Application of tumor-derived monocytes

Abstract

The present invention relates to the field of biomedicine technology, and mainly to the use of tumor-derived monocytes in the preparation of drugs for treating Alzheimer's disease. The present invention applies tumor-derived monocytes to the treatment of diseases for the first time. Experiments of the present invention have shown that tumor-derived monocytes can improve and treat Alzheimer's disease, and can also play a role in improving cognitive ability and effectively improve the pathological phenotype of Alzheimer's disease. At the same time, experiments have shown that tumor-derived monocytes only need to be injected once to maintain the therapeutic effect, and will not cause abnormal immune response and tumor occurrence. The tumor-derived monocytes proposed by the present invention have great application prospects in the treatment and prevention of AD.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and relates to the application of tumor-derived monocytes, in particular to the application of tumor-derived monocytes in the preparation of anti-Alzheimer's disease drugs. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset and neuronal loss as its main feature. Clinically, it is characterized by global dementia manifestations such as memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes. Studies have shown that a series of changes have occurred in the brains of AD patients, among which the main macroscopic changes in the brain are the general atrophy of the cerebral cortex, neurofibrillary tangles in the form of double-stranded helical filaments often appear in nerve cells, and senile plaques formed by the extracellular deposition of amyloid protein often appear in the hippocampus and frontal cortex, and have also been found in the basal ganglia, thalamus, cerebellum and other brain regions, but the exact cause has not been identified so far. At present, cholinesterase inhibitors (such as donepezil, rivastigmine, galantamine) and glutamate receptor antagonists (memantine) are mostly used in clinical treatment of AD. However, the pathogenesis of AD is diverse, and these drugs mostly target a single target, which can delay the progression of the disease to a certain extent, but it is difficult to cure AD fundamentally. [1] In addition, conventional anti-AD drugs require long-term medication and have disadvantages such as poor efficacy in the late stage.

[0003] The loss of neurons and their synapses is the core pathological change of AD, in which cell proliferation and apoptosis-related pathways play an important role. [2] The occurrence and development of cancer is inseparable from the abnormal proliferation and apoptosis of cells. [3] There are also a lot of literature suggesting that AD regulatory factors also play a regulatory role in cancer. For example, the inhibition of the BDNF / TrkB pathway in AD can lead to the loss of a large number of neuronal cells. [4] , and activation of this pathway in cancer can promote tumor cell growth and metastasis [5] ; Reactive oxygen species (ROS) cause cellular oxidative stress and promote the occurrence of AD [6] At the same time, ROS can damage DNA, leading to genomic instability and promoting tumor cell proliferation. [7] More importantly, there is a clear negative correlation between cancer and AD incidence [8,9] Clinical epidemiological data indicate that the risk of developing AD in people with cancer is at least 33% lower than that in people without a history of cancer.

[10] . These include reproductive system cancers such as prostate cancer, breast cancer, etc.

[11] , skin-related cancers such as squamous cell carcinoma, basal cell carcinoma, etc.

[12] , other cancers such as lung cancer, colorectal cancer, etc.

[13] In particular, the incidence of dementia and breast cancer has increased significantly among women. [14-16] At the same time, a longitudinal population study conducted in South Korea also verified that breast cancer (BC) was associated with a significantly reduced risk of dementia.

[17] , and studies have shown that breast cancer patients who receive chemotherapy are more likely to develop dementia [18,19] However, some studies have pointed out that lymphoma has a significant positive correlation with AD and can promote the occurrence of AD.

[20] In addition, the study also pointed out that the correlation between cancer and AD may be due to the fact that cancer patients have a lower average life expectancy and often die before AD occurs, so the negative correlation between cancer and AD does not really exist.

[21] . Breast cancer is a cancer with a long survival period and currently has relatively mature treatment methods. Patients with cancer can survive until the onset of AD. Therefore, clinical epidemiology suggests that the negative correlation between breast cancer and AD may be true. In summary, joint analysis of breast cancer and AD to explore the mechanism of the negative correlation between the incidence of the two can help us find effective ways to improve the AD phenotype and provide an innovative treatment for AD.

[0004] In recent years, immunotherapy has attracted much attention. New tumor treatment methods represented by CAR-T therapy have better efficacy than other treatments targeting a single target.

[22] . Therefore, applying immune cell-related therapies to the treatment of AD, which has a similarly complex pathogenesis and high heterogeneity, may achieve good results. Studies have shown that in AD transgenic mouse models (APP / PS1 and 5XFAD), circulating monocytes (CD14 / CD16+-) can infiltrate the brain to reduce Aβ load and improve cognitive ability. However, the phagocytic ability and phenotype of monocytes in AD patients or model animals are limited, and the effect on improving AD pathological changes is obviously insufficient.

[23] , cannot be used for AD treatment. Bone marrow-derived monocytes, i.e. monocytes (BMMs) produced by differentiation of bone marrow stem cells that have not yet completed final maturation to enter the blood, exhibit inflammatory responses comparable to microglia and peripheral blood monocytes. They have been shown to reduce the Aβ burden in AD mouse models and alleviate the pathological phenotype of AD, and are considered the best cell-based treatment for AD.

[24] However, BMMs are difficult to obtain and need to be obtained through the induction and differentiation of bone marrow stem cells. Therefore, finding a monocyte with similar abilities to BMMs and easy to obtain may be helpful for the prevention and treatment of AD.

[0005] Studies have found that most tumor cells exhibit certain stem cell characteristics. So will the blood monocytes of tumor patients be more similar to BMM? As breast cancer is a solid tumor, can its peripheral blood monocytes be used to prevent and treat AD? Will the injection of allogeneic monocytes cause a complex immune response? Tumor cells injected into the body are considered to have a carcinogenic risk. Does the injection of monocytes from breast cancer individuals to improve AD also have a carcinogenic risk? The above questions are not clear yet, so it is unknown whether breast cancer-derived monocytes can be used to treat AD.

[0006] After searching, it was found that currently, tumor-derived monocytes have not been used to treat related diseases, and there are no reports of using them to treat Alzheimer's disease.

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[0055] In view of the above problems, the present invention aims to provide a use of tumor-derived monocytes, in particular, the use of tumor-derived monocytes in the preparation of anti-Alzheimer's disease drugs.

[0056] The invention protects the use of tumor-derived mononuclear cells in the preparation of anti-Alzheimer's disease drugs.

[0057] The present invention also protects the use of the tumor-derived monocytes in the preparation of drugs for improving neuronal function, cognitive impairment and cognitive function.

[0058] Furthermore, the injection method of the tumor-derived monocytes is tail vein injection.

[0059] Furthermore, the tumor is breast cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, lung cancer, or colorectal cancer.

[0060] The present invention demonstrates that the reduction in the incidence of AD in BC patients is likely related to monocytes, and breast cancer-derived monocytes may be used to treat AD. According to the present invention, in order to explore the molecular mechanism of the negative correlation between AD and BC, the inventors obtained the following information from the Gene Expression Omnibus database (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ).

[25] Peripheral blood single-cell RNA sequencing (scRNA-seq) data were collected (Sequence numbers: GSE181279, GSE139495) [26,27] The samples included 3 AD cases, 11 BC cases and 3 normal controls (NC).

[28] The data were merged, quality-controlled, cell type identified and clustered. The final quality-control results are as follows: Figure 1 As shown, the inventors identified seven different cell populations ( Figure 2 A), the distribution of each cell population is divided into UMAP images according to different groups ( Figure 2 B), among the 7 cell populations, T cells accounted for the largest proportion, followed by monocytes, and pDCs accounted for the smallest number. It is worth noting that the proportion of monocytes in the BC group was significantly greater than that in the AD group and the NC group, and showed a trend of BC>NC>AD ( Figure 2 B) Literature indicates that circulating monocytes can infiltrate the brain tissue of AD transgenic mouse models (APP / PS1 and 5XFAD), reduce Aβ load, and improve cognitive ability, but monocytes in AD patients are often ineffective and difficult to improve their pathological changes [23,29] , while monocytes in breast cancer tissue are often hyperactive

[30] .

[0061] The inventors further determined the differentially expressed genes (DEGs) between the AD group and the NC group, and between the BC group and the NC group using the Findmarker function of the Seurat (4.2.1) software package, and filtered the DEGs with |log fold change|≥0.25 and p_val_adj<0.05 as the cutoff values; the clusterProfiler software package in R language was used

[31] and the metascape database (https: / / metascape.org / gp / index.html# / main / step1)

[32] Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed, and the DAVID database (https: / / david.ncifcrf.gov / )

[33] Biological Process (BP) enrichment analysis was performed in the BC group and the NC group, and the AD group and the NC group had 534 common differentially expressed genes in monocytes ( Figure 2 C), and after functional enrichment analysis of these differentially expressed genes, it was found that they were mainly enriched in multiple neurodegenerative disease-related pathways such as hsa05010: Alzheimer disease ( Figure 2 D) The above results suggest that the reduced incidence of AD in BC patients is very likely related to monocytes, and breast cancer-derived monocytes may be used for the treatment of AD.

[0062] The present invention demonstrates that tumor-derived monocytes can improve the function of human hippocampal neurons (HPPNCs) and AD model cells (HPPNCs) that overexpress the key pathogenic gene APOE4 of sporadic AD. APOE4 According to an embodiment of the present invention, blood samples of breast cancer patients are collected clinically, and blood mononuclear cells (BC-monocytes) are separated according to the method of Yang M et al.

[34] Human breast cancer-derived monocytes and HPPNCs or HPPNCs APOE4 When the cells were in the logarithmic growth phase, the CCK8 method was used to detect changes in cell activity. The results showed that when human breast cancer-derived monocytes and HPPNCs or HPPNCs APOE4 When the ratio is lower than 1:20, it will not affect the cell activity ( Figure 3 A). At the same time, the inventors analyzed the morphological changes of neurons and found that when the co-culture ratio of the two was 1:20, the number of protrusions of HPPNCs increased significantly; at the same time, HPPNCs APOE4 The number of neuronal processes and shortening of processes caused by APOE4 overexpression were inhibited to a certain extent after co-culture ( Figure 3 B). Cell processes are the extensions of the neuron cell body, which participate in the process of information transmission and play an important role in the normal function of neurons. This suggests that human breast cancer-derived monocytes can improve neuronal function.

[0063] NeuN is a neuron-specific nuclear protein that is only expressed in the nuclei of differentiated and mature neurons. Its reduced fluorescence intensity indicates neuronal damage; the postsynaptic marker PSD-95 is generally upregulated in the early stages of neuronal growth and development and is related to synaptic maturation. Its reduced fluorescence intensity indicates a decrease in the level of synaptic maturation. According to the embodiments of the present invention, the inventors found through cell slides and immunofluorescence detection that compared with HPPNCs, HPPNCs APOE4 The fluorescence of NeuN and PSD-95 in the culture medium was weakened; human breast cancer-derived monocytes were mixed with HPPNCs or HPPNCs APOE4 Co-culture at a ratio of 1:20 (hereinafter referred to as co-culture) did not affect the fluorescence intensity of NeuN and PSD-95 of HPPNCs; at the same time, HPPNCs APOE4 The fluorescence intensity of PSD-95 in the two neuronal cells was enhanced, and the fluorescence intensity of NeuN was reduced to a certain extent; finally, compared with donepezil (Dnp, 0.5 μM), a first-line drug for AD treatment, co-culture can significantly enhance the fluorescence intensity of PSD-95 of the two neuronal cells, while there is no significant difference in the fluorescence intensity of NeuN between the two ( Figure 3C) In conclusion, co-culture can enhance synaptic maturation and improve neuronal damage to a certain extent. APOE4 From the cell morphology, the cells treated with 0.5 μM Dnp were oval, while the cell morphology after co-culture was closer to normal neurons ( Figure 3 C). Mature synapses are the key to information transmission. Dnp increased the number of cells but failed to completely restore the synaptic morphology. Neurons may still not be able to function normally. That is, monocyte treatment is better than Dnp in promoting neuronal function recovery.

[0064] The above results suggest that co-culture of human breast cancer-derived monocytes and neurons (ratio of 1:20) has no effect on normal neurons and can significantly improve the cell phenotype of the AD model; compared with donepezil, the first-line AD drug, co-culture of human breast cancer-derived monocytes and neurons can also enhance neuronal maturation, that is, human breast cancer-derived monocytes can be used for the prevention and treatment of AD with a better effect.

[0065] The present invention also demonstrates the use of tumor-derived monocytes in the preparation of drugs for improving cognitive impairment and cognitive function. According to the present invention, wild-type mice and APP / PS1 double-transfected Alzheimer's disease model mice were selected. Since the number of blood monocytes is limited and it is not easy to separate, we refer to the previous research [35,36] At the same time, according to the actual extraction of monocytes, human breast cancer-derived monocytes were finally injected into the mouse tail vein at a concentration of 2×10 7 / mL, 200μL / mouse, and performed a Y-maze novel arm exploration experiment before and 7 days after treatment, recording: ① the number of entries into the starting arm, novel arm, and other arms; ② the activity time in the starting arm, novel arm, and other arms. After the experiment, the number of entries into the novel arm, the proportion of time and distance in the novel arm were counted. The results showed that 7 days after the tail vein injection of human breast cancer-derived monocytes in APP / PS1 mice, the number of entries into the novel arm, the time and distance of activities in the novel arm were significantly increased ( Figure 4 .). This indicates that human breast cancer-derived monocytes can improve behavioral changes in AD model APP / PS1 mice.

[0066] The examples of the present invention also prove that breast cancer-derived monocytes have better therapeutic effects than normal-derived monocytes in the same species, and the therapeutic effect is equivalent to donepezil. According to the examples of the present invention, the inventors selected 7-week-old C57BL / 6 mice and injected E0771 cells (at a concentration of 1×10 7 The breast cancer model (BC) was constructed by PCR with 100 μL / mouse. 7-week-old normal mice were selected as the control group (N). After 3 weeks of feeding in an SPF environment, the spleens were removed by dissection. TMThe Mouse Monocyte Isolation Kit (STEMCELL, Catalog No. 19861) was used to separate monocytes into breast cancer-derived monocytes (BC mon) and normal-derived monocytes (Nmon). The cells were prepared with PBS at 1×10 7 The neuronal degeneration, behavioral and memory disorders that occur in mice after aging are similar to those in clinical patients. Therefore, naturally aged C57BL / 6 mice aged 11-20 months were selected for tail vein injection of mononuclear cells of the same species (concentration of 1×10 7 The mice were treated with 100 μL of monocytes / mL, 200 μL / mouse) once. According to the behavioral performance of the mice before treatment, they were divided into: ① normal control group (NC), ② normal-breast cancer derived monocytes treatment group (NC-BC mon), ③ dementia control group (AD-control), ④ dementia-normal mouse monocytes treatment group (AD-N mon), ⑤ dementia-breast cancer derived monocytes treatment group (AD-BC mon), ⑤ dementia-donepezil treatment group (Donepezil). Water maze test was performed to evaluate the learning and memory ability of mice 1 week and 4 weeks after treatment. The results showed that after 4 weeks of treatment ( Figure 6 A), the movement distance of the AD-BC mon treatment group in the target quadrant of the water maze was significantly increased compared with the AD-control group and the AD-N mon group, and the residence time in the target quadrant was also improved to a certain extent, and there was no difference between the AD-BC mon treatment group and the Donepezil treatment group. Comparison of behavioral changes in mice in each group before and after 1 week of treatment ( Figure 6 B), compared with the AD-N mon group and the Donepezil group, the AD-BC mon group stayed in the target quadrant for a longer distance and a longer time, and the difference was more significant (P<0.001), suggesting that breast cancer-derived monocytes may be more effective in treating AD than monocytes from normal mice. Comparison of changes in the water maze test of each group of mice within 1 month ( Figure 6B), the time and distance of the NC group and the AD-control group staying in the target quadrant did not differ significantly before and after; while the injection of breast cancer-derived monocytes had no effect on the behavioral performance of normal mice (NC-BC mon group); interestingly, the time and distance of the dementia phenotype mice staying in the target quadrant were prolonged after injection of monocytes from normal mice (AD-N mon group), monocytes from breast cancer model mice (AD-BC mon group) and continuous intraperitoneal gavage of donepezil for 1 month (Donepezil group); but the improvement of the behavioral phenotype of the AD-N mon group mice only lasted for 1 week, while the improvement of the behavioral phenotype of the AD-BC mon group and the Donepezil group lasted for 1 month, which was more stable. That is, the tail vein injection of breast cancer-derived monocytes of the same species has the function of improving the cognitive level of mice, and the effect is better than that of monocytes from normal mice, and is equivalent to that of donepezil. At the same time, donepezil requires continuous gavage to maintain the therapeutic effect, while the tail vein injection of breast cancer-derived monocytes only requires one time to maintain the therapeutic effect. That is, breast cancer-derived monocytes can be used for the prevention, treatment and research of AD, and are more convenient.

[0067] Literature suggests that administration of donepezil to APP / PS1 and other AD model mice for 4 consecutive weeks can alleviate cognitive impairment

[37] , the duration of treatment required for memantine administration is longer [38,39] At the same time, these drugs also have disadvantages such as individual heterogeneity and unstable efficacy. [1] . However, breast cancer-derived monocytes only needed a single injection to significantly improve cognitive impairment in mice one week later, and the effect continued one month later. The improvement in behavioral phenotypes was superior to that of monocytes derived from ordinary mice ( Figure 4 , Figure 6 ). Donepezil requires continuous intragastric administration to improve the water maze phenotype of mice. Tail vein injection of breast cancer-derived monocytes is more convenient and quicker. That is, tail vein injection of breast cancer-derived monocytes has the ability to improve the cognitive level of mice for a long time. Breast cancer-derived monocytes can be used for the prevention and treatment of AD and are easier to use. In addition, a large number of studies have shown that allogeneic transplantation is often accompanied by severe immune rejection reactions, and may even lead to the death of patients and experimental animals. [40-42] Moreover, tumors can form within 7-10 days after tumor cells are injected into the tail vein of mice. [43-45] According to the embodiments of the present invention, AD model mice treated with human breast cancer-derived monocytes or mouse breast cancer-derived monocytes via tail vein injection showed no obvious abnormal immune response except for behavioral changes, and no obvious tumor nodules were found in the main organs of the body ( Figure 5 , Figure 7 ), that is, breast cancer-derived monocytes therapy does not cause abnormal immune responses and tumors.

[0068] The breast cancer monocytes provided by the present invention can not only significantly improve HPPNCs and HPPNCs APOE4 The level of cell synaptic growth can also improve the cognitive level of two AD model mice, and will not cause abnormal immune response and tumor occurrence in mice. This proves that breast cancer-derived monocytes can be used for the treatment of Alzheimer's disease and related research work, and can be used in the development of AD drugs.

[0069] The present invention uses tumor-derived monocytes for the treatment of AD for the first time, and has achieved good technical effects. The present invention experiments have shown that tumor-derived monocytes can improve and treat Alzheimer's disease, and can also play a role in improving cognitive ability and effectively improve the pathological phenotype of Alzheimer's disease; at the same time, experiments have shown that tumor-derived monocytes only need to be injected once to maintain the therapeutic effect, which is more convenient than intragastric administration of napiprofen; and tumor-derived monocytes will not cause abnormal immune responses and tumorigenesis. The tumor-derived monocytes proposed in the present invention have great application prospects in the treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the specific implementation methods and prior art solutions of the present invention, the following briefly introduces the drawings involved in the present invention. For ordinary technicians in this field, similar results can be obtained without any creative work.

[0071] Figure 1 For quality control of scRNA-seq data, Figure 1 A in the middle shows a Violin graph showing the amount of mRNA in each sample before quality control. Figure 1 B shows the Violin plot of the mRNA reads of each sample before quality control. Figure 1 C in the middle shows the Violin plot of the percentage of mitochondrial genes in each sample before quality control. Figure 1 D in the figure shows the Violin plot of the amount of mRNA in each sample after quality control. Figure 1 The Violin graph in E shows the mRNA readings of each sample after quality control. Figure 1 F is a Violin plot showing the percentage of mitochondrial genes in each sample after quality control.

[0072] Figure 2 Single cell subpopulations and differentially expressed genes, Figure 2 A in the middle represents cell type identification based on scRNA-seq data, and a total of 7 major cell types were identified. Figure 2 B in the middle indicates that the distribution of each cell population is divided into UMAP images according to different groups. Figure 2 C in the middle represents the differential gene intersection of monocytes between BC group and NC group and between AD group and NC group. Figure 2 Middle D shows the bubble chart of differential gene function enrichment analysis.

[0073] Figure 3 The effect of co-culture of breast cancer-derived monocytes and neurons on neuronal phenotypes. Figure 3 A in the middle shows the changes in the activity of nerve cells after cell co-culture. Figure 3 B in the middle shows the changes in the morphology and number of nerve cells after cell co-culture. Figure 3 C in the figure shows that donepezil (Dnp, 0.5 μM) was used as a positive control. APOE4 Changes in cell immunofluorescence staining after co-culture at a ratio of 1:20.

[0074] Figure 4 Changes in the Y-maze novel arm exploration experiment before and after injection of breast cancer-derived monocytes into the APP / PS1 animal model.

[0075] Figure 5 This is a comparison of the morphology of major organs in the APP / PS1 animal model after injection of breast cancer-derived monocytes.

[0076] Figure 6 The water maze test changes of naturally aged C57BL / 6 mice before and after injection of mononuclear cells derived from breast cancer of the same species into the tail vein. Figure 6 A in the middle is the result of water maze test after 4 weeks of treatment (* compared with AD-BC mon group, # compared with Donepezil group, * / #P<0.05, ** / ##P<0.01), Figure 6 Middle B is a comparison of the water maze conditions of mice from 0 to 4 weeks (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0077] Figure 7 This is a comparison of the morphology of major organs in C57BL / 6 mice, a naturally aging animal model, 2 months after tail vein injection of mononuclear cells derived from the same species of mouse breast cancer. DETAILED DESCRIPTION

[0078] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention. It should be noted that if specific techniques or conditions are not specified in the embodiments, the experimental results can be obtained according to the description of relevant literature in the field or product instructions. The reagents, instruments, cells and animal models used are all conventional products that can be purchased on the market if the manufacturer is not specified.

[0079] Example 1: Single-cell transcriptome analysis of breast cancer-AD

[0080] In order to explore the molecular mechanism of the negative correlation between AD and BC, the inventors collected peripheral blood single-cell RNA sequencing (scRNA-seq) data (sequence number: GSE181279, GSE139495) from the Gene Expression Omnibus database (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ), with a total of 3 AD cases, 11 BC cases and 3 normal control groups (NC). Then, the inventors used the Seurat (4.2.1) software package in R language to

[28] The data were merged, quality-controlled, cell type identified and clustered. The final quality-control results are as follows: Figure 1 As shown, the inventors identified seven different cell populations ( Figure 2 A). Then the distribution of each cell population is divided into UMAP images according to different groups ( Figure 2 B), among the 7 cell populations, T cells accounted for the largest proportion, followed by monocytes, and pDCs accounted for the least. It is worth noting that the proportion of monocytes in the BC group was significantly greater than that in the AD group and the NC group, and showed a trend of BC>NC>AD ( Figure 2 B).

[0081] The inventors further determined the differentially expressed genes (DEGs) between the AD group and the NC group, and between the BC group and the NC group using the Findmarker software package of Seurat (4.2.1), and filtered the DEGs with |log fold change|≥0.25 and p_val_adj<0.05 as the cut off values; the clusterProfiler software package of R language was used

[31] and the metascape database (https: / / metascape.org / gp / index.html# / main / step1)

[32] Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed, and the DAVID database (https: / / david.ncifcrf.gov / )

[33] Biological Process (BP) enrichment analysis was performed in the BC group and the NC group, and the AD group and the NC group had 534 common differentially expressed genes in monocytes ( Figure 2 C), and after functional enrichment analysis of these differentially expressed genes, it was found that they were mainly enriched in multiple neurodegenerative disease-related pathways such as hsa05010: Alzheimer disease ( Figure 2 D). The above results suggest that the reduced incidence of AD in BC patients is likely related to monocytes, and breast cancer-derived monocytes may be used for the treatment of AD.

[0082] Example 2: Co-culture experiment of human tumor-derived monocytes and neural cells

[0083] Cancers known to have a significant negative correlation with the incidence of AD include reproductive system cancers such as prostate cancer and breast cancer, skin-related cancers such as squamous cell carcinoma and basal cell carcinoma, and other cancers such as lung cancer and colorectal cancer. In this example, mononuclear cells derived from breast cancer were used.

[0084] 1. Blood sample collection: 5 ml of peripheral venous blood from breast cancer patients was collected clinically in a heparin anticoagulant tube, diluted with an equal volume of PBS and mixed thoroughly, and the mononuclear cells in the peripheral blood were separated using the Ficoll layering method. The cell pellets were frozen in a -80°C refrigerator, and each cryopreserved tube was numbered according to the numbering rules (patient information, batch, date).

[0085] 2. Extract monocytes: Mix the diluted blood with Ficoll separation solution, centrifuge at 400×g for 30 minutes at room temperature, and you can see obvious stratification; carefully remove the monocytes (white film layer) with a pipette, resuspend them in PBS, and centrifuge at 250g for 5 minutes to wash the cells. Discard the supernatant, resuspend the cells in PBS, and count the cell concentration.

[46] .

[0086] 3. CCK8 method to analyze cell activity: Human breast cancer-derived monocytes were mixed with human hippocampal neurons (HPPNCs) or AD model cells overexpressing the key pathogenic gene APOE4 of sporadic AD (HPPNCs APOE4 ) were inoculated in 96-well plates according to different ratios (1:1, 1:5, 1:10, 1:20, 1:50, 1:100), 3000 cells were inoculated in each well, 3 duplicate wells were set up for each group, and an equal volume of PBS solution was added to the edge to prevent evaporation, and then co-cultured in a constant temperature incubator (37°C, 5% CO2). After 24 hours, 10 μL of CCK8 detection reagent was added to each well, placed in the incubator for 2 hours, and the culture plate was taken out, protected from light, and the OD value of each group of cells was detected at a wavelength of 450nm on an ELISA reader, and the values ​​were recorded for statistics.

[0087] 4. Neuronal morphology detection and neurite analysis: Human breast cancer-derived monocytes were mixed with HPPNCs or HPPNCs APOE4According to different ratios (1:20, 1:50, 1:100), they were inoculated in 24-well plates, 10,000 cells were inoculated in each well, and 3 replicates were set up in each group. Co-culture was carried out in a constant temperature incubator (37°C, 5% CO2). After 24 hours, the cells in the logarithmic growth phase and in good condition were placed on an OLYMPUS inverted microscope, and the magnification and focal length were adjusted to ensure that the cells under the microscope were clearly visible, and then the cell morphology was recorded using cellSens imaging software. Cell processes are extensions of the neuronal cell body, which participate in the process of information transmission and play an important role in the normal function of neurons. Therefore, the length and number of neurites were calculated using the NeuronJ plug-in of ImageJ software.

[0088] 5. Cell Slides and Immunofluorescence Staining: Using donepezil (Dnp, 0.5 μM), a first-line drug for AD treatment, as a positive control, human breast cancer-derived monocytes were cultured with HPPNCs or HPPNCs APOE4 After co-culture, cell climbing experiments were performed. After 24 hours of culture, PSD-95 and NeuN were immunofluorescently stained.

[0089] (1) Cell culture: ① After trypsin digestion of neural cells, count and resuspend the cells in complete medium and adjust the cell density to 2×10 4 cells / ml. ②Wash the slide: Wash the 14mm diameter slide in 75% ethanol and serum-free basal medium in turn, carefully pick it up with tweezers and place it in a 24-well plate. ③Wash the slide twice: Add serum-free basal culture medium to the position where the slide is placed. ④Add complete culture medium: Add 0.5mL of complete cell culture medium to each well of the 24-well plate, and then, by applying the tension of the culture medium, make the glass slide and the culture dish adhere tightly together. ⑤Evenly drop the cell suspension into the 24-well plate, shake it well and observe it under the microscope, and then place it in the incubator for overnight culture. ⑥Once the cells are attached, add culture medium containing human breast cancer-derived monocytes or 0.5μM Dnp, shake it well and observe it under the microscope, and then place it in the incubator for 24h. ⑦Fix: Discard the supernatant with a pipette, add 0.5mL of 4% paraformaldehyde to each well for 20min, discard the supernatant, and add 0.5mL of PBS solution to each well.

[0090] (2) Immunofluorescence detection: ① Cell permeabilization: Slightly shake the stem cell slide, then use a histochemical pen to draw a circle on the coverslip, the position should be in the center area where the cells are evenly distributed. Next, add 50μL of permeabilization working solution and incubate at room temperature for 20 minutes, then wash with PBS 3 times for 5 minutes each time. ② Serum blocking: 3% BSA is evenly coated on the slide and blocked at room temperature for 30 minutes. ③ Add a mixed reagent of NeuN and PSD-95 primary antibodies: Mix the two primary antibodies from different sources, add the prepared primary antibodies to the cell well plate, and incubate at 4℃ in a wet box overnight. If it is a single label, skip step ④. ④ Add secondary antibody: Place the cell well plate on a decolorization shaker and shake and wash 3 times, 5 minutes each time. Add HRP-labeled goat anti-rabbit IgG and incubate at room temperature for 50 minutes. ⑤ DAPI counterstaining of cell nuclei: Place the slide in PBS solution and shake and wash 3 times on a decolorization shaker, 5 minutes each time. After the slide is slightly dry, add DAPI dye solution and incubate at room temperature in the dark for 10 minutes. ⑥ Seal the slide: Put the slide into PBS solution and shake it on a decolorizing shaker for 3 times, 5 minutes each time. After drying slightly, use anti-fluorescence quenching sealant to seal the slide. ⑦ Collect cell images under a fluorescence microscope: DAPI channel cell nucleus is blue, 488 channel positive is green, and CY3 channel positive is red. The excitation wavelength of DAPI is 330-380nm, and the emission wavelength is 420nm; the excitation wavelength of 488 is 465nm-495nm, and the emission wavelength is 515-555nm; the emission wavelength of CY3 is 590nm, and the excitation wavelength is 510-560nm. ⑧ Use ImageJ software to calculate the cell immunofluorescence intensity and record the data.

[0091] 6. Data statistics and drawing: All data were statistically analyzed using SPSS 26.0, and the significance of differences was compared by variance analysis. The test results were considered statistically significant when P < 0.05. Statistical graphs were drawn in GraphPad prism 8 software.

[0092] The results showed that when human breast cancer-derived monocytes were combined with HPPNCs or HPPNCs APOE4 When the ratio is lower than 1:20, it will not affect cell activity ( Figure 3 A). When the co-culture ratio of the two was 1:20, the number of HPPNCs protrusions increased significantly, and the reduction in the number of neuronal protrusions caused by APOE4 overexpression was reversed; at the same time, HPPNCs APOE4 The shortening of neurites was significantly inhibited ( Figure 3 B).

[0093] Immunofluorescence analysis showed that HPPNCs APOE4The fluorescence of NeuN and PSD-95 in the culture medium was weakened; human breast cancer-derived monocytes were mixed with HPPNCs or HPPNCs APOE4 Co-culture at a ratio of 1:20 (hereinafter referred to as co-culture) did not affect the fluorescence intensity of NeuN and PSD-95 of HPPNCs, but also reversed the weakening of PSD-95 fluorescence of APOE4 and improved the HPPNCs to a certain extent. APOE4 The NeuN fluorescence weakened.

[0094] Compared with donepezil (Dnp, 0.5 μM), a first-line drug for AD treatment, human breast cancer-derived monocytes can significantly enhance the PSD-95 fluorescence intensity of both neuronal cells, while there is no significant difference in the NeuN fluorescence intensity between the two ( Figure 3 C) In conclusion, human breast cancer-derived monocytes can enhance the synaptic maturation of neuronal cells and improve neuronal damage to a certain extent. APOE4 The cell morphology Figure 3 C), cells treated with 0.5 μM Dnp were oval in shape, while the morphology of neurons co-cultured with human breast cancer-derived monocytes was closer to normal neurons.

[0095] The above results suggest that co-culture of human breast cancer-derived monocytes and neurons (ratio of 1:20) has no effect on normal neurons and can significantly improve the phenotype of AD model cells; compared with donepezil, the first-line AD drug, breast cancer-derived monocytes can also enhance neuronal maturity, that is, breast cancer-derived monocytes can be used for the prevention, treatment and research of AD.

[0096] Example 3: Effects of human breast cancer-derived monocytes on AD model mice

[0097] 1. Animal grouping: APP / PS1 double-transfected Alzheimer's disease model mice (APP / PS1) showed a large number of senile plaques at 12 months of age, which had a pathological phenotype similar to AD. Therefore, APP / PS1 mice were selected for tail vein injection of human breast cancer-derived mononuclear cells (concentration of 2×10 7 The cells were divided into: ① wild type (WT) + PBS group, ② wild type (WT) + human breast cancer derived monocytes group, and ③ APP / PS1 + human breast cancer derived monocytes group.

[0098] 2. A Y-maze novel arm exploration experiment was performed before and 7 days after the injection of human breast cancer-derived monocytes. The three exploration arms were randomly set as the starting arm, the novel arm, and the other arms.

[0099] (1) Experimental process: In the first stage of the experiment, the novel arm was closed with a partition, and the novel arm was closed with a baffle. The mouse was placed in the starting arm and allowed to explore freely for 5 minutes. After taking out the mouse for 2 hours, the mouse was placed in the second stage of the experiment. The novel arm and the starting arm were opened, and the mouse was allowed to explore freely for 5 minutes and any observations were recorded. Before starting the experiment with the next animal, the Y maze needs to be cleaned and the odor needs to be eliminated with 75% alcohol.

[0100] (2) Detection index: ① the number of times entering the starting arm, novel arm and other arms; ② the activity time in the starting arm, novel arm and other arms. The statistical and drawing methods are the same as those in Example 2.

[0101] 3. After completing the behavioral test, the mice were immediately killed by intraperitoneal injection of 1% sodium pentobarbital solution, and the brain, heart, liver, and kidney specimens were taken to observe with the naked eye whether there were cancer metastases.

[0102] The results showed that after the tail vein injection of human breast cancer-derived monocytes, the number of times APP / PS1 mice and wild-type mice entered the novel arm, the time spent in the novel arm, and the proportion of the distance traveled were significantly increased ( Figure 4 ). That is, tail vein injection of breast cancer-derived monocytes has the purpose of improving the cognitive level of mice, and breast cancer-derived monocytes can be used for the prevention, treatment and research of AD.

[0103] In addition to behavioral changes, the mice did not show obvious abnormal immune responses, and no obvious tumor nodules were found in the main organs of the mice ( Figure 5 ), that is, xenogeneic breast cancer-derived monocytes therapy may not cause abnormal immune responses and tumors.

[0104] Example 4: Effects of mononuclear cells derived from breast cancer of the same species on AD model mice

[0105] 1. Construction of breast cancer model: 7-week-old C57BL / 6 mice were injected with E0771 cells (concentration of 1×10 7 The breast cancer model (BC) was constructed by PCR with 100 μL / mouse. At the same time, 7-week-old normal mice were selected as the control group (N). After 3 weeks of feeding in an SPF environment, the spleen was removed by dissection and the expression of proteins was analyzed using EasySep TM The Mouse Monocyte Isolation Kit (STEMCELL, Cat. No. 19861) was used to separate monocytes into breast cancer-derived monocytes (BC mon) and normal-derived monocytes (Nmon). The cells were prepared with PBS at 1×10 7 / mL of cell suspension.

[0106] 2. Animal grouping: Aging is a key factor in the pathogenesis of AD. Neuronal degeneration, behavioral and memory impairment in aged mice are similar to those in clinical patients. Therefore, naturally aged C57BL / 6 mice aged 11-20 months were selected for tail vein injection of mononuclear cells from the same species of mice (concentration of 1×10 7 / mL, 200μL / mouse). According to the behavioral performance of mice before treatment, they were divided into: ① normal control group (NC), ② normal-breast cancer model monocyte treatment group (NC-BC mon), ③ dementia control group (AD-control), ④ dementia-normal mouse monocyte treatment group (AD-N mon), ⑤ dementia-breast cancer model monocyte treatment group (AD-BC mon), ⑤ dementia-donepezil treatment group (Donepezil).

[0107] 2. Water maze test was performed before, 1 week and 4 weeks after injection of the same type of monocytes.

[0108] (1) Experimental apparatus and environment: The water maze experiment is an experiment that uses the animal's natural aversion to water to force the animal to find a hidden platform in the water on which it can stand. It is used to determine the animal's memory ability for spatial position and direction. The water maze consists of a pool with a diameter of 100 cm and a height of 38 cm, a platform with a diameter of 6 cm and a height of 14 cm, and an automatic image acquisition system. The parameters are set using a computer processing system. The center of the pool is divided into four quadrants of equal area. The quadrants are marked as the first to fourth quadrants in a counterclockwise direction, and the platform is located in the third quadrant. The height of the platform is adjusted to 1 cm below the water surface. In order to avoid light interference with the camera's image collection, blackout cloth is hung around the water maze to block it. The water temperature is maintained at 22-25°C, and the environment is kept quiet during the experiment.

[0109] (2) Hidden platform training: This stage requires continuous training for 5 days, 4 times a day. Set the monitoring time on the computer analysis system to 60 seconds, and put the animal's head into the water in quadrants 1, 2, 3, and 4 with its back facing the pool wall. If the mouse has not climbed onto the platform for more than 60 seconds, guide the mouse to the platform and stay there for 10 seconds. When the mouse finds the platform after entering the water and stays there for more than 2 seconds, it is the escape latency. After the training, wipe the mouse dry and put it back into the cage, record the escape latency of all mice for 5 days, and calculate the average escape latency of each mouse every day. The statistical and drawing methods are the same as in Example 2.

[0110] (3) Spatial exploration experiment: On the 6th day, the platform was removed and the mice were first placed in the water from the first quadrant (the opposite side of the platform) and allowed to freely explore the water for 60 seconds. The activity time of the animals in the third quadrant (the quadrant where the platform was placed, i.e., the target quadrant) and the number of times they crossed the platform quadrant were recorded. The statistical and drawing methods were the same as those in Example 2.

[0111] 3. After the treatment, the mice were raised for 2 months and then killed by intraperitoneal injection of 1% sodium pentobarbital solution. The brain, heart, lung, liver and kidney specimens were taken and observed with the naked eye for the presence of cancer metastases.

[0112] The results showed that after 4 weeks of treatment ( Figure 6 A), the movement distance of the AD-BC mon treatment group in the target quadrant of the water maze was significantly increased compared with the AD-control group and the AD-N mon group, and the residence time in the target quadrant was also improved to a certain extent, and there was no difference between the AD-BC mon treatment group and the Donepezil treatment group. Comparison of behavioral changes in mice in each group before and after 1 week of treatment ( Figure 6 B), compared with the AD-N mon group and the Donepezil group, the AD-BC mon group stayed in the target quadrant for a longer distance and a longer time, and the difference was more significant (P<0.001), suggesting that breast cancer-derived monocytes may be more effective in treating AD. Comparison of the changes in the water maze test of each group of mice within 1 month ( Figure 6 B), the time and distance that the NC group and the AD-control group stayed in the target quadrant did not differ significantly before and after; while the injection of breast cancer-derived monocytes had no effect on the behavioral performance of normal mice (NC-BC mon group); interestingly, the time and distance that the mice with dementia phenotype stayed in the target quadrant were prolonged after injection of monocytes from normal mice (AD-N mon group), monocytes from breast cancer model mice (AD-BC mon group) and continuous intraperitoneal gavage of donepezil (Donepezil group) for 1 month; but the improvement of the behavioral phenotype of mice in the AD-N mon group only lasted for 1 week, while the improvement of the behavioral phenotype in the AD-BC mon group and the Donepezil group lasted for 1 month, which was more stable. That is, the tail vein injection of breast cancer-derived monocytes of the same species has the function of improving the cognitive level of mice, and the effect is better than that of monocytes from normal mice, and is equivalent to that of donepezil. At the same time, donepezil requires continuous gavage to maintain the therapeutic effect, while the tail vein injection of breast cancer-derived monocytes only requires 1 time to maintain the therapeutic effect. That is, breast cancer-derived monocytes can be used for the prevention, treatment and research of AD, and are more convenient.

[0113] Two months after treatment, the mice were observed. The naturally aged mice injected with the same type of mouse breast cancer-derived monocytes showed no obvious abnormal immune response except behavioral changes. Dissection of the mice revealed no obvious tumor nodules in their main organs ( Figure 7 ), indicating that tail vein injection of monocytes derived from breast cancer of the same species may not cause abnormal immune responses and tumors.

Claims

1. The use of tumor-derived monocytes in the preparation of anti-Alzheimer's disease drugs, characterized in that: The tumor is breast cancer.

2. The use according to claim 1, characterized in that: The tumor-derived monocytes are used in the preparation of drugs for improving neuronal function of Alzheimer's disease, cognitive impairment and cognitive function.

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