1. Metastasis is a multi-step process by which tumor cells spread from a primary tumor to distant sites in the body and form secondary tumors. 2. The document discusses factors that drive tumor cell invasion and metastasis, including hypoxia, inflammation, escaping apoptosis and senescence, self-renewal ability, and coupling tumorigenesis with metastasis initiation and progression. 3. It also describes the key steps in metastasis including intravasation, survival in circulation, arrest and extravasation in distant organs, and growth of metastases.

1. Invasion and Metastasis
DR.KIRAN KUMAR BR

2. Metastasis is the process by which a tumor cell leaves the primary
tumor, travels to a distant site and establishes a secondary tumor.
Metastasis a/w several clinical and pathological characteristics :
• Tumour size
• Regional lymph node involvement.
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3. Metastasis pre-1900
LeDran 1757: Noted that malignant tumors begin as localized disease,
then spread to regional lymph nodes and then enter the circulation to
subsequently appear in the lung.
Bichat 1801: Tumors contain both parenchyma and stroma.
Recamier 1829 : Used the term “Metastases”
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4. 28/11/16

5. TISSUE TROPISM AND THE SEED AND SOIL
HYPOTHESIS
“Seed and soil” theory: the provision of a fertile environment
in which compatible tumor cells could grow.
Despite apparent similarities in clinical and histologic features
different cancer types do not exhibit the same predilection to
metastasize to the same organs.
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6. In contrast, James Ewing  argued that tissue tropism could be
accounted for mechanical factors and circulatory patterns of the primary
tumor.
Mechanistic theory: determined by the pattern of blood flow.
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7. EXAMPLE:
Colorectal cancer can enter the hepatic-portal system  propensity for
liver metastasis.
Prostate cancer  traverse presacral plexus that connects the
periprostatic and vertebral veins  propensity for metastases to the
lower spine and pelvis.
Current understanding  suggest that both seed and soil factors and
anatomic considerations contribute to metastatic tropism.

8. 5 major steps in metastasis:
1. Invasion and infiltration of surrounding normal host tissue with
penetration of small lymphatic or vascular channels.
2. Release of neoplastic cells into the circulation.
3. Survival in the circulation.
4. Arrest in the capillary beds of distant organs.
5. Penetration of the lymphatic or blood vessel walls followed by
growth of the disseminated tumor cells – Growth in distant organs.

9. Figure 14.4 The Biology of Cancer (© Garland Science 2014) p. 591; Figure 14.4, p. 644, 2nd Edition
Invasion-Metastasis Cascade Adapted from Fidler, Nat. Rev. Cancer 3: 453-458, 2003

10. Invasion and Infiltration
Tumor cells
Diminished cellular adhesion
Become motile Tumor cells
Use their migratory and invasive properties in order to burrow
through surrounding extracellular stroma.
Gain entry into blood vessels and lymphatics.
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11. 2. Intravasation and survival in the circulation:
• Tumor cells enter the circulation or intravasate  must be able to
withstand the physical shear forces and the hostility of sentinel
immune cells.
• Solid tumors  not accustomed to surviving as single cells 
interact with each other or blood elements to form intravascular tumor
emboli.
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12. Arrest and extravasation:
•Once arrested in the capillary system of distant organs  tumor cells
must extravasate into foreign parenchyma.
Growth in distant organs:
•Successful adaptation to the new microenvironment  sustained
growth.
•Ability to grow in distant organs  core of the seed and soil
hypothesis.
Rate-limiting.
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13. Figure 14.4 The Biology of Cancer (© Garland Science 2014) p. 591; Figure 14.4, p. 644, 2nd Edition
Invasion-Metastasis Cascade Adapted from Fidler, Nat. Rev. Cancer 3: 453-458, 2003

14. SELECTIVE PRESSURES AT THE PRIMARY TUMOR DRIVING
ACQUISITION OF METASTASIS FUNCTIONS
HYPOXIA
Oxygen and glucose can only diffuse 100 to 150 microns- Preinvasive tumor growth
Hypoxia
Portions of the expanding mass becoming hypoxic.
Seen in comedo-type DCIS of the breast  necrotic center characterizes these preinvasive breast tumors.
DCIS can take years to progress to invasive cancer or never progresses to cancer  hypoxia significant
barrier.

15. Under hypoxic conditions
Hypoxia-inducible factor (HIF) transcription factors HIF-1α and HIF-2α
become stabilized.
Transcription of over 100 HIF-α regulated genes: VEGF & PDGF
Cause quiescent blood vessels to undergo remodeling  laying down of a matrix that
activated endothelial cells use to form newly vascularized areas.

16. Various glycolysis genes expressed and their metabolic by-products .
Acidification of the extracellular space: normally toxic to cells and
requires further adaptation either by up-regulation of H+ transporters
or acquired resistance to apoptosis.
Invasion toward newly vascularized areas,
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17. HIF-α up-regulates :
1. Matrix metalloproteinase 1 & 2(MMP1,MMP2),
2. Lysyl oxidase (LOX) and
3. Chemokine receptor CXCR4.
MMP2  Degradation of the basement membrane.
MMP1  Alteration of the extracellular matrix (ECM) .
LOX  Clears away a barrier to migration.
CXCR4  Stimulates cancer cells to migrate to regions of angiogenesis.
Preinvasive tumors successfully deal with hypoxia and invade through the
basement membrane.

19. INFLAMMATION
Normal tissue homeostasis and architecture disrupted:
Lead to : Vessel injury.
Hypoxic zones
Extravasation of blood proteins
Entry of foreign pathogens.
Rapid response is mounted by a front line composed of immune and bone marrow-derived cells (BMDCs):
lymphocytes, neutrophils, macrophages, dendritic cells, eosinophil's and NK cells.
Purpose  To restore homeostasis through: Inflammation,
-Inflammation. Tissue formation, Tissue remodeling.
-

20. Tissue breakdown
Attracts neutrophils to infiltrate the wounded area.
Release various proinflammatory cytokines
Interleukin (IL)-8
IL-1β, and
TNF-α.
+
Reactive oxygen species and
Proteases : urokinase-type plasminogen activator(uPA)
Fight pathogens and debride devitalized tissue.
Neutrophils replaced by macrophages.

21. Activated macrophages
Provide :
- matrix remodeling capabilities (uPA, MMP9).
- synthesis of growth factors : FGF, PDGF,TGF-β.
- production of angiogenesis factors (VEGF).
Activate fibroblasts to synthesize new ECM and promote
neovascularization in the formation of granulation tissue.

22. • Cancer cells are surrounded by activated fibroblasts and BMDCs.
• Immune system actively fights the cancer as it does with invading bacterial or viral
pathogens.
• The inflammatory response apply significant selective pressure on the tumor to evade
immune-mediated attack.
• Tumors that progress produce an immunosuppressive environment
Immunoediting.

23. Tumor microenvironment selects for cells that favor production of immunomodulatory factors :
- TGF-β, cycoloxygenase-2 (COX2), CSF-1 (macrophage growth factor, colony-stimulating factor-
1), IL-10, and IL-6. These cytokines
Inhibit maturation of dendritic cells and promote tumor-associated
macrophages (TAMs)
+
Signaling events that involve stromal cell-derived factor-1 (SDF-1 or
CXCL12) and its ligand CXCR4 and CXCL5/CXCR2 (chemokine/receptor pair).
Recruit BMDCs that have immunosuppressive properties such as myeloid-
derived suppressor cells (MDSCs)
MDSCs increase local production of TGF-β, block T-lymphocyte function, and
inhibit the activation of NK cells.

25. • Rather than simply suppress the inflammatory response, cancer cells actually develop
mechanisms to both adopt and sustain it.
• MDSCs contributes to immunosuppression and also facilitate tumor invasion by
residing at the invasive front and secreting MMPs.
• TAMs are found at points of BM breakdown and help tumors degrade extracellular
proteins using uPA and MMPs or stimulate tumor growth and motility through EGF
receptor ligands and PDGF

26. • Growth factors secreted by the TAMs activate fibroblasts.
• Activated fibroblasts become carcinoma-associated fibroblasts (CAFs)
and promote primary tumor growth by secreting CXCL12 to stimulate
CXCR4 on tumor cells.
• Angiogenesis is also aided by the action of CAFs through recruitment of
endothelial progenitor cells by CXCL12 and by the action of TAMs that
are recruited to areas of hypoxia to produce VEGF.

27. ESCAPING APOPTOSIS AND SENESCENCE
• Intrinsic triggers for apoptosis include oncogene activation or tumor suppressor gene
loss.
• Inappropriate activation of c-MYC or the loss of Rb results in programmed cell death
that must be countered by :
Overexpression of antiapoptosis genes such as Bcl-2 or
Loss of proapoptotic regulators like p53.
• Extrinsic triggers for apoptosis  hypoxia, low pH, reactive oxygen species, loss of cell
contact, and immune-mediated killing.
• Cancer cells  ignore these cues  resist cell death  establishment of tumors.

28. SELF-RENEWAL ABILITY
• Normal tissues result from the differentiation of precursor cells called stem cells  multipotent cells
with self-renewal ability.
• In the adult, mature differentiated cells serve specialized tasks and have limited proliferative potential.
• One daughter cell  maintains the stem cell pool by self-renewal.
• Other daughter cell  starts the process of terminal differentiation
for tissue maintenance.
• Only a limited subset of cells in a cancer is capable of self-renewal is called Cancer stem cells.

29. COUPLING TUMORIGENESIS WITH METASTASIS
INITIATION
EPITHELIAL-TO-MESENCHYMAL TRANSITION
• One of metastasis initiation functions.
• Generation of many adult tissues and organs results from a series of EMT events and
the reverse process, a mesenchymal-to-epithelial transition (MET).
• Both characterized by
a) epithelial cells loosening their cell-cell adhesion,
b) losing cell polarity
c) gaining the ability to invade and migrate under controlled cues.

30. • Important regulators include
a) Notch and Wnt/β-catenin pathways,
b) TGF-β family members, and
c) FGF proteins
Set up regulatory networks involving EMT transcription
factors such as Snail and Twist.
• EMT is an important characteristic of metastasis-prone cancers.

31. • Hypoxia can induce Snail and Twist  direct target of HIF-1α.
Enhances β-catenin activity
Inhibits activity of glycogen synthase kinase-3β normally
induces the destruction of β-catenin
Enhanced β-catenin signaling
Promotes Snail expression
EMT
• Activation of Snail represses E-cadherin  further enhance β-catenin and reinforce Snail expression.

32. • Inflammatory microenvironment also promote EMT.
• TNF-α  inflammatory mediator secreted by TAMs  sets into motion a signaling
cascade that includes NF-κB and glycogen synthase kinase-3β  stabilize Snail and
β-catenin  enhances cancer cell migration.
• Cells that have undergone EMT are associated with increased resistance to apoptosis
 through prosurvival activity conferred by Snail and Twist.
• EMT help cancer overcome oncogene-induced senescence.

33. COUPLING TUMORIGENESIS WITH METASTASIS PROGRESSION
PREMETASTATIC NICHE
• Even before tumor cells colonize distant organs  help prepare foreign soil for the subsequent arrival
of disseminated tumor cells (DTCs)  remotely coordinating a “premetastatic niche” from the
primary tumor.
• Located within distant organs around terminal veins.
• Characterized by newly recruited hematopoietic progenitor cells of the myeloid lineage and by stromal
cells.
• Niche provides an array of cytokines, growth factors, and adhesion molecules to help support
metastatic cells on their arrival.

34. Primary tumor cells secrete of cytokines: VEGF and placental
growth factor.
Direct VEGFR myeloid cells to mobilize from the bone marrow
Localize to areas in target organs with increased fibronectin.
• If the primary tumor secretes VEGF  promotes fibronectin deposition in the lung  directs
construction of the premetastatic niche there.
• Combination of VEGF and placental growth factor  more widespread pattern of niche assembly.

35. VEGF, TGF-β, and TNF-α produced at the primary tumor site
Signal production of inflammatory proteins like S100A8 and
S100A9 specifically within the lung parenchyma.
Infiltration of myeloid cells into the lung and subsequent
formation of the niche.
• LOX produced by a hypoxic primary tumor environment can also direct formation of a
premetastatic niche.

37. • Once myeloid and activated stromal cells form the premetastatic niche  local
environment in the distant organ is altered by the production of inflammatory
cytokines and MMPs  begins bearing an evolving resemblance to the primary site.
• Primary tumor cells start wandering in the circulation  target organs with an
established premetastatic niche become a better soil in which to attach, survive, and
grow.

38. SURVIVAL IN THE CIRCULATION
• After intravasation into the circulation from the primary tumor  tumor cells
encounter significant physical stress due to shear forces or mechanical arrest in
small-diameter vessels.
• Hepatic sinusoids  activated by the mechanical restriction of tumor cells to secrete
nitric oxide.
• Nitric oxide can cause apoptosis of arrested tumor cells.
• Endothelial cells can also guard against wandering tumor cells

39. • Growth at the primary tumor site involves a selection for increased
resistance to apoptosis.
a) Antiapoptosis genes such as BCL2 or BCL-XL.
b)Loss of proapoptotic genes and genes downstream of the TNF-related
receptor family result in increased metastasis.

40. • Both circulating tumor cells (CTCs) and platelets can also express the αvβ3 integrin
 promote aggregation of these cells to form tumor emboli.
• This aggregation  facilitates arrest + protect against shear forces and NK cell-
mediated killing.
• Activation of αvβ3 INTEGRIN  result from CXCL12/CXCR4 signaling 
required for formation of tumor emboli and metastasis.

41. EXTRAVASATION AND COLONIZATION
• After arresting in capillaries  tumor cells that are able to survive grow
intravascularly  lead to a physical disruption of the vessels.
• Cancer cells can mimic leukocytes and bind to endothelial E- and P-selectins.
Ezrin - cytoskeletal anchoring protein  links the cell membrane to the actin
cytoskeleton and engages the cell with its microenvironment.
• VEGF expression by the tumor  lead to disruptions in endothelial cell junctions
and facilitate extravasation of cancer cells through enhanced vascular permeability.

42. • Expression of hypoxia-induced CXCR4 on CTCs allows for the selective
extravasation into certain organs.
• This selectivity is due to the expression of its ligand CXCL12 by certain organs that
include the lung, liver, bone, and lymph nodes.

43. TUMOR SELF-SEEDING
• Primary tumor impose the least resistance to colonization.
• CTCs can seed the primary tumor and contribute to its mass.
• Ability to self-seed promoted by IL-6 and IL-8  prometastatic
cytokines found in the tumor microenvironment.
• Expression of MMP1 and Fascin-1  facilitates transendothelial
migration and tumor self-seeding.

44. FROM METASTASIS PROGRESSION TO MACROMETASTATIC
COLONIZATION
DORMANCY
• A major limiting step in metastasis is acquiring the ability to sustain growth within a
distant site after extravasation.
• Vast majority of extravasated cancer cells do not form macrometastasis  latency
are referred to as metastatic dormacy
• Cellular dormancy  when cancer cells enter a state of growth arrest.

45. • Clinical evidence from one study  breast cancer patients with no evidence of
disease years after mastectomy and considered candidates for having metastatic
dormancy  detectable CTCs.
• Later acquisition of angiogenic properties allow such micrometastases to become
vascularized and emerge from their occult state.
• Mechanisms that contribute to cellular dormancy  balance between the RAF-
MEK-ERK pathway and the p38 MAPK pathway.
• Inhibition of the former and activation of the latter is associated with cellular
quiescence in a G0-G1 state.

46. ORGAN SELECTIVE GROWTH
BONE
• Homeostasis of the bone is maintained by osteoclasts & osteoblasts.
• The mineralized bone matrix is reabsorbed by osteoclasts and filled in by osteoblasts.
• The differentiation of osteoclasts from bone marrow  controlled by CSF-1 and the RANK receptor.
• RANK interacts with its ligand RANKL produced by osteoblasts, leading to a tight coupling of these
two cells with opposing actions.
• Osteoprotegerin (OPG) is a secreted antagonist of RANKL and prevents interaction with RANK and
resulting osteoclastogenesis.

47. • Osteoblasts differentiation  under the control of regulators insulin-like growth factor (IGF),
endothelin-1, bone morphogenetic proteins, and WNT proteins.
• Bone is one of the most common sites of distant spread.
• Latency period that precedes the development of gross osseous metastasis can be years.
• SRC kinase – provides latency period for development of gross osseous metastasis.
• SRC kinase  required for CXCL12- and IGF-mediated survival signals  protect indolent cancer
cells in the bone marrow from TRAIL-mediated apoptosis

48. • When the dormancy period expires  gross osseous metastases are characterized by two basic types:
osteoblastic and osteolytic.
• Tumors such as lung, kidney, and breast carcinomas produce osteolytic lesions.
• Breast cancer cells achieve osteolytic metastasis  secreting factors including parathyroid hormone-
related protein (PTHrP), TNF-α, IL-1, IL-6, IL-8, and IL-11  enhanced osteoclast activation,
degradation of bone matrix, and the release of matrix-associated cytokines that stimulate the cancer
cells.
• Secretion of PTHrP  production of the membrane-bound RANKL on osteoblasts  RANK-
mediated osteoclast activation.
• On degradation of the bone matrix, embedded growth factors are released, including TGF-β and IGF
 stimulate the tumor cells and enhance the entire vicious cycle.

49. • Genes involved in breast cancer bone metastasis IL-11, MMP1, ADAMTS1,
CXCR4, connective tissue growth factor (CTGF), and OPN  mediators of
osteolytic bone metastasis.
• Osteoblastic lesions result from the preferential stimulation of osteoblasts and/or the
inhibition of osteoclasts  typified by prostate cancer.
• Paracrine factors secreted by prostate cancer cells can regulate osteoblast
proliferation or differentiation, including bone morphogenetic proteins, WNT, TGF-
β, IGF, PDGF, FGF, and VEGF.

50. LUNG
• Extensive study on metastasis and invasion  direct inoculation of tumor cells into
venous circulation.
• Lung is the first & may be only organ encountered because of entrapment
by lung capillary bed.
• In a study seeking to gain insight into organ-selective metastasis genes  single cell-
derived clones from a human breast cancer cell line were discovered  exhibit
varying degrees of metastatic ability to the bone and to the lung.
• These observations lead to the discovery of an LMS

51. • LMS group of genes:
- Secreted factors  EREG, CXCL1, ANGPTL4 & SPARC
- Cell surface receptors  VCAM1 & IL13Rα2
- Extracellular matrix protein  TNC
- Proteases  MMP 1 & 2
- Intracellular effectors  ID1, Fascin 1, COX 2.
• Patients with LMS-expressing tumors are at a higher risk for lung metastasis.
• LMS-expressing tumors are larger at the time of diagnosis compared with LMS-
negative tumors.

52. BRAIN
• Principal sources of brain metastasis  lung and breast.
• Melanoma, colorectal, and renal cell carcinomas also can relapse in the
brain.
• Vascular access  restricted because of blood–brain barrier.
• Composed of tightly adjoined endothelial cells that are lined by basal
lamina & astrocyte foot processes.

53. • Melanoma cells that metastasize to the brain  high STAT3 transcriptional activity
compared with cutaneous metastases or primary melanoma specimens.
• Several genes found to mediate metastasis to the brain:
COX2, EGFR ligand HBEGF, α2,6 sialyltransferase,
ST6GALNAC5.
• COX2 & HBEGF  passage through the nonfenestrated
capillaries of brain & lungs
ST6GALNAC5  enhances ability of cancer cells to pass
through blood–brain barrier

54. LYMPHATICS
• Lymphatics  low shear force vessels  single layer of
endothelial cells with little or no basement membrane
and sparsely coated with pericytes.
• Lymphangiogenesis involves VEGF-C, VEGF-D and their receptor VEGFR-3.
• Non-VEGF family members can also induce lymphangiogenesis : FGF2, PDGF & angiopoietin
proteins
• Like the lung parenchyma and the bone marrow, lymph nodes secrete CXCL12, which can interact
with tumor-expressing CXCR4.
• Other chemokine receptors such as CXCR3 also play a role in lymph node metatasis

55. MICRO-RNAS AND METASTASIS
• MicroRNAs  approximately 22 nucleotide-long small ncRNAs  have been discovered to have
important effects on metastasis.
• MicroRNAs function by regulating the expression of hundreds of target genes.
• Sequence-specific binding between the microRNA and the 3′ untranslated regions of mRNAs 
results in either the degradation of the target mRNA or the inhibition of protein translation  can
influence a large number of genes.
• MicroRNAs can function both as metastasis activators and metastasis suppressors by influencing
numerous genes involved in metastasis initiation, progression, and colonization.

57. THANK YOU

58. Metastatic deposit in brain

59. Figure 14.1 The Biology of Cancer (© Garland Science 2007). P. 588; p. 642 2nd edition
Metastatic non-Hodgkins Lymphoma
CT Scan and PET Scan (positron emission tomography) of
incorporated radioactively-labelled deoxyfluoroglucose.
(Brain activity is normal, abdominal active is pathological)

60. Colon Carcinoma Metastatic to Liver Breast Carcinoma Metastatic to Brain
Fig. 2.2b and c
Weinberg
p. 27;
p. 33 2nd edition

61. Multiple Metastatic Lesions of Gastric Adenocarcinoma to Liver
See next slide

62. Primary Glioblastoma Compared to Breast Carcinoma
Metastasis to the Brain

63. Figure 14.2c The Biology of Cancer (© Garland Science 2007). P. 589
Carcinoma Metastatic to Bone. Stained for Epithelial Cell Markers
To Here March 22, 2016 Thursday