Introduction: Neoadjuvant endocrine therapy (NAE) offers a breast-conserving surgery rate and clinical response rate similar to those of neoadjuvant chemotherapy (NAC), while presenting fewer adverse events and lower pathological complete response rates. The assessment of pathological response determines degenerative changes and predicts the prognosis of breast cancer treated with NAC. This study clarified the degenerative changes occurring in breast cancer following NAE. Methods: Our study encompassed two groups: NAE, consisting of 15 patients, and NAC, comprising 18 patients. Tissue samples were obtained from core needle biopsies and surgeries. Nuclear and cell areas were calculated using Autocell analysis. Furthermore, we assessed markers associated with microtubule depolymerization (KIF2A) and initiators of apoptosis (caspase-9). Results: In the NAC group, we observed significant increases in both cytoplasmic and cell areas. These changes in cytoplasm and cells were notably more pronounced in the NAC group compared to the NAE group. After treatment, KIF2A exhibited a decrease, with the magnitude of change being greater in the NET group than in the NAC group. However, no discernible differences were found in caspase-9 expression between the two groups. Conclusion: Our findings indicate that NAE induces condensation in cancer cells via cell cycle arrest or apoptosis. Conversely, NAC leads to cell enlargement due to the absence of microtubule depolymerization. These discrepancies underscore the importance of accounting for these distinctions when establishing criteria for evaluating pathological responses.

Endocrine therapy for breast cancer relies on the estrogen receptor (ER) as a pivotal target of treatment, serving as an absolute predictive factor. While normal breast mature luminal cells exhibit a 20–30% ER expression, breast cancer cells have been reported to boast over 70% ER expression [1].

In 1990, neoadjuvant chemotherapy (NAC) combined with tamoxifen (TAM) yielded lower progressive free survival and overall survival rates compared to chemotherapy alone [2]. However, more recent research, especially in postmenopausal women, has indicated that neoadjuvant endocrine therapy (NAE) exhibits breast-conserving surgery (BCS) rates and clinical response rates (CRRs) similar to those of NAC. Notably, NAE promotes fewer adverse events and a lower pathological complete response rate (pCR) than NAC [3‒9]. Achieving pCR with NAC has been linked to prolonged disease-free survival and overall survival in triple-negative or human epidermal growth factor receptor 2 (HER2)-amplified breast cancer [10], whereas the significance of pCR for NAE remains unknown.

A study using the National Cancer Database uncovered a correlation between a longer NAE and a higher BCS rate [6]; however, the optimal administration period remains unknown. Considering the type of neoadjuvant, aromatase inhibitors (AIs) have shown superior CRR or BCS rate when compared to TAM [11‒14], with no significant differences observed among the administration of each AI [15, 16] or switching from one AI to another [17, 18]. Similarly, the combination of AIs with other drugs, such as selective ER down-regulator [19, 20], epidermal growth factor receptor inhibitor [21], mammalian target of rapamycin inhibitor [22], cyclin-dependent kinase 4/6 inhibitor [23‒25], multi-kinase inhibitor [26], and phosphatidylinositol-3 kinase inhibitor [27], have failed to yield significant differences from AI monotherapy. In premenopausal women, some studies showed higher CRR and BCS rate with NAC [4, 8], while a few limited studies employing ovarian function suppression (OFS) combined with AI have indicated a higher CRR compared to TAM with OFS [28]. Using NAE for 3 months, the preoperative endocrine prognostic index score has been introduced as a prognostic factor, defined by the pathological tumor size, node status, Ki67 levels, and ER status after treatment [29].

The assessment of the pathologic response to neoadjuvant systemic therapy has uncovered degenerative changes in NAC and predicted the prognosis of breast cancer [30]. Nonetheless, there has been a notable surge in the employment of NAE. These degenerative changes have typically been characterized by swelling or ballooning of the cytoplasm and nuclear or cell membrane rupture, indicative of necrosis [31]. Further, one of the inhibitors of breast cell apoptosis is estrogen, a hormone associated with menopausal atrophy that is also employed in the therapy of ER-positive breast cancer [32]. Apoptosis causes the cells to sharply circumscribe, condense the cytoplasm, and convolute the nuclear outline and cell surface [33]. In this study, we explored the therapeutic response of NAE and, more specifically, aimed to clarify the degenerative changes in breast cancer following NAE.

Patient Selection

Patients diagnosed with primary operable invasive breast cancer at Nippon Medical School Hospital who received either NAE or NAC followed by surgery were included in this study. “Group NAE” comprised patients who received at least 1 month of NAE followed by surgery between 2014 and 2019, while “group NAC” included those who received at least six cycles of NAC followed by surgery between 2018 and 2019. Patient characteristics such as menopausal status, age, HER2 status, and gender were not exclusion criteria. Ineligible patients were those with only non-invasive disease, metastases (except for lymph nodes), who experienced disease progression during treatment, or who exhibited pCR included in ypTis.

Tissue Collection and Processing

Tissues were obtained from core needle biopsy and surgery in both groups. These were fixed in buffered formalin for up to 18 h and subsequently embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. For quantification of nuclear and cytoplasmic areas, we measured and compared five regions of interest (ROIs), each measuring 0.07 mm2 within the tumor area, using HistoQuest cell analysis software (TissueGnostics, version 4.0.4.0158). The ROI area measured 300 μm. Digital hematoxylin and eosin slides were imported into the HistoQuest software, with the same threshold applied uniformly for the automated identification of tumor cells. The threshold value was determined using multiple tests. Nuclear and cytoplasmic areas were measured in five ROIs, and the cell area and the nuclear-cytoplasmic (N/C) ratio were calculated. The areas and N/C ratios were averaged for each sample. The amount of change was defined as the subtraction of cell count in the surgery sample minus that in the biopsy sample count, and the averages for each sample were calculated. The change ratio was calculated by dividing the amount of change by the biopsy sample count.

Immunohistochemistry

All tissue samples were subjected to immunohistochemistry for ER, PgR, HER2, Ki67, and kinesin motor protein (KIF) 2A as the microtubule depolymerization marker and caspase-9 as the initiator apoptosis marker. Tissue sections with a 4 μm thickness were deparaffinized, and those intended for staining with Ki67 were pretreated at 121°C for 15 min in 10 mm citrate buffer solution (pH 6.0). Endogenous peroxidase was blocked with 100% methanol (Wako, Tokyo, Japan) containing 0.3% hydrogen peroxidase (Wako) for 30 min at 21–25°C. Primary antibodies were then incubated in phosphate-buffered saline containing 1% bovine serum albumin at 4°C overnight. The primary antibodies used were as follows: Ki67 (clone: MIB-1, mouse monoclonal, #M7240, dilution 1:100; Dako, Denmark), KIF2A (rabbit polyclonal, #27218-1-AP, dilution 1:100; Proteintech, USA), and caspase-9 (Cleaved Asp330, #PA5-105272, dilution 1:300; Invitrogen, Carlsbad, CA, USA). Sections were then treated with Histofine Simple Stain MAX PO (mouse, Ki67; rabbit, KIF2 and cleaved 9; Nichirei Biosciences, Inc., Tokyo, Japan) for 40 min at 21–25°C, and peroxidase activity was visualized using 3,3′ diaminobenzidine (Dojindo laboratories, Kumamoto, Japan). Sections were counterstained with Mayer’s hematoxylin at 21–25°C for 1 min.

ER and PgR positivity was defined as >1% nuclear staining. The expression of HER2 was examined using HercepTest (Dako, Tokyo, Japan, with HER2 positivity defined as either 3+ or 2+ with HER2 gene amplification by fluorescence in situ hybridization). Ki67 positivity was defined as >10% nuclear staining, automatically counted at hot spots using e-Count (e-Path, Japan), and expressed as the mean percentage of positive cells. KIF2A positivity was measured in the cytoplasm and compared with the averages of five ROI (0.07 mm2) within the tumor area using HistoQuest. Caspase-9 positivity was defined as >1% nuclear staining, automatically counted using e-Count.

Statistical Analysis

All the statistical analyses were performed using SPSS version 28 (IBM Corp., Armonk, NY, USA). Quantitative variables were presented as mean and standard deviation, while categorical variables were described using absolute and relative frequencies. All p values were two sided, with statistical significance set at p < 0.05.

Patient and Treatment Characteristics

The mean age of patients at diagnosis was 57.9 years (SD 11.86, range 40–82). The NAE and NAC groups comprised 15 and 18 patients, respectively. Detailed characteristics of each group are presented in Table 1. The mean age at diagnosis for the net group NAE was 59.6 years (SD 12.13, range 45–82) and 56.4 years (SD 11.7, range 40–70) for the NAC group (p = 0.12). There were more lymph metastases in NAC than in NAE (p = 0.01). The expression of ER and PgR was significantly higher in the NAE group than in NAC (p = 0.03, 0.0006). One patient in each group exhibited HER2 amplification. Conversely, Ki67 expression was higher in the NAC group than in NAE (p = 0.00027). There were no significant differences in the menopausal status (p = 0.57) between groups. In the NAE group, 6 patients were treated with AI, 9 patients with TAM, and 3 patients received a combination of AI with OFS during 60.4 ± 11.8 days (8.6 weeks). In the NAC group, 15 patients were administered anthracycline, all patients were administered taxanes for 7.3 ± 1.3 cycles, and 1 patient was administered HER2 antibody. Residual cancer burden (RCB) score [34] was significantly higher in NAC group than in NAE group (p = 0.03); however, RCB classification had no difference between both groups.

Table 1.

Characteristics of the patients

NAE, n = 15NAC, n = 18p value
Age, years 59.6±12.13 56.39±11.78 0.18 
cT stage 
 T1 0.53 
 T2 11 11  
 T3  
cN stage 
 N0 15 0.01* 
 N1 13  
 N2  
Histology 
 Invasive NST 13 16 0.50 
 ILC  
 Others  
ER 
 Positive 15 13 0.02* 
 Negative  
PgR 
 Positive 15 0.000* 
 Negative 10  
Her2 
 Positive 0.88 
 Negative 14 17  
Ki67 
 Positive 16  
 Negative 11 0.000* 
Menopause 
 Pre 0.57 
 Post  
Period during treatment 60.4±11.8 days 7.33±1.33 cycles  
Medication TAM 6 Anthracycline 15  
 TAM+OFS 3 Taxane 18  
 AI 6 Anti-Her2 1  
RCB score 2.65±1.42 3.22±0.93 0.03* 
RCB classification    
 Class II 10  
 Class III 11 0.28 
NAE, n = 15NAC, n = 18p value
Age, years 59.6±12.13 56.39±11.78 0.18 
cT stage 
 T1 0.53 
 T2 11 11  
 T3  
cN stage 
 N0 15 0.01* 
 N1 13  
 N2  
Histology 
 Invasive NST 13 16 0.50 
 ILC  
 Others  
ER 
 Positive 15 13 0.02* 
 Negative  
PgR 
 Positive 15 0.000* 
 Negative 10  
Her2 
 Positive 0.88 
 Negative 14 17  
Ki67 
 Positive 16  
 Negative 11 0.000* 
Menopause 
 Pre 0.57 
 Post  
Period during treatment 60.4±11.8 days 7.33±1.33 cycles  
Medication TAM 6 Anthracycline 15  
 TAM+OFS 3 Taxane 18  
 AI 6 Anti-Her2 1  
RCB score 2.65±1.42 3.22±0.93 0.03* 
RCB classification    
 Class II 10  
 Class III 11 0.28 

*There was a significant difference.

Histopathological Analyses

Table 2 illustrates the measurements of nuclear, cytoplasmic, and cell areas, as well as the N/C rate. In the NAE group, there were differences observed in the nuclear area and N/C ratio between pre-and post-treatment, while in the NAC group, differences were noted in cytoplasmic area, cell area, and N/C ratio. Additionally, pre-treatment measurements revealed differences between the NAE and NAC groups in terms of nuclear, cytoplasmic, and cell areas, as well as N/C ratio. Key findings are summarized in Figure 1.

Table 2.

Contrast of each area and N/C ratio between pre- and post-treatment, NAE, and NAC

NAENACPre-NAE versus pre-NAC
prepostp valueprepostp valuep value
Nuclear area, μm2 26.40 31.70 0.03* 41.14 42.19 0.68 0.00* 
Cytoplasmic area, μm2 181.50 157.16 0.21 122.31 158.65 0.00* 0.00* 
Cell area, μm2 207.89 188.86 0.57 163.44 200.83 0.00* 0.00* 
N/C rate 0.14 0.19 0.00* 0.26 0.22 0.00* 0.00* 
NAENACPre-NAE versus pre-NAC
prepostp valueprepostp valuep value
Nuclear area, μm2 26.40 31.70 0.03* 41.14 42.19 0.68 0.00* 
Cytoplasmic area, μm2 181.50 157.16 0.21 122.31 158.65 0.00* 0.00* 
Cell area, μm2 207.89 188.86 0.57 163.44 200.83 0.00* 0.00* 
N/C rate 0.14 0.19 0.00* 0.26 0.22 0.00* 0.00* 

*There was a significant difference.

Fig. 1.

HE staining of pre- and post-treatment samples in NAE and NAC groups (×400). Representative images illustrating the pre-NAE (a, c) and post-NAE (b, d), as well as pre-NAC (e, g) and post-NAC (f, h). In NAE group, case 12 is shown (a, b) and case 7 is shown (c, d). In NAC, case 21 is shown (e, f) and case 3 is shown (g, h). Scale bar, 20 μm.

Fig. 1.

HE staining of pre- and post-treatment samples in NAE and NAC groups (×400). Representative images illustrating the pre-NAE (a, c) and post-NAE (b, d), as well as pre-NAC (e, g) and post-NAC (f, h). In NAE group, case 12 is shown (a, b) and case 7 is shown (c, d). In NAC, case 21 is shown (e, f) and case 3 is shown (g, h). Scale bar, 20 μm.

Close modal

Table 3 presents the changes in cytoplasmic and cell areas, which are decreased in NAE and increased in NAC (p = 0.007, 0.008). NAE increased the nuclear area by 20.1% (p = 0.001), and NAC increased the cytoplasmic and cell areas by 29.7% and 22.9%, respectively (p = 0.000, 0.000). Ki67 expression was significantly decreased following NAC treatment (Fig. 2).

Table 3.

Change of area between NAE and NAC

NAE, n = 15NAC, n = 18p value
Nuclear area 
 Amount of change, μm2 5.30 1.05 0.21 
 Change ratio, % 20.1 2.6 0.001* 
Cytoplasmic area 
 Amount of change, μm2 −24.34 36.34 0.007* 
 Change ratio, % −13.4 29.7 0.000* 
Cell area 
 Amount of change, μm2 −19.03 37.39 0.008* 
 Change ratio, % −9.2 22.9 0.000* 
NAE, n = 15NAC, n = 18p value
Nuclear area 
 Amount of change, μm2 5.30 1.05 0.21 
 Change ratio, % 20.1 2.6 0.001* 
Cytoplasmic area 
 Amount of change, μm2 −24.34 36.34 0.007* 
 Change ratio, % −13.4 29.7 0.000* 
Cell area 
 Amount of change, μm2 −19.03 37.39 0.008* 
 Change ratio, % −9.2 22.9 0.000* 

*There was a significant difference.

Fig. 2.

Changes in Ki67 expression between pre- and post-treatment. Ki67 in NAE (a) exhibited no differences (p = 0.27), while Ki67 expression in NAC (b) significantly decreased following treatment (p = 0.00).

Fig. 2.

Changes in Ki67 expression between pre- and post-treatment. Ki67 in NAE (a) exhibited no differences (p = 0.27), while Ki67 expression in NAC (b) significantly decreased following treatment (p = 0.00).

Close modal

Additional immunohistochemistry was performed on 12 patients in the NAE group and 11 patients in NAC due to the absence of invasive cancer in the residual tissue sections (Table 4). Both groups exhibited a decrease in KIF2A and caspase-9 decreased from pre-treatment to post-treatment (Fig. 3). The change in KIF2A expression was significantly greater in NET (p = 0.04), while caspase-9 showed no differences between groups.

Table 4.

Positivity and change of KIF2A and caspase-9 between NAE and NAC

NAENACp value
prepostp valueprepostp value
KIF2A positive rate, % 27.95 15.53 0.00* 13.10 8.06 0.05*  
Caspase-9 positive rate, % 2.05 32.37 0.00* 11.30 40.64 0.00*  
KIF2A area change, μm2 −13.60   −4.46   0.04* 
Caspase-9 area change, μm2 31.05   28.41   0.84 
NAENACp value
prepostp valueprepostp value
KIF2A positive rate, % 27.95 15.53 0.00* 13.10 8.06 0.05*  
Caspase-9 positive rate, % 2.05 32.37 0.00* 11.30 40.64 0.00*  
KIF2A area change, μm2 −13.60   −4.46   0.04* 
Caspase-9 area change, μm2 31.05   28.41   0.84 

*There was a significant difference.

Fig. 3.

Immunostaining of KIF2A and caspase-9 in pre- and post-treatment samples from NET and NAC (×400). KIF2A is shown in pre-NAE (a), post-NAE (b), pre-NAC (c), and post-NAC (d). Caspase-9 is shown in pre-NAE (e), post-NAE (f), pre-NAC (h), and post-NAC (g). The cytoplasmic expression of KIF2A decreased after both treatments, NAE (a, b) and NAC (c, d). The nuclear expression of caspase-9 increased following both treatments, NAE (e, f) and NAC (h, g). Scale bar, 20 μm.

Fig. 3.

Immunostaining of KIF2A and caspase-9 in pre- and post-treatment samples from NET and NAC (×400). KIF2A is shown in pre-NAE (a), post-NAE (b), pre-NAC (c), and post-NAC (d). Caspase-9 is shown in pre-NAE (e), post-NAE (f), pre-NAC (h), and post-NAC (g). The cytoplasmic expression of KIF2A decreased after both treatments, NAE (a, b) and NAC (c, d). The nuclear expression of caspase-9 increased following both treatments, NAE (e, f) and NAC (h, g). Scale bar, 20 μm.

Close modal

This study examined two distinct subgroups: one receiving hormone therapy (NAE) and the other chemotherapy (NAC). In the NAE group, all patients had ER- and PgR-positive breast cancer, with lower Ki67 labeling than those in the NAC group. This discrepancy was expected since NAC is primarily used for cases with higher Ki67 labeling indexes, HER2 positive, or hormone receptor-negative breast cancer. Ki67 expression significantly decreased in the NAC group compared to NAE due to NAC’s capacity to arrest the cell cycle and inhibit tumor proliferation [35, 36]. Consequently, we observed differences in nuclear, cytoplasmic, and cellular areas in the two groups before treatment. Instead of analyzing absolute values, we assessed changes between post-treatment and pre-treatment measurements. Before treatment, the N/C ratio was higher in the NAC group compared to NAE. When comparing pre- and post-treatment, NAC increased cytoplasmic and cell areas while decreasing the N/C ratio, similar to the assessment of pathologic response. Conversely, NAE promoted a tendency for cytoplasmic and cell areas to shrink, resulting in an increased N/C ratio. Changes in cytoplasmic and cellular areas were more pronounced in NAC than in NAE. Notably, NAC induced swelling in cytoplasmic and cellular areas, which was not observed in NAE.

One contributing factor to differences in cell size changes between NAC and NAE is the impact of medication on cell morphology. In the NAC group, all the patients received chemotherapy with a taxane-combined regimen, known to inhibit microtubule depolymerization. This regimen is often used for breast cancer with or without anthracycline and anti-HER2 antibodies [37]. KIF2, effective in dephosphorylation, binds to tubulin dimers, depolymerizes microtubules from the peeled protofilament end during ATP hydrolysis [38], and regulates spindle assembly [39]. Its higher expression in pre-NAE cases may be linked to their larger cytoplasmic area compared to pre-NAC cases. KIF2A expression also decreased following treatment in both groups. It seemed that NAC maintained microtubule polymerization and caused the cytoplasm of cancer cells to spindle and swell because of ineffective phosphorylation of KIF2.

Conversely, the change in KIF2A expression in NAE likely results from the inability of cancer cells to progress to the M phase of the cell cycle. While NAE decreased KIF2A, it had no effect on Ki67, which is expressed in phases other than the G0 phase. This suggests that cancer cells in the G1, S, G2, and M phases at pre-treatment remained in each cell cycle. The checkpoints governing cell cycle progression in each phase involve cyclin and cyclin-dependent kinase [40, 41]. The non-genomic action of ER regulates cyclin D via the PI3K/Akt pathway [42, 43]. Cyclin D combined with CDK 4 and CDK 6 activates the cyclin E-CDK 2 complex and accelerates the G1/S cell cycle checkpoint and the start of the S phase together [44, 45]. The selective CDK 4 and CDK 6 inhibitors potently inhibit the cell cycle inhibition [46]. Endocrine therapy induced cell cycle arrest without increasing cell size by preventing microtubule polymerization. Although some studies have reported a significant decrease in Ki67 expression by AI or AI with a CDK4/6 inhibitor over 16–24 weeks [8, 23], our study had a shorter NAE period (average 8.6 weeks). Longer NAE administration may result in increased cell cycle arrest. Moreover, patients with pTis and pCR were excluded from this study, potentially accounting for the lack of difference in Ki67 levels between pre- and post-treatment in NAE.

The second reason for differences in cell size between NAC and NAE is the variation in cell death types. Cell death can be accidental or programmed, with necrosis causing morphological changes, such as swelling [31]. In contrast, apoptosis, a programmed cell death, results in cytoplasmic condensation [31‒33], often associated with hormonal control [32]. Caspases are a family of genes that regulate cell death and inflammation, and caspase-9 is an initiator of apoptosis [47]. Here, we found no differences in caspase-9 expression between NAE and NAC; both groups showed increased caspase-9 levels after treatment. NAC in breast cancer induces necrosis and increases the N/C ratio [48]; distinguishing necrosis from apoptosis based solely on morphological changes can be challenging. Chemotherapy targets cells susceptible to apoptosis [49], and in endocrine therapy, cell cycle arrest activates p53, inducing apoptosis [50] and cell condensation. The increased nuclear area after NAE is likely due to the unchanged ratio of cell cycle G1-M, in contrast to NAC, which decreased Ki67 and increased the G0 cycle ratio. Additionally, the process of nuclear fragmentation into apoptotic bodies may contribute to this phenomenon [51] because uneven nuclear surfaces might be overestimated by HistoQuest. Caspase-9 is also related to autophagy [31, 45, 51] and the crosstalk between autophagy and apoptosis [52, 53]. Ueno et al. reported that NAE promoted increased autophagy markers (beclin 1) but not apoptosis markers (TUNEL) [54]. Therefore, further studies are required to better understand this interplay.

This study revealed significant differences in size changes between NAC and NAE after treatment. The assessment of pathological responses should consider these disparities. RCB after neoadjuvant systemic therapy is correlative with long-term survival outcomes [55]. Pearson’s correlation coefficient was calculated and revealing no significant correlation between the change of cell area and RCB (r = 0.298 for NAE and r = −0.075 for NAC). Thus, cytomorphological disparities following neoadjuvant treatment might not currently serve as a prognostic predictor.

Nevertheless, our study has limitations, including a relatively small sample size, a short duration of NAE treatment, and a retrospective study. Moreover, except for pCR and ypTis, there was limited observation for residual invasive disease after treatment. Due to the retrospective nature of this study, the varied medications used for chemotherapy and endocrine therapy might have also introduced some variability. Furthermore, it remains unknown whether NAC without a taxane regimen yields similar results. Moreover, image analysis using automatic calculations has inherent measurement limits. Further research is warranted to deepen our understanding of these findings.

This study has confirmed that NAE induces the condensation of cancer cells through cell cycle arrest and apoptosis, while NAC results in the enlargement of cancer cells due to the absence of microtubule depolymerization. These findings underscore the importance of considering these distinctions when establishing evaluation criteria for pathological responses. Furthermore, future research should delve into the mechanisms underlying cancer cell shrinkage in NAE, offering further insights into this treatment approach.

We would like to thank Editage (www.editage.jp) for English language editing.

This study protocol was reviewed and approved by the Ethics Committee of Nippon Medical School Hospital, approval number B-2020-190, date of decision November 4, 2020. This retrospective study involving pathological samples adhered to the principles outlined in the Declaration of Helsinki. Opt-out informed consent protocol was used for use of participant data for research purposes. This consent procedure was reviewed and approved by the Ethics Committee of Nippon Medical School Hospital, approval number B-2020-190, date of decision November 4, 2020. Informed consent was obtained by their surgeons. The patient’s data would be included in the registry. Patients had the option to opt out and have their information removed from the registry, which was facilitated through the Nippon Medical School Hospital Website.

The authors have no conflicts of interest to declare.

This work was supported by Grants-in-Aid for Scientific Research (JSPS KAKENHI, Grant No. JP22K07218).

Takashi Sakatani conceived and initiated the study. Hideko Hoshina contributed to the manuscript’s design, description, and data collection and analysis. Hiroyuki Takei edited and supervised the manuscript preparation. Takashi Sakatani and Ryuji Ohashi supervised manuscript preparation. Yoko Kawamoto conducted immunostaining and collected relevant data. All the authors have read and approved the final version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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