Vestibular schwannomas (VS) are benign tumors of the eighth cranial nerve caused by Schwann cell proliferation (1). A major characteristic in VS pathogenesis is the loss of merlin protein function. Merlin is a transmembrane protein that links cell membrane and cytoskeleton. It is activated by intercellular adhesion and by attachment to the extracellular matrix (2). In patients with NF2-related schwannomatosis (NF2), one allele for merlin on chromosome 22 is defective at birth, and a second hit during lifetime may result in loss of protein function. As hallmark tumors these patients develop bilateral VS at a young age. Later on, several other tumors, such as meningioma and ependymoma develop in addition in most cases (3, 4). Surgery, radiosurgery and “watch & wait” are the currently established therapeutic options for VS. However, in patients with NF2, these treatment options are very limited due to the higher invasiveness of the tumors and cumulative morbidity after multiple interventions and because of tumor burden. Bevacizumab might be an additional therapeutic choice. However, for this subgroup of VS as well as for older patients with high comorbidity, a better systemic therapeutic approach is urgently needed (3).

To understand genesis, sustenance and growth of VS, the surroundings of the tumor have to be taken into account. Inflammation and tumor microenvironment (TME) might play a crucial role in tumor formation and maintenance, in nerve infiltration, dysfunction and neuropathy. Understanding the composition of the TME will also benefit in the research of a potential systemic therapeutic approach not only for patients suffering from VS, who are not suitable for surgery due to age, comorbidity, or size of the tumor, but even more importantly for patients suffering from NF2. Identifying different cellular components of the TME, their quantity and localization might give us a better idea of the VS-TME interaction. The immune cell population consists of tumor-associated macrophages (TAMs) as well as CD4+ and CD8+ T-lymphocytes, regulatory T-cells (CD25+) and Natural Killer (NK) cells (CD7+). TAMs are derived from circulating monocytes and can be polarized into different functional phenotypes, primarily classified as M1 and M2. CCR2 identifies macrophages that immigrated to the central nervous system. Immune cell infiltration tends to happen in rather loosely cellular Antoni B areas (5–7). TAMs play an important role in solid tumors (8, 9) and drive regulating processes like growth and tumor invasion (8, 9). Depending on surrounding cytokine expression, macrophages show a broad variety of functional stages. Here, we focus on the investigation of the pro-inflammatory M1-macrophages and the immune-regulating M2-macrophages (10). While M1-macrophages show anti-tumor effects and are associated with a better prognosis in many solid tumors (11), M2-macrophages promote tumor growth and angiogenesis (12). Macrophage expression in VS had a significant impact on tumor growth (13) and tumor vessel density (14). Information on the role of lymphocytes in the VS TME is scarce. An investigation of 10 NF2-associated VS showed perivascular expression of lymphocytes (15). However, their role remains unclear.

Nuclear c-jun is a marker for Büngner cells, a Schwann cell in repair mode, which are an additional part of the TME beside immune cells (16). It is expressed in immature Schwann cells but downregulated in mature myelinating cells. Following injury, c-jun is upregulated and phosphorylated in Schwann cells, driving a demyelination program. Recent studies have highlighted the significance of c-jun in nerve injury response, as its conditional ablation in Schwann cells leads to increased neuronal death, impaired axonal regeneration, and diminished functional recovery (17). Molecularly, c-jun regulates the expression of myelin structural genes such as MBP and P0, as well as trophic factors like BDNF and GDNF, essential for regeneration (17). Additionally, c-jun activation induces expression of genes distinct from those in immature Schwann cells, indicating a specialized role in injury response. Paracrine signaling mediated by c-jun-induced GDNF and Artemin is crucial for neuronal survival post-injury (17). Importantly, c-jun’s involvement appears specific to the injury response, suggesting its essential role in peripheral nerve regeneration. Moreover, aberrant c-jun activation in peripheral neuropathy patients underscores its potential pathological significance. C-jun has been implicated in the context of Büngner cells within VS. These specialized Schwann cells contribute to the tumor microenvironment and play a pivotal role in tumor progression. C-jun has been shown to modulate various cellular processes, including proliferation, differentiation, and survival, and its aberrant activation has been associated with tumor growth and invasion. In VS, c-jun may influence the behavior of Büngner cells by regulating their response to local signaling cues and promoting their participation in tumor-stromal interactions. Understanding the role of c-jun in Büngner cells could shed light on the mechanisms underlying tumor-stromal crosstalk and provide insights into potential therapeutic targets. Further investigation is warranted to unravel the precise molecular pathways through which j-jun influences Büngner cells and its implications for VS progression (16). The aim of this retrospective analysis was to investigate the composition of the cellular TME and c-jun expression in VS in 40 patients and to determine possible correlation with clinical data. Differences between sporadic and NF2-associated VS will be investigated. Certain types of immune cells, present specifically in VS, could serve as targets for immunotherapeutic approaches in the future.

The present analysis offers a comprehensive examination of the TME in sporadic and NF2-associated VS, encompassing some clinical parameters and a wide array of immune cell markers. Emerging evidence underscores the increasing importance of immune cells within the microenvironment. Contrary to the conventional notion that only mutated Schwann cells bear relevance in VS pathogenesis, our findings reveal a substantial presence of immune cells, particularly macrophages, in these benign tumors (22).

Macrophages (CD14+, CD68+) are notably abundant in VS, particularly in growing tumors, larger lesions, those displaying a higher proliferation index, and cases characterized by prolonged symptom duration (23). M2 macrophages (CD163+) have a pivotal mechanistic role in tumor growth, angiogenesis, and tumor invasion, primarily through their capacity to degrade the extracellular matrix (8, 24–26). In our cohort, we found a significant higher expression of macrophage markers in both NF2-related and sporadic VS, which is mirrored by an analysis of bulk transcriptomic data of three microarray data sets comparing the gene expression profiles of sporadic and NF2-related VS (27). In this investigation, no significant differences in signaling pathways, gene expression, cell type abundance or imaging mass cytometry staining between NF2-related and sporadic VS could be found. Intriguingly, our cohort identified a substantial TAM marker presence not only in NF2 patients but also in sporadic cases when compared to healthy vestibular nerves. Expression of macrophage markers was lower (although not significantly) in sporadic compared to NF2-related VS, indicating potentially different macrophage infiltration between these tumor types in line with the findings provided by Lewis et al. (28). This study aimed to conduct a comparative analysis of the TME of sporadic and NF2-related VS using a comprehensive approach involving imaging and tissue analysis. Further examination revealed a notable increase of proliferation marker Ki67 regions characterized by high TAM density compared to those with low-density regions highlighting a potential link between macrophage infiltration and cellular proliferation. Several other Studies have linked higher TAM density with increased microvessel density and elevated levels of macrophage colony stimulating factor (M-CSF), suggesting potential therapeutic targets (29). Additionally, there’s evidence of spatial correlation between TAM density and expression of vascular endothelial growth (VEGF) and its receptor VEGFR-1, indicating a role for VEGF pathway in TAM recruitment. Investigation into sporadic VS growth has shown a positive correlation between immune cell factors such as CD45 and CD68 expression and tumor size and growth rate, as well as elevated CD163 expression in rapidly growing tumors (29). These findings underscore the significance of TAMs in VS progression and suggest potential therapeutic strategies targeting TAMs and angiogenesis pathways. However, our analysis could not replicate a correlation with tumor growth dynamics, constrained by the limitations of sample size and the retrospective nature of the study. None of the immune markers demonstrated a correlation with hearing function as an indicator of tumor induced neuropathy, a finding consistent with the majority of published literature, which highlights only isolated targets such as NLRP3 inflammasomes as correlating with hearing function (23). Regulatory T-cells, though infrequently investigated in the context of VS (30) exhibit a correlation with tumor growth. Our analysis identified CD25, a marker for regulatory T-cells, as highly expressed in rapidly growing sporadic VS in our cohort.

The significance of immune cells as novel targets for systemic interventions is gaining prominence. Potential avenues include the use of aspirin as a COX2 inhibitor, re-education of M2 macrophages to M1 macrophages, antagonists of IL-1 and TNFα, checkpoint inhibitors, PD-L1 inhibitors, or Bevacizumab as an angiogenesis inhibitor. Programmed death-1 (PD-1) is a surface protein found on cytotoxic T-cells, while its counterpart, Programmed death-ligand 1 (PD-L1), is frequently present on the surface of tumor cells in various malignancies. Binding of PD-L1 to PD-1 inhibits the immune response mediated by cytotoxic T-cells (30). Although PD-L1 expression was not evaluated in VS in our study, however, cytotoxic T-cells were present in both sporadic and NF2-associated VS. Targeting this interaction could be a promising avenue for future therapeutic interventions in VS. Regulatory T cells, identified by the expression of CD4, CD25, and Foxp3 proteins, play a pivotal role in tumor progression by suppressing immune responses specifically targeting tumors. In patients with NF2, the abundance of Foxp3-positive regulatory T cells in progressing schwannomas significantly exceeded that in non-progressing cases, indicating a potential link between the presence of these cells and tumor growth (30). While our analysis did not reveal a significant difference in CD25 expression between rapidly and slowly growing VS, there is a noticeable trend towards increased CD25 expression in rapidly growing sporadic VS. This suggests the possibility of targeting CD25-positive regulatory T cells as a potential treatment strategy for rapidly growing sporadic VS. Furthermore, immune cells could serve as prognostic biomarkers if imaging methods capable of in vivo quantification, such as PET-CT for Iba 1 (an M2 marker), can be developed. Such biomarkers could aid in assessing disease progression, especially in cases of partial resection (23). Notably, Schwann cells in repair mode, as indicated by c-jun expression, may also play a pertinent role in pathogenesis. Schulz and colleagues observed a modest increase in c-jun expression, indicative of Schwann cell immaturity, in crushed nerves within an orthotopic mouse model featuring both homozygous and heterozygous nf-2 knock-out mice, as compared to wild-type animals (31). In our investigation, we witnessed a significant overexpression in rapidly growing sporadic VS, indicating repair cells in rapidly growing tumors. Interestingly, these findings are mirrored by the expression of regulatory T-cells. The therapeutic implications and functional roles of these Schwann cells remain an enigma, awaiting further investigation.

Our study represents a comprehensive investigation into the cellular expression of TME within VS, encompassing both NF2-associated and sporadic cases. By analyzing a substantial cohort of 40 patients, we aimed to provide a thorough comparative analysis of the TME in these distinct clinical contexts. It is important to note that while our study contributes valuable insights to basic research, it does not extrapolate findings directly applicable to clinical diagnostic, prognostic, or therapeutic decisions. Although our primary focus was not on clinical applications, we acknowledge the potential therapeutic implications of targeting the TME for patients. However, our study did not specifically investigate therapeutic interventions. Our research involved an in-depth examination of various clinical parameters by assessing immune components of the TME through immunohistochemistry. This included the evaluation of T-cell markers (CD4, CD8), NK cells (CD7), regulatory T-cells (CD25), and monocytes/macrophages (CD14, CD68, CD163), alongside innovative markers such as iNOS (as a macrophage marker) and c-jun (as a Büngner cell marker). Through meticulous analysis, we were able to establish significant correlations between immune cell populations and various clinical parameters. However, it is important to acknowledge the limitations of our study. Analysis was performed using paraffin-embedded tissue, which may introduce changes compared to fresh tissue samples. Additionally, immunohistochemistry is a semi-quantitative method, and despite our efforts to ensure methodological rigor, inherent limitations and potential for error exist. While alternative methods such as flow cytometry could have been considered, they would have resulted in the loss of spatial distribution information for the markers within the tissue. In summary, our study contributes to the understanding of the TME in VS, emphasizing the complexity of immune cell interactions and their potential implications for clinical outcomes. However, further research incorporating complementary methodologies and larger patient cohorts is warranted to elucidate the clinical relevance of our findings.

In conclusion, immune cells play a pivotal role in the pathogenesis of VS, both in the context of NF2-associated and sporadic cases, with a particular emphasis on monocytes and macrophages. Notably, our analysis revealed significant differences in the expression of CCR2 and CD163 between NF2-associated and sporadic VS. Downregulation of iNOS, a marker associated with M1 macrophages, was observed in VS, although statistical significance was not reached. Furthermore, CD25, a marker indicative for regulatory T-cells, exhibited significant differences between slowly and rapidly progressing VS, suggesting its potential utility as a biomarker for assessing tumor growth dynamics. These findings underscore the emerging significance of immune cells as promising candidates for both biomarker development and systemic therapeutic approaches in the context of VS. Further prospective investigations are warranted to elucidate the functional implications of these immune cell markers and their potential as targets for therapeutic intervention.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Ethikkommission der Universität Würzburg. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

VN: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing. DZ: Formal analysis, Investigation, Validation, Writing – review & editing. CR: Investigation, Visualization, Writing – original draft. DK: Conceptualization, Methodology, Writing – review & editing. RN: Formal analysis, Validation, Writing – review & editing. ER: Investigation, Methodology, Writing – review & editing. CMM: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – review & editing. R-IE: Conceptualization, Methodology, Resources, Supervision, Writing – review & editing. CM: Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review & editing. ML: Conceptualization, Methodology, Supervision, Writing – review & editing. CH: Conceptualization, Methodology, Resources, Supervision, Writing – original draft. MB: Writing – original draft, Formal analysis, Funding acquisition, Investigation, Resources, Supervision, Visualization, Writing – review & editing.