A total of 90 cases of primary PA patients who received surgical resection and 50 cases of healthy volunteers were enrolled at the First Affiliated Hospital (Xijing Hospital) of the Air Force Medical University between June 2006 and May 2010. The inclusion criteria were as follows (1): the primary tumor (2); both radiological and pathological diagnosis verified as PA (3); no surgical or other special treatment; and (4) with complete clinicopathological information. The following were the exclusion criteria (1): together with severe liver, kidney, heart, and lung insufficiency (2); combined with other cancer; and (3) lost to follow-up. All patients were followed up by radiographical and clinical examinations till July 2022, and the median follow-up time is 101.6 months. The follow-up interval began on the date of the patient’s first tumor resection and ended on the date of tumor recurrence or last follow-up. Tumor recurrence was confirmed by clinical and imaging findings or histology analysis of specimens from the second surgery. Furthermore, another 15 normal human anterior pituitary glands were acquired from the donation process as the normal group. This study was approved by the ethics committee of First Affiliated Hospital (Xijing Hospital) belonging to the Air Force Medical University (No. 81627806). Informed consent was acquired from all patients, and the study was performed in accordance with the principles of the Declaration of Helsinki.

Serum samples were obtained from each patient before and on the 7th day after surgery. The changes in exosomal circCCDC66 between post-operation and recurrence from 30 recurrent patients were monitored. For serum specimens, peripheral venous blood (5 mL each) was stored at 4°C within 1 h after collection and centrifuged at 3000 g for 10 min at 4°C. Next, the supernatant was further centrifuged at 12,000 g for 10 min at 4°C to remove cell debris. Lastly, the supernatant fluids were transferred to RNase-free centrifuge tubes, and stored at −80°C for further analyses.

All tissue specimens were carried out and made anonymous by complying with the ethical and legal standards. The collected clinical specimens were first instantly frozen in liquid nitrogen and then stored in a −80°C refrigerator before examination. Frozen tissue specimens of PA were subjected to RNA examining after operation.

Enrichment and purification of exosomes from serum were conducted using the ExoQuick™ ULTRA EV Isolation Kit (cat: EQULTRA-20A-1, System Biosciences, Mountain View, CA, USA) according to the manufacturer’s instructions. All exosomes were stored at −80°C.

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphological characteristics of the purified exosomes as described previously (11).

Expression of exosomal markers such as protein CD63 and TSG101 was analyzed by Western blotting. RIPA lysis buffer (cat: 89901, Thermo Scientific) was used to extract total proteins. Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis, followed by transferring onto the polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Following blockage using 5% skimmed milk for 1 h, the PVDF membranes were incubated overnight with primary antibodies at 4°C. Finally, the combined signals were detected by Chemistar™ High-sig ECL Western Blot Substrate (cat: P10200, Tanon, Shanghai, China) after incubation of corresponding HPR-labeled secondary antibody for 2 h at room temperature. The antibodies CD63 (1:2,000, ab134045), TSG101 (1:2,000, ab30871), and HRP-conjugated IgG anti-rabbit (1:6,000, ab205718) were commercially acquired from Abcam (Cambridge, MA, USA).

The total RNA from tissue specimens was extracted using the TRIzol (Invitrogen, Carlsbad CA, USA) method. Exosomal RNA was isolated from serum utilizing the exoRNeasy Serum/Plasma Maxi Kit (Qiagen, Germany) following the manufacturer’s protocol. Next, the purity and concentration of RNA samples were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and the OD260/OD280 ratio that ranged from 1.8 to 2.0 indicated high purity. Total RNA was reverse transcribed to synthesize cDNA with random primers using a Prime Script RT Reagent Kit (TaKaRa, Dalian, China). Thereafter, the qPCR was executed by using Master Mix SYBR Green RT-PCR Super Mix (Tiangen Biotech Co., Ltd.) on the ABI 7500 Real-time PCR system (Applied Biosystems, CA, USA). The Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference of mRNAs. PCR primers are listed as follows: circCCDC66 F: 5′-ACC TAC AAC CGG AAG CCA G-3′, R: 5′-AGC AGT ACT GTT TCC TGA TGC-3′. GAPDH F: 5′-GGA GCG AGA TCC CTC CAA AAT-3′, R: 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′. The relative expression levels of circCCDC66 were calculated using the 2^−ΔΔCt method. All experiments were repeated three times.

Statistical Program for Social Sciences (SPSS) 22.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 8.0 Software (GraphPad Software, La Jolla, CA, USA) were used for all of the statistical analyses. The Student’s t-test or one-way ANOVA was used to analyze qRT‐PCR results between the PA and control groups. The chi-square or Fisher’s exact test was used to analyze the correlations between exosomal circCCDC66 expression and the clinicopathological factors of PA patients. The correlation of circCCDC66 expression levels in PA tissues and serum samples was performed by Spearman correlation coefficient. The receiver operating characteristic (ROC) curve and the Kaplan–Meier survival plot were established to evaluate the diagnostic and prognostic capacity of exosomal circCCDC66. Statistical significance was determined as p < 0.05 (*) or p < 0.01 (**).

Serum exosomes were extracted from PA patients and healthy control group samples to explore the potentially diagnostic biomarkers of exosomal circRNA in PA. As shown in Figure 1A , the exosomes characterized as cup-shaped morphology were observed with an atypical size of 30–160 nm. The diameters of exosomes in the healthy controls and PA patients were 73.02 and 74.12 nm, respectively ( Figure 1B ). Western blotting was applied to detect the expression of exosomal markers CD63 and TSG101, and the results proved that the two markers were all highly enriched in the extracted exosomes whether from healthy controls or PA patients ( Figure 1C ). Taken together, the above results, which confirmed the validity and purity of exosome extraction from serum in this study, served as a good foundation for further exploration.

In order to evaluate the diagnostic value and prognostic significance of circCCDC66 in PA, quantitative real-time PCR (qRT-PCR) was initially applied to examine the expression of circCCDC66 in serum exosomes. Levels of serum exosomal circCCDC66 were examined in Figure 2A to compare the difference between PA patients (n = 90) and healthy controls (n = 50). Analysis of the expression of exosomal circCCDC66 revealed an obviously significant upregulation in PA patients in contrast to healthy controls after normalization (p < 0.01, Figure 2A ). The expression of circCCDC66 in PA tissue samples was also measured, as shown by the results in serum exosome, and circCCDC66 was significantly upregulated in PA specimens ( Figure 2B ). Pearson’s correlation coefficient analysis revealed a significant positive correlation between the expression of PA tissues and serum samples (r = 0.864, p < 0.01, Figure 2C ). CircCCDC66 expression was totally increased in the serum exosome and tissue specimens of PA patients.

To further investigate whether serum exosomal circCCDC66 expressions are associated with tumor dynamics, we analyzed serum exosomal circCCDC66 expressions in 30 recurrent patients at different time periods. As observed, serum exosomal circCCDC66 expressions were statistically downregulated following resection of the primary tumor (p < 0.01, Figure 3A ). However, the expression levels of exosomal circCCDC66 were remarkably increased at the time of PA recurrence (p < 0.01, Figure 3B ). Collectively, serum exosomal circCCDC66, which was secreted and released by PA cells, could monitor tumor dynamics and serve as a potentially prognostic biomarker during treatment.

The receiver operating characteristic (ROC) curve analysis and the corresponding area under the curve (AUC) values were performed to confirm the ability of serum exosomal circCCDC66 as a potentially diagnostic and prognostic biomarker for PA patients. As demonstrated in Figure 4 , the AUC value of serum exosomal circCCDC66 levels was 0.8719 (95% CI: 0.8134–0.9304), which suggested that exosomal circCCDC66 could discriminate PA patients from healthy controls (p < 0.01). At the optimal cutoff value, the sensitivity, specificity, and positive and negative predictive values to distinguish PA patients were 80.00%, 84.00%, 86.64%, and 79.53%, respectively. These findings indicated that serum exosomal circCCDC66 could be a promising diagnostic biomarker to effectively monitor the PA occurrence.

In this cohort, we were engaged to confirm the prognostic function of serum exosomal circCCDC66 in PA patients. The clinical characteristics of the recruited PA patients are summarized in Table 1 according to the median expression of serum exosomal circCCDC66. The follow-up information of the patients was updated to July 2022. There were no statistically significant differences in age and gender distribution, while Knosp classification, tumor size, and recurrence were associated with the expression of serum exosomal circCCDC66 in PA (p < 0.01). Interestingly, the progression-free survival (PFS) was much longer in the low serum exosomal circCCDC66 group compared with the high serum exosomal circCCDC66 group (p < 0.01, Figure 5 ). Totally, these results provided lines of evidence that serum exosomal circCCDC66 may serve as a potential factor for prognosis prediction.