Application of cell spheroids in constructing human breast cancer immunohistochemical quality control

Abstract

The application provides application of cell balls in construction of human breast cancer immunohistochemical quality control products, and belongs to the technical field of pathological examination. The application solves the problems of scattered distribution of tumor cells in the immunohistochemical quality control products in the prior art, lack of morphological structure, and significant difference from human breast cancer tissue samples to be detected; the quality control sheet prepared by the application is more stable, can ensure that the tissue morphology on all used quality control sheets remains consistent, the quality control sheet can be mass-produced, and does not exist ethical problems, can provide breast cancer ER, PR, HER2 and Ki67 immunohistochemical quality control products for daily detection of clinical pathology departments, simplifies the procedure of setting immunohistochemical controls in clinical work, and only a single wax block can meet the quality control needs of human breast cancer immunohistochemical detection, is convenient and stable.

Description

Technical Field

[0001] This invention relates to the field of pathological examination technology, and in particular to the application of cell spheres in the construction of immunohistochemical quality control products for human breast cancer. Background Technology

[0002] Detecting the expression levels of ER, PR, HER2, and Ki67 proteins in breast cancer using immunohistochemistry is crucial for guiding breast cancer treatment. Using known commercial antibodies to detect the expression of ER, PR, HER2, and Ki67 proteins in paraffin-embedded breast cancer tissue samples is routine diagnostic practice, and the accuracy of this detection affects patient treatment decisions and prognosis.

[0003] Due to batch-to-batch variations in antibodies and related reagents, or the influence of immunohistochemical operators, it is routinely necessary to set up immunohistochemical quality control tissues during ER, PR, HER2, and Ki67 immunohistochemical testing for breast cancer to ensure correct immunohistochemical procedures. Through quality control tissues, operators can observe the spatial and subcellular localization of stained tissues and assess the difference between the actual staining intensity and the preset intensity.

[0004] Currently, the commonly used quality control tissues for ER, PR, HER2, and Ki67 immunohistochemistry are human breast cancer tissue samples and human breast cancer cell lines. Both types of quality control tissues have their advantages and disadvantages. Using human breast cancer tissue samples as quality control samples has limitations: the number of tissue samples that can be obtained after selection is limited, leading to the need to change quality control tissues during large-scale use, resulting in inconsistencies in the quality control tissues used for clinical samples. Furthermore, due to the heterogeneity of ER, PR, HER2, and Ki67 expression within human breast cancer tissue samples, the protein levels of ER, PR, HER2, and Ki67 detected on different tissue sections may be unstable. While using human breast cancer cell lines as quality control tissues can ensure the consistency of ER, PR, HER2, and Ki67 expression levels across different sections and meet the clinical demand for quality control tissues, these quality control tissues contain scattered tumor cells, lack morphological structure, and differ significantly from the human breast cancer tissue samples being tested.

[0005] Chinese patent CN104569397A discloses a quality control material for breast cancer detection and its preparation method, specifically a quality control material and its preparation method for HER2 gene amplification FISH detection, belonging to the fields of clinical laboratory science, pathology, and biotechnology. The breast cancer detection quality control material is a paraffin-embedded specimen slide made from cultured breast cancer cell lines. The advantages of this invention are: the quality control material made from in vitro cultured breast cancer cell lines can serve as a substitute for quality control materials made from real tissue samples; it allows for large sample volumes and high reproducibility; and by selecting different cell lines, different HER2 gene levels can be detected. The preparation method is simple, allows for mass production, is easy to store, and can be used for internal quality control and external quality assessment.

[0006] However, the aforementioned patented technology does not solve the defects of quality control products in the prior art. Summary of the Invention

[0007] The purpose of this invention is to provide the application of cell spheres in the construction of immunohistochemical quality control products for human breast cancer, which can provide immunohistochemical quality control products for ER, PR, HER2 and Ki67 in breast cancer for routine testing in clinical pathology departments.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of cell spheres in constructing immunohistochemical quality control materials for human breast cancer. The cell spheres are used to directly prepare paraffin blocks, which are then sliced ​​and used as immunohistochemical quality control materials for human breast cancer.

[0009] Preferably, the cell spheroids are prepared by a method comprising the following steps: The cells were cultured into cell spheres with intercellular adhesion.

[0010] Preferably, the cell spheres are derived from human breast cancer cell lines.

[0011] Preferably, the human breast cancer cell lines include BT474, BT20, T47D, or HCC1806.

[0012] This invention also provides a method for constructing a human breast cancer immunohistochemical quality control, comprising the following steps: Human breast cancer cell spheres with intercellular adhesion were sequentially fixed, molded, dehydrated, cleared, impregnated with paraffin, embedded, and sectioned to obtain human breast cancer immunohistochemical quality control material.

[0013] Preferably, the fixation is performed using 5-20% neutral buffered formalin as the fixative.

[0014] Preferably, the fixed time is more than 20 hours.

[0015] Preferably, the encapsulating material used in the molding process is agarose.

[0016] Preferably, the thickness of the slice is 3~5μm.

[0017] The present invention also provides immunohistochemical quality control products for human breast cancer obtained by the above-described construction method.

[0018] The beneficial effects of this invention are: This invention demonstrates that cell spheres made from human breast cancer cell lines can effectively replace human breast cancer samples and quality control slides made from cell lines, with staining effects that are not inferior to those of human breast cancer samples and quality control slides made from cell lines. The materials are readily available on the market and can be used for commercial production.

[0019] This invention describes the preparation of quality control materials according to the requirements of pathological tissue preparation, using 10% neutral buffered formalin fixative and a tissue dehydrator. The quality control material preparation can be performed during routine pathological tissue preparation without additional procedures, making it simple and easy to implement.

[0020] This invention allows the use of this quality control material in routine immunohistochemical detection of breast cancer ER, PR, HER2, and Ki67. It can be used together with human breast cancer samples for machine staining or manual staining, which is beneficial for quality control during the detection process.

[0021] In HER2 testing for human breast cancer, staining intensity, membrane integrity, and positive rate need to be evaluated. In this invention, the quality control samples contain four different staining results: 3+ staining indicates strong and intact cell membrane staining (100%); 2+ staining indicates moderately strong and intact cell membrane staining (100%); 1+ staining indicates weak and incomplete cell membrane staining (greater than 10%); and 0 staining indicates unstained cells (100%). These preset staining results fully comply with the guidelines for HER2 testing in human breast cancer (Arch Pathol Lab Med (2023) 147 (9): 993–1000). The four gradient staining results in this invention can meet the clinical testing requirements for HER2 in human breast cancer.

[0022] For ER staining in human breast cancer, it is often necessary to exclude false positives and false negatives caused by improper staining procedures. Clinically, normal ductal epithelium of the breast is commonly used as a positive internal control for ER, germinal centers of the tonsils are used as a positive external control, and mantle B cells of the tonsils are used as a negative external control (J Clin Oncol. 2020 Apr 20;38(12):1346-1366.). For PR staining in human breast cancer, similar to ER, it is also necessary to exclude false positives and false negatives caused by improper staining procedures. Clinically, columnar epithelial cells of the cervical canal are commonly used as a positive control, but suitable negative control tissues are lacking. In this invention, we provide suitable cell spheres as positive and negative controls for ER and PR staining. For Ki67 staining in human breast cancer, suitable control tissues are currently lacking. In this invention, we provide suitable cell spheres as Ki67 low-expression controls and high-expression controls.

[0023] The immunohistochemical quality control tissues for human breast cancer ER, PR, HER2, and Ki67 provided in this invention can simplify the procedure for setting up immunohistochemical controls in clinical work. Only a single paraffin block is needed to meet the quality control requirements for immunohistochemical detection of human breast cancer, which is convenient and stable. Attached Figure Description

[0024] Figure 1 This is a flowchart of the quality control product manufacturing process in this invention; Figure 2 This is a diagram of cell spheroid collection according to a specific embodiment of the present invention; Figure 3 This is a diagram illustrating the fabrication of a cell sphere model according to a specific embodiment of the present invention; Figure 4 This is a quality control sample image produced according to a specific embodiment of the present invention; Figure 5 This is a comparison of the HER2 staining pattern of the quality control sample with the staining patterns of the quality control sample made from patient breast cancer tissue and human breast cancer cell line in a specific embodiment of the present invention. Figure 6 This is a staining image of ER and PR samples for quality control products in a specific embodiment of the present invention; Figure 7 This is a staining image of Ki67, a quality control sample, in a specific embodiment of the present invention. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 This embodiment uses quality control materials based on commercially available cell lines. These quality control materials can be mass-grown and supplied in large quantities. At the same time, these quality control materials are paraffin blocks based on standardized pathological tissue processing procedures, which can meet the production needs of standardized quality control slides.

[0027] refer to Figure 1 The quality control production process includes culturing commercial human breast cancer cells and collecting and fixing cell spheres. After molding the fixed cell spheres, they enter the standardized paraffin tissue production process. The produced paraffin blocks include quality control tissues with four different HER2 expression levels and two different ER, PR and Ki67 expression levels.

[0028] This invention uses a commercially available human breast cancer cell line. Compared to commonly used human breast cancer tissue derived from patients, the quality control material in this invention exhibits more stable staining, enabling the long-term, large-scale, and consistent use of quality control materials. Furthermore, this quality control material has a tissue structure similar to human breast cancer tissue, resulting in more accurate interpretation of ER, PR, HER2, and Ki67 staining.

[0029] refer to Figure 2 A key step in the preparation of quality control slides lies in enabling cells from conventionally cultured human breast cancer cell lines to adhere and grow together, forming cell spheres that are less prone to disintegration. This invention utilizes a shaker to create these cell spheres, which remain stable during centrifugation, fixation, and paraffin block preparation. In tissue staining, their morphology and structure resemble ex vivo tissue samples. Compared to quality control slides made using patient tissue samples, those made with cell spheres are more stable, ensuring consistent tissue morphology across all slides used. These control slides can be mass-produced without ethical concerns.

[0030] refer to Figure 3 After centrifuging and agglomerating the collected cell spheres, they are molded with agarose gel, preventing them from dispersing during the preparation of paraffin tissue. This molding method is simple and easy to implement, and does not affect tissue fixation. With a constant cell quantity, it ensures consistent cell sphere density in each quality control slide, reducing batch-to-batch variations and making it more suitable for commercial production.

[0031] refer to Figure 4 The present invention comprises four quality control tissues, all produced within a single paraffin block, facilitating the fabrication of quality control slides and meeting the requirements for quality control slides under various staining conditions. In this invention, the paraffin block fabrication process can be performed entirely according to the conventional tissue paraffin block fabrication process, without the need for separate program or condition settings, thus completely simulating a paraffin block fabrication process closely resembling that of ex vivo tissue samples.

[0032] refer to Figure 5(AD) The HER2 control sheet for human breast cancer in this invention contains four different modes of HER2 staining. According to the guidelines for HER2 detection in human breast cancer (Arch Pathol Lab Med (2023) 147 (9): 993–1000.), the preset values ​​are: 3+ stained tissue with strong and intact cell membrane staining (100%); 2+ stained tissue with moderate and intact cell membrane staining (100%); 1+ stained tissue with weak and incomplete cell membrane staining (greater than 10%); and 0 stained tissue with unstained cells (100%). As can be seen in this figure, the cells in the control sheet adhere to each other, which is close to the growth pattern of tumor cells in patient tissue samples, making it convenient for users to monitor abnormalities in the HER2 staining process using this control sheet.

[0033] The quality control slides of this invention have similar tissue morphology to patient breast cancer tissue samples (IL), with cells adhering and growing together. Compared with currently used cell line (EH) controls, the quality control slides of this invention have the same staining effect, and even better results in the assessment of low expression.

[0034] refer to Figure 6 (A and B) Negative and positive staining in the human breast cancer ER quality control slides of this invention. It can be seen that the negative control slide has no nuclear staining at all; the positive control slide shows strong nuclear staining in 50% of the cells. (C and D) Negative and positive staining in the human breast cancer PR quality control slides of this invention. It can be seen that the negative control slide has no nuclear staining at all; the positive control slide shows strong nuclear staining in 80% of the cells.

[0035] refer to Figure 7 (A) Low expression staining in the Ki67 quality control slide of human breast cancer in this invention, with an expression rate of 15%. (B) High expression staining in the Ki67 quality control slide of human breast cancer in this invention, with an expression rate of 90%.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a human breast cancer immunohistochemical quality control product, characterized in that, Includes the following steps: Human breast cancer cell spheres with intercellular adhesion were sequentially fixed, molded, dehydrated, cleared, impregnated with paraffin, embedded, and sectioned to obtain human breast cancer immunohistochemical quality control material; The fixation process uses 5-20% neutral buffered formalin as the fixative. The fixed time is more than 20 hours; The encapsulating material used in the molding process is agarose; The thickness of the slice is 3~5μm; Human breast cancer cell lines were selected from BT474, BT20, T47D, or HCC1806; The cell spheres are manufactured using a shaker, and no scaffold material is used in the preparation of the cell spheres. The method for constructing the immunohistochemical quality control material for human breast cancer does not use scaffold materials.

2. Immunohistochemical quality control material for human breast cancer obtained by the construction method described in claim 1.

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