Name: quantification of tumor cell adhesion in lymph node cryosections
Uploaded: 2020-02-09T12:00:00
Description: Watch this Scientific Journal Video about Quantification of Tumor Cell Adhesion in Lymph Node Cryosections at JoVE.com

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.
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<ol>
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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.

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	<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 <strong clas...

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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 ...

The most frequent sites for metastasis are the original lymph nodes. Somatids can be used to detect the adhesive affinity between tumor cells'lymph node sections in vitro. One advantage of this technique is that fresh lymph node sections provide a natural complexity of the original tissue, which wouldn't be possible to recreate using purified proteins.This method could be useful for molecular oncology studies that seek to identify genes or therapies that interfere with tumor cells'adhesion at metastatic target organs, like lymph nodes. To harvest the lymph nodes within thirty minutes of sacrifice, place the adult Wistar rat in dorsal recumbency on a clean dissection board at room temperature and spray the fur with 70%isopropyl alcohol. Using sterilized instruments, lift the abdominal skin and make a medial skin incision to expose the abdominal viscera.Extract the intestine until the thoracic and abdominal lymph nodes become visible. Using blunt-tip scissors, carefully excise the lymph nodes without injuring the underlying superior mesenteric artery, and place the lymph node into a 15 mL tube containing 5 mL of sterile PBS. When all of the lymph nodes have been harvested, place one lymph node per cryomold face down on the base of each mold and add just enough optimal cutting temperature medium to cover the tissues.After snap freezing, use a cryostat to acquire 5 to 8 micrometer thick sections at 22 degrees Celsius and collect the sections onto glass microscope slides. Good quality cryosections from consecutive slices of the same lymph node are critical for minimizing differences between slices, and facilitating consistent cell adhesion rates. For tumor cell labeling, harvest the cells of interest from an appropriately staged cell culture and re-suspend the cells at a 1 x 10 to the 6 cells per mL concentration and serum-free medium.Transfer 1 mL of cells into a new 15 mL conical tube and label the cells with 2 micrograms per mL DiI(C18)for ten minutes at 37 degrees Celsius, with gentle agitation at five minutes to avoid cell sedimentation. At the end of the incubation, pellet the cells by centrifugation and wash the labeled cells two times with 10 mL of fresh serum-free medium to remove any excess dye. After the second wash, re-suspend the cells at a 1 x 10 to the 6 cells per mL of serum-free medium, supplemented with 0.1%bovine serum albumin concentration.Proceeding of the labeled tumor cells onto the harvested lymph nodes tissue sections, gently wash the sections two times in PBS, followed by re-hydration in fresh PBS for fifteen minutes at room temperature. At the end of the incubation, block any non-specific binding with 2.5%bovine serum albumin for thirty minutes at 37 degrees Celsius, before using a cotton swab to dry the slides. Use a hydrophobic pen to draw a barrier around each section and add 100 microliters of labeled cells onto each encircled lymph node.After one to two hours at 37 degrees Celsius in a humidified chamber, remove any non-adherence cells with four gentle washes in PBS and fix the remaining adherent fluorescence cells with 3.7%formaldehyde in PBS for fifteen minutes at room temperature. For quantification of the adhesive index, use the 10x objective on a fluorescence microscope to obtain individual TIFF images of each section in the bright and red fluorescent fields. Then, name and save the images systematically before opening the images in Fiji.After setting the scale, use the polygonal tool to select the region of interest to be quantified. Select to quantify lymph node area. The data will be expressed in square millimeters.Next, select Plugins, Analyze, Cell Counter and click the photo to be quantified. Click Initialize and select Counter Type. Then, click on the cells in the photo.The lymph node adhesion index will be expressed as the number of adherent tumor cells per lymph node covered area. To initialize the next photo, open the next photo and repeat the quantification as demonstrated. As observed, the morphology of the adherent cells is rounded in shape, and the cells are heterogeneously dispersed throughout the lymph node of interest.The lymph node adhesion index is two to three fold higher in NDRG4 negative breast cancer cell lines, compared to that measured for corresponding NDRG4 positive cells. This procedure can be adapted to assist adhesion rates in other metastatic target tissues, like brain or lungs, to evaluate the secondary preferential sites of adhesion for inseminated tumor cells. Remember that both fresh and frozen tissues should be considered biohazardous and should be handled using appropriate bio-safety precautions.

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