背景与目的

垂体神经内分泌肿瘤（pituitary neuroendocrine tumors，PitNETs）是第二常见的原发性脑肿瘤，其中生长激素瘤（somatotroph PitNET/adenoma）是一类起源于腺垂体分泌生长激素（growth hormone，GH）从而导致全身多系统并发症的PitNETs。侵袭性生长激素瘤低治愈率和高复发率严重影响患者的生活质量和预期寿命，是临床诊疗的难点，其生物学行为复杂多变，分子机制和可靠的预测生物标志物仍未明确。近年来，PitNETs的肿瘤微环境（tumor microenvironment，TME）研究进展显著，TME成分在侵袭性或复发性肿瘤生物学行为中起重要作用，目前对于PitNETs 的TME生物学功能和作用机制的研究仍然有限。因此，本研究旨在通过垂体生长激素瘤肢端肥大症患者的回顾性临床队列、肿瘤组织样本和体外作用及机制研究，探究侵袭性垂体生长激素瘤患者临床和预后特征、侵袭性生物标志物及分子机制，为临床制定个体化治疗方案、改善患者预后提供理论依据。

研究方法

本研究分为四个部分，分别从不同层面探究侵袭性垂体生长激素瘤患者的临床特征、预后指标、侵袭机制及其与TME的相互作用。

第一部分回顾性纳入于北京协和医院接受手术治疗的72例垂体生长激素瘤患者，分析其临床特征、预后指标及影响因素，重点关注年龄、性别、病程、临床表现、激素水平、影像学特征、分子病理特征等，并探讨术后3个月和长期生化缓解率与起病情况、肿瘤大小、侵袭性、激素水平、Ki-67指数等因素的关系。

第二部分聚焦于罕见的侵袭性垂体生长激素巨腺瘤（肿瘤最大径>40mm）的临床特征和治疗策略。回顾性分析67例生长激素瘤巨腺瘤患者的最大单中心队列，并与同期年龄和性别匹配的生长激素大腺瘤（10～40mm）患者进行比较，总结其临床表现和预后特征。

第三部分连续纳入10例侵袭性和10例非侵袭性垂体生长激素瘤患者肿瘤组织，采用基于数据独立采集（data-independent acquisition ，DIA）蛋白组学技术，对侵袭性和非侵袭性垂体生长激素瘤组织的蛋白质组学特征进行分析，发现潜在的生物标志物组织蛋白酶Z（cathepsin Z，CTSZ）。通过实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction, RT-qPCR）、免疫组化、多重免疫荧光、蛋白印记法在肿瘤组织中验证并探讨其与肿瘤侵袭性和临床预后的关系。通过质粒转染过表达GH3细胞中Ctsz进行体外验证。

第四部分进一步深入研究CTSZ在垂体生长激素瘤中的侵袭性机制，特别是其与巨噬细胞的协同作用。通过结合公共数据库中CTSZ的表达与预后特征，和巨噬细胞上清与GH3建立的共培养体系以及Ctsz重组蛋白处理实验，探究巨噬细胞来源因子对GH3细胞增殖、激素分泌、侵袭和迁移的影响，并通过RNA测序揭示相关调控机制。

研究结果

第一部分研究发现与非侵袭性相比，侵袭性垂体生长激素瘤患者的首诊年龄更小，激素分泌水平更高，肿瘤生长速度明显更快，且长期生化缓解率更低（38.5% vs 80.6%，P=0.002）。术后3个月和长期生化缓解率与术后空腹GH水平、口服葡萄糖耐量试验（oral glucose tolerance test，OGTT）GH谷值、肿瘤最大径和Knosp分级等因素密切相关，且术后1周内的空腹GH水平是术后3个月生化缓解的独立相关因素。

第二部分结果显示大多数垂体生长激素巨腺瘤（74.6%）和大腺瘤（78.8%）患者均以手足增大和面容改变为首发症状。视野缺损和低促性腺激素型性腺功能低减在巨腺瘤组较大腺瘤组更多见。巨腺瘤患者呈现显著激素高分泌和侵袭性生长特征（P值均<0.05）。随着腺瘤大小的增大，肿瘤全切率降低，术后并发症和所需要治疗方式总量显著增加（P值均<0.05）。中位随访36个月后，巨腺瘤和大腺瘤组患者的生化缓解率相当，分别为36.4%（12/33）和36.2%（17/47），而巨腺瘤组患者垂体前叶功能减退的发生率显著高于大腺瘤组（P<0.001）。其他因素包括发病年龄、诊断年龄、延迟诊断时间、代谢并发症、P53阳性率、Ki-67指数两组间差异无统计学意义。

第三部分蛋白质组学研究结果显示侵袭性与非侵袭性垂体生长激素瘤表现出不同的蛋白质组特征。与非侵袭组相比，侵袭组中与肿瘤增殖、迁移和侵袭相关的经典通路，包括IL-4、PDGF、PTEN、VEGF、PI3K/AKT、FAK等被显著激活，且差异表达蛋白与DNA修复、肿瘤细胞聚集、增殖、侵袭等生物学功能显著相关。首次发现CTSZ在侵袭性垂体生长激素瘤中显著高表达（倍数变化 = 13.893, P = 0.003），随后在72例肿瘤组织样本验证[免疫组化评分：侵袭组17.8（12.4-383.3），非侵袭组15.2（9.6–28.0），P = 0.036]，且其表达与肿瘤侵袭性和生长参数显著正相关。细胞水平功能验证表明，Ctsz过表达促进了GH3细胞的增殖、迁移和侵袭。

第四部分研究发现，CTSZ在泛癌中显著高表达，且与多种癌症不良预后及巨噬细胞浸润相关。多重免疫荧光显示在垂体生长激素瘤组织中CTSZ与巨噬细胞共定位，且与CD163的表达率呈显著正相关关系（R²=0.422，P=0.036），表明巨噬细胞来源的CTSZ可能在垂体生长激素瘤的侵袭性中发挥作用。由于GH3细胞自身Ctsz的转录水平和蛋白含量极低，因此，本研究一方面通过采用Raw264.7巨噬细胞上清或PMA活化后Raw264.7细胞上清处理GH3细胞，酶联免疫吸附试验检测证实两种上清中Ctsz含量均显著高于GH3自身分泌水平。另一方面，采用Ctsz重组蛋白单独处理GH3细胞。结果显示，巨噬细胞Raw264.7条件培养基促进GH3细胞的增殖、迁移、侵袭和GH含量的增加，而Ctsz重组蛋白对细胞增殖和GH分泌无显著影响。此外，巨噬细胞来源因子还促进GH3细胞面积、周长和Feret's直径显著增加，以及在透射电镜下观察到激素囊泡的外分泌增加证实了巨噬细胞来源因子对GH的促分泌作用。电镜下对照组GH3细胞中自噬溶酶体的数量最多，提示基础条件下细胞自噬活动较为活跃，而巨噬细胞上清和Ctsz重组蛋白处理后自噬溶酶体数量显著减少。蛋白印记显示巨噬细胞上清处理后自噬标志物Lc3-II显著下降，p62蛋白累积量减少，提示自噬降解明显恢复，细胞状态明显改善。RNA-seq结果提示巨噬细胞分泌因子可能通过调控TGF-β信号、钙信号以及多种免疫抑制相关通路发挥促肿瘤作用。RT-qPCR和蛋白印记验证Dcn/TGF-β/p15信号通路中Dcn下调减少了对Tgf-β1的抑制，导致Tgf-β1表达上调，而Tgf-β1对p15调节失控，其表达下调解除了对Cdk4的抑制，促进肿瘤细胞进展。

结论

本研究揭示了侵袭性垂体生长激素瘤患者的临床特征、预后指标及其分子机制，巨噬细胞分泌因子通过调控自噬活动和TGF-β信号通路，促进了GH3细胞的增殖、GH分泌、迁移和侵袭，特别是巨噬细胞来源CTSZ可能在其中发挥作用。研究结果为侵袭性垂体生长激素瘤的诊断、治疗及预后评估提供了理论依据，深化了对垂体生长激素瘤侵袭机制的理解，为未来的治疗策略提供了新的思路。

Background and Purpose

Pituitary neuroendocrine tumors (PitNETs) are the second most common primary brain tumors. Among them, somatotroph PitNET/adenoma are a type of neuroendocrine tumor originating from the adenohypophysis, which secretes growth hormone (GH) and leads to systemic complications. Invasive somatotroph PitNETs, characterized by low cure rates and high recurrence rates, significantly impact patients' quality of life and life expectancy, posing a challenging clinical issue and a hot research topic. The biological behavior of these tumors is complex and variable, and their molecular mechanisms and reliable predictive biomarkers remain unclear. In recent years, significant progress has been made in the study of the tumor microenvironment (TME) of PitNETs. TME components play a crucial role in the biological behavior of invasive or recurrent tumors. However, research on the biological functions and mechanisms of the TME in PitNETs is still limited. Therefore, this study aims to investigate the clinical and prognostic characteristics, invasive biomarkers, and molecular mechanisms of invasive somatotroph PitNETs through retrospective clinical cohorts, tumor tissue samples, and in vitro functional and mechanistic studies. The findings are expected to provide a theoretical basis for developing individualized treatment strategies and improving patient outcomes.

Methods

This study is divided into four parts, each exploring the clinical characteristics, prognostic indicators, molecular mechanisms, and interactions with the TME of invasive somatotroph PitNETs from different perspectives.

Part 1: A retrospective analysis was conducted on 72 patients with somatotroph PitNETs who underwent transsphenoidal surgery at Peking Union Medical College Hospital. Clinical characteristics and prognostic indicators were analyzed, with a focus on age, gender, disease duration, clinical manifestations, hormone levels, imaging features, and molecular pathological characteristics. The relationship between biochemical remission rates at 3 months post-surgery and long-term outcomes with factors such as disease onset, tumor size, invasiveness, hormone levels, and Ki-67 index was investigated.

Part 2: This section focused on the clinical characteristics and treatment strategies of rare invasive somatotroph giant adenomas (tumor diameter >40 mm). A retrospective analysis of 67 patients with somatotroph giant-adenomas, the largest single-center cohort to date, was performed. These patients were compared with age- and gender-matched patients with somatotroph macroadenomas (10–40 mm) from the same period. Clinical manifestations and prognostic features were summarized.

Part 3: Tumor tissues from 10 invasive and 10 non-invasive somatotroph PitNETs patients were consecutively included. Data-independent acquisition (DIA) proteomics technology was used to analyze the proteomic characteristics of invasive and non-invasive somatotroph PitNETs, identifying cathepsin Z (CTSZ) as a potential biomarker. Validation of CTSZ in tumor tissues was performed using real-time quantitative polymerase chain reaction, RT-qPCR), immunohistochemistry, immunofluorescence, and Western blotting to explore its relationship with tumor invasiveness and clinical prognosis. In vitro validation was conducted by overexpressing Ctsz in GH3 cells via plasmid transfection.

Part 4: The role of CTSZ in invasive somatotroph PitNETs, particularly its synergistic effects with macrophages, was further investigated. By combining CTSZ expression and prognostic features from public databases, as well as co-culture systems of macrophage supernatant with GH3 cells and Ctsz recombinant protein intervention experiments, the impact of macrophage-derived factors on GH3 cell proliferation, hormone secretion, invasion, and migration was explored. RNA sequencing was used to reveal the related regulatory mechanisms.

Results

Part 1: Compared to non-invasive cases, patients with invasive somatotroph PitNETs had a younger age at initial diagnosis, higher hormone secretion levels, significantly faster tumor growth rates, and lower long-term biochemical remission rates (38.5% vs. 80.6%, P = 0.002). The biochemical remission rates at 3 months post-surgery and long-term outcomes were closely associated with postoperative fasting GH levels, GH nadir during the oral glucose tolerance test (OGTT), maximum tumor diameter, and Knosp grading. Fasting GH levels within the first week after surgery were identified as an independent factor for biochemical remission at 3 months post-surgery.

Part 2: The results showed that most patients with somatotroph giant-adenomas and macroadenomas presented with acromegaly and facial changes as initial symptoms. Visual field defects and gonadal axis dysfunction were more common in the giant-adenoma group than in the macroadenoma group. Patients with giant-adenomas exhibited significantly higher hormone secretion and invasive growth characteristics (all P < 0.05). As tumor size increased, the total resection rate decreased, and postoperative complications and the total number of required treatments significantly increased (all P < 0.05). After a median follow-up of 36 months, the biochemical remission rates were comparable between the giant-giant-adenoma and macroadenoma groups [36.4% (12/33) vs. 36.2% (17/47)], while the incidence of anterior pituitary dysfunction was significantly higher in the macroadenoma group (P < 0.001). Other factors, including age at onset, age at diagnosis, delayed diagnosis age, metabolic complications, P53 positivity rate, and Ki-67 index, showed no significant differences between the two groups.

Part 3: Proteomic analysis revealed distinct proteomic profiles between invasive and non-invasive somatotroph PitNETs. Compared to the non-invasive group, classic pathways related to tumor proliferation, migration, and invasion, including IL-4, PDGF, PTEN, VEGF, PI3K/AKT, and FAK, were significantly activated in the invasive group. Differentially expressed proteins were significantly associated with biological functions such as DNA repair, tumor cell aggregation, proliferation, and invasion. CTSZ was identified for the first time as significantly overexpressed in invasive somatotroph PitNETs (Fold change = 13.893, P = 0.003). Validation in 72 tumor tissue samples [IHC score: invasive group 17.8 (12.4~383.3), non-invasive group 15.2 (9.6–28.0), P = 0.036] confirmed its positive correlation with tumor invasiveness and growth parameters. In vitro experiments demonstrated that Ctsz overexpression enhanced the proliferation, migration, and invasion capabilities of GH3 cells.

Part 4: CTSZ was found to be significantly overexpressed in pan-cancer and associated with poor prognosis and macrophage infiltration in various cancers. Immunofluorescence revealed co-localization of CTSZ with macrophages in somatotroph PitNETs tumor tissues, with a significant positive correlation between CTSZ and CD163 expression (R² = 0.422, P = 0.036), suggesting that macrophage derived CTSZ may play a role in the invasiveness of somatotroph PitNETs. Due to the extremely low transcriptional level and protein concentration of Ctsz in GH3 cells themselves, this study adopted two approaches. On one hand, Raw264.7 macrophage supernatant or PMA-activated Raw264.7 cell supernatant was used to treat GH3 cells, and enzyme-linked immunosorbent assay confirmed that the concentration of Ctsz in both supernatants was significantly higher than the secretion level of GH3 cells themselves. On the other hand, recombinant Ctsz protein was used to treat GH3 cells alone. The results showed that Raw264.7 macrophage-conditioned medium promoted the proliferation, migration, invasion, and increased the concentration of GH in the supernatants of GH3 cells, while the Ctsz recombinant protein had no significant effect on cell proliferation and GH secretion. Additionally, macrophage-derived factors also significantly increased the cell area, perimeter, and Feret's diameter of GH3 cells, and the increased exocytosis of hormone vesicles observed under transmission electron microscopy confirmed the pro-secretory effect of macrophage-derived factors on GH. Under electron microscopy, the control group of GH3 cells exhibited the highest number of autolysosomes, suggesting active autophagic activity under basal conditions, while the number of autolysosomes significantly decreased after intervention with macrophage supernatant and Ctsz recombinant protein. Western blot analysis showed that macrophage supernatant intervention led to a significant decrease in the autophagy marker Lc3-II and reduced accumulation of p62 protein, indicating a significant recovery of autophagic degradation and improved cell state. RNA-seq results revealed that macrophage-secreted factors exert pro-tumor effects by regulating the TGF-β pathway, calcium signaling pathway, and multiple immune suppression-related pathways. RT-qPCR and Western blot validation demonstrated that downregulation of Dcn in the Dcn/TGF-β/p15 signaling pathway reduced the inhibition of Tgf-β1, leading to upregulation of Tgf-β1. The dysregulation of Tgf-β1 on p15 resulted in downregulation of p15 expression, which lifted the inhibition on Cdk4 and promoted tumor progression.

Conclusion

This study elucidates the clinical characteristics, prognostic indicators, and molecular mechanisms of invasive somatotroph PitNETs. Macrophage-secreted factors promote the proliferation, GH secretion, migration, and invasion of GH3 cells by regulating autophagy and the TGF-β pathway, with macrophage-derived CTSZ potentially playing a significant role. The findings provide a theoretical foundation for the diagnosis, treatment, and prognostic evaluation of invasive somatotroph PitNETs, deepen the understanding of the invasive mechanisms, and offer new insights for future therapeutic strategies.