WELCOME TO THE LECTURE IN THE
BIOMEDICAL GROUP.
PLEASE ASK ANY QUESTIONS VIA THE
NIH VIDEOCAST SYSTEM, LIVE
FEEDBACK BUTTON, QUESTIONS
ROUTED TO SPEAKER AT THE END.
I'LL HAND IT OVER TO OUR
CO-HOST, DR. MATT WOLF, FOR
INTRODUCTION OF OUR SPEAKER.
>>ALL RIGHT.
THANK YOU VERY MUCH, CAITLIN.
TODAY IT IS MY PLEASURE TO
INTRODUCE DR. DAVE MOONEY.
DR. MOONEY IS THE FAMILY
PROFESSOR OF BIOENGINEERING AT
HARVARD UNIVERSITY, A FOUNDING
CORE FACULTY AND LEAD OF THE
IMMUNOMATERIALS FOCUS AREA AT
THE WYSS INSTITUTE FOR
BIOLOGICALLY INSPIRED
ENGINEERING, PUBLISHED HUNDREDS
OF PAPERS, AMASSED NUMEROUS
HONORS OVER HIS CAREER,
INCLUDING MEMBERSHIP IN SEVERAL
OF THE NATIONAL ACADEMIES,
INCLUDING ACADEMIES OF
ENGINEERING, MEDICINE,
INVENTORS.
HIS WORK HAS HAD TRANSLATIONAL
IMPACT, WITH NUMEROUS PATENTS,
AND INVENTIONS THAT HAVE BEEN
LICENSED BY OVER 15 COMPANIES
AND LEADING TO COMMERCIALIZED
PRODUCTS.
SO, DR. MOONEY'S LABORATORY IS
MULTI-DISCIPLINARY IN THEIR
APPROACH TO USE BIOMATERIALS TO
MANIPULATE BIOLOGICAL SYSTEMS.
HOW YOU'VE HEARD OF HIM DEPENDS
ON YOUR FIELD OF BIOENGINEERING
STUDY, HE'S HAD AN IMPACT.
I FIRST GOT TO LEARN ABOUT HIS
WORK IN MUSCULOSKELETAL ISSUE
ENGINEERING AND REGENERATIVE
MEDICINE.
LATER ON, I GOT TO KNOW MORE
ABOUT HIS WORK IN IMMUNE
ENGINEERING, CANCER
IMMUNOTHERAPY, MECHANIC -- MECH.
HE WILL BE TALKING ABOUT A
DIVERSE SET OF INTERESTS TODAY
AND I'M EXCITED TO HAND THAT
OVER AND START HIS TALK.
THANK YOU VERY MUCH FOR THE
OPPORTUNITY TO COME AND
PARTICIPATE IN A SEMINAR SERIES,
I GREATLY APPRECIATE THE
INVESTIGATOR FOR TAKING TIME TO
TALK WITH ME ABOUT THE RESEARCH,
A LOT OF EXCITING RESEARCH AND
DEVELOPMENT IN BIOENGINEERING
UNDERWAY AT THE NIH.
IT'S GREAT TO SEE.
I'M GOING TO TALK ABOUT ONE
ELEMENT OF THE WORK IN THE
LABORATORY TODAY.
THAT RELATES TO EXTRACELLULAR
MATRIX VISCOELASTICITY AND
INTERSECTION WITH BIOLOGY OF
CANCER.
BEFORE I GET STARTED I SHOULD
ACKNOWLEDGE THE WORK I'LL
DESCRIBE TODAY HAS BEEN LARGELY
FUNDED BY THE NCI NETWORK, NIDCR
FUNDED SOME WORK I'LL DESCRIBE
TODAY, AND THE NSF THROUGH THE
CENTER AT HARVARD.
TO START WITH THERE'S PEOPLE
THAT AREN'T ENGINEERS, SO THE
TERM VISCOELASTICITY MAY NOT BE
SO FAMILIAR WITH YOU.
IT RELATED TO MECHANICAL
PROPERTY OF MATERIALS, AND MANY
OF US ARE FAMILIAR WITH THE IDEA
OF STIFFNESS WHICH STIFFNESS
RELATES TO HOW MUCH RESISTANCE
MATERIAL PROVIDES.
YOU CAN THINK ABOUT A RUBBER
BAND, STIFFER OR SOFTER.
VISCOELASTICITY RELATES TO
SOMETHING DISTINCT, THE BEHAVIOR
OF A MATERIAL.
YOU CAN SEE THIS DEFORMS, AS
SOON AS THE METAL GOES BACK UP
AGAIN, THE MATERIAL SPRINGS BACK
TO ITS ORIGINAL SIZE AND SHAPE.
THAT'S SOILED-LIKE BEHAVIOR,
PURE -- SOLID-LIKE BEE HAIR
HEIFER.
LIQUIDS ARE VISCOUS OR
VISCOELASTIC, THE SAME
EXPERIMENT.
IN THIS CASE WITH BRAIN TUSH,
YOU CAN -- BRAIN TISSUE.
IT REFERS TO LIQUID-LIKE VERSUS
SOLID-LIKE BEHAVIOR OF THE
MATERIAL.
MOST MATERIALS HAVE SOME
ELEMENTS BOTH VISCOUS BEHAVIOR
AND ELASTIC, SO I WILL
FREQUENTLY TALK ABOUT MORE
ELASTIC MATERIALS OR MORE
VISCOUS MATERIALS TODAY, IF WE
MONITOR THE FORCE OR STRESS TO
APPLY, A MORE LIQUID-LIKE
MATERIAL ABOUT DISSIPATE -- WIL
DISSIPATE THE STRESS OVER TIME,
MORE ELASTIC MATERIAL WILL NOT,
THAT'S VISCOELASTICITY.
THE REASON WHY WE'RE INTERESTED
IN PHYSICAL PROPERTIES AND
MATERIALS IN CONTEXT OF CANCER
IS THAT AS I'M SURE ALL OF YOU
ARE AWARE, THAT SOLID TUMORS ARE
ASSOCIATED WITH CHANGES IN THE
EXTRACELLULAR MATRIX, INCLUDING
FIBROSIS, LEADS TO INCREASED
CROSS-LINKING OF THE
EXTRACELLULAR MATRIX AND
INCREASED DEPOSITION OF THE
MATRIX, THERE ARE ALTERATIONS IN
THE PHYSICAL PROPERTIES OF THE
EXTRACELLULAR MATRIX SURROUNDING
CELLS WITHIN THE TUMOR
MICROENVIRONMENT AS CANCER
PROGRESSES.
CELLS IN THE MICROENVIRONMENT OF
THE CANCER CAN ACTIVELY PROBE
MECHANICAL PROPERTIES AND SO
WHAT THIS VIDEO IS SHOWING YOU
IS A REMINDER TO EVERYONE THAT
CELL ADHESION AND ENGAGEMENT
WITH EXTRACELLULAR MATRIX IS AN
ACTIVE EVENT, NOT PASSIVE.
THE IMMUNE CELL IS MIGRATING
THROUGH THE COLLAGEN MATRIX,
ACTIVELY PUSHING AND PULLING AND
YOU CAN SEE THE INDIVIDUAL
COLLAGEN FIBRILS BEING FORMED BY
THE CELL.
CELLS CAN SENSE HOW MUCH
RESISTANCE THE EXTRACELLULAR
MATRIX PROVIDES TO THEIR OWN
FORCES, THIS CAN FEEDBACK AND
ALTER CELL BEHAVIOR.
THE IMPORTANCE OF THIS STIFFNESS
OF THE EXTRACELLULAR MATRIX AND
CHANGES IN STIFFNESS IN THE
CONTEXT OF CANCER HAS BEEN
APPRECIATED, THIS IS A COUPLE
CLASSIC PAPERS FROM THE FIELD,
FROM ABOUT 15, 20 YEARS AGO,
DEMONSTRATING IN THE CONTEXT OF
BREAST CANCER CELLS, THAT THEY
RESPOND QUITE SIGNIFICANTLY TO
ALTERATIONS IN STIFFNESS.
SO THAT'S SOMETHING THAT'S BEEN
KNOWN FOR SOME PERIOD OF TIME.
HOWEVER, THE FOCUS IN THIS
GENERAL SPACE AND MORE BROADLY
MECHANOTRANSDUCTION OUTSIDE
CANCER HAS BEEN FOCUSED ON THE
QUESTION OF STIFFNESS.
IN ESSENCE ACTING LIKE THE
TISSUES IN OUR BODY AND
EXTRACELLULAR MATRICES ARE
ELASTIC AND CAN BE CHARACTERIZED
BY STIFFNESS MEASURE.
I ALREADY MENTIONED TISSUES IN
OUR BODY, EXTRACELLULAR MATRIX
MOLECULES ARE ACTUALLY
VISCOELASTIC INSTEAD.
ON THIS GRAPH WHAT WE'RE LOOKING
AT IS THE Y-AXIS IS INDICATION
OF THE FLUID-LIKE PROPERTIES,
THE LOSS MODULUS, AND X-AXIS
STORAGE MODULUS, INDICATION OF
SOLID-LIKE PROPERTIES.
ALL THE SKELETAL AND SOFT
TISSUES, EVEN RECONSTITUTE
EXTRACELLULAR MATRICES HAVE
PROPERTIES INTERMEDIATE, BETWEEN
A SOLID AND FLUID, SO THEY ARE
ALL VISCOELASTIC.
SO OF COURSE THIS RAISES THE
QUESTION, IF CELLS ARE
SURROUNDED BY VISCOELASTIC
SUBSTRATES AND THE
VISCOELASTICITY MAY CHANGE, DO
CELLS RESPOND SIMILARLY OR
DIFFERENTLY, IS THIS A CONTROL
FEATURE SIMILAR TO HOW CELLS
RESPOND TO STIFFNESS.
NOW AS I MENTIONED A MOMENT AGO,
ONE WAY ONE CAN QUANTIFY HOW
VISCOELASTIC A MATERIAL IS BY
SOMETHING CALLED STRESS
RELAXATION TEST WHERE WE APPLY A
GIVEN DEFORMATION, MONITOR HOW
MUCH STRESS OR FORCE WE HAVE TO
CONTINUE TO APPLY TO MAINTAIN
DEFORMATION.
A PURELY ELASTIC SOLID, THE LINE
WOULD GO STRAIGHT ACROSS, BUT
VARIOUS TISSUES LINE DROPS
INDICATING VISCOELASTIC, THE
MORE RAPIDLY THE MORE VISCOUS
THE MATERIAL.
NOW, THERE IS INDICATIONS THAT
NOT ONLY THE STIFFNESS BUT
VISCOELASTICITY OF SOLID TUMORS
IS DIFFERENT FROM SURROUNDING
TISSUE.
IN THE CONTEXT OF BREAST CANCER
AND GLIOBLASTOMA THE
VISCOELASTICITY OF THE MATRIX
CHANGES WITH TUMOR DEVELOPMENT,
MAKES SENSE BECAUSE YOU'RE
GETTING MORE CROSS-LINKING WHICH
WILL MAKE THE TISSUE MORE
SOLID-LIKE.
SO WE WANTED TO BEGIN TO EXPLORE
THE IMPACT OF VISCOELASTICITY,
I'LL DESCRIBE HOW WE EXAMINED IN
CONTEXT OF IMMUNE CELLS, BUT WE
NEEDED AN EXPERIMENTAL SYSTEM TO
EXPLORE.
IT'S HASHED TO -- HARD TO DO IE
BODY BECAUSE IT'S GOING TO ALTER
MANY FEATURES SIMULTANEOUSLY.
SO WE INSTEAD HAVE REALLY RELIED
ON USING CELL CULTURE MODELS, 3D
CELL CULTURE MODELS INSTEAD,
USING MATERIALS WITH HIGHLY
DEFINED PHYSICAL PROPERTIES.
AND A LOT OF THE DATA THAT I'LL
DESCRIBE FOR YOU TODAY HAS BEEN
GENERATED WITH A PARTICULAR
MODEL SYSTEM WHICH IS HYDRAGELS
THAT WE CREATE FROM A
POLYSACCHARIDE ALGINATE,
NATURALLY DERIVED, TWO SUGAR
UNITS, G SUGAR UNITS ON THE
LEFT, M SUGAR UNITS ON THE
RIGHT.
AND ALGINATE CAN FORM A
SPACE-FILLING ALHYDROGEL BY G
SUGARS AND POLYMER CHAINS BELOW
COMBINING SUCH AS CALCIUM.
ONLY G BLOCKS WITH PARTICIPATE
IN THE CROSS-LINKING, NOT THE M
BLOCKS.
WE HAVE G SUGARS IN BLOCKS, MANY
IN A ROW, TYPICALLY ABOUT 20 IN
A ROW.
SO WITH THE SYSTEM WE CAN CREATE
GELS VARY THE STIFFNESS BY
VARYING THE CROSS-LINKER
CONCENTRATION, AND THEN AS WE
USE DIFFERENT MOLECULAR WEIGHT
POLYMERS CAN CONTROL STIFFNESS.
THE REASON WHY WE SPECIFICALLY
USE THESE ALGINATE
POLYSACCHARIDE GELS IS BECAUSE
THEY ALLOW US TO INDEPENDENTLY
CONTROL THE STIFFNESS OF THE
GEL, VISCOELASTICITY, THE MESH
SIDE AND PRESENTATION OF CELL
ADHESION LIGANDS.
THINKING ABOUT THE TYPICAL GEL
USED IN BIOLOGY, COLLAGEN GELS,
FIBRIN GELS, ONE CAN CHANGE THE
STIFFNESS BY INCREASING
CONCENTRATION OF POLYMER IN THE
GEL.
SO THIS DOES INCREASE STIFFNESS
BUT AT THE SAME TIME IT CHANGES
THE AVAILABILITY AND NUMBER OF
CELL ADHESION LIGANDS, SHOWN
HERE, CHANGES THE SIZE OF THE
SPACINGS BETWEEN THE POLYMER
CHAINS, WHICH CAN ALTER THE
DIFFUSION OF MACROMOLECULES LIKE
MORPHOGENS OR GROWTH FACTORS.
WE CAN MOVE TO SYNTHETIC
POLYMERS THAT DON'T HAVE CELLED
A LEASER -- ADHESION PROPERTIES.
WE CAN CONTROL THE STIFFNESS,
AND DECOUPLE CELL ADHESION LIKE
AND DENSITY BY CONJUGATING SMALL
PEPTIDES LIKE RGD-CONTAINING
PEPTIDES BUT ALTER THE MESH
SIZE, CHANGING DIFFUSIONAL
TRANSPORT OF MACROMOLECULES.
THE INTERESTING THING ABOUT
ALGINATE BECAUSE OF THIS BLOCK
STRUCTURE I MENTIONED BEFORE,
WE'RE ONLY THE G SUGAR -- WHERE
ONLY THE G SUGAR BLOCKS
PARTICIPATE IN CROSS-LINKING, IF
YOU ADD CROSS-LINKING AGENT TO
POLYMERS YOU'LL GET SOME
CROSS-LINKING INDICATED BY
ORANGE DOTS BUT THE WHOLE
JUNCTIONAL ZONE WILL GET ALIGNED
WITHIN THE ALHYDROGEL, WE CAN
VARY STIFFNESS WITHOUT CHANGING
NANOSCALE ARCHITECTURE, THE MESH
SIZE STAYS CONSTANT.
WE BY CONTROLLING THE MOLECULAR
WEIGHT OF THE POLYMER CHAINS
CONTROL HOW ENTANGLED THEY ARE
AND CAN ALTER VISCOELASTICITY
WHETHER THEY FLOW READILY.
THE EARLY WORK WAS DONE BY GU,
NOW AT HOPKINS, AND CHADHURI,
NOW AT STANFORD, CELLS CAN BE
INTACT IMPACTED BY
VISCOELASTICITY.
PUT ON A SUBSTRATE YOU CAN
APPRECIATE THE CELLS EXHIBIT
QUITE A BIT MORE SPREADING AND
FOCAL ADHESION FORMATION ON THE
VISCOELASTIC GEL AS CONTRASTED
TO PURELY ELASTIC HERE.
LOOKING AT ALKALINE PHOSPHATASE,
WE VARY RATE OF STRESS
RELAXATION BASICALLY GET GREATER
OSTEOGENESIS OF THESE CELLS.
WE PUBLISHED THIS WORK SEVERAL
YEARS AGO.
AND WHAT WE'VE BEEN MORE
RECENTLY INTERESTED IN, THIS IS
ALBERTO, NOW RUNNING HIS OWN
LABORATORY AT THE CRICK
INSTITUTE IN LONDON, INTERESTED
IN HOW VISCOELASTICITY MIGHT
ALTER COLLECTIVE CELL BEHAVIOR,
USING BREAST CELLS THAT YOU MAY
BE FAMILIAR WITH.
HE MADE AGGREGATES, EMBEDDED
INTO THE GELS ON THE LEFT, THAT
WERE MORE PURELY ELASTIC, ON THE
RIGHT MORE VISCOELASTIC, WHAT
YOU CAN APPRECIATE IS THE
EXPANSION OF THESE AGGREGATES IN
ESSENCE TISSUE DEVELOPMENT HERE
IS DRAMATICALLY DIFFERENT.
WE GET A SLOW GROWTH ON THE LEFT
AND MORE PURELY ELASTIC
MATERIAL, THE TISSUE MAINTAINS
SYMMETRY, WHILE ON THE RIGHT WE
ACTUALLY HAVE MUCH MORE RAPID
GROWTH.
WE HAVE BRANCH FORMATION, WE
BREAK SYMMETRY, SO DRAMATICALLY
DIFFERENT CELLULAR BEHAVIOR JUST
BECAUSE ON THE RIGHT IT'S MORE
VISCOELASTIC, ON LEFT MORE
ELASTIC.
SO WE GET MORPHOLOGICALLY STABLE
TISSUE GROWTH OR MORPHOLOGICALLY
UNSTABLE TISSUE GROWTH.
WE CONJUGATE SMALL RPG PEPTIDES
AND MATCHED STORED MODULUS, THE
STIFFNESS, BUT WE VARY THE RATE
OF STRESS RELAXATION BY CHANGING
THE MOLECULAR WEIGHT OF THE
POLYMER.
IF WE LOOK AT THE
CROSS-SECTIONS, YOU CAN
APPRECIATE HOW DRAMATICALLY
DIFFERENT GROWTH OF THESE
ORGANOIDS IS AS WE VARY THE --
PUT THEM IN ELASTIC OR
VISCOELASTIC SUBSTRATE, YOU CAN
APPRECIATE DRAMATIC DIFFERENCE
HERE.
NOW, THIS CHANGE IN
VISCOELASTICITY DRIVES AN
EPITHELIAL TO MESENCHYMAL
TRANSITION, EMT, OF THE CELLS.
IF WE LOOK AT THE VIMENTIN
EXPRESSION, THE BRANCHES ARE
PENETRATING INTO THE SURROUNDING
VISCOELASTIC GEL.
CELLS IN THE ELASTIC SUBSTRATE
EXPRESS CYTOKERATIN FORMATION.
WE SEE EXPRESSION OF SNAIL,
SLUG, ZEB1 CELLS, THIS IS AN
EFFECT IN TERMS OF IMPACT ON
TISSUE GROWTH, NOT JUST SEEN IN
VITRO, BUT WE ALSO SEE THIS IN
VIVO.
IF WE TAKE THE CELLS, WE
TRANSPLANT THEM INTO GELS MORE
VISCOELASTIC, ELASTIC, SEE MUCH
MORE DRAMATIC GROWTH, AND
BASICALLY INVASION INTO THE
SURROUNDING TISSUE IN THE
VISCOELASTIC SUBSTRATE.
NOW, WE'VE BEEN INTERESTED IN
TRYING TO MATHEMATICALLY MODEL
THIS BEHAVIOR TO GET A BETTER
UNDERSTANDING, ALSO TO ENABLE US
TO QUICKLY DO A WIDE VARIETY OF
DIFFERENT EXPERIMENTS BECAUSE WE
HAVE A BIG VARIABLE SPACE HERE.
SO IN COLLABORATION WITH
MAHADEVAN AT HARVARD WE'RE
MODELING THE MATRIX USING
CLASSIC VISCOELASTIC AND ELASTIC
APPROACHES, CONSIDERING THE
MATRIX TO BE PASSIVE, AND FOR
THE TISSUE WE MODEL IT AS
BASICALLY BEING A COLLECTION OF
INDIVIDUAL CELLS, EACH OF THESE
CELLS HAS THE CAPACITY TO EITHER
PROLIFERATE OR MIGRATE.
THOSE ARE THE ONLY TWO BEHAVIORS
CELLS WITH EXHIBIT.
WHAT YOU CAN APPRECIATE IS THAT
THESE SIMULATIONS REPLICATE THE
BEAVER -- BEHAVIOR, INNOVATING
AND GROWING MORE RAPIDLY.
THIS IS QUANTIFICATION OVER
MULTIPLE SIMULATIONS, PART OF
THE REASON WE WANT TO UNDERSTAND
FROM THIS PERSPECTIVE IS GAIN
INSIGHT INTO WHAT'S CONTROLLING
THIS PROCESS THAT WE'RE
EXAMINING, ALSO TO MORE RAPIDLY
EXPLORE THE EXPERIMENTAL SPACE.
SO, FOR EXAMPLE, HERE WITH THIS
3D CONSTRUCTION, IT RECOMMENDS
EACH ONE OF THESE DOTS IS A
SIMULATION WHERE WE VARIED
PARAMETERS IN OUR EXPERIMENTAL
SYSTEM, AND THEN RUN A
MATHEMATICAL SIMULATION.
SO WE CAN VERY RAPIDLY DO, YOU
KNOW, HUNDREDS OF SIMULATIONS
WHILE DOING HUNDREDS OF
EXPERIMENTS WOULD TAKE US YEARS.
AN INTERESTING OUTCOME OF THIS
IS WE'RE ABLE TO IDENTIFY
REGIONS IN THIS VARIABLE SPACE
THAT LEAD TO THIS UNSTABLE
GROWTH, THESE ARE ALL THE RED
DOTS, AND THEN CONVERSELY A
SPACE WHERE WE ACTUALLY GET THE
STABLE GROWTH, BASICALLY
ENCOMPASSED IN GREEN REGION IN
THE THREE DIMENSIONAL SPACE.
WE FOUND BASICALLY THIS
TRANSITION BETWEEN STABLE AND
UNSTABLE GROWTH IS REGULATED BY
THREE DIMENSIONAL PARAMETERS,
FIRST IS A, WHICH IS THE RATIO
OF BASICALLY DEPENDENT DEPENDEF
MOTILITY, THE SECOND IS BETWEEN
THE TISSUE AND MATRIX VISCOSITY.
WE THINK THIS COULD BE BROADLY
USEFUL TO EXPLORE MORPHOGENESIS
AND TUMORIGENESIS IN A NUMBER OF
CONTEXTS.
WE'VE ALSO BEEN INTERESTED IN
DETERMINING WHETHER THIS IDEA OF
VISCOELASTICITY CONTROLLING
ORGANOGENESIS IS BROADLY
RELEVANT OR SPECIFIC TO CELL
SYSTEM I DESCRIBED.
WE CAN TAKE THIS AND CREATE
INTERPENETRATING NETWORKS, FOR
EXAMPLE MATRIGEL TO DECOUPLE
FROM OTHER FEATURE, HERE
CONTROLLING PHYSICAL PROPERTIES
TO KEEP THE MATRIGEL CONSTANT.
AND WE CAN SEE FOR EXAMPLE THAT
IF WE DO THIS WITH IPNs CAN
KEEP TRANSPORTERS CONSTANT,
INCREASING CONCENTRATION TO MAKE
IT STIFFER, SIMULTANEOUSLY
DIMINISHING DIFFUSION.
WITH THESE KINDS OF NETWORKS CAN
EXAMINE OTHER SYSTEMS.
FOR EXAMPLE, WE'VE LOOKED AT
INTESTINAL ORGANOID DEVELOPMENT.
IN THE ELASTIC EXTRACELLULAR
MATRICES IS SLOW.
ON THE VISCOELASTIC MATRICES WE
GET DIFFERENTIATION OF STEM
CELLS INTO DAUGHTER CELLS ONE
WOULD EXPECT TO SEE, INTESTINAL
TISSUE, WE SEE MAINTENANCE OF
THE STEM CELL POPULATION
SPECIFICALLY AT REGIONS OF HIGH
CURVATURE AS OTHERS HAVE
PREVIOUSLY DESCRIBED.
SO, WE BASICALLY THINK MATRIX
ELASTICITY IS A FEATURE OF HOW
TUMORS MAY GROW, CONTROLS
PROLIFERATION, INVASION,
SYMMETRY BREAKING,
MECHANOTRANSDUCTION.
I WON'T GO THROUGH THE
MECHANOTRANSDUCTION PATHWAYS BUT
WHAT OUR STUDIES HAVE
DEMONSTRATED IS THAT THESE
EFFECTS ARE DEPENDENT ON ABILITY
OF THE CELLS TO ENGAGE WITH THE
MATERIAL, SPECIFICALLY RGD
LIGAND IN THESE GELS VIA
INTEGRIN RECEPTORS, WE SEE
SIGNALING THAT REGULATES
ASSEMBLY OF ACT ALLOWS TO PROBE
PHYSICAL PROPERTIES, MEDIATING
RESPONSIVENESS OF CELLS TO THE
MATRIX VISCOELASTICITY.
THOSE CELLS RESPOND.
WHAT KYLE FOUND, HE MOVED TO U
PENN TO TAKE AN ACADEMIC
POSITION THERE, BECAME
INTERESTED IN SOMETHING THAT
COULD BE QUITE IMPORTANT WHICH
IS HE BEGAN TO ASK WHETHER
IMMUNE CELLS MIGHT ACTUALLY ALSO
RESPOND TO THESE CHANGES IN THE
VISCOELASTICITY OF THE
EXTRACELLULAR MATRIX, INTERESTED
IN CON MYELOFIBROSIS, WHICH
INVOLVES BASICALLY MUTATIONS IN
HEMATOPOIETIC STEM CELLS AND IS
ASSOCIATED WITH BASICALLY
CHANGES IN FIBROSIS WITHIN THE
BONE MARROW, AND THEN
ALTERATIONS IN MYELOID CELLS.
SPECIFICALLY IN THIS DISEASE AS
INDICATED BY THIS STAINING ONE
GETS DEPOSITION OF EXTRACELLULAR
MATRIX, CHANGES IN PHYSICAL
PROPERTIES.
WHAT KYLE PROPOSED WAS THAT THE
VISCOELASTICITY WAS PROBABLY
ALSO CHANGING IN THE CONTEXT OF
THESE DISEASES AND THIS MIGHT BE
PLAYING AN IMPORTANT ROLE IN
MYELOID BIOLOGY.
AND THIS WORK I'LL DESCRIBE NEXT
HAS BEEN DONE IN COLLABORATION
WITH THE DANA FAR BORE, AND
CHARITE IN BERLIN.
TO START WITH KYLE WANTED SOME
SENSE OF WHAT KIND OF PHYSICAL
PROPERTIES HE SHOULD MODEL IN
HIS CELL CULTURE SYSTEM, WE
CHARACTERIZED NORMAL BONE
MARROW, THE NORMAL RANGE IS 1 TO
10-KPAs.
FRACTURE HEMATOMAS FROM
PATIENTS, WERE ABLE TO
CHARACTERIZE THEIR PROPERTIES
AND THEY WERE VISCOELASTIC WITH
HALFTIME OF RELAXATION OF 400
SECONDS.
THEN KYLE SET ABOUT DESIGNING A
ALHYDROGEL SYSTEM FOR CELL
CULTURE SYSTEM TO MIMIC
TRANSITION FROM NORMAL PHYSICAL
PROPERTIES TO ABNORMAL PHYSICAL
PROPERTIES.
HE AGAIN WENT TO USE ALGINATE,
POLYSACCHARIDE I DESCRIBED, TO
ACCESS LONG RELAXATION TIMES
BASED ON WHAT WAS HAPPENING IN
THE BONE MARROW WITH ALL THE
FIBROSIS.
SO, IN ADDITION TO THE ICONIC
CROSS-LINKING I DESCRIBED
BEFORE, HE BUILT IN SOME
COVALENT CROSS-LINKING USING
CLICK CHEMISTRY, THE BASIC IDEA
HERE IS HE WOULD PUT SMALL CLICK
FUNCTIONAL GROUPS ON THE
ALGINATE, THESE WOULD NOT HAVE A
LONG SPACER, THEY COULD ONLY
REACT WITH EACH OTHER IF
ADJACENT POLYMER CHAINS WERE
ALREADY QUITE CLOSE.
DO IONIC CROSS-LINKING, THAT
BRINGS POLYMER CHANGES IN CLOSE
PROXIMITY, THEN THE CLICK
CHEMICAL GROUPS HERE WOULD THEN
HAVE THE OPPORTUNITY TO REACT IN
MORE OR LESS BASICALLY CREATING
A MORE ELASTIC COMPONENT TO
THESE CROSS-LINKING JUNCTIONAL
ZONES.
AND HE DID DEMONSTRATE HE COULD
CREATE BASICALLY MORE
VISCOELASTIC OR MORE ELASTIC
MATERIALS BY COMBINING THIS
ICONIC AND COVALENT
CROSS-LINKING.
A KEY FEATURE AS WELL IS CONTEXT
OF FIBROSIS.
OBVIOUSLY COLLAGEN TYPE 1 IS A
KEY COMPONENT, SO WE WANTED TO
HAVE THAT ASPECT TO THIS MODEL
SYSTEM.
HE MADE INTERPENETRATING
NETWORKS AS I DESCRIBED EARLIER
BUT THIS TIME WITH TYPE 1
COLLAGEN, DEMONSTRATING THE TYPE
1 COLLAGEN RED READILY ASSEMBL,
MEDIATING CELL ENGAGEMENT AND
THE ALGINATE HERE BASICALLY TO
CHANGE THE PHYSICAL PROPERTIES
OF THE GELS, INDEPENDENT MANNER.
NOW HE TOOK MUSEUM MONOCYTES,
ENCAPSULATED IN GELS OF VARYING
STIFFNESS OR VARYING LEVELS OF
VISCOELASTICITY, CHARACTERIZED
CYTOKINE SECRETED, YOU CAN
APPRECIATE ONE GETS SIGNIFICANT
UPREGULATION OF SECRETION OF A
LOT OF INFLAMMATORY CYTOKINES,
FROM THE CELLS, WHEN THEY ARE IN
THE MORE ELASTIC STIFF MATERIAL
AS CONTRASTED TO ANY OF THE
OTHER CONDITIONS.
NOW IF WE LOOK AT THE CELLS
THEMSELVES, LOOK AT CELL SURFACE
MARKERS, ONE SEES, FOR EXAMPLE,
IN THIS TOP PLOT A SIGNIFICANT
INCREASE IN EXPRESSION IN
ELASTIC VERSUS SUBSTRATE,
SIGNIFICANT UPREGULATION OF
HLA-DR, CD80, TDL 1, WE'RE
INDUCING DIFFERENTATION, DRIVEN
BY CHANGE IN VISCOELASTICITY.
NOW, WE'VE DONE SOME GENE
EXPRESSION ANALYSIS OF THESE
CELLS, AND FOUND SIGNIFICANT
ALTERATIONS IN GENE EXPRESSION,
FOR THE CELLS IN THE MORE
VISCOUS GELS HAVE A PATTERN OF
GENE EXPRESSION REPRESENTATIVE
OF MONOCYTES, MATURE DENDRITIC
CELLS, MORE INFLAMMATORY PATTERN
IN THE ELASTIC SUBSTRATES.
NOW, THIS IS ALL IN A CELL
CULTURE MODEL.
IT'S DIFFICULT TO COMPARE
DIRECTLY TO WHAT'S HAPPENING IN
VIVO.
BUT KYLE WAS ABLE TO ACCESS
THROUGH SOME COLLABORATORS A
DATASET OF CHANGES IN GENE
EXPRESSION OF PATIENTS WITH
PRIMARY MYELOFIBROSIS,
TRANSITION FROM EARLY TO LATE
STAGE DISEASE WHICH ENCOMPASSES
CHANGES IN FIBROSIS AND CHANGING
IN STIFFENING AND CHANGES IN
VISCOELASTICITY, AND SO IF YOU
LOOKED AT EXPRESSION, CHANGES IN
EXPRESSION OF A NUMBER OF GENES,
AS WE WENT FROM ELASTIC STIFF TO
VISCOELASTIC MATERIAL, COMPARED
THAT TO THE SAME GENES AS THEY
WERE ALTERED IN EXPRESSION FROM
EARLY TO LATE STAGE ONE SEES
GOOD CORRELATION BETWEEN THESE.
IT LOOKS LIKE CHANGES WE'RE
SEEING IN MODEL SYSTEM ARE
SIMILAR TO THE CHANGES ONE SEES
IN PATIENTS AS THE DISEASE
PROGRESSES.
NOW, FIBROSIS ALSO OCCURS IN A
VARIETY OF OTHER SETTINGS.
SO KYLE ALSO WENT AND LOOKED AT
SINGLE CELL RNAseq DATA THAT
HAD PREVIOUSLY BEEN PUBLISHED,
IN THIS CASE WITH PATIENTS WITH
PULMONARY FIBROSIS OR PEOPLE
WITH NORMAL LUNG TISSUE.
AND LOOKED AT BASICALLY MONOCYTE
EXPRESSION OF THE GENE SETS WE
IDENTIFIED WERE REPRESENTATIVE
OF CELLS BASICALLY IN THESE
ELASTIC MATERIALS.
AND WHAT ONE SEES IS ELASTIC
GENE SIGNATURE DRAMATICALLY
UPREGULATED IN MONOCYTES AND
PATIENTS WITH PULMONARY FIBROSIS
VERSUS NORMAL DONOR INDICATING
PHYSIOLOGIC RELEVANCE IN TERMS
OF REFLECTING WHAT WE SEE IN THE
BODY AS WELL.
FURTHERMORE, WHAT KYLE WAS ABLE
TO DEMONSTRATE THAT GENE
EXPRESSION OF MONOCYTES IN
VISCOUS GELS ARE ACTUALLY
ASSOCIATED WITH POOR OVERALL
SURVIVAL FROM TCGA DATA.
YOU CAN SEE HERE HUMAN TUMORS,
PATIENT DATA, CUTANEOUS MELANOMA
AND SQUAMOUS CARS CARCINOMA.
FINDINGS ARE PATHOLOGICALLY
RELEVANT.
WE'D LIKE TO UNDERSTAND
MECHANISM BY WHICH THESE CELLS
ARE SENSING, THESE CHANGING
PHYSICAL PROPERTIES, POTENTIALLY
COULD CREATE TARGETS FOR
INTERVENTION IN THE FUTURE.
PI3 KINASE GAMMA, MYELOID
SPECIFIC VERSION OF PI3 KINASE
IS WELL DESCRIBED TO GENERATE
PUSHING FORCES IN THESE TYPES OF
CELLS, HERE YOU ARE SEEING IN A
NEUTROPHIL BASICALLY THE BRIGHT
INDICATES HIGH PI3 KINASE
ACTIVITY, YOU CAN APPRECIATE THE
CELLS USING THIS BASICALLY
MIGRATES DIRECTIONALLY.
WE KNOW THAT AS I MENTIONED THAT
THIS IS VERY IMPORTANT IN
DEVELOPMENT OF PUSHING FORCES,
THAT COULD ENABLE THE CELLS TO
ACTUALLY SENSE THE SURROUNDING
PHYSICAL PROPERTIES.
SO KYLE DID A SCREEN WHERE HE
LOOKED AT A VARIETY OF
INHIBITORS OF DIFFERENT
POTENTIAL MECHANOTRANSDUCTION
PATHWAYS, IN THIS SCREEN WE DID
A SIMPLE READOUT INITIALLY,
WHICH IS JUST THE ASSEMBLY OF A
CORTICAL F-ACTIN NETWORK.
NORMAL CELLS WHEN WE PUT THEM
INSIDE THE VISCOUS GELS WE DID
NOT GET SIGNIFICANT ASSEMBLY OF
F-ACTIN BUT IN GELS DID, USED
THIS AS A SIMPLE SCREENING.
WHEN WE USED AN INHIBITOR TO
MYELOID KINASE, IN CLINICAL
TRIALS FOR OTHER THINGS, IT
ABROGATED ACTIN ASSEMBLY.
WHEN WE TREATED IN VISCOUS OR
ELASTIC GELS WITH INHIBITOR
HERE, WITHOUT INHIBITOR WE GET
UPREGULATION AS I PREVIOUSLY
NOTED, AND ADDING INHIBITOR
SIGNIFICANTLY ABROGATED EFFECTS
OF THE CHANGE IN
VISCOELASTICITY, SUGGESTING THIS
IS A MECHANISM BY WHICH THE
CELLS ARE SENSING
VISCOELASTICITY AND TRANSDUCING
THIS INTO CHANGES IN GENE
EXPRESSION AND FATE.
NOW, IF WE GO BACK TO THE
STARTING POINT FOR THE STORY FOR
KYLE, WHICH IS MYELOFIBROSIS,
THIS IS A THE MOST AIMPRESSIVE
FORM OF A -- AGGRESSIVE FORM,
MEDIAN PATIENT, MEDIAN SURVIVAL
WITH PRIMARY MF IS ABOUT 6
YEARS.
SO THERE'S CLEARLY AN UNMET NEED
TO DEVELOP IMPROVED TREATMENTS
FOR THESE PATIENTS.
AND SO KYLE WANTED TO EXPLORE
WHETHER THIS KNOWLEDGE WE GAINED
COULD PROVIDE A TARGETABLE
OUTCOME IN THIS CONTEXT.
TO EXPLORE THIS WE WENT TO AN
ANIMAL MODEL OF MYELOFIBROSIS IN
COLLABORATION WITHMAN MULLALLY
AT DANA-FARBER, DISEASES CAUSED
BY MUTATION IN HEMATOPOIETIC
STEM CELLS.
SO WE TALK HEMATOPOIETIC STEM
CELLS THAT WERE MUTATED,
INTRODUCED INTO LETHAL IRRATE
RADIATED RECIPIENTS TO TAKE UP
RESIDENTS IN THE BONE MARROW,
OVER TIME COURSE 6 TO 7 MONTHS
LEADS TO MYELOFIBROSIS, AND THEN
THE HALLMARKS OF THE DISEASE,
FOR EXAMPLE, CHANGES IN BLOOD
CELL COUNTS.
WE WERE ABLE TO TAKE THE BONE
MARROW AT THE ADVANCED STAGE,
CHARACTERIZE MECHANICAL, WHEN WE
INTRODUCE MUTATED CELLS AND
CREATE MYELOFIBROSIS GET
INCREASED STIFFNESS, WE GET
DECREASE IN VISCOELASTICITY OF
THE BONE MARROW INDICATING WE'RE
INDEED GETTING SIGNIFICANT
PHYSICAL CHANGES, NOW WHEN WE
TOOK BASICALLY HISTOLOGICAL
SAMPLES AND PROVIDED THOSE TO A
PATHOLOGIST IN A BLINDED MANNER
AND HAD THEM GRADE DEGREE OF
MYELOFIBROSIS EVEN THOUGH
STIFFNESS CHANGES WITH
DEVELOPMENT THERE'S NO
CORRELATION WITH THE MF GREAT.
IN CONTRAST, THERE WAS A REALLY
STRONG CORRELATION BETWEEN MF
GRADE AND CHANGES IN
VISCOELASTICITY.
SO EXPERIMENT, WENT BACK TO THIS
MODEL, PROVIDED BASICALLY THIS
INHIBITOR, TO THE ANIMALS, WHAT
WE FOUND IS WHEN THE INHIBITOR
WAS PROVIDED TO THESE ANIMALS WE
HAD A SIGNIFICANT DOWNREGULATION
IN THE CIRCULATING INFLAMMATORY
MONOCYTES AND DENDRITIC CELLS
INDICATING ABROGATING ASPECTS OF
THE DISEASE BY INTERVENING IN
THE SIGNALING PATHWAY.
SO HOPEFULLY WHAT I WILL SHOW IN
THIS PART OF THE TALK IS THAT
WHEN WE HAVE IMMATURE MONOCYTES
AND THEY ARE IN A MORE
VISCOELASTIC MATRIX WHAT WE
BELIEVE HAPPENS IS THEY GENERATE
PUSHING FORCES, BUT THE MATERIAL
ITSELF WILL BASICALLY FLOW,
DISSIPATE THE STRESSES, AND SO
THE CELL DOESN'T BASICALLY GET
STRESSED MECHANICALLY.
IN CONTRAST IN THE MORE ELASTIC
MATRIX WHEN THE CELL PUSHES, IT
DOESN'T DISSIPATE, INSTEAD
STORES THEM, BASICALLY PUSHES
BACK ON THE CELL.
THE CELL ACTUALLY SENSES THIS
THROUGH THIS ONE PARTICULAR
PATHWAY.
SO WE THINK WE'VE IDENTIFIED A
TARGETABLE MECHANICAL CHECKPOINT
IN THE CONTEXT OF AT LEAST THIS
PARTICULAR DISEASE.
NOW, THE LAST PART OF WHAT I'D
LIKE TO TALK TO YOU A LITTLE BIT
ABOUT IS KIND OF THE MOST RECENT
FORAY INTO THIS SPACE, NOW
UNPUBLISHED DATA.
WHERE WE'VE BEEN INTERESTED IN
WHETHER ACTUALLY OTHER IMMUNE
CELLS, PARTICULARLY T CELLS, MAY
ALSO BE IMPACTED BY CHANGES IN
MATRIX VISCOELASTICITY.
WE'RE ALL FAMILIAR WITH
LIMITATION OF T CELL
IMMUNOTHERAPY IN CONTEXT OF
SOLID TUMORS, BELIEVED TO BE A
NUMBER OF BARRIERS TO THE
EFFECTIVE INSIDE -- EFFECTIVEN,
BASICALLY DIFFICULTIES IN CELLS
ACCESSING TUMORS.
YOU KNOW, DIVERSE
IMMUNOSUPPRESSION THAT HAPPENS
WITHIN THE TUMOR
MICROENVIRONMENT, AS WELL AS
ANTIGEN HETEROGENEITY.
WHAT WE BEGAN TO EXPLORE,
WHETHER ADDITIONAL FEATURE OF
THIS COULD BE CHANGES IN THE
MATRIX VISCOELASTICITY MAY ALSO
ALTER T CELL PHENOTYPE AND
EFFECTIVENESS DIRECTLY.
TO DO A REALITY CHECK ON THIS
CONCEPT, TO START WITH, A
Ph.D. STUDENT IN THE
LABORATORY SHOWN HERE WHO HAS
TAKEN A STAFF POSITION AT THE
WYSS INSTITUTE STARTED BASICALLY
BY GOING BACK TO PATIENT DATA,
SINGLE CELL RNAseq DATA, AND
HE JUST DID SOMETHING RELATIVELY
STRAIGHTFORWARD WHICH IS LOOKED
AT BASICALLY THE GENE EXPRESSION
OF T CELLS IN THREE CANCERS WE
COULD OBTAIN DATA FOR, AND
COMPARED THE GENE EXPRESSION OF
THE T CELLS IN BLOOD, WHICH
WOULD BE VISCOUS FLUID, AND
NORMAL TISSUE, WHICH WE WOULD
EXPECT TO BE A VISCOELASTIC
SOLID, AND THEN IN TUMORS WHICH
WE WOULD THINK WOULD BE MORE
ELASTIC.
AND WHAT HE FOUND WAS THAT
BASICALLY THE PATTERNS OF GENE
EXPRESSION WERE DRAMATICALLY
ALTERED FOR T CELLS IN THESE
THREE DIFFERENT ENVIRONMENTS.
SO FOR EXAMPLE HERE WE'RE
LOOKING AT ONE BASIC EXPRESSION
OF A BASICALLY THE GENE SET
INDICATIVE OF CELLS AND TUMORS,
YOU CAN SEE IT'S UPREGULATED FOR
ALL THREE OF THESE BASICALLY
TYPE OFS CANCER CAN -- TYPES O.
THE ONLY DIFFERENCE BETWEEN
BLOOD AND TUMOR TISSUE, THERE'S
A LOT OF DIFFERENCE IN PHYSICAL
PROPERTIES BUT THIS SUGGESTS
PERHAPS CHANGING PHYSICAL
PROPERTIES COULD RELATE TO SOME
ALTERATIONS IN T CELL PHENOTYPE.
TO GET AT THIS MECHANISTICALLY
WENT BACK TO 3D MODEL SYSTEM,
AND HERE HE TOOK WHAT KYLE HAD
DONE AND TOOK IT ONE STEP
FURTHER FORWARD, WHICH IS HE
USED THIS IDEA OF SMALL COVALENT
CROSS-LINKING MOLECULES THAT
COULD ONLY CROSS-LINK POLYMERS
THAT WERE ALREADY IN CLOSE
PROXIMITY, FOR EXAMPLE ALREADY
ASSEMBLED, AND HE CREATED PURELY
COLLAGEN GELS HERE, LET THE
COLLAGEN BASICALLY FIBRIL
ASSEMBLY OCCUR, WOULD DIFFUSE IN
BASICALLY A VERY SMALL DIVALENT
CROSS-LINKER TO CROSS-LINK
COLLAGEN IN CLOSE PROXIMITY
CREATING FAST OR SLOW GELS.
THE ARCHITECTURE WAS UNALTERED.
AND THE BASICALLY PORE SIZE WAS
UNALTERED INDICATED BY IMAGING.
HERE YOU'RE LOOKING AT
QUANTIFICATION OF THICKNESS AND
PORE DIAMETER SO WE CAN CHANGE
JUST THE VISCOELASTICITY WITHOUT
CHANGING THOSE OTHER FEATURES,
AND WE CAN MATCH BASICALLY THE
STIFFNESS AND HAVE VARYING RATES
OF STRESS RELAXATION.
HE WOULD ADD MORE COLLAGEN TO
CHANGE STIFFNESS, COMPARING
STIFFNESS LEVELS THERE'S THIS
ADDITIONAL FEATURE BUT ONLY
COMPARED MORE OR LESS VISCOUS
THAT IS ACTUALLY INDEPENDENT OF
ANY OTHER VARIABLES.
AND THEN IN COLLABORATION WITH A
CURRENT Ph.D. STUDENT IN THE
LABORATORY ENCAPSULATED T CELLS
WITHIN THESE GELS THAT WOULD BE
EITHER SOFT OR STIFF, AND EITHER
BASICALLY FAST RELAXING, MEANING
MORE VISCOELASTIC, OR SLOW
RELAXING, AND WHAT YOU CAN
APPRECIATE FROM THESE PLOTS IS
THEY NOTICE SIGNIFICANT
ALTERATION IN PHENOTYPE OF T
CELLS, AS HERE INDICATED BY
EXPRESSION OF CD45 RA, CD62L,
AND INDEED IF THEY LOOK AT A
NUMBER OF DIFFERENT KEY FEATURES
OF T CELL BIOLOGY, AND THEN
LOOKED AT HOW THEY WERE
EXPRESSED RELATIVELY IN THESE
DIFFERENT SETTINGS, ONE CAN SEE
ONE GETS DRAMATIC ALTERATION IN
EXPRESSION OF THESE MARKERS AS
ONE TRANSITIONS BETWEEN THE MORE
ELASTIC AND MORE VISCOELASTIC
SUBSTRATES.
ACTUALLY EFFECTS OF
VISCOELASTICITY WERE MUCH MORE
SIGNIFICANT THAN EFFECTS OF
CHANGING STIFFNESS.
NOW, THEY ALSO DID SOME SINGLE
CELL RNAseq, YOU CAN
APPRECIATE THE T CELLS IN THE
FAST RELAXING AND SLOW RELAXING
GELS OCCUPY DIFFERENT LOCATIONS
IN THIS UMAP REPRESENTATION.
IF WE PULL EXPRESSION IMPORTANT
IN T CELL BIOLOGY AND EFFECTOR
FUNCTION YOU CAN SEE THERE IS A
SIGNIFICANT ALTERATION IN GENE
EXPRESSION AS WE GO FROM FAST
RELAXING TO GELS.
IF WE DO CONSENSUS
FACTORIZATION, YOU CAN SEE WE
HAVE A VARIETY OF PATHWAYS
UPREGULATED, T-CELL RECEPTOR
SIGNALING, CYTOTOXIC RESPONSE,
MEDIATED CELL DEATH, WE'RE
GETTING ALTERATIONS IN MANY
GENES WE WOULD EXPECT WOULD BE
RELEVANT TO THE EFFECTIVENESS OF
T CELLS IN CONTEXT OF CANCER.
NOW, WE CAN ACTUALLY GENERATE
BASICALLY GENE EXPRESSION
MODULES, THAT REPRESENT CELLS IN
THE FAST OR SLOW RELAXING GELS,
FOR EXAMPLE THESE TWO MODULES
BASICALLY REPRESENT CELLS IN THE
FAST RELAXING GEL, THESE IN THE
SLOW.
WE CAN SEE THEY ARE
DIFFERENTIALLY EXPRESSED IN
POPULATIONS IN THE UMAP
REPRESENTATION, BUT THEN MORE
IMPORTANTLY, HE TOOK GENE
MODULES AND WENT BACK TO THAT
PRIMARY PATIENT DATA, WITH
DIFFERENT TYPES OF CANCER, IN
THIS CASE EXPANDED INCLUDING
BREAST CANCER DATASET THAT
BECAME AVAILABLE.
HE LOOKED FOR EXPRESSION OF THIS
SLOW MODULE IN THESE PATIENT
DERIVED T CELLS.
WHAT YOU CAN APPRECIATE IN ALL
FOUR TYPES OF CANCER THAT THIS
MODULE OF GENE EXPRESSION THAT
WE FOUND IN OUR IN VITRO MODEL
IN THE MORE ELASTIC GELS WAS
SIGNIFICANTLY OVEREXPRESSED IN T
CELLS WITHIN TUMORS THAT WE ALSO
WOULD EXPECT TO BE MORE ELASTIC
THAN IN THE BLOOD OR NORMAL
TISSUE.
AGAIN, SUPPORTING THE IDEA THAT
THERE MAY BE SOME REAL
PHYSIOLOGIC OR PATHOLOGICAL
RELEVANCE TO THESE CELL CULTURE
MODELS THAT WE'RE THEN
EXPLORING.
ANOTHER POSSIBILITY HERE FOR
THIS DATA, FOR ALL DATA
ACTUALLY, IS THAT INSTEAD OF
CHANGING THE GENE EXPRESSION OF
T CELLS, WE MIGHT BE SELECTING
FOR CERTAIN CLONES OF T CELLS IN
THE DIFFERENT MICROENVIRONMENTAL
CONDITIONS.
SO IF ONE REDOES THIS ANALYSIS
ON THE LEFT, BUT ACTUALLY DOES
IT ONLY FOR T CELLS OF SHARED
CLONOTYPE ONE SEES THE SAME
RESULT, NOT JUST A SELECTION
PROCESS FOR CERTAIN BASICALLY
CLONOTYPES OF T CELLS, I WON'T
GO THROUGH THE DATA, BUT NOW
THIS IS GOING BACK TO OUR CELL
CULTURE MODEL WITH T CELLS IN
THE FAST AND SLOW RELAXING GELS,
AND NET OUTCOME HERE IS THAT
WE'RE NOT GETTING ANY EFFECTS ON
SPECIFIC T CELL CLONES BY
ENCAPSULATING CELLS IN THESE
DIFFERENT GELS.
AGAIN, DOES NOT APPEAR WE'RE
CHANGING CLONOTYPES OR
PREFERRING CERTAIN CLONOTYPES
BUT ALTERING GENE EXPRESSION
ACROSS THE BOARD OF THESE T CELL
POPULATIONS.
WHAT WE'RE INTERESTED IN
UNDERSTANDING WHETHER THIS HAS
IMPACT ON FUNCTION OF T CELLS N
CONTEXT OF CANCER.
WE DID KILLING STUDDIE, T CELLS,
ACTIVATED, PUT INTO FAST OR SLOW
RELAXING GELS, LOOKED AT ABILITY
TO KILL THEIR TARGET CANCER
CELL, THIS IS IN CELL CULTURE
INITIALLY.
HERE LOOKING AT PERCENT
CYTOLYSIS.
THE CELLS BASICALLY IN THE STIFF
SLOW-RELAXING GELS WERE VERY
EFFECTIVE AT KILLING THE CANCER
CELLS, REGARDLESS OF THE LEVEL
OF STIMULATION, WHILE THE CELLS
BASICALLY IN THE FAST-RELAXING
GELS WERE LESS EFFECTIVE AT
KILLING.
IF WE LOOKED AT EXPRESSION OF A
NUMBER OF MARKERS OF THESE
CELLS, BASICALLY IN THIS ASSAY,
COMPARE AGAIN BASICALLY THE SLOW
AND FAST-RELAXING GELS, DURING
LEVELS OF STIMULATION, YOU CAN
AGAIN SEE DRAMATIC ALTERATIONS.
FOR EXAMPLE IN THE STIFF SLOW
RELAXING AND MORE ELASTIC GELS
YOU SEE UPREGULATION, AS
COMPARED TO THE CELLS BASICALLY
IN THE SAME STIFFNESS BUT FAST
RELAXING GEL.
AGAIN, SUPPORTING THAT BASICALLY
THE CYTOLYSIS DATA.
TO SEE IF THIS TRANSLATED IN
VIVO WE TOOK T CELLS, PUT THEM
INTO EITHER FAST OR SLOW
RELAXING GELS, FOR SEVERAL DAYS,
REMOVED THEM, AND DID AN
ADOPTIVE TRANSFER.
THIS WAS IN THE CONTEXT OF CAR T
CELLS.
HUMAN CAR T CELLS.
WE'RE BASICALLY PUTTING IN
IMMUNOCOMPROMISE THE ANIMALS
THAT RECEIVED RAW CELLS, CLASSIC
CAR T MODEL SYSTEM FOR
DEVELOPMENT OF THOSE PRODUCTS,
AND YOU CAN SEE IF WE DON'T DO
ANY ADOPTIVE TRANSFER OF THE CAR
T CELLS THE ANIMALS ALL RAPIDLY
SUCCUMB TO DISEASE, IF WE
INTRODUCE T CELLS THAT WERE IN A
MORE VISCOELASTIC SUBSTRATE,
THEY PROVIDE SOME CONTROL, BUT
THE CONTROL IS LOST, ANIMALS
WILL SUCCUMB.
IF THE HAVE THE SAME NUMBER OF T
CELLS IN A MORE ELASTIC
MICROENVIRONMENT BEFORE ADOPTIVE
TRANSFER THEY DO A MUCH BETTER
JOB OF CONTROLLING DISEASE
PROGRESSION AND ENHANCEMENT OF
SURVIVAL IN THIS SETTINGS.
CHANGES DO SEEM TO HAVE IMPACT
ON ABILITY OF T CELLS TO
ACTUALLY HAVE EFFECTIVE
PHENOTYPE AND FUNCTION IN AN
EFFECTIVE MANNER.
SO I'M GOING TO WRAP UP HERE.
HOPEFULLY WHAT I'VE BEEN ABLE TO
DEMONSTRATE IS THAT MATRIX
VISCOELASTICITY CAN INDEED
IMPACT THE FATE AND PHENOTYPE OF
MULTIPLE CELL TYPES THAT ARE
RELATIVE, RELEVANT FOR CANCER,
INCLUDING MANY FOUND IN TUMORS.
REMINDER, THIS IS WHAT WE MEAN
BY MORE VISCOUS, PERMANENT
DEFORMATION OF MATERIAL BECAUSE
THE MATERIAL FLOWS WHEN WE
BASICALLY PUSH OR PULL ON IT.
SO WHAT I'VE BEEN ABLE TO SHOW
HOPEFULLY IN THE DIFFERENT
ELEMENTS OF THE TALK IS THAT
MONOCYTE DIFFERENTIATION,
PRE-CANCEROUS CELLS AND CANCER
CELLS, ONE PIECE OF DATA, THEY
ARE IMPACTED AS WELL.
I DID NOT SHOW BUT WE'VE SEEN
SIMILAR EFFECTS WITH MSCs,
DRAMATIC CHANGE IN IMMUNOMONO
PHENOTYPE AS WE CHANGE
VISCOELASTICITY AND FINISHED BY
DEMONSTRATING HOW T EFFECTOR
CELL BIOLOGY IS ALSO
DRAMATICALLY IMPACTED BY THIS
ALTERATION IN MATRIX
VISCOELASTICITY.
I'LL FINISH HERE AND HOPEFULLY
THE TAKEHOME MESSAGE IS THAT
MATRIX VISCOELASTIC SUBSTRATE I
S -- MATRIX VISCOELASTICITY IS
SOMETHING WE WANT TO CONSIDER IN
CANCER BIOLOGY.
THANK YOU FOR YOUR ATTENTION,
AND I'D BE HAPPY TO TAKE ANY
QUESTIONS.
>>ALL RIGHT.
THANK YOU VERY MUCH.
EXCELLENT TALK.
SO LET'S GET TO IT FOR THE Q&A
PART OF OUR SESSION.
SO, IN YOUR TUNEABLE ALGINATE
MODEL TO WHAT EXTENT CAN YOU
VARY CALCIUM CONCENTRATION TO
NOT AFFECT THE CELLS WHETHER
CALCIUM IS TIGHTLY REGULATED FOR
ELECTRICAL ACTIVITY IN MANY CELL
TYPES?
>>YEAH, THAT'S ALWAYS A
QUESTION THAT COMES UP WITH THE
SYSTEM.
YEAH, SO IF ONE VARIES CALCIUM,
THAT CAN IMPACT CELLS, MANY
DIFFERENT WAYS.
IN ALL THESE EXPERIMENTS I'M
DESCRIBING TODAY, WE HAVE A
VARIETY OF CONTROL CONDITIONS I
DIDN'T SHOW HERE.
SO, FOR EXAMPLE, WE ADD
BASICALLY MORE CALCIUM TO THE
MEDIA TO SHOW BASICALLY OVER THE
RANGES WE'RE TALKING ABOUT HERE
WE DON'T SEE IMPACT OF THE
CALCIUM ITSELF.
ONE THING TO KEEP IN MIND IS
CALCIUM HERE IS BOUND BY
POLYSACCHARIDES, NOT FREE
CALCIUM AVAILABLE FOR UPTAKE.
WE BASICALLY FIND THAT THESE
EFFECTS WE'RE DESCRIBE ARE ARE
INDEPENDENT OF ANY MINOR CHANGES
IN CALCIUM, IN THE MEDIA.
BUT IT'S ALWAYS SOMETHING ONE
DOES NEED TO CHECK, ONE DOES
NEED TO CONFIRM.
>>GREAT.
SO I HAVE A QUESTION FOR THE
COLLAGEN INCORPORATING MODEL.
NOW, HOW IS THAT INTERACTING
WITH YOUR ALGINATE HYDRAGEL
NETWORK, IS IT ASSEMBLING,
FORMING FIBRILS, ARE THEY
INTERCONGESTION -- INTERDIG
TATE?
>>IN THAT CONFECTION AS LONG AS
WE CONTROL THE KINETICS IT'S
COMPLICATED BECAUSE WE HAVE
KINETICS OF THE ALGINATE
CROSS-LINKING, ALSO HAVE
KINETICS OF THE COLLAGEN FIBRIL
ASSEMBLY, FORMATION HAPPENS
EQUIVALENTLY IN ALL THE GELS
BEFORE YOU GET THE ALGINATE
CROSS-LINKING HAPPENING AROUND
IT.
IF WE HAVE COVALENT FIRST THAT
CAN INHIBIT COLLAGEN ASSEMBLY,
SO WE CONTROL THE KINETICS OF
THESE THREE ASPECTS, THE
QUESTION EQUIVALENT FIBRIL
COLLAGEN FORMATION, IT'S
EQUIVALENT, BUT ALGINATE MORE OR
LESS MECHANICALLY SUPPORTS THE
COLLAGEN, AND IT DICTATES
CHANGES IN STIFFNESS AND
VISCOELASTICITY BY CONTROLLING
THE CROSS-LINKING, IONIC VERSUS
COVALENT CROSS-LINKING.
>>BASICALLY ALGINATE IS
DOMINATING MECHANICAL PROPERTIES
IN THAT INSTANT?
>>YES, ABSOLUTELY.
YEP.
AND THAT'S ONE -- IN RELATION TO
EARLIER QUESTION, IN THOSE
STUDIES AS WE VARY THE EXTENT OF
VISCOELASTICITY ACTUALLY THE
CALCIUM CONTENT IN GELS IS
EXACTLY THE SAME BETWEEN ONE AND
THE NEXT, WE'RE BASICALLY
VARYING -- ADDING A LITTLE BIT
OF CLICK CHEMISTRY TO ALTER THE
BASICALLY VISCOELASTICITY.
>>ANOTHER QUESTION REGARDING
THE DYNAMICS BETWEEN YOUR HYDRA
G
EL SYSTEM ARE THEY
CHANGING THE MECHANICS OF THE
MATRIX, IF YOU TEST PROPERTIES
AFTER CELLS HAVE BEEN IN THERE,
DOES THAT CHANGE?
>>IT'S A HARD THING TO
CHARACTERIZE, LOOKING AT VERY
LOCALIZED EFFECTS.
WE KNOW THERE'S NO GROSS EFFECT.
IF WE MONITOR MACROSCALE
PROPERTIES OVER TIME, THEY DON'T
CHANGE.
BUT WE WOULDN'T EXPECT THAT.
I THINK THE QUESTION IS MORE
LOCALLY.
NOW, ONE THING WE DO KNOW, IT'S
VERY IMPORTANT, IS THE CELLS ARE
ACTIVELY REMODELING THE
MATERIALS.
FOR EXAMPLE, ACTUALLY SOME VERY
EARLY WORK WITH THESE SYSTEMS
GOING BACK TO LIKE 2015 OR
SOMETHING LIKE THIS, WE
DEMONSTRATED THAT THE RGD
LIGANDS ON POLYMERS GET
CLUSTERED BY CELLS, REACHING OUT
AND GRABBING, PULLING, AND SINCE
THE MATERIAL CAN FLOW, CELLS
DEFORM IT AND DO CHANGE IT.
ONE EARLY QUESTION WAS, SO IF
THEY ARE REMODELING, ARE THEY
REALLY JUST PULLING ENOUGH
POLYMER AROUND THEMSELVES THAT
MAYBE THEY ARE STIFFENING, MAYBE
WE'RE LIKING AT STIFFENING VIA
DIFFERENT MECHANISM.
SOME OTHER DATA I SHOWED YOU
TODAY INDICATES THAT THAT'S
LIKELY NOT -- STRONGLY INDICATES
IT'S NOT HAPPENING.
WE'VE DONE SOME INDIRECT
ANALYSIS OF THAT.
FOR EXAMPLE, WE LOOK AT OVERALL
GENE EXPRESSION, RNAseq, THAT
IF THE EFFECTS OF
VISCOELASTICITY WERE REALLY TO
ALLOW LOCAL STIFFENING, THEN YOU
WOULD EXPECT THE PATTERN OF GENE
EXPRESSION TO BE SIMILAR WHETHER
WE START WITH GELS THAT HAVE
DIFFERENT STIFFNESS OR WHETHER
THE CELLS MAKE THEM INTO
SOMETHING THAT HAS DIFFERENT
STIFFNESS AND FIND PATTERNS OF
GENE EXPRESSION AND DIFFERENTIAL
GENE EXPRESSION INDUCED BY
CHANGES IN STIFFNESS AND CHANGES
IN VISCOELASTICITY ARE LARGELY
ORTHOGONAL TO EACH OTHER.
WE'RE NOT GETTING MUCH OVERLAP
IN TERMS OF GENES GETTING
IMPACTED, HOW THEY ARE GETTING
IMPACTED, YOU KNOW, AGAIN
SUPPORTING VISCOELASTICITY IS
REALLY ACTING BY A DIFFERENT
MECHANISM, AND ONE OF THE FORMER
POSTDOCS HAS DOCUMENTED
SIGNALING PATHWAYS RELATED TO
VISCOELASTICITY THAT ARE
DISTINCT FROM CHANGES IN
STIFFNESS.
>>YEAH, A RELATED QUESTION THAT
JUST CAME IN ACTUALLY.
THE CELLS THEMSELVES ARE
VISCOELASTIC, CORRECT?
>>OH, YEAH.
YES.
>>YOU KNOW, HOW DOES THAT
COMPLICATE THIS MODELING OF
THIS -- TRYING TO UNDERSTAND
WHAT'S GOING ON HERE?
>>YEAH, SO THE CELLS THEMSELVES
ARE VISCOELASTIC, AND WE KNOW
THAT CELL STIFFNESS IS ALTERED
AS ONE PUTS CELLS INTO
ENVIRONMENTS, MATRICES.
TO THE BEST OF MY KNOWLEDGE NO
ONE LOOKED AT VISCOELASTIC
BEHAVIOR BUT I EXPECT IT WILL
BE.
AT THIS POINT IT'S UNKNOWN.
>>GOTCHA.
OKAY.
AND SO A QUESTION FOR THE
MONOCYTE DIFFERENTIATION
EXPERIMENTS, ELASTIC VERSUS
VISCOUS ENVIRONMENTS, WAS THERE
ANY PUSH TOWARDS THAT M1/M2
PHENOTYPE IN THE CELLS?
>>YEAH, NO, WE REALLY DIDN'T
SEE MUCH MACROPHAGE
DIFFERENTIATION IN THAT SETTING.
>>GOTCHA.
AND -- GREAT.
SO A QUESTION LATER ABOUT THE T
CELLS AND SPECIFICALLY THE HUMAN
DATASETS THAT YOU COMPARED.
IS THERE A WAY TO DISSECT OUT
THE OTHER MICROENVIRONMENTAL
FACTORS SUCH AS CYTOKINE MILIEU
WHEN COMPARING DIFFERENT TISSUES
LIKE THAT?
>>SO, WE MADE NO EFFORT TO DO
IT.
AS I HAD MENTIONED DURING THE
TALK THERE'S A LOT OF
DIFFERENCES BETWEEN BLOOD AND
NORMAL TISSUE, TUMORS.
SO WE DON'T TAKE THAT DATA AS
SHOWING CAUSE AND EFFECT.
BUT IT'S QUITE STRIKING THAT THE
CHANGES IN GENE EXPRESSION WE
SEE WHEN WE KEEP EVERYTHING
CONSTANT, ONLY CHANGE THE
VISCOELASTICITY, WE GET SIMILAR
CHANGES IN GENE EXPRESSION AS
YOU LOOK AT CELLS TRANSITIONING
BETWEEN THE THREE COMPARTMENTS
IN THE BODY.
CERTAINLY A LOT OF THINGS IMPACT
THE CELLS.
BUT THAT DATA SUGGESTS THAT
VISCOELASTICITY, EFFECTS OF
VISCOELASTICITY, YOU KNOW, MAY
BE MIRRORED IN PATIENTS AND
PATIENT SAMPLES AS WELL.
IN TERMS OF HOW YOU CAN DO THAT,
YOU CERTAINLY COULD ATTEMPT TO
DO THAT, YOU KNOW, WITH THE
HUMAN PATIENT DATA, I DON'T
THINK YOU COULD, BUT YOU COULD
CERTAINLY, YOU KNOW, TRY TO DO
THAT KIND OF DECOUPLING IN
MURINE MODELS, YOU KNOW, WITH
VARIOUS TRANSGENICS.
>>THAT'S THE BEAUTY OF IN VITRO
MODEL, RIGHT?
YOU CAN TAKE OUT A LOT OF THOSE
VARIABLES.
>>YES.
AND REALLY THE NEED HERE IS
ESPECIALLY WHEN TALKING ABOUT
IMPACT OF PHYSICAL PROPERTIES,
EXTRACELLULAR MATRIX, NO WAY I'M
AWARE OF TO PERTURB
VISCOELASTICITY WITHOUT
SIMULTANEOUSLY CHANGING
STIFFNESS AND MOST LIKELY
IMPACTING BIOCHEMICAL
COMPOSITION OF EXTRACELLULAR
MATRIX.
SO IT'S NOT THAT WE PREFER IN
THESE IN VITRO MODELS OVER IN
VIVO, BUT IF YOU WANT TO GET A
CAUSE AND EFFECT I THINK THE
ONLY WAY YOU CAN DO THAT IS BY
USING THESE TYPES OF IN VITRO
MOD
MODELS WITH TIGHT AND
INDEPENDENT CONTROL OVER
VARIABLES, LOOK TO SEE IF
THERE'S SUPPORT FOR FINDINGS IN
PATIENT-DERIVED SAMPLES AND IN
VIVO MODELS.
>>GREAT.
WE HAVE TIME FOR ONE MORE
QUESTION.
I'LL TAKE THIS OPPORTUNITY AND
BEAR WITH ME AS I ACCESS OLD
COURSE KNOWLEDGE.
>>OKAY.
>>SO, HAVE YOU THOUGHT ABOUT
OTHER VISCOELASTIC PROPERTIES
SUCH AS CREEP AND THING LIKE
THAT WHERE, YOU KNOW, WHEN YOU
THINK, AS I'M THINKING ABOUT
THIS, YOU KNOW, IS THE CELL
TRYING TO KEEP TENSION CONSTANT
OR SHAPE OR COMBINATION OF BOTH?
WHAT ARE YOUR THOUGHTS ON THOSE
OTHER PROPERTIES?
>>YEAH, NO, IT'S A REALLY
INTERESTING AND IMPORTANT
QUESTION.
YOU KNOW, CREEP IS BASICALLY FOR
THOSE WHO ARE NOT FAMILIAR,
CREEP IS THE FLIP SIDE OF STRESS
RELAXATION.
IN A CREEP STUDY YOU MAINTAIN
STRESS AND FOLLOW DEFORMATION.
THEN IN A VISCOELASTIC MATERIAL
THE DEFORMATION WILL THEN
CONTINUE TO INCREASE WITH TIME
IN ESSENCE AS MATERIAL FLOWS
UNDER THAT STRESS OR FORCE.
YEAH, THE QUESTION IS DO CELLS
KEEP A CONSTANT -- DO THEY APPLY
CONSTANT FORCE OR CONSTANT
STRAIN?
IT'S NOT CLEAR.
YOU KNOW, WHICH IS BASICALLY
PROBABLY MORE RELEVANT.
IT'S QUITE STRAIGHTFORWARD.
ACTUALLY I MEAN THE BEHAVIOR IS
LARGELY MIRRORED BETWEEN THE
STRESS RELAXATION IN A CREEP
TEST.
SO WE FIND BASICALLY
CHARACTERIZATION VIA HALFTIME
FOR STRESS RELAXATION TO BE A
VERY CONVENIENT WAY OF
CHARACTERIZING VISCOELASTIC
BEHAVIOR AND COMPARING IT.
SO WE DO THAT BUT WHETHER IT
TRULY IS THE APPROPRIATE METRIC
OR WHETHER CREEP MIGHT BE MORE
RELEVANT I THINK WE DON'T KNOW.
THERE'S ACTUALLY SOME DATA, YOU
KNOW, IN CELL CULTURE STUDIES
FOR BOTH BEING RELEVANT.
>>ALL RIGHT.
WELL, THAT'S ABOUT IT FOR TIME.
THANK YOU AGAIN.
I REALLY ENJOYED THE TALK TODAY.
AND A ROUND OF APPLAUSE TO YOU.
I'M SURE EVERYONE WATCHING FROM
YOUR LAB.
>>MY PLEASURE.