Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Summary

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Abstract

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Introduction

Despite being a highly complex and inefficient process¹, metastasis is a significant contributor to the morbidity and mortality of cancer patients². In fact, most cancer-related deaths are attributed to metastatic spread of disease^3,4. In order for tumor cells to successfully metastasize, they must detach from the primary site, invade through adjoining stroma, intravasate into blood circulation or lymphatics, travel to the capillary bed of a secondary site, extravasate into the secondary tissue, and proliferate or grow to form metastatic lesions⁵. The use of mouse models has been critical to furthering the understanding of the molecular mechanisms responsible for metastatic seeding and growth^6,7. Herein, we focus on breast cancer metastasis, for which both genetically modified mouse models as well as methods of transplantation are often used – each with their own set of advantages and limitations.

Genetically engineered mammary tumor models make use of mammary gland specific promoters, including MMTV-LTR (mouse mammary tumor virus long terminal repeat) and WAP (Whey Acidic Protein), to drive expression of transgenes in the mammary epithelium⁸. Oncogenes including polyoma middle T antigen (PyMT), ErbB2/Neu, c-Myc, Wnt-1, and simian virus 40 (SV40) have been expressed in this manner^{9,10,11,12,13}, and while these genetic models are useful for studying primary tumor initiation and progression, few readily metastasize to distant organs. Furthermore, these genetic mouse models are often more time and cost prohibitive than spontaneous or experimental metastasis models. Given the limitation of most genetically engineered mammary tumor models to study metastasis, transplantation techniques have become attractive methods to study this complex process. This includes orthotopic, tail-vein, intracardiac, and intracranial injection of suitable cell lines.

Although several breast cancer cell lines readily metastasize following orthotopic injection into the mammary fat pad^14,15, the consistency and reproducibility of metastatic tumor burden can be a challenge, and the duration of such studies can be on the order of several months. For evaluating lung metastasis, in particular, intravenous injection into the tail-vein is often a more reproducible and time-effective method with metastatic spread typically occurring within the span of a few weeks. However, since the intravenous injection model bypasses initial steps of the metastatic cascade, care must be taken in interpreting the results of these studies. In this demonstration, we show tail-vein injection of mammary tumor cells along with an accurate and comprehensive method of analysis.

Even though the research community has made significant progress in understanding the complex process of breast cancer metastasis, it is estimated that over 150,000 women currently have metastatic breast cancer¹⁶. Of those with stage IV breast cancer, >36% of patients have lung metastasis¹⁷; however, the site-specific pattern and incidence of metastases can vary based on molecular subtype^18,19,20,21. Patients with breast cancer-associated lung metastases have a median survival of only 21 months highlighting the need to identify effective treatments and novel biomarkers for this disease¹⁷. The use of experimental metastasis models, including the intravenous injection of tumor cells, will continue to advance our knowledge of this important clinical challenge. When combined with digital imaging pathology and the method of metastatic lung tumor burden analysis described within this protocol, tail-vein injections are a valuable tool for breast cancer metastasis research.

Protocol

Animal use followed University Laboratory Animal Resources (ULAR) regulations under the OSU Institutional Animal Care and Use Committee (IACUC)–approved protocol 2007A0120-R4 (PI: Dr. Gina Sizemore).

1. Tail-vein injection of breast cancer cells

1. Preparation of cells and syringe for injection
   1. Plate an appropriate number of cells based on the number of mice and cell concentration to be used.
      NOTE: The number of cells injected and time to the development of metastases will depend on the cell line used and will need to be optimized. In this demonstration, 1 x 10⁶ MDA-MB-231 cells are injected intravenously into NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice, and macroscopic lung lesions are observed no later than 24 days post-injection. For the MVT1 murine mammary tumor cell line¹⁷, 3 x 10⁶ cells are injected into immune-competent FVB/N mice with numerous lung metastases observed by 14 days^22,23.
   2. Aspirate media and rinse cell plates with 1x PBS. Trypsinize cells in minimal volume, add appropriate volume of media, and count cells using a hematocytometer or another preferred method. Trypan blue (0.4%) or other live/dead cell dyes can be used to determine viable cell counts.
   3. Pellet cells by spinning at 180 x g for 5 min.
   4. Resuspend appropriate number of cells in sterile 1x PBS such that a volume of 100 µL is injected per mouse. Keep cell suspension on ice to maintain viability.
   5. Prior to injection, thoroughly resuspend cells with a 200 µL or 1 mL pipette to avoid clumping. Draw up 100 µL in a 28 G insulin syringe (see Table of Materials).
   6. Eliminate any air bubbles by keeping the syringe vertical, tapping on the syringe, and slowly adjusting the plunger. Injection of air bubbles into the vein is likely to cause an air/gas embolism that can be fatal.
2. Lateral tail-vein injection
   NOTE: For experimental breast cancer metastasis assays, injections are performed on > 6 weeks old female mice.
   1. Handle the mouse by the tail and slide animal into a slotted tube/restraint device of an appropriate size (see the Table of Materials for restraint device used).
   2. Insert the plug portion of the restraint device and position the mouse on its side such that its lateral tail vein is easy to view. The mouse has a ventral artery in line with the genitalia, a dorsal vein, and two lateral caudal veins.
   3. Clean the surface of the mouse’s tail with an aseptic wipe. Grasp the tail between index finger and thumb with non-dominant hand and apply slight tension.
   4. Beginning at the distal portion of the tail, insert the needle parallel to the vein with the bevel end up.
   5. If allowed, carefully recap the needle and bend to a 20-30° angle. A single-handed approach or needle recapping device is highly recommended.
      NOTE: It is not necessary to aspirate as this may cause the vein to collapse. However, a small flash of blood may be seen when first placed. The needle will advance smoothly into the vein with proper placement.
   6. Slowly dispense the complete volume into the vein. There should not be resistance when the plunger is pushed.
   7. If any resistance is felt, promptly remove the syringe needle. If needed, re-insert the needle (ideally no more than 3 attempts) moving toward the proximal end of the tail or opposing lateral vein.
   8. A small volume of blood will likely be displaced after injection. Apply gentle pressure with sterile gauze and wipe with aseptic wipe.
   9. Promptly dispose of syringe in appropriate sharps container.
   10. Return the mouse to a clean, ventilated cage and monitor for signs of distress.
   11. Monitor mice 2-3x/weekly for signs of metastasis formation (labored breathing, hunched posture, weight loss) and general distress. The time to development of metastasis will depend on cell line and mouse strain.
   12. If using an in vivo live animal imaging device, image mice immediately after tail-vein injection to confirm successful injection of cells and obtain time “zero” data (specific details on in vivo bioluminescence imaging are not included herein, but are described by Yang et al.²⁴).

2. Lung tissue fixation and analysis of metastatic lung tumor burden

1. Lung tissue inflation to maintain the structural format of the lungs for histopathology
   1. After approved euthanasia procedures (e.g. carbon dioxide at 30 - 70% displacement of the chamber volume/min) are followed, secure the mouse carcass to a dissecting board with pins. Either spray or apply 70% ethanol to keep the mouse’s fur out of the way during dissection. 
      NOTE: Carbon dioxide asphyxiation can cause pulmonary hemorhhage as an expected background lesion, especially at slower flow rates.
   2. Open the thorax with a midline incision, extend the incision cranially/caudally through the peritoneum, and cut away the diaphragm by grasping the xyphoid process.
   3. Using a separate set of scissors to not dull the blades, cut the ribs along each side of the sternum and carefully remove rib cage leaving room for the lungs to expand.
   4. Isolate the trachea by removing submandibular salivary glands and infrahyoid musculature. Placing pins on either side of the trachea can prevent unwanted movement during needle insertion.
   5. Fill a 26 G syringe with 2-3 mL of 10% neutral buffered formalin and insert into the trachea.
   6. Slowly inject formalin and watch for the lungs to expand (usually requires ~1.5 mL of formalin).
   7. Once formalin begins leaking out of the lungs (avoid over inflation), pinch off the trachea with a pair of forceps, remove syringe needle and detach the entire respiratory apparatus. Place lungs, heart, etc. directly into formalin as additional trimming of tissue can be done post-fixation.
   8. Complete processing, embedding, sectioning of tissue, and hematoxylin and eosin (H&E) staining using standard methods.
2. Analysis of metastatic lung tumor burden
   1. Scan H&E-stained lung sections on a high-resolution, slide scanner at 40x magnification (Figure 3).
   2. Import images into image analysis software (e.g., Visiopharm Image Analysis) for quantification of lung metastases.
      NOTE: We recommend that new users either obtain onsite or online training to use the image analysis software. Numerous webinars are also available through the commercial webpage.
   3. Select the Visiopharm 10118 H&E Lung Metastasis App from the software’s app library.
      NOTE: The purpose of this app is to label and quantify lung metastasis on H&E-stained slides. As part of the 10118 H&E Lung Metastasis App, the first image processing step segments the lung tissue with the Tissue Detection App. The second image processing step uses the Metastasis Detect App which identifies the metastases inside the lung tissue. Metastases are identified via shape together with regions that are either too misshaped, too red or too sparse for being identified as metastases.
   4. Adjust the parameters defining shape and sparseness to best fit representative images. Segmented areas of tumor metastases and normal lung tissue can be displayed using different color labels for each tissue type.
      NOTE: In the event that the app cannot accurately separate metastases from normal lung tissue, a custom app using the Visiopharm Decision Forest program may need to be written as was done for the experiments (see Figure 2 and Figure 3). Details for writing a custom algorithm follow below. Otherwise, proceed to step 2.2.9.
   5. Open the Decision Forest program, which works by training multiple Classes [i.e., lung tissue (non-neoplastic), metastases, red blood cells, epithelium, and/or white space] on a desired image. In Figure 2, tumor metastases are blue, normal tissue is green, and bronchiolar epithelium is yellow. Also, red blood cells are in red and air spaces in pink.
   6. Follow the prompted series of yes or no questions to appropriately train each Class for an image. The accuracy of the algorithm will determine the number of yes/no questions. For the analysis, the custom algorithm/app was written with accuracy set to 50 (range 0-100).
   7. Adjust Features for each class by applying filters to sharpen, blur, sort by shape, etc. to enhance the accuracy of the algorithm/App. Visiopharm views each Class through one or multiple lenses known as Features. Features change how the Class sees the image to bring out certain colors or intensities.
      NOTE: For the custom algorithm, metastases measuring 8500 µm² and above are labeled and measured as metastases. This accounts for size variance and metastases too small to detect. Small misshaped areas and small metastatic areas under 8500 µm² were included in the normal tissue quantification.
   8. Save the modified settings from either the app or custom algorithm and then, apply the algorithm/app to an entire set or series of H&E-stained tissues.
   9. Finally, export all output variables, which includes those listed in Table 1. Area in microns squared (µm²) can be quantified for each tissue type and percentages are derived from specimen total net tissue area (i.e., total tissue minus air space).
   10. When creating a custom algorithm, review tissue markups in consultation with a veterinary pathologist board-certified by the American College of Veterinary Pathologists to ensure accurate measurements and differentiate between tissue types.

Results

If using unlabeled cells for tail-vein injection, it may be difficult to confirm lung colonization until (1) the time of necropsy if macrometastases can be observed or (2) following histological analysis if microscopic metastases exist. With extensive metastatic lung tumor burden, mice will have labored breathing. As with any tumor study, mice should be carefully monitored throughout the study duration. The use of labeled cells is an easy way to confirm successful tail-vein injection; hence the use of luciferase-tagged M...

Discussion

As researchers continue to use intravenous injection of tumor cells as an experimental model for metastasis, standard practices to analyze the resulting metastatic tumor burden are lacking. In some cases, significant differences in metastatic tumor burden upon manipulation of particular cell lines and/or use of chemical compounds can be observed macroscopically. However, in other instances, subtle differences in metastatic seeding and growth may be overlooked or misinterpreted without thorough pathological analysis. This...

Materials

Name	Company	Catalog Number	Comments
alcohol prep pads	Fisher Scientific	22-363-750	for cleaning tail prior to injection
dissection scissors	Fisher Scientific	08-951-5	for mouse dissection and lung tissue inflation
DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate	Fisher Scientific	MT10013CV	cell culture media base for MDA-MB-231 and MVT1 cell lines
Dulbecco's Phosphate-Buffered Salt Solution 1x	Fisher Scientific	MT21030CV	used for resuspending tumor cells for injection
ethanol (70 % solution)	OSU		used to minimize mouse's fur during dissection; use caution - flammable
Evan's blue dye	Millipore Sigma	E2129	used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent
Fetal Bovine Serum	Millipore Sigma	F4135	cell culture media additive; used at 10% in DMEM
forceps	Fisher Scientific	10-270	for dissection and lung tissue inflation
FVB/NJ mice	The Jackson Laboratory	001800	syngeneic mouse strain for MVT1 cells
hemacytometer (Bright-Line)	Millipore Sigma	Z359629	for use in cell culture to obtain cell counts
insulin syringe (28 G)	Fisher Scientific	14-829-1B	for tail-vein injections (BD 329424)
MDA-MB-231 cells	ATCC		human breast cancer cell line
MVT1 cells			mouse mammary tumor cells
needles (26 G)	Fisher Scientific	14-826-15	used to inflate the mouse's lungs
neutral buffered formalin (10%)	Fisher Scientific	245685	used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice	The Jackson Laboratory	005557	maintained by OSUCCC Target Validation Shared Resource
Penicillin Streptomycin 100x	ThermoFisher	15140163	cell culture media additive
sterile gauze	Fisher Scientific	NC9379092	for applying pressue to mouse's tail if bleeding occurs
syringe (5 mL)	Fisher Scientific	14-955-458	used to inflate mouse lung tissue
tail-vein restrainer	Braintree Scientific, Inc.	TV-150 STD	used to restrain mouse for tail-vein injections
Trypan blue (0.4 %)	ThermoFisher	15250061	used in cell culture to assess viability
Trypsin-EDTA 0.25 %	ThermoFisher	25200-114	used in cell culture to detach tumor cells from plate