TGF-β1 and Its Signal Transduction Pathways: Are They Corelated With The Elastic Characteristics of Breast Lesions?

Background: Shear wave elastography (SWE) can evaluate the tissue stiffness. Previous studies showed that the elastic characteristics of breast lesions were related to the components of extracellular matrix (ECM) which was directly or indirectly regulated by transforming growth factor beta 1(TGF-β1). However, it is rarely reported whether there is a correlation between TGF-β1 and the elastic characteristics of breast lesions. The purpose of this study was to investigate the relationship between TGF-β1 with its signal transduction pathways and the elastic characteristics of breast lesions. Methods: 135 breast lesions in 130 patients were included. Before operation or biopsy, SWE was performed. Elastic characteristics such as the maximum, mean, minimum and standard deviation (SD) of elastic modulus (Emax, Emean, Emin, Esd), the elastic ratio of the lesions to the peripheral tissue (Eratio) and the "stiff rim sign" were recorded. The expression levels of TGF-β1, Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT were detected by immunohistochemistry. The elastic characteristics and the expression levels of the above-mentioned indexes of benign lesions were were analyzed. Results: Emax, Emean, Esd, Eratio, “stiff rim sign” detection rate and the expression levels of TGF- β 1, et al. of benign were lower than those of malignant lesions (P(cid:0)0.0001). The expression levels of TGF- β 1, Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT were correlated with Emax, Emean, Esd, Eratio of breast lesions, the expression levels of TGF- β 1, et al. of lesions with “stiff rim sign” were higher than those of lesions without “stiff rim sign” (P(cid:0)0.05). And the expression levels of Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT were corelated with that of TGF- β 1 (r=0.678, 0.633, 0.645, 0.611, 0.589, 0.663, P(cid:0)0.0001). Conclusions: The expression levels of TGF-β1, et al. of breast lesions were corelated with the elastic characteristics, the expression levels of Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT were corelated with that of TGF- β 1, which speculated that TGF- β 1 might play an important role in the stiffness regulation of breast lesions through multiple signal transduction pathways. sign" and axillary lymph node metastasis, and the expression level of TGF-β1 was the main factor affecting the Emax, Emean, Esd, Eratio, "stiff rim sign", the expression levels of Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT were also correlated with the expression level of TGF-β1, which speculated that TGF-β1 might play an important role in the stiffness regulation of breast lesions through multiple signal transduction pathways.


Introduction
A number of criteria were considered when including patients: I. Pathological results obtained by VAB or surgical excision; II. Clear and de nite pathological diagnosis; III. Lesions haven't undergone neoadjuvant chemotherapy or radiotherapy; IV. No serious diseases of heart, lung, liver, kidney, etc. or malignant lesions; V. Patients can tolerate biopsy or surgical excision procedure; VI. Comprehensive information of clinical, ultrasound, pathology prognosis and follow-up; VII. Written informed consent was obtained.

SWE examination
Aixplorer ultrasound system (SuperSonic Imagine, Aix en Provence, France) with a L15-4 linear array probe was used for SWE examination. Ultrasound examination was performed at the site of the lesion rstly, and the best section to show the lesion was selected, and then switched to the SWE mode. Keep the patient to breathe smoothly, and the probe was placed at the site of the lesion with no pressure for 3 seconds to stabilize the image, then froze and store it. And after that the region of interest (ROI) was chosen. When choosing ROI, try to keep ROI to cover all parts of the lesion, especially the stiffest part.
Then the maximum elastic modulus (Emax), mean elastic modulus (Emean), minimum elastic modulus (Emin), the standard deviation of elastic modulus (Esd) was recorded, and the elasticity ratio (Eratio) of the lesion shown by ROI to the peripheral normal breast tissue at the same depth was also recorded.
Repeat the above steps in ve different sections of the lesion. Calculate and record the mean value of Emax, Emean, Emin, Esd and Eratio in these ve sets of data. Besides, the appearance of the red area of increased stiffness with or without open or closed ring at the edge of the lesion ("stiff rim sign") was observed.

Immunohistochemistry
The samples were xed in formalin and embedded in para n, and then cut into sections with a thickness of 4 μm, and then TGF-β1, Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT expression was evaluated by immunohistochemistry. Image-Pro Plus 6.0 was used for semi-quantitative analysis of immunohistochemical results. Five ROIs were randomly selected from each sample under the 400x eld of view and photographed to measure the integrated optical density (IOD) and area. The yellow area is the positive expression area. Expression levels of TGF-β 1 and other factors were expressed by average optical density (IOD/area).
Statistical analysis SPSS 26.0, standard version (SPSS Inc., Chicago, IL, USA) statistical software was used for statistical analysis. The quantitative data were expressed as (x±s) and the qualitative data were expressed as %. Student's t test was used to compare the differences between groups of quantitative data, and c 2 test was used to compare the differences of qualitative data. Taking the pathological results as the "gold standard", the receiver operator characteristic curve (ROC) of each factor were drawn respectively, and the e cacy was evaluated by the area under the curve (AUC), and the cutoff value, sensitivity and speci city were analyzed, and the differences among AUC were compared by Z test. Spearman rank correlation test was used for correlation analysis. P 0.05 was considered as the difference was statistically signi cant.

Analysis of elastic characteristics of breast lesions
The elastic characteristics of benign and malignant breast lesions were seen in Table 1, Figure 1A and Figure 2A. The Emax, Emean, Esd and Eratio of benign breast lesions was signi cantly lower than those of malignant lesions (P < 0.001), but there was no signi cant difference in Emin between benign and malignant breast lesions (P 0.202). Besides, the detection rate of "stiff rim sign" in malignant lesions was signi cantly higher than that of benign lesions (P < 0.001).

Expression levels of TGF-β 1 and other factors in breast lesions
The expression levels of TGF-β 1 and other factors in breast lesions were seen in Table 2, Figure 1B~H and Figure 2B~H. Immunohistochemical staining showed that TGF-β 1 and PI3K were mainly expressed in the cytoplasm, while Smad2/3, Erk1/2, p38 MAPK, JNK2 and AKT were mainly expressed in the cytoplasm and nucleus. And the expression levels of TGF-β 1 and other factors in malignant breast lesions were signi cantly higher than those in benign lesions (P < 0.001).
When using the expression levels of TGF-β1 and other factors as an index respectively for the differential diagnosis of benign and malignant breast lesions, the ROC curve was shown in Figure 3

Correlation analysis
The correlation between the expression levels of TGF-β1 and other factors and elastic characteristics in breast lesions was shown in Figure 4~7. Spearman test showed that the expression levels of TGF-β1 and other factors were positively correlated with Emax, Emean, Esd and Eratio in breast lesions ( Expression levels of TGF-β1 and other factors in breast lesions with and without "stiff rim sign" could be seen in Table 3. Immunohistochemical staining showed that the expression levels of TGF-β1 and other factors in breast lesions with "stiff rim sign" were signi cantly higher than those in breast lesions without "stiff rim sign" (P 0.001).

Axillary lymph node metastasis
Expression levels of TGF-β1 and other factors in malignant breast lesions with and without axillary lymph node metastasis could be seen in Table 4. The difference between malignant breast lesions with axillary lymph node metastasis and malignant breast lesions without axillary lymph node metastasis lied in that the expression levels of TGF-β 1 and other factors of former were signi cantly higher than those of the latter (P < 0.05).
Basing on the expression levels of TGF-β 1 and other factors in malignant breast lesions, ROC curves of which for differential diagnosis of malignant breast lesions with and without axillary lymph node metastasis were shown in Figure 9. 0.2348 respectively, the sensitivity and speci city for differential diagnosis of malignant breast lesions with or without axillary lymph node metastasis were 86.7% and 83.3%, 80.0% and 62.5%, 80.0% and 58.3%, 69.2% and 65.2%, 50.0% and 80.0%, 86.7% and 40.9%, 42.9% and 91.3%, respectively.

Quantile regression analysis
As shown in Table 5~8, the percentile regression analysis was performed with the expression levels of TGF-β 1, Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT in breast lesions as independent variables and Emax, Emean, Esd and Eratio as target variables respectively. At different quantiles, only the expression level of TGF-β1 always had a signi cant positive effect on Emax, Emean, Esd and Eratio, while Smad2/3 only had a certain effect on Emean at the point of 0.75th quartile, a negative effect on Eratio at the point of 0.45th quartile. Erk1/2 only had a certain effect on Emean at the 0.75th quartile. That is, the expression level of TGF-β1 is the most important factor to determine the Emax, Emean, Esd and Eratio of breast lesions.

Logistic regression analysis
Taking the expression levels of TGF-β1, Smad2/3, Erk1/2, p38MAPK, JNK2, PI3K and AKT in breast lesions as independent variables and "stiff rim sign" as dependent variable, logistic regression analysis was performed. The logistic regression equation was established as follows: Logit (P) =-5.977 17.049 X 1 X 1 represented the expression level of TGF-β1, that is, the expression level of TGF-β1 was the main factor determining the presence or absence of "stiff rim sign", as shown in Table 9.
Discussion SWE could quantitatively evaluate the elastic characteristics of breast lesions and more accurately judge the benign and malignant breast lesions. Our study showed that there were signi cant differences in elastic characteristics between benign and malignant breast lesions, which were the same as those of previous studies [8,20]. For malignant breast lesions, a large number of cancer cells proliferated, invaded and in ltrated the normal breast stroma and adipose tissue, and interacted with the tumor microenvironment, causing complex connective tissue hyperplasia and brosis, which were important typical pathological changes in the process of tumor development and invasion, they could lead to overexpression and accumulation of ECM components, rearrangement and cross-linking of ECM structure [21,22], and increase the stiffness of the lesions. At the same time, the proliferation and brosis of connective tissue caused by the continuous invasion of cancer cells made the broblasts grow to the edge of the lesion [23], according to the number of collagen and elastic bers and the degree of brosis, the lesions exerted different degrees of tension on the surrounding tissue [24], and elastin and brin would harden under stretching [25], thus increasing the stiffness of the tissue around the lesion by 2-10 times. As a result, the "stiff rim sign" appeared.
Our study found that the expression levels of TGF-β1 and other factors in malignant breast lesions were signi cantly higher than those in benign breast lesions. It was not di cult to understand such results. TGF-β had both inhibitory and promoting effects on tumor cells [26]. In the early stage of tumorigenesis, TGF-β could induce tumor cell apoptosis and inhibit tumor growth through the TGF-β/Smad signal pathway, but with the occurrence and development of tumor, most cancer cells could secrete TGF-β, and the elevated level of TGF-β could promote the occurrence and development of tumor [27]. The proliferation and invasion of cancer cells led to the activation of TGF-β1 and the increase of TGF-β1 expression, which affected the growth of cancer cells and promoted the transformation of normal broblasts into cancer associated broblasts (CAFs) [28,29]. CAFs could interact with cancer ECM to promote the growth, development, invasion and metastasis of cancer, thus further increasing the expression level of TGF-β1. For TGF-β1 could participate in almost the whole process of occurrence, development, invasion and metastasis of breast lesions through TGF-β1/Smad, TGF-β1/MAPK, PI3K/AKT and other signal transduction pathways, the expression levels of TGF-β1 and other factors in malignant breast lesions were naturally higher than those in benign lesions. These also explained another result of this study: the expression levels of TGF-β1 and other factors in malignant breast lesions with or without axillary lymph node metastasis were signi cantly different.
The study also showed that the expression levels of TGF-β1 and other factors in breast lesions had a certain value in the differential diagnosis of benign and malignant breast lesions, suggesting that TGF-β1 and other factors might be used as new indexes for differential diagnosis of benign and malignant breast lesions and new breakthrough points for clinical diagnosis and treatment.
In our study, the expression level of TGF-β1 was found to be correlated with Emax, Emean, Esd, Eratio, and the expression level of TGF-β1 in breast lesions with "stiff rim sign" was signi cantly higher than those in breast lesions without "stiff rim sign". According to the previous studies, on the one hand, TGF-β1 could promote the activation and production of CAFs. CAFs mainly synthesizes and secretes ECM proteins and proteins related to ECM remodeling, which in turn promotes the excessive accumulation of ECM components and the remodeling of ECM structure[18, 29,30]. On the other hand, TGF-β1 could directly stimulate the synthesis and cross-linking rearrangement of collagen, elastin and laminin, and inhibit the activity of enzymes that degrade ECM components to inhibit their degradation, lead to excessive accumulation of ECM components and structural changes of ECM, and increased ECM stiffness [22,31]. In addition, TGF-β1 could also improve cell adhesion by promoting cancer cell synthesis and secretion of a variety of proteases, resulting in cancer cells adhering to the surrounding breast stroma and adipose tissue, resulting in reduced lesion activity and increased lesion stiffness[32].
In the process of carcinogenesis and development, the signal transmitted by TGF-β1 could be transmitted to the nucleus through TGF-β1/Smad signal The study also found that the expression levels of Smad2/3, Erk1/2, p38MAPK, JNK2, PI3K and AKT in breast lesions were correlated with that of TGF-β1, which further suggested that TGF-β1 might indeed participate in almost the whole process of occurrence, development, invasion and metastasis of breast lesions through signal transduction pathways such as TGF-β1/Smad, TGF-β1/MAPK, PI3K/AKT. It was also found in the study that the expression level of TGF-β1 was the main factor affecting the elastic characteristics of breast lesions, such as Emax, Emean, Esd, Eratio, and the expression level of TGF-β1 was the independent risk factor for the presence of "stiff rim sign", which suggested that TGF-β1 might play an important role in the regulation of elastic characteristics of breast lesions, or it might become a new breakthrough point for differential diagnosis of benign and malignant breast lesions and a potential target for treatment.
There are some limitations in this study. In this study, the main components of ECM such as collagen bers and elastic bers were not detected, so the relationships between collagen bers, elastic bers and the expression levels of TGF-β1 and other factors and the elastic characteristics of breast lesions were not analyzed, so as to further explore whether TGF-β 1 regulates the stiffness of breast lesions by regulating ECM, and whether conventional ultrasound features were related to the expression levels of TGF-β1 and other factors were not analyzed, either. Therefore, the next step is to carry out the above two aspects of research, in order to make the study more in-depth and comprehensive.
In conclusion, the expression levels of TGF-β1 and other factors in breast lesions were correlated with Emax, Emean, Esd, Eratio, "stiff rim sign" and axillary lymph node metastasis, and the expression level of TGF-β1 was the main factor affecting the Emax, Emean, Esd, Eratio, "stiff rim sign", the expression levels of Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT were also correlated with the expression level of TGF-β1, which speculated that TGF-β1 might play an important role in the stiffness regulation of breast lesions through multiple signal transduction pathways.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
We declare that we have no nancial and personal relationships with other people or organizations that can inappropriately in uence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as in uencing the position presented in, or the review of, the manuscript entitled.        Note: the values in parentheses were t value, *P 0.05 **P 0.01 Table 9 The results of logistic regression analysis for determining the presence or absence of "stiff rim sign"  Figure 1 SWE and immunohistochemical images of invasive breast cancer in a 38-year-old woman. A: SWE showed that the Emax was 226.0 kPa, the Emean was 159.3 kPa, the Esd was 37.4 kPa, and the Eratio was 20.0, and the "stiff rim sign" (the white arrow) can be seen; B~H: Immunohistochemical staining showed that the expression of TGF-β1, Smad2/3, Erk1/2, p38 MAPK, JNK2, PI3K and AKT was strong or moderate positive, and the average optical density was 0.349, 0.342, 0.355, 0.172, 0.296, 0.373, 0.324, respectively (× 400).