scholarly journals Immune Characterization of Metastatic Colorectal Cancer Patients Post Reovirus Administration

2020 ◽  
Author(s):  
Ruwan Parakrama ◽  
Elisha Fogel ◽  
Carol Chandy ◽  
Titto Augustine ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Sequencing Analysis ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract Background: KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome.Methods: Reovirus was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay, was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001.Results:Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p=0.05); day 15 for GM-CSF (3.56; p=0.009), IFN-Ƴ (1.86; p=0.0004) and IL-12p70 (2.42; p=0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p=0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p=0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98- fold; 98% increase, p=0.00007). APCs were stimulated within 48 hours and activated (CD8+ CD70+) T cells within 168 hours as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x) , IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (>0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days.Conclusions: Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi- layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.

scholarly journals Immune Characterization Of Metastatic Colorectal Cancer Patients Post Reovirus Administration

2020 ◽  
Author(s):  
Ruwan Parakrama ◽  
Elisha Fogel ◽  
Carol Chandy ◽  
Titto Augustine ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Sequencing Analysis ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract Background KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome.Methods Reovirus was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay, was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001. Results Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p=0.05); day 15 for GM-CSF (3.56; p=0.009), IFN-Ƴ (1.86; p=0.0004) and IL-12p70 (2.42; p=0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p=0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p=0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98-fold; 98% increase, p=0.00007). APCs were stimulated within 48 hours and activated (CD8+ CD70+) T cells within 168 hours as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x) , IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (>0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days.Conclusions Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi-layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.

scholarly journals α-Particle-induced DNA damage tracks in peripheral blood mononuclear cells of [223Ra]RaCl2-treated prostate cancer patients

2021 ◽  
Author(s):  
S. Schumann ◽  
U. Eberlein ◽  
C. Lapa ◽  
J. Müller ◽  
S. Serfling ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Patients ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Absorbed Dose ◽  
Peripheral Blood Mononuclear ◽  
Absorbed Doses ◽  
Blood Mononuclear Cells

Abstract Purpose One therapy option for prostate cancer patients with bone metastases is the use of [223Ra]RaCl2. The α-emitter 223Ra creates DNA damage tracks along α-particle trajectories (α-tracks) in exposed cells that can be revealed by immunofluorescent staining of γ-H2AX+53BP1 DNA double-strand break markers. We investigated the time- and absorbed dose-dependency of the number of α-tracks in peripheral blood mononuclear cells (PBMCs) of patients undergoing their first therapy with [223Ra]RaCl2. Methods Multiple blood samples from nine prostate cancer patients were collected before and after administration of [223Ra]RaCl2, up to 4 weeks after treatment. γ-H2AX- and 53BP1-positive α-tracks were microscopically quantified in isolated and immuno-stained PBMCs. Results The absorbed doses to the blood were less than 6 mGy up to 4 h after administration and maximally 16 mGy in total. Up to 4 h after administration, the α-track frequency was significantly increased relative to baseline and correlated with the absorbed dose to the blood in the dose range < 3 mGy. In most of the late samples (24 h – 4 weeks after administration), the α-track frequency remained elevated. Conclusion The γ-H2AX+53BP1 assay is a potent method for detection of α-particle-induced DNA damages during treatment with or after accidental incorporation of radionuclides even at low absorbed doses. It may serve as a biomarker discriminating α- from β-emitters based on damage geometry.

scholarly journals Human Autologous In Vitro Models of Glioma Immunogene Therapy Using B7-2, GM-CSF, and IL12

2002 ◽  
Vol 29 (3) ◽  
pp. 267-275 ◽  
Author(s):  
Ian F. Parney ◽  
Maxine A. Farr-Jones ◽  
Kevin Kane ◽  
Lung-Ji Chang ◽  
Kenneth C. Petruk
Keyword(s):  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Gm Csf ◽  
Blood Mononuclear Cells ◽  
Immunogene Therapy ◽  
Tumor Cytotoxicity

Background:Cancer immunogene therapy is based on vaccination with radiated, autologous tumor cells transduced with immunostimulatory genes. To help determine an optimal glioma immunogene therapy strategy, we stimulated lymphocytes with autologous human glioma cells transduced with B7-2 (CD86), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin-12 (IL12).Methods:A human glioma-derived cell culture (Ed147.BT) was transduced with B7-2, GM-CSF, and/or IL12 using retroviral vectors. Autologous peripheral blood mononuclear cells (PBMC) were co-cultured with irradiated gene-transduced tumor alone or a combination of radiated wild type and gene-transduced cells. Peripheral blood mononuclear cells proliferation was determined by serial cell counts. Peripheral blood mononuclear cells phenotype was assessed by flow cytometry for CD4, CD8, and CD16. Anti-tumor cytotoxicity was determined by chromium-51 (51Cr) release assay.Results:Peripheral blood mononuclear cells cell numbers all decreased during primary stimulation but tumor cells expressing B7-2 or GM-CSF consistently caused secondary proliferation. Tumors expressing B7-2 and GM-CSF or B7-2, GM-CSF, and IL12 consistently increased PBMC CD8+ (cytotoxic T) and CD16+ (natural killer) percentages. Interestingly, anti-tumor cytotoxicity only exceeded that of PBMC stimulated with wild type tumor alone when peripheral blood mononuclear cells were stimulated with both wild type tumor and B7-2/GM-CSF- (but not IL12) transduced cells.Conclusion:PBMC proliferation and phenotype is altered as expected by exposure to immunostimulatory gene-transduced tumor. However, transduced tumor cells alone do not stimulate greater anti-tumor cytotoxicity than wild type tumor. Only B7-2/GM-CSF-transduced cells combined with wild type produced increased cytotoxicity. This may reflect selection of tumor subclones with limited antigenic spectra during retrovirus-mediated gene transfer.

scholarly journals Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

2021 ◽  
Author(s):  
Bo Li ◽  
Chunmei Yang ◽  
Gui Ja ◽  
Yansheng Liu ◽  
Na Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Effective Utilization ◽  
Blood Mononuclear Cells ◽  
Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

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