Marker Of Tumor Angiogenesis Research Articles

Caspase-3 in glioma indicates an unfavorable prognosis by involving surrounding angiogenesis and tumor cell repopulation.

Effective biomarkers for estimating glioma prognosis are deficient. Canonically, caspase-3 acts as an "apoptosis executioner". However, its prognostic role in glioma and mechanistic effects on prognosis remain unclear. With glioma tissue microarrays, the prognostic roles of cleaved caspase-3 and its association with angiogenesis were explored. Next, by analyzing the mRNA microarray data from the CGGA, the prognostic role of CASP3 expression and correlations between CASP3 and markers of glioma angiogenesis and proliferation were investigated. To biologically interpret the prognostic role of caspase-3 in glioma, the influence of caspase-3 on surrounding angiogenesis and glioma cell repopulation was investigated with an in vitro cell co-culture model, which comprises irradiated U87 cells and un-irradiated firefly luciferase (Fluc)-labeled HUVEC (HUVEC-Fluc) or U87 (U87-Fluc) cells. The over-expressed dominant-negative caspase-3 was used to suppress normal caspase-3 activity. High levels of cleaved caspase-3 expression were associated with poor survival outcomes in glioma patients. Higher microvessel density was observed in patients with high levels of cleaved caspase-3 expression. By mining the microarray data in CGGA, it was revealed that higher CASP3 expression was found in glioma patients with lower Karnofsky Performance score, higher WHO grade, malignant histological subtype, wild-type IDH. Higher CASP3 expression indicated a worse survival rate in glioma patients. Patients with high CASP3 expression and negative IDH mutation showed the worst survival rate. Positive correlations were found between CASP3 and markers of tumor angiogenesis and proliferation. Subsequent data based on an in vitro cell co-culture model revealed that caspase-3 in irradiated glioma cells mediated pro-angiogenic and repopulation-promoting effects via regulating COX-2 signaling. With glioma tissue microarrays, high levels of COX-2 expression showed inferior survival outcomes in glioma patients. Glioma patients with high levels of cleaved caspase-3 and COX-2 expression showed the worst survival outcomes. This study innovatively identified an unfavorable prognostic role of caspase-3 in glioma. The pro-angiogenic and repopulation-prompting effects of caspase-3/COX-2 signaling may explain its unfavorable prognostic role and offer novel insights into therapy sensitization and curative effect prediction of glioma.

Honokiol inhibits interleukin-induced angiogenesis in the NSCLC microenvironment through the NF-κB signaling pathway

Tumor angiogenesis, which may be affected by microenvironmental inflammation and promotes tumor development and metastasis, is one of the key reasons contributing to increased mortality. The goal of this study is to investigate how lignin analogs, specifically honokiol (HNK), block angiogenesis induced by the inflammatory milieu of lung cancer. The human lung cancer cell lines A549 and H460 were treated with HNK. Interleukin-1 was employed to mimic an inflammatory tumor microenvironment. Findings demonstrated that HNK drastically decreased the cell viability of A549 and H460 cells. In A549 and H460 cells, HNK also reduced the production of vascular endothelial growth factor (VEGF), the most important marker of tumor angiogenesis. Signal pathway studies revealed that HNK blocked the NF-κB signaling pathway. This effect, in turn, prevented the expression of VEGF by inhibiting the NF-κB signaling pathway. Human umbilical vein endothelial cells (HUVECs) from A549-conditioned medium cultures were subjected to HNK treatment, which decreased tubulogenesis, horizontal and vertical migration, and cell proliferation in HUVECs. Overall, HNK inhibited the NF–κB pathway. This effect resulted in the downregulation of VEGF, thus reducing the viability and angiogenesis of human lung cancer cell lines. In A549 cell xenografts, HNK decreased VEGF expression, tumor angiogenesis, and tumor development. Our research shows that HNK is a potential antiangiogenic molecule for the treatment of lung cancer.

Relevance of renal cell carcinoma angiogenesis and tumor stem cells

Objective: To investigate the relationship between tumor angiogenesis and renal cancer stem cells. Methods: The primary cell culture method was used to extract and isolate RCSCs, and then qRT-PCR and Western blot methods were used to detect the expression levels of the kidney cancer stem cell markers CD105 and Sox2 genes and proteins from different MVD kidney cancer tissues. Using TCGA database, analyze the correlation between tumor angiogenesis markers and tumor stem cell regulatory genes. Results: The stem cell markers CD105 and Sox2 genes in RCSCs derived from high MVD kidney cancer tissues were respectively increased by (2.34±1.77) times and (3.92±1.41) times (PCD105<0.01, PSox2<0.05)and protein levels were increased by (5.12±3.31) times and (4.90±3.30) times(PCD105<0.05, PSox2<0.01).Meanwhile,up to 30% of stem cell promoting stemness regulatory genes are positively correlated with angiogenesis genes CD31/PECAM1 and KDR, and 64 genes are also strongly positively correlated with CD31/PECAM1 and KDR genes. Conclusion: The high microvessel density of kidney cancer is strongly correlated with the existence of renal carcinoma stem cells.

Interactive effect of 6 weeks of High-intensity interval training and quercetin supplementation on expression of some tumor angiogenesis markers in female mice with breast cancer

Background: About angiogenesis, metastasis of breast cancer and exercise, Many studies have been done in recent decades to better understand of regulatory mechanisms. The purpose of this study was to investigate the Interactive effect of high-intensity interval training (HIIT) and quercetin supplementation (Q) on the expression of key angiogenic factors in tumor tissue of mouse with breast cancer. Methods: Twenty-four female Balb/C mouse were injected with estrogen receptor-dependent breast cancer cells MC4L2 and then divided into three groups of tumor (T), tumor + of high-intensity interval training (TH) and tumor + high-intensity interval training + quercetin (THQ). The TH and THQ groups ran the treadmill 3 days a week for 6 weeks and each session 1 hour. The THQ group received 110 mg / kg quercetin solution for 6 weeks, 3 days a week with training. After completion of the work, the mouse were sacrificed and their tumor tissue removed and frozen in liquid nitrogen And stored at -70 ° C. Expression of TIE-2 and VEGF-A genes was measured by REAL TIME-PCR. ΔCt, ΔΔCt, and Fold change were calculated with the relevant statistical tests at the significant level (P &lt;0.05) by GENEX software. Results: The results showed that THQ interaction significantly decreased the expression of TIE-2 and VEGF-A genes in TH and THQ groups compared to T group. In addition, quercetin in THQ group significantly decreased TIE-2 and VEGF-A gene expression compared to TH group. Conclusion: interaction of HIIT and Q supplementation are probably effective in inhibiting tumor angiogenesis.

Hyperdense Pancreatic Ductal Adenocarcinoma: Clinical Characteristics and Proteomic Landscape.

PurposeHypodensity of pancreatic ductal adenocarcinoma (PDAC) during contrast-enhanced computed tomography (CECT) examination is common, but a minority of PDAC patients exhibit hyperdense images. The present study examined the clinical characteristics and protein landscape of PDAC with hyperdensity.Materials and MethodsA total of 844 pathologically confirmed PDAC patients who underwent CECT before surgery were included. During the parenchymal phase of CECT, patients were assigned to the hyperdense or hypodense group based on CT values. Clinical and CT characteristics for predicting relapse-free survival (RFS) and overall survival (OS) were analyzed using the Kaplan–Meier method and Cox proportional hazards model. The expression of the tumor angiogenesis marker CD31 and stroma-related protein CTHRC1 were analyzed using immunohistochemistry (IHC) assay to evaluate differences between the two groups. Proteomics was performed to compare the possible mechanisms underlying the differential enhancement on CT scans.ResultsBased on CECT, 43 and 801 PDAC patients had hyperdense and hypodense lesions, respectively. All 43 patients presented a hyperdense lesion in the parenchymal phase. The mean CECT values of the hyperdense group were higher than the hypodense group (102.5 ± 17.4 and 53.7 ± 18.7, respectively, P < 0.001). The hyperdense group had a better prognosis than the hypodense group (median RFS, 19.97 vs. 12.34 months, P = 0.0176; median OS, 33.6 vs. 20.3 months, P = 0.047). Multivariate analysis showed that age, higher CA19-9 levels (> 300 U/ml), tumor stage, tumor differentiation, tumor CT density, and adjuvant chemotherapy were significant independent prognostic factors for OS. CD31 immunohistochemical staining showed that the hyperdense PDACs had a higher microvessel density than the hypodense group (P < 0.001). CTHRC1 expression was higher in the hypodense group (P = 0.019). Sixty-eight differentially expressed proteins were found using the tandem mass tag labeling-based quantification of the proteomes of PDAC tissue samples, and 7 proteins (POFUT1, PKP2, P0DOX4, ITPR1, HBG2, IGLC3, SAA2) were related to angiogenesis.ConclusionPatients who presented with a hyperdense mass on CECT had a higher microvessel density and better prognosis. Anti-angiogenic therapy may be suitable for these patients.

Effect of 6 Weeks of Quercetin Supplementation on Expression of Some Tumor Angiogenesis Markers in Female Mice with Breast Cancer

Effect of 6 Weeks of Quercetin Supplementation on Expression of Some Tumor Angiogenesis Markers in Female Mice with Breast Cancer

Newly crocin-coated magnetite nanoparticles induce apoptosis and decrease VEGF expression in breast carcinoma cells

Nanoparticles can act on tumor cells at the nanoscale, causing biological therapeutic responses. In the current work, magnetic nanoparticles (MNPs) were obtained by means of a co-precipitation method and functionalized with dextran (D) and crocin, a saffron metabolite with potential antiproliferative properties. These MNPs were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission-scanning electron microscopy (FESEM), and Fourier transform infrared (FTIR) techniques. The crystalline structure of the functionalized MNPs was confirmed by XRD with a crystallite size of ca. 12 nm. The successful coating of MNPs surface with dextran and crocin was confirmed by FTIR spectroscopy. The functionalized MNPs were then used in order to elucidate their antiproliferative and apoptosis-inducing activities in the MCF-7 breast tumor cell line using the tetrazolium-based colorimetric (MTT) and Annexin V/PI flow cytometric assays, respectively. Real time-PCR analysis was used to determine the mRNA levels of VEGF as a marker of tumor angiogenesis. The antitumoral effects of crocin-coated D-MNPs were significantly higher than those of free crocin after 24 (P = 0.01) and 48 h (P < 0.0001) of incubation. Results of Annexin V/PI staining indicated that the rate of early apoptosis in MCF-7 cells treated with crocin-coated D-MNPs was higher than the cell fraction undergoing late apoptosis after 6 (P = 0.002), 12 (P < 0.0001), 24 (P < 0.0001), and 48 h (P < 0.0001) of incubation when compared to administration of free crocin. These results indicate that crocin-coated D-MNPs are a much successful drug nanocarrier with enhanced therapeutic effects than the free administration of the bioactive compound, crocin, becoming an attractive alternative option for the therapeutic use of this class of bioactive principles.

Abstract 1472: Paraoxonase 3 is a novel tumor suppressor protein in xenograft mouse model

Abstract Introduction: Dysfunction of mitochondrial respiratory chains or its associated machinery favors the aerobic glycolysis leading to upregulation of genes related to cell proliferation, metastasis, drug resistant and cancer cell stemness, i.e., the Warburg effect, which has a major role in the pathogenesis of tumor formation. Various studies have documented that perturbation of mitochondria leads to apoptosis suggesting that the precise spatiotemporal regulation of mitochondrial function is a critical regulator of both cell survival and death, whcih determine tumorigenesis. Paraoxonase 3 (PON3) is an intracellular protein localized to the mitochondrial associated membrane. PON3 binds coenzymes Q10 and regulates the respiratory complex activity and prevents the ubisemiquinone-mediated mitochondrial superoxide levels in response to stress conditions. PON3 deficiency leads to dysfunction of mitochondria, which along with an increase in mitochondrial oxidative stress causes systemic inflammation, atherosclerosis, obesity and gall stone formation. PON3 is upregulated in ovarian cancer tissues. However, it's expression in various stages of ovarian cancer and its role on cancer development have not been studied. Hypothesis: PON3 expression may regulate the ovarian cancer development by enhancing the mitochondrial function. Methods and Results: Using human ovarian carcinoma tissue array, we identified that PON3 expression was higher in both early and late stages of ovarian cancer when compared to normal tissue. Interestingly, we demonstrated that overexpression of PON3 prevented tumor formation in a mouse ovarian cancer xenograft model. Overexpression of PON3, (i) reduced the tumor angiogenesis marker, CD31 levels in the tumor microenvironment and (ii) inhibited ovarian cancer cell proliferation in both normoxic and hypoxic condition in vitro. Moreover, PON3 reduced VEGF levels (but not IGF-1 levels) under hypoxic conditions. Furthermore, mechanistically, overexpression of PON3 decreased the VEGF level by accelerating the proteasome-dependent protein degradation of hypoxia inducible factor-1 α (HIF-1α) under hypoxic conditions. Finally, we demonstrate that the inhibitory effect of PON3 on HIF-1α levels is, in part, mediated by mitochondrial function. Conclusion: We report for the first time that PON3 functions as a tumor suppressor in ovarian cancer possibly by suppressing VEGF signaling via HIF-1α by enhancing the mitochondrial function suggesting that PON3 might be a successful strategy to inhibit the ovarian cancer development. Citation Format: Asokan Devarajan, Feng Su, Dawoud Sulaiman, Dorothy Nguyen, Robin Farias-Eisner, Srinivasa T. Reddy. Paraoxonase 3 is a novel tumor suppressor protein in xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1472.

Expression of the ghrelin/growth hormone secretagogue receptor axis and its functional role in promoting tumor growth in primary central nervous system lymphomas

Ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), have been found in a variety of malignant tumor tissues, suggesting a biological function of the ghrelin/GHS-R axis in tumor growth and progression. Among central nervous system tumors, primary central nervous system lymphomas (PCNSLs) are relatively rare and characterized by a rapid progression and poor prognosis. In order to clarify ghrelin expression and its functional role in promoting tumor growth and progression in PCNSLs, we undertook an immunohistochemical investigation for ghrelin and GHS-R expression in 43 patients and tested the effect of ghrelin inhibition on lymphoma cells. Furthermore, we investigated the expression of CD105, a marker for tumor angiogenesis, to explore its association with the ghrelin/GHS-R axis. The Kaplan-Meier method and Cox's proportional hazards regression model were used to determine the association of ghrelin/GHS-R expression with overall survival rate. The immunohistochemical study showed moderate/strong immunostaining of cells for ghrelin and GHS-R in 40 patients (93.0%) and 39 patients (90.7%), respectively. A ghrelin inhibitor did not affect tumor cell proliferation in vitro. Expression levels of ghrelin and GHS-R were divided into high and low groups by the rate of moderate-strong staining cells to tumor cells. The survival rate was significantly lower in patients with high GHS-R expression (P = 0.0368 by log-rank test; P = 0.0219 by Wilcoxon test). In addition, multivariate analysis of overall survival using Cox's proportional hazards regression model indicated that GHS-R was a significant independent prognostic factor (P = 0.0426). CD105 expression on tumor vessels was positive in 33 patients (33/37, 89.2%). There was a positive correlation between the moderate-strong staining rate of ghrelin and CD105-positive vessel count. These results indicated that the ghrelin/GHS-R axis plays a potential role in promoting tumor growth and progression through neoangiogenesis, rather than the proliferation of tumor cells.

Lagerstroemia speciosa (L.) Pers Leaf Extract Attenuates Lung Tumorigenesis via Alleviating Oxidative Stress, Inflammation and Apoptosis.

One of the major etiological factors that account for lung cancer is tobacco use. Benzo(a)pyrene [B(a)P], one of the main constituents of tobacco smoke, has a key role in lung carcinogenesis. The present study was conducted to investigate the cytotoxicity of an aqueous ethanolic extract of Lagerstroemia speciosa (L.) Pers leaves (LLE) on human lung adenocarcinoma cells (A549), as well as its in vivo antitumor effect on a lung tumorigenesis mice model. Our results revealed that LLE possesses cytotoxic activity against the A549 cell line. Mice orally administered B(a)P (50 mg/kg body weight) showed an increase in relative lung weight with subsequent decrease in final body weight. Serum levels of tumor marker enzymes AHH, ADA and LDH and the inflammatory mediator NF-κB increased, while total antioxidant capacity (TAC) decreased. In addition, we observed the increased activity of metalloproteinases (MMP-2 and MMP-12) and levels of the tumor angiogenesis marker VEFG and the lipid peroxidation marker MDA, as well as decreased levels of the non-enzymatic antioxidant GSH and enzymatic antioxidants CAT and GSH-Px in lung tissues. Moreover, B(a)P administration up-regulated the expression of the COX-2 gene, pro-inflammatory cytokines TNF-α and IL-6, and an anti-apoptotic gene Bcl-2, and at the same time down-regulated expression of pro-apoptotic genes BAX and caspase-3 and the p53 gene. Pre- and post-treatment with LLE (250 mg/kg body weight) attenuated all these abnormalities. Histopathological observations verified the protective effect of LLE. Overall, the present data positively confirm the potent antitumor effect of L. speciosa leaves against lung tumorigenesis.

CAF-induced placental growth factor facilitates neoangiogenesis in hepatocellular carcinoma.

As a highly malignant tumor, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. In most HCC patients, the development of HCC begins with hepatitis, which is followed by fibrosis and cirrhosis before progressing to HCC. Cancer-associated fibroblasts (CAFs), which are generally believed to be derived from activated hepatic stellate cells (HSCs), are highly involved in the development of HCC through the secretion of cytokines and angiogenic factors. The results of our study showed that a considerable number of CAFs highly expressed CD90 and were enriched in HCC tissues. Bioinformatics analysis of the transcriptome of HCC tissues revealed that placental growth factor (PlGF) is significantly correlated with CD90 expression. The isolated primary CAFs and activated HSCs overexpressed PlGF and CD90. In addition, the results of gene expression profiling interactive analysis based on The Cancer Genome Atlas showed that high levels of both PlGF and CD90 are correlated with tumor angiogenesis markers (CD31, CD34, and CD105) and predict poor HCC patient prognosis. In summary, our results suggest that CAFs can generate PlGF and may provide an effective target for CAFs-regulated neoangiogenesis in HCC.

High CTHRC1 expression may be closely associated with angiogenesis and indicates poor prognosis in lung adenocarcinoma patients

BackgroundThis study aimed to investigate the prognostic value of the potential biomarker collagen triple helix repeat containing 1 (CTHRC1) in lung adenocarcinoma (LUAD) patients.MethodsA total of 210 LUAD patients diagnosed between 2003 and 2016 in the Department of Pathology of the First Affiliated Hospital of Sun Yat-sen University were included in this study. The expression of CTHRC1 and vascular endothelial growth factor (VEGF), and microvessel density (MVD, determined by CD34 immunostaining) were evaluated by immunohistochemistry in LUAD tissues. The association between the expression of these proteins and clinicopathological features or clinical outcomes was analyzed.ResultsHere, we confirmed that CTHRC1 expression was associated with prognosis and can serve as a significant predictor for overall survival (OS) and progression-free survival (PFS) in LUAD. Additionally, we observed that CTHRC1 expression was positively associated with tumor angiogenesis markers, such as VEGF expression (P < 0.001) and MVD (P < 0.01). Then, we performed gene set enrichment analysis (GESA) and cell experiments to confirm that enhanced CTHRC1 expression can promote VEGF levels. Based on and cox regression analysis, a predictive model that included CTHRC1, VEGF and MVD was constructed and confirmed as a more accurate independent predictor for OS (P = 0.001) and PFS (P < 0.001) in LUAD than other parameters.ConclusionsThese results demonstrated that high CTHRC1 expression may be closely related to tumor angiogenesis and poor prognosis in LUAD. The predictive model based on the CTHRC1 level and tumor angiogenesis markers can be used to predict LUAD patient prognosis more accurately.

CD44(+) tumor cells promote early angiogenesis in head and neck squamous cell carcinoma

The role of CD44 in progression of head and neck squamous cell carcinoma (HNSCC) has been controversial. The goal of this study was to study the effects of CD44(+) tumor cells on the initial stages of tumor angiogenesis and to evaluate CD44 as a potential marker of tumor angiogenesis. The CD44 gene expression was studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer data base. Expression levels of CD44 and of microvascular density (MVD) markers were assessed by immunohistochemistry performed with tissue microarrays in a cohort of 49 HNSCC patients, 11 patients with dysplasia and 12 control oral mucosa tissues. The 4-nitroquinoline-1-oxide oral carcinogenesis mouse model was used to study CD44 expression during carcinogenesis. Gelatin sponges seeded with CD44(+), CD44(-) and unsorted cancer cells suspended in Matrigel were implanted in NOD/SCID mice into a dorsal skinfold chamber and compared to non-seeded sponges as controls. Angiogenic response was assessed by intravital microscopy. In the TCGA analysis, CD44 gene expression correlated with various pro-angiogenic genes. In human HNSCC tissues, CD44 expression was upregulated and was associated with blood vessels, although no correlation between MVD and CD44 expression was found. During oral carcinogenesis CD44 expression was upregulated. In dorsal skinfold chambers, CD44(+) cells showed a significantly higher MVD than CD44(-) or unsorted cells (p < 0.001). The results indicate that CD44(+) cells contain pro-angiogenic factors and stimulate tumor angiogenesis in HNSCC. Thus, CD44 might emerge as a potential angiogenic biomarker and a therapeutic target for anti-angiogenic therapies.

Expression Profile of Endoglin in Different Grades of Endometrial Cancer.

Endoglin is a marker of active, proliferating endothelial cells of blood vessels. In many cancers, it is present in both peripheral vessels and vessels located inside the tumor. Endoglin is more specific and sensitive compared to other tumor angiogenesis markers. It is suggested that endoglin can be considered a reliable marker of disease outcome. The aim of the study was to assess the expression of endoglin and to determine its potential usefulness as a complementary molecular marker of endometrial cancer. The study included 60 women who underwent hysterectomy: 45 with endometrioid endometrial cancer (study group) and 15 without neoplastic changes (control group). The study group was further divided according to the degree of histological differentiation: G1, 17; G2, 15; and G3, 13. The expression of endoglin was determined immunohistochemically with mouse anti-Endoglin monoclonal antibody. The obtained reactions were evaluated using light microscopy. Analysis of endoglin expression in endothelium showed that it reached 145% of the control. In G2, we observed that the endoglin level decreased and was similar to the control, while in G3 it increased and was even higher than in G1. In cancer cells, endoglin expression increased with the grade of endometrial cancer. Endoglin can be considered a valuable complementary molecular marker, allowing to visualize the advancement of the cancer process, including endometrial cancer.

Role of circulating endothelial progenitor cells in patients with breast cancer

Objective The aim was to assess the role of the circulating endothelial progenitor cells (CEPCs) in stages of breast cancer (BC) in patients. Materials and Methods We reviewed papers on the relation between CEPCs and stages of BC from Medline databases (PubMed, Medscape, Science Direct, and EMF-Portal) and all materials available on the internet from 1994 to 2016. The initial search presented 100 articles, of which, 30 met the inclusion criteria. The articles studied the relation between endothelial progenitor cells and stages of BC. If the studies did not fulfill the inclusion criteria, they were excluded. Study quality assessment included whether ethical approval was gained, eligibility criteria specified, appropriate controls, adequate information, and defined assessment measures. Comparisons were made by structured review with the results tabulated. Findings Endothelial progenitor cells counts were significantly higher in advanced stages of BC compared with those with early stages and controls in almost all the studied publications. Conclusion Our review concludes CEPCs elevation in the blood of patients is a useful marker of tumor angiogenesis and progression and early predictor of metastasis in patients with BC.

Abstract 5563: Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins

Abstract Cytokines are able to mediate a potent anticancer activity but have significant side effects that prevent dose escalation to therapeutically-active regimens. Targeting the cytokines to the site of disease helps to improve their therapeutic index while decreasing associated toxicities. However, peak concentrations in blood are still associated with side-effects. We have developed a novel technology (termed “CORK Technology”), which consists in masking the undesired activity of a cytokine in circulation. The “cork” can be either an antibody fragment or a small molecule, which specifically binds to the cytokine thus preventing the interaction with its receptor. Once the complex accumulates in the tumor, the cork dissociate from the cytokine allowing it to exert its function. Capitalizing on the fact that the kinetic dissociation constant koff of the complex is identical in different species, this technology can be easily translated from mouse to man with comparable results. In this poster we describe the experimental implementation of CORK Technology to improve the therapeutic index of the clinical stage L19-IL2 (L19 is an antibody fragment specific to the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis). Two classes of IL2 inhibitors were discovered and characterized: (i) a fully human scFv fragment, and (ii) a small organic ligand, featuring a modified methylindole moiety. Both types of agents reduced IL2 activity in vitro, were able to dissociate from the cognate cytokine in a kinetically-tuned process and allowed the administration of the L19-IL2 fusion protein at higher doses, both in normal and in tumor-bearing mice. The technology is applicable to other cytokine payloads and facilitates the development of targeted cytokine products with “activity on demand” (i.e., with increased activity at the tumor site, promoting an activation and proliferation of tumor-resident lymphocytes). Albeit still in its infancy, this elegant approach provides promising results and a strong rationale to further investigate this therapeutic power of the CORK Technology. Citation Format: Tiziano Ongaro, Martina Bigatti, Filippo Sladojevich, Etienne Donckele, Alessandra Micaela Villa, Dario Neri. Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5563.

Salidroside inhibits migration, invasion and angiogenesis of MDA‑MB231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2.

The major hallmarks of tumor progression are angiogenesis, migration and metastasis. Among the components of Rhodiola rosea, salidroside (p‑hydroxyphenethyl-β‑d-glucoside) is one of the most potent, and is present in all Rhodiola species. Recent data have revealed the anticancer effects of salidroside; however, the mechanism underlying its ability to inhibit tumor angiogenesis remains unknown. The present study aimed to analyze how salidroside affects major factors involved in breast cancer, and to elucidate its ability to inhibit angiogenesis and invasion. Signal transducer and activator of transcription3 (STAT3) is a marker for tumor angiogenesis and migration, which interacts with matrix metalloproteinases (MMPs). Specifically, MMPs act as a downstream target for STAT3. Using western blotting and reverse transcription-quantitative polymerase chain reaction analysis, the present study demonstrated that treatment of MDA‑MB231 triple-negative breast cancer (TNBC) cells with salidroside led to inhibition of invasion and migration markers, and of STAT3 signaling. Furthermore, in vitro angiogenesis analyses in human umbilical vein endothelial cells confirmed the anti-angiogenic activity of salidroside. An electrophoretic mobility shift assay also demonstrated that salidroside may inhibit the DNA-binding activity of STAT3, preventing STAT3 from binding to a novel binding site of the MMP2 gene promoter. In conclusion, the present results demonstrated that salidroside may downregulate the STAT3 signaling pathway, and inhibit cell viability, migration and invasion through MMPs in breast cancer cells.

Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER⁺ Breast Cancer Cells.

Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER+) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. We also investigated the migration and invasive ability of nobiletin in ER+ cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER+ breast cancer cells.

The role of microvessel density, lymph node metastasis, and tumor size as prognostic factors of distant metastasis in colorectal cancer.

Angiogenesis is essential for tumor growth and metastasis. CD105 is reportedly a specific marker for tumor angiogenesis. It has been demonstrated that monoclonal antibodies to CD105 have high affinity for activated endothelial cells. A relationship between metastasis and microvessel density (MVD), as an indicator of neovascularization, has been identified in patients with colorectal cancer as shown by the presence of monoclonal antibodies to CD105. However, data on potentially confounding factors such as lymphatic and vascular infiltration and tumor size are lacking. We further investigated the relationship between MVD and distant metastasis, along with potentially confounding clinicopathological factors, to more precisely characterize this relationship. In this retrospective study, we analyzed colorectal cancer specimens surgically or endoscopically resected from January to September 2009. We defined MVD as the number of microvessels stained by monoclonal antibodies to CD105 per ×400 field. Selected clinicopathological factors were analyzed and stepwise multivariate logistic regression was performed to identify independent risk factors for distant metastasis. We analyzed 129 lesions. The median follow-up time was 34 months (range, 6-85 months) in patients with distant metastasis and 61 months (range, 60-86 months) in those without distant metastasis. At the time of resection or during subsequent follow-up, 32 patients had distant metastases. The MVD was significantly greater in patients with than without distant metastases (mean ± standard deviation: 10.4±4.9 vs. 7.6±3.3, P=0.008; Welch's t-test). Stepwise multivariate logistic regression indicated that MVD, regional lymph node metastasis, and tumor size were independent risk factors for distant metastases. Combining assessment of monoclonal antibodies to CD105-positive MVD with assessment of regional lymph node metastasis and tumor size may help to identify patients who need more intensive surveillance after surgery for colorectal cancer.

STEAP-1: A Potential Biomarker, Promising Target?

STEAP1 is one of six-transmembrane epithelial antigen of prostate (STEAP) highly expressed in human prostate cancer and is up-regulated in multiple cancers, including bladder, colon, breast, Ewing and lung cancers. Immunohistochemical analysis demonstrates significant STEAP1 expression at the intercellular communication between adjacent cells suggesting that this antigen must be a channel, or a transport protein indicating its potential role in tumor cell intercellular communication increasing the potential of STEAP1 as a diagnostic, prognostic, prophylactic and/or therapeutic target for new immunotherapeutic strategies. In prostate cancer, overexpression relates adenocarcinoma and prostatic intraepithelial neoplasia scores suggest that STEAP1 could be involved in tumor initiation and progression and may be of clinical usefulness in early disease diagnosis. Also, STEAP1 can serve as a biomarker of worse prognosis because of its association with higher Gleason score, seminal vesicle invasion, biochemical recurrence, and worse outcome. Therapeutically, STEAP1 is one of a few prostate cancer antigens that meet the appropriate criteria and represent an attractive target for antibody cancer therapy. It can be a target of anti-tumor CD8, or serve as a tool for vaccination as shown by xenograft models and phases I/II studies. STEAP1 could also serve as a new marker of tumor angiogenesis in lung cancer, indicate for occult residual tumor cells in patients with Ewing Sarcoma and also be used as a cell surface antigen for the development of breast cancer immunotherapy. The standardization of its evaluation as well as the validation through randomized trials would be necessary.