Modulation of the "blood-tumor" barrier improves immunotherapy

Blood vessels inside tumors are crucial for cancer survival and progression but equally contribute to the tumor's intrinsic resistance to therapy. Abnormal blood flow in the local tumor environment acts as a physiological barrier to the delivery of chemotherapeutic agents. Furthermore, tumor vasculature can also act as a barrier for immune cell migration into the tumor parenchyma. Much has been made of anti-angiogenic therapies that specifically inhibit vessel growth. However, recent findings demonstrate that the chaotic architecture of tumor blood vessels can be reversed which in turn normalizes blood flow and physical parameters in the tumor environment. Importantly, vessel normalization also improves lymphocyte migration into tumor tissue and immune destruction. Identification of regulator of G protein signaling 5 (RGS5) as a key modulator of the vascular barrier in tumor progression and regression has brought new insights into the molecular basis of vessel normalization and opens new therapeutic opportunities.     

Targeting tumor micro-environment for design and development of novel anti-angiogenic agents arresting tumor growth

Angiogenesis: a process of generation of new blood vessels has been proved to be necessary for sustained tumor growth and cancer progression. Inhibiting angiogenesis pathway has long been remained a significant hope for the development of novel, effective and target orientated antitumor agents arresting the tumor proliferation and metastasis. The process of neoangiogenesis as a biological process is regulated by several pro- and anti-angiogenic factors, especially vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, hypoxia inducible factor 1 and transforming growth factor. Every endothelial cell destined for vessel formation is equipped with receptors for these angiogenic peptides. Moreover, numerous other angiogenic cytokines such as platelet derived growth factor (PGDF), placenta growth factor (PGF), nerve growth factor (NGF), stem-cell factor (SCF), and interleukins-2, 4, 6 etc. These molecular players performs critical role in regulating the angiogenic switch. Couple of decade's research in molecular aspects of tumor biology has unraveled numerous structural and functional mysteries of these angiogenic peptides. In present article, a detailed update on the functional and structural peculiarities of the various angiogenic peptides is described focusing on structural opportunities made available that has potential to be used to modulate function of these angiogenic peptides in developing therapeutic agents targeting neoplastic angiogenesis. The data may be useful in the mainstream of developing novel anticancer agents targeting tumor angiogenesis. We also discuss major therapeutic agents that are currently used in angiogenesis associated therapies as well as those are subject of active research or are in clinical trials.     

Sensitivity of MRI tumor biomarkers to VEGFR inhibitor therapy in an orthotopic mouse glioma model

MRI biomarkers of tumor edema, vascular permeability, blood volume, and average vessel caliber are increasingly being employed to assess the efficacy of tumor therapies. However, the dependence of these biomarkers on a number of physiological factors can compromise their sensitivity and complicate the assessment of therapeutic efficacy. Here we examine the response of these MRI tumor biomarkers to cediranib, a potent vascular endothelial growth factor receptor (VEGFR) inhibitor, in an orthotopic mouse glioma model. A significant increase in the tumor volume and relative vessel caliber index (rVCI) and a slight decrease in the water apparent diffusion coefficient (ADC) were observed for both control and cediranib treated animals. This contrasts with a clinical study that observed a significant decrease in tumor rVCI, ADC and volume with cediranib therapy. While the lack of a difference between control and cediranib treated animals in these biomarker responses might suggest that cediranib has no therapeutic benefit, cediranib treated mice had a significantly increased survival. The increased survival benefit of cediranib treated animals is consistent with the significant decrease observed for cediranib treated animals in the relative cerebral blood volume (rCBV), relative microvascular blood volume (rMBV), transverse relaxation time (T2), blood vessel permeability (K(trans)), and extravascular-extracellular space (ν(e)). The differential response of pre-clinical and clinical tumors to cediranib therapy, along with the lack of a positive response for some biomarkers, indicates the importance of evaluating the whole spectrum of different tumor biomarkers to properly assess the therapeutic response and identify and interpret the therapy-induced changes in the tumor physiology.     

Angiogenesis in gynecological cancers and the options for anti-angiogenesis therapy

Angiogenesis is required in cancer, including gynecological cancers, for the growth of primary tumors and secondary metastases. Development of anti-angiogenesis therapy in gynecological cancers and improvement of its efficacy have been a major focus of fundamental and clinical research. However, survival benefits of current anti-angiogenic agents, such as bevacizumab, in patients with gynecological cancer, are modest. Therefore, a better understanding of angiogenesis and the tumor microenvironment in gynecological cancers is urgently needed to develop more effective anti-angiogenic therapies, either or not in combination with other therapeutic approaches. We describe the molecular aspects of (tumor) blood vessel formation and the tumor microenvironment and provide an extensive clinical overview of current anti-angiogenic therapies for gynecological cancers. We discuss the different phenotypes of angiogenic endothelial cells as potential therapeutic targets, strategies aimed at intervention in their metabolism, and approaches targeting their (inflammatory) tumor microenvironment.     

肿瘤血管靶向药物的研究进展

肿瘤血管在实体瘤的发生发展中具有重要的作用,靶向肿瘤血管的新药研发已成为一个热点领域.抗肿瘤血管的治疗策略分为肿瘤新生血管生成抑制剂（tumor angiogenesis inhibitor,TAI）和肿瘤血管靶向药物（vascular targeting agents,VTAs）两方面的研究.肿瘤新生血管生成抑制剂旨在抑制肿瘤新生血管生成的过程,而肿瘤血管靶向药物则是通过快速而有选择性地破坏肿瘤血管功能,使肿瘤血供受阻,导致肿瘤坏死.VTA类药物分为两类：一是小分子抑制剂（small molecule agents）,利用肿瘤血管和正常组织血管存在的差别选择性地破坏肿瘤血管;另一种是生物制剂（biological agents）,借助能够特异结合肿瘤血管的配体将毒素、凝血诱导剂、凋亡诱导分子等运送到肿瘤血管,引起血管阻塞使肿瘤坏死.     

Screening assay for blood vessel maturation inhibitors

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.     

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.     

Diverse multidrug-resistance-modification agents inhibit cytolytic activity of natural killer cells

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells selected for resistance to one chemotherapeutic agent simultaneously acquire resistance to several apparently unrelated drugs. MDR in tumor cells is associated with the over-expression of P-glycoprotein, an ATP-dependent cell-membrane transport molecule. P-glycoprotein is also expressed in several normal tissues but its physiological role(s) is unknown. We recently observed that a hierarchy of MDR-like activity exists among human peripheral blood lymphocytes in the order CD8>CD4>CD20 (cytoxic/suppressor T cells, helper T cells and B cells respectively). In this study, we report that natural killer (NK) cells also express MDR-like activity. This activity could be inhibited with verapamil or solutol HS-15, two agents that reverse MDR in tumor cells. These, and four additional reversing agents, were used to investigate the possible role of P-glycoprotein in NK cells. We observed that at 10% of their IC₅₀, five of six reversing agents inhibited NK-cell-mediated cytotoxicity; at higher (but non-toxic) doses, all six agents were inhibitory. These data suggest that NK-cell-mediated cytotoxicity may require the functional expression of an efflux molecule similar or identical to P-glycoprotein.This work was supported in part by grants from the National Cancer Institute (CA 57 470), the American Cancer Association — Illinois Division (no. 91-48), The Arthur Andersen Foundation and the Rayman, Freidman, Ditore and Rosenmutter Funds.     

Biological and anti-vascular activity evaluation of ethoxy-erianin phosphate as a vascular disrupting agent

The effects of ethoxy-erianin phosphate (EBTP) on cell proliferation, mitotic cell arrest, migration, infiltration, and endothelial tubular structures were evaluated in this study. The antiproliferative activity of EBTP and combretastatin A-4P (CA4P) was analyzed on several tumor cells (including MCF-7, HeLa, 2LL, and 2LL-IDO) and on an endothelial cell (human umbilical vein endothelial cells [HUVECs]) as well as a human normal liver cell (L02). The results showed that EBTP possessed antiproliferative activity in the micromole range and was relatively less toxic than CA4P. Treating HUVECs with EBTP caused cell accumulation in the G2/M phase, and wound-healing assays indicated that EBTP inhibited cell migration. Furthermore, EBTP and CA4P destroyed the vasculature in endothelial cells and showed vascular disrupting activity of the chorioallantoic membrane in fertilized chicken eggs. In addition, we found that EBTP suppressed the expression of indoleamine 2,3-dioxygenase (IDO) and significantly inhibited IDO-induced migration and infiltration of 2LL-IDO cells. Administration of EBTP blocked vasculogenic mimicry in 2LL-IDO cells, which was typically observed in tube formation assays of 2LL-IDO cells. Moreover, the results of Lewis lung carcinoma in mice showed a high inhibition rate of EBTP. EBTP is an effective vascular disrupting agent that is superior to CA4P and may prevent and treat malignancy by inhibiting the expression of IDO.     

高能聚焦超声在治疗晚期胰腺癌中的疗效分析

目的：通过对晚期胰腺癌患者行高强度聚焦超声治疗,观察其近期疗效,为晚期胰腺癌患者的临床治疗提供一种新的选择。方法：对15例晚期胰腺癌患者行高强度聚焦超声治疗,对比治疗前后的KPS评分,疼痛感受评分,CA199,三大常规,生化检查,B超观察肿瘤回声及血供,CT观察肿瘤大小改变。结果：HIFU治疗后,患者KPS评分升高,疼痛评分下降,肿瘤标志物下降,B超观察肿瘤回声,其中10例肿瘤回声增强,11例肿瘤血供减少或消失,CT示大部分患者治疗后肿瘤体积缩小或不变,治疗后三大常规、生化和电解质无明显改变。结论：运用高强度聚焦超声治疗晚期胰腺癌患者,在改善患者临床症状方面有明显疗效,并且能缩小肿瘤体积,减少或中断肿瘤血供,是一种很有发展前景的无创治疗方法。     

Growing tumor vessels: more than one way to skin a cat - implications for angiogenesis targeted cancer therapies

The establishment of a functional, integrated vascular system is instrumental for tissue growth and homeostasis. Without blood vessels no adequate nutrition and oxygen would be provided to cells, nor could the undesired waste products be efficiently removed. Blood vessels constitute therefore one of the largest and most complex body network whose assembly depends on the precise balance of growth factors acting in a complementary and coordinated manner with cells of several identities. However, the vessels that are crucial for life can also foster death, given their involvement in cancer progression towards malignancy and metastasis. Targeting tumor vasculature has thus arisen as an appealing anti-cancer therapeutic approach. Since the milestone achievements that vascular endothelial growth factor (VEGF) blockade suppressed angiogenesis and tumor growth in mice and prolonged the survival of cancer patients when administered in combination with chemotherapy, the clinical development of anti-VEGF(R) drugs has accelerated remarkably. FDA has approved the use of bevacizumab – a humanized monoclonal antibody against VEGF – in colorectal, lung and metastatic breast cancers in combination with standard chemotherapy. Additional broad-spectrum VEGF receptor tyrosine kinase inhibitors, such as sunitinib and sorafenib, are used in monotherapy for metastatic renal carcinoma, while sunitinib is also approved for imatinib resistant gastrointestinal stromal tumors and sorafenib for advanced stage hepatocellular carcinoma. Nevertheless, the survival benefit offered by VEGF(R) blockers, either as single agents or in combination with chemotherapy, is calculated merely in the order of months. Posterior studies in preclinical models have reported that despite reducing primary tumor growth, the inhibition of VEGF increased tumor invasiveness and metastasis. The clinical implications of these findings urge the need to reconcile these conflicting results. Anti-angiogenic therapy represents a significant step forth in cancer therapy and in our understanding of cancer biology, but it is also clear that we need to learn how to use it. What is the biological consequence of VEGF-blockade? Does VEGF inhibition starve the tumor to death – as initially postulated – or does it rather foster malignancy? Can anti-VEGF(R) therapy favor tumor vessel formation by VEGF-independent means? Tumors are very diverse and plastic entities, able to adapt to the harshest conditions; this is also reflected by the tumor vasculature. Lessons from the bench to the bedside and vice versa have taught us that the diversity of signals underlying tumor vessel growth will likely be responsive (or resistant) to distinct therapeutic approaches. In this review, we propose a reflection of the different strategies tumors use to grow blood vessels and how these can have impact on the (un)success of current anti-angiogenic therapies.     

Design,synthesis, and biological evaluation of water-soluble amino acid prodrug conjugates derived from combretastatin,dihydronaphthalene, and benzosuberene-based parent vascular disrupting agents

Targeting tumor vasculature represents an intriguing therapeutic strategy in the treatment of cancer. In an effort to discover new vascular disrupting agents with improved water solubility and potentially greater bioavailability, various amino acid prodrug conjugates (AAPCs) of potent amino combretastatin, amino dihydronaphthalene, and amino benzosuberene analogs were synthesized along with their corresponding water-soluble hydrochloride salts. These compounds were evaluated for their ability to inhibit tubulin polymerization and for their cytotoxicity against selected human cancer cell lines. The amino-based parent anticancer agents 7, 8, 32 (also referred to as KGP05) and 33 (also referred to as KGP156) demonstrated potent cytotoxicity (GI₅₀ = 0.11–40 nM) across all evaluated cell lines, and they were strong inhibitors of tubulin polymerization (IC₅₀ = 0.62–1.5 μM). The various prodrug conjugates and their corresponding salts were investigated for cleavage by the enzyme leucine aminopeptidase (LAP). Four of the glycine water-soluble AAPCs (16, 18, 44 and 45) showed quantitative cleavage by LAP, resulting in the release of the highly cytotoxic parent drug, whereas partial cleavage (<10–90%) was observed for other prodrugs (15, 17, 24, 38 and 39). Eight of the nineteen AAPCs (13–16, 42–45) showed significant cytotoxicity against selected human cancer cell lines. The previously reported CA1-diamine analog and its corresponding hydrochloride salt (8 and 10, respectively) caused extensive disruption (at a concentration of 1.0 μM) of human umbilical vein endothelial cells growing in a two-dimensional tubular network on matrigel. In addition, compound 10 exhibited pronounced reduction in bioluminescence (greater than 95% compared to saline control) in a tumor bearing (MDA-MB-231-luc) SCID mouse model 2 h post treatment (80 mg/kg), with similar results observed upon treatment (15 mg/kg) with the glycine amino-dihydronaphthalene AAPC (compound 44). Collectively, these results support the further pre-clinical development of the most active members of this structurally diverse collection of water-soluble prodrugs as promising anticancer agents functioning through a mechanism involving vascular disruption.     

Endpoints,patient selection,and biomarkers in the design of clinical trials for cancer vaccines

Therapeutic cancer vaccines are an emerging and potentially effective treatment modality. Cancer vaccines are usually very well tolerated, with minimal toxicity compared with chemotherapy. Unlike conventional cytotoxic therapies, immunotherapy does not result in immediate tumor shrinkage but may alter growth rate and thus prolong survival. Multiple randomized controlled trials of various immunotherapeutic agents have shown a delayed separation in Kaplan–Meier survival curves, with no evidence of clinical benefit within the first 6–12 months of vaccine treatment. Overall survival benefit is seen in patients with lower disease burden who are not expected to die within those initial 6–12 months. The concept of improved overall survival without marked initial tumor reduction represents a significant shift from the current paradigms established by standard cytotoxic therapies. Future clinical studies of therapeutic vaccines should enroll patients with either lower tumor burden, more indolent disease or both, and must seek to identify early markers of clinical benefit that may correlate with survival. Until then, improved overall survival is the only clear, discriminatory endpoint for therapeutic vaccines as monotherapies.     

肿瘤精准医学知识数据库的设计与构建

目的:整合现有前沿的大量而分散的精准医学知识以形成系统完整的知识数据库,为个体组学数据的临床应用提供依据,旨在最终实现基于组学特征的精准用药推荐。方法:采用MySQL数据库管理系统构建数据库,从FDA伴随诊断、NCCN指南、My Cancer Genome、GDSC四大权威医学资源中手动收集精准用药知识,并将原始数据标准化、结构化后以统一的格式存储。结果:成功设计并构建了肿瘤精准医学知识库,目前共收录1 940条精准用药指导,涵盖了基因突变等14种不同类型的组学特征。结论:精准医学知识数据库收录了肿瘤分子组学特征和治疗策略的关联信息,可为临床上个体化治疗方案的制订提供参考依据。数据库的建立为精准医疗临床决策支持系统的开发奠定了基础。     

Tumor microenvironment abnormalities: causes, consequences, and strategies to normalize

A solid tumor is an organ-like entity comprised of neoplastic cells and non-transformed host stromal cells embedded in an extracellular matrix. The expression of various genes is influenced by interactions among these cells, surrounding matrix, and their local physical and biochemical microenvironment. The products encoded by these genes, in turn, control the pathophysiological characteristics of the tumor, and give rise to the abnormal organization, structure, and function of tumor blood vessels. These abnormalities contribute to heterogeneous blood flow, vascular permeability, and microenvironment. Proliferating tumor cells produce solid stress which compresses blood and lymphatic vessels. As a result of vessel leakiness and lack of functional lymphatics, interstitial fluid pressure is significantly elevated in solid tumors. Each of these abnormalities forms a physiological barrier to the delivery of therapeutic agents to tumors. Furthermore, the metabolic microenvironment in tumors such as hypoxia and acidosis hinder the efficacy of anti-tumor treatments such as radiation therapy and chemotherapy. A judicious application of anti-angiogenic therapy has the potential to overcome these problems by normalizing the tumor vessels and making them more efficient for delivery of oxygen and drugs. Combined anti-angiogenic and conventional therapies have shown promise in the clinic.     

Receptor cross-talk in angiogenesis: Mapping environmental cues to cell phenotype using a stochastic, Boolean signaling network model

Cancer invasion and metastasis depend on tumor-induced angiogenesis, the means by which cancer cells attract and maintain a blood supply. During angiogenesis, cellular processes are tightly coordinated by signaling molecules and their receptors. Understanding how endothelial cells synthesize multiple biochemical signals can catalyze the development of novel therapeutic strategies to combat cancer. This study is the first to propose a signal transduction model highlighting the cross-talk between key receptors involved in angiogenesis, namely the VEGF, integrin, and cadherin receptors. From experimental data, we construct a network model of receptor cross-talk and analyze its dynamics. We identify relationships between receptor activation combinations and cellular function, and show that cross-talk is crucial to phenotype determination. The network converges to a unique set of output states that correspond to known cell phenotypes: migratory, proliferating, quiescent, apoptotic, and it predicts one phenotype that challenges the “go or grow” hypothesis. Finally, we use the model to study protein inhibition and to suggest molecular targets for anti-angiogenic therapies.     

Perspective on the dynamics of cancer

Background

The genetic diversity of cancer and the dynamic interactions between heterogeneous tumor cells, the stroma and immune cells present daunting challenges to the development of effective cancer therapies. Although cancer biology is more understood than ever, this has not translated into therapies that overcome drug resistance, cancer recurrence and metastasis. The future development of effective therapies will require more understanding of the dynamics of homeostatic dysregulation that drives cancer growth and progression.

Results

Cancer dynamics are explored using a model involving genes mediating the regulatory interactions between the signaling and metabolic pathways. The exploration is informed by a proposed genetic dysregulation measure of cellular processes. The analysis of the interaction dynamics between cancer cells, cancer associated fibroblasts, and tumor associate macrophages suggests that the mutual dependence of these cells promotes cancer growth and proliferation. In particular, MTOR and AMPK are hypothesized to be concurrently activated in cancer cells by amino acids recycled from the stroma. This leads to a proliferative growth supported by an upregulated glycolysis and a tricarboxylic acid cycle driven by glutamine sourced from the stroma. In other words, while genetic aberrations ignite carcinogenesis and lead to the dysregulation of key cellular processes, it is postulated that the dysregulation of metabolism locks cancer cells in a state of mutual dependence with the tumor microenvironment and deepens the tumor’s inflammation and immunosuppressive state which perpetuates as a result the growth and proliferation dynamics of cancer.

Conclusions

Cancer therapies should aim for a progressive disruption of the dynamics of interactions between cancer cells and the tumor microenvironment by targeting metabolic dysregulation and inflammation to partially restore tissue homeostasis and turn on the immune cancer kill switch. One potentially effective cancer therapeutic strategy is to induce the reduction of lactate and steer the tumor microenvironment to a state of reduced inflammation so as to enable an effective intervention of the immune system. The translation of this therapeutic approach into treatment regimens would however require more understanding of the adaptive complexity of cancer resulting from the interactions of cancer cells with the tumor microenvironment and the immune system.

     

A potential antitumor peptide therapeutic derived from antineoplastic urinary protein

New therapies in cancer treatment are focusing on multifaceted approaches to starve and kill tumors utilizing both antiangiogenic and chemotherapeutic compounds. Antineoplastic Urinary Protein (ANUP), a 32k Da protein normally secreted in human urine, has been previously described as a molecule possessing both antiproliferative and antiangiogenic activities. Two synthetic peptides complimentary to the N-terminus of ANUP were designed to test their ability to reproduce these beneficial effects but ultimately to provide a more useful small molecule therapeutic. The results show that the peptides reduced tumor burden by up to 70% in a nude mouse model and demonstrated the ability to inhibit blood vessel formation in a chick chorioallantoic membrane assay (CAM).