A breast cancer classification method based on Piezo1 expression

Abstract

This invention discloses a breast cancer classification method based on Piezo1 expression. After obtaining tumor tissue samples from patients, the method detects the Piezo1 protein expression level using immunohistochemistry and classifies them into high-expression and low-expression types according to a validated scoring standard. This classification result guides individualized treatment, with high-expression patients recommended for treatment regimens including inhibitors of mechanotransmission pathways. By integrating tumor mechanotransmission characteristics into the clinical classification system, this invention can identify a subgroup of breast cancer driven by an abnormal mechanotransmission microenvironment that cannot be distinguished by traditional molecular classification. This not only improves and supplements the existing classification framework, making the understanding of breast cancer heterogeneity more comprehensive, but also more accurately predicts the invasiveness, metastasis potential, and poor prognostic risk of these patients.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a method for breast cancer typing based on Piezo1 expression. Background Technology

[0002] Breast cancer is a highly heterogeneous malignant tumor. Currently, molecular subtyping systems based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the proliferation index Ki-67 (such as Luminal A, Luminal B, HER2 overexpression, and triple-negative types) have become the core standard for guiding the selection of treatment regimens (such as endocrine therapy and anti-HER2 targeted therapy) and prognostic assessment.

[0003] The aforementioned classification methods primarily focus on growth factor signaling, hormone dependence, and proliferative activity of tumor cells, failing to adequately incorporate another crucial biological dimension of tumorigenesis and development: mechanotransduction. Studies have shown that mechanical stresses in the tumor microenvironment (such as stromal stiffness and fluid pressure) can influence malignant behavior of tumor cells through specific mechanosensitive ion channels, including proliferation, invasion, metastasis, and treatment resistance. Therefore, existing classification systems may inherently omit subgroups of breast cancer driven by mechanotransduction, possessing unique biological characteristics and clinical outcomes. This could lead to biased prognostic predictions for some patients (especially those insensitive to current standard treatments or prone to recurrence) and miss potential therapeutic opportunities for precise intervention targeting this pathway.

[0004] Therefore, in response to the problems mentioned above, this invention proposes a breast cancer classification method based on Piezo1 expression. Summary of the Invention

[0005] To overcome the problem that existing molecular classification systems for breast cancer fail to integrate tumor biomechanical signal transduction characteristics, which may affect the accuracy of classification and the effectiveness of treatment guidance, this invention proposes a breast cancer classification method based on Piezo1 expression. This method is used to identify and define a subtype of breast cancer driven by abnormal biomechanical signal transduction, thereby making up for the shortcomings of existing classification systems and providing a more comprehensive basis for clinical prognosis and individualized treatment (especially targeted therapy against biomechanical signaling pathways).

[0006] The technical solution of this invention is: a breast cancer typing method based on Piezo1 expression, comprising the following steps:

[0007] S1, Obtain a tumor tissue sample from a breast cancer patient, which is either formalin-fixed paraffin-embedded tissue or a fresh frozen tissue sample;

[0008] S2, The expression level of Piezo1 protein in the tumor tissue sample was detected by immunohistochemistry, specifically including staining with a validated anti-Piezo1 monoclonal antibody and scoring by two or more pathologists in a double-blind manner;

[0009] S3, based on the Piezo1 protein expression score, classifies breast cancer into two subtypes:

[0010] Piezo1 high expression type, immunohistochemical score ≥4 points (based on the product of positive cell percentage and staining intensity, where positive cell percentage >50% and staining intensity is strong positive is 3 points, positive cell percentage 30-50% and staining intensity is medium to strong positive is 2 points, positive cell percentage <30% and staining intensity is weak positive is 1 point, no staining is 0 points, the total score range is 0-12 points, and the threshold is set at ≥4 points).

[0011] Piezo1 low expression type, immunohistochemical score <4:

[0012] S4. The above classification results are integrated with clinicopathological parameters to guide individualized treatment decisions. Among them, patients with high Piezo1 expression are given priority for treatment regimens that include inhibitors of mechanotransmission pathways or integrin-targeting drugs.

[0013] Preferably, the anti-Piezo1 monoclonal antibody used in the immunohistochemical method is a cross-validated equivalent antibody.

[0014] Preferably, the method further includes simultaneously detecting the expression of estrogen receptor, progesterone receptor, HER2 and Ki-67 in the same tumor tissue sample, and integrating the Piezo1 expression subtype with the traditional molecular subtype to form a composite subtype.

[0015] As a preferred option, when the tumor is classified as having high Piezo1 expression and is also triple-negative breast cancer, a combination of Piezo1 pathway inhibitors and immune checkpoint inhibitors is used.

[0016] Preferably, the determination of the Piezo1 overexpression genotype is further verified by qPCR or Western Blot, wherein the relative expression level of Piezo1 mRNA in qPCR is more than 3 times higher than that in adjacent normal tissue, and / or the expression level of Piezo1 protein in Western Blot is more than 2 times higher than that in internal reference protein.

[0017] Preferably, the method further includes simultaneously detecting the colocalization of Piezo1 and tumor microenvironment markers on the same tissue section using multiplex immunofluorescence technology. Cases with high Piezo1 expression and colocalization ≥20% with the tumor-associated fibroblast marker α-SMA are further classified as "mechanically activated microenvironment".

[0018] Preferably, the typing results are associated with patient prognostic assessment, wherein the independent hazard ratio of disease-free survival to overall survival corresponding to Piezo1 high expression is ≥1.5, and this hazard ratio remains statistically significant after adjusting for age, stage and traditional molecular typing using a Cox proportional hazards model.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention integrates tumor biomechanical signal transduction characteristics into the clinical classification system, thereby enabling the identification of a subgroup of breast cancer driven by abnormal biomechanical microenvironment (Piezo1 high expression type) that cannot be distinguished by traditional molecular classification (such as classification based on ER, PR, HER2). This not only improves and supplements the existing classification framework, making the understanding of breast cancer heterogeneity more comprehensive, but also enables more accurate prediction of the invasion and metastasis potential and poor prognostic risk of this group of patients.

[0021] 2. Based on the direct correlation between Piezo1 expression subtypes and specific treatment recommendations, this invention can provide clinical guidance for precise interventions that go beyond traditional treatment modalities. In particular, for patients with high Piezo1 expression, including those who are insensitive to conventional endocrine therapy or targeted therapy (such as some triple-negative breast cancer), it suggests that they may benefit from mechanotransmitter inhibitors, integrin-targeted drugs, or their combination with existing therapies (such as chemotherapy and immunotherapy), thereby potentially overcoming treatment resistance mediated by mechanotransmitters and opening up new personalized treatment pathways. Attached Figure Description

[0022] Figure 1 The diagram shown is a schematic representation of the method flow of the present invention.

[0023] Figure 2 The diagram illustrates the process of establishing and validating the Piezo1 immunohistochemical detection method of the present invention.

[0024] Figure 3 The diagram shows the workflow of the correlation analysis between Piezo1 typing and traditional clinicopathological features of the present invention.

[0025] Figure 4 The diagram illustrates the verification process of the Piezo1 typing as an independent prognostic marker according to the present invention.

[0026] Figure 5 The diagram shows the survival curves for high and low expression types of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Please see Figure 1 This invention provides an embodiment of a breast cancer classification method based on Piezo1 expression:

[0029] (1) Formalin-fixed paraffin-embedded tissue samples were used to ensure seamless integration with existing pathological diagnostic procedures. The heat-retrieval antigen retrieval method was employed, and IHC staining was performed using a validated anti-Piezo1 monoclonal antibody with high specificity and high affinity. Positive and negative controls were established (including a tissue control with known high expression and a negative control using PBS instead of the primary antibody) to ensure the accuracy and comparability of each test. This step transforms the abstract "mechanical signal transduction activity" into a clearly visible and reproducible brown precipitate signal in the tumor cell membrane / cytoplasm, realizing the visualization and operationalization of the mechanobiological phenotype.

[0030] (2) The scoring was conducted independently by two or more senior pathologists who were unaware of the patient's clinical information under an optical microscope. The scoring system combined the percentage of positive tumor cells and the staining intensity, using a product method (Histo-score, H-score). For example: based on the percentage of positive cells (P): 0 (<5%), 1 (5-30%), 2 (31-50%), 3 (>50%); based on the average staining intensity (I): 0 (no staining), 1 (weakly positive, pale yellow), 2 (moderately positive, brownish-yellow), 3 (strongly positive, brownish-red). The final H-score = P × I, with a theoretical range of 0-9. The optimal prognostic threshold was determined using receiver operating characteristic (ROC) curves through survival analysis of a large number of retrospective samples. For example, an H-score ≥ 4 was defined as "high Piezo1 expression", and an H-score < 4 was defined as "low expression". This scoring and typing method transforms continuous protein expression data into discrete categorical variables with clear clinical significance, achieving a reproducibility of over 90%.

[0031] (3) Cross-integration analysis was performed on the Piezo1 classification results with traditional clinicopathological parameters (such as TNM staging, histological grade) and molecular classifications (Luminal A, Luminal B, HER2+, TNBC). For example, a subgroup with special biological behavior, "TNBC with high Piezo1 expression", was identified. Through large-sample cohort studies, multivariate Cox regression models have confirmed that high Piezo1 expression is an independent risk factor affecting disease-free survival and overall survival. Based on this, and combined with the theory revealed by basic research that Piezo1 channel activation can promote intracellular calcium ion influx and activate downstream signaling pathways such as ROCK, YAP / TAZ, and ERK, thereby driving tumor cell invasion, metastasis, and drug resistance, this invention proposes a targeted treatment navigation: For patients with high Piezo1 expression, especially those with poor prognosis after standard treatment, it is recommended to consider participating in clinical trials targeting mechanical signaling pathways (such as Piezo1 itself, integrins, FAK, ROCK, or YAP / TAZ inhibitors) in addition to conventional treatment, or to explore combination strategies of these targeted drugs with chemotherapy and immunotherapy.

[0032] Please see Figure 2 The present invention provides Embodiment 1:

[0033] (1) From 50 randomly selected breast cancer patients and their paired adjacent normal breast tissue, standard tissue sections with a thickness of 4 micrometers were continuously cut from formalin-fixed paraffin-embedded blocks using a microtome. These sections were then pasted onto glass slides treated with poly-L-lysine or silanization to prevent detachment. The sections were then baked in a constant temperature oven at 65°C for 2 hours to enhance tissue adhesion. The glass slides were then immersed in fresh xylene I and xylene II for 10 minutes each to completely dissolve the paraffin. They were then immersed in a gradient of ethanol (100%, 95%, 85%, 75%) for 5 minutes each to gradually increase tissue moisture. Finally, the slides were rinsed with deionized water or phosphate buffer to complete the hydration process.

[0034] (2) Place the dewaxed and hydrated slides into a repair box filled with pH 6.0 citrate antigen repair buffer, ensuring that the liquid completely submerges the tissue. Place the repair box into an autoclave, cover it, and place it on a hot plate to heat until the steam valve sprays out stable steam. Start timing and maintain the high pressure and high heat state for 3 minutes to fully open the protein structure that has been cross-linked due to fixation and expose the masked Piezo1 antigen epitopes. After the repair is completed, allow it to cool naturally to room temperature. Then, gently rinse the slides three times with phosphate buffer (PBS, pH 7.4) for 3 minutes each time to remove residual repair solution.

[0035] (3) On the tissue sections after antigen retrieval, add sufficient 3% hydrogen peroxide aqueous solution and incubate at room temperature in the dark for 15 minutes to completely quench the endogenous peroxidase activity of the tissue and prevent non-specific background from being generated during subsequent color development. After rinsing with PBS, add 5% bovine serum albumin or normal goat serum blocking solution and incubate at room temperature for 30 minutes to block non-specific binding sites on the tissue. After discarding the blocking solution, do not rinse, and directly add the validated anti-Piezo1 monoclonal antibody working solution prepared according to the predetermined dilution ratio (1:200) to ensure that it completely covers the tissue. Place the sections horizontally in a humidified chamber and incubate stably in a refrigerator at 4°C (about 16-18 hours) to ensure that the antibody and antigen are fully bound.

[0036] (4) Remove the humidified chamber from the 4°C refrigerator and allow it to equilibrate at room temperature for 30 minutes. Rinse the slides three times with PBS buffer to remove unbound primary antibodies. Then, add working solution of polymer-enhanced horseradish peroxidase-labeled secondary antibody that matches the species of the primary antibody. Incubate at room temperature for 30 minutes. Rinse with PBS. Then, add freshly prepared diaminobenzidine chromogenic solution. Closely monitor the chromogenic process under a microscope. When the positive signal (brownish) is clearly visible and the background is not stained (usually about 1-5 minutes), immediately immerse the slides in deionized water to stop the reaction. Counterstain the cell nuclei with hematoxylin for about 1 minute. Differentiate with hydrochloric acid alcohol. Turn blue with running water. Finally, dehydrate with graded ethanol and clear with xylene. Mount with neutral resin to prepare pathological slides that can be used for permanent observation and scoring.

[0037] (5) To confirm the specificity of the immunohistochemical staining signal, this embodiment sets up a control, including using tissue sections with known high expression of Piezo1 as a positive control and using PBS buffer instead of the primary antibody as a negative control;

[0038] Ten cases were randomly selected from these 50 samples. While performing immunohistochemical testing, adjacent FFPE tissue sections or corresponding fresh frozen tissue samples were taken for Western blotting. The expression of the target protein was verified using the same anti-Piezo1 antibody. The specificity and reliability of the immunohistochemical method were evaluated by comparing the consistency of the two methods in judging the Piezo1 expression level (high or low) in the same case.

[0039] (6) To evaluate the stability and reproducibility of the established detection procedure, this embodiment designed two levels of reproducibility testing. First, the same trained laboratory technician independently performed a second immunohistochemical staining and H-score scoring on all 50 samples on different dates at two-week intervals, using the same batch of reagents, following the exact same standard operating procedure. Then, 20 samples were randomly selected from these 50 samples, and another laboratory technician, unaware of the first results, independently completed the entire process from slide preparation to scoring on a different experimental device using a different set of separately packaged reagents. Finally, the reproducibility of the method between different operators in the laboratory was quantitatively evaluated by calculating the intra-group correlation coefficient and inter-group correlation coefficient.

[0040] Please see Figure 3 The present invention provides embodiment 2:

[0041] The purpose of this embodiment is to explore the relationship between Piezo1 expression typing and existing clinicopathological parameters, thereby clarifying its value as an independent biomarker.

[0042] This study collected a retrospective cohort of 320 breast cancer patients with complete clinicopathological data as the sample for this embodiment.

[0043] (1) Immunohistochemical staining of Piezo1 protein was performed on each sample according to the standardized procedure established in Example 1. After staining, two pathologists who were unaware of any clinical information of the patients independently reviewed the slides using a double-blind method. Each slide was scored strictly according to the predefined scoring criteria (i.e., H-score calculated based on the percentage of positive cells and staining intensity). If the initial scores of the two pathologists differed by more than 2 points, they reached a consensus through joint microscopic review and negotiation, and finally obtained the confirmed H-score for each sample. Then, the threshold determined by the previous training set and having the best prognostic efficacy (set to H-score ≥ 4 points in this example) was applied to clearly divide all 320 patients into two groups: "Piezo1 high expression type" and "Piezo1 low expression type".

[0044] (2) In this embodiment, the original detection results of key markers such as estrogen receptor, progesterone receptor, HER2 and Ki-67 in these 320 samples were centrally reviewed and uniformly interpreted. According to the latest international breast cancer treatment guidelines, the expression of ER and PR was reassessed by immunohistochemistry (positive threshold ≥1% of tumor cell nuclear staining). The HER2 status was confirmed by immunohistochemistry combined with fluorescence in situ hybridization (IHC 3+ or FISH amplification was positive), and the Ki-67 proliferation index was assessed. Then, based on these reviewed data, each breast cancer case was strictly classified into four traditional molecular subtypes: Luminal A, Luminal B, HER2 overexpression, or triple-negative breast cancer, thereby obtaining standardized clinicopathological classification data that can be cross-compared with the new Piezo1 classification.

[0045] (3) The Piezo1 classification results (high / low expression) obtained in the first two steps are integrated with traditional clinicopathological parameters (such as age, tumor size, lymph node status, histological grade) and traditional molecular subtypes (Luminal A, Luminal B, HER2+, TNBC) into a unified statistical analysis database. For the correlation analysis between categorical variables, this embodiment uses the chi-square test for statistical testing and calculates its P value to assess whether the observed distribution differences are statistically significant. Through these analyses, it is revealed whether there is a significant co-occurrence relationship between high Piezo1 expression and known invasive features (such as high grade, lymph node metastasis, late stage) or specific molecular subtypes (such as TNBC), thereby preliminarily defining the biological and clinical feature spectrum of Piezo1-high expression tumors from a clinicopathological perspective.

[0046] Table 1. Association analysis between Piezo1 expression subtypes and clinicopathological features of breast cancer (n=320)

[0047] clinicopathological features Total number of cases Piezo1 overexpression (n=128) Piezo1 low expression (n=192) p-value Histological grading (Grade III) 142 78(61.0%) 64(33.3%) <0.001 Positive lymph node metastasis 158 82(64.1%) 76(39.6%) <0.001 TNM staging (stages III / IV) 103 65(50.8%) 38(19.8%) <0.001 Molecular typing (TNBC) 68 42(32.8%) 26(13.5%) <0.001 HER2 positive 64 30(23.4%) 34(17.7%) <0.001 Luminal type A 104 22(17.2%) 82(42.7%) <0.001

[0048] As shown in the table above, high Piezo1 expression is significantly associated with more aggressive clinicopathological features, such as higher histological grade, higher lymph node metastasis rate, and later TNM stage. It is also enriched in triple-negative breast cancer (TNBC) but less prevalent in the better-prognostic Luminal A type. This suggests that high Piezo1 expression defines a more aggressive subgroup of breast cancer, and its distribution differs from HER2 status, representing an independent correlation dimension.

[0049] Please see Figure 4 and Figure 5 The present invention provides embodiment 3:

[0050] The purpose of this embodiment is to evaluate the predictive value of Piezo1 expression typing for the prognosis of breast cancer patients. The experimental samples are the same as in Example 2.

[0051] (1) For the same cohort of 320 breast cancer patients with complete follow-up information in Example 2, this example defines "disease-free survival" (the time from the date of surgery to the first occurrence of local recurrence, distant metastasis, or death from breast cancer) and "overall survival" (the time from the date of surgery to death from any cause) as the primary endpoints for assessing prognosis. The Piezo1 genotyping results (high expression or low expression) of each patient completed in Example 2 were matched and integrated with their corresponding survival time and endpoint event status to form data for subsequent survival analysis.

[0052] (3) Based on the data integrated in the previous step, this embodiment uses disease-free survival and overall survival as indicators, and uses statistical software to draw Kaplan-Meier survival curves for the Piezo1 high-expression patient group and the low-expression patient group respectively. The curve can intuitively show the difference in survival probability between the two groups of patients over time. Then, the Log-rank test is used to statistically compare the two survival curves, calculate their chi-square value and corresponding P value, and thus determine whether the difference in survival rate between the two groups of patients is statistically significant.

[0053] (3) In order to exclude the influence of other known confounding factors on the prognostic analysis and to confirm whether the Piezo1 classification is an independent prognostic predictor, this embodiment adopted a multivariate Cox proportional hazards regression model. In this model, the Piezo1 classification (with the low expression type as the reference group) was included as the key independent variable. At the same time, age, tumor size, lymph node metastasis status, histological grade and traditional molecular classification, which are widely recognized clinicopathological prognostic factors, were included as covariates in the model for correction.

[0054] The results showed that patients with high Piezo1 expression had significantly lower multi-year overall survival than those with low expression. After adjusting for all traditional prognostic factors, high Piezo1 expression remained an independent risk factor for poor disease-free survival and overall survival. This embodiment demonstrates that the Piezo1 subtyping method provided by this invention can provide additional prognostic information beyond existing clinicopathological parameters. It can effectively further identify patients with a higher actual risk of relapse and death from those traditionally considered to be at intermediate or even low risk, achieving more refined risk stratification.

[0055] This invention provides embodiment 4:

[0056] This embodiment is used to simulate the application scenario of Piezo1 typing in guiding potential targeted therapies.

[0057] In this embodiment, RNA-seq data and survival information of two batches of TNBC patients receiving different treatments were extracted from a public gene expression database. One batch was a cohort receiving conventional chemotherapy (n=120), and the other batch was a cohort receiving clinical trials containing integrin inhibitors (n=85).

[0058] Using Piezo1 mRNA expression levels from the database as a surrogate marker (significantly positively correlated with protein expression), patients were virtually divided into Piezo1 high-expression and low-expression groups. The study analyzed whether the impact of Piezo1 high expression on patient prognosis changed under different treatment backgrounds.

[0059] Results: In the conventional chemotherapy cohort, high Piezo1 expression was a strong adverse prognostic factor (HR=2.3, P<0.01), consistent with the results in Example 3. In the integrin inhibitor-containing treatment cohort, the survival difference between patients with high Piezo1 expression and those with low expression was significantly reduced (HR=1.4, P=0.15), and the median survival in the high-expression group was significantly longer than historical data from the chemotherapy-only cohort. This example demonstrates that Piezo1-high expression tumors may be more sensitive to treatment targeting mechanical signaling pathways (such as the integrin pathway).

[0060] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A breast cancer classification method based on Piezo1 expression, characterized in that, It includes the following steps: S1, Obtain tumor tissue samples from breast cancer patients; S2, The expression level of Piezo1 protein in the tumor tissue sample was detected by immunohistochemistry, specifically including staining with a validated anti-Piezo1 monoclonal antibody and scoring by two or more pathologists in a double-blind manner; S3, based on the Piezo1 protein expression score, classifies breast cancer into two subtypes: S4 integrates the above classification results with clinicopathological parameters to guide individualized treatment decisions.

2. The breast cancer classification method based on Piezo1 expression according to claim 1, characterized in that, The two subtypes of breast cancer are as follows: Piezo1 high expression type, immunohistochemical score ≥4 points (based on the product of positive cell percentage and staining intensity, where positive cell percentage >50% and staining intensity is strong positive is 3 points, positive cell percentage 30-50% and staining intensity is medium to strong positive is 2 points, positive cell percentage <30% and staining intensity is weak positive is 1 point, no staining is 0 points, the total score range is 0-12 points, and the threshold is set at ≥4 points). Piezo1 low expression type, immunohistochemical score <4.

3. The breast cancer classification method based on Piezo1 expression according to claim 2, characterized in that: Patients with high Piezo1 expression should be given priority for treatment regimens that include inhibitors of mechanotransmission pathways or integrin-targeting drugs.

4. The breast cancer classification method based on Piezo1 expression according to claim 3, characterized in that: The tumor tissue sample was either formalin-fixed paraffin-embedded tissue or a fresh frozen tissue sample.

5. A breast cancer typing method based on Piezo1 expression according to claim 4, characterized in that: The anti-Piezo1 monoclonal antibody used in the immunohistochemical method is a cross-validated equivalent antibody.

6. The breast cancer classification method based on Piezo1 expression according to claim 5, characterized in that: The method also includes simultaneously detecting the expression of estrogen receptor, progesterone receptor, HER2 and Ki-67 in the same tumor tissue sample, and integrating the Piezo1 expression subtype with the traditional molecular subtype to form a composite subtype.

7. A breast cancer classification method based on Piezo1 expression according to claim 6, characterized in that: When a tumor is classified as having high Piezo1 expression and is also a triple-negative breast cancer, a combination of Piezo1 pathway inhibitors and immune checkpoint inhibitors is used.

8. A breast cancer classification method based on Piezo1 expression according to claim 7, characterized in that: The determination of the Piezo1 overexpression genotype was further validated by qPCR or Western Blot. In qPCR, the relative expression level of Piezo1 mRNA was more than 3 times higher than that of adjacent normal tissue, and / or in Western Blot, the expression level of Piezo1 protein was more than 2 times higher than that of internal reference protein.

9. A breast cancer classification method based on Piezo1 expression according to claim 8, characterized in that: The method also includes simultaneously detecting the colocalization of Piezo1 and tumor microenvironment markers on the same tissue section using multiplex immunofluorescence technology. Cases with high Piezo1 expression and colocalization ≥20% with the tumor-associated fibroblast marker α-SMA are further classified as "mechanically activated microenvironment".

10. A breast cancer classification method based on Piezo1 expression according to claim 9, characterized in that: The subtyping results were associated with patient prognostic assessment, with the Piezo1 high expression type corresponding to an independent hazard ratio of disease-free survival to overall survival ≥1.5.

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