Fibroblast-like macrophage and application thereof in tumor metastasis prediction and prognosis evaluation

Abstract

The invention belongs to the technical field of biology, and particularly relates to fibroblast-like macrophages and application thereof in tumor metastasis prediction and prognosis evaluation. According to the invention, tumor focuses are subjected to single cell sequencing, and a cell subset of co-expression collagen genes and macrophage-related genes is found to be remarkably enriched in the tumor focuses of various tumors, and is named as collagen-rich macrophages / fibroblast-like macrophages. The collagen-rich macrophage / fibroblast-like macrophage provided by the invention can be detected in tumor focuses and peripheral blood, can be used as a marker to be applied to prediction, diagnosis and prognosis of various tumors, especially osteosarcoma, and is helpful for timely judging whether tumor cells have chemotherapy drug resistance or not; therefore, a proper treatment strategy can be timely adjusted and given, invalid or low-effect chemotherapy is avoided, other effective treatment strategies are timely adopted, and the lifetime of a patient is prolonged.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a fibroblast-like macrophage and its application in tumor metastasis prediction and prognosis evaluation. Background Art

[0002] Tumors are a type of disease caused by abnormal proliferation of cells in the body. There are many types, including benign tumors and malignant tumors (i.e., cancer). Malignant tumors pose a serious threat to patients' lives. Most patients with early-stage malignant tumors can undergo surgical resection of tumor tissues. However, due to the heterogeneity of cancer and individual differences, local recurrence or metastasis is prone to occur after tumor tissue resection. Some patients will also experience in situ recurrence, ectopic metastasis, and chemotherapy resistance after surgery. Therefore, timely judgment of the condition of patients with malignant tumors and understanding of the patient's prognosis and survival will help to adopt personalized treatment plans for different patients. In addition, patients with recurrence, metastasis, and patients with advanced tumors often need to receive chemotherapy, radiotherapy, molecular targeted therapy, and immunotherapy. Among them, chemotherapy is one of the traditional tumor treatment methods, mainly including targeted therapy with clear targets and conventional chemotherapy with unclear targets. However, during chemotherapy, many patients will develop chemotherapy resistance, which affects the treatment effect of malignant tumors. In addition, as the medication cycle increases, tumor cells will gradually develop resistance to chemotherapy drugs, making the originally effective treatment plan ineffective, leading to tumor recurrence or progression.

[0003] Osteosarcoma is the most common primary malignant tumor of bone. Most patients develop the disease between the ages of 10 and 30, and it is prone to occur in the distal femur, proximal tibia, and proximal humerus. Osteosarcoma patients have a short course of disease and rapid progression. Local skip lesions may occur and invade adjacent soft tissues. Osteosarcoma is prone to metastasize to the lungs through the bloodstream and to other bones in the late stage of the disease. At present, osteosarcoma adopts a comprehensive treatment strategy of preoperative chemotherapy-surgery-postoperative chemotherapy. After regular surgical resection and neoadjuvant chemotherapy, the limb-saving surgery rate of patients with limb osteosarcoma can be increased to 90%-95%, and the 5-year survival rate can reach 60%-80%. However, the incidence of postoperative lung metastasis of osteosarcoma can reach about 30%, especially in patients who are not sensitive to preoperative chemotherapy, and their recurrence and metastasis rates are higher. In addition, osteosarcoma cases are prone to resistance to intensive chemotherapy, leading to severe toxicity and increased incidence of neutropenia, infectious complications, and thrombocytopenia. At present, there is no effective and rapid method for early prediction of chemotherapy resistance in cancer patients, especially osteosarcoma patients, at home and abroad.

[0004] Whether tumor cells are sensitive to chemotherapy drugs depends not only on whether their genes mutate, but also on the surrounding environment to a great extent. In addition, the drug-resistant mutations of tumor cells themselves will also be affected to a certain extent by the surrounding environment. The tumor microenvironment (TME) refers to the internal and external environment in which the occurrence, growth and metastasis of tumors are closely related to tumor cells. In addition to tumor cells, the tumor microenvironment also contains various types of cells such as immune cells and fibroblasts, as well as various extracellular components such as growth factors. The tumor microenvironment is affected by the various types of cells and various extracellular components it contains, and can help these cells and extracellular components promote the occurrence, progression and metastasis of tumors. In addition, the tumor microenvironment may also play an important role in the formation of chemotherapy resistance.

[0005] In addition, after receiving standardized treatment, osteosarcoma patients need to undergo regular lung CT and whole-body bone scans to monitor tumor recurrence and metastasis. Patients cannot undergo the above examinations frequently, and the long-term mortality risk cannot be effectively assessed when the patient visits the doctor. Therefore, the development of a marker that can be used to detect chemotherapy resistance of malignant tumors, especially osteosarcoma, and predict its prognosis and survival when the patient visits the doctor has important clinical significance and practical value. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a fibroblast-like macrophage subset and its application in tumor metastasis prediction and prognosis assessment.

[0007] The above-mentioned object of the present invention is achieved through the following technical scheme: use of a fibroblast-like macrophage subpopulation as a biomarker in the preparation of products for diagnosing tumors, predicting tumor metastasis and / or prognosis.

[0008] Compared with the prior art, the collagen-rich macrophages / fibroblast-like macrophages provided by the present invention are significantly enriched in a variety of tumor types, especially in osteosarcoma, and have the potential to be used as a poor prognosis indicator for tumors. In addition, collagen-rich macrophages / fibroblast-like macrophages can be detected in tumor lesions and peripheral blood, and collagen-rich macrophages / fibroblast-like macrophages in peripheral blood have excellent predictive performance. Therefore, collagen-rich macrophages / fibroblast-like macrophages can be used as a marker for the prediction, diagnosis, and prognosis of tumors, especially osteosarcoma, which helps to timely determine whether tumor cells have chemotherapy resistance, so as to timely adjust and give appropriate treatment strategies, avoid ineffective or low-effect chemotherapy, and promptly adopt other effective treatment strategies to prolong patient survival.

[0009] Furthermore, the fibroblast-like macrophages express macrophage-related genes and collagen genes, the macrophage-related genes include at least one of CD14, CD45, CD11B, CD163, CD68, and MARCO, and the collagen genes include at least one of COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91.

[0010] Furthermore, the tumor is from any one of esophageal cancer, nasopharyngeal carcinoma, synovial sarcoma, pancreatic cancer, ependymoma, gastric cancer, Ewing sarcoma, colorectal cancer, liver cancer, malignant peripheral nerve sheath tumor, serous ovarian cancer, neurofibroma, gastrointestinal stromal tumor, gastrointestinal neuroendocrine cancer, prostate cancer, rhabdomyosarcoma, lung cancer, T-cell lymphoma, squamous cell carcinoma, breast cancer, osteosarcoma, etc.

[0011] Furthermore, the fibroblast-like macrophage subpopulation is derived from peripheral blood or tumor tissue.

[0012] Furthermore, the products include reagents, chips, and test kits.

[0013] Furthermore, the subject with high expression of the fibroblast-like macrophage subpopulation is prone to or has already developed cancer metastasis, or has a poor prognosis, or has already suffered from cancer.

[0014] The present invention also provides a use of a fibroblast-like macrophage detection reagent in preparing a product for diagnosing osteosarcoma, predicting the risk of osteosarcoma lung metastasis, osteosarcoma chemotherapy resistance and / or osteosarcoma survival prognosis.

[0015] Furthermore, the fibroblast-like macrophages express macrophage-related genes and collagen genes, the macrophage-related genes include at least one of CD14, CD45, CD11B, CD163, CD68, and MARCO, and the collagen genes include at least one of COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91.

[0016] Furthermore, the detection method of the fibroblast-like macrophage subpopulation is selected from one or more of the following detection technologies or methods: single-cell sequencing technology, flow cytometry staining, and immunofluorescence staining.

[0017] Furthermore, the proportion of the fibroblast-like macrophage subpopulation in peripheral blood mononuclear cells or total myeloid cells in tumor tissue is higher than 10%, indicating that the tumor patient is prone to or has already developed cancer metastasis, or has a poor prognosis, or has already developed cancer.

[0018] The present invention also provides a reagent for detecting the expression products of macrophage-related genes and collagen genes for use in preparing products for diagnosing osteosarcoma, predicting the risk of osteosarcoma lung metastasis, osteosarcoma chemotherapy resistance and / or osteosarcoma prognosis assessment.

[0019] Furthermore, the macrophage-related genes include at least one of CD14, CD45, CD11B, CD163, CD68, and MARCO, and the collagen genes include at least one of COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the cell dimensionality reduction information distribution map and gene expression distribution map of the pan-cancer single-cell sequencing in Example 1.

[0021] Figure 2A This is UMAP and identification of cell subpopulations from single-cell sequencing of osteosarcoma clinical samples in Example 2.

[0022] Figure 2B The cell ratios of collagen-rich macrophages / fibroblast-like macrophages in different lesions.

[0023] Figure 2C The expression of collagen genes and macrophage-related genes in collagen-rich macrophages / fibroblast-like macrophages.

[0024] Figure 3A This is the flow cytometric staining result of the osteosarcoma clinical sample in Example 3 of the present invention.

[0025] Figure 3B The cell ratios of collagen-rich macrophages / fibroblast-like macrophages in different lesions.

[0026] Figure 4 This is the multispectral (TSA) detection result of the osteosarcoma clinical sample in Example 3.

[0027] Figure 5A These are the prognostic analysis results of the osteosarcoma clinical samples in Example 5.

[0028] Figure 5B This is the prognostic analysis result of the osteosarcoma public data in Example 5.

[0029] Figure 6 This is the peripheral blood single cell data of the osteosarcoma patient in Example 6.

[0030] Fig. 7AThe results of probe in situ hybridization of peripheral blood of tumor patients in Example 7, wherein a is a peripheral blood sample of a primary drug-resistant patient; b is a peripheral blood sample of a primary chemotherapy-sensitive patient; and c is a peripheral blood sample of a patient with lung metastasis.

[0031] Figure 7B for Fig. 7A Statistical results.

[0032] Figure 7C This is the predicted performance of collagen-rich macrophages / fibroblast-like macrophages in Example 7.

[0033] Fig.7D This is the effect of the number of collagen-rich macrophages / fibroblast-like macrophages on the prediction performance in Example 7. DETAILED DESCRIPTION

[0034] In order to overcome the deficiencies of existing chemotherapy resistance detection and prognosis assessment of malignant tumors, the present invention aims to find a tumor marker for more convenient early warning of the long-term survival of patients with malignant tumors, so as to better guide the determination of tumor treatment plans and comprehensive detection of disease progression.

[0035] Based on this, the present invention is effective for a variety of cancers, such as esophageal carcinoma, nasopharyngeal carcinoma, synovial sarcoma, pancreatic cancer, ependymoma, gastric cancer, Ewing sarcoma, colorectal cancer, liver cancer, malignant peripheral nerve sheath tumor, serous ovarian cancer, neurofibromas, gastrointestinal stromal tumor, gastrointestinal neuroendocrine cancer, prostate cancer, rhabdomyosarcoma, lung cancer, T cell lymphoma, squamous cell carcinoma, breast cancer, We performed single-cell sequencing analysis on tumor lesions of mitochondria, carcinoma, and osteosarcoma, and then used the Seurat software package to perform dimensionality reduction clustering on the single-cell sequencing data of various tumors to screen for cell subpopulations that were significantly enriched in each tumor lesion, as well as tumors with a high level of enrichment of this cell subpopulation.

[0036] Furthermore, taking tumors with a high enrichment level of this cell subpopulation as an example, the cell proportion of this cell subpopulation in the chemotherapy-resistant lesions, chemotherapy-sensitive lesions, and lung metastatic lesions of the tumor, as well as the cell quantity in the peripheral blood of tumor patients, were detected to explore the role of this cell subpopulation in tumor chemotherapy resistance detection, tumor diagnosis, and prognosis.

[0037] The present invention will be further described in detail below in conjunction with specific examples, which are only used to illustrate the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0038] The term "pan-cancer" refers to cancer types in which cellular telomerase is abnormally elevated. This type of cancer accounts for more than 90% of human cancer types. Most of them originate from the carcinogenesis of cells in epithelial tissues, and also include sarcomas, blastomas, etc.

[0039] The term "single-cell sequencing" refers to a new technology for amplifying and sequencing the whole transcriptome at the single-cell level, and its principle is to amplify the trace whole transcriptome RNA of the isolated single cell and then perform high-throughput sequencing. In the present invention, any single-cell transcriptome sequencing technology that has been disclosed can be used for single-cell sequencing.

[0040] The term "multispectral imaging technology" refers to an imaging method that uses multiple monochromatic light sources (different wavelengths) to project onto the target object, and collects images of reflected or transmitted monochromatic light of different wavelengths based on the differences in spectral absorption of different tissues and substances. This technology is widely used in many fields, including medicine (such as fundus examination), remote sensing, agriculture, geological exploration, etc. In the medical field, multispectral imaging technology can provide detailed images of different depths of the fundus, which is helpful for early diagnosis and treatment of diseases.

[0041] The term "tyramide signal amplification (TSA) technology" is a staining technique based on enzymatic detection methods, mainly used in multiplex fluorescent immunohistochemistry. This technique uses horseradish peroxidase (HRP) to perform high-density in situ labeling of target proteins or nucleic acids. 2 O 2 Under the environment, the fluorescently labeled tyramide molecules are activated by HRP catalysis, resulting in a large number of enzymatic reactions, which deposit the fluorescent pigment at the site of close binding with the antigen, thereby achieving significant signal amplification. This technology allows the simultaneous detection of multiple target points on the same tissue section, and comprehensively studies the cell composition, cell function and cell-to-cell interactions.

[0042] The term "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single-chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

[0043] The term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probe can be directly or indirectly labeled, including primers. Hybridization methods include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

[0044] Primer or probe of the present invention can use phosphoramidite solid phase support method or other well-known method chemical synthesis.Also can use many means known in the art to modify the nucleotide sequence.Non-limiting examples of these modifications are methylation, capping, displacement and modification between nucleotides with one or more analogs of natural nucleotides, for example, modification of uncharged linker (for example, methyl phosphate, phosphotriester, phosphoramidite, carbamate etc.), or modification of charged linker (for example, phosphorothioate, phosphorodithioate etc.).

[0045] CIBERSORT deconvolution algorithm: Developed by Stanford University, this algorithm directly infers and quantifies the composition and proportion of 22 infiltrating immune cells in tumor tissues based on gene expression data. It can simultaneously determine 22 immune cell types, overcoming the tediousness of conventional immunohistochemistry and immunofluorescence experiments and the difficulty of detecting a small number of markers. It also overcomes the disadvantages of clinical specimens being scarce and difficult to obtain, and being unable to meet the needs of multi-omics experiments. The 22 immune types include naive B cells, memory B cells, plasma cells, CD8+T cells, naive CD4+T cells, resting memory CD4+T cells, activated memory CD4+T cells, follicular helper T cells, regulatory T cells, gamma delta T cells, resting NK cells, activated NK cells, monocytes, M0 macrophages, M1 macrophages, M2 macrophages, resting dendritic cells, activated dendritic cells, and resting mast cells. resting), activated mast cells (Mast cellsactivated), eosinophils (Eosinophils) and neutrophils (Neutrophils).

[0046] MARCO refers to macrophage receptor for collagen structure; DCN refers to decorin; COL1A1 refers to type I collagen alpha 1; COL1A2 refers to type I collagen alpha 2; COL3A1 refers to type III collagen alpha 1; LUM refers to basement membrane polysaccharide; THY1 refers to thymocyte differentiation antigen 1.

[0047] Example 1: Pan-cancer single-cell sequencing analysis

[0048] Single-cell sequencing was performed on tumor lesions of esophageal cancer, nasopharyngeal carcinoma, synovial sarcoma, pancreatic cancer, ependymoma, gastric cancer, Ewing sarcoma, colorectal cancer, liver cancer, malignant peripheral nerve sheath tumor, serous ovarian cancer, neurofibroma, gastrointestinal stromal tumor, gastrointestinal neuroendocrine carcinoma, prostate cancer, rhabdomyosarcoma, lung cancer, T-cell lymphoma, squamous cell carcinoma, breast cancer, and osteosarcoma, and the obtained single-cell sequencing analysis data were analyzed using the Seurat software package.

[0049] To exclude low-quality cells, cells with less than 200 detected genes and mitochondrial gene counts exceeding 15% were deleted. For each tumor dataset, the filtered expression matrix was normalized by the Seurat software package, and the cells were divided into multiple clusters, i.e., cell clusters. The gene expression obtained by cell clustering was then visualized using the unified manifold approximation projection (UMAP).

[0050] See also Figure 1 In the pan-cancer single-cell sequencing analysis, it was found that a special type of macrophage subpopulation was significantly enriched in tumor lesions. This subpopulation has the potential to serve as an indicator of poor tumor prognosis. Further analysis found that this subpopulation expressed both macrophage markers, such as CD14, CD45, CD11B, CD163, CD68, and MARCO, and collagen markers, such as COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91. Therefore, this subpopulation was named: Fibroblast like macrophage, FLMs, or fibroblast-rich macrophage (Collagen-rich macrophage, CRMs). In addition, Figure 1 It shows that the proportion of collagen-rich macrophages / fibroblast-like macrophages in osteosarcoma tumor lesions is higher than that in most groups of tumors. Therefore, subsequent studies will use osteosarcoma as an example to further analyze the application of collagen macrophages / fibroblast-like macrophages in tumor prognosis and chemotherapy resistance detection.

[0051] Example 2: Single-cell sequencing analysis of osteosarcoma clinical samples

[0052] In this study, 10 cases of chemotherapy-resistant lesions (tumor necrosis rate <90% and recurrence or metastasis within six months after surgery), 10 cases of pulmonary metastatic lesions, and 10 cases of chemotherapy-sensitive lesions (tumor necrosis rate >90% and no recurrence or metastasis within 2 years of follow-up after surgery) were strictly matched in the osteosarcoma tissue sample cohort of the Department of Orthopedic Oncology of the First Affiliated Hospital of Sun Yat-sen University. Single-cell sequencing (10X Genomics) was performed on 10 cases in each group. The Seurat software package was used to perform dimensionality reduction clustering on the single-cell sequencing data to obtain a visual UMAP, and the genes specifically expressed for each cell subpopulation were calculated.

[0053] See also Figure 2A-2C , collagen-rich macrophages / fibroblast-like macrophages are significantly enriched in chemotherapy-resistant lesions and lung metastatic lesions, and the proportion of collagen-rich macrophages / fibroblast-like macrophages in the total myeloid cells in tumor tissue is about 15% to 35%, while the proportion of collagen-rich macrophages / fibroblast-like macrophages in the total myeloid cells in tumor tissue is significantly reduced in chemotherapy-sensitive lesions, and their proportion in the total myeloid cells in tumor tissue is <3%. Therefore, collagen-rich macrophages / fibroblast-like macrophages have the potential to be used for osteosarcoma chemotherapy resistance detection, lung metastasis prediction, and prognosis evaluation.

[0054] Example 3: Flow cytometric staining analysis in osteosarcoma clinical samples

[0055] Methods: In this study, fresh tumor tissues from patients undergoing osteosarcoma surgery were collected from the Department of Orthopedics at the First Affiliated Hospital of Sun Yat-sen University: 20 cases of chemotherapy-resistant lesions (tumor necrosis rate <90%) and 20 cases of chemotherapy-sensitive lesions (tumor necrosis rate >90%). Tumor tissues were dissociated using the Miltenyi tumor tissue dissociation kit, and single cells from the dissociated tissues were collected for live and dead cell staining (Zombie) and antibody (CD45, CD14, CD11B, COL1A1, CD91) staining and incubation, and detected by flow cytometry.

[0056] See also Figure 3A and 3B Flow cytometric staining results showed that collagen-rich macrophages / fibroblast-like macrophages (Zombie-&CD45+&CD14+&CD11B&COL1A1+&CD91+) were significantly enriched in chemotherapy-resistant lesions, but significantly reduced in chemotherapy-sensitive lesions. This further proves that collagen-rich macrophages / fibroblast-like macrophages have the potential to be used for osteosarcoma chemotherapy resistance detection and prognosis assessment.

[0057] Example 4: Multispectral (TSA) analysis in osteosarcoma clinical samples

[0058] The fresh tumor tissue in Example 3 was used to prepare paraffin tissue sections, and the sections were subjected to multicolor fluorescent immunohistochemistry staining (MARCO, COL1A1) and tyramide signal amplification. The sections were observed and images were collected under a multispectral imaging system, and the positive cells were counted. The differences in the expression of MARCO and COL1A1 between chemotherapy-resistant lesions and chemotherapy-sensitive lesions of osteosarcoma were analyzed.

[0059] See also Figure 4 , collagen-rich macrophages / fibroblast-like macrophages are significantly enriched in chemotherapy-resistant lesions, but significantly reduced in chemotherapy-sensitive lesions. Therefore, collagen-rich macrophages / fibroblast-like macrophages have the potential to be used for the detection of chemotherapy resistance and prognosis assessment in osteosarcoma.

[0060] Example 5: Prognostic analysis in osteosarcoma clinical samples

[0061] To clarify the clinical significance of collagen-rich macrophages / fibroblast-like macrophages in osteosarcoma, this study collected 75 osteosarcoma tissue RNA sequencing cohorts from the First Affiliated Hospital of Sun Yat-sen University and osteosarcoma public data (TARGETDATA, 83 cases), and used CIBERSORT deconvolution analysis technology to perform prognostic analysis on RNA transcriptome data of osteosarcoma tumor tissues.

[0062] See also Figure 5A and Figure 5B The higher the content of collagen-rich macrophages / fibroblast-like macrophages in osteosarcoma tissue, the faster the patient will develop lung metastasis and the lower the patient's progression-free survival (PFS).

[0063] Example 6: Single-cell sequencing analysis of peripheral blood samples from patients with osteosarcoma

[0064] Peripheral blood samples from three patients with osteosarcoma were collected for single-cell sequencing analysis, and the sequencing results were subjected to dimensionality reduction and clustering. Figure 6 , there are collagen-rich macrophages / fibroblast-like macrophages in peripheral blood samples. Therefore, the presence of collagen-rich macrophages / fibroblast-like macrophages in the peripheral blood of tumor patients has the potential to quickly, simply and effectively predict the prognosis of tumor patients.

[0065] Example 7: Fluorescence in situ hybridization analysis of tumor peripheral blood samples

[0066] 5 mL peripheral blood samples were collected from patients with osteosarcoma and synovial sarcoma and filtered with a nanomembrane. Then, multiple sets of nucleic acid probes were used for in situ hybridization (DAPI, CD163, COL1A2, COL3A1) to label and detect cells in the peripheral blood, and predictive performance analysis was performed.

[0067] See also Figures 7A-7D, CD163+&COL1A2+&COL3A1+&THY1+ macrophages (collagen-rich macrophages / fibroblast-like macrophages) exist in the peripheral blood of tumor patients. The number of collagen-rich macrophages / fibroblast-like macrophages in the peripheral blood samples of chemotherapy-resistant patients and patients with lung metastasis is significantly higher than that of chemotherapy-sensitive patients. The results of the prediction performance analysis showed that the prediction performance (AUC) of collagen-rich macrophages / fibroblast-like macrophages was 0.794. The prediction performance of collagen-rich macrophages / fibroblast-like macrophages in peripheral blood was comprehensively evaluated, and it was found that 4 collagen-rich macrophages / fibroblast-like macrophages / 5mL can well balance the sensitivity and specificity of the prediction.

[0068] In summary, compared with the prior art, the present invention has the following advantages:

[0069] 1. The collagen-rich macrophages / fibroblast-like macrophages provided by the present invention are significantly enriched in various tumor types, especially in osteosarcoma, so the collagen-rich macrophages / fibroblast-like macrophages have the potential to serve as an indicator of poor prognosis of tumors.

[0070] 2. In clinical samples of osteosarcoma, collagen-rich macrophages / fibroblast-like macrophages are significantly enriched in chemotherapy-resistant lesions and lung metastatic lesions, but significantly reduced in chemotherapy-sensitive lesions. This suggests that collagen-rich macrophages / fibroblast-like macrophages can be used as effective biomarkers for chemotherapy resistance in osteosarcoma, helping clinicians to make efficacy assessments earlier, timely change treatment plans for chemotherapy-resistant patients, and improve patient survival rates.

[0071] 3. The high content of collagen-rich macrophages / fibroblast-like macrophages is positively correlated with the risk of lung metastasis in osteosarcoma patients, indicating that collagen-rich macrophages / fibroblast-like macrophages can predict tumor prognosis and treatment effects earlier, providing clinicians with more sufficient decision-making basis and helping clinicians develop more reasonable treatment plans.

[0072] 4. The present invention detected collagen-rich macrophages / fibroblast-like macrophages in peripheral blood samples, and the number of collagen-rich macrophages / fibroblast-like macrophages in peripheral blood samples of chemotherapy-resistant patients and patients with lung metastasis was significantly higher than that of chemotherapy-sensitive patients. Comprehensive evaluation of the prediction performance of peripheral blood collagen-rich macrophages / fibroblast-like macrophages showed that 4 collagen-rich macrophages / fibroblast-like macrophages / 5mL can well balance the sensitivity and specificity of prediction.

[0073] 5. The present invention can collect only the patient's peripheral blood to detect tumor chemotherapy resistance, predict lung metastasis, and predict prognosis assessment. It has little trauma to the patient, high sensitivity, and is highly operable, stable, and repeatable.

[0074] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of a fibroblast-like macrophage subset as a biomarker in the preparation of products for diagnosing tumors, predicting tumor metastasis and / or prognosis.

2. The application according to claim 1, characterized in that: The fibroblast-like macrophages express macrophage-related genes and collagen genes, the macrophage-related genes include at least one of CD14, CD45, CD11B, CD163, CD68, and MARCO, and the collagen genes include at least one of COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91.

3. The application according to claim 2, characterized in that: The tumor is from any one of esophageal cancer, nasopharyngeal cancer, synovial sarcoma, pancreatic cancer, ependymoma, gastric cancer, Ewing sarcoma, colorectal cancer, liver cancer, malignant peripheral nerve sheath tumor, serous ovarian cancer, neurofibroma, gastrointestinal stromal tumor, gastrointestinal neuroendocrine cancer, prostate cancer, rhabdomyosarcoma, lung cancer, T-cell lymphoma, squamous cell carcinoma, breast cancer, and osteosarcoma.

4. The use according to claim 3, characterized in that: The fibroblast-like macrophages are derived from peripheral blood or tumor tissue.

5. The use according to claim 4, characterized in that: The subject with high expression of the fibroblast-like macrophages is prone to or has already developed cancer metastasis, or has a poor prognosis, or has already suffered from cancer.

6. Use of a fibroblast-like macrophage detection reagent in the preparation of a product for diagnosing osteosarcoma, predicting the risk of osteosarcoma lung metastasis, osteosarcoma chemotherapy resistance and / or osteosarcoma survival prognosis.

7. The use according to claim 6, characterized in that: The fibroblast-like macrophages express macrophage-related genes and collagen genes, the macrophage-related genes include at least one of CD14, CD45, CD11B, CD163, CD68, and MARCO, and the collagen genes include at least one of COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91.

8. The use according to claim 7, characterized in that: The high proportion of the fibroblast-like macrophage subpopulation in peripheral blood mononuclear cells or total myeloid cells in tumor tissue indicates that the tumor patient is prone to or has already developed cancer metastasis, or has a poor prognosis, or has already developed cancer.

9. Use of a reagent for detecting the expression products of macrophage-related genes and collagen genes in the preparation of products for diagnosing osteosarcoma, predicting the risk of osteosarcoma lung metastasis, osteosarcoma chemotherapy resistance and / or osteosarcoma prognosis assessment.

10. The use according to claim 9, characterized in that: The macrophage-related genes include at least one of CD14, CD45, CD11B, CD163, CD68, and MARCO, and the collagen genes include at least one of COL1A1, COL1A2, COL3A1, LUM, DCN, THY1, and CD91.

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