Expression of Periostin in Benign Salivary Gland Tumors.

Parotid tumors present a wide range of histological features, from benign to malignant. Periostin, an extracellular matrix protein specifically expressed in the periosteum and periodontal ligament, is isolated from osteoblast cell lines. It regulates fibrosis and collagen deposition and plays an important role in myocardial repair after myocardial infarction. It is also known to be involved in otorhinolaryngological-diseases. This study included 36 patients [38 specimens; 16 men and 20 women, mean age 59.2 (range 26-82) years] who underwent parotid tumor resection at the Division of Otorhinolaryngology, Tohoku Medical and Pharmaceutical University, between April 2017 and March 2022 and were clinically and pathologically diagnosed as having benign parotid tumors. Formalin-fixed, paraffin-embedded sections from the surgical specimens were autoclaved and immunostained with anti-periostin antibodies to evaluate the expression and distribution of periostin. Histologically, the tumors were diagnosed as pleomorphic adenomas in 15 cases (15 specimens), Warthin's tumors in 13 cases (15 specimens), basal cell adenomas in 2 cases (2 specimens), oncocytomas in 4 cases (4 specimens), and myoepitheliomas in 2 cases (2 specimens). An increased expression of periostin was found in 32 of 38 samples (84.2%) in the stroma of benign parotid tumors. Four distinct patterns of periostin expression were observed in benign parotid gland tumors: negative, superficial, infiltrative, and diffuse. Statistically significant differences were found between periostin expression patterns and histological classification of the tumors. Our results suggest that periostin may be involved in the pathogenesis of benign parotid tumors and could serve as a new biomarker for these tumors.

Immunohistochemical Localization of D-ß-Aspartic Acid and Periostin in Vocal Fold Polyps.

Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. D-ß-aspartic acid is the major isomer of D-aspartic acid found in elderly tissue. In this study we investigated the expression and the role of D-ß-aspartic acid and periostin in the formation of vocal fold polyps. The expression patterns of D-ß-aspartic acid and periostin in 36 surgical specimens of vocal fold polyps from 36 patients were investigated immunohistochemically. In the epithelium of vocal polyps, D-ß-aspartic acid was expressed in all cases. Expression of D-ß-aspartic acid was detected in 25 samples obtained from patients with vocal fold polyps stroma. Expression of periostin was detected in 28 samples obtained from patients with vocal fold polyps. Two patterns of D-ß-aspartic acid expression were observed in vocal fold polyps stroma: positive type and negative type. The following four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between D-ß-aspartic acid expression patterns and periostin expression patterns. From these findings we speculate that periostin and D-ß-aspartic acid participate in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and the formation and development of vocal fold polyps.

Expression of Periostin in Vocal Fold Polyps.

Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. The aim of this article was to investigate the expression and the role of periostin in the formation of vocal fold polyps. The expression patterns of periostin in 59 surgical specimens of vocal fold polyps from 54 patients were investigated immunohistochemically. Normal vocal fold mucosa specimens from 5 patients who had undergone total laryngectomy were used as the control group. Retrospective study with planned data collection was conducted at Tohoku Medical and Pharmaceutical University. Expression of periostin was detected in 43 (72.9%) samples and four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between periostin expression patterns and the histological subtypes of vocal fold polyps. The infiltrative pattern of periostin expression was significantly dominant in vascular-hyaline types. Expression of transforming growth factor-ß (TGF-ß) was also detected in the vocal fold polyps. Our results confirmed that periostin might be involved in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and formation and development of vocal fold polyps.

YM750, an ACAT Inhibitor, Acts on Adrenocortical Cells to Inhibit Aldosterone Secretion Due to Depolarization.

Primary aldosteronism (PA) is considered the most common form of secondary hypertension, which is associated with excessive aldosterone secretion in the adrenal cortex. The cause of excessive aldosterone secretion is the induction of aldosterone synthase gene (CYP11B2) expression by depolarization of adrenocortical cells. In this study, we found that YM750, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, acts on adrenocortical cells to suppress CYP11B2 gene expression and aldosterone secretion. YM750 inhibited the induction of CYP11B2 gene expression by KCl stimulation, but not by angiotensin II and forskolin stimulation. Interestingly, YM750 did not inhibit KCl-stimulated depolarization via an increase in intracellular calcium ion concentration. Moreover, ACAT1 expression was relatively abundant in the zona glomerulosa (ZG) including these CYP11B2-positive cells. Thus, YM750 suppresses CYP11B2 gene expression by suppressing intracellular signaling activated by depolarization. In addition, ACAT1 was suggested to play an important role in steroidogenesis in the ZG. YM750 suppresses CYP11B2 gene expression and aldosterone secretion in the adrenal cortex, suggesting that it may be a potential therapeutic agent for PA.

Expression and clinicopathological significance of glucocorticoid receptor, SGK1, and NDRG1 in hormone-naïve prostate carcinoma.

Glucocorticoid receptor (GR) has been implicated in prostate carcinoma growth and progression. Glucocorticoid receptor beta (GRß) acts as an inhibitor of GR; however, its function is not well understood. Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a GR-responsive gene that phosphorylates N-myc downstream-regulated gene 1 (NDRG1) and is involved in cancer growth and invasion. However, the expression of GR, GRß, SGK1, and NDRG1 in prostate cancer and their relationship with clinicopathological and functional significance remain unknown. The association between the status of GR, GRß, SGK1, and NDRG1 immunoreactivity and clinicopathological variables was analyzed in patients with prostate carcinoma to explore their clinical significance. In prostate carcinoma cases, the relative abundance of GR and NDRG1 immunoreactivity was inversely and significantly associated with the primary tumor stage (pT), while GR immunoreactivity was inversely and significantly associated with the Ki-67 score. The relative expression status of NDRG1 was significantly associated with that of GR. However, no significant correlation was observed between any of the clinicopathological parameters and GRß and SGK1 expression. Our findings indicate that GR and NDRG1 expression status is correlated with clinicopathological features in patients with prostate cancer.

An activation of LC3A-mediated autophagy contributes to de novo and acquired resistance to EGFR tyrosine kinase inhibitors in lung adenocarcinoma.

The development of therapeutic resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs, ie erlotinib or gefitinib) has been the major clinical problem when treating lung adenocarcinoma patients with these agents. However, its mechanisms have not necessarily been well studied to this date. Autophagy has been recently considered to play pivotal roles in escaping from the effects of anti-neoplastic agents. Therefore, in this study, we examined its roles in the development of resistance to EGFR-TKIs in lung adenocarcinoma. We first established erlotinib-resistant cell lines (PC9/ER) from parental PC9 cells by exposing the cells to erlotinib. In PC9/ER, autophagy-related LC3A expression came to be up-regulated and constitutive activation of LC3A-mediated autophagy became more pronounced through the process of acquiring therapeutic resistance. In addition, inhibition of LC3A or autophagy restores sensitivity to EGFR-TKIs in PC9/ER. LC3A was also activated at the transcriptional level in de novo resistant cells via demethylation of the MAP1LC3A gene. We then evaluated the status of LC3A in 169 lung adenocarcinoma patients using immunohistochemistry. LC3A immunoreactivity was only detected in carcinoma cells (89/169 patients), not in non-tumoural cells. In addition, LC3A immunoreactivity was significantly correlated with progression-free survival (p = 0.0039) and overall survival (p = 0.0040) of 35 patients treated with EGFR-TKIs. The results of our present study demonstrated that LC3A-mediated autophagy in carcinoma cells was involved in the development of resistance to EGFR-TKIs, and that LC3A could serve as a promising therapeutic target for overcoming resistance to EGFR-TKIs and a novel predictor of response to EGFR-TKIs in lung adenocarcinoma patients.

GATA6, SF1, NGFIB and DAX1 in the remodeled subcapsular zones in primary aldosteronism.

The majority of the cases diagnosed as primary aldosteronism (PA) are caused by aldosterone-producing adenoma (APA) or idiopathic hyperaldosteronism (IHA). Histopathologically, both IHA and adjacent adrenal glands of APA demonstrate remodeled subcapsular zone (RSZ) but these zones in two disorders are markedly different in terms of steroidogenesis. 3ß-Hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3ß-HSD) expression has been known to be activated synergistically by GATA6 and SF1, and repressed by DAX1 through abolishing the activation. Nerve growth factor-induced clone B (NGFIB) is also known as one of the transcription factors to bind to and activate 3ß-HSD promoter. The results of our immunohistochemical analysis demonstrated the expression levels of 3ß-HSD in RSZ of IHA were higher than in RSZ of adjacent adrenals of APA, while those in the zona glomerulosa (ZG) of normal adrenal gland (NA) were in between these two RSZs. The expression levels of GATA6, SF1 and DAX1 did not prominently differ among these three types of adrenals, especially between in RSZs of IHA and APA cases, indicating the marked difference of 3ß-HSD expression was unlikely to be explained by the levels of these three factors. However, the levels of NGFIB expression were significantly higher in RSZ of IHA than in RSZ of adjacent adrenals of APA and the ZG of NA (P<0.05), which may partly account for the expression levels of 3ß-HSD among the three groups of adrenals. These results may imply NGFIB plays important roles in the marked differences in steroidogenic functions in the two distinct types of RSZ of PA cases.

Immunohistochemical localization of CD31, CD34, and periostin in vocal fold polyps.

BACKGROUND: Long-term voice-abuse or sudden vocal fold microvascular disruption can lead to injury and subsequent repair/remodeling of the vocal fold mucosa. Periostin is known to be involved in airway remodeling and in various otolaryngological diseases. In ischemic heart disease, increased CD31 expression has been observed around cardiomyocytes during remodeling, and endothelial proliferation has been reported to occur at these sites. OBJECTIVES: We investigated the expression and the roles of CD31, CD34, and periostin in the formation of vocal fold polyps. MATERIALS AND METHODS: Fifty-seven samples of vocal fold polyps were investigate histopathologically and immunohistochemically. RESULT: Expression of CD31 and CD34 was detected in 41 (71.9%) and 53 (93.0%) samples, respectively, obtained from patients with vocal fold polyp. Expression of periostin was detected in 41 (71.9%) samples obtained from patients with vocal polyps. The vocal polyp samples could be classified into three histological subtypes. Three patterns of CD31 and CD34 expression were observed in the vocal polyp. Four patterns of periostin expression were observed in vocal polyps. An association was observed between the CD31 expression pattern and the histological subtype of vocal fold polyps. CONCLUSION AND SIGNIFICANCE: In vocal fold polyps, evaluation of vascular endothelial markers may be useful for staging.

Murine Double Minute 2 Antagonist Nutlin-3 Enhanced Chemosensitivity in Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: Murine double minute 2 (MDM2) is well known to inhibit p53 function and its over-expression is associated with poor prognosis in several human malignancies. Nutlin-3, a small-molecule inhibitor of MDM2, exerts antitumor effects on various solid tumors harboring wild-type p53. We aimed to clarify its effects on esophageal cancer. MATERIALS AND METHODS: We first examined the potential antitumor effects of nutlin-3 according to MDM2 status using esophageal carcinoma cell lines (KYSE 170/180). We then immunolocalized MDM2 immunoreactivity in 62 surgical cases of esophageal squamous cell carcinoma undergoing neoadjuvant chemotherapy followed by esophagectomy and correlated the findings with clinicopathological variables. RESULTS: MDM2 mRNA expression in KYSE 170 was significantly higher than that in KYSE 180 cells. No significant changes were detected in both cell lines when nutlin-3 was added. However, cell proliferation was significantly decreased in KYSE 170 cells treated with nutlin-3 and cisplatin compared to cisplatin alone but not in KYSE 180. MDM2 immunoreactivity was also significantly associated with poor sensitivity to neoadjuvant chemotherapy in the cases examined. CONCLUSION: The combination of nutlin-3 with chemotherapeutic agents may become a novel therapeutic strategy in esophageal cancer over-expressing MDM2.

Cervical Liposarcoma Revisited: A Case Report and Scoping Literature Review.

The commonest sites for liposarcoma are the retroperitoneum and lower extremities. Liposarcoma of the head and neck region is a rare and potentially life-threatening malignancy. Tumors originating in the right cervical space cause special diagnostic and therapeutic difficulties. In the present report, we describe a case of differentiated liposarcoma of the right cervical region. The tumor continued to grow slowly over 3 years before a definitive diagnosis was established. Extended extirpation of the tumor was performed and proved efficacious in that no recurrence has been observed for 4 years. Recommendations for earlier and accurate diagnosis and treatment of this rare neoplasm are discussed.

Immunohistochemical localization of d-ß-aspartic acid in congenital and acquired middle ear cholesteatoma.

Objective/Hypothesis: Middle ear cholesteatoma is characterized by abnormal growth of the keratinizing squamous epithelium of the temporal bone. d-ß-aspartic acid is the major isomer of d-aspartic acid found in elderly tissue. We assessed the immunoreactivity to k-ß-aspartic acid of congenital and acquired middle ear cholesteatomas. Study Design: Case-control studies. Material and Methods: Tissue samples were collected from 21 patients comprising 21 ears with congenital middle ear cholesteatoma and 26 patients comprising 29 ears with acquired type. Their clinical and histopathological features were investigated. We divided the middle ear cholesteatoma samples into three layers: the perimatrix, matrix, and cystic contents. The patterns of immunoreactivity to d-ß-aspartic acid expression were then assessed immunohistochemically. Results: Two patterns of immunoreactivity to d-ß-aspartic acid were detected in middle ear cholesteatoma: infiltrative and diffuse. In congenital middle ear cholesteatoma, d-ß-aspartic acid expression was observed throughout all the layers (perimatrix, matrix, and cystic contents), and immunoreactivity to d-ß-aspartic acid was dramatically strong in all layers. The expression levels of d-ß-aspartic acid to the cystic content and perimatrix were significantly higher in congenital middle ear cholesteatoma than in the acquired type. Conclusions: This study showed the expression levels of d-ß-aspartic acid in middle ear cholesteatoma to differ significantly between congenital and acquired middle ear cholesteatoma. Our results indicate that overexpression of d-ß-aspartic acid is likely to be involved in the pathogenesis of cholesteatoma, and we speculate that d-ß-aspartic acid could be a novel biomarker for, and a therapeutic target in, congenital and acquired middle ear cholesteatoma. Level of Evidence: 4.

Clinicopathological Significance of Estrogen Receptor ß and Estrogen Synthesizing/Metabolizing Enzymes in Urothelial Carcinoma of Urinary Bladder.

Sex-specific differences in the incidence of urinary bladder carcinomas are well known, and the possible involvement of sex steroids has been proposed. We previously reported the association of the loss of androgen receptors and androgen-producing enzymes with tumor progression of urinary bladder cancer patients. Clinically, the selective estrogen receptor modulators (SERMs) were reported to suppress the progression of these tumors but the status of estrogen receptors (ERs) has not been well studied in patients with bladder urinary cancer. Moreover, not only ERs but also estrogen-related enzymes, such as aromatase, steroid sulfatase (STS), and estrogen sulfotransferase (EST), have been reported in the biological/clinical behavior of various hormone-dependent carcinomas but not studied in urinary bladder carcinoma. Therefore, in this study, we immunolocalized ERs as well as estrogen metabolizing enzymes in urinary bladder carcinoma and performed immunoblotting and cell proliferation assays using the bladder urothelial carcinoma cell line, T24. The results revealed that the loss of STS and aromatase was significantly correlated with advanced stages of the carcinoma. In vitro studies also revealed that T24 cell proliferation rates were significantly ameliorated after treatment with estradiol or diarylpropionitrile (DPN). EST and aromatase were also significantly correlated with the nuclear grade of the carcinoma. The results of our present study, for the first time, demonstrated that biologically active estrogens that bind to ERs could suppress tumor progression and the inactive ones could promote its progression and the potential clinical utility of SERM treatment in selective patients with urinary bladder carcinoma.

Endoscopic Treatment of Sinonasal Leiomyosarcoma: A Case Report in Light of the Literature.

A 49-year-old Japanese man presented with a rare case of sinonasal leiomyosarcoma with left nasal bleeding for 12 months. He had no history of irradiation or malignancies, including retinoblastoma. Preoperative histological examination suggested vascular leiomyoma. Complete resection with endoscopic surgery was performed. Histological examination during the operation suggested that the tumor was a leiomyoma. However, immunohistochemical staining for α smooth muscle actin and desmin were helpful in establishing a definitive diagnosis of sinonasal leiomyosarcoma. The resection margins were positive for tumor cells. Staging with CT of the neck and thorax, ultrasound of the abdomen, and MRI of the head ruled out metastases. Second endoscopic tumor resection surgery was performed for positive resection margins. The patient's condition was successfully managed with additional excision, and he remains well with no evidence of recurrence and metastasis 36 months after treatment. Endoscopic management should be considered in suitable cases.

Metastatic Renal Cell Carcinoma to the Left Sphenoid Sinus: A Case Report in Light of the Literature.

A 79-year-old Japanese woman presented with a rare case of metastatic renal cell carcinoma to the left sphenoid sinus with left nasal bleeding. She had previously had right radical nephrectomy for renal cell carcinoma at the age of 64 years and brain and spinal cord infarction at 74 years. Endoscopic examination revealed no mass in the nasal cavity. CT and MRI revealed a tumor in the left sphenoid sinus. The size of the tumor increased gradually from 12 to 15 years after the radical nephrectomy. Complete resection with endoscopic surgery was performed without preoperative embolization. The tumor cells had clear cytoplasm and were arranged in a trabecular pattern lined by a layer of endothelial cells. These findings were identical to the pathological findings of the surgical specimen of the renal cell carcinoma from 15 years previous. A pathological diagnosis of metastatic renal cell carcinoma of clear cell type (grade 1) was made. PET-CT demonstrated no metastasis. The patient's condition was successfully managed with excision of the tumor, and she remains well with no evidence of recurrence and metastasis 36 months after treatment. Metastatic renal cell carcinoma to the sphenoid sinus is rare, but it might be considered in the differential diagnosis of masses in the paranasal sinus even long after initial treatment of renal cancer.

The expression of sex steroid receptors and sex steroid-synthesizing/metabolizing enzymes in metastasized lymph nodes of prostate cancer.

The expression statuses of sex steroid receptors and sex steroid-synthesizing/metabolizing enzymes have been reported in primary prostate cancer lesions, but that in metastatic lymph nodes has remained unknown. Therefore, in this study, we immunolocalized these proteins in primary tumors and paired metastatic lymph nodes of prostate cancer and correlated the findings with clinicopathological factors of individual patients. The expression statuses of AR and ER ß was significantly increased in metastatic lymph nodes compared with primary lesions, whereas that of 17ßHSD1, 17ßHSD2, 17ßHSD5, and STS immunoreactivity was decreased in metastatic lymph nodes. In metastatic lymph nodes, the status of 5α2 was significantly correlated with that of AR. In addition, 17ßHSD5-, 5α1-, STS-, and EST-positive cases were significantly associated with Gleason score (GS) status (GS > 8 versus GS < 7) in metastatic lymph nodes. Results of our present study did demonstrate that in situ androgen and estrogen metabolism and action play roles in pathophysiology of prostate cancer in metastatic lymph nodes, but these steroidogenic effects could be different from those in primary lesions.

Endoscopic Treatment of Sinonasal Glomangiopericytoma: A Case Report in Light of the Literature.

A 71-year-old Japanese male patient presented with a rare case of Glomangiopericytoma (GPC) of the left nasal with obstruction. Complete resection with endoscopic surgery was performed. Immunohistochemical staining for smooth muscle actin, ß catenin, cyclin D1, vimentin, and factor 13 were helpful in establishing a definitive diagnosis. Extranasal treatment has been traditionally performed for successful management. However, recent advances in endoscopic treatment have enabled complete endoscopic resection of GPC, minimizing morbidity and facilitating subsequent surveillance for recurrence. Endoscopic management should be considered in suitable cases.

A Gata3 3' Distal Otic Vesicle Enhancer Directs Inner Ear-Specific Gata3 Expression.

Transcription factor GATA3 plays vital roles in inner ear development, while regulatory mechanisms controlling its inner ear-specific expression are undefined. We demonstrate that a cis-regulatory element lying 571 kb 3' to the Gata3 gene directs inner ear-specific Gata3 expression, which we refer to as the Gata3 otic vesicle enhancer (OVE). In transgenic murine embryos, a 1.5-kb OVE-directed lacZ reporter (TgOVE-LacZ) exhibited robust lacZ expression specifically in the otic vesicle (OV), an inner ear primordial tissue, and its derivative semicircular canal. To further define the regulatory activity of this OVE, we generated Cre transgenic mice in which Cre expression was directed by a 246-bp core sequence within the OVE element (TgcoreOVE-Cre). TgcoreOVE-Cre successfully marked the OV-derived inner ear tissues, including cochlea, semicircular canal and spiral ganglion, when crossed with ROSA26 lacZ reporter mice. Furthermore, Gata3 conditionally mutant mice, when crossed with the TgcoreOVE-Cre, showed hypoplasia throughout the inner ear tissues. These results demonstrate that OVE has a sufficient regulatory activity to direct Gata3 expression specifically in the otic vesicle and semicircular canal and that Gata3 expression driven by the OVE is crucial for normal inner ear development.

Progesterone arrested cell cycle progression through progesterone receptor isoform A in pancreatic neuroendocrine neoplasm.

In pancreatic neuroendocrine neoplasms (Pan-NEN) progesterone signaling has been shown to have both inhibitory and stimulatory effects on cell proliferation. The ability of progesterone to inhibit tumor proliferation is of particular interest and is suggested to be mediated through the less abundantly expressed progesterone receptor (PR) isoform A (PRA). To date the mechanistic processes underlying this inhibition of proliferation remain unclear. To examine the mechanism of PRA actions, the human Pan-NEN cell line QGP-1, that endogenously expresses PR isoform B (PRB) without PRA, was transfected with PRA. PRA transfection suppressed the majority of cell cycle related genes increased by progesterone including cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2). Importantly, following progesterone administration cell cycle distribution was shifted to S and G2/M phases in the naïve cell line but in PRA-transfected cells, this effect was suppressed. To see if these mechanistic insights were confirmed in patient samples PRA, PRB, CCNA2, CCNB, CDK1 and CDK2 immunoreactivities were assessed in Pan-NEN cases. Higher levels of cell cycle markers were associated with higher WHO grade tumors and correlations between the markers suggested formation of cyclin/CDK activated complexes in S and G2/M phases. PRA expression was associated with inverse correlation of all cell cycle markers. Collectively, these results indicate that progesterone signals through PRA negatively regulates cell cycle progression through suppressing S and G2/M phases and downregulation of cell cycle phases specific cyclins/CDKs.

Tumor microenvironment in functional adrenocortical adenomas: immune cell infiltration in cortisol-producing adrenocortical adenoma.

The tumor microenvironment plays pivotal roles in various human neoplasms. However, that of benign tumor, particularly hormone-secreting endocrine tumors, has remained virtually unknown. Therefore, we firstly attempted to analyze the tumor microenvironment of autonomous hormone-secreting adrenocortical adenomas. We first histologically evaluated 21 cortisol-producing adrenocortical adenoma (CPA) and 13 aldosterone-producing adrenocortical adenoma (APA) cases. Quantitative histologic analysis revealed that intratumoral immune cell infiltration (ICI) was more pronounced in CPAs than in APAs. We then evaluated the cytokine and chemokine profiles using polymerase chain reaction arrays in APAs and CPAs. Angiogenic chemokines, C-X-C motif chemokine ligand (CXCL) 1 and CXCL2, were significantly more abundant in CPAs than in APAs using subsequent quantitative polymerase chain reaction and immunohistochemical analyses. We then examined the vascular density between these 2 adenomas, and the density was significantly higher in overt CPAs than in APAs. Of particular interest, CXCL12-positive vessels were detected predominantly in CPAs, and their infiltrating immune cells were C-X-C motif chemokine receptor 4 (CXCR4) positive. These results above indicated that CXCL12-CXCR4 signaling could partly account for ICI detected predominantly in CPAs. We then further explored the etiology of ICI in CPAs by evaluating the senescence of tumor cells possibly caused by excessive cortisol in CPAs. The status of senescence markers, p16 and p21, was significantly more abundant in CPAs than in APAs. In addition, all CPA cases examined were positive for senescence-associated ß-galactosidase. These results all indicated that exposure to local excessive cortisol could result in senescence of tumors cells and play essential roles in constituting the characteristic tissue microenvironment of CPAs.