Lecture 18 - Invasion and Metastasis Part 2

how to visualize metastasis in lymphatic system?

Tg mice-Rip-Tag transgenic mice H&E staining

Intravasation occurs where?

between or across endothelial cells *multiple signals and processes lead to endothelial retraction or contraction

Usually CTC's are single cells, but occasionally, CTC clusters are detected in patient blood. Why is this BAD?

clusters show high expression of adhesion protein plakoglobin-->correlated with worse patient prognosis. CTC clusters have a greater potential to form lung metastases than single CTCs, owing to a survival advantage in the lungs.

What does cytoskeletal and membrane remodeling do in intravasation?

creates a transitory pore-like structure

Function of ADAM12 on ECs in intravasation?

induce cleavage of vascular endothelial cadherin and angiopoietin 1 receptor (TIE2) which leads to disruption of endothelial junctions

Role of TGFB1 in intravasation?

induces the retraction of endothelial junctions

EMT dynamics during tumor progression

induction of a partial-EMT optimizes tumour-initiating potential while still maintaining cell plasticity, i.e. the ability to reverse the EMT process and thus undergo an MET, thereby generating more epithelial progeny, whose presence greatly increases the success of metastatic colony formation.

many CTC's die en route. How does this happen?

loss off attachment-anoikis loss of stromal support shear forces in the cirulation NK cell attack

metastatic cancer cells may travel through lymph ducts to

lymph nodes

organotropism

propensity to affect a particular organ system-usually a system that is especially vital for the survival of the host-like the nervous system or the circulatory system.

pN-range adhesion forces favor

rapid and stable intravascular adhesion

What does par-1 do in intravasation?

remodels endothelial junctions

How do macrophages attract cancer cells toward blood vessels during intravasation?

secrete EGF and TNF1a which induces the retraction of endothelial junctions and thus facilitates cancer cell transendothelial migration (TEM)

draining lymph nodes are

sentinels of metastasis

Three prerequisites for metastatic colonization

A. Seeding and maintenance of cancer stem cells B. Adaptive organ specific programs C. Metastatic Niche

Transendothelial migration (TEM)

Cancer cells then transmigrate through the endothelial barrier during this process, and after this they invade the basement membrane that surrounds the blood vessels. Cells can then enter a state of dormancy or proliferate within this new microenvironment, where a few of them will give rise to micrometastases and then macrometastases. However, most of the cancer cells that extravasate will not colonize these new tissues but will undergo cell death instead

Extravasation

Cancer cells use the bloodstream to spread throughout the body, and then cells leave the circulation through this process. *involves the specific interaction of cancer cells with vascular endothelial cells via cell adhesion and chemokine-related processes, during which cognate ligands or receptors are expressed on cancer cells and endothelial cells and/or the initial trapping of cancer cells within the blood vessels, leads to their specific adhesion.

How to detect micrometastases?

IHC *using cytokeratin-specific antibodies makes it possible to detect a single-cell micrometastasis among 10^5 or even 10^6 surrounding mesenchymal cells in the blood, bone marrow, or lymph node (cytokeratin marks epithelial cells) H&E staining

What governs where CTC's go?

In some cases, the tendencies of a tumor to spawn metastases in one or another tumor reflect the abilities of the cancer cells from the primary tumor cells to adapt to (and thus colonize) the microenvironment of distant tissues; this is likely to explain the strong tendencies of prostate and breast cancers to generate metastases in the bone marrow. In other cases, the layout of the circulation may strongly influence the site of metastasis. For example, the high proportion of liver metastases deriving from primary colon cancers may reflect the drainage via the portal vein of blood from the colon directly into the liver

pre-metastatic niche

Primary tumors create a favorable microenvironment, namely, pre-metastatic niche, in secondary organs and tissue sites for subsequent metastases. The pre-metastatic niche can be primed and established through a complex interplay among primary tumor-derived factors, tumor-mobilized bone marrow-derived cells, and local stromal components.

CTCs can function as

a useful surrogate marker -liquid biopsy -early detection -help identify molecular mechanisms

Role of Ca2+-calmodulin complex in transcellular intravasation?

activates myosin light chain (MLC) kinase (MLCK) at the sites of cancer cell attachment, which leads to local phosphorylation (P) of MLC and to actomyosin contraction. In turn, this leads to rapid cytoskeletal and membrane remodelling, which creates a transitory pore-like structure for the cancer cell to cross the EC.

What can happen if CTC's get stuck within the vessel?

can proliferate within the vessel

The diverse functions of EMT/MET in cancer progression and metastasis

cancer stemness chemoresistance immune evasion altered metabolism blocked senescence

small metastatic deposits in lymph nodes probably represent

dead ends fo cancer cells *function as surrogate markers that reveal the extent of dissemination from the primary tumor into the general circulation

How can dormancy be activated in cells that have metastasized?

either through an active response to signals encountered in the new tissue microenvironment from an absence of contextual cues that carcinoma cells previously depended on while residing in their sites of origin within primary tumors

Flow-mediated endothelial remodeling drives

extravasation of tumor cells

intravasation

how cancer cells enter the bloodstream. Cells migrate through endothelial cell junctions.

six characteristics that define the pre-metastatic niche which enable tumor cell colonization and promote metastasis

immunosuppression inflammation angiogenesis lymphangiogenesis organotropism reprogramming

Amoeboid migration associated with

minimal cell matrix adhesions and rounded morphology

Dormant Niches

perivascular niche or hematopoietic stem cell niche

arrest of circulating tumor cells occurs in blood vessels with

permissive flow profiles

Alternative view on metastasis?

primary tumors send out tiny, protein and RNA packed vesicles (exosomes) that prepare distant sites for tumor cells to take root and recruit bone marrow cells to assist. This "pre metastatic niche" nourishes the tumor cells that arrive later

mesenchymal migration associated with

strong cell matrix adhesions and elongated morphology

The strong tendency of colon carcinomas to metastasize to the liver may reflect

the fact that these cancer cells leave the gut via the portal vein and the liver is the "first pass organ" (blood from spleen and gut go through the heart first before going back to the heart)

Evidence for solitary dormant tumor cells?

the fluorescence intensity of many of these cells did not differ significantly from the intensity of cells shortly after labeling, indicating that these cells had not divided even once following labeling. Following isolation and in vitro culturing, the descendants of many of these cells were tumorigenic (Moreover, the dormant cancer cells were found to be fully resistant to a chemotherapy that reduced by 75% the size of metastases that were growing rapidly in the same mice

colonization

the most complex and challenging step of the invasion-metastasis cascade *colonization is rate limiting

hemodynamic forces

tune the arrest, adhesion, and extravasation of circulating tumor cells *hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth.

Once CTCs extravasate, more problems arise. These are:

1. cell death 2. dormancy

How are CTC's protected as they travel through the blood stream?

1. coating by platelets 2. neutrophils protect from NK cell attack

Frequency of metastases to an organ is governed by two parameters:

1. frequency with which metastasizing cells are physically trapped in an organ 2. ease with which they can adapt to the microenvironment of that organ, thereby colonizing it

Common biological principles of metastasis

1.Metastasis occurs mainly through a sequential, multi-step process that can be conceptualized as the invasion-metastasis cascade. 2.In the case of carcinomas, the EMT program enables primary tumor cells to accomplish most if not all of the steps involved in the physical dissemination of tumor cells to a distant site. 3.The fate of disseminating carcinoma cells is strongly influenced by interactions that they experience during transit through the circulatory system. 4.Disseminated carcinoma cells must escape clearance by the arms of the immune system and subvert the cellular programs that impose a state of dormancy. 5.The process of active metastatic colonization is contingent upon the dissemination of cancer stem cells that can re-initiate tumor growth; the ability of their progeny to assemble adaptive, organ-specific colonization programs; and the establishment of a microenvironment conducive to metastasis.

Explain the colonization prerequisite of adaptive organ specific programs

Although cancer stem cells are endowed with the potential to re-initiate tumor growth, the proliferative expansion to an overt metastatic colony is dependent on the ability to contrive organ-specific colonization programs that allow these cells to thrive in a foreign tissue microenvironment. An array of organ-specific metastatic programs has been described in the literature but there is also evidence for the existence of colonization programs that confer multi-organ metastatic potential.

What type of ECM allows several types of carcinoma cells (Breast being a main one) to thrive?

Bone ECM is an unusually rich source of the mitogenic and trophic factors that allow several types of carcinoma cells (e.g. breast) to thrive. Consequently, by provoking the demineralization of bone, cancer cells gain access to the storehouse of factors sequestered in the bone ECM and use them to support their own proliferation and survival

What's more efficient, single CTC's or CTC clusters?

CTC clusters introduced experimentally into the venous circulation are far more efficient than individual carcinoma cells in seeding metastatic colonies, ostensibly because, relative to single CTCs, they are more resistant to apoptosis and may have an advantage in physically lodging in the lumia of vessels

How to identify a sentinel node?

Can usually be identified among all of the lymph nodes draining the breast by injecting a dye (or a weak radioactive solution) into the tumor

Role of notch signaling in intravasation?

Cancer cells can use Notch receptors to bind to Notch ligands on ECs and thereby transmigrate through the endothelial junctions

Circulating tumor cells (CTCs)

Carcinoma cells escaping from primary tumors can intravasate into the circulation, either as single circulating tumor cells (CTCs) or as multicellular CTC clusters. The bloodstream represents a hostile environment for CTCs, exposing them to rapid clearance by natural killer (NK) cells or fragmentation due to the physical stresses encountered in transit through the circulation. Carcinoma cells gain physical and immune protection through the actions of platelets, which coat CTCs shortly after intravasation.

dormant disseminated tumor cells (DTCs)

Carcinoma cells that have disseminated prior to the surgical removal of the primary tumor may persist in distant tissue environments as dormant disseminated tumor cells (DTCs). Patients harboring such reservoirs of occult carcinoma cells are considered to have minimal residual disease and are at increased risk of eventual metastatic recurrence. Although DTCs are most frequently examined in the bone, the delayed outgrowth of metastases in other organs suggests that they, too, can harbor dormant DTCs

Where do DTCs reside?

DTCs may reside in dormant niches such as the hematopoietic stem cell niche or the perivascular niche. Thrombospondin-1 (TSP1), present in the basement membrane surrounding mature blood vessels, promotes dormancy.

How to DTCs survive?

Dormant DTCs rely on unique biochemical signaling pathways that sustain their survival and impose programs of quiescence. Signals from the microenvironment, such as CXCL12, can activate SRC and AKT to promote DTC survival. Reduced integrin-mediated mitogenic signaling, coupled with the actions of certain dormancy-inducing cytokines, enacts a quiescent program in DTCs that is associated with an ERKlow/p38high signaling state.

How to DTCs evade detection?

Dormant cells can evade detection by NK cells through the repression of NK cell-activating ligands and are likely subject to surveillance by the adaptive immune system, which may keep cancer cells in a dormant state through the actions of IFNγ.

Explain the colonization prerequisite of metastatic niche

During many stages of metastatic growth, cancer cells depend on interactions with their microenvironmental niche and cross talk with various stromal cells, including endothelial cells, fibroblasts, and cells of the innate and adaptive immune system. The ECM is also an important component of the niche and can be modified in ways that support metastatic colonization. In some cases the formation of a metastatic niche may actually precede the arrival of cancer cells, in what is referred to as a pre-metastatic niche *secretion of TGFB by blood vessel. Inhibition of T cells and NK cells. Communication with metastasis-associated macrophages and fibroblasts

Why is it difficult for CTCs to travel in blood and lymph vessels?

Most cancer cells are larger than erythrocytes (>20 vs. 7 microns) and are not deformable, they are unable to negotiate narrow passages, such as the lumen of capillaries **May pinch off large amounts of cytoplasm or bypass capillaries by traveling through arterial-venous shunts to get to other places

Why does drug resistance occur with EMT?

Mouse breast cancer model RFP+ epithelial tumour cells undergoing EMT permanently convert into GFP+ cells following activation of Fsp1-Cre. Sorted RFP+ and GFP+ cells and performed RNA seq analysis CTX-treated GFP+ cells elevated their expression of many drug-metabolizing enzymes including drug transporters *FSp1 is a mesenchymal gene

How does extravasation occur?

Once lodged in a capillary, activated platelets and carcinoma cells secrete a number of bioactive factors that can act on monocytes, endothelial cells, and the carcinoma cells themselves. The collective effects of these interactions promote the transendothelial migration (TEM) of carcinoma cells, which can be aided by metastasis-associated macrophages (MAMs) in the target parenchyma. In lieu of TEM, arrested carcinoma cells may also proliferate intraluminally (not shown) or induce necroptosis in endothelial cells.

Seed and soil hypothesis

Stephen Paget the ability of a disseminated cancer cell to successfully found a metastasis depends on whether a distant tissue offers it a hospitable environment to survive and proliferate *but contralateral metastases are relatively rare so not the whole story

EMT can be induced by

TGF-B and other signals

Bone specific metastasis

The bone microenvironment (ME) secretome generated by osteoblasts, osteoclasts, or other cells may promote bone metastasis, while tumor cells can produce factors such as LOX to induce pre-metastatic niche formation. Interactions between tumor cells and osteoblasts through adherens junctions (via E-cadherin/N-cadherin, JAG1/Notch) and gap junctions (via CX43) also facilitate bone metastasis. Colonizing tumor cells express osteoblast-specific markers such as ALP and RUNX2 to escape immunosurveillance tumor cells secrete factors promoting bone turnover to induce osteolysis, which in turn produces factors to stimulate tumor growth, creating a "vicious cycle."

Explain the colonization prerequisite of seeding and maintenance of cancer stem cells

The capacity to seed and maintain a population of cancer stem cells, which are competent to re-initiate tumor growth, appears to be an initial prerequisite for metastatic growth. Dormant DTCs also exhibit key cancer stem cell attributes that probably contribute to their prolonged persistence in a quiescent state and their ability to eventually spawn a metastatic colony.

How does intravasation occur?

invading cancer cells first need to move to blood vessels, which can be within the tumour (neovasculature) or close to the tumour. Invasion through the stroma to blood vessels can be promoted by tumour-associated macrophages. Cancer cells can then enter the circulation by transmigrating either paracellularly through the endothelial cell (EC) junctions or transcellularly through the EC body. Matrix metalloproteinase 1 (MMP1) seems to be required for paracellular intravasation in regions where protease-activated receptor 1 (PAR1) on ECs mediates the remodelling of endothelial junctions. Alternatively, a disintegrin and metalloproteinase 12 (ADAM12) on ECs can induce cleavage of vascular endothelial cadherin (VE-cadherin) and angiopoietin 1 receptor (TIE2), which leads to disruption of endothelial junctions. macrophages can attract cancer cells towards blood vessels by secreting epidermal growth factor (EGF) and can also secrete tumour necrosis factor 1α (TNF1α), which induces the retraction of endothelial junctions, thus facilitating cancer cell transendothelial migration (TEM). Cancer cells can use Notch receptors to bind to Notch ligands on ECs and thereby transmigrate through the endothelial junctions they can also secrete transforming growth factor β1 (TGFβ1), which induces the retraction of endothelial junctions. During transcellular intravasation, the Ca2+ -calmodulin complex in an EC activates myosin light chain (MLC) kinase (MLCK) at the sites of cancer cell attachment, which leads to local phosphorylation (P) of MLC and to actomyosin contraction. In turn, this leads to rapid cytoskeletal and membrane remodelling, which creates a transitory pore-like structure for the cancer cell to cross the EC.

Induction and formation of the pre-metastatic niche

involves tumor-derived secreted factors (TDSFs), extracellular vesicles (EVs) TDSFs and EVs induce the mobilization and recruitment of several cell populations to secondary organ sites, including BMDCs such as VEGFR1+ HPCs and CD11b+ myeloid cells, and regulatory/suppressive immune cells including MDSCs, Treg cells, TAMs, and tumor-associated neutrophils (TANs). The interaction among these TDSFs, tumor-recruited cells, and local stroma may create a suitable niche microenvironment for metastatic tumor cell colonization. In addition, hypoxia and ECM remodeling also promote the formation of pre-metastatic niche.

spread of cancer occurs via

lymphatic and blood vessels mainly

What type of spread is responsible for the great majority of distant metastases?

spread via the blood circulation