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 adhesive interactions between tumor cells and frozen LNs.
Structurally, LNs are discrete sponge-like masses of dense and extensive networks of ECM fibers, frequently referred to as &#34;reticular fibers&#34;, which act as paths for cell migration and as conduits for rapid delivery of soluble factors (antigens and/or chemokines) within the LN parenchyma27. The preserved reticular fibers of the frozen LNs of the assay support haptotactic signals and provide scaffolds for tumor cell adhesion in vitro. These fibers are made up primarily of structural proteins, such as collagens I and III, and by secondary ECM elements, such as fibronectin, tenascin, laminin, vitronectin and heparan sulfate proteoglycans28,29. Following cell adhesion, most of these LN-derived ECM factors provide molecular cues that determine cell survival (proliferative or dormancy states) or cell death (anoikis) through integrin-mediated signals.
Here, we demonstrate the assay using xenogeneic rat LNs and a human breast tumor cell line. Alternatively, other sources of LNs could be used. The composition of rat, mouse or human LNs includes the same structural and functional proteins that are part of native mammalian ECM, all preserving similar binding sites that are necessary for cell adhesion23. Importantly, the only critical step is to use consecutive slices of the same LN per experiment to minimize regional variations in ECM composition of each LN section, which in turn could dictate the cell adhesion rate.
A drawback of the assay is that it does not recapitulate the first steps of lymphatic dissemination, only reflecting the adhesive strength of tumor cells to LNs. For example, seeding less aggressive breast tumor cells on LN sections, like the MCF-7 (Figure 2) or the T47D tumor cell lines24, lead to a strong adhesion to LN sections in vitro, at similar levels than the observed for the high aggressive MDA-MB-231 tumor cells (data not shown). However, it is well known that orthotopic MCF-7 xenograft tumors cannot reach sentinel LNs, while MDA-MB-231 tumors spontaneously metastasize to them30. Clearly, the main bottleneck for MCF-7 cells LN-metastasis formation occur in steps before they reach and adhere to LNs, like the inability of MCF-7 cells efficiently escape from the primary tumors. So, the strength of the assay described here is not establish direct correlations with LN-metastatic potential, but is a simple method to quantify the adhesive properties of a tumor cell in a more realistic ECM in vitro. By using frozen tissues, the cryosections represent the natural complexity of LNs in terms of structure and composition, which would be impossible to recreate using synthetic techniques, particularly those using purified ECM proteins.
Additional limitations of the method are (1) it does not allow the evaluation of the chemotactic potential of factors secreted by LNs and that (2) it does not inform on whether the cell-specific adhesion to LN sections is a result of preferential binding to ECM proteins, cells or any other structures present in LN sections. However, we felt that this approach could be relevant and must be seriously considered for particular applications, but were beyond the scope of this particular manuscript. For example, in a recent study, we identified the N-Myc downstream-regulated gene 4 (NDGR4) as a mechanistic biomarker of LN metastasis in breast tumors24. Mechanistically, tumor cells lacking NDRG4 expression increase adhesion to cryosections of LNs by favoring the assembly of &#946;1-integrin receptors at the leading edge of breast tumor cells. Furthermore, using additional controls, like dishes coated with purified ECM proteins, we uncovered that differential adhesion to LN sections is a result of selective association with vitronectin24.
Finally, it is worth noting that this method is not restricted to LNs sections and could be set-up to assess cell adhesion to different living organs, like cryosections of spleen or lungs. Metastatic cells exhibit organotropism and measurements of adhesive strength in frozen sections of different organs in vitro, could be a useful mean for predict organ-specific cancer dissemination.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 cm2</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 CO2 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&#225;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 &#34;OCT&#34; 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 Figure 2B, the morphology of adherent cells is rounded in shaped, and they are heterogeneously dispersed throughout the LN. The LN adhesive index is 2-fold higher in NDRG4-negative MCF-7 cells (877 &#177; 124 cells/mm2 of LN) compared to that in corresponding NDRG4-positive MCF-7 cells (412 &#177; 76 cells/mm2 of LN, p = 0.03) (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60531/60531fig1a.jpg" /> 
Figure 1: Stepwise procedure for the isolation of the rat mesenteric LNs. (A) Ventral midline skin incision: euthanized rats were placed in dorsal recumbency position and a 30-50 mm midline incision was made in the skin overlying the mid abdomen, exposing the abdominal viscera (liver, small intestine, cecum and bladder). (B) The small intestine was gently pulled out from abdominal cavity exposing rat mesenteric LNs embedded in visceral adipose tissue. (C) Gross anatomy of the dissected gastrointestinal tract after removal. (D) Dissected mesenteric lymph nodes from the connecting adipose tissue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60531/60531fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60531/60531fig2a.jpg" /> 
Figure 2: Representative results of tumor cell adhesion to rat lymph node sections. (A) Illustrative flow cytometry analysis showing the intensity of DiI(C18) labeling (upper quadrant) compared with nonlabelled cells (lower quadrant). (B) Light (left) and fluorescent (right) microscopy images of red-labeled MCF-7 cells adherent to LN sections after the washing step. (C) After adhesion assay, attached cells on coverslips are manually quantified by using a calibration scale to estimate the lymph node area and fluorescent microscopy to direct cell counting. (D) NDRG4 knockdown in MCF-7 breast tumor cells increases lymph node adhesion. Representative images of red fluorescent MCF-7 cells (NDRG4-positive or NDRG4-negative DiIC18-labelled cells) 30 min after seeding on 5 &#181;m rat lymph node sections. The LN adhesive index is expressed as the number of adherent tumor cells per LN covered area (cells/mm2). Scale bar = 200 &#181;m. *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60531/60531fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.7K Views.  Hospital Sírio-Libanês. 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.

Video: Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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 ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Summary

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Chapters in this video

Lung Tumor Cell Recruitment Assay

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

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

In Vivo EPR Assessment of pH, pO₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8⁺ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells