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Targeting the vasculature in hepatocellular carcinoma treatment: Starving versus normalizing blood supply

Targeting the vasculature in hepatocellular carcinoma treatment: Starving versus normalizing... Downloaded from http://journals.lww.com/ctg by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/05/2020 Citation: Clinical and Translational Gastroenterology (2017) 8, e98; doi:10.1038/ctg.2017.28 Official journal of the American College of Gastroenterology www.nature.com/ctg CLINICAL AND SYSTEMATIC REVIEWS Targeting the vasculature in hepatocellular carcinoma treatment: Starving versus normalizing blood supply 1,2,3,4 1,2,3 1,2,3 4 1,2,3 5 4 Ken Liu , Xiang Zhang , Weiqi Xu , Jinbiao Chen ,Jun Yu , Jennifer R. Gamble and Geoffrey W. McCaughan Traditional treatments for intermediate or advanced stage hepatocellular carcinoma (HCC) such as transarterial chemoemboliza- tion (TACE) and anti-angiogenesis therapies were developed to starve tumor blood supply. A new approach of normalizing structurally and functionally abnormal tumor vasculature is emerging. While TACE improves survival in selected patients, the resulting tumor hypoxia stimulates proliferation, angiogenesis, treatment resistance and metastasis, which limits its overall efficacy. Vessel normalization decreases hypoxia and improves anti-tumor immune infiltrate and drug delivery. Several pre-clinical agents aimed at normalizing tumor vasculature in HCC appear promising. Although anti-angiogenic agents with vessel normalizing potential have been trialed in advanced HCC with modest results, to date their primary intention had been to starve the tumor. Judicious use of anti-angiogenic therapies is required to achieve vessel normalization yet avoid excessive pruning of vessels. This balance, termed the normalization window, is yet uncharacterized in HCC. However, the optimal class, dose and schedule of vascular normalization agents, alone or in combination with other therapies needs to be explored further. Clinical and Translational Gastroenterology (2017) 8, e98; doi:10.1038/ctg.2017.28; published online 15 June 2017 Subject Category: Review INTRODUCTION conventional treatments such as arterial embolization and sorafenib aim to starve the tumor of its blood supply (and Hepatocellular carcinoma (HCC) is the fifth most common cancer therefore oxygen and nutrients). In contrast, an emerging in men and ninth most common in women worldwide. The concept in cancer treatment is the “normalization hypothesis” disease carries a high mortality rate and represents the third most where tumor vessels, which are aberrant both in structure and frequent cause of cancer death globally. The median survival function, are normalized to improve tumor perfusion and 2,3 following diagnosis is poor, ranging from four to 20 months. oxygenation. Such approaches have been associated with While potentially curative therapies such as surgical reduced metastasis and improved delivery of chemo-, radio- resection, liver transplantation or ablation can result in and immune therapies. 5-year overall survival rates of 470%, they are applicable In this review, we describe the structural and functional to less than 30% of patients with HCC. Currently, treatment abnormalities in HCC blood vessels. We will then discuss the options for patients with intermediate and advanced HCC treatment of HCC by targeting the vasculature through two remain limited and are considered palliative. Transarterial opposing approaches: the traditional method of starving the chemoembolization (TACE) which combines injection of blood supply and the new paradigm of vasculature normalization. chemotherapy and occlusion of the tumor blood supply, has been shown to improve survival in some randomized CHANGES IN VASCULATURE IN HCC controlled trials of patients with unresectable HCC but not 7–9 others. After an initial objective tumoral response in Like other solid tumors, HCC cannot grow beyond a few approximately 25–40% of patients, treated tumors can millimeters in size without angiogenesis. Through a process revascularize and require retreatment until the capacity to of angiogenic switch, an HCC is able to evolve from a keep the cancer under control is lost. For patients with dysplastic nodule and grow in size by acquiring an increasing advanced HCC, the only therapy with proven benefit is the number and density of unpaired arteries (i.e., not accompa- multi-kinase inhibitor sorafenib which extends median overall nied by bile ducts) supplying it. This switch is the rate-limiting survival by two to three months. step in hepatocarcinogenesis and is stimulated by an Amongst its anti-tumor properties, sorafenib also exerts imbalance of angiogenic factors in favor of those that are anti-angiogenic effects by inhibiting vascular endothelial proangiogenic. Both tumor cells and adjacent cells secrete growth factor (VEGF) receptor tyrosine kinases. Hence, VEGF, basic fibroblast growth factors (bFGF), angiopoietins, 1 2 Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Department of Medicine and Therapeutics, The Chinese University of Hong 3 4 Kong, Hong Kong, China; State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Centenary Institute and AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, University of Sydney, Sydney, New South Wales, Australia and Centre for the Endothelium, Vascular Biology Program, Centenary Institute, and University of Sydney, Sydney, New South Wales, Australia Correspondence: Geoffrey W. McCaughan, MBBS, FRACP, PhD, Centenary Institute and AW Morrow Gastroenterology and Liver Center, Royal Prince Alfred Hospital, University of Sydney, Sydney, New South Wales, Australia. E-mail: g.mccaughan@centenary.usyd.edu.au Received 9 February 2017; accepted 19 April 2017 Downloaded from http://journals.lww.com/ctg by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/05/2020 Starving vs. normalizing blood supply to treat HCC Liu et al. Figure 1 Structure of normal vessels vs. tumor vessels in the liver sinusoid. (a) In healthy liver sinusoids, the endothelium is regular, fenestrated and lacks a basement membrane. Hepatic stellate cells remain in a quiescent state. (b) In hepatocellular carcinoma, the endothelium is thickened and loses its fenestrations while a discontinuous basement membrane is formed through a process called capillarization. Tumor cells form the vessel wall in some areas. Hepatic stellate cells become activated and release vascular endothelial growth factor as well as other angiogenic factors. These vessels are structurally and functionally abnormal. platelet derived growth factor (PDGF), placental growth factor can lose their polarity and detach from the BM causing them (PlGF) and transforming growth factor among others. The to stack upon each other (stratification) and protrude into the tumor subsequently becomes hypervascular and draws blood lumen. Quantitation of these abnormal tumor vessels by from ectopic arteries to obtain nutrients for growth and to immunostaining for endothelial cell markers such as CD34 15,16 metastasize to distant organs. However, these vessels are (i.e., the tumor microvessel density), has been shown to be both structurally and functionally abnormal (Figure 1). independent poor prognostic factors for disease-free and overall patient survival in HCC. Furthermore, liver tumor cells themselves actively participate in the formation of new Tumor vessel structure. Macroscopically, tumor vessels vessels either partially by occupying the vessel wall in mosaic are tortuous, with uneven diameters and irregular branching 21,22 vessels or completely in vasculogenic mimicry, which is patterns. While normal liver sinusoidal endothelium is associated with high tumor grade, invasion and metastasis, fenestrated and lacks a basement membrane (BM), HCC and shortened survival. sinusoidal endothelium is thicker, has fewer fenestrations, The BM of the capillarized sinusoids is affected to varying shows BM formation and expresses the phenotype of degrees ranging from relatively intact BM in differentiated capillary blood vessels. Hence this process is termed HCCs to sharply defective BM in more anaplastic HCCs. capillarization. Endothelial cell proliferation is dramatically This degradation of BM is mediated by matrix metalloprotei- increased and circulating bone marrow-derived endothelial progenitor cells and hematopoietic stem cells are recruited to nases (MMP) which are highly expressed in HCC cells. aid tumor angiogenesis. Endothelial cells of HCC vessels Moreover, degradation of the BM by MMP2 and MMP9 Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. mobilizes proangiogenic factors such as VEGF sequestered in due to alterations in leukocyte-endothelium adhesion 26 43 the BM, thus further potentiating tumor angiogenesis. Both molecule and chemokine expression. MMP2 and MMP9 expression have been demonstrated to be Third, the rapid proliferation of tumor and non-tumor cells, 27,28 predictors of poor prognosis in HCC patients. leaky vessels and regional hypoperfusion all result in hypoxia Capillaries are enveloped by mural pericytes. In the liver, the which is a potent stimulator of angiogenesis mediated by the hepatic stellate cell (HSC) expresses multiple smooth muscle expression of hypoxia-inducible factor 1 (HIF-1). This leads cell markers and are considered the pericyte equivalent. In to the formation of more non-productive HCC vessels which HCC, HSCs become activated by secretion of cytokines such further aggravates hypoxia thereby establishing a vicious as PDGF and transforming growth factor-β by tumor cells. cycle (Table 1 and Figure 2). Tumor hypoxia has numerous Tumor-activated HSCs in turn create a proangiogenic, other cancer-promoting effects in HCC which will be discussed prometastatic microenvironment by facilitating endothelial in the following section. proliferation and survival through release of VEGF as well as other angiogenic factors. While pericytes are deficient in the TRADITIONAL TREATMENT APPROACH—STARVING abnormal vessels of non-HCC tumors, activated HSCs HCC OF BLOOD SUPPLY proliferate and enhance their coverage of the sinusoids in The recognition of the classical model of tumor angiogenesis cirrhosis and HCC. Increased numbers of HSCs in the HCC as a therapeutic target was made in the 1970s by Folkman. microenvironment is associated with cell migration and Simplistically, it was thought that limiting or obliterating the invasion. angiogenic response could improve outcomes, essentially Abnormalities of tumor vasculature are also seen at the starving the tumor to death. As HCCs typically have arterial ultrastructural and molecular level. Schmitt et al. demon- hypervascularity, it would therefore seem logical to starve the strated VEGF-induced disruption of occludin-delineated tight tumor of its blood supply as a therapeutic approach. First junctions in HCCs and peritumoral normal liver parenchyma, described by Doyon et al. in 1974, transarterial embolization thus facilitating a possible mechanism for tumor invasion. (TAE) achieves angiographic occlusion of the HCC blood The aberrant expression of claudins, which are integral supply using embolizing agents. It capitalizes on the unique structural and functional components of tight junctions, is situation of HCC acquiring its blood supply from the hepatic observed in HCC and may have a causal role in tumor artery while the surrounding liver (with dual blood supply) formation and progression by inducing epithelial–mesenchy- receives blood predominantly from the portal vein. The mal transition (EMT). In addition, vascular endothelial selective arterial occlusion by embolic agents such as gel (VE)-cadherin (a key protein in endothelial adherens junc- foam or polyvinyl alcohol results in tumor ischemia, hypoxia tions) is endocytosed and uncoupled from catenin-associated and ultimately necrosis while minimizing damage to liver proteins in response to VEGF. As a consequence, endothelial tissue. The addition of regional chemotherapy (TACE) with cell-cell junctions are loosened and vascular permeability is lipiodol (theoretically) enhances anti-tumor effects as che- increased in VEGF-induced tumor angiogenesis. motherapeutic agents can be given at higher concentrations and remain localized in the tumor for longer periods. As Tumor vessel function. These abnormalities in endothelial radioembolic agents do not work by creating ischemia from cells, BM and cell junctions collectively contribute to tumor vessel occlusion, they will not be discussed here. vessels which are excessively leaky in HCC with several 35,36 consequences arising from this. First, the extravasation of proteins and fluid into tumor interstitium leads to peritumor Efficacy of arterial embolization. Over the past decade, edema and interstitial hypertension via increases in oncotic TACE has become standard of care for patients with Barcelona clinic liver cancer (BCLC) stage B (intermediate) pressure and hydrostatic pressure. The impaired diffusion of HCC. Although early randomized controlled trials (RCTs) molecules and vascular collapse caused by capillarized demonstrated strong anti-tumor effects in TAE or TACE sinusoids, peritumoral edema and interstitial hypertension compared with conservative or suboptimal treatments (e.g., reduces delivery of oxygen and therapeutic agents into the 37,38 tamoxifen or intravenous 5-fluorouracil), all failed to show a tumor. This results in a tumor microenvironment of survival benefit. It was not until 2002, that two RCTs hypoxia, acidosis and potentially reduced efficacy of anti- 7,9 demonstrated improved survival. Results from meta- cancer treatments. Prognostically, tumor pressure in HCC has analyses and systematic reviews are overall in favor of TACE been shown to correlate with differential grade, presence of vascular invasion and intrahepatic metastasis, as well as local over non-active treatment. Two meta-analyses which 39,40 and distant recurrence rates after treatment. included almost identical studies (five out of six RCTs in Second, the poorly organized tumor vasculature with common) both found TAE or TACE improved the two-year tortuous, irregularly shaped, and leaky vessels is less survival compared with non-active treatment in patients with 51,52 responsive to vasoactive signals and unable to support unresectable HCC. Overall, the improvement observed in efficient blood flow. There is considerable heterogeneity in survival with arterial embolization was 46–47%. On the other tumor blood flow which is brisk in some areas and sluggish in hand, a more recent Cochrane meta-analysis of nine RCTs 42 53 others. This variation is not only observed spatially, but also revealed no difference in survival. However, the inclusion of temporally as blood flow changes with continuous vessel studies of patients with either early stage HCC (who benefit remodeling. This patchy perfusion leads to non-uniform most from curative therapies) or advanced HCC (who have delivery of oxygen, nutrients and drugs to the tumor. The poor response and less tolerance to TACE) may have influx of immune effector cells into the tumor is also impaired biased these results. While studies of TACE may differ Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. procedurally in use of chemotherapy agents (or lack thereof), Limitations of TACE and the effect of tumor hypoxia. embolizing material and number of repeated treatments, it is Aside from the aforementioned restrictions on patient selec- tion, TACE holds other drawbacks in HCC treatment. Although clear that patient selection is important. The most ideal the intention of TACE is to starve the HCC of its blood supply, candidates for TACE are patients with liver confined tumors (unresectable, without vascular invasion), preserved liver the resulting hypoxia in the tumor has subsequently been function (Child-Pugh class A or B) and the absence of portal shown to stimulate dedifferentiation, proliferation, angiogen- 60 61 vein thrombosis. esis and metastasis of the cancer itself. Recently, Lai et al. demonstrated a significant association between hypoxia in TACE treated HCCs and the induction of CK19, a marker for TAE vs. TACE. Although TACE is the more widely accepted an aggressive tumor phenotype. A histological study of 24 treatment approach, it remains doubtful whether it is superior HCCs treated with TAE in patients undergoing surgical to TAE alone. Several RCTs and two meta-analyses have resection found the proliferative activity of tumor cells and indicated no survival difference between the two intratumoral endothelial cells was increased after TAE com- 51,55–59 treatments. These results suggest TACE derives its pared to untreated tumors. Accordingly, the rate of local anti-tumor effects predominantly from the ischemic effect due recurrence after initial TACE is upwards of 80% and recurrent to embolization rather than the addition of chemotherapy. tumors have significantly shorter doubling times compared to primary HCCs. Central to these processes is the role of HIF-1α,a Table 1 Detrimental effects of hypoxia in carcinogenesis heterodimer transcription factor, which induces the expression of genes involved in cell survival, proliferation and Detrimental effects of hypoxia in carcinogenesis angiogenesis. During normoxia HIF-1α is hydroxylated, Induction of a more aggressive tumor phenotype ubiquinated and rapidly degraded by proteosomes. In response Upregulation of growth factors to hypoxia, HIF-1α binds to the promoter region of VEGF and Induction of apoptosis resistance DNA hypermethylation of tumor suppressor genes induces its transcription. Protein levels of both activated HIF-1α 64,65 Induction of EMT and VEGF are significantly increased following TACE. During hypoxia, HIF-1α and VEGF through the modulation of Impaired anti-tumor immune response Impaired entry of anti-tumor immune cells, e.g., CD8+ T cells other proteins (such as myeloid cell factor 1 and Bcl-2) create 80,83 Recruitment of immunosuppressive Treg cells and MDSCs an environment of apoptosis-blocking and tumor cell Increased expression of immune checkpoints 66,67 survival. Specifically, arterial embolization has been shown to upregulate the anti-apoptotic protein Bcl-2 which causes Stimulation of angiogenesis Induction of chemoresistance and radioresistance HCC cells to escape apoptosis induced by anoxic injury, rendering them resistant to further embolization treatments. In EMT, epithelial-mesenchymal transition; MDSC, myeloid-derived suppressor cell. addition, hypoxia can contribute to carcinogenesis via HIF-1α Figure 2 Vicious cycle of hypoxia, non-productive angiogenesis and tumor growth. Angiogenesis is required for a tumor to grow beyond a few millimeters. However, this neovascularisation produces abnormal leaky vessels which give rise to interstitial hypertension, edema and tumor hypoxia. Although some treatments (e.g., TACE) aim to achieve hypoxia in order to kill the tumor by starvation, hypoxia has been demonstrated to stimulate further angiogenesis and tumor growth through a variety of mechanisms (see text: Limitations of TACE and the effect of tumor hypoxia). HIF-1α, hypoxia-inducible factor 1α; IGF-2, insulin-like growth factor-2; MDSC, myeloid-derived suppressor cell; MMP, matrix metalloproteinases; TACE, transarterial chemoembolization; VEGF, vascular endothelial growth factor. Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. independent processes such as DNA hypermethylation of molecule tyrosine kinases also do this to a certain extent. tumor suppressor genes. The upregulation of HIF-1α and Sorafenib, regorafenib, lenvatinib, sunitinib, cediranib and VEGF along with other angiogenic factors such as insulin-like axitinib are multi-targeted tyrosine kinase inhibitors trialed in growth factor-2 (IGF-2) induced by hypoxia play a major role in HCC with activity against receptor tyrosine kinases involved 69 70 the stimulation of neovascularisation and EMT which in neovascularization and tumor progression including VEGF facilitate tumor progression and metastasis. Moreover, receptors (VEGFR) 1–3 and/or PDGF receptors (PDGFR)-α reciprocal positive regulation exists between these factors as and -β. Both VEGF and PDGF pathways are key mediators of IGF-2 increases HIF-1α stability which in turn induces VEGF angiogenesis which are overexpressed and play significant expression. Prognostically, increased plasma levels of roles during hepatocarcinogenesis. When VEGF interacts HIF-1α, VEGF and IGF-2 are all associated with the develop- with VEGFRs on the endothelial cell surface, it causes ment of metastasis and poor outcomes in post-TACE patients autophosphorylation of its intracellular tyrosine kinase and 72–75 and HCC patients in general. activation of downstream proteins resulting in a mitogenic Tumor hypoxia is also a known driver of chemoresistance in effect on endothelial cells. The binding of PDGF to its HCC. Through both HIF-1α-mediated and HIF-1α-independent receptors, after dimerization and activation of the intracellular pathways, hypoxia can protect tumor cells from chemotherapy tyrosine kinase, stimulates endothelial cell migration (rather 76,77 induced apoptosis. In addition, hypoxia elicits the expres- than proliferation) as well as survival and migratory signals to sion of multidrug resistance-related genes such as multidrug pericytes that provide support to vascular endothelial cells. resistance protein 1 and lung resistance protein in HCC cell Inhibition of these intracellular tyrosine kinase receptors has lines. An autocrine signaling loop involving PDGF-BB, Akt been shown to reduce endothelial cell proliferation, tubule and HIF-1α which confers cisplatin resistance in HCC cell lines formation, microvascular area and density in tumors (i.e., 11,92 under hypoxic conditions has also been discovered. Con- starving the tumor by inhibiting angiogenesis). Multi- versely, HIF-1α downregulation by antisense gene therapy kinase agents which target both VEGFRs and PDGFRs have enhances the therapeutic efficacy of doxorubicin against additive effects on limiting angiogenesis as inhibiting HCC. These mechanisms could explain the lack of an PDGFRs has been shown to cause pericyte detachment additive effect seen in chemoembolization over bland emoliza- from the endothelium, leaving endothelial cells more suscep- tion alone, as discussed previously, while the use of systemic tible to VEGF inhibition. Furthermore, some multi-targeted chemotherapy in HCC has similarly been ineffective. have direct anti-cancer effects. For example, sorafenib The tumor microenvironment is altered by hypoxia in its inhibits Raf kinase which is part of the Raf/MEK/ERK immune status. Although not yet extensively studied in HCC, signaling cascade involved in cell growth and survival and the effect of hypoxia on the intrahepatic immune infiltrate may is overactivated in HCC. Hence multi-kinase inhibition may be crucial. Hypoxia has been shown, in other cancers, to enable these small molecules to be used as monotherapies, promote chemokine-mediated recruitment of immunosup- something that has not proved effective with the pure VEGF 80,81 pressive Treg cells and myeloid-derived suppressor antagonist bevacizumab. The efficacy of these anti- cells. Furthermore, recruited monocytes and resident angiogenic therapies in clinical trials is discussed below. macrophages in the hypoxic tumor microenvironment differ- entiate into tumor associated macrophages (TAMs). The NOVEL TREATMENT APPROACH—NORMALIZATION OF polarization of these TAMs favor a tumor-promoting M2-like VASCULATURE IN HCC phenotype over a tumor-suppressive M1-like phenotype. This concept that hypoxia converts the tumor microenviron- The normalization hypothesis. Realizing the limitations of ment from immunosupportive to immunosuppressive appears 83 starving tumor vasculature due to the adverse consequences to also apply in HCC. Indeed, both the presence and balance of hypoxia, a growing number of pre-clinical and clinical (CD8+ effector cells vs. Treg) of within tumor infiltrating studies have explored the emerging (yet counterintuitive) lymphocytes have proved to be independent prognostic 84 paradigm of normalizing vasculature to treat cancer. Normal- factors in HCC. Furthermore, programmed death-ligand 1 ization of the vasculature adopts approaches that reverse the (PD-L1) expression is increased by HIF-1α in hypoxia and 85,86 classical phenotype of tumor blood vessels at the cellular and facilitates the evasion of anti-tumor immunity by HCCs. molecular level as opposed to simply obliterating the Thus, through the exacerbation of hypoxia (in an already vasculature. This essentially means a change to the tumor hypoxic microenvironment), the anti-tumor effects of TAE or microenviroment with less hypoxia, less vascular leak, TACE are modest and self-limiting. Arterial embolization may, increased pericyte numbers together with an increase in the in fact, paradoxically promote the HCC to become more infiltration of CD8+ T cells and a decrease in the neutrophil to aggressive and evasive leading to progression and metas- lymphocyte ratio (i.e., the opposite to changes seen in new tasis. The limitation of starving tumor blood supply to treat HCC tumor vessels as described above). Such changes HCC is further highlighted by the lack of efficacy seen when have the potential increase delivery of other cancer directed combining TACE with anti-angiogenic therapies such as 87 88 therapies and may be synergic with immune directed sorafenib and bevacizumab. Clearly other approaches therapies such as checkpoint inhibition. need to be explored. Mechanisms of action of current anti-angiogenic Pre-clinical studies. Examples of effective vessel normal- therapies. Although TACE is the par excellence example of ization have been now been demonstrated at the experimental targeting HCC via vascular starvation the current small level. Hamzah et al. observed that vessel normalization in Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. Table 2 Pre-clinical agents that have potential normalization activity Table 3 Features of vessel normalization Pre-clinical agents that have potential normalization activity Features of vessel normalization 96,97 PlGF inhibitors Reduced vessel diameter and tortuosity 102–104 Some traditional Chinese medicine compounds Decrease in vascular permeability HIF-1α inhibitors Decrease in tissue hypoxia VE-Cadherin modulator (e.g., Blockmir to inhibit miR-27a) Decrease in interstitial pressure and edema Increase in pericyte coverage around blood vessels Improvement in number and function of intratumoral immune cells HIF-1α, hypoxia-inducible factor 1α; PlGF, placental growth factor; VE-Cadherin, vascular endothelial cadherin. As the benefits of vascular normalization extend beyond tumor-bearing mice deficient in Rgs5 (a protein overexpressed cancer, novel targets also arise from non-oncological studies by pericytes in aberrant tumor vasculature) was associated of vascular biology. We have developed a novel Blockmir to with significant increases in infiltrating tumor-specific CD4+ inhibit miR-27a, a microRNA which targets VE-cadherin and CD8+ T cells and prolonged survival. In a separate study resulting in its downregulation. This led to vascular normal- of mice treated with a designer angiostatic peptide, anginex, ization and potently enhanced recovery from ischemic limb increased leukocyte infiltration was also seen through impro- injury in mice. Early experience with the same Blockmir in a ved leukocyte-vessel wall interactions in tumor vessels. subcutaneous isograft HCC model demonstrated an inhibition Several in vitro and in vivo studies of vessel normalizing of tumor growth (Zhao et al. Accepted Cancer Res. 2017). In agents have now also documented anti-tumor activity against the B16F10 melanoma model, the Blockmir decreased HCC (Table 2). Placental growth factor is a member of the vascular leak and tissue hypoxia, increased pericyte numbers, VEGF subfamily. After binding to its receptor VEGFR-1, it induced greater infiltration of T cells into the interior of the induces pro-cancer responses in endothelial, malignant, and tumor and showed a reduction in tumor growth by 60% (Zhao immune cells. Increased expression of PlGF is associated with et al. Accepted Cancer Res. 2017). Interestingly, such poor prognosis in HCC. Importantly, PlGF inhibition reduces vascular normalization effects plus the effect of an anti PD-1 tumor growth and induces vessel normalization in experi- monoclonal antibody showed a synergistic anti-tumor effect. 96,97 mental HCC models. Histidine-rich glycoprotein (HRG) is a host-produced protein deposited in the tumor stroma which can induce a change in polarization TAMs in favor of the M1- CURRENT CLINICAL ANTI-ANGIOGENIC THERAPIES: like phenotype. This effect of HRG on TAM polarization also DO THEY HAVE VESSEL NORMALIZING POTENTIAL? indirectly resulted in tumor vessel normalization. Both these Non-HCC directed therapies. The normalization hypoth- processes are PlGF-mediated. Furthermore, Vandewynckel 98 esis was initially proposed in an attempt to explain the et al. recently demonstrated that PlGF inhibition possibly observation that some anti-angiogenic therapies potentiate exerts its anti-tumor effects by improving intratumor hypoxia the effects of chemotherapy and radiotherapy. Multiple which is a potent activator of the pro-survival, PKR-like clinical trials have shown that combination therapy with endoplasmic reticulum kinase (PERK) pathway in HCC cells. bevacizumab (humanized anti-VEGF monoclonal antibody) A number of pharmacological agents that target the driver of and conventional chemotherapy improved patient survival abnormal vasculature, HIF-1α, have also been linked to and response rates over either therapy alone. This effect was vasculature normalization. EZN-2968 is a RNA antagonist observed across multiple advanced stage (metastatic) that specifically binds HIF-1α mRNA and reduces its expres- cancers: colorectal cancer, non-small cell lung cancer, breast sion by 80% and the expression of VEGF mRNA by 50% in 107–110 cancer and renal cancer. Given that anti-VEGF mice livers. A phase I study presented results demonstrating therapy aims to starve tumor blood supply and is associated its anti-tumor activity in patients with advanced malignancy with vessel pruning while chemotherapy relies on this same including HCC. Another suppresser of HIF-1α is NVP- blood supply for drug delivery, these findings were unex- BEZ235, a dual PI3K/mTOR inhibitor which induces apoptosis pected. It was hypothesized by Jain et al. that anti-VEGF of hypoxic cells. This novel agent has exhibited promising therapy augmented chemotherapy delivery and efficacy by activity against HCC. transiently reversing vessel abnormalities (and hence the Several vasoactive traditional Chinese medicine com- tumor microenvironment) without destroying them. In a pounds have exhibited anti-tumor efficacy against HCC in landmark study, the same group went on to demonstrate mouse xenograft models. Sinomenine hydrochloride, a known that within 12 days of infusion, a single dose of bevacizumab inducer of vascular normalization, has also been shown to in rectal carcinoma patients reduced microvascular density inhibit HCC growth by promoting cell cycle arrest and and improved vessel permeability, pericyte coverage and caspase-dependent apoptosis. Tanshinone IIA, a herbal interstitial hypertension. These are all markers of vascular extract from Chinese sage (Salvia miltiorrhiza), inhibited HCC normalization (Table 3). Conversely, Yang et al. recently metastasis and improved survival after palliative resection showed that discontinuation of anti-VEGF treatment in mice through the promotion of VEGFR-1/PDGFR-related vascular created a period of profound structural abnormality in liver normalization. This anti-metastatic potential was also seen sinusoidal capillaries with enlargement of pore sizes, loss of 105 112 in a study of Buyang Huanwu decoction. VE-cadherin and hyper-permeability. These changes led Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. Table 4 Potential approaches using vessel normalization in hepatocellular carcinoma Potential approaches using vessel normalization in hepatocellular carcinoma Normalization alone Normalization and established chemotherapy drugs (increase drug delivery) Normalization and multi-kinase inhibitors (increase drug delivery) Normalization and immunotherapy e.g., checkpoint inhibitors (increase entry and activity of anti-tumor immune cells) to tumor extravasation and marked promoted liver properties. An exploratory phase II RCT of sorafenib plus metastases in a mouse colorectal cancer model. doxorubicin vs. doxorubicin alone in advanced HCC found It is important to recognize that beneficial effects of anti- encouraging results with greater median PFS and OS seen in VEGF therapy may be dose dependent. According to Jain and combination therapy. However, the same authors could not colleagues, a paradox exists with anti-angiogenic therapy confirm the superiority of sorafenib plus doxorubicin compared where judicious application leads to selective pruning of with sorafenib in a phase III trial of 346 patients. No survival immature tumor vessels leaving a relatively normalized benefit was seen at the cost of higher toxicity. Trials of sunitinib network of vessels. While on the other hand, sustained or with reduced dosing showed modest activity with 2.9% RR, high doses may result in excessive regression of vasculature 3.9 months PFS, and 9.8 months OS. Interestingly, a trans leading to the same adverse effects of hypoxia seen in greater decrease in tumor vascular permeability (K , treatment attempts to starve the tumor. This delicate measured by dynamic contrast-enhanced magnetic reso- balance has been termed the “normalization window”. This nance imaging) at day 14 after treatment was associated with refers to a transient pharmacologically induced time period partial response or stable disease. This suggests that degree after the commencement of anti-angiogenic therapy during of vascular normalization may be a determinant of HCC which tumor vessels exhibit features of normalization and response to sunitinib. Nevertheless, a phase III trial of sunitinib improved functionality (reduced hypoxia) resulting in increa- vs. sorafenib in untreated patients with advanced HCC was sed vulnerability of cancer cells to cytotoxic therapies. The terminated early due to significantly worse survival (7.9 vs. commencement and duration of this window has been studied 10.2 months median OS) and higher toxicity. across different cancers using different vessel normalizing In recent developments, another multi-kinase inhibitor agents and can vary widely. For example, the time period of lenvatinib has been reported to have non-inferior overall increased oxygenation in mice melanoma, breast carcinoma survival compared to sorafenib for first-line treatment in and ovarian carcinoma models treated with bevacizumab was patients with unresectable HCC. In the second-line setting, demonstrated to be between day two and day four after regorafenib was also found to significantly improve OS in 115 134 starting treatment. In comparison, mice with human glioma patients with HCC who progressed on sorafenib. However, xenografts treated with anti-angiogenic agent suramin showed the effects of these treatments on vessel normalization are improved oxygenation compared to controls for up to 5 weeks not known. afterwards. The normalization window in human HCC is Targeting the epidermal growth factor signaling pathway via currently not known. erlotinib can reduce vascular permeability, tumor hypoxia and enhance responses to chemotherapy and radiation. Phase II studies in advanced HCC have demonstrated 0–9% RR, HCC directed therapies. In contrast to the aforementioned 136,137 3–4 months PFS and 10–13 months OS. However, the studies in other cancers, the synergistic effects of anti- combination of erlotinib with other anti-angiogenesis agents angiogenic therapies have not been reproduced in HCC. such as sorafenib and bevacizumab failed to show any Phase II trials of bevacizumab combined with systemic 138,139 117,118 additional benefit. chemotherapy did not show numerically superior Other agents directed against VEGFR such as cediranib, response rates (RR), progression-free survival (PFS), or axitinib and ramucirumab have all demonstrated normal- overall survival (OS) compared to bevacizumab alone or 119,120 ization properties in other cancers but have not been shown chemotherapy alone. to benefit HCC patients beyond the effect seen with Thalidomide exerts anti-angiogenic effects via inhibition of 140–145 sorafenib. Thus, although normalization effects are VEGF, bFGF and HIF-1α to improve tumor hypoxia and seen with some of the above agents, synergistic effects in interstitial hypertension—changes associated with vessel 121–123 human HCC either with chemotherapy or anti-angiogenic normalization. However, clinical trials of thalidomide 124,125 126 therapies have not so far been seen. either alone or in combination with chemotherapy or radiotherapy in unresectable HCC have been met with disappointing results (o5% RR). FUTURE PERSPECTIVES Although sorafenib and sunitinib can destroy tumor vascu- lature, they also both demonstrate the ability to normalize Vascular normalization with anti-angiogenic agents is now an tumor vasculature. Currently, sorafenib is licensed for use emerging approach to treat many cancers. In human HCC as monotherapy but it might prove effective when combined clinical trials, current anti-angiogenic therapies (beyond with chemotherapy on the basis of its vascular normalizing sorafenib) have not found the “normalization window” in order Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. to improve outcomes. It is likely that the optimal class, dose the results have failed to demonstrate comparable efficacy and schedule of these agents required to achieve normal- with the current standard of care, sorafenib. A better under- ization and yet avoid excessive pruning is not known. standing of the normalization window is required to guide Moreover, the exact timing of combination chemotherapy or dosing of anti-angiogenic therapy in relation to concomitant radiotherapy in order to capitalize on the normalization window therapies. The development of biomarkers may help in when their anti-tumor effects are enhanced is similarly unclear. selecting patients who benefit from these targeted therapies. These unknowns may partially explain why benefits seen in pre-clinical studies have not translated into clinically signifi- CONFLICT OF INTEREST cant improvements. Another unanswered question is whether and how this transient normalization window can be Guarantor of article: Geoffrey W McCaughan. prolonged. Clearly further characterization of the normal- Specific author contributions: Literature search, drafting of ization window is needed. Current approaches to treat HCC the article, critical revision of the article: Ken Liu, Geoffrey W using anti-VEGF therapies and multi-targeted tyrosine kinase McCaughan; contributed to draft, critical revision of the article: inhibitors are unlikely to be sufficient even if used appro- Xiang Zhang, Jinbiao Chen, Jun Yu, Jennifer R Gamble; priately. Therefore, a multipronged approach involving several created the figures, critical revision of the article: Weiqi Xu; anti-angiogenic pathways is likely to be required. Other All authors have read and approved the final version. relevant mediators of vessel normalization and their drug Financial support: None. targets also need to be explored. Novel strategies such as Potential conflicts of interest: None. targeting VE-cadherin particularly in combination with checkpoint inhibitors appear to be promising (Table 4). 1. Ferlay J, Soerjomataram I, Ervik M et al. GLOBOCAN 2012 v10, Cancer Incidence and Contributing to this challenge is a lack of validated surrogate Mortality Worldwide: IARC CancerBase No 11 [Internet] International Agency for Research biomarkers to signify when, or indeed if, vascular normal- on Cancer: Lyon, France, 2013. Available at http://globocan.iarc.fr/. Accessed on 5 February 2017. ization has occurred. In addition to guiding appropriate dosing 2. 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A phase II and biomarker study of ramucirumab, other third party material in this article are included in the article’s a human monoclonal antibody targeting the VEGF receptor-2, as first-line Creative Commons license, unless indicated otherwise in the credit line; monotherapy in patients with advanced hepatocellular cancer. Clin Cancer Res 2013; 19: 6614–6623. if the material is not included under the Creative Commons license, 144. Zhu AX, Park JO, Ryoo BY et al. Ramucirumab versus placebo as second-line treatment in users will need to obtain permission from the license holder to patients with advanced hepatocellular carcinoma following first-line therapy with sorafenib reproduce the material. To view a copy of this license, visit http:// (REACH): a randomised, double-blind, multicentre, phase 3 trial. Lancet Oncol 2015; 16: 859–870. creativecommons.org/licenses/by-nc-nd/4.0/ 145. Kang YK, Yau T, Park JW et al. Randomized phase II study of axitinib versus placebo plus best supportive care in second-line treatment of advanced hepatocellular carcinoma. Ann Oncol 2015; 26: 2457–2463. r The Author(s) 2017 Clinical and Translational Gastroenterology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical and Translational Gastroenterology Wolters Kluwer Health

Targeting the vasculature in hepatocellular carcinoma treatment: Starving versus normalizing blood supply

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Downloaded from http://journals.lww.com/ctg by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/05/2020 Citation: Clinical and Translational Gastroenterology (2017) 8, e98; doi:10.1038/ctg.2017.28 Official journal of the American College of Gastroenterology www.nature.com/ctg CLINICAL AND SYSTEMATIC REVIEWS Targeting the vasculature in hepatocellular carcinoma treatment: Starving versus normalizing blood supply 1,2,3,4 1,2,3 1,2,3 4 1,2,3 5 4 Ken Liu , Xiang Zhang , Weiqi Xu , Jinbiao Chen ,Jun Yu , Jennifer R. Gamble and Geoffrey W. McCaughan Traditional treatments for intermediate or advanced stage hepatocellular carcinoma (HCC) such as transarterial chemoemboliza- tion (TACE) and anti-angiogenesis therapies were developed to starve tumor blood supply. A new approach of normalizing structurally and functionally abnormal tumor vasculature is emerging. While TACE improves survival in selected patients, the resulting tumor hypoxia stimulates proliferation, angiogenesis, treatment resistance and metastasis, which limits its overall efficacy. Vessel normalization decreases hypoxia and improves anti-tumor immune infiltrate and drug delivery. Several pre-clinical agents aimed at normalizing tumor vasculature in HCC appear promising. Although anti-angiogenic agents with vessel normalizing potential have been trialed in advanced HCC with modest results, to date their primary intention had been to starve the tumor. Judicious use of anti-angiogenic therapies is required to achieve vessel normalization yet avoid excessive pruning of vessels. This balance, termed the normalization window, is yet uncharacterized in HCC. However, the optimal class, dose and schedule of vascular normalization agents, alone or in combination with other therapies needs to be explored further. Clinical and Translational Gastroenterology (2017) 8, e98; doi:10.1038/ctg.2017.28; published online 15 June 2017 Subject Category: Review INTRODUCTION conventional treatments such as arterial embolization and sorafenib aim to starve the tumor of its blood supply (and Hepatocellular carcinoma (HCC) is the fifth most common cancer therefore oxygen and nutrients). In contrast, an emerging in men and ninth most common in women worldwide. The concept in cancer treatment is the “normalization hypothesis” disease carries a high mortality rate and represents the third most where tumor vessels, which are aberrant both in structure and frequent cause of cancer death globally. The median survival function, are normalized to improve tumor perfusion and 2,3 following diagnosis is poor, ranging from four to 20 months. oxygenation. Such approaches have been associated with While potentially curative therapies such as surgical reduced metastasis and improved delivery of chemo-, radio- resection, liver transplantation or ablation can result in and immune therapies. 5-year overall survival rates of 470%, they are applicable In this review, we describe the structural and functional to less than 30% of patients with HCC. Currently, treatment abnormalities in HCC blood vessels. We will then discuss the options for patients with intermediate and advanced HCC treatment of HCC by targeting the vasculature through two remain limited and are considered palliative. Transarterial opposing approaches: the traditional method of starving the chemoembolization (TACE) which combines injection of blood supply and the new paradigm of vasculature normalization. chemotherapy and occlusion of the tumor blood supply, has been shown to improve survival in some randomized CHANGES IN VASCULATURE IN HCC controlled trials of patients with unresectable HCC but not 7–9 others. After an initial objective tumoral response in Like other solid tumors, HCC cannot grow beyond a few approximately 25–40% of patients, treated tumors can millimeters in size without angiogenesis. Through a process revascularize and require retreatment until the capacity to of angiogenic switch, an HCC is able to evolve from a keep the cancer under control is lost. For patients with dysplastic nodule and grow in size by acquiring an increasing advanced HCC, the only therapy with proven benefit is the number and density of unpaired arteries (i.e., not accompa- multi-kinase inhibitor sorafenib which extends median overall nied by bile ducts) supplying it. This switch is the rate-limiting survival by two to three months. step in hepatocarcinogenesis and is stimulated by an Amongst its anti-tumor properties, sorafenib also exerts imbalance of angiogenic factors in favor of those that are anti-angiogenic effects by inhibiting vascular endothelial proangiogenic. Both tumor cells and adjacent cells secrete growth factor (VEGF) receptor tyrosine kinases. Hence, VEGF, basic fibroblast growth factors (bFGF), angiopoietins, 1 2 Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Department of Medicine and Therapeutics, The Chinese University of Hong 3 4 Kong, Hong Kong, China; State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Centenary Institute and AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, University of Sydney, Sydney, New South Wales, Australia and Centre for the Endothelium, Vascular Biology Program, Centenary Institute, and University of Sydney, Sydney, New South Wales, Australia Correspondence: Geoffrey W. McCaughan, MBBS, FRACP, PhD, Centenary Institute and AW Morrow Gastroenterology and Liver Center, Royal Prince Alfred Hospital, University of Sydney, Sydney, New South Wales, Australia. E-mail: g.mccaughan@centenary.usyd.edu.au Received 9 February 2017; accepted 19 April 2017 Downloaded from http://journals.lww.com/ctg by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/05/2020 Starving vs. normalizing blood supply to treat HCC Liu et al. Figure 1 Structure of normal vessels vs. tumor vessels in the liver sinusoid. (a) In healthy liver sinusoids, the endothelium is regular, fenestrated and lacks a basement membrane. Hepatic stellate cells remain in a quiescent state. (b) In hepatocellular carcinoma, the endothelium is thickened and loses its fenestrations while a discontinuous basement membrane is formed through a process called capillarization. Tumor cells form the vessel wall in some areas. Hepatic stellate cells become activated and release vascular endothelial growth factor as well as other angiogenic factors. These vessels are structurally and functionally abnormal. platelet derived growth factor (PDGF), placental growth factor can lose their polarity and detach from the BM causing them (PlGF) and transforming growth factor among others. The to stack upon each other (stratification) and protrude into the tumor subsequently becomes hypervascular and draws blood lumen. Quantitation of these abnormal tumor vessels by from ectopic arteries to obtain nutrients for growth and to immunostaining for endothelial cell markers such as CD34 15,16 metastasize to distant organs. However, these vessels are (i.e., the tumor microvessel density), has been shown to be both structurally and functionally abnormal (Figure 1). independent poor prognostic factors for disease-free and overall patient survival in HCC. Furthermore, liver tumor cells themselves actively participate in the formation of new Tumor vessel structure. Macroscopically, tumor vessels vessels either partially by occupying the vessel wall in mosaic are tortuous, with uneven diameters and irregular branching 21,22 vessels or completely in vasculogenic mimicry, which is patterns. While normal liver sinusoidal endothelium is associated with high tumor grade, invasion and metastasis, fenestrated and lacks a basement membrane (BM), HCC and shortened survival. sinusoidal endothelium is thicker, has fewer fenestrations, The BM of the capillarized sinusoids is affected to varying shows BM formation and expresses the phenotype of degrees ranging from relatively intact BM in differentiated capillary blood vessels. Hence this process is termed HCCs to sharply defective BM in more anaplastic HCCs. capillarization. Endothelial cell proliferation is dramatically This degradation of BM is mediated by matrix metalloprotei- increased and circulating bone marrow-derived endothelial progenitor cells and hematopoietic stem cells are recruited to nases (MMP) which are highly expressed in HCC cells. aid tumor angiogenesis. Endothelial cells of HCC vessels Moreover, degradation of the BM by MMP2 and MMP9 Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. mobilizes proangiogenic factors such as VEGF sequestered in due to alterations in leukocyte-endothelium adhesion 26 43 the BM, thus further potentiating tumor angiogenesis. Both molecule and chemokine expression. MMP2 and MMP9 expression have been demonstrated to be Third, the rapid proliferation of tumor and non-tumor cells, 27,28 predictors of poor prognosis in HCC patients. leaky vessels and regional hypoperfusion all result in hypoxia Capillaries are enveloped by mural pericytes. In the liver, the which is a potent stimulator of angiogenesis mediated by the hepatic stellate cell (HSC) expresses multiple smooth muscle expression of hypoxia-inducible factor 1 (HIF-1). This leads cell markers and are considered the pericyte equivalent. In to the formation of more non-productive HCC vessels which HCC, HSCs become activated by secretion of cytokines such further aggravates hypoxia thereby establishing a vicious as PDGF and transforming growth factor-β by tumor cells. cycle (Table 1 and Figure 2). Tumor hypoxia has numerous Tumor-activated HSCs in turn create a proangiogenic, other cancer-promoting effects in HCC which will be discussed prometastatic microenvironment by facilitating endothelial in the following section. proliferation and survival through release of VEGF as well as other angiogenic factors. While pericytes are deficient in the TRADITIONAL TREATMENT APPROACH—STARVING abnormal vessels of non-HCC tumors, activated HSCs HCC OF BLOOD SUPPLY proliferate and enhance their coverage of the sinusoids in The recognition of the classical model of tumor angiogenesis cirrhosis and HCC. Increased numbers of HSCs in the HCC as a therapeutic target was made in the 1970s by Folkman. microenvironment is associated with cell migration and Simplistically, it was thought that limiting or obliterating the invasion. angiogenic response could improve outcomes, essentially Abnormalities of tumor vasculature are also seen at the starving the tumor to death. As HCCs typically have arterial ultrastructural and molecular level. Schmitt et al. demon- hypervascularity, it would therefore seem logical to starve the strated VEGF-induced disruption of occludin-delineated tight tumor of its blood supply as a therapeutic approach. First junctions in HCCs and peritumoral normal liver parenchyma, described by Doyon et al. in 1974, transarterial embolization thus facilitating a possible mechanism for tumor invasion. (TAE) achieves angiographic occlusion of the HCC blood The aberrant expression of claudins, which are integral supply using embolizing agents. It capitalizes on the unique structural and functional components of tight junctions, is situation of HCC acquiring its blood supply from the hepatic observed in HCC and may have a causal role in tumor artery while the surrounding liver (with dual blood supply) formation and progression by inducing epithelial–mesenchy- receives blood predominantly from the portal vein. The mal transition (EMT). In addition, vascular endothelial selective arterial occlusion by embolic agents such as gel (VE)-cadherin (a key protein in endothelial adherens junc- foam or polyvinyl alcohol results in tumor ischemia, hypoxia tions) is endocytosed and uncoupled from catenin-associated and ultimately necrosis while minimizing damage to liver proteins in response to VEGF. As a consequence, endothelial tissue. The addition of regional chemotherapy (TACE) with cell-cell junctions are loosened and vascular permeability is lipiodol (theoretically) enhances anti-tumor effects as che- increased in VEGF-induced tumor angiogenesis. motherapeutic agents can be given at higher concentrations and remain localized in the tumor for longer periods. As Tumor vessel function. These abnormalities in endothelial radioembolic agents do not work by creating ischemia from cells, BM and cell junctions collectively contribute to tumor vessel occlusion, they will not be discussed here. vessels which are excessively leaky in HCC with several 35,36 consequences arising from this. First, the extravasation of proteins and fluid into tumor interstitium leads to peritumor Efficacy of arterial embolization. Over the past decade, edema and interstitial hypertension via increases in oncotic TACE has become standard of care for patients with Barcelona clinic liver cancer (BCLC) stage B (intermediate) pressure and hydrostatic pressure. The impaired diffusion of HCC. Although early randomized controlled trials (RCTs) molecules and vascular collapse caused by capillarized demonstrated strong anti-tumor effects in TAE or TACE sinusoids, peritumoral edema and interstitial hypertension compared with conservative or suboptimal treatments (e.g., reduces delivery of oxygen and therapeutic agents into the 37,38 tamoxifen or intravenous 5-fluorouracil), all failed to show a tumor. This results in a tumor microenvironment of survival benefit. It was not until 2002, that two RCTs hypoxia, acidosis and potentially reduced efficacy of anti- 7,9 demonstrated improved survival. Results from meta- cancer treatments. Prognostically, tumor pressure in HCC has analyses and systematic reviews are overall in favor of TACE been shown to correlate with differential grade, presence of vascular invasion and intrahepatic metastasis, as well as local over non-active treatment. Two meta-analyses which 39,40 and distant recurrence rates after treatment. included almost identical studies (five out of six RCTs in Second, the poorly organized tumor vasculature with common) both found TAE or TACE improved the two-year tortuous, irregularly shaped, and leaky vessels is less survival compared with non-active treatment in patients with 51,52 responsive to vasoactive signals and unable to support unresectable HCC. Overall, the improvement observed in efficient blood flow. There is considerable heterogeneity in survival with arterial embolization was 46–47%. On the other tumor blood flow which is brisk in some areas and sluggish in hand, a more recent Cochrane meta-analysis of nine RCTs 42 53 others. This variation is not only observed spatially, but also revealed no difference in survival. However, the inclusion of temporally as blood flow changes with continuous vessel studies of patients with either early stage HCC (who benefit remodeling. This patchy perfusion leads to non-uniform most from curative therapies) or advanced HCC (who have delivery of oxygen, nutrients and drugs to the tumor. The poor response and less tolerance to TACE) may have influx of immune effector cells into the tumor is also impaired biased these results. While studies of TACE may differ Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. procedurally in use of chemotherapy agents (or lack thereof), Limitations of TACE and the effect of tumor hypoxia. embolizing material and number of repeated treatments, it is Aside from the aforementioned restrictions on patient selec- tion, TACE holds other drawbacks in HCC treatment. Although clear that patient selection is important. The most ideal the intention of TACE is to starve the HCC of its blood supply, candidates for TACE are patients with liver confined tumors (unresectable, without vascular invasion), preserved liver the resulting hypoxia in the tumor has subsequently been function (Child-Pugh class A or B) and the absence of portal shown to stimulate dedifferentiation, proliferation, angiogen- 60 61 vein thrombosis. esis and metastasis of the cancer itself. Recently, Lai et al. demonstrated a significant association between hypoxia in TACE treated HCCs and the induction of CK19, a marker for TAE vs. TACE. Although TACE is the more widely accepted an aggressive tumor phenotype. A histological study of 24 treatment approach, it remains doubtful whether it is superior HCCs treated with TAE in patients undergoing surgical to TAE alone. Several RCTs and two meta-analyses have resection found the proliferative activity of tumor cells and indicated no survival difference between the two intratumoral endothelial cells was increased after TAE com- 51,55–59 treatments. These results suggest TACE derives its pared to untreated tumors. Accordingly, the rate of local anti-tumor effects predominantly from the ischemic effect due recurrence after initial TACE is upwards of 80% and recurrent to embolization rather than the addition of chemotherapy. tumors have significantly shorter doubling times compared to primary HCCs. Central to these processes is the role of HIF-1α,a Table 1 Detrimental effects of hypoxia in carcinogenesis heterodimer transcription factor, which induces the expression of genes involved in cell survival, proliferation and Detrimental effects of hypoxia in carcinogenesis angiogenesis. During normoxia HIF-1α is hydroxylated, Induction of a more aggressive tumor phenotype ubiquinated and rapidly degraded by proteosomes. In response Upregulation of growth factors to hypoxia, HIF-1α binds to the promoter region of VEGF and Induction of apoptosis resistance DNA hypermethylation of tumor suppressor genes induces its transcription. Protein levels of both activated HIF-1α 64,65 Induction of EMT and VEGF are significantly increased following TACE. During hypoxia, HIF-1α and VEGF through the modulation of Impaired anti-tumor immune response Impaired entry of anti-tumor immune cells, e.g., CD8+ T cells other proteins (such as myeloid cell factor 1 and Bcl-2) create 80,83 Recruitment of immunosuppressive Treg cells and MDSCs an environment of apoptosis-blocking and tumor cell Increased expression of immune checkpoints 66,67 survival. Specifically, arterial embolization has been shown to upregulate the anti-apoptotic protein Bcl-2 which causes Stimulation of angiogenesis Induction of chemoresistance and radioresistance HCC cells to escape apoptosis induced by anoxic injury, rendering them resistant to further embolization treatments. In EMT, epithelial-mesenchymal transition; MDSC, myeloid-derived suppressor cell. addition, hypoxia can contribute to carcinogenesis via HIF-1α Figure 2 Vicious cycle of hypoxia, non-productive angiogenesis and tumor growth. Angiogenesis is required for a tumor to grow beyond a few millimeters. However, this neovascularisation produces abnormal leaky vessels which give rise to interstitial hypertension, edema and tumor hypoxia. Although some treatments (e.g., TACE) aim to achieve hypoxia in order to kill the tumor by starvation, hypoxia has been demonstrated to stimulate further angiogenesis and tumor growth through a variety of mechanisms (see text: Limitations of TACE and the effect of tumor hypoxia). HIF-1α, hypoxia-inducible factor 1α; IGF-2, insulin-like growth factor-2; MDSC, myeloid-derived suppressor cell; MMP, matrix metalloproteinases; TACE, transarterial chemoembolization; VEGF, vascular endothelial growth factor. Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. independent processes such as DNA hypermethylation of molecule tyrosine kinases also do this to a certain extent. tumor suppressor genes. The upregulation of HIF-1α and Sorafenib, regorafenib, lenvatinib, sunitinib, cediranib and VEGF along with other angiogenic factors such as insulin-like axitinib are multi-targeted tyrosine kinase inhibitors trialed in growth factor-2 (IGF-2) induced by hypoxia play a major role in HCC with activity against receptor tyrosine kinases involved 69 70 the stimulation of neovascularisation and EMT which in neovascularization and tumor progression including VEGF facilitate tumor progression and metastasis. Moreover, receptors (VEGFR) 1–3 and/or PDGF receptors (PDGFR)-α reciprocal positive regulation exists between these factors as and -β. Both VEGF and PDGF pathways are key mediators of IGF-2 increases HIF-1α stability which in turn induces VEGF angiogenesis which are overexpressed and play significant expression. Prognostically, increased plasma levels of roles during hepatocarcinogenesis. When VEGF interacts HIF-1α, VEGF and IGF-2 are all associated with the develop- with VEGFRs on the endothelial cell surface, it causes ment of metastasis and poor outcomes in post-TACE patients autophosphorylation of its intracellular tyrosine kinase and 72–75 and HCC patients in general. activation of downstream proteins resulting in a mitogenic Tumor hypoxia is also a known driver of chemoresistance in effect on endothelial cells. The binding of PDGF to its HCC. Through both HIF-1α-mediated and HIF-1α-independent receptors, after dimerization and activation of the intracellular pathways, hypoxia can protect tumor cells from chemotherapy tyrosine kinase, stimulates endothelial cell migration (rather 76,77 induced apoptosis. In addition, hypoxia elicits the expres- than proliferation) as well as survival and migratory signals to sion of multidrug resistance-related genes such as multidrug pericytes that provide support to vascular endothelial cells. resistance protein 1 and lung resistance protein in HCC cell Inhibition of these intracellular tyrosine kinase receptors has lines. An autocrine signaling loop involving PDGF-BB, Akt been shown to reduce endothelial cell proliferation, tubule and HIF-1α which confers cisplatin resistance in HCC cell lines formation, microvascular area and density in tumors (i.e., 11,92 under hypoxic conditions has also been discovered. Con- starving the tumor by inhibiting angiogenesis). Multi- versely, HIF-1α downregulation by antisense gene therapy kinase agents which target both VEGFRs and PDGFRs have enhances the therapeutic efficacy of doxorubicin against additive effects on limiting angiogenesis as inhibiting HCC. These mechanisms could explain the lack of an PDGFRs has been shown to cause pericyte detachment additive effect seen in chemoembolization over bland emoliza- from the endothelium, leaving endothelial cells more suscep- tion alone, as discussed previously, while the use of systemic tible to VEGF inhibition. Furthermore, some multi-targeted chemotherapy in HCC has similarly been ineffective. have direct anti-cancer effects. For example, sorafenib The tumor microenvironment is altered by hypoxia in its inhibits Raf kinase which is part of the Raf/MEK/ERK immune status. Although not yet extensively studied in HCC, signaling cascade involved in cell growth and survival and the effect of hypoxia on the intrahepatic immune infiltrate may is overactivated in HCC. Hence multi-kinase inhibition may be crucial. Hypoxia has been shown, in other cancers, to enable these small molecules to be used as monotherapies, promote chemokine-mediated recruitment of immunosup- something that has not proved effective with the pure VEGF 80,81 pressive Treg cells and myeloid-derived suppressor antagonist bevacizumab. The efficacy of these anti- cells. Furthermore, recruited monocytes and resident angiogenic therapies in clinical trials is discussed below. macrophages in the hypoxic tumor microenvironment differ- entiate into tumor associated macrophages (TAMs). The NOVEL TREATMENT APPROACH—NORMALIZATION OF polarization of these TAMs favor a tumor-promoting M2-like VASCULATURE IN HCC phenotype over a tumor-suppressive M1-like phenotype. This concept that hypoxia converts the tumor microenviron- The normalization hypothesis. Realizing the limitations of ment from immunosupportive to immunosuppressive appears 83 starving tumor vasculature due to the adverse consequences to also apply in HCC. Indeed, both the presence and balance of hypoxia, a growing number of pre-clinical and clinical (CD8+ effector cells vs. Treg) of within tumor infiltrating studies have explored the emerging (yet counterintuitive) lymphocytes have proved to be independent prognostic 84 paradigm of normalizing vasculature to treat cancer. Normal- factors in HCC. Furthermore, programmed death-ligand 1 ization of the vasculature adopts approaches that reverse the (PD-L1) expression is increased by HIF-1α in hypoxia and 85,86 classical phenotype of tumor blood vessels at the cellular and facilitates the evasion of anti-tumor immunity by HCCs. molecular level as opposed to simply obliterating the Thus, through the exacerbation of hypoxia (in an already vasculature. This essentially means a change to the tumor hypoxic microenvironment), the anti-tumor effects of TAE or microenviroment with less hypoxia, less vascular leak, TACE are modest and self-limiting. Arterial embolization may, increased pericyte numbers together with an increase in the in fact, paradoxically promote the HCC to become more infiltration of CD8+ T cells and a decrease in the neutrophil to aggressive and evasive leading to progression and metas- lymphocyte ratio (i.e., the opposite to changes seen in new tasis. The limitation of starving tumor blood supply to treat HCC tumor vessels as described above). Such changes HCC is further highlighted by the lack of efficacy seen when have the potential increase delivery of other cancer directed combining TACE with anti-angiogenic therapies such as 87 88 therapies and may be synergic with immune directed sorafenib and bevacizumab. Clearly other approaches therapies such as checkpoint inhibition. need to be explored. Mechanisms of action of current anti-angiogenic Pre-clinical studies. Examples of effective vessel normal- therapies. Although TACE is the par excellence example of ization have been now been demonstrated at the experimental targeting HCC via vascular starvation the current small level. Hamzah et al. observed that vessel normalization in Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. Table 2 Pre-clinical agents that have potential normalization activity Table 3 Features of vessel normalization Pre-clinical agents that have potential normalization activity Features of vessel normalization 96,97 PlGF inhibitors Reduced vessel diameter and tortuosity 102–104 Some traditional Chinese medicine compounds Decrease in vascular permeability HIF-1α inhibitors Decrease in tissue hypoxia VE-Cadherin modulator (e.g., Blockmir to inhibit miR-27a) Decrease in interstitial pressure and edema Increase in pericyte coverage around blood vessels Improvement in number and function of intratumoral immune cells HIF-1α, hypoxia-inducible factor 1α; PlGF, placental growth factor; VE-Cadherin, vascular endothelial cadherin. As the benefits of vascular normalization extend beyond tumor-bearing mice deficient in Rgs5 (a protein overexpressed cancer, novel targets also arise from non-oncological studies by pericytes in aberrant tumor vasculature) was associated of vascular biology. We have developed a novel Blockmir to with significant increases in infiltrating tumor-specific CD4+ inhibit miR-27a, a microRNA which targets VE-cadherin and CD8+ T cells and prolonged survival. In a separate study resulting in its downregulation. This led to vascular normal- of mice treated with a designer angiostatic peptide, anginex, ization and potently enhanced recovery from ischemic limb increased leukocyte infiltration was also seen through impro- injury in mice. Early experience with the same Blockmir in a ved leukocyte-vessel wall interactions in tumor vessels. subcutaneous isograft HCC model demonstrated an inhibition Several in vitro and in vivo studies of vessel normalizing of tumor growth (Zhao et al. Accepted Cancer Res. 2017). In agents have now also documented anti-tumor activity against the B16F10 melanoma model, the Blockmir decreased HCC (Table 2). Placental growth factor is a member of the vascular leak and tissue hypoxia, increased pericyte numbers, VEGF subfamily. After binding to its receptor VEGFR-1, it induced greater infiltration of T cells into the interior of the induces pro-cancer responses in endothelial, malignant, and tumor and showed a reduction in tumor growth by 60% (Zhao immune cells. Increased expression of PlGF is associated with et al. Accepted Cancer Res. 2017). Interestingly, such poor prognosis in HCC. Importantly, PlGF inhibition reduces vascular normalization effects plus the effect of an anti PD-1 tumor growth and induces vessel normalization in experi- monoclonal antibody showed a synergistic anti-tumor effect. 96,97 mental HCC models. Histidine-rich glycoprotein (HRG) is a host-produced protein deposited in the tumor stroma which can induce a change in polarization TAMs in favor of the M1- CURRENT CLINICAL ANTI-ANGIOGENIC THERAPIES: like phenotype. This effect of HRG on TAM polarization also DO THEY HAVE VESSEL NORMALIZING POTENTIAL? indirectly resulted in tumor vessel normalization. Both these Non-HCC directed therapies. The normalization hypoth- processes are PlGF-mediated. Furthermore, Vandewynckel 98 esis was initially proposed in an attempt to explain the et al. recently demonstrated that PlGF inhibition possibly observation that some anti-angiogenic therapies potentiate exerts its anti-tumor effects by improving intratumor hypoxia the effects of chemotherapy and radiotherapy. Multiple which is a potent activator of the pro-survival, PKR-like clinical trials have shown that combination therapy with endoplasmic reticulum kinase (PERK) pathway in HCC cells. bevacizumab (humanized anti-VEGF monoclonal antibody) A number of pharmacological agents that target the driver of and conventional chemotherapy improved patient survival abnormal vasculature, HIF-1α, have also been linked to and response rates over either therapy alone. This effect was vasculature normalization. EZN-2968 is a RNA antagonist observed across multiple advanced stage (metastatic) that specifically binds HIF-1α mRNA and reduces its expres- cancers: colorectal cancer, non-small cell lung cancer, breast sion by 80% and the expression of VEGF mRNA by 50% in 107–110 cancer and renal cancer. Given that anti-VEGF mice livers. A phase I study presented results demonstrating therapy aims to starve tumor blood supply and is associated its anti-tumor activity in patients with advanced malignancy with vessel pruning while chemotherapy relies on this same including HCC. Another suppresser of HIF-1α is NVP- blood supply for drug delivery, these findings were unex- BEZ235, a dual PI3K/mTOR inhibitor which induces apoptosis pected. It was hypothesized by Jain et al. that anti-VEGF of hypoxic cells. This novel agent has exhibited promising therapy augmented chemotherapy delivery and efficacy by activity against HCC. transiently reversing vessel abnormalities (and hence the Several vasoactive traditional Chinese medicine com- tumor microenvironment) without destroying them. In a pounds have exhibited anti-tumor efficacy against HCC in landmark study, the same group went on to demonstrate mouse xenograft models. Sinomenine hydrochloride, a known that within 12 days of infusion, a single dose of bevacizumab inducer of vascular normalization, has also been shown to in rectal carcinoma patients reduced microvascular density inhibit HCC growth by promoting cell cycle arrest and and improved vessel permeability, pericyte coverage and caspase-dependent apoptosis. Tanshinone IIA, a herbal interstitial hypertension. These are all markers of vascular extract from Chinese sage (Salvia miltiorrhiza), inhibited HCC normalization (Table 3). Conversely, Yang et al. recently metastasis and improved survival after palliative resection showed that discontinuation of anti-VEGF treatment in mice through the promotion of VEGFR-1/PDGFR-related vascular created a period of profound structural abnormality in liver normalization. This anti-metastatic potential was also seen sinusoidal capillaries with enlargement of pore sizes, loss of 105 112 in a study of Buyang Huanwu decoction. VE-cadherin and hyper-permeability. These changes led Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. Table 4 Potential approaches using vessel normalization in hepatocellular carcinoma Potential approaches using vessel normalization in hepatocellular carcinoma Normalization alone Normalization and established chemotherapy drugs (increase drug delivery) Normalization and multi-kinase inhibitors (increase drug delivery) Normalization and immunotherapy e.g., checkpoint inhibitors (increase entry and activity of anti-tumor immune cells) to tumor extravasation and marked promoted liver properties. An exploratory phase II RCT of sorafenib plus metastases in a mouse colorectal cancer model. doxorubicin vs. doxorubicin alone in advanced HCC found It is important to recognize that beneficial effects of anti- encouraging results with greater median PFS and OS seen in VEGF therapy may be dose dependent. According to Jain and combination therapy. However, the same authors could not colleagues, a paradox exists with anti-angiogenic therapy confirm the superiority of sorafenib plus doxorubicin compared where judicious application leads to selective pruning of with sorafenib in a phase III trial of 346 patients. No survival immature tumor vessels leaving a relatively normalized benefit was seen at the cost of higher toxicity. Trials of sunitinib network of vessels. While on the other hand, sustained or with reduced dosing showed modest activity with 2.9% RR, high doses may result in excessive regression of vasculature 3.9 months PFS, and 9.8 months OS. Interestingly, a trans leading to the same adverse effects of hypoxia seen in greater decrease in tumor vascular permeability (K , treatment attempts to starve the tumor. This delicate measured by dynamic contrast-enhanced magnetic reso- balance has been termed the “normalization window”. This nance imaging) at day 14 after treatment was associated with refers to a transient pharmacologically induced time period partial response or stable disease. This suggests that degree after the commencement of anti-angiogenic therapy during of vascular normalization may be a determinant of HCC which tumor vessels exhibit features of normalization and response to sunitinib. Nevertheless, a phase III trial of sunitinib improved functionality (reduced hypoxia) resulting in increa- vs. sorafenib in untreated patients with advanced HCC was sed vulnerability of cancer cells to cytotoxic therapies. The terminated early due to significantly worse survival (7.9 vs. commencement and duration of this window has been studied 10.2 months median OS) and higher toxicity. across different cancers using different vessel normalizing In recent developments, another multi-kinase inhibitor agents and can vary widely. For example, the time period of lenvatinib has been reported to have non-inferior overall increased oxygenation in mice melanoma, breast carcinoma survival compared to sorafenib for first-line treatment in and ovarian carcinoma models treated with bevacizumab was patients with unresectable HCC. In the second-line setting, demonstrated to be between day two and day four after regorafenib was also found to significantly improve OS in 115 134 starting treatment. In comparison, mice with human glioma patients with HCC who progressed on sorafenib. However, xenografts treated with anti-angiogenic agent suramin showed the effects of these treatments on vessel normalization are improved oxygenation compared to controls for up to 5 weeks not known. afterwards. The normalization window in human HCC is Targeting the epidermal growth factor signaling pathway via currently not known. erlotinib can reduce vascular permeability, tumor hypoxia and enhance responses to chemotherapy and radiation. Phase II studies in advanced HCC have demonstrated 0–9% RR, HCC directed therapies. In contrast to the aforementioned 136,137 3–4 months PFS and 10–13 months OS. However, the studies in other cancers, the synergistic effects of anti- combination of erlotinib with other anti-angiogenesis agents angiogenic therapies have not been reproduced in HCC. such as sorafenib and bevacizumab failed to show any Phase II trials of bevacizumab combined with systemic 138,139 117,118 additional benefit. chemotherapy did not show numerically superior Other agents directed against VEGFR such as cediranib, response rates (RR), progression-free survival (PFS), or axitinib and ramucirumab have all demonstrated normal- overall survival (OS) compared to bevacizumab alone or 119,120 ization properties in other cancers but have not been shown chemotherapy alone. to benefit HCC patients beyond the effect seen with Thalidomide exerts anti-angiogenic effects via inhibition of 140–145 sorafenib. Thus, although normalization effects are VEGF, bFGF and HIF-1α to improve tumor hypoxia and seen with some of the above agents, synergistic effects in interstitial hypertension—changes associated with vessel 121–123 human HCC either with chemotherapy or anti-angiogenic normalization. However, clinical trials of thalidomide 124,125 126 therapies have not so far been seen. either alone or in combination with chemotherapy or radiotherapy in unresectable HCC have been met with disappointing results (o5% RR). FUTURE PERSPECTIVES Although sorafenib and sunitinib can destroy tumor vascu- lature, they also both demonstrate the ability to normalize Vascular normalization with anti-angiogenic agents is now an tumor vasculature. Currently, sorafenib is licensed for use emerging approach to treat many cancers. In human HCC as monotherapy but it might prove effective when combined clinical trials, current anti-angiogenic therapies (beyond with chemotherapy on the basis of its vascular normalizing sorafenib) have not found the “normalization window” in order Clinical and Translational Gastroenterology Starving vs. normalizing blood supply to treat HCC Liu et al. to improve outcomes. It is likely that the optimal class, dose the results have failed to demonstrate comparable efficacy and schedule of these agents required to achieve normal- with the current standard of care, sorafenib. A better under- ization and yet avoid excessive pruning is not known. standing of the normalization window is required to guide Moreover, the exact timing of combination chemotherapy or dosing of anti-angiogenic therapy in relation to concomitant radiotherapy in order to capitalize on the normalization window therapies. The development of biomarkers may help in when their anti-tumor effects are enhanced is similarly unclear. selecting patients who benefit from these targeted therapies. These unknowns may partially explain why benefits seen in pre-clinical studies have not translated into clinically signifi- CONFLICT OF INTEREST cant improvements. Another unanswered question is whether and how this transient normalization window can be Guarantor of article: Geoffrey W McCaughan. prolonged. Clearly further characterization of the normal- Specific author contributions: Literature search, drafting of ization window is needed. Current approaches to treat HCC the article, critical revision of the article: Ken Liu, Geoffrey W using anti-VEGF therapies and multi-targeted tyrosine kinase McCaughan; contributed to draft, critical revision of the article: inhibitors are unlikely to be sufficient even if used appro- Xiang Zhang, Jinbiao Chen, Jun Yu, Jennifer R Gamble; priately. Therefore, a multipronged approach involving several created the figures, critical revision of the article: Weiqi Xu; anti-angiogenic pathways is likely to be required. Other All authors have read and approved the final version. relevant mediators of vessel normalization and their drug Financial support: None. targets also need to be explored. Novel strategies such as Potential conflicts of interest: None. targeting VE-cadherin particularly in combination with checkpoint inhibitors appear to be promising (Table 4). 1. Ferlay J, Soerjomataram I, Ervik M et al. GLOBOCAN 2012 v10, Cancer Incidence and Contributing to this challenge is a lack of validated surrogate Mortality Worldwide: IARC CancerBase No 11 [Internet] International Agency for Research biomarkers to signify when, or indeed if, vascular normal- on Cancer: Lyon, France, 2013. Available at http://globocan.iarc.fr/. Accessed on 5 February 2017. ization has occurred. In addition to guiding appropriate dosing 2. 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Published: Jun 1, 2017

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