Meningiomas are the most commonly occurring CNS tumors in adults (42.6% of all tumors) (1). The overall proportion of WHO grades 1, 2, and 3 intracranial meningiomas was 94.6%, 4.2%, and 1.2%, respectively (2); however, these data may differ across studies. Tumor recurrence represents a major challenge in the management of meningioma patients (3–8). The main prognostic factors are the extent of surgical resection and meningioma grade (2, 7). Favorable outcomes are typically achieved with radical resection (i.e., in non-skull base meningiomas without infiltration of nerves, vessels, dural venous sinuses or bone) and in benign meningiomas treated with surgery and radiotherapy. The overall rate of meningioma recurrence is 14-33% according to different authors, and it can occur despite treatment with total resection and subsequent radiotherapy (3–8). The biological aggressiveness of meningiomas, defined by their locally destructive growth that precludes radical resection, involves invasion of the dura mater, brain, blood vessels, dural venous sinuses, skull bones, and is more commonly associated with grade 2 and 3 tumors (9, 10). The 5-year recurrence rate is 0-2.36% for grade 1 meningiomas and 7.35-11.46% for grade 2 meningiomas. In contrast, grade 3 meningiomas have the most aggressive behavior, demonstrating a recurrence rate of 50–94% (5).

Recurrences predominantly are located within or near the surgical bed (11). Pharmacotherapy can be used in meningioma, especially in malignant and other recurrent tumors; however, no effective standard pharmacological regimens are established for these patients (2, 7, 12). Therefore, local tumor control represents a critical focus in the meningioma clinical management. Despite being the primary modalities for tumor local control, both surgical resection and radiosurgery demonstrate suboptimal effectiveness against malignant and recurrent meningiomas (2, 6–9). Therefore, there is a pressing clinical need to develop and refine intraoperative local therapeutic strategies that can improve the radicality of resection for atypical and anaplastic meningiomas while simultaneously targeting residual tumor cells. Photodynamic therapy represents a promising modality for the local control of meningiomas.

Photodynamic therapy is a modern oncological treatment; however, its clinical application in neuro-oncology is still at the clinical study stage, primarily for gliomas and less frequently for meningiomas (13–17). PDT was developed as an adjuvant to surgery to improve its efficacy via a photochemical reaction, which relies on three components: a photosensitizer (PS), light, and oxygen. The antitumor effect of PDT involves three key mechanisms: direct cell death induced by reactive oxygen species (ROS), vascular damage, and the induction of an acute inflammatory response that mobilizes the immune system (18). Furthermore, some PSs, such as 5-aminolevulinic acid (5-ALA), are also used in intraoperative fluorescence diagnostics. Research on the application of PDT for meningiomas is scarce and predominantly based on cell line studies. Nonetheless, these preliminary investigations demonstrate the feasibility of this approach (19–22).

5-Animolevulinic acid (5-ALA) is one of the most useful photosensitizers for intraoperative application. It is a highly selective theranostic agent with a short biological half-life (14). The rationale for using 5-ALA in PDT lies in its role as a precursor in heme biosynthesis. Following exogenous administration, 5-ALA is metabolized to the photoactive compound protoporphyrin IX (PpIX). Under normal conditions, PpIX is converted to heme by ferrochelatase in an iron-dependent reaction. However, the limited availability of iron and reduced ferrochelatase activity in tumor cells leads to selective PpIX accumulation, enabling PDT. PpIX accumulation in meningioma cells upon 5-ALA administration has been reported in several studies (23, 24). Additionally, in vitro experiments confirmed the cytotoxic efficacy of 5-ALA-based photodynamic therapy against meningioma cell lines (20, 21). This PS is particularly valuable for visualizing superficially located infiltrative tumor parts and residual tissue during surgery. However, visual assessment of 5-ALA-induced PpIX fluorescence intensity is inherently subjective and operator-dependent. This limitation underscores the necessity for implementing objective methods to quantify PpIX accumulation intraoperatively.

There is no established single standard for brain tumors PDT in modern guidelines and scientific works, and no studies have investigated intraoperative fluorescence diagnostics and PDT in recurrent meningiomas. Thus, we conducted a prospective, single-center pilot study to evaluate the feasibility of combined fluorescence-guided surgery and photodynamic therapy for the treatment of in recurrent intracranial atypical and anaplastic meningiomas.

In this single-center, prospective cohort study, we evaluated the feasibility of a novel integrated approach combining 5-aminolevulinic acid fluorescence-guided surgery with intraoperative photodynamic therapy for recurrent atypical and anaplastic intracranial meningiomas. The management of these recurrent lesions remains a formidable neurosurgical challenge, as they often exhibit aggressive local growth and are difficult to further interventions after exhausting standard treatments of surgery and radiotherapy (4–6). Our findings demonstrate that this combined strategy is not only feasible and safe but also provides improvements in surgical radicality and elicits profound biological effects at the cellular level.

A cornerstone finding of our investigation is the consistent and universal accumulation of 5-ALA-induced protoporphyrin IX in recurrent atypical and anaplastic meningiomas. Visible fluorescence was observed in 100% of our prospective cohort, a result that strongly validates the applicability of fluorescence-guided surgery for this specific pathology. This finding gains particular significance in the context of the existing literature. A review by Dijkstra et al. (25) reported high but variable sensitivity (92–98%) and specificity (95%) for 5-ALA across studies, predominantly involving mixed cohorts of primary and recurrent meningiomas, the majority being WHO grade 1 (25). Similarly, a large prospective study by Potapov et al. (26), which included 101 patients with meningiomas (WHO grades 1-3), contained only 9% recurrent cases (26). Consistent with our results, these researches did not found correlation between fluorescence intensity and WHO grade or histological subtype, noting frequent heterogeneity in fluorescence patterns. Large-scale studies focusing exclusively on recurrent meningiomas are absent from the literature, with evidence limited primarily to individual case reports (27–29). Consequently, our results from a homogeneous cohort of 23 recurrent tumors provide unique evidence that the process of recurrence itself, potentially associated with altered cellular metabolism and disruption of the blood-brain barrier, may facilitate consistent and reliable PpIX synthesis. This high rate of PpIX accumulation is a critical enabler, allowing neurosurgeons to leverage a well-established tool from glioma surgery (30) for this new clinical scenario.

The utility of this fluorescence was directly translated into a significantly higher rate of gross-total resection (Simpson Grades I-II) in the FGS group compared to controls group (95.6% vs. 77.1%). However, this observation showed only a borderline statistical trend (p = 0.07). While not reaching the conventional 0.05 threshold, this finding is clinically encouraging and likely reflects the added value of intraoperative fluorescence in the challenging surgical landscape of recurrent disease. It is probable that the relatively small cohort size in this feasibility trial limited the statistical power to definitively confirm this improvement. Future larger-scale studies are necessary to determine if this trend translates into a statistically significant clinical advantage.

Recurrent meningiomas are often embedded in post-operative scar tissue, making the discrimination between tumor and adhered brain parenchyma or fibrotic planes exceptionally difficult under conventional white light (9, 10). Beyond our study, which specifically compared the extent of meningioma resection using 5-ALA with a control group, similar controlled investigations are absent from the literature. In a retrospective study by Cornelius et al. (31), which included 31 patients with primary WHO grade I (61%) meningiomas, the improvement in the radicality of resection was assessed subjectively by the operating surgeon without comparison to a control group of patients (31). This underscores the methodological uniqueness of our work. Our experience confirms that 5-ALA fluorescence effectively highlights superficial infiltrative nests and residual tumor tissue within these adhesions, thereby improving the surgeon’s ability to achieve a more radical and anatomically precise resection, which is a key prognostic factor for delaying further recurrence.

The most significant methodological insight from our study is the demonstrated discordance between subjective visual fluorescence assessment and objective, quantitative measurement of protoporphyrin IX (PpIX). We found no statistically significant correlation between the Visual Fluorescence Score (FIS) and the quantitative Fluorescence Index (FI) obtained via biospectroscopy. Heterogeneity in visible fluorescence has also been reported in the works of Dijkstra et al. (25) and Potapov et al. (26) (25, 26). In a study by Scheichel et al. (2019), which included 11 patients, fluorescence was observed in 100% of meningiomas with bone and soft tissue infiltration (32). However, the intensity of visible fluorescence was not uniform—strong and homogeneous fluorescence was noted in 81.8% of cases, while vague and heterogeneous fluorescence was present in 18.2%. This reinforces the conclusion that the human eye is a poor judge of the actual PpIX concentration in tissue. This subjectivity can be attributed to several factors, including variable background illumination, the surgeon’s individual perception, and optical properties of the tissue, such as blood content and the thickness of the overlying meninges. These findings underscore the necessity of employing more objective methods for quantifying PpIX accumulation in meningioma tissue. Supporting this notion, a study by Knipps et al. (33) involving 13 patients with primary meningiomas demonstrated that a spectrometer exhibited high sensitivity in detecting meningioma cells, whereas the surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases (33). This finding has profound implications, especially for PDT.

Furthermore, the absence of a significant association between fluorescence intensity and various clinical-pathological factors such as WHO grade, tumor location, or prior treatment history is, paradoxically, a positive clinical outcome. It suggests that the ability to accumulate 5-ALA is a ubiquitous property of recurrent meningiomas, independent of their specific subtype or history. This universality greatly enhances the clinical applicability of the technique, as neurosurgeons can anticipate visible fluorescence and plan for FGS and subsequent PDT without the need for complex pre-operative patient selection protocols.

A critical distinction of our PDT protocol lies in its personalized dosimetric approach. While the majority of the clinical literature on 5-ALA PDT in neurosurgery is dedicated to glioblastomas—and in contrast to those studies which have typically employed fixed light doses, such as the 200 J/cm² used in the work by Vermandel et al. (2021) (34), we utilized fluorescence index measurements to adapt the therapy. Our data strongly advocate for the integration of quantitative biospectroscopy as an indispensable tool to guide PDT dosimetry. By providing a real-time, objective measure of PpIX accumulation, biospectroscopy ensures that light delivery is tailored to the actual target concentration, thereby maximizing the potential for a potent cytotoxic effect while establishing a standardized endpoint for therapy (photobleaching). This methodology represents a significant paradigm shift from a one-size-fits-all dose to a biologically guided, patient-specific treatment. The significant post-PDT reduction in FI, achieving levels comparable to normal brain tissue, confirms the biological activity of the treatment and the successful achievement of target photobleaching. This objective endpoint is a major advancement over subjective visual assessment and is supported by in vitro evidence that PpIX concentration directly correlates with PDT efficacy in meningioma cells (20, 21, 35). We believe this methodology is highly translatable to other biospectroscopy platforms. By using an in-patient internal control (normalization to the intact dura mater) rather than an absolute numerical threshold, the protocol accounts for inter-device variability and differences in light-tissue interaction. Other platforms can implement a similar relative thresholding strategy by calibrating their signal-to-background ratio against healthy intracranial structures, which remains a robust and reproducible approach across different fluorescence detection systems.

The clinical feasibility and safety of our protocol of combination of FGS and FDT is objectively reflected in the stability of the Karnofsky Performance Status scores. The fact that all 23 patients maintained a KPS score of 70% or higher postoperatively demonstrates that the PDT-induced apoptotic changes did not compromise the patients’ functional independence. This preservation of high-level neurological function is a key safety indicator, especially given the aggressive nature of recurrent high-grade meningiomas and the targeted application of PDT at the infiltrative margins.

At a median follow-up of 16 months, no cases of local recurrence were observed in the experimental group. However, this finding should be interpreted strictly as a preliminary observation rather than definitive proof of long-term local control. Given the aggressive biological behavior and high recurrence rates inherent to atypical and anaplastic meningiomas, the current follow-up duration is insufficient to draw final conclusions regarding the long-term oncological efficacy of the integrated PDT protocol. Continuous long-term surveillance of this cohort is essential to evaluate the durability of the observed clinical response. Also Takahashi et al. (36) reported that prior radiotherapy could limit the effectiveness of PDT with talaporfin sodium (36), our results with 5-ALA in a population where many had received prior radiotherapy (39.1%) suggest a potent cytotoxic effect.

A central question remains whether the observed clinical benefits stem from the improved visualization provided by FGS or the direct cytotoxic effects of PDT. Our findings suggest that these two modalities are inherently complementary and synergistic. Notably, even after achieving macroscopically total resection, intraoperative biospectroscopy revealed a high fluorescence index (FI) in the resection bed (median 20.0 a.u. vs. 1.2 a.u. in intact dura), confirming the presence of residual PpIX-accumulating tumor cells. These “hotspots” of microscopic infiltration, particularly within the tumor matrix of skull base lesions or the peritumoral zone, represent the primary targets for PDT. By inducing apoptosis in these spectroscopically identified but surgically inaccessible residuals, PDT provides an additional layer of local control that exceeds the limits of standard microsurgical radicality. This combinatorial strategy is further supported by in vitro evidence indicating that the susceptibility of meningioma cells to 5-ALA-PDT is highly variable and dependent on intrinsic factors, such as ferrochelatase activity (35). Such variability underscores the necessity of a robust initial resection to reduce the overall tumor burden, followed by targeted PDT to address the remaining invasive cells. Ultimately, these findings provide a strong rationale for future randomized controlled trials to definitively establish the therapeutic impact of this synergistic approach compared to the current standard of care.

Our study provides also comprehensive histopathological and cellular-level characterization of the effects of 5-ALA-mediated photodynamic therapy in recurrent intracranial meningiomas. The data reveal that PDT induces a multi-mechanistic cytotoxic program, effectively targeting both the tumor matrix and the critical peritumoral niche, thereby providing a biological basis for the observed clinical outcomes.

The total ablation of progesterone receptor expression following PDT represents a significant alteration in the tumor’s biological phenotype. PR signaling is a key pathway implicated in meningioma proliferation (9). At the same time the study by Ren et al. (37) demonstrate that negative PR expression was a radiosensitive biomarker on progression free survival for de novo atypical meningiomas patients after gross-total resection (37). The complete loss of this receptor signature suggests that PDT inflicts profound damage that disrupts fundamental cellular identity and function, potentially pushing tumor cells towards a non-proliferative state.

The most therapeutically significant finding is the robust induction of apoptosis within the peritumoral brain tissue. The significant increase in caspase-3-positive glial cells (36.3 ± 9.6 vs. 14.8 ± 2.2 cells/mm², p<0.0001), and notably, the novel appearance of apoptosis in neuronal nuclei, provides direct evidence that PDT’s effect extends beyond the macroscopic tumor margin. Caspase-3 is a central executioner protease in the intrinsic apoptotic pathway, and its activation is a well-documented mark of PDT-induced cell death across various cancer models (38–40). The induction of apoptosis in the peritumoral zone represents a delicate balance between achieving maximal local tumor control and preserving functional brain tissue. From a biological perspective, the peritumoral microenvironment (including neuronal and glial elements) is known to undergo significant remodeling, often forming a supportive niche for residual tumor cells (41). In aggressive, recurrent meningiomas, targeting this infiltrative margin is crucial. We hypothesize that the observed apoptotic shifts in the peritumoral zone may reflect the disruption of this tumor-supportive niche, which is an expected and potentially beneficial aspect of the photodynamic effect, as long as it remains spatially restricted and clinically asymptomatic. As previously mentioned, our results showed no new or worsening neurological deficits in the experimental group postoperatively, suggesting that the observed microscopic apoptotic changes did not translate into clinical morbidity.

Our confocal microscopy data elucidates the precise subcellular chronology of PDT-induced death: initial oxidative damage leads to proteotoxic stress (Hsp70 upregulation), followed by mitochondrial dysfunction (TMRM loss), and culminates in nuclear degradation. This sequence confirms the activation of the intrinsic apoptotic pathway, initiated by massive oxidative injury. The strong induction of Hsp70 is a known cellular response to PDT-induced protein damage and serves as a clear marker of irreversible cellular stress (42). It is also worth noting the immunomodulatory antitumor effect of Hsp70, which, under stress, can be exposed on the surface of the plasma membrane of tumor cells on the one hand (43), and released into the extracellular space on the other (44). It has previously been shown that membrane-bound Hsp70 can activate the cytolytic and migratory anti-cancer activity of natural killer cells (through interaction with a CD94 receptor) (45, 46). Thus, local induction of chaperone expression can lead to stimulation of the immune response; however, this aspect was not addressed in the present work. The correlation of these profound morphological changes with the quantitative biospectroscopy data — where PpIX photobleaching served as the procedural endpoint — validates biospectroscopy as a reliable real-time surrogate for predicting ultimate biological effect.

At the same time, when analyzing morphological and immunohistochemical alterations in the areas after PDT, several potential confounding factors, such as surgical manipulation, localized ischemia, and photothermal effects, must be considered. To minimize the impact of these variables, we obtained paired biopsy specimens from the identical tumor zones strictly before and after irradiation, while the use of bipolar coagulation was avoided in these sampling areas to prevent artifactual tissue damage. Furthermore, the PDT procedure was standardized with a fixed laser power of 2.0 W and included real-time intraoperative thermometry to ensure that no significant thermal injury occurred. Although the precise independent contribution of each perioperative factor remains difficult to quantify, these methodological precautions suggest that the observed induction of apoptosis, loss of PR expression and increase Hsp70 expression are primarily associated with the photodynamic effect. Consequently, these histological shifts should be viewed as the net outcome of the integrated surgical and photodynamic intervention.

While this study provides promising evidence for the feasibility and biological activity of combined FGS and PDT in recurrent meningiomas, several limitations must be acknowledged.

First, the non-randomized design with a retrospective control group introduces potential for selection bias and confounding. Despite our efforts to ensure comparability through strict inclusion criteria and baseline analysis, unmeasured factors could have influenced the outcomes. The decision to deny randomization was primarily dictated by the exploratory and pilot nature of this research. Recurrent atypical (WHO grade 2) and anaplastic (WHO grade 3) meningiomas represent a rare and heterogeneous patient population with limited salvage treatment options. Randomized allocation was considered premature and statistically underpowered at this stage, as the primary goal was to determine if the combined intervention could be safely integrated into the neurosurgical workflow without increasing perioperative morbidity. Consequently, the observed differences in the extent of resection and recurrence rates, while encouraging, must be interpreted with caution and require validation in a randomized controlled trial. Second, the relatively modest cohort size (n=23 in the experimental group) and the single-center design of the study may constrain the external validity of our findings. The cohort, while representative of our institution’s patient population, may not fully capture the heterogeneity of recurrent meningiomas across broader clinical settings.

Third, the study is limited by a relatively short follow-up period (median 16 months; IQR 11.0–22.0) for patients in the experimental group. While preliminary clinical data were observed, the feasibility-oriented nature of this work precludes definitive conclusions regarding therapeutic superiority. Further studies with extended follow-up periods are mandatory to confirm the impact of this combined approach on long-term progression-free and overall survival.

A notable limitation of this feasibility study is the lack of a dedicated control arm utilizing 5-ALA fluorescence-guided surgery without photodynamic therapy. Consequently, it is not possible to statistically decouple the independent effect of PDT from the improved extent of resection achieved through FGS. The superior outcomes observed in the experimental group should, therefore, be viewed as a result of the integrated synergistic protocol rather than the effect of PDT as a stand-alone modality.

Finally, the quantitative biospectroscopy and PDT light delivery systems used represent a specific technological setup. The generalizability of our dosimetric parameters and fluorescence index thresholds to other hardware and software platforms requires further investigation.

Despite these limitations, this study establishes a critical foundation for future research and provides the first comprehensive histopathological evidence of PDT’s mechanism of action in meningiomas.

Our prospective, single-center pilot cohort study established that the integrated use of 5-ALA-guided resection and intraoperative PDT appears to be a safe and feasible strategy for managing recurrent high-grade meningiomas. Moreover, we identified and provided a solution for a key limitation of visual fluorescence assessment by validating quantitative biospectroscopy as a necessary companion for objective measurement, particularly when bridging from diagnostic fluorescence to PDT. The personalized, biospectroscopy-guided PDT protocol represents a significant evolution from fixed-dose paradigms. While our preliminary data suggest a potential improvement in local control compared to standard microsurgical resection, the exploratory nature of these comparative efficacy claims must be acknowledged. These findings justify further investigation in multi-center randomized controlled trials to definitively establish the independent therapeutic impact of PDT on long-term survival in this challenging patient population.