L-RNA aptamer-based CXCL12 inhibition combined with radiotherapy and bevacizumab in newly-diagnosed glioblastoma: expansion of the phase I/II GLORIA trial

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: minor_revision

Abstract
Rapid vascular recovery is a key feature preceding glioblastoma (GBM) recurrence after radiotherapy (RT). We performed spatial expression analyses, providing a rationale for dual inhibition of two non-redundant, spatially distinct acting factors, CXCL12 and VEGF. Subsequently, we expanded a multi-centric phase 1/2 trial (NCT04121455), which initially combined RT and the CXCL12-neutralizing L-RNA-aptamer olaptesed pegol (NOX-A12) in patients with incompletely resected, newly-diagnosed GBM lacking MGMT promoter methylation. The primary endpoint was safety, secondary endpoints included maximum tolerable dose, recommended phase 2 dose, NOX-A12 plasma levels, topography of recurrence, tumor vascularization, neurologic assessment in neuro-oncology (NANO), quality of life, median progression-free survival (PFS), 6-months PFS and overall survival (OS). For the expansion arm, six patients were included that additionally received the VEGF-targeting antibody bevacizumab (BEV) to RT and NOX-A12. Combinatory treatment was well-tolerated and safe with no treatment-related deaths, resulting in abrogated tumor perfusion (rCBV, FTB high) and delayed tumor regrowth as per mRANO. Median progression-free (PFS) and overall survival (OS) after RT + BEV + NOX-A12 were 9.1 and 19.9 months, respectively, significantly outperforming RT + NOX-A12 (p = 0.009; p = 0.021) in a post-hoc comparative analysis, with two patients exceeding 2-year OS. These findings establish proof-of-principle for dual inhibition of CXCL12 and VEGF in patients with newly-diagnosed GBM following RT.

Introduction
Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and remains associated with a severely limited prognosis1. Standard-of-care (SOC) treatment consists of maximum-feasible resection, external-beam radiotherapy (RT) and adjuvant chemotherapy with temozolomide (TMZ)2,3. Roughly 60% of the tumors harbor an unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoter4, which confers resistance to alkylating chemotherapy. These patients face a dismal median progression-free survival (PFS) of 4–6 months and a median overall survival (OS) of 10-15 months5–8. Complete tumor resection is an independent prognostic factor9,10 with a significant impact on OS11,12.

A key feature of GBM recurrence is the efficient modulation of the tumor microenvironment (TME) to restore the microvasculature13, which provides residual tumor cells with vital nutrients and oxygen by exploiting two distinct mechanistic pathways, angiogenesis and vasculogenesis.

Angiogenesis is characterized by hypoxia-induced autocrine and paracrine synthesis of vascular endothelial growth factor A (VEGFA)14, which mediates local sprouting of pre-existing vessels15,16. Following promising preclinical data indicating a synergistic effect with RT17, the VEGFA-targeting monoclonal antibody bevacizumab (BEV) has been previously investigated as a potential therapeutic agent in GBM and yielded improved response rates, prolonged PFS and reduced glucocorticoid requirements in recurrent GBM18,19. However, while providing significantly improved PFS, its addition to chemoradiotherapy repeatedly failed to demonstrate an OS benefit in phase 220 and phase 321,22 trials.

In contrast, vasculogenesis describes de novo vessel formation mediated by bone marrow-derived progenitor cells (BMDCs)23–27. Radiation-induced loss of endothelial cells, accumulation of reactive oxygen species, and aggressive tumor growth with aberrant neovascularization collectively lead to impaired tumor perfusion, triggering TGF-β- augmented induction of the hypoxia-related factor HIF1α. This, in turn, attracts CXCR4+ BMDCs along a gradient of the CXCR4 ligand CXCL1224,28,29. Elevated CXCL12 expression facilitates not only BMDC migration but also their adhesion and homing to hypoxic tumor regions24. CXCL12 has furthermore been reported to repel and sequester T cells30–32, promote GBM cell invasion and decrease apoptosis33,34. Pre-clinical studies have demonstrated improved intracranial tumor control in orthotopic GBM models after RT and subsequent treatment with the CXCR4-inhibiting bicyclam derivative plerixafor35. In a phase 1/2 trial with 20 high-grade glioma patients, plerixafor administration was shown to be safe and yielded favorable clinical outcomes. In this trial, the unusually frequent occurrence of out-of-field recurrences suggests a pivotal role of CXCR4+ cells in locally confined post-radiogenic neovascularization36.

Combined treatment with RT and the CXCL12-inhibiting pegylated L-RNA aptamer olaptesed pegol (NOX-A12) demonstrated significant tumor reduction and sustained complete regressions in an autochthonous rat brain tumor model mimicking highly treatment-refractory GBM37. We previously reported on safety and subgroup-definable clinical efficacy for RT and NOX-A12 in newly-diagnosed, MGMT-unmethylated and incompletely resected GBM38. In this dose-escalation part of the multicentric GLORIA trial (NCT04121455), a higher frequency of CXCL12+ endothelial and glioma cells (EG12) was significantly associated with prolonged PFS under NOX-A12, but not in a SOC cohort. GLORIA patients with EG12 high GBM derived the greatest benefit from treatment. The inferior outcome of EG12 low GBM and the lack of translation into a significant OS benefit raise questions about the individual TME composition and factors driving drug resistance, both at baseline and recurrence. To date, no clinical trial has investigated dual targeting of CXCL12 and VEGF as two key mediators of post-radiogenic revascularization in GBM.

We here report on a spatially resolved mechanistic concept of GBM vascularization, providing a rationale for a combinatorial first-line treatment with RT plus dual inhibition of post-radiogenic angiogenesis and vasculogenesis, demonstrating early signs of improved clinical efficacy for this approach in the multicentric phase 1/2 GLORIA trial.

Results
Characterization of compartment-specific vascularization patterns
In a stepwise approach with increasing spatial resolution (Fig. 1a), we first aimed to investigate the relationship between hypoxia, VEGFA and CXCL12 expression in GBM using the publicly available bulk RNA sequencing dataset (n = 159) from The Cancer Genome Atlas (TCGA)39. VEGFA expression was positively correlated (rs = 0.59, p < 0.001) with the Winter hypoxia score40, a widely used hypoxia gene signature, but this was not the case for CXCL12 (rs = −0.07, p = 0.358). Carboanhydrase 9 (CA9), a hypoxia-inducible gene and well-established marker for detection of hypoxic regions by immunohistochemistry41, also showed a strong positive correlation with VEGFA (rs = 0.715, p < 0.001), but not CXCL12 (rs = −0.16; p = 0.043; Fig. 1b).

Next, we assessed regional mRNA expression patterns of VEGFA and CXCL12 in human GBM samples (n = 122) from the Ivy Glioblastoma Atlas Project42 with available neuropathology-based tissue mapping (Fig. 1c). CXCL12 was significantly higher expressed than both VEGFA and CA9 in the infiltration zone and hypervascularized tumor compartments (p < 0.001, Supplementary Fig. 1a), while VEGFA and CA9 were particularly abundant in necrotic tumor areas (p < 0.001, Supplementary Fig. 1b). Confirming the RNA sequencing data from bulk GBM39, CA9 and VEGFA were significantly positively correlated (rs = 0.783; p < 0.001), in contrast to CXCL12 and VEGFA (rs = −0.474; p < 0.001) (Supplementary Fig. 1c).

Aiming for single-cell spatial resolution, we performed multiplex-immunofluorescence (mIF) stainings on human GBM tissue samples (n = 5, Fig. 1d and Supplementary Fig. 2). Consistent with our previous findings38, we detected the highest frequency of CXCL12 positivity in endothelial cells, particularly in the microvascular proliferation zone (Fig. 1e). The hypoxia marker CA9, which exhibited one of the strongest positive correlations with VEGFA in previous analyses, was primarily expressed around necrotic tumor areas. Although co-expression of CXCL12 and CA9 was observed, CXCL12 expression was largely independent of CA9 positivity (Fig. 1f). This finding supports the notion that CXCL12 is regulated not solely by hypoxia but also by additional mechanisms across diverse cellular sources. In summary, these findings suggest both shared and distinct regulatory mechanisms of vascularization in GBM, providing a strong rationale for dual therapeutic targeting of CXCL12 and VEGFA (Fig. 2).

To further test and validate this hypothesis, we next generated patient-derived glioblastoma organoids (GBOs) from surgical specimens cultured on human brain slices (Fig. 3a) and performed spatial transcriptomics on a representative model. The GBO tumor tissue displayed extensive hypoxic areas with pronounced VEGF expression at the core, whereas CXCL12 expression was sparse and, when present, colocalized predominantly with few endothelial cells, and, to a lesser extent, pericyte-, macrophage- and glioma-related transcripts (Fig. 3b). Notably, CXCL12 expression was detected in normal brain vasculature of the surrounding, the GBO embedding tissue (Fig. 3c). To overcome the mechanistic limitations of revascularization assessment in a non-perfused GBO model, we conducted parallel spatial transcriptomics analyses on a representative GBM patient specimen with well-defined zonal architecture (Fig. 3d). Consistent with our previously reported observations, CXCL12 expression localized to endothelial cells, pericytes, macrophages/microglia and glioma cells. In contrast, VEGF expression strongly correlated with hypoxia-related genes, while CXCL12 showed only a weak association with hypoxia but a robust link to the described cellular compartments (Fig. 3e).

Taken together with our preceding analyses, these findings further supported the spatially and mechanically distinct involvement of both VEGF- and CXCL12-mediated pathways in GBM vascular remodeling. Consequently, within the GLORIA trial, we evaluated the potential additional clinical benefit of VEGFA-targeting with BEV in combination with NOX-A12.

Trial design, enrollment and patient characteristics
The GLORIA trial (NCT04121455) is a multicentric phase 1/2 trial conducted at six major neuro-oncology centers in Germany. It was designed to evaluate the safety and efficacy of RT combined with continuous intravenous (i.v). treatment with NOX-A12 in newly-diagnosed, incompletely resected IDH-wildtype GBM (CNS WHO grade 4) lacking MGMT promoter methylation (Fig. 4a). The primary endpoint was safety, secondary endpoints included maximum tolerable dose (MTD), recommended phase II dose (RP2D), NOX-A12 plasma levels, topography of recurrence, tumor vascularization, neurologic assessment in neuro-oncology (NANO), quality of life (QOL), median PFS, 6-months PFS and OS. In the dose escalation part of the trial, NOX-A12 was administered at escalating dose levels (DLs) of 200, 400 and 600 mg per week. Patients in the first expansion arm received 600 mg NOX-A12 per week combined with biweekly 10 mg/kg body weight BEV.

Between September 2019 and September 2021, three patients were enrolled at each DL of the dose escalation part of the trial (Supplementary Fig. 3) as previously reported38. One patient of DL3 dropped out and was replaced, resulting in a total of ten patients treated with RT and NOX-A12. Between November 2021 and May 2022, six additional patients were enrolled in the expansion arm receiving RT, NOX-A12, and BEV. The median age at diagnosis of the full trial population (n = 16) was 63 (range 43 to 79) years. Of these, 14 patients had undergone partial resection; two were not amenable to resection and received biopsy only. No patient had undergone gross total resection. The median residual tumor volume at baseline was 6.2 (range 0.9–34.1) cm3 and the median highly-perfused tumor fraction (FTB high) at baseline was 26.4% (range 1.8–67.5%). Additional baseline characteristics are shown in Fig. 4b, with a complete tabular summary provided in Supplementary Data 1. The median time to permanent discontinuation of the experimental drug was 5.4 (range 2.9–11.1) months for RT + NOX-A12 and 9.1 (4.1–25.7) months for RT + NOX-A12 + BEV.

Pharmacokinetics
NOX-A12 plasma levels were not influenced by additional BEV treatment, reaching a stable steady-state after approximately one week in all patients and surpassing 1.5 µM, which was considered to be the minimum plasma level required for disrupting CXCL12-mediated migration while minimizing bone marrow cell mobilization43. With increasing NOX-A12 dosage, plasma levels increased correspondingly (p = 0.007), exceeding the stable CXCL12 levels (p = 0.116; Supplementary Fig. 4). NOX-A12 plasma levels were significantly higher from DL1 to DL2 (p = 0.020) and DL3 (p = 0.020), but did not differ significantly with additional BEV treatment (p = 0.270).

Safety
The favorable safety profile of RT and NOX-A1238 remained unchanged with the addition of BEV in the expansion arm (Fig. 5a). Triple therapy (RT + NOX-A12 + BEV) was safe and well tolerated, with no patient discontinuing treatment due to adverse events (AEs). No dose-limiting toxicities or treatment-related deaths were observed. Treatment was discontinued per protocol at the scheduled end of treatment (EOT) after 26 weeks in one patient. Five patients were treated beyond progression, and treatment was stopped as per the investigator’s decision due to suspected decline in patient benefit or deterioration of general condition. Among 139 AEs reported in the expansion arm receiving RT + NOX-A12 + BEV, only two (1.5%) were considered solely related to NOX-A12 (Fig. 5b). Of all grade ≥ 2 AEs (n = 97), 4 grade 2 events (4.1%) were at least attributed to NOX-A12. The majority of AEs were of grade 1 or 2 (91.4%) and were either unrelated or associated to the tumor or RT. No grade 4 or 5 events occurred. The most common treatment-emergent AE (TEAEs, n = 139) was alopecia, reported in five patients with a maximum severity of grade 2 (Supplementary Fig. 5). Four patients experienced hypertension, likely related to BEV, which was effectively managed with pharmaceutical interventions (Supplementary Fig. 6). Equally, four patients each reported headache and fatigue. An increase in alanine aminotransferase, the most concerning treatment-related AE (TRAE) under RT + NOX-A12, occurred only once in the expansion arm (grade 2). Complete listings of all AEs, TEAEs and TRAEs for both RT + NOX-A12 and RT + NOX-A12 + BEV are available in Supplementary Data 1, Supplementary Data 2 and Supplementary Data 3, respectively. MTD and RP2D have been reported previously38, with no additional dose-defining findings arising from the expansion arm.

Radiographic and clinical response
All 16 patients enrolled were included in the response analysis (Fig. 6a). Among patients treated with NOX-A12 + BEV (n = 6), one patient achieved a confirmed complete response (CR), four partial response (PR) and one stable disease, resulting in an overall response rate of 83.3%. In addition, two patients (33.3%) surpassed 2-year OS. In direct comparison, responses were deeper and more durable with the addition of BEV to NOX-A12. Under NOX-A12 only (n = 10), nine patients (90%) exhibited radiographic responses in terms of reduced magnetic resonance imaging (MRI) lesion sizes in at least one time point of follow-up. Of these, four patients (40%) achieved PR based on mRANO criteria (≥50% reduction in sum of the products of the longest perpendicular diameters, SPD).

Under NOX-A12 + BEV, all six patients achieved at least radiographic PR (Fig. 6b). In a post hoc analysis, the median best change from baseline of the highly-perfused GBM fraction (FTB high) was −98.2 (−61.3 to −100)% under NOX-A12 + BEV compared to only one patient achieving >50% reduction in relative cerebral blood volume (rCBV) under NOX-A12 alone (Fig. 6c). Additional treatment with BEV resulted in significantly deeper tumor (p = 0.003) and perfusion responses (p < 0.001). Although apparent diffusion coefficient (ADC) responses were comparable, advanced imaging of FTB high again highlighted pronounced effects under NOX-A12 + BEV (Supplementary Fig. 7). Figure 6d depicts an exemplary response pattern of a patient.

Clinical outcomes
While a PFS event was definable for 15 of the GLORIA patients, one patient needed to be censored for PFS according to the pre-defined statistical analysis plan (see patient narratives in Suppl. Note 1).

Exploratory comparative analyses were performed to compare clinical outcomes between the initial dose-escalation cohorts and the expansion arm receiving additional BEV. With a median PFS of 5.7 (range 1.9–8.5, 95% confidence interval (CI) 1.9–6.0) months vs. 9.1 (range 3.9–17.0, 95% CI 7.8-not estimable) months, patients receiving triple treatment with RT + NOX-A12 + BEV showed a significantly longer PFS than patients receiving RT + NOX-A12 only (HR 0.14 (95% CI 0.02-0.60); log-rank test, p = 0.009; Fig. 7a). Compared to patients receiving SOC, PFS with NOX-A12 + BEV was also significantly superior to an internal histology-matched SOC cohort (Supplementary Data 2) with a median PFS of 4.0 months (HR 0.11 (95% CI 0.02-0.41); log-rank test, p = 0.001). Compared to an external, contemporary multicentric SOC cohort derived from Drexler et al.44 (Supplementary Data 3) with patient characteristics matching the GLORIA inclusion criteria, PFS with NOX-A12 + BEV was longer, but did not reach statistical significance (median PFS 6.0 months; HR 0.48 (95% CI 0.16-1.15); log-rank test, p = 0.104).

Importantly, patients with additional BEV treatment had a significantly longer OS (HR 0.23 (95% CI 0.05-0.79); log-rank test, p = 0.021; Fig. 7b) with a median OS of 12.7 (range 4.7–18.4, 95% CI 4.7-15.8) months for RT + NOX-A12 and 19.9 (range 4.1–28.1, 95% CI 4.1-not estimable) months for the triple treatment. OS of patients receiving RT + NOX-A12 + BEV also significantly outperformed patients undergoing SOC treatment in both the internal histology-matched cohort (median OS 9.5 months; HR 0.17 (95% CI 0.04-0.53); log-rank test, p = 0.003) and the external SOC cohort (median OS 11.0 months; HR 0.32 (95% CI 0.09-0.88); log-rank test, p = 0.039).

To enhance comparability with the SOC cohorts and existing literature, we additionally calculated GLORIA survival outcomes from the date of initial surgery. Both PFS and OS of patients receiving NOX-A12 + BEV were approximately 1.1 months longer, without affecting the statistical significance of comparisons with the SOC cohorts (Supplementary Fig. 8).

Given the frequent inclusion of patients with gross total resection and incomplete molecular pathology assessment in contemporary trials and real-world cohorts, the PFS and OS of GLORIA participants receiving triple therapy surpass previously reported outcomes in these populations5,20,44–46 in the most unfavorable patient subgroup of unmethylated and incompletely resected GBM (Supplementary Fig. 9).

Correlation of outcomes with CXCL12 expression
We next assessed in an exploratory analysis whether outcomes of patients receiving RT + NOX-A12 + BEV correlated with CXCL12 expression. A higher frequency of CXCL12+ endothelial and glioma cells (EG12) may serve as a predictive marker for CXCL12-targeted therapy, as it was significantly associated with prolonged PFS under NOX-A12, but not in a SOC cohort38. In the dose-escalation part of the trial with patients receiving RT + NOX-A12, EG12 correlated significantly with PFS (rs = 0.865, p = 0.005). This correlation was lost in patients receiving RT + NOX-A12 + BEV (Suppl. Fig. 10), with a negative trend between EG12 and both PFS (rs = −0.714, p = 0.136) and OS (rs = −0.371, p = 0.497).

Methods
Trial design and oversight
GLORIA (SNOXA12C401, 2024-510964-21, NCT04121455) is a multicentric phase 1/2 study of RT in combination with NOX-A12 in first-line partially resected or unresected GBM (CNS WHO grade 4) patients with unmethylated MGMT promoter. The trial consisted of an initial dose-escalation arm and an additional expansion arm that evaluates NOX-A12 in combination with BEV. GLORIA was conducted at six academic centers in Germany, whereas the protocol was approved by the ethics committees at each participating site (ethics committees of the university hospitals of Mannheim, Bonn, Leipzig, Essen, Tübingen, and Münster). The study design and conduct complied with all relevant regulations regarding the use of human study participants. The trial followed the guidelines of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practices Guidelines. Each patient provided written informed consent in accordance with established guidelines. The trial was reviewed by an independent data safety and monitoring committee. No trial participant received financial compensation. The first patient was enrolled on October 07, 2019. The last patient of the dose-escalation arm was enrolled on September 2, 2021. The last patient of the expansion arm with BEV was enrolled on May 23, 2022. The trial was first registered on EudraCT upon approval by the German authority (Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM)) in May 2019 and transitioned to CTIS on August 2024. The dose escalation was designed as a modified 3+3 rule-based design according to Le Tourneau et al.55 with three successional cohorts consisting of three patients each. Patients of DL1 were to be treated with a weekly dose of 200 mg, DL2 with a weekly dose of 400 mg, and DL3 with a weekly dose of 600 mg NOX-A12. After four weeks of treatment of the first patient of DL1, the data safety monitoring board (DSMB) reviewed all DLTs, AEs, and relevant laboratory values. During the following ten weeks of treatment, the DSMB was kept continuously informed about all DLTs and SAEs, and, at the end of this period, reviewed all DLTs, AEs, and relevant laboratory values, including NOX-A12 plasma concentrations prior to enrollment of the next two patients of this DL. The evaluation was repeated prior to enrolling patients in DL2 and after patients 2 and 3 received at least four weeks of treatment. The same procedures were performed prior to the enrollment of further patients in DL2 and DL3. If none of the three patients in any DL experienced a DLT, another three patients were to be treated at the next higher DL. However, if one of the three patients in a DL experienced a DLT, three more patients were to be treated at the same DL. The dose escalation was planned to be continued until at least two patients among a cohort of three to six patients experienced DLT (i.e., ≥33% of patients with a DLT at that DL), but the dose would not be escalated beyond 600 mg/week. The recommended phase 2 dose (RP2D) was defined as the DL just below this toxic DL, or 600 mg/week, if this DL is not toxic. Six patients were to be included into the expansion arm. After 4 weeks of treatment of the first patient with NOX-A12 in combination with BEV, the DSMB reviewed all DLTs, all AEs, and relevant laboratory values. DLTs, according to the common terminology criteria for adverse events (CTCAE, version 5.0), were defined as any grade 3–4 non-hematological toxicities (excluding grade 3 vomiting and/or nausea, if encountered without adequate and optimal prophylactic therapy), at any DL, assessed by the investigator and/or the sponsor as related to NOX-A12.

Inclusion criteria of the dose-escalation arm of the trial were age ≥18 years, incompletely resected or biopsied GBM (detectable postoperative residual tumor), absence of MGMT promoter (hyper) methylation, Eastern Cooperative Oncology Group (ECOG) performance score ≤2, estimated life expectancy ≥3 months, stable or decreasing dose of corticosteroids and adequate hepatic and renal function. Inclusion criteria of the expansion arm with BEV (Arm A) additionally allowed the inclusion of patients with fully resected tumors. The full listing of inclusion and exclusion criteria are provided in Supplementary Note 2. Sex was determined based on self-report. However, no sex-specific analyses were performed because the study was not powered to detect sex-related differences, and no a priori hypotheses regarding sex effects were defined. All patients were neuropathologically confirmed as GBM, IDH-wildtype (CNS WHO grade 4) according to the 2021 WHO classification for CNS tumors. If patients were ≤54 years of age, IDH mutations were additionally assessed by pyrosequencing of the IDH1 and IDH2 loci. A minimally redacted version of the study protocol is provided in Supplementary Note 2.

Treatment and endpoints
Following adequate cranial wound healing and implantation of a venous port catheter, treatment with NOX-A12 was initiated within six weeks post cranial surgery. In the dose escalation cohorts, after an initial dose of 70, 160 or 230 mg per day, respectively on day 1, patients were administered a fixed dose of 200, 400 or 600 mg NOX-A12 per week (DL 1, DL 2, DL 3) by continuous (24h) i.v. infusion over a commercially-available closed pump system (CADD®-Solis VIP Ambulatory Infusion Pump by Smiths Medical) starting on day 1. Treatment with NOX-A12 ended after 26 weeks. In Expansion Arm A, patients received BEV by i.v. infusion at doses of 10 mg/kg every 2 weeks for 26 weeks. Patients with disease progression during the 26-week treatment period continued treatment with all assessments if deemed appropriate by the investigator. Continuation of treatment with NOX-A12 and, if applicable, also BEV beyond 26 weeks was allowed as per each investigator’s decision, if the patient had a clear clinical benefit. No simultaneous systemic oncologic treatment was permitted. Baseline patient and treatment characteristics are enlisted in Supplementary Data 1. Clinical and radiographic follow-up assessments included standard and advanced MRI sequences. The primary endpoint of the trial was safety as per the incidence of AEs. Secondary endpoints included NOX-A12 plasma levels, MTD, RP2D, imaging parameters with a specific emphasis on monitoring re-vascularization, topography of recurrence, PFS, OS and clinician/patient reported outcomes (CRO/PRO). Post hoc comparative analyses were performed to evaluate radiographic and clinical outcomes, as well as baseline CXCL12 expression profiles, between the initial dose-escalation cohorts and the expansion arm receiving additional BEV. Topography of recurrence as well as clinician- and patient-reported outcomes (NANO, QoL) were collected but are not included in the present analysis and will be reported separately. RT was initiated on day 2 after the start of NOX-A12 (and BEV) and administered as intensity-modulated, image-guided RT. Patients in the dose escalation arm received RT in a normofractionated (2 Gy per fraction) or hypofractionated (2.67 Gy per fraction) fashion up to cumulative doses of 60 or 40.05 Gy, respectively. For the expansion arm, all patients underwent normofractionated RT with 2 Gy per fraction to a cumulative dose of 60 Gy. For treatment planning, pre- and post-surgery MRI scans were co-registered on planning computed tomography (CT) scans. Gross tumor volumes (GTV), clinical target volumes (CTV) and planning target volumes (PTV) were defined as per current guidelines56.

Assessment of clinical and radiographic response
Patients visited the study site once weekly when presenting for the change of the medication cassette of the pump. Clinical routine follow-up visits included regular AE monitoring, physical and neurological assessments, vital signs, ECG and blood tests. AEs were assessed and graded by the investigators according to the National Cancer Institute CTCAE, version 5.0. Baseline MRIs were obtained within a week prior to treatment initiation and up to six weeks post-surgery or post-biopsy. Follow-up MRIs were obtained every eight weeks under treatment and at EOT. MRI imaging sequences included: 3D T1-weighted volumetric imaging (3D T1), T2-fluid-attenuated inversion recovery (FLAIR) imaging, diffusion-weighted imaging (DWI), T1-weighted dynamic contrast-enhanced perfusion imaging (DCE), T2-weighted turbo spin-echo imaging (T2 TSE), T2-weighted dynamic susceptibility contrast-enhanced perfusion imaging (DSC), and post-contrast 3D T1 imaging. The following additional advanced imaging parameters were calculated: DWI-derived ADC; diffusion susceptibility contrast (DSC)-derived leakage-corrected normalized rCBV and threshold-calculated FTB high (rCBV > 1.75); dynamic contrast-enhanced (DCE)-derived transfer constant of contrast agent (Ktrans) between the blood and the extravascular extracellular space (EES), fractional EES volume (ve), and fractional plasma volume (vp). All image post-processing and interpretation were performed using IB NeuroTM (Imaging Biometrics), Olea Sphere (Olea Medical), and Mint LesionTM (Mint Medical GmbH) software and assessed by a central reader not involved in the treatment of the patients (S.B.). MRI response values for all patients can be found in the Source Data File.

Outcome assessment
All MRI images were uploaded to an imaging database and the outcome was centrally assessed by a board-certified radiologist with expertise in the field, blinded for study site and clinical status. Target lesions (TLs) and non-target lesions (NTLs) were identified, validated and assessed in regard to tumor size (SPD) and corresponding time-point tumor response according to the modified Response Assessment in Neuro-Oncology (mRANO)57. Two patients enrolled had a singular residual tumor lesion meeting the inclusion criteria, while not qualifying for a target lesion (<10 mm in at least one diameter as per mRANO), thus documented as NTL. New non-measurable contrast-enhancing lesions only constituted progression in the case of a complete response (CR). NTLs only impacted the response assessment in the case of a complete response of TLs. Preliminary tumor progression (PD) or regression (PR) required confirmation in a successive scan after eight weeks. PFS was calculated as the time (in days) between the first day of treatment with NOX-A12 to the day of PD. The PFS event was defined as the first date at which progression criteria had been met, i.e., (1) the date of the first sequentially confirmed MRI assessment resulting in preliminary PD or (2) the date of the radiographic assessment irrespective of its outcome in case of simultaneous investigator-assessed clinical progression attributable to no other cause apart from the tumor or; (3) the date of death by any cause if the patient died before clinical or radiographic progression. If preliminary PD was not confirmed in a sequential MRI and there was no subsequent SD, PR, or CR, the date of preliminary PD was still considered as an event for PFS if (1) the patient stopped protocol treatment due to clinical progression; (2) no further response assessments were done; or (3) the patient died due to any cause. For patients without a clinical or confirmed radiographic progression prior to a change of systemic therapy, PFS was censored at the date of initiation of a new anticancer treatment. Independent of MRI or clinical assessment, the diagnosis of PD was not established in the case of pathologically confirmed pseudo-progression after re-surgery, which was the case in one patient, where treatment with NOX-A12 was continued afterwards. OS was calculated as the time from the first day of treatment with NOX-A12 until death by any cause. All survival data were calculated in months, rounded to one decimal place. The individual clinical courses, therapies, investigator decisions, and definitions of PFS and OS events for all patients are described in detailed narratives provided in Supplementary Note 1.

Internal and external standard-of-care (SOC) cohorts
We benchmarked oncological outcomes of the GLORIA trial to an internal (University Hospital Bonn)38 and a recently published external German multicenter cohort44 of GBM patients with comparable patient characteristics resembling the inclusion criteria of the GLORIA trial treated with standard RT and optional TMZ. The procedures were approved by the Ethics Committee of the University Hospital Bonn (approval number: 222/23-EP). The local reference cohort includes patients treated at the University Hospital Bonn between 2010 and 2023. Histological criteria for selection of reference patients were: newly-diagnosed GBM, IDH-wildtype (CNS WHO grade 4) according to the valid WHO classification for CNS tumors, absence of MGMT promoter methylation as confirmed by pyrosequencing58. Clinical criteria were: ECOG of 0-2, status post biopsy or incomplete resection, and first-line therapy with RT (and optionally TMZ). Data to establish the external German multicenter SOC cohort was extracted from publicly available annotated data44, which provided data of IDH-wild type glioblastoma patients undergoing surgical resection and sequential chemoradiotherapy between 2016 and 2023 from three German neuro-oncological centers (Hamburg, Frankfurt, Berlin). The patients selected for the SOC cohort had the following characteristics closely resembling the GLORIA trial: IDH-wildtype glioblastoma, ECOG 0-2, incomplete resection (<90% removal), unmethylated MGMT promoter. The patient characteristics of both cohorts are provided in Supplementary Data 2 and 3 and the Source Data File.

Plasma NOX-A12 and CXCL12 concentrations
A Liquid Chromatography-UV assay, based on an anion-exchange chromatography analysis coupled to a UV detector, was used to detect and quantify the analyte NOX-A12 in patient plasma samples. The comparable calibration curve, generated from a standard of dilution series, corresponded to a linear range of NOX-A12 concentrations in human plasma from 0.5 to 200 µg/mL (0.034–13.6 µM). Quantification of CXCL12 concentrations in patient plasma samples was performed by a contractor (Swiss BioQuant AG) using HPLC-MS/MS bioanalytics. The comparable calibration curve corresponded to a linear range of CXCL12 (human CXCL12α and CXCL12β) concentrations in human plasma from 25 to 2500 nM.

Multiplexed immunofluorescence of tumor tissue
To assess cellular distribution patterns of hypoxia and CXCL12, human GBM samples were assessed by multiplexed immunofluorescence. Histological criteria for selection of these samples were: newly-diagnosed GBM, IDH-wildtype (CNS WHO grade 4) according to the valid WHO classification for CNS tumors, absence of MGMT promoter methylation as confirmed by pyrosequencing58. All tumor samples were obtained following informed consent as part of SOC surgical procedures, and all patients had consented to analyses of their tissue. The scientific procedures were approved by the Ethics Committee of the University Hospital Bonn (approval number: 222/23-EP). Formalin-fixed paraffin-embedded (FFPE) tumor samples were sliced by standard procedures at 3 µm slice thickness and adhered onto poly-L-lysine-coated coverslips. Antibody conjugation, tissue staining, and mIF imaging were performed (with modifications) as described elsewhere59,60. In short, purified, carrier-free antibodies were conjugated to maleimide-modified oligonucleotides (Biomers), concentrated, reduced, and washed with buffer. Maleimide-modified oligonucleotides were first dissolved on 1x DPBS, then added to the reduced antibody and incubated at room temperature for two hours in a 2:1 (w/w) ratio with the antibodies. Next, the conjugated antibodies were washed in high-salt PBS three times and then eluted by centrifugation at 3000 × g for 2 min in the CODEX® antibody stabilizer solution. The conjugated antibodies were stored at 4°C until usage. Prepared FFPE tissues were baked at 55°C for 30 min, and rehydrated by immersion in fresh xylene twice, for 5 min and in descending concentrations of ethanol, each step for 5 min (100% twice, 95% twice, 70%, ddH2O twice). Heat-induced epitope retrieval was performed using 1x Dako target retrieval solution, pH 9 (Agilent) at high pressure, for 20 min. Tissues were then washed for 10 min in 1x TBS IHC wash buffer with Tween 20 (Thermo Fisher, 28360). Tissues were blocked for 1 h at room temperature using 100 µl of blocking buffer. Conjugated antibodies were added to the blocking buffer, concentrated through a 50 kDa Amicon Ultra Filter and resolved in the blocking buffer. Tissues were incubated with the antibody staining solution in a humidity chamber overnight at 4°C. The following antibodies were used: Ki-67, clone B56, BD Biosciences, Cat.# 556003 (RRID: AB_396287); SDF1/CXCL12, clone 79018, Thermo Fisher, Cat.# MA5-23759 (RRID: AB_2608711); α-SMA, clone 1A4, Thermo Fisher, Cat.# 14-9760-82 (RRID: AB_2572996); CD31, clone EP3095, Abcam, Cat.# ab226157; GFAP, clone 2.2B10, Thermo Fisher Scientific, Cat.# 13-0300 (RRID: AB_2532994); CD68, clone KP-1, Biolegend, Cat.# 916104 (RRID: AB_2616797); CA9, polyclonal, NovusBio, Cat.# NB100-417. After staining, tissues were washed twice in S2 buffer and fixed with a three-step fixation process. First, tissues were fixed in S4 containing 1.6% paraformaldehyde (Electron Microscopy Science, 15710-S) for 10 min, followed by a 15 min long incubation in 100% ice-cold methanol (Sigma, 34860-1L-R) for 5 min, and a final fixation with BS3 fixative solution dissolved in 1x PBS at room temperature for 20 min. Tissues were stored in S4 in a 6-well plate at 4°C for up to two weeks, or further processed for imaging. 400 nM fluorescent oligonucleotide working solution was aliquoted in CorningTM black 96-well plates (Merk, CLS3925-100EA) in 250 µl of plate buffer, according to the multi-cycle reaction panel. Image acquisition was performed on Zeiss Axio Observer 7 microscope equipped with a Colibri 7 LED Light source (Carl Zeiss), and a Prime BSI PCIe camera (Teledyne Photometrics). Imaging cycles were performed using an Akoya Phenocycler™ instrument and CODEX® instrument manager software (Akoya Biosciences). Automated images were acquired with the Plan-Apochromat 20X/0,8 M27 (a = 0.55 mm) objective (Carl Zeiss), and the imaging pipeline was controlled by a focus strategy with autofocus for each support point created, with a three z-stack image with a distance of 1.5 µm. DAPI (1:300 final concentration) was imaged in each cycle at an exposure time of 20 milliseconds and an LED intensity of 40%. The images were processed with CODEX® Processor (Akoya Biosciences, v. 1.8.3.14) and analyzed with HALO® Image Analysis software (Indica Labs, v. 3.3.2541.256). After each multi-cycle reaction, standard H&E staining was performed on the same tissue slice to confirm histopathological features. The H&E staining was analyzed independently by a neuropathologist with 5 years of experience and a board-certified neuropathologist with more than 30 years of experience in the field. With consensus, pathological features of GBM (CNS WHO grade 4) were again confirmed, and zonal characteristics within the tumor tissue were depicted. Adjacent regions like leptomeninges, hemorrhage or healthy brain tissue were excluded and only confirmed tumorous tissue parts were considered for the following analyses. Annotated H&E stainings can be found in Supplementary Fig. 11 and Supplementary Fig. 12. Analyses were performed using the Highplex FL module (v. 4.1.2) from HALO®. A nucleus/cytoplasm membrane % completeness threshold for positivity was set as follows: DAPI, Nucleus % Completeness Threshold 15%; CXCL12, Nucleus and Cytoplasm % Completeness Threshold 30%; CD68, Nucleus and Cytoplasm % Completeness Threshold 30%; CD31, Nucleus and Cytoplasm % Completeness Threshold 30%; Ki-67, Nucleus % Completeness Threshold 35%, α-SMA, Nucleus and Cytoplasm % Completeness Threshold 35%; GFAP, Nucleus and Cytoplasm % Completeness Threshold 25%%; CA9, Nucleus and Cytoplasm % Completeness Threshold 20%. Cellular phenotypes were defined as follows: endothelial cells (DAPI+, CD31+), pericytes (DAPI+, α-SMA+, CD31-), macrophages/microglia (DAPI+, CD68+), tumor cells (DAPI+, GFAP+, CD68-, CD31-, α-SMA-). All phenotypes were also assessed for CXCL12 and CA9 expression positivity. Nuclear segmentation was based on DAPI with a custom-trained deep learning neural network algorithm, with nuclear segmentation aggressiveness of 0.5 and nuclear size for positivity set between 12 and 1000 µm2. All buffers and solutions for multiplexed immunofluorescence are provided in Table 1 below. Details on the multi-cycle reactions and oligo sequences can be found in Supplementary Data 4.

Generation of brain slice-implanted glioblastoma organoids (Core2Edge)
The Core2Edge model involves the implantation of patient-derived GBOs into human cortical brain slices. The procedures were approved by the ethics committee of the University of Bonn (approval no. 162/10 and 504/20), and patients provided written informed consent. Human organotypic brain slices were generated from tissue obtained during epilepsy surgery. After trimming, tissue blocks were mounted on a vibratome and sectioned into 300 µm slices in ice-cold, carbogenated brain slice preparation medium (see Supplementary Table 4). Intact slices were transferred onto Millicell® inserts (Millipore., cat. no. PICM03050) positioned at the air–liquid interface and cultured in brain slice growth medium (see Supplementary Table 4) at 37°C and 5% CO₂ for 24 h. For the Core2Edge model, GBOs from a stable pool were selected for implantation. GBOs were generated from patient-derived tumor tissue with confirmed glioblastoma, IDH-wildtype, WHO grade 4, according to the protocol by Jacob et al.61 Prior to implantation, GBOs were stained with CellBrite™ Green to enable visualization during the implantation (see Supplementary Table 4). Implantation was performed under a fluorescence stereomicroscope. Therefore, a small incision was created in the white matter of each organotypic slice, and a single stained GBO was gently positioned into the tissue using a microdissector. Slices were then returned to the air–liquid interface and maintained for 10 days, with medium changes every 48 h.

Spatial transcriptomics
Following co-culture, Core2Edge slices were fixed in 4% buffered formalin, dehydrated through graded ethanol and xylene, and embedded in paraffin using standard procedures. In addition, FFPE tissue from a glioblastoma patient was processed according to the criteria described in the section Multiplexed immunofluorescence. FFPE blocks from both the Core2Edge samples and the clinical tumor specimen were sectioned at 5 µm and prepared for spatial transcriptomics using the Xenium In Situ platform (10x Genomics). Sections were mounted within the defined sample area and processed following the Tissue Preparation Guide (CG000578, 10x Genomics), employing the Human Brain Panel (266 genes) together with 100 custom-designed probes. After probe hybridization, ligation, rolling-circle amplification, DAPI staining, and cyclic fluorescent imaging, spatial gene expression data were acquired using the Xenium Analyzer. Spatial transcriptomics data were processed and visualized using Xenium Explorer (version 4.1.0, 10x Genomics). Cell-type and pathway signatures were defined a priori based on curated gene sets derived from established literature. For each spatial feature, log-normalized expression values of the genes within a given signature were averaged to obtain a composite signature score. Cell-type signatures were defined as follows: glioma cells: AQP4, OLIG1, OLIG2, POSTN, SOX2; endothelial cells: CD34, CLDN5, PECAM1, PLVAP, VWF; pericytes: ABCC9, ACTA2, CSPG4, PDGFRB, RGS5, TGFBI; macrophages: CD14, CD163, FCGR3A, CD68; microglia: AIF1, CX3CR1, CXCL10, IL1B, P2RY12, TMEM119, TREM2; hypoxia signature: ANGPTL4, CA9, DDIT3, HIF1A, LDHA, MIF, PGK1, SLC2A1.

To generate spatial density maps, the tissue was divided into 10×10 µm bins, and local signal intensity was computed using kernel-smoothed density estimation. Density values were visualized using the Inferno color scheme (low: black/dark violet; high: bright yellow/white). Heatmaps display either individual gene expression or composite signature scores across spatial coordinates. All spatial plots, overlay visualizations, and region annotations were generated in Xenium Explorer.

Public data RNA sequencing analysis
The bulk tumor dataset Glioblastoma Multiforme from the TCGA Research Network was downloaded from cbioportal62 under https://www.cbioportal.org/study/summary?id=gbm_tcga_pan_can_atlas_2018. The GBM dataset with regional tissue mapping from Puchalski et al.42 was downloaded from the IVY GAP Glioblastoma Atlas Project (https://glioblastoma.alleninstitute.org/static/home). Data was analyzed and visualized using R version 4.2.2 and GraphPad Prism 10.

Software and statistical analysis
No formal sample size calculations were performed for this trial. The dose escalation part was designed as a modified 3+3 rule-based design as described above and in the study protocol provided in Supplementary Note 2. No data were excluded from the analyses. The experiments were not randomized, and thus, investigators were not blinded to treatment during experiments and outcome assessment. The independent central reader was blinded to all clinical aspects of the trial. Similarly, spatial gene expression profiling and mIF were conducted in a blinded manner, with investigators unaware of clinical trial data and patient identities.

Graphical elements were generated using R version 4.2.2, GraphPad Prism 10 (GraphPad Software) and Adobe Illustrator 2023 (Adobe Inc.). Illustrations in this manuscript were in part created using Servier Medical Art, provided by Servier, and licensed under a Creative Commons Attribution 4.0 Unported License (https://smart.servier.com). Database management (eCRF) was carried out using Viedoc version 4.66 eCRF (Viedoc Technologies) and Microsoft Excel 2019 (Microsoft Corporation). Statistical tests were performed using GraphPad Prism 10 and R version 4.2.2 as specified in the figure legends. Descriptive statistics were applied to characterize the patient collectives, treatment response, and observed toxicity. Survival rates were estimated using the Kaplan–Meier method and compared by the log-rank test. Exploratory Cox proportional hazards regression analyses were additionally performed, and the proportional hazards assumption was verified using log-log survival plots and Schoenfeld residuals. Group differences for continuous variables were evaluated using the unpaired two-tailed Mann–Whitney U test. Spearman’s rank correlation was used for correlation analysis. Group comparisons of drug and target concentrations at identical time points were performed using the Kruskal–Wallis test, followed by pairwise Mann–Whitney tests. Proportional data were transformed using the arcsine square-root transformation to better meet assumptions of normality required for parametric testing. For integrated pharmacokinetic evaluation, the area under the curve (AUC) was calculated for each patient. Between-group differences in AUCs were assessed using Welch ANOVA.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a well-written follow up manuscript of the GLORIA trial reporting a 6 patient safety expansion cohort of CXCL12 inhibition combined with Bevacizumab and radiotherapy in newly diagnosed GBM.\nThe authors provide the rationale of the combination with spatially resolved data obtained from two publically available data sources (TCGA and the Ivy Glioblastoma Atlas Project). This suggests distinct regulatory mechanisms of vascularization in GBM which the authors then explore using multi-colour IF in 5 human GBM samples obtained via SOC surgical procedures.\nMulti-colour IF does confirm spatially distinct expression of CXCL12 and CA9 (surrogate of hypoxia) supporting their hypothesis.\nThe authors then report the clinical course of the additional 6 patients treated on the expansion cohort (the escalation data having been previously already published - Giordano 2024. Bevacizumab was given at 10mg/kg -the dose previously used in the Avaglio studies. No additional safety signals were seen, albeit numbers too small to make conclusions. The outcomes of these 6 patients due appear to be better than expected with standard of care, in particular given the poor prognosis patient population enrolled (incomplete resection, PS<2) although I note steroid doses are not reported. No translational data from these 6 patients is actually presented - have the authors got access to these samples as baseline multi-colour IF on these samples would have significantly strengthened the paper.\nIn conclusion, the authors provided a solid rationale for the exploration of their combination hypothesis - however I am disappointed that there were no mechanistic attempts to explore this in vitro (for example using brain slice cultures from their SOC surgical samples) which would have provided proof of concept given their spatial IF assays are already established.\nThe preliminary clinical data from 6 patients treated in an expansion cohort is intriguing but too small to draw any firm conclusions.", "reviewer_2": "Overall the manuscript is very well written and quite interesting. I have the following questions:\nAre there differences in the clinical and surgical traits between the external contemporary multicentric SOC cohort and the internal histology-matched SOC? Why was progression-free survival (PFS) data only available for 39 out of 64 patients in the external cohort? Could this skew the results, making the median PFS appear higher?\nIt appears that there is a significant confounder, as patients in Arm B of the 6-arm expansion may have undergone a gross total resection (GTR). Why was this permitted?\nWhat was the approach to censoring patient C2-001? It appears that medians and ranges were calculated using an Excel spreadsheet. Was this the method for conducting the survival analysis? It's unusual to report ranges for median survival; generally, the median from a Kaplan-Meier estimate should include the corresponding 95% confidence interval.\nSince patients were recruited before the release of the WHO 2021 guidelines and there does not appear to be any IDH testing for enrollment criteria, it would be helpful to specify whether any IDH-MT patients were recruited, how many, and whether they received treatment.\nWhy are survival estimates not calculated from the day of diagnosis (i.e., surgery)? This method would ease comparisons with existing literature.\nWhen were Cox models utilized, and were the underlying assumptions verified?", "reviewer_3": "The manuscript is well-written, and the figures are presented clearly.\nThe extended arm of the GLORIA trial clearly demonstrates significant improvements in PFS and OS in patients with GBM who did not receive multiple salvage therapy interventions, compared to the dose-escalation phase of the GLORIA trial. The lack of comparison group with RT+BEV would make it difficult to determine the impact of NOX. Overall the study is structured to test toxicity and a possible initial signal of efficacy. The PFS and OS as described, given historical controls, limit enthusiasm for this compound/approach. Additional SOC control groups should be provided that include clinical trials and/or BEV administration. Similarly, to have a radiographic response in the non-Bev group could be problematic given the effect of bev on the MRI. We agree with the authors that an expanded cohort will be necessary to determine ultimate efficacy, but should include a bev control group.\nIntroduction:\nLine 94 – This section lacks sufficient background information on the Hypoxia-VEGF-CXCL12 axis, which is crucial for readers to fully understand the manuscript. The rationale behind VEGF-CXCL12-mediated dual-inhibition therapy should be stated more clearly.\nResults:\n• Figure 1: I suggest including immunostaining images for each cell type analyzed (endothelial cells, pericytes, TAM, glioma cells), along with their colocalization with CA9 and CXCL12.\n• Did the authors examine the correlation between EG12high/low expression and PFS in the RT+NOX-A12+ BEV arm, as was done in the dose-escalation phase of the trial? This should be addressed in the text.\nDiscussion:\nLine 341 – The statement \"CXCL12 is a central factor driving pro-tumorigenic remodeling of the TME\" is a strong claim. Consider rephrasing this for clarity.\nMethods:\nFor clarity, present the “Buffers and Solutions for Multiplexed Immunofluorescence” section in a table format.\nSupplementary Figures:\n• Supplementary Figure 3: Please include the title of the figure in the figure legend.\n• Perform statistical analysis and report the results in the text."}, "author_response": "We thank you and the reviewers for the thoughtful and constructive feedback on our\nmanuscript.\nWe have carefully considered all comments and revised the manuscript accordingly. As part\nof this process, particularly extensive time- and resource-consuming spatial tissue analyses\nof trial and SOC patients were additionally performed to furthermore enhance mechanistic\ninsights as suggested by reviewers #1 and #3.\nBelow, we provide a detailed, point-by-point response to each reviewer’s comment. All\nchanges are highlighted in the revised manuscript, and page/paragraph numbers are provided\nin our responses for clarity.\nWe believe these revisions have significantly further improved the manuscript meeting the\nstandards for publication in Nature Communications.\nReviewer #1\nThis is a well-written follow up manuscript of the GLORIA trial reporting a 6 patient safety\nexpansion cohort of CXCL12 inhibition combined with Bevacizumab and radiotherapy in newly\ndiagnosed GBM.\nAnswer: We thank the reviewer for these positive and encouraging comments regarding the\nquality and clarity of our manuscript.\nThe authors provide the rationale of the combination with spatially resolved data obtained from\ntwo publically available data sources (TCGA and the Ivy Glioblastoma Atlas Project). This\nsuggests distinct regulatory mechanisms of vascularization in GBM which the authors then\nexplore using multi-colour IF in 5 human GBM samples obtained via SOC surgical procedures.\nMulti-colour IF does confirm spatially distinct expression of CXCL12 and CA9 (surrogate of\nhypoxia) supporting their hypothesis.\nThe authors then report the clinical course of the additional 6 patients treated on the expansion\ncohort (the escalation data having been previously already published - Giordano 2024.\nBevacizumab was given at 10mg/kg -the dose previously used in the Avaglio studies. No\nadditional safety signals were seen, albeit numbers too small to make conclusions. The\noutcomes of these 6 patients due appear to be better than expected with standard of care, in\nparticular given the poor prognosis patient population enrolled (incomplete resection, PS<2)\nalthough I note steroid doses are not reported. No translational data from these 6 patients is\nactually presented - have the authors got access to these samples as baseline multi-colour IF\non these samples would have significantly strengthened the paper.\nAnswer: We appreciate the reviewer’s comment. To strengthen the translational aspect of our\nwork, we have now gained access to the samples and performed extensive further analyses\nfor all the six GLORIA expansion arm patients. The results of these new analyses have been\nincorporated into the revised version of the manuscript in Results (page 17) and Discussion\n(page 18).\nWe were able to confirm our previous findings for the spatial distribution of CXCL12 (Fig. 1\ne, f) also in these samples. The previously established EG12 score, which quantifies CXCL12\nexpression in the endothelial and glioma cell compartment, was not associated with response\nin patients receiving combined CXCL12 and VEGF inhibition, in contrast to those treated with\nCXCL12 inhibition only (as shown previously in Giordano et al., Nat Comm 2024). Collectively,\nthese findings support our model that VEGF and CXCL12 pathways cooperate in\nrevascularization, yet remain functionally and spatially distinct. Thus, predictive biomarkers for\nCXCL12 inhibition may not be applicable to a combination therapy. However, we acknowledge\nthat the number of patients in the combination arm is low and limits inference. Accordingly, we\npresent these data as exploratory and hypothesis-generating and have included the analyses\nas a new Supplementary figure (Suppl. Fig. 10).\nResults, page 17, paragraph 1: We next assessed whether outcomes of patients receiving\nRT+NOX-A12+BEV correlated with CXCL12 expression. A higher frequency of CXCL12+\nendothelial and glioma cells (EG12) may serve as a predictive marker for CXCL12-targeted\ntherapy, as it was significantly associated with prolonged PFS under NOX-A12, but not in a\nSOC cohort.38 In the dose-escalation part of the trial with patients receiving RT+NOX-A12,\nEG12 correlated significantly with PFS (r = 0.865, p = 0.005). This correlation was lost in\ns\npatients receiving RT+NOX-A12+BEV (Suppl. Fig. 10), with a negative trend between EG12\nand both PFS (rs = -0.714, p = 0.136) and OS (r = -0.371, p = 0.497).\ns\nDiscussion, page 18, paragraph 3: We also detected physiological CXCL12 expression in\nhealthy human brain vessels, in line with an emerging role of the CXCL12-CXCR4 axis for\nearly-response to cerebral ischemia in conditions like cerebral insults, as shown in mouse\nmodels.47,48 It appears likely that comparable damage and repair mechanisms are activated\nin GBM, where angiogenesis and vasculogenesis appear to be simultaneous, yet spatially\ndistinct and likely complementary mechanisms. This is supported by our findings in publicly\navailable GBM datasets and multi-omics analyses of representative GBM patients, as well as\nby the translational assessment of tissue from GLORIA patients. The frequency of CXCL12\nexpression in endothelial and glioma cells was prognostic for therapeutic benefit exclusively\nin patients receiving CXCL12-targeted therapy only. This association was no longer observed\nin patients who also received VEGF-targeted treatment, supporting the concept of\ncooperative, but functionally and spatially distinct signaling between the CXCL12 and VEGF\npathways, suggesting that predictive biomarkers for CXCL12 inhibition may not be applicable\nto a combination therapy.\nSupplementary Figures, page 45: A new Suppl. Fig. 10 was added, showing the correlation\nof oncologic outcomes with compartmental CXCL12 expression.\nSupplementary Figures, page 47: A new Suppl. Fig. 12 was added, providing side-by-side\nillustration of tumor areas analyzed in multiplexed immunofluorescence staining and\ncorresponding H&E staining of GLORIA expansion arm A patients.\nThe outcomes of these 6 patients due appear to be better than expected with standard of care,\nin particular given the poor prognosis patient population enrolled (incomplete resection, PS<2)\nalthough I note steroid doses are not reported.\nAnswer: Baseline steroid exposure has now been added to the manuscript and incorporated\ninto Fig. 3b (formerly Fig. 2). Three patients received dexamethasone at baseline, defined as\ntime of NOX-A12 initiation. We note, however, that patients in the GLORIA trial started\ntreatment with NOX-A12 after a median of 1.1 months post-surgery; at which point ongoing\ncorticosteroid use is generally uncommon (Mistry, Front Oncol 2023).\nResults, page 11, Fig. 3b: Baseline steroid doses were added to the figure for all patients.\nIn conclusion, the authors provided a solid rationale for the exploration of their combination\nhypothesis - however I am disappointed that there were no mechanistic attempts to explore\nthis in vitro (for example using brain slice cultures from their SOC surgical samples) which\nwould have provided proof of concept given their spatial IF assays are already established.\nThe preliminary clinical data from 6 patients treated in an expansion cohort is intriguing but\ntoo small to draw any firm conclusions.\nAnswer: In the revised manuscript, we now provide substantial new experimental data that\noffer mechanistic insights, including studies using GBM-derived organoids (GBOs) cultured\non human brain slices, as suggested, and spatial transcriptomics at single-cell resolution\n(Xenium® platform) from the brain slices, normal brain tissue, and GBM specimens (new Fig.\n2).\nFirst, GBO cultured on human brain slices demonstrated extensive hypoxic areas with strong\nactivation of hypoxia-driven gene programs including VEGFA at the core, whereas CXCL12\nexpression was sparse and, when present, colocalized predominantly with few endothelial\ncells, and, to a lesser extent, pericyte-, macrophage- and glioma-related transcripts (new Fig.\n2a,b). Notably, CXCL12 expression was detected in normal brain vasculature of the\nsurrounding, the GBO embedding tissue (new Fig. 2c). Due to inherent limitations of the GBO\nhuman brain slice culture system, such as the absence of circulation and the restricted culture\nduration, it was not feasible to investigate angiogenesis, immune cell recruitment, or the\nrecruitment of endothelial progenitor cells.\nTaken together, these findings corroborate our results from our immunofluorescence studies\non a mechanistic level and transcript level, showing that the induction of VEGFA and CXCL12\nappears to be driven by largely complementary mechanisms, whereby hypoxia is the main\ndriver for VEGFA in GBM but not so much or at all for CXCL12. In contrast, CXCL12\nexpression is already present in vessels of normal human brain and thus appears to be\naugmented in the microvascular proliferation zones of GBM as also shown by additional\nspatial transcriptomics of GBM (new Fig. 2d,e).\nWe have edited the manuscript as follows:\nResults, page 8, paragraph 1: To further test and validate this hypothesis, we next\ngenerated patient-derived glioblastoma organoids (GBOs) from surgical specimens cultured\non human brain slices (Fig. 2a) and performed spatial transcriptomics on a representative\nmodel. The GBO tumor tissue displayed extensive hypoxic areas with pronounced VEGF\nexpression at the core, whereas CXCL12 expression was sparse and, when present,\ncolocalized predominantly with few endothelial cells, and, to a lesser extent, pericyte-,\nmacrophage- and glioma-related transcripts (Fig. 2b). Notably, CXCL12 expression was\ndetected in normal brain vasculature of the surrounding, the GBO embedding tissue (Fig. 2c).\nTo overcome the mechanistical limitations of revascularization assessment in a non-perfused\nGBO model, we conducted parallel spatial transcriptomics analyses on a representative GBM\npatient specimen with well-defined zonal architecture (Fig. 2d). Consistent with our previously\nreported observations, CXCL12 expression localized to endothelial cells, pericytes,\nmacrophages/microglia and glioma cells. In contrast, VEGF expression strongly correlated\nwith hypoxia-related genes, while CXCL12 showed only a weak association with hypoxia but\na robust link to the described cellular compartments (Fig. 2e).\nTaken together with our preceding analyses, these findings further supported the spatially and\nmechanistically distinct involvement of both VEGF- and CXCL12-mediated pathways in GBM\nvascular remodeling. Consequently, within the GLORIA trial, we evaluated the potential\nadditional clinical benefit of VEGFA-targeting with BEV in combination with NOX-A12.\nResults, page 9, Figure 2: A new Figure 2 was introduced presenting the results of the spatial\ntranscriptomics from patient-derived glioblastoma organoid brain slices and freshly resected\nglioblastoma specimen.\nDiscussion, page 18, paragraph 3: We also detected physiological CXCL12 expression in\nhealthy human brain vessels, in line with an emerging role of the CXCL12-CXCR4 axis for\nearly-response to cerebral ischemia in conditions like cerebral insults, as shown in mouse\nmodels.47,48 It appears likely that comparable damage and repair mechanisms are activated in\nGBM, where angiogenesis and vasculogenesis appear to be simultaneous, yet spatially\ndistinct and likely complementary mechanisms. This is supported by our findings in publicly\navailable GBM datasets and multi-omics analyses of representative GBM patients, as well as\nby the translational assessment of tissue from GLORIA patients. The frequency of CXCL12\nexpression in endothelial and glioma cells was associated with therapeutic benefit exclusively\nin patients receiving CXCL12-targeted therapy only. This association was no longer observed\nin patients who also received VEGF-targeted treatment, supporting the concept of\ncooperative, but functionally and spatially distinct signaling between the CXCL12 and VEGF\npathways, suggesting that potential predictive biomarkers for CXCL12 inhibition may not be\napplicable to a combination therapy.\nMethods, page 27, 28 and 34: Information was added for the newly introduced techniques.\nReviewer #2:\nOverall the manuscript is very well written and quite interesting. I have the following questions:\nAnswer: We thank the reviewer for the positive assessment of our work and for the thoughtful\nquestions, which we address in detail below.\nAre there differences in the clinical and surgical traits between the external contemporary\nmulticentric SOC cohort and the internal histology-matched SOC?\nAnswer: Ensuring optimal comparability between our trial cohort and the internal and external\nSOC cohorts was of highest importance.\nThe internal SOC cohort included patients treated at the neuro-oncological center of the\nUniversity Hospital Bonn, Germany between 2010 and 2023. Histological criteria for selection\nof reference patients were: newly-diagnosed GBM, IDH-wildtype (CNS WHO grade 4),\nabsence of MGMT promoter methylation. Clinical criteria were: ECOG of 0-2, status post\nbiopsy or incomplete resection, and first line therapy with RT (and optionally TMZ).\nThe external SOC cohort was derived from a publicly available multicenter dataset with\ncomparable patient characteristics (Drexler et al., 2024). Histological criteria were: newly-\ndiagnosed GBM, IDH-wildtype, absence of MGMT promoter methylation. Clinical criteria were:\nECOG of 0-2, status post incomplete resection (<90% removal), and first line therapy with RT\n(and optionally TMZ).\nBoth cohorts thus represent contemporary patient populations from German neuro-\noncological centers treated with SOC, with only minor differences summarized in Extended\nData Table 2 and 3 (primarily slight variations in RT fractionation and adjuvant temozolomide\nadministration). For full transparency, the patient characteristics of both cohorts are provided\nin the Source Data File. The Methods section was revised accordingly to clarify cohort\ncomparability.\nMethods, page 23, paragraph 2: We benchmarked oncological outcomes of the GLORIA\ntrial to an internal (University Hospital Bonn)38 and a recently published external German\nmulticenter cohort44 of GBM patients with comparable patient characteristics resembling\ninclusion criteria of the GLORIA trial treated with standard RT and optional TMZ.\nMethods, page 23, paragraph 2: The patient characteristics of both cohorts are provided in\nExtended Data Table 2 and 3 and the Source Data File.\nWhy was progression-free survival (PFS) data only available for 39 out of 64 patients in the\nexternal cohort? Could this skew the results, making the median PFS appear higher?\nAnswer: We cannot comment on why PFS data were available for only a subset of patients\nin the external SOC cohort, as none of our authors were involved in that analysis. However,\nloss to follow-up causing PFS censoring is common in oncological standard-of-care cohorts\ntreated in a routine setting. Given that said data set has been published very recently (Drexler\net al., Nat Med, 2024) we consider the data to be robust and representative of current SOC\noutcomes for this GBM patient subgroup. PFS is well-known to be influenced by the number\nof informatively censored patients. We acknowledge that the high frequency of PFS censoring\nmay have introduced bias, potentially leading to an overestimation of the true PFS in this\ncohort. However, this would rather disadvantage the GLORIA cohort in direct comparison, and\nwe have now addressed this potential effect in the Discussion.\nDiscussion, page 18, paragraph 2: Both PFS and OS in GLORIA patients receiving NOX-\nA12 + BEV appear favorable to comparable contemporary external SOC cohorts. However,\nthe high frequency of PFS censoring in these44 may have introduced bias, potentially leading\nto an overestimation of the true PFS, thereby disadvantaging the GLORIA cohort in direct\ncomparison.\nIt appears that there is a significant confounder, as patients in Arm B of the 6-arm expansion\nmay have undergone a gross total resection (GTR). Why was this permitted?\nAnswer In the initial trial protocol, only patients with residual tumor were eligible for the trial.\nFollowing a change of the protocol under active trial recruitment, GTR was then permitted in\nall arms of the trial. This decision was made as there was no obvious reason to question the\ndrugs’ mode of action in patients lacking MRI contrast-enhancing tumor and the sponsor\nwished to further explore the treatment also in this clinical setting.\nHowever, none of the patients that eventually were recruited, had undergone GTR. All\nGLORIA patients in all arms had an incomplete resection or were not amenable to resection.\nOf note, arm B of the expansion was never recruited, as the sponsor decided to focus on the\ncombinatory treatment of RT+NOX-A12+BEV after considering the first interim results in arm\nA.\nWe added a statement in the Results section to clarify this aspect.\nResults, page 10, paragraph 3: No patient had undergone gross total resection.\nWhat was the approach to censoring patient C2-001?\nAnswer: According to the pre-defined statistical analysis plan and after careful consideration\nwith the statistician and the sponsor, C2-001 required censoring for PFS due to the individual\npatient course (see below). To ensure full transparency, we exceed journal requirements and\ndecided to provide patient narratives as a Supplementary Note for all trial patients:\nSupplementary Note 1 (patient narratives), page 2, paragraph 2: This is a patient with a\nfrontotemporal GBM. The patient underwent partial resection on May 22, 2020. After proper\ndiagnosis, the patient was included in GLORIA. The patient was started on 400 mg/week NOX-\nA12 on June 22, 2020 and was treated with hypofractionated RT. The first scan in week 9\nshowed stable disease. The scan performed on October 19, 2020 (week 18) suggested tumor\nrecurrence (pPD) which was not confirmed in a further scan. Of note, at the time point of this\nMRI no AEs were reported and the time point NANO score remained 0. Despite this, the PI\ndecided to discontinue NOX-A12 and initiate salvage therapy with temozolomide on November\n18, 2020 without performing an MRI scan. On January 6, 2021 the patient received an infusion\nof bevacizumab. Although a scan performed 3 weeks later showed PR, while previous pseudo-\nprogression cannot be excluded, we considered the overall time point response as “not\nevaluable” due to “bevacizumab blinding”. (…)\nSince salvage therapy was i) initiated in the absence of confirmed PD and ii) no clinical\ndeterioration was noted at the previous scan date, the patient is censored for PFS at the time\npoint of the scan preceeding the initiation of salvage therapy (October 19, 2020).\nFinal interpretation:\n- PD as per RANO is indeterminable, resulting in PFS being censored at 120 days\n- The overall survival time was 287 days\nIt appears that medians and ranges were calculated using an Excel spreadsheet. Was this the\nmethod for conducting the survival analysis?\nAnswer: No calculations and statistical analyses were conducted using Microsoft Excel. The\nprovided Excel spreadsheet is for information of the reader only as an obligatory submission\nrequirement by the Editorial Office of Nature Communications to assure access to the raw\ndata for the public.\nAll statistical tests, including descriptive statistics, were performed using GraphPad Prism 10\nand R version 4.2.2 as specified in the figure legends and the Methods section.\nIt's unusual to report ranges for median survival; generally, the median from a Kaplan-Meier\nestimate should include the corresponding 95% confidence interval.\nAnswer: After consulting with our in-house statistician, we initially decided to provide ranges\ninstead of 95% confidence intervals to avoid potentially misleading associations given the\noverall number of patients in the expansion arm is only six individuals. We considered the\ncombination of ranges and median values to best reflect the data’s distribution in this relatively\nsmall patient collective. Following the reviewer’s recommendation, we have now added 95%\nconfidence intervals. Please note that, due to the small number of events, the upper\nconfidence limit could not be estimated in some cases, this is then clearly indicated in the text.\nResults, page 15, paragraph 2 and page 16, paragraph 2: 95% confidence intervals have\nbeen added for survival outcomes.\nSince patients were recruited before the release of the WHO 2021 guidelines and there does\nnot appear to be any IDH testing for enrollment criteria, it would be helpful to specify whether\nany IDH-MT patients were recruited, how many, and whether they received treatment.\nAnswer: All patients were confirmed as glioblastoma, IDH-wildtype (CNS WHO grade 4)\naccording to the WHO 2021 classification for CNS tumors by immunohistochemistry. If\npatients were younger than 54 years, they were assessed additionally by pyrosequencing for\nIDH1 and IDH2.\nTo clarify, we made the following modification to the Results and Methods sections of\nthe manuscript:\nResults, page 10, paragraph 2: The GLORIA trial (NCT04121455) is a multicentric phase\n1/2 trial conducted at six major neuro-oncology centers in Germany. It was designed to\nevaluate the safety and efficacy of RT combined with continuous intravenous (i.v). treatment\nwith NOX-A12 in newly-diagnosed, incompletely resected IDH-wildtype GBM (CNS WHO\ngrade 4) lacking MGMT promoter methylation (Fig. 3a).\nResults, page 11, Fig. 3b: IDH wildtype status was added to the figure for all patients.\nMethods, page 21, paragraph 1: All patients were neuropathologically confirmed as GBM,\nIDH-wildtype (CNS WHO grade 4) according to the 2021 WHO classification for CNS tumors.\nIf patients were ≤ 54 years of age, IDH mutations were additionally assessed by\npyrosequencing of the IDH1 and IDH2 loci.\nWhy are survival estimates not calculated from the day of diagnosis (i.e., surgery)? This\nmethod would ease comparisons with existing literature.\nAnswer: To enhance comparability, we have now additionally provided survival estimates\ncalculated from the date of initial surgery.\nWe agree that most trials rather report survival estimates calculated from the day of surgery.\nHowever, several recent trials, such as EF14 (Stupp et al., 2017), defined survival from the\ninitiation of experimental treatment. In accordance with the predefined trial protocol, survival\nin our analysis was calculated from the start of experimental treatment with the CXCL12\ninhibitor. We acknowledge that this approach is indeed disadvantageous for the GLORIA\ncohorts and this limitation is now emphasized more prominently in the Discussion. Please note\nthat the per-protocol analysis remains unchanged, as it was predefined in the trial protocol as\na secondary endpoint.\nResults, page 16, paragraph 3: To enhance comparability with the SOC cohorts and\nexisting literature, we additionally calculated GLORIA survival outcomes from the date of initial\nsurgery. Both PFS and OS of patients receiving NOX-A12+BEV were approximately 1.1\nmonths longer, without affecting the statistical significance of comparisons with the SOC\ncohorts (Suppl. Fig. 8).\nSupplementary Figures, page 43: A new Suppl. Fig. 8 was added, showing GLORIA\nsurvival outcomes from the date of initial surgery in comparison to the external and the internal\nhistology-matched SOC cohort.\nSupplementary Figures, page 44: Suppl. Fig. 9 (previously 7) was modified, now\nadditionally showing GLORIA survival outcomes from the date of initial surgery in comparison\nto SOC and literature cohorts.\nDiscussion, page 18, paragraph 2: In contrast, many studies initiate treatment earlier or\ncommonly define the date of surgery as starting point for outcome assessment. Consequently,\nthe per protocol GLORIA survival data should be interpreted with caution, as it likely\nunderestimates survival compared with most contemporary trials.\nWhen were Cox models utilized, and were the underlying assumptions verified?\nAnswer: The primary outcome analysis was performed using the Kaplan–Meier method and\nlog-rank tests, which do not rely on proportional hazards assumptions. We agree that the\nadditional Cox proportional hazards regression analyses have limited interpretability given the\nsmall sample size in this trial. However, hazard ratios were included for completeness and to\nfacilitate comparison with other clinical reports, in line with common reporting standards. The\nproportional hazard assumption was evaluated graphically using log-log survival plots and\nSchoenfeld residuals and appeared reasonable within the limitations of the dataset. The\ncorresponding Methods section was revised accordingly, and the Discussion expanded to\nfurther emphasize the limited statistical power of the survival analyses.\nDiscussion, page 19, paragraph 2: While the small sample size limits the statistical\ninterpretability and power to evaluate the survival impact of compartmental CXCL12\nexpression within this subset, the results still provide important hypothesis-generating insights.\nMethods, page 28, paragraph 1: Survival rates were estimated using the Kaplan–Meier\nmethod and compared by log-rank test. Exploratory Cox proportional hazards regression\nanalyses were additionally performed, and the proportional hazards assumption was verified\nusing log-log survival plots and Schoenfeld residuals.\nReviewer #3:\nThe manuscript is well-written, and the figures are presented clearly.\nAnswer: We thank the reviewer for this positive assessment of our work and for the thoughtful\nquestions. Please find our remarks below.\nThe extended arm of the GLORIA trial clearly demonstrates significant improvements in PFS\nand OS in patients with GBM who did not receive multiple salvage therapy interventions,\ncompared to the dose-escalation phase of the GLORIA trial. The lack of comparison group\nwith RT+BEV would make it difficult to determine the impact of NOX. Overall the study is\nstructured to test toxicity and a possible initial signal of efficacy. The PFS and OS as described,\ngiven historical controls, limit enthusiasm for this compound/approach. Additional SOC control\ngroups should be provided that include clinical trials and/or BEV administration. Similarly, to\nhave a radiographic response in the non-Bev group could be problematic given the effect of\nbev on the MRI. We agree with the authors that an expanded cohort will be necessary to\ndetermine ultimate efficacy, but should include a bev control group.\nAnswer: We agree that the triple treatment in the GLORIA expansion arm with addition of\nBEV clearly improved outcomes at unchanged good tolerability. Indeed, ultimate\ninterpretations of efficacy requires a randomized control group which is planned for the\nupcoming phase 2/3 trial currently in preparation. The characteristics of the control groups will\nbe ultimately determined by the official FDA requests. Unfortunately, SOC patients treated\nwith BEV in the first line are not available in Europe, as BEV was never approved for GBM by\nthe EMA. Therefore, historical high-quality outcome data for BEV only are available from the\ntwo phase 3 trials by Gilbert et al. and Chinot et al. which are referenced and discussed in the\nmanuscript. Additionally, Ulrich Herrlinger, co-author (co-senior) of this manuscript, has\nshared his extensive expertise on BEV treatment as former PI of the GLARIUS trial. Carefully\ntaking the obvious limitation of a phase 1/2 trial into consideration, we believe that the survival\noutcome for RT+NOX-A12+BEV warrants further assessment in a follow-up trial on efficacy.\nDirect comparison clearly requires caution, but the outcome of GLORIA expansion arm A\nexceeds our SOC cohorts as well as historical controls with comparable patient\ncharacteristics. An overview and comparison of these cohorts is provided in the revised Suppl.\nFig. 9. We also revised the manuscript following this reviewer comment to tone down our\nstatements and strengthen said aspects in the Discussion. Notably, in GLORIA, survival\nendpoints were not calculated from day of diagnosis (as is common in most of the historical\nSOC and BEV cohorts), but from the day of initiation of experimental treatment, which is\naround 1.1 months later. Consequently, the per protocol GLORIA survival data likely\nunderestimates survival compared with most contemporary trials.\nOur trial results also emphasize the importance of advanced MRI analyses to prevent\nmisinterpretation of radiographic pseudoresponse and pseudoprogression. We expanded the\nDiscussion section to address the points raised by the reviewer.\nDiscussion, page 18, paragraph 2: While the PFS observed in GLORIA patients receiving\nBEV is consistent with outcomes reported in recent (larger-scaled) clinical trials of the VEGFA-\ntargeting drug,20–22 the combinatory approach resulted in a median OS of 19.9 months from\nNOX-A12 initiation and 2/6 patients surpassing 2 year-OS, suggesting a trend toward\nimproved long-term outcomes. Direct comparisons remain challenging, as the referenced\ntrials enrolled overall more prognostically favorable patient populations. We consider the here\nreported GLORIA results a robust OS signal, especially since we only included GBM patients\nwith highly unfavorable prognosis.\nDiscussion, page 19, paragraph 2: In a future phase 2/3 trial, particular emphasis should be\nplaced on the inclusion of adequate control arms and rigorous radiographic response\nassessment. Given the vascular mechanism of the therapeutic approach presented here,\nadvanced MRI techniques will be essential to reliably distinguish true response from true\nprogression.\nIntroduction: Line 94 – This section lacks sufficient background information on the Hypoxia-\nVEGF-CXCL12 axis, which is crucial for readers to fully understand the manuscript. The\nrationale behind VEGF-CXCL12-mediated dual-inhibition therapy should be stated more\nclearly.\nAnswer: We have expanded the Introduction to include additional background on the\nHypoxia–CXCL12 signaling axis. We furthermore clarified the rationale for VEGF–CXCL12\ndual inhibition in GBM.\nIntroduction, page 4, paragraph 4: Radiation-induced loss of endothelial cells, accumulation\nof reactive oxygen species, and aggressive tumor growth with aberrant neovascularization\ncollectively lead to impaired tumor perfusion triggering TGF-β-augmented induction of the\nhypoxia-related factor HIF1α. This, in turn, attracts CXCR4+ BMDCs along a gradient of the\nCXCR4 ligand CXCL12.24,28,29 Elevated CXCL12 expression facilitates not only BMDC\nmigration but also their adhesion and homing to hypoxic tumor regions.24\nIntroduction, page 5, paragraph 1: To date, no clinical trial has investigated dual targeting\nof CXCL12 and VEGF as two key mediators of post-radiogenic revascularization in GBM.\nResults: Figure 1: I suggest including immunostaining images for each cell type analyzed\n(endothelial cells, pericytes, TAM, glioma cells), along with their colocalization with CA9 and\nCXCL12.\nAnswer: We have now included representative immunostaining images for endothelial cells,\npericytes, TAMs, and glioma cells, showing respective colocalization with CA9 and CXCL12\nin Figure 1d. Additional images are provided in a new Suppl. Fig. 2.\nResults, page 6, paragraph 3: Aiming for single-cell spatial resolution, we performed\nmultiplex-immunofluorescence (mIF) stainings on human GBM tissue samples (n = 5, Fig. 1d\nand Suppl. Fig. 2).\nResults, page 7, Figure 1: Immunostaining images for each cell type analyzed and their\ncolocalization with CA9 and CXCL12 have been added in Fig. 1d.\nSupplementary Figures, page 36: A new Suppl. Fig. 2 was additionally provided, showing\nmore representative immunostaining images for all the cell types and respective colocalization\nwith CA9 and CXCL12.\nDid the authors examine the correlation between EG12high/low expression and PFS in the\nRT+NOX-A12+ BEV arm, as was done in the dose-escalation phase of the trial? This should\nbe addressed in the text.\nAnswer: Following the reviewers’ comment, we have gained access to the tissue of all\nexpansion arm patients and now performed the requested analysis correlating EG12\nexpression levels with survival outcomes in the RT+NOX-A12+BEV arm (see also Reviewer\n#1). The results are included in the revised manuscript and discussed accordingly.\nWe were able to confirm our previous findings for the spatial distribution of CXCL12 also in\nthe expansion arm patient samples. The previously established EG12 score, which quantifies\nCXCL12 expression in the endothelial and glioma cell compartment, was not associated with\nresponse in patients receiving combined CXCL12 and VEGF inhibition, in contrast to those\ntreated with\nCXCL12 inhibition only (as shown previously in Giordano et al., 2024). These\nfindings support our concept that VEGF and CXCL12 pathways cooperate in\nrevascularization but are also functionally and spatially distinct. Thus, predictive biomarkers for\nCXCL12 inhibition may not be applicable to a combination therapy. However, we are also aware that\nthe number of patients is low and thus scientific value remains limited as also pointed out in\nthe manuscript. We added the analyses in an additional Supplementary figure.\nResults, page 17, paragraph 1: We next assessed whether outcomes of patients receiving\nRT+NOX-A12+BEV correlated with CXCL12 expression. A higher frequency of CXCL12+\nendothelial and glioma cells (EG12) may serve as a predictive marker for CXCL12-targeted\ntherapy, as it was significantly associated with prolonged PFS under NOX-A12, but not in a\nSOC cohort.38 In the dose-escalation part of the trial with patients receiving RT+NOX-A12,\nEG12 correlated significantly with PFS (r = 0.865, p = 0.005). This correlation was lost in\ns\npatients receiving RT+NOX-A12+BEV (Suppl. Fig. 10), with a negative trend between EG12\nand both PFS (rs = -0.714, p = 0.136) and OS (r = -0.371, p = 0.497).\ns\nDiscussion, page 18, paragraph 3: We also detected physiological CXCL12 expression in\nhealthy human brain vessels, in line with an emerging role of the CXCL12-CXCR4 axis for\nearly-response to cerebral ischemia in conditions like cerebral insults, as shown in mouse\nmodels.47,48 It appears likely that comparable damage and repair mechanisms are activated in\nGBM, where angiogenesis and vasculogenesis appear to be simultaneous, yet spatially\ndistinct and likely complementary mechanisms. This is supported by our findings in publicly\navailable GBM datasets and multi-omics analyses of representative GBM patients, as well as\nby the translational assessment of tissue from GLORIA patients. The frequency of CXCL12\nexpression in endothelial and glioma cells was associated with therapeutic benefit exclusively\nin patients receiving CXCL12-targeted therapy only. This association was no longer observed\nin patients who also received VEGF-targeted treatment, supporting the concept of\ncooperative, but functionally and spatially distinct signaling between the CXCL12 and VEGF\npathways, suggesting that potential predictive biomarkers for CXCL12 inhibition may not be\napplicable to a combination therapy.\nSupplementary Figures, page 45: A new Suppl. Fig. 10 was added, showing the correlation\nof oncologic outcomes with compartmental CXCL12 expression in the respective GLORIA\ncohorts.\nSupplementary Figures, page 47: A new Suppl. Fig. 12 was added, providing side-by-side\nillustration of tumor areas analyzed in multiplexed immunofluorescence staining and\ncorresponding H&E staining of GLORIA expansion arm A.\nDiscussion: Line 341 – The statement \"CXCL12 is a central factor driving pro-tumorigenic\nremodeling of the TME\" is a strong claim. Consider rephrasing this for clarity.\nAnswer: We have rephrased and toned down this statement for clarity.\nDiscussion, page 19, paragraph 1: Our findings support this hypothesis, suggesting that\nCXCL12 contributes substantially to pro-tumorigenic remodeling of the TME and, ultimately,\nGBM recurrence after RT.\nMethods: For clarity, present the “Buffers and Solutions for Multiplexed Immunofluorescence”\nsection in a table format.\nAnswer: We have reformatted this section into a table to improve clarity and readability.\nMethods, page 26: Table 1: Buffers and solutions for multiplexed immunofluorescence\nSupplementary Figures: Supplementary Figure 3: Please include the title of the figure in the\nfigure legend.\nAnswer: We have now added the title to the legend of Supplementary Figure 4 (previously\nSuppl. Fig. 3) as requested.\nPerform statistical analysis and report the results in the text.\nAnswer: Statistical analyses have been performed for the relevant comparisons and added\nto the Methods. The corresponding results are now reported in the Results section and\nSupplementary Fig. 4 (previously Suppl. Fig. 3).\nResults, page 12, paragraph 1: With increasing NOX-A12 dosage, plasma levels increased\ncorrespondingly (p = 0.007), exceeding the stable CXCL12 levels ((p = 0.116; Suppl. Fig. 4).\nNOX-A12 plasma levels were significantly higher from DL 1 to DL 2 (p = 0.020) and DL3 (p =\n0.020), but did not differ significantly with additional BEV treatment (p = 0.270).\nMethods, page 28, paragraph 4: Group comparisons of drug and target concentrations at\nidentical time points were performed using the Kruskal–Wallis test, followed by pairwise\nMann–Whitney tests. For integrated pharmacokinetic evaluation, the area under the curve\n(AUC) was calculated for each patient. Between-group differences in AUCs were assessed\nusing Welch ANOVA.\nSupplementary Figures, page 39: Suppl. Fig. 4 (previously Suppl. Fig. 3) was modified, now\nadditionally providing statistical analyses."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a really good job in thoughtfully addressing the reviewers comments, providing further translational data supporting their hypotheses. I have no further comments - this is an important area of unmet need.", "reviewer_2": "My concerns have been addressed", "reviewer_3": "Concerns adequately addressed"}, "author_response": ""}]

Decision letter