Metastasis and Routes of Spread

Key Concept: Why Metastasis is Inefficient

The Three Pathways (Routes) of Metastatic Spread

1. Seeding of Body Cavities and Surfaces

• Most commonly involves the peritoneal cavity
• Other surfaces affected: pleura, pericardium, meninges, and joints
• Classic example: Ovarian carcinoma - spreads widely over peritoneal surfaces yet may not invade the underlying tissues; often covers the peritoneal surfaces with a heavy cancerous coating
• Mucus-secreting appendiceal or ovarian carcinomas may fill the peritoneal cavity with gelatinous neoplastic mass = pseudomyxoma peritonei
• CNS tumors (e.g., medulloblastoma, ependymoma) may penetrate cerebral ventricles and be carried by cerebrospinal fluid to implant on meningeal surfaces

2. Lymphatic Spread

• Tumors do not contain functional lymphatic vessels; lymphatics at the tumor margins are sufficient for spread
• The pattern of spread follows natural routes of lymphatic drainage
• Skip metastasis can occur - cancer cells bypass the immediately proximal nodes and are trapped in subsequent nodes (possibly due to variation in normal lymphatic drainage patterns)
• Sentinel lymph node = the first regional lymph node receiving lymph flow from a primary tumor; can be identified by injection of dyes or radiolabeled tracers near the tumor; biopsy determines extent of spread

Note: Enlarged lymph nodes near a primary neoplasm do not always indicate cancerous involvement - necrotic tumor cells and tumor antigens can evoke reactive hyperplasia (lymphadenitis). Biopsy is necessary.

3. Hematogenous Spread

• Tumor cells preferentially penetrate thin-walled veins rather than thick-walled arteries
• Cells arrest in the first capillary bed encountered
• Portal blood flows to liver → liver is most common site
• Caval blood flows to lungs → lungs are most common site
• Renal cell carcinoma and hepatocellular carcinoma have a propensity to grow within veins in a snakelike fashion, extending up the IVC to the right heart

• Skeletal muscle, despite being rich in capillaries, is rarely a site of metastasis

The Metastatic Cascade (Hematogenous Route - Step by Step)

Phase 1: Invasion of the Extracellular Matrix (ECM)

• E-cadherin (transmembrane glycoproteins) function is lost due to mutations
• Epithelial-Mesenchymal Transition (EMT) - silences E-cadherin expression
  • Controlled by transcription factors SNAIL and TWIST
  • Downregulates epithelial markers (E-cadherin)
  • Upregulates mesenchymal markers (vimentin, smooth muscle actin)
  • Creates a promigratory phenotype

• Tumor cells secrete proteolytic enzymes OR induce stromal cells (fibroblasts, inflammatory cells) to produce them
• Key enzymes: Matrix metalloproteinases (MMPs), cathepsin D, urokinase plasminogen activator
• MMP9 (gelatinase): cleaves type IV collagen in basement membranes; also releases VEGF from ECM-sequestered pools
• Benign tumors have little MMP9 activity vs. malignant counterparts which overexpress it
• Metalloproteinase inhibitor concentrations are also reduced in many cancers

• Tumor cells bind to newly exposed ECM binding sites created by protease cleavage

• Tumor cells move through degraded basement membrane and interstitial connective tissue
• Guided by chemotactic factors produced by inflammatory and stromal cells

Phase 2: Vascular Dissemination, Tissue Homing, and Colonization

1. Intravasation - tumor cells penetrate blood vessel/lymphatic walls
2. Circulation as tumor cell emboli - often coated with platelets (protective)
3. Interaction with host lymphoid cells - surviving immune surveillance
4. Arrest in distant capillary bed - adhesion to basement membrane
5. Extravasation - exit from blood vessel
6. Formation of metastatic deposit - colonization of new tissue
7. Angiogenesis and growth at metastatic site

Liver Studded with Metastatic Cancer (Gross Pathology)

Tumors That Rarely Metastasize

• Gliomas (malignant CNS tumors) - rarely metastasize
• Basal cell carcinomas of skin - rarely metastasize
• This demonstrates that local invasiveness and metastatic potential are separable properties

Special Case: Leukemias and Lymphomas ("Liquid Tumors")

• Derived from blood-forming cells that normally circulate
• Assumed to be disseminated at diagnosis by definition
• Always considered malignant regardless of spread

Molecular Basis of Carcinogenesis

The Hallmarks of Cancer

I. Types of Cancer Genes

A. Proto-Oncogenes → Oncogenes (Gain-of-function)

• Proto-oncogenes: Normal cellular genes whose products promote cell proliferation
• Oncogenes: Mutated or overexpressed versions of proto-oncogenes that function autonomously - having lost dependence on normal growth-promoting signals
• Oncoproteins: Proteins encoded by oncogenes that drive increased cancer cell proliferation

How Proto-Oncogenes Become Oncogenes:

• BCR::ABL fusion (t(9;22), Philadelphia chromosome) → Chronic Myeloid Leukemia (CML)
• MYC overexpression via t(8;14) → Burkitt lymphoma
• BCL2 overexpression via t(14;18) → Follicular lymphoma (anti-apoptotic)

Key Oncoproteins by Category:

• PDGF-β → astrocytoma
• TGF-α → many carcinomas (autocrine loop)

• EGFR - point mutations → lung adenocarcinoma (targetable with gefitinib/erlotinib)
• HER2/ERBB2 - gene amplification → breast cancer (targetable with trastuzumab)
• RET - point mutations → multiple endocrine neoplasia (MEN) type 2
• Oncogenic RTK mutations cause constitutive, growth factor-independent tyrosine kinase activity

• ABL → BCR::ABL fusion → CML (targetable with imatinib)
• JAK2 → point mutations → myeloproliferative neoplasms

• RAS (KRAS, NRAS, HRAS) → mutated in ~30% of all cancers
  • Normally: GDP-bound (inactive) ↔ GTP-bound (active)
  • Oncogenic mutation: blocks GTPase activity → constitutively GTP-bound = constitutively active
  • Activates downstream MAPK and PI3K/AKT pathways
  • BRAF (downstream of RAS) → point mutations → melanoma, colorectal cancer

• MYC - master transcription factor; regulates genes for rapid cell growth
  • Overexpressed via chromosomal translocations (Burkitt), gene amplification (neuroblastoma)
  • Drives expression of cyclins → cell cycle progression

• CDK4 overexpression/mutation → accelerates G1→S transition (many cancers)
• Cyclin D1 amplification → breast, esophageal cancers

B. Tumor Suppressor Genes (Loss-of-function)

• Familial retinoblastoma: inherit one defective RB allele (1st hit) + acquire somatic mutation in normal allele (2nd hit) → tumor
• Sporadic retinoblastoma: two independent somatic mutations required → rarer, usually unilateral

The RB Gene (Retinoblastoma protein)

• Hypophosphorylated RB (active form): binds and inhibits the E2F transcription factor → blocks cell cycle progression in G1
• Growth factor signaling activates CDK4/6 + cyclin D → phosphorylates RB → releases E2F → transcription of S-phase genes → cell cycle entry
• Oncogene mutations (CDK4, cyclin D amplification) or loss of CDK inhibitors (p16/INK4a) all converge on RB inactivation

The TP53 Gene ("Guardian of the Genome")

• In non-stressed cells: p53 is bound and ubiquitinated by MDM2 → proteasomal degradation (virtually undetectable)
• MDM2 gene is amplified in 33% of sarcomas → functional p53 deficiency without TP53 mutation
• HPV E6 protein binds p53 and promotes its degradation (mechanism in cervical carcinoma)

• DNA damage/hypoxia → ATM/ATR kinases phosphorylate p53 and MDM2 → disrupts p53-MDM2 binding → p53 accumulates
• Oncogenic stress (RAS activation) → p14/ARF (encoded by CDKN2A) → binds MDM2 → displaces p53

1. Transient cell cycle arrest → DNA repair → resume division (if repair successful)
2. Senescence (permanent cell cycle arrest)
3. Apoptosis (programmed cell death) - via transcription of pro-apoptotic genes (BAX, PUMA)

Other Key Tumor Suppressor Genes:

II. Mechanisms of Oncogene Activation - Key Signaling Pathways

1. Growth factor binds receptor
2. Transient receptor activation → cytoplasmic signal transducers (RAS, PI3K)
3. Signal transmission to nucleus
4. Transcription factor activation
5. Gene expression → cell cycle entry + proliferation

RAS-MAPK Pathway

• RAS mutation → constitutive MAPK activation → transcription of pro-proliferative genes (FOS, JUN, MYC)

PI3K-AKT-mTOR Pathway

• PI3K mutations/PTEN loss → constitutive AKT activation → promotes cell survival (inhibits apoptosis), protein synthesis, Warburg effect

Wnt-β-catenin Pathway

• APC loss or β-catenin mutations → β-catenin accumulates in nucleus → activates MYC, cyclin D1 → proliferation

III. Molecular Basis of Multistep Carcinogenesis

Normal epithelium
     ↓ APC mutation (early)
Small adenoma
     ↓ KRAS mutation
Larger adenoma
     ↓ SMAD4/TGF-β loss
     ↓ TP53 mutation (late)
Adenocarcinoma

• Certain mutations occur early (APC in colon cancer)
• Others occur late (TP53)
• Similar stepwise progression documented for cervical dysplasia, endometrial hyperplasia, epidermal dysplasia

IV. Epigenetic Contributions

• DNA methylation (especially at CpG islands of tumor suppressor promoters) → gene silencing
• Histone modifications → alter chromatin accessibility → activate oncogenes or silence suppressors
• Cancer cells have abnormal methylation patterns - global hypomethylation (genomic instability) + focal hypermethylation (silencing tumor suppressors)

V. Genomic Instability - The Enabling Hallmark

• Driver mutations: directly contribute to malignant phenotype
• Passenger mutations: neutral, accumulated due to mutator phenotype (hundreds to thousands in carcinogen-induced tumors)
• By clinical detection (~10⁹ cells, ≥30 doublings), most tumors are genetically heterogeneous across subclones
• Tumor subclones undergo Darwinian selection - the fittest subclones dominate, explaining tumor progression and therapy resistance

VI. Tumor Initiation and Promotion (Chemical Carcinogenesis Model)

• Initiation: permanent DNA damage (mutation) from carcinogenic agent - irreversible
• Promotion: stimulation of initiated cell proliferation by promoters (not mutagenic alone); drives clonal expansion of mutated cells → further mutations → fully malignant clone
• Promoters are reversible (removing them stops progression at early stages)

Mechanism of Metastasis

Why is Metastasis so Inefficient?

PHASE 1: Invasion of the Extracellular Matrix

• Basement membrane - surrounds epithelium and vascular endothelium
• Interstitial connective tissue - fills spaces between cells

Step 1 - Loosening of Tumor Cell-Tumor Cell Interactions

• Direct mutation - loss-of-function mutations in CDH1 gene (e.g., gastric adenocarcinomas, lobular breast carcinoma)
• Epithelial-Mesenchymal Transition (EMT) - E-cadherin is transcriptionally silenced, at least transiently

• Controlled by transcription factors SNAIL and TWIST
• Not just E-cadherin loss - a full phenotypic reprogramming:

Step 2 - Degradation of the Basement Membrane and ECM

• Release of VEGF sequestered in ECM → promotes angiogenesis
• Generation of collagen/proteoglycan cleavage products with chemotactic, angiogenic, and growth-promoting effects
• Creation of novel ECM binding sites that draw tumor cells forward (Step 3)

Step 3 - Attachment to Remodeled ECM Components

• In normal epithelial cells: integrins binding basement membrane laminin and collagens are restricted to the basal surface → keeps cells resting and polarized
• Loss of ECM adhesion normally triggers anoikis (apoptosis from loss of anchorage; literally "without a home")
• Tumor cells resist anoikis by expressing alternative integrins that transmit survival signals despite ECM detachment

Step 4 - Migration and Invasion

Attach at leading edge → detach at trailing edge → contract actin cytoskeleton → move forward

1. Tumor cell-derived autocrine motility factors - chemokines and growth factors (e.g., insulin-like growth factors, IGF)
2. ECM cleavage products - collagen and laminin fragments acting as chemotactic gradients
3. Stromal paracrine factors - most importantly hepatocyte growth factor/scatter factor (HGF/SF), which binds the receptor tyrosine kinase MET on tumor cells → potent stimulation of motility

PHASE 2: Vascular Dissemination, Homing, and Colonization

Survival in Circulation

• Mechanical shear stress
• Anoikis (apoptosis from loss of anchorage)
• Innate and adaptive immune destruction

• Tumor cell emboli/aggregates are far more likely to establish metastases than single cells
• Platelet coating - tumor cells associate with platelets via homotypic and heterotypic interactions; platelet aggregates stabilize emboli and enhance survival
• Tumor cells may express anionic substances (e.g., polyphosphate) that activate factor XII → fibrin deposition → further stabilizes emboli
• Circulating as a group provides collective competence - collectively more likely to possess all properties needed for metastasis, including stem cell-like properties for plasticity in a new microenvironment

Organ Tropism - Where Metastases Settle

• Portal venous drainage → liver (colon cancer)
• Systemic venous drainage → lungs
• Paravertebral venous plexus (Batson's plexus) → spine (prostate, thyroid)

• Adhesion molecules - tumor cells express adhesion molecules (e.g., CD44) whose ligands are preferentially expressed on endothelial cells of target organs. CD44 normally guides T lymphocytes to lymphoid tissues by binding hyaluronate on high endothelial venules; solid tumors express CD44 to exploit the same mechanism
• Chemokine receptors - some cancer cells express chemokine receptors (e.g., CXCR4) that guide them toward tissues expressing the matching chemokine (CXCL12), mimicking how immune cells home to tissues
• Seed-soil hypothesis (Paget) - certain tissues provide a favorable "soil" for specific tumor "seeds"; tissues with high vascularity but rare metastases (skeletal muscle, spleen) represent "unfavorable soil"

Extravasation

• Tumor cells undergo transmigration between endothelial cells + egress through basement membrane
• Requires: integrins, laminin receptors, proteolytic enzymes, and chemokines (from tumor cells and innate immune cells like monocytes and neutrophils)
• Mechanisms differ based on endothelium type: fenestrated endothelium (liver, bone marrow) vs. tight-junction endothelium (brain)

Colonization and Tumor Dormancy

Tumor cells secrete cytokines, growth factors, and ECM molecules → modify resident stromal cells → stromal cells make the metastatic site habitable ("pre-metastatic niche")

1. Breast cancer cells secrete PTHrP (parathyroid hormone-related protein)
2. PTHrP stimulates osteoblasts to produce RANK ligand (RANKL)
3. RANKL activates osteoclasts → bone matrix degradation
4. Bone matrix releases embedded growth factors: IGF, TGF-β
5. IGF and TGF-β bind receptors on cancer cells → activate survival/proliferation signaling

Summary: The Metastatic Cascade at a Glance

Primary tumor growth → angiogenesis
         ↓
Selection of metastatic subclone (EMT: SNAIL/TWIST activation, E-cadherin loss)
         ↓
STEP 1: Loosening of cell-cell contacts (E-cadherin loss / EMT)
         ↓
STEP 2: ECM degradation (MMP2, MMP9, uPA, cathepsin D)
         ↓
STEP 3: Attachment to remodeled ECM (altered integrins, anoikis resistance)
         ↓
STEP 4: Migration (autocrine IGF, HGF/MET, actin cytoskeleton ratcheting)
         ↓
INTRAVASATION (penetrate vascular basement membrane)
         ↓
CIRCULATION (platelet coating, fibrin stabilization of tumor emboli)
         ↓
Immune evasion in bloodstream
         ↓
ARREST in capillary bed (organ-specific adhesion: CD44, CXCR4)
         ↓
EXTRAVASATION (integrins, proteases, chemokines)
         ↓
COLONIZATION (stromal remodeling, pre-metastatic niche, PTHrP-RANKL-osteoclast axis)
         ↓
Angiogenesis + Growth = METASTATIC TUMOR

• Invasion occurs in 4 steps: loosening of cell-cell contacts → ECM degradation → attachment to remodeled ECM → migration
• E-cadherin inactivation (mutations or EMT via SNAIL/TWIST) is the first step
• MMPs and cathepsins mediate ECM degradation; also release pro-tumorigenic growth factors and generate chemotactic fragments
• Metastatic site is predicted by vascular drainage (first-pass) AND organ-specific tropism
• CD44 and chemokine receptors (CXCR4) mediate organ-specific homing
• EMT transcription factors SNAIL and TWIST are the master regulators of the invasive phenotype