Tumor Hematogenous Metastasis Research Articles

Synergistic potentiation of the anti-metastatic effect of a Ginseng-Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NET formation.

The combination of the roots of ginseng and Salvia miltiorrhiza is an effective approach for treating metastatic cancer in patients with Qi stagnation and blood stasis patterns. However, the molecular mechanism underlying the combined use of ginseng and Salvia miltiorrhiza is unknown. This study unveils the pharmacological foundation of ginseng and Salvia miltiorrhiza by examining the involvement of neutrophils in the critical process of tumor hematogenous metastasis. Additionally, by employing a reverse pharmacology research model (effect-target-constituent), potential potent components were screened, and the dominant component formulations were determined. An experimental lung metastatic model was constructed to compare the antitumor effects of ginseng and Salvia miltiorrhiza. RNA sequencing was employed to identify pivotal biological events and key targets, while the detection of CD62E expression and neutrophil extracellular traps (NETs) release was used to screen for effective substances in ginseng and Salvia miltiorrhiza. In addition, a comprehensive array of in vitro and in vivo experiments was conducted to explore the underlying mechanisms and therapeutic significance. Compared with single-herb use, the use of ginseng or Salvia miltiorrhiza significantly reduced tumor metastasis, which was accompanied by reduced neutrophil infiltration into the lungs. Cryptotanshinone (CPT), an active constituent of Salvia miltiorrhiza, can inhibit neutrophil adhesion and recruitment to lung tissue by downregulating the expression of E-selectin (CD62E) in endothelial cells. Moreover, the ginseng -derived ginsenoside Rg1 mitigated the formation of NETs in lung tissues and reversed the protumor effects of NETs. We further explored the efficacy of combination therapy with Rg1 and CPT, which also reduced tumor metastasis in vivo. Ginseng and Salvia miltiorrhiza exhibited a mutual potentiation of the anti-metastatic effect by suppressing both early and late stages of neutrophil-initiated metastasis cascade. Rg1 and CPT represent the synergistic ingredients from ginseng and Salvia miltiorrhiza, respectively.

Direct contact between tumor cells and platelets initiates a FAK-dependent F3/TGF-β positive feedback loop that promotes tumor progression and EMT in osteosarcoma

Platelets have received growing attention for their roles in hematogenous tumor metastasis. However, the tumor-platelet interaction in osteosarcoma (OS) remains poorly understood. Here, using platelet-specific focal adhesion kinase (FAK)-deficient mice, we uncover a FAK-dependent F3/TGF-β positive feedback loop in OS. Disruption of the feedback loop by inhibition of F3, TGF-β, or FAK significantly suppresses OS progression. We demonstrate that OS F3 initiated the feedback loop by increasing platelet TGF-β secretion, and platelet-derived TGF-β promoted OS F3 expression in turn and modulated OS EMT process. Immunofluorescence results indicate platelet infiltration in OS niche and we verified it was mediated by platelet FAK. In addition, platelet FAK was proved to mediate platelet adhesion to OS cells, which was vital for the initiation of F3/TGF-β feedback loop. Collectively, these findings provide a rationale for novel therapeutic strategies targeting tumor-platelet interplay in metastatic OS.

The role of tumor-platelet interplay and micro tumor thrombi during hematogenous tumor metastasis.

In addition to their pivotal roles in coagulation and thrombosis, platelets are crucial in tumor progression, with plenty of clinical and experimental data demonstrating that the interplay of platelets and tumor cells is essential for hematogenous tumor metastasis. After detach from primary sites, tumor cells intravasate into the blood circulation becoming circulating tumor cells and induce platelet activation, aggregation and encasement around tumor cells to form micro tumor thrombi, which create a permissive tumor microenvironment for metastasis. Platelets in micro tumor thrombi protect tumor cells from immune surveillance and anoikis (detachment-triggered apoptosis) through various pathways, which are significant for tumor cell survival in the bloodstream. Moreover, platelets can facilitate tumor metastasis by expediting epithelial-mesenchymal transition (EMT), adhesion to the endothelium, angiogenesis, tumor proliferation processes and platelet-derived microvesicle (PMV) formation. Here, we provide a synopsis of the current understanding of the formation of micro tumor thrombi and the role of micro tumor thrombi in tumor hematogenous metastasis based on the tumor-platelet interplay. We also highlight potential therapeutic strategies targeting platelets for tumor treatment, including cancer-associated platelet-targeted nanomedicines.

A Supramolecular Nitric Oxide Nanodelivery System for Prevention of Tumor Metastasis by Inhibiting Platelet Activation and Aggregation.

Tumor cell-induced platelet aggregation (TCIPA) is known as a critical step in hematogenous tumor metastasis. The endogenous nitric oxide (NO) plays an important role in anticoagulation, which might have great potential to inhibit TCIPA. Herein, a glutathione-sensitive supramolecular nanocarrier is prepared via host-guest interaction for effective delivery of NO and chemotherapeutic agent gemcitabine (GEM). NO could be effectively released in tumor cells and inhibits platelet activation and aggregation. The inhibition of TCIPA by NO could effectively attenuate the migration and invasion of tumor cells in vitro. Furthermore, the in vivo experiments demonstrate that the NO and GEM co-delivered supramolecular nanocarriers can suppress the growth of primary tumor. More importantly, although NO-containing nanocarriers cannot inhibit the growth of primary tumors effectively, they can significantly inhibit tumor metastasis. This NO-based nano-delivery system not only provides new inspiration for multifunctional applications of NO in cancer therapy but also shows great potential in clinical antimetastatic applications.

Targeting HECTD3-IKKα axis inhibits inflammation-related metastasis

Metastasis is the leading cause of cancer-related death. The interactions between circulating tumor cells and endothelial adhesion molecules in distant organs is a key step during extravasation in hematogenous metastasis. Surgery is a common intervention for most primary solid tumors. However, surgical trauma-related systemic inflammation facilitates distant tumor metastasis by increasing the spread and adhesion of tumor cells to vascular endothelial cells (ECs). Currently, there are no effective interventions to prevent distant metastasis. Here, we show that HECTD3 deficiency in ECs significantly reduces tumor metastasis in multiple mouse models. HECTD3 depletion downregulates expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, in mouse primary ECs and HUVECs stimulated by inflammatory factors and inhibits adhesion of tumor cells to ECs both in vitro and in vivo. We demonstrate that HECTD3 promotes stabilization, nuclear localization and kinase activity of IKKα by ubiquitinating IKKα with K27- and K63-linked polyubiquitin chains at K296, increasing phosphorylation of histone H3 to promote NF-κB target gene transcription. Knockout of HECTD3 in endothelium significantly inhibits tumor cells lung colonization, while conditional knockin promotes that. IKKα kinase inhibitors prevented LPS-induced pulmonary metastasis. These findings reveal the promotional role of the HECTD3-IKKα axis in tumor hematogenous metastasis and provide a potential strategy for tumor metastasis prevention.

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

A Relatively Small Gradient of Extracellular pH Directs Migration of MDA-MB-231 Cells In Vitro.

Hematogenous tumor metastasis begins with the invasion and spread of primary tumor cells in the local tissue leading to intravasation. We hypothesized that tumor cells might actively migrate toward intratumor vessels with the extracellular metabolic gradient acting as a guiding cue. Here, we determined in vitro whether the extracellular gradient of pH can act as a cue for directional migration in MDA-MB-231 cells. Cell migration was determined by the wound-healing assay under gradients of extracellular pH (~0.2 units/mm) and oxygen concentration (~6% O2/mm) that were produced by a microfluidic device, gap cover glass (GCG). Without GCG, the migration of cells was spatially homogeneous; the same number of cells migrated to the rectangular wound space from the left and right boundaries. In contrast, when GCG generated pH/O2 gradients across the wound space, the number of cells migrating to the wound space from the boundary with higher pH/O2 values was considerably decreased, indicating a preferential movement of cells toward the region of higher pH/O2 in the gradient. The addition of hepes in the extracellular medium abolished both the extracellular pH gradient and the directional cell migration under GCG. We conclude that relatively small gradients of pH in the extracellular medium compared to those found in Na+/H+ exchanger-driven cell migration were sufficient to guide MDA-MB-231 cells. The directional cell migration as guided by the metabolic gradient could effectively elevate the probability of intravasation and, ultimately, hematogenous metastasis.

Dihydrodiosgenin inhibits endothelial cell-derived factor VIII and platelet-mediated hepatocellular carcinoma metastasis.

Background: Our previous studies have demonstrated that diosgenin and diosgenin derivatives exhibit excellent antithrombotic activity via regulating platelet function and coagulation factor level. Platelets and blood coagulation system are highly associated with tumor hematogenous metastasis. Therefore, the purpose of this study was to evaluate whether dihydrodiosgenin (dydio) mediated-platelet inhibition or coagulation factor level modulation is involved in hepatocellular carcinoma cell (HCC) metastasis.Methods: Cell viability was examined by MTT and colony formation assays. Platelet aggregation text and morphology were used to assess dydio’s role on tumor cell-induced platelet activation (TCIPA). Scratch assay, adhesion assay and Western blot were used to evaluate dydio’s role on platelet-mediated metastasis. Western blot and fluorescence detection were performed to clarify dydio's role on endothelial cell (EC) function. The mice lung metastasis model was constructed to investigated dydio’s function on coagulation factor and platelet-mediated metastasis.Results: This study found that pretreatment with dydio caused a significant inhibition of TCIPA. Platelets exposed to dydio significantly inhibited their adhesion to tumor cells, meanwhile, releasates of platelets that pretreated with dydio led to diminished cancer cell proliferation and migration along with the increase of epithelial markers E-cadherin and loss of mesenchymal phenotype. Additionally, ECs pretreated with dydio suppressed factor VIII (FVIII) level which in turn restrained the activation of platelets and the adhesion of cancer cells or platelets to ECs. Interestingly, our study demonstrated that FVIII could promote HCC proliferation. In vivo study revealed that mice intragastrical (i.g.) administration with dydio significantly inhibited the lung metastasis of hepal-6 cells which is highly correlated with the altered platelet function and coagulation level.Conclusion: Taken together, these results demonstrated that dydio altered platelet function and coagulation FVIII level, resulting in decreased metastatic potential of HCC. Thus, our study reveals that dydio exerts novel mechanisms of antitumor action beside its direct antitumor activity.

C‐type lectin‐like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor‐bearing mice

Background C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2-podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.

Real - time monitoring of circulation tumor cells in a surgical orthotopic implantation model of human metastatic prostate cancer

Objective To study the relationship of circulation tumor cells (CTCs) and metastatic process, we use subcutaneous model and surgical orthotopic implantation model of human metastatic prostate cancer and detected by in vivo flow cytometry (IVFC). Methods The human prostate cancer cell line PC3 cells were transduced with pLVX-EGFP-3FLAG lentivirus vector containing the green fluorescent protein (GFP) and puromycin resistance gene. PC3 cells, high-expression of GFP, were obtained by screening of stably transfected clones. PC3-GFP cells or unlabeled tumor cells were implanted into the right armpit of 10 nude mice to build up subcutaneous xenograft prostate cancer mode1, which provided tumor fragments to implant the orthotopic prostate tumor mode1. Tumor tissue with high fluorescent from the subcutaneous tumor was harvested and cut into pieces of 1 mm3, and grafted into the anterior prostates of 10 nude mice. The number of CTCs monitored by IVFC real-time detecting fluorescent signal noninvasively at 5 d, 10 d and 15 d after tumor established. Results A PC3-GFP cell line was established which had the same growth pattern as well as tumorigenesis ability as mock-transfected PC3 cells. IVFC detected 2.37, 6.25, 99.75 CTCs/h at 5, 10, and 15 days. Conclusion The orthotopic prostate cancer models are better to assess the morphology, growth, metastatic pattern and development characteristics of clinical disease. They are also more representative of a primary tumor with respect to tumor site and metastasis, especially CTCs dynamics. The in vivo flow cytometry technique combined with orthotopic tumor models might provide insights into tumor hematogenous metastasis and guidance to cancer therapy. Key words: Prostate cancer; Metastasis; Circulating tumor cells; In vivo flow cytometry

Melanoma cell metastasis via P-selectin-mediated activation of acid sphingomyelinase in platelets

Metastatic dissemination of cancer cells is one of the hallmarks of malignancy and accounts for approximately 90% of human cancer deaths. Within the blood vasculature, tumor cells may aggregate with platelets to form clots, adhere to and spread onto endothelial cells, and finally extravasate to form metastatic colonies. We have previously shown that sphingolipids play a central role in the interaction of tumor cells with platelets; this interaction is a prerequisite for hematogenous tumor metastasis in at least some tumor models. Here we show that the interaction between melanoma cells and platelets results in rapid and transient activation and secretion of acid sphingomyelinase (Asm) in WT but not in P-selectin-deficient platelets. Stimulation of P-selectin resulted in activation of p38 MAPK, and inhibition of p38 MAPK in platelets prevented the secretion of Asm after interaction with tumor cells. Intravenous injection of melanoma cells into WT mice resulted in multiple lung metastases, while in P-selectin-deficient mice pulmonary tumor metastasis and trapping of tumor cells in the lung was significantly reduced. Pre-incubation of tumor cells with recombinant ASM restored trapping of B16F10 melanoma cells in the lung in P-selectin-deficient mice. These findings indicate a novel pathway in tumor metastasis, i.e., tumor cell mediated activation of P-selectin in platelets, followed by activation and secretion of Asm and in turn release of ceramide and tumor metastasis. The data suggest that p38 MAPK acts downstream from P-selectin and is necessary for the secretion of Asm.

Numerical simulation of a single cell passing through a narrow slit

The narrow slit between endothelial cells that line the microvessel wall is the principal pathway for tumor cell extravasation to the surrounding tissue. To understand this crucial step for tumor hematogenous metastasis, we used dissipative particle dynamics method to investigate an individual cell passing through a narrow slit numerically. The cell membrane was simulated by a spring-based network model which can separate the internal cytoplasm and surrounding fluid. The effects of the cell elasticity, cell shape, nucleus and slit size on the cell transmigration through the slit were investigated. Under a fixed driving force, the cell with higher elasticity can be elongated more and pass faster through the slit. When the slit width decreases to 2/3 of the cell diameter, the spherical cell becomes jammed despite reducing its elasticity modulus by 10 times. However, transforming the cell from a spherical to ellipsoidal shape and increasing the cell surface area by merely 9.3% can enable the cell to pass through the narrow slit. Therefore, the cell shape and surface area increase play a more important role than the cell elasticity in cell passing through the narrow slit. In addition, the simulation results indicate that the cell migration velocity decreases during entrance but increases during exit of the slit, which is qualitatively in agreement with the experimental observation.

Reinforcing endothelial junctions prevents microvessel permeability increase and tumor cell adhesion in microvessels in vivo.

Tumor cell adhesion to the microvessel wall is a critical step during tumor metastasis. Vascular endothelial growth factor (VEGF), a secretion of tumor cells, can increase microvessel permeability and tumor cell adhesion in the microvessel. To test the hypothesis that inhibiting permeability increase can reduce tumor cell adhesion, we used in vivo fluorescence microscopy to measure both microvessel permeability and adhesion rates of human mammary carcinoma MDA-MB-231 cells in post-capillary venules of rat mesentery under the treatment of VEGF and a cAMP analog, 8-bromo-cAMP, which can decrease microvessel permeability. By immunostaining adherens junction proteins between endothelial cells forming the microvessel wall, we further investigated the structural mechanism by which cAMP abolishes VEGF-induced increase in microvessel permeability and tumor cell adhesion. Our results demonstrate that 1) Pretreatment of microvessels with cAMP can abolish VEGF-enhanced microvessel permeability and tumor cell adhesion; 2) Tumor cells prefer to adhere to the endothelial cell junctions instead of cell bodies; 3) VEGF increases microvessel permeability and tumor cell adhesion by compromising endothelial junctions while cAMP abolishes these effects of VEGF by reinforcing the junctions. These results suggest that strengthening the microvessel wall integrity can be a potential approach to inhibiting hematogenous tumor metastasis.

Arresting the Colonial Destiny of Metastatic Seeds with DNA Aptamers

In recent years, several studies have identified a class of carbohydrate-binding proteins known as (endothelial) E- and (platelet) P-selectins as potential therapeutic targets for inhibiting hematogenous tumor metastasis, owing to their critical role in the metastatic process. Selectins enable tethering and rolling of circulating tumor cells or cancer stem cells (CTC/CSCs) to the postcapillary venules, promoting their transmigration through the endothelium and subsequent homing to metastatic sites. In this issue of Molecular Therapy, Kang et al. describe the preclinical characterization of a nucleic acid DNA aptamer to E-selectin as a safe and effective therapeutic option for preventing this process and inhibiting breast cancer metastasis.1

Regulation of hematogenous tumor metastasis by acid sphingomyelinase.

Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1−/− mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of α5β1 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing β1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis.

Circulation long non-coding RNAs act as biomarkers for predicting tumorigenesis and metastasis in hepatocellular carcinoma.

Alpha Fetal Protein (AFP) was one of the traditional biomarker for diagnosis of Hepatocellular carcinoma (HCC) clinically, however, with the low specificity of AFP, the early diagnosis or the metastasis prediction of HCC is inferior. A new, minimally invasive and more specificity biomarker for the diagnosis or metastasis prediction of HCC are necessary. In this study, we applied an lncRNA microarray to screen the potential biomarker for HCC. The multi-stage validation and risk score formula detection was used for validation. We discovered three lncRNA, RP11-160H22.5, XLOC_014172 and LOC149086, which were up-regulated in HCC comparing with the cancer-free controls with the merged area under curve (AUC) in training set and validation set of 0.999 and 0.896. Furthermore, XLOC_014172 and LOC149086 was confirmed highly increased in metastasis HCC patients with the merged AUC in training set and validation set of 0.900 and 0.934. Besides, most patients presented a decreased level of the three lncRNAs after operation, while the patients with secondary increased level might be associated with tumor hematogenous metastasis. RP11-160H22.5, XLOC_014172 and LOC149086 might be the potential biomarker for the tumorigenesis prediction and XLOC_014172 and LOC149086 for metastasis prediction in the future.

Abstract B25: ASK1 is involved in tumor lung metastasis through regulation of platelet functions

Abstract Apoptosis signal-regulating kinase 1 (ASK1) is a MAP3K in the JNK and p38 MAPK pathways and responds to various stresses. Accumulating evidence indicates that ASK1 plays important roles in tumorigenesis by regulating apoptosis and inflammation. However, little is known about its role in tumor metastasis. Here we examined the roles of ASK1 in lung metastasis by intravenous injection of Lewis lung carcinoma cells constitutively expressing luciferase (3LL-Luc2). 14 days after injection of 3LL-Luc2 cells, ASK1-/- mice showed remarkably reduced number of tumor nodules and decreased luciferase activity of the lung lysates than wild-type mice. Taken together with the data obtained by in vivo imaging system (IVIS), ASK1 deficiency appeared to result in the attenuation of lung metastasis in this model. Hematogenous tumor metastasis is known to consist of multiple steps such as invasion, intravasation, extravasation and colonization. We next measured the time-course-dependent luciferase activity of the lung lysates after injection from 10 minutes to 7 days in order to investigate which stages of tumor metastasis ASK1 is involved in. ASK1-/- mice had markedly lower luciferase activity of the lung lysates than wild-type mice as early as 3 hours after injection. Moreover, ASK1 phosphorylation in lungs of wild-type mice was accelerated at that point; hence ASK1 is suggested to be involved in the very early stage called seeding stage of lung metastasis, which is the time point prior to the extravasation of tumor cells. Myeloid cells such as platelets and innate immune cells are known to participate in the seeding stage and we next investigated whether ASK1 in myeloid cells is involved in tumor metastasis by analyzing bone marrow chimeric mice. Wild-type mice with bone marrow from ASK1-/- mice showed significant decrease in luciferase activity of the lung lysates compared to wild-type mice with bone marrow from wild-type mice, indicating that ASK1 in myeloid cells plays an important role in tumor metastasis. Among various myeloid cell types, we first focused on platelets. Platelets adhere and aggregate to surround tumor cells in the seeding stage and are known to positively regulate hematogenous tumor metastasis. Transcriptional analysis of platelets revealed that the mRNA expression of some genes related to platelet functions was reduced in ASK1-/- mice. In addition, analysis by western blot revealed that platelets of ASK1-/- mice exhibit markedly reduced phosphorylation of JNK and p38, both of which are well-known downstream signaling molecules of ASK1 and also are indicated to participate in platelet functions. Moreover, ASK1-/- mice showed bleeding tendency in tail bleeding assay. These data collectively indicate that ASK1 deficiency may attenuate platelet functions. Defect in platelet functions of ASK1-/- mice may result in attenuated interaction between platelets and tumor cells. In conclusion, ASK1 deficiency is suggested to attenuate lung metastasis through impaired platelet functions. Citation Format: Miki Kamiyama, Isao Naguro, Hidenori Ichijo. ASK1 is involved in tumor lung metastasis through regulation of platelet functions. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B25. doi:10.1158/1538-7445.CHTME14-B25

Automatic Classification and Quantification of Cell Adhesion Locations on the Endothelium.

To target tumor hematogenous metastasis and to understand how leukocytes cross the microvessel wall to perform immune functions, it is necessary to elucidate the adhesion location and transmigration pathway of tumor cells and leukocytes on/across the endothelial cells forming the microvessel wall. We developed an algorithm to classify and quantify cell adhesion locations from photomicrographs taken from the experiments of tumor cell/leukocyte adhesion in individual microvessels. The first step is to identify the microvessel by a novel gravity-field dynamic programming (DP) procedure. Next, an anisotropic image smoothing suppresses noises without unduly mitigating crucial visual features. After an adaptive thresholding process further tackles uneven lighting conditions during the imaging process, a series of local mathematical morphological operators and eigenanalysis identify tumor cells or leukocytes. Finally, a novel double component labeling procedure categorizes the cell adhesion locations. This algorithm has generated consistently encouraging performances on photomicrographs obtained from in vivo experiments for tumor cell and leukocyte adhesion locations on the endothelium forming the microvessel wall. Compared with human experts, this algorithm used 1/500-1/200 of the time without having the errors due to human subjectivity. Our automatic classification and quantification method provides a reliable and cost effective approach for biomedical image processing.

Endothelial Delta-like 4 (DLL4) promotes renal cell carcinoma hematogenous metastasis.

The Notch ligand Delta-like 4 (DLL4) plays an important role in tumor angiogenesis, which is required for tumor invasion and metastasis. Here we showed that DLL4 was elevated in endothelium and Notch signaling was activated in renal cell carcinoma (RCC). Exogenous DLL4 induced RCC cell migration and invasion by activating intercellular Notch signaling. Importantly, the DLL4/Notch/Hey1/MMP9 cascades connecting the endothelium to the cancer cells in metastasis were identified. Knockdown of Hey1 decreased expression of MMP9 and attenuated tumor invasion. The clinical investigation on 120 cases of RCC specimens indicated that expressions of Hey1 and MMP9 correlated with DLL4 density. Moreover, univariate and multivariate analyses showed that tumor hematogenous metastasis not only was depended on microvessel density but was also associated with tumor size and DLL4 density. During 4-year surveillance, high-level of DLL4 density was associated with a higher probability of developing metastasis and being sensitive to target therapies. Our data suggest that RCC progression is caused in part by activated DLL4/Notch signaling, interaction of endothelium and cells, which can be therapeutically targeted.

Anticoagulation Inhibits Tumor Cell-Mediated Release Of Platelet Angiogenic Proteins and Disrupts The Platelet Angiogenic Potential

Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. While the involvement of platelets in hematogenous tumor metastasis has long been recognized, the cause and effect relationship linking the two remains unclear. Due to the propensity for clotting, patients with malignancy are often anti-coagulated with heparin products, which paradoxically offer a survival benefit by an unknown mechanism. We hypothesized that anti-thrombotic agents alter the release of angiogenesis regulatory proteins from platelets. We have previously shown that we can manipulate the angiogenic potential of the platelet releasate through physiological (platelet agonists) and pathological activation (MCF-7 tumor cells) (Battinelli et al., 2011). Our data reveals that platelets exposed to heparin (UFH) or its derivative low molecular weight heparin (LMWH) release statistically significant decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells). The angiogenic potential from ADP or tumor cell generated platelet releasate is also significantly decreased as evidenced by dramatically diminished capillary tube branch point formation and endothelial cell migration. To explore the impact of these anticoagulants on the angiogenic protein contents of the releasate, we analyzed the releasate from platelets exposed to LMWH alone or activated with ADP or MCF-7 cells in conjunction with LMWH. Using a angiogenesis protein array to simultaneously sample the angiogenic content of the platelet releasate, we found that exposure to LMWH resulted in a decrease in pro-angiogenic protein content. Fondaparinux (Xa inhibitor) demonstrated similar impact on the platelet angiogenic potential. Since MCF-7 cells are known to secrete thrombin; the main target of anticoagulants, we hypothesized that these drugs disrupt thrombin signaling through the platelet PAR1 receptor. Addition of PAR1 antagonists to platelets decreased VEGF release and angiogenic potential. Exposure to a PAR1 agonist in the presence of anticoagulants rescued the angiogenic potential. Direct evidence of disrupted PAR1 signaling was demonstrated by decreased cleavage (activation) of the PAR1 receptor on anticoagulated platelets in the presence of MCF-7 cells (as measured by SPAN12 using Flow Cytometry). This data establishes a mechanism by which anticoagulants can decrease the angiogenic potential of platelets through impaired PAR1 activation. The impact of anticoagulants was also observed in platelet releasate of patients. We measured the VEGF concentration in the platelet releasate of patients currently medicated with LMWH or Fondaparinux and observed a statistically significant decrease in VEGF release after exposure to MCF-7 cells. Taken together, these data underscore the pivotal role of platelets in regulating tumor angiogenesis and point to a potential source for development of therapeutic intervention targeting the PAR1 receptor. Disclosures:No relevant conflicts of interest to declare.