Extracellular vesicles released by glioblastoma cells: saboteurs, biomarkers and therapeutics


>>GOOD AFTERNOON, EVERYONE. WELCOME TO THE WEDNESDAY AFTERNOON LECTURE. TODAY TO BE PRESENTED BY A FRIEND OF MINE, WHO I HAVE KNOWN FOR MORE YEARS THAN I WILL PROBABLY WANT TO DISCLOSE THINK I MET DR. BREAKEFIELD WHEN I WAS A POSTDOC AND HAD A WONDERFUL OPPORTUNITY TO LEARN FROM HER THEN AND HAVE CONTINUED TO SINCE THAT TIME IN THE FIELD THAT SHE’S BEEN WORKING ON, WHICH IS IN THE AREA OF NEUROSCIENCE, AND AS YOU WILL HEAR TODAY A PARTICULAR ASPECT OF THAT AN EXPLORATION OF HOW EXTRACELLULAR VESICLES RELEASEDDED BY GLIOBLASTOMA CELLS CAN PLAY AN IMPORTANT ROLE IN UNDERSTANDING BIOLOGY AND PERHAPS IN A THERAPEUTIC WAY. SHE’S HERE BECAUSE OF HER ROLE IN THE COMMON FUND EXTRACELLULAR RNA COMMUNICATION PROGRAM, ONE OF THOSE BOLD INITIATIVES THE COMMON FUND SUPPORTS AND THAT XANDRA PLAYED AN IMPORTANT ROLE IN. HER CURRICULUM IS BEGINNING WITH AN UNDERGRADUATE DEGREE AT WILSON COLLEGE, GOING ON TO GET A Ph.D. RIGHT AROUND THE CORNER AT GEORGETOWN UNIVERSITY, AND THEN SHE CAME HERE, THE NIH, IN THE EARLY ’70s WHERE SHE WAS A POSTDOC WITH NONE OTHER THAN MARSHAL NIRENBERG AND CONTINUED AS STAFF FELLOW FOR ANOTHER YEAR AND WENT TO THE IVY LEAGUE, INITIALLY AT YALE, WHICH IS WHERE I ENCOUNTERED HER IN THE HUMAN GENETICS DEPARTMENT, AND THEN IN 1984 MOVED FROM YALE TO ANOTHER IVY LEAGUE PLACE YOU’VE HEARD OF CALLED HARVARD WHERE SHE CURRENTLY IS PROFESSOR OF NEUROLOGY AND ALSO IS GENETICIST AT MASS GENERAL HOSPITAL, AN ELECTED MEMBER OF THE AMERICAN ACADEMY OF ARTS AND SCIENCES, SHE WON A LIFETIME ACHIEVEMENT AWARD FROM THE SOCIETY FOR NEUROSCIENCE, AND A SPECIAL ACHIEVEMENT AWARD RELEVANT TO TODAY’S PRESENTATION FROM THE INTERNATIONAL SOCIETY FOR EXTRACELLULAR VESICLES, ISEV, IN 2015. HER RESEARCH HAS FOCUSED ON THE NERVOUS SYSTEM BUT IN A WIDE VARIETY OF INTERESTING AND INNOVATIVE WAYS, AND AS I MENTIONED SHE’S HERE TODAY TO TALK TO YOU ABOUT ONE OF THOSE, NAMELY WHAT CAN WE LEARN FROM EXTRACELLULAR VESICLES ABOUT GLIOBLASTOMA IN TERMS OF WHAT THEY ARE UP TO, INTERESTING SUBTITLE, SABOTEUR OF BIOMARKERS AND THERAPEUTICS JOIN ME IN WELCOMING TO THE PODIUM PROFESSOR XANDRA BREAKFIELD. [APPLAUSE]>>I CAN BARELY SEE YOU ABOVE THE PODIUM. USUALLY THEY BRING A BOX FOR ME TO STAND ON. I MIGHT HAVE TO VEER THIS WAY. IF YOU CAN’T HEAR ME, I DON’T THINK YOU CAN SEE ME — YOU LEFT OUT AFTER I GRADUATED FROM WILSON COLLEGE I CAME HERE AND WORKED AS A TECHNICIAN FOR TWO YEARS WITH ANDY LEWIS IN VIROLOGY, AND I WOULD LIKE TO ADD I WAS UPSTAIRS, IT WAS THE LAB AT YALE AND YOU WERE DOWNSTAIRS AND CAUGHT A COURSE IN MOLECULAR BIOLOGY, BUT AT THE TIME I DIDN’T KNOW ANYTHING, I WAS A SINGLE PARENT AND DIDN’T HAVE TIME TO GO. I HAVE ALWAYS REGRETTED THAT. I COULD HAVE GONE ON FASTER, I DID GO INTO MOLECULAR BIOLOGY EVENTUALLY. TODAY WE’LL TRY TO GIVE YOU DIFFERENT ASPECTS, IF WE RUN OUT OF TIME YOU MAY CARE LESS ABOUT THERAPEUTICS. THE MAJOR FOCUS IS SABOTEURS, PASSED ON BIOMARKERS TO A LOT OF PEOPLE, THE VESICLES MAKE FANTASTIC BIOMARKERS. THIS IS MY DISCLOSURE STATEMENT. I GOT INTO IT BY ACCIDENT, WORKING ON VIRUS VECTORS, TOXICITIES, MY OFFICE WAS NEXT TO A NEUROSURGEON, I WAS TELLING HIM HOW FRUSTRATING IT WAS, GETTING TOXICITY WHEN TRYING TO CURE THE BRAIN. WITH GLIOBLASTOMA, WHEN WE TAKE THE TUMOR OUT, WE TAKE OUT NORMAL TISSUE SO A LITTLE TOXICITY WE CAN LIVE WITH. WE STARTED WORKING TOGETHER ON ONCOLYTIC HERPES VIRUSES BUT THAT’S HOW I GOT STARTED WORKING ON IT. I WOULD SAY IN THE TIME I’VE BEEN WORKING ON IT, THERAPY FOR THIS DISEASE HAS NOT CHANGED, NOR HAS PROGNOSIS. THIS IS REALLY ONE OF YOUR MORE DISMAL CANCERS, SOMETIMES I WONDER WHY WE KEEP WORKING ON IT BECAUSE NOTHING HAS CHANGED IN 20 YEARS, BASICALLY I THINK THEY HAVE GONE FROM A LIFESPAN OF 12 MONTHS FROM THE TIME OF DIAGNOSIS TO 15 MONTHS IN THAT TIME. WE HAVE — I WOULD SAY IT’S BEEN DIFFICULT TO IMPROVE THERAPEUTIC INTERVENTION, FOR A LOT OF REASONS. WHICH I DON’T NEED TO GO INTO. THAT WAS ONE OF THE THINGS THAT DROVE US INTO THE BIOMARKER FIELD TO TRY TO TRACK THE TUMORS WITHOUT HAVING TO GO BACK INTO THE BRAIN. THE TUMORS ARE VERY HETEROGENEOUS WITH CANCER STEM CELLS, EVEN THOUGH THERE HAVE BEEN FOUR TRANSCRIPTIONAL CLASSES WHEN THEY DID SINGLE CELL RNA SEQUENCING ON TUMORS FROM INDIVIDUAL HUMANS THEY FIND EVERYTHING IS IN THERE. THEY ARE VERY, VERY GENETICALLY HETEROGENEOUS. THESE ARE PICTURES, THIS IS THE TYPICAL PICTURE THEY SHOW YOU, A TENNIS BALL IN YOUR BRAIN, BUT HERE YOU CAN SEE THE VERY ACTIVE METABOLISM, AND VERY ACTIVE DNA SYNTHESIS. AND YOU THINK, OH, HERE IT IS, AND WE’LL JUST TAKE IT OUT, BUT REALLY ANOTHER FEATURE OF THESE TUMORS IS THEY SEND INVASIVE CELLS JUST THROUGHOUT THE BRAIN SO AS SOON AS YOU TAKE THIS GUY OFF, ANOTHER GUY POPS UP HERE. SO WE WERE STUDYING GLIOBLASTOMA IN THE LAB, BASIC RESEARCHERS, AND WE HAD BEEN USING DIFFERENT MODELS IN MOUSE MODELS, BUT THE TUMORS THAT GROW IN MOUSE MODELS, TYPICAL MOUSE, THEY ARE NOT INVASIVE. YOU KNOW, THEY ARE NOT VERY HETEROGENEOUS, DON’T HAVE MUTATIONS AS THE HUMANS HAVE, SO I SAID TO MY POSTDOC AT THE TIME, WE NEED TO GET FRESH TISSUE FROM THESE GLIOBLASTOMA PATIENTS AND GET IT IN THE LAB AND LET’S SEE WHAT WE FIND OUT. AND I’VE TOLD THE STORY A COUPLE TIMES TODAY, BUT INITIALLY WE’RE PhDs, WE DO WORK RIGHT NEXT TO A HOSPITAL, BUT WE ASKED COULD WE GET SOME FRESH GLIOBLASTOMA TISSUE? AND THE FIRST TWO PEOPLE WE TALKED TO SAID THERE’S NO WAY. YOU KNOW, WE COULD GET IT TO YOU WITHIN A FEW HOURS, NO, WE WANT IT RIGHT AWAY. BOB CARTER HAS COME BACK TO BE CHIEF OF NEUROSURGERY I. HE SAID NO PROBLEM, WE’LL SCRUB THEM IN. WE STARTED GETTING FRESH GLIOBLASTOMA BRAIN TUMOR TISSUE, PUT IN CULTURE, AFTER THE THIRD TIME JOHAN SAID THE CELLS ARE VERY WEIRD. AND YOU CAN SEE ALL THESE PROTRUSIONS FROM THE SURFACE, AND WE WERE LIKE, ARE THEY RELEASING THESE THINGS INTO THE MEDIA AND SURE ENOUGH YOU SEE THEY RELEASE DIFFERENT SIZE VESICLES, DOWN TO 50 NANOMETERS UP TO TUMORS RELEASE A REALLY BROAD RANGE OF SIZE VESICLES. AND SO WE FOUND THERE WAS LITERATURE, A GREAT DEBATE BETWEEN WHETHER THESE WERE VESICLES, WERE CELLULAR DEBRIS IN CELL CULTURE AND TRIED TO FIGURE OUT HOW MANY WERE RELEASED PER DAY, IT WAS PRETTY RAPID. THEN BECAUSE WE’RE GENETICISTS, WE LOOKED AT RNA, THEY DO CONTAIN RNA. MOST UNDER 200 NUCLEOTIDES. YOU SEE RIBOSOMAL PEAKS, WE KNEW WE HAD A SOURCE OF RNAs FROM TUMORS BUT WONDERED, WELL, LET ME TALK ABOUT THESE VESICLES. THIS IS FROM LATER WORK. KIND OF REVIEWED BY OTHER PEOPLE. IN ADDITION TO WHAT WE SAW, WHICH WERE RNAs, WHICH THE MESSENGER RNA IS THE microRNA, WE ALSO FOUND SOME DNA, THERE’S A LOT OF DIFFERENT KINDS OF PROTEIN INVOLVD IN ANTIGEN PRESERVATION — PRESENTATION, THERE’S ENZYMES, THERE’S TRANSCRIPTION FACTORS, A WHOLE BODY LOAD OF STUFF INTO WHICH THERE’S A LITTLE RNA. AND PROBABLY I THINK YOU HAVE A LOT OF LECTURES ON VESICLES, I DON’T THINK I NEED TO GO INTO IT MUCH BUT THERE’S AT LEAST TWO ROUTES OF PRODUCTION, ONE IS THROUGH MULTI-VESICULAR BODIES THAT FUSE WITH THE PLASMA MEMBRANE, AND DUMP THEM OUT, EXOSOMES, AND LIKE RETROVIRUS, CALLED MICRO VESICLES, THEY CAN PRESENT ANTIGENS, STIMULATE CELL SIGNALING FROM THE OUTSIDE. THEY CAN ENTER AND CONTENTS CAN BE ACTIVE IN CELLS, EVEN MESSENGER RNA, TYPICAL SMALL ONES, MICRORNAs CAN BE ACTIVE, ENDOCYTOSIS, AND CAN STIMULATE MHC MOLECULES. IT’S LIKE TAKING A LITTLE BIOPSY OF THE CELL. YOU HAVE A LOT OF COMPONENTS OF THE CELL THERE, AND TRYING TO FACTOR OUT WHAT’S DOING WHAT IS ONE OF THE ISSUES IN THE FIELD. JUST SUFFICE IT TO SAY HERE EARLY ON IT WAS RECOGNIZED THAT THIS COULD BE A NEW FORM OF INTRACELLULAR COMMUNICATION BECAUSE YOU WOULD BE ABLE TO TRANSFER THESE RNAs AND DNA AND PROTEINS, EVEN MEMBRANE PROTEINS NOT TYPICALLY IN THE SECRETOME OF THE CELL, NOT NORMALLY SECRETED, BUT YOU CAN TRANSFER OVER THESE PACKAGES. AND PEOPLE HAVE SHOWN THE PROTEINS ON THE SURFACE CAN BE ACTIVE IN SIGNALING. THERE’S PROTEINS, TRANSCRIPTION FACTORS THAT CAN TURN ON GENES. RNAs CAN MAKE PROTEINS. RNAs CAN POTENTIALLY BECOME DOUBLE STRAND AND INSERT INTO THE GENOME. JUST IT’S ALMOST LIKE ANYTHING COULD HAPPEN, OR WHAT WAS THAT WHACKY WEDNESDAY OR WHATEVER. SO IT’S ALMOST — EVERYBODY GOT EXCITED BECAUSE OF ALL THE POTENTIAL THERE WAS HERE, BUT IT’S — WE’RE STILL IN THE PROCESS OF TRYING TO DECIPHER. I LOOK AT IT LIKE THIS IS A NEW LANGUAGE OF COMMUNICATION BETWEEN CELLS. THE WORDS, A LOT OF COMPONENTS I TALKED ABOUT, BUT THESE COMPONENTS MAY ACT IN CONCERT IN WAYS THAT YOU MAY NOT ANTICIPATE. AND IT’S ALWAYS COMMUNICATED AS A MASS. OKAY? PEOPLE — YOU ALWAYS SEE THE PEOPLE, OH, WE DO A LITTLE TOO, THERE’S MIR 21 IN THE VESICLES, KNOCKS DOWN MESSAGES IN THE CELLS, THAT’S WHAT THE EXOSOMES ARE DOING BUT IN FACT IT’S A COLLECTIVE PASS. WHATEVER IS HAPPENING IS HAPPENING THROUGH A LOT OF PROTEINS, RNAs, ET CETERA, INTERACTION TOGETHER. AND THIS IS AN EVEN HARDER THING TO STUDY, ITS BIODIRECTIONAL, ALL CELLS ARE RELEASING THEM AND TAKING THEM UP. IT’S NOT A SIMPLE ONE-TO-ONE ERROR GOING OUT. THERE’S VESICLES, PARTICLES, SOME ARE PROBABLY JUNK MAIL. AND DIFFERENT VESICLES AND CARGOES PROBABLY HAVE DIFFERENT MESSAGES. ANOTHER ISSUE WE’RE STILL TRYING TO FIGURE OUT IS WHO TALKS TO WHO, IF THIS IS REALLY A COMMUNICATION SYSTEM IN THE BODY, WHICH CELLS ARE TRYING TO TELL WHICH CELLS TO DO WHAT. SO BACK TO BRAIN TUMORS, I’VE BEEN INTERESTED IN THE POTENTIAL FOR CELLULAR COMMUNICATION AND CONTEXT WITH BRAIN TUMOR. THAT’S WHERE I COME INTO SABOTEURS, BECAUSE THEY ARE NOT IN THERE TO BE NICE GUYS, FOR SURE. YOUR TUMOR DEVELOPS AND THERE’S A LOT OF THINGS THAT HAVE BEEN ASSOCIATED WITH VESICLES RELEASED BY TUMOR. IMMUNE SUPPRESSION, STIMULATE OF MIGRATION AND INVASION, EPIGENETIC CHANGES, ANGIOGENESIS, ET CETERA. SO WE’RE INTERESTED IN BRAIN TUMORS. WE’RE TRYING TO FIGURE OUT WHY THEY HAVE SUCH AN ADVANTAGE, WHAT ARE THEIR ACHILLES HEELS. WE WANTED TO TRY TO UNDERSTAND THIS BETTER. ONE OF THE THINGS WE DID WAS TO LABEL THE CELLULAR MEMBRANES. OTHER PEOPLE HAVE DONE THIS. IT’S NOT UNIQUE WITH US. WITH A PALMITOLYATION SIGNAL. YOU CAN SEE IT LABELS VESICLES DISTINCTLY SO THIS GIVES US A WAY TO FOLLOW THEM. OH, WHAT HAPPENED? I DON’T THINK I DID ANYTHING WRONG BUT I HAVE A FRIEND BACK THERE WHO IS GOING TO HELP ME. SO USING THESE LABELED CELLS IN CULTURE YOU CAN SEE THESE USED TO BE MOVIES, I GOT SO FRUSTRATED SHOWING MOVIES. ANYWAY YOU WOULD SEE HERE IS THE CELL, FORMS THESE VESICLES, THESE ARE TUMOR CELLS THAT GET RELEASED. YOU CAN ALMOST SEE IT LIKE IN REAL TIME. SEEMS LIKE IT WOULD BE ENERGETICALLY PROHIBITIVE BUT THEY SEEM TO KEEP RELEASING THEM. THIS IS WITH MICROSCOPY, ANOTHER TUMOR THAT’S LABELED, IN VIVO YOU SEE THESE VESICLES JUST BUDDING OFF AND BEING RELEASED INTO THE ENVIRONMENT. AND THE PERSON THAT DID THIS INTRAVITAL MICROSCOPY IS THORSTEN MEMPEL. HERE THE TUMOR CELLS ARE LABELED WITH RED FLUORESCENT PROTEIN. MYELOID SAYS, MICRO GLIA AND MACROPHAGES ARE GREEN. YOU CAN SEE IN REAL TIME VESICLES GET RELEASE AND TAKEN UP BY MYELOID CELLS. SO THESE ARE RELEASED IN VIVO, TAKEN UP BY SURROUNDING CELLS. NOW, WE’LL GET BACK TO WHAT CONSTITUTES A TUMOR AND HOW DOES IT SUSTAIN ITSELF IN THE BRAIN. SO WE KNOW THAT IT BASICALLY MOLDS ITS MICROENVIRONMENT. IT’S GOING TO TAKE OVER THE BRAIN LITERALLY. IT DOES THIS BY SECRETING CYTOKINES. IT DOES IT BY SIGNALING THROUGH, YOU KNOW, PROTEINS AND RECEPTORS ON THE CELL SURFACE. AND YOU KNOW, DOES IT DO ALSO BY EXTRACELLULAR VESICLES RELEASED AND TAKEN UP BY NORMAL CELLS? SO IN THIS ENVIRONMENT YOU THINK OF ALL THE NORMAL CONSTITUENTS OF THE BRAIN, LIKE NEURONS. BUT REALLY IF YOU ACTUALLY LOOK AT THE BRAIN TUMOR ITSELF, MOST OF IT CONSISTS OF MICRO GLIA AND MACROPHAGES, REACTING TO PRESENCE OF TUMOR IN A BIG WAY. THEY ARE ATTRACT THE TO THE TUMOR, AND THE TUMOR IS GOING TO SUBJUGATE THEM IS A GOOD WAY TO PUT IT. SO WE DECIDED INITIALLY AGAIN TAKING THE PRIMITIVE APPROACH BECAUSE THAT WAS THE WAY WE WERE LOOKING AT IT, YOU KNOW, IT WAS microRNAs, WHAT ARE THE HIGHEST UPREGULATED microRNAs IN GLIOBLASTOMA AND CAN THEY BE TRANSFERRED TO THESE MICROGLIA, IN THIS CASE WE PICKED MICROGLIA BECAUSE THEY WERE EASIER TO GROW THE MACROPHAGES, NO GREAT INSIGHT THERE. GLIOBLASTOMAS TYPICALLY HAVE HIGH LEVELS OF MIR 21, AND IT’S VERY HIGH IN THE VESICLES. WE THOUGHT THIS MIR-451 WAS OUR GUY. WE WERE TRACKING IT BUT THAT’S AN ARTIFACT OF SERUM. THIS IS A — THERE’S NO DIFFERENCE IN THE SEQUENCE BETWEEN HUMAN AND BOVINE 451, BUT EVEN IF YOU HAVE WHAT THEY CALL VESICLE DEPLETED SERUM, YOU HAVE A LOT, A TON OF THIS MIR-451 PRESENT. THESE ARE THINGS WE’RE LEARNING ALONG THE WAY. YOU’LL FIND A LOT OF PAPERS ON MIR-451 AND HOW IT HELPS SUPPRESS THINGS IN MICROGLIA BUT IT’S PRESENCE IN THE SERUM OF THE CULTURE ITSELF. WE PICKED MICROGLIA BECAUSE THEY WERE RELATIVELY EASY TO GROW AND THEY WERE MAJOR CONSTITUENT OF THE TUMOR. WE WERE VERY FORTUNATE LATER TO DISCOVER THAT ONE OF OUR COLLEAGUES ON THE FLOOR, JOE AND HIS WIFE SUSAN HICKMAN WERE MICROGLIA EXPERTS. THEY ARE SENTINELS IN THE BRAIN AND SEND OUT PROCESSES, FEELING AROUND, TRYING TO FIGURE OUT WHAT’S GOING ON. HERE SUZANNE DID LASER-INDUCED INJURY, GREEN ARE MICROGLIA. WHERE SHE INTRODUCED THE INJURY THEY ALL JUST RUSHED INTO THAT SPOT. THEY ARE VERY, VERY SENSITIVE TO CHANGES IN THEIR ENVIRONMENT AND IN THE CASE OF INFECTION ARE GOING TO TRY TO GET RID OF THE INFECTION. SO WE SET UP A MODEL SYSTEM INITIALLY, WE KNEW THE GLIOBLASTOMA CELLS HAD HIGH LEVELS OF MIR-21, MICROGLIA HAD LOW LEVELS, VESICLES HAD HIGH LEVELS. WHEN WE LOOKED IN THE MICROGLIA EXPOSED TO THE VESICLES IN CULTURE, WE SAW WE DID RAISE MIR21 LEVELS, CELLS STARTED PROLIFERATING RAPIDLY, TARGET MESSAGES WENT DOWN AND THERE WERE OTHER CHANGES IN THE CELLS. NOW WE PUBLISHED THAT PAPER. EVERYONE SAID SINCE THEN, WELL, IT’S NOT — AGAIN, THE MIR21 YOU CAN’T DISTINGUISH EVEN HUMAN AND HOUSE, THE SAME SEQUENCE, MAYBE THE MIR-21 DIDN’T COME FROM THE EVs, MAYBE YOU CHANGED THE PHYSIOLOGY OF THE CELL. BUT I JUST HEARD TODAY FROM ONE OF MY STUDENTS THAT WE NOW HAVE ACTUALLY DONE THIS IN MICE, I’LL SHOW YOU HOW WE’VE DONE THOSE EXPERIMENTS, KNOCKED OUT FOR MIR21, YES, THE MIR21 LEVELS GO UP, SINCE THE MOUSE IS KNOCKED OUT FOR MIR21 THEY HAVE TO BE COMING FROM TUMOR. BUT THAT STUDY IS STILL A WORK IN PROGRESS. WE SET UP — NICK MAAS, I TALKED TO SOMEONE HERE WHO KNOWS HIM, HE’S FROM THE NETHERLANDS WE DECIDED TO SEE WITHIN THE CONTEXT OF THE BRAIN, WITHIN THE CONTEXT OF A BRAIN TUMOR, DO MICROGLIA WHO HAVE TAKEN UP MORE TUMOR VESICLES HAVE A DIFFERENT PHENOTYPE THAN MICROGLIA THAT HAVE NOT TAKEN UP VERY MANY TUMOR VESICLES? SO THE TUMOR IS LABELED. THIS IS A MOUSE GLIOMA IN A SYNGENEIC MODEL, IMPLANTED IN THE BRAIN, WE WAIT ABOUT A MONTH, SACRIFICE, DO HISTOCHEMISTRY AND SEPARATE BY FACS ANALYSIS. I TOLD STUDENTS THIS IS NOT A GOOD IDEA, IF YOU TRY TO DISASSOCIATE, ALL THE PROCESS IS GOING TO GET SHEARED OFF, EVERYBODY IS GOING TO DIE, THIS ISN’T GOING TO WORK. AS TYPICAL IN MY LAB, THEY DID IT ANYWAY BECAUSE THAT’S WHAT THEY WANTED TO DO. FIRST OF ALL, HERE IS A PICTURE OF TUMOR. THE TUMOR IS LABELED WITH GFP. MACROPHAGES ARE LABELED WITH CCR2, AND THEN IBA1 TO SHOW YOU IN THE TUMOR HOW DENSE MICROGLIA AND MACROPHAGES ARE. PEOPLE HAVE KNOWN THE MORE MICROGLIA AND MICROGLIA, THE WORSE THE PROGNOSIS. THEY ARE CAUSING TROUBLE. THIS IS THE TUMOR-BEARING BRAIN, DIFFERENT CONTROLS. SEPARATED MICROGLIA HAVING HIGH CD11B AND INTERMEDIATE CD45 AND YOU HAVE TO COME DOWN. THESE ARE CONTROLS, IN THE END WE TAKE THE MICROGLIA AND SEPARATE THEM BASED ON THE LEVEL OF GFP. THE MORE THEY HAVE TAKEN UP TUMOR VESICLES THE MORE GFP THEY SHOULD HAVE. THERE’S NOT A LOT WE ISOLATED BUT WE WERE FORTUNATE TO BE DOWN THE HALL FROM DAVID TWO DID THE MESSENGER RNA SEQUENCING AND SAID THIS WAS NO PROBLEM. I SHOW THIS BECAUSE AT ONE TALK SOMEBODY QUESTIONED WITH THE DEEP RNA SEQUENCING ANALYSIS OF THE MITOCHONDRIA WE ISOLATEDDED BY FACS THEY DO SHOW TRANSCRIPTOME OF MICROGLIA. WE DID ISOLATE MICROGLIA. AND IF YOU LOOK AT ALL THE DIFFERENT MESSAGES THAT YOU SEE EXPRESSED, ONE THING WE NOTICED IS THAT IN MICROGLIA IF YOU COMPARE NOW THE ONES THAT TOOK UP THE GFP VESICLES VERSUS ONCE THAT DIDN’T, IT’S NOT TO SAY THEY DIDN’T TAKE UP SOME. THEY TOOK UP MORE FOR SURE, DEFINITELY A DIFFERENTIAL. THE BIG DIFFERENCE IN RNA LEVELS, SPECIFIC RNAs IN THE MICROGLIA THAT TOOK UP THE VESICLES, COMPARED TO MACROPHAGES WHICH SEEMED TO HAVE EATEN THEM FOR DINNER. THE VESICLES DON’T SEEM TO HAVE MADE A BIG EFFECT ON TRANSCRIPTOME OF MACROPHAGES IN VICINITIES OF THE TUMOR WHEREAS THE MICROGLIA WERE STRONGLY AFFECTED. AND THIS IS JUST SHOWING UNSUPERVISED CLUSTERING. AT FIRST WHEN YOU LOOK AT IT YOU DON’T GO, AHA AND AHO, BUT THESE ARE CONTROL MICROGLIA, THE ONES IN THE TUMOR-BEARING BRAIN ARE DIFFERENT AND THERE ARE SUBTLE DIFFERENCES BETWEEN THE ONES THAT WE HAD TAKEN TUMOR VESICLES AND ONES THAT HADN’T. THIS SLIDE JUST IS TO SHOW THAT, YOU KNOW, WHEN WE LOOK AT THE MESSENGER RNA EXPRESSION ESPECIALLY FOR PROTEINS THAT GO UP IN RESPONSE TO TUMOR BEING PRESENT, THEY GO UP WITH THE RNA LEVEL AND THEY GO UP AT THE PROTEIN LEVEL, JUST ANOTHER KIND OF CONTROL. SO, JOEL KORI IS A BIG EXPERT ON TRANSCRIPTOME OF MICROGLIA AND DIVIDED TRANSCRIPTS INTO DIFFERENT FUNCTIONAL PATHWAYS. THE FAVORITE IS THE SENSOME, THE PATHWAYS USED TO SENSE THERE’S A PROBLEM IN THE BRAIN LIKE YOU STUCK A LASER BEAM AND DID NEUROSURGERY OR GOT AN INFECTION. AND THE THING THAT YOU CAN LOOK AT THESE EGF NEGATIVE VERSUS POSITIVE, AND WHAT YOU TEND TO SEE IS THAT MOST OF THESE TRANSCRIPTOMES ARE GOING DOWN AND THEY ARE GOING DOWN MORE WHEN THE MICROGLIA WERE POSITIVE FOR TUMOR-DERIVED GFP. SOME GO UP BUT MOSTLY IT’S ALMOST LIKE THEY ARE PUTTING A BLINDFOLD ON. LIKE THE TUMOR PUTS A BLINDFOLD ON THESE AND SAYS I’M NOT HERE BASICALLY. THEN WE LOOKED AT OTHER PATHWAYS THAT HE ASSOCIATED THROUGH OTHER STUDIES WITH DEVELOPMENT OF THE BRAIN AND WITH TISSUE REPAIR, AND YOU CAN SEE THOSE ARE GOING UP. SO IT’S ALMOST LIKE THE MICROGLIA FIRST THEY PUT BLINDERS ON AND THEN IT’S LIKE, YOU KNOW, HAIL FRIENDS, WELL MET, THEY TRY TO SUPPORT THE TUMOR. AND THEN IF YOU LOOK AT SOME OF THE TRANSCRIPTS THAT ARE INVOLVED IN IMMUNE FUNCTIONS, YOU SEE A LOT OF UPREGULATION OF ONES THAT ARE INVOLVED IN INHIBITION, AND IN THE BRAIN, TUMOR ENVIRONMENT, THERE IS A LOT OF IMMUNE INHIBITION, AND ONE HERE THAT’S DOWNREGULATED THAT WOULD HAVE HELPED WITH IMMUNE ACTIVATION. SO JUST TO CAP THAT, THIS IS A SLIDE JOE PUT TOGETHER, IT’S HARD TO READ BUT IT WILL GET BETTER. MICROGLIA FUNCTION UNDER THE INFLUENTIAL OF GLIOBLASTOMA. NORMAL FUNCTIONS ARE WHERE A SENTINEL AND NURTURER — WHY ISN’T THIS GOING? SO WHAT HAPPENS TO THE WARRIOR FUNCTION? THIS IS WHERE THEY WOULD BE FIGHTING BACK INFECTION OR SOMETHING. IT’S WEAKENED. DECREASE IN PHAGOCYTOSIS, NO INCLINATION TO FIGHT THE TUMOR. SENTINEL FUNCTIONS ARE DISABLED AS IF THEY ARE BLINDED TO THE INTRUDER. AND NURTURER FUNCTION IS SUPPOSED TO PROMOTE TUMOR GROWTH, IMMUNE SUPPRESSION, THESE NURTURE FUNCTIONS SEEM LIKE THEY ARE TRYING TO — IT’S A DEVELOPMENTAL SCENARIO AND THEY ARE TRYING TO HELP THE TUMOR DEVELOP. I TRY TO DESCRIBE THIS, LIKE THEY DECIDED THEY HAVE A LITTLE BABY THAT’S GROWING AND THEY ARE GOING TO TAKE CARE OF IT. THAT’S KINDS OF WHERE WE ARE ON THE STORY WITH THE STORY WITH THE SABOTEURS BUT WE’RE TRYING TO DEVICE METHODS TO TRY TO GET RID OF THESE MICROGLIA USING MORE OF A GENE THERAPY APPROACH AND USING THE PROMOTERS THAT ARE UPREGULATED IN THESE MICROGLIA COMPARED TO NORMAL MICROGLIA. WE’LL SEE HOW THAT GOES. THE ONLY THING THAT — AFTER 20 YEARS OF STUDYING GLIOBLASTOMA, WE KEEP FINDING OUT NEW THINGS, THERE MAY BE AN ACHILLES HEEL. WE KEEP PERTURBING IT. WHAT ABOUT EXTRACELLULAR VESICLES AS BIOMARKERS? THIS NO LONGER NEEDS TO BE STATED. IT’S HAPPENING. THERE’S EVEN PRODUCTS EMERGING. ALL CELLS RELEASE THEM TO VARYING DEGREES INCLUDING TUMOR CELLS, THEY ARE FOUND IN ALL BIOFLUIDS, ESPECIALLY IMPORTANT IN BRAIN TUMOR WHERE YOU DON’T WANT TO GO IN AND TAKE A BIOPSY. A MIXED ARRAY OF VESICLES, THEY COULD BE THANE CONTAIN A LOT OF INFORMATION, PROTEIN, ET CETERA. WE ISOLATED FROM SERUM OF PATIENTS, AND LEFT IT OUT ON THE BENCH FOR TWO DAYS, MEASURED THE RNA WITH A BIOANALYZER AND PERFECTLY INTACT. IT’S COMPLETELY PROTECTED IN THAT — WITHIN THAT VESICLE. YOU CAN USE PROTEINS THAT EXPRESS ON THE CELL SURFACE OF YOUR WHATEVER TISSUE YOU’RE WANTING TO ANALYZE, AND USE THEM TO ENRICH FOR VESICLES, THAT INCREASES YOUR SENSITIVITY AND SELECTIVITY FOR MARKERS. I WON’T TALK ABOUT THAT NOW. I MENTION THE INFORMATION. SO WHEN WE FIRST THOUGHT ABOUT THIS, THIS WAS LIKE OUR — I HAVE A SLIDE I WANT TO PUT IN HERE BECAUSE A FEW TIMES IN YOUR CAREER WE HAVE ONE OF THESE LIGHT BULB MOMENTS, SO WE FOUND THE VESICLES, THAT SHOWED WE ARE RNA. WE’RE LIKE, YOU KNOW, THE BLOOD-TUMOR BARRIER IS WEAK, WHAT IF SOME GETS IN THE BLOODSTREAM, WHAT COULD WE LOOK AT THAT WOULD BE TUMOR SPECIFIC THAT, YOU KNOW, WOULDN’T EVER BE IN THE BLOODSTREAM IF YOU DIDN’T HAVE A TUMOR, AND WHERE WE COULD HAVE PRIMERS THAT WE COULD NEVER PICK UP THE NORMAL MESSAGE, WE WERE ON A ROLL. BUT THE PROBLEM WITH BRAIN TUMORS IS THAT AS I SAID, ALL THE CELLS ARE RELEASING VESICLES, FROM ENDOTHELIAL CELLS, MACROPHAGES, LYMPHOCYTES, AND NOW YOU’RE GOING TO SPRINKLE IN A FEW EVs, AND THAT COMPARED TO WHAT — PEOPLE HAVE MADE MUCH GREATER PROGRESS WITH PERIPERAL TUMORS BECAUSE THERE’S A LOT MORE TUMORS FROM THOSE — I MEAN VESICLES FROM TUMORS IN THE BLOOD BUT EVEN FOR GLIOBLASTOMA WE WERE SUCCESSFUL. SO WE TARGETED INITIALLY EGFRB 3, A DELETION THAT’S QUITE COMMON IN GLIOBLASTOMAS, HAS A POOR PROGNOSIS BUT THERE’S A LOT OF AGENTS BEING DEVELOPED INCLUDING VACCINES TO TARGET THIS PARTICULAR MUTATION. AND THEN THERE’S MUTANT IDH 1 AND 2, THIS IS A VERY CRITICAL MUTATION, NOW THE FIRST DIVIDER IN THE HUGE CLASSIFICATION OF TUMORS BECAUSE YOU’RE A DIFFERENT ENTITY IF YOU HAVE THIS MUTATION IN ISOCRIT RATE DEHIGH DODGE NOWS, LOWER GRADE, BETTER PROGNOSIS, THEY ARE DEVELOPING DRUGS TO TARGET THOSE MUTANTS, MUTANT SPECIFIC. THIS IS BOB CARTER AGAIN, HE USES CSF FROM PATIENTS, HE GETS THE STATUS OF THE EGFRB 3 FROM TUMORS AND LOOKS IN THE CSF, WHICH WAS MORE SENSITIVE THAN THE SERUM, AND BASICALLY HE GETS VERY HIGH SPECIFICITY, AND MODEST SENSITIVITY. SO THIS IS SOMETHING STILL A WORK IN PROGRESS TO PICK THIS UP. WE HAVE PICKED IT UP FROM SERUM BUT OUR PICKUP RATE IS EVEN LOWER IN SERUM. AND THEN SOMEONE, I DON’T KNOW WHO INITIALLY HAD THE IDEA, SAID, YOU KNOW, PEOPLE ARE LOOKING AT CIRCULATING TUMOR CELLS. THEY ARE LOOKING AT CELL-FREE DNA, LOOKING AT — YOU KNOW, EXTRACELLULAR RNA IN SERUM. AND WHY SHOULD YOU JUST LOOK AT ONE? THEY ALL HAVE INFORMATION, RIGHT? SO THIS PARTICULAR STUDY THEY SAID, MAYBE THE FREE CIRCULATING DNA COMES FROM DYING TUMOR CELLS SO WE WANT TO SEE WHO IS DYING, AND THEN THE LIVING CELLS PRODUCING VESICLES CAN GIVE US A PORTRAIT OF WHAT’S — OF THE MUTATIONAL STATUS OF THE TUMOR CELLS THAT ARE STILL LIVING. BUT BASICALLY YOU’RE GETTING A DOUBLE WHAMMY, COLLECTING TWO INFORMATIVE SETS OF GENETIC DNA FROM THE SERUM AND HERE WHEN WE — THE LAB DID THAT FOR IDH 1 MUTANTS IN PLASMA SHE WAS ABLE TO DISTINGUISH IDH 1 MUTANT CARRIERS FROM THE HEALTHY ONES. SO I THINK THAT HAS A LIFE OF ITS OWN NOW, JOHAN HAS A COMPANY, I SHOULDN’T SAY THE NAME, (INDISCERNIBLE), WE’RE VERY PROUD OF HIM. I MIGHT ADD AS I MENTIONED BEFORE THIS EXTRACELLULAR RNA, THAT CAN BE IN RNB PARTICLES IN A STABLE FORM, BEING USED AS BIOMARKERS FOR NEUROLOGIC DISEASES, MYOCARDIAL INFARCTION, FUNDED BY THE EXTRACELLULAR RNA CONSORTIUM, KIDNEY DISEASE, I MEAN THERE’S A HUGE EFFORT OF LABORATORIES, THEY FINDS MARKERS. THE ERCC IS STANDARDIZING PROTOCOLS TO GET LARGE REFERENCE PEDIGREES AND PUT THEM IN DATABASES TO SEE WHAT’S REAL AND WHAT ISN’T REAL. NOW I WANTED TO TALK ABOUT THE POTENTIAL USE AS THERAPEUTIC VEHICLES BECAUSE THAT’S — SOMEHOW I ALWAYS GET BACK TO THERAPY. AND THERE’S A LOT OF EFFORT IN THIS AREA NOW. I’M NOT GOING TO DESCRIBE IN DETAIL, MOST PEOPLE ARE JUST TRYING TO ISOLATE VESICLES FROM MESENCHYMAL STEM CELLS, LARGER MOUSE AND LOAD THEM, USE THEM. AND THAT LOOKS VERY PROMISING. NOT THE STRIDES WE’RE TAKING BUT HOPEFULLY THE IDEA. SO MANY CELL TYPES, EVERYBODY IS RELEASING THEM. YOU CAN USE MESENCHYMAL, DENDRITIC, WHATEVER CELLS YOU HAVE. THEY ARE PROTECTED, RELATIVELY STABLE. ONCE YOU PUT THEM INTO THE BODY THEY GET TAKEN UP PRIMARILY BY THE LIVER AND GOBS, BUT OFFICIALLY TAKEN UP BY ALL CELLS AND THEY CAN POTENTIALLY BE TARGETED TO SPECIFIC CELLS IN VIVO ALTHOUGH THAT REMAINS SOMETHING OF A CHALLENGE. THIS IS JUST A SLIDE FROM SUMMER, AND LUCY IS INVOLVED IN THIS, TO SHOW YOU SOME OTHER THINGS PEOPLE ARE USING, USING THEM FOR ANTIGEN PRESENTATION, FOR VACCINATION, THEY ARE USING THEM FOR IMMUNE MODULATION, TISSUE REPAIR, YOU’LL SEE LOTS OF PAPERS COMING OUT OF THIS AND I THINK IT’S GOING TO TAKE A LONG TIME TILL WE SORT THROUGH AND SEE WHAT WORKS AND DOESN’T WORK. THIS IS JUST AN EXAMPLE OF LOADING. YOU CAN PUT SEQUENCES INTO YOUR RNAs THAT WILL FACILITATE THEIR GOING INTO THE VESICLES, YOU CAN GET PROTEINS, OVEREXPRESS RNA, PUT VIRUSES IN THEM, OR ISOLATE VESICLES AND PUT IN RNAs OR PROTEINS BY OTHER METHODS. METHODS ARE BEING WORKED OUT TO LOAD THEM WITH SPECIFIC THERAPEUTICS. I WANTED TO TALK ABOUT OUR WORK ON SCHWANNOMAS, AFTER 20 YEARS WORKING ON GLIOBLASTOMAS AND HAVING MANY THERAPIES GOING INTO CLINICAL TRIALS HAVING NO BENEFIT I WAS LIKE MAYBE I SHOULD TRY A BENIGN TUMOR. I MIGHT HAVE BETTER CHANCES. I PICKED SCHWANNOMAS, AND BASICALLY THERE’S THREE DISEASES, HEREDITARY DISEASES PRIMARILY WHERE SCHWANN CELLS IF THEY LOSE A TUMOR SUPPRESSOR GENE WILL FORM TUMORS ON THE CELLS OR NERVES, USUALLY BENIGN. BUT THEY CAN CAUSE MOTOR DYSFUNCTION, PAIN, HEARING LOSS. THIS ONE NF1 IS VERY COMMON IN THE HUMAN POPULATION WITH 1 IN 3,000 CARRIERS. AND THE CURRENT TREATMENT IS BASICALLY SURGICAL RESECTION. BUT THAT CAN, AS YOU MIGHT IMAGINE, CAUSE NERVE DAMAGE AND IS NOT EVEN USUALLY DONE. HERE IS SCOTT PLOTKIN, DIRECTOR OF OUR CLINIC, SHOWING IN AN MRI IN AN NF2 PATIENT, 50% HAVE TUMORS, THE AVERAGE VOLUME IS 83 MILLIMETERS, THEY FORM ALL ALONG THE NERVE FIBERS, AT THE BASE OF THE SPINE, ET CETERA. THEY CAN CAUSE VERY SERIOUS PROBLEMS BUT THEY ARE NOT MALIGNANT, SO ALL WE HAVE TO DO IS REDUCE THEIR SIZE. THIS JUST ACTUALLY SHOWS YOU SOME MORE SERIOUS ONES, THE VESTIBULAR SCHWANNOMAS ON THE 8th NERVE THAT CAUSE DEAFNESS. SO WE MADE A MODEL WHERE WE TOOK A HUMAN SCHWANN CELL FROM NF2 PATIENT, IMPLANT THEM IN NUDE MICE, THEY FORM TUMORS AND WE WORKED OUT A SCHEDULE WHERE THE TUMORS WOULD REPRODUCIBLY FORM OVER A PERIOD OF TIME. THESE SCHWANNOMA CELLS HAVE BEEN IMMORTALIZED, E6, E7, MORE TRANSFORMED, NOT A GREAT MODEL BUT THE VESSEL WE HAVE FOR NOW. WE HAVE THE IDEA WE SHOULD TAKE — THIS IS LIKE A WIMPY CASPASE, CASPASE 1, NORMALLY INVOLVED IN DEVELOPMENT. HOOK IT UP WITH A PROMOTER, P ZERO ACTIVE IN SCHWANN CELLS AND MAKE AN AV VECTOR, INJECT IN THE TUMOR TO SHRINK THEM. ONE THING I WANT TO POINT OUT, I’VE DONE THIS WORK WITH BRYNNER AND FULCI, IF YOU TOOK AN AV VECTOR, THIS IS LIKE A GOOD SEROTYPE AND EXPRESS GFP WE ONLY HIT ABOUT — IF WE’RE LUCKY 10% ACTUALLY OF THE CELLS IN THE TUMOR. BUT IF WE INJECT THIS P-0 ICE, WE GET A LARGE NUMBER OF APOPTOTIC BODIES. THERE IS BYSTANDER EFFECT, WHEN THESE CELLS — WHEN A SUBSET OF CELLS GETS THIS CASPASE 1, OTHER CELLS AROUND IT START DYING OFF TOO. SO IN THE TUMOR MODEL, HERE WE USE THE GFP CONTROL. THE TUMOR GROWS, IF YOU PUT GFP IN IT. IF YOU PUT IN P0-ICE. ITS NEVER GROWS HERE, HERE IT REGRESSES. IT LOOKS A LITTLE PROMISING. IF YOU LOOK AT THE NERVES THEMSELVES AFTER THIS TREATMENT, THEY ACTUALLY LOOK NORMAL, WHICH SURPRISED US BUT ONCE YOU GET RID OF THE TUMOR THEY SEEM TO NORMALIZE. THIS IS JUST TO SHOW KIND OF A THERAPEUTIC EFFECT. HERE IS THE GREEN, THE TUMOR SIGNAL. WE USE BIOLUMINESCENCE. HERE IS THE TUMORS GROWING. INJECT VECTOR, TUMOR SHRINKS. HERE IS THE MEASURE OF PAIN. SO THE HIGHER — THEY BASICALLY TAKE DIFFERENT FIBERS AND PRESS THEM AGAINST THE PALM OF THE ANIMAL. IF THEY ARE SENSITIZED TO PAIN, THEY GET A REACTION SOONER. SO AS THE TUMOR STARTS GROWING, THE ANIMALS BECOME MORE SENSITIVE TO THIS TYPE OF PAIN BUT ONCE YOU REGRESS THE TUMOR THEY NORMALIZE. SO ALL THIS LOOKS KIND OF PROMISING. AND WHERE DO EVs COME INTO THIS? WELL, WE HAVEN’T SPECIFICALLY PROVEN BUT THIS IS OUR THEORY. CASPASE IS KNOWN BY OTHER INVESTIGATORS, CASPASE 1, YOU COULD INCORPORATE EVs, HERE IF WE TOOK LIKE CELL-FREE CONDITIONED MEDIUM FROM CELLS WE INFECT WITH OUR VECTOR, IT KILLS SHAWAN CELLS. IF YOU MIX, TRANSFECT, YOU CAN KILL THEM. WE’VE HYPOTHESIZED EV TRANSFER OF CASPASE 1, BECAUSE IT’S NOT NORMALLY SECRETED, YOU HAVE TO GET IT OUT IN EV, AND THIS IS OUR THEORETICAL MODEL, SO HERE IS THE NERVE FIBER, SURROUNDED BY SCHWANN CELL SHEATH. WE HAVE A SCHWANNOME A FORMING, INJECT VECTOR, WE HAVE MORE EVIDENCE BUT FOR NOW WE’RE GOING TO LET IT GO HERE. THE VESICLES NOW CONTAIN, THAT ARE RELEASED BY INFECTED CELLS NOW CONTAIN CASPASE 1, LEADING TO DEATH OF OTHER SCHWANN CELLS AND YOU GET TUMOR REGRESSION. SO THAT COVERED THE THREE TOPICS. I WANT TO THANK THE NCATS AND ERCC. I THINK THEY HAVE DONE AN AMAZING JOB OF HELPING THIS INFORMATION ESPECIALLY ON EXTRACELLULAR RNA GO FORWARD IN ALL THREE DIRECTIONS. YOU KNOW, THE NORMAL BIOLOGY, THE BIOMARKERS AND THE THERAPY, THEY HAVE A GREAT WEBSITE, XRNA.ORG, WHERE THEY PUT ALL KINDS OF DATA AND PROTOCOLS, IT’S A VALUABLE RESOURCE. THIS IS JUST A PICTURE OF SOME OF THE PEOPLE IN MY LAB, CHARLES, NICK, ERIC, CHRISTINA, JUST A FEW FACES. THERE’S LEONARDO, BENCE, AND THEN HERE IS THE LIST OF THE PEOPLE, PROBABLY DOESN’T LIST EVERYBODY BUT I’VE HAD A LOT OF PEOPLE FROM THE NETHERLANDS IN& MY LAB. I WOULD SAY AT LEAST HALF, I BECAME A POPULAR SPOT FOR VISITORS. JOEL KHOURY AND DAVID TINGE, DEEP SEQUENCING. MY FUNDING SOURCES ARE ALL NIH. AND I DO WANT TO THANK TWO PROGRAM OFFICERS, I THINK BECAUSE OF THEIR REAL VISION AND WISDOM, BILL TIMER AND KEVIN HALKOFF, IN THE OLD DAYS WHEN I CALLED MY PROGRAM OFFICER I THOUGHT I WAS TALKING TO A RECORDING. HOW DID IT GO. I WOULD GET A RECORDED ANSWER. PRESS 1, PRESS 2. BUT IT SEEMS TO HAVE CHANGED. THE PROGRAM OFFICERS NOW ACTUALLY UNDERSTAND THE SCIENCE AND THEY CAN KIND OF GUIDE YOU, AND THAT’S BEEN A BIG BREAKTHROUGH I THINK. SO THANK YOU. [APPLAUSE] >>THANKS FOR AN INTERESTING ROMP THROUGH THREE ASPECTS OF EXTRACELLULAR VESICLES AND THOUSAND THEY MIGHT APPLY IN NEUROSCIENCE. WE HAVE TIME FOR QUESTIONS. WE’LL START HERE.>>THIS IS MORE OF A COMMENT REGARDING MIR21 LEVELS, ENDOGENOUS OR COMING FROM THE VESICLES, SO THE WAY WE HAD THE SAME ISSUE, AND THE WAY WE TACKLED IT WAS TO MEASURE THE PREMIER RECIPIENT CELLS ASSUMING UPREGULATION ALSO IN THE PREMIER, ONCE YOU DON’T SEE THAT I A SUM YOU ASSUME THE VESICLES.>>INTERESTING.>>MY QUESTION IS YOU SAY TUMOR CELLS RELEASE EXTRACELLULAR VESICLES RECOGNIZED BY NORMAL, INCORPORATE THE IN THE NORMAL CELLS, BECOME TUMOR-LIKE. YOU ALSO SAID THAT IT’S IN BIOFLUID LIKE BREAST MILK, WHAT IF CANCER PATIENTS, A MOTHER WHO IS GIVING — BREASTFEEDING, DOES THAT MEAN THEIR BABIES ARE ACTUALLY BEING, YOU KNOW, –>>YEAH, I DIDN’T ACTUALLY SAY THE VESICLES FROM THE TUMOR CELLS IMMORTALIZE OTHER CELLS BUT OTHER PAPERS SAY THAT. AND I WOULD SAY THIS HAS BEEN A CONCERN FOR SOME PEOPLE THAT YOU MIGHT BE ABLE TO TRANSMIT IMMORTALIZING GENES OR RNA, ESPECIALLY BREASTFEEDING OR SOMETHING LIKE THAT. I HAD THE FDA ASK ME ONCE, WHEN WE MAKE A VACCINE, IT’S FILLED WITH VESICLES AND CELLS WHEN YOU PUT THE VACCINE ARE IMMORTALIZED, ARE THESE IMMORTALIZING PROTEINS, YES, GOING INTO THE VESICLES, WHETHER THAT’S ENOUGH TO CAUSE ANYTHING IS ANOTHER ISSUE. INTERESTING POINT. A LOT OF PEOPLE ARE INTERESTED IN THAT. IT’S A REASON I WOULD SAY, YOU KNOW, PEOPLE TALK ABOUT — WHAT CELL LINE ARE WE GOES TO USE TO ISOLATE VESICLES FROM THEY ARE THERAPEUTIC CELLS, NUMBER OF GROUPS ARE GONE TO THOSE LOADED WITH ONCOPROTEINS AND YOU SHOULD USE MESENCHYMAL STEM CELLS. I DON’T THINK I’M ONE IN THAT AREA, I HAVE MY OPINION, I AGREE WITH YOU.>>A FOLLOW-UP OF THAT, THE QUESTIONER PROPOSED A CIRCUMSTANCE WHERE THESE WOULD BE PRESUMABLY DELIVERED ORALLY, IN THIS CASE MOTHER TO BABY. THERE WAS A PAPER FOUR OR FIVE YEARS AGO THAT GOT EVERYBODY ALL VERY EXCITED THAT PURPORTED TO SHOW YOU COULD IN FACT DETECT VESICLES IN THE BLOOD OF PEOPLE WHO WERE VEGETARIANS AND THAT THOSE VESICLES CONTAINED MICRORNA OF PLANT ORIGIN AND AFFECTED GENE EXPRESSION IN LIVER OF PEOPLE EATING A LOT OF THOSE PARTICULAR PLANTS, ENORMOUSLY EXCITING IF TRUE BECAUSE IT SUGGESTED A WAY OF GENE ENVIRONMENT INTERACTION. BUT I GATHER THERE HAS BEEN SOME QUESTION ABOUT WHETHER THAT CAN BE REPRODUCED, WHERE DO WE STAND? IF YOU’RE GOING TO USE THESE THERAPEUTICALLY AND COULD TAKE A LIQUID SOLUTION AS OPPOSED TO INJECTED THAT WOULD MAKE LIFE –>>PEOPLE PUT THEM UP INTRANASALLY. ORALLY, PATIENTS HOLD IT IN THEIR MOUTH. I THINK INEFFICIENT BUT YOU’VE HEARD FROM JOHAN THAT CAN FIND BOVINE — I THINK MESSENGER RNA FRAGMENTS, VERY SMALL AMOUNTS IN MEAT EATERS COMPARED TO VEGETARIANS SO THERE IS SOME TRANSFER BUT WHETHER IT’S ENOUGH TO BE FUNCTIONAL EVERYBODY IS IN DOUBT ABOUT.>>IT BLOWS YOU AWAY THINKING THIS WOULD IMMEDIATELY GET DIGESTED IN THE G.I. TRACT AND YOU WOULD NEVER GET RNA INTO THE CIRCULATION.>>EVERYBODY IS TAKING IT UP ALONG THE WAY, MAYBE TAKEN UP AS IT GOES ALONG THE EESOPHAGUS, MAYBE WHEN IT HITS THE STOMACH –>>ACROSS THE EPITHELIUM YOU WOULDN’T HAVE THOUGHT WOULD BE FRIENDLY TO RECEIPT OF THIS KIND OF MATERIAL.>>YEAH, YEAH.>>OVER HERE.>>XANDRA, THANKS FOR DECONFLATING MICROGLIA AND MACROPHAGES, BECAUSE THAT HAPPENS A LOT. BUT WHAT I’M ESPECIALLY INTERESTED IN, THIS NOVEL FUNCTION OF MICROGLIA IN THE TUMOR, MICROGLIA RESPOND AS YOU POINTED OUT TO A LOT OF DIFFERENT STIMULI. SO WHEN YOU LOOK AT THE TRANSCRIPTOME OF THE MICROGLIA HOW DOES IT COMPARE TO TUMOR DERIVED MICROGLIA VERSUS MICROGLIA ACTIVATED BY OTHER INSULT?>>IT’S VERY DIFFERENT. THEY USED TO SAY IT WAS THE M1 AND M2 CLASS.>>RIGHT.>>NOW JOE KHOURY SAYS AT LEAST 13 DIFFERENT STATES OF MICROGLIA CAN ENTER, THE GLIOBLASTOMA STATE IS ANOTHER ONE AGAIN. THEY ARE — WHEN THE MICROGLIA ARE IN THE CONTEXT OF THE BRAIN TUMOR AND WHETHER IT’S BECAUSE THEY ARE VERY CLOSE TO THE TUMOR CELLS OR WHETHER THEY — MAYBE THAT’S WHY THEY TOOK UP MORE VESICLES, I DON’T KNOW, WE CAN’T BLAME THE VESICLES AT THIS POINT BUT THEY BECOME PROPHETIC IN THEIR, YOU KNOW, WHAT THEY ARE SUPPOSED TO BE DOING. THEY DON’T DO ANYTHING EXCEPT FOR SUPPORT THE TUMOR.>>OVER HERE.>>JUST A POINT IN TERMS OF HAVING DONE PATHOLOGY SOMETIME AGO, DEALING WITH GLIOBLASTOMA, ALL THESE TYPE OF THINGS, I REMEMBER HOW DIFFICULT IT WAS TO MAKE A DIAGNOSIS AFTER TREATMENT THAT IT HAD ACTUALLY REGRESSED OR YOU’VE BEEN SUCCESS SO THE MICRO VESICLES MIGHT BE A GOOD FOLLOW-UP TO MAKE SURE YOU TREATED SUCCESSFULLY, TO THAT EXTENT A VERY SUCCESSFUL — IT’S A REAL STEP FORWARD FOR PATHOLOGISTS SCRATCHING THEIR HEADS, WHETHER THIS IS NECROSIS, RADIATION OR GLIOBLASTOMA. THERE’S A GOOD DEAL OF LITERATURE GOING TOWARD SYNTHETIC BIOLOGY, CREATING CELLS, WONDERING HOW THAT WOULD APPLY TO VESICLES, IS THAT WHAT THEY ARE PRINCIPALLY USING NOW OR GOING TO –>>A BIG MOVE, A LOT OF PEOPLE INVOLVED IN NANOPARTICLE OR LIPOSOME DELIVERY, TRYING TO INCORPORATE SOME FEATURES, SOME PROTEINS, SOME LIPIDS THAT DISTINGUISH THE EXTRACELLULAR VESICLES INTO THE LIPOSOMES ESPECIALLY. ONE TALK TODAY, THEY TRIED TO FUSE THEM TOGETHER. LIPOSOMES WITH EVs IN HOPES OF GAINING PROPERTIES WHICH ARE, WELL, EVs ARE NOT TOXIC, NORMALLY THEY ARE VERY STABLE. SO AVIDLY TAKE THEM UP, YOU KNOW, THEY SEEM TO HAVE A WAY OF GETTING CONTENTS INTO THE CYTOPLASM. YEAH, A LOT OF PEOPLE SEE THAT AS THE FUTURE. TO LEARN FROM THE EVs AND TO FIGURE OUT WHAT COMPONENTS OF THEM CAN BE INCLUDED IN A SYNTHETIC VESICLE.>>ONE PAPER OUT WHERE I FORGOT THE NAME OF THE GUY, THE OTHER GUY WHO DID THE GENOME, DR. VENTER, CREATED ONE THAT MIMICS THE SIMPLEST MICROORGANISM. I FORGOT THE NAME. THE SIMPLEST OF THE SIMPLEST, THEY HAVE MIMICKED IT PRETTY WELL.>>AMAZING.>>THANK YOU.>>HI. JUST WONDERING IN ADDITION TO DELIVERING TOXIC CHEMICALS OR MOLECULES WHAT ABOUT TARGETING BIOGENESIS OR BIOSYNTHESIS IN THE TUMOR CELLS.>>PEOPLE WOULD LOVE TO DO THAT, JUST TO DO COX POSTULUS, I THINK IT’S THE VESICLES CAUSING THIS YOU’D LIKE STOP VESICLE WE LEASE AND SEE IF YOU STILL GET IT, BUT FIRST OF ALL NOBODY HAS FIGURED OUT HOW TO DO IT, THEY SEEM TO GET AROUND IT ONE WAY OR THE OTHER, THE CELL DOESN’T LIKE IT, RIGHT? IT’S VERY HARD. IT’S EASIER TOE PREVENT UPTAKE, BUT VERY HARD TO BLOCK RELEASE. I FORGET EXACTLY WHAT YOUR QUESTION IS. YOU HAVE ANOTHER ASPECT OF THAT?>>COME TO THE MIC.>>IT SEEMS BIOGENESIS PATHWAYS (INDISCERNIBLE) ESCORT COMPLEX, WOULD THAT HELP IN TERMS OF SPECIFIC TARGETING, THOSE COMPONENTS IN THE — I MEAN LIKE THE GBM CELLS OR OTHER CANCER CELL TYPES?>>FOR INSTANCE, WE TRIED THAT. THE MULTI-VESICULAR PATHWAY USES THIS RAD 27A TO RELEASE, THAT WAS WORK SHOWN IN MY OTHER PAPER, AND SO WE TRIED TO BLOCK THAT PATHWAY IN THE GLIOBLASTOMA CELLS TO SEE IF THEY WOULD GROW AS WELL AS TUMORS, AND THEY ACTUALLY SEEMED TO GROW AS WELL AS TUMORS, THEY MAY NOT INVADE AS MUCH. IT’S VERY — YEAH, RATHER DICEY.>>I HAD A QUESTION ABOUT LIPID (INDISCERNIBLE) VESICLES, THICKER THAN THE GOLGI, TAKES PLACE BECAUSE OF (INDISCERNIBLE) RETICULUM, LONGER ONES GO INTO THE PLASMA MEMBRANE. WHAT IS THE THICKNESS OF THE VESICLES AND SORTING OF PROTEINS TAKING PLACE.>>THAT’S A HUGELY IMPORTANT QUESTION. AND I CAN’T PERSONALLY ANSWER IT. WE HAVE A TALK AT THE AMERICAN SOCIETY OF GENE CELL THERAPY BY ANASTASIA, IT’S DIFFERENT FROM PLASMA MEMBRANE, THEY SHIFT PHOSPHO-SEARING OUT, HIGHER CHOLESTEROL. THE LIPID THING IS DIFFERENT IN THEM. NOT MANY PEOPLE STUDIED THAT TO DATE BUT IT’S A CRITICAL ISSUE.>>GREAT. ONE FINAL QUESTION FOR YOU, IN TERMS OF THIS WHOLE FIELD OF EXTRACELLULAR VESICLES. THE COMMON FUND IS SUPPORTING, WHAT THIS MIGHT DO IN CANCER AND NEW FORM OF ENDOCRINOLOGY SENDING SIGNALS FROM ONE PART OF THE BODY TO ANOTHER. MAYBE EVEN A DISTANCE, NOT JUST LOCALLY. WHAT DO YOU SEE AS THE MOST IMPORTANT NEXT STEPS FOR THIS PARTICULAR FIELD TO BRING IT FORWARD INTO WHEREVER IT NEEDS TO GO, TO FULLY UNDERSTAND WHAT THESE VESICLES ARE DOING AND HOW DO UTILIZE THOSE FOR MEDICAL PURPOSES?>>YEAH, I MEAN I THINK RIGHT NOW, THE BIGGEST EMBARRASSMENT IS FIRST OF ALL WE DON’T KNOW HOW MANY DIFFERENT TYPES OF VESICLES THEY ARE AND WHAT THEIR CONTENTS ARE, QUANTIFICATION, WHAT JENNIFER JONES WORKED ON, WE CAN’T COUNT THEM PROPERLY. IT’S VERY HARD TO LABEL THEM PROPERLY. LABELS THAT — FIRST OF ALL IF YOU LABEL THEM LIKE FLUORESCENT PROTEINS ONCE THEY GET TAKEN INTO THE CELL AND RELEASED YOU CAN’T SEE THE FLUORESCENT PROTEIN ANYMORE. DYES AGGREGATE. WE HAVEN’T GOT GOOD WAYS TO LABEL THEM OR SEPARATE THEM. UNTIL WE CAN DO THAT, YOU KNOW, FOR INSTANCE WE KNOW NOW THAT ON AVERAGE THERE’S ONE MICRORNA PER VESICLE BUT IT LOOK 100 VESICLES, IS IT ALL IN ONE, IF SO WHAT KIND — IT’S ALMOST EMBARRASSING REALLY WHAT WE DON’T KNOW ABOUT THESE VESICLES.>>A LOT OF TECHNOLOGY DEVELOPMENT NEEDED. SOUNDS LIKE A LOT OF PROBLEM IS THIS IS VERY HETEROGENEOUS, YOU CAN’T SAY NOW WE’VE FIGURED OUT WHAT VESICLES ARE DOING BECAUSE THERE’S ALL DIFFERENT TYPES WITH DIFFERENT POTENTIAL FUNCTIONS.>>JUST TO GIVE ONE EXAMPLE OF A LONG DISTANCE EFFECT, IF SOMEBODY HAS HIV, THERE’S DATA SHOWING THEIR VESICLES CAN SENSITIZE THE LIVER, VESICLES PRODUCED BY INFECTED CELLS BUT NOT THE VIRUS ITSELF CAN SENSITIZE THE LIVER TO HIV INFECTION, SO THERE ARE INDICATIONS THAT THEY DO TRAVEL GREAT DISTANCES.>>MUCH TO STAY TUNED.>>YEAH.>>XANDRA, THANK YOU FOR WALKING US THROUGH INTERESTING APPLICATIONS AND RAISING OUR CONSCIOUSNESS ABOUT AN INTERESTING FIELD. PLEASE, LET’S THANK DR. BREAKEFIELD AGAIN. AND YOU’RE ALL INVITED NOW TO A RECEPTION IN THE MEDICAL LIBRARY, IF YOU WISH TO CONTINUE THE CONVERSATION, OR TO HAVE COOKIES AND COFFEE, OR BOTH. [END OF PROGRAM]