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Atherosclerosis is the major pathology causing death in the developed world and, although risk factor modification has improved outcomes over the last decade, there is no cure. The role of the vasa vasora (VV) in the pathogenesis of atherosclerotic plaque is unclear but must relate to the predictability of diseased sites in the arterial tree. VV are small vessels found on major arteries and veins which supply nutrients and oxygen to the vessel wall itself while removing waste. Numerous studies have been carried out to investigate the anatomy and function of the VV as well as their significance in vascular disease. There is convincing evidence that VV are related to atherosclerotic plaque progression and vessel thrombosis, however, their link to the pathology of plaque initiation remains an interesting but neglected topic. We aim to present the evidence on the anatomy and functional behaviour of VV as well as their relationship to the initiation of atherosclerosis. At the same time, we wish to highlight inconsistencies in, and limitations of, the evidence available.

Diagnostic imaging of aggressive cancer with a high stroma content may benefit from the use of imaging contrast agents targeted with peptides that have high binding affinity to the extracellular matrix (ECM). In this study, we report the use of superparamagnetic iron-oxide nanoparticles (IO-NP) conjugated to a nonapeptide, CSGRRSSKC (CSG), which specifically binds to the laminin-nidogen-1 complex in tumours. We show that CSG-IO-NP accumulate in tumours, predominantly in the tumour ECM, following intravenous injection into a murine model of pancreatic neuroendocrine tumour (PNET). In contrast, a control untargeted IO-NP consistently show poor tumour uptake, and IO-NP conjugated to a pentapeptide. CREKA that bind fibrin clots in blood vessels show restricted uptake in the angiogenic vessels of the tumours. CSG-IO-NP show three-fold higher intratumoral accumulation compared to CREKA-IO-NP. Magnetic resonance imaging (MRI) T2-weighted scans and T2 relaxation times indicate significant uptake of CSG-IO-NP irrespective of tumour size, whereas the uptake of CREKA-IO-NP is only consistent in small tumours of less than 3 mm in diameter. Larger tumours with significantly reduced tumour blood vessels show a lack of CREKA-IO-NP uptake. Our data suggest CSG-IO-NP are particularly useful for detecting stroma in early and advanced solid tumours.

High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune‐modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin–nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα‐CSG fusion protein to tumour ECM in tumour‐bearing mice. Intravenously injected TNFα‐CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM‐rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour‐bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα., This study establishes a new approach to treat solid tumours by immune modulation and ECM depletion. The developed agent, TNFα‐CSG, has dual capacity as an immunotherapeutic and ECM reducing agent which improves tumour perfusion and drug delivery.

Compression optical coherence elastography (OCE) enables rapid acquisition with high resolution over fields of view relevant to many clinical applications. Compression OCE typically provides a relative measure of mechanical properties; however, we have recently demonstrated a technique which quantifies stiffness via a compliant layer, termed quantitative OCE. In quantitative OCE, stiffness is reported as a tangent modulus, which is a surrogate for Young’s modulus at a given preload in non-linear elastic material. In biological tissues, which are typically non-linear elastic, values of stiffness reported through quantitative OCE could be over- or under-estimated, and are heavily biased by the arbitrary bulk preload applied to that region. We present a method to measure tissue nonlinearity locally, by preforming compression OCE at multiple preloads ranging from 2% to 40%. We show, through presentation of 2D quantitative elastograms, that compression OCE has the potential to measure the non-linear stiffness in tissue mimicking phantoms and biological tissue. Further, intrinsic mechanical contrast in tissue is dependent upon its preload. By tailoring tissue preload, we demonstrate improved contrast between benign and tumor tissue in a murine liver carcinoma model.

In many applications of optical coherence elastography (OCE), it is necessary to rapidly acquire images in vivo, or within intraoperative timeframes, over fields-of-view far greater than can be achieved in one OCT image acquisition. For example, tumour margin assessment in breast cancer requires acquisition over linear dimensions of 4-5 centimetres in under 20 minutes. However, the majority of existing techniques are not compatible with these requirements, which may present a hurdle to the effective translation of OCE. To increase throughput, we have designed and developed an OCE system that simultaneously captures two 3D elastograms from opposite sides of a sample. The optical system comprises two interferometers: a common-path interferometer on one side of the sample and a dual-arm interferometer on the other side. This optical system is combined with scanning mechanisms and compression loading techniques to realize dual-scanning OCE. The optical signals scattered from two volumes are simultaneously detected on a single spectrometer by depth-encoding the interference signal from each interferometer. To demonstrate dual-scanning OCE, we performed measurements on tissue-mimicking phantoms containing rigid inclusions and freshly isolated samples of murine hepatocellular carcinoma, highlighting the use of this technique to visualise 3D tumour stiffness. These findings indicate that our technique holds promise for in vivo and intraoperative applications.

The ability to detect and quantify macrophage accumulation can provide important diagnostic and prognostic information for atherosclerotic plaque. We have previously shown that LyP-1, a cyclic 9-amino acid peptide, binds to p32 proteins on activated macrophages, facilitating the visualization of atherosclerotic plaque with PET. Yet, the in vivo plaque accumulation of monomeric [(18)F]FBA-LyP-1 was low (0.31 ± 0.05%ID/g). To increase the avidity of LyP-1 constructs to p32, we synthesized a dendritic form of LyP-1 on solid phase using lysine as the core structural element. Imaging probes (FAM or 6-BAT) were conjugated to a lysine or cysteine on the dendrimer for optical and PET studies. The N-terminus of the dendrimer was further modified with an aminooxy group in order to conjugate LyP-1 and ARAL peptides bearing a ketone. Oxime ligation of peptides to both dendrimers resulted in (LyP-1)4- and (ARAL)4-dendrimers with optical (FAM) and PET probes (6-BAT). For PET-CT studies, (LyP-1)4- and (ARAL)4-dendrimer-6-BAT were labeled with (64)Cu (t1/2 = 12.7 h) and intravenously injected into the atherosclerotic (ApoE(-/-)) mice. After two hours of circulation, PET-CT coregistered images demonstrated greater uptake of the (LyP-1)4-dendrimer-(64)Cu than the (ARAL)4-dendrimer-(64)Cu in the aortic root and descending aorta. Ex vivo images and the biodistribution acquired at three hours after injection also demonstrated a significantly higher uptake of the (LyP-1)4-dendrimer-(64)Cu (1.1 ± 0.26%ID/g) than the (ARAL)4-dendrimer-(64)Cu (0.22 ± 0.05%ID/g) in the aorta. Similarly, subcutaneous injection of the LyP-1-dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. In the same model system, ex vivo fluorescence images within aortic plaque depict an increased accumulation and penetration of the (LyP-1)4-dendrimer-FAM as compared to the (ARAL)4-dendrimer-FAM. Taken together, the results suggest that the (LyP-1)4-dendrimer can be applied for in vivo PET imaging of plaque and that LyP-1 could be further exploited for the delivery of therapeutics with multivalent carriers or nanoparticles.

Stroma is an integral part of solid tumors and plays a key role in growth promotion and immune suppression. Most current therapies focus on destroying tumors and/or abnormal vasculature. However, evidence is emerging that anticancer efficacy improves with vessel normalization rather than destruction. Specific targeting of cytokines into tumors provides proof-of-concept that tumor stroma is dynamic and can be remodeled to increase drug access and alleviate immune suppression. Changing the inflammatory milieu 'opens' tumors for therapy and thus provides a license for destruction. This involves reprogramming of paracrine signaling networks between multiple stromal components to break the vicious cycle of angiogenesis and immune suppression. With active immunotherapy rapidly moving into the clinic, local cytokine delivery emerges as an attractive adjuvant.

Atherosclerosis treatments are generally aimed at altering systemic lipid metabolism such that atherogenesis, the formation of plaque, is curtailed. The plaques themselves offer some potential therapeutic targets. For example, selective depletion of macrophages, which play a key role in atherogenesis, inhibits plaque formation. However, it has not been possible to take advantage of these targets because the drugs that have been tested have not been sufficiently selective. We have developed a peptide, LyP-1, which specifically targets atherosclerotic plaques, penetrates into plaque interior, and accumulates in plaque macrophages. In tumors, LyP-1 can cause apoptosis in cells that take up the peptide. Here we show, using three different atherosclerosis models in ApoE null mice that prolonged systemic treatment with LyP-1 triggers apoptosis of plaque macrophages and reduces plaque in advanced hypoxic plaques, and that it does so without increasing necrotic core of plaques or causing detectable side effects. We also show that LyP-1 recognizes human plaque. These findings suggest that LyP-1 could serve as a lead compound for the development of a new class of anti-atherosclerosis drugs.

We have recently described a class of peptides that improve drug delivery by increasing penetration of drugs into solid tumors. These peptides contain a C-terminal C-end Rule (CendR) sequence motif (R/K)XX(R/K), which is responsible for cell internalization and tissue-penetration activity. Tumor-specific CendR peptides contain both a tumor-homing motif and a cryptic CendR motif that is proteolytically unmasked in tumor tissue. A previously described cyclic tumor-homing peptide, LyP-1 (sequence: CGNKRTRGC), contains a CendR element and is capable of tissue penetration. We use here the truncated form of LyP-1, in which the CendR motif is exposed (CGNKRTR; tLyP-1), and show that both LyP-1 and tLyP-1 internalize into cells through the neuropilin-1-dependent CendR internalization pathway. Moreover, we show that neuropilin-2 also binds tLyP-1 and that this binding equally activates the CendR pathway. Fluorescein-labeled tLyP-1 peptide and tLyP-1-conjugated nanoparticles show robust and selective homing to tumors, penetrating from the blood vessels into the tumor parenchyma. The truncated peptide is more potent in this regard than the parent peptide LyP-1. tLyP-1 furthermore improves extravasation of a co-injected nanoparticle into the tumor tissue. These properties make tLyP-1 a promising tool for targeted delivery of therapeutic and diagnostic agents to breast cancers and perhaps other types of tumors.

The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1 + cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1–coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[ 18 F]fluorobenzoic acid ([ 18 F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [ 18 F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [ 18 F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

Accessibility of tumors for highly effective local treatment represents a major challenge for anticancer therapy. Immunostimulatory oligodeoxynucleotides (ODN) with CpG motifs are ligands of TLR9, which prime spontaneous antitumor immunity, but are less effective when applied systemically. We therefore developed a liposome-based agent for selective delivery of CpG-ODN into the tumor environment. A peptide that specifically targets angiogenic endothelial cells in a transgenic tumor model for islet cell carcinogenesis was engrafted into CpG-ODN containing liposomes. Intravenous injection of these liposomes resulted in specific accumulation around tumor vessels, increased uptake by tumor-resident macrophages, and retention over time. In contrast, nontargeted liposomes did not localize to the tumor vasculature. Consequently, only vascular targeting of CpG-ODN liposomes provoked a marked inflammatory response at vessel walls with enhanced CD8+ and CD4+ T cell infiltration and, importantly, activation of spontaneous, tumor-specific cytotoxicity. In a therapeutic setting, 40% of tumor-bearing, transgenic mice survived beyond week 45 after systemic administration of vascular-directed CpG-ODN liposomes. In contrast, control mice survived up to 30 wk. Therapeutic efficacy was further improved by increasing the frequency of tumor-specific effector cells through adoptive transfers. NK cells and CD8+ T cells were major effectors which induced tumor cell death and acted in conjunction with antivascular effects. Thus, tumor homing with CpG-ODN-loaded liposomes is as potent as direct injection of free CpG-ODN and has the potential to overcome some major limitations of conventional CpG-ODN monotherapy.

Current anticancer therapy is a delicate balance between elimination of malignant cells and harmful side effects for the host. In this study, we used a tumor-homing peptide to engineer anti-CD40 agonist antibodies and recombinant IL-2 such that they were selectively delivered into spontaneously arising tumors in a transgenic mouse model of islet cell carcinogenesis. Intravenous injection of these agents, either separately or together, led to accumulation in the vicinity of tumor neovessels without toxic side effects. Although both molecules are critical for adaptive immunity, the most profound effects were seen in endothelial cells. Combined, local anti-CD40 and IL-2 therapy reduced tumor vascularity and significantly delayed tumor growth in mice. Remarkably, tumor-bearing mice remained disease-free long-term when targeted anti-CD40 and IL-2 were combined with transfers of preactivated antitumor immune cells. In this therapeutic setting, triggering of CD40 on endothelial cells induced an inflammatory response of the vessel wall and facilitated effector cell accumulation in the tumor parenchyma while IL-2 promoted antigen-specific immune cell persistence. We believe this is a novel and highly effective anticancer approach, whereby tumor stroma is “conditioned” for enhanced immune cell entry and survival, facilitating immune-mediated tumor destruction and leading to a sustained antitumor response.

Current clinical success with anti-cancer immunotherapy provides exciting new treatment opportunities. While encouraging, more needs to be done to induce durable effects in a higher proportion of patients. Increasing anti-tumor effector T cell quantity or quality alone does not necessarily correlate with therapeutic outcome. Instead, the tumor microenvironment is a critical determinant of anti-cancer responsiveness to immunotherapy and can confer profound resistance. Yet, the tumor-promoting environment - due to its enormous plasticity - also delivers the best opportunities for adjuvant therapy aiming at recruiting, priming and sustaining anti-tumor cytotoxicity. While the tumor environment as an entity is increasingly well understood, current interventions are still broad and often systemic. In contrast, tumors grow in a highly compartmentalized environment which includes the vascular/perivascular niche, extracellular matrix components and in some tumors lymph node aggregates; all of these structures harbor and instruct subsets of immune cells. Targeting and re-programming specific compartments may provide better opportunities for adjuvant immunotherapy.

Drug treatment of severe malaria must be rapidly effective. Suppositories may be valuable for childhood malaria when circumstances prevent oral or parenteral therapy. We compared artesunate suppositories ( n = 41; 8 to 16 mg/kg of body weight at 0 and 12 h and then daily) with intramuscular (i.m.) artemether ( n = 38; 3.2 mg/kg at 0 h and then 1.6 mg/kg daily) in an open-label, randomized trial with children with severe Plasmodium falciparum malaria in Papua New Guinea (PNG). Parasite density and temperature were measured every 6 h for ≥72 h. Primary endpoints included times to 50% and 90% parasite clearance (PCT 50 and PCT 90 ) and the time to per os status. In a subset of 29 patients, plasma levels of artemether, artesunate, and their common active metabolite dihydroartemisinin were measured during the first 12 h. One suppository-treated patient with multiple complications died within 2 h of admission, but the remaining 78 recovered uneventfully. Compared to the artemether-treated children, those receiving artesunate suppositories had a significantly earlier mean PCT 50 (9.1 versus 13.8 h; P = 0.008) and PCT 90 (15.6 versus 20.4 h; P = 0.011). Mean time to per os status was similar for each group. Plasma concentrations of primary drug plus active metabolite were significantly higher in the artesunate suppository group at 2 h postdose. The earlier initial fall in parasitemia with artesunate is clinically advantageous and mirrors higher initial plasma concentrations of active drug/metabolite. In severely ill children with malaria in PNG, artesunate suppositories were at least as effective as i.m. artemether and may, therefore, be useful in settings where parenteral therapy cannot be given.

A preliminary study from our laboratory found retinol (vitamin A alcohol) to have in vitro activity against Plasmodium falciparum at concentrations close to those in normal human serum (1-3 muM). To characterize the antimalarial potential of retinol in more detail, the 3D7 and K1 laboratory strains of P. falciparum were maintained in continuous culture and [H-3]hypoxanthine incorporation and microscopy were used to assess the effect of retinol against asexual stages of the parasite life-cycle. Losses of retinol and retinol-associated hemolysis were also quantified in the in vitro culture system. There were retinol losses of >50% but no hemolysis was observed with added retinol concentrations up to 100 M. All stages of parasite development showed comparable sensitivity to retinol including merozoite invasion (range of mean IC50 values 10.1-21.4 muM after adjustment for losses). Retinol pre-treatment of uninfected RBC did not inhibit merozoite invasion. Retinol treatment was associated with increased vacuolization within the parasite food vacuole and evidence of parasite membrane rupture. These appearances were similar to those seen with quinoline and artemisinin compounds. Although these data do not support a role for acute retinol supplementation in the treatment of falciparum malaria, they add to knowledge regarding potential antimalarial therapies and justify assessment of more potent synthetic retinoids and their metabolites. (C) 2004 Elsevier Inc. All rights reserved.

In this paper, we describe a technique capable of visualizing mechanical properties at the cellular scale deep in living tissue, by incorporating a gradient-index (GRIN)-lens micro-endoscope into an ultrahigh-resolution optical coherence elastography system. The optical system, after the endoscope, has a lateral resolution of 1.6 µm and an axial resolution of 2.2 µm. Bessel beam illumination and Gaussian mode detection are used to provide an extended depth-of-field of 80 µm, which is a 4-fold improvement over a fully Gaussian beam case with the same lateral resolution. Using this system, we demonstrate quantitative elasticity imaging of a soft silicone phantom containing a stiff inclusion and a freshly excised malignant murine pancreatic tumor. We also demonstrate qualitative strain imaging below the tissue surface on in situ murine muscle. The approach we introduce here can provide high-quality extended-focus images through a micro-endoscope with potential to measure cellular-scale mechanics deep in tissue. We believe this tool is promising for studying biological processes and disease progression in vivo.

Depth-encoded optical coherence elastography (OCE) enables simultaneous acquisition of two three-dimensional (3D) elastograms from opposite sides of a sample. By the choice of suitable path-length differences in each of two interferometers, the detected carrier frequencies are separated, allowing depth-ranging from each interferometer to be performed simultaneously using a single spectrometer. We demonstrate depth-encoded OCE on a silicone phantom and a freshly excised sample of mouse liver. This technique minimizes the required spectral detection hardware and halves the total scan time. Depth-encoded OCE may expedite clinical translation in time-sensitive applications requiring rapid 3D imaging of multiple tissue surfaces, such as tumor margin assessment in breast-conserving surgery.

Trovafloxacin, a recently-developed fourth-generation fluoroquinolone, is more potent than other quinolone drugs against a wide range of organisms including Toxoplasma gondii . We assessed the in vitro antimalarial activity of trovafloxacin against three laboratory-adapted Plasmodium falciparum isolates and compared the results with those of ciprofloxacin and norfloxacin. Synchronous and asynchronous cultures were exposed to a range of drug concentrations, and growth inhibition was assessed using 3 H-hypoxanthine incorporation. All isolates, both synchronous and asynchronous, exhibited comparable sensitivities with trovafloxacin (EC 50 range, 1.8×10 −5 to 3.7×10 −5 mol/l) and ciprofloxacin (2.0×10 −5 to 3.9×10 −5 mol/l), but were less sensitive to norfloxacin (5.4×10 −5 to 6.6×10 −4 mol/l). These results confirm that ciprofloxacin is twice as potent as norfloxacin against P. falciparum in vitro, but also show that trovafloxacin and ciprofloxacin have similar antimalarial potency. The EC 50 concentrations of all three drugs were generally higher than those achieved after conventional doses in humans, suggesting that their clinical application may be limited to combination therapy. Recent reports of hepatotoxicity with trovafloxacin may also prevent the use of this drug in humans. However, newer fourth-generation quinolones may prove safer and have similar antimalarial potency.

In an in vitro assessment of antimalarial combinations, dihydroartemisinin (DHA) showed no interaction or was mildly antagonistic when combined with piperaquine, pyronaridine, or naphthoquine. Interactions between 4-aminoquinolines and related drugs were also indifferent/antagonistic. The clinical significance of mildly antagonistic DHA combinations is uncertain but may become important if parasite drug sensitivity declines.

As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded a 200-fold increase in binding affinity and restored anti-VCAM-1 binding in tissue sections from ApoE(−/−) mice ex vivo. The pro-antibody was efficiently activated by native proteases in aorta tissue extracts from ApoE(−/−), but not from normal mice, and accumulated in aortic plaques in vivo with enhanced selectivity when compared to the unmodified antibody. Pro-antibody accumulation in aortic plaques was MMP-dependant, and significantly inhibited by a broad-spectrum MMP inhibitor. These results demonstrate that the activity of disease-associated proteases can be exploited to site-specifically target antibody activity in vivo.

G protein–coupled biological processes are important for an ever-increasing number of human diseases and require fine-tuning through accessory molecules such as the regulators of G protein signaling (RGS). RGS5, a marker for tumor-resident pericytes, was recently established as playing a pivotal role in vascular maturation and vessel remodeling during carcinogenesis. Remarkably, tumors arising in a RGS5-deficient background display vessels with normalized morphology and an overall improved blood flow. Furthermore, these morphologic changes also lead to dramatic improvements in lymphocyte access to tumors and success of antitumor immunotherapy. Here, we consider the implications of these findings, and how they contribute to enhancing our understanding of remodeling angiogenic vessels as means for improving anticancer therapies. [Cancer Res 2009;69(2):396–9]

The vasculature of solid tumours is morphologically aberrant and characterized by dilated and fragile vessels, intensive vessel sprouting and loss of hierarchical architecture. Constant vessel remodelling leads to spontaneous haemorrhages and increased interstitial fluid pressure in the tumour environment. Tumour-related angiogenesis supports tumour growth and is also a major obstacle for successful immune therapy as it prevents migration of immune effector cells into established tumour parenchyma. The molecular mechanisms for these angiogenic alterations are largely unknown. Here we identify regulator of G-protein signalling 5 (Rgs5) as a master gene responsible for the abnormal tumour vascular morphology in mice. Loss of Rgs5 results in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. These vascular and intratumoral changes enhance influx of immune effector cells into tumour parenchyma and markedly prolong survival of tumour-bearing mice. This is the first demonstration, to our knowledge, of reduced tumour angiogenesis and improved immune therapeutic outcome on loss of a vascular gene function and establishes a previously unrecognized role of G-protein signalling in tumour angiogenesis.

Reduced plasma retinol concentrations occur in human malaria but the benefits of supplementation remain uncertain. We assessed the in vivo efficacy of retinol administration, and its effect on lipid peroxidation, in a Plasmodium berghei murine model. Animals received vehicle (n = 17) or retinol (i) before P. berghei inoculation (four doses), (ii) at parasitaemia 10–15% (three to four doses) or (iii) before and after inoculation (six to seven doses; n = 15 in each group), with euthanasia on day 8 post-inoculation or when the parasitaemia exceeded 50%. Multiple-dose pre-inoculation retinol reduced endpoint parasitaemia by 24% (P = 0.001 versus controls). A reduction of 18% (P = 0.042) was observed when retinol was given to parasitaemic animals. Retinol was ineffective when given both before and after infection (11% reduction; P = 0.47). Although retinol supplementation did not change plasma retinol concentrations, liver retinol content increased and correlated inversely with endpoint parasitaemia (r = −0.45, P = 0.001). Malaria infection augmented concentrations of the free radical lipid peroxidation end-product F2-isoprostanes in plasma, erythrocytes and liver by 1.8-, 2.8- and 4.9-fold, respectively, but retinol supplementation had no effect on these increases. Consistent with some human malaria studies, prophylactic retinol reduces P. berghei parasitaemia. This effect relates to augmentation of tissue retinol stores rather than to retinol-associated changes in oxidant status.

To determine whether travellers from malaria-free countries can develop unrecognised or asymptomatic malaria after visiting endemic areas, we analysed data from 751 prospective blood donors who had visited or resided in malaria-risk areas in the previous 3 years. Malarial antibodies were measured using an established commercial enzyme-linked immunoassay incorporating four recombinant blood-stage plasmodial antigens and with published sensitivity ≥83% and specificity 100%. Details of countries visited and antimalarial chemoprophylaxis used were obtained by questionnaire. Among the 606 subjects resident in malaria-free countries and with no past history of malaria, 176 had visited high-risk countries as categorised by the World Health Organisation (WHO). Of these, 89 took no chemoprophylaxis including 6 (6.7%) who were antibody-positive; there were no antibody-positive subjects in the 87 who took chemoprophylaxis ( P = 0.029), which was that recommended by WHO in 84% of cases. These data underscore the value of effective antimalarial prophylaxis in non-immune travellers visiting high malaria-risk areas but also suggest that unrecognised infections can occur in those unprotected by chemoprophylaxis.

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0+/-0.9)x10(-3) mL mol(-1). The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88+/-0.60)x10(-6) relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.

Solid tumors are frequently resistant to immunotherapy. We demonstrated that low-dose tumor necrosis factorα (TNFα), when directly targeted to the tumor environment, exerts dual effects by improving vessel functionality and activating immune cells. This vascular remodeling in an inflammatory context enhances active immunotherapy and promotes tumor regression.

Solid tumors are intrinsically resistant to immune rejection. Abnormal tumor vasculature can act as a barrier for immune cell migration into tumors. We tested whether targeting IFNγ and/or TNFα into pancreatic neuroendocrine tumors can alleviate immune suppression. We found that intratumoral IFNγ causes rapid vessel loss, which does not support anti-tumor immunity. In contrast, low-dose TNFα enhances T-cell infiltration and overall survival, an effect that is exclusively mediated by CD8 + effector cells. Intriguingly, lymphocyte influx does not correlate with increased vessel leakiness. Instead, low-dose TNFα stabilizes the vascular network and improves vessel perfusion. Inflammatory vessel remodeling is, at least in part, mediated by tumor-resident macrophages that are reprogrammed to secrete immune and angiogenic modulators. Moreover, inflammatory vessel remodeling with low-dose TNFα substantially improves antitumor vaccination or adoptive T-cell therapy. Thus, low-dose TNFα promotes both vessel remodeling and antitumor immune responses and acts as a potent adjuvant for active immunotherapy.