Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Summary

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.

Abstract

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.

Introduction

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 "seeds") acquire specific biological abilities that allow them to evade primary sites and establish malignant growth at distant sites (the "soil")¹. Recently, several novel concepts regarding the "seed and soil" relations have emerged, such as the induction of premetastatic niches (conceptualized as a "fertile soil" needed for "seeds" to thrive), self-seeding of primary tumors by DTCs, "seed" 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 neck^4,5, breast⁶, prostate⁷, lung⁸, gastric⁹, colorectal^10,11 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 "seeds" 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.

Protocol

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 Sírio-Libanês 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

1. 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.
2. Spray the rat carcass with 70% isopropyl alcohol and use sterilized instruments for LN harvesting.
3. 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).
4. 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.
5. Harvest LNs into 15 mL conical tubes containing 5 mL of sterile phosphate buffered saline (PBS).
6. Properly discard the rat carcasses.
7. 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.
8. 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.
9. 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 °C until sectioning.
10. Section the LN with a cryostat adjusting section thickness to 5-8 µm. Transfer crysections onto microscope slides.
    NOTE: Before sectioning, remove the frozen samples from the -80 °C freezer and allow them to equilibrate to the temperature in the cryostat microtome chamber at -22 °C for approximately 30 min. LN-containing slides can be stored at -80 °C for up to one month.

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 properties^25,26.

1. Dissociate cells growing under ideal conditions (i.e., in complete medium) and resuspend in serum-free medium at a density of 10⁶ cells/mL.
2. Add 1 mL of cell suspension (10⁶ cells) to a15 mL conical tube and label with Dil(C₁₈) (2 µg/mL) for 10 min at 37 °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).
3. Centrifuge the labeled suspension tubes at 300 x g for 4 min.
4. 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).

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.

1. Under sterile conditions, to prepare PLL- or BSA-coated wells, add 300 µL 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 °C.
2. 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.

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.

1. Gently wash the cryosections twice with PBS and rehydrate with PBS for 15 min at room temperature.
2. Block unspecific adhesion to cryosections with 2.5% BSA for 30 min at 37 °C. Use immunohistochemistry wash chambers and lamina cradles to ensure that the entire O.C.T was removed during washes and incubations.
3. 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.
4. For the tumor cell adhesion assay, add 100 µL 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 °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.
5. 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.

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's ImageJ/FIJI software).

1. 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.
2. 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).
3. 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.
4. 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).
5. Click on Global (this calibration applies to all images opened in this ImageJ/FIJI session) and press OK.
6. 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.
7. 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).

Results

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

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL Conical Tubes	Corning	352096
2-propanol	Merck	109634
Benchtop Laminar Flow	Esco Cell Culture
Bin for Disc	Leica	14020139126
Bovine Serum Albumin	Sigma-Aldrich	A9647-100
Cell culture flask T-25 cm2	Corning	430372
Cryostat	Leica	CM1860 UV
Cryostat-Brush with magnet	Leica	14018340426
DiIC18 Cell Traker Dye	Molecular Probes	V-22885
Fetal Bovine Serum (FBS)	Life Technologies	12657-029
Fluorescence microscope	Nikon Eclipse 80
Forma Series II CO2 incubator	Thermo Scientific
Formaldehyde	Sigma-Aldrich	252549
High Profile Disposable Razor	Leica	14035838926
Incubation Cube (IHC)	KASVI	K560030
Inverted microscope	Olympus	CKX31
Isofluran 100 mL	Cristália
Liquid Bloquer Super Pap Pen	Abcam, Life Science Reagents	ab2601
Optimal Cutting Temperature "OCT" compound	Sakura	4583
Phosphate-buffered Saline (PBS)	Life Technologies	70011-044
Poly-L-lysine	Sigma-Aldrich	P8920
RPMI	Gibco	31800-022
Serological Pipettes 1 mL	Jet Biofil	GSP010001
Serological Pipettes 10 mL	Jet Biofil	GSP010010
Serological Pipettes 2 mL	Jet Biofil	GSP010002
Serological Pipettes 5 mL	Jet Biofil	GSP010005
Serological Pipettes 50 mL	Jet Biofil	GSP010050
Serological Pipettor Easypet 3	Eppendorf
Tissue-Tek cryomold	Sakura	4557
Trypan Blue 0.4%	Invitrogen	T10282
Trypsin	Instituto Adolfo Lutz	ATV