Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Interfering with Gal-1-mediated angiogenesis contributes to the pathogenesis of preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disorder characterized by sudden onset of hypertension and proteinuria in the second half of pregnancy (>20 wk). PE is strongly associated with abnormal placentation and an excessive maternal inflammatory response. Galectin-1 (Gal-1), a member of a family of carbohydrate-binding proteins, has been shown to modulate several processes associated with placentation and to promote maternal tolerance toward fetal antigens. Here, we show that Gal-1 exhibits proangiogenic functions during early stages of pregnancy, promoting decidual vascular expansion through VEGF receptor 2 signaling. Blocking Gal-1-mediated angiogenesis or lectin, galactoside-binding, soluble, 1 deficiency results in a spontaneous PE-like syndrome in mice, mainly by deregulating processes associated with good placentation and maternal spiral artery remodeling. Consistent with these findings, we observed a down-regulation of Gal-1 in patients suffering from early onset PE. Collectively, these results strengthen the notion that Gal-1 is required for healthy gestation and highlight Gal-1 as a valuable biomarker for early PE diagnosis.

Vascular galectins in tumor angiogenesis and cancer immunity.

Sustained tumor angiogenesis, i.e., the induction and maintenance of blood vessel growth by tumor cells, is one of the hallmarks of cancer. The vascularization of malignant tissues not only facilitates tumor growth and metastasis, but also contributes to immune evasion. Important players in all these processes are the endothelial cells which line the luminal side of blood vessel. In the tumor vasculature, these cells are actively involved in angiogenesis as well in the hampered recruitment of immune cells. This is the result of the abnormal tumor microenvironment which triggers both angiostimulatory and immune inhibitory gene expression profiles in endothelial cells. In recent years, it has become evident that galectins constitute a protein family that is expressed in the tumor endothelium. Moreover, several members of this glycan-binding protein family have been found to facilitate tumor angiogenesis and stimulate immune suppression. All this has identified galectins as potential therapeutic targets to simultaneously hamper tumor angiogenesis and alleviate immune suppression. The current review provides a brief introduction in the human galectin protein family. The current knowledge regarding the expression and regulation of galectins in endothelial cells is summarized. Furthermore, an overview of the role that endothelial galectins play in tumor angiogenesis and tumor immunomodulation is provided. Finally, some outstanding questions are discussed that should be addressed by future research efforts. This will help to fully understand the contribution of endothelial galectins to tumor progression and to exploit endothelial galectins for cancer therapy.

A Comparative Analysis of Local and Systemic Immunological Biomarkers in Females With Breast Implants and Capsular Contracture.

Background: The etiology of capsular contracture (CC), the most common complication following breast augmentation, remains unclear. Chronic, fibrotic inflammation resulting in excessive fibrosis has been proposed as a potential mechanism. Objectives: In this study, we aimed to investigate the relation between biomarkers that are associated with inflammation and fibrosis and the severity of CC. Methods: Fifty healthy females were categorized into 3 groups: females with no-to-mild CC (Baker 1-2; n = 15), females with severe CC (Baker 3-4; n = 20), and a control group awaiting breast augmentation (n = 15). We assessed 5 biomarkers (galectin-1 [Gal-1], interferon-ß [INF-ß], interferon-γ [INF-γ], interleukin-6 [IL-6], and tumor necrosis factor-α [TNF-α]) in breast implant capsules and serum samples. Results: No significant differences in intracapsular cytokine levels were observed between the Baker 1-2 and the Baker 3-4 groups, as the levels were generally low and, in some cases, almost undetectable. In the blood samples, no significant differences in Gal-1, INF-γ, IL-6, or TNF-α levels were found within the 3 groups. We identified significantly increased levels of INF-ß (P = .009) in the blood samples of females with severe CC, driven mainly by 3 extremely high values. Conclusions: The cytokines assessed in this study did not reflect the degree of CC among females with silicone breast implants. However, 3 females with severe CC, who all had prolonged silicone exposure, showed extremely elevated levels of INF-ß in their serum samples. This possible association between prolonged silicone exposure and systemic inflammation in some females should be further investigated.

Phenotypic immune characterization of gastric and esophageal adenocarcinomas reveals profound immune suppression in esophageal tumor locations.

Background: Tumors in the distal esophagus (EAC), gastro-esophageal junction including cardia (GEJAC), and stomach (GAC) develop in close proximity and show strong similarities on a molecular and cellular level. However, recent clinical data showed that the effectiveness of chemo-immunotherapy is limited to a subset of GEAC patients and that EACs and GEJACs generally benefit less from checkpoint inhibition compared to GACs. As the composition of the tumor immune microenvironment drives response to (immuno)therapy we here performed a detailed immune analysis of a large series of GEACs to facilitate the development of a more individualized immunomodulatory strategy. Methods: Extensive immunophenotyping was performed by 14-color flow cytometry in a prospective study to detail the immune composition of untreated gastro-esophageal cancers (n=104) using fresh tumor biopsies of 35 EACs, 38 GEJACs and 31 GACs. The immune cell composition of GEACs was characterized and correlated with clinicopathologic features such as tumor location, MSI and HER2 status. The spatial immune architecture of a subset of tumors (n=30) was evaluated using multiplex immunohistochemistry (mIHC) which allowed us to determine the tumor infiltration status of CD3+, CD8+, FoxP3+, CD163+ and Ki67+ cells. Results: Immunophenotyping revealed that the tumor immune microenvironment of GEACs is heterogeneous and that immune suppressive cell populations such as monocytic myeloid-derived suppressor cells (mMDSC) are more abundant in EACs compared to GACs (p<0.001). In contrast, GACs indicated a proinflammatory microenvironment with elevated frequencies of proliferating (Ki67+) CD4 Th cells (p<0.001), Ki67+ CD8 T cells (p=0.002), and CD8 effector memory-T cells (p=0.024). Differences between EACs and GACs were confirmed by mIHC analyses showing lower densities of tumor- and stroma-infiltrating Ki67+ CD8 T cells in EAC compared to GAC (both p=0.021). Discussions: This comprehensive immune phenotype study of a large series of untreated GEACs, identified that tumors with an esophageal tumor location have more immune suppressive features compared to tumors in the gastro-esophageal junction or stomach which might explain the location-specific responses to checkpoint inhibitors in this disease. These findings provide an important rationale for stratification according to tumor location in clinical studies and the development of location-dependent immunomodulatory treatment approaches.

CXCR4(+) dendritic cells promote angiogenesis during embryo implantation in mice.

Early pregnancy is characterized by decidual adaption to the developing embryo involving angiogenesis and vascular growth. Failure of decidual vascular expansion is linked to diseases of pregnancy. Dendritic cells (DC) have been associated with vascular growth during early gestation, though it is unknown whether their capacity to modulate angiogenesis is ubiquitous to all DC subsets. Here, we show that DC normally found associated with the decidual vasculature co-express the C-X-C chemokine receptor type 4 (CXCR4). In addition, we demonstrate that impaired homing of CXCR4(+)DC during early gestation provoked a disorganized decidual vasculature with impaired spiral artery remodeling later in gestation. In contrast, adoptive transfer experiments provided evidence that CXCR4(+)DC are able to rescue early pregnancy by normalizing decidual vascular growth and delivery of pro-angiogenic factors, which results in adequate remodeling of the spiral arteries during placental development. Taken together, our results indicate an important role of CXCR4(+)DC in the regulation of decidual angiogenesis and highlight the importance of the CXCL12/CXCR4 pathway during this process, suggesting that this may represent a key pathway to evaluate during pregnancy pathologies associated with impaired vascular expansion.

Profiling Lgals9 splice variant expression at the fetal-maternal interface: implications in normal and pathological human pregnancy.

Disruption of fetal-maternal tolerance mechanisms can contribute to pregnancy complications, including spontaneous abortion. Galectin-9 (LGALS9), a tandem repeat lectin associated with immune modulation, is expressed in the endometrium during the mid and late secretory phases and in decidua during human early pregnancy. However, the role of LGALS9 during pregnancy remains poorly understood. We used real-time PCR and immunohistochemical staining to analyze the expression of Lgals9/LGALS9 during mouse gestation as well as in human tissues obtained from normal pregnancy and spontaneous abortions. In mice, three Lgals9 splice variants were detected, the expression of which was differentially regulated during gestation. Furthermore, decidual Lgals9 expression was deregulated in a mouse model of spontaneous abortion, whereas placental levels did not change. We further found that the LGALS9 D5 isoform suppresses interferon gamma production by decidual natural killer cells. In human patients, six Lgals9 splice variants were detected, and a decrease in Lgals9 D5/10 was associated with spontaneous abortion. Altogether, these results show a differential regulation of Lgals9 isoform expression during normal and pathological pregnancies and designate Lgals9 as a potential marker for adverse pregnancy outcomes.

Blocking of frizzled signaling with a homologous peptide fragment of wnt3a/wnt5a reduces infarct expansion and prevents the development of heart failure after myocardial infarction.

BACKGROUND: The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. METHODS AND RESULTS: Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. CONCLUSIONS: Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.

Method to Study the Role of Galectins in Angiogenesis In Vivo Using the Chick Chorioallantoic Membrane Assay.

Angiogenesis is a complex multi-step process involving various activities of endothelial cells. These activities are influenced in vivo by environmental conditions like interactions with other cell types and the microenvironment. Galectins play a role in several of these interactions and are therefore required for proper execution of in vivo angiogenesis. This chapter describes a method to study galectins during physiologic and pathophysiologic angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay.

Examination of the Role of Galectins and Galectin Inhibitors in Endothelial Cell Biology.

The growth of new blood vessels is a key event in many (patho) physiological processes, including embryogenesis, wound healing, inflammatory diseases, and cancer. Neovascularization requires different, well-coordinated actions of endothelial cells, i.e., the cells lining the luminal side of all blood vessels. Galectins are involved in several of these activities. In this chapter, we describe methods to study galectins in three key functions of endothelial cells during angiogenesis, i.e., endothelial cell migration, endothelial cell sprouting, and endothelial cell network formation.

Galectins in Esophageal Cancer: Current Knowledge and Future Perspectives.

Esophageal cancer is a disease with poor overall survival. Despite advancements in therapeutic options, the treatment outcome of esophageal cancer patients remains dismal with an overall 5-year survival rate of approximately 20 percent. To improve treatment efficacy and patient survival, efforts are being made to identify the factors that underlie disease progression and that contribute to poor therapeutic responses. It has become clear that some of these factors reside in the tumor micro-environment. In particular, the tumor vasculature and the tumor immune micro-environment have been implicated in esophageal cancer progression and treatment response. Interestingly, galectins represent a family of glycan-binding proteins that has been linked to both tumor angiogenesis and tumor immunosuppression. Indeed, in several cancer types, galectins have been identified as diagnostic and/or prognostic markers. However, the role of galectins in esophageal cancer is still poorly understood. Here, we summarize the current literature with regard to the expression and potential functions of galectins in esophageal cancer. In addition, we highlight the gaps in the current knowledge and we propose directions for future research in order to reveal whether galectins contribute to esophageal cancer progression and provide opportunities to improve the treatment and survival of esophageal cancer patients.

Galectokines: The Promiscuous Relationship between Galectins and Cytokines.

Galectins, a family of glycan-binding proteins, are well-known for their role in shaping the immune microenvironment. They can directly affect the activity and survival of different immune cell subtypes. Recent evidence suggests that galectins also indirectly affect the immune response by binding to members of another immunoregulatory protein family, i.e., cytokines. Such galectin-cytokine heterodimers, here referred to as galectokines, add a new layer of complexity to the regulation of immune homeostasis. Here, we summarize the current knowledge with regard to galectokine formation and function. We describe the known and potential mechanisms by which galectokines can help to shape the immune microenvironment. Finally, the outstanding questions and challenges for future research regarding the role of galectokines in immunomodulation are discussed.

C/EBPδ Suppresses Motility-Associated Gene Signatures and Reduces PDAC Cell Migration.

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most aggressive human cancers and occurs globally at an increasing incidence. Metastases are the primary cause of cancer-related death and, in the majority of cases, PDAC is accompanied by metastatic disease at the time of diagnosis, making it a particularly lethal cancer. Regrettably, to date, no curative treatment has been developed for patients with metastatic disease, resulting in a 5-year survival rate of only 11%. We previously found that the protein expression of the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) negatively correlates with lymph node involvement in PDAC patients. To better comprehend the etiology of metastatic PDAC, we explored the role of C/EBPδ at different steps of the metastatic cascade, using established in vitro models. We found that C/EBPδ has a major impact on cell motility, an important prerequisite for tumor cells to leave the primary tumor and to reach distant sites. Our data suggest that C/EBPδ induces downstream pathways that modulate actin cytoskeleton dynamics to reduce cell migration and to induce a more epithelial-like cellular phenotype. Understanding the mechanisms dictating epithelial and mesenchymal features holds great promise for improving the treatment of PDAC.

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro.

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbß3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbß3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

Chemokines modulate glycan binding and the immunoregulatory activity of galectins.

Galectins are versatile glycan-binding proteins involved in immunomodulation. Evidence suggests that galectins can control the immunoregulatory function of cytokines and chemokines through direct binding. Here, we report on an inverse mechanism in which chemokines control the immunomodulatory functions of galectins. We show the existence of several specific galectin-chemokine binding pairs, including galectin-1/CXCL4. NMR analyses show that CXCL4 binding induces changes in the galectin-1 carbohydrate binding site. Consequently, CXCL4 alters the glycan-binding affinity and specificity of galectin-1. Regarding immunomodulation, CXCL4 significantly increases the apoptotic activity of galectin-1 on activated CD8+ T cells, while no effect is observed in CD4+ T cells. The opposite is found for another galectin-chemokine pair, i.e., galectin-9/CCL5. This heterodimer significantly reduces the galectin-9 induced apoptosis of CD4+ T cells and not of CD8+ T cells. Collectively, the current study describes an immunomodulatory mechanism in which specific galectin-chemokine interactions control the glycan-binding activity and immunoregulatory function of galectins.

Molecular profiles of response to neoadjuvant chemoradiotherapy in oesophageal cancers to develop personalized treatment strategies.

Identification of molecular predictive markers of response to neoadjuvant chemoradiation could aid clinical decision-making in patients with localized oesophageal cancer. Therefore, we subjected pretreatment biopsies of 75 adenocarcinoma (OAC) and 16 squamous cell carcinoma (OSCC) patients to targeted next-generation DNA sequencing, as well as biopsies of 85 OAC and 20 OSCC patients to promoter methylation analysis of eight GI-specific genes, and subsequently searched for associations with histopathological response and disease-free (DFS) and overall survival (OS). Thereby, we found that in OAC, CSMD1 deletion (8%) and ETV4 amplification (5%) were associated with a favourable histopathological response, whereas SMURF1 amplification (5%) and SMARCA4 mutation (7%) were associated with an unfavourable histopathological response. KRAS (15%) and GATA4 (7%) amplification were associated with shorter OS. In OSCC, TP63 amplification (25%) and TFPI2 (10%) gene promoter methylation were associated with an unfavourable histopathological response and shorter DFS (TP63) and OS (TFPI2), whereas CDKN2A deletion (38%) was associated with prolonged OS. In conclusion, this study identified candidate genetic biomarkers associated with response to neoadjuvant chemoradiotherapy in patients with localized oesophageal cancer.

Combining Radiation Therapy With Interferons: Back to the Future.

Radiation therapy has been linked to the induction of an intratumoral type I interferon (IFN) response, which positively affects the response to treatment. This has spiked the interest to combine radiation therapy with IFN-based treatment. Interestingly, this combination treatment has been considered previously, since preclinical studies demonstrated a radiosensitizing effect of interferons. As a result, multiple clinical trials have been performed combining radiation therapy with interferons in different tumor types. Although potential benefit has been suggested, the outcomes of the trials are diverse and challenging to interpret. In addition, increased grade ≥3 toxicity frequently resulted in a negative recommendation regarding the combination therapy. The latter appears premature because many studies were small and several aspects of the combination treatment have not yet been sufficiently explored to justify such a definite conclusion. This review summarizes the available literature on this combination therapy, with a focus on IFN-α and IFN-ß. Based on preclinical studies and clinical trials, we evaluated the potential opportunities and describe the current challenges. In addition, we identify several issues that should be addressed to fully exploit the potential benefit of this combinatorial treatment approach.

Development of transient radioresistance during fractionated irradiation in vitro.

BACKGROUND AND PURPOSE: Effective combination treatments with fractionated radiotherapy rely on a proper understanding of the dynamic responses that occur during treatment. We explored the effect of clinical fractionated radiotherapy on the development and timing of radioresistance in tumor cells. METHODS AND MATERIALS: Different colon (HT29/HCT116/COLO320/SW480/RKO) and high-grade astrocytoma (D384/U-251MG) cancer cell lines were treated for 6 weeks with daily fractions of 2 Gy, 5 days per week. Clonogenic survival was determined throughout the treatment period. In addition, the radiosensitivity of irradiated and non-irradiated was compared. Finally, the effect of different dose fractions on the development of radioresistance was determined. RESULTS: All cell lines developed radioresistance within 2-3 weeks during fractionated radiotherapy. This was characterized by the occurrence of a steady state phase of clonogenic survival. In U-251MG cells this was accompanied by increased cell senescence and stemness. After recovering from six weeks of treatment, the radiosensitivity of fractionally irradiated and non-irradiated cells was similar. Including transient radioresistance, described as (α/ß)-(d+1), as a factor in the classic LQ model resulted in a perfect fit with the experimental data observed during fractionated radiotherapy. This was confirmed when different dose fractions were applied. CONCLUSIONS: Fractionated irradiation of cancer cells in vitro following clinical radiation schedules induces a reversible radioresistance response. This adaptive response can be included in the LQ model as a function of the dose fraction and the alpha/beta-ratio of a given cell line. These findings warrant further investigation of the mechanisms and clinical relevance of adaptive radioresistance.

Targeting Tumor Vascular CD99 Inhibits Tumor Growth.

CD99 (MIC2; single-chain type-1 glycoprotein) is a heavily O-glycosylated transmembrane protein (32 kDa) present on leukocytes and activated endothelium. Expression of CD99 on endothelium is important in lymphocyte diapedesis. CD99 is a diagnostic marker for Ewing's Sarcoma (EWS), as it is highly expressed by these tumors. It has been reported that CD99 can affect the migration, invasion and metastasis of tumor cells. Our results show that CD99 is also highly expressed in the tumor vasculature of most solid tumors. Furthermore, we found that in vitro CD99 expression in cultured endothelial cells is induced by starvation. Targeting of murine CD99 by a conjugate vaccine, which induced antibodies against CD99 in mice, resulted in inhibition of tumor growth in both a tumor model with high CD99 (Os-P0109 osteosarcoma) and low CD99 (CT26 colon carcinoma) expression. We demonstrated that vaccination against CD99 is safe, since no toxicity was observed in mice with high antibody titers against CD99 in their sera during a period of almost 11 months. Targeting of CD99 in humans is more complicated due to the fact that the human and mouse CD99 protein are not identical. We are the first to show that growth factor activated endothelial cells express a distinct human CD99 isoform. We conclude that our observations provide an opportunity for specific targeting of CD99 isoforms in human tumor vasculature.

Targeted gene-delivery strategies for angiostatic cancer treatment.

Gene therapy is one of the promising strategies in cancer treatment. Recent studies identified molecular targets on angiogenically activated endothelial cells that can be used to deliver gene-transfer vehicles to the tumor site specifically. Furthermore, non-viral vehicles are emerging as an alternative for traditional viral gene-therapy approaches. Here, we describe how viral and non-viral gene-transfer vehicles have been and can be modified to target tumor endothelial cells for anti-angiogenesis gene therapy. Improving the specificity and safety of existing gene-therapy vehicles will make angiogenesis-targeted cancer gene therapy a valuable tool in the clinical setting.