Saket Jain
Eric J. Chalif
Manish K. Aghi- Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, United States
Glioblastoma is the most aggressive brain tumor with a median survival ranging from 6.2 to 16.7 months. The complex interactions between the tumor and the cells of tumor microenvironment leads to tumor evolution which ultimately results in treatment failure. Immunotherapy has shown great potential in the treatment of solid tumors but has been less effective in treating glioblastoma. Failure of immunotherapy in glioblastoma has been attributed to low T-cell infiltration in glioblastoma and dysfunction of the T-cells that are present in the glioblastoma microenvironment. Recent advances in single-cell sequencing have increased our understanding of the transcriptional changes in the tumor microenvironment pre and post-treatment. Another treatment modality targeting the tumor microenvironment that has failed in glioblastoma has been anti-angiogenic therapy such as the VEGF neutralizing antibody bevacizumab, which did not improve survival in randomized clinical trials. Interestingly, the immunosuppressed microenvironment and abnormal vasculature of glioblastoma interact in ways that suggest the potential for synergy between these two therapeutic modalities that have failed individually. Abnormal tumor vasculature has been associated with immune evasion and the creation of an immunosuppressive microenvironment, suggesting that inhibiting pro-angiogenic factors like VEGF can increase infiltration of effector immune cells into the tumor microenvironment. Remodeling of the tumor vasculature by inhibiting VEGFR2 has also been shown to improve the efficacy of PDL1 cancer immunotherapy in mouse models of different cancers. In this review, we discuss the recent developments in our understanding of the glioblastoma tumor microenvironment specially the tumor vasculature and its interactions with the immune cells, and opportunities to target these interactions therapeutically. Combining anti-angiogenic and immunotherapy in glioblastoma has the potential to unlock these therapeutic modalities and impact the survival of patients with this devastating cancer.
Introduction
Glioblastoma (GBM) is the most common primary brain malignancy in adults, comprising nearly 50% of all primary central nervous system (CNS) cancer, with an average annual incidence of 3.22 per 100,000 people (1). Despite decades of research that has improved our functional understanding of the molecular and genetic characteristics of GBM, there has been minimal improvement in overall survival, as evidenced by the dismal long-term survival ranging from 6.2 to 16.7 months in patients receiving trimodal therapy (2, 3). Unfortunately, new classes of medications that have revolutionized treatment for cancer outside of the CNS have so far been unsuccessful in clinical trials for GBM. Two classes of drugs that have failed phase III trials in GBM are checkpoint inhibitors and vascular endothelial growth factor (VEGF) inhibitors. These failures occurred despite the fact that targeting the immune system and angiogenesis were particularly promising candidates for the treatment of GBM due to its marked local immunosuppression and propensity for angiogenesis. Interestingly, recent studies have suggested that substantial interactions exist between immunotherapies and anti-angiogenic therapies in many cancers, including GBM. Understanding this interplay may lead to the development of improved and synergistic combinatorial therapies. In this review, we summarize the latest insights researchers have produce on the immunologic and angiogenic components of the GBM microenvironment with a particular emphasis on how immune and anti-angiogenic therapies might interact in GBM. We also review existing agents that are currently undergoing investigation as targeted unimodal or combinatorial therapy in GBM.
The Immune Microenvironment of GBM
The microenvironment of the normal brain is generally immunosuppressive and was formally considered an immune privileged organ before the discovery of lymphatic vasculature lining murine dural sinuses (4). Despite this, the brain remains an immunologically unique organ as peripheral immune cells will only rarely patrol through the functional blood brain barrier (BBB). This BBB is composed of endothelial cells held together by intercellular tight junctions that restrict entry of most immune cells, and those cells that do cross will rapidly exit unless they have recognized a local antigen. As GBM grow beyond 1-2mm in diameter, the BBB becomes compromised allowing for a more robust infiltration of immune cells (5). Despite BBB breakdown and increased immune cell entry, GBM avoids targeting by immune cell through a number of mechanisms including local T-cell exhaustion, low tumor mutation burden, high heterogeneity among tumor cells, as well as release of a variety of soluble factors that lead to low levels of local and circulating immune cells.
CD8+ T cells in GBM are characteristically exhausted as a result of persistent stimulation. Exhaustion represents a unique transcriptomal profile that leads to an up-regulation of inhibitory immune checkpoints that ultimately leads to cell senescence (6). These dysfunctional T cells are classically identified by downregulated CD27/28 and upregulated CD57 and immune checkpoint receptors, which are accompanied molecularly by a decrease in proliferation and cellular metabolism, impaired response to cytokines, and eventual apoptotic death (7–10). One mechanism by which tumor cells provoke T cell death is through expression of Fas-L, which binds to Fas death receptor on T cells and leads to a caspase-mediated apoptotic pathway (11). GBM tumor cells often also express such checkpoint proteins as programmed death ligand 1 (PD-L1) and CTLA-4 for which increased levels of expression predict a worse clinical prognosis (12). Various transcription factors have been implicated in T cell exhaustion such as PBX3, Prdm1, Eomes family, CD122, and others that collectively contribute to changes in phenotype towards loss of effector function (6).
Tumor mutational burden (TMB) is defined as the total number of protein-altering mutations in coding regions of genes. In many cancers, a high TMB is associated with an immune-reactive phenotype and substantial local cytotoxic CD8+ cell population (13). GBM has a lower TMB phenotype than most other solid malignancies, which likely contributes to its poor prognosis due to fewer immunogenic neoantigens to provoke immune response. Unexpectedly, a higher relative TMB among patients with GBM confers a worse prognosis on survival and worse response to immunotherapy, which opposes the trend seen in most other cancers (14–16).
GBM also has profound intratumoral heterogeneity as characterized by intercellular genetic diversity within the tumor. Previously canonical subtypes of GBM (proneural, neural, mesenchymal, classical) have recently been challenged as evidence from single cell sequencing data reveals that these subtypes are all variably expressed within the same GBM sample, which reflects the heterogeneity of different spatial compartments in the tumor with molecular classifications that likely exist on a continuum rather than binary form (17, 18). High intratumoral heterogeneity results in inconsistent molecular targets whereby divergent tumor cells will not respond similarly to certain therapies.
GBM tumor cells release various cytokines that contribute to the immunosuppressive milieu including IL-1/IL-6/IL-10 (suppresses activity of CD8+ and Th cells) (19–21), chemokine CCL22 (attracts CD25+ FoxP3+ regulatory T cells to the tumor niche) (22, 23), and TGF-β (facilitates epithelial−to−mesenchymal transition and impedes transmigration of T cells to the tumor via the downregulation of ICAM expression on the endothelial cell surface) (24–26). In addition to local immunosuppression, there is systemic immune impairment as indicated by decreased levels of circulating T cells and increased proportion of regulatory T cells measured in the peripheral blood (27). Systemic immunosuppression as measured by high neutrophil to lymphocyte ratio (NLR) is a negative prognostic factor on overall survival and progression-free survival (28).
The Vascular Microenvironment of GBM
Extensive angiogenesis is characteristic of glioblastoma and is controlled by a number of converging pathways. Glial stem cells are one of the main driver of angiogenesis. They serve vital functions in providing blood supply and are identified by the fraction of GBM expressing CD133+. One mechanism by which glial stem cells route blood to tumor is through upregulation of genes involved in angiogenesis such as release of vascular endothelial growth factor (VEGF) (29). Endothelial cells, in turn, promote adjacent phenotypic change towards tumor stems cell via NOTCH ligand expression as well as release of nitric oxide to activate notch signaling (30, 31). This results in a positive feedback loop between stem cells and the blood vessel wall, promoting rapid angiogenesis. Additionally, tumor stem cells may miraculously themselves differentiate to endothelium, functionally assisting in the formation of competent microvessels (32, 33). Interestingly, tumor-derived endothelial cells are more prevalent within the core of the glioblastoma than the tumor periphery. This likely speaks to adaptation responses allowing these cells to survive in more stressful conditions than normally derived vasculature. Pericytes have also been reported to derive from the same cell lineage as GBM stem cells (34). GBM has intensive metabolic demands, and there is often local tissue hypoxia due to insufficient oxygen supply. Hypoxia drives expression of tumor stem cells genes such as those involved in the Notch pathway and calcineurin pathway (35). Hypoxia-inducible factor 2 alpha (HIF-2a) is the driver of stem cell change in response to hypoxia, and unlike HIF-a, which promotes apoptosis, HIF-2a promotes resilience in low oxygen conditions (36). HIF-2a also leads to upregulated transcription of VEG-F.
These studies collectively provide intriguing evidence that tumor blood vessels themselves are neoplastic and capable of actively remodeling the perivascular niche.
Vessel co-option is another means by which GBM cells can gain access to oxygen and nutrients. In this process, GBM cells grow towards and then along existing vasculature within the brain. In particular, GBM grow in areas where there is large surface area for tumor to endothelial cell contact, such as between micro vessels that run parallel to each other, among capillary loops, or near dilated capillaries (37). One essential driver of vessel co-option is WNT-7 expression, a pathway promoted within Oligodendrocyte-precursor stem cells (38). An important chemokine for co-option is bradykinin, which is released by endothelial cells and serves as a chemoattract to tumor cells (39).
Current Immunotherapies for GBM
A variety of immunotherapies for have been tested in phase I, II, and III clinical trials. These therapies generally fit into the following categories: targeted molecular inhibitors, vaccine-based therapies, viral therapies, and adoptive T-cell therapies. While no individual or combination of immunotherapy for GBM has so far been successful in phase III testing, a number show promise in certain subgroups of patients and these are currently being further investigated. One difficulty in testing new therapies is the relative few number of patients that present with GBM in comparison to the total number of therapies on trial. To remedy this, many trials have begun to use historical control groups and may combine clinical phase I and II or phase II and III testing in certain cases (40–43).
Targeted Molecular Inhibitors
Immune checkpoint inhibitors (CPI) are the most well studied molecular inhibitors in GBM and they have shown impressive increases in survival for a number of other cancer types (44). These drugs target inhibitory receptors expressed by immune cells or their ligands. The most well-studied CPIs target programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). T-cell immunoglobulin and mucin domain 3 (Tim-3) and Lymphocyte-activation gene 3 (LAG-3) are more recently investigated CPIs that have shown to be co-expressed with classic CPIs and show promise as additional targets in clinical testing (45–50).
The failure so far for CPI to promote survival in GBM, despite its efficacy in other cancer types, is likely a combination of the many unique facets of the GBM immune landscape as described above (51). Briefly, it is a heterogenous tumor with low tumor mutational burden and persistently exhausted tumor-associated lymphocytes. As such, an incomplete immune response is mounted with subsequent selection of tumor cells best able to respond to CPI. There also exists an intricate balance between pro and antitumoral immune regulation, whereby targeting one immune checkpoint receptor results in immediate recalibration of other signaling pathways to re-center the balance and prevent immune overactivity. To overcome varied methods of resistance, CPI in combination with other targeted molecular inhibitors holds future promise as these therapies may work synergistically to target select pathways that GBM utilizes to overcome CPI monotherapy.
A tryptophan metabolic enzyme, IDO is also considered a contributing factor for immune resistance in GBM through tryptophan metabolism. A recent study has shown that IDO can have shown that IDO can suppress immune response by inducing the expression of compliment factor H (CFH) independent of tryptophan metabolism and could act as a potential target for therapy (52).
Interactions of the tumor cells with the cells of the tumor microenvironment has led to the discovery of novel targets for therapies. A recent preclinical study showed that targeting tumor associated macrophages (TAMs) using a colony stimulating factor 1 receptor (CSF-1R) combined with radiation results in increased survival of mice (53). A recent study from our lab identified cancer associated fibroblasts (CAFs) in GBM and showed that pro-tumoral effects of CAFs are mediated through osteopontin and HGF pathway in GBM (54). STAT3, a member of STAT family of transcription factors has been shown to have an important role in regulating the GBM tumor microenvironment and is considered a promising target (55).
Vaccine-Based Therapies
Peptide, dendritic cell, and heat shock protein vaccines are the primary vaccine types in GBM treatment. Peptide vaccines consist of the direct inoculation of tumor associated antigens (TAA). These peptides can be extracted from patient tumor tissue, or from synthetic production of canonical GBM epitopes. Commonly targeted GBM TAAs are epidermal growth factor receptor variant III (EGFRvIII), gp100, survivin, TRP-2, AIM-2, MAGE-1 (56). One of the difficulties of peptide vaccines is that individual use is restricted to certain HLA haplotypes, which limits generalizability of these agents, and creates a substantial hurdle to their testing in clinical trials (57).
Among the challenges of vaccine therapy are the heterogeneity of tumor cell populations that may not all hold the same mutations. As such, there will be a selection towards those cells that do not express selected TAA. Furthermore, there is a large population of MHC I−absent GBM tumor cells that will not respond to vaccine approaches because they do not present antigens. Lastly, the local GBM landscape is T cell depleted, so developing therapies to enhance T cell infiltration to tumor will be necessary in combinatorial approaches to augment the efficacy of vaccine approaches. In general, because of their minimal toxicity, there is little risk in adding vaccines on top of other chemotherapeutic or targeted modalities.
Viral Therapies
Viral based therapies tackle the immunosuppressive GBM microenvironment through direct oncolysis and delivery of therapeutic payload or gene therapy to the tumor. These are generally delivered intratumorally or postoperatively into the resection cavity, and specifically target GBM due to its high metabolic activity and rapid cell cycle progression in comparison to surrounding brain parenchyma. This approach has the unique benefit that the viral vector itself will often stimulate an immune response that an immunostimulatory transgene within the virus can further potentiate. Subsequent tumor cell death in presence of activated immune cells will theoretically allow tumor cells antigens to be processed and subsequently targeted by immune cells. A variety of virus types have reached clinical trials including retrovirus, adenovirus, lentivirus, herpes simplex virus, and reovirus, parvovirus, measles virus, poliovirus, and others (58, 59). The few viral therapies that have reached phase 3 trials have failed to demonstrate positive effect on overall survival (60–63). Additionally, the most thoroughly investigated therapies delivered the suicide genes thymidine kinase or cytosine deaminase (toca 511) rather than agents that may more directly modulate the local immune landscape. There are promising agents on the horizon in preclinical, phase 1, and phase 2 studies that directly deliver immunomodulatory agents such as ad-RTS-hil-12, interferon beta, VB111 (discussed below), tesurpaterev (64), RLI, and others (59, 62, 65).
Adoptive T-Cell Therapies
Adoptive T cell therapy (ATC) is a setup by which autologous T cells are extracted from patient, expanded in vitro, and subsequently returned to patient in larger numbers. More recently, there have been efforts to genetically modify extracted T cells to express specific antigen or tumor receptors. Clinical trials for ATC lag behind other approaches and none have yet reached phase III testing.
Current Anti-Angiogenic Therapies for GBM
Most anti-angiogenic therapies target ligands, their receptors, or downstream signaling pathways that are implicated in vessel growth. The primary driver of angiogenesis in GBM is VEGF-A, which is secreted by tumor cells and binds to receptor VEGFR-2 on the endothelial cell surface, resulting in the activation of PI3K–Akt and MAP kinase pathways that promote endothelial cell proliferation and survival. Weaker proangiogenic growth factors are platelet derived growth factor (PDGF) that binds to PDGFRa/b and fetal growth factor (FGF) that binds to FGFR, as well as cell surface targets such as Notch and αvβ3/αvβ5 integrins. Targeting the above proteins or their implicated intracellular signaling proteins has been an active area of investigation.
Antibody Therapies
Bevacizumab, a monoclonal antibody that targets free VEGF-A, is the only targeted therapy that has received FDA approval for GBM. It was originally granted accelerated approval in 2009 for recurrent GBM based on the success in prolonging patient survival in two phase II clinical trials (66, 67). Thereafter, bevacizumab had disappointing results for newly diagnosed GBM in randomized phase III clinical trials AVAglio and RTOG 0825, in which there was no improvement in overall survival (68, 69). Despite this, FDA converted bevacizumab to full approval for recurrent GBM due to a reduction in disease progression based on findings from another phase III study, EORTC 26101 (70). Bevacizumab’s failure to improve OS despite its prolongation of progression free survival is likely due to impressive improvements in imaging that are merely artifact changes in blood flow (via rapid reduction in vessel permeability and contrast extravasation) rather than true treatment effect on tumor biology. However, bevacizumab has been shown to result in reduction in the use of corticosteroids to treat brain edema. There are many other antibody therapies that are being investigated, including those targeting other growth factors (HGH, PDGF, PGF, etc), their receptors (VEGFR-2, EGFR, PDGFR, etc), as well as decoy receptors (VEGF-trap), but none so far have had successful phase three trials.
Resistant Mechanisms to Anti-Angiogenic Therapies
Mechanisms of resistance to antibody therapy are manifold and include converging adaptive and intrinsic mechanisms centered on upregulation of alternative or redundant angiogenic pathways, protection of tumor vasculature by recruiting proangiogenic cells such as pericytes, increased invasiveness of tumor cells that further co-opt normal brain vasculature, increased metastatic seeding, selection and propagation of those tumor subpopulations that avoid inhibition, and myeloid cells that release alternative proangiogenic factors (71, 72). Furthermore, one study found that about 20% of primary GBM do not express VEGFA and as such would likely not at all respond to anti-VEGF treatment (73). Microarray and single-cell sequencing of bevacizumab-resistant patient glioblastoma specimens demonstrates upregulated mesenchymal genes, particularly β1 integrin glycoprotein, receptor tyrosine kinase c-Met, YKL-40, and transcription factor ZEB1 (74–76). Glucose transporter 3 (GLUT3) also appears to play a vital role in antiangiogenic therapy resistance, and inhibiting this protein resulted in cell death in bevacizumab-resistant GBM cells (77). To combat rapid resistance, a number of other targets have been developed including tyrosine kinase inhibitors, signal pathway inhibitors, and novel targeted therapies that can be used singly or in combination to target vasculature in a multifaceted approach. These have begun to be employed in combination with bevacizumab to target tumor invasion and angiogenesis (78–81).
Tyrosine Kinase Inhibitors
Small molecule inhibitors are an alternate way to target growth factor ligands and their receptors. Unlike the fine selection that antibody therapy has on its target, small molecule inhibitors of tyrosine kinase will variably target several tyrosine kinase receptors that together impact vessel growth. For instance, the best studied tyrosine kinase inhibitor cediranib targets VEGFR-1/2/3, PDGFR- α/β, FGFR-1, EGFR, as well as the stem cell factor c-kit receptor (82–84). However, cediranib has failed phase III clinical testing in prolonging progression free survival in patients with recurrent GBM (84). One of the particular difficulties of small molecule inhibitors in the treatment of GBM is the relatively impermeable BBB that heavily restricts delivery of these molecules to the tumor. It has been demonstrated that many tyrosine kinase inhibitors are indeed substrates of P-glycoproteins and other resistance proteins that are highly expressed on capillary endothelial cells and are involved in active efflux of drugs out of the CNS. Additionally, while small molecule inhibitors often inhibit multiple types of tyrosine kinase, in general they are insufficient to block all receptor signaling, and as a result GBM may simply respond by upregulating or activating these same tyrosine kinase receptors (85).
Miscellaneous Agents
A diverse set of other agents have been developed to target vascular growth via unique mechanisms. Some targets for inhibition include signaling pathways that are downstream of tyrosine kinase such as protein kinase C, mTOR, Ras, and others, which have proven successful in multiple other cancer types. For instance, thalidomide is being tested in glioma and it has been shown to inhibit EGF-induced phosphorylation of extracellular signal regulated kinase (ERK), as well as EGF-induced Ras activation by preventing transition to GTP-bound active Ras (86). There are intriguing other agents with mechanisms that function outside of the tyrosine kinase signaling pathway framework. These include cytokines and other soluble factors, extracellular ligands, as well as intracellular cell machinery with diverse and sometimes converging pathways. One such agent is celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, which has been shown to reduce vascularization and subsequently suppress expression of proteins VEGF and HIF-1α (87). In a phase 2 study, however, the combination of thalidomide and celecoxib in addition to standard of care failed to meet the primary endpoint in reducing progression free survival, and also failed to correlate treatment response with a reduction in angiogenic peptides including VEGF (88). There are also hormonal therapies such as 2-methoxyestradiol, an estradiol metabolite, which downregulates HIF-1a at the posttranscriptional level and results in decreased HIF-1α-mediated VEGF expression. Results of a phase 2 trial showed modest anti-tumor effect (89).Another HIF factor HIF-2α has been studied in GBM and is associated with poor patient outcome (90). Recently, FDA approved a HIF-2α inhibitor belzutifan for hemangioblastomas a different central nervous system tumor.
Matrix metalloproteinases are found in tumor cells and are implicated in cell invasion by means of proteolytic degradation of extracellular proteins. There is evidence that these metalloproteinases facilitate the specific invasion associated with vessel cooption (91). In addition, matrix metalloproteinases are able to activate various cytokines, such as TGF-B and VEGF through direct interaction (92). Inhibitors of these metalloproteinases hold promise as anti-angiogenic agents in a variety of cancers, however one such agent marimastat failed phase 2 testing in newly diagnosed GBM patients (93).
Another promising agent is enzastaurin, an inhibitor of protein kinase Cβ (PKC-β). Anti-angiogenic effect of this drug is based around an alternate downstream VEGF signaling pathway that is essential for endothelial proliferation and migration. Inhibition of PKC-β by enzastaurin has been demonstrated to decrease microvascular density and VEGF expression in human tumor xenografts (94). The drug also causes direct cytotoxicity to tumor cells. After results in a phase 2 trial in which a germline polymorphism on chromosome 8 (DGM1) was found post hoc to be associated with a significant increase in response to enzastaurin in newly diagnosed GBM patients, it has been granted fast track approval for phase 3 testing in biomarker positive patients (95, 96).
Integrins are yet another molecular vascular target. These are highly expressed on the endothelial surface and interact with extracellular matrix proteins to promote endothelial cell migration. They also interact and with immunoglobulin superfamily molecules to promote pro-angiogenic macrophage trafficking to tumors (97). However, in phase 3 clinical testing, the addition of cilengitide—a cyclic RGD pentapeptide that selectively inhibits the integrins αvβ3, αvβ5 and α5β1—to temozolomide did not improve outcomes (98). Additionally, combination trials of cilengitide with cediranib, a VEGFR inhibitor, failed to produce good results (99).
Synergy in Immunotherapy and Antiangiogenic Agents in GBM
Despite decades of developing new antiangiogenic agents and immunotherapies, none so far have successfully prolonged overall survival for newly diagnosed or recurrent GBM. However, some show promising results in certain subgroups (e.g. enzastaurin in GBM patients with the DGM1 polymorphism). While these results have been disappointing, there is optimism that combination therapies between agents that target the immune and vascular systems could be more successful. It has been demonstrated that there exists substantial crosstalk between the vascular and immune systems. Understanding how these interactions may potentiate drug effects will likely lead to the development of successful therapies for GBM in the future.
Soluble Factors With Dual Immunologic and Angiogenic Functions
A variety of soluble factors have been demonstrated to influence both the immunologic and angiogenic aspects of the tumor microenvironment. One of these is VEGF, the primary driver of angiogenesis, which is also a potent immunosuppressive factor that promotes tumor growth by modulating the adaptive and innate immune compartments. VEGF affects the ability of CD34+ hemopoietic progenitor cells to differentiate into functional dendritic cells (DC) in an NF-kB signaling-dependent manner, thus contributing to evasion of immune survelience (100, 101). Those DCs that do develop in setting of VEGF have dramatically reduced functional capacity in presenting antigen to allogenic T cells or in stimulating a primary immune response with a presented antigen. Interestingly, VEGF does not affect function of already-mature DCs (102). These findings are corroborated in a report on GBM where VEGF blockade likewise led to more differentiated and less active DCs in the brain (103). VEGF enhances a number of inhibitory checkpoints involved in T cell exhaustion including PD-1, as Tim-3, CTLA-4, and Lag-3 (104). Data from colorectal cancer reveals that VEGF induces the expression of transcription factor TOX in T cells to drive an exhaustion-specific transcription program (105). VEGF also suppresses immune cell trafficking through the downregulation of various cell adhesion molecules including ICAM-1 and VCAM-1 (106, 107). VEGF has been demonstrated to promote the recruitment and proliferation of several immunosuppressive cells, including regulatory T cells and M2-like pro-tumoral macrophages (108, 109). VEGF may also effect systemic immune system, as demonstrated in mice subjected to VEGF infusion have decreased overall quantity of systemic DCs, T-cells, and B-cells as measured in spleen and lymph nodes (102). Other growth factors are also implicated dually in the immune and vascular compartments, such as FGF, which in addition to its potent anti-angiogenic properties, also attracts immunosuppressive immune cells such as myeloid-derived suppressor cells (MDSCs) as demonstrated in breast cancer (110). FGF also promotes M2 polarization (110).
TGF-β is another multifunctional cytokine that is implicated in immune and vascular escape mechanisms in GBM (111). TGF-β signaling stimulates production of VEGF and a number of other pro-angiogenic factors including HIF-1, FGF (112). TGF-β is in a signaling loop with proangiogenic metalloproteinases released by cancer cells, that lead to mutual upregulation and facilitates tumor progression, vessel cooptation, and proangiogenic state (113). Interestingly, TGF-β is also implicated in anti-angiogenic pathways, and it appears that competing mechanisms result in a fine balance in angiogenic signaling which is finely dependent on cell content (114). GBM and other malignancies predominantly exploit the pro-angiogenic signaling pathway (115).
TGF-β exerts strong immunosuppressive pro-tumoral effects on all cells in the immune system. TGF-β1 in particular has been demonstrated to potently block differentiation of immune cells to cytotoxic CD8+ cells or CD4+ cells, and also inhibits their function by suppressing the release of killing enzymes such as granzyme and perforin from CD8+ cells (111). It also directly inhibits MHC class I expression on glioma cells. Because of its broad implications in many pro-tumoral mechanisms, there are a number of inhibitors of TGF-β that are being tested as therapies for GBM (111).
Immune Cells Influencing the Tumor Vasculature
Immune cells may regulate tumor angiogenesis by releasing soluble factors that generally promote vascular genesis. M2 macrophages produce a number of proangiogenic factors including growth factors (VEGF, EGF, FGF, PDGF, TGF-b), CXC/CCL chemokines, and ANGPT2 (116, 117). Likewise, CD8+ T-cells have been shown to upregulate a number of chemokines including CXCL9, CXCL10, and CXCL11, which collectively enhancing pericyte recruitment into the tumor microenvironment (118). In ovarian cancer, tumor-associated plasmacytoid dendritic cells induce angiogenesis in vivo through production of TNF-alpha and IL-8 (119). MDSCs and neutrophils may promote angiogenesis by producing matrix metalloproteinase 9 as well as Bv8, of which both have been demonstrated to promote release of VEGF (116, 120). Bv8 inhibition resulted in reduced tumor vasculature in several solid malignancies (121). MDSCs can also integrate into the vasculature itself, helping to create a stable and proliferative vessel wall (122). Regulatory T cells have been implicated as pro-angiogenic forces, and their depletion in ovarian cancer resulted in robust reduction of the VEGF as measured in tumor microenvironment (123). A number of other immune cells have been reported to release VEGF including several types of natural killer cells and B cells (117).
One intriguing report from patients with recurrent GBM shows that an increase in infiltrating tumor-associated macrophages after bevacizumab is associated with poor survival, which suggests that entry of these macrophages from peripheral blood to tumor may represent an escape mechanism from antiangiogenic therapy (124). Another report reveals that a relative downregulation of macrophage migration inhibitory factor exists in bevacizumab-resistant GBM xenografts compared to bevacizumab-naïve xenografts (125). The apparent difference in these findings likely speaks to the complex interplay between M1 and M2 differentiated phenotypes that are implicated in mechanisms of bevacizumab resistance, likely by a downregulation in total migrating macrophages, but relative proliferative expansion of M2 macrophages to promote tumor growth (125).
Effects of the Abnormal Tumor Vasculature on Immune Cells
The dysfunctional vasculature present in most cancers generally prevents the activation of immune cells. Indeed, a “tumor-endothelial barrier” has been described by which tumor endothelial cells suppress T cells, target them for destruction, and block them from entering the tumor (126). As part of this barrier, tumor endothelial cells will downregulate a variety of integrins and other adhesion molecules necessary for immune cell margination and subsequent extravasation (107). Specifically, endothelin 1 was found to be upregulated in numerous immunosuppressed tumors, and mechanistically blocks T cell adhesion to the endothelium through production of nitric oxide resulting in the suppression of ICAM1 (127). The immunosuppressive mediator IDO expressed in endothelial cells can cause dilation of vessels mediated via nitric oxide in CNS tumors. While tumor vasculature suppresses entry of most immune cells, it has been demonstrated that immunosuppressive cells such as regulatory T cells are better able to migrate through endothelium, though mechanisms by which tumor selectively allows entry are still being investigated (128).
Those pro-inflammatory immune cells that manage to attach to endothelium are immunologically suppressed by a number of ligands on the endothelial surface, including inhibitory checkpoints and a reduction in MHC class I-presenting complexes. Specifically, endothelial cells have been found to express PD-L1 and PD-L2 that retain their function in downregulating CD8+ T cell activation and cytoxicity (129). In GBM, PD-L1 levels positively correlate with VEGF (130). Expression of TIM-3 has also been described to be upregulated in a number of cancer associated endothelium, including lymphoma, where it functioned to inhibit activation of CD4+ T cells and Th1 phenoytype polarization (131). Fas ligand has been demonstrated to be functionally competent on tumor endothelium. In one intriguing study, inhibition of VEGF resulted in tumor growth suppression by CD8+ T cells in manner that was dependent on the attenuation of FasL (132). The tumor endothelium also produces a number of anti-inflammatory cytokines including endothelin-1, FGF, TGF-beta, IL-6, IL-8, PDGF, G-CSF, and others (133).
Individual Therapies That Target Both the Immune and Vascular Compartments of GBM
As is evident from the substantial crosstalk that exists between the immune environment and vasculature, any one targeted therapy will likely be implicated in a variety of mechanisms that have unintended effects on cancer biology. For instance, by blocking VEGF-A, bevacizumab may inhibit VEGF-mediated immune suppression by suppressing regulatory T cells or expression of immune checkpoints. Likewise, immune checkpoint inhibitors may suppress M2 phenotypic change, resulting in a decreased M2-mediated angiogenesis. In a similar manner, some therapeutic agents for GBM have been specifically designed as dual agents to target angiogenesis and activate the immune system. Chief among these is ofranergene obadenovec (VB-111), a replication-deficient adenovirus vector that carries a transgene for a chimeric death receptor composed of TNFα receptor connected to intracellular Fas (62). When TNFα binds to the chimeric receptor, Fas pathway leads to cell quiescence and death. This transgene is restricted however to angiogenic endothelial cells which nearly exclusively have an activated pre-proendothelin 1 (PPE-1)–3x promoter (63). Initiation of this therapy has shown to result in dramatic infiltration of CD8+ T cells in tumor tissue with subsequent cell apoptosis, which likely results dually from pathways downstream of chimeric death receptor in addition to immunogenic viral epitopes that stimulate immune targeting (134). While a phase III trial failed to demonstrate survival benefit of VB-111, the patients enrolled here did not receive a ‘priming’ dose of VB-111 that may prove necessary in synergistic success with bevacizumab, as demonstrated with the good results in prior phase II study that used such a ‘priming’ dose in the study design (135). Another treatment with overlapping effects are Ang-2 inhibitors, which have been developed after GBM treated with bevacizumab were shown to express higher Ang-2 levels (136). Intriguing results from preclinical glioma studies demonstrate the reprogramming of tumor associated macrophages from M2 to M1 phenotype that co-occurs with vessel density reduction during treatment with an Ang-2 inhibitor (136–138).
However, other drugs are being developed that accommodate obviously competing immunologic and vascular pathways in the tumor microenvironment. One such example is ABT-510, a thrombospondin-1 (TSP-1) mimetic drug that competes with TSP-1 and inhibits glioma angiogenesis in vivo (139). The receptor for TSP-1, CD36, is upregulated in antigen presenting cells such as tumor associated macrophages and dendritic cells (140). Targeting this receptor for inhibition may therefore inadvertently augment the immunosuppressive local milieu in GBM.
Combining Immunotherapies With Anti-Angiogenic Therapies
There is good preclinical and clinical evidence in a variety of cancer types demonstrating improved survival when combining immunotherapies with agents that target vasculature. For instance, bevacizumab plus interferon-alpha—an immunostimulatory cytokine— is first line therapy in renal cell carcinoma and has been shown to nearly double progression free survival from 5 months to 9-10 months in two phase III clinical trials, as well as objectively increase overall survival (141, 142). In 2018, the pivotal IMpower150 study demonstrated in non-small cell lung cancer that the addition of the PD-L1 inhibitor atezolizumab to bevacizumab and chemotherapy resulted in a 22% reduction of risk of death and a 38% reduction in disease progression compared to bevacizumab and chemotherapy alone (143). There also exists successful phase III data for atezolizumab in combination with bevacizumab for unresectable hepatocellular carcinoma, which demonstrates a 42% reduction in risk of death and 41% reduction in progression when compared to the tyrosine kinase inhibitor sorafenib (144).
There are also a number of clinical trials that have combined immunotherapy and anti-angiogenic agents in GBM (Table 1). The recent appreciation of the profound effect that bevacizumab has on tumor biology has resulted in many newer clinical trials stratifying patients that have previously failed bevacizumab into a separate treatment arm than bevacizumab-naïve patients. Some trials evaluate the effectiveness of a certain therapy with and without bevacizumab. Despite the promise of combinatorial therapy, clinical trials for GBM are generally conservative in their approach, as evidenced by bevacizumab being the only anti-angiogenic agent that has so far been trialed with immunotherapy. Bevacizumab does have the theoretical advantage in indirectly promoting an immune response through the reduction in use of corticosteroids (149). But there have been no successful phase III trials yet in immune therapy and anti-VEGF combinatorial treatment for GBM. The two best studied combinations are immune checkpoint inhibitors with bevacizumab and vaccine-based therapies with bevacizumab. However, the development of new regimens will be necessary for future success.
Table 1 Clinical trials for glioblastoma with combination immunotherapy and anti-angiogenic therapy.
Checkpoint inhibitors that have been tested with bevacizumab in clinical trials are monoclonal antibody inhibitors of PD-1 or PD-L1, including camrelizumab, bembrolizumab, durvalumab, and nivolumab. Two of these trials have completed phase II testing. In one study, pembrolizumab in combination with bevacizumab was ineffective in prolonging overall survival or progression free survival, and no tumor immune biomarkers that were collected (including tumor PD-L1 expression, tumor-infiltrating lymphocyte density, immune activation gene expression signature, and plasma cytokines) predicted outcomes (148). Interestingly, poor survival correlated with increased baseline dexamethasone use and increased posttherapy plasma VEGF, which should be carefully evaluated as potential markers in future combinatorial studies. In a second study, durvalumab in combination with bevacizumab and radiotherapy showed promise among a subgroup of patients with unmethylated MGMT tumors, however full results have yet to be posted (43). An intriguing study that is currently enrolling patients investigates the effect of retifanlimab, a PD-1 inhibitor, with or without epacadostat, an indoleamine 2,3-dioxygenase (IDO) inhibitor, in combination with bevacizumab and radiation in recurrent glioblastoma (150). IDO is an enzyme that catalyzes the rate limiting step of tryptophan (Trp) catabolism, converting Trp to kynurenine (Kyn). It has been demonstrated that Trp depletion and Kyn accumulation leads to immunosuppression by functional inhibition of CD8+ and NK cells, and functional stimulation regulatory T cells (151). The addition of epacadostat may result in a necessary reduction of the immunosuppressive milieu of GBM that enables efficacy of a PD-1 inhibitor with bevacizumab.
There are a number of vaccine therapies that have undergone clinical testing with bevacizumab, including TAA, HSP, and DC vaccines. Rindopepimut, an EGFRvIII-targeted vaccine that consists of a peptide with homology to EGFRvIII that is conjugated to keyhole limpet hemocyanin, is one promising agent that in combination with bevacizumab has completed phase II testing in patients with relapsed EGFRvIII-expressing GBM (145). Although there was a relatively small sample size of 36 patients in the experimental arm, these patients had an improved overall survival compared to control arm (hazard ratio of 0.53), and 33% of patients were able to discontinue steroids compared to 0% in control arm. Another vaccine in combination with bevacizumab that has reached phase II clinical trial is an autologous HSP, HSPPC-96, generated from patient resected tumors (146). However, the study was terminated after interim analysis surprisingly showed worse overall survival in experimental group compared to the control group, and complete results have not yet been published.
ERC1671 (gliovac) is an intriguing immunotherapy that has been tested in combination with bevacizumab, and it consists of autologous inactivated tumor cells lysate from the patient to be treated, inactivated tumor cells and lysate from three other GBM patients, cyclophosphamide to inhibit local immunosuppression, and GM-CSF as an adjuvant to enhance the immune response (147). Interim results show improved median overall survival of 12 months in ERC1671 plus bevacizumab arm, compared to 7.5 months in bevacizumab alone. Additionally, CD4+ T-lymphocyte counts correlated with overall survival. Full results are pending.
SL-701 is a vaccine therapy with adjuvants GM-CSF and imiquimod that has completed phase 2 testing. This vaccine is comprised of synthetic peptides designed to elicit an immune response against interleukin-13 receptor alpha-2, ephrinA2 and survivin (40). Although an initial report suggested a possible survival tail in refractory GBM patients, full data has not been released.
Table 2 includes the targeted therapies that have been combined with bevacizumab in clinical trials and which involve the selective inhibition of intercellular pathways that are partially implicated in immune signaling. Many of these have unfortunately resulted in disappointing trial results. VB-111’s effect on the immune system is discussed above. One promising agent is abemaciclib, a CDK 4/6 inhibitor, that induces a T-cell inflamed tumor microenvironment, and may also potentiate the effects of bevacizumab through reduction in metabolic invasiveness (154–156).
Table 2 Clinical trials for glioblastoma with combination anti-angiogenic therapy and targeted inhibitors implicated in immunity.
Future Directions
Despite the failure so far in individual immunotherapies and anti-angiogenic therapies in GBM, translational experiments have recently shed new light on the crosstalk between the immune and vascular systems in GBM. One study demonstrated that successful treatment of combined anti-VEGFR2 and anti–PD-L1 in breast cancer and pancreatic cancer was correlated with the induction of high endothelial venules (HEV) that resulted in lymphocyte infiltration through activation of lymphotoxin β receptor (LTβR) signaling (157). While combinatorial therapy with anti-VEGFR2 and anti–PD-L1 showed no induction of HEV in a GBM line, LTβR agonists were then trialed which induced HEVs and enhanced function of CD8+ T cells in GBM (157). This study provides good mechanistic evidence of the utility of combinatory therapy, introduces a new therapeutic target, and underscores that possible biomarkers may exist for treatment response, which will likely lead to the further investigations to those factors that may predispose certain therapies to induce formation of HEV with subsequent lymphocyte infiltration. An intriguing report that utilizes the syngeneic GL261 glioma line demonstrates that anti-VEGF therapy in combination with a picornavirus vaccine (that expresses epitope OVA257–264 to enhance antigen specific CD8+ T-cell) resulted in a synergistic treatment response with prolonged overall survival and delayed disease progression compared to the additive individual effects of these therapies (158). Another intriguing report details a screen for immune mutations in response to anti-VEGF treatment in GL261 and KR158B murine glioma lines that revealed a dose-dependent upregulation of immunosuppressive regulatory T-cell genes in response to anti-VEGF (159). Subsequently, Anti-CD25 to eliminate regulatory T-Cells was injected prior to initiation of anti-VEGF therapy and resulted in improved overall survival compared to either therapy alone (159).
Future challenges include the development of new and rational combinations of treatments, utilization of biomarkers for improved allocation of patients to clinical trials with improved therapeutic monitoring, as well as the broadening of agents that target the vasculature in addition to bevacizumab. While it is likely that some strategies to reduce angiogenesis will also decrease immune cell access, optimal synergistic approaches will generate a robust anti-tumor immune response while simultaneously inhibiting vascular growth.
Conclusion
The GBM immune and vascular landscape is incredibly complex, and it is likely that a ‘magic bullet’ treatment does not exist. Indeed, a more attainable solution is a shift in perspective to view GBM as a chronic disease, in which combinations of therapies are used on multiple fronts to suppress tumor cell invasion, impair delivery of nutrients, and promote an anti-tumor immune response. Combining immunotherapy and antiangiogenic therapy has shown promise in preclinical models and subsets of real-world patients. Further understanding how these agents interact with one another as well as clinical validation of these results will be essential for further progress in GBM treatment.
Author Contributions
SJ and EC gathered the ideas together and wrote the review. MA provided ideas for the subtopics, edited the manuscript, and provided overall supervision for the manuscript. All authors contributed to the article and approved the submitted version.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s Note
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References
1. Ostrom QT, Cioffi G, Gittleman H, Patil N, Waite K, Kruchko C, et al. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2012-2016. Neuro Oncol (2019) 21(S5):V1–V100. doi: 10.1093/neuonc/noz150
2. Stupp R, Taillibert S, Kanner A, Read W, Steinberg DM, Lhermitte B, et al. Effect of Tumor-Treating Fields Plus Maintenance Temozolomide vs Maintenance Temozolomide Alone on Survival in Patients With Glioblastoma a Randomized Clinical Trial. JAMA - J Am Med Assoc (2017) 318(23):2306–16. doi: 10.1001/jama.2017.18718
3. Koshy M, Villano JL, Dolecek TA, Howard A, Mahmood U, Chmura SJ, et al. Improved Survival Time Trends for Glioblastoma Using the SEER 17 Population-Based Registries. J Neurooncol (2012) 107(1):207–12. doi: 10.1007/s11060-011-0738-7
4. Ahn JH, Cho H, Kim JH, Kim SH, Ham JS, Park I, et al. Meningeal Lymphatic Vessels at the Skull Base Drain Cerebrospinal Fluid. Nature (2019) 572(7767):62–6. doi: 10.1038/s41586-019-1419-5
5. Jain RK, Di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in Brain Tumours. Nat Rev Neurosci (2007) 8(8):610–22. doi: 10.1038/nrn2175
6. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular Signature of CD8+ T Cell Exhaustion During Chronic Viral Infection. Immunity (2007) 27(4):670–84. doi: 10.1016/j.immuni.2007.09.006
7. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, et al. Expression of CD57 Defines Replicative Senescence and Antigen-Induced Apoptotic Death of CD8+ T Cells. Blood (2003) 101(7):2711–20. doi: 10.1182/blood-2002-07-2103
8. Dey M, Huff WX, Kwon JH, Henriquez M, Fetcko K. The Evolving Role of CD8+CD28- Immunosenescent T Cells in Cancer Immunology. Int J Mol Sci (2019) 20(11):2810. doi: 10.3390/ijms20112810
9. Carosella ED, Rouas-Freiss N, Tronik-Le Roux D, Moreau P, LeMaoult J. HLA-G. An Immune Checkpoint Molecule. Adv Immunol (2015) 127:33–144. doi: 10.1016/bs.ai.2015.04.001
10. Frimpong A, Kusi KA, Adu-Gyasi D, Amponsah J, Ofori MF, Ndifon W. Phenotypic Evidence of T Cell Exhaustion and Senescence During Symptomatic Plasmodium Falciparum Malaria. Front Immunol (2019) 10:1345(JUN). doi: 10.3389/fimmu.2019.01345
11. Didenko VV, Ngo HN, Minchew C, Baskin DS. Apoptosis of T Lymphocytes Invading Glioblastomas Multiforme: A Possible Tumor Defense Mechanism. J Neurosurg (2002) 96(3):580–4. doi: 10.3171/jns.2002.96.3.0580
12. Hao C, Chen G, Zhao H, Li Y, Chen J, Zhang H, et al. PD-L1 Expression in Glioblastoma, the Clinical and Prognostic Significance: A Systematic Literature Review and Meta-Analysis. Front Oncol (2020) 10:1015. doi: 10.3389/fonc.2020.01015
13. Khasraw M, Walsh KM, Heimberger AB, Ashley DM. What is the Burden of Proof for Tumor Mutational Burden in Gliomas? Neuro Oncol (2021) 23(1):17–22. doi: 10.1093/neuonc/noaa256
14. Touat M, Li YY, Boynton AN, Spurr LF, Iorgulescu JB, Bohrson CL, et al. Mechanisms and Therapeutic Implications of Hypermutation in Gliomas. Nature (2020) 580:517–23. doi: 10.1038/s41586-020-2209-9
15. Wang L, Ge J, Lan Y, Shi Y, Luo Y, Tan Y, et al. Tumor Mutational Burden is Associated With Poor Outcomes in Diffuse Glioma. BMC Cancer (2020) 20(1):1–12. doi: 10.1186/s12885-020-6658-1
16. Riviere P, Goodman AM, Okamura R, Barkauskas DA, Whitchurch TJ, Lee S, et al. High Tumor Mutational Burden Correlates With Longer Survival in Immunotherapy-Naïve Patients With Diverse Cancers. Mol Cancer Ther (2020) 19(10):2139–45. doi: 10.1158/1535-7163.MCT-20-0161
17. Wang Q, Hu B, Hu X, Kim H, Squatrito M, Scarpace L, et al. Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates With Immunological Changes in the Microenvironment. Cancer Cell (2017) 32(1):42–56.e6. doi: 10.1016/j.ccell.2017.06.003
18. Patel AP, Tirosh I, Trombetta JJ, Shalek AK, Gillespie SM, Wakimoto H, et al. Single-Cell RNA-Seq Highlights Intratumoral Heterogeneity in Primary Glioblastoma. Science (80- ) (2014) 344(6190):1396–401. doi: 10.1126/science.1254257
19. Oft M. IL-10: Master Switch From Tumor-Promoting Inflammation to Antitumor Immunity. Cancer Immunol Res (2014) 2(3):194–9. doi: 10.1158/2326-6066.CIR-13-0214
20. Fontana A, Hengartner H, De Tribolet N, Weber E. Glioblastoma Cells Release Interleukin 1 and Factors Inhibiting Interleukin 2-Mediated Effects. J Immunol (1984) 132(4):1837–44.
21. Kumari N, Dwarakanath BS, Das A, Bhatt AN. Role of Interleukin-6 in Cancer Progression and Therapeutic Resistance. Tumor Biol (2016) 37(9):11553–72. doi: 10.1007/s13277-016-5098-7
22. Kang S, Xie J, Ma S, Liao W, Zhang J, Luo R. Targeted Knock Down of CCL22 and CCL17 by siRNA During DC Differentiation and Maturation Affects the Recruitment of T Subsets. Immunobiology (2010) 215(2):153–62. doi: 10.1016/j.imbio.2009.03.001
23. Crane CA, Ahn BJ, Han SJ, Parsa AT. Soluble Factors Secreted by Glioblastoma Cell Lines Facilitate Recruitment, Survival, and Expansion of Regulatory T Cells: Implications for Immunotherapy. Neuro Oncol (2012) 14(5):584–95. doi: 10.1093/neuonc/nos014
24. Wrann M, Bodmer S, De Martin R, Siepl C, Hofer-Warbinek R, Frei K, et al. T Cell Suppressor Factor From Human Glioblastoma Cells is a 12.5-Kd Protein Closely Related to Transforming Growth Factor-β. EMBO J (1987) 6(6):1633–6. doi: 10.1002/j.1460-2075.1987.tb02411.x
25. Lohr J, Ratliff T, Huppertz A, Ge Y, Dictus C, Ahmadi R, et al. Effector T-Cell Infiltration Positively Impacts Survival of Glioblastoma Patients and is Impaired by Tumor-Derived TGF-β. Clin Cancer Res (2011) 17(13):4296–308. doi: 10.1158/1078-0432.CCR-10-2557
26. Bryukhovetskiy I, Shevchenko V. Molecular Mechanisms of the Effect of TGF-β1 on U87 Human Glioblastoma Cells. Oncol Lett (2016) 12(2):1581–90. doi: 10.3892/ol.2016.4756
27. Fecci PE, Mitchell DA, Whitesides JF, Xie W, Friedman AH, Archer GE, et al. Increased Regulatory T-Cell Fraction Amidst a Diminished CD4 Compartment Explains Cellular Immune Defects in Patients With Malignant Glioma. Cancer Res (2006) 66(6):3294–302. doi: 10.1158/0008-5472.CAN-05-3773
28. Han S, Liu Y, Li Q, Li Z, Hou H, Wu A. Pre-Treatment Neutrophil-To-Lymphocyte Ratio is Associated With Neutrophil and T-Cell Infiltration and Predicts Clinical Outcome in Patients With Glioblastoma. BMC Cancer (2015) 15(1):1–10. doi: 10.1186/s12885-015-1629-7
29. Garcia JL, Perez-Caro M, Gomez-Moreta JA, Gonzalez F, Ortiz J, Blanco O, et al. Molecular Analysis of Ex-Vivo CD133+ GBM Cells Revealed a Common Invasive and Angiogenic Profile But Different Proliferative Signatures Among High Grade Gliomas. BMC Cancer (2010) 10. doi: 10.1186/1471-2407-10-454
30. Zhu TS, Costello MA, Talsma CE, Flack CG, Crowley JG, Hamm LL, et al. Endothelial Cells Create a Stem Cell Niche in Glioblastoma by Providing NOTCH Ligands That Nurture Self-Renewal of Cancer Stem-Like Cells. Cancer Res (2011) 71(18):6061–72. doi: 10.1158/0008-5472.CAN-10-4269
31. Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW, Hambardzumyan D, et al. Perivascular Nitric Oxide Activates Notch Signaling and Promotes Stem-Like Character in PDGF-Induced Glioma Cells. Cell Stem Cell (2010) 6(2):141–52. doi: 10.1016/j.stem.2010.01.001
32. Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, et al. Glioblastoma Stem-Like Cells Give Rise to Tumour Endothelium. Nature (2010) 468(7325):829–35. doi: 10.1038/nature09624
33. Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, et al. Tumour Vascularization via Endothelial Differentiation of Glioblastoma Stem-Like Cells. Nature (2010) 468(7325):824–30. doi: 10.1038/nature09557
34. Cheng L, Huang Z, Zhou W, Wu Q, Donnola S, Liu JK, et al. Glioblastoma Stem Cells Generate Vascular Pericytes to Support Vessel Function and Tumor Growth. Cell (2013) 153(1):139–52. doi: 10.1016/j.cell.2013.02.021
35. Seidel S, Garvalov BK, Wirta V, Von Stechow L, Schänzer A, Meletis K, et al. A Hypoxic Niche Regulates Glioblastoma Stem Cells Through Hypoxia Inducible Factor 2α. Brain (2010) 133(4):983–95. doi: 10.1093/brain/awq042
36. Carmeliet P, Dor Y, Herber JM, Fukumura D, Brusselmans K, Dewerchin M, et al. Role of HIF-1α in Hypoxiamediated Apoptosis, Cell Proliferation and Tumour Angiogenesis. Nature (1998) 394(6692):485–90. doi: 10.1038/28867
37. Winkler F, Kienast Y, Fuhrmann M, Von Baumgarten L, Burgold S, Mitteregger G, et al, et al. Imaging Glioma Cell Invasion In Vivo Reveals Mechanisms of Dissemination and Peritumoral Angiogenesis. Glia (2009) 57(12):1306–15. doi: 10.1002/glia.20850
38. Griveau A, Seano G, Shelton SJ, Kupp R, Jahangiri A, Obernier K, et al. A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment. Cancer Cell (2018) 33(5):874–889.e7. doi: 10.1016/j.ccell.2018.03.020
39. Montana V, Sontheimer H. Bradykinin Promotes the Chemotactic Invasion of Primary Brain Tumors. J Neurosci (2011) 31(13):4858–67. doi: 10.1523/JNEUROSCI.3825-10.2011
40. Peereboom DM, Nabors LB, Kumthekar P, Badruddoja MA, Fink KL, Lieberman FS, et al. Phase 2 Trial of SL-701 in Relapsed/Refractory (R/R) Glioblastoma (GBM): Correlation of Immune Response With Longer-Term Survival. J Clin Oncol (2018) 36(15_suppl):2058–8. doi: 10.1200/jco.2018.36.15_suppl.2058
41. Shih KC, Chowdhary SA, Becker KP, Baehring JM, Liggett WH, Burris HA, et al. A Phase II Study of the Combination of BKM120 (Buparlisib) and Bevacizumab in Patients With Relapsed/Refractory Glioblastoma Multiforme (GBM). J Clin Oncol (2015) 33(15_suppl):2065–5. doi: 10.1200/jco.2015.33.15_suppl.2065
42. Butowski N, Lamborn KR, Berger MS, Prados MD, Chang SM. Historical Controls for Phase II Surgically Based Trials Requiring Gross Total Resection of Glioblastoma Multiforme. J Neurooncol (2007) 85(1):87–94. doi: 10.1007/s11060-007-9388-1
43. Reardon DA, Kaley TJ, Dietrich J, Clarke JL, Dunn G, Lim M, et al. Phase II Study to Evaluate Safety and Efficacy of MEDI4736 (Durvalumab) + Radiotherapy in Patients With Newly Diagnosed Unmethylated MGMT Glioblastoma (New Unmeth GBM). J Clin Oncol (2019) 37(15_suppl):2032–2. doi: 10.1200/jco.2019.37.15_suppl.2032
44. Ribas A, Wolchok JD. Cancer Immunotherapy Using Checkpoint Blockade. Science (80- ) (2018) 359(6382):1350–5. doi: 10.1126/science.aar4060
45. Goldberg MV, Drake CG. LAG-3 in Cancer Immunotherapy. In: Current Topics in Microbiology and Immunology, vol. 344. (2010). p. 269–78. New York City: Springer Link. doi: 10.1007/82_2010_114
46. Wolf Y, Anderson AC, Kuchroo VK. TIM3 Comes of Age as an Inhibitory Receptor. Nat Rev Immunol (2020) 20(3):173–85. doi: 10.1038/s41577-019-0224-6
47. Kim JE, Patel MA, Mangraviti A, Kim ES, Theodros D, Velarde E, et al. Combination Therapy With Anti-PD-1, Anti-TIM-3, and Focal Radiation Results in Regression of Murine Gliomas. Clin Cancer Res (2017) 23(1):124–36. doi: 10.1158/1078-0432.CCR-15-1535
48. Harris-Bookman S, Mathios D, Martin AM, Xia Y, Kim E, Xu H, et al. Expression of LAG-3 and Efficacy of Combination Treatment With Anti-LAG-3 and Anti-PD-1 Monoclonal Antibodies in Glioblastoma. Int J Cancer (2018) 143(12):3201–8. doi: 10.1002/ijc.31661
49. Konovalov AN, Pitskhelauri DI. Principles of Treatment of the Pineal Region Tumors. Surg Neurol (2003) 59(4):250–68. doi: 10.1016/S0090-3019(03)00223-4
50. Lim M, Ye X, Piotrowski AF, Desai AS, Ahluwalia MS, Walbert T, et al. Updated Phase I Trial of Anti-LAG-3 or Anti-CD137 Alone and in Combination With Anti-PD-1 in Patients With Recurrent GBM. J Clin Oncol (2019) 37(15_suppl):2017–7. doi: 10.1200/jco.2019.37.15_suppl.2017
51. Reardon DA, Brandes AA, Omuro A, Mulholland P, Lim M, Wick A, et al. Effect of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: The CheckMate 143 Phase 3 Randomized Clinical Trial. JAMA Oncol (2020) 6(7):1003–10. doi: 10.1001/jamaoncol.2020.1024
52. Zhai L, Bell A, Ladomersky E, Lauing KL, Bollu L, Nguyen B, et al. Tumor Cell IDO Enhances Immune Suppression and Decreases Survival Independent of Tryptophan Metabolism in Glioblastoma. Clin Cancer Res (2021) 27:6514–28. doi: 10.1158/1078-0432.ccr-21-1392
53. Akkari L, Bowman RL, Tessier J, Klemm F, Handgraaf SM, de Groot M, et al. Dynamic Changes in Glioma Macrophage Populations After Radiotherapy Reveal CSF-1r Inhibition as a Strategy to Overcome Resistance. Sci Transl Med (2020) 12(552). doi: 10.1126/scitranslmed.aaw7843
54. Jain S, Rick JW, Joshi R, Beniwal A, Spatz J, Chang ACC, et al. Identification of Cancer-Associated Fibroblasts in Glioblastoma and Defining Their Pro-Tumoral Effects. bioRxiv (2021) 2021.05.08.443250. doi: 10.1101/2021.05.08.443250
55. Piperi C, Papavassiliou KA, Papavassiliou AG. Pivotal Role of STAT3 in Shaping Glioblastoma Immune Microenvironment. Cells (2019) 8:1398. doi: 10.3390/cells8111398
56. Kamran N, Calinescu A, Candolfi M, Chandran M, Mineharu Y, Asad AS, et al. Recent Advances and Future of Immunotherapy for Glioblastoma. Expert Opin Biol Ther (2016) 16(10):1245–64. doi: 10.1080/14712598.2016.1212012
57. Zhao L, Zhang M, Cong H. Advances in the Study of HLA-Restricted Epitope Vaccines. Hum Vaccines Immunother (2013) 9(12):2566–77. doi: 10.4161/hv.26088
58. Martikainen M, Essand M. Virus-Based Immunotherapy of Glioblastoma. Cancers (Basel) (2019) 11(2). doi: 10.3390/cancers11020186
59. Haddad AF, Young JS, Aghi MK. Using Viral Vectors to Deliver Local Immunotherapy to Glioblastoma. Neurosurg Focus (2021) 50(2):1–7. doi: 10.3171/2020.11.FOCUS20859
60. Westphal M, Ylä-Herttuala S, Martin J, Warnke P, Menei P, Eckland D, et al. Adenovirus-Mediated Gene Therapy With Sitimagene Ceradenovec Followed by Intravenous Ganciclovir for Patients With Operable High-Grade Glioma (ASPECT): A Randomised, Open-Label, Phase 3 Trial. Lancet Oncol (2013) 14(9):823–33. doi: 10.1016/S1470-2045(13)70274-2
61. Rainov NG. A Phase III Clinical Evaluation of Herpes Simplex Virus Type 1 Thymidine Kinase and Ganciclovir Gene Therapy as an Adjuvant to Surgical Resection and Radiation in Adults With Previously Untreated Glioblastoma Multiforme. Hum Gene Ther (2000) 11(17):2389–401. doi: 10.1089/104303400750038499
62. Cloughesy TF, Brenner A, De Groot JF, Butowski N, Zach L, Campian JL, et al. A Randomized Controlled Phase III Study of VB-111 Combined With Bevacizumab vs Bevacizumab Monotherapy in Patients With Recurrent Glioblastoma (GLOBE). Neuro Oncol (2020) 22(5):705–17. doi: 10.1093/neuonc/noz232
63. Cloughesy TF, Petrecca K, Walbert T, Butowski N, Salacz M, Perry J, et al. Effect of Vocimagene Amiretrorepvec in Combination With Flucytosine vs Standard of Care on Survival Following Tumor Resection in Patients With Recurrent High-Grade Glioma: A Randomized Clinical Trial. JAMA Oncol (2020) 6(12):1939–46. doi: 10.1001/jamaoncol.2020.3161
64. Zeng J, Li X, Sander M, Zhang H, Yan G, Lin Y. Oncolytic Viro-Immunotherapy: An Emerging Option in the Treatment of Gliomas. Front Immunol (2021). doi: 10.3389/fimmu.2021.721830
65. Chiocca EA, Lukas R, Yu J, Oberheim Bush NA, Buck J, Demars N, et al. ATIM-15. A Phase 1 Study of Ad-RTS-hIL-12 + Veledimex in Adults With Recurrent Glioblastoma: Dose Determination With Updated Overall Survival. J Clin Oncol (2017) 35(15):2044. doi: 10.1200/JCO.2017.35.15_suppl.2044
66. Friedman HS, Prados MD, Wen PY, Mikkelsen T, Schiff D, Abrey LE, et al. Bevacizumab Alone and in Combination With Irinotecan in Recurrent Glioblastoma. J Clin Oncol (2009) 27(28):4733–40. doi: 10.1200/JCO.2008.19.8721
67. Kreisl TN, Kim L, Moore K, Duic P, Royce C, Stroud I, et al. Phase II Trial of Single-Agent Bevacizumab Followed by Bevacizumab Plus Irinotecan at Tumor Progression in Recurrent Glioblastoma. J Clin Oncol (2009) 27(5):740–5. doi: 10.1200/JCO.2008.16.3055
68. Chinot OL, Wick W, Mason W, Henriksson R, Saran F, Nishikawa R, et al. Bevacizumab Plus Radiotherapy–Temozolomide for Newly Diagnosed Glioblastoma. N Engl J Med (2014) 370(8):709–22. doi: 10.1056/nejmoa1308345
69. Gilbert MR, Dignam JJ, Armstrong TS, Wefel JS, Blumenthal DT, Vogelbaum MA, et al. A Randomized Trial of Bevacizumab for Newly Diagnosed Glioblastoma. N Engl J Med (2014) 370(8):699–708. doi: 10.1056/nejmoa1308573
70. Wick W, Gorlia T, Bendszus M, Taphoorn M, Sahm F, Harting I, et al. Lomustine and Bevacizumab in Progressive Glioblastoma. N Engl J Med (2017) 377(20):1954–63. doi: 10.1056/nejmoa1707358
71. Bergers G, Hanahan D. Modes of Resistance to Anti-Angiogenic Therapy. Nat Rev Cancer (2008) 8(8):592–603. doi: 10.1038/nrc2442
72. Blank A, Kremenetskaia I, Urbantat RM, Acker G, Turkowski K, Radke J, et al. Microglia/macrophages Express Alternative Proangiogenic Factors Depending on Granulocyte Content in Human Glioblastoma. J Pathol (2021) 253(2):160–73. doi: 10.1002/path.5569
73. García-Romero N, García-Romero N, Palacín-Aliana I, Palacín-Aliana I, Madurga R, Madurga R, et al. Bevacizumab Dose Adjustment to Improve Clinical Outcomes of Glioblastoma. BMC Med (2020) 18(1):1–16. doi: 10.1186/s12916-020-01610-0
74. Chandra A, Jahangiri A, Chen W, Nguyen AT, Yagnik G, Pereira MP, et al. Clonal ZEB1-Driven Mesenchymal Transition Promotes Targetable Oncologic Antiangiogenic Therapy Resistance. Cancer Res (2020) 80(7):1498–511. doi: 10.1158/0008-5472.CAN-19-1305
75. Carbonell WS, Delay M, Jahangiri A, Park CC, Aghi MK. β1 Integrin Targeting Potentiates Antiangiogenic Therapy and Inhibits the Growth of Bevacizumab-Resistant Glioblastoma. Cancer Res (2013) 73(10):3145–54. doi: 10.1158/0008-5472.CAN-13-0011
76. Jahangiri A, De Lay M, Miller LM, Shawn Carbonell W, Hu YL, Lu K, et al. Gene Expression Profile Identifies Tyrosine Kinase C-Met as a Targetable Mediator of Antiangiogenic Therapy Resistance. Clin Cancer Res (2013) 19(7):1773–83. doi: 10.1158/1078-0432.CCR-12-1281
77. Kuang R, Jahangiri A, Mascharak S, Nguyen A, Chandra A, Flanigan PM, et al. GLUT3 Upregulation Promotes Metabolic Reprogramming Associated With Antiangiogenic Therapy Resistance. JCI Insight (2017) 2(2). doi: 10.1172/jci.insight.88815
78. Lee EQ, Zhang P, Wen PY, Gerstner ER, Reardon DA, Aldape KD, et al. NRG/RTOG 1122: A Phase 2, Double-Blinded, Placebo-Controlled Study of Bevacizumab With and Without Trebananib in Patients With Recurrent Glioblastoma or Gliosarcoma. Cancer (2020) 126(12):2821–8. doi: 10.1002/cncr.32811
79. Affronti ML, Jackman JG, McSherry F, Herndon JE, Massey EC, Lipp E, et al. Phase II Study to Evaluate the Efficacy and Safety of Rilotumumab and Bevacizumab in Subjects With Recurrent Malignant Glioma. Oncologist (2018) 23(8):889. doi: 10.1634/theoncologist.2018-0149
80. Galanis E, Anderson SK, Butowski NA, Hormigo A, Schiff D, Tran DD, et al. NCCTG N1174: Phase I/comparative Randomized Phase (Ph) II Trial of TRC105 Plus Bevacizumab Versus Bevacizumab in Recurrent Glioblastoma (GBM) (Alliance). J Clin Oncol (2017) 35(15_suppl):2023–3. doi: 10.1200/jco.2017.35.15_suppl.2023
81. Pan E, Supko JG, Kaley TJ, Butowski NA, Cloughesy T, Jung J, et al. Phase I Study of RO4929097 With Bevacizumab in Patients With Recurrent Malignant Glioma. J Neurooncol (2016) 130(3):571–9. doi: 10.1007/s11060-016-2263-1
82. Wedge SR, Kendrew J, Hennequin LF, Valentine PJ, Barry ST, Brave SR, et al. AZD2171: A Highly Potent, Orally Bioavailable, Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor for the Treatment of Cancer. Cancer Res (2005) 65(10):4389–400. doi: 10.1158/0008-5472.CAN-04-4409
83. Batchelor TT, Sorensen AG, di Tomaso E, Zhang WT, Duda DGG, Cohen KS, et al. AZD2171, a Pan-VEGF Receptor Tyrosine Kinase Inhibitor, Normalizes Tumor Vasculature and Alleviates Edema in Glioblastoma Patients. Cancer Cell (2007) 11(1):83–95. doi: 10.1016/j.ccr.2006.11.021
84. Batchelor TT, Mulholland P, Neyns B, Nabors LB, Campone M, Wick A, et al. Phase III Randomized Trial Comparing the Efficacy of Cediranib as Monotherapy, and in Combination With Lomustine, Versus Lomustine Alone in Patients With Recurrent Glioblastoma. J Clin Oncol (2013) 31(26):3212–8. doi: 10.1200/JCO.2012.47.2464
85. Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH, Wiedemeyer R, et al. Coactivation of Receptor Tyrosine Kinases Affects the Response of Tumor Cells to Targeted Therapies. Science (80- ) (2007) 318(5848):287–90. doi: 10.1126/science.1142946
86. Noman ASM, Koide N, Khuda IIE, Dagvadorj J, Tumurkhuu G, Naiki Y, et al. Thalidomide Inhibits Epidermal Growth Factor-Induced Cell Growth in Mouse and Human Monocytic Leukemia Cells via Ras Inactivation. Biochem Biophys Res Commun (2008) 374(4):683–7. doi: 10.1016/j.bbrc.2008.07.090
87. Sun YZ, Cai N, Liu NN. Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1α and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells. Cell Physiol Biochem (2017) 44(4):1640–50. doi: 10.1159/000485764
88. Kesari S, Schiff D, Henson JW, Muzikansky A, Gigas DC, Doherty L, et al. Phase II Study of Temozolomide, Thalidomide, and Celecoxib for Newly Diagnosed Glioblastoma in Adults. Neuro Oncol (2008) 10(3):300–8. doi: 10.1215/15228517-2008-005
89. Kirkpatrick J, Desjardins A, Quinn J, Rich J, Vredenburgh J, Sathornsumetee S, et al. Phase II Open-Label, Safety, Pharmacokinetic and Efficacy Study of 2-Methoxyestradiol Nanocrystal Colloidal Dispersion Administered Orally to Patients With Recurrent Glioblastoma Multiforme. J Clin Oncol (2007) 25(18_suppl):2065–5. doi: 10.1200/jco.2007.25.18_suppl.2065
90. Renfrow JJ, Soike MH, West JL, Ramkissoon SH, Metheny-Barlow L, Mott RT, et al. Attenuating Hypoxia Driven Malignant Behavior in Glioblastoma With a Novel Hypoxia-Inducible Factor 2 Alpha Inhibitor. Sci Rep (2020) 10:15195. doi: 10.1038/s41598-020-72290-2
91. Rao JS, Sawaya R, Gokaslan ZL, Yung WKA, Goldstein GW, Laterra J. Modulation of Serine Proteinases and Metalloproteinases During Morphogenic Glial-Endothelial Interactions. J Neurochem (1996) 66(4):1657–64. doi: 10.1046/j.1471-4159.1996.66041657.x
92. Wick W, Platten M, Weller M. Glioma Cell Invasion: Regulation of Metalloproteinase Activity by TGF-β. J Neurooncol (2001) 53(2):177–85. doi: 10.1023/A:1012209518843
93. Levin VA, Phuphanich S, Yung WKA, Forsyth PA, Del Maestro R, Perry JR, et al. Randomized, Double-Blind, Placebo-Controlled Trial of Marimastat in Glioblastoma Multiforme Patients Following Surgery and Irradiation. J Neurooncol (2006) 78(3):295–302. doi: 10.1007/s11060-005-9098-5
94. Kreisl TN, Kotliarova S, Butman JA, Albert PS, Kim L, Musib L, et al. A Phase I/II Trial of Enzastaurin in Patients With Recurrent High-Grade Gliomas. Neuro Oncol (2010) 12(2):181–9. doi: 10.1093/neuonc/nop042
95. Butowski N, Chang SM, Lamborn KR, Polley MY, Pieper R, Costello JF, et al. Phase II and Pharmacogenomics Study of Enzastaurin Plus Temozolomide During and Following Radiation Therapy in Patients With Newly Diagnosed Glioblastoma Multiforme and Gliosarcoma. Neuro Oncol (2011) 13(12):1331–8. doi: 10.1093/neuonc/nor130
96. Butowski NA, Shazer RL, Sun H, Han I, Jivani MA, Luo W. DGM1 may Serve as a Novel Genetic Biomarker of Response to Enzastaurin in Glioblastoma. J Clin Oncol (2019) 37(15_suppl):2023–3. doi: 10.1200/jco.2019.37.15_suppl.2023
97. Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in Angiogenesis and Lymphangiogenesis. Nat Rev Cancer (2008) 8(8):604–17. doi: 10.1038/nrc2353
98. Stupp R, Hegi ME, Gorlia T, Erridge SC, Perry J, Hong YK, et al. Cilengitide Combined With Standard Treatment for Patients With Newly Diagnosed Glioblastoma With Methylated MGMT Promoter (CENTRIC EORTC 26071-22072 Study): A Multicentre, Randomised, Open-Label, Phase 3 Trial. Lancet Oncol (2014) 15(10):1100–8. doi: 10.1016/S1470-2045(14)70379-1
99. Gerstner ER, Ye X, Duda DG, Levine MA, Mikkelsen T, Kaley TJ, et al. A Phase I Study of Cediranib in Combination With Cilengitide in Patients With Recurrent Glioblastoma. Neuro Oncol (2015) 17(10):1386–92. doi: 10.1093/neuonc/nov085
100. Ohm JE, Carborne DP. VEGF as a Mediator of Tumor-Associated Immunodeficiency. Immunol Res (2001) 23(2-3):263–72. doi: 10.1385/IR:23:2-3:263
101. Dikov MM, Ohm JE, Ray N, Tchekneva EE, Burlison J, Moghanaki D, et al. Differential Roles of Vascular Endothelial Growth Factor Receptors 1 and 2 in Dendritic Cell Differentiation. J Immunol (2005) 174(1):215–22. doi: 10.4049/jimmunol.174.1.215
102. Gabrilovich D, Ishida T, Oyama T, Ran S, Kravtsov V, Nadaf S, et al. Vascular Endothelial Growth Factor Inhibits the Development of Dendritic Cells and Dramatically Affects the Differentiation of Multiple Hematopoietic Lineages In Vivo. Blood (1998) 92(11):4150–66. doi: 10.1182/blood.v92.11.4150
103. Malo CS, Khadka RH, Ayasoufi K, Jin F, AbouChehade JE, Hansen MJ, et al. Immunomodulation Mediated by Anti-Angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma. Front Oncol (2018) 8:320(AUG). doi: 10.3389/fonc.2018.00320
104. Voron T, Colussi O, Marcheteau E, Pernot S, Nizard M, Pointet AL, et al. VEGF-A Modulates Expression of Inhibitory Checkpoints on CD8++ T Cells in Tumors. J Exp Med (2015) 212(2):139–48. doi: 10.1084/jem.20140559
105. Kim CG, Jang M, Kim Y, Leem G, Kim KH, Lee H, et al. VEGF-A Drives TOX-Dependent T Cell Exhaustion in Anti-PD-1-Resistant Microsatellite Stable Colorectal Cancers. Sci Immunol (2019) 4(41):555. doi: 10.1126/sciimmunol.aay0555
106. Campesato LF, Merghoub T. Antiangiogenic Therapy and Immune Checkpoint Blockade Go Hand in Hand. Ann Transl Med (2017) 5(24). doi: 10.21037/atm.2017.10.12
107. Griffioen AW, Damen CA, Blijham GH, Groenewegen G. Tumor Angiogenesis is Accompanied by a Decreased Inflammatory Response of Tumor-Associated Endothelium. Blood (1996) 88(2):667–73. doi: 10.1182/blood.v88.2.667.bloodjournal882667
108. Fukumura D, Kloepper J, Amoozgar Z, Duda DG, Jain RK. Enhancing Cancer Immunotherapy Using Antiangiogenics: Opportunities and Challenges. Nat Rev Clin Oncol (2018) 15(5):325–40. doi: 10.1038/nrclinonc.2018.29
109. Lai YS, Wahyuningtyas R, Aui SP, Chang KT. Autocrine VEGF Signalling on M2 Macrophages Regulates PD-L1 Expression for Immunomodulation of T Cells. J Cell Mol Med (2019) 23(2):1257–67. doi: 10.1111/jcmm.14027
110. Chen W, Shen L, Jiang J, Zhang L, Zhang Z, Pan J, et al. Antiangiogenic Therapy Reverses the Immunosuppressive Breast Cancer Microenvironment. Biomark Res (2021) 9(1):1–16. doi: 10.1186/s40364-021-00312-w
111. Kaminska B, Kocyk M, Kijewska M. TGF Beta Signaling and its Role in Glioma Pathogenesis. Adv Exp Med Biol (2013) 986:171–87. doi: 10.1007/978-94-007-4719-7_9
112. Zagzag D, Salnikow K, Chiriboga L, Yee H, Lan L, Ali MA, et al. Downregulation of Major Histocompatibility Complex Antigens in Invading Glioma Cells: Stealth Invasion of the Brain. Lab Investig (2005) 85(3):328–41. doi: 10.1038/labinvest.3700233
113. Krstic J, Santibanez JF. Transforming Growth Factor-Beta and Matrix Metalloproteinases: Functional Interactions in Tumor Stroma-Infiltrating Myeloid Cells. Sci World J (2014) 2014:2014. doi: 10.1155/2014/521754
114. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, Ten Dijke P. Balancing the Activation State of the Endothelium via Two Distinct TGF-β Type I Receptors. EMBO J (2002) 21(7):1743–53. doi: 10.1093/emboj/21.7.1743
115. Seystahl K, Papachristodoulou A, Burghardt I, Schneider H, Hasenbach K, Janicot M, et al. Biological Role and Therapeutic Targeting of TGF-B3 in Glioblastoma. Mol Cancer Ther (2017) 16(6):1177–86. doi: 10.1158/1535-7163.MCT-16-0465
116. Lee WS, Yang H, Chon HJ, Kim C. Combination of Anti-Angiogenic Therapy and Immune Checkpoint Blockade Normalizes Vascular-Immune Crosstalk to Potentiate Cancer Immunity. Exp Mol Med (2020) 52(9):1475–85. doi: 10.1038/s12276-020-00500-y
117. Motz GT, Coukos G. The Parallel Lives of Angiogenesis and Immunosuppression: Cancer and Other Tales. Nat Rev Immunol (2011) 11(10):702–11. doi: 10.1038/nri3064
118. Bromley SK, Mempel TR, Luster AD. Orchestrating the Orchestrators: Chemokines in Control of T Cell Traffic. Nat Immunol (2008) 9(9):970–80. doi: 10.1038/ni.f.213
119. Curiel TJ, Cheng P, Mottram P, Alvarez X, Moons L, Evdemon-Hogan M, et al. Dendritic Cell Subsets Differentially Regulate Angiogenesis in Human Ovarian Cancer. Cancer Res (2004) 64(16):5535–8. doi: 10.1158/0008-5472.CAN-04-1272
120. Bruno A, Pagani A, Pulze L, Albini A, Dallaglio K, Noonan MDM, et al. Orchestration of Angiogenesis by Immune Cells. Front Oncol (2014) 4 JUL:131. doi: 10.3389/fonc.2014.00131
121. Shojaei F, Wu X, Zhong C, Yu L, Liang XH, Yao J, et al. Bv8 Regulates Myeloid-Cell-Dependent Tumour Angiogenesis. Nature (2007) 450(7171):825–31. doi: 10.1038/nature06348
122. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The Role of Myeloid Cells in the Promotion of Tumour Angiogenesis. Nat Rev Cancer (2008) 8(8):618–31. doi: 10.1038/nrc2444
123. Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang LP, et al. Tumour Hypoxia Promotes Tolerance and Angiogenesis via CCL28 and T Reg Cells. Nature (2011) 475(7355):226–30. doi: 10.1038/nature10169
124. Lu-Emerson C, Snuderl M, Kirkpatrick ND, Goveia J, Davidson C, Huang Y, et al. Increase in Tumor-Associated Macrophages After Antiangiogenic Therapy is Associated With Poor Survival Among Patients With Recurrent Glioblastoma. Neuro Oncol (2013) 15(8):1079–87. doi: 10.1093/neuonc/not082
125. Castro BA, Flanigan P, Jahangiri A, Hoffman D, Chen W, Kuang R, et al. Macrophage Migration Inhibitory Factor Downregulation: A Novel Mechanism of Resistance to Anti-Angiogenic Therapy. Oncogene (2017) 36(26):3749–59. doi: 10.1038/onc.2017.1
126. Lanitis E, Irving M, Coukos G. Targeting the Tumor Vasculature to Enhance T Cell Activity. Curr Opin Immunol (2015) 33:55–63. doi: 10.1016/j.coi.2015.01.011
127. Buckanovich RJ, Facciabene A, Kim S, Benencia F, Sasaroli D, Balint K, et al. Endothelin B Receptor Mediates the Endothelial Barrier to T Cell Homing to Tumors and Disables Immune Therapy. Nat Med (2008) 14(1):28–36. doi: 10.1038/nm1699
128. Nummer D., ES-P-J of the Role of Tumor Endothelium in CD4+CD25+ Regulatory T Cell Infiltration of Human Pancreatic Carcinoma. (2007) Oxford: Oxford University press, academic.oup.com.
129. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, Chernova T, et al. Endothelial Expression of PD-L1 and PD-L2 Down-Regulates CD8+ T Cell Activation and Cytolysis. Eur J Immunol (2003) 33(11):3117–26. doi: 10.1002/eji.200324270
130. Xue S, Hu M, Li P, Ma J, Xie L, Teng F, et al. Relationship Between Expression of PD-L1 and Tumor Angiogenesis, Proliferation, and Invasion in Glioma. Oncotarget (2017) 8(30):49702–12. doi: 10.18632/oncotarget.17922
131. Huang X, Bai X, Cao Y, Wu J, Huang M, Tang D, et al. Lymphoma Endothelium Preferentially Expresses Tim-3 and Facilitates the Progression of Lymphoma by Mediating Immune Evasion. J Exp Med (2010) 207(3):505–20. doi: 10.1084/jem.20090397
132. Motz GT, Santoro SP, Wang LP, Garrabrant T, Lastra RR, Hagemann IS, et al. Tumor Endothelium FasL Establishes a Selective Immune Barrier Promoting Tolerance in Tumors. Nat Med (2014) 20(6):607–15. doi: 10.1038/nm.3541
133. Pirtskhalaishvili G, Nelson JB. Endothelium-Derived Factors as Paracrine Mediators of Prostate Cancer Progression. Prostate (2000) 44(1):77–87. doi: 10.1002/1097-0045(20000615)44:1<77::AID-PROS10>3.0.CO;2-G
134. Arend RC, Beer HM, Cohen YC, Berlin S, Birrer MJ, Campos SM, et al. Ofranergene Obadenovec (VB-111) in Platinum-Resistant Ovarian Cancer; Favorable Response Rates in a Phase I/II Study are Associated With an Immunotherapeutic Effect. Gynecol Oncol (2020) 157(3):578–84. doi: 10.1016/j.ygyno.2020.02.034
135. Cloughesy TF, Brenner A, De Groot JF, Butowski N, Zach L, Campian JL, et al. A Randomized Controlled Phase III Study of VB-111 Combined With Bevacizumab vs Bevacizumab Monotherapy in Patients With Recurrent Glioblastoma (GLOBE). Neuro Oncol (2020) 22(5):705–17. doi: 10.1093/neuonc/noz232
136. Scholz A, Harter PN, Cremer S, Yalcin BH, Gurnik S, Yamaji M, et al. Endothelial Cell-Derived Angiopoietin-2 is a Therapeutic Target in Treatment-Naive and Bevacizumab-Resistant Glioblastoma. EMBO Mol Med (2016) 8(1):39–57. doi: 10.15252/emmm.201505505
137. Peterson TE, Kirkpatrick ND, Huang Y, Farrar CT, Marijt KA, Kloepper J, et al. Dual Inhibition of Ang-2 and VEGF Receptors Normalizes Tumor Vasculature and Prolongs Survival in Glioblastoma by Altering Macrophages. Proc Natl Acad Sci USA (2016) 113(16):4470–5. doi: 10.1073/pnas.1525349113
138. Kloepper J, Riedemann L, Amoozgar Z, Seano G, Susek K, Yu V, et al. Ang-2/VEGF Bispecific Antibody Reprograms Macrophages and Resident Microglia to Anti-Tumor Phenotype and Prolongs Glioblastoma Survival. Proc Natl Acad Sci USA (2016) 113(16):4476–81. doi: 10.1073/pnas.1525360113
139. Nabors LB, Fiveash JB, Markert JM, Kekan MS, Gillespie GY, Huang Z, et al. A Phase 1 Trial of ABT-510 Concurrent With Standard Chemoradiation for Patients With Newly Diagnosed Glioblastoma. Arch Neurol (2010) 67(3):313–9. doi: 10.1001/archneurol.2010.16
140. Silverstein RL, Febbraio M. CD36, a Scavenger Receptor Involved in Immunity, Metabolism, Angiogenesis, and Behavior. Sci Signal (2009) 2(72):re3. doi: 10.1126/scisignal.272re3
141. Harshman LC, Srinivas S. The Bevacizumab Experience in Advanced Renal Cell Carcinoma. Onco Targets Ther (2010) 3:179–89. doi: 10.2147/OTT.S8157
142. Escudier B, Bellmunt J, Négrier S, Bajetta E, Melichar B, Bracarda S, et al. Phase III Trial of Bevacizumab Plus Interferon Alfa-2a in Patients With Metastatic Renal Cell Carcinoma (AVOREN): Final Analysis of Overall Survival. J Clin Oncol (2010) 28(13):2144–50. doi: 10.1200/JCO.2009.26.7849
143. Socinski MA, Jotte RM, Cappuzzo F, Orlandi F, Stroyakovskiy D, Nogami N, et al. Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC. N Engl J Med (2018) 378(24):2288–301. doi: 10.1056/nejmoa1716948
144. Cheng A-L, Qin S, Ikeda M, Galle P, Ducreux M, Zhu A, et al. IMbrave150: Efficacy and Safety Results From a Ph III Study Evaluating Atezolizumab (Atezo) + Bevacizumab (Bev) vs Sorafenib (Sor) as First Treatment (Tx) for Patients (Pts) With Unresectable Hepatocellular Carcinoma (HCC). Ann Oncol (2019) 30:ix186–7. doi: 10.1093/annonc/mdz446.002
145. Reardon DA, Desjardins A, Vredenburgh JJ, O’Rourke DM, Tran DD, Fink KL, et al. Rindopepimut With Bevacizumab for Patients With Relapsed EGFRvIII-Expressing Glioblastoma (REACT): Results of a Double-Blind Randomized Phase II Trial. Clin Cancer Res (2020) 26(7):1586–94. doi: 10.1158/1078-0432.CCR-18-1140
146. Bloch O, Shi Q, Anderson SK, Knopp M, Raizer J, Clarke J, et al. ATIM-14. Alliance A071101: A Phase II Randomized Trial Comparing the Efficacy of Heat Shock Protein Peptide Complex-96 (HSPPC-96) Vaccine Given With Bevacizumab Versus Bevacizumab Alone in the Treatment of Surgically Resectable Recurrent Glioblastoma. Neuro Oncol (2017) 19(suppl_6):vi29–9. doi: 10.1093/neuonc/nox168.110
147. Bota DA, Chung J, Dandekar M, Carrillo JA, Kong XT, Fu BD, et al. Phase II Study of ERC1671 Plus Bevacizumab Versus Bevacizumab Plus Placebo in Recurrent Glioblastoma: Interim Results and Correlations With CD4+ T-Lymphocyte Counts. CNS Oncol (2018) 7(3):CNS22. doi: 10.2217/cns-2018-0009
148. Nayak L, Molinaro AM, Peters K, Clarke JL, Jordan JT, de Groot J, et al. Randomized Phase II and Biomarker Study of Pembrolizumab Plus Bevacizumab Versus Pembrolizumab Alone for Patients With Recurrent Glioblastoma. Clin Cancer Res (2021) 27(4):1048–57. doi: 10.1158/1078-0432.CCR-20-2500
149. Vredenburgh JJ, Cloughesy T, Samant M, Prados M, Wen PY, Mikkelsen T, et al. Corticosteroid Use in Patients With Glioblastoma at First or Second Relapse Treated With Bevacizumab in the BRAIN Study. Oncologist (2010) 15(12):1329–34. doi: 10.1634/theoncologist.2010-0105
150. Campian JL, Abraham C, Luo J, Talcott G, Katumba R, Kim AH, et al. Safety and Efficacy Study of Retifanlimab and Epacadostat in Combination With Radiation and Bevacizumab in Patients With Recurrent Glioblastoma. J Clin Oncol (2021) 39(15_suppl):TPS2070–TPS2070. doi: 10.1200/jco.2021.39.15_suppl.tps2070
151. Hornyák L, Dobos N, Koncz G, Karányi Z, Páll D, Szabó Z, et al. The Role of Indoleamine-2,3-Dioxygenase in Cancer Development, Diagnostics, and Therapy. Front Immunol (2018) 9:151(JAN). doi: 10.3389/fimmu.2018.00151
152. Odia Y, Sul J, Shih JH, Kreisl TN, Butman JA, Iwamoto FM, et al. A Phase II Trial of Tandutinib (MLN 518) in Combination With Bevacizumab for Patients With Recurrent Glioblastoma. CNS Oncol (2016) 5(2):59–67. doi: 10.2217/cns-2015-0010
153. Hainsworth JD, Shih KC, Shepard GC, Tillinghast GW, Brinker BT, Spigel DR. Phase II Study of Concurrent Radiation Therapy, Temozolomide, and Bevacizumab Followed by Bevacizumab/Everolimus as First-Line Treatment for Patients With Glioblastoma. Clin Adv Hematol Oncol (2012) 10(4):240–6.
154. Teh JLF, Aplin AE. Arrested Developments: CDK4/6 Inhibitor Resistance and Alterations in the Tumor Immune Microenvironment. Clin Cancer Res (2019) 25(3):921–7. doi: 10.1158/1078-0432.CCR-18-1967
155. Sweid A, Daou BJ, Weinberg JH, Starke RM, Sergott RC, Schaefer J, et al. Experience With Ventriculoperitoneal and Lumboperitoneal Shunting for the Treatment of Idiopathic Intracranial Hypertension: A Single Institution Series. Oper Neurosurg (2021) 21(2):57–62. doi: 10.1093/ons/opab106
156. Schaer DA, Beckmann RP, Dempsey JA, Huber L, Forest A, Amaladas N, et al. The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade. Cell Rep (2018) 22(11):2978–94. doi: 10.1016/j.celrep.2018.02.053
157. Allen E, Jabouille A, Rivera LB, Lodewijckx I, Missiaen R, Steri V, et al. Combined Antiangiogenic and Anti-PD-L1 Therapy Stimulates Tumor Immunity Through HEV Formation. Sci Transl Med (2017) 9(385):9679. doi: 10.1126/scitranslmed.aak9679
158. Renner DN, Malo CS, Jin F, Parney IF, Pavelko KD, Johnson AJ. Improved Treatment Efficacy of Antiangiogenic Therapy When Combined With Picornavirus Vaccination in the GL261 Glioma Model. Neurotherapeutics (2016) 13(1):226–36. doi: 10.1007/s13311-015-0407-1
Keywords: glioblastoma, glioma, anti-angiogenic therapy, bevacizumab, immunotherapy, checkpoint inhibitors, combinatorial therapy
Citation: Jain S, Chalif EJ and Aghi MK (2022) Interactions Between Anti-Angiogenic Therapy and Immunotherapy in Glioblastoma. Front. Oncol. 11:812916. doi: 10.3389/fonc.2021.812916
Received: 10 November 2021; Accepted: 17 December 2021;
Published: 12 January 2022.
Edited by:
Andreas Pircher, Innsbruck Medical University, AustriaReviewed by:
Sadhak Sengupta, Triumvira Immunologics, Inc., United StatesRimas Vincas Lukas, Northwestern University, United States
Copyright © 2022 Jain, Chalif and Aghi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Manish K. Aghi, Manish.Aghi@ucsf.edu
†These authors have contributed equally to this work and share first authorship