Invasion and Metastasis - Robbins Pathology

1. Invasion of the ECM
2. Vascular dissemination, tissue homing, and colonization

Phase 1: Invasion of the Extracellular Matrix

• Breach the underlying basement membrane
• Traverse the interstitial connective tissue
• Penetrate the vascular basement membrane to enter circulation

Step 1 - Loosening of Tumor Cell-Tumor Cell Interactions

• Mutational inactivation of E-cadherin genes
• Activation of beta-catenin genes
• Epithelial-Mesenchymal Transition (EMT) - controlled by transcription factors SNAIL and TWIST, which suppress E-cadherin expression

Step 2 - Degradation of the Basement Membrane and Interstitial Matrix

• Matrix Metalloproteinases (MMPs) - especially MMP-9 (gelatinase), which cleaves Type IV collagen of the basement membrane
• Cathepsin D
• Urokinase plasminogen activator

• Release VEGF sequestered within the ECM (promoting angiogenesis)
• Generate chemotactic fragments from ECM glycoproteins
• Create angiogenic and growth-promoting cleavage products

Step 3 - Attachment to Novel ECM Components

• Laminin receptors on tumor cells bind to laminin in the basement membrane - these are overexpressed in many aggressive carcinomas
• Fibronectin receptors facilitate attachment to interstitial matrix

Step 4 - Migration and Invasion of Tumor Cells

• Autocrine motility factors secreted by tumor cells themselves
• Paracrine factors from stromal/inflammatory cells (chemotactic gradients)
• Cleavage products of ECM components (collagen, laminin fragments) which are chemotactic
• Growth factors like IGF-I and IGF-II released from degraded ECM

Phase 2: Vascular Dissemination, Tissue Homing, and Colonization

Intravasation and Circulation

• Tumor cells enter blood vessels or lymphatics
• In the circulation, they are vulnerable to immune destruction (NK cells, T cells)
• They may form tumor cell emboli protected by aggregates of platelets and fibrin - this shields them from immune attack
• Most circulating tumor cells are destroyed; only rare cells produce metastases, explaining why the metastatic process is highly inefficient

Arrest and Extravasation

• Tumor cells arrest in the first capillary bed encountered - most commonly lungs and liver
  • Liver is the first capillary bed for portal venous drainage
  • Lungs are the first capillary bed for systemic venous drainage
• Once arrested, cells adhere to the vascular endothelium (via adhesion molecules) and then extravasate using the same mechanisms as intravasation

Organ Tropism

• Expression of chemokine receptors on tumor cells whose ligands are expressed by endothelial cells at the target site
  • Example: Breast cancer cells express CXCR4, whose ligand CXCL12 (SDF-1) is abundantly expressed in lungs, liver, and bone marrow
• Expression of specific adhesion molecules on target organ endothelium

• Prostate cancer - bone
• Breast cancer - lung, liver, bone, brain
• Colon cancer - liver
• Neuroblastoma - adrenal/liver

Colonization - Formation of Macroscopic Metastases

• Tumor cells first form micrometastases
• To grow into macroscopic tumors, they must adapt to a foreign microenvironment
• This requires:
  • Evasion of local immune responses
  • Induction of angiogenesis at the new site
  • Modulation of stromal cells in the new niche
  • Possible dormancy - micrometastases may remain dormant for years before reactivating

Summary Table

Why is Metastasis so Inefficient?

1. Mechanical destruction in the circulation
2. Immune attack by NK cells and CTLs
3. Failure to adhere and extravasate successfully
4. Inability to adapt to a foreign microenvironment
5. Failure to induce angiogenesis at the new site

• Robbins & Kumar Basic Pathology (Robbins Pathology), Chapter 6 - Neoplasia
• Robbins, Cotran & Kumar Pathologic Basis of Disease, Chapter 7 - Neoplasia

Acute Inflammation - Robbins, Cotran & Kumar Pathologic Basis of Disease

Definition and Overview

1. Dilation of small vessels leading to increased blood flow
2. Increased permeability of the microvasculature - allowing plasma proteins and leukocytes to leave the circulation
3. Emigration of leukocytes from the microcirculation, their accumulation at the focus of injury, and activation to eliminate the offending agent

Cardinal Signs of Inflammation

Phase 1: Vascular Reactions

A. Changes in Vascular Flow and Caliber

1. Transient vasoconstriction (seconds) - initial neurogenic reflex
2. Vasodilation - induced by histamine and other mediators acting on vascular smooth muscle; involves mainly postcapillary venules; causes heat and redness
3. Increased permeability - protein-rich fluid pours into the extravascular tissues
4. Stasis - loss of fluid + increased vessel diameter = slower blood flow and red cell concentration in small vessels (vascular congestion)
5. Margination - as stasis develops, neutrophils accumulate along the endothelium

B. Exudate vs. Transudate

C. Increased Vascular Permeability (Vascular Leakage)

1. Contraction of endothelial cells - opening of interendothelial gaps; elicited by histamine, bradykinin, leukotrienes; called the immediate transient response (15-30 minutes); the most common mechanism
   • A delayed prolonged form (2-12 hours, lasting days) may occur with mild injury (e.g., sunburn) due to continued endothelial contraction or mild endothelial damage
2. Endothelial injury - necrosis and detachment from severe physical injuries, thermal burns, microbial toxins, or neutrophil-induced damage; leakage starts immediately and is sustained until vessels are repaired

D. Lymphatic Response

• Lymph flow increases to drain excess edema fluid
• Leukocytes, cell debris, and microbes may enter lymphatics
• Lymphangitis (inflamed lymphatics) and lymphadenitis (enlarged, inflamed draining lymph nodes) may follow
• Red streaks near a skin wound = inflamed lymphatic channels = diagnostic of lymphangitis

Phase 2: Cellular Events - Leukocyte Recruitment

Steps of Leukocyte Recruitment

• As blood flow slows (stasis), leukocytes (especially neutrophils) accumulate along the vessel wall (margination)
• They then roll along the endothelium via selectins
  • P-selectin (stored in Weibel-Palade bodies of endothelium and platelet alpha granules; rapidly mobilized by histamine/thrombin)
  • E-selectin (expressed by activated endothelium; induced by TNF, IL-1)
  • L-selectin on leukocytes binds to ligands on endothelium

• Rolling leukocytes are activated by chemokines on the endothelial surface
• Chemokines trigger conformational change in integrins on leukocytes - increasing their affinity for endothelial ligands
• Integrins (LFA-1, Mac-1 on leukocytes) bind to ICAM-1 on endothelium (induced by TNF and IL-1)
• This results in firm, stable adhesion

• Leukocytes squeeze between endothelial cells
• PECAM-1 (CD31) on leukocytes and endothelial cells mediates this migration
• After crossing the endothelium, leukocytes penetrate the basement membrane using collagenases

• Leukocytes migrate through tissues toward the site of injury guided by a chemotactic gradient
• Exogenous chemotactic agents: bacterial products (especially formyl-methionine peptides - f-Met-Leu-Phe)
• Endogenous chemotactic agents:
  • Complement fragment C5a
  • Leukotriene B4 (LTB4)
  • IL-8 (CXCL8) / other chemokines

Sequence of Leukocytes at the Site

• 0-6 hours: Neutrophils dominate (first responders; short-lived in tissues - die by apoptosis within hours to a few days)
• 24-48 hours: Monocytes arrive and differentiate into macrophages (more long-lived, produce cytokines and growth factors for repair)

Phase 3: Phagocytosis and Destruction

Phagocytosis - Three Steps

1. Recognition and attachment - enhanced by opsonins:
   • IgG antibodies (Fc receptors on phagocytes)
   • C3b complement fragment (complement receptors)
2. Engulfment - pseudopods enclose the particle, forming a phagosome, which fuses with lysosomes to form a phagolysosome
3. Intracellular killing:
   Oxygen-dependent (most important):
   • Respiratory burst - activation of NADPH oxidase converts O₂ to superoxide (O₂⁻), then to H₂O₂, then HOCl (hypochlorous acid via myeloperoxidase) - the most potent bactericidal agent
   • Nitric oxide (NO) - produced by macrophages via iNOS
   Oxygen-independent:
   • Lysozyme - degrades bacterial cell walls
   • Lactoferrin - chelates iron
   • Major basic protein (eosinophils) - against parasites
   • Defensins - antimicrobial peptides

Neutrophil Extracellular Traps (NETs)

• Neutrophils can extrude their nuclear chromatin decorated with antimicrobial proteins (elastase, histones, MPO)
• These NETs trap and kill extracellular microbes
• Also seen in sepsis where they can cause vascular damage

Mediators of Inflammation

Arachidonic Acid Metabolism

• Cyclooxygenase (COX) pathway → Prostaglandins + Thromboxane
  • Aspirin/NSAIDs block COX (both COX-1 and COX-2)
  • Selective COX-2 inhibitors (celecoxib) spare gastric mucosa
• 5-Lipoxygenase pathway → Leukotrienes (LTB4, LTC4, LTD4, LTE4)
  • Leukotriene receptor antagonists (montelukast) used in asthma
• Lipoxins - produced via the 5-LOX pathway; anti-inflammatory - they suppress neutrophil recruitment (switch from LT to lipoxin production is one active termination mechanism)

Termination of Acute Inflammation

1. Mediators have short half-lives and are degraded after release
2. Neutrophils die by apoptosis within hours to days
3. Switch from leukotrienes → lipoxins (antiinflammatory)
4. Liberation of TGF-beta and IL-10 from macrophages
5. Cholinergic neural impulses inhibit TNF production in macrophages

Morphologic Patterns of Acute Inflammation

1. Serous Inflammation

• Accumulation of protein-rich but cell-poor fluid (watery exudate) in body cavities or into tissue
• Example: blister formation in skin burns; pleural effusion in early pleuritis
• The fluid originates from serum or mesothelial cell secretion

2. Fibrinous Inflammation

• More severe vascular leakage allows large proteins (fibrinogen) to pass out → deposited as fibrin
• Occurs on organ surfaces, especially pericardium ("bread-and-butter pericarditis") and pleura
• May be resolved by fibrinolysis (resolution) or organized into scar (organization)

3. Purulent (Suppurative) Inflammation

• Production of large amounts of pus (neutrophils + dead cells + microbes)
• Caused by pyogenic bacteria (e.g., staphylococci)
• Abscess = localized collection of pus with central necrosis surrounded by neutrophils, then granulation tissue

4. Ulcers

• A local defect (excavation) of the surface of an organ or tissue, caused by sloughing of inflamed necrotic tissue
• Typical sites: stomach, duodenum, lower legs (vascular insufficiency)
• Characterized by acute and chronic inflammation at the base and margins

Outcomes of Acute Inflammation

1. Resolution - complete restoration of normal structure and function; occurs when injury is limited and tissue is capable of regeneration (e.g., lobar pneumonia resolving)
2. Healing by fibrosis (scarring) - when substantial tissue destruction occurs or in non-regenerating tissues; inflammation resolves but is replaced by connective tissue scar
3. Progression to chronic inflammation - when the injurious agent cannot be eliminated or there is interference with normal healing; leads to persistent inflammation with ongoing tissue damage and repair

Summary

"When a host encounters an injurious agent, resident phagocytes try to eliminate it. Sentinel cells react by liberating cytokines, lipid messengers, and other mediators. These act on small blood vessels, promoting efflux of plasma proteins and recruitment of circulating leukocytes to the site. Recruited leukocytes are activated and try to remove the offending agent by phagocytosis. As the injurious agent is eliminated and antiinflammatory mechanisms become active, the process subsides and the host returns to a normal state of health."

• Robbins, Cotran & Kumar Pathologic Basis of Disease, Chapter 3