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 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 "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.
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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
- 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. - Spray the rat carcass with 70% isopropyl alcohol and use sterilized instruments for LN harvesting.
- 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).
- 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. - Harvest LNs into 15 mL conical tubes containing 5 mL of sterile phosphate buffered saline (PBS).
- Properly discard the rat carcasses.
- 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.
- 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.
- 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.
- 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 properties25,26.
- Dissociate cells growing under ideal conditions (i.e., in complete medium) and resuspend in serum-free medium at a density of 106 cells/mL.
- 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). - Centrifuge the labeled suspension tubes at 300 x g for 4 min.
- 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.
- 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.
- 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.
- Gently wash the cryosections twice with PBS and rehydrate with PBS for 15 min at room temperature.
- 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.
- 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.
- 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. - 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).
- 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.
- 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).
- 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.
- 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).
- Click on Global (this calibration applies to all images opened in this ImageJ/FIJI session) and press OK.
- 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.
- 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).
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Representative 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 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 ± 124 cells/mm2 of LN) compared to that in corresponding NDRG4-positive MCF-7 cells (412 ± 76 cells/mm2 of LN, p = 0.03) (Figure 2D).
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. Please click here to view a larger version of this figure.
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 µm 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 µm. *p < 0.05. Please click here to view a larger version of this figure.
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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 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 "reticular fibers", 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 β1-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.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
We thank Dr. Rosana De Lima Pagano and Ana Carolina Pinheiro Campos for technical assistance. This work was supported by grants from: FAPESP - São Paulo Research Foundation (2016/07463-4) and Ludwig Institute for Cancer Research (LICR).
Materials
| 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 |
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