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Claire M. Wells and Maddy Parsons (eds.), Cell Migration: Developmental Methods and Protocols, Methods in Molecular Biology, vol. 769, DOI 10.1007/978-1-61779-207-6_21, © Springer Science+Business Media, LLC 2011

Chapter 21

Simple Experimental and Spontaneous Metastasis Assays in Mice

Gary M. Box and Suzanne A. Eccles

Abstract

Many steps of the metastatic cascade can be reproduced in simple in vitro assays such as tumour cell interactions with matrix proteins, proteolysis, chemotaxis, haptotaxis, and invasion into matrices or explanted tissues. Nevertheless, there are no fully adequate substitutes for the complexity of the in vivo process. Here, we describe two “experimental” metastasis assays to yield lung or liver colonies (mimick-ing established micrometastatic disease), and two spontaneous metastasis assays for breast and prostate carcinomas. Examples include either murine tumour cell lines in syngeneic immunocompetent mice or human tumour xenografts in immunodeprived mice.

Key words: Lung metastasis, Liver metastasis, Lymphatic metastasis, Mouse, Organ colonisation, Breast cancer, Prostate cancer, Orthotopic

Metastasis is the major cause of treatment failure for most solid cancers and a greater understanding of the underlying molecular mechanisms is needed if we are to develop effective new treat-ments (1–3). Simple in vivo assays can mimic the late stages of metastasis (tumour cell circulation, lodgement, and organ coloni-sation) and may be appropriate for evaluation of therapies designed to target established disseminated disease.

These so-called experimental metastasis assays involve the introduction of tissue cultured tumour cells into the peripheral circulation. Most tumour cells (except those of lymphoreticular origin) arrest in the first capillary bed encountered, hence any superficial vein can be used to deliver cells to the lung with high

1. Introduction

312 G.M. Box and S.A. Eccles

efficiency based on the venous return to the lungs for oxygenation. For convenience, the lateral tail veins of the mouse are generally used. Similarly, liver metastases can selectively be generated by introducing appropriate tumour cell types (e.g. colon or pancre-atic carcinomas) into the hepatic portal circulation. This can be achieved by injection into a mesenteric vein, but an easier method (described here) is injection into the spleen, from which blood flow is rapid and direct to the liver. This allows almost immediate resection of the spleen (with no adverse effects to the mouse) to prevent extensive local tumour growth.

However, the “gold standard” is the development of “spon-taneous metastasis” from an orthotopic tumour (i.e. grown in the correct anatomical location), particularly for the evaluation of novel therapeutic agents destined for clinical use (4, 5). It has been shown that the correct tissue microenvironment (especially for human tumour xenografts) allows expression of enhanced metastatic potential compared with subcutaneous implants. Here, we give examples of breast carcinomas grown in the mammary fat pad (which, in the case of human tumour xenografts, can be excised to allow time for metastases to manifest) and carcinomas injected into the prostate gland (which are left in situ) and from which lymph node metastases develop.

Cells injected as a bolus into the circulation of a normal mouse may not experience the same microenvironment, or behave the same as those released gradually from an animal bearing an established primary tumour. For example it has recently been shown that primary tumours can selectively condition “pre-metastatic niches” in organs to dictate the sites of secondary disease (6, 7). All methods are, therefore, a balance of pragmatism and accuracy of reflection of the clinical reality, and each model should be selected according to the primary aims of the experimental study. In all cases, the highest standards of animal care and welfare should be adhered to, with the mildest possible procedure adopted. This is entirely consistent with high-quality preclinical cancer research (8).

1. Dulbecco’s modified Eagle’s medium and RPMI-1640 medium, both supplemented with heat-inactivated 10% foetal bovine serum.

2. Versene and TrypLE™ (Gibco®, UK). 3. Hank’s balanced salt solution (HBSS) without calcium chlo-

ride and magnesium chloride.

2. Materials

2.1. Cell Culture

31321 Simple Experimental and Spontaneous Metastasis Assays in Mice

4. Cell lines selected from the following (according to the model required, Table 1):(a) Mouse melanoma, B16 F10 (CRL-6475, American Type

Culture Collection®).(b) Human fibrosarcoma, HT-1080 (CCL-121, American

Type Culture Collection®).(c) Mouse mammary tumour, 4T1 (CRL-2539, American

Type Culture Collection®).(d) Human colorectal adenocarcinoma, LS 174T (CL-188,

American Type Culture Collection®).(e) Human prostate adenocarcinoma, PC-3 (CRL-1435,

American Type Culture Collection®) (see Note 1).

1. Mouse restrainer/platform designed for tail vein injections. 2. Sterile needle (gauge 25–27) and sterile 1-mL syringe. 3. 70% Isopropyl alcohol (swabbing disinfectant). 4. Sterile gauze swab. 5. Physical or chemical vasodilator (e.g. incandescent lamp or

warm water (~43°C)); neat isopropyl alcohol or Vasolate (VetTec Solutions Ltd).

2.2. Lung Colonisation

Table 1 Examples of cell lines from an authenticated source used as metastatic models

Name RouteMetastatic site

Mouse strain

Selected reference

ATCC® number

Mouse tumour cell lines4T1 breast carcinoma m.f.p. l, lv, bn BALB/c (37) CRL-2539B16 F10 melanoma i.v. l C57BL/6 (38) CRL-6475CT26 colon carcinoma i.v. l C57BL/6 (39) CRL-2638,

CRL-2639Lewis LL2 lung carcinoma i.v. l C57BL/6J (40) CRL-1642

Human tumour cell linesMDA-MB-231 breast

carcinomai.v., m.f.p, ic l, lv, ln, bn Athymic or

SCID(41) HTB-26

MDA-MB-435a i.v. and m.f.p l, lv, ln Athymic (41) HTB-129HT-1080 sarcoma i.v. l Athymic (42) CCL-121PC-3 prostate carcinoma Prostate l, lv, ln, bn Athymic (28, 43) CRL-1435LS 174T colon carcinoma isp lv Athymic (44) CL-188HCT 116 colon carcinoma isp lv Athymic (45) CCL-247

m.f.p mammary fat pad, i.v. intravenous, isp intra-splenic, ic intra-cardiac (beyond the scope of this chapter), l lung, lv liver, ln lymph node, bn bonea Some controversy as to whether this cell line is a breast carcinoma or melanoma, but it is nevertheless a useful and reliable model (46–50)

314 G.M. Box and S.A. Eccles

6. B16 F10 single cell suspension, at 0.1 million in 100 mL, to female C57BL/6 mice (syngeneic recipients) or HT-1080 single cell suspension, at 0.1 million in 100 mL, to female CrTac:NCr-Foxn1nu athymic mice (human tumour xenograft).

1. Sterile needle (gauge 30) and sterile 1-mL syringe. 2. 70% Isopropyl alcohol (swabbing disinfectant). 3. Sterile gauze swab. 4. Sterile cotton buds. 5. Gas anaesthetic (e.g. isofluorane) and vapouriser (both

VetTech Solutions Ltd). 6. Sterile 4/0 mersilk suture (Ethicon, UK). 7. Sterile surgical instruments (e.g. scissors, forceps, wound

clips, and applicator). 8. 10% Iodine solution. 9. LS 174T human colon carcinoma cell suspension at one mil-

lion per100 mL. 10. Post-operative analgesic (e.g. buprenorphine). 11. Female CrTac:NCr-Foxn1nu athymic nude mice.

1. Sterile needle (gauge 25) and sterile 1-mL syringe. 2. 70% Isopropyl alcohol (swabbing disinfectant). 3. Sterile gauze swab. 4. Hair removal clippers or depilatory cream. 5. Gas anaesthetic (e.g. isofluorane) and vapouriser (both

VetTech Solutions Ltd). 6. Sterile 4/0 mersilk suture. 7. Sterile surgical instruments (e.g. scissors, forceps, wound

clips, and applicator). 8. 10% Iodine solution. 9. 4T1 cell suspension at 0.1 million/10 mL. 10. Post-operative analgesic (e.g. buprenorphine). 11. Female syngeneic Balb/c mice.

1. Sterile needle (gauge 30) and sterile 1-mL syringe. 2. 70% Isopropyl alcohol (swabbing disinfectant). 3. Sterile gauze swab. 4. Gas anaesthetic (e.g. isofluorane) and vapouriser (both

VetTech Solutions Ltd). 5. Sterile 4/0 mersilk suture. 6. Sterile surgical instruments (e.g. scissors, forceps, wound

clips, and applicator).

2.3. Liver Colonisation

2.4. Mammary Fat Pad Tumour-Derived Metastasis

2.5. Prostate Tumour-Derived Metastasis

31521 Simple Experimental and Spontaneous Metastasis Assays in Mice

7. 10% Iodine solution. 8. PC-3 cell suspension at 0.5 million/20 mL. 9. Post-operative analgesic (e.g. buprenorphine). 10. Male CrTac:NCr-Foxn1nu athymic mice.

1. Bouin’s fixative Add 15 volumes of saturated picric acid to 5 volumes of for-

malin, and then add 1 volume of glacial acetic acid. 2. India ink stain

India ink 15 mL

Water 85 mL

Ammonium hydroxide 150 mL

3. Feket’s fixative

70% Ethanol 60 mL

Formalin 6 mL

Glacial acetic acid 1 mL

4. 10% Formol saline fixative (neutral buffered)

Formalin 10 mL

Water 90 mL

Sodium chloride 0.90 g

Sodium dihydrogen orthophosphate 0.40 g

Disodium hydrogen orthophosphate 0.65 g

All tissue-culture procedures should be performed in a class II laminar flow cabinet, applying strict aseptic techniques. Given the frequency of misidentification and cross-contamination (9, 10), it is essential that all cell lines are rigorously checked for their origins and genetic identity (11, 12). It is also important that cell lines are free from contamination with infectious agents such as myco-plasma, which can influence their biological behaviour and present a risk to handlers and animals (13, 14). Cells must, therefore, be routinely screened for mycoplasma using a highly sensitive poly-merase chain reaction (PCR) method (15, 16). It is not good practice to maintain cells in antibiotics to ensure sterility , as this can mask low-level bacterial contamination, lead to outgrowth of

2.6. Histology Reagents

3. Methods

316 G.M. Box and S.A. Eccles

antibiotic-resistant pathogens, and may also affect the behaviour of cells. Cells are maintained in a humidified incubator set at 37°C in an atmosphere of 5% carbon dioxide in air. The preferred cul-ture flasks have a screw-cap lid with a 0.2-mm filter. Growth medium is replaced every 2–4 days depending on the growth rate and density of the cells. Cells should be harvested whilst in an exponential log phase of growth (50–70% confluent).

It is important to ensure that cells for intravenous delivery are not clumped, otherwise potentially lethal pulmonary embolism is a possibility. Numerous washings of cells are required to rid them of sticky residual serum components or DNA released from dam-aged cells. Some cells such as colon carcinomas also secrete a mucinous glycocalyx which can cause clumping and must be removed by vigorous washing. The harvesting of sub-confluent populations and their final resuspension in a vehicle free of both calcium and magnesium ions will help to ensure single cell prepa-rations. Furthermore, mice that survive delivery of clumped cells develop more pulmonary foci than cohorts receiving the same total in a single cell preparation (17), and the variability between mice can be greater.

Athymic nude mice CrTac:NCr-Foxn1nu (Taconic, USA), and C57BL/6 and BALB/c mice (Charles River, UK) at 6–8 weeks old are housed in individually ventilated cages and given sterile water and fed ad libitum.

1. Pre-warm tissue culture reagents. 2. Decant all medium from culture vessel. 3. Remove traces of serum by rinsing with Versene using gentle

rocking for 20 s and decant. 4. Add TrypLE™ dissociation solution (1 mL/75-cm2 flask)

and gently rock so that the entire monolayer is bathed. 5. Incubate for 5 min. 6. Dislodge cells from vessel by gentle tapping. 7. Gather cells by adding growth medium (containing serum to

inactivate remaining trypsin) and decant to a tube for centri-fuging (500 × g for 5 min)

8. Resuspend the pelleted cells in HBSS to attain a single cell suspension and determine cell concentration using a cell counting chamber.

9. Adjust the concentration for the appropriate inoculum vol-ume by centrifugation and resuspension in a small volume.

10. Keep cells on ice to maintain viability and prevent aggregation.

1. Place a mouse in the restrainer. 2. Disinfect the whole tail using an alcohol-dampened swab.

3.1. Cell Culture

3.2. Animal Procedure for Lung Colonisation

31721 Simple Experimental and Spontaneous Metastasis Assays in Mice

3. Mix the tumour cell suspension with a sterile syringe and load with an excess volume.

4. Attach a sterile needle and, importantly, expel any air bubbles, leaving the intended volume for injection.

5. Rotate the tail to locate one of the two lateral tail veins. 6. Vasodilate the tail vein (see Note 2). 7. With the needle bevel facing upward and on the same plane

as the tail vein, slide the needle in 2 mm. A slight pull on the syringe plunger should reveal a flash of blood in the needle hub confirming correct entry (see Note 3).

8. Push the plunger in slowly and deliver the cell suspension. The vein will change from dark to light as the cell suspension temporarily replaces the blood (Fig. 1). Any resistance or blanching will indicate the needle is not in the vein.

9. Remove the needle from the vein and with a sterile gauze swab, apply slight pressure to the injection site until bleeding has stopped.

10. Remove the mouse from the restrainer and return to its cage. 11. Observe the mouse for 5–10 min to ensure no recurrent

bleeding or other ill effects. 12. Repeat using a fresh needle and new cell suspension for each

mouse (loading a syringe with sufficient cells for multiple mice can lead to blood clotting in the needle between injec-tions, and the delivery of variable numbers of cells; this applies to all such procedures).

Fig. 1. Tail vein inoculation for lung metastases.

318 G.M. Box and S.A. Eccles

1. Anaesthetize a mouse (see Note 4) and turn to the right lateral position.

2. If necessary, shave or depilate fur. 3. Disinfect the skin above the spleen with an alcohol-dampened

swab. 4. Make an 8-mm cutaneous incision, perpendicular to the length

of the spleen, and carry down through the abdominal wall. 5. Exteriorise the spleen onto a sterile swab saturated with ster-

ile saline, blunt dissecting any pancreatic tissue. 6. Mix the tumour cell suspension with a sterile syringe and load

with an excess volume. 7. Attach a sterile needle and, importantly, expel any air bubbles,

leaving the intended volume for injection. 8. Slowly inject the single cell suspension under the spleen cap-

sule, marked by a visible pale wheal (Fig. 2). 9. Remove the needle and stop injection site bleeding by apply-

ing gentle pressure with a sterile cotton bud for 30 s. 10. Ligate the splenic blood vessels with suture. 11. Remove the spleen by cutting distal to the ligatures. 12. Close the abdominal muscle with suture and swab the wound

with iodine solution. Close the skin incision with surgical clips. 13. Administer post-operative analgesic according to the manu-

facturer’s instructions. 14. Soak surgical instruments in a multipurpose disinfectant.

Wash and rinse prior to autoclave sterilisation. 15. Observe daily for signs of post-operative ill health, inflamma-

tion, or infection and remove surgical clips after 7 days.

3.3. Animal Procedure for Liver Colonisation

Fig. 2. Intra-splenic inoculation for liver metastases.

31921 Simple Experimental and Spontaneous Metastasis Assays in Mice

1. Anaesthetize a mouse and turn to the supine position. 2. If necessary, shave or depilate fur. 3. Disinfect the mammary glands with a dampened swab (see

Note 5). 4. Make a 5-mm skin incision adjacent to a gland and identify

the fatty tissue (Fig. 3). 5. Mix the tumour cell suspension with a sterile syringe and load

with an excess volume. Attach a sterile needle and expel any air bubbles.

6. Direct the bevel of the needle upwards and inject slowly. 7. Close the incision with suture. 8. Soak surgical instruments in a multipurpose disinfectant.

Wash and rinse prior to autoclave sterilisation. 9. As the tumour grows, monitor routinely for potential skin

ulceration.

1. Do not allow tumours to grow to excessive sizes (max 8–10 mm mean diameter).

2. Anaesthetise a mouse and turn to the supine position. 3. If necessary, shave or depilate fur. 4. Disinfect the area of excision with an alcohol-dampened

swab.

3.4. Animal Procedure for Mammary Fat Pad Tumour-Derived Metastasis

3.4.1. Injection of Tumour Cells

3.4.2. Excision (Mastectomy) of Mammary Tumour

Fig. 3. Mammary fat pad inoculation.

320 G.M. Box and S.A. Eccles

5. With the aid of sterile surgical instruments, cut around the whole tumour mass, removing an adequate margin of skin tissue (see Note 6).

6. Swab the wound with iodine solution and repair with surgical clips.

7. Administer post-operative analgesic according to the manu-facturer’s instructions.

8. Soak surgical instruments in a multipurpose disinfectant. Wash and rinse prior to autoclave sterilisation.

9. Observe daily for signs of inflammation or infection and remove surgical clips after 7 days.

10. Continue observation for signs of ill health – lung metastases may manifest by shortness of breath or laboured breathing. Palpate regional (axillary) lymph nodes and abdomen for signs of metastasis.

1. Anaesthetise a male mouse and turn to the supine position. 2. If necessary, shave or depilate fur. 3. Disinfect the area of excision with an alcohol-dampened

swab. 4. Make an 8-mm midline incision above the pubic symphysis. 5. Mix the tumour cell suspension with a sterile syringe and load

with an excess volume. 6. Attach a sterile needle and, importantly, expel any air bubbles,

leaving the intended volume for injection. 7. Locate the bladder and exteriorise onto a sterile swab satu-

rated with sterile saline, cutting away connective tissue if nec-essary to aid release (Fig. 4).

8. Insert the needle into the ventral lobe of the prostate gland, exit into the dorsolateral lobe, and inject the cell suspension (see Note 7).

9. Check that there is no internal bleeding and then lift the abdominal muscle, allowing the bladder to retract.

10. Close the abdominal muscle with suture and swab the wound with iodine solution. Close the skin incision with surgical clips.

11. Administer post-operative analgesic according to the manu-facturer’s instructions.

12. Soak surgical instruments in a multipurpose disinfectant. Wash and rinse prior to autoclave sterilisation.

13. Observe daily for signs of post-operative ill health, inflamma-tion, or infection and remove surgical clips after 7 days.

14. Palpate the abdomen for signs of lymph node metastases.

3.5. Animal Procedure for Prostate-Derived Metastasis

32121 Simple Experimental and Spontaneous Metastasis Assays in Mice

1. A mouse is overdosed with anaesthetic and turned to the supine position.

2. Starting from the diaphragm, an incision is made upwards to the neck, exposing the thoracic cavity.

3. Hold the top of the trachea with forceps and dissect the lung, trachea, and heart en bloc and lay onto a saline-moistened sterile gauze swab.

4. Remove the heart and wash the lung in sterile water and return to the moistened swab.

5. Insert a large gauge needle (19 G) into the open end of the trachea and deliver approximately 1 mL of staining solution or fixative.

1. Bouin’s methodImmediately after sacrifice, remove the lungs and trachea en bloc.Wash the lungs in water.Using a wide gauge needle (19 G) and syringe, deliver fixative into the lung via the trachea.Fix overnight (<24 h) at room temperature by gently rotating in a 30-mL glass universal tube.

3.6. Thoracotomy and Removal of Lung and Trachea En Bloc

3.7. Staining of Lung Colonies for Quantitation

Fig. 4. Intra-prostatic injection.

322 G.M. Box and S.A. Eccles

Remove fixed tissue to 70% ethanol for long-term storage at room temperature.

2. India ink and Feket’s methodImmediately after sacrifice, remove the lungs and trachea en bloc.Wash the lungs in water.Using a wide gauge needle (19 G) and syringe, deliver ink into the lungs via the trachea.Wash the stained lung in Feket’s solution.Fix overnight (<24 h) in Feket’s solution at room tempera-ture by gently rotating in a 30-mL plastic universal tube.Remove fixed tissue to 70% ethanol for long-term storage at room temperature.

3. Formol saline fixationImmediately after sacrifice, remove the lung with the trachea intact.Wash the lung in water.Using a wide gauge needle (19 G) and syringe, fix the lung via the trachea.Fix for 4–24 h (optimise depending on colony size) at room temperature by gently rotating in a 30-mL plastic universal tube.Remove fixed tissue into sterile saline for short-term storage or to 70% ethanol for longer periods at room temperature.

The principal organ for development of “experimental metasta-ses” following intravenous delivery is the lung. However, extra-pulmonary disease is possible, so full autopsy examining the viscera, lymph nodes, and brain is advised. Mice showing abnor-mal balance or behaviour in particular merit sectioning and his-tology of the brain.

The time taken to develop metastases and the expected distri-bution of disease may differ from published information. The variability will come from different mouse strains; age and sex of individuals; their housing conditions; and health status. Variations in culture conditions and handling of cell lines can significantly alter their behaviour, and each operator should establish their own standard procedures and note the in vivo growth and meta-static rates for each cell line, so that any deviations can be carefully monitored. Changes in serum batches can lead to alterations in cell behaviour. Tumorigenicity and metastatic phenotype can change during long-term passage, or by altering harvesting proce-dures. Establish the “baseline” properties of the cell line and bank multiple early passage aliquots to avoid genetic drift or adaptation to tissue culture, which can reduce in vivo malignant potential.

3.8. Quantitation of Experimental and Spontaneous Metastases

32321 Simple Experimental and Spontaneous Metastasis Assays in Mice

A time course study to assess the rate and extent of metastasis development provides the observer the opportunity to standardise operating procedures and to define humane end points for termi-nation, prior to animals developing severe symptoms. Death as an end point (survival study) is unacceptable (18).

For experimental studies, cohorts of 8–10 mice or fewer are generally sufficient to apply nonparametric analysis (e.g. Mann–Whitney rank sum test) for comparative analysis of groups, e.g. to test the effects of therapeutic agents (19).

Tumour lung colony quantitation is performed by careful gross examination. For animals with large numbers of lesions, one lobe can be examined and the total number approximated by multiplica-tion. Surface lesions can be counted under a dissecting microscope or removed with a micro curve-ended forceps for counting, sizing, and weighing. Most tumours develop at the surface of the lung, and this can give a representative reflection of total tumour burden even if there are additional tumours deep within the lung parenchyma.

Discrimination of tumours is assisted by the use of fixation and staining (see Subheading 3.6): Bouin’s fluid provides a con-trast of white lesions against yellow lung tissue (20), whereas India ink stains the lung blue (21, 22). The B16 melanoma is a popular choice for experimental lung metastasis assays, as the melanin content of the tumour renders the black lesions easily visible against the normal lung without the need for staining (although non-pigmented lesions may also occur) (23). However, in all cases, microscopic lesions also need to be considered (see Note 8). An alternative is to section formalin-fixed lungs, take 5-mM sections, and stain with haematoxylin and eosin (H&E) for scoring by light microscopy (Fig. 5).

3.8.1. Lung Metastases

Fig. 5. Lung metastases.

324 G.M. Box and S.A. Eccles

A crude method, when tumour burden is extensive or colonies have coalesced and cannot be individually discriminated, is to remove and weigh the lungs of each mouse and express the results as average lung weight ± standard deviation. The lung weight of an average 25-g mouse is approximately 200 mg (although this should be determined from age-matched normal mice of the same age and strain) and hence some estimate of the total tumour burden can be adduced. However, this method is unreliable and not recom-mended since the presence of tumours can cause inflammation, oedema, and congestion, which can add to the mass of the tissue.

Quantitative PCR offers a precise method of detection and quantification of metastatic tumour burden in human xenograft models where microscopic lesions are common. Human and mouse primers can be designed to amplify a unique, species-specific, conserved, and non-transcribed sequence in the mouse and human genome (24). Alternatively, amplifying a specific mRNA transcript using real-time TaqMan PCR (25) offers another approach.

Computer-assisted video imaging is a technique that utilises mathematical models sensitive to subtle changes in size, shape, position, and tissue characteristics affected by disease (26). Increasingly, cells engineered to express fluorescent proteins or luciferase are being used to allow semi-quantitative, dynamic, non-invasive optical imaging of internal tumours, e.g. (22) and see Chapters 22 and 23, this issue.

Serial sectioning and H&E staining are an option for microscopic analysis but a quantitative RT-PCR TaqMan approach offers a more sensitive and cost-effective approach (27). It is highly repro-ducible and quantitative, eliminating associated risks encountered with other PCR-based detection methods. Inokuchi et al. (25) utilised this approach and highlighted the sensitivity compared to conventional H&E staining and immunohistochemical staining by amplifying cytokeratin-19 mRNA transcripts from breast can-cer patients. Havens et al. identified early microscopic lodgings and metastatic homing events through real-time PCR using human Alu sequence TaqMan probes (28).

Liver can be handled as for lung and surface or internal tumours scored by eye, dissection, or microscopic means (see Fig. 6). No staining is required as the pale tumour colonies are easily distin-guishable from the reddish-brown liver parenchyma. Invasive end points, as described above, are an option but non-invasive (e.g. bioluminescent or fluorescent optical) imaging modalities should be considered. Developments in magnetic resonance imaging (MRI) (29), small-animal computed tomography (30), and posi-tron emission tomography (31) offer a different end point, and multimodality imaging, combining both molecular and anatomi-cal data, is the favoured approach (32, 33).

3.8.2. Lymph Node Metastases (Breast and Prostate Models)

3.8.3. Liver Metastases and Other Sites

32521 Simple Experimental and Spontaneous Metastasis Assays in Mice

1. Recent genetic testing has revealed a high frequency of mis-identification and cross-contamination of commonly used cell lines (9, 10). It is, therefore, essential that all human and mouse cell lines are sourced from a reputable establishment rigorously checked for their genetic identity (11, 12). Cell line authenticity is an increasing prerequisite by publishers who now often require short tandem repeat profiling as the measure for confirmation (34). It is also important that cell lines are free from contamination with infectious agents such as mycoplasma and pathogenic viruses which can influence their biological behaviour (35) and present a risk to handlers and animals (13, 14, 16, 36).

2. Incandescent lamp – place a well-ventilated mouse cage approximately 20 cm from a lamp, with a 150 W light bulb, for approximately 5 min.Warm water – take the tail of a restrained mouse and immerse it in a 15-mL tube containing water at approximately 43°C (<50°C) for 30–60 s.Vasolate or swabbing alcohol – dampen a gauze swab with the liquid and rub a lateral tail vein of a restrained mouse. Vasodilation is instant and remains so for several minutes.Mouse tail illuminators – specially designed restrainers sit on a light source that has an illuminated slot for the tail to be placed.

3. A prudent method is to first puncture the tail vein close to the tip, and if unsuccessful, continue towards the base of the tail

4. Notes

Fig. 6. Liver metastasis.

326 G.M. Box and S.A. Eccles

to a proximal point. If the chosen lateral vein collapses, move to the contralateral vein.

4. A mouse is restrained manually and gently, but firmly, held to a gas mask passing a fresh flow of oxygen (2 L/min). The vapouriser is initially set to 5% to induce (approximately 30–60 s) and lowered to 2% to maintain anaesthesia through-out the surgical procedure. Tail and footpad reflexes are checked before any incision is performed. Throughout the procedure, a constant vigil is paramount, watching for any indication related to under-sedation or laboured breathing if over-sedated.A mammary fat pad injection procedure will require 5–8 min of anaesthetic, with the mastectomy requiring between 5 and 15 min, the time depending on whether the tumour has invaded the muscle wall.An intra-prostatic procedure will require between 7 and 12 min, the time dictated by the amount of fat and connec-tive tissue needed to be cut away.An intra-splenic procedure will require 10–12 min, the time lengthening with persistent bleeding.

5. Mice have ten mammary glands: four inguinal (posterior) and six thoracic (anterior). The best policy is to inject either the lower thoracic fat pads (closest to the inguinal set) or the highest inguinal fat pads since these are furthest from local lymph nodes. The more anterior site is reported to be more favourable for growth. This procedure ensures, at macroscopic examination, that lesions that develop post-excision of the primary tumour are genuine local lymph node metastases and not due to recurrence of primary tumour remnants.

6. Failure to remove the entire primary tumour will result in regrowth. Some animal project licences may not allow the opportunity to repeat the excision and force an unnecessary cull.

7. The dorsal and lateral (dorsolateral) lobes of the prostate gland are found between the ventral and anterior lobes, at the junction of the urinary bladder, urethra, and seminal vesicles. The inoculum site should “bleb”, confirming correct deliv-ery. No bleeding will occur for this injection.

8. The method of euthanasia should be considered. Carbon dioxide causes minute haemorrhaging (petechiae) in the lungs, potentially adding confusion when identifying small tumour foci during histological examination. Cervical dislo-cation causes clots in the lung. The cleanest method is to overdose with anaesthetic.

32721 Simple Experimental and Spontaneous Metastasis Assays in Mice

All animal procedures should be performed in accordance with UK Home Office regulations under the Animals (Scientific Procedures) Act 1986 or other national guidelines and the recently published NCRI guidelines for animal experimentation (18), which all major publishers have endorsed. This publication offers clear guidance on maximum volumes for inoculation at dif-ferent sites, further examples of robust metastasis models, proce-dures, and humane end points. A further useful reference (regarding stringent reporting of animal experiments) is also rec-ommended (www.nc3rs.org.uk/reportingguidelines).

Acknowledgements

This work was funded by Cancer Research UK Grants CA309/A2187, CA309/A8274. We acknowledge NHS funding to the NIHR Biomedical Research Centre.

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