Highligts in the Management of

Urogenital CancerMediterranean School of Oncology

CINBO - Rome, May 9-10, 2008

Animal models for the study of prostate cancerMauro Piantelli

University-Foundation “G D’Annunzio”, Chieti

ANIMAL TUMOR MODELS

• Spontaneous Tumors

• Carcinogen-induced tumors (chemical; UV…)

• Mutant, transgenic, knockout mouse models (GEM)

• Transplantable (rodent & human): solid tumors & leukemias

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Tumor Grafts

• Xenograft: across species

• Allograft: to another animal of the same species

• Syngeneic: inbred strain (99% homozygosity)

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Immunodeficient rodent models as xenograft recipients

**Use of X-irradiated mice

• Nude (nu mutation, 1962)

• SCID (loss of V(D)J recombinase)

• bg/nu/xid (combined mutations)

• Additional mutations:– RAG-1 and RAG-2 (RAGnull mutation)– NOD (non-obese diabetes mutation)

A nude mouse is a genetic mutant that has a deteriorated or removed thymus gland, resulting in an inhibited immune system due to a greatly reduced number of T cells. The phenotype, or main outward appearance of the mouse is a lack of body hair, which gives it the "nude" nickname. The nude mouse is valuable to research because it can receive many different types of tissue and tumor grafts, as it mounts no rejection response. These xenografts are commonly used in research to test new methods of imaging and treating tumors. The genetic basis of the nude mouse mutation is a disruption of the FORKHEAD BOX N1 (FOXN1) gene

Severe Combined Immunodeficiency (SCID) is a severe immunodeficiency genetic disorder that is characterised by the complete inability of the adaptive immune system mount, coordinate, and sustain an appropriate immune response, usually due to absent or atypical T and B lymphocytes.

Several forms of SCID occur in animal species. SCID mice are routinely used as model organisms for research into the basic biology of the immune system and the effects of disease on mammalian systems.They have been extensively used as hosts for normal and malignant tissue transplants. In addition, they are useful for testing the safety of new vaccines or therapeutic agents in immunocompromised individuals.Like other forms of SCID, SCID mice present without the ability to make T or B lymphocytes. As such the mice cannot fight infections, nor reject tumours or transplants.The condition is due to an exceedingly rare recessive mutation on Chromosome 16 responsible for deficient activity of an enzyme involved in DNA repair (Prkdc or "protein kinase, DNA activated, catalytic polypeptide"). Because V(D)J recombination does not occur, the humoral and cellular immune systems fail to mature.

Human Cell Lines (e.g.)

LNCaP: Prostate-ADK metastasized to the supraclavicular lymph-node;

androgen-dependent.

PC-3: Prostate-ADK metastasized to the bone;

androgen-independent.

DU-145: Prostate-ADK metastasized to the brain;

androgen-independent.

Sunitinib malate (SU-11248) aloneor in combination with low-dose docetaxel

inhibits the growth of DU-145 prostate cancer xenograftsCumashi A. et al. 2008, submitted

The treatment of reference in hormone refractory prostate cancer consists of chemotherapy with docetaxel combined with prednisone.

Sunitinib malate (SU-11248, Sutent) was developed as an oral multitargeted receptor tyrosine kinase (RTK) inhibitor of PDGF-R, VEGF-R, KIT, and FLT3. (Renal-cell carcinoma, GIST)

Angiogenesis is directly correlated with Gleason score, tumor stage, progression, metastasis and survival

DU-145 cells were selected as they are not detectably hormone sensitive and show high level of KDR and Flt-1

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Metastasis Models

Hunter Nature Reviews Cancer 6, 141–146 (2006)

Sequential Model

Spontaneous Metastasis: Formation of a tumor at the site of implantation followed by distant metastasis

Artificial Metastasis:Colonies formed in distant organs by tumor cells directly injected in the circulationi.v. lungi.s. liveri.c. bone, others

PC-3 growing in the tibia 

GENETICALLY ENGINEERED ANIMAL

MODELS (GEMM)

Early…………...

•Tumor prone inbred strains (i.e associated to the expression of a mouse virus)

•Mouse mutants generated by random germ-line mutagenesis, followed by screening for specific tumor phenotype

Lobund-Wistar ratSpontaneous PCa is very unusual in rat other than this

strain.Spontaneous PCa arose at 25% incidence, in anterior and

dorso-lateral lobes, at 26-28 months (mean age).

Advantages of Lobund-Wistar model are:• Tumor can be induced in high (90%) incidence after

tumor initiation (single dose of methyl-nitrosuurea, NMU), followed by repeated implantation of testosterone propionate (tumor promoter).

• Tumor are adenocarcinoma and can metastasize.• Early hyperplastic epithelial lesions appear prior to

palpable cancers.• Hormonal dependence of this model:• testosterone is the promoter and estradiol,

dihydroxytestosterone, and castration inhibit cancer formation.

Copenhagen rat has an incidence of microscopic PCa of only 10% by the end of life, and an incidence of macroscopic cancer of 1-2%.

In contrast, the ACI/Seg strain (derived by crossing an AUG strain with an inbred COP rat) has an incidence of microscopic PCa >80%, and of macroscopic cancer of 15%.

Dunning rat• The R-3327 Dunning rat adenocarcinoma was

derived from a spontaneous papillary adenocarcinoma of the dorsa prostate of an aging Copenhagen rat.

• A number of sublines have been derived from R-3327 Dunning cancer, including G and H sublines, and these have given rise to sub-sublines.

• There are a wide variety of sublines that vary from well differentiated, diploid, androgen-sensitive, and non-metastatic to anaplastic, aneuploid, androgen-independent, and metastatic

Prostate, rat, ventral aspect

Lateral lobes not visible, dorsal lobe: only caudal part visible

Prostate, rat, dorsal aspect

Rat, abdominal cavity, ventral aspect.

Cg: coagulation gland, Dd: deferent duct, Sv: seminal vesicle,Ub: urinary bladder, Vl: ventral lobe of prostate

The urethra is dissected.(Cg: coagulation gland, Sv: seminal vesicle,

Ub: urinary bladder,Ur: urethra)

Removal of prostate, urinary bladder, and seminal vesicles as a unit.

Cg: coagulation gland, Dl: dorsolateral lobe of prostate, Sv: seminal vesicle,Ub: urinary bladder, Ur: urethra, Vl: ventral lobe of prostate

Outer" aspects of freshly dissected (left) and fixed (right) prostate.

Cg: coagulation gland, Dl: dorsolateral lobe of prostate,Sv: seminal vesicle, Ur: urethra, Vl: ventral lobe of prostate

Inner" aspects of fixed (left) and freshly

dissected (right) prostate.

Compared to the situation in the living animal (in situ), the ventral prostate is flipped back.Cg: coagulation gland, Dl: dorsolateral lobe of prostate, Dsv: duct of seminal vesicle,Sv: seminal vesicle, Ub: urinary bladder, Ur: urethra, Vl: ventral lobe of prostate

The prostate lobes are embedded with the "outer" aspects down, i.e. the dorsolateral lobes with the dorsal surface down, and the ventral prostate with

the ventral surface down, because this part was flipped back.

Dd: deferent duct, Dl: dorsolateral lobe of prostate, Sv: seminal vesicle, Ur: urethra, Vl: ventral lobe of prostate

White solid arrows: primary tumorWhite hollow arrows: mesenteric lymph nodesPink arrows: inguinal lymph nodes,Green arrow: axial lymph node

GENETICALLY ENGINEERED ANIMAL

MODELS (GEMM)

Clin Cancer Res, 12, 2006Carver B.S. and Pandolfi P.P.

GENETICALLY ENGINEERED ANIMAL MODELS (GEMM)

Engineering the mouse genome: a powerful tool for gene discovery and validation in cancer genetics

Mouse models to study initiation, progression and treatment of cancer

Early…………...

•Tumor prone inbred strains (i.e associated to the expression of a mouse virus)

•Mouse mutants generated by random germ-line mutagenesis, followed by screening for specific tumor phenotype

………..80’: Conventional transgenic and knock out mice

•Mice produced by oocytes injected with oncogenes (transgene) were tumor prone

•Possibility of introducing specific mutations into endogenous genes and transmitting these through the mouse germ line (gene targeting):

•knock out •knock inn

……..to-day: Conditional mouse tumor model

Conditional gene mutation (or controllable oncogene expression), allows to induce somatic mutations in tissue specific (target gene under a tissue specific promoter) and time controlled fashion (drug-inducible system)

(Jokers and Berns :Nature Reviews Cancer, 2:251,2002

The last decade has seen a remarkable shift in animal-based cancer research from xenografted tumor models toward transgenic models of disease.

The intellectual motivation behind this transformation is the belief that transgenic models will recapitulate the complete course of carcinogenesis more accurately.

Transgenic model of prostate cancer:the TRAMP mouse

This hypothesis is rooted in the recognition of several advantages that transgenic models offer when compared to xenograft systems.

Among these are that the process of carcinogenesis begins with normal cells, progresses through distinct genetic and histological stages, occurs in an immuno-competent host, occurs in its proper cellular microenvironment, and that metastasis can occur along routes and to sites relevant to clinical disease.

Transgenic model of prostate cancer:the TRAMP mouse

In the TRAMP model, expression of the SV40 early genes (T and t antigen, Tag) are driven by the prostate-specific promoter probasin that leads to cell transformation within the prostate.

One hundred percent of male TRAMP mice develop PCa without any chemical or hormonal treatment, and, as in man, these mice develop Prostatic Intraepithelial neoplasia (PIN), well- or moderately-differentiated adeno-carcinomas that express androgen receptors, undifferentiated carcinomas that do not express it, and phyllodes tumors in the seminal vescicles.

TheTRAMP mouse is a model for PCa in which progressive forms of the disease occurs in a manner similar to the human disease and, therefore, findings of chemopreventive efficacy of a given treatment in this model have potential clinical significance

The TRAMP mouse has become a popular preclinical model for studying chemoprevention of PCa, and has been used to elucidate the antitumorigenic effects of several classes of chemopreventive regimens, including antiandrogen, ornithine decarboxylase inhibitors, green tea polyphenols, COX-2 inhibitors, phytoestrogens, retinoic acid, apigenin and flavonolignans.

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IL-6-type cytokine family

4. Leukemia inhibitory factor (ns)5. Ciliary neurotrophic factor (nd)6. Cardiotrophin-1 (nd)7. Cardiotrophin like cytokine (nd)

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Angiogenic cytokines

Anti-angiogenic cytokines

IL-8: ns

In germ-free Lobund-Wistar rats,

the incidence of spontaneous cancers

is only 20% that of conventional L-W rats.

LEGISLATIONAnimal right organisation continue to

campaign for elimination of in vivo studies

Governments have enacted legislation to control more carefully the use of animals in research

Use validated alternative when available

Use animals rationally and responsibly

Follow specific information on animal care and welfare