The Fibroblast in Tumorigenesis

Stroma

• Structural• Supportive• Dynamic regulatory role• Epithelial-Stromal/Mesenchymal interactions• Developmentally: esp higher• Postdevelopmentally• Pathologically

Stroma and Cancer• Initial focus on cell-autonomous aspects of cancer• Increasing awareness of non-cell autonomous

mechanisms• Tumors not pure: intimate admixture of cells

suggests symbiosis• “Who is invading who?”• From early stages, tumors seam to be able to

recruit / subvert adjacent tissues• Also important for metastasis: e.g. sc vs ortho• Additional consideration: is adjacent tissue normal?

Stroma and Cancer contd

• In terms of volume, stroma may predominate (breast, colon, stomach, panc)

• Stromal response, Desmoplastic response, Desmoplasia…desmos (Gr): a band

• Characterized by marked ECM changes: collagens, FN, PGs, GAGs

• Infiltration of vessels, inflammatory cells

“Tumors as wounds that do not heal”

Fibroblasts and Myofibroblasts

• The major stromal cell type in most cancers• Slender fusiform smooth nucleus• Well developed Golgi• Numerous RER cisternae• Usu smooth cell contour / few short extensions• No plasmalemmal attachments, plaques, dense

patches, basal lamina, IC junctions or cell-to-stroma attachment sites

Fibroblast Ultrastructure

Myofibroblast• Irregular cellular outlines, numerous long

extensions• Connected by adherens and gap junctions• Partly enveloped by basal lamina with

attachment plaques, dense patches, pinocytotic vesicles

• Cell stromal attachment sites• Cytoplasmic stress fibers

Myofibroblast ultrastructure

Myofibroblast phenotype

• FBMFB: Activation of a partial smooth muscle differentiation program: express vimentin, alpha sm actin, sm myosin heavy chain, desmin, calponin, alpha1 integrin

• Altered function:• Increased proliferation rate• Altered ECM protein expression: increased

collagens III, V, decreased Laminin, increased MMPs, TIMPs, GFs

Induction of the Phenotype• Stromal response to diverse injurious stimuli:

injury/wounding, radiation, cancer, pulmonary hypertension/fibrosis, bleomycin lung toxicity

• Desmoplasia mainly due to myofibroblasts: large amounts of collagen and contractile ability

• Some observations in Breast cancer: Higher proportion in invasive vs in situ In invasive ca: seen mainly at invasive front, the younger areas (vs central zone) In in situ: seen in immediate peripheryMechanism…

In Vitro Phenotype of “Carcinoma Associated Fibroblasts”

• Enhanced collagen, hyaluronate synthesis

• Disorganized growth patterns

• Uncontrolled growth, altered proliferation potential, abnormal migratory behaviour

• Increased growth factors: IGF I II, PDGF, TGF beta, HGF, KGF

• Essentially equivalent to myofibroblasts

Expression of “fetal phenotypes” in Cancer patients and Hereditary Cancer patients

(Schor 1986, 1988)

• Migratory behavior on 3D collagen gels• “Cell density migration index” (migration of normal vs transformed fibroblasts differentially

affected by plating density with an inverse correlation in normals)

• Ranges of values for adult, fetal, transformed cells• Fetal phenotype in 17/34 Patients with no previous family history breast cancer (11 benign disease, 23 Cancer) 15/16 patients with +family history of breast cancer (9 benign, 7 cancer) 2/2 1st degree (healthy) relatives of breast cancer patients

Antecol (1986)• Skin fibroblasts from patients with hereditary

cancer syndromes (including HNPCC) show Disorganized actin

Disorganized growth patterns

Decreased requirements for serum in vitro

• Thus far: tumors associated with fibroblasts with altered morphology and behaviour

• Epiphenomenon vs facilitative (even causitive)?• Do CAFs promote tumor growth?

Can CAFs direct progression of initiated human prostate epithelium? (Olumi 1999)

• Hypothesis: CAFs may affect tumor progression in non-tumorigenic cells

• Experiments equivalent to study of genetic determinants of tumorigenesis (initiation, promotion etc)

• Grew CAFs with initiated (TAg) or normal PECs• Looked at change in tumorigenesis and indices

(morphology, RIP, proliferation)• In vivo strategy: mixed ECs and CAFs and set in

collagen gel, then under renal capsule of athymic mice

CAFs cause dramatic tumor progression

when grafted as tissue recombinants with

initiated ECs in vivo

Experimental Manipulation of Stroma to become “Oncogenic” (in absence of tumor)

• Carcinogen-treated stroma heterotypically grafted with untreated ECs (skin, bladder) increased tumorigenicity

• Barcellos-Hoff (2000) looked at the effects of irradiated vs non-irradiated (epithelium-free) mammary stroma on promotion of tumorigenesis

• COMMA-D cells (p53 mutations, yet non-tumorigenic and retain developmental potential)

• Cell transplanted into epithelium-free mammary fat pads (irradiated and non-irradiated)

• Tumor incidence at 6 weeks 81%+/-12SE (Irrad) vs 19%+/-2SE (Sham)• Tumor Size 243mm+/-61.3 (Irrad) vs 30.8mm+/-8.7 (Sham)• Speed of appearance• 100% by 6wks (Irrad) vs 39% by 10wks (Sham)

• Local vs global effects assessed by hemibody radiation: no tumors on non-irradiated side

Mouse models of stromal contribution to tumor progression

• HPV 16-Ker 14 Mice: develop multistage squamous neoplasia: EHADSCCMet

• MMP 9 upregulated early in neoplasia• Crossed to MMP 9 KO mice: delay in dysplasia

formation and fewer carcinomas• Restriction in keratinocyte hyperproliferation• Marrow ablation and transplantion of MMP 9+

hematopoietic cells restores the hyperproliferative phenotype and rate of carcinoma formation

Mouse models of stroma aiding progression (contd)

• mmtv-PyMT x (Csf-1op /Csf-1op)• Recessive null mutation in Csf-1• Early stages of tumorigenesis are preserved• Attenuation of late stages of invasion/metastasis• Difference appears to be in macrophage

recruitment rather than a direct proliferative effect on tumor

• Targeting Csf-1 to mammary epithelium restores MPh recruitment and metastatic potential

• (But ?autocrine effects)

• Clear stromal role in aiding progression• Role in Initiation? Hints from “fetal fibroblasts,

stromal genetic changes..

Sternlicht 1999, Thomasset 1998• Experimental evidence for initiating effects:• Autoactivated MMP-3/Str-1 in mammary

epithelium (WAP promoter) – see the following• Reactive stroma• Mulistage neoplastic progression• TIMP-1 double transgenic: decrease tumorigenesis• Tumors show non-random chromosomal changes

Reactive Stroma in Stl-1 Transgenic mice

Aetiology of “Oncogenic stroma”

1. Cross talk from tumor: co-culture experiments have shown tumor induced FBMFB (gradated pattern of myogenic differentiation) (Ronnov-Jessen, Bissell)

2. Carcinogens (as before)

3.Wound healing: Increased tumor risk at areas of scarring, also areas of chronic inflammation. TGF-beta, DNA damage

4. Stromal Senescence

5.Viral

5. Viral effect on stroma

• Interleukin-6 promotes growth in Multiple Myeloma

• HHV8 (Kaposi’s associated) encodes Interleukin-6

• Rettig examined MM samples for viral sequences

• Found them…

6. Stromal Genetic changes

1. Concurrent, Independent LOH in stroma as well as epithelium in DCIS

2. Mutations in stromal cells: Juvenile Polyposis TSG locus at 10q22

3. Haploinsufficiency of stromal cells

• Neurofibromas: benign tumors of peripheral nerve sheath, but heterogenous (Schwann cells, axons, perineurial cells, fibroblasts), thought reactive hyperplasias in past

• NF1: familial cancer syndrome: germline mutation in neurofibromin, a member of the RasGAP family

• Nf1-/- die before birth. Nf1+/- don’t get neurofibromas

• Conditional Nf1 model: floxed Nf1 allele deleted by Cre transgene under control of Schwann cell-specific promoter Krox-20

• Nf1flox/-;Krox20-cre develop neurofibromas: SC origin

• To assess the effects of heterozygosity for Nf1 in the neighboring cells, compared

Nf1flox/-;Krox20-cre (heterozygous stroma)

Nf1flox/flox;Krox20-cre (wild type stroma)

• ?Heterozygosity in which cell type

Epithelial-Fibroblast Heterotypic signaling

1. TGF-beta

• Protean effects, contribution is not straight forward - supressive at early stages, facilitative in late stages; elevated in many cancers, yet tumors often have mutated pathway (TGFbRI,II, SMAD2 4, Rb) ?suggests a paracrine effect; can have tumor autonomous effects (EMT, SC cell lines more “fibroblastoid” in vivo)

• Important in desmoplastic response

• Preferentially exp at advancing edges and in LN suggests involvement in tumor – stromal interaction

• In vitro: TGFb FB MFB

• Role in bone metastasis..

2. Epithelial PDGF expression• Potent mitogen, chemoattractant for mesenchymal cells• PDGF autocrine signaling loops can cause tumorigenesis

(FB, Glia), but not usually in carcinomas (no R)• Suggests contribution is via paracrine mechanisms• Essential for desmoplastic response in a Human BrCa

xenograft model: abrogated by dom neg PDGF-A• Mechanism of promoting desmoplasia not clear: mitogenic

to FB but does not induce FB MFB: maybe works via MPh recruitment

3.VEGF / Angiogenesis• VEGFs may play an indirect role in early stages of

desmoplasia via increased vascular permeability: fibrin clots infiltration of inflammatory vessels and blood vessels (correlation: VEGF and desmoplasia in some tumors)

• 3. Fibroblast produced ECM• ECM tends to promote tumorigenesis• Coinjection of Matrigel with tumor cells enhances efficiency, decreases

latency, even when stripped of GF• Coinjection of killed fibroblasts with tumor cells: enhanced efficiency of

tumor establishment, but did not increase the rate of growth (vs live FB) – early effect

• 4. Fibroblast produced Growth Factors• May have paracrine effect on tumors, tumors may be dependent on these

in early stages, then become independent (?difficulty in culturing primary human breast cancers)

• IGF I, II, HGF, EGF family (Strong stromal expression of AR, HRG is the prognostic factor)

• Another mechanism for growth factor regulation: Regulation of accessibility of GFs in the ECM (EGF family, TGFb, PDGF, b-FGF etc)

• Fibroblasts may be key gatekeepers e.g. via protease production

• 1. Stand in for oncogenic signaling (need only be temporary)

• 2. Increase in genomic instability

• 3. Supports / regulates angiogenesis

• 4. Selective pressure

• 4. Protect from host response

• etc