We thank Dr. Rosana De Lima Pagano and Ana Carolina Pinheiro Campos for technical assistance. This work was supported by grants from: FAPESP - S&#227;o Paulo Research Foundation (2016/07463-4) and Ludwig Institute for Cancer Research (LICR).

LNs were recovered from fresh carcasses of healthy adult Wistar rats sacrificed by cervical dislocation. We followed the NIH Guidelines for Pain and Distress in Laboratory Animals and all procedures were approved by the Ethics Committee and Animal Research of the Research and Education Institute of the S&#237;rio-Liban&#234;s hospital (CEUA P 2016-04).
NOTE: All fresh frozen tissues are considered biohazardous and should be handled using appropriate biosafety precautions.
1. Lymphadenectomy and Cryosectioning
<ol>
	<li>Place fresh carcasses of adult Wistar rats (180-220 g) lying in dorsal recumbency on a clean dissection board at room temperature. 
	NOTE: LNs must be collected up to 30 min post euthanasia.</li>
	<li>Spray the rat carcass with 70% isopropyl alcohol and use sterilized instruments for LN harvesting.</li>
	<li>Lift the abdominal skin with the aid of tweezers and open a cavity with a medial incision without damaging the underlying tissue, exposing the abdominal viscera. Pull out the intestine and the thoracic and abdominal LNs become visible (Figure 1).</li>
	<li>Carefully excise the LNs from each rat with the use of blunt tip scissors to avoid injuring the superior mesenteric artery lying behind. 
	NOTE: Depending on the location of the resected lymph node, it is necessary to clean other tissues adhered to it, such as mesenteric tissue.</li>
	<li>Harvest LNs into 15 mL conical tubes containing 5 mL of sterile phosphate buffered saline (PBS).</li>
	<li>Properly discard the rat carcasses.</li>
	<li>Remove fresh LNs from the PBS, roll and dry the node on a dry filter paper. Place it in a small Petri dish and add embedding solution for frozen tissue specimens (O.C.T.) for 2 min.</li>
	<li>Transfer and orientate the LN face down in a desired position in the base of a cryomold, with just enough O.C.T to cover it. Avoid bubbles near the tissue. The sectioning surface is the bottom of the cryomold.</li>
	<li>Immediately snap-freeze the cryomold in a Styrofoam cooler with dry ice. When there is still a small part of unfrozen O.C.T. (~20-35 s), transfer the sample to aluminum foil and place it in a cooler with dry ice while continuing to freeze other samples. At the end, store all samples at -80 &#176;C until sectioning.</li>
	<li>Section the LN with a cryostat adjusting section thickness to 5-8 &#181;m. Transfer crysections onto microscope slides. 
	NOTE: Before sectioning, remove the frozen samples from the -80 &#176;C freezer and allow them to equilibrate to the temperature in the cryostat microtome chamber at -22 &#176;C for approximately 30 min. LN-containing slides can be stored at -80 &#176;C for up to one month.</li>
</ol>
2. Cellular Labeling with Fluorescent Dyes
NOTE: Fluorescent dyes are widely used in cell biology. We prefer to use the long-chain dialkylcarbocyanines labeling (DiI(C18), excitation 549 nm, Emission 565 nm) because they are bright, stable and can be added directly to culture media, does not affecting cell viability or cell adhesive properties25,26.
<ol>
	<li>Dissociate cells growing under ideal conditions (i.e., in complete medium) and resuspend in serum-free medium at a density of 106 cells/mL.</li>
	<li>Add 1 mL of cell suspension (106 cells) to a15 mL conical tube and label with Dil(C18) (2 &#181;g/mL) for 10 min at 37 &#176;C. 
	NOTE: After 5 min, gently agitate the tubes to avoid cell sedimentation and understaining of sedimented cells. Larger densities require longer incubation times for uniform staining. An optimal incubation time for cell staining varies with cell line. It can be better quantified using the conventional FL2 flow cytometry detection channel (Figure 2A).</li>
	<li>Centrifuge the labeled suspension tubes at 300 x g for 4 min.</li>
	<li>Remove the supernatant and wash twice in 10 mL of serum-free medium. Recover the cells as red pellets. Resuspend the cells at 106 cells/mL in serum-free medium with 0.1% bovine serum albumin (BSA).</li>
</ol>
3. Precoating Dishes with Poly-L-lysine Solution or BSA as a Seeding Control (Optional)
NOTE: We used cell culture dishes precoated with PLL as positive loading-control surfaces to ensure that different experimental groups of tumor cells were seeded at the same number, as well as BSA-coated surfaces as negative controls.
<ol>
	<li>Under sterile conditions, to prepare PLL- or BSA-coated wells, add 300 &#181;L of PLL (0.1% w/v in H2O) or BSA (diluted at 2.5% w/v in H2O) directly to the 24-well plate and incubate overnight at 4 &#176;C.</li>
	<li>Remove solution by aspiration, gently rinse the surface with sterile PBS and air-dry the plate at room temperature in the tissue-culture hood before cell seeding. 
	NOTE: The final volumes of PLL or BSA must be adjusted according to the area of different well plates.</li>
</ol>
4. Seeding Fluorescent-labeled Tumor Cells on LN Cryosections or PLL/BSA-coated Wells
NOTE: As experimental controls, we used (1) cell culture dishes precoated with PLL or BSA and (2) consecutive sections of the same LN per experiment (see this detail in Figure 2D), where the latter will minimize regional variations in extracellular matrix (ECM) composition of each LN section, which in turn can dictate the cell adhesion rate. For the following tumor cell adhesion assay, select high quality and sequential LN cryosections.
<ol>
	<li>Gently wash the cryosections twice with PBS and rehydrate with PBS for 15 min at room temperature.</li>
	<li>Block unspecific adhesion to cryosections with 2.5% BSA for 30 min at 37 &#176;C. Use immunohistochemistry wash chambers and lamina cradles to ensure that the entire O.C.T was removed during washes and incubations.</li>
	<li>Drain the excess BSA on a dry paper towel, dry the outline of LN sections with a cotton swab and encircle the sections using a PAP pen.</li>
	<li>For the tumor cell adhesion assay, add 100 &#181;L of cell suspension (from step 2.4) to each encircled LN section or well in the 24-well PLL-coated plates and place it in a humidified chamber rack for 1-2 h at 37 &#176;C in the conventional cell culture incubator. 
	NOTE: The final volume of cell suspension needs be adjusted according to the area of different encircled LNs.</li>
	<li>Gently wash off non-adherent cells four times with PBS. Fix the remaining adherent fluorescent cells with 3.7% formaldehyde in PBS for 15 min at room temperature.</li>
</ol>
5. Manual Quantification of the Adhesive Index
NOTE: The adhesive index (i.e., tumor cells/LN mm2) was achieved using a 10X objective and manually counting the number of tumor cells, readily identified by light microscopy and confirmed by a fluorescence microscopy (Figure 2D), per lymph node areas of several independent fields (obtained using National Institute of Health&#39;s ImageJ/FIJI software).
<ol>
	<li>Use a fluorescent microscope with a 10x objective to take separate TIFF images in two channels corresponding to the bright and red-fluorescent fields (Figure 2D). Name and save these images systematically.</li>
	<li>Start ImageJ/FIJI, open the images and set the scale. It is necessary to use a calibration scale (e.g., a micrometric ruler 1 mm) (Figure 2C).</li>
	<li>Open the photo of the micrometric ruler (or a stage micrometer), select the Straight line tool and draw a straight line that defines a known distance.</li>
	<li>In the Analyze menu, select Set Scale. The Distance in pixels will be filled based on the length (in pixels) of the line drawn in step 5.3. The Known distance will be filled with the real distance (in this case, in millimeters) and the unit of length in the Unit of length field (in this case, in millimeters).</li>
	<li>Click on Global (this calibration applies to all images opened in this ImageJ/FIJI session) and press OK.</li>
	<li>Lymph node area quantification: Select the Wand tool and by double clicking, open the Wand tool settings. Set mode to 8-connected. Click in the photo and set the tolerance until select all lymph nodes in the photo and press OK. To measure the area, open Analyze | Measure (CTRL + M). The area is expressed in the units set earlier.</li>
	<li>Tumor cell quantification: Open the light microscopy/fluorescence images in FIJI software. Select Plugins | Analyze | Cell Counter | Cell Counter. Click on the photo to be quantified and press Initialize button in the cell counter window. Select counter type (1-8) and click on the cells in the photo. To initialize the next photo, press the Reset button in the cell counter window, open the new photo and repeat all steps. 
	NOTE: LN adhesion index is expressed as the number of adherent tumor cells per LN covered area (cells/mm2).</li>
</ol>

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The authors have nothing to disclose.

Lymphatic system dissemination of cancer cells requires a variety of complex cell-driven events. They initiate with cell detachment from primary tumor and the remodeling of the extracellular matrix (ECM) architecture, and are supported by persistent chemotaxis and active migration through the afferent lymphatics en route to the sentinel LNs. If cancer cells adhere and survive in LNs, they can easily spread to other secondary organs. Here we describe an easy method for rapid and low-cost functional analysis of specific ad...

Cancer metastasis is the main reason for treatment failure and the dominant life-threatening aspect of cancer. As postulated 130 years ago, the metastatic spread results when an elite of disseminated tumor cells (DTCs, the &#34;seeds&#34;) acquire specific biological abilities that allow them to evade primary sites and establish malignant growth at distant sites (the &#34;soil&#34;)1. Recently, several novel concepts regarding the &#34;seed and soil&#34; relations have emerged, such as the induction of premetastatic niches (conceptualized as a &#34;fertile soil&#34; needed for &#34;seeds&#34; to thrive), self-seeding of primary tumors by DTCs, &#34;seed&#34; dormancy at secondary organs and the parallel progression model of metastasis2.
For most solid malignancies, DTCs can reside and be detected in many mesenchymal organs, such as bone marrow and lymph nodes (LNs) in patients with or without evidence of clinical metastasis. Because tumor-draining LNs are the first location of the regional spread of DTCs, LN status is an important prognostic indicator and is often associated with adjuvant therapy decisions3. For some tumor types, the correlation between LN status and worse outcomes is strong, including head and neck4,5, breast6, prostate7, lung8, gastric9, colorectal10,11 and thyroid cancers12.
LNs are small ovoid organs of the lymphatic system, that are covered with reticular cells and enclosed with lymphatic vessels. These organs are absolutely necessary for the functioning of the immune system13. LNs act as attractant platforms for immune circulating cells, bringing the lymphocytes and antigen-presenting cells together14. However, LNs also attract circulating tumor cells. Over decades, LNs were pictured as passive routes of transportation for metastatic tumor cells. However, recent studies have indicated that tumor cells may also be guided towards LNs by chemotactic (chemokines) and/or haptotactic (extracellular matrix elements) cues secreted by the lymphatic endothelium15. As examples, overexpression of the CCR7 receptor in tumor cells facilitates the guidance of metastatic melanoma cells towards tumor-draining LNs16. In addition, extracellular LN proteins provide an adhesive scaffold for the recruitment and survival of circulating tumor cells17. In fact, tumor-draining LNs provide fertile soil for the seeding of DTCs, which can be maintained in proliferative or dormant states by specific LN microenvironmental signals18. The final fate of these LN-residing DTCs is controversial; some works suggest that these cells are passive indicators of metastatic progression19, while others propose that they are more likely founders of resistance (by self-seeding primary sites) and/or act as cellular reservoirs for metastases (spreading &#34;seeds&#34; for tertiary cancer growth)20,21. Recently, using preclinical models, it has been demonstrated that a fraction of these LN-residing DTCs actively invaded blood vessels, entered into the blood circulation and colonized the lungs21.
Considering that the presence of cancer cells in LNs is a marker for cancer aggressiveness and invasiveness, in this study, we optimized a classic method developed by Brodt22 to quantitatively measure tumor cell adhesion to LNs in vitro. The use of a fluorescence-based assay allowed us to develop a low-cost, rapid, sensitive and environmentally friendly (nonradioactive) protocol for the detection of adhesive alterations between tumor cells and LN cryosections. Using the MCF-7 breast cancer cells expressing different levels of NDRG4 gene expression and rat LN frozen sections to exemplify the method, we showed that this protocol allowed a good correlation between tumor cell adhesion to LNs in vitro and LN metastasis observed in breast cancer patients24.

<table><tbody><tr><td>15 mL Conical Tubes</td><td>Corning</td><td>352096</td><td></td></tr><tr><td>2-propanol</td><td>Merck</td><td>109634</td><td></td></tr><tr><td>Benchtop Laminar Flow</td><td>Esco Cell Culture</td><td></td><td></td></tr><tr><td>Bin for Disc</td><td>Leica</td><td>14020139126</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9647-100</td><td></td></tr><tr><td>Cell culture flask T-25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Corning</td><td>430372</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1860 UV</td><td></td></tr><tr><td>Cryostat-Brush with magnet</td><td>Leica</td><td>14018340426</td><td></td></tr><tr><td>DiIC18 Cell Traker Dye</td><td>Molecular Probes</td><td>V-22885</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>12657-029</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon Eclipse 80</td><td></td><td></td></tr><tr><td>Forma Series II CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td></td></tr><tr><td>High Profile Disposable Razor</td><td>Leica</td><td>14035838926</td><td></td></tr><tr><td>Incubation Cube (IHC)</td><td>KASVI</td><td>K560030</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>CKX31</td><td></td></tr><tr><td>Isofluran 100 mL</td><td>Crist&amp;aacute;lia</td><td></td><td></td></tr><tr><td>Liquid Bloquer Super Pap Pen</td><td>Abcam, Life Science Reagents</td><td>ab2601</td><td></td></tr><tr><td>Optimal Cutting Temperature &quot;OCT&quot; compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>31800-022</td><td></td></tr><tr><td>Serological Pipettes 1 mL</td><td>Jet Biofil</td><td>GSP010001</td><td></td></tr><tr><td>Serological Pipettes 10 mL</td><td>Jet Biofil</td><td>GSP010010</td><td></td></tr><tr><td>Serological Pipettes 2 mL</td><td>Jet Biofil</td><td>GSP010002</td><td></td></tr><tr><td>Serological Pipettes 5 mL</td><td>Jet Biofil</td><td>GSP010005</td><td></td></tr><tr><td>Serological Pipettes 50 mL</td><td>Jet Biofil</td><td>GSP010050</td><td></td></tr><tr><td>Serological Pipettor Easypet 3</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Tissue-Tek cryomold</td><td>Sakura</td><td>4557</td><td></td></tr><tr><td>Trypan Blue 0.4%</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>Trypsin</td><td>Instituto Adolfo Lutz</td><td>ATV</td><td></td></tr></tbody></table>

quantification of tumor cell adhesion in lymph node cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

We illustrate the assay by evaluating the LN adhesive potential of red fluorescent MCF-7 breast cancer cells expressing different levels of the NDRG4 gene (referred to as NDRG4-positive and NDRG4-negative cells), a negative modulator of beta1-integrin clustering at the cell surface24, by examining the fractions of rat LN-adherent tumor cells. Examples of the raw images of this protocol are shown in Figure 2. As observed in <strong clas...

Watch this Scientific Journal Video about Quantification of Tumor Cell Adhesion in Lymph Node Cryosections at JoVE.com

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

quantification-of-tumor-cell-adhesion-in-lymph-node-cryosections

Research

JoVE Journal

Cancer Research

11.9K Views.  Hospital Sírio-Libanês. De meest voorkomende sites voor metastase zijn de oorspronkelijke lymfeklieren. Somatids kunnen worden gebruikt om de kleefaffiniteit tussen de lymfekliersecties van tumorcellen in vitro te detecteren. Een voordeel van deze techniek is dat verse lymfekliersecties een natuurlijke complexiteit van het oorspronkelijke weefsel bieden, wat niet mogelijk zou zijn om te recreëren met behulp van gezuiverde eiwitten.Deze methode kan nuttig zijn voor moleculaire oncologiestudies die genen of th...

Video: Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Immunology and Infection

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Biology

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Source: Ali, M. Y et al., <a href="https://www.jove.com/v/52532"> Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J. Vis. Exp. (2015)
This video describes the technique of studying the cytoskeleton and focal adhesions in primary human colon cancer cells to understand its variations depending on substrate rigidity. This culture of cancer cells on soft and hard substrates allows various downstream biophysical measurements for cancer prognosis.

Source: Ali, M. Y et al.,  Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

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