eoe sub articles

EoE Sub Articles

1. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model
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	<li>Differentiation of Human Promyelocytic (HL60) Cells into Neutrophil-like Cells
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		<li>Adjust HL60 cell number to 3 x 105 cells/ml then add 1 &#181;M retinoic acid (RA) and 1.25% DMSO to induce their differentiation. Seed each 3 x 105&#160;HL60 cells per well in 48-well plate then incubate for 48 hr at 37 &#176;C with 5% CO2</...

determining the role of neutrophil-like cells in lymphoma cell sensitivity to a test drug

Source: Hirz, T., et al. <a href="https://app.jove.com/v/53846">Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents.</a> J. Vis. Exp. (2016).
This video demonstrates an assay investigating the role of neutrophil-like cells in safeguarding lymphoma cells against the cytotoxicity of chemotherapeutic agents. A 3D co-culture, consisting of lymphoma and neutrophil-like cells, is established and exposed to vincristine&#8212;a chemotherapy drug. Direct contact with the neutrophil-like cells induces an upregulation of anti-apoptotic proteins in the lymphoma cells, thereby promoting their survival in the presence of vincristine.

<img alt="Figure 1" src="/files/ftp_upload/22011/22011_Figure1.jpg" /> 
Figure 1.&#160;Structural Parameters of Differentiated HL60 Cells. (A)&#160;Forward-scatter (FSC) and side-scatter (SSC) plots represent HL60 and differentiated HL60 cells (HL60diff).&#160;(B)&#160;HL60 and HL60diff&#160;cells were labeled with anti-CD11b-AF700 or anti-CD38-APC followed by flow cytometry analysis. After gating on each cell popul...

Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

1. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model

	Differentiation of Human ...

Take a multi-well plate containing 3D spheroid monocultures of lymphoma cells, cancerous white blood cells, and co-cultures of lymphoma and neutrophil-like cells in a basement membrane matrix.
Add a test drug that binds to intracellular tubulin and disrupts microtubule polymerization, resulting in cellular apoptosis.
In the co-culture, direct lymphoma cell contact activates the neutrophil-like cells, inducing cytokine secretion.
These cytokines interact with lymphoma cell receptors, triggering downstream signaling cascades and upregulating anti-apoptotic protein expression, promoting cell survival.
Discard the media. Add a dissociation buffer to disrupt cellular adhesion.
Scrape the spheroids and incubate them under agitation. Transfer the suspension to tubes and continue agitation to form single-cell suspensions.
Introduce a fluorophore-conjugated antibody cocktail that labels lymphoma and neutrophil-like cells.
Add fluorophore-tagged annexin V and propidium iodide to label apoptotic and dead cells.
Using flow cytometry, detect increased lymphoma cell survival in the co-culture, indicating neutrophil-mediated protection against the drug.

determining-role-neutrophil-like-cells-lymphoma-cell-sensitivity-to

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

JoVE Journal

Cancer Research

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Immunology and Infection

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

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