Tumor-derived Endothelial Cells Research Articles

The Presence of Small-Size Circulating Tumor Cells Predicts Worse Prognosis in Non-Small Cell Lung Cancer Patients.

Most patients with non-small cell lung cancers (NSCLC) are diagnosed at advanced stages. The 5-year survival rate of patients with advanced lung cancer is less than 20%, which makes lung cancer the leading cause of cancer-related deaths worldwide. To identify indicators that can predict the prognosis of lung cancer patients. To determine the correlation between circulating tumor cells (CTCs), circulating tumor-derived endothelial cells (CTECs), and their subtypes and the prognosis of patients with NSCLC, 80 patients with lung cancer were recruited and 48 patients who met the enrollment criteria were selected in this study. Peripheral blood was collected from the enrolled patients before any treatment and analyzed by the subtraction enrichment and immunostaining-fluorescence in situ hybridization technique to determine the correlation between CTCs and CTECs and lung cancer disease progression and to identify prognostic indicators. In all patients, the positive rate of CTCs was 100% and the positive rate of CTECs was 81.3%. The CTEC positivity rate was higher in late-stage patients than in early-stage patients (P = .03). Patients with advanced or lymph node metastases had a higher rate of small-size CTC positivity than those with early or no lymph node metastases. Large-size CTEC positivity was higher in patients with advanced NSCLC than in early-stage patients. Patients with ≥1 small-size CTC had shorter progression-free survival, and it was an independent prognostic factor. Small-size CTCs are a reliable prognostic indicator and a probable predictor of the severity of disease in NSCLC patients.

Abstract 4212: Mimicking HCC complex biology and diverse treatment response in a patient-derived microfluidic model

Abstract Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is closely associated with progressive stages of liver diseases. Its transformation, survival, progression and metastasis have been associated with regulation mechanisms orchestrated by the tumor stroma. Patient differences in tumor composition and cellular signaling are reflected in the diversity of therapy response. Hence, comprehensive in vitro models that capture patient-specific tumor and stromal components are necessary to elucidate and evaluate anticancer therapy. Patient-derived dissociated HCC cells from eight different patients were combined with tumor-derived fibroblasts and endothelial cells within a microfluidic setup comprising 64 parallel chips in a microtiter plate format. Cultures were challenged with an individual or combinatorial treatment from a panel of tool compounds for 72 hours in an automated setup. We used immunofluorescence and high content confocal imaging to assess the structural and biological interaction between cancer cells and the associated stroma. Viability assessment, artificial intelligence-based vascular morphology characterization and multiplexed chemokine/cytokine release measurements were used to elucidate patient and drug-dependent effects of single or combinatorial treatments. Cellular interaction within the microfluidic platform led to a vascularized tumor construct, with hepatocellular carcinoma aggregates being enveloped and traversed by a lumenized and interconnected vascular plexus, in association with tumor-derived fibroblasts. Compound treatment led to profound differences across the tested parameters, indicating different anticancer effects in conjunction with the biological diversity of the patient material. Finally, morphological assessment revealed distinct compound-induced effects on the tumor’s vascular organization. We present a vascularized patient-derived HCC model that includes relevant cellular players of the tumor microenvironment found in vivo. These co-cultures are highly suitable for studying specific cell types as well as patient-specific responses. We envision that this system has the potential to provide a platform for understanding the interplay between different cell types present in hepatocellular carcinomas, with sufficient scalability ease of use for industrial and clinical implementation. Citation Format: Orsola Mocellin, Abbie Robinson, Aleksandra Olczyk, Stephane Treillard, Thomas Olivier, Chee P. Ng, Jeroen Heijmans, Arthur Stok, Gilles van Tienderen, Monique Verstegen, Sebastian J. Trietsch, Henriëtte L. Lanz, Paul Vulto, Jos Joore, Karla Queiroz. Mimicking HCC complex biology and diverse treatment response in a patient-derived microfluidic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4212.

Identification of GB3 as a Novel Biomarker of Tumor-Derived Vasculature in Neuroblastoma Using a Stiffness-Based Model.

Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

Microfluidic model of the alternative vasculature in neuroblastoma

Neuroblastoma (NB) is a highly vascularized pediatric tumor arising from undifferentiated neural crest cells early in life, exhibiting both traditional endothelial-cell-driven vasculature and an intriguing alternative vasculature. The alternative vasculature can arise from cancer cells undergoing transdifferentiation into tumor-derived endothelial cells (TEC), a trait associated with drug resistance and tumor relapse. The lack of effective treatments targeting NB vasculature primarily arises from the challenge of establishing predictive in vitro models that faithfully replicate the alternative vasculature phenomenon. In this study, we aim to recreate the intricate vascular system of NB in an in vitro context, encompassing both types of vascularization, by developing a novel neuroblastoma-on-a-chip model. We designed a collagen I/fibrin-based hydrogel closely mirroring NB’s physiological composition and tumor stiffness. This biomaterial created a supportive environment for the viability of NB and endothelial cells. Implementing a physiological shear stress value, aligned with the observed range in arteries and capillaries, within the microfluidic chip facilitated the successful development of vessel-like structures and triggered transdifferentiation of NB cells into TECs. The vascularized neuroblastoma-on-a-chip model introduced here presents a promising and complementary strategy to animal-based research with a significant capacity for delving into NB tumor biology and vascular targeting therapy.

10038-ANGI-2 NEW ANTI-ANGIOGENIC THERAPY WITH THE ANTI-DEPRESSANT TOWARD TUMOR DERIVED ENDOTHELIAL CELLS (TDECS)

Abstract BACKGROUND Anti-angiogenic therapy has been reported to prolong patient survival in some malignancies, but has not been shown to be effective in glioblastoma. We focused on the relationship between the differentiation of glioma stem-like cells (GSCs) into tumor-derived endothelial cells (TDECs) and resistance to anti-angiogenic therapy. In particular, we elucidated the mechanism of drug resistance of TDECs to anti-angiogenesis inhibitors, and identified and elucidated the efficacy of novel anti-angiogenesis inhibitors with potential clinical application. METHODS 005 mouse GSCs were differentiated into TDECs under hypoxic conditions and analyzed by RNA-Seq over time. Furthermore, novel angiogenesis inhibitors for glioblastoma were evaluated by tube formation assay and existing blood-brain barrier permeable drugs. Novel angiogenesis inhibitors were administered in vitro to differentiated TDECs and in vivo to the 005 mouse brain tumor model and evaluated by RNA-Seq, immunohistochemistry, MRI imaging, and survival. FINDINGS In the GSCs005 brain tumor model, inhibition of the Vascular Endothelial Growth Factor (VEGF) pathway had no anti-tumor effect and conversely increased TDECs. A drug screening by Existing Drugs Redevelopment showed that the antidepressant sertraline inhibited TDEC tube formation. Sertraline decreased the expression of Lama4 and Ang2 in TDECs, which play an important role in non-VEGF-mediated angiogenesis in tumors. sertraline in combination with VEGF pathway inhibitors showed prolonged survival in the 005 model. CONCLUSION Diversity of tumor vascular endothelial cells across VEGF and non-VEGF pathways was shown to induce anti-angiogenic resistance. The combination of sertraline and VEGF pathway inhibitors with antidepressants was an effective anti-angiogenic therapy for glioblastoma and could be applied safely and cost-effectively.

A comparative study on ctDNA and tumor DNA mutations in lung cancer and benign cases with a high number of CTCs and CTECs

BackgroundLiquid biopsy provides a non-invasive approach that enables detecting circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) using blood specimens and theoretically benefits early finding primary tumor or monitoring treatment response as well as tumor recurrence. Despite many studies on these novel biomarkers, their clinical relevance remains controversial. This study aims to investigate the correlation between ctDNA, CTCs, and circulating tumor-derived endothelial cells (CTECs) while also evaluating whether mutation profiling in ctDNA is consistent with that in tumor tissue from lung cancer patients. These findings will help the evaluation and utilization of these approaches in clinical practice.Methods104 participants (49 with lung cancer and 31 with benign lesions) underwent CTCs and CTECs detection using integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy. The circulating cell-free DNA (cfDNA) concentration was measured and the mutational profiles of ctDNA were examined by Roche AVENIO ctDNA Expanded Kit (targeted total of 77 genes) by next generation sequencing (NGS) in 28 patients (20 with lung cancer and 8 with benign lesions) with highest numbers of CTCs and CTECs. Mutation validation in matched tumor tissue DNA was then performed in 9 patients with ctDNA mutations using a customized xGen pan-solid tumor kit (targeted total of 474 genes) by NGS.ResultsThe sensitivity and specificity of total number of CTCs and CTECs for the diagnosis of NSCLC were 67.3% and 77.6% [AUC (95%CI): 0.815 (0.722–0.907)], 83.9% and 77.4% [AUC (95%CI): 0.739 (0.618–0.860)]. The concentration of cfDNA in plasma was statistically correlated with the size of the primary tumor (r = 0.430, P = 0.022) and CYFRA 21–1 (r = 0.411, P = 0.041), but not with the numbers of CTCs and CTECs. In this study, mutations were found to be poorly consistent between ctDNA and tumor DNA (tDNA) in patients, even when numerous CTCs and CTECs were present.ConclusionDetection of CTCs and CTECs could be the potential adjunct tool for the early finding of lung cancer. The cfDNA levels are associated with the tumor burden, rather than the CTCs or CTECs counts. Moreover, the poorly consistent mutations between ctDNA and tDNA require further exploration.

Tumor-derived Endothelial Cell: Important Etiological Factors in Endometriosis

Endometriosis (EMS) is a very complex disease with high heterogeneity. Recently, single-cell RNA sequencing (scRNA-seq) has been applied to comprehensively characterize cellular heterogeneity. Here, we built a new transcriptomic profile of EMS cellular signatures. Three women diagnosed with endometriosis were recruited. Their fresh eutopic endometrium (EM) and ectopic endometrium (EC) tissues were sampled during surgery. ScRNA-seq was performed on 10x Genomics Chromium. Thirty cell clusters were identified as more than ten different cell types using cell type-specific marker genes. Re-clustering analysis revealed five subtypes of endothelial cells (ECs). Compared to EM, the proportion of tumor-derived ECs (IGFBP3+) was significantly increased in EC (43.8 vs. 16.0%). 63 differentially expressed genes (DEGs) between tumor-derived ECs and normal ECs were enriched in "angiogenesis", such as EFNB2, DLL4, and THSD7A. Subsequently, 114 retrospective EMS cases were included in clinical validation studies of EFNB2. It was co-expressed with PECAM1 and IGFBP3 and significantly increased in EC. Meanwhile, the recurrence rate of women with EFNB2++ expression was significantly higher than that of EFNB2+ cases (p <0.05). The significant increase in tumor-derived ECs characterized by neovascularization may be an important pathological feature of EMS. In addition, EFNB2 plays an important role and is closely related to the recurrence of EMS.

Aneuploid subtypes of circulating tumor cells and circulating tumor-derived endothelial cells predict the overall survival of advanced lung cancer.

This study aimed to detect circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) in patients with advanced lung cancer, for describing the distribution characteristics of CTC and CTEC subtypes, exploring the correlation between CTC/CTEC subtypes and novel prognostic biomarkers. A total of 52 patients with advanced lung cancer were enrolled in this study. Using the subtraction enrichment-immunofluorescence in situ hybridization (SE-iFISH) system, CTCs and CTECs derived from these patients were identified. Based on cell size, there were 49.3% small and 50.7% large CTCs, and 23.0% small and 77.0% large CTECs. Triploidy, tetraploidy, and multiploidy varied in the small and large CTCs/CTECs. Besides these three aneuploid subtypes, monoploidy was found in the small and large CTECs. Triploid and multiploid small CTCs and tetraploid large CTCs were associated with shorter overall survival (OS) in patients with advanced lung cancer. However, none of the CTECs subtypes showed a significant correlation with patient prognosis. In addition, we found strong positive correlations (P<0.0001) in the four groups including triploid small cell size CTCs and multiploid small cell size CTECs, and multiploid small cell size CTCs and monoploid small cell size CTECs. Furthermore, combined detection of the specific subtypes, including triploid small CTC and monoploid small CTEC, triploid small CTC and triploid small CTEC, and multiploid small CTC and monoploid small CTEC, were associated with poor prognosis in advanced lung cancer. Aneuploid small CTCs are associated with the outcome of patients with advanced lung cancer. In particular, the combined detection of triploid small CTCs and monoploid small CTECs, triploid small CTCs and triploid small CTECs, and multiploid small CTCs and monoploid small CTECs has clinical significance for predicting prognosis in patients with advanced lung cancer.

IMMU-22. REPROGRAMMING OF MICROGLIA/MACROPHAGES IN GLIOBLASTOMA IMPROVES ANTI-TUMOR T CELL RESPONSES

Abstract Glioma-infiltrating myeloid cells such microglia and macrophages (GAM) are a major component of the immunosuppressive tumor microenvironment in glioblastoma (GBM). Especially their anti-inflammatory M2-like activation state might counteract efficacy of immunotherapeutic interventions by impairing effector T cell infiltration and function. Therefore, we studied the ability of the small molecule inhibitors (SMI) PLX3397, BLZ945, and GW2580 that target the M2-associated colony stimulating factor-1 receptor (CSF1R) to shape GAM phenotype towards a pro-inflammatory M1-like phenotype and how this affects the T cell compartment.Upon treatment of freshly isolated CD11b-sorted GBM patient-derived GAMs (n = 15) a decreased expression of CD163 (M2-like) and an increased expression of HLA-DR (M1-like) as well as an increased nitrite production as an indirect measure for iNOS activation (M1-like) was observed which was most pronounced for BLZ945, and GW2580. Transcriptome analysis of treated vs. untreated GAMs supported a reprogramming towards a pro-inflammatory phenotype. In addition, treatment of GAMs in a GBM patient-derived tumor organoid model confirmed a substantial decrease of CD163+ M2-polarized GAM together with a reduced number of proliferating tumor cells. Finally, treatment of patient-derived GAMs led to an increased T cell transmigration through a dense barrier of autologous tumor-derived endothelial cells and increased the ability of T cells to kill autologous tumor cells. Our results support CSF1R blockade is able to reduce the immunosuppressive and pro-tumorigenic functions of GAM substantially and thereby could enhance the effectiveness of T cell-based immunotherapy.

Circulating Tumor-Derived Endothelial Cells: An Effective Biomarker for Breast Cancer Screening and Prognosis Prediction.

Background Circulating tumor-derived endothelial cell (CTEC) is a new potential tumor biomarker to be associated with cancer development and treatment efficacy. However, few evidences are available for breast cancer. Methods Eighty-nine breast cancer patients were recruited, and preoperative and postoperative blood samples were collected. Besides, 20 noncancer persons were enrolled as controls. An improved subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) method was adopted to codetect CD31+ aneuploid CTEC and CD31− aneuploid circulating tumor cell (CTC). Then, the clinical significance of CTCs and CTECs on breast cancer screening and prognosis prediction was evaluated and compared. Results The positive rate of CTCs and CTECs in newly diagnosed breast cancer patients was 68.75% and 71.88%. Among detected aneuploid circulating rare cells, CTEC accounts for a greater proportion than CTC in breast cancer patients. CTEC-positive rate and level were significantly higher in breast cancer patients with lymph node metastasis (LNM) than those without LNM (P=0.043), while there was no significant difference in CTC. CTEC (area under the curve, AUC = 0.859) had better performance than CTC (AUC = 0.795) to distinguish breast cancer patients from controls by receiver operator characteristic curve analysis. Preoperative CTEC count ≥ 2 was a significant risk factor for reducing PFS of breast cancer patients. Conclusions CTECs may function as a reliable supplementary biomarker in breast cancer screening and prognosis prediction.

Nonhematogenic circulating aneuploid cells confer inferior prognosis and therapeutic resistance in gliomas

Aneuploidy is the hallmark of malignancy. Our previous study successfully detected nonhematogenic circulating aneuploidy cells (CACs) in types of gliomas. The current prospective clinical study aims to further precisely subcategorize aneuploid CACs, including CD31− circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells, and thoroughly investigate the clinical utilities of these different subtypes of cells. Co‐detection and analysis of CTCs and circulating tumor‐derived endothelial cells (CTECs) expressing CD133, glial fibrillary acidic protein (GFAP), or epidermal growth factor receptor variant III (EGFR vIII) were performed by integrated subtraction enrichment and immunostaining fluorescence in situ hybridization (SE‐iFISH) in 111 preoperative primary diffuse glioma patients. Aneuploid CACs could be detected in most de novo glioma patients. Among detected CACs, 45.6% were CD31−/CD45− aneuploid CTCs and the remaining 54.4% were CD31+/CD45− aneuploid CTECs. Positive detection of CTECs significantly correlated with disruption of the blood–brain barrier. The median number of large CTCs (LCTCs, >5 μm, 2) in low‐grade glioma (WHO grade 2) was less than high‐grade glioma (WHO grades 3 and 4) (3, p = 0.044), but this difference was not observed in small CTCs (SCTCs, ≤5 μm), CTECs or CACs (CTCs + CTECs). The numbers of CTCs, CTECs, or CACs in patients with contrast‐enhancing (CE) lesions considerably exceeded that of non‐CE lesions (p < 0.05). Receiver operating characteristic curves demonstrated that CD31+ CTECs, especially LCTECs, exhibited a close positive relationship with CE lesions. Survival analysis revealed that the high number of CD31− CTCs could be an adverse factor for compromised progression‐free survival and overall survival. Longitudinal surveillance of CD31− CTCs was suitable for evaluating the therapeutic response and for monitoring potential emerging treatment resistance.

P2X Purinergic Receptors Are Multisensory Detectors for Micro-Environmental Stimuli That Control Migration of Tumoral Endothelium.

Simple SummaryExtracellular ATP is highly concentrated in tumor stroma. In this study, we investigated the effects of the synthetic ATP analog Benzoylbenzoyl-ATP, 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), an agonist for P2X receptors, on tumor-derived endothelial cells (TEC) obtained from three different human tumors (breast, kidney and prostate carcinomas, respectively, BTEC, RTEC and PTEC). Treatment with high BzATP concentrations (100 µM) significantly reduced migration of all TEC types, resulting ineffective on human normal microvascular endothelium (HMEC); intriguingly, both the functional effect and associated calcium signals are sensitive to some key biological parameters of tumor stroma that include pH, Ca2+ and Zn2+. The lack of calcium signals selectively observed in PTEC, in which BzATP still retains its functional effect, suggests variability of intracellular signaling among TEC. These findings provide novel insights into the role of extracellular ATP as a multisensory regulator of migratory potential in tumoral endothelium.The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.

Intelligent Nanodelivery System-Generated 1 O2 Mediates Tumor Vessel Normalization by Activating Endothelial TRPV4-eNOS Signaling.

Tumor microenvironment (TME)-responsive intelligent photodynamic therapy (PDT) systems have attracted increasing interest in anticancer therapy, due to their potential to address significant and unsatisfactory therapeutic issues, such as limited tissue penetration, inevitable normal tissue damage, and excessive impaired vessels. Here, an H2 O2 -triggered intelligent LCL/ZnO PDT nanodelivery system is elaborately designed. LCL/ZnO can selectively regulate tumor-derived endothelial cells (TECs) and specifically kill tumor cells, by responding to different H2 O2 gradients in TECs and tumor cells. The LCL/ZnO is able to normalize tumor vessels, thereby resulting in decreased metastases, and ameliorating the immunosuppressive TME. Further analysis demonstrates that singlet oxygen (1 O2 )-activated transient receptor potential vanilloid-4-endothelial nitric oxide synthase signals generated in TECs by LCL/ZnO induce tumor vascular normalization, which is identified as a novel mechanism contributing to the increased ability of PDT to promote cancer therapy. In conclusion, designing an intelligent PDT nanodelivery system response to the TME, that includes both selective TECs regulation and specific tumor-killing, will facilitate the development of effective interventions for future clinical applications.

Comprehensive Atlas of Circulating Rare Cells Detected by SE-iFISH and Image Scanning Platform in Patients With Various Diseases.

ObjectiveCirculating rare cells (CRCs) are known as a crucial nucleated cellular response to pathological conditions, yet the landscape of cell types across a wide variety of diseases lacks comprehensive understanding. This study aimed at detecting and presenting a full spectrum of highly heterogeneous CRCs in clinical practice and further explored the characterization of CRC subtypes in distinct biomarker combinations and aneuploid chromosomes among various disease groups.MethodsPeripheral blood was obtained from 2,360 patients with different cancers and non-neoplastic diseases. CRC capture and identification were accomplished using a novel platform integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy with a high-throughput automated image scanning system, on which hemocyte, tumor, epithelial, endothelial, mesenchymal, and stemness biomarkers were immunostained and displayed simultaneously. Double chromosome enumeration probe (CEP8 and CEP12) co-detection was performed on isolated CRCs from an extended trial for two chromosome ploidy patterns.ResultsA comprehensive atlas categorizing the diverse CRCs into 71 subtypes outlining was mapped out. The presence of epithelial–mesenchymal transition (EMT) or endothelial–mesenchymal transition (EndoMT), the cells with progenitor property, hematologic CRCs expressing multiple biomarkers, CRCs at “naked nuclei” status, and the rarely reported aneuploid mesenchymal epithelial–endothelial fusion cluster were described. Circulating tumor cells (CTCs) were detected in 2,157 (91.4%) patients; the total numbers of CTCs and circulating tumor-derived endothelial cells (CTECs) were relatively higher in several digestive system cancer types and non-neoplastic infectious diseases (p < 0.05). Co-detection combining CEP8 and CEP12 showed a higher diagnostic specificity on account of 57.27% false negativity of CRC detection through a single probe of CEP8.ConclusionsThe alternative biomarkers and chromosomes to be targeted by SE-iFISH and the image scanning platform, along with the comprehensive atlas, offer insight into the heterogeneity of CRCs and reveal potential contributions to specific disease diagnosis and therapeutic target cell discovery.

Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis

Aneuploid Circulating Tumor-Derived Endothelial Cell (CTEC): A Novel Versatile Player in Tumor Neovascularization and Cancer Metastasis

Detection of circulating rare cells benefitted the diagnosis of malignant solitary pulmonary nodules.

Solitary pulmonary nodules (SPNs) are challenging in differentiating between benignancy and malignancy. Therefore, more effective non-invasive biomarkers are urgently needed. The purpose of this investigation was to examine whether circulating rare cells (CRCs) could facilitate the differentiation between benign and malignant SPNs as well as its sensitivity and specificity. 164 patients diagnosed with SPNs, 24 healthy volunteers, and 25 patients diagnosed with advanced-stage lung cancer were included. CT/PET-CT images, serum tumor markers, and biopsy results were collected. The CRCs were examined using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) and their relationship with malignant or benign SPNs was analyzed. The total CRC numbers from patients with malignant SPNs diagnosed by biopsy were significantly greater compared to those with benign SPNs (P < 0.0001), but not significantly different from patients with advanced lung cancer (P > 0.05). The total CRCs, with a cut-off value of 21.5 units, showed 67.6% sensitivity and 73.3% specificity [area under curve (AUC) 95% CI, 0.778 (0.666-0.889)] in discriminating benign and malignant SPNs and the triploid CRCs exhibited a high positive likelihood ratio of 8.4, which suggested that CRCs appeared to have a distinct advantage in discriminating benign and malignant SPNs compared to CT/PET-CT images and serum tumor markers and could be a potential screening indicator for lung cancer in the high-risk population. SE-iFISH could effectively detect CRCs including circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) and the detection of CRCs could benefit the differentiation of patients with benign and malignant SPNs.

IMMU-07. REPROGRAMMING OF MICROGLIA/MACROPHAGES IN GLIOBLASTOMA IMPROVES ANTI-TUMOR T CELL RESPONSES

Abstract Glioblastoma (GBM) still remains incurable. In search for new treatment modalities immunotherapy might be attractive but highly depends on a sufficient infiltration and function of effector T cells in an immunosuppressive microenvironment. In this study, we analyzed if blocking or reprogramming M2polarized glioma-associated microglia/macrophages (GAMs) could improve spontaneous effector T cell responses and thus enhance the effectiveness of immunotherapy. This was tested by sorting patient-derived CD11b+ cells from GBM tissues and treating these GAMs with small molecule inhibitors (SMI) targeting the colony stimulating factor1 receptor (CSF1R). Especially CD11b+ cells treated with the SMI GW2580 presented with a reduced expression of the M2 marker CD163 (p &amp;lt; 0.01) and an increased expression of HLA-DR (p &amp;lt; 0.05). Conditioned media of SMI-treated GAMs also contained significantly higher levels of nitrite (p &amp;lt; 0.001) and a reduced concentration of the immunosuppressive cytokine IL-6 (p &amp;lt; 0.05). Moreover, gene expression profiles of GW2580-treated GAMs showed a shift from an immunosuppressive towards a pro-inflammatory phenotype. Most importantly, the addition of conditioned media of GW2580-treated GAMs to a co-culture of autologous tumor and T cells significantly reduced the number of live tumor cells as compared to the use of conditioned media of untreated GAMs (p &amp;lt; 0.05). Interestingly, in some cases the ability of T cells to transmigrate through a dense barrier of autologous tumor-derived endothelial cells could also be increased. In summary, we showed that CSF-1R blockade with the SMI GW2580 can reprogram GAM phenotype and thereby improve T cell activation. This strongly suggests further studies on the use of GW2580 in combination with immunotherapeutic approaches for the treatment of GBM.

Ionizing radiation induces endothelial transdifferentiation of glioblastoma stem-like cells through the Tie2 signaling pathway

Glioblastomas (GBM) are brain tumors with a poor prognosis despite treatment that combines surgical resection and radio-chemotherapy. These tumors are characterized by abundant vascularization and significant cellular heterogeneity including GBM stem-like cells (GSC) which contribute to tumor aggressiveness, resistance, and recurrence. Recent data has demonstrated that GSC are directly involved in the formation of new vessels via their transdifferentiation into Tumor Derived Endothelial Cells (TDEC). We postulate that cellular stress such as ionizing radiation (IR) could enhance the transdifferentiation of GSC into TDEC. GSC neurospheres isolated from 3 different patients were irradiated or not and were then transdifferentiated into TDEC. In fact, TDEC obtained from irradiated GSC (TDEC IR + ) migrate more towards VEGF, form more pseudotubes in MatrigelTM in vitro and develop more functional blood vessels in MatrigelTM plugs implanted in Nude mice than TDEC obtained from non-irradiated GSC. Transcriptomic analysis allows us to highlight an overexpression of Tie2 in TDEC IR+. All IR-induced effects on TDEC were abolished by using a Tie2 kinase inhibitor, which confirms the role of the Tie2 signaling pathway in this process. Finally, by analyzing Tie2 expression in patient GBMs by immunohistochemistry, we demonstrated that the number of Tie2+ vessels increases in recurrent GBM compared with matched untreated tumors. In conclusion, we demonstrate that IR potentiates proangiogenic features of TDEC through the Tie2 signaling pathway, which indicates a new pathway of treatment-induced tumor adaptation. New therapeutic strategies that associate standard treatment and a Tie2 signaling pathway inhibitor should be considered for future trials.

An Essential NRP1-Mediated Role for Tagln2 in Gastric Cancer Angiogenesis.

Knowledge about the precise biological role and underlying mechanism of Tagln2 in tumor progression is relatively limited, especially in angiogenesis focused on tumor derived endothelial cells (ECs) has rarely been reported. Here, the function, molecular mechanism and potential clinical value of Tagln2 in gastric cancer (GC) angiogenesis were investigated. GC tissue microarrays were used to assess the expression of Tagln2 in ECs. The relationships between expression and clinicopathological features were analyzed to evaluate the clinical value of Tagln2. Gain- and loss-of-function approaches were performed in ECs to investigate the functions of Tagln2 in angiogenesis. A combination of angiogenesis antibody array, RNA-Seq analyses and a series of in vitro experiments were performed to reveal the proangiogenic mechanism mediated by NRP1. Immunohistochemistry performed on an independent tissue chip (n=75) revealed significant upregulation of Tagln2 in tumor-derived ECs which were specifically immunolabeled with CD34. Additionally, high Tagln2 levels correlated significantly with the presence of lymph node as well as distant metastases. Gain- and loss-of-function approaches highlighted the function of Tagln2 in promoting EC proliferation, motility, and capillary-like tube formation and in reducing apoptosis. Tagln2 upregulation led to significantly increased mRNA and protein levels of NRP1 and subsequently activated the NRP1/VEGFR2 and downstream MAPK signaling pathways. These data indicate the importance of Tagln2 in angiogenesis, as a potential therapeutic target, and as a candidate prognostic marker in GC.

AXL Overexpression in Tumor-Derived Endothelial Cells Promotes Vessel Metastasis in Patients With Hepatocellular Carcinoma.

Portal vein tumor thrombus (PVTT) is one of the most serious forms of hepatocellular carcinoma (HCC) vessel metastasis and has a poor survival rate. However, the molecular mechanism of PVTT has not yet been elucidated. In this study, the molecular mechanism of AXL expressed in tumor-derived endothelial cells (TECs) in vessel metastasis was investigated. High AXL expression was observed in TECs, but not in the tumor cells of HCC patients with PVTT and this was associated with poor overall survival (OS) and disease-free survival (DFS). AXL overexpression was positively associated with CD 31 expression both in vitro and in vivo. AXL promoted the cell proliferation, tube formation, and migration of both TECs and normal endothelial cells (NECs). High expression of AXL in TECs promoted the cell migration, but not the proliferation of HCC cells. Further studies demonstrated that AXL promoted cell migration and tube formation through activation of the PI3K/AKT/SOX2/DKK-1 axis. AXL overexpression in HUVECs promoted tumor growth and liver or vessel metastasis of HCC in xenograft nude mice, which could be counteracted by treatment with R428, an AXL inhibitor. R428 reduced tumor growth and CD 31 expression in HCC in PDX xenograft nude mice. Therefore, AXL over-expression in TECs promotes vessel metastasis of HCC, which indicates that AXL in TECs could be a potential therapeutic target in HCC patients with PVTT.