I. also been applied for probing the mechanical

I.  Relevance of nano-biomechanics in monitoring diseases

Nanobiomechanics, as an emerging powerful technology to explore
mechanical aspects of biological matter at the nanoscale, has recently opened a
new horizon in scientific research by generating significant contribution in
the study of human diseases. Besides being instrumental
in increasing our
understanding of cellular behavior and cellular manifestations behind disease condition
and progression,
biomechanical research of physiological and pathological processes of different
diseases provided valuable knowledge for the development of therapies 25. During
the last two decades great progress has also been made in elasticity imaging at
whole organ level with the development of ultrasonic elasticity imaging and
magnetic resonance elastography (MRE) 26,27 having a wide application area from  cancer detection in almost all major organs 28, through the investigation of chronic liver diseases, but it has
also been applied for probing the mechanical properties of  intracranial tumors 29 and  muscle stiffness of
patients with neuromuscular disorders 30 as well. In contrast with the clinical use of whole organ
elasticity imaging methods, the tools of nano-biomechanics hold the advantage
of providing valuable information about mechanical changes at the cellular-level.
Changes in cells’ mechanical phenotype, such as adhesion or elasticity, affects
a wide range of human diseases and thus may contribute to the understanding of the
pathophysiology and the progress of arthritis 31, asthma 32, vascular disorders 33, malaria 34,35, sickle cell anemia 36, muscular dystrophies 37–39, cancer 35,40, and others.

AFM
is a powerful tool for identification and diagnosis of pathologically altered
cells or tissues in various human samples

This work will particularly focus on
nanomechnical measurements addressing brain metastasis formation and
amyothrophic lateral sclerosis.


reading Suresh et al 2005 acta biomater

1. Brain
metastasis formation

Cancer is still the second leading cause of
morbidity and mortality worldwide in countries of all income levels, with
approximately 14.1 million new cancer cases and responsible for 8.2 million deaths
in 2012 worldwide 41. Globally, nearly every 6th death is due to cancer. The
most life-threatening aspect of cancer is metastasis, being the main cause of
cancer patient mortality. Among all, the presence of brain metastasis (BM) is
one with the poorest prognosis, often causing life-impairing neurological
symptoms, where the median survival time can be counted in months, rarely few
years 42. While the different cancers show different propensities to form
BM, the majority of BMs originate from the primary tumor of lung cancer
(40%–50%), breast cancer (15%–25%) and malignant melanoma (5%–20%) 43.  Although melanoma is only 1
to 2% of all cancers, it invades the brain with one of the highest frequencies 44. Autopsy studies showed that up to 90% of patients with end-stage
melanoma had BM 45. Metastasis to
brain is difficult to treat; local surgery, whole brain radio therapy,
stereotactic radiosurgery have been the only treatment approaches for a long
time 46. Recently, incorporation
of systemic treatments such as molecularly targeted therapies and
immunotherapies have emerged as alternatives 47. Although results of
these therapies seem to be promising, along with this, prevention might be important as
well. Despite
our growing knowledge about biology of BM formation, the precise details that
trigger and guide tumor cell toward the brain are still under debate. In order to find effective
prevention strategies, lacking details of brain metastasis formation needs to
be elucidated.

1.1. Malignant
melanoma

Melanoma is among the most aggressive and
therapy-resistant human cancers. Occurrence of melanoma has increased
worldwide, being responsible for over 80% of skin cancer deaths 48. Despite that metastatic melanoma has relatively low abundance, it
shows high resistance to conventional therapies 49,50. The main reason for this is the high level of its genetic
heterogeneity. Among all human cancers melanomas carry the highest number of
mutations, generally more than 10 per Mb 51, resulting tumors with differently mutated domains that necessitate
different treatments 52. In some cases even the metastases could show different genotype
compared to its primary tumor 53. The most affected signaling cascade by melanoma is the
mitogen-activated protein kinase (MAPK) pathway, which includes the signaling molecules
Ras, Raf, MEK and ERK, and plays an important role in the regulation of
cellular growth, migration, and survival. BRAF (~50%) and NRAS (20-25%) are the
two major mutations in melanoma, causing constitutive activation of the MAPK
pathway which then leads to the expression of genes encoding proteins that
regulate cell proliferation and survival.

1.2. The
process of metastasis formation

Cancer cells can use blood or lymphatic
transport to form metastases in distant organs. Since the central nervous
system (CNS) lacks a lymphatic system, the only way for cancer cells to reach
the brain is via the blood stream. However, a tumor cell to form a metastasis,
especially in the brain, needs to go through several challenging processes,
which requires a high degree of motility and adaptability to the different
environments. After the acquisition of a motile phenotype the cell must escape from
primary tumor site, invade nearby tissues, brake through blood vessel wall and enter
into the blood circulation, referred to as intravasation. Here the cancer cells
are exposed to a turbulent blood flow and exposed to the offensive of immune
cells that recognize them as abnormal. If a cancer cell is able to survive in
this extreme environment and escape detection by immune cells, it can
disseminate and reach distant organs, like the brain. Through extravasation,
after firmly attached to the blood vessel wall the cell transmigrate through
the cerebral endothelial layer into the brain. Here interacts with the
microenvironment, which could result in a survival, proliferation and lastly
the formation of a secondary tumor 54.

1.3. Blood-Brain
Barrier

The
first challenge for hematogenously disseminated melanoma cells to reach
the brain parenchyma is to
break through the tight layer
of brain endothelial cells, forming the morphological basis of the blood-brain barrier (BBB)
55. This precisely
regulated barrier between the systemic circulation and the CNS protects the neuronal
tissue by restricting
the free movement of solutes and cells between the two compartments. Since
several environmental and
molecular factors play crucial role in to prevent the penetration into
the CNS and arrest melanoma
cells at the luminal surface of the blood vessels,  these must have special attributes to overcome these
obstacles and facilitate breaching the BBB 56. However, after a successful transmigration BBB may have some
supportive role as well by protecting the metastatic cells from the immune
surveillance of the organism or releasing substances favorable for metastasis
growth 55. This and its neural crest origin are among the main causes for the
high brain metastasis formation tendency of melanoma.  

1.4. The use
of AFM in cancer research

Nanomechanical cellular information provided
by atomic force microscopy can identify morphological variations, cellular binding forces, cell
stiffness changes and
surface adhesion behaviors that could enhances our understanding of cancer at the cellular and
tissue level and thus can lead to development of advanced clinical solutions 57. The
first pioneering comparative study on the elastic properties of cancerous cells
reported a one order of magnitude decrease in the Young’s modulus of cancerous
cells (Hu456, T24, BC3726) in contrast to normal cells (Hu609 and HCV29) 58, focusing the attention on the importance of cancer cell mechanics
investigations. This led to the initiation of numerous studies providing quantitative
biomechanical fingerprints of cancer-related changes and contributing
greatly to cancer research that may even facilitate the development of future
cancer therapies 59,60. Data accumulated so far clearly demonstrated that individual
cancer cells are more deformable than their healthy counterparts in the
majority of cancer cell types, including bladder, breast, prostate, ovarian,
colon, thyroid, kidney, and other cancers 61,62. A few studies went further by raising the question whether
invasiveness can be reflected in the nanomechanical parameters of cancer cells.
and obtained decreased elastic modulus along with higher metastatic potential 63–65.

Changes in cell adhesion are defining in the
development of cancer. There
is growing evidence showing that cancer cells, by changing their structure and behavior,
can alter their mechanical phenotype thereby facilitating their dissemination 56. It has been reported that malignant breast cancer cells show
reduced inter-homocellular
adhesion which decreases with metastatic potential, hence promoting their
escape from the primary site 66. In addition, some other studies obtained that cancer cells display
an increased adherence to the ECM, apparently due to the same reason (ref). Moreover, in case
of melanoma a modulation of cell-cell
interaction strength was observed depending on the extracellular
protonation 67.

A crucial step in the multistage process of
cancer metastasis, especially in case of BM formation, is the firm adhesion of blood-travelling
tumor cells to the blood vessel lining endothelial layer. This can be
addressed directly by AFM
based single-cell force spectroscopy to investigate cellular mechanics and the
dynamics of intercellular interactions throughout tumor cell
transmigration 57,68. Excluding the
immobilization of the cancer cell, during these measurements there is no need
for any staining or other cell-life impairing preparation. In vitro cell co-culture
models dealing with the whole transmigration process have already been
successfully established 69. A detailed understanding of
tumor cell – endohetial cell interaction could lead to the identification of
mechanisms which could reduce transmigration and thus BM formation. A recent
study showed how the expression
of cell adhesion molecules in endothelial cells influences their adhesive properties
to bladder cancer cells 70. Thickening of the outer
glycocalyx layer of the endothelial cells, induced by short term hyperglycaemia,
resulted in higher adhesion between lung carcinoma and human aorta endothelial
cells 71. The accumulated knowledge about the contribution of biomechanical
properties such as
elasticity, adhesion dynamics, or strength to the cell’s mechanical phenotype are essential for improvement
of early cancer identification, thus could be substantial in future
cancer therapeutics.

Short
term (up to a few tens of seconds) adhesive properties can be studied upon
analysis of the detaching process of individual cells.

Basically
every active and passive connection might contribute to the established
adhesive strength between a blood travelling tumor cell and the endothelial
cells.

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