Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS).

OBJECTIVE: This study is to explore the correlation between the contrast-enhanced ultrasound (CEUS) characteristics of breast cancer and the epithelial-mesenchyme transformation (EMT).
METHODS: Totally 119 patients of breast cancer underwent CEUS. Tissues in the active area were collected and subjected to the immunohistochemical detection, PT-PCR and Western blot. Correlation analysis was conducted between the clinical pathological parameters and the CEUS indicators.
RESULTS: The expression levels of CD44, N-cadherin, and β-catenin in breast cancer tissues were higher than those in adjacent tissues (P<0.05). However, the expression levels of CD24 and E-cadherin in breast cancer tissues were lower than those in adjacent tissues (P<0.05). There was no significant difference in E-cadherin mRNA and Vimentin levels between cancer and adjacent tissues (P>0.05). The expressions were up-regulated in the CSCs, with higher histological grade, lymph node metastasis, and negative estrogen receptor (ER) expression. Smaller breast tumors, with no lymph node metastasis, lower clinical stage, and positive ER expression, tended to exhibit the up-regulated epithelial phenotype. Breast tumors, with high histological grade, lymph node metastasis, high clinical staging grade, and negative ER expression, tended to exhibit the up-regulated interstitial phenotype. The peak intensity of the time-intensity curve (TIC) for the CEUS was positively correlated with the CSC marker CD44 and the interstitial phenotype marker N-cadherin. The starting time of enhancement was negatively correlated with the N-cadherin. Area under the curve was positively correlated with the expression of CD44 and N-cadherin, while negatively correlated with the epithelial phenotype marker β-catenin. The time to peak was negatively correlated with the interstitial phenotypes Vimentin and N-cadherin, with no correlation with the E-cadherin or β-catenin.
CONCLUSION: Breast cancers show the enlarged lesions after enlargement and perfusion defect for the CEUS. The fast-in pattern, high enhancement, and high perfusion in the TIC are correlated with the CSCs and EMT expressions, suggesting poor disease prognosis.

Introduction

Breast cancer accounts for 23% of the malignant tumors, and early screening and prognosis assessment is of great significance for the breast cancers [1]. At present, the puncture biopsy has been commonly used in the diagnosis of breast cancer. However, due to the tumor tissue heterogeneity, the pathological results of the punctured tumor tissue might be limited [2], and the puncture biopsy is usually invasive. Therefore, non-invasive assessments for breast are needed for the prognosis of breast cancers. The relationship between the ultrasound features and the biological features of breast cancers has been quantitatively evaluated based on the radiomics, and the accuracy of predicting the hormone receptor expression in breast cancer is as high as 67.7%, indicating that the tumor features could be obtained at the genetic and cellular levels through the ultrasound images [3].

Contrast-enhanced ultrasound (CEUS) can display the microvascular structure within the tumor in real time, with a high spatial and temporal resolution. CEUS can visually exhibit the characteristics of the microcirculation within the tumor, which can be quantitatively analyzed besides the qualitative evaluation [4]. In our previous study, the qualitative CEUS indicators well correlated with the prognostic factors (i.e., the tumor size, histological grade, and clinical stage) have been screened and selected, including the perfusion defect, enhancement range, enhancement degree, and time to peak, peak intensity, starting time of enhancement, and area under the curve [5].

Cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) are key regulators for the breast cancer aggressiveness [6]. Breast cancer CSCs have the self-renewal and multi-directional differentiation abilities, which are closely related to the biological behavior of tumorigenesis, proliferation, metastasis, and drug resistance. The EMT refers to the loss of tight junctions between cells, loss of polarity, and gaining the mesenchymal cell characteristics, accompanied by the up-regulated N-cadherin expression and down-regulated E-cadherin expression, which has been shown to be related to the tumor invasion, metastasis, and treatment resistance [6]. CSCs and EMT cells are closely related with each other, which share similar characteristics.

It has been shown that CD44 and VEGF are co-expressed in cancer tissues [7], suggesting synergistic effects on tumor microangiogenesis and metastasis. The occurrence of EMT in the CSCs would promote the proliferation and migration of fibroblasts and induce angiogenesis, also supporting the association between the CSCs and breast cancer microvessels. CEUS based on microcirculation perfusion can not only display the tumor blood vessels, but also provide the evidence concerning the microcirculation hemodynamics, which might contribute to the investigation of the aggressiveness of breast cancer [8]. However, the extent to which the CEUS characteristics of breast cancer can reflect the distribution of CSCs and the EMT extension is still unclear.

In this study, the macroscopic imaging-based CEUS technology was used to reflect the tumor microcirculation perfusion for the imaging analysis of breast cancer lesions. More active cancerous tissues were obtained through puncture under the real-time guidance of CEUS. The presence of CSCs and the extension of EMT in these tissues were analyzed, and its correlation with CEUS characteristics was also studied, in order to screen for the CEUS indicators that better reflect breast cancer aggressiveness and prognosis.

Materials and methods

Study subjects

A total of 119 females with breast cancer who were admitted to our hospital from January 2017 to October 2018 were included in this study, with a median age of 46 years. Inclusion criteria were as follows: patients meeting the diagnostic criteria for breast cancer, who had received CEUS, without mental disorders. Exclusion criteria included: patients with pregnancy; with moderate to severe anemia; in menstrual periods; with vaginal bleeding of unknown reason; or having previously received radiation, chemotherapy, and/or surgical treatment. All patients underwent CEUS examination before puncture. The specimens were collected from the most representative plane under the guidance of CEUS. The obtained tissue specimens were subjected to immunohistochemical examination, PT-PCR and Western blot analysis. The study was approved by the Ethics Committee of the Tumor Hospital Affiliated to the Xinjiang Medical University, and the written informed consent was obtained from each patient.

CEUS analysis

The CEUS analysis was performed with the Logic E9 color Doppler ultrasound system (GE, Boston, USA), with a 9L-4 probe and the mechanical index of 0.16. For detection, the patient was lying on his back, and the morphology, size, and edge of the tumor was checked with the 2D ultrasound, with the probe position maintained. Totally 5 ml contrast agent was quickly injected into the elbow vein of the healthy upper limb, followed by 5 ml 0.9% sodium chloride injection. After the contrast agent injection, the dynamic images were recorded for 2 min 30 s, until the enhanced lesion image was reduced. The qualitative indicators of contrast agent perfusion were analyzed, including whether the lesion range was enlarged after enhancement compared with the 2D ultrasound, and whether there was perfusion defect within the lesion. The regions of interest (ROIs) covered the entire lesion area, and the normal breast tissue of the same depth was used as the control area, to obtain the time intensity curve (TIC). The indicators were observed, including the peak intensity, time to peak, starting time of enhancement, and area under the curve (Fig 1). The time to peak was the time between the start of CEUS enhancement and the peak intensity within the ROIs. The peak intensity was the maximum enhanced intensity of the ROIs. The area under the curve was defined as total volume of contrast medium (or blood) traversing the region of interest. The representative sections of CEUS were marked before surgery.

Fig 1

Time intensity curve analysis of contrast-enhanced ultrasound.

The peak intensity, time to peak, rising time, and, the area under the curve were indicated in the figure.

Immunohistochemistry

After surgery, the lesion was dissected and sliced, in consistency of the CEUS sections. The immunohistochemistry was performed with the Envision two-step method, to detect the CSCs markers (i.e., the CD44 and CD24) and the EMT markers (i.e., the E-cadherin, β-catenin, vimentin, N-cadherin). The following primary antibodies were used herein: rabbit anti-E Cadherin monoclonal antibody (1:100 dilution; ab40772; Abcam, Cambridge, MA, USA), rabbit anti-beta Catenin monoclonal antibody (1:100 dilution; ab32572; Abcam), mouse anti-Vimentin monoclonal antibody (1:100 dilution; ab8978; Abcam), rabbit anti-N Cadherin monoclonal antibody (1:100 dilution; ab76011; Abcam), rabbit anti-CD44 polyclonal antibody (1:100 dilution; ab157107; Abcam), and mouse anti-CD24 monoclonal antibody (1:100 dilution; MA5-11833; Invitrogen, Carlsbad, CA, USA). Criteria for immunohistochemical results were as follows: CD44 and E-cadherin were positively expressed in the cell membrane and cytoplasm of tumor cells; CD24 was positively expressed in the cytoplasm of tumor cells; N-cadherin and β-catenin were positively expressed in the cell membrane of tumor cells; Vimentin was positively expressed in the cytoplasm of the tissue around the cancer nest. The scoring criteria were as follows: 5 high-power fields were randomly observed in each section, and 100 tumor cells were counted. The staining results were evaluated by the Fromowitz comprehensive scoring method. The percentage of positive tumor cells was used to obtain a semi-quantitative score, indicating the relative intensity of protein expression. According to the Sinicrope modified method, the percentage of positive tumor cells was calculated as follows: < 5%, 0; 5%-25%, 1; 25%-50%, 2; 50%-75%, 3; and >75%, 4.

Quantitative real-time PCR

Totally 100 mg breast cancer tissue was ground with liquid nitrogen, and total RNA was extracted with TRLZOL. After determining the concentrations, the cDNA was obtained with the revere transcription PCR. Quantitative real-time PCR was performed with the SYBR Green Select Mix on the PCR machine. Primer sequences were as follows: E-cadherin, forward 5’-CGAGAGCTACACGTTCACGG-3’ and reverse 5’-GGGTGTCGAGGGAAAAATAGG-3’; β-catenin, forward 5’-AGCTTCCAGACACGCTATCAT-3’ and 5’-CGGTACAACGAGCTGTTTCTAC-3’; and Vimentin, forward 5’-AGTCCACTGAGTACCGGAGAC-3’ and 5’-CATTTCACGCATCTGGCGTTC-3’; N-cadherin, forward 5’-AGCCAACCTTAACTGAGGAGT-3’ and reverse 5’-GGCAAGTTGATTGGAGGGATG-3’; CD44, forward 5’-CTGCCGCTTTGCAGGTGTA-3’ and reverse 5’-CATTGTGGGCAAGGTGCTATT-3’; CD24, forward 5’-CTCCTACCCACGCAGATTTATTC-3’ and 5’-AGAGTGAGACCACGAAGAGAC-3’; and hsa actin, forward 5’-ACAGAGCCTCGCCTTTGCC-3’ and reverse 5’-GAGGATGCCTCTCTTGCTCTG-3’. The 10-μl PCR reaction system consisted of 1 μl cDNA template, 5 μl Mix, 0.7 μl primer each, 0.05 μl ROX, and 2.55 μl RNase-free water. The reaction conditions were as follows: 95°C for 2 min; 95°C for 30 s for totally 40 cycles; and 60°C for 30 s for totally 40 cycles.

Western blot analysis

Cancer tissues and para-cancer tissues were subjected to RIPA lysis. After centrifugation, total proteins were obtained. The protein concentration was determined by the BCA method. Then the proteins were separated by SDS-PAGE and transferred onto the PVDF membrane (Cat# IPVH00010; Millipore). After blocking, the membrane was incubated with primary antibodies: anti-E-cadherin (1:10000 dilution; ab40772; Abcam), anti-β-catenin (1:5000 dilution; ab32572; Abcam), anti-vimentin (1:1000 dilution; ab8978; Abcam), anti-N-cadherin (1:5000 dilution; ab76011; Abcam), anti-CD44 (1:2000 dilution; ab157107; Abcam), anti-CD24 (1:200 dilution; MA5-11833; Invitrogen), and anti-β-actin (1:800 dilution; D110001; Shanghai Health Worker, Shanghai, China) at 4°C overnight. After washing, the membranes were incubated with the secondary antibody at room temperature for 1 h. After washing, the signals were detected using Chemiluminescence Imaging System (ChemiScope Mini 3300; Shanghai Qinxiang Scientific Instrument Co., Ltd., Shanghai, China).

Clinical pathological data collection

The subjected were divided into the group younger than 46 years and the group no younger than 46 years. According to the tumor sizes, the lesions were divided into the ≤1 cm, 2–5 cm, and ≥5 cm groups. According to the histological grades, the lesions were divided into the 1–2 grade and 3 grade groups. Moreover, the subjects were divided into the lymph node metastasis and none lymph node metastasis groups. According to the clinical stages, they were divided into the ≤2 stage group and the ≥3 stage group. Moreover, the subjects with the Luminal A and B types were classified as the hormone receptor-dependent group, while the subjects with the her-2 over-expression and triple-negative breast cancer were classified as the non-hormone receptor-dependent group.

Statistical analysis

Data were expressed as mean±SD. The SPSS 21.0 software was used for statistical analysis. If the data conformed to the normal distribution, the single-factor analysis of variance was performed. Multiple comparisons were performed with the sidak method for the data with homogeneity of variance, while the Tamhane method was used for the data with the heterogeneity of variance. The data without normal distribution were first subjected to the logarithmical transform for normalization, and then the above-mentioned one-way analysis of variance method was performed for statistical analysis, or the Wilcoxon rank sum test was used. Correlation analysis of the expression index was performed with the Pearson correlation. P < 0.05 was considered as statistically significant.

Results

Analysis of clinicopathological characteristics of study subjects

The included 119 patients aged from 29 to 81 years, with a median age of 46 years. The premenopausal patients accounted for 60.5% (n = 72), and the postmenopausal patients accounted for 39.5% (n = 47). Moreover, 53.8% of patients had menarche later than 13 years old. All 119 specimens were confirmed by pathology, including 12 cases (10.1%) of in situ ductal carcinoma, 91 cases (76.5%) of invasive ductal carcinoma and invasive lobular carcinoma, and 16 cases (13.4%) of invasive ductal carcinoma with in situ carcinoma and invasive lobular carcinoma with in situ carcinoma. According to the histological grades, in these 119 specimens, there were 5 cases of grade 0, 10 cases of grade I, 71 cases of grade II, and 33 cases of grade III. All patients underwent the axillary lymphadenopathy, and 29 of them showed metastases. Moreover, there were 15 cases of Luminal A type, 42 cases of Luminal B type, 30 cases of her-2 over-expression type, and 32 cases of triple negative breast cancer.

Expression characteristics of CSCs and EMT in these subjects

The representative sections of CEUS were marked before surgery and tissue specimens were collected from these marked sections. The expression levels of CSCs and EMT proteins in these specimens were investigated with the immunohistochemistry. Our results from the immunohistochemical analysis showed that the expression levels of CD44, β-catenin, and N-cadherin in the breast cancer tissues were up-regulated compared with the normal tissues. Moreover, the expression levels of CD24 and E-cadherin in the breast cancer tissues were down-regulated compared with the normal tissues. However, there were no significant differences in the expression levels of vimentin (Fig 2 and Table 1).

Fig 2

Epithelial-mesenchymal transition (EMT) of breast cancer stem cells.

The expression levels EMT-related factors were detected with immunohistochemistry (Magnification 200× and 40×), including the CD44 (4 vs 2 points for the breast cancer and normal surrounding tissue groups, respectively), CD24 (2 vs 4points), E-cadherin (1 vs 3 points), N-cadherin (4 vs 0 points), β-catenin (3 vs 1 points), and Vimentin (3 vs 3 points).

Table 1

Immunohistochemical score of CSCs and EMT in breast cancer tissues (n = 56).

	CD24	CD44	E-cadherin	N-cadherin	β-catenin	Vimentin
Breast cancer	1.333±1.047	2.931±1.241	1.250±0.500	2.017±1.235	2.586±1.200	3.431±0.797
Normal tissues	2.259±1.278	1.533±1.060	2.948±1.343	0.200±0.561	1.533±1.125	3.250±0.957
Z	-2.531	-3.828	-4.681	-4.634	-2.984	-1.284
P	0.011	<0.001	<0.001	<0.001	0.003	0.199

On the other hand, our results from the quantitative fluorescence analysis showed that, in the breast cancer tissues, the mRNA expression levels of CD44, N-cadherin, and β-catenin were significantly up-regulated, while the mRNA expression levels of CD24 were significantly down-regulated, compared with the normal tissues adjacent to the cancer (Fig 3). There were no significant differences in the mRNA expression levels of vimentin and E-cadherin between the breast cancer tissues and adjacent normal tissues (Fig 3). Moreover, compared with normal tissues adjacent to cancer, the breast cancer tissues had significantly up-regulated protein expression levels of CD44, N-cadherin, and β-catenin, but significantly down-regulated protein expression levels of E-cadherin and CD24 (Fig 4A and 4B). No significant difference was observed in the Vimentin protein expression levels between the cancer and adjacent tissues.

Fig 3

The mRNA expression levels of CSC-related and EMT-related genes in breast cancer tissues (n = 63).

The mRNA expression levels of CD44 (A), CD24 (B), E-cadherin (C), N-cadherin (D), β-catenin (E), and Vimentin (F) were detected with the quantitative real-time PCR. Compared with the normal surrounding tissues, * P < 0.05, ** P < 0.01.

Fig 4

The expression levels of CSCs-related and EMT-related proteins in breast cancer tissues (n = 9).

The protein expression levels of CD44, CD24, E-cadherin, N-cadherin, β-catenin, and Vimentin were detected with the Western blot analysis (A). Statistical analysis of the protein expression levels (B). Compared with the normal surrounding tissues, * P < 0.05.

Correlation between CSC- and EMT-related markers and disease prognostic factors

Correlation between the relative expression levels of CSC- and EMT-related genes and the clinicopathological indicators of breast cancer was investigated. As shown in Table 2, our results showed that the CSCs expression was related to the histological grade, lymph node status, and hormone receptor status, with no relation with the age, tumor size, and clinical stage. Our results showed that the CSCs expression was elevated in the breast cancer tissues with higher histological grade, lymph node metastasis, and ER negative expression. Moreover, the EMT was related to the tumor size, histological grade, lymph node metastasis, clinical stage, and hormone receptor status, with no relation to the patient age. Furthermore, the breast cancer epithelial phenotype expression was up-regulated for the relatively small tumors, as well as the cases with no lymph node metastasis, lower clinical stage, and ER positive expression. However, the expression of interstitial phenotype tended to increase for the breast cancers with higher histological grade, lymph node metastasis, higher clinical stage, and ER negative expression.

Table 2

Relationship between the relative mRNA expression levels of CSC- and EMT -related genes and clinicopathological indicators in breast cancer tissues.

	Age		Statistical analysis	Lesion size			Statistical analysis	Histological grade			Statistical analysis	Lymph node metastasis		Statistical analysis	Clinical stage		Statistical analysis	Molecular pathology typing		Statistical analysis
	< 46 years	≥ 46 years		≤1 cm	2–5 cm	≥5 cm		Grade I	Grade II	Grade III		Yes	No		≤ 2 stage	≥3 stage		Hormone receptor dependent	Non-hormone receptor dependent
CD44	1.18±0.223	1.20±0.236	T = -0.334	1.213±0.113	1.267±0.242	1.221±0.179	F = -0.312	1.07±0.127	1.20±0.260	1.25±0.227	F = -3.160	1.200±0.234	1.015±0.215	T = 3.473	1.161±0.223	1.228±0.231	T = -1.248	1.081±0.210	1.281±0.267	T = -2.202
			P = 0.739				P = 0.733				P = 0.049			P = 0.001			P = 0.216			P = 0.031
E-cadherin	0.87±0.247	0.89±0.316	T = -0.236	1.033±0.392	0.789±0.243	0.759±0.290	F = -3.662	0.87±0.318	0.85±0.199	0.95±0.342	F = -0.745	0.888±0.283	1.088±0.245	T = 2.412	0.827±0.246	0.942±0.310	T = -1.741	1.041±0.310	0.830±0.246	T = -2.438
			P = 0.814				P = 0.033				P = 0.479			P = 0.019			P = 0.086			P = 0.018
β-catenin	1.13±0.214	1.15±0.179	T = -0.265	1.302±0.072	1.135±0.156	1.237±0.248	F = -3.241	1.12±0.176	1.09±0.218	1.19±0.175	F = -1.900	1.144±0.178	1.289±0.247	T = 2.464	1.200±0.212	1.088±0.164	T = 2.464	1.149±0.204	1.127±0.182	T = -0.464
			P = 0.792				P = 0.047				P = 0.158			P = 0.016			P = 0.016			P = 0.644
Vimentin	0.99±0.314	0.94±0.264	T = -0.705	1.137±0.469	0.920±0.267	0.936±0.290	F = -1.374	1.00±0.157	1.00±0.371	0.91±0.253	F = -0.691	0.958±0.295	0.979±0.282	T = 2.249	0.965±0.310	0.962±0.268	T = 0.034	0.987±0.331	0.926±0.202	T = -0.860
			P = 0.483				P = 0.262				P = 0.505			P = 0.804			P = 0.973			P = 0.393
N-cadherin	1.13±0.179	1.23±0.365	T = -1.389	1.419±0.635	1.187±0.219	1.286±0.298	F = -1.713	0.97±0.123	1.09±0.125	1.41±0.361	F = -18.647	1.201±0.319	1.004±0.147	T = 2.323	1.105±0.172	1.260±0.366	T = -2.323	1.005±0.171	1.260±0.366	T = -2.260
			P = 0.169				P = 0.191				P<0.001			P = 0.023			P = 0.023			P = 0.029
CD24	0.88±0.159	0.92±.280	T = -0.744	0.776±0.125	0.836±0.236	0.967±0.236	F = -2.035	0.86±0.174	0.89±0.207	0.93±0.272	F = -0.454	0.881±0.228	0.984±0.220	T = 1.569	0.906±0.256	0.898±0.196	T = 0.135	0.905±0.177	0.897±0.296	T = -0.152
			P = 0.460				P = 0.141				P = 0.637			P = 0.121			P = 0.893			P = 0.880

Correlation between tumor microcirculation perfusion characteristics and CSCs/EMT

The correlation between the CEUS performance of breast cancers and CSCs/EMT was investigated. The range of lesions on CEUS was beyond the range of two-dimensional ultrasound in 91 cases (76.5%) of breast cancer. Additionally, 37 cases (31.1%) of breast cancer showed perfusion defects. Using the surrounding normal tissue as control, the peak intensity, time to peak, starting time of enhancement, and the area under the curve were obtained. The time intensity curve of 90 cases (75.6%) showed fast-in mode and high enhancement (Fig 5). Our results showed that, the perfusion defect was observed after CEUS in the breast cancers, suggesting the morphological changes of ischemic necrosis in these breast cancer lesions, which was related to the expression of interstitial phenotype, while not related to the CEUS quantitative parameters. Moreover, the breast cancer lesions with perfusion defect tended to have up-regulated expression of the interstitial marker N-cadherin, while there were no significant differences in the CSCs or the epithelial markers between these groups (Table 3). For the breast cancer with enlarged lesions after enhancement, the TIC tended to be fast-in and high-enhanced, with down-regulated expression of epithelial E-cadherin and up -regulated expression of interstitial N-cadherin (Table 4). Correlation analysis between the CEUS quantitative parameters of breast cancers and the relative expression levels of CSC- and EMT-related mRNAs showed that the peak intensity was positively related to the expression levels of the CSCs marker CD44 and the interstitial marker N-cadherin, while the starting time of enhancement was negatively related to the expression of interstitial marker N-cadherin. Moreover, the area under the curve was positively correlated with the expression levels of the CSCs marker CD44 and the interstitial marker N-cadherin, which was negatively correlated with the expression of the epithelial marker β-catenin. The time to peak was related to the expression levels of the interstitial markers Vimentin and N-cadherin, with no correlation with the epithelial marker E-cadherin or β-catenin (Table 5).

Fig 5

Contrast-enhanced ultrasound (CEUS) of breast cancer.

The representative image of the CEUS in breast cancer was shown, with enlarged lesion range (expanding beyond that of the 2D ultrasound). Perfusion defect could be seen in the central area. Compared with the normal surrounding tissue, the TIC showed the fast-in pattern and high enhancement.

Table 3

Correlation between perfusion defect after breast cancer imaging and relative expression of CSC- and EMT-related genes.

	E-cadherin	β-catenin	Vimentin	N-cadherin	CD44	CD24	Peak intensity	Starting time of enhancement	Area under the curve	Time to peak
No perfusion defect	0.780±0.223	1.151±0.197	0.974±0.366	1.159±0.199	1.264±0.248	0.822±0.160	20.670±7.968	15.833±4.594	1122.021±552.451	23.633±8.177
Perfusion defect	0.925±0.371	1.212±0.165	0.915±0.191	1.334±0.388	1.233±0.171	0.910±0.296	24.692±7.827	13.333±4.733	1275.142±533.454	20.958±6.617
T/Z value	-1.688	-1.204	-0.122	-2.142	0.506	-1.036	-1.858	1.960	-1.028	1.298
P	0.100	0.234	0.903	0.037	0.615	0.300	0.069	0.055	0.309	0.200

Table 4

Relationship between the enhancement and expansion of breast cancer after angiography and the relative mRNA expression of CSC- and EMT-related genes.

	E-cadherin	β-catenin	Vimentin	N-cadherin	CD44	CD24	Peak intensity	Starting time of enhancement	Area under the curve	Time to peak
No enhancement enlargment	0.923±0.325	1.198±0.193	1.022±0.389	1.055±0.198	1.265±0.196	0.783±0.108	19.357±6.955	16.739±4.191	1027.495±556.650	25.913±8.597
Enhancement enlargment	0.738±0.240	1.164±0.180	0.893±0.202	1.297±0.360	1.239±0.232	0.919±0.280	24.759±8.198	13.226±4.695	1310.699±510.972	19.871±5.578
T/Z value	2.306	0.666	-0.875	-2.424	0.436	-1.854	-2.550	2.844	-1.939	3.129
P	0.025	0.508	0.382	0.019	0.665	0.064	0.014	0.006	0.058	0.003

Table 5

Correlation analysis of CEUS parameters of breast cancer with relative mRNA expressions of CSC- and EMT-related genes.

	Correlation coefficient
	E-cadherin	β-catenin	Vimentin	N-cadherin	CD44	CD24
Peak intensity	0.023	0.256	-0.091	0.384	0.445	0.032
P	0.826	0.014	0.389	<0.001	<0.001	0.766
Starting time of enhancement	-0.090	-0.024	0.170	-0.288	-0.164	-0.093
P	0.395	0.823	0.106	0.005	0.117	0.375
Area under the curve	-0.132	-0.402	0.066	0.500	0.496	-0.044
P	0.208	<0.001	0.530	<0.001	<0.001	0.680
Time to peak	-0.042	-0.009	-0.232	-0.246	-0.094	-0.161
P	0.689	0.936	0.026	0.018	0.373	0.126

Discussion

At present, the genetic tests of the penetrating tumor tissues have been commonly used in the disease diagnose and prognosis prediction. However, the heterogeneity of tumor structure often interferes with the accuracy of puncture [9], and CSCs often differentiate into cells with different heterogeneities [10]. The occurrence, development and metastasis of tumors all depend on the formation of microvessels, and the heterogeneity of tumors is also reflected in the microcirculation construction [11]. The 2D ultrasound combined with CEUS can reflect the microcirculation construction of breast cancer [12-14]. Breast cancer cells would cause severe damages to the original normal blood vessels and tissues, which would in turn induce the production of turbulent blood vessels [15]. The microvessel density of the lesions would be significantly increased, which significantly increases the blood flow to the cancer tissues, leading to a significant increase in the flow of the contrast agent in the cancer tissues in the early stage of perfusion [16]. The flow rate is significantly accelerated, the initial perfusion time and the peak time appear earlier, and the peak intensity is significantly increased [14]. However, the vascular wall in cancer tissue is often abnormally absent, resulting in a significant increase in the permeability of the blood vessel. A large number of tumor thrombi form in veins and lymphatic ducts, aggravating the degree of interstitial edema, and the tortuosity of blood vessels makes the contrast agent in the blood vessels. There is a large amount of retention in the vascular bed, so the clearance time of contrast agent in breast cancer is significantly extended, and the area under the curve is significantly increased [14]. In our previous study, many qualitative indicators and quantitative parameters of CEUS had been investigated, which had been shown to reflect the tumor heterogeneity of breast cancer microcirculation construction, therefore screening out the most valuable prognostic factors for breast cancer [4]. In this study, according to the macroscopic images, the representative cancer tissues were screened out based on the CEUS indicators, and subjected to the molecular biological test, and the correlation between the imaging indicators with the breast cancer CSCs and EMT indicators was analyzed, further evaluating the ability of CEUS for the assessment of breast cancer prognosis.

It is indicated that CSCs and the EMT may be linked, and their key signal transduction pathways are intersecting, both playing important roles in tumor metastasis and recurrence [17]. Moreover, the EMT is closely also related to the function of CSCs [17]. In this study, our results showed that, β-catenin, as an epithelial phenotype, had a higher expression rate in the study cases, presumably related to the higher proportion of triple-negative breast cancer in these cases. It has been shown that the β-catenin would be abnormally increased in the tumor invasion and metastasis, as well as over-expressed in the triple negative breast cancers [18]. Among the prognostic factors, the breast cancers with large tumor diameter, high histological grade, cerebral fossa lymph node metastasis, high clinical stage, and negativeness for hormone receptor tended to have up-regulated EMT expression. Moreover, the breast cancers with high histological grade, cerebral fossa lymph node metastasis, and negative hormone receptors expression tended to have up-regulated expression of CSCs. These results suggest that, the up-regulated CSC and EMT expressions might be associated with the rapid proliferation of cancer cells, enlarged tumor lesions, lymphatic and hematogenous metastases, and poor prognosis. It has been shown that the increased expressions of CD44 in the breast cancer tissues determine the tumor progression and metastasis, and the CD44 could weaken the adhesion between tumor cells and enhance the matrix adhesion, promoting the tumor cell migration and metastasis [19]. The expression of E-cadherin would be inhibited by certain factors, leading to the destruction of the interepithelial connection and the weakening of the adhesion between cancer cells. The expression of N-cadherin would lead to the interstitial characteristics, which makes it easy to escape from the primary site and metastasize. Tumor cells would acquire the stem cell-like characteristics during the EMT process, which also proves the close relationship between these two processes [20]. These results suggest that the expression of CSCs and EMT are closely related to the disease prognosis.

The proliferation, differentiation, and metastasis of breast cancers are associated with the tumor blood vessels, and the qualitative and quantitative parameters of CEUS are mainly the macroscopic manifestations of tumor blood vessels [4]. In this study, the correlation between CEUS performance of breast cancer lesions and CSC/EMT was investigated, thus suggesting the relationship between tumor microcirculation perfusion characteristics and CSC/EMT. Perfusion defect in breast cancers after CEUS is also a manifestation of structural heterogeneity. The structural and functional defects of blood vessels in tumor tissues would cause blood flow disturbances and uneven perfusion pattern in the tumor lesions. The rapid proliferation of tumor cells would lead to increased demand for oxygen, finally resulting in hypoxia [20]. Hypoxia can induce tumor cells to develop EMT, and at the same time acquire the characteristics of CSCs, synergistically contributing to the tumor invasion and metastasis [21]. Hypoxia often occurs in larger breast cancers with necrosis, which would be manifested as perfusion defect in the CEUS features. In this study, our results confirmed the high expression levels of interstitial phenotype N-cadherin in the breast cancer with perfusion defect. It has also been confirmed in a previous study that the perfusion defects are related to older age, higher histological grade, and later clinical stage [5], suggesting that it might be related to poor disease prognosis. In the solid tumors, the CSC production, their ability to self-renew, and the undifferentiated state maintenance require the activation of tumor cells under hypoxic conditions to acquire new functional properties [21]. Hypoxia plays an important role in the phenotype and function of CSCs. However, in this study, our results showed that the perfusion defect was not related to other CEUS parameters, suggesting that there may be no difference in the incidence of hypoxic necrosis between breast cancer with rich blood supply and with poor blood supply, that is, breast cancer with perfusion defect is not necessarily the breast cancer with deficient blood supply.

On the contrary, it has been shown that the enhancement and expansion after CEUS often occur in breast cancers with high perfusion and blood supply. The part of the enhancement beyond the lesion area of 2D ultrasound is the marginal zone of breast cancer, i.e., the proliferation and infiltration area. In these proliferation and infiltration areas, the structure is abnormally arranged, and the tumors intersperses into the normal tissues, often accompanied by the hyperplastic fibrous tissue, lymphatic vessels, and blood vessels, resulting in unclear mass boundary [18]. Due to the large number of tortuous and irregularly nourishing blood vessels, vigorously dividing vascular endothelial cells, and arteriovenous fistulas, the CEUS enhancement shows that this area is the most aggressive area in the breast cancer [22]. In this study, our results showed that the breast cancer with enlarged lesions after enhancement was more likely to undergo the EMT. Because EMT is usually associated with poor prognosis, the results of this study also indirectly indicate that enlarged lesions after enhancement are related to poor prognosis of breast cancer.

It has been shown that the TICs for the breast cancer characterized by the fast-in pattern, high enhancement, and high perfusion, are characterized by the high expressions of CSCs and EMT. Moreover, the correlation between the TIC and the interstitial phenotype is stronger than with the epithelial phenotype. When CSCs and EMT are highly expressed, the breast tumors would proliferate rapidly and aggressively. The occurrence of EMT in CSCs will promote the proliferation and migration of fibroblasts, thus inducing the angiogenesis, abundant tumor blood supply, fast blood flow, and elevated average blood volume, with thin tumor vessel wall. Moreover, the arteriovenous fistula is easy to form, which would cause the abnormal high-speed blood flow, as indicated by the fast-in pattern, high enhancement and high perfusion in the TIC.

Conclusions

In conclusion, our results showed that the CEUS performance of breast cancers had a certain correlation with CSCs and EMT. Some of the CEUS indicators may contribute to the prognosis assessment of breast cancer. The enlarged lesions after enhancement, perfusion defect, fast-in pattern, and high enhancement might indirectly indicate the poor prognosis of breast cancer. However, further in-depth studies concerning the application of CEUS imaging omics in clinic, with enlarged sample size and standardized operating procedures, are still needed in the future.

STROBE statement—checklist of items that should be included in reports of observational studies.

(DOCX)

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16 Nov 2020

PONE-D-20-18825

Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)

PLOS ONE

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Reviewer #1: The authors have demonstrated that the correlation between the parameters of less invasive CEUS and the expression level of CSC/EMT markers in breast cancer by IHC and RT-PCR. However, the experimental results are not sufficient to state their conclusions. The authors must evaluate IHC either in the same cell (double staining) or by the staining using serial sections. RT-PCR should use RNA extracted from cancer cells or normal cells recovered by laser microdissection, rather than RNA extracted from whole tissues. Furthermore, it is necessary to use the TaqMan probe method, which has a high quantitative yield, instead of the cybr green method, which is a semi-quantitative method.

Minor points:

1) Please insert scale bars in Fig.1.

2)The information of antibodies of IHC should be written down in Materials and Methods.

3)Please describe the detailed statistical analysis methods in Materials and Methods.

4)There are inappropriate citations, such as quoting a lung cancer paper even though it is a breast cancer citation. Please review the selection of cited papers.

5) There are many misspellings. Please check the spelling.

Reviewer #2: Contrast-enhanced ultrasound (CEUS) procedure carried out with new medical equipment and new-generation micro-bubble contrast reagents has been suggested to increase the accuracy in providing information on the microcirculation. The overall aim of the research described in this manuscript is to assess whether CEUS-based features may provide prognostic type of information. In particular, CEUS-based microcirculation features are examined for correlation with cancer-stem cell (CSC)-like subpopulations including those undergoing epithelial-mesenchymal transition (EMT). The rationale for focusing on tumor cells with CSC and EMT-like characteristics relates to potential roles in tumor heterogeneity and aggressiveness. The manuscript including the background, methods, and results would benefit from better language use and inclusion of information in terms of providing a clearer understanding of the rationale and the objectives of the study, the methodologies used, data interpretation including implications.

Specific Comments-

1. Some of the information presented in the discussion should be considered to be moved to the background to help provide a basis for the study.

2. Several published studies (eg Ji et al, 2017. Clin Hemorheol Microcirc, Zhao et al, 2017 Onco Targets Ther, Cao et al, 2014. Ultrasound Med Biol, Wang et al, 2017, PLoS One, and Vraka et al, 2918 In Vivo) have assessed the correlation between CEUS-based parameters and prognostic markers in breast cancer should be alluded to and referenced in the background and/or discussion.

3. The reason for use of 2D US prior to the CEUS should be included in the method.

4. A figure that depicts the various parameters obtained related to the CEUS describe on page 6 in the methods should be included (see ). The functional implications of these parameters in terms of circulation/perfusion should be clearly stated to help understand their significance when described in the result sections. Use of full names (and not abbreviations) is recommended throughout the manuscript.

5. A rationale should be provided for the criteria used to follow the various biomarkers described in the IHC section of the method. The nature of the antibodies used what they recognize on target proteins should be included. What is the rationale for looking for cytoplasmic localization of CD24? β-catenin localization in cytoplasmic/nucleus can relate to WNT-pathway activation and potentially EMT, given this was this aspect inspected? How epithelial/carcinoma vs stromal cells distinguished? Where antibodies for cytokeratins used?

6. EMT induction requires the expression of transcription factors. Thus, to better show the relationship of the biomarkers used, it will be important to assess the expression (correlation) of some of EMT-TF with the biomarkers and the CEUS-features tested in the current study.

**********

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11 May 2021

Dear editor,

On behalf of all co-authors, I would like to thank you for your letter concerning our manuscript. We greatly appreciate you and the reviewers for the critical reading of our manuscript and giving us the favorable comments and instructive suggestions.

We have carefully proof-read and revised the manuscript according to the reviewers’ comments and the editorial notes. Here we submit the revised manuscript. In the following pages, the responses to the comments are described point-by-point.

Once again, we want to extend our appreciation to you and the reviewers for the valuable and helpful comments. We sincerely hope that the manuscript has been revised to your satisfaction, and we would be grateful if the manuscript could be considered for publication.

In addition, we would like to update our Funding Statement as follows:

This work was supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region (2018D01C276).

Sincerely yours,

Jianbing Ding

Basic Medical College, Xinjiang Medical University, No. 393 Xinyi Road, Urumqi 830011, Xinjiang, China. Tel: 86-18999230065. Email: dingjb1234@ aliyun.com & 1601379937@qq.com

The reviewer’s comments and the authors’ responses:

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have demonstrated that the correlation between the parameters of less invasive CEUS and the expression level of CSC/EMT markers in breast cancer by IHC and RT-PCR. However, the experimental results are not sufficient to state their conclusions.

Response: To validate our conclusions, we have further performed Western blot analysis of CSC-related and EMT-related markers in tumor and tumor adjacent tissues. The results showed that compared with normal tissues adjacent to cancer, the breast cancer tissues had significantly up-regulated protein expression levels of CD44, N-cadherin, and β-catenin, but significantly down-regulated protein expression levels of E-cadherin and CD24 (Fig. 3A and 3B). No significant difference was observed in the Vimentin protein expression levels between the cancer and adjacent tissues. We have added this result to the revised Fig. 3 and modified the revised manuscript accordingly.

However, further studies are still needed to validate our results.

The authors must evaluate IHC either in the same cell (double staining) or by the staining using serial sections.

Response: In immunohistochemistry, the staining color position and depth indicated the expression levels and patterns, and it was difficult to perform double staining on the very one section. Therefore, these indicators were subjected to IHC staining on serial sections.

However, the reviewer raised a very good point. We will do as suggested in future studies.

RT-PCR should use RNA extracted from cancer cells or normal cells recovered by laser microdissection, rather than RNA extracted from whole tissues.

Response: The tumor samples were obtained under the guidance of B-ultrasound. The hyperplastic gland tissues were used as control. Due to limited funding, we did not recover the cells by laser microdissection. However, the reviewer raised a very good point. We will do as suggested in future studies.

Furthermore, it is necessary to use the TaqMan probe method, which has a high quantitative yield, instead of the cybr green method, which is a semi-quantitative method.

Response: The quantitative real-time PCR was performed to detect the mRNA expression levels of the target genes. However, the reviewer raised a very good point. We will do as suggested in future studies.

Minor points:

1) Please insert scale bars in Fig.1.

Response: In fact, the figures were taken at the magnification of 200×, which has been added in the figure legends.

2) The information of antibodies of IHC should be written down in Materials and Methods.

Response: According to the comment, we have added the information of IHC antibodies in Materials and Methods section of the revised manuscript.

3) Please describe the detailed statistical analysis methods in Materials and Methods.

Response: If the data conformed to the normal distribution, the single-factor analysis of variance was performed. Multiple comparisons were performed with the sidak method for the data with homogeneity of variance, while the Tamhane method was used for the data with the heterogeneity of variance. The data without normal distribution were first subjected to the logarithmical transform for normalization, and then the above-mentioned one-way analysis of variance method was performed for statistical analysis, or the Wilcoxon rank sum test was used. Correlation analysis of the expression index was performed with the Pearson correlation.

4) There are inappropriate citations, such as quoting a lung cancer paper even though it is a breast cancer citation. Please review the selection of cited papers.

Response: Sorry for the inappropriate citations. According to the comment, corresponding changes have been made herein.

5) There are many misspellings. Please check the spelling.

Response: According to the comment, we have further gotten editorial help to improve the English writing of the revised manuscript. Please check!

Reviewer #2: Contrast-enhanced ultrasound (CEUS) procedure carried out with new medical equipment and new-generation micro-bubble contrast reagents has been suggested to increase the accuracy in providing information on the microcirculation. The overall aim of the research described in this manuscript is to assess whether CEUS-based features may provide prognostic type of information. In particular, CEUS-based microcirculation features are examined for correlation with cancer-stem cell (CSC)-like subpopulations including those undergoing epithelial-mesenchymal transition (EMT). The rationale for focusing on tumor cells with CSC and EMT-like characteristics relates to potential roles in tumor heterogeneity and aggressiveness. The manuscript including the background, methods, and results would benefit from better language use and inclusion of information in terms of providing a clearer understanding of the rationale and the objectives of the study, the methodologies used, data interpretation including implications.

Specific Comments-

1. Some of the information presented in the discussion should be considered to be moved to the background to help provide a basis for the study.

Response: According to the comment, corresponding changes have been made herein.

2. Several published studies (eg Ji et al, 2017. Clin Hemorheol Microcirc, Zhao et al, 2017 Onco Targets Ther, Cao et al, 2014. Ultrasound Med Biol, Wang et al, 2017, PLoS One, and Vraka et al, 2918 In Vivo) have assessed the correlation between CEUS-based parameters and prognostic markers in breast cancer should be alluded to and referenced in the background and/or discussion.

Response: According to the comment, we have added the following reference to the Discussion section. Please check!

Reference:

Vraka I, Panourgias E, Sifakis E, Koureas A, Galanis P, Dellaportas D, Gouliamos A, Antoniou A. In Vivo.Correlation Between Contrast-enhanced Ultrasound Characteristics (Qualitative and Quantitative) and Pathological Prognostic Factors in Breast Cancer. 2018;32(4):945-954. Epub 2018/07/30. doi: 10.21873/invivo.11333. PubMed PMID: 29936484;PubMed Central PMCID: PMC6117754.

3. The reason for use of 2D US prior to the CEUS should be included in the method.

Response: In fact, for the CEUS, the contrast mode should be conducted on the basis of a clear two-dimensional ultrasound image. Although the results needed not to be analyzed for the two-dimensional ultrasound, the operation method would be necessary, which represented the diagnostic rule for contrast ultrasound. However, these methodological information needed not to be explained in the study.

4. A figure that depicts the various parameters obtained related to the CEUS describe on page 6 in the methods should be included (see ). The functional implications of these parameters in terms of circulation/perfusion should be clearly stated to help understand their significance when described in the result sections. Use of full names (and not abbreviations) is recommended throughout the manuscript.

Response: According to the comment, corresponding information has been added herein. For example, we have used the full names instead of abbreviations according to the suggestion.

5. A rationale should be provided for the criteria used to follow the various biomarkers described in the IHC section of the method. The nature of the antibodies used what they recognize on target proteins should be included. What is the rationale for looking for cytoplasmic localization of CD24? β-catenin localization in cytoplasmic/nucleus can relate to WNT-pathway activation and potentially EMT, given this was this aspect inspected? How epithelial/carcinoma vs stromal cells distinguished? Where antibodies for cytokeratins used?

Response: According to the comment, corresponding information concerning the primary antibodies and the criteria for expression evaluation have been added in the revised manuscript.

Moreover, based on the Human-Protein-Atlas database, CD24 is located in the cytoplasm. The activation of the WNT pathway would cause the translocation of β-catenin from the cytoplasm to the nucleus [1]. In the nucleus, β-catenin combines with the LEF/TCF transcription factor to replace the auxiliary inhibitor, and this combination recruits the additional auxiliary activators of the Wnt target genes to promote the process of EMT [2]. There have been articles [3] reporting that CD24 is the target of Wnt in colorectal cancers. Therefore, from database predictions and previous literature, the WNT pathway activation and potential EMT would affect the CD24 cytoplasmic localization.

In addition, through information retrieving, the keratin (epithelial cells) and vimentin (stromal cells) are used to distinguish the epithelial cells from the stromal cells.

References:

[1] Borgal L, Habbig S, Hatzold J, Liebau MC, Dafinger C, Sacarea I, Hammerschmidt M, Benzing T, Schermer B. The ciliary protein nephrocystin-4 translocates the canonical Wnt regulator Jade-1 to the nucleus to negatively regulate β-catenin signaling. J Biol Chem. 2012 Jul 20;287(30):25370-80.

[2] Kobayashi W, Ozawa M. The epithelial-mesenchymal transition induced by transcription factor LEF-1 is independent of β-catenin. Biochem Biophys Rep. 2018 Jun 12;15:13-18.

[3] Chen YC, Lee TH, Tzeng SL. Reduced DAXX Expression Is Associated with Reduced CD24 Expression in Colorectal Cancer. Cells. 2019 Oct 12;8(10):1242.

6. EMT induction requires the expression of transcription factors. Thus, to better show the relationship of the biomarkers used, it will be important to assess the expression (correlation) of some of EMT-TF with the biomarkers and the CEUS-features tested in the current study.

Response: Based on the PROMO database, Genecards database and UCSC database, the transcription factors related to EMT indicators were predicted. The common transcription factors included the SP1, YY-1, etc. Through literature searching, the transcription factors were all related to EMT, which regulated the process of EMT through modulating the gene transcription. It has been reported that the prepared nanobubbles showed high affinity and specificity to breast cancer cells and tumors with the neuropeptide YY 1 receptors, with minimal toxicity and damages to the organs [1]. Biodegradable photoluminescent nanobubbles were used as ultrasound contrast agents for targeted breast cancer imaging. The transcription factor prediction results were obtained as attached files.

Reference:

[1] Li J, Tian Y, Shan D, Gong A, Zeng L, Ren W, Xiang L, Gerhard E, Zhao J, Yang J, Wu A. Neuropeptide Y Y1 receptor-mediated biodegradable photoluminescent nanobubbles as ultrasound contrast agents for targeted breast cancer imaging. Biomaterials. 2017 Feb;116:106-117.

Submitted filename: 2021 04 30 Response letter revised.docx

Click here for additional data file.

12 Jul 2021

PONE-D-20-18825R1

Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)

PLOS ONE

Dear Dr. Ding,

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Stella E. Tsirka

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

As evident from the Reviewer's comments, it is critical to re-address the images in Figure 1. It is very hard to see how the images provided come from serial sections, or how they correlate with each other, so that one can extract meaningful conclusions. Please provide a revised Figure 1 with appropriate images, and/or low magnifications of the images to

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: They have not essentially revised for my major concerns.

My biggest concerns are below. Although authors described that they use the serial section for IHC, but as far as I can see in the picture in Figure 1, I can not think that they have observed the same tumor or normal site in the continuous section. The photos should be shown the same tumor or normal site in the serial section.

In figure4, the amounts of N-cadherin, Beta-catenin, and Vimentin proteins in these representative photos are not consistent with these quantitative graphs.

Thus, I think that their experimental results are inadequate to draw their conclusions.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Aug 2021

Dear editor,

We are resubmitting the Manuscript ID PONE-D-20-18825R1 entitled Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS) to PLOS ONE. Our responses to the comments by the reviewers are outlined below. Please also see the revised manuscript for details. For easily reading, we use the TRACK function of MICROSOFT WORD. Please simply select “Accept changes” to get rid of the TRACK markers if you do not like the tracks.

In addition, we would like to update our Funding Statement as follows:

This work was supported by the Supporting Project from Xinjiang Science and Technology Department (No.2020E0269).

Additional Editor Comments:

As evident from the Reviewer's comments, it is critical to re-address the images in Figure 1. It is very hard to see how the images provided come from serial sections, or how they correlate with each other, so that one can extract meaningful conclusions. Please provide a revised Figure 1 with appropriate images, and/or low magnifications of the images to allow for evaluation of location of panels and potential co-localizations, as well as the assessment of the same tumor.

Response: As suggested, we have further provided a revised Figure 2 (the original Figure 1) with high and low magnifications of the same tumor or normal sites. We have also made corresponding changes in the revised manuscript. Please check!

Reviewers' comments:

Reviewer #1: They have not essentially revised for my major concerns.

My biggest concerns are below. Although authors described that they use the serial section for IHC, but as far as I can see in the picture in Figure 1, I can not think that they have observed the same tumor or normal site in the continuous section. The photos should be shown the same tumor or normal site in the serial section.

Response: As suggested, we have further provided a revised Figure 2 (the original Figure 1) with high and low magnifications of the same tumor or normal sites. We have also made corresponding changes in the revised manuscript. Please check!

In figure4, the amounts of N-cadherin, Beta-catenin, and Vimentin proteins in these representative photos are not consistent with these quantitative graphs.

Response: As suggested, we have modified the representative and quantitative Western blot results in the revised Figure 4 to ensure that the amounts of N-cadherin, Beta-catenin, and Vimentin proteins in these representative photos were consistent with these quantitative graphs. We have also made corresponding changes in the revised manuscript. Please check!

Thus, I think that their experimental results are inadequate to draw their conclusions.

Response: As suggested, we have modified some results and some conclusion to ensure that the conclusions were supported by the experimental results. For example, the overstated conclusions have been modified. Please check the revised manuscript for details.

Submitted filename: 2021 08 25 Response letter revised.docx.docx

Click here for additional data file.

18 Oct 2021

PONE-D-20-18825R2Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)PLOS ONE

Dear Dr. Ding,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please fix the figure legends as indicated by the reviewers. Please submit your revised manuscript by Dec 02 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Stella E. Tsirka

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I think it has improved. The IHC analysis result, which was a major concern, seems to be a mixture of those that can be compared at the same point in the continuous section and those that are not. However, since the tumor sample is very small, I understand that it is difficult to analyze. No more peer-reviewed rounds are needed.

Minor points.

1) The square part of the low-magnification photo of CD24 and the square part of the magnifying magnification are 90 degrees out of alignment. Please correct it as it is confusing.

2) The word Breast is missing in the graph on the upper right of Figure 4B.

3) There is a misspelling in the upper left corner of Table 1. The term of "cancer" is "caner".

4) The value of N-cadherin in Table 5 is 0.000. Please enter an appropriate value.

Reviewer #2: Figure legends are non-existence or very brief. Keys of various symbols and color codes should be explained in the legends to allow a better appreciation of the results and hence the conclusions. The relationship between figures 1 and 2 needs to clearly stated. It will be helpful to indicate n values for various analyses when possible.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

22 Nov 2021

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: We confirm that the reference list is complete and correct. No further changes to the reference list are needed. We did not cite any retracted papers.

Reviewers' comments:

Reviewer's Responses to Questions

Reviewer #1: I think it has improved. The IHC analysis result, which was a major concern, seems to be a mixture of those that can be compared at the same point in the continuous section and those that are not. However, since the tumor sample is very small, I understand that it is difficult to analyze. No more peer-reviewed rounds are needed.

Minor points.

1) The square part of the low-magnification photo of CD24 and the square part of the magnifying magnification are 90 degrees out of alignment. Please correct it as it is confusing.

Response: Sorry for the confusion. We have corrected the low-magnification photo of CD24 in the revised Figure 2. Please check!

2) The word Breast is missing in the graph on the upper right of Figure 4B.

Response: Sorry for the typo. We have corrected this in the revised Figure 4. Please check!

3) There is a misspelling in the upper left corner of Table 1. The term of "cancer" is "caner".

Response: Sorry for the typo. We have corrected "caner" into "cancer" in the revised Table 1. Please check!

4) The value of N-cadherin in Table 5 is 0.000. Please enter an appropriate value.

Response: To be more clear, we have changed P=0.000 into P<0.001. Please check the revised Table 5 for details.

Reviewer #2: Figure legends are non-existence or very brief. Keys of various symbols and color codes should be explained in the legends to allow a better appreciation of the results and hence the conclusions. The relationship between figures 1 and 2 needs to clearly stated. It will be helpful to indicate n values for various analyses when possible.

Response: As suggested, we have further provided more detailed information to the figure legends. Figure 1 shows the time intensity curve analysis of contrast-enhanced ultrasound (CEUS). The representative sections of CEUS were marked before surgery. Then, the tissue specimens were collected from these marked sections and subjected to immunohistochemical analysis of CSCs and EMT proteins (Figure 2). We have further clarified this in the revised manuscript. Please check!

Additionally, the n values have been indicated in the figure legends and table titles. Please check!

Submitted filename: 2021 11 19 response to reviewer letter revised.docx

Click here for additional data file.

25 Nov 2021

Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)

PONE-D-20-18825R3

Dear Dr. Ding,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stella E. Tsirka

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

13 Dec 2021

PONE-D-20-18825R3

Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)

Dear Dr. Ding:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stella E. Tsirka

Academic Editor

PLOS ONE