the PD-MTX + αPD-L1 group, indicating the restoration of bone structure stability. This indicates that PD-MTX + αPD-L1 can effectively promote the restoration of bone structure, further confirming its multiple roles in anti-bone metastasis and bone protection.

[0205] Summary: Figure 6 The experimental results show that PD-MTX + αPD-L1 has therapeutic advantages in prostate cancer bone metastasis:

[0206] 1. Significantly inhibit bone metastatic tumors: effectively reduce tumor volume, extend the survival period of mice, and down-regulate key factors such as PSA, VEGF, and RANKL.

[0207] 2. Protect and reshape the bone microenvironment: Micro-CT and bone metabolism index analysis (BMD, BV / TV, BS / BV, Tb.Th) showed that PD-MTX + αPD-L1 can reduce bone destruction and maintain / improve bone structure.

[0208] 3. Bidirectional regulation of immunity and osteogenesis: By regulating inflammatory factors such as TNF-α, IL-10, and IL-12, and molecules such as Runx2 and PD-L1, PD-MTX + αPD-L1 can promote anti-tumor immunity and intervene in abnormal bone remodeling.

[0209] Figure 7Shows the effects of PD-MTX + αPD-L1 combination therapy on immune-related markers in the tumor microenvironment. Specifically divided into nineteen parts from (a) to (s) as follows:

[0210] (a) Schematic diagram of the experiment to detect the effects of PD-MTX on immune-related markers in the tumor microenvironment;

[0211] In tumor-bearing mice, PD-MTX, αPD-L1, or the combination therapy group (PD-MTX + αPD-L1) were injected respectively, and a control group (NC) and other controls (such as PD-MSCs or PD-MSCs + αPD-L1) were set up to compare the curative effects. After treatment for a period of time, tumor tissues and related lymphoid organs were sampled, and key immune markers (T cell subsets, DCs, macrophages, NK cells, etc.) were detected by flow cytometry, immunofluorescence staining and other methods.

[0212] The schematic diagram shows how the combination therapy regulates the tumor microenvironment: PD-MTX induces immunogenic cell death (ICD), releases tumor antigens and activates innate immunity.

[0213] (b) Flow cytometry analysis of the expression of CD4 and CD8;

[0214] The tumor tissues were digested into single-cell suspensions; flow cytometry was performed using fluorescently labeled antibodies against CD4 and CD8 to detect the proportion of tumor-infiltrating T cells. The results showed that the proportion of CD8 + T cells in the PD-MTX + αPD-L1 group increased significantly, and the proportion of CD4 + T cells remained or changed slightly; compared with other groups, the infiltration of CD8 + T cells was more obvious. The number of CD8 + T cells in the NC group was relatively low, and CD4 + T cells may exist in a certain proportion but are mostly in a functionally inhibited state. This indicates that the combination therapy is beneficial to enhancing the infiltration and activation of CD8 + T cells in the tumor microenvironment, laying the foundation for subsequent cytotoxic killing.

[0215] (c) Ratio changes of CD8 + / CD4 + T cell subsets;

[0216] The flow cytometry data in (b) were further statistically analyzed to calculate the ratio of CD8 + / CD4 + T cells. This ratio reflects to a certain extent the proportion of tumor-killing T cells to helper T cells. Generally, the higher the ratio, the stronger the anti-tumor effect. The results showed that the CD8 + / CD4+ The ratio is the highest, showing a significant difference compared with the NC group or single-treatment groups. This indicates that the combination therapy significantly enhances the dominant position of cytotoxic T cells in the tumor microenvironment, thereby enhancing the overall anti-tumor immune response.

[0217] (d) Flow cytometry analysis of the mean fluorescence intensity distribution of FOXP3;

[0218] While detecting T cell subsets, regulatory T cells (Tregs) were labeled with anti-FOXP3 antibody. The expression level of FOXP3 in T cells was measured to evaluate the activation or enrichment of Tregs. The results showed that the expression of FOXP3 in the PD-MTX + αPD-L1 group was relatively decreased, suggesting that Tregs were reduced or their function was inhibited. The high expression of FOXP3 in the NC group indicated that Tregs were active in the tumor microenvironment, suppressing the immune response. This shows that by inhibiting the PD-L1 pathway and inducing ICD, the combination therapy can reduce the enrichment of Tregs, thereby relieving the immunosuppression of effector T cells.

[0219] (e) Quantitative results of panel (d);

[0220] Statistical analysis was performed on the FOXP3 fluorescence intensity or the proportion of FOXP3 + cells in (d), and a bar graph was plotted. The results showed that the mean fluorescence intensity (MFI) of FOXP3 in the PD-MTX + αPD-L1 group was significantly lower than that in the single-treatment groups or the NC group, with statistical differences. The quantitative analysis and the flow cytometry peak distribution corroborated each other, further confirming that the combination therapy effectively inhibited the expansion or activation of Tregs.

[0221] (f) Immunofluorescence staining results of markers CD11c and CD86;

[0222] Immunofluorescence double staining was performed on tumor tissue sections to detect the dendritic cell (DC) marker CD11c and the co-stimulatory molecule CD86 respectively. The distribution and activation degree of DCs in tumor tissues were observed. The results showed that there was an increase in CD11c + DC infiltration and stronger expression of the co-stimulatory molecule CD86 in the PD-MTX + αPD-L1 group, indicating an increased activation level of DCs. The number of DCs was scarce and the CD86 signal was weak in the NC group. This shows that the PD-MTX + αPD-L1 group promoted the activation and distribution of DC cells.

[0223] (g) Flow cytometry analysis of the expression of MHC II-positive cells;

[0224] The single-cell suspension of tumor tissue was detected using a fluorescently labeled antibody against MHC II; the maturation of antigen-presenting cells such as DCs was evaluated (high expression of MHC II usually means stronger antigen presentation ability). The results showed that the proportion of MHC II + cells was significantly increased in the PD-MTX + αPD-L1 group, indicating the emergence of a large number of mature DCs in the tumor microenvironment. Echoing the results in (f), the combination therapy significantly enhanced the antigen presentation function, providing a key link for T cell activation. This indicates that the combination therapy not only enhanced T cell activation but also promoted the maturation and activation of antigen-presenting cells (DCs), contributing to the continuous amplification of the immune response.

[0225] (h) Diagram for detecting the activation of DCs by CD86 expression and the percentage of MHC II-positive cells;

[0226] Combining the results of CD86 and MHC II in (f) and (g), the proportion of positive cells was calculated; the data was visualized as a bar chart or... to measure the degree of DC activation. The results showed that the PD-MTX + αPD-L1 group had a significant increase in the proportion of CD86 + MHC II + cells, which was significantly higher than that of the single-drug treatment group or the NC group. Quantitative analysis further confirmed that PD-MTX + αPD-L1 significantly promoted the maturation and activation of DCs and enhanced the anti-tumor immunogenicity.

[0227] (i) Immunofluorescence staining results of ICD markers F4 / 80 and iNOS;

[0228] F4 / 80 is a specific marker for mouse macrophages, and iNOS (inducible nitric oxide synthase) is a typical functional molecule of M1 macrophages; immunofluorescence double staining of tumor tissue was performed to observe the distribution of M1 macrophages in the tumor microenvironment. The results showed that the number of F4 / 80 + macrophages was significantly increased in the PD-MTX + αPD-L1 group, and the iNOS staining intensity was higher, indicating that more macrophages were in the M1 polarization state (pro-inflammatory, tumor-killing). In the NC group, there were few F4 / 80-positive cells or mainly M2 macrophages, and the iNOS signal was weak. This indicates that the combination therapy significantly promoted the polarization of M1 macrophages and further enhanced the killing ability in the tumor microenvironment.

[0229] (j) Flow cytometry analysis of CD86 expression;

[0230] Following the same idea as in (i), after gating the single cells in the tumor tissue with F4 / 80 + the CD86 expression was detected; CD86 is a common co-stimulatory molecule of M1 macrophages and can also be used to evaluate the activation level of macrophages. The results showed that the CD86 in the PD-MTX + αPD-L1 group+ The highest proportion of cells indicates that macrophages are significantly activated and tend to polarize towards M1. The indicators such as CD86 and iNOS corroborate each other, proving that PD-MTX + αPD-L1 can reshape the macrophage phenotype and enhance its anti-tumor function.

[0231] (k) Expression of the M1 macrophage polarization markers iNOS and CD86;

[0232] The results of (i) and (j) were comprehensively analyzed, and iNOS and CD86 were detected simultaneously in the same sample; the proportion of double-positive (M1 type) macrophages in different groups was compared. The results showed that the iNOS + CD86 + in the PD-MTX + αPD-L1 group was significantly increased, and functional M1 macrophages dominated. This indicates that the combination therapy not only enhances DC maturation but also significantly promotes M1 polarization, inhibits tumor immune escape, and accelerates tumor cell clearance.

[0233] (l) Flow cytometry analysis of the expression of CD69 to detect the activation of cytotoxic T (NKT) cells;

[0234] CD69 is an early activation marker of T cells; the expression level of CD69 was detected to evaluate the activation degree of cytotoxic T cells. The results showed that the proportion of CD69 + T cells in the PD-MTX + αPD-L1 group increased significantly; the expression level of CD69 in the NC group was low, and the T cells were in a weakly activated state. This indicates that the combination therapy can make more T cells in an activated killing state, which helps to strengthen the elimination of tumor cells.

[0235] (m) Quantitative results of Fig. (l);

[0236] The flow cytometry data in (l) were statistically analyzed, and a bar chart was drawn to compare the proportion or mean fluorescence intensity of NKT cells in each group. The results showed that the indicators in the PD-MTX + αPD-L1 group were the highest, with significant differences compared with the NC group or the single treatment group. Quantitative analysis confirmed that the combination of drugs had an obvious beneficial effect in activating T cells.

[0237] (n) Flow cytometry analysis of the expression of NK1.1 in NK cells;

[0238] NK1.1 is a common marker for mouse NK cells; the proportion of NK1.1 + cells was detected to evaluate the infiltration and quantitative changes of NK cells. The results showed that the NK1.1 +The proportion of cells increased significantly, indicating an increase in NK cells in the tumor microenvironment; the number of NK cells in the NC group was relatively insufficient, and the inhibitory microenvironment was more obvious. This shows that the combination therapy not only enhances T cell responses but also facilitates the recruitment or expansion of NK cells, synergistically killing tumors.

[0239] (o) Quantitative results of figure (n);

[0240] For NK1.1 of (n) + The proportion of cells or MFI of NK cells was statistically analyzed and visualized graphs were plotted. The results showed that the PD-MTX + αPD-L1 group was also significantly higher than other groups at the NK cell level, with statistically significant differences. This indicates that PD-MTX + αPD-L1 can activate innate immune components (NK cells), further improving the anti-tumor efficacy of the overall immune network.

[0241] (p) Immunofluorescence staining results of CD8 and GranZyme B;

[0242] Double fluorescence labeling of CD8 + T cells and the cytotoxic factor GranZyme B were performed on tumor tissue sections; the distribution and activation status of cytotoxic T cells in tumors were observed. The results showed that the number of CD8 + T cells in the PD-MTX + αPD-L1 group increased significantly, and the staining intensity of GranZyme B was higher, indicating that these T cells were activated and had strong cytotoxicity. The number of CD8 + cells in the NC group was scarce, and the GranZyme B signal was weak or absent. This shows that the combination therapy can effectively induce tumor-infiltrating CD8 + T cells to produce cytotoxic molecules such as GranZyme B, achieving efficient tumor killing.

[0243] (q) Quantitative results of figure (p);

[0244] Count the CD8 + GranZyme B + cells or analyze the fluorescence intensity in immunofluorescence sections to compare the differences between groups. The results showed that the number of CD8 + GranZyme B + double-positive cells in the PD-MTX + αPD-L1 group was much higher than other groups, with statistically significant differences. Echoing the aforementioned T cell activation indicators, it further confirmed that PD-MTX + αPD-L1 significantly enhanced the tumor-specific cytotoxic immune response.

[0245] (r) Immunofluorescence staining results of Perforin protein expression;

[0246] Perforin is one of the core molecules for cytotoxic killing by T cells and NK cells. An increase in the expression level of Perforin protein indicates that combination therapy can significantly enhance the killing activity of effector immune cells in the tumor microenvironment, thereby enhancing the overall anti-tumor immune effect. Tumor tissue sections were selected, and immunofluorescence labeling was performed on the key killing factor perforin secreted by cytotoxic lymphocytes (such as CD8 + T cells, NK cells, etc.); the localization and fluorescence intensity of Perforin were observed under a fluorescence microscope to evaluate the functional state of cytotoxic immune cells in the tumor microenvironment. The results showed that more Perforin-positive cells or stronger Perforin fluorescence signals were visible in the tumor tissues of the PD-MTX + αPD-L1 group, indicating that a large number of cytotoxic immune cells (especially activated CD8 + T cells, NK cells) had fully expressed Perforin and possessed stronger cytotoxic functions. The Perforin expression in the NC group and single-treatment groups was relatively low, suggesting that the activation degree of cytotoxic immune cells was limited.

[0247] (s) Figure 7 Quantification results in (r);

[0248] Combined with the immunofluorescence images of (r), an image analysis software was used to quantify the proportion or fluorescence intensity of Perforin-positive cells; a bar graph was drawn to compare the differences between different treatment groups. The results showed that the quantitative indicators of Perforin (such as the number of positive cells, average fluorescence intensity) in the PD-MTX + αPD-L1 group were significantly higher than those in the NC group or single-drug treatment groups, and the differences were statistically significant. Echoing the microscopic observations of (r), the quantitative data further confirmed that the combination therapy could effectively up-regulate Perforin expression, enhance tumor-specific immune killing, and provide a solid experimental basis for the combined immunotherapy of prostate cancer and other solid tumors.

[0249] Summary: Figure 7 The experimental results of show that PD-MTX provides a key prerequisite for the activation of antigen-presenting cells (DCs, macrophages, etc.) by inducing immunogenic cell death (ICD) and effectively releasing tumor antigens; while αPD-L1 blocks the PD-L1 / PD-1 signaling pathway and further relieves the inhibition of T cells. The synergistic effect of the two produces a significant gain effect on multiple immune pathways:

[0250] 1. T cell subset remodeling: The CD8 + / CD4 + ratio increased significantly, and CD8 +T cell infiltration and activation (expression of cytotoxic molecules such as CD69, Granzyme B, Perforin, etc.) were significantly enhanced; the expression level of FOXP3 in regulatory T cells (Tregs) decreased, reducing immunosuppression in the tumor microenvironment.

[0251] 2. Enhanced innate immunity:

[0252] ① Activation of dendritic cells (DCs): Markers of maturation such as CD11c, MHC II, CD86, etc. were significantly upregulated, improving antigen presentation and T cell priming efficiency;

[0253] ② Macrophage M1 polarization: In F4 / 80 + cells, the expression of iNOS and CD86 increased, and the proportion of pro-inflammatory and cytotoxic M1 macrophages increased;

[0254] ③ Increased number of NK cells (NK1.1 + ), enhancing the surveillance and clearance of tumors by innate immunity.

[0255] 3. Overall improvement in cytotoxicity:

[0256] ① CD8 + T cells and NK cells jointly secrete cytotoxic factors such as Granzyme B and Perforin, forming a strong killing effect on tumor cells;

[0257] ② The synergistic effect between immune cells breaks multiple immune escape barriers in the tumor microenvironment.

[0258] Overall, the combination therapy of PD-MTX + αPD-L1 promotes each other in both "activating anti-tumor immunity" and "relieving immunosuppression", significantly enhancing the infiltration and function of local immune cells in tumors, and finally achieving an efficient and long-lasting anti-tumor effect. This strategy provides strong experimental evidence for the clinical treatment of prostate cancer and other solid tumors, and also opens up a broader application prospect for subsequent combination immunotherapy and personalized cancer therapy.

[0259] In summary, the present invention utilizes the excellent drug-loading capacity of PD-MSCs to load the chemotherapeutic drug MTX into the cells, resulting in the preparation of PD-MTX. The special cell membrane and internal structure of PD-MSCs endow it with pH sensitivity, enabling precise and continuous drug release in the acidic tumor microenvironment, thereby increasing the drug concentration in tumors and reducing the toxic side effects on normal tissues. By combining PD-MTX with αPD-L1, the tumor microenvironment can be reshaped, the anti-tumor immune response can be activated, and a synergistic effect of "chemotherapy + immunotherapy" can be formed, achieving precise, continuous, and safe treatment of prostate cancer and its bone metastases, thus breaking through the limitations of existing treatment regimens. It is worth mentioning that the high-temperature treatment method of the present invention is not limited to the application of human umbilical cord mesenchymal stem cells, but can also be applied to other types of cells; in terms of drug loading, it is not limited to loading MTX, but can also be compatible with other drugs other than MTX. Therefore, the present invention not only brings a revolutionary breakthrough to tumor treatment, but also opens up new avenues for the treatment of other diseases.

[0260] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. A preparation method for highly heat-treated cells, characterized in that, The high-heat treated cells are PD-MSCs, and the preparation method thereof is as follows: Pretreatment of hUC-MSCs: hUC-MSCs were trypsinized and collected into 15 mL centrifuge tubes. The tubes were centrifuged at 500 RCF for 5 min and the supernatant was discarded. Preparation of PD-MSCs: The pretreated hUC-MSCs were heat-treated at 45°C for 1 h to convert them into high-heat-treated derived mesenchymal stem cells, namely PD-MSCs.

2. A hyperthermia-treated cell prepared according to the preparation method of claim 1.

3. Use of the hyperthermia-treated cells according to claim 2 in loading drugs.

4. A preparation method of a high-temperature treatment cell drug delivery system, characterized in that, The high-heat treatment cell drug delivery system is PD-MTX, and its preparation method is as follows: The hyperthermia-treated cells according to claim 2 were co-incubated with 1 mL of mitoxantrone at a concentration of 500 μg / mL at 4° C. for 24 h, and the supernatant was discarded to obtain hyperthermia-treated derived stem cells loaded with mitoxantrone, i.e., PD-MTX; PBS was added to PD-MTX and stored at 4°C.

5. A high-heat-treated cell drug delivery system prepared according to the preparation method according to claim 4.

6. Use of the hyperthermia-treated cell drug delivery system according to claim 5 in disease treatment.

7. Use of the hyperthermia-treated cell drug delivery system according to claim 5 in combination with αPD-L1 in disease treatment.

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