Aerolysin Cytotoxicity Can Serve as a Surrogate Marker for Immune Sensitivity of Tumor Cells to Granule-Exocytosis Pathway in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3864-3864
Author(s):  
Oner Ozdemir ◽  
Mustafa Buyukavci ◽  
Yaddanapudi Ravindranath ◽  
Sureyya Savasan
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Surrogate Marker ◽  
Granule Exocytosis ◽  
Flow Cytometric ◽  
Lak Cell ◽  
Cell Kill

Abstract Background: Aerolysin is a pore-forming toxin produced by Aeromonas hydrophila and interacts with target mammalian cells through binding to glycosylphosphatidyl inositol (GPI)-anchored proteins, subsequently forming a pore in the plasma membrane. Both osmotic lysis of the cell secondary to fluid influx thru pores and a rapid increase in intracellular calcium, which may have been the signal for apoptosis, when a small number of channels were opened appear to be operational in aerolysin cytotoxicity. Perforin (PFP), a prototype of pore-forming proteins is closely linked to granzyme B effect and the central element of natural killer (NK) and T cell mediated cytotoxicity, and induces apoptosis of target cells through granule-exocytosis pathway. Since perforin/granzyme B pathway is pivotal to immune mediated elimination and due to similarities between PFP and aerolysin action, we investigated the relationship between aerolysin cytotoxicity and lymphokine activated killer (LAK) cell-mediated elimination of human tumor cells. Material and Methods: We studied the effect of active aerolysin (2x10−10M) against eight tumor cell lines and five acute myeloid leukemia patient samples. Aerolysin cytotoxicity was assessed at eight different time points: first at ½ to 4 hours of incubation consistent with the known optimal cell kill mediated by PFP in NK cytotoxicity, then lastly at 18 hours to be able see overall effect. Cytotoxicity was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6 (3) /PI staining and further quantification of fluorosphere-adjusted events. Tumor cell type selection was based on their sensitivity against LAK cell-mediated cytotoxicity. Acute myeloid leukemia cells K562, U937, CMK, Meg-01, HL-60 and resistant central nervous system tumor cells (HTB-11, HTB-12 and HTB-14) were included in this study. LAK cell-mediated cytotoxicity against tumor cell lines and patient samples was measured by flow cytometric cell mediated cytotoxicity assay. Drug cytotoxicity was also evaluated in all cell lines and patient samples. Results: In timing studies, aerolysin treatment reached peak cytotoxicity around 2 hours of incubation. U937 was the most sensitive cell type to both LAK cells and aerolysin. Some cells were very resistant to LAK killing and aerolysin at the concentration used. There was significant correlation between aerolysin cytotoxicity and LAK cell-mediated kill at 2 hours in cell lines and patient samples (r = 0.99; p < 0.001, r = 0.85; p = 0.035, respectively). There was also a statistically significant correlation between optimal dose aerolysin killing at 18 hours of incubation and LAK cell-mediated cytotoxicity (r = 0.94; p = 0.04). However, there was not any correlation between drug cytotoxicity and LAK cell-mediated cytotoxicity or aerolysin-induced cell kill. Conclusion: Aerolysin sensitivity may be used as a surrogate marker for the assessment of in vitro LAK cell-mediated tumor cell kill through granule/exocytosis pathway.

scholarly journals Pre-Clinical Combination of AO-176, a Highly Differentiated Clinical Stage CD47 Antibody, with Either Azacitidine or Venetoclax Significantly Enhances DAMP Induction and Phagocytosis of Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Mike J Donio ◽  
W. Casey Wilson ◽  
Isra Darwech ◽  
Gabriela Andrejeva ◽  
Benjamin J Capoccia ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Surface ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Innate Immune ◽  
Private Company ◽  
Acute Myeloid

While T cell checkpoint inhibitors are mainstays of cancer immunotherapy, therapies that direct innate immune responses against cancer are lacking. CD47, a "don't eat me" signal, is an innate immune cell checkpoint which binds SIRPα on macrophages and dendritic cells to limit phagocytosis and its upregulation on tumor cells leads to evasion of immune detection and clearance. Therapeutic antibodies have previously been developed to block CD47 and induce phagocytosis of tumor cells, thus validating the pathway. AO-176, a next generation humanized IgG2 anti-CD47 antibody, was developed to block the CD47/SIRPα interaction and induce tumor cell phagocytosis. Moreover, AO-176 directly kills tumor cells through a non-ADCC-dependent mechanism via induction of programmed cell death type III. In addition to these tumor eliminating properties, AO-176 has the potential for a strong safety profile as a result of its preferential binding to tumor versus normal cells, lack of RBC binding, and enhanced binding to tumor cells at acidic pH. CD47 has previously been shown to be upregulated on acute myeloid leukemia (AML) leukemic stem cells (LSC), enabling their expansion through evasion from phagocytic clearance. As a result, patients with increased CD47 on AML LSCs have worse overall survival. In this study, AO-176 efficacy was evaluated in AML cell lines as a single agent and in combination with azacitidine and venetoclax which are approved therapies for AML. Azacitidine is a cytosine analogue which acts to inhibit DNA methylation, and venetoclax is a potent Bcl-2 inhibitor. Previous studies have shown that azacitidine induces apoptosis of tumor cells and increases cell surface exposure of calreticulin, a DAMP (Damage Associate Molecular Pattern) which provides a strong pro-phagocytic signal. From these findings, it was hypothesized that azacitidine would enable increased tumor cell phagocytosis when combined with AO-176. The potential for a similar enhancement with a combination of AO-176 and venetoclax was also explored. The ability of AO-176, with or without azacitidine or venetoclax, to induce DAMPs on the surface of AML cells was assessed. Cell surface expression of DAMPs, calreticulin and PDIA3, were measured by flow cytometry. AO-176, azacitidine, and venetoclax as single agents potently increased both calreticulin and PDIA3 in a dose-dependent manner on AML cell lines such as HL60 This is the first time, to our knowledge, that venetoclax has been shown to induce DAMPs. To better understand the functional implications of these findings, in vitro phagocytosis assays were performed. Azacitidine and venetoclax significantly enhanced AO-176-mediated phagocytosis of AML cells compared to any of the agents alone. Moreover, when AO-176 was combined with azacitidine in direct tumor cell killing assays, enhanced activity was observed in a subset of AML cell lines. In conclusion, AO-176 combined with either azacitidine or venetoclax, resulted in significant enhancement of phagocytic AML cell clearance in vitro which also correlated with the ability of these agents to induce DAMPs. In vivo treatment with AO-176 in combination with these agents is in progress. AO-176 is being evaluated in phase 1 clinical trials for the treatment of patients with solid tumors (NCT03834948) and multiple myeloma (NCT04445701). Disclosures Donio: Arch Oncology: Current Employment, Current equity holder in private company. Wilson:Arch Oncology: Current Employment, Current equity holder in private company. Darwech:Arch Oncology: Current Employment, Current equity holder in private company. Andrejeva:Arch Oncology: Current Employment, Current equity holder in private company. Capoccia:Arch Oncology: Current Employment, Current equity holder in private company. Puro:Arch Oncology: Current Employment, Current equity holder in private company. Kashyap:Arch Oncology: Current Employment, Current equity holder in private company. Pereira:Arch Oncology: Current Employment, Current equity holder in private company.

scholarly journals Breast Tumor Cell Lines From Pleural Effusions2

1974 ◽  
Vol 53 (3) ◽  
pp. 661-674 ◽  
Author(s):  
R. Cailleau ◽  
R. Young ◽  
M. Olivé ◽  
W. J. Reeves
Keyword(s):  
Chromosome Number ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Breast Tumor ◽  
Tumor Cell Lines ◽  
Breast Cancer Patients ◽  
Pleural Effusions ◽  
Epithelial Tumor

Summary During 1973, 4 new epithelial tumor cell lines were isolated from pleural effusions from breast cancer patients. We describe 3 of these lines: MDA-MB-134, with a mean chromosome number of 43; MDA-MB-175, with a mean chromosome number of 49; and MDA-MB-231, with a mean chromosome number between 65 and 69. We isolated the same cell type from 4 of 10 effusions from MDA-MB-134 and from 6 of 8 effusions from MDA-MB-175. We found that pleural effusions as a source of breast tumor cells to be cultured and studied in vitro have the following advantages: 1) large amounts of material and the possibility of obtaining sequential samples from the same patient; 2) high viability of tumor cells; 3) scarcity or absence of fibroblasts; and 4) the possibility of separating the tumor cells from other “contaminating” cell types by differences in their speed or degree of attachment to the flask. All lines from different patients differed, as seen grossly and microscopically. All lines from sequential pleural effusions from the same patient were apparently alike. No viruses or mycoplasmas were detected in any line.

Defibrotide (DF) Targets Tumor-Microenvironmental Interactions and Sensitizes Multiple Myeloma and Solid Tumor Cells to Cytotoxic Chemotherapeutics.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 286-286 ◽  
Author(s):  
Constantine S. Mitsiades ◽  
Cecile Rouleau ◽  
Krishna Menon ◽  
Beverly Teicher ◽  
Massimo Iacobelli ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Single Agent ◽  
Conventional Chemotherapy ◽  
Adhesive Properties

Abstract Introduction: Defibrotide (DF) is a polydisperse oligonucleotide with anti-thrombotic, thrombolytic, anti-ischemic, and anti-adhesive properties, which selectively targets the microvasculature and has minimal hemorrhagic risk. DF is an effective treatment for veno-occlusive disease (VOD), an important regimen-related toxicity in stem cell transplantation characterized by endothelial cell injury. DF also augments stem cell mobilization by modulating adhesion in vivo. Because of its cytoprotective effect on the endothelium, we specifically investigated whether DF protects tumor cells from cytotoxic anti-tumor agents. Further, because of its broad anti-adhesive properties, we evaluated whether DF modulates the interaction of MM cells with bone marrow stromal cells (BMSCs), which confers growth, survival and drug resistance in the BM milieu. Methods: In vitro studies in isogenic dexamethasone (Dex)-sensitive and resistant MM cell lines (MM-1S and MM1R, respectively) showed that DF does not attenuate the sensitivity of MM cells to Dex, the proteasome inhibitor bortezomib (PS-341), melphalan (MEL), vinca alkaloids (vincristine, vinblastine), taxanes (paclitaxel) or platinum (cisplatin), but does decrease their sensitivity to doxorubicin. These selective effects in vitro of DF in protecting tumor cells against doxorubicin and modestly sensitizing MM cells to platinum was also confirmed in solid tumor breast (MCF-7) and colon (HT-29) carcinoma cell lines. Although DF had minimal in vitro inhibitory effect on MM or solid tumor cell growth in vitro, it showed in vivo activity as a single agent and enhanced the responsiveness of MM tumors to cytotoxic chemotherapeutics, such as MEL or cyclophosphamide, in human MM xenografts in SCID/NOD mice. The in vivo single-agent activity and chemosensitizing properties of DF, coupled with its lack of major in vitro activity, suggested that DF may not directly target tumor cells, but rather modulate tumor cell interaction with BMSCs. In an ex vivo model of co-culture of primary MM tumor cells with BMSCs (which protects MM cells against conventional chemotherapy), DF alone had a only modest effect on tumor cell viability, but it significantly enhanced MM cell sensitivity to cytotoxic chemotherapy (e.g. MEL), suggesting that a major component of the biological effects of DF may be attributable not to direct targeting of tumor cells, but to modulation of the interactions that tumor cells develop with the local stromal milieu. Conclusion: Our studies show that DF mediates in vivo anti-MM activity by abrogating interactions of MM cells with their BM milieu, thereby enhancing sensitivity and overcoming resistance to conventional chemotherapy. These data support future clinical trials of DF, in combination with both conventional and novel therapies, to improve patient outcome in MM.

Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2468-2468
Author(s):  
Eugen Dhimolea ◽  
Jana Jakubikova ◽  
Richard W.J. Groen ◽  
Jake E. Delmore ◽  
Hannah M. Jacobs ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Response ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Bortezomib Treatment ◽  
Rpmi 8226

Abstract Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. Disclosures: Groen: Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

scholarly journals STUDY OF EFFECTS OF CIRCADIAN RHYTHMS ON SOLID TUMOR CELLS PROLIFERATION AND CHEMORESISTANCE

2020 ◽  
Vol 66 (5) ◽  
pp. 563-571
Author(s):  
Anna Danilova ◽  
N. Avdonkina ◽  
Ye. Gubareva ◽  
I. Baldueva ◽  
Anton Zozulya ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Lung Tumor ◽  
Morphological Characteristics ◽  
Biological Properties ◽  
Physiological Processes

Circadian clock is a complex mechanism regulating many different physiological processes. Preclinical, epidemiological and clinical studies demonstrate association between circadian rhythms disruption and tumor initiation. Study of modulation of solid tumor cells biological properties through enhancement of clock mechanisms could attribute to the development of more effective chemo- and hormone therapy approaches. Aim: Evaluate the effects of ovarian and lung tumor cells synchronization with dexamethasone in vitro on cells sensitivity to cisplatin. Materials and methods: Metastatic ovarian cancer (n=3) and lung cancer (n=3) cell lines were obtained from patients tumors. Tumor cell cultivation was performed in accordance with the protocol. Artificial synchronization was performed with dexamethasone 200 nM introduction to the cell cultures. Doses of cisplatin used were 1.5 and 3.0 mg/ml. xCELLigence Real-Time Cell Analysis and Cell-IQ was used to measure proliferation and chemoresistance of tumor cells. Results: Each cell-line had individual morphological characteristics and proliferation parameters. Preliminary incubation with dexamethasone (2 h) had a stimulating effect on proliferation of all tumor cell lines (Slope min -4.3(0.3)хЕ ‘х10-3 - max 36.8(0.6)хЫх10'3, min 2.2(0.2)хЕ1х10'3- max 50.4(0.8)хЕ1х10'3), and increased their sensitivity to cisplatin (min -43(2.6)хЕ1х10-3 - max 57.5(0.6)хЕ1х10-3 и min -217,3(2,2) -1,9(0,1)хч-1х10-3 - max -1,9(0,1)хч'1х10'3, respectively. Conclusion: These results should be the platform for future studies of the interaction of clock mechanisms, cell cycle regulation and viability of tumor cells.

Virus like Particles in a Human Lung Tumor

1974 ◽  
Vol 32 ◽  
pp. 18-19
Author(s):  
R.E. Nordquist ◽  
R.E. Coalson ◽  
J.A. Mohr ◽  
E.R. Rhoades ◽  
J.J. Coalson ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Lung Tumor ◽  
Biological Agent ◽  
Virus Like Particles ◽  
Human Lung Tumor ◽  
Ultrastructural Studies ◽  
Indicator Cell

Ultrastructural studies of pulmonary needle biopsies from patients with alveolar cell carcinoma (ACC) revealed the presence of a virus-like material in the nucleus and cytoplasm of tumor cells. Stinson et. al. reported the presence of filamentous virus-like particles in four of six cases in an ultrastructural study of ACC. In vitro studies by Coalson et. al. confirmed the presence of a biological agent and demonstrated that cell-free supernate from tumor cell cultures could induce cytopathic effect when applied to indicator cell lines. It was also shown in this report that tumor cell lines derived from this tumor produced the filterable biological agent until the 18-20 passage in culture. An extension of this investigation on the cell lines derived from ACC demonstrated that a unique antigen was associated with ACC tumor cells and that this antigenicity could be induced in indicator cell lines following treatment with cell free extracts.

Antiproliferative and Antitumor Activity of the 2-Cyanoaziridine Compound Imexon on Tumor Cell Lines and Fresh Tumor Cells In Vitro

1992 ◽  
Vol 84 (16) ◽  
pp. 1238-1244 ◽  
Author(s):  
E. M. Hersh ◽  
C. R. Gschwind ◽  
C. W. Taylor ◽  
R. T. Dorr ◽  
R. Taetle ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Tumor Cell Lines

Treatment of leptomeningeal metastases in a rat model using a recombinant adenovirus containing the HSV-tk gene

1996 ◽  
Vol 85 (4) ◽  
pp. 648-654 ◽  
Author(s):  
Arnaud J. P. E. Vincent ◽  
Maria del C. Esandi ◽  
Gerry van Someren ◽  
Juus L. Noteboom ◽  
Cees J. J. Avezaat ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Reporter Gene ◽  
Rat Model ◽  
Recombinant Adenovirus ◽  
Human Tumor ◽  
Leptomeningeal Metastases ◽  
Cell Injection

✓ The authors constructed recombinant adenoviral vectors to investigate their potential for gene therapy treatment of leptomeningeal metastases. Several human cell lines that were derived from tumors occurring as leptomeningeal metastases and that were infected in vitro with major late promoter recombinant adenovirus containing the luciferase (luc) gene (IG.Ad.MLP.luc.) showed high levels of expression. When these human tumor cell lines were infected in vitro with recombinant adenovirus harboring the herpes simplex virus—thymidine kinase (HSV-tk) gene (IG.Ad.MLP.TK), they were highly sensitive to the killing effects of ganciclovir (GCV). Transduction efficiency of leptomeningeal tumor cells in vivo was assessed by injecting 9-L rat brain tumor cells into the cerebrospinal fluid of Fischer rats via the cisterna magna. After 3 days, recombinant adenovirus containing the lacZ reporter gene (IG.Ad.MLP.lacZ) was injected via the same route. Six days after tumor cell injection, expression of the reporter gene was observed in tumor cells along the total neural axis. Subsequently, rats with leptomeningeal metastases were treated 3 days after tumor cell injection with HSV-tk. Beginning on the next day, GCV was injected intraperitoneally for 10 days. The rats that developed neurological symptoms were killed immediately. The symptom-free latency of every rat was determined. The rats treated with HSV-tk and subsequent GCV had significantly longer (p < 0.01) symptom-free latency than all control groups. This study demonstrates the feasibility and efficacy of this therapeutic approach in a rat model. Clinically, it should be used in the palliative treatment of patients with leptomeningeal metastases.

High affinity Fc receptor binding and potent induction of antibody-dependent cellular cytotoxicity (ADCC) in vitro by anti-epidermal growth factor receptor antibody cetuximab

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3041-3041 ◽  
Author(s):  
X. Kang ◽  
D. Patel ◽  
S. Ng ◽  
M. Melchior ◽  
D. Ludwig ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Growth Factor Receptor ◽  
Tumor Cell Lines ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Effector Cells ◽  
Epidermal Growth

3041 Background: Cetuximab is an IgG1 monoclonal antibody specific for human epidermal growth factor receptor (EGFR). Cetuximab acts as a functional antagonist by blocking ligand (EGF and TGFa) binding to EGFR and, therefore, inhibits EGFR activation and downstream signaling in tumor cells. Methods: In the present study, we analyzed the binding activity of cetuximab to human Fc receptors and tested whether cetuximab can elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro using tumor cell lines expressing various levels of EGFR. Results: Cetuximab bound to human FcRI and FcRIII with high affinity (EC50 of 0.13 nM and 6 nM, respectively), whereas an aglycosylated form of cetuximab or an IgG2 antibody to EGFR (panitumumab) did not bind to FcRI and FcRIII. A panel of head and neck, colon, and breast tumor cell lines were tested for cetuximab-mediated ADCC using human peripheral blood mononuclear cells (PBMC) from normal donors as effector cells. We found that cetuximab-induced ADCC to these tumor cell lines ranging from 20–90% specific killing. Interestingly, the extent of ADCC induced by cetuximab correlated with the level of EGFR expression on cell surface. The aglycosylated form of cetuximab or panitumumab, did not elicit ADCC of EGFR expressing tumor cells, even though cetuximab and panitumumab bind to EGFR with similar affinity (Kd=87 pM and 83 pM, respectively). To identify the immune cell population responsible for ADCC, NK cells were isolated from PBMC and tested in an ADCC assay. Purified NK cells elicited high levels of specific killing of cetuximab bound tumor cells, suggesting that NK cells are one of effector cell populations in human PBMCs responsible for the observed ADCC activity. Conclusions: Thus, cetuximab can effectively link effector cells to EGFR expressing tumor cells and mediate potent ADCC against EGFR-expressing human tumors. [Table: see text]

Essential experimental steps and estimates of renal carcinoma initiating cells.

2014 ◽  
Vol 32 (15_suppl) ◽  
pp. 11127-11127
Author(s):  
Craig Gedye ◽  
Danylo Sirskyj ◽  
Nazleen Carol Lobo ◽  
Ella Hyatt ◽  
Andrew Evans ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Rock Inhibitor ◽  
Cell Renal Cell Carcinoma ◽  
Cell Potential

11127 Background: Rare cancer stem cells (CSC), proposed to be solely responsible for tumor propagation and re-initiation, are functionally identified as tumor-initiating cells (TIC) from ex vivo tumors using xenotransplantation and clonogenic limiting dilution assays (LDA). TIC have not previously been described from ex vivohuman clear cell renal cell carcinoma (ccRCC). Methods: Primary human ccRCC samples (n=120) from patients undergoing nephrectomy were processed and implanted as subcapsular fragments or cell suspension injection LDAs with Matrigel in NOD/SCID/IL2Rγ-/- (NSG) mice, and observed for at least 6 months. In vitro clonogenic LDAs assays were performed from primary cell suspensions and ccRCC cell lines. LDAs were supplemented with human stromal cells and proteins, and the Y-26732 ROCK inhibitor. Multiparametric flow cytometry and immunofluorescence were used to investigate tumor heterogeneity and cell viability. Results: ccRCC TIC appeared rare from injected suspensions, but xenografts engrafted frequently from tiny fragments, and clonogenic frequencies were 103-104greater than TIC frequencies, suggesting that LDAs underestimated ccRCC tumor cell potential. We systematically identified multiple methodological steps that distort quantitation and identification of ccRCC TIC. For example cell viability was highly variable prior to processing, disaggregation itself destroyed up to 99% of tumor cells, standard assays substantially overestimated tumor cell viability in suspensions, and supplementation with human extracellular cells or proteins, or inhibition of anoikis by Y-26732 increased clonogenic and TIC frequencies in cell lines and primary ccRCC suspensions. Annexin-V staining revealed that tumor cells were more apoptotic then normal stromal cells, and that tumor cells positive for CD44 (a putative CSC marker) were more viable than CD44- tumor cells. Conclusions: We describe multiple, unappreciated and largely unavoidable observational errors in essential methods used to study TIC in ccRCC. ccRCC TIC may be more common than appreciated. Re-examination of the CSC hypothesis in other solid tumors is warranted in view of these previously unexplored methodological biases.

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