CN116819066A - Liver cancer microenvironment detection kit - Google Patents
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Abstract
本发明公开了一种肝癌微环境检测试剂盒，其包括如下成分：用于制备肝癌组织冰冻切片所需的表面活性剂、封闭剂、固定剂、包埋剂；CD68第一抗体，偶联有荧光染料一的荧光抗体一；PPT1第一抗体；偶联有荧光染料二的荧光抗体二，其中，荧光染料一和荧光染料二是不同颜色的荧光染料，荧光抗体一中的抗体与荧光抗体二中的抗体不相同。该试剂盒能够检测肝肿瘤组织内的PPT1表达水平和巨噬细胞浸润水平，反映肝细胞癌微环境免疫特征和术后总体生存风险，在临床实践中有助于指导肝癌患者免疫治疗决策制定，实施肝癌精准治疗。The invention discloses a liver cancer microenvironment detection kit, which includes the following components: surfactant, sealing agent, fixative, and embedding agent required for preparing frozen sections of liver cancer tissue; CD68 first antibody, coupled with Fluorescent antibody one of fluorescent dye one; PPT1 first antibody; fluorescent antibody two coupled with fluorescent dye two, wherein fluorescent dye one and fluorescent dye two are fluorescent dyes of different colors, and the antibody in fluorescent antibody one is the same as fluorescent antibody two. The antibodies in are not the same. This kit can detect the expression level of PPT1 and the level of macrophage infiltration in liver tumor tissue, reflecting the immune characteristics of the microenvironment of hepatocellular carcinoma and the overall survival risk after surgery. It can help guide the decision-making of immunotherapy for liver cancer patients in clinical practice. Implement precision treatment of liver cancer.
Description
Technical Field
The invention belongs to the field of clinical medical diagnostic tools, relates to a liver cancer microenvironment detection kit, and in particular relates to an immunofluorescence kit for detecting the expression level of CD68 and PPT1 in tumors and the infiltration level of macrophages.
Background
Liver cancer has a sixth rank in incidence among various cancers, and a third rank in mortality. Hepatocellular carcinoma is the most common histological type of primary liver cancer, accounting for about 80-90% of total cases. Although a few patients meet the criteria of radical surgery, most patients who have recurrent or metastatic disease after surgery require further treatment. Multiple kinase inhibitors as a first-line treatment regimen for advanced hepatocellular carcinoma only slightly extend the overall survival of the patient. The emergence of immunotherapy such as immune checkpoint blockers (PD-1) has changed the treatment pattern of liver cancer, but its objective remission rate is still less than 20%. In addition, prognostic evaluation systems such as BCLC staging and TNM staging have failed to meet the demands placed on accurate stratification of patients by new therapeutic strategies. Therefore, there is a need to identify effective means by which the prognosis risk and therapeutic effect of hepatocellular carcinoma can be predicted.
The deep knowledge of the complex cellular and molecular networks of the immune microenvironment of hepatocellular carcinoma tumors has helped to develop new anti-tumor therapeutic strategies based on immune responses. The functional balance between immune effector and immunosuppressive cells and the immune evasion capacity of tumor cells determine the response rate of immunotherapy. In hepatocellular carcinoma, resident Kupffer cells and macrophages migrating from the blood circulation are largely enriched in the tumor microenvironment. They may exert antitumor effects such as phagocytosis and antigen presentation, but are also readily induced by tumor cells into tumor-associated macrophages. The highly heterogeneous and plastic phenotype of macrophages can have a significant impact on surrounding cells, thereby affecting the balance between anti-tumor immunity and immune evasion in hepatocellular carcinoma. Therefore, in order to reverse the hepatocellular carcinoma immunosuppressive microenvironment, improve the immunotherapeutic effect and predict prognosis, it is desirable to better characterize the molecular landscape of tumor-associated macrophages in the hepatocellular carcinoma microenvironment and accurately identify key macrophage subpopulations. However, macrophage markers that can be used as stable prognosis classifiers or microenvironment signature revealers remain an unmet need.
Palmitoyl protein thioesterase 1 (PPT 1) is an enzyme that catalyzes the process of depalmitoylation, and has been reported to be up-regulated in a variety of cancers and is associated with poor prognosis, including hepatocellular carcinoma, oral squamous cell carcinoma, and esophageal carcinoma. Notably, PPT1 has been identified as a molecular target for antimalarial drugs such as DQ661 and DC661, and these PPT1 inhibitors can impair lysosomal catabolism by accumulation of palmitoylated proteins, thereby inhibiting tumor growth and sensitizing tumor cells to targeted and immunotherapeutic therapies. However, previous studies focused on the biological effects of PPT1 in tumor cells, whose expression and primary role in complex immune microenvironments of hepatocellular carcinoma, especially macrophages, remain to be explored further.
Disclosure of Invention
In the research of liver cancer tissues, based on numerous characterization information statistics of microenvironments, including expression level changes of proteins PPT1, CD45, CD8, CD68, CD4, PD-1 (CD 279) and the like, and state/content changes of T cells, B cells, macrophages, NK cells, plasma cells, neutrophils, eosinophils, DNT cells and the like, it is found that proteins such as PPT1 and the like cannot be independently used as biomarkers to reflect the liver cancer microenvironment conditions. However, it has also been unexpectedly found that the combination of the protein PPT1 and CD68, and further the combination of the protein PPT1 and macrophages, can accurately characterize the liver cancer microenvironment, and further evaluate the overall risk of survival of a liver cancer patient after surgery. Based on the research result, the invention comprises the following technical scheme.
A liver cancer microenvironment detection kit is an immunofluorescence kit and mainly comprises the following components:
the preparation method is used for preparing a surfactant, a blocking agent and a fixing agent required by frozen sections of liver cancer tissues;
CD68 primary antibody (also known as CD68 primary antibody);
a fluorescent antibody I coupled with a fluorescent dye I, wherein the fluorescent antibody I can be combined with a CD68 primary antibody-CD 68 conjugate, and corresponds to or is abbreviated as a CD68 secondary antibody;
PPT1 primary antibodies (also known as PPT1 primary antibodies),
a fluorescent antibody II coupled with a fluorescent dye II, wherein the fluorescent antibody II can be combined with a PPT1 primary antibody-PPT 1 conjugate, and is equivalent to or abbreviated as a PPT1 secondary antibody,
wherein the fluorescent dye I and the fluorescent dye II are fluorescent dyes with different colors, namely different fluorescent wavelengths; the antibodies in the fluorescent antibody I are different from the antibodies in the fluorescent antibody II.
For example, the antibodies in the first fluorescent antibody and the antibodies in the second fluorescent antibody are IgG. For example, the IgG antibody is selected from goat anti-mouse IgG, goat anti-rabbit IgG, rabbit anti-mouse IgG, and the like.
Preferably, the fluorescent antibody is Alexa Fluor 488 (Cat. Ab150113, abcam) goat anti-mouse IgG H&L(Alexa 488 (ab 150113); the second fluorescent antibody is CY3 (Cat.ab6939, abcam), namely goat anti-rabbit IgG H&LPre-adsorbing secondary antibody (ab 6939).
In a narrow sense, the liver cancer microenvironment refers to the CD68, PPT1 expression levels and macrophage infiltration levels within liver tissue. Clinical studies show that the PPT1 expression level and macrophage infiltration level can accurately reflect the micro-environmental immunity characteristics of the hepatocellular carcinoma and are used for predicting the overall risk of poor survival after operation.
One skilled in the art will readily appreciate that CD68 and PPT1 expression levels in liver cancer tissue samples refer to the protein content or concentration of CD68 and PPT 1.
In the present invention, CD68 and PPT1 biscationic cells may be represented as "ppt1+ macrophages", wherein the symbol "+" represents positive, expression, as will be readily understood by those skilled in the art. Similarly, cd8+ T cells represent positive T cells with CD8 expression, and so on.
Macrophage infiltration levels were generally a proportion of the number of cd68+ cells to the cd45+ immune cells in random 5 fields, with greater than 50% high infiltration.
Further, the liver cancer microenvironment detection kit also comprises the following components:
an embedding agent selected from OCT embedding agents or paraffin waxes for submerging liver cancer tissue samples;
EDTA antigen retrieval buffer solution, which is used for antigen retrieval before formalin fixation and paraffin embedding tissue section immunohistochemical staining, and increases the display effect of the immunohistochemical experiment;
an anti-fluorescence quenching caplet containing a nuclear dye (also known as a DNA dye) is used to caplet a slice after incubation with an antibody.
In one embodiment, the nuclear dye may be DAPI (4', 6-diamidino-2-phenylindole); the surfactant may be 0.5% Triton-X100 buffer; the blocking agent may be a BSA blocking solution, for example, a 5% BSA blocking buffer; the fixative is formalin, for example 4% paraformaldehyde.
The liver cancer microenvironment detection kit can implement liver cancer microenvironment detection through the following steps:
i. preparing frozen sections of liver cancer tissues;
incubating the frozen section with a first antibody of CD68 (first antibody of CD 68) and a first antibody of PPT1 (first antibody of PPT 1) overnight after the frozen section is penetrated by a surfactant and blocked by a blocking agent, and then incubating and blocking the frozen section with a first fluorescent antibody and a second fluorescent antibody;
determining the infiltration level of PPT1+ macrophages in liver cancer tissues according to the result of fluorescence co-localization of CD68 and PPT1, and dividing patients into a high PPT1+ macrophage infiltration group and a low PPT1+ macrophage infiltration group according to the infiltration level.
In one embodiment, the frozen sections described in step i may be prepared by: fresh tissue is frozen in liquid nitrogen after embedding with an embedding agent such as OCT, then frozen in a cryostat microtome and fixed with a fixing agent such as 4% paraformaldehyde.
In step iii, cell infiltration can be determined by flow cytometry in combination with TSNE dimension reduction maps.
In step iii, the method for assessing ppt1+ macrophage infiltration level may be: and (3) identifying cells with green fluorescence carried by a macrophage marker CD68 and red fluorescence carried by PPT1 overlapped (merge) as CD68 and PPT1 double-positive cells, namely PPT1+ macrophages, randomly selecting 5 fields in a tissue slice for cell counting, and obtaining the infiltration quantity of the PPT1+ macrophages.
Preferably, the above formula (mathematical model) for determining the level of ppt1+ macrophage infiltration is: f=the sum of the numbers of cd68+ppt1+ macrophages in 5 fields/the sum of the numbers of cd68+ cells in 5 fields, where F is the infiltration level, F < 0.5 is considered low ppt1+ macrophage infiltration, F > 0.5 is considered high ppt1+ macrophage infiltration.
In one embodiment, when liver cancer tissue has high ppt1+ macrophage infiltration, tumor tissue has more depleted PD-1+cd8+ t cell infiltration and indicates poor overall survival and poor prognosis after surgery. That is, high ppt1+ macrophage infiltration correlated positively with high infiltration of depleted cd8+ T cells.
Obviously, the detection result of the liver cancer microenvironment detection kit can be used for evaluating the overall risk of poor survival (prognosis risk) of a liver cancer patient after operation, and the overall survival time of a liver cancer patient with high PPT1+ macrophage infiltration of tumor tissues is obviously shorter than that of a liver cancer patient with low PPT1+ macrophage infiltration.
In a preferred embodiment, the liver cancer microenvironment detection kit further comprises a description of the use of the kit, wherein the description describes steps and a determination formula (mathematical model) for performing liver cancer microenvironment detection.
For example, the above description can be written on a bottle, test tube and the like, a board, or on a single piece of paper, or outside or inside the container, for example paper with an operating demonstration video APP download window such as a two-dimensional code, or in the form of multimedia such as a CD, a U-disc, a netdisk, etc.
The invention discovers for the first time that the expression level of CD68 and PPT1 and the infiltration level of macrophages can reflect the micro-environmental immunity characteristics of liver cancer tissues and the overall risk of poor survival of liver cancer patients after operation, and CD68+PPT1+macrophages are effective biomarkers for prognosis of liver cancer. Based on the detection, the immunofluorescence kit for detecting the liver cancer microenvironment is provided, the liver cancer microenvironment detection standardization is realized, the liver cancer detection flow is simplified, and the immunofluorescence kit has important clinical value for promoting accurate treatment of liver cancer and improving prognosis of patients.
Drawings
FIG. 1 shows representative immunofluorescent co-localized images of CD68 and PPT1 in example liver cancer tissues. Wherein image merge shows cells with green fluorescence carried by CD68 coincident with red fluorescence carried by PPT 1.
Figure 2 shows the analytical cooling of tumor microenvironment immune cell infiltration based on ppt1+ macrophage infiltration levels in tumor tissue in examples. Wherein, the A graph is a statistical curve of TSNE dimension reduction images and the proportion of various cells in CD45+ cells, the proportion reflects the infiltration proportion of various immune cell types in a high PPT1+ macrophage infiltration group and a low PPT1+ macrophage infiltration group, in the graph, except that CD8+ T cells are enriched in the high PPT1+ macrophage infiltration group, the infiltration proportion of other immune cells including B cells, macrophages, NK cells, plasma cells, neutrophils, eosinophils and DNT cells among the high PPT1+ macrophage infiltration group has no statistical difference; panel B is a plot comparing PD-1 expression levels on CD8+ T cells for a high PPT1+ macrophage infiltration group and a low PPT1+ macrophage infiltration group.
FIG. 3 shows a prognostic assay profile for overall survival of high PPT1+ macrophage infiltration groups and low PPT1+ macrophage infiltration groups in the examples.
Detailed Description
According to the immunofluorescence kit, through detecting the co-expression level of CD68 and PPT1 of a section of a liver cancer tissue sample and combining a flow cytometry analysis technology, the infiltration level of CD68+PPT1+macrophages in tumors is measured, and the obtained immune cell infiltration condition and functional characteristics in the tumor microenvironment can predict the overall risk of poor survival of a liver cancer patient after operation, so that the immunofluorescence kit is beneficial to guiding decision making of immunotherapy of the liver cancer patient in clinical practice and promoting accurate treatment of the liver cancer.
In recent years, clinical immunotherapy against tumors has been advanced in breakthrough, and the complex composition of tumor microenvironment (Tumor microenvironment, TME) has also limited accurate diagnosis of tumors. TME is composed of tumor cells, immune cells, vascular and endothelial cells, fibroblasts, and various stroma, and is continuously in the process of dynamic changes, thus posing a serious challenge to tumor treatment. For accurate delineation of complex tumor microenvironments (and other biological microenvironments), this cannot be achieved by conventional IHC (immunohistochemical detection), relying on polychromatic, or even supercolour imaging solutions.
In view of this challenge, a plurality of solutions have been proposed, for example Akoya Biosciences proposes a rapid 9-color imaging, 40-color depth imaging analysis and the like, which provides reliable technical support for oncology and other biological analysis research, and three-in-one fully analyzes tissue immune microenvironment, thereby providing an accurate solution for oncology and biological research.
The liver cancer microenvironment detection kit detects a liver cancer tissue sample by a multicolor immunofluorescence method, and determines the tumor microenvironment by means of imaging analysis of frozen sections and flow cytometry.
The term "liver cancer microenvironment detection kit" herein also refers to an "immunofluorescence kit", which may be abbreviated as "kit", which means the same meaning and may be used interchangeably.
The kit comprises two different antibodies aiming at each antigen to be detected, wherein one of the antibodies is a capture antibody for capturing the antigen, namely a first antibody for short, and the other is a detection antibody for detecting the captured antigen, namely a combined antibody or a second antibody for short. The two antibodies are combined with different antigen epitopes (antigenic determinants) of antigen protein in sequence to form a first antibody-antigen conjugate, and then the first antibody-antigen conjugate is combined with a second antibody to generate a sandwich structure of the first antibody-antigen-second antibody so as to detect the antigen protein by a double-antibody sandwich method. IgG antibodies may be used as secondary antibodies, and in embodiments of the invention IgG antibodies conjugated with fluorescent dyes are specifically used as secondary antibodies.
For the detection of more than two of different antigen proteins PPT1, CD45, CD8, CD68, CD4 and PD-1 (CD 279), the immunological detection can be carried out by using fluorescent dyes with different colors, namely, the fluorescent dyes are coupled to the secondary antibodies of the fluorescent dyes, so that multicolor depth imaging analysis of fluorescence of more than two colors is realized.
The CD68 primary antibody may be any antibody or antibody fragment capable of binding to CD68 protein, and may be recombinant, chimeric, humanized and murine. The antibody may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody, preferably using commercially available antibodies, examples of which include cat.ab283654, abcam; cat.29176 (Cell Signaling Technology).
The PPT1 primary antibody may be any antibody or antibody fragment capable of binding PPT1 protein, and may be recombinant, chimeric, humanized and murine. The antibody may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody, preferably a commercially available PPT1 antibody is used, examples of which include cat.ab89022, abcam, for example.
The fluorescent dye I and the fluorescent dye II respectively coupled on the IgG antibody in the fluorescent antibody I and the fluorescent antibody II are different, and are respectively selected from the following groups: including but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, cy3TM, cy5TM, allophycocyanin, texas red, champagne chlorophyll (peridenin chlorophyll), cyanines, tandem conjugates, such as phycoerythrin-Cy 5TM, green fluorescin, rhodamine, fluorescein Isothiocyanate (FITC) and Oregon GreenTM, rhodamine and its derivatives (such as Texas red and tetrarhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, cyDyesTM, 6-carboxymethylfluorescein (commonly known abbreviated as FAM and F), 6-carboxy-2 ',4',7',4, 7-Hexachlorofluorescein (HEX), 6-carboxy-4', 5 '-dichloro-2', 7 '-dimethoxyfluorescein (JOE or J), N' -tetramethyl-6 carboxyrhodamine (TAMRA or T), 6-and-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G 5 or G5), 6-carboxyrhodamine-6G (R6G 6 or G6), and rhodamine 110; cyanine dyes, such as Cy3, cy5, and Cy7 dyes; coumarin, such as umbelliferone; a benzamide dye, for example Hoechst33258; phenanthridine dyes, such as texas red; ethidium dye; an acridine dye; carbazole dyes; a phenoxazine dye; porphyrin dyes; polymethine dyes such as cyanine dyes, e.g., cy3, cy5, etc.; BODIPY dyes and quinoline dyes.
The kit also comprises necessary reagents for preparing liver cancer tissue sample slices, such as a surfactant, a blocking agent, a fixing agent, an embedding agent, EDTA antigen repair buffer solution, an anti-fluorescence quenching sealing tablet containing a cell nucleus dye (also called DNA dye) and the like, and all the reagents can be commercially available.
Embedding agents are used to submerge the tissue sample, and are typically selected from OCT embedding agents or paraffin waxes. OCT embedding agents are water-soluble mixtures of polyethylene glycol and polyvinyl alcohol, which have been widely used in immunohistochemical laboratories to support tissue during frozen sections to increase tissue continuity and reduce wrinkling and fragmentation. The OCT is used to pre-infiltrate the tissue and then the cryostat section is performed, resulting in an improved quality of the section.
Formalin is a commonly used fixative that facilitates subsequent staining, immunohistochemistry, etc. by fixing tissue specimen sections, for example, formalin may be 4% paraformaldehyde.
The surfactant is a cell permeabilizer, which may be 0.5% Triton-X100 buffer.
Blocking agents are used for blocking of sections or cell samples during immunofluorescence. After blocking, the subsequent non-specific binding of the primary antibody or the secondary antibody to the sample can be reduced, the background can be reduced, and the signal-to-noise ratio can be enhanced. Bovine serum albumin (Bovine Serum Albumin, BSA) solution, which is commonly used in the biological arts, may be used as a blocking agent.
EDTA antigen retrieval buffer solution, which is used for antigen retrieval before formalin fixation and embedded tissue section immunohistochemical staining, and increases the display effect of the immunohistochemical experiment;
the anti-fluorescence quenching caplets containing the nuclear dye can be used to cap sections after incubation with antibodies. Effective encapsulation is critical to immunofluorescence imaging observations. Fluorophores are very sensitive to excitation light in fluorescence imaging and photobleaching and light quenching during storage, etc., and longer storage of samples can be achieved with the use of cappers.
In an embodiment, the cell infiltration situation may be described by a TSNE dimension reduction map, which may be generated by FlowJo software. The software can be installed in a full-automatic immunofluorescence microscopic image analyzer, a flow cytometer and the like.
The kit can reflect the immune characteristics of tumor microenvironment by performing immunofluorescence detection in tumor tissues, determine markers or marker events which can effectively judge the prognosis risk of a liver cancer patient, effectively evaluate the prognosis risk of the patient, provide guidance for decision making of liver cancer immunotherapy and prolong the survival time of the liver cancer patient.
The invention will be further described with reference to specific examples and drawings. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the examples of the present invention, if no specific explanation is given for the experimental operating temperature, this temperature is usually referred to as room temperature (10-40 ℃).
The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to weight percentages unless otherwise indicated.
Examples
1. Study object inclusion
The study subjects were 169 tumor tissues of hepatocellular carcinoma patients provided by the secondary mountain hospital of the university of double denier, and the inclusion and exclusion criteria were:
(1) The liver cancer is subjected to radical excision and the pathology is clearly diagnosed as liver cell cancer;
(2) No other cancer-related treatments have been received prior to surgery;
(3) No history of other malignancy;
(4) Has perfect clinical pathology data and follow-up information.
2. The research method comprises the following steps:
(1) The 169 patients with hepatocellular carcinoma were harvested from their surgically resected tumor tissue specimens and frozen sections were performed while fresh tissue was harvested and flow-tested using a flow cytometer (FACSCalibur III, BD Co.). The postoperative follow-up content of the patient comprises blood routine, liver function examination, serum tumor marker detection, abdominal ultrasound, chest radiography and the like. The overall post-operative survival time is defined as the time interval from the date of surgery to death or last follow-up.
(2) Immunofluorescent staining of tumor tissue
The sections were immersed in EDTA repair buffer and heated to boiling, cooled naturally at room temperature, and then 0.5% Triton-X100 blocking solution was added dropwise at room temperature for 30min, followed by blocking with 1% BSA at room temperature for 2h. Appropriately diluted CD68 primary antibodies cat.ab283654, abcam and PPT1 primary antibodies cat.ab89022, abcam, respectively, were added dropwise according to the manufacturer's instructions, washed 3 times with PBS after overnight at 4 ℃, the corresponding fluorescent antibodies Alexa Fluor 488 (cat.ab150113, abcam) and fluorescent antibodies dicyy 3 (cat.ab6939, abcam) were added, incubated for 2h at room temperature, and finally DAPI-containing anti-fluorescence quenching cappers were added for capping.
The seal was analyzed using a fully automated immunofluorescence microscopy image analyzer (BX 53, oympus Co.) and the results are shown in FIG. 1. In the figure, green fluorescence shows the expression of CD68, red fluorescence shows the expression of PPT1, and Merge shows macrophages coexpressing CD68 and PPT1, which are abbreviated as CD68+PPT1+macrophages or PPT1+macrophages.
(3) Analysis of relation between tumor PPT1+ macrophage infiltration level and hepatocellular carcinoma microenvironment immune characteristics and prognosis of patient
According to the immunofluorescence microscopic image in fig. 1, ppt1+ macrophage infiltration height grouping is carried out, 5 visual fields are randomly selected in a tissue section to carry out ppt1+ cd68+ cell counting, flow data obtained from fresh tissues are visualized as TSNE two-dimensional images, and a judgment formula of the ppt1+ macrophage infiltration level is obtained through statistics: the sum of the numbers of cd68+ ppt1+ macrophages in f=5 fields/the sum of the numbers of cd68+ cells in 5 fields, where F is the infiltration level, F < 0.5 is considered low ppt1+ macrophage infiltration, F > 0.5 is considered high ppt1+ macrophage infiltration, and the difference in the immune cell infiltration ratios in the tumors of the two groups of patients is compared, the results are shown in fig. 2 a.
The figure also shows that, except for enrichment of CD8+ T cells in the high PPT1+ macrophage infiltration group, infiltration ratios of other immune cells including B cells, macrophages, NK cells, plasma cells, neutrophils, eosinophils, DNT cells and the like between the high PPT1+ macrophage infiltration group and the low PPT1+ macrophage infiltration group have no statistical difference.
(4) The overall survival time after the operation was plotted and Kaplan-Meier survival analysis was performed, and the results are shown in fig. 3.
3. Experimental results:
immunofluorescence results of liver cancer tissues show that PPT1 is mainly expressed in cd68+ macrophages in liver cancer tissues, i.e., PPT1 and CD68 are expressed in macrophages in liver cancer tissues (as shown in fig. 1). The cell is called a cd68+ppt1+ macrophage or ppt1+ macrophage.
Flow results show that the high ppt1+ macrophage infiltration group has more cd8+ T cell infiltration than the low ppt1+ macrophage infiltration group tumor (as shown in fig. 2 a), but the functional depletion marker PD-1 molecule expression level of cd8+ T cells is significantly increased (as shown in fig. 2B), suggesting that the high ppt1+ macrophage infiltration liver cancer tissue has more depleted cd8+ T cells, which may be more sensitive to immunotherapy.
Kaplan-Meier survival analysis shows that liver cancer patients with high PPT1+ macrophage infiltration group have shorter postoperative overall survival time (as shown in figure 3), suggesting that PPT1+ macrophages are effective biomarkers for liver cancer prognosis.
In summary, PPT1 is mainly expressed in macrophages and is positively correlated with high infiltration of depleted cd8+ T cells in liver cancer tissues. PPT1+ macrophages are used as effective prognosis prediction indexes, are helpful for evaluating immune characteristics and prognosis risks of hepatocellular carcinoma, and can be used for guiding the immune treatment management of liver cancer patients and predicting the postoperative survival time of the hepatocellular carcinoma patients in clinical practice.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art will appreciate that, in light of the principles of the present invention, improvements and modifications can be made without departing from the scope of the invention.
Claims (10)
1. The liver cancer microenvironment detection kit is characterized by comprising the following components:
the preparation method is used for preparing a surfactant, a blocking agent and a fixing agent required by frozen sections of liver cancer tissues;
a CD68 primary antibody;
a fluorescent antibody one conjugated with a fluorescent dye one, the fluorescent antibody one being capable of binding to a CD68 primary antibody-CD 68 conjugate;
PPT1 primary antibody;
a fluorescent antibody II coupled with a fluorescent dye II, wherein the fluorescent antibody II can be combined with a PPT1 primary antibody-PPT 1 conjugate,
the fluorescent dye I and the fluorescent dye II are fluorescent dyes with different colors, and the antibodies in the fluorescent antibody I are different from the antibodies in the fluorescent antibody II.
2. The liver cancer microenvironment detection kit according to claim 1, wherein the antibodies in the fluorescent antibody one and the antibody in the fluorescent antibody two are IgG.
3. The liver cancer microenvironment detection kit according to claim 1 or 2, further comprising the following components:
embedding agent selected from OCT embedding agent or paraffin;
EDTA antigen retrieval buffer;
an anti-fluorescence quenching caplet containing a nuclear dye.
4. The liver cancer microenvironment detection kit according to claim 1 or 2, wherein the fluorescent antibody is Alexa Fluor 488 (cat. Ab150113, abcam); the second fluorescent antibody is CY3 (cat.ab6939, abcam); the nuclear dye is DAPI; the surfactant is 0.5% Triton-X100 buffer; the blocking agent is BSA blocking solution; the fixative is formalin.
5. The liver cancer microenvironment detection kit according to claim 1 or 2, wherein the liver cancer microenvironment detection is performed by the steps of:
i. preparing frozen sections of liver cancer tissues;
incubating the frozen sections with a first antibody of CD68 and a first antibody of PPT1 overnight after the frozen sections are permeabilized with a surfactant and blocked with a blocking agent, and then incubating and blocking the frozen sections with a first fluorescent antibody and a second fluorescent antibody;
and determining that the CD68 and PPT1 double positive cells in liver cancer tissues are PPT1+ macrophages according to the result of fluorescence co-localization of the CD68 and the PPT1, and dividing patients into a high PPT1+ macrophage infiltration group and a low PPT1+ macrophage infiltration group according to infiltration levels of the cells.
6. The liver cancer microenvironment detection kit according to claim 5, wherein in step i, the preparation process of the frozen section is as follows: fresh tissue is frozen in liquid nitrogen after being embedded by an embedding agent, then frozen and sliced in a cryostat slicer, and fixed by a fixing agent.
7. The liver cancer microenvironment detection kit according to claim 5, wherein in step iii, the cell infiltration is determined by flow cytometry in combination with TSNE dimension reduction map.
8. The liver cancer microenvironment detection kit according to claim 5, wherein in step iii, the ppt1+ macrophage infiltration level is evaluated by: and identifying cells with green fluorescence carried by a macrophage marker CD68 and red fluorescence carried by PPT1 as PPT1+ macrophages, randomly selecting 5 fields of view in the tissue section for cell counting, and obtaining the infiltration quantity of the PPT1+ macrophages.
9. The liver cancer microenvironment detection kit according to claim 8, wherein the determination formula of ppt1+ macrophage infiltration level is: f=the sum of the numbers of cd68+ppt1+ macrophages in 5 fields/the sum of the numbers of cd68+ cells in 5 fields, where F is the infiltration level, F < 0.5 is considered low ppt1+ macrophage infiltration, F > 0.5 is considered high ppt1+ macrophage infiltration.
10. The liver cancer microenvironment detection kit of claim 9, wherein when liver cancer tissue has high ppt1+ macrophage infiltration, tumor tissue has high infiltration of depleted PD-1+cd8+ t cells and indicates poor overall survival after surgery.
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| CN117741148A (en) * | 2024-02-19 | 2024-03-22 | 苏州颐坤生物科技有限公司 | Marker combination for predicting curative effect of immunotherapy and model construction method and application |
| CN119086911A (en) * | 2024-08-22 | 2024-12-06 | 中山大学 | Application of reagents for determining CD93 protein and CD68 protein levels in adjacent tissues of patients with liver cancer in the preparation of a liver cancer prognosis prediction kit |
| CN119290542A (en) * | 2024-12-13 | 2025-01-10 | 天津医科大学总医院空港医院 | A thymic tumor microenvironment antigen detection kit and its application in myasthenia gravis evaluation |
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