Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen

Abstract Background Glomus tumors are subcutaneous tumors arising from glomus bodies, thermoregulatory components of the skin. These tumors could occur in visceral organs where glomus bodies are not normally present. Herein, we report a case of primary pancreatic glomus tumor with aggressive direct invasion into the superior mesenteric vein (SMV). To the best of our knowledge, this is the second case report of a glomus tumor arising in the pancreas. Case presentation A 46-year-old woman was referred to our hospital due to vomiting with epigastric and back pain. Dynamic-CT revealed a well-circumscribed hypervascular mass, measuring 37 mm in its maximal diameter involving the pancreatic head. Both CT and endoscopic ultrasonography (EUS) revealed direct invasion into the SMV and radiologically suspected tumor thrombus. Biopsy sample obtained by EUS-guided fine needle aspiration revealed proliferation of small cells, round-to-oval tumor cells with round nuclei and scant cytoplasm. A histological diagnosis of pancreatic neuroendocrine tumor, G1 was initially considered. Therefore, subtotal stomach-preserving pancreatoduodenectomy using Child-II reconstruction was subsequently performed. Her SMV was resected and reconstructed due to extensive tumor involvement. Subsequent histopathological analysis revealed solid tumor cells proliferation that comprised oval-shaped nuclei and scant cytoplasm around disorganized or slit-shaped vessels in hematoxylin–eosin-stained slides. Immunohistochemical analysis then demonstrated positive immunoreactivity for smooth muscle actin, vimentin, and CD34, but negative for chromogranin A, synaptophysin, CD56, and signal transducer and activator of transcription 6. Based on these histological findings of resected specimens, the lesion was subsequently diagnosed as a primary pancreatic glomus tumor harboring direct invasion into the SMV. Her postoperative course was uneventful and annual surveys for the following 4 years post-op detected no clinical signs of recurrence. Conclusions We report a very rare case of glomus tumor of the pancreas accompanied by venous invasion. Curative surgical resection is the best treatment option for pancreatic glomus tumors. Although pancreatic glomus tumor is rare, it should be taken into consideration in the differential diagnosis of a pancreatic solid tumor with hypervascularity.

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MODL-12. DEVELOPMENT OF A NOVEL IMMUNOCOMPETENT MOUSE MODEL FOR DIFFUSE INTRINSIC PONTINE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.586 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii413-iii413

Author(s):

Maggie Seblani ◽

Markella Zannikou ◽

Katarzyna Pituch ◽

Liliana Ilut ◽

Oren Becher ◽

...

Keyword(s):

Flow Cytometry ◽

Mouse Model ◽

Growth Factor Receptor ◽

Diffuse Intrinsic Pontine Glioma ◽

Pontine Glioma ◽

Time Of Application ◽

Tumor Associated Antigen ◽

Sarcoma Virus

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor affecting young children. Immunotherapies hold promise however the lack of immunocompetent models recreating a faithful tumor microenvironment (TME) remains a challenge for development of targeted immunotherapeutics. We propose to generate an immunocompetent DIPG mouse model through induced overexpression of interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen overexpressed by glioma cells. A model with an intact TME permits comprehensive preclinical assessment of IL13Rα2-targeted immunotherapeutics. Our novel model uses the retroviral avian leucosis and sarcoma virus (RCAS) for in vivo gene delivery leading to IL13Rα2 expression in proliferating progenitor cells. Transfected cells expressing IL13Rα2 and PDGFB, a ligand for platelet derived growth factor receptor, alongside induced p53 loss via the Cre-Lox system are injected in the fourth ventricle in postnatal pups. We validated the expression of PDGFB and IL13Rα2 transgenes in vitro and in vivo and will characterize the TME through evaluation of the peripheral and tumor immunologic compartments using immunohistochemistry and flow cytometry. We confirmed expression of transgenes via flow cytometry and western blotting. Comparison of survival dynamics in mice inoculated with PDGFB alone with PDGFB+IL13Rα2 demonstrated that co-expression of IL13Rα2 did not significantly affect mice survival compared to the PDGFB model. At time of application, we initiated experiments to characterize the TME. Preliminary data demonstrate establishment of tumors within and adjacent to the brainstem and expression of target transgenes. Preclinical findings in a model recapitulating the TME may provide better insight into outcomes upon translation to clinical application.

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Hsp90 inhibitors deplete key anti-apoptotic proteins in pediatric solid tumor cells and demonstrate synergistic anticancer activity with cisplatin

International Journal of Cancer ◽

10.1002/ijc.20611 ◽

2004 ◽

Vol 113 (2) ◽

pp. 179-188 ◽

Cited By ~ 55

Author(s):

Rochelle Bagatell ◽

Jason Beliakoff ◽

Cynthia L. David ◽

Marilyn T. Marron ◽

Luke Whitesell

Keyword(s):

Anticancer Activity ◽

Tumor Cells ◽

Solid Tumor ◽

Hsp90 Inhibitors ◽

Pediatric Solid Tumor ◽

Apoptotic Proteins

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Postoperative metastasis prediction based on portal vein circulating tumor cells detected by flow cytometry in periampullary or pancreatic cancer

Cancer Management and Research ◽

10.2147/cmar.s210332 ◽

2019 ◽

Vol Volume 11 ◽

pp. 7405-7425 ◽

Cited By ~ 2

Author(s):

Lianyuan Tao ◽

Li Su ◽

Chunhui Yuan ◽

Zhaolai Ma ◽

Lingfu Zhang ◽

...

Keyword(s):

Pancreatic Cancer ◽

Flow Cytometry ◽

Portal Vein ◽

Tumor Cells ◽

Circulating Tumor Cells

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Tumor-associated antigen-based personalized dendritic cell vaccine in solid tumor patients

Cancer Immunology Immunotherapy ◽

10.1007/s00262-020-02496-w ◽

2020 ◽

Vol 69 (7) ◽

pp. 1375-1387

Author(s):

Qian-Ting Wang ◽

Ying Nie ◽

Sheng-Nan Sun ◽

Tao Lin ◽

Ru-Jin Han ◽

...

Keyword(s):

Dendritic Cell ◽

Solid Tumor ◽

Dendritic Cell Vaccine ◽

Cell Vaccine ◽

Tumor Patients ◽

Tumor Associated Antigen

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In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0703875104 ◽

2007 ◽

Vol 104 (28) ◽

pp. 11760-11765 ◽

Cited By ~ 209

Author(s):

W. He ◽

H. Wang ◽

L. C. Hartmann ◽

J.-X. Cheng ◽

P. S. Low

Keyword(s):

Flow Cytometry ◽

Tumor Cells ◽

Circulating Tumor Cells

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Determination of Circulating Tumor Cells by Flow Cytometry in the Bladder Cancer Patients

Urology and Andrology - Open Journal ◽

10.17140/uaoj-2-115 ◽

2018 ◽

Vol 2 (1) ◽

pp. 25-30 ◽

Cited By ~ 1

Author(s):

Elif Ercan ◽

◽

Ender Sımsek ◽

Ozen Ozensoy Guler ◽

Abdullah Erdem Canda ◽

...

Keyword(s):

Flow Cytometry ◽

Bladder Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells

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163 Improved anti-tumor activity of next-generation TCR-engineered T cells through CD8 co-expression

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.163 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A173-A173

Author(s):

Gagan Bajwa ◽

Justin Gunesch ◽

Inbar Azoulay-Alfaguter ◽

Melinda Mata ◽

Ali Mohamed ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Solid Tumor ◽

Cd4 T Cells ◽

Tumor Response ◽

Hla Class I ◽

Next Generation ◽

Engineered T Cells ◽

Anti Tumor Activity

BackgroundSuccessful targeting of solid tumors with TCR-engineered T cells (TCR-T) will require eliciting of antigen-specific, multi-dimensional, sustained anti-tumor immune response by infused T cells while overcoming the suppressive tumor microenvironment. First-generation TCR-T approaches have demonstrated clinical efficacy in some solid cancers. However, effective treatment across several solid tumor indications may require engineered T cells with enhanced anti-tumor activity. Here, we show pre-clinical data from one of the engineering approaches currently being developed for next-generation ACTengine® TCR-T product candidates. We evaluated the impact of co-expression of different CD8 co-receptors on functionality of CD4+ and CD8+ T cells genetically modified with an HLA class I-restricted TCR and determined the depth and durability of anti-tumor response in vitro.MethodsHere, we used a PRAME-specific TCR currently being tested in the ACTengine® IMA203 clinical trial. T cells expressing either the TCR alone or co-expressing the TCR and CD8α homodimer (TCR.CD8α) or CD8αβ heterodimer (TCR.CD8αβ) were characterized for transgene expression, antigen-recognition, and functional efficacy in vitro. Comprehensive evaluation of CD4+ T cells expressing TCR.CD8α or TCR.CD8αβ was performed focusing on cytotoxic potential and the breadth of cytokine response against target-positive tumor cell lines.ResultsIntroduction of CD8α or CD8αβ enabled detection of transgenic TCR on the surface of CD4+ T cells via HLA multimer-guided flow cytometry otherwise lacking in the TCR only transduced T cells. Co-expression of either form of CD8 co-receptor endowed CD4+ T cells with the ability to recognize and kill target positive tumor cells; however, genetic modification with TCR.CD8αβ led to more pronounced CD4+ T cell activation as compared to TCR.CD8α. Most distinct differences were observed in the breadth and magnitude of cytokine responses, less in cytotoxic activity against tumor cells. T cells expressing TCR.CD8αβ showed superior induction of Th1 cytokines e.g. IFNγ, TNFα, IL-2, GM-CSF in vitro upon antigen stimulation as compared to TCR.CD8α-T cells. Additionally, TCR.CD8αβ T cells demonstrated more efficient engagement with antigen-presenting cells and consequently, modulation of cytokine response than TCR.CD8α-T cells.ConclusionsOur findings illustrate that engaging CD4+ T cells via CD8 co-expression potentiates anti-tumor activity of HLA class I restricted TCR-T cells in vitro. The pleiotropic effects mediated by activated CD4+ T cells including acquired cytotoxicity may potentially improve outcomes in solid tumor patients when applied clinically. In addition, the differential functional profile of TCR-T cells co-expressing either CD8α or CD8αβ suggests that optimizing the type of co-receptor is relevant to maximize anti-tumor response.

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Antitumor activity of L-asparaginase from Erwinia Carotovora from against different leukemic and solid tumours cell lines

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20135905498 ◽

2013 ◽

Vol 59 (5) ◽

pp. 498-513 ◽

Cited By ~ 9

Author(s):

O.Yu. Abakumova ◽

O.V. Podobed ◽

P.A. Karalkin ◽

L.I. Kondakova ◽

N.N. Sokolov

Keyword(s):

Dna Synthesis ◽

Tumor Cells ◽

Solid Tumor ◽

Human Fibroblasts ◽

Erwinia Carotovora ◽

Cell Number ◽

Jurkat Cells ◽

Leukemic Cells ◽

Cycle Phase ◽

Tumor Cell Death

We have studied dose- and time-dependent antitumor and cytotoxic effects of Erwinia carotovora L-asparaginase (ECAR LANS) and Escherichia coli L-asparaginase (MEDAC) on human leukemic cells and human and animal solid tumor cells. We determined the sensitivity of tumor cells to L-asparaginases, as well the effect L-asparaginases on cell growth rate, protein and DNA synthesis per se and with addition of different cytostatics. The data obtained demonstrated that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division and had no effect on protein and DNA synthesis. Cytofluorometric study of solid and leukemic cells demonstrated that the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. The HL-60 cell line was only exemption. At the same time, cells treatment with L-asparaginase and doxorubicin combination leaded to increase of apoptotypical cell number to 60% for MCF7 cells, to 40% for Jurkat cells and to 99% for HL-60 cells. We have excluded apoptosis as main reason for tumor cell death after asparaginase treatment because multi resistant Jurkat/A4 cells have been asparaginase sensitive. We have not found ECAR LANS L-asparaginase effect on normal human fibroblasts growth ability and we had come to conclusion that enzyme cytotoxcisity related only with asparagine deficiency.

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Flow Cytometry Immunophenotyping for Diagnostic Orientation and Classification of Pediatric Cancer Based on the EuroFlow Solid Tumor Orientation Tube (STOT)

Cancers ◽

10.3390/cancers13194945 ◽

2021 ◽

Vol 13 (19) ◽

pp. 4945

Author(s):

Cristiane de Sá de Sá Ferreira-Facio ◽

Vitor Botafogo ◽

Patrícia Mello Ferrão ◽

Maria Clara Canellas ◽

Cristiane B. Milito ◽

...

Keyword(s):

Flow Cytometry ◽

Solid Tumors ◽

Solid Tumor ◽

Pediatric Cancer ◽

Diagnostic Procedures ◽

Hematologic Malignancies ◽

Multiparameter Flow Cytometry ◽

Diagnostic Screening ◽

Pediatric Solid Tumors

Early diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and classification of hematologic malignancies, its application to non-hematopoietic pediatric tumors remains limited. Here we designed and prospectively validated a new single eight-color antibody combination—solid tumor orientation tube, STOT—for diagnostic screening of pediatric cancer by MFC. A total of 476 samples (139 tumor mass, 138 bone marrow, 86 lymph node, 58 peripheral blood, and 55 other body fluid samples) from 296 patients with diagnostic suspicion of pediatric cancer were analyzed by MFC vs. conventional diagnostic procedures. STOT was designed after several design–test–evaluate–redesign cycles based on a large panel of monoclonal antibody combinations tested on 301 samples. In its final version, STOT consists of a single 8-color/12-marker antibody combination (CD99-CD8/numyogenin/CD4-EpCAM/CD56/GD2/smCD3-CD19/cyCD3-CD271/CD45). Prospective validation of STOT in 149 samples showed concordant results with the patient WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). These included: 63/63 (100%) reactive/disease-free samples, 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors together with 28/38 (74%) leukemia/lymphoma cases; the only exception was Hodgkin lymphoma that required additional markers to be stained. In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45− CD56++ non-hematopoietic solid tumors: 13/13 (GD2++ numyogenin− CD271−/+ nuMyoD1− CD99− EpCAM−) neuroblastoma samples, 5/5 (GD2− numyogenin++ CD271++ nuMyoD1++ CD99−/+ EpCAM−) rhabdomyosarcomas, 2/2 (GD2−/+ numyogenin− CD271+ nuMyoD1− CD99+ EpCAM−) Ewing sarcoma family of tumors, and 7/7 (GD2− numyogenin− CD271+ nuMyoD1− CD99− EpCAM+) Wilms tumors. In summary, here we designed and validated a new standardized antibody combination and MFC assay for diagnostic screening of pediatric solid tumors that might contribute to fast and accurate diagnostic orientation and classification of pediatric cancer in routine clinical practice.

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