>>THANK YOU SO MUCH FOR COMING
OUT TODAY.
THIS IS FANTASTIC TO SEE SO MANY
PEOPLE HERE IN PERSON AND WE
REALLY APPRECIATE THAT.
I'M VERY HAPPY TO INTRODUCE
SENTHIL MUTHUSWAMY TODAY.
HE GOT HIS PH.D. FROM MCMASTER
UNIVERSITY IN CANADA.
HE DID A POSTDOCTORAL FELLOWSHIP
WITH JOAN BRUGGE AND HE HAD
SEVERAL FACULTY POSITIONS BEFORE
COMING HERE, ONE COLDSPRING
HARBOR, ANOTHER ONE, HE BECAME
THE LEE K. AND MARGARET LAU
CHAIR IN BREAST CANCER RESEARCH
IN TORONTO, THEN HE BECAME THE
DIRECTOR OF THE CELL BIOLOGY
PROGRAM IN THE DEPARTMENT OF
MEDICINE AT HARVARD MEDICAL.
AND HE JOINED THE NCI IN 2022,
WHERE HE BECAME MY LAB CHIEF AND
MANY OTHER PEOPLE IN THE ROOM
HERE TODAY.
THE LABORATORY OF CANCER BIOLOGY
AND GENETICS, AND WE'RE VERY
HAPPY TO HAVE HIM HERE.
HE'S RECEIVED NUMEROUS SCHOLAR
AWARDS FOR HIS RESEARCH,
INCLUDING ONE FROM MARIA ALLEN V
FOUNDATION U.S. ARMY AIR OF
HOPE, AND HE WAS AMONG THE FIRST
TO ESTABLISH ORGANOID CULTURES
TO EXPLORE 3D CELL-CELL
INTERACTIONS AND THEIR
TRANSLATIONAL POTENTIAL FOR
UNDERSTANDING THE ROLE OF CELL
POLARITY IN CANCER, THE ROLE OF
DRUG RESISTANCE, AND IDENTIFYING
NEW THERAPEUTIC MODALITIES.
AND I HOPE IT OKAY TO SAY HE
PLANS TO LAUNCH A CCR ORGANOID
FACILITY WITH CATHY KELLY TO
DEVELOP CLINICALLY A ANNOTATED
ORGANOID LINES.
SO TODAY, HE WILL SPEAK TO US ON
ORGANOIDS AND CELL POLARITY IN
TRANSLATIONAL AND MECHANISTIC
CANCER RESEARCH.
THANK YOU, SENTHIL.
[APPLAUSE]
THANK YOU VERY MUCH, BEV, FOR
THAT NICE INTRODUCTION.
IT'S ALWAYS A LITTLE BIT
EXCITING AND NERVE-WRACKING TO
PRESENT IN FRONT OF YOUR
COLLEAGUES BECAUSE YOU'RE GOING
TO SEE THEM AGAIN AND AGAIN.
AND SO THEY WILL TEND TO
REMEMBER THE MISTAKES YOU MADE,
NOT THE POSITIVE THINGS, SO BEAR
WITH ME AND FORGIVE ME IF I MAKE
ANY MISTAKES.
SO I WANT TO FIRST SAY THAT I'M
VERY EXCITED AND THRILLED TO
BECOME PART OF THE INTRAMURAL
PROGRAM HERE AT NCI.
IT WAS AN EYE-OPENING EXPERIENCE
DURING THE INTERVIEW PROCESS,
AND AFTER COMING HERE, IT'S ONLY
GROWN ON ME MORE THAN IT DID
BEFORE.
SO I WANT TO THANK THE COMMITTEE
AND TOM FOR TRUSTING IN ME AND
RECRUITING ME HERE BEFORE I
START TALKING ABOUT WHAT WE DO.
SO WHAT I THOUGHT I'LL DO IS TO
SUMMARIZE SOME OF THE FINDINGS
FROM MY LAB THAT GIVES A FLAVOR
FOR THE KIND OF RESEARCH WE DO,
AND I WANT TO START HERE SAYING
THAT THE OVERALL -- IN PRINCIPLE
THE OVERALL RESEARCH PROGRAM IN
MY LAB REALLY IS AN INTEGRATION
BETWEEN BASIC AND TRANSLATIONAL
SCIENCE.
THE BASIC SCIENCE SIDE OF IT
REALLY DEALS WITH CELL PRO LAIRT
PROPOLARITYPROTEINS AND THE ROLG
RESISTANCE AND THE TRANSLATION
SIDE DEALS WITH 3D CULTURE AND
ORGANOID MODELS IN THE CONTEXT
OF TRANSLATION RESEARCH.
SO FOR THE SAKE OF STRUCTURE AND
ORGANIZATION, I'M GOING TO
REALLY GROUP MY TALK IN TWO
GROUPS, ON TWO TOPIC.
THE TOPIC ONE DEALS WITH THE
MODELS WE GENERATE AND HOW WE
USE THEM FOR TRANSLATION
RESEARCH, AND THEN THE TOPIC TWO
DEALS WITH THE POLARITY AND
MECHANISMS.
AND FEEL FREE TO STOP ME BETWEEN
THE TWO IF YOU NEED TO, BUT I
WILL CONTINUE ON AS IF YOU DON'T
HAVE ANY QUESTIONS AT THAT
POINT.
SO MY INTEREST IN 3D CULTURE
MODELS STARTED WHEN I WAS A GRAD
STUDENT WHEN I REALIZED I -- ANA
POSTDOC AND I REALIZED
EPITHELIAL CELLS AND THE CANCER
THAT COMES WITH IT IN A PATIENT
EXISTS IN A THREE DIMENSIONAL
ORGANIZATION AND GROWING CELLS
IN A PLASTIC DISH DOES NOT
RE-CREATE THE BIOLOGY THAT A
TYPICAL CELL SEES.
SO WE BEGAN A PROJECT TO TAKE
HUMAN MAMMARY CELL LINE CALLED
MCF-10A.
AND DEVELOPED CONDITIONS WHERE
WE CAN PLATE THEM ON MATRIX AND
THEY ACTUALLY OVER A PERIOD OF
ABOUT TWO WEEKS, THEY GROW AND
UNDERGO MORPHOGENESIS TO
ESTABLISH STRUCTURES THAT HAVE A
LOT OF SIMILARITY WITH THAT OF A
HUMAN BREAST ACINI.
THE TOP PART WHERE IT TALKS
ABOUT HOW AN ACINUS LOOKS LIKE
AND A CROSS-SECTION IS VERY
SIMILAR TO WHAT YOU SEE WHEN YOU
GROW CELLS IN 3D CULTURE.
THE LOGIC WAS TO GENERATE THESE
STRUCTURES AND THEN ACTIVATE
ONCOGENES IN THEM SO WE BEGIN TO
UNDERSTAND HOW THE CELLS WILL
RESPOND IN A THREE DIMENSIONAL
CONTEXT AS OPPOSED TO A REGULAR
MONO LAYER.
THE RESULTS WERE QUITE
INTERESTING AND REALLY
FASCINATING FOR US PAU PAUSE WHE
TAKE THESE CELLS AND ACTIVATE AN
ONCOGENE, ERBB2, ONE OF THE
DOMINANT ONCOGENES IN BREAST
CANCER AND THERE ARE MANY DRUGS
THAT ARE CURRENTLY USED TO TREAT
PATIENTS WITH THIS POSITIVE
DISEASE THAT EXPRESSES THE
ERBB2.
WHAT WE NOTED WAS THAT WHEN YOU
ACTIVATE ERBB2 THESE THREE
DIMENSIONAL STRUCTURES
COMPLETELY LOSE CONTROL AND GROW
INTO AN AMORPHOUS SORT OF SHAPE
VERY SIMILAR TO HOW CANCEROUS
TISSUES WOULD BE GROWING IN
VIVO.
SO THIS PROMPTED US TO REALLY
THINK ABOUT HOW WE CAN USE THESE
THREE DIMENSIONAL CULTURE MODELS
FOR UNDERSTANDING VARIOUS
ASPECTS OF BIOLOGY.
I WOULD ADD THAT THE SAME CELLS,
WHEN YOU PLATE THEM AS A MONO
LAYER AND ACTIVATE ERBB2, YOU
DON'T REALLY SEE MUCH OF AN
EFFECT OTHER THAN A SMALL AMOUNT
OF PROLIFERATIVE ADVANTAGE.
SO THE BIOLOGY REVEALS ITSELF IN
THE CORRECT CONTEXT THAT IS NOT
APPARENT OTHERWISE.
SO WITH THIS IN MIND, WHEN I
STARTED THE LAB, WE BEGAN USING
THESE MODELS FOR VARIOUS
APPLICATIONS BUT OVER THE YEAR,
WE BEGAN TO THINK ABOUT OTHER
CONTEXTS IN WHICH WE CAN USE
THIS.
AND THAT'S WHEN WE BEGAN
THINKING ABOUT DEVELOPING MODELS
FOR PANCREATIC CANCER USING
EMBRYONIC STEM CELLS.
THE REASON BEING THERE ARE NOT
MANY MODELS AVAILABLE TO STUDY
PANCREATIC CANCER AT THAT TIME,
AND THERE WERE A LOT OF INTEREST
IN GENERATING HUMAN EMBRYONIC
STEM CELLS TO INDUCE THEM TO
DIFFERENTIATE TOWARDS THE
ENDOCRINE PANCREAS.
THESE ARE THE CELLS IN THE
PANCREAS THAT SECRETE INSULIN
AND SO FORTH.
THERE'S A REGION IN THE MEDICINE
FIELD VERY INTERESTED IN
TREATING THEM FOR INSULIN
INSUFFICIENCY.
EXOCRINE PANCREAS IS MADE UP OF
DUCTAL AND EPITHELIAL CELLS, THE
CONTEXT WHERE MOST OF THE
PANCREATIC CANCER ORIGINATES
FROM.
SO LING HUANG IS AT THE FORD
INSTITUTE IN DETROIT,
COORDINATEED WITH A STEM CELL
BIOLOGIST AND SPENT A LOT OF
TIME IN TAKING PANCREATIC
PROGENITOR CELLS AND SLOWLY
COERCING THEM.
THE PROCESS WAS HIGHLY
SUCCESSFUL, IT TOOK A LONG TIME,
BUT IT WAS SUCCESSFUL AND THAT
ACTUALLY LED US INTO REALLY
THINKING THAT WE CAN POTENTIALLY
TAKE STEM CELLS AND PUSH THEM
INTO A LINEAGE OF INTEREST.
THE PROCESS OF DOING THIS REALLY
EDUCATED US IN HOW WE CAN USE
THESE CONDITIONS TO GROW PATIENT
TUMORS DIRECTLY FROM THE
PATIENT, AND THAT LED TO THE
DEVELOPMENT OF A TUMOR ORGANOID
CULTURE CONTEXT FOR PANCREATIC
CANCER WHERE WE CAN TAKE
SURGICALLY RESECTED PANCREAS
TUMORS AND THEN GENERATE TUMOR
ORGANOIDS FROM THEM.
WE NOW USE THESE PLATFORMS QUITE
ROBUSTLY.
THE ORGANOID PLATFORM IS USED
MULTIPLE TIMES, FOR THE STEM
CELL WE STILL STICK TO
PANCREATIC CANCER ORGANOIDS.
FOR THE FIRST ONE, OUR GOAL IS
TO INDUCE DIFFERENTIATION OF
CELLS AND FACILITATE
ORGANIZATION THAT RESEMBLES A
NORMAL EPITHELIAL STRUCTURE.
IN THE TUMOR ORGANOID CASE, THE
GOAL IS TO MAINTAIN
DIFFERENTIATION ON ORGANIZATION
SO THE TUMOR ORGANOIDS WE GROW
IN CULTURE RESEMBLE AS MUCH AS
POSSIBLE THE PATIENT TUMOR IT
CAME FROM.
SO TO MAKE THE POINT VERY CLEAR,
SO IN THE TOP PANEL, WHAT YOU
SEE IS THAT THESE ARE EMBRYONIC
STEM CELLS INDUCED TO
DIFFERENTIATE INTO DUCTAL
ACINUS, THEY'RE MUCH SMALLER
THAN THE DUCTAL ORGANOIDS.
THIS IS WHAT YOU SEE IN VIVO,
AND THEY HAVE PROPER APICAL
BASAL POLARIZATION OF BASE MEMO
BRAINGS IN THE BASAL SIDE AND
TIGHT JUNCTIONS IN THE MIDDLE
SURROUNDING THE LUMEN.
THE RIGHT PANEL IS AN ELECTRON
MICROGRAPH SHOWING YOU HOW
THEY'RE ORGANIZED.
TUMORS ARE ALSO THE SAME.
YOU CAN TAKE A PATIENT TUMOR,
GROW THEM IN ORGANOIDS, AS
ORGANOIDS, THEN IF YOU OBSERVE
THE HISTOPATHOLOGY OF THE
ORGANOID THAT WE GROW, THE
ARCHITECTURE OF THE ORGANOID HAS
AN UNCANNY RESEMBLANCE TO WHERE
THE PATIENT TUMOR CAME FROM.
SO THAT GAVE US THE CONFIDENCE
THAT THE CULTURE CONDITIONS WE
HAVE GENERATED ALLOWS THE CELLS
TO EXIST IN A STRUCTURE AND
FUNCTION FORMAT THAT IS SIMILAR
TO WHAT IS EXISTING IN VIVO,
WHICH MEANS THEY COULD BE REALLY
POWERFUL TOOLS TO REALLY
UNDERSTAND CANCER BIOLOGY MORE.
SO WITH THESE MODELS IN MIND, WE
HAVE DONE A SERIES OF
EXPERIMENTS WHERE WE USE THEM
FOR VARIOUS APPLICATIONS, BOTH
THE DISCOVERY AND FOR
TRANSLATION RESEARCH, AND FOR
THE SAKE OF CLARITY, I'M GOING
TO GROUP THE RESULTS IN THREE
CATEGORIES AND PRESENT TO YOU
AND SUMMARIZE FOR THE MOST PART
THE RESULTS WE HAVE OBTAINED TO
MAKE THE POINTS ON HOW WE USE
STEM CELL ORGANOIDS FOR MODELING
CANCER INITIATION, HOW WE USE
TUMOR ORGANOIDS IN THE CONTEXT
OF PRE-CLINICAL AND CLINICAL
STUDIES, AND LASTLY, ONE OF THE
MORE EXCITING AVENUES FOR US IS
HOW WE CAN USE ORGANOIDS TO
IDENTIFY AND DISCOVER NEW CLASS
OF TUMOR TARGETING T-CELL
RECEPTORS.
SO FOR THE FIRST ONE, OUR FOCUS
WAS ON PANCREATIC CANCER BECAUSE
IT IS AN UNMET NEED.
AS YOU KNOW, THAT MOST OF THE
PANCREATIC CANCER PATIENTS ARE
DIAGNOSED LATE.
WE DO NOT KNOW HOW TO DETECT
THEM EARLY SO UNDERSTANDING HOW
CANCER ORIGINATES IN PANCREATIC
CANCER CAN LIKELY PROVIDE NEW
INSIGHTS AN WITH THAT IN MIND,
WE WENT BACK TO OUR STEM CELL
BASED EXOCRINE PANCREAS ORGANOID
MODEL AND BEGAN USING THEM.
I'LL JUST SHOW YOU ONE PIECE OF
DATA TO COMMUNICATE TO YOU THAT
IN IN FACT THESE LINEAGE
COMMITTED ORGANOIDS IN FACT ARE
TRULY LINEAGE COMMITTED.
WE HAVE DONE EXTENSIVE ANALYSIS
BY GENE EXPRESSION COMPARISON
BETWEEN ACINAL AND DUCTAL BUT TO
MAKE THE POINT, I'LL SHOW YOU
SOME OF THE READOUTS THAT WE
HAVE USED AS WELL.
IF YOU LOOK AT MOLECULAR
MARKERS, FOR EXAMPLE, DUCTAL
CELLS ARE KNOWN TO EXPRESS SOCKS
SOX9 AND -- WHICH IS
UNDETECTABLE IN THE ACINAR
LINEAGE.
THE OPPOSITE IS TRUE -- YOU WILL
SEE THE ACINUS ORGANOIDS EXPRESS
THEM QUITE READILY WHEREAS THE
DUCTAL ONES DO NOT.
FUNCTIONALLY THESE ORGANOIDS
ALSO RETAIN THEIR
LINEAGE-SPECIFIC FUNCTION
BECAUSE IF YOU MEASURE CARBONIC
OR LIPASE ACTIVITY, ACINAR CELLS
ARE KNOWN TO SECRETE -- IN OUR
BODY, HIGH IN THE DUCTAL AND LOW
IN ACINAR, THE OPPOSITE IS TRUE
FOR LIGHT-BASED ACTIVITY.
THESE ORGANS WE GENERATE DO HAVE
A LINEAGE COMMITMENT WI BUT FROA
MOLECULAR AND FUNCTIONAL POINT
OF VIEW.
THE QUESTION IS WHAT CAN WE USE
THESE FOR?
NOW THERE ARE TWO TYPES OF
PANCREATIC PRE-MALIGNANT
LESIONS.
ONE OF THEM WHICH IS THE MORE
COMMON ONE IS CALLED THE
PANCREATIC INTRA -- IT'S THOUGHT
TO ORIGINATE FROM DUCTAL OR
ACINAR OR CENTROACINAR CELLS --
THESE CELLS WHEN THEY'RE
TRANSFORMED AND THE DRIVER
ONCOGENE IS MUTANT -- SO WE
DESIGNED A STRATEGY SO WE CAN
USE AN INDUCIBLE -- KRAS, ALLOW
TO DIFFERENTIATE INTO DIFFERENT
LINEAGES AND THEN YOU ACTIVATE
KRAS AND ASK WHAT HAPPENS TO THE
PHENOTYPE.
THE RESULTS ARE PUBLISHED, I
WON'T GO INTO THE DETAILS.
WHAT WE FOUND WAS THAT BOTH
ACINAR AND DUCTAL CELLS DO
RESPOND TO KRAS -- UNDERGOING
DISRUPTION OF THE THREE
DIMENSIONAL STRUCTURE.
INTERESTINGLY, ACINAR CELLS WHEN
YOU ACTIVATE KRAS UNDERGO
METAPLASIA.
IT'S THE WAY THE ACINAR CELLS
WILL NOW BECOME TUMORS.
THEY WILL GO ON TO TRANSFORM TO
BECOME DUCTAL CARCINOMA.
WE OBSERVED THE SAME THING IN
CULTURE AS WELL.
MORE INTERESTINGLY IF YOU TAKE
THESE CELLS AND YOU TRANSPLANT
THEM INTO THE PANCREAS OF MICE,
THE KRAS G12D EXPRESSING ACINAR
CELLS -- WHEREAS THE SAME
ONCOGENE EXPRESSING ACINAR CELLS
WERE ONLY 11% EFFICIENT.
THIS DICHOTOMY IN THE WAY IN
WHICH KRAS WORKS IN ACINAR
VERSUS DUCTAL IS EXACTLY WHAT
IT'S BEEN REPORTED IN MULTIPLE
STUDIES IN MOUSE MODELS, AND
THOSE ARE STUDIES THAT I POINT
HERE -- I THINK THIS REALLY
CONFIRMS TO US THESE TYPE OF
LINEAGE COMMITTED ORGANOIDS ARE
POWERFUL TOOL TO REALLY
UNDERSTAND HOW THE CANCER
INITIATES IN THIS MODEL SYSTEM.
THE ADVANTAGE BETWEEN MOUSE AND
HERE IS WE KNOW THERE IS
SIGNIFICANT DIFFERENCE BETWEEN
MOUSE AND HUMAN CELL LINES SO
HERE WE HAVE A HUMAN CELL LINE
BASED MODEL THAT WE CAN USE FOR
UNDERSTANDING EARLY STAGES.
SO LING HUANG, WHO IS NOW IN
HENRY FORD HEALTH, WILL NOW BE
PURSUING MANY OF THESE STUDIES
IN HIS OWN GROUP IN DETROIT.
SO THE SECOND FORM OF LESION
THAT IS LESS FREQUENT BUT IT IS
OBSERVED IN PATIENTS IN A
SIGNIFICANT NUMBER ARE CALLED
INTRAPAPILLARY MUCINOUS
NEOPLASMS.
UNLIKE IPM AND THOSE ARE
MICROSCOPIC IN THE PANCREAS,
THESE ARE LARGE CYSTIC
DILATIONS.
THAT MEANS THAT THESE ARE
DETECTABLE BY MOLECULAR IMAGING
IN A PATIENT, SO THERE ARE MANY
PATIENTS, HUNDREDS AND THOUSANDS
OF PATIENTS TODAY WALKING AROUND
WITH AN IPMN LESION WHO ARE JUST
MONITORED CLINICALLY TO SEE IF
AND WHEN THEY WILL PROGRESS.
SO IF YOU CAN UNDERSTAND HOW
THEY'RE REGULATED YOU CAN
ACTUALLY HAVE A THERAPEUTIC
IMPACT TO PREVENT PROGRESSION OF
PATIENTS WITH IPMN LESIONS.
THE CELL OF ORIGIN FOR -- IS
THOUGHT TO BE DUCTAL ALTHOUGH
THERE'S NO CLEAR UNTIL OUR STUDY
WAS PUBLISHED.
THERE IS A MUTATION CALLED GNAS,
ALWAYS SEEN IN EARLY IPMN
LESIONS.
SO WE DECIDED TO TAKE THE GNAS
MUTANT AND DO THE SAME LOGIC AS
WE DID IN KRAS AND ASK WHAT
HAPPENS.
SO I W A BOA POSTDOC IN THE LABD
THESE EXPERIMENTS USING THE MU
TAN GNAS IN DUCTAL OR ACINAR
CELLS.
WHAT YOU NOTICE ON THE LEFT IS
WHEN YOU ACTIVATE GNAS IN THE
DUCTAL CELLS YOU GET THESE LARGE
CYSTIC DILATIONS IN GROWTH.
THERE'S INCREASE IN CELL NUMBER
AND STRUCTURE VO VOLUME.
WHEREAS ACINAR CELLS ON THE
OTHER HAND SHOW A MODEST
PHENOTYPE.
THE SIZE DIFFERENCE IS RATHER
SMALL.
YOU CAN QUANTITATIVELY OBSERVE
THERE'S AN EIGHT FOLD DIFFERENCE
IN CELL SIZE IN RESPONSE TO GNAS
IN DUCTAL WHERE IT'S LESS THAN
THREE FOLD IN ACINAR ORGANOIDS.
SO THIS DIFFERENTIAL RESPONSE TO
IT WAS ACTUALLY QUITE
INTERESTING.
EVEN MORE INTERESTING WAS THAT
IF YOU LOOK AT EXPRESSION OF
MOLECULAR MARKER THAT IS
ROUTINELY USED TO DIAGNOSE IPMN
IN THE CLINIC.
SO EXPRESSION OF MUC2, THE DUCTS
THAT ARE DILATED, IS A MARKER
FOR IPMN LESIONS.
WHEN WE ACTIVATED GNAS IN THE
DUCTAL ORGANOIDS, WE CAN SEE
EXPRESSION OF MUC2 WHEREAS THAT
IS NOT OBSERVED WHEN YOU
ACTIVATE THEM IN ACINAR
ORGANOIDS.
WE CLEARLY DEMONSTRATE IN THIS
THAT ACINAR CELLS CANNOT
POSSIBLY GO DOWN THE PATH OF
IPMN UNDER THESE CONDITIONS.
IF YOU FORCE THEM, ACINAR DUCTAL
METAPLASIA AND THEN EXPRESS MUCM
THIS WHOLE PROCESS.
SO WE REALLY THOUGHT WITH ABOUT
OKAY HOW DO WE REALLY UNDERSTAND
THIS BIOLOGY AND WHAT ARE THE
MOLECULAR MECHANISMS BY WHICH
THIS IS BEING REGULATED.
SO IT'S A G PROTEIN COUPLED
RECEPTOR.
ALMOST ALL KNOWN FUNCTIONS OF
GNAS IS THOUGHT TO GO THROUGH
ACTIVATION OF PKA, SO WE THOUGHT
OKAY IF WE INHIBIT PKA WE SHOULD
BE ABLE TO INHIBIT ALL THE
BIOLOGY DOWNSTREAM OF GNAS.
SO WE USED A FEW DIFFERENT
INHIBITORS, I'M SHOWING DATA FOR
ONE OF THEM.
YOU LOOK FOR GNAS-INDUCED
SIGNALING BY READING OUT EITHER
PAN PKA SUBSTRATE ANTIBODY ON
THE TOP OR A SPECIFIC SUBSTRATE
OR PVASP ON THE BOTTOM, YOU CAN
COMPLETELY SHUT DOWN PK
SIGNALING IN THESE CELLS.
SO WHAT HAPPENS IF YOU ADD THE
INHIBITOR AND THEN ACTIVATE IN
THESE DUCTAL LESIONS?
SURPRISINGLY IT HAD NO EFFECT.
SO I REALLY WANTED TO TEST
WHETHER THIS IS ACTUALLY -- THIS
IS A PROLIFERATION-DRIVEN OR
IT'S CYSTIC DILATION BECAUSE
OF -- ACTIVITY.
SO WE DID SOME EDU LABELING AND
THEN ASKED IS THE LABELING
HAPPENING IN RESPONSE TO GNAS
ACTIVATION IN THE ABSENCE AND
PRESENCE OF THE INHIBITOR, AND
IN BOTH CASES -- WAS ABLE TO SEE
INITIATION OF DNA SYNTHESIS IN
BOTH -- IN THE DUCTAL ORGANOIDS.
AND WHEN SHE QUANTITY TAITS IT,
QUANTITATES IT, ONTHE LEFT, IN T
HAD NO EFFECT IN THE FUNCTION OF
DNA SYNTHESIS.
SO THIS GAVE US AN IDEA THAT
THERE IS DEFINITELY A
PK-INDEPENDENT ARM OF REGULATING
CELL CYCLE BY GNAS ACTIVATION.
BY CONTRAST, THE ACINAR
ORGANOIDS HAS A WEAK PHENOTYPE,
AND IF YOU'VE SEEN THE BOTTOM IN
THE PRESENCE OF DOX, THAT SMALL
INCREASE IS COMPLETELY SHUT DOWN
WHEN YOU ADD THE PK INHIBITOR.
SO THIS MEANS GNAS IS
EXCLUSIVELY USING DIFFERENT
SIGNALING PATHWAYS AND DIFFERENT
LINEAGES IN HOW IT INITIATES
PROLIFERATION.
THEN WE SAID WHAT HAPPENS TO MUC
MUC2, THIS WAS CONFUSING AND
INTERESTING AT THE SAME TIME.
DUCTAL ORGANOIDS, WAS SET DOWN
BY PKA SIGNALING.
SO THIS IS REALLY PLACED IN A
VERY INTERESTING POSITION
BECAUSE WHAT IT SUGGESTS IS THAT
DEPENDING ON THE LINEAGE AND
DEPENDING ON CONTEXT AND
DEPENDING ON THE READOUT, YOU
MAY -- GNAS'S DIFFERENT
SIGNALING MECHANISMS.
PROLIFERATION IN THE DUCTAL
CELLS IS REALLY DRIVING
PROLIFERATION IN A
PK-INDEPENDENT MANNER WHEREAS
EVERYTHING ELSE WE HAVE READ SO
FAR IS A PK-INDEPENDENT PROCESS.
SO WE NEED TO ASK A SERIES OF
QUESTIONS LIKE HOW DOES GNAS
INDUCE PROLIFERATION IN DUCTAL
CELLS IN PK-INDEPENDENT MANNER.
THE REASON THE ANSWER WOULD BE
QUITE POWERFUL BECAUSE IF YOU
WANT TO INHIBIT -- HAVE GNAS
INDUCED IPMN IN PATIENTS YOU
WANT AN ARM THAT IS GENERALLY
NOT INHIBITING PK TOTAL.
SO IF YOU WANT TO FIND A
SPECIFIC PATHWAY USED TO BIND
GNAS IN DUCTAL CELLS TO INDUCE
PROLIFERATION, THAT COULD HAVE
SIGNIFICANT THERAPEUTIC
POTENTIAL.
AND SHE'S ALSO INTERESTED IN
FINDING OUT WHAT IT TAKES FOR
THESE GNS LESIONS TO BECOME
FULLY MALIGNANT AND SO FORTH.
SHE'S ALSO INTERESTED IN USING
THIS PLATFORM TO IDENTIFY NEW
BIOMARKERS.
IN HER OWN LAB.
SO I HOPE I CONVINCED YOU THAT
THE STEM CELL BASED ORGANOIDS
ARE VERY POWERFUL TOOLS TO
REALLY THINK ABOUT MODELING
DISEASE IN GENERAL, BUT MODELING
CANCER IN OUR CASE.
SO IN THE NEXT FEW MINUTES, I'LL
TALK ABOUT THE WAY IN WHICH WE
USE TUMOR ORGANOIDS FOR VARIOUS
DISCOVERY AND TRANSLATION
APPLICATIONS.
SO WE DID A SERIES OF
EXPERIMENTS THAT I'LL SUMMARIZE
IN THIS BECAUSE I
WANT TO FOCUS IN THE SECOND PART
OF THE RESULTS THAT WE GENERATED
TO UNDERSTAND WHETHER ORGANOIDS
ACTUALLY ARE ABLE TO HAVE
BIOLOGY SIMILAR TO THAT IN VIVO.
WE HAD A SERIES OF PDX TUMORS
AND ORGANOIDS, AND DID A SERIES
OF ANALYSIS INCLUDING GENE
EXPRESSION AND MUTATION PROFILE
ANALYSIS, BUT IN ADDITION TO
THAT, WE DID ALSO A SERIES OF
CELL BIOLOGICAL STUDIES, WHERE
WE COMPARED DRUG SENSITIVITIES,
WE COMPARED THE SECRETORY SYSTEM
IN VIVO COMPARED TO CULTURE AND
ALSO GLYCOMICS, HOW THE SURFACE
PROTEIN ARE CONSERVED BETWEEN
THE ORGANOIDS AND THE PDX
MODELS.
THE RESULTS WERE ACTUALLY QUITE
INFORMATIVE.
WE FOUND THAT THE ORGANOIDS
PRETICKET IN VIVO DRUG RESPONSE
IN ABOUT 80 TO 90% ACCURACY IN
CULTURE.
AND WE ALSO REALIZED THAT
MEASURING DRUG SENSITIVITY IN
ORGANOIDS IS VERY TRICKY.
IT'S NOT EASY TO USE -- EVERY
ORGANOID HAS ITS OWN DOUBLING
TIME AND THERE ARE SLOW GROWING
AND FAST GROWING ONES SO IN
ORDER TO REALLY ACCOUNT FOR
THESE VARIATIONS, WE HAVE
DEVELOPED A NORMALIZED AREA
UNDER THE CURVE METHOD THAT
ALLOWS US TO REALLY OBJECTIVELY
COMPARE DRUG RESPONSE BETWEEN
ORGANOID LINES.
AND WE KNOW NOW THAT THE
VESICLES THAT ARE SECRETED BY
THE ORGANOIDS IN CULTURE CAN BE
SEEN IN THE PATIENT'S BLOOD,
WHICH MEANS THIS COULD BE A
POWERFUL TOOL TO FIND SECRETE --
I THINK THIS CAN BE HELPFUL
THERE.
AND AS I MENTIONED, THE SURFACE
GLYCAN PROFILE ARE ABOUT 80%
SIMILAR, WHICH GIVES US
CONFIDENCE THAT IN THE 3D
CULTURE, THEY'RE ABLE TO
MAINTAIN SOME ASPECT OF THE
SURFACEOME SIMILAR TO THAT IN
VIVO.
SO ARMED WITH THAT INFORMATION,
WE WANTED TO ASK, IS THIS REALLY
GOING TO BE USEFUL IN THE
CLINICAL SETTING?
SO FOR THIS WE NICHE WRA INITIAA
CLINICAL TRIAL CALLED HOPE
REASONS, WE HOPED IT WORKED FOR
THE SAKE OF PATIENTS, AND IT WAS
LED BY JOE GROSSMAN AND MANNY
HIDALGO IN THE CLINIC AND BY
LING HUANG AND -- IN THE LAB,
AND THE GOAL WAS TO GENERATE
ORGANOID MODELS FROM PATIENTS
AND THEN TREAT THE ORGANOID
MODELS WITH THE SAME DRUG
THEY'RE GOING TO RECEIVE AFTER
THE DRUG AND THEN COMPARE THE
RESULTS.
THE END POINTS WERE ACTUALLY
VERY SIMPLE.
WE FIRST WANTED TO KNOW IF THIS
IS FEASIBLE AND SECOND WE WANTED
TO SEE IF THERE WAS A
CORRELATION BETWEEN ORGANOID
RESPONSE IN THE PDO AND PATIENT
RESPONSE IN THE CLINIC.
THE EQUALLY REPRESENTED FROM
MALE AND FEMALE AND ALSO EQUALLY
REPRESENTED FROM ALL STAGES OF
THE DISEASE.
THERE ARE TWO SUBTYPES OF
PANCREATIC CANCER, BASAL AND
CLASSICAL AND WE HAVE ORGANOID
MODELS THAT ARE EITHER CLASSICAL
OR BASAL THAT MATCHES WITH THE
TUMOR AS WELL AND THE CLASSIC
TUMOR RESULTS IN A CLASSIC
ORGANOID AND BASAL PATIENT TUMOR
RESULTS IN A BASAL PART OF OR
THE NOID.
SO IN ORDER TO REALLY UNDERSTAND
HOW THIS IS USEFUL, WE TOOK A
DEEP DIVE APPROACH ON 14
PATIENTS.
NOW THESE 14 PATIENTS RECEIVED
42 DIFFERENT DRUGS, BECAUSE MOST
PATIENTS ARE TREATED WITH
COMBINATIONS AT THIS STAGE, AS
THE COMBINATION LISTED HERE SO
WE DECIDED TO TAKE THESE
ORGANOID MODELS AND GENERATE
42 DIFFERENT AUC VALUES.
THEN WE WENT BACK TO THE CLINIC
AND ASKED WHAT IS THE CLINICAL
RESPONSE OF THIS PATIENTS AS
PERVERSE US TO CRITERIA.
USUALLY A QI NATION OF IMAGING,
BIOMARKERS AND OTHER
CHARACTERISTIC THAT IS USED IN
THE CLINIC.
AND SO WHEN YOU COMPARE THE
PATIENT RESPONSE TO THE AUC
VALUES OF THE DRUG FOR THE
SPECIFIC ORGANOIDS, THERE WAS A
VERY INTERESTING SEGREGATION.
SO THE PATIENTS WHO ARE LABELED
IN BLUE ARE THOSE PATIENTS WHO
HAD YOU HAD EITHER A STABLE
DISEASE OR PARTIAL RESPONSE.
WHEREAS THE PATIENTS IN RED ARE
ALL PATIENTS WHO PROGRESSED ON
THE TREATMENT.
IF YOU LOOK AT ALL THE AUC
VALUES, WHAT WE REALIZED WAS
THAT THERE IS AT LEAST ONE DRUG
IN THE COCKTAIL THAT HAS AN AUC
VALUE OF LESS THAN .55, THE
PAYABLE EITHER HAD STABLE
DISEASE OR PARTIAL RESPONSE.
WHEREAS IF THE AUC VALUE IS
HIGHER THAN THAT, THEN THE
PATIENT PROGRESSED.
WE REALIZED THAT IDENTIFYING A
DRUG THAT HAS AN AUC VALUELESS
THAN .55 IN THE ORGANOID CULTURE
GIVES US ABOUT 80 TO 85%
PREDICTIVE POWER TO TELL WHETHER
THE DRUG IS GOING TO WORK OR
NOT.
THE SECOND INSIGHT WE GAINED, IN
A DRUG COCKTAIL, PRESENCE WITH
AUC -- WAS SUFFICIENT TO PROVIDE
POSITIVE RESPONSE.
SO THIS HAS SEVERAL
IMPLICATIONS.
THERE'S A COUNTERINTUITIVE
APPLICATION WHICH IS IDENTIFY
THE DRUGS THAT DO NOT WORK IN
THE ORGANOID, YOU CAN EXCLUDE
THEM IN THE CLINIC AND YOU MAY
ACTUALLY EXPERIENCE THAT AS
WELL.
PERHAPS WE CAN USE THEM AS A WAY
TO UNDERSTAND WHY PATIENT TO
PATIENT DIFFERENCES IN DRUG
TERRITORIMENT DO OCCUR.
SO I'M HOPING THESE ARE THE KIND
OF QUESTIONS WE WILL BE ABLE TO
ANSWER IN OUR LAB HERE IN LCBG
IN COLLABORATION WITH THE
CLINICAL TEAM HERE.
SO THE LAST RESULTS I WANT TO
SUMMARIZE IS HOW WE HAVE USED
ORGANOIDS IN THE CONTEXT OF
DISCOVERING T-CELL RECEPTORS,
TUMOR TARGETING T-CELL
RECEPTORS.
IN THIS CONTEXT, WE ACTUALLY
TOOK A COMPLETELY DIFFERENT
APPROACH, AND WE DECIDED NOT TO
FOCUS ON TUMOR INFILTRATING
LYMPHOCYTES BUT REALLY TO FOCUS
ON PERIPHERAL BLOOD DERIVED
T-CELLS.
AND THE REASON IS WE THINK THIS
MAY ALLOW US TO FIND T-CELL
RECEPTORS THAT ARE DIFFICULT TO
FIND BECAUSE FINDING T-CELL
RECEPTORS IN THE PERIPHERAL
BLOOD IS USUALLY LIKE FINDING A
NEEDLE IN A HAYSTACK.
WE DON'T WANT BIAS TOWARDS ANY
ANTIGENS OR NEOANTIGENS, WE JUST
WANT NATURE TO SELECT ITSELF.
SO THE WAY IT'S DONE IS WE
GENERATE ORGANOID FROM A
PATIENT, AT THE SAME TIME WE
ALSO GET PERIPHERAL BLOOD, WE
EXPAND THE T-CELLS FROM THE
PATIENT AND WE CO-CULTURE THEM
TOGETHER, TWO WEEKS, A SELECTIVE
NUMBER OF T-CELLS WILL NOW
EXPAND, ORGANOID PRIMED T-CELLS.
NOW THE BIOLOGY WORKS
BEAUTIFULLY SO HERE IS AN
ORGANOID AND A PBMC DIRECT
T-CELL FROM THE SAME PATIENT.
YOU PUT IT TO THE AND THEY START
KILLING AND ATTACKING THE
ORGANOID AND IN A PERIOD OF 14
DAYS, THEY COMPLETELY KILL ALL
THE TUMORS, AND IN THE PROCESS,
THE T-CELLS GET ACTIVATED AND
EXPAND IN NUMBER.
ONCE YOU GENERATE A POPULATION
OF CELLS, THEY'RE REALLY ROBUST
IN KILLING THE TUMOR.
IF YOU CHALLENGED -- THEY
ACTUALLY WILL KILL IT WITHIN 10
HOURS.
WHEN YOU PUT THEM TOGETHER, THEY
COMPLETELY OBLITERATE THE
ORGANORGANIZE QUITE EFFICIENTLYD
THAT'S HOW THEY SEEM TO WORK IN
ALL THE CASES WE'VE TESTED SO
FAR.
NOW THESE T-CELLS HAVE ALL THE
CYTOTOXIC T-CELL SIGNATURES IN
RESPONSE TO STIMULATION OR
EXPOSURE TO ORGANOIDS, THEY
UNDERGO PROLIFERATION, THEY
SECRETE IN DIFFERENT -- IT
REALLY TOLD US WE CAN NOW
ESTABLISH A POPULATION OF
T-CELLS BY CO-CULTURING WITH
ORGANOIDS.
SO THE QUESTION IS, IS THERE
CLONAL EXPANSION GOING ON,
BECAUSE THAT IS CENTRAL FOR US
TO REALLY BE ABLE TO TAKE TO THE
NEXT LEVEL.
IN ORDER TO DO THAT, WE TOOK
MATCHED OBMC AND OP T-CELLS FROM
THE SAME PATIENT, THEN WE ASKED
WHAT FRACTION OF THE TCR YOU
FIND IS REPRESENTATIVE OF THE
ENTIRE POPULATION OF THAT
CULTURE.
THE RESULTS WERE QUITE
FASCINATING AND SURPRISING.
FOR THE PBMCs JUST AS
EVERYBODY WOULD EXPECT, MOST
TCRs WERE PRESENT IN .1%.
THERE WERE SOME TCRs THAT WERE
5 TO 6% BUT MOST OF THEM VERY
LOW IN NUMBER.
BY CONTRAST, THE OP T-CELLS, --
SOMETIMES ONE TCR WAS ABOUT 70
TO 90%.
THAT TOLD US THAT THERE IS
SEVERAL ORDERS OF MAGNITUDE OF
EXPANSION HAPPENING IN THIS
CO-CULTURE SETTING, EMPOWERING
US TO THINK PERHAPS THIS IS A
WAY WE CAN FISH OUT THE T-CELL
RECEPTORS THAT ARE ABLE TO
TARGET THESE TUMOR CELLS FROM
THE CIRCULATION.
SO WE WANTED TO KNOW IF THESE
TCRs ARE ACTUALLY THE ONES
THAT ARE DRIVING THE PHENOTYPE,
AND IF THEY ARE TRANSFERABLE TO
OTHER CELLS.
TO DO THAT, WE SEQUENCE THE
VARIABLE CHAINS AND THEN CREATED
A CHIMERIC TCR EXPRESSING THE
CONSTANT REGION FOR MOUSE.
NOW YOU CAN EXPRESS THIS WITH
THIS KIE ME TICK RECEPTOR AND
THEN ASK WILL THIS T-CELL HAVE
THE ABILITY TO ATTACK AND KILL
THE ORGANOID THAT THE T-CELL
RECEPTOR WAS GENERATED FROM?
AND THE ANSWER IS YES.
SO WE TOOK A PATIENT 10 DERIVED
T-CELL RECEPTOR SO THAT'S
AUTOLOGOUS, THAT SHOULD KILL,
WHEREAS A PERSON 38 ORGANOID
WHICH IS ALLOGENEIC SO WE DON'T
KNOW WHETHER IT WILL KILL OR
NOT.
SO WHEN YOU CO-CULTURE THAT,
WHEN -- IT QUITE EFFECTIVELY
KILLS WITHIN 24 HOURS AND 38 ON
THE OTHER HAND IS QUITE WEEK.
THIS KILLING IS STILL MEDIATED
BY ANTIGEN PRESENTATION BY THE
TUMOR CELLS BECAUSE IF YOU THROW
IN A FUNCTION BLOCKING ANTIBODY,
YOU COMPLETELY BLOCK THIS
PHENOTYPE.
SO THESE ENGINEERED T-CELLS SEE
SOMETHING, WE DON'T KNOW WHAT
THAT IS, ON THE TUMOR CELL, AND
THEN KILLS THE TUMOR CELLS.
SO THIS HAS REALLY ALLOWED US
INTO A NEW ERA OF THINKING THAT
PERHAPS THIS WAY OF DOING THIS
CAN BE ONE WAY OF DOING ADAPTIVE
CELL THERAPY, WHICH IS
LABORIOUS, MAYBE TI TIME-CONSUM,
BUT ON THE OTHER HAND USING THIS
PLATFORM TO IDENTIFY TUMOR
TARGETING TCRs IS QUITE A
POWERFUL STRATEGY AND THAT'S
SOMETHING WE'RE GOING TO HEAVILY
INVEST ON, SO HOPING TO BE ABLE
TO DO A LOT OF THESE THINGS HERE
AND AT NCI.
SO BEYOND THESE THREE STORIES,
WE HAVE USED ORGANOID PLATFORM
FOR OTHER MULTIPLE ORGANIZATIONS
BUT I DON'T WANT TO -- WE
PUBLISHED MOST OF THEM AND
YOU'LL BE ABLE TO FIND THEM.
SO GIVEN THE POWER OF THIS, WHEN
I MOVED HERE WITH THE SUPPORT OF
THOMAS TELLY AND CATHY KELLY,
CATHY AND I FACILITATED -- ONE
IS TO GENERATE ORGANOID MODELS
BY COLLABORATING WITH THE
CLINICAL TEAM HERE AT NCI FROM
THE PATIENTS WHO UNDERGO SURGERY
HERE, AND POTENTIALLY MAKE THEM
AS A RESOURCE FOR THE COMMUNITY
HERE.
THE SECOND IS TO SERVE AS A
TRAINING AND KNOWLEDGE BASE, TO
REALLY PROVIDE HANDS-ON TRAINING
FOR THOSE WHO MAY BE NEEDING
TRAINING AND MENTOR THEM THROUGH
THE TRANSFER OF KNOWLEDGE TO
THEIR OWN SPACE.
AND WHATEVER MEDIA WE HAVE
GENERATED FOR SOME ORGANOIDS,
WE'LL PROVIDE -- THE RECIPES,
WILL PROVIDE THEM TO THE LAB WHO
MAY BE INTERESTED IN USING THEM.
SO I'M HOPING THIS WILL TAKE
OFF -- IT'S ALREADY TAKING OFF,
ACTUALLY, THANKS TO THE CLINICAL
TEAM, JONATHAN IS SITTING THERE,
WHERE WE GET A LOT OF SAMPLES
AND WE'RE GENERATING ORGANOIDS
ON A ROUTINE BASIS.
SO NOW I'M GOING TO SHIFT GEAR
AND GO INTO THE SECOND TOPIC OF
HOW WE ARE REALLY INVESTIGATING
THE ROLE PLAYED BY CELL POLARITY
PROTEINS IN THE CONTEXT OF DRUG
RESISTANCE.
SO I'M NOT SURE HOW MANY OF YOU
KNOW OR HEARD TOO MUCH ABOUT
CELL POLARITY BUT FOR THE SAKE
OF THE TRAINEES IN THE AUDIENCE,
THE CELL POLARITY IS ACTUALLY A
FUNDAMENTAL PROPERTY OF ALL
CELLS.
IT IS THE MECHANISM BY WHICH
ASYMMETRY IS CREATED WITHIN A
CELL.
THE FRONT OF A CELL LOOKS
DIFFERENT FROM THE REAR OF A
SELL IN A MIGRATING CELL.
THE PORTION OF THE CELL THAT
FACES THE LUMINAL SPACE OF THE
EMPTY DUCTAL SPACE IS DIFFERENT
FROM WHAT IS IN THE BASE.
ALL THAT ASYMMETRY IS CREATED BY
THE CELL POLARITY PROTEINS.
NOW THIS POLARITY IS A
FUNDAMENTALLY IMPORTANT FOR
PHYSIOLOGY.
POLARITY DICTATES HOW YOU ABSORB
NUTRIENTS IN THE GUT AND YOU
SECRETE MILK IN THE DUCT, IN THE
MAMMARY GLAND.
THOSE ARE ALL PROCESSES STRICTLY
REGULATED BY THESE PROTEINS.
SO DISRUPTION OF THIS POLARITY
OF PROTEINS ALMOST ALWAYS LEADS
TO THE DIRECTIONAL PROCESSES
WHICH LEAD TO DISEASE.
NOW THERE'S BEEN A LOT OF
STUDIES IN MODEL ORGANISMS
TRYING TO UNDERSTAND THIS,
MOSTLY FROM DROSOPHILA C.
ELEGANS -- POLARITY PROTEINS,
THEY'RE SCAFFOLDING MOLECULES
AND SOME KINASES.
I WON'T GO INTO THE DETAILS OF
ALL THESE MOLECULES AT THIS
POINT.
THE REASON I GOT INTERESTED IN
POLARITY PROTEINS A LONG TIME
AGO IS SOME OF THE STUDIES
PUBLISHED IN DROSOPHILA.
IF YOU HAVE A LOSS OF FUNCTION
MUTATION OF A POLARITY PROTEIN,
NOT ONLY DO YOU LOSE CELL
STRUCTURE, BUT THE CELL GAINS
UNCONTROLLABLE GROWTH ADVANTAGE.
IT ACTUALLY GROWS TO FORM A
TUMOR-LIKE PHENOTYPE IN -- AND
THESE FLIES ARE IN THE LARVAE.
SO LED TO GENETICIST TO COIN THE
TERM THAT THESE ARE NEOPLASTIC
TUMOR SUPPRESSORS.
BUT IT WAS NOT KNOWN AT THAT
TIME WHETHER THEY ARE TUMOR
SUPPRESSORS IN MAMMALIAN CELLS
OR NOT.
SO THAT'S HOW WE STARTED IN MY
LAB ASKING THE QUESTION, ARE
THESE TUMOR SUPPRESSORS IN
MAMMALIAN CONTEXT?
HE PUBLISHES -- LOSS OF FUNCTION
OF POLARITY PROTEINS WILL
COOPERATE WITH ONCOGENES TO
INDUCE TUMOR, TRANSFORM CELLS TO
INDUCE METASTATIC PHENOTYPE AND
SO FORTH.
ALONG THE WAY WE MADE SOME
POLARITY PROTEINS THAT, WHEN
OVEREXPRESSED, ACTUALLY PROMOTE
PROLIFERATION.
SOME MIGHT SHOW THAT EVEN IF YOU
MAKE A TRANSGENIC MOUSE MODEL,
WE CAN INDUCE GENERATION OF
TUMORS.
SO THIS REALLY PUZZLED US FOR A
WHILE AND ALSO WHEN YOU LOOK AT
THE GENOMICS DATABASE, THERE WAS
ACTUALLY A CLASS OF POLARITY
PROTEINS, A GROUP OF THEM, A FEW
THAT ARE FREQUENTLY
OVEREXPRESSED AND AMPLIFIED IN
MULTIPLE CANCER TYPES.
SO THIS MADE US THINK THAT MAYBE
NOT ALL POLARITY PROTEINS ARE
THE SAME, MAYBE SOME OF THES ARE
POSITIVE REGULATORS OF BIOLOGY.
AND THE REASON -- WE WANTED TO
REALLY PURSUE THIS BECAUSE IF
THEY ARE POSITIVE REGULATORS,
THEY MAY USE A NEW CLASS OF
TARGETS THAT YOU CAN GO AFTER,
IT'S NOT A DNA DAMAGE REPAIR OR
ANY OF THOSE PROCESSES THAT ARE
ROUTINE STUDIES THERAPEUTIC
ADVANTAGE.
WITH THAT IN MIND WE INITIATED A
PROGRAM IN THE LAB TO ASK --
PROMOTERS OF CANCER BIOLOGY.
THE GENE WE FOCUSED ON WAS
CALLED LETHAL GIANT LARVAE,
RECENTLY DISCOVERED IN
DROSOPHILA.
LOSS OF MUTATION RESULTS IN
LETHAL GIANT LARVAE BECAUSE --
GROW OUT OF CONTROL.
AT THAT TIME THE LITERATURE WAS
REALLY VAGUE IN IS IT REALLY A
TUMOR SUPPRESSOR OR PROMOTER.
THERE WERE RESULTS THAT WERE
PUBLISHED, BOTH CAN GO IN BOTH
DIRECTIONS.
SO BECAUSE OF THAT, WE ACTUALLY
TOOK THIS MOLECULE AND YOU ALSO
SAY TO -- IT WAS SO COMPLICATED
THAT IT WAS CALLED THE TAR BABY,
BECAUSE ANYBODY THAT TOUCHES LGL
CANNOT REALLY COMPLETE THEIR
STUDY BECAUSE IT GOES BOTH WAYS,
DEPENDING ON THE TOPIC, SO
NOBODY COULD REALLY UNDERSTAND
HOW LGL WAS WORKING WELL AT THAT
TIME.
SO SATO NOW HAS HIS OWN LAB IN
JAPAN AND LOOKED AT LLGL EGGS
PRETION IN CANCER AS A FUNCTION
OF CLINICAL OUTCOME.
IF HE LOOKS, HIGH EXPRESSION
CORRELATED WITH GOOD OUTCOME IN
ERPR POSITIVE CANCERS.
THIS IS CONSISTENT WITH THE
REASONING THAT LLGL1 MAY BE A
ACTUAL MORE SUPPRESSOR, SO HIGH
IS GOOD.
THE RESULTS WERE OPPOSITE WHEN
YOU LOOKED AT LGL2.
WHEN YOU SEE HIGHER LEVELS OF
LGL2, IT HAD A STRONG
CORRELATION WITH POOR OUTCOME.
SO YOU'LL SEE AT THE RED LINE
HERE WHICH IS HIGH, THE PATIENTS
DIE FASTER COMPARED TO THE BLACK
ONE.
AND THIS BIOLOGY WAS MORE
SPECIFIC TO ERPR POSITIVE, NOT
NEGATIVE.
SO YOU TOOK A PANEL AND STARTED
LOOKING FOR LGL1 AND LGL2.
LGL2 EXPRESSION WAS HIGHER IN
ERP POSITIVE COMPARED TO
NEGATIVE.
LGL1 IS MORE NEGATIVE THAN ALL
THAT HE TSH THERE'S A SELECTION
FOR HIGHER LEVELS OF LGL2 IN
THESE CELLS.
TO TEST IT, HE KNOCKED DOWN --
I'M SORRY -- WHAT HE DID WAS HE
ACTUALLY GENERATED MCF 7 CELLS
THAT OVER -- AND ASK DO THEY
GIVE ANY SORT OF PROLIFERATIVE
ADVANTAGE.
THE RESULTS WERE CONFUSING FOR A
LONG TIME.
HE COULD NOT FIND ANY BIOLOGY
THAT WAS ACTUALLY SUGGESTING
THAT LGL1 AND L
GL2 OVEREXPRESSION WAS ACTUALLY
HELPFUL FOR THESE CELLS.
UNTIL HE CHALLENGED THESE CELLS
WITH THE NUTRIENT STRESS
CONDITIONS.
HE HE SWITCHED THESE, THE
CONTROL AND LGL1 WILD TYPE
EXPRESSING CELLS DIDN'T REALLY
LIKE IT, DIDN'T GROW WELL,
WHEREAS THE LGL2 CELLS CONTINUED
TO GROW WELL.
SO THIS GAVE US A CONNECTION
THAT MAYBE THERE'S A CONNECTION
BETWEEN NUTRIENT AND LGL2.
THIS IS SIMPLY REMOVING THE --
IF YOU DIAL -- YOU'LL HAVE --
THEN YOU GAIN THE PHENOTYPE.
AND WHAT YOU LOSE HERE ARE
THINGS LAKE AMINO ACI LIKE AMIN.
WE ALSO GENERATED LGL -- HE PUT
THEM IN XENOGRAPH TRANSPLANTS.
KNOCKDOWN OF LGL2 REALLY IMPAIRS
TUMOR -- IT IS A POSITIVE
REGULATOR OF GROWTH FOR THESE ER
POSITIVE BREAST CANCER CELLS.
SINCE THIS IS ALL NUTRITIONALLY
RELATED, WE ALSO VENTURED INTO
REALLY LOOKING AT THE
METABALOME, YOU SAID ANY -- WHEN
YOU ANALYZE ALL THE METABOLITES,
IT TURNS OUT THAT THE
INTRACELLULAR LEVELS WERE
DIFFERENTIALLY REGULATED BETWEEN
CONTROL AND KNOCK DOWN CELLS.
THE CELL CANNOT MAKE THEM SO
THEY RELY ON EXTRACELLULAR
SOURCE TO TAKE UP AMINO ACIDS
FOR BIOLOGICAL PROCESSES.
NOW AMONG THE NINE ESSENTIAL
AMINO ACIDS, SIGNIFICANT LOWER
IN KNOCK DOWN CELL THAN COMPARED
TO OTHERS.
SINCE THEY WERE MULTIPLE, YOU
ALSO WANTED TO KNOW IF ANY OF
THEM ARE SPECIFICALLY INVOLVED
OR A COLLECTION OF ALL OF THEM.
TO DO THAT HE LOWERED ALL THE
ESSENTIAL AMINO ACIDS AND THEN
ADDED BACK ONE AT A TIME TO SEE
WERE ANY OF THOSE RESCUE
PROLIFERATION.
THIS GAVE US A DIRECT INDICATION
COMPLETELY UNEXPECTED THAT
THERE'S SOME CONNECTION BETWEEN
A POLARITY PROTEIN LGL2 AND
LOSING UP TAKE BY THESE CELLS IN
THE WAY THEY USE IT.
SO THE QUESTION IS HOW DOES THIS
HAPPEN?
I'LL JUST SUMMARIZE IT SO I CAN
TELL YOU ABOUT SOME UNPUBLISHED
WORK.
WHAT HE FOUND WAS THAT THIS
POLARITY PROTEIN NOW FUNCTIONS
AS A TRAFFICKING REGULATOR.
IT BINDS TO AN AMINO ACID
TRANSPORTER ALSO KNOWN AS
LAT1 AND IT BINDS TO THE CYTOSOL
AND TRANSPORTS TO THE CELL
MEMBRANE.
IF YOU KNOCK DOWN LGL2, THE
LOCALIZATION CHANGES.
NOW IT ACCUMULATES IN THE
CYTOSOL, DOESN'T GET TO THE
MEMBRANE.
SO WHEN YOU HAVE HIGHER LEVELS
OF L TB. L2 -- AND THAT GIVES
THE CELL AN ADVANTAGE.
TO SUMMARIZE THESE, WHAT HE
FOUND -- IN THE MEMBRANE IT
BINDS TO A SNARE PROTEIN CALLED
YTK6 AND THAT FACILITATES IN
MEMBRANE INSERTION OF THESE
CELLS.
NOW THERE ARE A LOT OF QUESTIONS
HERE WE NEED TO UNDERSTAND
EXACTLY HOW THIS PROCESS WORKS,
AND THAT IS SOMETHING HE'S DOING
IN HIS OWN LAB IN JA JAPAN.
SO IN ADDITION TO REALLY
UNDERSTANDING THE ROLE FOR
LDR2 IN -- HE MADE AN
INTERESTING OBSERVATION, IT
TURNS OUT WHEN YOU HAVE HIGH
LEVELS OF LGL2, THESE DRUGS CAN
RESIST TAMOXIFEN.
SO THAT MEANT THAT MAYBE IT IS
INVOLVED IN SOME -- IN DRUG --
DEVELOPMENT OF DRUG RESISTANCE.
NOW FOR THOSE OF YOU THAT KNOW,
70% OF BREAST CANCERS ARE THE
ERPR POSITIVE SUBTYPE AND ABOUT
30% OF THEM WILL ACTUALLY
DEVELOP RESISTANCE TO TREATMENT
CURRENTLY USED.
IN THE CLINIC, THOSE 30% OF THE
PATIENTS RECEIVE CDK46 INHIBITOR
AS A TREATMENT PARADIGM FOR
THEM.
SO WE BEGAN ASKING IS THIS
REALLY RELEVANT IN THE --
CONTEXT AS WELL.
SO THESE ARE ALL ONGOING STUDIES
BEING DONE BY TALENTED POSTDOC
IN THE LAB, MIKE OLIPHANT, AND
I'M GOING TO SHARE WITH YOU AND
HAPPY TO TAKE ANY SUGGESTIONS ON
THIS PART OF THE PROJECT.
SO HE GENERATED A PANEL OF CELL
LINES THAT EXPRESS SLC7A5.
WHEN YOU OVEREXPRESS 7A5 THAT BY
ITSELF WAS SUFFICIENT TO GIVE A
THREE TO FIVE FOLD SHIFT IN
ISA50 VALUE, SO THE RED LINE
SHOWS THE OVEREXPRESSING CELLS
AND THE BLUE LINE IS THE CONTROL
CELLS, AND YOU CAN SEE THERE IS
APPROXIMATELY A FOUR PHOTO
INCREASE IN THE IC50 VALUES
MEANING THEY'RE -- THAN THE
REST.
THIS IS AN INDUCED RESISTANCE SO
THIS SUGGESTS 75 IS SUFFICIENT
TO CONFER RESISTANCE.
THEN THE QUESTION WAS IS IT
NATURALLY SELECTED.
SO WE WANTED TO LOOK AT SOME
SPECIFIC RESISTANT MODELS TO SEE
IS THERE -- SO WE COLLABORATED
WITH NICK TURNER TO GET THE CELL
LINES MADE WITH CDK46 AND WHEN
MIKE LOOKS AT THE SURFACE
LEVELS, THEY'RE SIGNIFICANTLY
HIGHER IN THESE MODELS COMPARED
TO THE SENSITIVE ONES,
SUGGESTING THE FACT THERE IS
PROBABLY SELECTION FOR IT DURING
THE DEVELOPMENT OF RESISTANCE IN
THESE CELL LINES.
THE MORE POWERFUL DATA CAME WHEN
BEGAN A COLLABORATION WITH NICK
WAGLEY'S GRAWP AT DANA FARBER.
NICK HAS BEEN COLLABORATING
SAMPLES AND ANALYZING THE --
WITH THE DEVELOPMENT OF C
CD46 INHIBITORS IN THE CLINIC.
IN THE FIVE PAIRS WE LOOKED AT,
IN ALL CASES, THERE WAS A
SIGNIFICANT INCREASE, A
NOTICEABLE INCREASE, I SHOULD
SAY, IT'S TOO SMALL A NUMBER TO
CALL IT SIGNIFICANT, INCREASE IN
THE LEVELS OF 7A5 PRE AND POST
TREATMENT.
ALTHOUGH THIS LOOKS LIKE FIVE
SAMPLES, THESE ARE VERY PRECIOUS
SAMPLES TO COME BY.
SO THIS IS REALLY REASSURING FOR
US TO KNOW THAT 7A5 MAY ACTUALLY
BE DRIVING RESISTANCE IN THESE
PATIENTS.
IT TURNS OUT IF YOU BREAK THEM
DOWN INTO PATIENTS WHO HAVE A
QUIET RESISTANCE THAN HAVE
PATIENT WHO HAVE -- THE INTR,
THE -- COMPARED TO THE SENSITIVE
PATIENTS.
SO ARMED BY THIS INFORMATION
THAT THIS IS PROBABLY A DRIVER
OF RESISTANCE, MIKE HAS BEGUN A
SERIES OF STUDIES TO REALLY GO
DOWN THE PATH OF UNDERSTANDING
THE MECHANISM BY WHICH THIS MAY
HAPPEN.
SO LEUCINE, AS SOME OF YOU MAY
KNOW, IS A BRANCH AMINO ACID SO
THERE ARE SOME SIMPLE KITS YOU
CAN USE TO MEASURE BRANCH AMINO
ACID LEVELS IN CELLS.
SO MIKE TOOK HIS OVEREXPRESSING
CELL LINES AND SPONTANEOUS
RESISTANT MODEL, IN BOTH CASES,
THE LEVELS GO UP.
NOT ONLY THE LEVELS TRANSPORTED
HIGHER IN THESE CELLS, THEY
FUNCTIONALLY ARE WORKING MORE BY
TAKING UP MORE AMINO ACIDS
INSIDE THE CELL.
IS THE INCREASING LEVEL OF AMINO
ACID, IS THAT WHAT IS DRIVING
THE PHENOTYPE?
TO TEST THAT, WHAT MIKE DID IS A
SIMPLE EXPERIMENT.
YOU COMPARE COMPLETE MEDIA
WHETHER IT'S NORMAL LEVELS OF
LEUCINE, TO THE DRUG RESPONSE IN
A MEDIA THAT HAS ONE-TENTH THE
AMOUNT OF LEUCINE.
SO IF YOU DROP THE LEUCINE
LEVELS THEN THE RESISTANCE
DROPS, THE CELLS BECOME A LITTLE
BIT MORE SENSITIVE, GIVING US
THE INDICATION THAT IN FACT
TOGGLING THE LEUCINE LEVELS
INSIDE THE CELL CAN TOGGLE IN
WHICH THE WAY THE CELLS CAN
FIGHT.
THOSE OF YOU, LEUCINE WHEN IT
GET INTO THE CELL IS AN
IMPORTANT INDICATOR OR MTOR
SIGNALING.
SO WE THOUGHT MAYBE THIS BIOLOGY
IS GOING TO MTOR.
IT'S TRUE WHEN YOU ADD LEUCINE,
WE ASKED IF YOU TREAT THESE
CELLS WITH INHIBITOR DO YOU SEE
A SENSITIVITY BETWEEN
OVEREXPRESSING CELLS,
DIFFERENTIAL BETWEEN -- AND
THE -- SO THEY ARE EQUALLY
SENSITIVE.
SO THIS TELLS US THAT THE
INCREASED CONCENTRATION OF
LEUCINE IN THESE RESISTANT CELLS
CONFERRED BY 7A5 OVEREXPRESSION
IS PROBABLY GOING THROUGH
SOMETHING ELSE OTHER THAN MTOR
BIOLOGY.
SO THE QUESTION IS WHAT ARE
THEY.
I THINK THIS IS AN EMERGING --
IT'S A PROJECT THAT IS ONGOING,
AND SO MIKE TOOK A TOTAL
METABALOME ANALYSIS, LOOKED AT
ALL THE ME TAB LOAN IN THESE
CELLS, AND SURPRISINGLY WHEN HE
LOOKED AT IT THE THE IPT
IMMEDIATES WERE ACTUALLY
UPREGULATED IN THESE CELLS, ALL
UP IN THESE CELLS.
BY CONTRAST, NONE OF THE TCA
CYCLE INTERMEDIATES WERE
ACTUALLY CHANGING IN THESE
CELLS.
SO THIS TOLD US THERE IS SOME
CONNECTION BETWEEN GLYCOLYSIS
AND THE AMINO ACID TRANSFORMER.
WE DON'T KNOW EXACTLY HOW IT
WORKS BUT WE'RE BEGINNING TO
UNDERSTAND IT.
SO THIS IS ALL IN CELL CULTURE.
SO SINCE WE HAVE HUGE AMOUNTS,
THIS IS DRIVEN BY CULTURE
CONDITIONS, SO WHAT MIKE DID WAS
HE USED THE SAME CCF7, GENERATED
XENOGRAPH TUMORS IN THE MAMMARY
FAT PAD AND THEN ANALYZED THE
METABALOME OF THOSE CELLS, AND
WHAT HE FOUND IS EXACTLY THE
SAME THING.
-- GO UP BUT NOT THE TCS CYCLE.
SO -- OR TAKING UP MORE GLUCOSE,
AND WE DON'T KNOW WHAT THAT IS.
TO TEST IT, MIKE DID A SERIES OF
EXPERIMENTS.
HE MEASURED THE INTRACELLULAR
LEVELS OF GLUCOSE BY USING A
LABELED GLUCOSE, IN THIS CASE
2DG, AND ASKED WHETHER THE CELLS
HAVE A DEFINITE ABILITY TO TAKE
UP MORE GLUCOSE AND THE ANSWER
IS YES.
SO BOTH THE OVEREXPRESSING CELLS
AND THE SPONTANEOUS RESISTANT
MODEL CAN TAKE UP MORE GLUCOSE.
WE ALSO KNOW THE
GLUT1 TRANSPORTERS ALSO GO UP SO
THERE IS A BIOLOGY HERE
EMERGING, IT NOW CONNECTED TO
GLYCOLYSIS.
SO THE WAY THE CELL USES THESE
INTERMEDIATES IS REALLY TO
CHANGE THE BIOENERGETICS OF
THESE CELLS, SO WE BEGAN TESTING
TO SEE IS THERE A REAL
DIFFERENCE BETWEEN THE CONTROL
AND OVEREXPRESSING CELLS.
WE PLEASURED USING SEAHORSE
ANALYSIS.
BOTH OCR, OXYGEN CONSUMPTION
RATE -- ARE INCREASED IN THESE
CELLS.
I CAN ANSWER QUESTIONS ABOUT WHY
THIS IS THE CASE BECAUSE YOU
WOULD EXPECT ECAR LEVELS TO GO
UP WITH GLYCOLYSIS BUT NOT
CONTROL LEVELS.
YOU CAN MAP THESE TWO VALUES AND
CREATE AN ENERGY MAP AND WHAT WE
NOTE IS WHEN WE CREATE AN ENERGY
MAP, THE 7FA WORKS
OVEREXPRESSING CELLS EXIST IN A
HIGH ENERGETIC STATE, WHEREAS
THE CONTROL WERE IN A LOWER AND
ENERGETIC -- AND THE WAY THEY
CAN RESIST TREATMENT.
SO WHAT MIKE DID WAS TO REALLY
TAKE THESE SOLES, AND THEN
MEASURE OXYGEN CONSUMPTION RATE
TO SEE DOES THE TREATMENT DO
ANYTHING TO THESE CELLS.
IT TURNS OUT THE RESULTS WERE
COUNTERINTUITIVE AND SURPRISING.
THEY ACTUALLY GO TO EVEN HIGHER
ENERGY STATE, AS IF THEY ARE
REVVING UP THEIR SYSTEM TO RUN
AWAY.
SO THIS REALLY PUTS US IN A VERY
CLEAR PATH.
MAYBE THE ENERGY STATE OF THE
CELL IS REALLY -- IF THAT'S THE
CASE, DO YOU NOW RESTORE THE
BIOLOGY.
TO DO THAT YOU CAN INHIBIT
GLUCOSE UPDATE BY USING A 2DG
GLUCOSE TO BLOCK GLYCOLYSIS.
WHEN YOU TREAT THESE CELLS WITH
2GD YOU SHUT DOWN THE ECAR AND
THE OCR BIG TIME AND YOU CREATE
AN ENERGY MAP.
UNDER THESE CONDITIONS,
RESISTANCE IS SIGNIFICANTLY
IMPAIRED.
SO WE KNOW WE CAN TOGGLE THESE
THINGS.
NOW THE BIG CONNECTION FOR US WE
NEED TO UNDERSTAND IS, HOW DOES
AP AMINO ACID TRANSPORTER,
CONNECT AND REINQUIRE THE
GLYCOLYSIS IN THOSE THEES CELLS
AND WE ARE NOW ACTIVELY WORKING
TO UNDERSTAND THIS BIOLOGY.
SO THESE STUDIES WITH LGL AND
7A5 HAS REALLY PUT NUSS A PATH
WITH HOW WE CAN STUDY POLARITY
PROTEINS IN THE CONTEXT OF
CELL -- AND POTENTIALLY EVEN THE
CELL SURFACE ONLY, AND ALL THESE
COMBINED CAN HAVE AN IMPACT IN
DRUG RESISTANCE, METASTASES AND
THE WAY THE CELL SEES THE
MICROENVIRONMENT.
THESE ARE SOME OF THE STUDIES
AND PROJECTS THAT WE ARE
CURRENTLY INITIATING IN THE LAB
HERE AT LCBG.
SO IN SUMMARY, AS I MENTIONED,
THE RESEARCH PROGRAM IN OUR LAB
REALLY IS AN INTEGRATION BETWEEN
BASIC AND TRANSLATION RESEARCH,
WE GO BACK AND FORTH.
AND THE CELL POLARITY STUDIES
REALLY UNDERSTANDING SIGN SIGNAG
SUR FA SOME IN ORGANOIDS, USING
THEM FOR EITHER MODELING
CLINICAL RESPONSE OR
UNDERSTANDING -- FINDING THERAPY
RESISTANCE IN THE CLINIC.
SO ALL OF THIS CANNOT BE DONE
WITHOUT POWERFUL COLLABORATIONS
AND THE RESEARCH GREUN.
SO THESE ARE THE COLLABORATORS
WHO HELPED US IN DIFFERENT PARTS
OF THE PROJECT IN THE PAST.
SO ALL THE ORGANOID WORK THAT
I'VE PRESENTED TODAY WAS DONE
BEFORE I GOT HERE.
IT'S FROM BIDMC HEAD AND NECK
AND ONCOLOGY WITH HELP FROM
MANUEL HIDALGO.
CELL PRO LA POLARITY AND DRUG
RESISTANCE WITH MARCIA HAGIS,
MYLES BROWN AND FORREST WHITE.
THE MEMBERS.
NEW TEAM ARE LISTED THERE, AND
MIKE IS STILL IN BOSTON FOR
FAMILY REASONS AND CONTINUING
THE PROJECT, AND WE HAVE NOW
INITIATED A STRONG COLLABORATION
HERE AND GETTING SAMPLES TO
GENERATE ORGANOID MODELS BOTH
FOR THE BIOBANK APPROACH AND FOR
THE TCR DISCOVERY STUDIES.
AND THE MEMBERS OF THE WORK I
PRESENTED, I MENTIONED THEM
ALONG THE WAY.
AND I CAN STOP HERE, AND BEFORE
I TAKE ANY QUESTIONS I WANT TO
THANK ALL OF YOU FOR BEING HERE
BECAUSE IT IS AMAZING TO TALK TO
LIVE AUDIENCE FACE-TO-FACE THAN
SEEING A MONITOR.
SO THANK YOU FOR MAKING THE
EFFORT OF BEING HERE.
[APPLAUSE]
>>WE'D LIKE TO OPEN IT UP TO
QUESTIONS AT THIS PARTICULAR
POINT IN TIME.
WHILE YOU'RE GATHERING YOUR
THOUGHTS ON THAT, YOU TALKED
ABOUT -- JUST ABOUT EVERYTHING
THAT YOU COULD DO WITH ORGANO
ORGANOIDS, BUT YOU DIDN'T
MENTION ANYTHING ABOUT
EPIGENETICS.
SO ARE YOU DOING ANYTHING OR
THINKING ABOUT WORKING IN THAT
AREA AT ALL?
>>NO, BECAUSE WE'RE NOT GOOD AT
IT.
SO BUT WE ARE HAPPY TO HELP
ANYBODY WHO WANTS TO DO.
>>OKAY.
>>HI.
THIS IS -- FROM SURGERY BRANCH.
THE PAST FEW YEARS WE'VE BEEN
GROWING FROM OUR RESECTIONS AND
WE'VE BEEN TESTING THEM ALONG
OUR NEOANTIGEN PIPELINE, RIGHT,
TO FIND THE REACTIVITIES.
THE CHALLENGE WE'RE CURRENTLY
FACING IS FINDING MANY
REACTIVITIES AGAINST ORGANOID
THAT ARE NOT NEW ANTIGENS, SO
THE TARGETS ARE UNKNOWN.
HAVE YOU WORKED ON WHAT --
IDENTIFYING THOSE TUMOR TARGETS
THAT THESE TCRs ARE SEEING?
>>THAT'S A MILLION DOLLAR
QUESTION BECAUSE IT IS SUCH A
DIFFICULT TASK, BUT WE HAVE
INITIATED A COLLABORATION USING
ONE OF THE TCRs, SO HE HAS A
DISCOVERY PIPELINE THAT HAS THE
ENTIRE CODED GENOME PRESENTED ON
THE SURFACE AS SHORT PEPTIDES.
IN A CELL-BASED ASSAY, YOU CAN
ENGINEER THESE TCR IN A T WELL
AND USE IT AS BAIT TO FIND OUT
WHAT IT BINDS TO, KEEP IN MIND
THESE ARE JUST PEPTIDES
PRESENTED.
IF THE PEPTIDE IS MODIFIED IN
ANY FORM OR SHAPE, THAT IS NOT
REPRESENTED HERE.
RIGHT?
SO WE CAN ALSO TAKE THE TCRs
AND DO COMPUTATIONALLY ASK WHAT
MAY BE THE LIKELY TARGET.
SO WE DID BOTH.
SO FOR ONE OF THE TCRs.
THE COMPUTATIONAL APPROACH GAVE
A PEPTIDE FOR AN ANTIGEN THAT
WAS A COMMON ONCOGENE, TO SEE IF
IT WOULD STIMULATE AND IT HAD NO
EFFECT.
THEN THROUGH STEVE'S GROUP HE
FOUND A PEPTIDE THAT IS
TRANSCRIBED IN THE REVERSE
DIRECTION OF A CODING GENE.
SO IT IS -- IT'S COMPLETELY
COUNTERINTUITIVE WHAT YOU WOULD
THINK ABOUT.
THIS PEPTIDE YOU WOULDN'T EVEN
THINK THAT WOULD EXIST BECAUSE
IT'S A SHORT PEPTIDE IN THE
REVERSE DIRECTION OF A GENE.
SO WHETHER THAT IS THE GENE OR
NOT, THE PEPTIDE OR NOT, WE
DON'T KNOW, BUT THAT WAS THE
STRONGEST HIT AND THE ONLY HIT
HE COULD FIND.
SO IT ONLY TELLS US THAT YOU
HAVE TO TAKE COMPLETELY UNBIASED
APPROACH TO GO AFTER THEM AND
EACH TCR WOULD BE A TASK ON ITS
OWN.
SO I DON'T HAVE AN ANSWER BUT I
THINK IT IS FEASIBLE.
>>ALL RIGHT.
THE OTHER THING WE'VE BEEN DOING
IS WHATEVER THE HARVEST SITE IS,
SAY IF WE TAKE COLORECTAL
METASTATIC TO THE LUNG, WE TRY
TO GROW THE NORMAL LUNG AS AN
ORGANOID AND USE AS A CONTROL.
DO YOU HAVE ANY EXPERIENCE IN
USING NORMAL CONTROLS OR DO YOU
THINK THEY'RE USELESS?
>>NO, I THINK FOR THE
CO-CULTURE NORMAL CONTROLS ARE
VERY POWERFUL.
SO WE HAVE DONE THEM, NOT FOR
ALL THE CANCERS.
WE'VE ONLY DONE THEM FOR BREAST
AND FOR -- IN THE MOUSE CONTEXT,
AND IN THE HUMAN CONTEXT, WE
HAVE SOME KIDNEY CANCERS.
AND WE CAN SEE SELECTIVITY
BETWEEN THE TUMOR -- IF YOU
SELECT THEM WITH THE TUMOR, IT
DOESN'T KILL THE --
>>CAN I HAVE ONE LAST QUESTION?
SORRY.
FOR THE TCRs WE KNOW ARE
NEOANTIGEN REACTIVE -- WE TRIED
KILLING ASSAYS BUT WE ALSO
TRY -- SO WE ALSO TRY TO
REPLICATE THAT IN A PDX MODEL.
ONE ISSUE WE HAD IS IMROAING
THOSE ORGANOIDS IN A MOUSE AS A
PDX MODEL, THEY HAVE BEEN
FAILING.
I DON'T KNOW IF YOU'VE
EXPERIENCED THAT, IF YOU HAVE
ANY ADVICE.
THE OTHER THING IS, DO YOU THINK
IT'S NECESSARY TO TEST IN A
MOUSE OR IN VITRO ASSAY --
>>I WOULDN'T TOUCH THE SECOND
QUESTION BECAUSE THAT'S ALMOST
LIKE AN FDA QUESTION SO LET'S
NOT TALK ABOUT THAT.
>>RIGHT.
>>FOR THE FIRST ONE, YES, IT
USUALLY WORKS, WE CAN TALK MORE,
WE CAN DISCUSS MORE.
BUT THERE IS PROBABLY A
DIFFERENCE BETWEEN THE WAY YOU
ENRICH THE T-CELLS AND THE WAY
WE DO IT.
SO WE CAN TALK MORE.
>>ALL RIGHT.
THANK YOU.
>>MORE DETAILS.
>>I WAS JUST WONDERING --
HETEROJE NATE DO YOU THINK YOU
OBTAIN IN YOUR CULTURES?
>>VERY LITTLE BECAUSE YOU'RE
GETTING A BIOPSY IN THE FIRST
PLACE OR A SMALL SECTION OF THE
TUMOR.
SO WHATEVER YOU GET BY ITSELF IS
NOT REPRESENTING THE ENTIRE
TUMOR.
WHAT YOU GET ALSO, THERE IS A
LOT OF ATTRITION IN THE CULTURE
AND WHATEVER GROWS, GROWS.
SO WE WILL NOT BE ABLE TO ANSWER
THAT QUESTION IN ANY CLEAR WAY
BECAUSE IT IS IMPOSSIBLE TO
ANSWER THAT QUESTION.
>>DO YOU FIND IT THEN
SURPRISING THAT YOU GET SUCH A
GOOD CORRELATION BETWEEN DRUG
RESPONSES IN THE ORGANOIDS AND
WHAT WAS SEEN IN THE CLINICAL
SETTING?
>>SO YES AND NO.
IT IS SURPRISING THAT YOU CAN
SEE, BUT PERHAPS THESE ARE --
KEEP IN MIND THE DRUGS WE TESTED
ARE ALL CHEMOTHERAPY DRUGS.
SO IF THEY ARE SENSITIVITIES
DICTATED BY SOME COMMON ELEMENTS
THAT ARE SHAPED BY MULTIPLE
CELLS, MAYBE A TRUNCAL MUTATION
OF SOME SORT, THEN MAYBE THAT'S
WHAT WE ARE LOOKING AT.
THERE MAY BE SOME INTRATUMOR
DIFFERENCES IN THE WAY THEY
WOULD RESPOND WHEN YOU GIVE IT
TO THE PATIENT AND THAT IS
LIKELY TO BE THE CASE.
BUT I THINK IT IS IMPOSSIBLE TO
REPRESENT THE ORGANOID OR A --
ANY MODEL FOR THAT MATTER TO
CLOSELY MIMIC THE PATIENT
TUMOR -- ENTIRE HETEROGENEITY SO
THAT'S A REALITY AND WE HAVE TO
WORK WITH WHAT WE HAVE.
>>OKAY.
ANOTHER ONE?
>>HI.
AMAZING JOB.
I HAVE A QUESTION.
WHEN I THINK ABOUT POLARITY, I
ONLY THINK ABOUT CELL
CYTOSKELETON.
DID YOU HAVE ANY EVIDENCE BY
MODIFYING THIS POLARITY
PROTEIN -- IF YOU CHANGE THE
MORPHOLOGY OF THE CELLS LIKE
EVEN THE -- HOW THE CELLS MOVE?
>>SO IN THE CONTEXT OF LGL2, WE
DID A SERIES OF EXPERIMENTS TO
SEE IF CELL POLARITY IS ALTERED,
AND WHEN WE OVEREXPRESSED LGL2,
WE DO NOT SEE A MAJOR CHANGE.
BECAUSE THERE ARE COMPENSATORY
MECHANISMS THAT KICK IN.
AND YOU DON'T SEE CHANGES IN
CELL-CELL JUNCTIONS OR
COMPOSITION OF THE --
CYTOSKELETON.
IF YOU KNOCK DOWN, THERE IS A
CHANCE YOU CHANGE YOU WILL SEE E
MORPHOLOGY, BUT IF YOU LOOK
AT -- JUNCTIONS AND SO FORTH,
IT'S NOT DRAMA IK IT ENOUGH.
THE GROWTH RATES ARE THE SAME,
THE STRUCTURE IS MORE OR LESS
CONSERVED.
IN PART THAT'S BECAUSE POLARITY
PROTEINS HAVE A HUGE -- PAUSE
YOU HAVE TO MAINTAIN THE
STRUCTURE AT ALL COSTS.
SO IT'S RARELY THAT IN A
MAMMALIAN CELL, YOU RUN A
DOMINANT PHENOTYPE -- YOU HAVE
TO TAKE OUT A CLASS, FOR
EXAMPLE.
>>ANY OTHER QUESTIONS?
OKAY.
>>THANK YOU.
[APPLAUSE]