Previous studies rarely focused on the difference in stiffness between invasive pituitary adenoma in the CS and SDL. Our current study demonstrated that the tumor texture was stiffer in the CS tumor that of the SDL tumor by DCE-MRI, AFM, and histological analysis. We also observed that stiffer matrix contributed to tumor invasion and EMT. Proteomic analysis further revealed that cancer associated fibroblasts could play a critical role in ECM reprogramming and was validated in PA tumor tissues and primary CAFs from CS and SDL. Additionally, we found that the mTOR pathway was activated in stiffer CS tumors and the everolimus inhibited this pathway. These findings provided valuable insights into the intratumor heterogeneity of the tumor texture, which could lead to improved treatment efficacy for invasive pituitary adenomas.
In the past, preoperative texture MRI evaluations of PA was all based on the overall evaluation of the adenoma. Our research represented an innovative approach for the analysis of stiffness by introducing regional quantitative measurements via DCE-MRI. Specifically, Ktrans is the permeability rate of the contrast agent from the microvessels to the extravascular ECM space, reflecting the local microvascular blood flow status and surface permeability. Kep represents the speed at which the contrast agent transferred from the extravascular space to microvessels, while Ve mainly reflects the percentage of contrast agent concentration in the extravascular extracellular space in the entire voxel [16]. These parameters have been predominantly utilized for indirect assessment of tumor heterogeneity in texture analysis in breast cancer and glioma studies. In our study, we observed higher Ktrans in the SDL compared to the CS, indicating the CS group have a lower contrast agent permeability, potentially associated with a greater stiffness density of the ECM. Furthermore, the higher value of Ve in the CS also suggested a higher contrast agent concentration, potentially indicative of soft ECM in this area. These conclusions were verified by subsequent endoscopic suction strength needed to aspirate tumors.
AFM has been widely utilized to investigate the mechanical profiles of cells, tissues, and macromolecules such as proteins. In particular, AFM in force mapping mode has been employed to directly measure the stiffness of tumor tissues and normal tissues [25, 26]. There are distinct stiffness peaks between malignant and benign tissues [27, 28]. Therefore, AFM holds significant promise in cancer research for both diagnostic and therapeutic purposes. In our present study, we discovered that pituitary tumor tissue located in the CS was significantly firmer than that located in the SDL. AFM provided a quantitative and direct method for determining the stiffness of pituitary tissues in different locations, and these findings were consistent with the results obtained from DCE-MRI.
Tumor stiffness is increasingly recognized as an important contributing factor to disease progression and invasion, and is primarily determined by the amount of ECM, particularly the amount of collagen and hyaluronan within the tumor [29]. The ECM comprises a three-dimensional network of macromolecules, such as proteins, proteoglycans, glycosaminoglycans, and collagens, within which soluble factors such as cytokines, chemokines and growth factors are embedded [30]. Tumor cells and stromal cells can remodel the ECM through the secretion of metalloproteinases (MMPs). Previous studies have shown that high expressions of MMP2 and MMP9 were associated with the invasion and recurrence of pituitary adenoma [31, 32]. In our study, we observed an increase in the expression levels of MMP2 and MMP9 as the ECM became stiffer, potentially contributing to tumor invasion. This highlights the intricate interplay between tumor stiffness, ECM composition, and the activity of MMPs in the context of pituitary adenoma progression.
EMT is a process related to tumorigenesis and tumor progression by which epithelial cells acquired the mesenchymal phenotypes, facilitating the migration, invasion, and metastasis [33]. Gao et al. reported that EMT was a significant hallmark associated with the stiffness of pituitary adenomas [14]. Additionally, stiff pituitary adenomas were found to contain more CAFs and endothelial cells. CAFs have been shown to promote EMT in pituitary adenomas by secreting cytokine factors [14]. This finding suggests a complex interplay between CAFs and EMT in promoting the stiffening of the pituitary adenoma texture. In our study, as the ECM stiffness increased, pituitary adenoma cells exhibited increased expression of mesenchymal markers. EMT and mechanotransduction are highly interactive and complex processes, which rely on several mechanisms, such as the Hippo/YAP, Piezo1, TGF-β signaling and so on [34–36]. Further exploration of the mechanism of stiffness-related EMT will aid in identifying potential treatment targets for invasive pituitary adenoma.
Our further investigations revealed that the mTOR pathway played an important role in the matrix stiffness of CS tumors. As the tumor volume increased, tumor cells can sense and transmit mechanical cues, which facilitated tumor growth, proliferation, invasion, and EMT [37]. The mTOR is at the central hub of signaling networks that regulate physical phenotype of cancer cells and transmit extracellular mechanical signals [38]. In our study, we observed the activation of the mTOR pathway in both CS tumors and GH3 cells cultured under conditions of high matrix stiffness, leading to downstream effects. CCND1 is a key regulator of cell cycle and proliferation [39]. c-Myc protein functions as a transcriptional regulator and modulates cell growth, apoptosis, and stem cell formation [40]. Bcl-2 regulates apoptosis, autophagy, and metabolism[41]. As matrix stiffness increased, the expression levels of CCND1, c-Myc and BCL-2 all elevated. This indicated that matrix stiffness could regulate multiple downstream effects and increase the invasion of PAs. Interestingly, altering matrix stiffness represents a new treatment option for pituitary adenoma. This highlights the potential therapeutic significance of targeting the mTOR pathway in the context of invasive pituitary adenoma.