scholarly journals Abstract 3837: Primary tumor cell lines derived from gastric PDX tumors displays differences in chemosensitivity

2017 ◽  
Author(s):  
Xiaomei Ge ◽  
Fulin Qiang ◽  
Yixin Zhang ◽  
Jibing Liu ◽  
Lei Yang ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Primary Tumor ◽  
Tumor Cell Lines ◽  
Primary Tumor Cell

Expression and secretion of VEGF in solid tumor cells is mediated by the mammalian target of rapamycin (mTOR)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14123-14123
Author(s):  
E. M. Lackner ◽  
M. T. Krauth ◽  
R. Kondo ◽  
L. Rebuzzi ◽  
K. Eigenberger ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Tumor Cell Lines ◽  
Neoplastic Cells ◽  
Malignant Effusions ◽  
Vegf Protein

14123 Background: Tumor progression and metastasis formation are often associated with enhanced angiogenesis and with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and a mediator of vascular permeability. We here describe that VEGF is produced and secreted by neoplastic cells in various solid tumors and its production mediated through mTOR. Methods and Results: As assessed by ELISA, the VEGF protein was detected in supernatants of cell lines derived from breast cancer (MDA-MB231), pancreatic carcinoma (BxPC-3), lung cancer (A-427), colon carcinoma (HCT8), and cholangiocellular carcinoma (EGI-1). In addition, VEGF was detected in supernatants of primary tumor cells obtained from malignant effusions in various malignancies (breast cancer, n=4; pancreatic cancer, n=1; ovarial cancer, n=1; parotic carcinoma, n=1; oesophageal carcinoma, n=1). In each case, VEGF protein was detectable in neoplastic cells by immunocytochemistry, and was found to accumulate in supernatants of cultured tumor cells over time, suggesting constant production and secretion. Correspondingly, as assessed by RT-PCR, primary tumor cells as well as the cell lines tested were found to express VEGF mRNA in a constitutive manner. Since mTOR is a well known regulator of VEGF synthesis, we applied rapamycin on primary neoplastic cells and on tumor cell lines. Rapamycin (20–200 nM) was found to counteract the production and secretion of VEGF in all tumor cells tested (VEGF in supernatants in cultures supplemented with rapamycin at 100 nM compared to control=100% on day 6: MDA-MB231: 11.8±0.2%; BxPC-3: 23.6±18.8%; A-427: 30.1±3.4%; HCT8 17.2±0.5%; EGI-1 28.4±1.1%; p<0.05). By contrast, neither rapamycin nor VEGF were found to modulate growth of primary tumor cells or the growth of the tumor cell lines tested. Conclusions: Various human tumor cells express and secrete VEGF. VEGF production is mediated through mTOR. These observations may have implications for the design of new treatment approaches attempting to counteract VEGF production/secretion and thus VEGF-dependent angiogenesis and effusion- formation in solid tumors. No significant financial relationships to disclose.

Patulin Acts Synergistically with Doxorubicin and Bortezomib in Cytotoxicity against Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2664-2664
Author(s):  
Alex Hessel ◽  
Malefa Tselanyane ◽  
Fengrong Wang ◽  
Ebenezer David ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Primary Tumor ◽  
Dose Effect ◽  
Tumor Cell Lines ◽  
Synergism And Antagonism

Abstract Patulin (4-Hydroxy-4H-furo 3,2-C-pyran-2(6H)-one) is a first-in-class mycotoxin under development as a novel chemotherapeutic agent. The mechanism of action of Patulin has been reported to include activation of mitogen activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). We have previously shown Patulin to have activity against myeloma, leukemia, and lymphoma cell lines, as well as primary tumor cells in clinical samples from patients with these diagnoses (Wang BLOOD 2007). Moreover, we have shown that Patulin specifically and potently targets tumor cells over normal cells and effectively killed primary tumor cells of patients with refractory illness. The aim of this study was to determine whether Patulin acts synergistically with the topoisomerase inhibitor doxorubicin or the proteasome inhibitor bortezomib. We hypothesize that drugs with non-cross-reactive modes of action could be complementary. Human tumor cell lines from B-cell lymphoma (DAUDI), T-Cell leukemia and lymphoma (Jurkat and H9, respectively), and myeloid leukemia (HL60) malignancies were tested for their sensitivity to single agents Patulin, bortezomib, and doxorubicin as well as Patulin in combination with the latter two agents (Patulin and bortezomib, P + B; Patulin and doxorubicin, P + D). Cells were treated with a range of concentrations of each single agent and the drugs in combination over 24 hours. Following treatment, cell metabolic activity was assessed using a microculture tetrazolium (MTT) assay and cell viability was assessed by flow cytometry using Annexin V and propidium iodide (PI) staining. Dose-effect curves, median effect plots, and combination index (CI) values were generated in the Compusyn software program for each target cell population. Median-effect doses (IC50s) of individual drugs were interpolated using the y-intercept of median-effect plots. Three dose-effect data points were used to create a range of CI values at different fractions of affect (fa). The lower and upper values of the CI range were used to characterize drug combinations as synergistic, antagonistic, or additive based on Chou’s Symbols for Synergism and Antagonism using CI analysis (Table 1). The role for ROS in the mechanism of action of Patulin was confirmed by flow cytometry showing increased levels of ROS in cell lines following Patulin exposure. Preincubation of cell lines with N-acetyl cysteine (NAC) or concurrent exposure to Patulin and NAC abrogated the cytotoxic activity of the mycotoxin. H9 cells were most sensitive to the effects of Patulin, with an IC50 of 1.2 μM. Combinations of P + B acted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cells; however, P + B also demonstrated moderate antagonism against the Jurkat and H9 cell lines (Table 2). Likewise, combinations of P + D interacted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cell lines while simultaneously demonstrating strong antagonism against the H9 cell line. Patulin kills leukemia and lymphoma cells via generation of intracellular ROS. Synergy of Patulin with either bortezomib or doxorubicin in leukemia and lymphoma cell lines indicates a distinct mechanism of action for the mycotoxin and compared to other chemotherapeutics and supports the rationale for continued development of Patulin as a novel chemotherapeutic mycotoxin. TABLE 1. Chou’s Symbols for Synergism and Antagonism using CI Analysis CI Description &lt; 0.1 Very Strong Synergism 0.1–0.3 Strong Synergism 0.3–0.7 Synergism 0.7–0.85 Moderate Synergism 0.85–0.90 Slight Synergism 0.90–1.10 Nearly Additive 1.10–1.20 Slight Antagonism 1.20–1.45 Moderate Antagonism 1.45–3.3 Antagonism 3.3–10 Strong Antagonism &gt; 10 Very Strong Antagonism TABLE 2. IC50 of single agents patulin, bortezomib, and doxorubicin and CI in hematological cancer cell lines Tumor cell line IC50 of patulin (μM) IC50 of bortezomib (nM) IC50 of doxorubicin (μM) CI: P + B CI: P + D T-Cell Jurkat 1.16 5600 5.2 0.6 -- 1.2 0.1 -- 1.2 H9 1.2 14 0.33 0.4 -- &gt; 1 0.3 -- &gt; 1 B-Cell DAUDI 0.98 1.1 0.19 &lt; 0.1 -- 0.1 &lt; 0.1 -- 0.3 Myeloid HL60 1.7 0.42 0.07 &lt; 0.1 -- 0.1 &lt; 0.1

JAK1 and JAK2 Modulate Tumor Cell Susceptibility To Natural Killer (NK) Cells Through Regulation Of PDL1 Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3472-3472
Author(s):  
Roberto Bellucci ◽  
Allison Martin ◽  
Davide Bommarito ◽  
Kathy S. Wang ◽  
Gordon J Freeman ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Nk Cell ◽  
Cell Lysis ◽  
Cell Killing ◽  
Tumor Cell Lines ◽  
Ifn Γ

Abstract NK cells are the primary effectors of the innate immune response against infections pathogens and malignant transformation through their efficient cytolytic activity and cytokine secretion. Nevertheless, tumor cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not yet completely understood. To identify novel pathways that modulate tumor cell resistance to NK cells, we previously developed a cell-cell interaction based screening approach using a large sub-set of a lentiviral shRNA library containing multiple independent shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 significantly increased secretion of INF-γ from NK cells and increased tumor cell susceptibility to NK cell lysis. To examine the role of the JAK signaling pathway in the modulation of tumor cell susceptibility to NK lysis, we analyzed down-stream signaling pathways in several cell lines (IM9, U937, K562, RPMI, MM1S KM12BM) and primary tumor cells (AML, MM, ALL). In the absence of NK cells, silencing JAK1 or JAK2 did not affect the basal activation of STAT proteins (STAT1(pY701), STAT1(pS727), STAT3(pY705), STAT3(pS727), STAT4(pY693), STAT5(pY694), STAT6(pY641)) or AKT(pS473) and ERK1/2(pT202/pY204) or expression of activating or inhibitory ligands on tumor cells. Because JAK1 and JAK2 transduce signals downstream of the IFN-γ receptor, we hypothesized that JAKs may play a role in tumor cell evasion of NK cell activities such as cytolysis and IFN-γ secretion. To test this hypothesis we pre-incubated various tumor cell lines or primary tumor cells with activated NK supernatant or recombinant human IFN-γ. Tumor cell activation in this fashion resulted in activation of STAT1 (pSTAT1(pY701)) but none of the other STATs, ERK or AKT. As expected, STAT1 activation was blocked when JAK1 or JAK2 were silenced or inhibited by a JAK inhibitor. Silencing of STAT1 with 2 independent shRNAs also resulted in increased tumor susceptibility to NK cell cytolysis in 3 different tumor cell lines tested. To confirm that IFN-γ secreted by activated NK cells induced resistance in tumor cell targets we used a blocking IFN-γ antibody (D9D10). 10μg/ml D9D10 completely blocked STAT1 phosphorylation and in different experiments using U937, IM-9, KM12BM, MM1S and RPMI we found that D9D10 significantly increased specific NK target cell lysis by 51.8%, 78.5%, 25.1%, 20.6% and 28.5% compared to IgG1 isotype controls. Similar results were obtained whit different primary tumor cells. To determine whether IFN-γ stimulation affected expression of ligands involved in NK cell recognition of tumor cells, we analyzed the effect of activated NK supernatant or IFN-γ on the expression of MHC Class I, β2M, HLA-C, HLA-A2, NKG2D, NKP44, NKP46, NKP30 ligands using chimeric FC proteins, MICA/B, DNAM-1 ligands (CD112, CD155), 2B4 ligand (CD48), TRAIL ligands (TRAIL-R1, TRAIL-R2), Fas ligand (CD95) and PD1 ligands (PDL1, PDL2, B7H3, B7H4). The basal expression of these ligands varied among the various tumor cell lines or primary tumors tested but the only ligand that was significantly up-regulated in every tumor sample tested was PDL1. PDL1 expression by tumor cells is known to inhibit T cell immunity. To test whether increased levels of PDL1 could also inhibit NK cell killing, we co-cultured primary NK cells with U937, IM9, KM12BM, RPMI, K562, MM1S, primary MM, AML and ALL cells with or without 10μg/ml anti-PDL1 antibody (recombinant mab with Fc mutated to eliminate FcR-mediated effects). Blocking PDL1 significantly increased NK cell killing of U937, IM9, KM12BM, RPMI, MM, AML and ALL (p=0.03, p=0.02, p=0.03, p=0.005, p=0.009, p=0.03 and p=0.02 respectively). NK cell killing activity did not further increase when a JAK inhibitor was added to the co-culture. These results show that NK cell secretion of IFN-γ results in IFN receptor signaling and activation of JAK1, JAK2 and STAT1 in the tumor cell targets, followed by rapid up-regulation of PDL1 expression and increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents subsequent PDL1 up-regulation resulting in increased susceptibility of tumor cells to NK cell activity suggesting that JAK pathway inhibitors may work synergistically with other immunotherapy regimens by eliminating IFN-induced PDL1 mediated immunoinhibition. Disclosures: Freeman: Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

Interaction of Human Tumor Cells with Human Platelets and the Coagulation System

1983 ◽  
Vol 50 (03) ◽  
pp. 726-730 ◽  
Author(s):  
Hamid Al-Mondhiry ◽  
Virginia McGarvey ◽  
Kim Leitzel
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Human Tumor ◽  
Human Platelets ◽  
Factor Vii ◽  
Coagulation System ◽  
Breast Adenocarcinoma ◽  
Activate Factor ◽  
Tumor Cell Lines

SummaryThis paper reports studies on the interaction between human platelets, the plasma coagulation system, and two human tumor cell lines grown in tissue culture: Melanoma and breast adenocarcinoma. The interaction was monitored through the use of 125I- labelled fibrinogen, which measures both thrombin activity generated by cell-plasma interaction and fibrin/fibrinogen binding to platelets and tumor cells. Each tumor cell line activates both the platelets and the coagulation system simultaneously resulting in the generation of thrombin or thrombin-like activity. The melanoma cells activate the coagulation system through “the extrinsic pathway” with a tissue factor-like effect on factor VII, but the breast tumor seems to activate factor X directly. Both tumor cell lines activate platelets to “make available” a platelet- derived procoagulant material necessary for the conversion of prothrombin to thrombin. The tumor-derived procoagulant activity and the platelet aggregating potential of cells do not seem to be inter-related, and they are not specific to malignant cells.

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