Tumor Tissue Factor Research Articles

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

Study on the mechanism of oral administration of tetrandrine during neoadjuvant chemotherapy for colon cancer.

Colon cancer is a digestive tract tumor with one of the highest frequencies worldwide, and with a high fatality rate. The present study aimed to investigate the expression and regulation of inflammatory factors in tumor tissues, monocytes and blood samples in patients with colon cancer (n=46) following treatment with neoadjuvant chemotherapy combined with tetrandrine. All patients underwent tumor resection after neoadjuvant chemotherapy. In the experimental group, 20 cases took tetrandrine during chemotherapy, while in the control group, 26 cases underwent chemotherapy without tetrandrine. Reverse transcription-quantitative PCR and western blotting were performed to detect the mRNA and protein expression levels of TNF-α. ELISA was used to detect the cytokine/chemokine expression levels [IL-15, IL-1β and IL-6, as well as chemokine ligand (CCL)2, CCL5, CCL20, chemokine (C-X-C motif) ligand CXCL1, CXCL2, CXCL3, CXCL5 and CXCL10 in the culture supernatant of colon cancer tissue]. Human blood mononuclear cells were cultured, and cytokine release was determined by ELISA. Cell proliferation ability was assessed using the MTT assay. Compared with the control group, the mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) were downregulated in tumor tissues and serum and the serum levels of IL-15, IL-1β and IL-6 were relatively low in the experimental group. The expression levels of CCL5, CXCL2 and CXCL10 in the supernatant of cancer tissue culture were relatively low, compared with the conditioned medium prepared from tumor tissues of patients not receiving tetrandrine. When the cultured blood mononuclear cells were stimulated by the tissue culture supernatant from the experimental group, less IL-15, IL-1β and IL-6 were released, compared with the medium of tumor tissues of patients not taking tetrandrine. Following stimulation with the tissue culture supernatant from the experimental group, the proliferation ability of HCT116 colon cancer cells significantly declined. During chemotherapy of patients with colon cancer, tetrandrine may inhibit the expression of TNF-α in cancer tissues and blood, reduce the release of inflammatory factors and chemokines and decrease cancer cell proliferation. These findings provide a theoretical basis for the treatment of colon cancer in the clinic.

Ginsenoside Rg3 liposomes regulate tumor microenvironment for the treatment of triple negative breast cancer

Objective To improve the solubility and targeting of Ginsenoside Rg3 (G-Rg3), in the current study, we constructed a novel targeting functional material folic acid -poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate (FA-PEOz-CHMC, FPC) modified G-Rg3 liposomes (FPC-Rg3-L). Methods FPC was synthesized by using folic acid (FA) as a targeted head coupling with acid-activated poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate. The inhibitory effects of the G-Rg3 preparations on mouse breast cancer cells (4T1) were investigated by CCK-8 assay. Paraffin sections of female BALB/c mice viscera were taken for hematoxylin-eosin (H&E) staining after continuous tail vein injection of G-Rg3 preparations. BALB/c mice bearing triple-negative breast cancer (TNBC) were used as animal models to investigate the inhibition of G-Rg3 preparations on tumor growth and improving quality of life. Transforming growth factor-β1 (TGF-β1) and α-smooth muscular actin (α-SMA) were used to investigate the expression of two fibrosis factors in tumor tissues by western blotting. Results Compared with G-Rg3 solution (Rg3-S) and Rg3-L, FPC-Rg3-L had a significant inhibitory effect on 4T1 cells (p < .01), and the half maximal inhibitory concentration (IC50) of FPC-Rg3-L was significantly lower (p < .01). The H&E results showed that the injection of FPC-Rg3-L and Rg3-S did not cause damage to the organs of mice. Compared with the control group, tumor growth was significantly inhibited in mice treated with FPC-Rg3-L and G-Rg3 solutions (p < .01). Conclusions This study presents a new and safe treatment for TNBC, reduces the toxic and side effects of the drug, and provides a reference for the efficient use of Chinese herbal medicine components.

Tissue Factor (TF) Is Upregulated in Tumor-Bearing Mice Treated with Immune Checkpoint Inhibitor (ICI)

Introduction: Venous thromboembolism (VTE) is an important adverse event associated with immune checkpoint inhibitors (ICI) in cancer patients (12-15% annual rate). We have developed a murine model of venous thrombosis associated with ICI therapy (referred to as ICI-CAT). We found that mice bearing CT26 colon carcinoma treated with ICI developed larger thrombi following IVC occlusion compared with mice treated with IgG. ICI did not increase thrombus weight in mice without tumors. Tissue factor (TF) plays an important role in cancer-associated thrombosis (CAT) in patients treated with conventional therapies, and TF+ extracellular vesicles (EV) were associated with the development of VTE in pancreatic cancer. However, little is known about the impact of ICI on TF expression in patients treated with ICI. To address the underlying mechanism of ICI-associated thrombosis (IAT), we have examined the expression of TF in tumor and immune cells, as well as circulating EV, in our murine CT26 model. Methods: A syngeneic colon carcinoma model (CT26) in BALB/c mice was utilized to evaluate ICI-CAT following inferior vena cava ligation (stasis). Tumor tissue was analyzed for TF expression by immunoblotting using an anti-TF antibody (ab189483, Abcam). Tumor tissue was also dissociated, and TF-positive cells were assessed by flow cytometry analysis with CD45+ and CD45- gating using antibodies for CD11b, Ly6C, Ly6G (BioLegend), and TF (R&D). EV were isolated from platelet poor plasma from ICI or control IgG-treated mice by centrifugation (20,000 x g). Procoagulant activity of EV TF was measured using a chromogenic substrate to detect FXa generation following incubation of EV with FVIIa in the presence or absence of an anti-TF antibody. Results: Treatment of tumor-bearing mice with ICI markedly induced TF expression in tumors (Fig. A, Leftupper). Furthermore, levels of EV TF antigen (Fig. A, Left bottom) and EV TF activity (Fig. A, Right) were increased in mice treated with ICI. To explore the cellular origin of TF, we performed flow cytometry with dissociated tumor cells. A greater percentage of TF-positive cells in both the CD45+ (37.8%) and CD45- (74.6%) populations were detected in tumors from ICI treated mice compared to those treated with control IgG (27.9% and 49.8%, respectively), suggesting both tumor and tumor-infiltrating immune cells contribute to the larger thrombi observed in ICI-treated mice (Fig. B). Further analysis of CD45+ TF+ cells by CD11b gating (data not shown) revealed that a TF expression in a monocyte population (CD11b+ Ly6C+) was increased 3-fold by ICI (46.6%) compared to control IgG (14.2%) (Fig. C). We also found that TF expression was increased in cultured CT26 cells in response to interferon-g, but not in response to anti-PD-1 and anti-CTLA-4 antibodies, suggesting that ICIs do not directly upregulate TF and that more complex cellular interactions and inflammatory changes may be involved. Conclusions: ICI treatment of tumor-bearing mice results in increased tumor TF expression and EV TF expression. TF was also upregulated in immune cells in the tumor microenvironment after ICI treatment, including both tumor/stromal cells (CD45-), and tumor-associated monocytes and neutrophils (CD45+). These results suggest that multiple underlying mechanisms and diverse cellular expression underlie TF expression in this model, and that these processes many contribute to the larger thrombi observed in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combination therapy with interleukin-15 and metformin as a synergistic treatment for pancreatic cancer.

To investigate the efficacies and mechanisms of combination therapy with interleukin-15 (IL-15) and metformin (Met) on suppressing pancreatic cell proliferation and protecting Panc02-bearing mice. MTT assays were applied to assess the inhibitory effects of IL-15 combined Met or Met and IL-15 alone on proliferation of normal HPDE6-C7 and Panc02 cells. After tumor grew up to 150 mm3, the Panc02-bearing xenograft model mice were randomly divided into saline group, IL-15 and Met alone group and combined treatment group (n=8) with the administration of each agent every other day for three weeks, and survival rates were recorded. The changes in tumor size, expression levels of the apoptosis, autophagy as well as Akt/mTOR/STAT3-related factors in tumor tissues were all measured. MTT assays demonstrated significantly inhibiting efficacy of combination therapy with IL-15 and Met on Panc02 cell proliferation compared to other groups (all p<0.05) with combination index<1 showing evident synergistic effect. Moreover, the apoptosis rate of Panc02 cell under combined treatment were 95.5±3.2% and significantly higher than those of others (all p<0.01). In addition, combined administration remarkably inhibited the growth of pancreatic carcinoma, and improved survival rate of Panc02-bearing model with less body weight loss. Furthermore, combined treatment significantly downregulated anti-apoptotic proteins as well as Akt/mTOR/STAT3 signaling pathway and upregulated autophagy related factors, respectively, compared with those of monotherapy groups in both Panc02 cells and tumor tissues. Combined treatment of IL-15 with Met showed synergistic anti-tumor efficacies on Panc02 cells attributing to promotion on apoptosis, autophagy and inhibition on Akt/mTOR/STAT3 signaling-transduction in Panc02-bearing model mice.

Tissue Factor and Extracellular Vesicles: Activation of Coagulation and Impact on Survival in Cancer

Simple SummaryThe tissue factor (TF)-factor VIIa complex is the major physiological initiator of blood coagulation. Tumors express TF and release TF-positive extracellular vesicles (EVs) into the circulation, and this is associated with the activation of coagulation. Circulating levels of EVTF activity may be a useful biomarker to identify patients at risk for thrombosis. Tumor TF and TF-positive EVs are also associated with reduced survival.Tissue factor (TF) is a transmembrane glycoprotein that functions as a receptor for FVII/FVIIa and initiates the extrinsic coagulation pathway. Tumors and cancer cells express TF that can be released in the form of TF positive (TF+) extracellular vesicles (EVs). In this review, we summarize the studies of tumor TF and TF + EVs, and their association with activation of coagulation and survival in cancer patients. We also summarize the role of tumor-derived TF + EVs in venous thrombosis in mouse models. Levels of tumor TF and TF + EVs are associated with venous thromboembolism in pancreatic cancer patients. In addition, levels of EVTF activity are associated with disseminated intravascular coagulation in cancer patients. Furthermore, tumor-derived TF + EVs enhance venous thrombosis in mice. Tumor TF and TF + EVs are also associated with worse survival in cancer patients, particularly in pancreatic cancer patients. These studies indicate that EVTF activity could be used as a biomarker to identify pancreatic cancer patients at risk for venous thrombosis and cancer patients at risk for disseminated intravascular coagulation. EVTF activity may also be a useful prognostic biomarker in cancer patients.

An Antibody-like Polymeric Nanoparticle Removes Intratumoral Galectin-1 to Enhance Antitumor T-Cell Responses in Cancer Immunotherapy.

Antibodies have shown potential to deplete immunosuppressive factors in tumor tissues. However, intrinsic drawbacks, including time-consuming processes in preparation, high cost, and short half-life time, greatly restrict their applications. In this work, we report an antibody-like polymeric nanoparticle (APN) that is capable of specifically capturing and removing galectin-1 in tumor tissues, thereby enhancing the antitumor T-cell responses. The APN is composed of an albumin-polymer hybrid nanoparticle (core) and an acid-responsive PEG shell. The core of the APN contains multiple recognition units and Tuftsin peptides to capture target factors and activate macrophage-mediated phagocytosis, respectively. By employing galactose as recognition units, the APN facilitated the phagocytosis of galectin-1 in tumor tissues, thereby improving the antitumor responses of tumor-infiltrating T cells. Since the recognition units in the APN can be further replaced to capture and remove other peptides/proteins, the APN provides a feasible approach for the development of synthetic nanoformulations to regulate biological systems and treat diseases.

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.

Radiation therapy enhanced therapeutic efficacy of anti-PD1 against gastric cancer.

ABSTRACTRadiation therapy is an important method in tumor treatment with distinct responses. This study aimed to investigate the immune effects of radiation therapy on the syngeneic gastric tumor model. Mouse forestomach carcinoma (MFC) cells were irradiated with different X-ray doses. Cell proliferation was determined by clonogenic assay. Gene and protein expression were determined by real-time quantitative PCR and western blot, respectively. The tumor model was established by subcutaneously injecting tumor cells in 615-(H-2 K) mice. Levels of immune-related factors in tumor tissues were determined by immunohistochemistry and flow cytometry. 5 Gy × 3 (three subfractions with 4 h interval) treatment significantly inhibited cell proliferation. Protein expression of stimulator of interferon genes (Sting) and gene expression of IFNB1, TNFα as well as CXCL-9 significantly increased in MFC cells after irradiation. In the MFC mouse model, no obvious tumor regression was observed after irradiation treatment. Further studies showed Sting protein expression, infiltration of dendritic cells and T cells, and significantly increased PD-1/PD-L1 expression in tumor tissues. Moreover, the irradiation treatment activated T cells and enhanced the therapeutic effects of anti-PD1 antibody against MFC tumor. Our data demonstrated that although the MFC tumor was not sensitive to radiation therapy, the tumor microenvironment could be primed after irradiation. Radiation therapy combined with immunotherapy can greatly improve anti-tumor activities in radiation therapy-insensitive tumor models.

Immunological functional study by use of the chemotherapy drug plus immunologic hapten like penicillin on the fibrosis of local tumor inflammatory reaction and the immunity of tumor-bearing mice.

e15196 Background: Observe the antitumor effect of intratumoral injection of chemotherapy drugs or combined with immune adjuvant on Balb/c and c57 tumor bearing mice and the local inflammatory response, fibrosis and immune effect of tumor. Methods: H22 ascites tumor was inoculated in the armpit of BABL / c mice left forelimb to establish subcutaneous solid tumor model of liver cancer, B16 melanoma tumor filtrate was inoculated in the armpit of C57 mice left forelimb to establish subcutaneous solid tumor model of melanoma. The mice were randomly divided into DNP control group, penicillin control group, I group, II group, III group, IV group, V group, VI group, DOX control group, PYM control group, BLM control group and control group when the longest tumor diameter was about 5-8mm. On the first day of the experiment, the mice in each group were injected with corresponding of different group drugs. Three mice in each group were killed on day 1, day 4 and day 7 respectively and then the tumors were taken The C57 mice were used for immunohistochemistry BABL/c mice to detect the content changes of various fibroproliferative, inflammatory and immune related factors. Results: I group: The expression of COL1A1, CD4, IL12A, TGFB1, elastin, NFkB, COX 2, CD11c, CD8 and TNFa in group I was higher than that in other factors at day 1. After 4 and 7 days of administration, all factors decreased significantly; The results of C57 Mouse immunohistochemistry showed that the expression of CD4, cd11bc, Foxp3 in tumor tissue of group V was significantly increased on the first day after administration, and the expression of CD4, CD8, CD86, CD11bc in tumor tissue of group V was significantly increased on the seventh day. Conclusions: The results of immunohistochemistry and PCR showed that the content of fibroproliferative, inflammatory and immune factors in tumor tissue changed significantly after administration og intratumoral injection of combination drugs and penicillin with H2O2. All the above results has showed that chemotherapy drug and hapten was released in the tumor through H2O2 and induced the inflammatory reaction inside tumor (including various fiber proliferation and tumor growth inhibition) as well as the systemic immune response, this also is called abscopal effect.

Antitumor and anti-angiogenic effects of artemisinin on breast tumor xenografts in nude mice

Breast cancer is a high incidence disease in humans. Artemisinin is an important extract that is widely used as an antimalarial drug which also serve as effective treatments for cancer. 32 nude mice were injected with 0.2 ml of MDA-MB-231 cell suspension of 2 × 107 cells/ml respectively. The nude mice models were randomly divided into four groups of 8 in each group. Each group was given daily gavage, high dose group: 200 mg/kg/0.1 ml, middle dose group 100 mg/kg/0.1 ml, low dose group 50 mg/kg/0.1 ml, control group: 0.1 ml vegetable oil was fed continuously for 21 days. ELISA was used to detect serum vascular endothelial growth the content of factor VEGF and hypoxia-inducible factor HIF-1α were detected. The expression of Notch pathway-related factors in tumor tissue was detected by fluorescence quantitative assay. ELISA results showed that the serum VEGF decreased significantly in the high dose group compared with the control group (p < .01), while the other dose groups did not have significant (p > .05). The serum HIF-1α in the high dose group compared with the control group, the decrease in HIF-1α was significant (p < .05), and the other groups were not significant (p > .05). The result of fluorescence quantitative section showed that artemisinin could down-regulate the expression of notch signaling related factors notch1, Dll4 and Jagged1, and 200 mg/kg dose group had the most significant effect. It may inhibit the development of tumors by reducing serum angiogenesis-related factors VEGF, HIF-1ɑ and inhibiting the activity of notch1 signaling pathway related factors.

Tissue Factor and Cancer: Regulation, Tumor Growth, and Metastasis.

There is a strong relationship between tissue factor (TF) and cancer. Many cancer cells express high levels of both full-length TF and alternatively spliced (as) TF. TF expression in cancer is associated with poor prognosis. In this review, the authors summarize the regulation of TF expression in cancer cells and the roles of TF and asTF in tumor growth and metastasis. A variety of different signaling pathways, transcription factors and micro ribonucleic acids regulate TF gene expression in cancer cells. The TF/factor VIIa complex enhances tumor growth by activating protease-activated receptor 2 signaling and by increasing the expression of angiogenic factors, such as vascular endothelial growth factor. AsTF increases tumor growth by enhancing integrin β1 signaling. TF and asTF also contribute to metastasis via multiple thrombin-dependent and independent mechanisms that include protecting tumor cells from natural killer cells. Finally, a novel anticancer therapy is using tumor TF as a target to deliver cytotoxic drugs to the tumor. TF may be useful in diagnosis, prognosis, and treatment of cancer.

MicroRNA-524-5p suppresses the progression of papillary thyroid carcinoma cells via targeting on FOXE1 and ITGA3 in cell autophagy and cycling pathways.

MicroRNAs are beneficial for cancer therapy as they can simultaneously downregulate multiple targets involved in diverse biological pathways related to tumor development. In papillary thyroid cancer, many microRNAs were identified as differentially expressed factors in tumor tissues. In another way, recent studies revealed cell proliferation, cell cycling, apoptosis, and autophagy are critical pathways controlling papillary thyroid cancer development and progression. As miR‐524‐5p was approved as a cancer suppressor targeting multiple genes in several types of cancer cells, this study aims to characterize the role of miR‐524‐5p in the thyroid cancer cell. The expression of miR‐524‐5p was decreased in the papillary thyroid cancer tissues and cell lines, while forkhead box E1 (FOXE1) and ITGA3 were increased. In the clinical case, expression of miR‐524‐5p, FOXE1, and ITGA3 were significantly correlated with papillary thyroid cancer development and progression. FOXE1 and ITGA3 were approved as direct targets of miR‐524‐5p. miR‐524‐5p could inhibit papillary thyroid cancer cell viability, migration, invasion, and apoptosis through targeting FOXE1 and ITGA3. Cell cycling and autophagy pathways were disturbed by downregulation of FOXE1 and ITGA3, respectively. Collectively, miR‐524‐5p targeting on FOXE1 and ITGA3 prevents thyroid cancer progression through different pathways including cell cycling and autophagy.

Quantitative assessment of tumor-associated tissue eosinophilia and mast cells in tumor proper and lymph nodes of oral squamous cell carcinoma.

Background:Oral squamous cell carcinoma (OSCC) is the most common cancer of the oral cavity. Tumor stage, thickness, lymph node metastasis (LNM), extranodal spread, perineural invasion, tumor differentiation, mutations, human papillomavirus infection and tumor microenvironment are independent prognostic indicators of OSCC. However, clinically, among all factors, LNM is considered an important prognostic factor in OSCC as it not only determines the stage of disease but also the strongest independent factor which predicts recurrence of disease. Further research proves that there are several biologically important factors in tumor tissue and LNs which promote or defend LNM. While it is proposed that tumor-associated tissue eosinophils (TATE) and mast cells (MCs) have “immuno-protective” effect, this remains unproven and various researchers have conflicting opinion.Aim:The aim is to determine the presence of TATE and MCs in OSCC and to evaluate if any association exists between them and LNM.Study Design:It is a comparative retrospective study between two groups including 35 OSCC cases positive and 35 negative for LNM.Materials and Methodology:Quantification of cells was done by counting total number of cells in 10 high-power fields under ×40 objective lens using “zigzag” method and dividing it by total number of fields. Eosinophils stained bright red with carbol chromotrope and MCs purple-violet with toluidine blue.Statistics:Independent t-test and Pearson's correlation were done using STATA IC 0.2 software. The level of significance was at 5%. Comparison of eosinophil and MC infiltration was done based on gender, metastatic, nonmetastatic LN and in tumor proper.Results and Conclusion:Our study showed weak positive correlation between mean eosinophils count in tumor and LNs which implies a definite association between the microenvironment of tumor, its progression and LNM. There was a significant association between MC density and decreased LNM also. We conclude that an increased number of immunological cells (TATE and MCs) are a favorable prognostic indicator in OSCC. There is evidence of reduction in LNM with increasing density of these immunological cells. Recognition of TATE and MCs as integral to tumor biology opens an avenue for novel approaches to cancer therapies.

Quantitative assessment of tumor-associated tissue eosinophilia and mast cells in tumor proper and lymph nodes of oral squamous cell carcinoma.

Background:Oral squamous cell carcinoma (OSCC) is the most common cancer of oral cavity. Tumor stage, thickness, lymph node metastasis (LNM), extranodal spread, perineural invasion, tumor differentiation, mutations, human papillomavirus infection, and tumor microenvironment are independent prognostic indicators of OSCC. However, clinically, among all factors, LNM is considered an important prognostic factor in OSCC as it not only determines the stage of disease but also the strongest independent factor which predicts recurrence of disease. Further research proves that there are several biologically important factors in tumor tissue and LNs which promote or defend LNM. While it is proposed that tumor-associated tissue eosinophils (TATE) and mast cells (MCs) have “immuno-protective” effect, this remains unproven and various researchers have conflicting opinion.Aim:The aim is to determine the presence of TATE and MCs in OSCC and to evaluate if any association exists between them and LNM.Study Design:It is a comparative-retrospective study between 2 groups including 35 OSCC cases positive and 35 negative for LNM.Materials and Methodology:Quantification of cells was done by counting total number of cells in 10 high-power fields under ×40 objective lens using “zigzag” method and dividing it by total number of fields. Eosinophils stained bright red with carbol chromotrope and MCs purple-violet with toluidine blue.Statistics:Independent t-test and Pearson's correlation were done using STATA IC 0.2 software. Level of significance was at 5%. Comparison of eosinophil and MC infiltration was done based on gender, metastatic, nonmetastatic LN, and in tumor proper.Results and Conclusion:This study showed weak positive correlation between mean eosinophils count in tumor and LNs. Recognition of TATE and MCs as integral to tumor biology opens an avenue for novel approaches to cancer therapies. We conclude that an increased number of immunological cells are a favorable prognostic indicator in OSCC.

Abstract 5203: Tissue factor targeted radionuclide therapy with 177Lu-FVIIai inhibits tumor growth of human pancreatic cancer xenografts

Abstract Introduction: Tissue factor (TF) is involved in cancer processes responsible for aggressiveness and invasiveness and there is a correlation between tumor TF expression, metastatic potential, and patient outcome. The aim of the study was to develop a novel compound for localized TF targeted radionuclide therapy based on Factor VII (FVII), the natural ligand to TF. In the current study, we investigated the biodistribution and therapeutic potential of 177Lu labeled active site inhibited FVIIa (177Lu-FVIIai) for radionuclide therapy targeting TF in an experimental mouse model of pancreatic cancer. Methods: p-SCN-Bn-CHX-A’’-DTPA was conjugated to FVIIai followed by radiolabeling with 177Lu (177Lu-CHX-A’’-DTPA-FVIIai). A pancreas xenograft mouse model (BxPC3) was used to assess the therapeutic potential of 177Lu-FVIIai. NMRI nude mice were injected subcutaneously with BxPC3 cells. When tumors reached 50 mm3, the mice were randomized into groups receiving 177Lu-FVIIai, FVIIai, or vehicle. 177Lu-FVIIai was administered in doses of 15 MBq, 7.5 MBq or 2 x 7.5 MBq (n=8 mice/group). Tumor growth was monitored by 3 weekly measurements. Ex vivo biodistribution of 177Lu-FVIIai was studied in several organs at 1, 4, 24, 72 and 168 hours after injection. The in vivo biodistribution was evaluated by SPECT/CT imaging. Furthermore, competition and dose escalation experiments (1-30 MBq) were performed. Toxicity effects of the treatment with 177Lu-FVIIai were evaluated by hematology. Results: FVIIai was successfully radiolabeled with 177Lu with a high specific activity of 10-25 GBq/µmol after EDTA scavenging and PD-10 purification. No difference in tumor growth was observed between the FVIIai and vehicle groups. Mice receiving 15 MBq 177Lu-FVIIai had a significantly reduced tumor growth from day 0 to day 19 compared with mice from the control groups (425.5±44.8% versus 614.2±49.1%; p=0.02). No significant difference in tumor growth was observed in the groups receiving 7.5 MBq or 2 x 7.5 MBq compared with controls on day 19. There was a significant increase in the survival of mice treated with 7.5 MBq 177Lu-FVIIai compared with the controls (p=0.007). Treatment with 15 and 2 x 7.5 MBq 177Lu-FVIIai did not influence survival. Ex vivo tumor uptake of 177Lu-FVIIai was 1.16±0.04, 1.97±0.18, 1.95±0.07, 1.01±0.06, 0.31±0.02 percent injected dose per gram (%ID/g) at 1, 4, 24, 72 and 168 hours post-injection, respectively. Competition with unlabeled FVIIai 10 minutes before 177Lu-FVIIai injection significantly reduced tumor uptake of 177Lu-FVIIai (from 2.5±0.16 %ID/g to 1.7±0.05 %ID/g; p&amp;lt;0.05). Tumor uptake (%ID/g) of 177Lu-FVIIai was unchanged when varying the dose from 1-30 MBq. A transient decrease in leucocyte counts was observed for the mice receiving 15 and 7.5 MBq 177Lu-FVIIai. Conclusion: FVIIai was successfully radiolabeled with 177Lu. 177Lu-FVIIai showed therapeutic potential in a mouse model of human pancreatic cancer. Citation Format: Mette Munk Jensen, Jesper Fonslet, Camilla S. Knudsen, Troels E. Jeppesen, Andreas I. Jensen, Gregory W. Severin, Carsten H. Nielsen, Andreas Kjær. Tissue factor targeted radionuclide therapy with 177Lu-FVIIai inhibits tumor growth of human pancreatic cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2017-5203

Evaluation of venous thrombosis and tissue factor in epithelial ovarian cancer

ObjectiveOvarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population. MethodsWe performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF. Results35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75–6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001. ConclusionsTFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Interleukin‑6 induces an epithelial‑mesenchymal transition phenotype in human adamantinomatous craniopharyngioma cells and promotes tumor cell migration.

Total resection of adamantinomatous craniopharyngioma (ACP) is complex and often leads to postoperative recurrence. This is due to the tendency of the tumor to invade the surrounding brain tissue and the generation of a local inflammatory state between the tumor cells and parenchyma. While there is evidence to suggest that interleukin-6 (IL-6) induces craniopharyngioma (CP)-associated inflammation, particularly in ACP, the role of IL-6 in the progression of ACP remains unclear. The results of the present study demonstrated that CP inflammation was associated with pathological classification, extent of surgery, degree of calcification and postoperative hypothalamic status scale. Cytokine antibody arrays were conducted to measure the expression of IL-6 and other inflammatory factors in tumor tissues in response to various levels of inflammatory exposure. IL-6, IL-6 receptor (IL-6R) and glycoprotein 130 expression was detected by immunohistochemistry. In addition, an ELISA was performed to quantify the levels of soluble IL-6R (sIL-6R) in the cystic fluid and supernatants of ACP cells and tumor-associated fibroblasts. These measurements demonstrated that ACP cells produce IL-6 and its associated proteins. In addition, the results revealed that while the viability of ACP cells was not affected, the migration of ACP cells was promoted by IL-6 treatment in a concentration-dependent manner. Conversely, treatment with an IL-6-blocking monoclonal antibody significantly decreased the migration of ACP cells. In addition, IL-6 treatment increased the expression of vimentin and decreased the expression of E-cadherin in a dose-dependent manner. The findings of the present study demonstrate that IL-6 may promote migration in vitro via the classic- and trans-signaling pathways by inducing epithelial-mesenchymal transition in ACP cell cultures.

Angiogenesis features in patients with melanoma with different BRAf status

Neoangiogenesis is an important factor in the development and progression of cancer. Neoangiogenesis in tumors is necessary for its further growth and subsequent metastasis. Intensity ofangiogenesis depends on a balance ofproangiogenic and antiangiogenic factors. One of these angiogenic factors is matrix metalloproteinase-9 (MMP-9) enzyme family endopeptidases participating in the extracellular matrix degradation and remodeling of vessels. It is known that the BRAF V600E mutation can affect the expression ofpro-angiogenic factors in tumor tissue. The purpose of this study was to investigate the microvasculature density in the tumor andperitumoral area in patients with melanoma with different BRAF-status using counting CD-31 positive stained vascular endothelial cells, as well as detection of the expression of matrix metalloprotieinase-9 in tumor cells and cells of microenvironment, followed by analysis of the relationships among these indicators. The material for the study is based on samples of tumor (n = 57) obtainedfrom patients with melanoma. The study revealed that there was a trend to increased angiogenesis in 2-fold (p &lt; 0.05) with the BRAF-positive lentigo melanoma, compared with BRAF-negative patients lentigo melanoma. Vascularization level change was detected, depending on the primary tumor site: the level of vascularization was statistically higher in BRAF-positive patients with localized tumors on the skin of the trunk (p &lt; 0.05), with no significant differences for other important morphologic parameters such as ulceration of the tumor, the severity of lymphocytic infiltration and tumor thickness by Breslow (p &gt; 0.05). No statistically significant differences in the expression of MMP-9 depending on the BRAF-status in the tumor cells and stromal cells in microenvironment (p &gt; 0.05) were detected. Nevertheless, a tendency to an increase in the expression of MMP-9 in the surrounding stroma cells (fibroblasts, lymphocytes, endothelial cells, polymorph nuclear leukocytes, regardless of BRAF-status) was shown. Despite some features of tumor angiogenesis in the skin melanoma patients with different BRAF status angiogenesis in the tumor is influenced by a variety of proangiogenic and antiangiogenic stimulation that are common patterns regardless of BRAF-status.

Predictive model of thrombospondin-1 and vascular endothelial growth factor in breast tumor tissue.

Angiogenesis, the formation of new blood capillaries from pre-existing vessels, is a hallmark of cancer. Thus far, strategies for reducing tumor angiogenesis have focused on inhibiting pro-angiogenic factors, and less is known about the therapeutic effects of mimicking the actions of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important endogenous inhibitor of angiogenesis that has been investigated as an anti-angiogenic agent. TSP1 impedes the growth of new blood vessels in many ways, including crosstalk with pro-angiogenic factors. Owing to the complexity of TSP1 signaling, a predictive systems biology model would provide quantitative understanding of the angiogenic balance in tumor tissue. Therefore, we have developed a molecular-detailed, mechanistic model of TSP1 and vascular endothelial growth factor (VEGF), a promoter of angiogenesis, in breast tumor tissue. The model predicts the distribution of the angiogenic factors in tumor tissue, revealing that TSP1 is primarily in an inactive, cleaved form owing to the action of proteases, rather than bound to its cellular receptors or to VEGF. The model also predicts the effects of enhancing TSP1’s interactions with its receptors and with VEGF. To provide additional predictions that can guide the development of new anti-angiogenic drugs, we simulate administration of exogenous TSP1 mimetics that bind specific targets. The model predicts that the CD47-binding TSP1 mimetic markedly decreases the ratio of receptor-bound VEGF to receptor-bound TSP1, in favor of anti-angiogenesis. Thus, we have established a model that provides a quantitative framework to study the response to TSP1 mimetics.