In our earlier lecture we have shown that
each and every parts of her body this is that
mechano transduction by different mechanical
forces. In this lecture we shall go through
that how that mechano transduction happening
in case of cancer and in case of bounties
of forming cells. Why cancer because in the
cancer metastasis? Mechanical force playing
the main role for the migration of cancer
cell from that source tumor to the distance
places through that blood circulation system.
And bone is a ah a mechanical sensitive system
and it is bone forming ability solely depends
upon that mechano signals, without these mechanical
signals bone forming cells will not work.
So, that is why we are concentrating on that
cancer metastasis and bone formation osteoblasts
mechano transduction in this lecture.
So, take home lesson of this lecture utility
of microfluidic platform for studying mechanobiology
particularly cancer milieu; because we know
that that focal adhesion and lipid rafts and
stress activated ion channels are the mechano
sensitive cubes or mechano sensitive ah parts
of the cells, and these are responsible of
various biological outcomes of the different
types of cells. Here we are focusing that
how that focal adhesion complex dissociation
and lipid raft micro domain internalization
mammalian cells happening in the microfluidic
confinements, and how that lipid raft micro
diamond organisation in stress adaptive mechano
transduction in circulating tumor cells. Basically
what I want to mean that circulating tumor
cells; when that tumor cells is in the blood
stream facing that shear stress of that blood.
And as already I have mentioned that bone
is the mechano sensitive organ, and how that
shear stress influences osteoblast cells means
your bone forming cell to work, in terms of
that calcium signaling ah .
Now, we are coming that tumor formation, but
how that cancer cells situation in the tumor.
So, cancer cell starts due to the genetic
aberration and due to this lot of signaling
pathways are starting. And one of the pathways
are epidermal growth factor Ras and ERK pathway
. So, this pathway stimulates the proliferation
of cancer cell, because we know that cancer
cells have a uncontrolled growth rate in that
sequence this helps that cytoskeleton arrangement
and it develops cytoskeleton stressor.
So, which stimulates that that rho rock and
myosin light chain phosphorylation then actin
myosin interactions. So, these two pathways
interact with each other, to stimulate that
extracellular matrix stiffness. As extracellular
matrix stiffness is increases, that stimulates
that focal adhesion which acts as a molecular
clutch it influences that integrin activation
focal adhesion kinase, then stimulate both
proliferation as well as cytoskeleton tensions.
So, what will happening in that process that
e c ms gets stiffer means rigid in the down.
Here we can see that in the normal cases that
is same is soft matrix, cells have a normal
size when cancer cells start growth uncontrolled
growth that matrix becomes rigid, and at the
same time lot of other events happening in
that situation, because as cancer cell growth
it invites that federalists that lot of immune
cell like a t cell b cells in that micro environment.
So, cell population is very much high within
this confinement. So, this you can compare
like say say one argon balloon is attached
to a thread, and when that argon balloon is
expanding that tension in the thread is increasing.
So, this type of mechanism is happening in
that cancer tumour case, and at the same time
that your blood capillaries becomes necrotic
means irregular your accessible matrix stiffness
are not regular means different places special
temporal is here matrix density is different
places is different.
. So, this is that multi cascades of how that
cancer cell metastasize or migrated from that
source place to the distant place. At the
source place means where that tumor develops
that metastatic cascades cancer cells within
a primary tumor acquire that invasive phenotype.
What is invasive phenotype means that initial
positions that initial positions cancer cells
are encouraged dependent means, there more
or less static. During the high population
of the cancer cell and other cells and due
to oxygen limitation and so, many other factors
that cancer cells, some cancer cell want to
escape from that site. So, for that escape
mechanism they have to developed some non
encourage dependent system by which will be
changing to metastatic cells means from that
primary site they will be invasing that nearby
blood vessel or lymphatic vessels.
So, as it enters the blood vessel, it occurs
the property of non anchorage dependent property
means they need not any anchorage for their
survival they will survive in the blood flow
and faces that shear stress of the bloods.
So, in that shear stress majority of the your
tumor cells will die, some will survive which
can withstand this ah shear strain. ah In
that way migrated to a distant place that
is a distant organ CTC exists in the circulation
and invade that micro environment of the foreign
tissue means the distance tissue; and they
are cancer cell must be able to evade the
innate immune system and also survive as a
single cell or as a cluster of cells then
again that tumors starts. Basically starting
from that source tumor to blood vessels, this
is totally devine by that mechanical force
.
So, mechano transduction is playing very vital
role in that migration process, and interstitial
flow is playing major role when that metastatic
cell migrates from that source and invade
that near about or near larger cell .
So, interstitial flow 
depends upon that pressure gradient develops
between arteriole interstitial space that
near about lymphatic vessel and that venules.
And as that tumor is very much heterogenic
with respect of ECM the interstitial pressure
difference is also different in different
places. So, microfluidic platforms are used
extensively to study the migration of the
cancer cell in that interstitial flow system.
So, what literature is saying that, in the
interstitial flow effects cell migration in
different ways local strain gradient induction
flow upregulated proteolysis by migrating
cells stress mediated cytokine activation
and autologous chemotaxis. Main findings are
that downstream migration means that in the
direction of the interstitial flow, that is
your cells are migrating according to the
chemotaxis means your growth factor or cytokine
gradient. Whereas, lot of cells also migrated
upstream of that interstitial flow and that
is driven by stress activated things, and
that is focal adhesion is involved for your
upstream movement means both type of movements
are available, in that is your from tumour
primary site to migration to near nearest
blood vessel .
And as that tumor is heterogenic population
of cells and cancer cells itself are heterogeneous
so; obviously, their migration will be of
ah heterogeneous. To understand that system
Hessler has developed utilized a new platform
for micro fluidic system where that two types
of interstitial gradients are exploited to
know the heterogeneity of that cancer cell
migration. . So, heterogeneity means some
ah response to flow by moving faster, but
randomly, some cells migrate in the flow direction
in two autologous chemotaxis and others may
move in the opposite direction due to the
local stress gradients which means there are
different types of cellular migration in interstitial
flow.
So, what Hessler finds that cell migration
within a population is heterogeneous that
is some cells migrate upstream and other downstream
of the flow and minority of the cell populations
are more random there may be more directed
more randomness more faster means these are
the around 5 to 10 percent of the cancer cell
populations are very much heterogeneous with
respect of their migration rate. So, these
cells might be the important for the metastatic
survival or metastatic potential.
So, with this study they have identified that
migrating potential or different cells with
respect to other majority cells which have
a different migration potential. So, with
this background or collaborative works Professor
Simon Chakraborty has revealed some unique
findings when that CTC means here at that
encourage independent stage, cancer cells
migrating from that source to that blood stream
like say ah focal addition and that lipid
wrap, how they are behaving and related processes.
So, challenge in that system is that it is
to be addressed whether environment of surrounding
the micro confinement plays a vital role in
the cell survival within the passage of vasculature
or tissue matrices. So, how they address this
problem? In our earlier lectures we have described
that, how to measure the traction force by
using traction force microscopy system and
membrane fluidity, and other microfluidic
platforms to reveal that augmented stress
responsive traits of cell lines in a micro
confinements . So, in that process we found
a new parameter like say we are telling that
cell response time T R is introduced to analyze
the stress adaptive properties of the mammalian
cells what is T R? So, it is a time lag between
focal adhesion disassembly and lipid raft
appearance in the same positions of the cell.
So, now we are going to details how that phenomenon
is happening.
. So, here is the image of one mammalian cell,
then this is the image of lipid raft in the
cellular membrane as well in the cellular
in ah inside of that. And these are the images
of that focal adhesion points and we concentrated
on one of the focal adhesion points, that
how it is dissociated. When you are giving
shear stress that focal adhesion points in
that where that shear stress is given, that
focal adhesion points dissociates. You see
that here this focal adhesion slowly dissociates
and and that dissociation for the full dissociation
it takes around 20 minute it is represented
by this curve at the same time we are looking
for that how lipid rafts behaviour in that
situation, here we see that in the same confocal
plane that lipid rafts internalization it
takes around around say 20 minutes or18 minutes
time. So, this is that time lag between the
two events and this is a characteristic property
of that mammalian cells, when you are giving
a some stress means in the condition of fluid
flow.
So, now you want to know. So, how that focal
adhesion is involved in this process and this
is that representation, where you are showing
that 50 percent of the lipid raft internalization
events happening at any given period of time
under that some shear stress. When we innovate
the focal adhesion by using focal adhesion
kinase inhibitor NSC 23766 we are finding
that that red pillars basically, they are
indicating that that lipid raft internalization
event are not happening.
So, we can correlate that focal adhesion dissociation
and that lipid raft internalization this is
a characteristics phenomenon in that confinement
and if this shear stress.
And that response time T R dependent upon
that two variables one is your confinement
confinement what I want to mean here that
height of the channel is two height of the
channel height of the cell is constant we
can vary the height of the channel. So, confinement
one parameter another each shear stress developed
by the different rates of fluid flow . So,
there as a critical conditions like say when
that channel height is 70 micrometer, and
that shear stress is more than 10 times per
square centimetre then we are finding that
sigma del decline of that T R, but in other
cases this is not behaving or open system
this is not behaving sigma del.
So, you are taking this critical conditions
and we measured that T R for that varieties
of cells starting from that normal cell lines
like say NIH 3T3 L929 and most evasive cancer
cell line like say MCF 7 B16F HeLa cell and
lot of other cells. And if you see that what
we are seeing in that micro confinement situation
had the shear stress 10 dynes per square centimetre
and T R is around 10 minutes for these cancer
cells and whereas, it is more than 20 to 30
minutes for the normal cells. And that indicates
that we have find a unique parameter response
time to differentiate between that normal
cells and cancer cells particularly in their
micro confinement, where that is critical
micro confinement and that critical shear
stress .
So, cosine arises how this factor is happening
in the molecular level. So, we know that epidermal
growth factor is a growth promoting one of
that growth factors and it binds with that
epidermal growth factor receptors, and pass
the signal means food factor receptor is phosphorylated
then signal is passing. And that lipid raft
is very much connected with this membrane
bone receptors and on shear that lipid rafts
get rearranged and and undergoes internalization.
So, what we find it here that, autocrine growth
factors that EGF works nicely in that confinement
with this western blot and the de magic techniques.
We saw that that in absence of EGF, only shear
stress suffice to activate that EGFR and which
helps the internalization of the lipid raft
and at the same time that is a focal addition
dissociation. So, we connect is whole these
things that in the micro confinement autocrine
effect of that EGF stimulates that chemical
signal transduction, which is influenced by
that mechanical signals of shear stress .
So, what we infer from these studies, sigmoidal
shaped decline of T R depends on confinement
and shear stress; that same T R occurs when
that channel height decreased below threshold
value of seventy micrometer and stress is
elevated beyond 10 dynes per square centimetre.
And this T R is due to augmentation of secreted
autocrine growth factor epidermal growth factor
that in EGF concentration and amplification
of the fluid shear stress the microfluidic
environment. And T R is less for invasive
cancer cells means cancer cells are stress
adoptive they can adopt which cells adopt
this shear during their blood flow they can
survive otherwise they will die. So, these
things indicating that cancer cells are more
adaptive to stressful situation in the micro
confinement of blood vessel by arranging their
cytoskeleton, during migration and extradition
.
With this context we are following that another
set of experiments where involvement of lipid
raft in mechano transduction is explored in
details, because lipid raft is that place
where it can gives the shelter of your membrane
bound enzymes, growth factor receptors, stretch
activated ion channels and it also activates
the downstream of mechano transduction that
is a MMP pathways .
So, here we are exploring that autophagy another
biological out means biological readouts or
it is a phenomenon of housekeeping mechanism
whereby whereby the cell degrades damaged
unnecessarily dysfunctional cellular components
through the action of lysosome. What is lysosome
already we have discussed this is a means
your garbage bag it recirculate or digest
that unwanted material and ah do its job.
So, autophagy is a phenomenon where that unutilized
organelles or molecules so, and so, forth
the digested in the lysosomes, and gives the
energy to the cells when the cells are in
stress. And different types of autophagy are
theres like say my to autophagy nick autophagy
macro autophagy etcetera now it is coming
. So, autophagy is a double edged sword means
when their cells are in stress condition like
say when that metastatic cells are filling
the stress in the bloodstream or cells are
in starvation, they utilize its own energy
sources and metabolize it or digested then
metabolize it to get the energies, but that
is your pro survival mechanism.
On the contrary when that autophagy is our
uncontrolled means self between things going
unrestricted, then it kills itself it triggers
that apoptosis that is program cell cell death
that is why that autophagy is a double edged
sword it acts as a pro survival or death inducing
mechanism according to the need of the cell.
So, now you are looking for that how that
mechano transduction or shear is involved
to stimulate that autophagy either pro survival
or death inducing. So, that is why you have
raised the questions whether physiological
shear stress elicits autophagy in cervical
cancer cells as immediate adaptive response
whether this response is protective towards
the cells or do membrane cytoskeleton components
like say lipid rafts, participate in the process
of shear mediated mechano transduction.
So, in that experiments we have two parts
means on chip part another is off chip part.
On chip part we are giving that cells with
shear stress and with that measuring autophagy
by reporter plasmid p t f LC 3 exposed to
pulses of steady flow, and off chip part we
are using that syringe pass model for the
application of shear stress to cells and collection
of cells, then after that assess the viability
and protein expression and etcetera .
So, what we are finding it here? So, in the
control set means when you are giving now
shear we are measuring that autophagy in terms
of puncta. Puncta means you can see that dots
actually say green dots and yellow dots and
a red dots . So, autophagy flux is the ratio
of that red and green dots with respect of
your total number of dots present in that
in one cell. Because we are measuring that
autophagy flux of single cells taking say
100 cell or 1000 cells together and we are
emerging their data.
So, these data reveals that when you are giving
two pascals shear that yellow dots means is
a mixture of your green and red dots and only
red dots are increasing with respect of your
total dots with green dots.
And when you are giving MBCD which disrupt
that your lipid rafts that with this two plus
pascal shear surprising now we are saying
that, the decrease of significant decrease
of your RFP and GFP means yellow dots. So,
this indicates that MBCD which disturbed that
lipid rafts definitely involved in autophagy
flask.
The next question we are arising how that
shear stress induces the autophagy flux whether
it is a pro death or pro survival. Here what
we find in a not cell from that slide; when
you are giving shear that autophagy is survival
inducing means that it induces cell to survive
in that stress does not mean stress adaptive.
And when you are disrupting that lipid rafts
your it is inducing more apoptosis more cell
death means that with this findings that lipid
rafts has a interesting role to withstand
the shear and give the particular biological
outcome with respect to autophagy whether
it is a pro survival or death inducing things
So, all these things is model in that way,
inhibition of protective autophagy by lipid
raft disruptions an invasion of P 38 map kinase
phosphorylation is the intermediate of that
you are ah signaling chemical signal processing,
shorter life span of tumor cells in circulation
and lack the metastatic ability .
Next we are coming in that bone system it
is a mechano sensitive system, where that
mechanical signal is necessary for that activity
of bone cell osteoblast. So, here we are saying
that this is the blood vessels surrounding
cells at osteoblast cells, then that bone
formation is going on when in the osteoblast
is confirming or converted to osteocytes.
During this whole the process that is mechanical
stimuli influences lot means when normal state
mechanical stimuli is there there is a equilibrium
between bone dissociation and bone formation
that is I used it.
When overused means when are using working
or more load in the bones um indicated by
these arrows then osteoblasts recruited and
it induces bone growth, then it is coming
to a steady state. Then when is underused
means not much mechanical signal is going
on, then osteoblast bone degrading cells are
activating then bone start degrade and then
is some steady state reaches. So, this indicates
that mechanical signal is very much responsible
for osteoblasts function that is bone forming
function, and it produces lot of your extracellular
matrix that is your calcium calcium hydroxyapatite
and etcetera .
So, whole literature is saying that, when
you are giving shear stress on osteoblast
in parallel channel on one chip intensity
of the intercellular calcium intensity because
proportional to intensity of the shear stress
between 0.03pascal to 0.24 pascal. And this
shear intercellular calcium concentration
such peak at the beginning and influence a
mechanical transduction. And when he removed
the shear then again calcium concentration
decreases and that calcium intensity peak,
it depends upon the shear stress. So, it was
reported by that then after that our group
with collaboration Professor Chakravarthy,
we are utilizing that fluidic platform to
explore that differ how the different types
of fluid flow influence that the osteoblast
function in terms of calcium flicker generation.
So, here in the fluidic channel we are using
different types of fluid flow like steady
state pulsatile flow and oscillatory flow,
in the osteoblast cells. What you are finding
that oscillatory fluid flow that is oscillated
fluid flow so, that maximum calcium flicker
concentration as well its calcium amplitude,
and this is measured by your calcium cytoplasmic
calcium flicker concentration and calcium
flicker is due to that you have calcium transporter
channels, which are present in the membrane
plasma membrane endoplasmic reticulum.
So, now question arises what type of that
is stress responsive channels or what type
of calcium channels are involved in that situation
of shear stress.
So, you are using ah . So, here we are saying
that that mechanic here oscillatory shear
stress is involved in the process and what
hot frequency. Here that point to hot frequency
that oscillated shear stress is a maximum
so, in that calcium flicker concentration
in that the osteoblast cells. So, to explore
that what type of calcium channels are involved
in the shear situation because varieties of
calcium channels are involved in that process.
So, we use different types of inhibitors for
a particular group of your calcium ah activated
channels like say voltage gated calcium channel,
store operated calcium channel, stage activated
calcium channel, calcium ATpase and phospholipids.
And there are specific inhibitors for these
type of channels and what our findings is
that, when you are using gadolinium chloride
which is inhibiting the stress activated calcium
channels that calcium flicker concentration
decreases drastically around say 80 percent
. So, we are telling that that stage activated
calcium channels are involved in this process
and we also know that calcium in that stage
activated calcium channels are harboring in
that liquid raft.
So, definitely lipid raft irresponsible or
activated or organized in that when you are
giving that oscillatory shear stress.
. So, to none of them to identify that which
type of stress activated I calcium channels
unresponsive. So, these group of calcium channels
are termed as transient receptor potential
super family member . So, there are different
members like say v 2 p 2 m 7 m 6 etcetera.
So, what we did we knock down the osteoblast
cells by s i r n a technology, then we are
looking for that or we are confirming there
whether cells are knock down or not by osteon
blot and we are saying that T RPM 7 all these
things which are successful knockdown for
the cells. And in the knockdown cells we measured
that calcium flasks in that oscillator shear
stress and we find that the T R PM 7 long
down cells there is no calcium flickers this
clearly indicates that T R P M 7 is really
responsible for that calcium flicker generation
due to oscillator shear stress .
So, as you mention that lipid raft is the
position or lipid raft is that ah means domain
where that the stress type of stress activator
and channels are present. So, you are looking
for that how that space. So, temporally in
the membrane that lipid rafts, and that calcium
flicker generation is occurring. So, what
we are saying it here at that same time point
that same time point and that same places,
calcium flicker generation as well as lipid
raft organization is happening. So obviously,
question arises. So, what is the role of your
lipid raft for calcium flicker generation.
So, if we disrupt that lipid raft by using
your methane cyclodextrin, what will happen?
So, if our hypothesis is correct then if we
disrupt that calcium flicker generation will
not be there for that is demonstrated here.
So, if we use AMECD and do that same shere
and calcium flicker generation is almost the
control area .
So, this indicates that that shear oscillatory
fluid flow induces that lipid raft rearrangement,
which induces TRPM 7 organization and when
that TRPM 7 circum closer together that induces
calcium flicker generation inside that side
of logic.
. So, all these things are possible using
that micro fluidics to understand that mechano
signal transduction during cancer metastasis
and osteoblasts in function. So, what we are
exploited here high resolution imaging at
single cell level. So, study that spatio temporal
dynamics of intricate membrane related cellular
events. So, what we find is here that, disassembly
of focal adhesion points due to shear triggers
localized lipid raft internalization and shear
stress triggers calcium flickers in shear
exposed bone cells.
Simulation of physical flow conditions mimicking
in vivo niche physiological laminar shear
stress induces EGFR activation making cancer
cell. Invasive induces protective autophagy
aiding cancer cell survival and oscillatory
shear stress of frequency for 0.2 hertz induces
optimal bone growth means, your calcium flicker
generation it will go.
And mimicking in vivo confinements possibly
replicate the natural cells confinement aspect
ratio and our finding is that cancer cells
are more active have lesser response time
means, they are more adaptive than non cancerous
cells in that confinement . So, such studies
in that importance of lipid raft mediated
mechano biological signaling cascades, as
a candidate drug targets in various physiological
ailments .
