(1) Cancer cells can metastasize by spreading through lymphatic vessels to nearby lymph nodes. (2) The process of lymphatic metastasis involves cancer cells degrading the extracellular matrix, entering lymphatic capillaries, and traveling through the lymphatic system to lymph nodes. (3) Lymphangiogenesis, the growth of new lymphatic vessels, is stimulated by VEGF-C and VEGF-D and can promote cancer cell entry into lymphatic vessels and spread to lymph nodes. Blocking these pathways may help prevent lymphatic metastasis.

1. Cancer Metastasis Lymphatic Spread

2. Metastasis ‘ Metastases’ are tumour implants discontinuous with the primary tumour Approximately 30% of newly diagnosed patients with solid tumours present with metastases (1) They are the major cause of cancer-related morbidity and mortality Pathways of Spread (1) (1) Seeding of the body cavities and surfaces When a malignant neoplasm penetrates into a natural ‘open field’ eg. peritoneal cavity (2) Lymphatic Spread Most common pathway for the initial dissemination of carcinomas Tends to follow the natural routes of lymphatic drainage (3) Haematogenous Spread Typical of sarcomas More readily via venous (than arterial) system

3. Invasion & Metastasis For tumour cells to break loose from the primary mass, enter the blood/lymph vessels and produce a secondary growth they must interact with the extracellular matrix (ECM) at several stages: Breach the underlying basement membrane Transverse the interstitial connective tissue Penetrate the vascular basement membrane Invasion of the ECM is an active process which involves: Detachment of tumour cells from each other Attachment to matrix components Degradation of the ECM Migration of tumour cells

4. Invasion & Metastasis cont. Detachment of Tumour Cells Normal cells are adhered to one another via transmembrane glycoproteins (E-cadherins) Downregulation of E-cadherin expression in adenocarcinoma of the colon Decreased ability for adherence and facilitation of detachment from the primary tumour Attachment of Matrix Components Expression of integrins by tumour cells serve as receptors for ECM components Receptor-mediated attachment of tumour cells to laminin & fibronectin Increased density of receptors = increased invasiveness

6. Invasion & Metastasis cont. Degradation of ECM Secretion of proteolytic enzymes by tumour cells Induction of host cell protease synthesis (eg. type IV collagenase) Cleavage of type IV collagen of the epithelial & vascular basement membranes Migration of Tumour Cells Cleavage products of matrix components have growth-promoting, angiogeneic and chemotactic activities Promotion of migration of tumour cells through loosened ECM, and through the degraded basement membrane

9. Lymph Node Metastasis Motility towards lymphatic capillaries aided by lower interstitial fluid pressures within the ECM, than in the tumour ~ ‘tide of fluid’ (2) Having reached the lymphatic capillaries, tumour cells move along external surface of the endothelium and invade into the lumen via interendothelial gaps (2) The composition of lymphatic vessels makes it easy for fluid, particles and cells to pass into the vessels (3) Having gained access to the capillaries, the tumour cells embolise singly or in clusters towards local lymph nodes Some cells do not reach the nodes, but adhere to the lymphatic endothelium and cause ‘in transit’ metastases (2) Tumour cells enter the subcapsular sinus of the lymph node through the afferent lymphatics and may either: Invade the cortex of the node Bypass the node via lymphpaticovenous connections Travel directly into the efferent lymphatics and spread to further local nodes

11. Lymphangiongenesis Until recently lymphatic metastasis was believed to be a ‘passive process’ – it has now become apparent that lymphangiogenesis can contribute actively to tumour metastasis (4) Studies describing lymphatic growth and development did not emerge until the late 1990s due to a lack of defined lymphatic endothelial markers (5) Widely established lymphatic endothelial markers include: Vascular Endothelial Growth Factor-3 (VEGFR-3)– the first lymphangiogenic growth factor identified Podoplanin LYVE-1 Prox-1 FOXC2 Current knowledge does not yet provide a full understanding of their exact role in lymphangiogenesis.

12. Lymphangiogenesis cont. While the mechanisms of tumour lymphangiogenesis are not fully understood or defined, multiple studies have established VEGF-C & -D as the main regulator of lymphangiogenesis (6, 7, 8, 9, 10) Over-expression of VEGF-C in animal tumour models has demonstrated strong increases in lymphatic vessel formation and significant promotion of lymph node metastases (7, 9). Recently VEGF-C has also been shown to promote further metastasis from regional to distal lymph nodes and organs (10) It is proposed that tumour lymphangiogenesis increases the lymphatic vascular area within or close to the tumour, and therefore increases contact between tumour cells and the lymphatics ~ facilitating entry of malignant cells into the lymphatic system, thereby promoting metastatic spread (5). Colorectal cancer data is conflicting in human models, but it seems likely that both VEGF-C/-D contribute to the disease course (5)

14. Therapeutic Strategies Anti-lymphangiogenic therapy is an important area for future research ~ assuming that restriction of lymphatic vessel growth associated with tumours will prevent lymph node metastases Lymph node metastases are a key event in colorectal tumour progression; With lymph node metastases 5-year survival is reduced from 90% to 68% VEGF-C/-D induced stimulation of VEGFR-3 represents a promising target for anti-lymphangiogenic therapy Blocking extracellular ligand-receptor interactions with neutralising monoclonal antibodies to either receptors or ligands ~ demonstrated in some animal models Soluble VEGFR-3-fc fusion protein has also been shown to inhibit lymphangiogenesis (5)

15. References (1) Kumar, Abbas, Fausto (eds) (2005). Robbins and Coltran ~ Pathological Basis of Disease. 7 th Ed. Elsevier Saunders, Pennsylvania. (2) Nathanson, SD. (2003). Insights into the Mechanisms of Lymph Node Metastasis. Cancer , 98 ; 2: 413-423. (3) Swartz, MA. & Skobe, M. (2001). Lymphatic Function, Lymphangiogenesis, and Cancer Metastasis. Microscopy Research and Technique , 55 :92-99. (4) Ji, RC. (2006). Lymphatic endothelial cells, tumour lymphangiogenesis and metastasis: New insights into intratumoral and peritumoral lymphatics. Cancer Metastasis Rev , 25 : 677-694. (5) Sundlisaeter, E. et al. (2007). Lymphangiogenesis in colorectal cancer – Prognostic and therapeutic aspects. Int. J Cancer , 121 : 1401-1409. (6) Yoo, PS. et al (2007). A Novel In Vitro Model of Lymphatic Metastasis from Colorectal Cancer . Journal of Surgical Research, ‘article in press’. (7) Kawakami, M. et al (2005). Vascular Endothelial Growth Factor C Promotes Lymph Node Metastasis in a Rectal Cancer Orthotopic Model. Surg Today , 35 : 131-138. (8) Akagi, K. et al (2000). Vascular endothelial growth factor C (VEGF-C) expression in human colorectal cancer tissues. Br J Cancer , 83 : 887-891. (9) Skobe, M. et al (2001). Induction of tumour lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med , 7 : 192-198. (10) Hirakawa, S. et al (2007). VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumour metastasis to distant sites. Blood , 109 : 1010-1017.