Current Protocols in Pharmacology || Preclinical Orthotopic and Intracardiac Injection Models of Human Breast Cancer Metastasis to Bone and Their Use in Drug Discovery

UNIT 14.18Preclinical Orthotopic and IntracardiacInjection Models of Human BreastCancer Metastasis to Bone and Their Usein Drug Discovery

Ellen Scepansky,1 Robert Goldstein,2 and Michael Rosenblatt3

1Tufts Medical Center, Division of Hematology/Oncology, Boston, Massachusetts2Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences,Boston, Massachusetts3Tufts University School of Medicine, Department of Physiology and Medicine, Boston,Massachusetts

ABSTRACT

Breast cancer is the most common malignancy in women, with the development ofdistant metastases rendering the condition incurable. Relatively little is known about thefactors governing the progression from primary tumor to metastasis, in part because ofthe difficulty in modeling what is a complex series of events. Detailed in this unit aredescriptions of two murine models of breast cancer metastasis to bone that can be usedto screen the effectiveness of new chemical entities on this disease process. Curr. Protoc.Pharmacol. 52:14.18.1-14.18.23. C© 2011 by John Wiley & Sons, Inc.

Keywords: breast cancer � bone metastasis � orthotopic model � intracardiac model �

bioluminescent imaging

INTRODUCTION

In the United States, nearly 200,000 new diagnoses of breast cancer and 40,000 breastcancer deaths occur annually (Jemal et al., 2009), accounting for more than 25% ofall cancer cases in women. The majority of patients with advanced breast cancer havethe disease metastatic to bone. In addition to the incurable status associated with theappearance of bone metastases, these lesions can cause significant morbidity from hy-percalcemia of malignancy, pathological fractures, spinal cord compression, and chronicpain. Currently the only drugs that specifically target bone metastases are the nitrogen-containing bisphosphonates (e.g., zoledronic acid) and bone-avid radiopharmaceuticals(e.g., samarium-153) (Lam et al., 2007). While the latter are used only for palliation ofpain, the bisphosphonates have a well-defined role in the clinical management of cancer-induced bone disease from solid tumors and multiple myeloma (Russell and Rogers, 1999;Coleman, 2008; Costa and Major, 2009). They have been shown to reduce the number ofskeletal-related events when used in the setting of established bone metastases. They mayalso reduce skeletal tumor burden when used prophylactically. Though bisphosphonates,particularly the potent intravenous agent zolendronic acid, have improved the quality oflife of patients with skeletal metastases, they do not have a cytotoxic effect on establishedlesions and therefore do not cure metastatic disease. Thus, there remains a pressing needto identify new bone-specific targets and to develop agents that interact with these sites.Within the past 5 years, some progress (Lev et al., 2005; Body et al., 2006; Feeley et al.,2006; Hoeppner et al., 2009) has been made toward these goals. Targets include RANKL(receptor activator of nuclear factor-κB ligand—see Background Information), growthfactors such as PDGF (platelet-derived growth factor) and the BMPs (bone morpho-genetic protein family), and the Wnt signaling pathway, which inhibits cancer-associatedosteolysis along with other regulatory functions.

Current Protocols in Pharmacology 14.18.1-14.18.23, March 2011Published online March 2011 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471141755.ph1418s52Copyright C© 2011 John Wiley & Sons, Inc.

Cellular andAnimal Modelsin Oncology andTumor Biology

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Supplement 52

Preclinical Modelsof Breast Cancer

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Supplement 52 Current Protocols in Pharmacology

A novel mouse model has been developed in which human bone is implanted ectopicallyin NOD/SCID mice (see Basic Protocol 1) and human breast cancer cells are injectedinto an orthotopic site, the mouse mammary fat pad (Kuperwasser et al., 2005; see BasicProtocol 2). This model facilitates the study of mechanisms by which metastatic cellslocalize to, and grow within, the human bone microenvironment. The orthotopic injectionof breast cancer cells is believed to more accurately reproduce early events in the processof tumor metastasis, including development of an invasive phenotype, detachment fromand degradation of extracellular matrix, development of motility, and extravasation intoblood vessels, than models wherein tumor cells are introduced directly into the circulation(Ottewell et al., 2006; Talmadge et al., 2007).

The orthotopic injection of breast cancer cells model also provides a mechanism fortesting potential therapeutic agents that may prevent the seeding of metastatic foci anddestruction of bone due to metastases. Furthermore, as an “all-human” system, thismodel provides a more accurate representation of molecular and cellular events involvedin the process of human breast cancer metastasis to bone than other models. Indeed, themetastasis gene signature observed with this model is different from the one previouslyobtained from an intracardiac injection model of cancer metastasis to murine bone (Kanget al., 2003; Goldstein et al., 2008). For example, three of the most highly overexpressedgenes in bone metastatic populations in the earlier model were MMP-1, CTGF, and IL1.Each represents a distinct biological activity, i.e., invasion, angiogenesis, and osteolysis,respectively. Key genes identified in the humanized model are MMP13, HUNK, and IL-17BR, which have functions analogous but not identical to those identified in the earliermodel. Additionally, osteopontin (OPN), which stimulates osteoclast adhesion to thebone matrix, is up-regulated in lesions within murine bone (as compared to the primarytumor) and down-regulated in metastasis to human bone engrafted in mice in the laterstudy, and IL-17BR overexpression alone is able to promote metastasis to human bone(R. Goldstein and M. Rosenblatt, unpub. observ.).

A relatively new strategy to longitudinally monitor tumor growth in live animals isnon-invasive bioluminescent imaging (see Basic Protocol 3). For this approach, cancercell lines transfected to stably overexpress the firefly luciferase gene are inoculated intothe host animal. After a period of cell growth, the animals are injected with luciferin,and tumors formed from the cells are visualized with specialized imaging equipmentthat measures the intensity of luminescence in photons/second (Klerk et al., 2007). Theresults from such studies correlate with external caliper measurements of tumor volume,but allow earlier detection of tumor growth (Jenkins et al., 2003). Furthermore, if animalsare injected prior to necropsy, metastases within organs of interest may be visualizedafter dissection. The results are obtained rapidly and the method is sensitive, althoughfalse positives occur.

This technique has been used extensively to identify metastasis to bone both in the murineskeleton and in the human bone implants (Basic Protocol 1), freeing the investigator fromhaving to rely solely on histological evaluation, which may overlook small metastaticdeposits unless the entire bone is sectioned and examined.

The human bone grafts used in the model are obtained from discarded tissue frompatients undergoing total hip replacement surgery, and implanted subcutaneously intoNOD/SCID mice. After 4 weeks, the bone graft becomes vascularized, and its structure,including the bone marrow, is morphologically similar to that of normal human bone.Endothelial cells in the vasculature supplying the graft are derived from both mouse andhuman cells. Several human breast cancer cell lines have been introduced systemicallyinto model animals. One of these, SUM 1315, derived from the chest wall metastasis of abreast cancer patient, reliably forms metastases in the human bone graft at 8 to 12 weeksfollowing mammary fat pad injection (Kuperwasser et al., 2005). On average, half of the


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Current Protocols in Pharmacology Supplement 52

bone grafts are found to contain tumors with this model. The bone-avid MDA-MB-231cell line was found in subsequent experiments to metastasize with approximately thesame frequency (R. Goldstein and M. Rosenblatt, unpub. observ).

Although this model replicates even the early steps of breast cancer metastasis to bone,like all xenograft models it requires an immunocompromised host. Therefore, the roleof the immune system in disease progression cannot be examined. Unfortunately, mostof the existing murine models of breast cancer demonstrate a low frequency of skeletalmetastasis, making them impractical for use in this area of study. One exception is the4T1 model (Pulaski and Ostrand-Rosenberg, 2001), which uses cell lines derived froma spontaneously arising BALB/c mammary tumor. However, because this is a syngeneicmodel it cannot be used to study human breast cancer. The 4T1 cells, when introducedorthotopically, are capable of relatively rapid metastasis to several organs affected inbreast cancer including lungs, liver, and brain, as well as bone. These cells also havebeen modified to express firefly luciferase, enabling the longitudinal examination of amurine model analogous to late-stage human breast cancer (Tao et al., 2008).

The model described in this unit has certain limitations. These include the 12 to 16 weeksneeded to complete an experiment and the fact that the procedure is labor intensive. Thismakes it unsuitable for screening multiple agents as potential candidate drugs. Directinoculation of cancer cells into the skeletons of immunocompromised mice by intra-tibial or femoral injection (Wang and Chang, 1997) provides a relatively simple meansfor evaluating the effect of an agent on breast cancer growing within bone, although itdoes not reproduce the process of metastasis.

The intracardiac injection model (see Alternate Protocol) can be used to generate lesionswithin the bone, liver, and other tissues following the introduction of human breastcancer cells into the left cardiac ventricle of an immunocompromised mouse (Kang et al.,2003). Although the early stages of metastasis, which include detachment, acquisition ofmotility, and invasion, are not represented, the model is comparatively simple to use andyields results quickly.

NOTE: All protocols using live animals must first be reviewed and approved by anInstitutional Animal Care and Use Committee (IACUC) or must conform to governmentalregulations regarding the care and use of laboratory animals.

BASICPROTOCOL 1

SUBCUTANEOUS IMPLANTATION OF HUMAN BONE INTOIMMUNOCOMPROMISED MICE

This protocol represents the initial steps required for use of the “all-human” murine modelof breast cancer metastasis to bone. The investigator cuts samples from human cancellousbone and subcutaneously implants them in anesthetized mice under aseptic conditions.

Materials

Human cancellous bone—femoral head(s) from total hip replacement surgerySterile phosphate-buffered saline (PBS; Fisher Scientific)6- to 8-week-old female NOD/SCID mice (Jackson Laboratories)Betadine surgical scrubIndividually packaged 70% alcohol prep padsBuprenorphrine for injection, 0.1 mg/kg per animal (e.g., Alstoe Animal Health;

please note that this can only be supplied to a veterinarian)Antibiotic suspension (sulfamethoxazole and trimethoprim 200 mg/40 mg per

5 ml, e.g., brand names Sulfatrim, septra; e.g., California Vet Supply,http://www.calvetsupply.com)

Bone cutting saw: tabletop model (Marmed) or hand-held saw (Styker Instruments)plus clamps to immobilize the sample


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Bone biopsy trephine cutter (DePuy Mitek) or other equivalent coaxial tool of 5- or6-mm diameter

Sterile towels and gauzeCirculating-water heating pad (part of the anesthesia unit)Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen and

isoflurane source, heated surgical stage; e.g., Colonial Medical Supply)Tape (paper surgical or clear office type)Electric clippersSterile gauzeSurgical gloves and masks, sterileAutoclaved surgical instruments: use small-sized (4.5-in.) operating room supplies

or items specifically manufactured for small-animal surgery (Roboz) including:Straight scissorsHemostat or needle holderTwo pairs of forceps

9-mm wound clips with matched stapler and removal tool (Becton Dickinson)Ear punch for identification of individual mice (Kent Scientific)Glass-bead autoclave (for sterilization of instruments between each animal’s

procedure; e.g., Cole-Parmer, cat. no. 10779-05)0.5- or 1.0-ml syringes equipped with 28-G needlesAnimal cages

Prepare the human bone1. Obtain the femoral head (which would otherwise be discarded) at the completion of

total hip replacement.

The bone should be handed off from the operating room table and placed in a sterile drycontainer.

A tissue-engineered bone construct (i.e., scaffolds made from silk fibroin protein sponges)can be used as a metastasis target (Li et al., 2006); however, such materials are notcommercially available

Timing should be such that the bone is prepared and implanted into mice within a maximumof 4 hr. The bone should be refrigerated when it is not being handled. The femoral headis the preferred material source as it provides a relatively large volume of bone and is acommon site of metastasis to the appendicular skeleton.

2. Secure and immobilize the femoral head using bone clamps or against the bandsawguide. With either a tabletop bandsaw or a hand-held surgical saw, cut the femoralhead into uniform slices of the desired thickness (minimum 5 mm, maximum 10 mm).Saw blades should be autoclaved and the supporting equipment and surfaces shouldbe as clean as is possible. Place bone slices into a sterile cup containing PBS.

Note that because this step requires some practice to master, the operators should fa-miliarize themselves with the equipment by testing it on bone samples prior to the dayplanned for surgery.

3. Place a bone slice on a new sterile surface. Using the bone trephine or other hollowcutting instrument, such as a 6-mm diameter Mitek COR autograph instrument, cutcylindrical cores of bone that are 5- to 6-mm diameter by 0.5- to 1.0-cm long fromeach slice. Place them in PBS (Fig. 14.18.1).

This must be performed manually, as using a power drill fitted with a cylindrical cuttingtool leads to excessive tissue destruction.

Prepare the animal for surgery4. Prepare a sterile field using towels in an animal procedure room, preferably one

dedicated to immunocompromised animals. Turn on the circulating-water heatingpad. If a dedicated sterile room is not available, work should be performed in alaminar flow hood.


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Figure 14.18.1 Cut cylindrical cores of bone.

5. Turn on the oxygen supply, vacuum, and isoflurane source (set to 2% to 3% initially).Place the mice in the induction chamber.

Once the investigator has become familiar with the surgical techniques that follow, threeto five mice housed in one cage can be anesthetized at the same time. Prior to that, miceshould be anesthetized individually.

6. Remove one mouse from the induction chamber and place it in a prone position inthe nose cone apparatus. Verify that the mouse is adequately anesthetized by testingthe pedal reflex.

The pedal reflex is tested by pinching a foot with forceps. Lack of a response to the pinchindicates adequate anesthesia. Level of anesthesia, respiration, and body temperatureshould be monitored throughout the procedure.

7. Secure each of the four limbs using tape, with the hind legs oriented towards theoperator.

The limbs should be extended radially before they are secured to keep some tension on theskin and subcutaneous tissues.

8. Using clippers, shave a 2.5-cm wide area of the dorsum, from just above the hindlegs to the mid-thorax. Prepare the shaved skin using Betadine scrub on sterile gauze,followed by alcohol applied with a sterile prep pad. Remove hair from the surgicalfield.

As the operator becomes more skilled, a smaller area can be shaved to minimize skinirritation.

Perform subcutaneous implantation9. Don mask and sterile surgical gloves. Use tissue forceps and straight scissors to make

two small (6- to 7-mm) incisions, one just above each flank (Fig. 14.18.2).


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Figure 14.18.2 Incisions for bone implantation.

Figure 14.18.3 Insertion of bone implant.

10. Using forceps and either scissors or a hemostat, dissect the skin from the underlyingfascia in a plane extending caudally on both sides. Place the instrument into theincision and gently spread its tips to remove skin from fascia, gradually advancingtowards the animal’s shoulders.

Adequate dissection is important to facilitate the next step.

11. Use a hemostat or needle driver to select a human bone segment, and then positionit firmly within the tips with the long axis parallel to the axis of the instrument. Useforceps in the other hand to access one of the two incisions (Fig. 14.18.3) and advancethe hemostat subcutaneously until the implant reaches the level of the forelegs. Openthe instrument tips slightly to disengage the bone, and remove the hemostat from theincision. Repeat this procedure for the second incision. Verify that excessive tensionhas not been placed on the skin overlying the implant; if it appears that this is thecase, use the hemostats to reposition the bone slightly. Use a finger placed externallywhile withdrawing the hemostat to secure the implant and prevent it from dislodging.


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12. Close the incision using two wound clips per incision. If individual mice within onecage will be receiving different treatments, mark for identification using an ear punch.

13. Resterilize the instruments by removing any adherent tissue with an alcohol pad andplacing them in a glass-bead autoclave for 10 sec.

Post-surgical care

14. Administer buprenorphrine (0.1 mg/kg i.v.) using a 0.5- or 1.0-ml syringe equippedwith a 28-G needle and place the mouse on a warmed surface until it has fully re-covered from anesthesia. Add 10 ml of the sulfamethoxazole/trimethoprim antibioticsuspension to the (full) water bottle of each cage for 7 days.

15. Check the mice at 4 hr post-procedure and observe daily for the first 7 days thereafterfor overall activity, discomfort, and wound problems, such as dehiscence or infection.Thereafter, and throughout the experimental period, carefully monitor the animals 2to 3 times each week. Remove the wound clips 7 to 10 days after surgery.

BASICPROTOCOL 2

ORTHOTOPIC IMPLANTATION OF HUMAN BREAST CANCER CELLSINTO IMMUNOCOMPROMISED MICE CONTAINING ECTOPIC HUMANBONE TISSUE

This procedure allows the investigator to inoculate mice with human breast cancercells in the anatomic location that is analogous to the human breast. It may follow thesubcutaneous implantation of human bone (Basic Protocol 1) or may be used alone toexamine metastasis to the mouse skeleton or other organs. This same procedure couldalso be used to implant murine breast cancer cells or with mice who do not have thehuman bone implanted (e.g., if studying metastasis to the mouse skeleton, the experimentcould be shortened by 4 weeks by skipping the bone implantation).

Materials

Firefly luciferase-expressing, bone-avid human breast cancer cell line, eitherMDA-MB-231-luc2 (Caliper Life Sciences) or SUM 1315 (Asterand)transfected with lentiviral particles containing the luc gene

Medium for cell growth: for MDA-MB-231 cells, RPMI 1640 supplemented with10% fetal bovine serum and penicillin/streptomycin; for SUM-1315 cells, F-12(Ham’s) supplemented with 5% fetal bovine serum, penicillin/streptomycin,5 μg/ml insulin, and 10 ng/ml epidermal growth factor (EGF)

Phosphate-buffered saline (PBS)0.05% (w/v) trypsin-EDTA (Invitrogen, cat. no. 25200-056)Matrigel basement membrane matrix (BD Biosciences)Female NOD/SCID mice with bone implants placed 4 weeks earlier (if

experimenter plans to examine metastasis to human bone; see Basic Protocol 1)Betadine surgical scrubIndividually packaged 70% alcohol prep padsAntibiotic suspension (sulfamethoxazole and trimethoprim 200 mg/40 mg per

5 ml, e.g., brand names Sulfatrim, septra; e.g., California Vet Supply,http://www.calvetsupply.com)

150-mm tissue culture plates37◦C, 5% CO2 incubator50-ml centrifuge tubesHemacytometer or automated cell counter2-ml microcentrifuge tubesSterile towels and gauzeCirculating-water heating pad (part of the anesthesia unit)


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Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen andisoflurane source, heated surgical stage)

Tape (paper surgical or clear office type)Electric clippersSurgical gloves and mask, sterileAutoclaved surgical instruments: use small-sized (4.5-in.) operating room supplies

or items specifically manufactured for small-animal surgery (Roboz) including:Straight scissorsHemostat or needle holderTwo pairs of forceps

50-μl Hamilton syringe with 22-G needle9-mm wound clips with matched stapler and removal tool (Becton-Dickinson)Glass-bead autoclave

NOTE: The 4-week interim period (for the mice bone implants) allows the graft to becomefully vascularized while ensuring that the animal is not excessively aged (>9 months) atthe end of the experiment. Active hematopoiesis and bone remodeling is present uponhistological examination of the implants at the end of a 16-week experimental period. Ingeneral, all implants remain viable, barring immediate post-operative complications.

Prepare the breast cancer cells for implantation1. Grow breast cancer cells to subconfluence in 150-mm diameter tissue culture plates

in a 37◦C, 5% CO2 incubator. For 80% confluence, estimate 3 to 4 million cells perplate to determine the number of plates required.

Cells that were previously frozen and thawed should be cultured for ∼2 weeks (or4 passages) prior to use. Do not use cells that are 100% confluent as they tend to formtumors more slowly. Viability of cells may be tested prior to implantation using one ofseveral commercially available assay kits (see Wilson, 2000), such as the MTT (methodof transcriptional and translational) assay, a colorimetric test measuring the activity ofenzymes that reduce yellow MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] to purple formazan in living cells (e.g., Roche Applied Science).

2. Wash the cells on each plate with 10 ml PBS, and then aspirate the solution. Pipet 2to 4 ml of trypsin onto each plate and return them to the incubator for 3 to 4 min. Tapthe side of each plate after applying trypsin to ensure that the entire surface of theplate is covered. Use a microscope to verify that cells are floating prior to the nextstep. If the cells remain adherent, return the plate to the incubator for an additionalminute.

3. Add 5 ml of growth medium to each plate to terminate the trypsin reaction. Collectall of the cells and the medium from the plates and place into one or two 50-mlcentrifuge tubes Centrifuge 5 min at 150 × g, room temperature, to form a pellet.Aspirate the supernatant.

4. Resuspend the cells in room-temperature PBS by pipetting up and down until thepellet has dissolved. If using a hemacytometer, it is convenient to add 5 ml of PBSper confluent plate. Use a hemacytometer or automated cell counter to determine thetotal number of cells in the sample.

If cells are too concentrated, they are difficult to count.

5. Centrifuge the samples again as in step 3. This time resuspend the cells in the precisevolume that will provide 500,000 cells for each 20 μl mammary fat pad injection,which is equivalent to a concentration of 25,000 cells/μl. Prepare a suspension thatis two parts PBS to one part Matrigel.

Matrigel should be stored on ice in a refrigerator prior to use. If it reaches room temper-ature, it becomes too viscous for use.


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Figure 14.18.4 Identification of fourth mammary fat pad.

6. Place the suspended cells in a 2-ml microcentrifuge tube on ice. Using towels,prepare a sterile field in an animal procedure room, preferably one dedicated toimmunocompromised animals. Turn on the circulating water heating pad.

If a dedicated sterile room is not available, work should be performed in a laminar flowhood.

The investigator should proceed immediately from the cell culture area to the procedureroom, as the total time the cell suspension is kept on ice should be <3 hr.

Prepare the animal for implantation7. Turn on the oxygen supply, vacuum, and isoflurane source (set to 2% to 3% initially).

Place the mice in the induction chamber.

Once the operator has become familiar with the surgical techniques that follow, three tofive mice housed in one cage can be anesthetized at the same time. Prior to that, miceshould be anesthetized individually.

8. Remove one mouse from the induction chamber and place it in a supine position inthe nose cone apparatus. Identify the 4th of 5 paired mammary fat pads (Fig. 14.18.4).Verify that the mouse is adequately anesthetized by testing the pedal reflex.

The pedal reflex is tested by pinching a foot with forceps. The anesthesia is consideredadequate when there is no response to the pinch. Level of anesthesia, respiration, andbody temperature should be monitored throughout the procedure.

9. Secure each of the four limbs using tape, with the hind legs oriented towards theexperimenter.

The limbs should be extended radially before they are secured to keep some tension on theskin and subcutaneous tissues.

10. Using clippers, shave a 2.5-cm wide area of the dorsum, from just above the hindlegs to the lower border of the ribs (Fig. 14.18.5). Prepare the shaved skin using aBetadine scrub on sterile gauze, followed by alcohol applied with a sterile prep pad.Remove hair from the surgical field.

11. Don a mask and sterile surgical gloves. Use tissue forceps and straight scissorsto make an inverted Y-shaped incision centered at the 4th mammary fat pad(Fig. 14.18.6). Each arm of the incision should be ∼7- to 8-mm in length.


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Figure 14.18.5 Preparation for mammary fat pad injection.

Figure 14.18.6 Incision for mammary fat pad injection.

Perform orthotopic implantation of the cancer cells12. Use forceps to lift the skin at either the left or right corner of the incision and

either scissors or a hemostat to dissect the skin from the underlying fascia in a planeextending laterally. Place the instrument into the incision and gently spread its tips toremove skin from fascia, gradually advancing until the 4th mammary fat pad (MFP)is clearly visible (Fig. 14.18.7).

The MFP is identifiable as a thin layer of tissue, which is pinker than the undersideof the surrounding skin, containing two small blood vessels that converge towards thenipple. Inexperienced operators may find it helpful to use a dissecting microscope for thisprocedure.

13. Gently tap the tube containing the cell suspension to mix it. Draw 20 μl into a 50-μlHamilton syringe. Carefully insert the needle between the skin and the MFP andadvance it 5 mm (Fig. 14.18.8). With the bevel of the needle turned towards the skin


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Figure 14.18.7 Identification of mammary fat pad.

Figure 14.18.8 Injection of cancer cells.

surface, slowly inject the cell suspension. Repeat this procedure for the contralateralMFP.

If the injection is performed correctly, a small bubble should appear within the tissueof the MFP. If liquid is visible on the inner surface of the MFP or the outer surface ofthe skin, the injection was performed incorrectly and a new animal should be selected toensure the accurate delivery of 500,000 cells. A 22-G needle is preferred for this step, asit minimizes shear injury to the cells.

14. Close the incision using wound clips (generally one per arm of the Y; see Fig. 14.18.9).

15. Resterilize the instruments by removing any adherent tissue with an alcohol pad andplacing them in a glass-bead autoclave for 10 sec.

16. Place the mouse on a warmed surface until it has recovered from anesthesia. Add10 ml of sulfamethoxazole/trimethoprim antibiotic suspension to the (full) waterbottle of each cage and maintain for 7 days.


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Figure 14.18.9 Closure of incision.

17. Check mice at 4 hr post-procedure and observe daily for the first 5 days thereafter, foroverall activity, discomfort, and wound problems, such as dehiscence or infection.Thereafter, and throughout the experimental period, carefully monitor the animals 2to 3 times each week. Remove wound clips 7 to 10 days after surgery.

18. Begin compound treatment on the day after cancer cell implantation for preventivemodels, or at a selected time point (2 weeks post-implantation for MDA-MB-231cells or 4 weeks for SUM-1315 cells) for treatment models.

For example, the bisphosphonate, zoledronic acid can be administered subcutaneously asa neutralized stock solution diluted with PBS. The maximum recommended dose for rodenttumor models is 100 μg/kg/week divided into 2 to 7 doses/week. A significant decrease inmetastasis frequency is observed when this compound is tested in the humanized modelusing a dose of 30 μg/kg/week (divided into 2 weekly doses) for 12 weeks, beginning day1 after tumor cell implantation.

Since the MDA cells grow/divide about twice as quickly, their treatment should be started2 weeks post-implantation (as indicated in the step).

19. If tumors are not palpable at 6 to 8 weeks after MFP injection, use bioluminescentimaging (Basic Protocol 3) to verify that cancer cells are present at the orthotopicsite.

If the techniques described are performed correctly, the rate of tumor take is 100%.

20. If any animal experiences significant decline in health, such as excessive loss of bodyweight, dehydration, inability to feed or groom, or if tumors become large enough toimpede the ability of the animal to move about its cage, it should be euthanized (e.g.,cervical dislocation or CO2 asphyxiation). Otherwise, carry out the experiment for aset period after which all animals are sacrificed.

BASICPROTOCOL 3

BIOLUMINESCENT IMAGING OF BREAST CANCER LESIONS IN MICE

Bioluminescent imaging using the IVIS Imaging Systems (Caliper Life Sciences) is ameans for localizing cancer cells expressing the firefly luciferase gene and monitoringtumor growth in situ. Histologic analysis alone may be inadequate for the study ofmetastasis, as lesions can be missed unless the entire tissue block is sectioned andexamined.


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Materials

Mice bearing tumors expressing luciferase (from Basic Protocol 2 or AlternateProtocol)

D-luciferin solution: dissolve powder (e.g., Caliper Life Sciences) in sterile PBS tomake concentration 10 mg/ml(aliquots should be protected from light and maybe stored indefinitely at −20◦ to −80◦C)

10% buffered formalin70% ethanol

IVIS Imaging Systems (Caliper Life Sciences)Anesthesia delivery system (induction chamber, nose cone apparatus, oxygen and

isoflurane source, heated surgical stage; e.g., Colonial Medical Supply)0.5- or 1.0-ml insulin syringes equipped 29-G needlesSurgical instruments for necropsy: scissors and forcepsPetri dishes

Additional reagents and equipment for euthanizing the animals (Donovan andBrown, 2006)

1. Prior to using the imaging equipment, complete a training program provided by thefacility housing the unit and/or the manufacturer.

2. Initialize the system, and turn on the oxygen supply, vacuum, and isoflurane supplyto induction chamber (set at 2% to 3%).

While up to five mice may be imaged simultaneously by an experienced investigator,inexperienced operators should begin by imaging one or two mice at a time.

3. Administer 100 μl (10 U on an insulin syringe) of luciferin i.p. to each mouse. Placemice in the induction chamber and wait 10 min.

Figure 14.18.10 Xenogen image of primary tumors. For the color version of this figure go tohttp://www.currentprotocols.com/protocol/ph1418.


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4. Turn on the isoflurane supply to the imaging unit and turn off the supply to the in-duction chamber. Place the mice supine into the nose cones inside the unit, beginningin the center. Set the field of view (E for five mice, D for three) and binning number(smaller for higher resolution).

5. Close the door and acquire an image. The default exposure time is 1 min. At theend of an experiment, primary tumors are generally visible at 1 sec exposure(Fig. 14.18.10). If the image is saturated, shorten the exposure time or decreasethe aperture by increasing the f-stop.

6. If the endpoint of the experiment has been reached, sacrifice the mice by cervicaldislocation or CO2 inhalation (Donovan and Brown, 2006).

7. Use scissors and forceps to dissect the bone implants and adherent skin, and thencarefully remove the skin and subcutaneous tissue from each mouse and place theimplants on petri dishes marked to identify individual animals. Position bone coressuch that the side that was superficial in situ is touching the dish.

8. Image bone cores beginning with the maximum imaging time (5 min) and shortenexposure time for saturated images. Record implants that display a luminescent focus(Fig. 14.18.11) as positive for metastasis.

The animals’ hind limbs and other organs of interest may also be imaged at this time.Efficiency is important as the concentration of luciferase begins to decrease by 30 minafter injection.

9. Remove each primary tumor and record its weight.

10. Place tissues in formalin for fixation and storage prior to any histological study.Decalcify the bone in formalin for 24 hr and then store indefinitely in 70% ethanol.

Figure 14.18.11 Xenogen imaging of metastatic lesions. For the color version of this figure goto http://www.currentprotocols.com/protocol/ph1418.


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ALTERNATEPROTOCOL

INTRACARDIAC INJECTION OF BREAST CANCER CELLS INTOIMMUNOCOMPROMISED MICE CONTAINING HUMAN BONE TISSUE

This procedure makes it possible to bypass the initial stages of tumor development andto shorten the experimental time by 4 weeks or more compared to the orthotopic model.It may result in metastasis to the mouse liver and brain, as well as the skeleton, and maybe used with Basic Protocol 1, or alone, to examine tumor growth in human or murinebone, respectively. The procedure described can be used for mice that either do or do nothave the human bone implanted.

Materials

Firefly luciferase-expressing, bone-avid human breast cancer cell line, e.g.,MDA-MB-231-luc2 (Caliper Life Sciences)

Medium for cell growth: for MDA-MB-231 cells, RPMI 1640 supplemented with10% fetal bovine serum and penicillin/streptomycin

Phosphate-buffered saline (PBS; Fisher Scientific)6- to 8-week-old female NOD/SCID or nude mice (Jackson Laboratory), or mice

that have had bone implanted (Basic Protocol 1) four weeks earlierBetadine surgical scrubIndividually packaged 70% alcohol prep pads

Hemacytometer or automated cell counterAnesthesia delivery system (induction chamber, nose cone apparatus, oxygen and

isoflurane source, heated surgical stage; e.g., Colonial Medical Supply)Tape (paper surgical or clear office type)Sterile towels and gauzeSurgeon’s mask and gloves, sterile1-ml insulin syringes with 26-G needles

1. Prepare the cells for implantation as in Basic Protocol 2, steps 1 to 6, but suspendthe cells in an appropriate volume of PBS to achieve a concentration of 1,000,000cells/ml.

Do not use Matrigel for intracardiac injection.

2. Turn on the oxygen supply, vacuum, and isoflurane source (set to 2% to 3% initially).Place the mice in the induction chamber.

Once the operator has become familiar with the surgical techniques that follow, three tofive mice housed in one cage can be anesthetized at the same time. Prior to then, miceshould be anesthetized individually.

3. Remove one mouse from the induction chamber and place it in a supine position inthe nose cone apparatus. Verify that the mouse is adequately anesthetized by testingthe pedal reflex.

The pedal reflex is tested by pinching the foot with forceps. The anesthesia is consideredadequate when there is no response to the pinch. Level of anesthesia, respiration, andbody temperature should be monitored throughout the procedure.

4. Secure each of the four limbs using tape, with the hind legs oriented towards theoperator. Prep the chest using Betadine scrub on sterile gauze followed by alcoholapplied with a sterile prep pad.

5. Don a mask and sterile surgical gloves. Gently tap the tube containing the cellsuspension to mix it. Draw 100 μl into a 1-ml syringe equipped with a 26-G needle.Leave an air bubble at the top of the syringe, towards the plunger.

6. While holding the needle angled towards the operator and to the right, insert it intothe second intercostal space, 3 mm to the left of the sternum. Advance 5 mm and


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turn the needle gently until the pulsatile flow of bright red arterial blood is observedentering the hub. Inject the cell suspension (but not the air bubble) over 30 sec.

7. Withdraw the needle and apply pressure on the injection site for 30 sec using analcohol wipe. If dark venous blood is encountered, withdraw the needle and applypressure.

The injection may be reattempted once, but not more than twice, using a fresh needle.

8. Place the mouse on a warmed surface until it has fully recovered from anesthesia.Perform bioluminescent imaging (see Basic Protocol 3) as soon as possible afterintracardiac injection to verify distribution of tumor cells throughout the animal’scirculation.

9. Check the mice at 4 hr post-procedure and observe daily for the next 3 days for overallactivity, discomfort, or wound problems, such as dehiscence or infection. Thereafter,carefully monitor the animals 2 to 3 times per week.

COMMENTARY

Background InformationMetastasis, from the Greek methistanai, “to

remove, change,” is the major cause of mor-tality in cancer and is directly involved in 90%of cancer deaths. The appearance of metastaticlesions usually implies that a patient’s cancerhas become incurable. Unfortunately, our un-derstanding of the factors that make a cancercell able to metastasize lags far behind ourcurrent understanding of the mechanisms oflocal invasion, in part because of the difficultyin modeling what is a complicated, multi-stepprocess. While many characteristics of a pri-mary tumor may be examined at least prelim-inarily by in vitro methods, such techniquescannot be readily adapted to the study of tu-mor metastasis to a secondary tissue. Animalmodels are essential to this endeavor. A briefoutline of the process of metastasis (below)illustrates the complexity of the undertaking.

Cancer develops when a cell gains a sur-vival advantage from genetic aberrations. Be-cause cancer cell genomes are unstable, theyhave a higher than normal rate of mutability,allowing some cells to develop advantageoustraits. Likewise, a population of cancer cellsunder selective pressures from various envi-ronments may evade multiple barriers that thebody has developed to maintain tissue home-ostasis, thus becoming capable of metastasis(Chambers et al., 2002; Nguyen et al., 2009;Tsuji et al., 2009).

At each step there must be a permis-sive interaction between the tumor cell andits environment (Joyce and Pollard, 2009).Specific mutations allow loss of cellular adhe-sion and development of a phenotype with in-

creased motility and invasiveness (Gupta andMassague, 2006). Tumor cells must detachfrom the extracellular matrix, degrade sur-rounding tissue, and be able to move in a di-rected manner (Somerville et al., 2003; Jin andVarner, 2004; Steeg, 2006). Cancer cell inva-sion of tumor-associated vasculature is facil-itated by the so-called “angiogenic switch,”whereby hypoxia or another metabolic stressdrives the up-regulation of genes codingfor proteins (most notably VEGF) associ-ated with the formation of new vascula-ture (Folkman, 2002). These new vessels arestructurally abnormal, being tortuous, dilated,and leaky due to widened intracellular junc-tions and a discontinuous basement membrane(Carmeliet and Jain, 2000; Hida et al., 2008;Baeriswyl and Christofori, 2009).

Some tumor cells enter the bloodstream,which is a harsh environment because of thehigh shear forces, the presence of immunecells, and the lack of a substratum. Death upondetachment (anoikis) contributes to metastaticinefficiency (Douma et al., 2004). Cells thatdo survive may arrest in a capillary bed. Hom-ing to specific target organs is mediated byendothelial cells, which express distinct sur-face proteins in different tissues (Brown andRuoslahti, 2004). The ability of a metastatic tu-mor to grow depends upon its interaction withthe microenvironment of the new tissue, boththe resident elements and cells recruited fromother sites (Kaplan, 2005). Each tissue placesdifferent requirements on invading tumor cells,and those able to successfully colonize a spe-cific location therefore must express all genesnecessary (Fokas et al., 2007) to adapt to their


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surroundings. Very few micrometastases over-come the barriers to becoming clinically rele-vant, macroscopic lesions.

The most common secondary tissue in-vaded by breast and prostate cancer is bone,with 70% of patients with these advancedmalignancies also having bone metastases.A large subset of human cancers, includ-ing myeloma, lung, colon, stomach, blad-der, uterus, rectum, thyroid, and kidney alsodemonstrate osteotropism, the ability to col-onize bone. Bone is an attractive target fortumor cells for several reasons (Boyce et al.,1999; Roodman, 2004). The volume of bloodflow is high in the skeleton (10% of car-diac output), especially in the red marrow.Blood moves sluggishly through marrow si-nusoids, allowing exposure to immune cellsand soluble factors, which promote tumorgrowth. Tumor cells frequently display adhe-sion molecules, which bind to marrow stromalcells and bone matrix (Chung, 1995). Notably,bone is a repository for immobilized growthfactors, which are released during its resorp-tion (Chantrain et al., 2008).

Osseous metastases are characterized as os-teolytic or osteoblastic based on the radio-graphic appearance of lesions, reflecting thedirection in which the normal bone remodel-ing process is dysregulated. In actuality, mostlesions have areas of both osteolytic and os-teoblastic activity (Coleman, 2001), with theinitial event in most malignancies being os-teolysis. Cancer cells that metastasize to bone“hijack” the physiologic mechanisms that con-trol normal bone remodeling (Roodman, 2004)rather than resorbing bone directly. Normalbone remodeling begins with activation of os-teoclasts, which arise from precursor cells ofthe monocyte-macrophage lineage. The keyinteraction appears to be between the recep-tor activator of NF-kappa B ligand (RANKL)on both stromal cells and osteoblasts andRANK, a receptor found on inactive osteo-clasts and dendritic cells. Binding leads toreceptor trimerization and activates multiplesignaling cascades resulting in osteoclast acti-vation, cytoskeletal reorganization, and anti-apoptosis signaling (Teitelbaum and Ross,2003). OPG is an osteoblast-secreted recep-tor that acts as a soluble “decoy receptor”for RANKL, decreasing osteoclastogenesis.The ratio of RANKL to OPG regulates theactivity of osteoclasts. Tumor cells secretea soluble isoform of RANKL in addi-tion to parathyroid-related hormone (Liao andMcCauley, 2006), interleukins, TNFα, andother factors that up-regulate the expression

of RANKL on osteoblasts. The end resultis disruption of the normal balance betweenbone formation and resorption. As osteoclastsare activated and resorb bone, previously im-mobilized growth factors, including TGFβ,BMP, and IGF, are released from the bonematrix (Virk and Lieberman, 2007), stimulat-ing both tumor growth and further bone de-struction. This is the so-called “vicious cycle”of pathogenesis and progression of osteolyticmetastases.

Critical Parameters andTroubleshooting

The success of each procedure described inthis unit is dependent upon the use of metic-ulous, aseptic surgical technique. Each facil-ity’s prerequisite animal handling and surgicaltraining sessions must be completed prior to at-tempting any of these interventions, and micemust be monitored continuously throughoutthe peri- and post-operative periods. For theduration of each experiment, mice should beobserved for loss of more than 15% bodyweight, respiratory difficulty, wound dehis-cence or infection, inability to feed or groomdue to lethargy, dehydration, or tumor growth.If any of these signs are observed, the ani-mal should be sacrificed for humane consider-ations.

Problems that may be encountered duringexperiments and possible explanations are asfollows:

• Inadequate primary tumor growth is likelydue to a problem with cancer cell prepara-tion. Ideally, the cells should have been thawedand plated 2 to 3 weeks prior to implanta-tion. Longer or shorter culture times can resultin decreased viability and poor tumor growth.Cells should be placed on ice immediately fol-lowing preparation and mixed prior to eachinjection. They should not be kept on ice formore than 3 hr and experiments should beplanned accordingly, with the technical skilland experience of the experimenter taken intoconsideration.

• Inadequate generation of bone metastasesmay also be due to problems with cell prepa-ration when the intracardiac injection model isused, or to inadequate primary tumor growthwhen an orthotopic model is used, or to useof nonviable bone implants in the humanizedorthotopic model. Bone segments should beimplanted within 2 to 4 hr of resection of thefemoral head.

• Wound problems are common followingthe bone implantation surgery, being much lessfrequent after mammary fat pad implantation.


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Figure 14.18.12 Connectivity density: number of nodes (points at which 3 or more trabecularelements join, represented by the red ovals), per cubic millimeter.

Figure 14.18.13 MicroCT images of bone implants without metastasis. Two representativesections—note the intact trabeculae.

Small (<5 mm) openings along the staple linewill heal by secondary intention, but ulcerationover the bone implants will not. These animalsmust be sacrificed. If there is any evidence thatskin breakdown is due to the mice fighting,they should be housed individually.

Anticipated ResultsOrthotopic implantation of cancer cells, as

described in this unit, should result in thedevelopment of primary tumors in >95% ofmammary fat pads. Untreated mice orthotopi-cally inoculated with SUM 1315 cells become


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moribund 12 to 14 weeks later, with MDA-MB-231 cells being more proliferative, limit-ing experiments with these cells to 8 weeks.Both cell lines will, on average, producemetastases to 50% of the implanted bone coreswithin those intervals. MDA-MB-231 cellstransplanted by intracardiac injection typi-cally produce widespread metastases to skele-ton, brain, and liver within 4 weeks. All ex-periments should be initiated using 20% moreanimals than are deemed necessary to achievestatistical significance. When planning anexperiment using the humanized orthotopicmodel, note that each femoral head typicallyyields 30 to 40 bone cores. If bone from morethan one patient is required for an experi-ment, the implants from each source should beevenly divided among the treatment groups.

The architecture of trabecular bone maybe analyzed using microCT (Chappard et al.,2008). This modality is particularly usefulwhen samples are uniform. Studies of thegrowth of cancer cells injected directly intothe mouse skeleton are often evaluated withmicroCT (Fritz et al., 2007), as the tibiae ofthe mice are all similar. Parameters measuredinclude bone volume fraction (bone volume/

total volume), trabecular number, trabecularthickness, trabecular spacing, and connectiv-ity density (Xiang et al., 2007). The last ofthese is defined as the number of nodes, orpoints at which three or more trabeculae arejoined, (Fig. 14.18.12) per square millime-ter of tissue volume. In the humanized ortho-topic model, the development of metastasesthat are detectable via bioluminescence is cor-related most closely with a decrease in con-nectivity density (compare Figs. 14.18.13 and14.18.14), but the variability among samples,particularly if bone from more than one patientis used, often prevents attainment of significantresults. MicroCT is also useful in directing thesectioning of fixed tissues, as histologic detec-tion of metastases within the bone implants(Fig. 14.18.15) might otherwise be difficult.

Example experimentThe following is an example of the results

that may be obtained from an experiment uti-lizing the humanized orthotopic model of bonemetastasis from breast cancer and the bispho-sphonate, zoledronic acid. The drugs testedwere anti-resorptive, bone-specific agentsbelieved to delay progression of skeletal

Figure 14.18.14 MicroCT images of bone implants with metastasis. Two representative mid-axialsections. Note the presence of gaps, consistent with infiltration by non-osseous tissue.


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Figure 14.18.15 H and E staining of tumor cells within human bone implant (10x). Two repre-sentative sections showing nests of adenocarcinoma within actively remodeling bone.

metastases. Drug A was a new compoundtargeting RANKL (see Background Informa-tion). The bisphosphonate zolendronic acid(Novartis) was used as a reference drug.

Three groups of twelve mice each were im-planted with human bone (Basic Protocol 1),and 4 weeks later with 500,000 SUM-1315cells via the fourth mammary fat pad (BasicProtocol 2). Beginning one day after mammaryfat pad inoculation, mice were injected subcu-taneously with the appropriate dose of zole-dronic acid [30 mg/kg/week (0.75 mg/mouse);50-μl injections, Monday and Friday] or anequal volume of PBS (control group). Doses

for novel compounds should be chosen basedon manufacturers’ recommendations and lit-erature review. Two to three mice per groupwere lost to post-operative complications orother illness.

After 12 weeks of treatment, biolumines-cent imaging (Basic Protocol 3) was per-formed and the mice sacrificed. Primarytumors were present in 100% of mammaryfat pads and did not differ in weight betweenthe two groups (Fig. 14.18.16). Nine of 18implants in the control group, 2 of 20 in theexperimental group treated with drug A, and3 of 18 in the experimental group treated


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0.001

0.10

0.05

0.15

0.20

0.25

0.30

2

exp 1: primary tumor size (g)

t-test data:p when comparing where

0.6400.6150.858

1 and 21 and 32 and 3

1 control2 drug A3 drug B

3

Figure 14.18.16 Primary tumor size.

01

0.2

0.1

0.3

0.4

0.5

0.6

2

exp 1: bone samples with metastases (proportion)

t-test data:p when comparing where

0.0080.0350.561

1 and 21 and 32 and 3

1 control2 drug A3 drug B

3

Figure 14.18.17 Proportion of bone samples with metastases.


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with zoledronic acid contained metastases(Fig. 14.18.17), indicating that both drugswere effective in decreasing metastasiscompared with the control (p = 0.008, drugA, and p = 0.035, zoledronic acid; all compar-isons were done using student’s t-test). Whilefewer metastases were seen with drug A thanwith zoledronic acid, this result did not reachstatistical significance.

Bone implants treated with drug A werefurther analyzed using microCT; the bonevolume fraction was unchanged by the an-tiresorptive drug, but connectivity density wassignificantly higher (5.5/mm3) in the treatedgroup compared to controls (3.9/mm3).

Time ConsiderationsEach of the three surgical procedures de-

scribed in this unit require at least 15 minper mouse for the new experimenter to com-plete. With practice, this may be decreased to5 min, allowing a study using 50 mice to beprepared in 1 day. Orthotopic models require8 to 12 weeks per experiment, with implanta-tion of human bone cores adding an additional4 weeks prior to cancer cell inoculation. Intrac-ardiac injection experiments can be completedwithin 4 to 6 weeks.

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Current Protocols in Pharmacology || Preclinical Orthotopic and Intracardiac Injection Models of Human Breast Cancer Metastasis to Bone and Their Use in Drug Discovery