Bevacizumab-Induced Hypertension in Glioblastoma Patients and Its Potential as a Modulator of Treatment Response

Published online: 21 June 2023 OnlinePublGlioblastoma invasion is the primary mechanism responsible for its dismal prognosis and is the direct result of interactions between glioblastoma cells and the tumor vasculature. The dysregulated microvasculature in glioblastoma tumors and vessels co-opted from surrounding brain tissue support rapid tumor growth and are utilized as pathways for invasive cancer cells. Attempts to target the glioblastoma vasculature with antiangiogenic agents (eg, bevacizumab) have nonetheless shown limited and inconsistent efficacy, and the underlying causes of such heterogeneous responses remain unknown. Several studies have identified that patients with glioblastoma who develop hypertension following treatment with bevacizumab show significant improvement in overall survival compared with normotensive nonresponders. Here we review these findings and discuss the potential of hypertension as a biomarker for glioblastoma treatment response in individual patients and the role of hypertension as a modulator of interactions between tumor cells and cells in the perivascular niche. We suggest that a better understanding of the actions of bevacizumab and hypertension at the cellular level will contribute to developing more effective personalized therapies that address glioblastoma tumor cell invasion.Kaitlin G. Scheer, Lisa M. Ebert, Michael S. Samuel, Claudine S. Bonder, Guillermo A. Gome

Increased expression of flightless I in cutaneous squamous cell carcinoma affects wnt/beta-catenin signaling pathway

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/β-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/β-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased β-catenin and a decrease in the expression of the downstream effector of β-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.Gink N. Yang, Xanthe L. Strudwick, Claudine S. Bonder, Zlatko Kopecki and Allison J. Cowi

Control of immune cell entry through the tumour vasculature: a missing link in optimising melanoma immunotherapy?

Metastatic melanoma remains a fatal disease to many worldwide, even after the breakthrough introduction of targeted therapies such as BRAF inhibitors and immune checkpoint blockade therapies such as CTLA-4 and PD-1 inhibitors. With advances in our understanding of this disease, as well as the increasing data gathered from patient studies, the significance of the host immune response to cancer progression and response to treatment is becoming clear. More specifically, the presence of intratumoral CD8⁺ cytotoxic T-cells correlates with better prognosis whereas the accumulation of monocytes/macrophages and neutrophils in the tumour is often associated with worse prognosis. Access and infiltration of circulating leukocytes into the tumour is governed by adhesion molecules and chemokines expressed by the endothelial cells of the vasculature. This review focuses on the adhesion molecules and chemokines which control the homing of CD8⁺ cytotoxic T-cells, monocytes and neutrophils to peripheral tissues, including tumours. We discuss the role of these leukocyte subsets in regulating melanoma growth, and detail the mechanisms used by tumours to selectively recruit or exclude these leukocytes for their own advantage. In doing so, we bring to light an underappreciated component of tumour biology which should be considered in combination with current treatments to selectively alter the leukocyte composition of tumours and ultimately enhance treatment outcome.Lih Yin Tan, Carmela Martini, Zvi G Fridlender, Claudine S Bonder, Michael P Brown and Lisa M Eber

An oncogenic role for sphingosine kinase 2

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an antiproliferative/ pro-apoptotic function for SK2, while others indicate it has a prosurvival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.Heidi A. Neubauer, Duyen H. Pham, Julia R. Zebol, Paul A.B. Moretti, Amanda L. Peterson, Tamara M. Leclercq, Huasheng Chan, Jason A. Powell, Melissa R. Pitman, Michael S. Samuel, Claudine S. Bonder, Darren J. Creek, Briony L. Gliddon and Stuart M. Pitso

CD36 promotes vasculogenic mimicry in melanoma by mediating adhesion to the extracellular matrix

Background: The formation of blood vessels within solid tumors directly contributes to cancer growth and metastasis. Until recently, tumor vasculature was thought to occur exclusively via endothelial cell (EC) lined structures (i.e. angiogenesis), but a second source of tumor vasculature arises from the cancer cells themselves, a process known as vasculogenic mimicry (VM). While it is generally understood that the function of VM vessels is the same as that of EC-lined vessels (i.e. to supply oxygen and nutrients to the proliferating cancer cells), the molecular mechanisms underpinning VM are yet to be fully elucidated. Methods: Human VM-competent melanoma cell lines were examined for their VM potential using the in vitro angiogenesis assays (Matrigel), together with inhibition studies using small interfering RNA and blocking monoclonal antibodies. Invasion assays and adhesion assays were used to examine cancer cell function. Results: Herein we demonstrate that CD36, a cell surface glycoprotein known to promote angiogenesis by ECs, also supports VM formation by human melanoma cancer cells. In silico analysis of CD36 expression within the melanoma cohort of The Cancer Genome Atlas suggests that melanoma patients with high expression of CD36 have a poorer clinical outcome. Using in vitro ‘angiogenesis’ assays and CD36-knockdown approaches, we reveal that CD36 supports VM formation by human melanoma cells as well as adhesion to, and invasion through, a cancer derived extracellular matrix substrate. Interestingly, thrombospondin-1 (TSP-1), a ligand for CD36 on ECs that inhibits angiogenesis, has no effect on VM formation. Further investigation revealed a role for laminin, but not collagen or fibronectin, as ligands for CD36 expressing melanoma cells. Conclusions: Taken together, this study suggests that CD36 is a novel regulator of VM by melanoma cancer cells that is facilitated, at least in part, via integrin-α3 and laminin. Unlike angiogenesis, VM is not perturbed by the presence of TSP-1, thus providing new information on differences between these two processes of tumor vascularization which may be exploited to combat cancer progression.Carmela Martini, Mark DeNichilo, Danielle P. King, Michaelia P. Cockshell, Brenton Ebert, Brian Dale, Lisa M. Ebert, Anthony Woods, and Claudine S. Bonde

Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.Heidi A. Neubauer, Melinda N. Tea, Julia R. Zebol, Briony L. Gliddon, Cassandra Stefanidis, Paul A.B. Moretti, Melissa R. Pitman, Maurizio Costabile, Jasreen Kular, Brett W. Stringer, Bryan W. Day, Michael S. Samuel, Claudine S. Bonder, Jason A. Powell, Stuart M. Pitso

Vasculogenic properties of adventitial Sca-1(+)CD45(+) progenitor cells in mice: a potential source of vasa vasorum in atherosclerosis

The cellular origins of vasa vasorum are ill-defined and may involve circulating or local progenitor cells. We previously discovered that murine aortic adventitia contains Sca-1⁺CD45⁺ progenitors that produce macrophages. Here we investigated whether they are also vasculogenic. In aortas of C57BL/6 mice, Sca-1⁺CD45⁺ cells were localised to adventitia and lacked surface expression of endothelial markers (<1% for CD31, CD144, TIE-2). In contrast, they did show expression of CD31, CD144, TIE-2 and VEGFR2 in atherosclerotic ApoE(-/-) aortas. Although Sca-1⁺CD45⁺ cells from C57BL/6 aorta did not express CD31, they formed CD31⁺ colonies in endothelial differentiation media and produced interconnecting vascular-like cords in Matrigel that contained both endothelial cells and a small population of macrophages, which were located at branch points. Transfer of aortic Sca-1⁺CD45⁺ cells generated endothelial cells and neovessels de novo in a hindlimb model of ischaemia and resulted in a 50% increase in perfusion compared to cell-free control. Similarly, their injection into the carotid adventitia of ApoE(-/-) mice produced donor-derived adventitial and peri-adventitial microvessels after atherogenic diet, suggestive of newly formed vasa vasorum. These findings show that beyond its content of macrophage progenitors, adventitial Sca-1⁺CD45⁺ cells are also vasculogenic and may be a source of vasa vasorum during atherogenesis.Deborah Toledo-Flores, Anna Williamson, Nisha Schwarz, Sanuja Fernando, Catherine Dimasi, Tyra A. Witt, Thao M. Nguyen, Amrutesh S . Puranik, Colin D. Chue, Sinny Delacroix, Daniel B. Spoon, Claudine S. Bonder, Christina A. Bursill, Belinda A. Di Bartolo, Stephen J. Nicholls, Robert D. Simari, Peter J. Psalti

Circulating human progenitor cells - the sentinels of vascular repair?

Abstract P.1.3Sarah L. Brice, Emma Thompson, Michaelia P. Cockshell, Shaundeep Sen, Claudine S. Bonde

BMPR2-expressing bone marrow-derived endothelial-like progenitor cells alleviate pulmonary arterial hypertension in vivo

BACKGROUND AND OBJECTIVE:Pulmonary arterial hypertension (PAH) is characterized by increased resistance in the distal pulmonary arteries, ultimately leading to right heart failure and, despite the available therapeutics, survival remains poor. Reduced expression of bone morphogenetic protein receptor type 2 (BMPR2) is strongly associated with PAH. Cell therapies are of interest in PAH, but whether this approach can upregulate BMPR2 is not known. Our objective was to evaluate a preclinical cell therapy approach based on upregulation of BMPR2. METHODS:We assessed the therapeutic effect of intravenously injected BMPR2-augmented rat bone marrow-derived endothelial-like progenitor cells (BMPR2-BM-ELPC) on PAH in the rat monocrotaline (MCT) model. RESULTS:The cells accumulate in the lungs with negligible systemic distribution, but the vast majority are lost from the lungs by 24 h. Lungs from rats treated with BMPR2-BM-ELPC exhibited an immediate increase in BMPR2 and related intracellular signalling proteins. Treatment with BMPR2-BM-ELPC attenuated PAH as demonstrated by a reduction in right ventricular hypertrophy as well as right ventricular systolic and mean pulmonary arterial pressures. In addition, this treatment reversed PAH-induced vascular remodelling with a significant reduction in vessel thickness and muscularization. In view of the short retention time of injected cells in the lungs, the mechanism for the effects seen may be intracellular communication via exosomes. In support of this hypothesis, we demonstrate that BMPR2-transduced outgrowth endothelial progenitor cells (OECs) release BMPR2-expressing exosomes. CONCLUSION:BMPR2-augmented ELPC demonstrate therapeutic benefits in the rat model and may have clinical translation potential.Rebecca L. Harper, Suzanne Maiolo, Rebekah J. Ward, Jemma Seyfang, Michaelia P. Cockshell, Claudine S. Bonder and Paul N. Reynold

Topical application of fingolimod perturbs cutaneous inflammation

The prevalence of allergies, including rhinitis, eczema, and anaphylaxis, is rising dramatically worldwide. This increase is especially problematic in children who bear the greatest burden of this rising trend. Increasing evidence identifies neutrophils as primary perpetrators of the more severe and difficult to manage forms of inflammation. A newly recognized mechanism by which neutrophils are recruited during the early phase of histamine-induced inflammation involves the sphingosine kinase (SK)/sphingosine-1-phosphate axis. This study examines whether topical application of fingolimod, an established SK/sphingosine-1-phosphate antagonist already in clinical use to treat multiple sclerosis, may be repurposed to treat cutaneous inflammation. Using two mouse models of ear skin inflammation (histamine- and IgE-mediated passive cutaneous anaphylaxis) we topically applied fingolimod prophylactically, as well as after establishment of the inflammatory response, and examined ear swelling, SK activity, vascular permeability, leukocyte recruitment, and production of proinflammatory mediators. The present study reveals that when applied topically, fingolimod attenuates both immediate and late-phase responses to histamine with reduced extravasation of fluid, SK-1 activity, proinflammatory cytokine and chemokine production, and neutrophil influx and prevents ear swelling. Intravital microscopy demonstrates that histamine-induced neutrophil rolling and adhesion to the postcapillary venules in the mouse ears is significantly attenuated even after 24 h. More importantly, these effects are achievable even once inflammation is established. Translation into humans was also accomplished with epicutaneous application of fingolimod resolving histamine-induced and allergen-induced inflammatory reactions in forearm skin. Overall, this study demonstrates, to our knowledge for the first time, that fingolimod may be repurposed to treat cutaneous inflammation.Wai Y. Sun, David P. Dimasi, Melissa R. Pitman, YiZhong Zhuang, Robert Heddle, Stuart M. Pitson, Michele A. Grimbaldeston, and Claudine S. Bonde