Welcome to the Clinical Center Grand Rounds, a weekly series of educational lectures for physicians and health care professionals broadcast from the Clinical Center at the National Institutes of Health in Bethesda, MD. The NIH Clinical Center is the world's largest hospital totally dedicated to investigational research and leads the global effort in training today's investigators and discovering tomorrow's cures. Learn more by visiting us online at http://clinicalcenter.nih.gov OUR SPEAKER TODAY IS DR. JENNIFER C JONES. AN NIH STADTMAN TENURE TRACK INVESTIGATOR AND A SCHOLAR IN THE CENTER FOR CANCER RESEARCH WHERE SHE LEADS THE TRANSLATIONAL AND NANOBIOLOGY SECTION IN THE LABORATORY OF PATHOLOGY. DR. JONES EARNED BOTH HER MEDICAL DEGREE AND Ph.D. IN CANCER BIOLOGY AT STANFORD UNIVERSITY IN PALO ALTO, CALIFORNIA. SHE RECEIVED A POST-DOCTORAL FELLOWSHIP IN IMMUNOLOGY AND INTERNSHIP IN INTERNAL MEDICINE AND IN RADIATION ONCOLOGY AND FELLOWSHIP IN RADIATION AND IMMUNOTHERAPY ALL AT STANFORD. SHE SUBSEQUENTLY DEPARTED CALIFORNIA TO COMPLETE AN ONCOLOGY RESEARCH FELLOWSHIP AT THE DANA FARBER INSTITUTE IN BOSTON BEFORE COMING TO THE NATIONAL INSTITUTES OF HEALTH AS A PHYSICIAN ONCOLOGY AND PHYSICIAN SCIENTIST IN THE NCI'S RADIATION ONCOLOGY BRANCH. DR. JONES' LAB FOCUSES ON TUMOR AND TRANSLATIONAL STUDIES. THE AIM OF HER RESEARCH IS TO DECODE THE BIOLOGICAL LEXICON OF CIRCULATI CIRCULATING EXTRACELLULAR VESICLES AND PARTICLES AND INTERESTED IN NEW APPROACH TO THE FUNCTIONAL STATUS OF EACH PATIENT'S INTERCONNECTED SYSTEMS TO IMPROVE PATIENT CARE, TREATMENT SELECTION AND QUALITY OF LIFE. DR. JONES HAS PUBLISHED MORE THAN 50 PEER-REVIEWED MANUSCRIPTS IN THE LITERATURE TO DATE AND CO-HOLDER OF NUMEROUS PATENTS. SHE HAS SERVED AS A RESEARCH MENTOR FOR POST-DOCTORAL STUDENTS AT BOTH STANFORD AND THE NIH. IN 2018, DR. JONES RECEIVED THE YOUNG INVESTIGATOR AWARD FROM THE PROSTATE CANCER FOUNDATION AND PAMELA AND KATHRYN'S RENAL CELL CARCINOMA AWARD. THE TITLE OF HER GRAND ROUNDS FREN PRESENTATION TODAY IS EXTRACELLULAR VESICLES IN LIQUID BIOPSIES AND FOCUSSING ON WHAT MATTERS. NOW WE'LL TURN IT OVER TO DR. JONES. IT'S A PLEASURE TO BE HERE WITH YOU AND SHARE THIS WITH YOU TODAY. I'M GOING TO TALK ABOUT EVAs AND LIQUID BIOPSIES AND STEER THEM TOWARDS THE FOCUS OF OUR LAB AND WHAT MARTS IN GETTING INFORMATION AND WHAT'S IMPORTANT IN GETTING INFORMATION FROM THE SCIENCE. THE STRUCTURE OF THE TALK I'LL FOLLOW TODAY IS I'LL START WITH THE BIG PICTURE IDEA. I'LL INTRODUCE SOME CHALLENGES AND TALK A LITTLE BIT ABOUT THE PROGRESS ON THE BASIC RESEARCH FRONT, SOME PROGRESS IN THE TRANSLATIONAL EXAMPLES AND A LITTLE BIT OF THE FUTURE DIRECTIONS. THE LEARNING OBJECTIVES FROM A CME POINT OF VIEW, I HOPE CLINICIANS AND RESEARCHERS ALIKE WILL BOTH LEAVE THIS TALK WITH A CLEARER UNDERSTANDING OF THE PLACE OF E ASSAYS AND LIQUID BIOPSIES AND HOPE IT WILL ENCOURAGE MORE THOUGHTFUL ANALYSES OF EV RESULTS AND STUDIES IN GENERAL THAT'S BEEN THE STATUS QUO IN THE LITERATURE TO DATE. AND I'LL PRESENT NEW METHODS TO IMPROVE THE ACCURACY OF EV STUDIES AND POINT YOU IN THE DIRECTION OF TOOLS AND NEXT STEPS FOR TRANSLATION OF THE EV TYPE STUDIES TO THE SETTING. AS MENTIONED, I HAVE SOME DISCLOSURES WHICH IS THE RESEARCH IS FUNDED BY THE NCI INTRAMURAL RESEARCH PROGRAM AND WE HAVE A COLLABORATIVE AGREEMENT AND HAVE A NUMBER OF INTELLECTUAL PROPERTY FILINGS NOTED ON THE SPECIFIC FILINGS AS PERTINENT. THERE'S A QUOTE I REMEMBER EVERY SPRINGTIME BY ADDLY STEVENSON. I THINK IT SETS THE TONE FOR WHAT I'VE BEEN THINKING AND PREPARING FOR THIS TALK. YOUR DAYS ARE SHORT HERE AND THIS IS THE LAST OF YOUR SPRINGS AND NOW IN THE SERENITY AND QUIET OF THIS LOVELY SPACE TOUCH THE DEPTHS OF TRUTH AND FEEL THE HEM OF HEAVEN YOU WILL GO AWAY WITH OLD GOOD FRIENDS AND DON'T FORGET WHEN YOU LEAVE HERE, WHY YOU CAME. THIS WAS QUOTED BY DONALD KENNEDY QUITE OFTEN AT STANFORD GRADUATIO GRADUATIONS AND AT THE COMMENCEMENT AT PRINCETON. I WANT TO START BY FOCUSSING ON THIS AND DON'T FORGET WHEN YOU LEAVE HERE WHY YOU CAME. IT LEADS ME TO THE WHY. WHY ARE WE HERE AND WHAT IS SO SPECIAL ABOUT THE RESEARCH WE'RE DOING AT THE CLINICAL CENTER AND NIH AND IN THIS PROGRAM? SO I THINK THERE ARE TWO QUESTIONS THAT MOST OF US WHO ARE DOING TRANSLATIONAL AND CLINICAL RESEARCH AT THE CLINICAL CENTER CONFRONT ON A VERY REGULAR BASIS AND THE FIRST IS WHAT IS WRONG. HOW CAN WE TELL WHAT'S WRONG WITH OUR PATIENT. AND HOW CAN WE HELP. CONVERSELY WE NEED TO KNOW WHAT IS HELPING THE PATIENT GET BETTER. THERE'S A MASSIVE EXPANSION IN THE DIAGNOSTIC TESTS AND MOLECULAR PROFILING AND ABILITY TO ANSWER THE QUESTIONS OF WHAT IS WRONG AND HOW CAN WE HELP. BROADLY SPEAKING WE BREAK THE TESTS AND THE AIMS OF THE TESTS INTO A COUPLE GROUPS WHICH IS DIAGNOSIS, RISK STRATIFICATION AND MONITORING. RISK STRATIFICATION AND MONITORING BY THE ADJUSTMENT AND SELECTION OF TREATMENT. THIS BRINGS ME TO REALLY THE OVERARCHING MIND SET OF THE IDEA HERE WHICH IS THAT IN THE MODERN CONTEXT HERE, WE'VE ALL GROWN UP AND LEARNED TO USE BASIC LAB IMAGING AND SOME MOLECULAR DIAGNOSIS WITH BIOPSIES. THERE'S AN EMERGING FIELD OF LIQUID BIOPSYING AND HAS GROWN TO INCLUDE CELL FREE NUCLEIC ACID ASSAYS AND BEGINNING TO INCLUDE A COUPLE YEARS AGO WHAT PEOPLE WERE CALLING EXOSOME ASSAYS MORPHED INTO TO WHAT PEOPLE ARE PEOPLE ARE CALLING EXTRACELLULAR VESICLE ASSAYS AND REALIZING THERE'S OTHER COMPLEXES INCLUDING ISLETS AND PROTEINS AND IN A URINE SAMPLE YOU CAN FIND THESE AFTER YOU REMOVE THE CELLS. EXTRACELLULAR CELLS ARE FRAGMENTS EITHER SYSTEMICALLY PRODUCED INSIDE THE CELL OR SHED FROM THE SURFACE. EITHER WAY THEY HAVE A LIPID BIOLATER WITH INTERNAL CARGO AND THE CONSTITUENT COMPONENTS CAN BE OF NUCLEIC ACIDS AND DIFFERENT THINGS. WHAT'S NICE ABOUT THE MULTI COMPONENT NATURE OF THESE VESICLES IS THEY HAVE ATTRIBUTES WHICH RELATE TO WHERE THEY CAME FROM. SOME OF THE EVs WILL HAVE ATTRIBUTES OF CERTAIN CELL TYPES OR CERTAIN ORGAN TYPES AND OTHER EV WES -- EVs WILL HAVE OTHER ATTRIBUTES TO APROVIDE THE ABILITY TO THINK OF SYSTEM-BASED PROFILING BASED ON COMPARTMENTS THAT EITHER RELATE TO THE CELLULAR OR ORGAN NETWORKS REALLY TO WHERE THE VESICLES COME FROM. ONCE YOU START CONSIDERING THE USE OF THESE AS COMPARTMENTALIZED PACKETS OF INFORMATION YOU CAN LEARN FROM IT QUICKLY BECOMES EXCITING TO CONSIDER COMPARING THESE PACKETS OF INFORMATION BETWEEN HEALTHY AND SITUATIONS WHERE YOU HAVE A DISEASE OR RESPONSE TO TREATMENT. AND THIS REALLY APPLIES TO ANY DISEASE YOU COULD CONCEIVE OF WHEREIN YOU CAN START WITH SOME ABILITY TO PULL OUT VESICLES THAT ARE ORIGINATE FROM THE DISEASE OR THE ORGAN OR RELATED TO THE TREATMENT. SO IT GETS TO THE FACT THAT IT'S NOT JUST THE SURFACE PHENOTYPE. IT'S NOT JUST THE PROTEIN RECEPTORS THAT SIGNIFY THE DISTINCTIVENESS OF THESE DIFFERENT COMPARTMENTS AND DIFFERENT GROUPS OF EXTRA CELLULAR VESICLES BUT THE OTHER CARGO THEY CARRY. AS I MENTIONED BEFORE, IT'S NOT JUST PROTEINS BUT VARIOUS FORMS OF NUCLEIC ACIDS MOSTLY NON-COATING NUCLEIC ACIDS AND ALSO LIPIDS AND GLYCOFORMS OF VARIOUS TYPES. SO THE OVER ARCHING HYPOTHESIS OF MY RESEARCH PROGRAM IS WE CAN BASICALLY COMPARTMENTALIZE TO STRATIFY THESE EV POPULATIONS IN WAYS WE CAN THEN INTERROGATE THEM TO LEARN WHAT'S HAPPENING IN THE DIFFERENT ORGANS AND DIFFERENT CELL TYPES UNDER DIFFERENT CONDITIONS AND BECAUSE THEY'RE PACKETS OF INFORMATION, THE DEPTH OF THAT INFORMATION WILL BE MORE INFORMATIVE THAN SAY A SINGLE MARKER. SO WE KNOW IN THE PATIENTS THAT I SEE WHO MY PATIENTS TEND TO BE PATIENTS WITH ADVANCED DISEASE AND ADAPTIVE STRATEGIES ARE VERY IMPORTANT AND VERY NECESSARY. ONE ADVANTAGE IS EVs ARE REAL TIME INFORMATION PACKET COME FROM ALL CELL TYPES. WE'RE WORKING HARD AT DEVELOPING EV BASED ASSAYS TO ALLOW FOR BETTER DIAGNOSIS OF TREATMENT AND WHAT I'LL PROCEED TO NEXT IS ADDRESSING MAKING THE ASSAYS PARTICULARLY IN THE TRANSLATIONAL SENSE GUIDING THE BASIC SCIENCE AND PROCESSES HAPPENING BUT IN CLINICAL DECISION UNDERSTANDING THE PATHOPHYSIOLOGY AND NEED INFORMATIVE AND REPRODUCIBLE ANALYSES AND THAT'S WHEN YOU TAKE A BLOOD TAM PAL IN A CANCER PATIENT IS VERY APPEALING TO BE ABLE TO LEARN LEARN WHERE THE PATIENT DOESN'T HAVE TO UNDER GO SURGERY OR INTERVENTIONAL PROCEDURE BUT IN THAT BLOOD SAMPLE WE MAY BE ABLE TO QUIN FER THINGS WITH THE TUMOR OR THE OTHER SYSTEMS INVOLVED. THIGHS ARE BROAD CLASSES OF LIQUID BIOPSIES APPROACHES WHEN IT COMES TO EXTRACELLULAR VESICLE TYPE ANALYSES. I WANT TO EMPHASIZE WE'VE IMPROVED AND THE FIELD IS DOING BETTER AT THESE TESTS. WHEN YOU LOOK AT THE ACCURACY OF PRECISION IN ALMOST ALL MANUSCRIPTS YOU PICK UP YOU NEED TO BE AWARE OF THE FACT THAT THE TOOLS WE HAVE ARE NOT YET EXTRAORDINARILY ACCURATE. SOME OF THE TOOLS WE USE MAY BE ABLE TO SEE A REPRODUCIBLE METHOD WI METHOD BUT WE DON'T HAVE VERY ACCURATE TOOLS THAT CAN CIRCUMSCRIBE THE EVs WITH GREAT PRECISION AND CLARITY YET AT LEAD IN THE PUBLISHED LITERATURE TO DATE. AND WHEN YOU TAKE A BLOOD SAMPLE WHAT IS IN THAT BLOOD SAMPLE? IN GENERAL, YOU MIGHT GET 10 OR SO COPIES OF CIRCULATING TUMOR CELLS IN A BLOOD BASED ASSAY. EVs, EXTRACELLULAR VESICLES PRODUCED BY THE TUMOR CELLS ARE BY OTHER TISSUES OF INTEREST. WE THINK ON THE ORDER OF A MILLION TO A BILLION PER MILLILITER AND SOUNDS LIKE A LOT AND SOUNDS WHEN YOU ISOLATE THINGS OF THAT CISE YOU SHOULD BE ABLE TO DO A GOOD JOB OF THAT AND ALSO IN THE BLOOD IS SOMEWHERE BETWEEN UP TO A BILLION EV LIKE PARTICLES AND CO CO-ISOLATES AND IF YOU'RE FAMILIAR WITH THE FIELD YOU'LL KNOW THIS EXAMPLE WELL. A PROMINENT MOLECULAR TESTING COMPANY DECIDED TO DO PILOT STUDIES AND LOOK INTO URINE IN MEN SCREENING FOR PROSTATE ANSWER IS AND WANTED TO SAY CAN YOU ISOLATE AN EXOSOME POPULATION WITH RNA WITH A SIGNATURE OF RISK FOR PROSTATE CANCER. THIS URINE TEST REPLACED BIOPSIES. THE URINE IS COLLECTED IN THE CLINIC AND CAN SIT IN THE RESTRING RATER FOR AN EXTENDED PERIOD OF TIME AND DAYS AND USE THE CLARIFICATION METHOD AND HAS A THREE GENE OR RNA SIGNATURE. THE USEFULNESS OF THE SIGNATURE HAS BEEN SHOWN AND UNDER GONE THROUGH CLINICAL VALIDATION SO FAR IN TERMS OF SHOWING A CORRELATION SHOWING COMPARED TO STANDARD OF CARE THAT USING THE GENE EXPRESSION ASSAY PLUS THE ST STANDARD OF CARE SIGNIFICANTLY IMPROVES THE OPERATING WAY IN A WAY THAT'S USEFUL AND WHEN YOU LOOK AT A WATER FALL PLOT OF THE PATIENTS IN THAT STUDY IT SUGGESTS THERE'S A GROUP OF PATIENTS WHO MAY ABLE TO AVOID A BIOPSY. WHY DO I MAKE THIS EXAMPLE? QUICKLY THEY REALIZED ACTUALLY THEY HAD STARTED WHAT WAS THEY THOUGHT WAS AN EXOSOME ISOLATION METHOD AND THEN REALIZED THE SIGNATURE THEY WERE SEEING DID NOT COME FROM EVs BUT ASSOCIATED WITH COISO LATES AND SOMETHING WHEN YOU PICK UP THE LITERATURE AND SOMEBODY REPORTS A MARKER AND SAYING IT'S ASSOCIATED WITH THIS OR THAT AND SAY IT'S AN EV MARKER, I THINK THERE'S SOME CAUTION WARRANTED IN INTERPRETING IT AS AN EV MARKER. IT'S A USEFUL MARKER BUT MAY NOT ACTUALLY BE AN EV MARKER. TO TRY TO MAKE THIS A LITTLE BIT MORE OF A CAREFUL APPROACH, WE STAYED WITH THE PROSTATE CANCER EXAMPLE AND THIS SAY PATIENT WHO HAD -- IS A PATIENT WHO HAD WIDELY METASTATIC PROS KATE CANCER AND YOU CAN SEE THE AREAS LIGHTING UP ON THE PET SCAN AND KNEW BECAUSE HE HAD PET SCANS THAT THE TUMOR WAS PSMA, POSITIVE SPECIFIC MEMBRANE ANTIGEN POSITIVE AND HYPOTHESIZED WE COULD SEE AN RNA SIGNATURE THAT RELATED TO PROSTATE CANCER AND SEE IT MORE ON THE PSMA POSITIVE EVs. COMPARED TO UNFRACTIONATED EVs AND WE FOUND THAT THE PSMA POSITIVE EVs DO HAVE A VERY STRONG SIGNATURE SET OF RNAs ASSOCIATED WITH ADVANCED MALIGNANCY AND MOST OF THOSE RNAs WOULD NOT HAVE BEEN IDENTIFIABLE IN THE UNFRACTIONATED EV SAMPLE. MEANING, JUST SIMPLY DOING AN EV PURIFICATION WOULD NOT ALLOW YOU TO SEE IT EASILY AND THIS IS THE ONE TAKE HOME PATIENT FOR PEOPLE WHO SAY I WANT TO STUDY EVs IN DISEASE X, Y AND Z. WHEN YOU PICK UP THE LITERATURE, ONE SEEMS TO INDICATE EVs ARE A GREAT SOURCE FOR OMIC MATERIAL AND FOR RNA SEQ AND OTHER METHODS TO STUDY THE EV OMICS. IT TURNS OUT MANY STUDIES DONE LACK THE RIGOR TO CLAIM OR DEMONSTRATE WITH THE STUDIES THEY'VE DONE THE RESULTS ARE ACTUALLY FROM EV. RECORDING IT FROM THE EV RESULT IS LEADING TO INACCURACY AND UNDERSTANDINGS IN THE LITERATURE. THE OTHER POINT OF CAUTION IS THAT MOST CONSUMER KITS ARE DRIVEN BY THE CONSUMPTION OF THE KITS AND I SHOWED MANY OF OUR METHODS ARE PRECISE AND COULD BE REPRODUCED CONSISTENTLY. IF YOU USE THE SAME KIT OVER AND OVER AGAIN YOU MAY GET THE SAME RESULT. BUT IT TURNS OUT IN SEVERAL STUDIES IF YOU USE DIFFERENT KITS WHICH ALL REPORTEDLY ISO LATE EVs ARE IN DIFFERENT WAYS THE ASSUMPTION YOU GET DIFFERENT RESULTS. YOU NEED GREAT CARE WHEN YOU USE KITS LIKE EXO THIS OR THAT AND WHEN YOU USE THE KITS YOU NEED TO DO YOUR DUE DILIGENCE TO EITHER SAY THIS IS MATERIAL THAT WAS IS ISOLATED OR SOME QUANTIFICATION OF WHAT THE MATERIAL ACTUALLY IS. SO, AS I MENTIONED BEFORE WE HAVE THE PROBLEM OF THE OVERLAPPING SIZE DISTRIBUTION AND HUGE NUMBER OF LIPO PROTEINS IN CIRCULATION. THIS HIGHLIGHTS DIFFERENCES IN DENSITY FOR STRATIFICATION OF DIFFERENT LIPO PROTEINS AND DIFFERENT VESICLES THAT COME FROM CELLS. AND WANTED TO SHOW AS BIOLOGISTS AND CLINICIANS WE HAVE A LOT OF TEST AND SKILLS AND CONFIDENCE IN THE TOOLS WE HAVE FOR DOING SMALL MOLECULE ASSAYS AND FOR STUDYING CELLS. IN THIS NANO MATERIAL SPACE IN BETWEEN, THE TOOLS ARE SOMETIMES QUITE GOOD FOR VERY HOMOGENUS TYPE OF MATERIALS FOR SPECIFIC CLEAR CUT MATERIALS BUT WE ARE SEVERELY LACKING TOOLS FOR ANALYZING THE COMPLEX EV PACKAGES IN THAT AREA. THAT WAS ONE OF THE MAIN FOCUSES OF OUR LAB IS TO GENERATE WAYS FOR US TO PRODUCE FOCUSSED ROBUST OMICS TYPE DATA. SO THE SMALLNESS OF THE EXTRACELLULAR VESICLES INCLUDING EXOSOME LIKE PARTICLES, THE SIZE IS ONE PROBLEM BUT THE SMALL SIZES THEN LEADS TO THE FACT THAT THERE ARE VERY FEW COPIES OF ANY MOLECULE ON THE SURFACE OF THOSE AND DETECTION OF THOSE CAN BE QUITE CHALLENGING. AND WHEN YOU'RE PROCESSING THEM IF YOU WANTED TO THINK ABOUT THE SIZE OF THE REGIONS. A VESICLE MAY BE APPROXIMATELY 70, 80, 100 NANOMETERS AND THE SIZE IS IN ISSUE AND YOU HAVE TO THINK ABOUT IF YOU'RE REMOVING DO YOU NEED TO REMOVE A PARTICULAR ASSAY. IF YOU'RE USING LIPIDS ARE THOSE IN EXTRACELLULAR VESICLES OR IN A LIPID LAYER AND WE MENTIONED QUANTITY. ONE THING I DIDN'T MENTION AND SPEAKS TO THE UNCERTAINTY OF THE FIELD IS THE NUMBERS I QUOTED TO YOU HAVE TO BE SORT OF PRESENTED WITH A CAVEAT OF A BUFFER OF IN CERTAINTY OF PLUS OR MINUS A COUPLE ORDERS OF MAGNITUDE. WHEN WE REPORT CELL NUMBERS FROM A BLOOD TEST OR OTHER THINGS, WE HAVE SOME CONFIDENCE WHAT THAT NUMBER ACTUALLY MEANS. THE TOOLS ARE SO PRIMITIVE WE'RE USING AT THE MOMENT THAT IF YOU LOOK HISTORICALLY OVER THE PAST 10 YEARS, THERE'S A GREAT RANGE IN THE NUMBERS OF REPORTED PARTICLES BECAUSE THE INSTRUMENTS AND METHODS HAVE VARIED. TRYING TO ADDRESS THIS AND WHAT WE QUICKLY REALIZED IS THE SCOPE OF THE PROBLEM THAT WE WERE FACING FELT TO US A LITTLE BIT LIKE WHAT IT FELT TO THE FOLKS AT NASA WHERE YOU KNOW WHERE YOU WANT TO GO, YOU WANT TO GET TO SPACE. WE WANT TO GET TO TRANSLATIONAL ASSAYS AND EVs AND KNOW WHAT IT MEANS AND WANT TO INTERPRET IT IN A MEANINGFUL WAY TO TRANSLATE DISEASE BUT NEED TO START AT A BASIC LEVEL IN THE SAME WAY TO GET TO SPACE THEY HAD TO START WITH BASIC RESEARCH, COME UP WITH PROTOTYPES AND TESTS AND PROOF OF CONCEPTS AND TEST THEM IN THE LAB AND TEST PROTOTYPES, BUT THIS IS REALLY IMPORTANT BECAUSE IF WE WANT TO GO INTO THIS EXPLORED SPACE, WE NEED TO DO THESE STEPS. SO IN THINKING ABOUT HOW THIS PROCESS LOOKS IN THE CONTEXT OF APPROACHING EV RESEARCH, I'VE ADAPTED THIS TO ULTIMATELY WANT TO GET TO CLINICAL USE OF PROVEN ASSAYS. BUT WE'RE BUILDING THROUGH THE BASIC RESEARCH UP TO AT THIS POINT WORKING ALSO ON RETROSPECTIVE COHORTS. THE GOAL OF THIS IS TO IMPROVE THE LIVES OF OUR PATIENTS AND TO BE ABLE TO DO THIS KIND OF THROUGH AND THROUGH GROUND UP TRANSLATIONAL WORK IN AN ENVIRONMENT WHERE IT'S POSSIBLE AND VERY DIFFICULT TO DO ON THE OUTSIDE. I'M SHOW SOME BASIC METHOD OF PIPELINE DEVELOPMENT AND THEN GET INTO TRANSLATIONAL CLINICAL ASSAYS HIGH THROUGHPUT COMPATIBLE. I'VE EXPLAINED THE CHALLENGES OF SMALL EVs WITH SMALL EPITOPES AND THE HETEROGENEITY OF THE THINGS WE WANT TO STUDY AND THE LABELS WE USE OR THE LIPO PROTEINS THAT COME WITH THEM. AND SORT OF EMPHASIZE WE NEED NEW NANOTECHNOLOGIES FOR THE RESEARCH AND NEW COMPATIBLE METHODS. SO FOR THE PROGRESS ON THE BASIC FRONT, SOME OF OUR EARLIEST WORK WAS HELP BY USING VIRUSES OF THE PROTOTYPES OF THE SAMPLE TYPES. AND HIV IS ROUGHLY THE SIZE OF AN AVERAGE 100 NANOMETER VESICLE. THIS IS WORK WE DID OVER A NUMBER OF YEARS LOOKING AT WHAT IS ACTUALLY HAPPENING IN FLOW CYTOMETRY AND WHAT HAS BEEN DESIGNED FOR CELLULAR WORK AND WHAT IT IS WE CAN SEE AND NOT SEE WITH THAT INSTRUMENT AND FOUND THAT THERE ARE EV POPULATIONS THAT WE CAN DISCRIMINATE ABOVE THE INSTRUMENT NOISE HERE AND WE DISCOVERED THAT IN MANY FLOW CYTOMETRY THE LASER HITTING THE FLUIDICS IS ENOUGH TO CREATE THE BACKGROUND NOISE YOU NEED TO MONITOR AS YOU'RE STUDYING IT. AND WE CAN TEST THE QUESTIONS NOT ONLY ARE WE ABLE TO SEE THE DIFFERENT POPULATIONS BUT CAN WE SORT THE POPULATIONS AND IN SORTING TO THE PARTICLES MAINTAIN FIDELITY. SO IF WE HAVE A GREEN OR RED LABELLED VIRUS AND MIX THOSE TWO SAMPLES AND SORT OF SAVE THE RED OR THE GREEN THAT THOSE ARE SORTED WITH FIDELITY AND WE DID CRISSCROSS TROPISM STUDIES WITH CELL LINES TO DEMONSTRATE THE SPECIFIC HIV VARIANTS WERE ABLE TO INFECT JUST THEIR COMPATIBLE PARTNER CELL LINE AND NOT THE OTHER. WE WERE ABLE TO DEMONSTRATE THE SORTING DOES WORK AND THE PARTICLES MAINTAIN THEIR STRUCTURAL INTEGRITY AND SO THIS HAS BEEN A USEFUL SIDE ENDEAVOR FOR US TO LOOK AT VIRUSES. AND THEN WITH COVID WE ALSO REALIZED THAT SOME OF THE TOOLS THAT WE HAD BEEN DEVELOPING WERE IMPORTANT FOR THE RESEARCH COMMUNITY SO WE MADE AN EARLY EFFORT TO SHARE EVERYTHING THAT WE HAD WITH THE VIRAL RESEARCH COMMUNITY TO FACILITATE VARIABILITY TO STUDY QUESTIONS ABOUT VIRAL VARIANTS. AND FOR THIS WE WERE ABLE TO CREATE A WEBSITE WHICH IS COMPLIANT WITH THE FEDERAL REPORTING OF REQUIREMENTS AND THIS HAS BEEN A GREAT HELP TO US WITH SHARING THE INFORMATION WITH THIS COMMUNITY. JOSHUA WELSH IN OUR LAB HAS DEVELOPED A NUMBER OF SOFTWARE TOOLS, SOME OF WHICH I'LL TALK ABOUT A LITTLE BIT THAT MAKE THESE KINDS OF ASSAYS MORE RIGOROUS, MORE REPRODUCIBLE AND ALSO CALIBRATED AND SHAREABLE IN A MEANINGFUL WAY TO BEGIN TO DATABASE AND COLLECT DATA IN A WAY THAT WILL BE INFORMATIVE MORE BROADLY TO THE COMMUNITY DOWN THE ROAD. WE ALSO HAVE ON THIS WEB PAGE TUTORIALS EXPLAINING THE MORE DIFFICULT TOPICS. WE ALSO, AS I MENTIONED FOR THE SAKE OF THE VIRUS RESEARCH COMMUNITY ALSO WE MADE PROTOCOLS AVAILABLE. SO WE HAVE A LINK TO OUR WEBSITE A WHOLE SET OF PROTOCOL LINKS TO ACCESS WHAT WE DO IN THE LAB. SO JUST TO INTRODUCE REALLY WITH AN EASY TO SEE VISIBLE EXAMPLE WHY DOES THE LIMIT OF DETECTION MATTER IN AN INSTRUMENT AND THE FOCUS ON WHAT YOU CAN AND CAN'T RESOLVE? THESE ARE TWO MATCHED SAMPLES RUN ON TWO DIFFERENT INSTRUMENTS. ONE IS SPECIFICALLY DESIGNED FOR NANOFLOW CYTOMETRY AND THE OTHER DESIGNED FOR LOOKING AT CELL. THIS IS WHERE WE WERE ABLE TO SORT HIV POPULATIONS INTO THEIR DIFFERENT TYPES AND MAINTAIN THEIR INTEGRITY. THIS IS SHOWING THAT IF YOU HAVE EVs FROM BONE MARROW DERIVED DENDRITIC CELLS AND LOOK AT THE POPULATION USING CLASS 2MHC YOU CAN'T DRAW THE LINE BETWEEN WHAT IS NEGATIVE AND POSITIVE AND WITH AN INSTRUMENT WITH DETECTION YOU SEE THIS HOMOGENOUS LOOKING POPULATION THAT IS FAIRLY DIM DIVIDES INTO POPULATIONS OF HIGHER, INTERMEDIATE AND LOW MCH ABUNDANCE ON THE EVs AND WE LOOKED CANCER EVs TA EXPRESSES PSMA AND YOU CAN'T DRAW A LINE BETWEEN THE POSITIVE AND NEGATIVE WHERE IT BECOMES EASIER TO DO ON AN INTERRUSTRUMENT THAT IS ABLE TO DETECT A PE MOLECULE. SO WHAT WE DISCOVERED EARLY ON IS THAT IF WE WANTED TO ENUMERATE THE VESSELS WE'RE LOOKING AT AND COUNT THE PSMA POSITIVE EVs PER MILLILITER IT REQUIRES YOU REQUIRE EVERY VESSEL THAT HAS EVEN ONE COPY OF PSMA ON IT. AND CONVENTIONAL FLOW CYTOMETERS WORK WELL AND YOU CAN LABEL ANTIBODY WITH THE GOLD AND SILVER BECAUSE THERE'S A PLASMA THAT LETS US DETECT JUST ONE WHEREAS WHEN WE DID THE CALCULATION TO SEE HOW MANY ANTIBODIES THE FLUORESCENT LABELS NEEDED FOR THE CONVENTIONAL FLOW CYTOMETER TENDED TO BE 2,000 LABELS NEEDED FOR DETECTION. AND THIS JUST DEMONSTRATE SOME OF THE FUN SPECTRAL PROPERTIES OF SILVER AND GOLD THAT MAKES THIS POSSIBLE. SO THIS IS SHOWING YOU A SILVER TO GOLD RATIO AND PLOTS FOR GOLD HEREBY AND SILVER HERE -- GOLD HERE AND SILVER HERE. YOU CAN SEE WHERE THERE'S THIS TALL BLUE ARROW AT ABOUT 405 NANOMETERS FROM THE LASER, YOU SEE A VERY CLEAR PEAK OF SCATTERED ABSORBED LIGHT WHERE YOU CAN SEE SILVER QUITE WELL AND LESS SO GOLD AND IT FLIPS AND THEN YOU CAN SEE THE GOLD BETTER BETWEEN 60 AND THE SILVER DIPS DOWN. SO WHAT THAT MEANS, THIS IS SHOWING DOT PLOTS OF SINGLE SILVER OR GOLD PARTICLES. THE SILVER, AND GOLD IN RED DETECTED AT 405 NANOMETERS FROM WAVE LENGTH AND THE OTHER MEASUREMENTS AND YOU CAN SEE THOUGH THE PARTICLES ARE STRICTLY SPEAKING THE SAME SIZE, THEY LOOK DIFFERENT AND HAVE DIFFERENT SCATTERED UNITS AT THE DIFFERENT WAVE LENGtTHS WITH THE PROPERTIES AND WE HAVE INSTRUMENTS TO FACILITATE SEEING THIS AND JOSH WHO HAS BEEN DEVELOPING OTHER INTERFACES FOR OTHER TYPES OF NANO MATERIAL MEASUREMENTS TO BE ABLE TO LOOK AT THE DATA MORE RIGOROUSLY. WINNING OF THE THING WE KNOW IS IF YOU DON'T REMOVE THE UNBOUND LABELS IT CAN BE DETECTED WITH THE EVs AND GIVE A FALSE POSITIVE SIGNAL. IN MANY CASES YOU NEED TO REMOVE THE UNBOUND LABEL TO HAVE A VALID SIGNATURE. SO WE DEVELOPED A WAY TO WAY TO REMOVE UNBOUND LABELS FROM EVs WHEN WE DO STAINING AND IF YOU WANT TO SEE THAT PROTOCOL AND SEE FIT HELPS YOUR -- IF IT HELPS YOUR PROCEDURES, THAT'S THERE. SO IN ALL OF THIS, THERE'S SO MANY TOOLS FOR SORTING AND FOR SUBSET SEPARATION AND PURE -- PURIFICATION AND WE MADE THE TOOLS AVAILABLE FOR PEOPLE. THIS REALLY CONSTITUTES THE BEGINNINGS OF THE VERY BASIC PARTS OF THE TECHNOLOGY. ONE THING WE THEN REALIZED IS WHEN YOU ARE LOOKING AT EVs FROM A SYSTEM OR EVEN IN A SIMPLE SYSTEM, THAT SAMPLE TYPE IS MUCH LESS COMPLEX AND MUCH LESS DIFFICULT TO TEASE OUT PARTICULARLY WHEN THE CELL TYPE IS LESS COMPLICATED TO TEASE OUT THAN THE BIO FLUID. WHAT WE REALIZED WHEN WE WENT TO SAMPLES IS WE NEEDED TO TIE THAT TOGETHER WITH A MULTIPLEX ASSAY OF DETECTION TO ALLOW US TO DO EV MARKER REPERTOIRE PROFILING. WE EXPANDED THE SCOPE OF THE PIPELINE TO RUN THESE TWO APPROACHES IN SOME WAY SIDE BY SIDE AND BACK TO BACK. SO THIS IS THE FLOW OF THE PIPELINE WHEREAS I MENTIONED WE DEVELOPED BETTER WAYS TO DO SIMPLE PROCESSING AND THE CONTE CONTEXT ANALYSIS. THIS IS THE EXAMPLE OF THE MULTI TEX REPERTOIRE PROFOILING -- PROFILING APPROACH AND IF YOU WANT TO SEE MORE OF THE DATA, WE HAVE THIS UNDER REVIEW RIGHT NOW. WHAT YOU CAN SEE IS WHEN WE DID THIS ASSAY AND LOOKED AT THE DIFFERENT MARKERS FOR THE CAPTURE AND DETECTION, YOU CAN SEE CLUSTERING CLEAR SIGNATURES WHEN THE SAMPLE WAS SORT FROM CSF AND THEY WERE TIGHT AND DISTINCTIVE FROM PLASMA AND SERUM. YOU CAN SEE THAT WHEN WE ANALYZED IT AND THERE WERE DISTINCT MARKER REPERTOIRES AND PROFILES AND CAN SEE AN INTERESTING SPLIT IN TERMS OF THE IN TERMS OF THE PURIFICATION METHOD AND WE ALSO HAVE A METHOD OF BASICALLY REMOVING UNBOUND LABELS OR REMOVING CONCURRENT PROTEINS AND WHAT WE FOUND IS THAT THAT METHOD OF USING THE RESIN TO REMOVE THE UNBOUND PROTEINS IS MUCH MORE SIMILAR TO THE PROFILES WE WOULD SEE IN THIS ALONE AND USING THE SIZE EXCLUSION COLUMN KIND OF TOSSES THE SURFACE MARKER PROFILE TO IT'S OWN CORNER HERE. AND REALIZED WE CAN ALSO FLIP THIS AROUND AND LINK THIS DATA TO THE PROFILES OF WHERE THOSE MARKERS ARE COMING FROM IN TERMS OF THEIR ASSOCIATED PHENOTYPES AND ORIGINS OF THE REPERTOIRE BEING DISPLAYED AND I HAVE A WEBSITE ON THE EV RESOLUTION AND THE TRANSLATIONAL EV STUDIES AND THIS NEW PIPELINE WE'RE STITCHING TOGETHER TO LOOK AT THE SINGLE EV STUDIES AND MULTI PARAMETRIC EV STUDIES. SINCE THE PURPOSE OF THIS TALK FROM THE BEGINNING IS TO PUT THE PROGRESS ON THE CLINICAL FRONT AND HOW WE'RE ABLE TO SEE WHAT'S HAPPENING WITH OUR PATIENTS? I'M GOING TO START WITH A COUPLE EXAMPLES OF CLINICAL CASES AND WHAT IS RESPONDING AND NOT RESPONDING AND AS A RADIATION ONCOLOGY THERE'S SOMETHING EXTRAORDINARY YOU LEARN IN TRAINING BUT ALMOST NEVER IN YOUR CAREER GET A CHANCE TO SEE. BUT INCREASINGLY IN THE CONTEXT OF IMMUNOTHERAPY AND HERE AT NIH WHERE IMMUNOTHERAPY IS A COMMON AREA OF STUDY, WE HAVE SEEN SOME CASES WHERE WE WILL IRRADIATE ONE AREA AND THE REMAINING AREAS OF DISEASE WILL MARKEDLY REGRESS AND THAT'S AN ABSCOPAL AFFECT. OUT OF THE FIELD OF RADIATION. SO YOU RADIATE ONE AREA AND THE OTHER SITES SHRINK. AND USUALLY THIS HAPPED -- HAPPENED IN THIS PATIENT WITHIN THE FIRST FIVE DAYS AND YOU DON'T USUALLY IMAGE AND RE-IMAGE SO FREQUENTLY DURING RADIATION YOU WOULDN'T SEE THEM BUT IN HER CASE THESE WERE CUTANEOUS LESIONS SO WE COULD SEE IT BUT DIDN'T KNOW THE MECHANISM AND WOULD LIKE TO KNOW THE MECHANISM SO WE CAN DRIVE THIS KIND OF FAVORABLE RESPONSE IN OTHER TREATMENTS. SO WHAT I'M SHOWING HERE IS A TIME LINE OF THREE OF THE TREATMENTS THAT WE DID FOR HER. THE FIRST IS WHEN SHE HAD THAT ABSCOPAL RESPONSE AND CAME BACK FOR TWO ADDITIONAL TREATMENTS WHERE SHE HAD RESPONSES IN THE FIELD BUT NOT OUT OF THE FIELD. THIS IS DOCUMENTING HOW MUCH THE OUT OF FIELD TUMORS SHRINK. AND MORE THAN 70% OF THE ONES THAT WERE OUT OF FIELD SHRANK WHICH IS REMARKABLE IN FIVE DAYS OF RADIATION. THE IL OF WENT UP AND IL8 WENT UP AND IL10 WENT DOWN. THERE'S NOT AN IMMUNE CELL TYPE WE CAN POINT TO WE CAN SAY IS RESPONSIBLE FOR THIS BUT WHEN WE LOOKED AT THE EVs AND DID THIS KIND OF REPERTOIRE PROFILING WE USED THE SAME CAPTURED MULTI PLEX APPROACH WITH ADDITIONAL ANTIBODIES AND HAD A MASSIVE SCREEN AND HER OF EVs DURING THE SAM PAZ -- SAMPLES WERE VERY DIFFERENT FROM THE EV REPERTOIRES DURING THE STANDARD RESPONSE TO TREATMENT OR EV. YOU CAN SEE THE KIND OF THING WE'RE HOPING TO BE ABLE TO LEARN TO SEE IN THESE EV POPULATIONS AND BUILD ON TO MORE INFORMATIVE ASSAYS. SO WHAT YOU CAN SEE DURING THE AB SCOPAL RESPONSE IS THERE'S A SHIFT TO THESE INNATE RESPONSES AND WHAT WAS ABSENT IS SIGNIFICA SIGNIFICANT T CELL RESPONSE OR MEMORY SIGNATURES AND GOES WITH THE FACT SHE DOESN'T HAVE PERMANENT ANYONE IMMUNE RESPONSE AND THIS IS A STUDY WITH ANALYSIS AND LOOKING AT WHETHER OR NOT WE CAN SEE THE DIFFERENCE BETWEEN RESPONDERS AND NON-RESPONDERS EARLY IN THE TREATMENT COURSE. WHEN TREATMENT STARTED COURSE 1 DAY 0 AND LOOKED AT ONE DAY 15 AND COURSE THREE DAY ONE. COURSE THREE DAY ONE MOST END POINT STUDIES WILL LOOK FOR RESPONSES AT THAT POINT AND WANTED TO SHOW IF THE EVs COULD SHOW SOMETHING. IT'S REASONABLE TO THINK THEY MIGHT. THAT'S WHEN WE THINK THE BIOLOGICAL RESPONSE IS GETTING STARTED. IN FACT YOU CAN SEE THEM SEPARATE INTO RESPONDERS AND NON-RESPONDERS. WE LOOKED AT THE SAEASSAYS AND WE SEE MORE ADVANCED MULTI PLEX ASSAYS TO SEE IF WE'LL SEE THIS ACROSS THE COURSE OF TREATMENT RESPONSE VERSUS NON-RESPONSE. I'LL SHOW YOU A COUPLE EXAMPLES OF DISEASE. FIRST IS A STUDY LOOKING AT SPECIFIC RENAL CELL ASSOCIATED MARKERS WE CAN USE TO PULL OUT REAL CELL ASSOCIATED EVs AND WE IDENTIFIED A CLUSTER OF ADDITIONAL MARKERS THAT WE DO HOPE TO DO FURTHER STUDIES WITH TO LOOK AT WHAT RNA AND OTHER COMPONENTS ARE ASSOCIATED WITH THESE MARKERS THAT WE HAVE FOUND TO BE RELATIVELY OVER EXPRESSED IN THESE UNIQUE CANCER CELL LINES FROM OUR GROUP. WE ALSO HAVE HAD THE PRIVILEGE TO BE ABLE TO LOOK AT SUBSETS OF CSF AND SAMPLES OF CSF AND THOSE SAMPLES HAVE GIVEN US CLEAR SIGNATURES OF DISTINCTIVE REPERTOIRES FROM SERUM. THIS IS A COUPLE OF SETHS -- SETS OF PATIENTS WITH HEALTHY VOLUNTEERS AND MULTIPLE SCLEROSIS AND A MYELOPATHY AND CAN YOU SEE THE SPECIFIC CORRELATIONS BETWEEN THE MARKER COMBINATIONS ARE FAIRLY DISTINCTIVE BETWEEN THE GROUPS. THERE'S SOME OVERLAP BUT THERE'S ALSO DISTINCTIVENESS. THAT BEGINS TO IDENTIFY FOR US WHICH ARE THE MARKERS OF INTEREST. DO WE WANT TO LOOK AT THE RELATIVE CARGO OF THIS DISEASE AND THIS PATHOLOGY AND THIS CORRELATIVE REPERTOIRES THIS PATTERN IS SUGGESTING WE GO WITH THE EVs OF THESE AND CARGO OF THOSE. I FEEL WE'RE AT THE 1960s COMPUTATIONAL LEVEL OF PUNCH CARDS BUT THIS IS THE BEGINNINGS OF BEING ABLE TO SEE THE LANDSCAPE OF THE DIFFERENT DISEASES THAT WE'RE LOOKING AT SO THAT WE CAN THEN GO AND LOOK AT HOW TO FURTHER LOOK AT THE CARGO IN THOSE DISEASES. I MENTIONED BEFORE AROUND I'LL BRING UP ONE SLIDE AGAIN, WHAT WE THINK IS GOING TO BE MOST INTERESTING IS THE REST OF THE PACKAGE WHEN HAVE YOU A MARKER THAT PULLS OF EVs FROM AN ORGAN OR CELL TYPE OR RESPONDING TISSUE AND AS THE CARGO AND THE REST OF THE MATERIAL CARRIED WITH IT WE THINK WILL BE INFORMATIVE PART OF THE INFORMATION AND THE REASON FOR THAT IS BECAUSE THESE PACKETS CARRY SO MANY DIFFERENT TYPES OF INFORMATION. RNAs AND DNAs AND LIPO PROTEINS AND LIPIDS AND THIS IS THE BEGINNING OF GETTING A HANDLE ON THOSE AND WE'RE WORKING WITH A COLLEAGUE IN BOSTON WHO HAS A TECHNIQUE FOR LOOKING AT MARKERS INSIDE THE CELL PENETRATING PEPTIDES AND WE MENTIONED SOME LABELS LOOKING AT OPTIMIZING AND BASICALLY THIS IS GIVING US A WAY OF APPROACHING EVs INSIDE AND OUTSIDE IN THESE WORK FLOWS. WE REALIZE WE HAVE A LOT OF DIFFERENT TYPES OF DATA BEING PRODUCED FROM ALL OF THESE DIFFERENT INSTRUMENTS, ALL THESE DIFFERENT DATA TYPES AND A WAY TO COMBINE THAT AND IN A WAY THAT NOT COMBINE THAT FROM WITHIN OUR GROUP BETWEEN DAY TO DAY AND INSTRUMENT TO INSTRUMENT, BUT A WAY TO CON BINE IT BETWEEN GROUPS. THERE'S A GROUP AT BAYLOR WE'RE WORKING WITH AS PART OFF THE U GRANT COMMON FUND PROJECT AND ANOTHER GROUP AT BAYLOR AND THEY HAVE DEVELOPED AN ATLAS AN EXTRACELLULAR RNA AXIS WERE WHERE THEY ABLE TO ADD IN SAMPLES AND THE CYTOMETRY PORTAL TO ADD IN THAT DATA AS THE BEGINNINGS OF WHAT IS NEEDED TO HAVE AN EV DATA STRUCTURE FOR ALL OF THE DIFFERENT TYPES OF EV DATA. WITHIN NCI, WE'RE DEVELOPING TOOLS AND PROTOCOLS IN DATA ALL OF WHICH IS BEING SHARED AND ALL THIS ANALYSIS IS ALSO BEING HELP BY THE GROUP IN THE CANCER DATA SCIENCE LAB FOLKS. AND NOT ONLY MANAGED SOFTWARE BUT INCORPORATED WAY TO CALIBRATE THE DATA. RATHER THAN CONVERTING EVERYTHING INTO STANDARDIZED CALIBRATED DATA TO COMPARE APPLES TO APPLES COMPARISONS. AND SHOULD GET AN ACADEMIC USE ON THE WEBSITE WHICH NOT ONLY PROVIDES OUTPUT BUT IS CLEAR ABOUT THE CALIBRATION AND CALIBRATION AND INSTRUMENT OPTIMIZATION. THIS WILL RADICALLY HELP DATA ACROSS THE GROUPS IN OUR FIELD. AND SHOULD BE HELPFUL FOR MANY OMICS IN MANY FIELD. I'VE SHOWN WE CAN DETECT TUMOR EVs AND THE MORE PRECISE YOU CAN BE THE MORE CLARITY YOU HAVE ON WHAT THE DATA NEEDS AND THIS CAN HELP UNDERSTAND NEW BIOLOGICAL MECHANISMS AN TARGETS. FUTURE DIRECTIONS ALL THIS INFRASTRUCTURE HERE IS LEADING AS I MENTIONED EARLY ON TO BEING ABLE TO DO THESE TRANSLATIONAL ASSAYS. ALSO WANTED TO BACK IT UP AND BE ABLE TO LOOK IN A BASIC WAY DISSECTING THE BEST WAY TO UNDERSTAND DISEASE AND WHAT'S HAPPENING AND IMPROVE OUR PATIENT'S RESPONSES TO TREATMENT. AS YOU KNOW, AS A RADIATION ONCOLOGIST, WE LIKE TO SEE RESPONSES QUICKLY. AND WE HAVE THE DESIRE TO KNOW AND AND WE CAN LOOK AT THE S PRODUCTS AND DEFINE THE Es FROM THE MICE IN THE SAME WAY AND WE STARTED TO LOOK FOR RADIATION SPECIFIC MARKERS AND THIS IS KIND OF EXCITING BECAUSE WE WHAT WE ARE HOPING TO ENABLE IS QUE AND WHERE YOU RADIATE ONE TUMOR YOU ARE INTERESTED IN THE OTHER SITES OF TUMOR. IF YOU HAVE EVs YOU CAN ISOLATE BY A TUMOR MARKER AND RADIATION MARKER YOU CAN SAY THIS IS CHANGING THE RADIATED TUMOR AND CHANGING OR NOT CHANGING IN THE UNIRRADIATED TUMOR AND WITHOUT HAVING TO DO BIOPSIES. WE'RE BUILDING ON THAT TO LOOK INTO THE WINDOWS INTO SYSTEMS BIOLOGY AND IT'S OBVIOUSLY NOT JUST RELATED TO CANCER AND RADIATION BUT ALSO DISEASE AND RESPONDER AND NON-RESPONDER SITUATIONS ANY CASE OF PATIENTS BEING CENTER AT THE CLINICAL CENTER OR ELSEWHERE IN MEDICINE. I JUST WANT TO COME BACK TO AND THANK AGAIN NCI FOR MAKING ALL THIS POSSIBLE AND I THINK IT WILL HELP ANSWER WHAT IS WRONG AND HOW TO HELP AND MAKE THINGS BETTER. THIS IS THE WHY I STARTED WITH. WHY WE CAME. I WANT TO CIRCLE BACK TO THE QUOTE WE STARTED WITH. IT DOESN'T JUST END WITH AND DON'T FORGET WHEN YOU LEAVE WHY YOU CAME BUT ALSO BECAUSE GOOD FRIENDS AND I HAVE TO THANK ALL THEE -- THE PEOPLE WHO MADE THIS POSSIBLE AND IT'S A MASSIVE GROUP INCLUDING MY ADVISING COMMITTEE AND PATHOLOGY AND RADIATION ONCOLOGY AND THE MALIGNANCY BRANCH AND CORNELL AND IN MY LAP WE HAVE [LISTING NAMES] AND ALL THE PEOPLE WHO HELPED IN THE EARLIER YEARS WITH THE DATA AND TOOLS WE'VE DEVELOPED. I THINK WITH ALL OF THAT, I'LL TAKE ANY QUESTIONS. >> THANK YOU FOR THAT. WE HAVE ONE QUESTION FROM THE AUDIENCE WE WANT TO ASK, IS THERE ANY RESOURCE WHERE WE HAVE INFORMATION ABOUT EXOSOMAL MICRO-RNAs IN DIFFERENT CANCERS? >> THAT'S INTERESTING AND SPEAKS TO THE EARLY MISCONCEPTION. THE RNA DATA I SHOWED YOU AND WE SPECIFICALLY LOOKED AT MICRO-RNA BECAUSE THE DOGMA AT THE TIME WAS EVs HAVE RNAs AND IT BECAME CLEAR MICRO RNAs TEND TO BE CO-ICE OWE -- CO-ISOLATES AND I POINT TO WORK DONE AT AMHERST. I THINK IN GENERAL THE MICRO-RNA SIGNATURES ASSOCIATED WITH CANCER ARE VERY INTERESTING AND VERY COMPELLING. I THINK AS WE LEARN MORE WHAT WE'RE GOING TO FIND IS MANY OF THOSE ARE REALLY ASSOCIATED WITH GLYC YO PROTEINS OR -- GLYCOPROTEINS OR OTHER PROTEIN MACULAR MOLECULAR PROTEINS AND THERE'S EXOMERES AND THERE'S EXTRA CELLULAR PARTICLES WE DON'T KNOW WHAT TO CALL YET AND THE MICRO-RNA TENDS TO BE CARRIED IN SOME OF THOSE AS WELL. WE DON'T YET HAVE THE HUMAN ATLAS PRESENT FOR PROTEINS IN DIFFERENT CELL TYPES. WE'RE HOPING OVER TIME, WE BEGIN TO INTEGRATE AND THIS IS WHY IT IS IMPORTANT PEOPLE START TO BE A LITTLE BIT MORE CON SEN -- CON CONSCIENTIOUS AND THINK WITH THE NOMENCLATURE TO NOT PROPAGATEMISUNDERSTANDING OF WHAT THE DATA MEANS. >> MY NAIVE QUESTION RELATED TO THE VESICLES THEMSELVES, I SUPPOSE AND MANY YEARS WHEN WE WERE STUDYING CELL BIOLOGY AND LEARNED VESICLES ARE WAYS FOR CELLS TO TRANSPORT STUFF SOME PLACE OR EXCRETE MAYBE BAD STUFF, FOR CANCER/TUMOR CELLS WHAT DO THE EXTRA CELLULAR VESICLES DO? WHAT ARE NAY -- THEY UP TO? >> THAT'S A GREAT QUESTION AND EARLY ON IN DOING PROTEOMICS WE SCRATCHED OUR HEAD AND DIDN'T FIND THE NICE FINDING MARKERS WIDE LIKE TO SEE BUT HAVE SEEN A LOT OF RELATED TO METABOLISM AND ENDOCYTOSIS AND PROCESSING OF PROTEINS. I THINK WE HAVE A MIND SET RIGHT NOW OF IDENTIFYING POPULATIONS BY PROTEINS THAT ARE IDENTIFYING REGARD TO THE ORIGINS OF THE EVs. AND I THINK BIOLOGICALLY THE EVs ARE PRODUCED WITH LESS CARE TO AN ADDRESS SIGNATURE OF WHERE THEY CAME FROM AND WHERE THEY'RE GOING AND MORE INFORMATION THAT'S RELATED TO THE FUNCTION OF THOSE EVs. LIKE SOME ARE RELATED TO TOSSING OUT TRASH. OTHERS ARE RELATED TO COMMUNICATING VERY SPECIFIC CARGO AND THIS IS REALLY THE FUN OF THE FIELD RIGHT NOW TO BEGIN TO DEFINE THESE AND DEFINING THEM BY FUNCTIONAL CLASSES IS NOT SOMETHING ON I FOCUSSED ON IN THE TALK TODAY BUT I THINK THAT'S WHERE WE'RE GOING TO GO IS THOUGH IT'S WONDERFUL TO BE ABLE TO PULL OUT BY ORGAN, I THINK THE FUN BIOLOGICAL AND TRANSLATIONAL AND MEDICAL INFORMATION'S GOING TO COME FROM THE FUNCTIONAL DATA. >> TERRIFIC. AND WE HAVE ONE FINAL QUESTION FROM THE AUDIENCE IS THERE WORK ON E ISOLATES FROM IS A LIE? >> -- SALIVA? >> THERE IS AND SOME ARE LOOKING SPECIFICALLY AT EVs AND SALIVA AND A GROUP AT UCLA AND THERE ARE SEVERAL LABS LOOKING AT THOSE KINDS OF THINGS. I THINK SALIVA PROBABLY LIKE CSF IS GOING TO BE A MORE CLEAR CUT WAY OF LOOKING AT EVs AND MICROBIOME IMPORTANT TOO BUT WE HAVEN'T STARTED ANY OF THAT. THERE'S GREAT WORK THERE. >> GREAT, OUR TIME IS NOW UP. AGAIN, THANK YOU FOR A FANTASTIC PRESENTATION. HIGHLY INFORMATIVE AND REALLY VERY CLEAR. HAVE A GREAT AFTERNOON. BYE-BYE. >> BYE.