Preclinical studies of TMZ and Bev combination therapy for glioma demonstrated anti-tumor activity through inhibition of angiogenesis [25, 26]; however, clinical results were disappointing. Adaptation of the TME that leads to activation of redundant angiogenesis pathways is one mechanism that can lead to acquired resistance to anti-angiogenic therapies that target VEGF and its receptors [27]. Changes in angiogenic factors and cytokines have been described in patients treated with anti-angiogenic agents that target VEGF and tyrosine kinase [17, 28, 29]; however, few studies have analyzed paired samples from the same patients who underwent surgical resection during both naive and Bev-resistance stages [8, 9, 21]. In addition, it should be unique that we focused on expression of angiogenic factors other than VEGF as a salvage angiogenesis pathway in tumor vessel including vascular endothelial cell.

Clinical trials of VEGF-targeted therapy for GBM have shown upregulated serum levels of ANGPT2, EphA2, and FGF2 in the refractory period [17, 20, 28], while changes in the expression level of PLGF were controversial [8, 10, 21, 29]. Here, we report that expressions of ANGPT2, EphA2, and PLGF are upregulated in tumors resistant to Bev therapy, while expression levels of ANGPT1 and FGF2 evaluated by immunohistochemistry remain stable (Fig. 2A, B). We previously showed that the TME becomes normoxic in the effective Bev stage and hypoxic in the refractory Bev stage, regardless of VEGF suppression [5]. This raises the question of whether changes in tumor oxygenation during VEGF-targeting therapy affect alternative angiogenesis pathways. The present study demonstrates changes in expression levels of angiogenic factors in heterogeneous TME during Bev therapy (Fig. 5).

Scheme representing changes in VEGF, CD34, and alternative angiogenic factors in tumor vessels at naïve, effective and refractory Bev stages. FGF2, EphA2, and PLGF levels increase in the effective and refractory Bev stages. ANGPT1 tends to decrease in the effective Bev stage compared with the naïve Bev stage, and increase in the refractory Bev stage again, while ANGPT2 tends to increase in the refractory stage compared with naïve and effective Bev stages

FGF2, EphA2, and PLGF levels were upregulated in effective and refractory Bev stages under reduced vascular density (Fig. 2C, D, E) [5], regardless of therapeutic response. PLGF, belonging to the VEGF-family, was not altered between effective and refractory Bev stages [30]. PLGF was observed in tumor cells and vascular endothelial cells in hypoxic GBM, indicating that the TME under hypoxic conditions is also a source of PLGF [21]. PLGF elevation is significant during Bev therapy response in patients with metastatic colorectal cancer [31] and GBM [10], making it a reliable as a predictive biomarker in clinical outcomes.

Upregulation of EphA2 has potential as a novel immunotherapy for patients that are refractory to Bev [18,19,20, 32, 33]; however, the low expression level of EphA2 correlate with a favorable prognosis in the Cancer Genome Atlas GBM database [20], suggesting that EphA2 could also be a predictive biomarker and an alternative target for salvage therapy after Bev failure.

FGF2 is crucial for tumor angiogenesis and alternative angiogenic pathways during Bev therapy [7]. Multiple kinase inhibitors target VEGF, FGF, and PDGF receptor pathways to overcome resistance. Okamoto et al. investigated vascular structures and expression levels of angiogenic factors, including FGF2 and PDGF by paired comparison of initial surgery and autopsied samples after Bev failure [9]. Our present data demonstrated FGF2 to be upregulated at Bev therapy commencement, while a phase II study showed no significant change before or after Bev therapy, indicating no impact on clinical outcome [28, 34]. Further studies are needed to understand the significance of FGF2 induction in Bev therapy for GBM.

In naïve Bev, ANGPT1 facilitates vascular normalization and maturation during angiogenesis, whereas ANGPT2 has antagonist properties towards ANGPT1 [15, 35]. After VEGF blockade, expression level of ANGPT1 increased in a narrow therapeutic window during tumor oxygenation [13], while ANGPT2 induced vascular remodeling and sprouting under hypoxic TME at Bev resistance [14, 15, 36,37,38]. Our data show that ANGPT1 was downregulated in the effective Bev stage compared with the naïve Bev stage, while ANGPT2 remained unchanged until the refractory stage. Our previous and present data [5, 13, 16, 37, 38] indicate that ANGPT1 and ANGPT2 might be reciprocally regulated during tumor oxygenation but upregulated together in the refractory period (Fig. 5). It is also known that ANGPT2 compensates for VEGF inhibition by recruiting perivascular myeloid-derived suppressive cells and M2 macrophages [39, 40]. Upregulation of ANGPT2 is associated with T-cell exclusion, and blocking it promotes CD8+ T-cell infiltration, resulting in anti-tumor effects [41]. These evidences might support results of a clinical trial for bispecific antibodies targeting VEGF and ANGPT2 [42, 43], potentially supporting ANGPT2-targeted therapy combined with anti-VEGF therapy as a second-line therapy for patients with refractoriness of Bev. It also suggests that inhibition of ANGPT2 may overcome Bev resistance, and that combined immunotherapy may avoid tumor recurrence in hypoxic and immunosuppressive TME.

Radiographic comparison between enhancement and non-enhancement patterns in refractory Bev showed that there were no differences in expression levels of various angiogenic factors [23]. In the present study of recurrence patterns on MRI classified as T1-flare up and T2-diffuse pattern as previously described [5, 24], expression of all angiogenic factors examined in the present study was elevated in recurrent tumors with the T2-diffuse pattern on MRI. This discrepancy might be due to selection bias of autopsy samples as well as surgical resection of from non-enhancement pattern MRI tissue specimens in refractory Bev.

All angiogenic factors examined in the present study were upregulated in T2-diffuse/circumscribed pattern patients compared with cT1-flare up patients in pair cases of naïve-refractory, indicating that alternative angiogenesis pathways might be therapeutic targets when the non-enhancement pattern of recurrence on MRI occurs in patients with refractory to Bev.

In summary, this study reveals differential activation of salvage angiogenic pathways in surgical specimens, with FGF2, EphA2, and PLGF upregulated in tumor vessels, while ANGPT1 and ANGPT2 were downregulated and upregulated, respectively. Upregulation of angiogenic factors in hypoxic TME may compensate for a reduced blood supply, providing alternative therapeutic targets for recurrent GBM after VEGF-targeted therapy failure.

The study has several limitations. We assessed expression levels of angiogenic factors with focusing on tumor vessels including vascular endothelial cells, but it is difficult to detect expression levels of vascular endothelial cells accurately and strictly. It might be possible that pericytes and macrophage around tumor vessels were also included, consisting of tumor vessels. In addition, the present study was retrospective, limited to paired tissues from the same patients, and restricted to naïve and refractory Bev stages. The rarity of salvage surgery for recurrent GBM after Bev failure, RT, and TMZ makes achieving statistical significance difficult. In addition, paired samples were restricted to patients who underwent surgery for newly diagnosed GBM after preoperative neoadjuvant Bev [3]. Comparing paired samples between effective and refractory Bev stages is necessary to increase the study’s significance. Further studies using a larger number of patients are needed.