>> OKAY, WELL, WE'LL GET STARTED AND WE HAVE A FEW ANNOUNCEMENTS TO MAKE. ONE, THE VIDEO CAST FROM LAST WEEK, SEPTEMBER 12th, IT'S NOW POSTED SO YOU COULD WATCH THAT IF YOU WANT TO. IT'S ON THE THE ARCHIVE. YOU CAN WATCH IT ON YOUR COMPUTER ANYTIME. THE WEB SITE IS HTT//VIDEOCAST.NIH.GOV. ALSO WE HAVE SOME OF THE POWER POINTS NOW POSTED THAT'S ON THE CCR WEB SITE THAT'S THE HTTP//CANCER .GOV. AND WE MENTIONED IN THE FIRST LECTURE THAT THE PROCESS OF CARCINOGENESIS, HAVE YOU SMALL CANCER CELLS LEADING TO TUMOR FORMATION, OUR SECOND SPEAKER TODAY,TZPhb ENRIQUE ZIDURI, WILL BE TALKING ABOUT ANGIOGENESIS, IT WILL HAVE SMALL TUMORS BECOME LARGE TUMORS AND THEN FROM LARGE TUMORS SOME CELLS FLAKE OFF AND AND THEY WORK IN THE BLOOD SYSTEM, AND SO WE HAVE KENT HUNTER OUR FIRST SPEAKER FOR BACKGROUND PURPOSES AND HE GOT A Ph.D. IN 1991 AT M. I.T. IN BOSTON, HE WAS AT THE FOX CHASE CENTER, HE'S STUDIED CANCER BIOLOGY AND GENETICS AND HIS THOUGHT TODAY IS SYSTEM GENETICS OF METASTATIC SUSCEPTIBILITY. KENT? OKAY, THANK YOU I GET MYSELF WIRED UP HERE. SO OBVIOUSLY MY LAB ARE TORSEINTERESTED IN THE TERMINAL STAGES OF CANCER METASTASIS AND THE REASON FOR THIS IS THAT YOU SHOULD KNOW, BEING HERE AT THE NCI OR THE NIH, THAT THE MAJORITY OF PATIENT WHO IS HAVE SOLID TUMORS, NOT HEMATO LOGICAL TUMORS BUT THE PATIENT WHO IS DIE OF CANC I--CANCER, 90% OF PATIENTS WHO HAVE TUMORS THAT ARE SOLID DIE OF METASTATIC TUMORS AND IF WE ACCOUNTED UNDERSTAND AND CONTROL THE THE METASTATIC PROCESS, WE HAVE A MUCH BETTER CHANCE OF HELPING PATIENTS WHO COME INTO OUR CLINICS, SO OBVIOUSLY, IF WE CAN PREVENT CANCER THAT WOULD BE THE BEST THING BUT EVEN--THERE'S ALWAYS GOING TO BE SOME PROPORTION OF THE POPULATION THAT WE DON'T PREVENT CANCER IN AND THEN ONCE WE HAVE THESE PATIENTS PREVENT THEM FROM PROGRESSING, WE'LL HAVE A MUCH BETTER CHANCE OF DOING WHAT IS OUR ULTIMATE GOAL WHICH IS TO HELP ALL OF THESE PATIENTS SURVIVE. SO JUST FOR FRAME OF REFERENCE, TERRY JUST KIND OF MENTIONED A LITTLE BIT OF THIS, THIS IS THE PROCESS OF METASTASIS, EVERYONE KNOWS IT BUT I LIKE TO SHOW IT JUST FOR A COUPLE REASONS AND ONE THAT'S BECAUSE EVERYONE KNOWS WHAT I'M TALKING ABOUT IS ALSO TO POINT OUT ANOTHER COUPLE OF THINGS SO METASTASIS STARTS WHERE YOU HAVE THE CELL ESCAPE PROGRESS THE THE PRIMARY TUMOR AND IT CAN ENTER INTO THE LIM FATTICS OR IT CAN CROSS INTO THE PERO TO NEAL CAVITIES OR FLUID FILLED SPACES AND THEN TRANSIT IN THE A PLACE IN THE BODY WHERE IT CAN ARREST BY ONE OF TWO BASIC MECHANISMS AND ONE IS TUMOR CELLS ARE RATHER LARGE SO THEY CAN ARREST IN CAPILLARY BEDS OR WHATEVER THE TUMOR CELL SYSTEM TOO LARGE TO GET THROUGH THE LUMEN OF THE CAPILLARY OR THERE'S ALSO EVIDENCE THAT TUMOR CELLS ARE CAPABLE OF SPECIFIC RECEPTOR MEDIATED ADHESION TO WITHIN CERTAIN PARTS OF THE VASCULATURE AND THEN THEY CAN GROW WITHIN THE VASCULATURE UNTIL A BURST THROUGH THE WALLS OR THEY CAN ACTUALLY EXTRACT THEM BEFORE THEY BEGIN GROWING AND EVENTUALLY GROW INTO THESE LIFE THREATENING LESIONS SO THAT THE OTHER REASON I WANTED TO POINT OUT THIS SLIDE, AND AND THEY'RE SPREADING THROUGHOUT THE WATER FROM ONE POINT TO ANOTHER, IT'S ALSO AN IMPORTANT PROBLEM WE HAVE TO CONSIDER, THIS PORTION OF THE CASCADE WHERE THE CELLS ACTUALLY ENTER INTO THE VASCULATURE INTO THE BODY AND, AND THEN MAKING A STRATEGY WITHIN A SHORT PERIOD OF TIME IN TERMS OF HOURS OR DAYS, BUT THE CLINICAL MANIFESTATION OF THIS SEIZE IS NOT SEEN FOR YEARS OR THE CASE OF BREAST CANCER SOMETIMES DECADES AFTER THE PRIMARY TUMOR HAS BEEN REMOVED. SO THESE CELLS EITHER SOMETHING LIKE THIS, OR IS SUBCLINICAL MICRO METASTASIS ARE PRESENT IN THE BODY AND THEY CAN BE PRESENT FOR VERY LONG PERIODS OF TIME AND WE HAVE TO FIGURE OUT SOME WAY OF DEALING WITH THESE UNTIL WE'RE GOING TO ACTUALLY SOLVE THIS PROBLEM ALTOGETHER. NOW, OBVIOUSLY METASTASIS HAS BEEN IMPORTANT AND BEEN A SUBJECT OF A GREAT DEAL OF RESEARCH FOR MANY, MANY YEARS AND THIS IS A REVIEW FROM A VERY RECENT ARTICLE THAT I FOUND AND MANY DIFFERENT BIOLOGICAL PROCESSES AND CELLULAR PROCESS VS BEEN IDENTIFIED ASSOCIATE WIDE METASTASIS, ANYWHERE FROM ANGIOGENESIS, MOTILITY, VASCULARIZATION, ET CETERA AND SO FORTH. SO IT'S NOT SIMPLY THAT THESE CELLS BREAK OFF FROM THE TUMOR AND FLOAT SOMEWHERE ELSE, THERE'S AN AWFUL LOT OF BIOCHEMISTRY INVOLVED IN THIS. OVER THE YEARS MANY PEOPLE HAVE IDENTIFIED GENES THATŤ— ARE ASSOCIATE WIDE METASTASIS, LIKE TUMOR SUPPRESSOR GENES OR GENS THAT ARE METASTASIS, SUPPRESSOR GENES AND HIRE'S GANNA ANOTHER REVIEW THAT WAS PUBLISHED A FEW YEARS AGO, THERE'S SOMETHING ON THE ORDER OF ABOUT 30-40 OF THESE GENES THAT EXIST THAT HAVE BEEN SHOWN IN THE LITERATURE. AND AGAIN, THESE ARE GENES THAT LIKE TUMOR SUPPRESSORS, REINTRODUCTION OF THESE GENES INTO A METASTATIC CELL LINE WILL SUPPRESS THESE TO METASTASIZE, IN THE CASE OF SUPPRESSORS THEY DON'T AFFECT THE PRIMARY TUMOR, SO THAT SUGGESTS IN ADDITION TO ALL THE COMPLEXITY THAT WE HAVE WITH WITH TUMOR GENESIS, THERE'S A WHOLE SECOND CLASS OF GENES THAT WE NEED TO DEAL WITH THAT ARE ASSOCIATE WIDE THE METASTATIC PROCESS, AND AS YOU SEEN HERE ALL OF THESE GENE VS BEEN IMPLICATED IN MANY, MANY DIFFERENT PROCESSES, SUGGEST THANKSGIVING IS AGAIN A VERY COMPLICATED PROCESS THAT INVOLVES NOT ALL OF THE DIFFERENT CELLULAR COMPONENTS OF THE CELL IN THE CELLULAR MACHINERY. BUT NOT ONLY IS METASTASIS A COMPLICATED CELLULAR PROCESS, BUT IT'S ALSO A SYSTEMIC DISEASE. THE PROCESS OF METASTASIS IS NOT JUST INVOLVED IN THE TUMOR CELLS, THE TUMOR IS ALSO ACTUALLY RECRUIT A NUMBER OF OF DIFFERENT CELL TYPES FROM THE BONE MARROW AND THESE ARE ALL INTIMATELY INVOLVED IN METASTATIC DISEASE AND WHAT IS NOW CALLED THE METASTATIC NICHE, TUMORS SECRETING FACTORS FROM THE TUMORS WILL RECRUIT VARIOUS CELLS FROM THE BONE MARROW, THEY WILL ACTUALLY GO TO THE SITE OF THE SECONDARY METASTASIS AND FORM PERMISSIVE NICHES IN THOSE ORGANS BEFORE THE PRIMARY TUMOR CELLS ARRIVE. SO, I WANT TO YOU THINK OF METASTASIS NOT AS LIKE WE THINK OF NECESSARILY, AT LEAST IN THE PAST OF TUMOR CELLS, ONLY THE FOLKS ON THE TUMOR CELLS BUT YOU HAVE TO THINK OF THE ENTIRE BODY, ALL OF THE CELLS IN THE BODY ARE INVOLVED, NOT OLDSMOBILE THE TUMOR CELLS BUT THE NORMAL CELLS, NOT ONLY IN THE SECONDARY ORGAN BUT ALSO JUST IN ORGANS LIKE BONE MARROW THAT CAN ACTUALLY BE INVOLVED IN THIS PROCESS. NOW, HAVING ADDED ALL THOSE LAYERS OF COMPLEXITY, I WILL ADD ANOTHER ONE AND THIS IS WHAT MY LABORATORY FOCUSES ON. AND THAT IS ACTUALLY METASTASIS IS NOT ONLY A SEMANTIC DISEASE ASSOCIATE WIDE MUTATIONS IN THE PRIMARY TUMOR, BUT IT'S ALSO AN INHERITED DISEASE. THERE IS PREDISPOSITION TO METASTASIS RUNNING THROUGH THE HUMAN POPULATION AND I AM GOING TO SHOW YOU THE DATA AS TO WHY WE BELIEVE THAT. SO, J*5:6 A LITTLE BIT OF BACKGROUND AND ALSO SOME CAUTIONARY ASIDES, MY LABORATORY IS A MOUSE GENETICS LABORATORY PRIMARILY, WE DO A LOT OF MOUSE MODELING AND WE DO MOUSE MODELING AND METASTASIS BECAUSE CAN YOU DO IT EXPERIMENTALLY IN THE ANIMALS EASILY AND ONE OF THE THINGS THAT HAS BEEN DONE OVER THE YEARS FOR MOUSE MODELING OF HUMAN CANCERS HAS BEEN STARTING WITH THINGS LIKE THIS. YOU HAVE A--IN THIS CASE, A CAREIO TYPE AND WE KNOW THAT SOLID TUMORS ARE MUTE O GENIC AND WE THEY DO NASTY THINGS TO THEIR GENOMES AND YOU SEE THE BREAK POINTS AND OVER THE YEARS PEOPLE HAVE BEEN NARROWING IN ORIGINALLY BY PHYSICAL MEANS AND NOW DAYS IT'S ALL NEXT GENERATION SEQUENCING AND YOU SEE THE GENOMES AND JEANS THAT OF INTEREST TO YOU AND SEE YOU THEM MUTATE INDEED COMMON TUMORS AND THEN YOU BUILD A MOUSE MODEL LIKE THIS AND THIS OF COURSE IS A KNOCK OUT MOUSE WHERE YOU'RE LOOKING AT A PARTICULAR GENE TO SEE HOW IT AFFECTS A TUMOR PHENOTYPE THAT YOU'RE INTERESTED IN. AND THESE GENETICALLY ENGINEERED MOUSE MODEL VS BEEN EXTREMELY IMPORTANT AND I DON'T WANT TO DENIGRATE THEM IN ANY WAY ALTHOUGH I'M GO TO SAY SOME UNPLEASANT THINGS ABOUT THEM, THEY TELL US A GREAT DEAL WITH MECHANISM, THEY ALLOW US TO MODEL MUTATIONS IN HUMANS IN A WAY THAT WE CAN ACTUALLY GET VERY GOOD INFORMATION ABOUT THE MECHANISM UNDERLYING THAT PARTICULAR MUTATION AND ITS ROLE IN THOSE CANCERS. THE PROBLEM WITH THESE MODELS IS THEY'RE ON INBRED AND I DON'T CARE HOW MANY MICE YOU HAVE IN A COHORT OF ANIMALS WHEN STUDY STDING A GENETICALLY ENGINEERED MODEL, THEY DO NOT REPRESENT THIS AND THAT IS THE HUMAN POPULATION BECAUSE ALL OF THIS PHYSICAL DIVERSITY THAT YOU SEE IN THIS TICKETTURE AND AROUND--PICTURE AND AROUND US IS ENCODED BY POLYMORPHISMS AND THESE GUYS DON'T HAVE ANY. THEY ARE INBRED. THEY HAVE BEEN INBRED SPECIFICALLY BECAUSE THEY REMOVE ALL OF THE VARIABILITY HERE IN THE POPULATION THAT RECOGNIZED A HUNDRED YEARS AGO TO CAUSE PROBLEMS IN OUR ABILITY TO UNDERSTAND TUMOR BIOLOGY, THAT'S--ANOTHER LITTLE ASIDE. THE FIRST INBRED MOUSE, DEVELOPMENTAL ENDOCRINOLOGYIATE, THE BROWN ALBINO MICE WAS MADE BY C C LITTLE TO GET RID OF THIS BECAUSE HE RECOGNIZED THAT HE WAS HAVING TROUBLE ACTUALLY INTERPRETING HIS RESULTS ABOUT THE CANCER ETIOLOGY BECAUSE THERE WAS TOO MUCH VALID AND RELIABLABILITY DUE TO POLYMORPHISMS SEGGREGATING IN THE MOUSE COLONIES. SO, THESE GUYS ARE VERY GOOD. THEY TELL US MECHANISM BUT THEY DON'T MODEL HUMAN POPULATIONS VERY WELL. AND THAT'S IMPORTANT BECAUSE WE KNOW FROM ALL SORTS OF GENETICS THAT OUR PARTICULAR INHERITANCE IS AN EXTREMELY IMPORTANT COMPONENT AND FACTOR ON WHETHER WE DEVELOP DISEASE. WE KNOW HEART DISEASE RUNS IN FAMILY, WE KNOW CANCERS RUNS IN FAMILIES AND ALSO, YOU KNOW, YOUR INHERITANCE REALLY IMPACTS BASICALLY ALL OF YOUR PHYSIOLOGY BUT OUR GENETICALLY ENGINEERED MODELS FOR THE MOST PART DON'T MODEL THAT. OKAY? SO, MY LABORATORY'S APPROACH IS ACTUALLY TO START ADDRESSING THAT. WE HAVE MANY, MANY, MANY EXCELLENT GENETICALLY REENGINEERED MOUSE MODELERS OUT THERE BUT WE'RE INTERESTED TO LOOK AT THIS SORT OF HOW GERM LINE POLYMORPHISM AFFECTS THE ABILITY OF TUMORS TO METASTASIZE IT, BECAUSE THE GENES THAT WERE ASSOCIATE WIDE IT WILL TELL YOU ABOUT THE PHYSIOLOGY OF THE METASTASIS AND ALSO THEY'RE NOT NECESSARILY THE GENES THAT GET MUTATED. SO DIFFERENCES IN NORMAL PHYSIOLOGY FOR EXAMPLE, IMMUNE FUNCTION CAN HAVE AN IMPORTANT ROLE IN METASTASIS, BUT YOU WON'T PICK THEM UP BY GWAS OR GENOME SEQUENCING BECAUSE THEY'RE NOT MUTATED. THEY'RE NORMAL PRESENCE IN THE POPULATION SO IF YOU'RE DOING A TUMOR NORMAL SEQUENCING, YOU DON'T SEE THEM BECAUSE THEY'RE NORMAL IN BOTH. AND NOT ONLY ARE WE INTERESTED TO SEE HOW IT AFFECTS THE PHYSIOLOGY THERE BUT WE'RE ALSO INTERESTED IN A LOST THE MOLECULAR MECHANISMS GOING UNDERLYING ALL THIS INCLUDING GENE EXPRESSION SIGNATURE ANALYSIS THAT I'LL TALK ABOUT SOME LATER. SO HOW DO WE DO THIS, WELL, I TOLD YOU MY LABORATORY IS A MOUSE GENETICS LABORATORY, WE USE THIS PARTICULAR MODEL. AND NOW THAT I'VE JUST SAID NASTY THINGS ABOUT THE GENETIC ENGINEERED MODELS, BECAUSE THEY'RE INBRED, THIS IS AN INBRED MOUSE MODEL. THIS IS A TRANSGENIC MODEL, CARRIES A MOUSE TUMOR VIRUS, ENHANCER PROMOTER, DRIVING THE MIDDLE T ANTIGEN WHICH RESULTS IN THE EXPRESSION OF THIS ANTIGEN IN THE MEMORY GLAND BY ABOUT FOUR WEEKS OF AGE. NOT MUCH WEIGHT, YOU GET HYPERž /– PLASSIAS AND THEN YOU GET THE NASTY FOCAL TUMORS BY NINE WEEKS OF AGE, ALL THE ANIMALS AND THE ALL THE ANIMALS WILL DEVELOP TUMORS AND BY THE TAME THEY HAVE TO BE EUTHANIZED DUE TO MASSIVE TUMOR BURREDIN MORE THAN 85% CLOSER TO 95% OF THESE, THEY GET PULMONARY METASTASIS. SO THIS IS A HIGHLY AGGRESSIVE MODEL. THIS HAPPENS VERY RAPIDLY AND THESE DON'T SURVIVE VERY LONG. HOW DO YOU SEE IF IT HAS AN EFFECT ON A METASTASIS AND THAT'S SHOWN ON THIS SLIDE HERE: YOU LET THE MOUSE DO THE WORK BY TAKING TRANSGENIC ANIMAL AND GET AHOLD OF THE COLLECTION OF INBRED MOUSE STRAINS AND EACH OF THESE DIFFERENT OUTCROSSES NOW BECAUSE OF THE MIRACLE OF MY O SIS, WILL BE ON A DIFFERENT GENETIC BACKGROUND BECAUSE THE POLYMORPHISMS ARE REDUCED BY THIS STRAIN HERE AND YOU CAN LOOK AT THE PROGENY OF THIS OUTCROSS AND YOU CAN SEE THE COMBINATIONS THAT ARE HIGHLY METASTATIC ARE THERE ANY THAT ARE INTERMEDIATE AND ARE THERE ANY TRAIN COMBINATIONS THAT E CULT IN LOW METASTATIC ABILITY AND WHEN YOU DO THAT YOU EPPED UP WITH THIS SLIDE. AND THIS IS ANOTHER ONE OF THE GREAT SLIDES WHERE IT'S 3000 MICE AND A YEAR AND HALF OR FOUR ON ONE HISTOGRAM PLOT. ON THE Y AXIS IS METASTATIC INDEX, THIS IS JUST OUR FANCY TERM FOR THE DENSITY OF PULL MONITOR PULMONARY--PULMONARY METASTASIS, THIS IS THE SECTION HERE AND THIS IS THE TRANSGENIC ANIMAL TWO. THE THE IMPORTANT THING HERE IS THIS GOLD BAR, THIS IS THE ORIGINAL FEB BACKGROUND, WHEN YOU BREED THAT MIDDLE ANIMAL TO THIS ANIMAL OVER HERE, AKRJ, WHAT YOU SEE IS A TWO AND HALF TO THREE FOLD INCREASE IN THE NUMBER OF METASTASIS THESE ANIMALS HAVE. COMPARED TO ANIMALS DOWN HERE WHICH OBLIGATIONS BLAT THE ANIMAL'S ABILITY TO METASTASIZE. SO THIS IS ALL INDUCED BY EXACTLY THE SAME TRANSGENE. IT IS TURNED ON EXACTLY THE SAME TIME AS FAR AS WE CAN TELL. IT IS EXPRESSED AT EXACTLY THE SAME LEVEL. THERE'S NO DIFFERENCE IN THE POST TRANSLATIONAL MODIFICATION. SO THAT WOULD SUGGEST, THAT WHAT WE'RE LOOKING AT HERE IS NOT AN EFFECT DIRECTLY ON THE TRANSGENE, BUT WE'RE LOOKING AT EFFECTS THAT ARE SOMEWHERE ELSE IN THE ANIMALS GENOME. THIS CURVE ALSO TELLS THE GENETICIST THAT YOU'RE PROBABLY NOT LOOKING AT A SINGLE GENE BECAUSE YOU'RE NOT SEEING A BIMODAL PEAK HERE YOU'RE SEE THANKSGIVING NICE CURVE. IT TELLS YOU THERE ARE PROBABLY A LOT OF DIFFERENT GENES PRESENT IN THE GENOME, WHICH IS NOT SURPRISING, IT'S A COMPLICATED GENOME. >> THIS IS COMPUTE OUD ON EFERAGE OF EACH PROGENY? >> IT'S A AVERAGE FROM THE NUMBER OF ANIMALS FROM THAT STRAIGHT ACROSS, YEAH. I'M CHEATING HERE A LITTLE BIT. I'M NOT SHOWING YOU THE ERROR BARS BECAUSE IN METASTASIS ASSAYS THERE'S A HUGE AMOUNT OF VALID AND RELIABLABILITY, AND IT'S FRIGHTENING IF YOU DON'T DO THE STATISTICS AS WELL. BUT EVERYTHING YOU SEE HERE IS OF INTEREST AND WE'VE DONE A LOT OF ANIMALS UNDER EACH OF THESE. AFTER GREAT QUESTION, THE ANSWER IS AS FAR WOOS KETELL, WE HAVEN'T SEEN ANYTHING THAT'S STATISTICALLY SIGNIFICANT SHOWING WHERE THE TUMORS ARISE IN THESE ANIMALS COMPARED TO OTHERS SO INGUINAL AND YOU GET THEM IN THE AX ILLEGALSARY BUT THE INTERESTING THING IS IN THIS MODEL AND MOST MODELS FOR MEMORY TUMORS IN THEM, YOU TEND TO SEE THEM IN THE AX ILLEGALSARY RATHER THAN THE INGUINALS FIRST AND THAT'S NOT JUST BECAUSE IT'S EASIER TO FEEL THEM IN THE RIB CAGE. IF YOU DO THE WORK, THEY ARISE EARLIER IN THEAC ILLEGALSARY MAMMARY GLANDS BUT WE DON'T SEE ANY DIFFERENCE HERE IN THESE STRAINS? ANYTHING ELSE BEFORE I--PLEASE STOP AND ASK ME QUESTIONS IF YOU HAVE ANYTHING AS A GO ALONG. I WOULD LIKE TO TO BE NICE AND INFORMAL. >> SO I'M CURIOUS YOU CLAIM THIS CANNOT BE THE WORK OF ADVANCED GENE? SO I BELIEVE THAT YOU ALREADY DONE THAT, THE PC R BALANCE ON THIS STRAINS, RIGHT? I MEAN-- >> YES. >> SO YOU SAY THAT YOU DON'T SEE ANY ANYWHERE? >> NO I SAID WE DON'T SEE ANY DIFFERENCE IN THE TRANSGENE THAT INDUCES THIS, WHEN YOU SIT DOWN AND DO THE MAPPING, THAT'S ABOUT WHAT YOU ASKED, YES. SO WE SET OUT AND THE NEXT SLIDE HERE IS JUST A DIAGRAM I'VE PUT TOGETHER MANY YEARS AGO, SHOWING THE VARIOUS WAYS CAN YOU DO GENETIC MAPPING AND THE MOUSE, AND WE EPPEDDED UP DOING A TOTAL OF SIX CROSSES TO DATE, LOOKING AT TWO DIFFERENT METHODS BACK ACROSS THE RECOMBINANT INBRED FOR THOSE WHO CARE, WE HAVE THREE MORE COOKING RIGHT NOW AND WE'VE BEEN ANALYZING THEM, LOOKING FOR REGIONS IN THE GENOME AND GENES IN THE GENOME THAT ARE ACTUALLY ASSOCIATED WITH METASTATIC SUSEPTIBILITY, BACK IN THE DAY WHEN WE DID THIS, YOU USED TO GET STUFF THAT LOOKED LIKE THIS. HERE A LONG SCORE PLOT SHOWING A MOUSE CHROMOSOME FOR THOSE OF YOU WHO DON'T KNOW, MOUSE CHROMOSOME IS THE LEAST INBRED IN THE LABORATORY ARE ACROUGH ATOM CENTRIC, HERE'S THE SEBT ROW MERE, THE TELOMERE, AND IS THIS A GREEN LINE PLOT WHICH IS A STATISTICAL PROBABILITY AFFECTING YOUR GENE AFFECTING ANYWHERE ALONG THE CHROMOSOME AND UP HERE YOU CAN SEE IT'S AWAY, IT'S HIGHLY SIGNIFICANT SUGGESTING THAT SOMEWHERE IN THIS INTERVAL OF 500 GENE THERE IS WAS A GENES OR GENE THAT WERE AFFECTING OUR PHENOTIME. EVEN I'M NOT CRAZY ENOUGH TO DO 500 GENES ALTHOUGH SOME OF MY PEOPLE IN MY LABORATORY MIGHT CLAIM THAT I AM. WE NARROW THD DOWN OVER A PERIOD OF YEARS TO THIS 110 KILOBASE HAPLOTYPE. WE DID COMPLETE EXON SEQUENCING OF THOSE GENES AND DISCOVERED IN THIS PARTICULAR GENE, A GENE CALLED SIPPA, ONE WHO IS A SINGLE POLYMORPHISM RIGHT HERE IN A COIL DOMAIN OF A--THIS ALPHA HELIX OF A PROTEIN INTERACTION, CALLED A PDZ DOMAIN AND THE ALLELE THAT WAS SEEN IN THE LOW METASTATIC ANIMAL SYSTEM PREDICTED TO UNWIND THAT ALPHA HELIX AND PRESUMABLY PREDICTED AT THAT TIME TO INTERFERE WITH IT'S ABILITY TO BIND WITH ANY OF IT'S PROINE PARTNERS. I'M NOT SHOWING YOU THE THE DATA, OF WHAT TURNED OUT TO BE THE CASE AND THIS REDUCES ENZYMATIC ACTIVITY OF THIS PROTEIN. IT'S A RAP MOLECULE, HERE'S THE GAP ACTIVITY AND HERE'S THE HIGHLY METASTATIC ALLELE AND LOW METASTATIC FROM DBA AND YOU CAN SEE THE ENZYMATIC ACTIVITY OF THAT PROTEIN BY ABOUT HALF. THAT ALLOWED US TO GO IN AND DO STANDARD--WHAT'S NOW A STANDARD ASSAY BUT IN THOSE DAYS WOULDN'T DO ANYTHING EXCITING WHICH WAS TO MODEL THIS BY TAKING A HIGHLY METASTATIC CELL LINE AND KNOCKING DOWN THE SIPA ONE USING SiRNA TECHNOLOGY AND PLANTING THOSE INTO ANIMALS TO SEE IF REDUCING THE ACTIVITY, BUT NOT ELIMINATING IT, A MODEST REDUCTION WOULD ALSO RESULT IN>Lm1 DECREASE IN METASTASIS AS WOULD BE PREDICTED. SO HERE'S THE EXAMPLE, WHAT YOU'RE SHOWING WITHOUT MODEST, THIS IS WHAT GET WITH REDUCTION OF RNA LEVELS, WE HAVE ABOUT A 30--THREE FOLD REDUCTION RNALEVELS, MAYBE A TWO FOLD REDUCTION OF PROTEIN LEVELS, TURNS OUT YOU CANNOT ELIMINATE THIS PROTEIN FROM THESE CELLS OTHERWISE THEY DIE. THIS MODEST WAS DUCKS WERE THEN IMPLANT INDEED THE ORTHOTOPIC SITE WHICH IS THE MEMORY FAT PADS AND WE LET THE THE TUMORS GROW AND THEK TO SEE THAT THEY METASTASIZED AND THIS IS WHAT WE GOT. HERE'S THE HIGHLY METASTATIC CELL LINE BY ITSELF, AS YOU CAN SEE, YOU GET THESE PINK METASTASIS, YOU REDUCE PROTEIN LEVEL BY ABOUT TWO FOLD, BASICALLY, THE ANIMALS STOP GETTING METASTASIS. IF YOU DO THE OPPOSITE EXPERIMENT WHICH IS OVER EXPRESSING THE HIGHLY METASTATIC ALLELE, WHAT YOU SEE IS YOU GET MANY MORE METASTASIS. SO, WE'RE TALKING ABOUT ONLY FROM HERE TO HERE, ABOUT A FOUR FOLD DIFFERENCE IN PROTEIN EXPRESSION, WHICH IS WELL WITHIN PHYSIOLOGICAL RANGES FOR MANY PROTEINS, BUT YOU'RE SEEING AN ENORMOUS ABILITY IN THE ABILITY3aQiJt THES E TUMORS TO METASTASIZE, SO THIS SUGGESTS IN ADDITION TO ALL OF THE CONSITTATIVE ACTIVATION YOU CAN SEE ONCA GENES OR CONSITTATIVE DELETION YOU SEE WITH TUMOR SUPPRESSOR GENES IF YOU SIT THERE AND MUCK AROUND A LITTLE BIT WITH FUNCTION, IN BY POLYMORPHISMS YOU CAN HAVE TREMENDOUS AFFECTS IN METASTATIC CAPACITY. BUT OF COURSE, BUT OF COURSE, THIS IS A MOUSE, THIS IS NOT HUMAN AND THERE ARE PHYSIOLOGICAL DIFFERENCES BETWEEN MOUSE AND HUMAN IN THE MAMMARY GLAND, THE STRUCK SURDIFFERENT AND THE CONCERN IS ALWAYS WHETHER OR NOT,--OH, ACTUALLY BEFORE I GET TO THAT, ONE OF THE--ONE OF THE CONCERNS YOU WOULD HAVE TO SAY IS YOU DEAL WITH IS THE FACT YOU MAY BE AFFECTING FOR METASTASIS ASSAY, YOU MAY AFFECT THE PRIMARY TUMORS ABILITY TO GROW, THAT TURNS OUT NOT TO BE THE CASE BECAUSE WHEN YOU LOOK AT THE TUMOR GROWTH, WHEN YOU KNOCK DOWN SIPA ONE, YOU ELIMINATE METASTASIS THAT THE PRIMARY TUMOR GROWS FASTER. SO, WHAT ARE WE DO SEE EXAMPLES WHERE YOU FIND INTERESTING GENES THAT WIPE OUT METASTASIS BUT THEY ALSO OBLIGATIONS BLAT THE PRIMARY TUMORS BUT THERE'S AN COLLECTION OF GENES LIKE THIS WHERE YOU ARE ACCELERATING PRIMARY TUMOR GROWTH OR NOT AFFECTING PRIMARY TUMOR GROWTH AND SUBTLE VARIATIONS ARE HAVING A MAJOR EFFECT ON METASTATIC DISEASE. >> YES. >> [INDISCERNIBLE]--KNOCK OUTS? >> THEY'RE STABLE. >> NEXT QUESTION, [INDISCERNIBLE] EXAMPLE WHERE POLYMORPHISMS [INDISCERNIBLE]--NUMEROUS OTHERS--[INDISCERNIBLE] >> DON'T JUMP AHEAD. SO THE QUESTION WE WANT TO ASK THIS WILL ANSWER YOUR QUESTION, AND SINCE WE'RE DOING GENETICS WE'RE LOOKING AT INHERITANCE, THERE'S PLENTY OF COHORTS WHERE PEOPLE HAVE INFORMATION ABOUT CLINICAL OUTCOME AND THE NUMBER OF COHORTS THAT WE COULD GENOTYPE AND WE WENT OUT THERE AND ASK THE QUESTION, ARE THERE POLYMORPHISMS IN THE HUMAN SIPA ONE THAT CORRELATE WITH DISEASE OUTCOME. AND THE ANSWER TO THAT IS, YES, HERE'S AN EXAMPLE, SIPA ONE IF YOU ARE CARRYING„i THE RARE ALLELE, HOMOZYGOUSLY YOU LESS LIKELY TO DEVELOP METASTASIS, EITHER HOMOSIGHINGOUS, THIS IS NOT A FUNCTIONAL, FUNCTIONAL THAT IT DOESN'T CHANGE IN THE AND IT'S THE COLOR OF THE GENE AND WAS PRESUMED TO AFFECT RNA LEVELS AND WE HAVE EXAMPLES NOW IN MULTIPLE SITUATIONS AND WE CAN--YOU KNOW WE SRO MOLLY MORPHISMS AND THE GENE TRANSCRIPTION OF THE MOUSE AND VICE VERSA, EVERYONE WITH ME SO FAR? IF THIS IS TRUE THEN THERE ARE OF COURSE MOUSE TRAIN TAG ARE MORE SUSEPTIBLE OF COURSE. THERE ARE TWO DIFFERENT GROUPS NOW WHO LOOKED AT BREAST CANCER USING THE SWEDISH CANCER REGISTRY WHICH IS A WONDERFUL REGISTRY AND THEY ALSO HAVE THE ADVANTAGES FOR EPITEEMIOLOGY THIS THE ADJUVANT THERAPY BECAME MORE COMMON IN EUROPE LATER THAN IN THE UNITED STATES AND THEY„i HAVE A LARGER PROPORTION OF PATIENT WHO IS DIDN'T RECEIVE ADJUVANT THERAPY SO YOU DON'T HAVE TO RETURN OF RESULTS WORRY ABOUT THAT AT THE COMPOUND, SO THE IMPORTANT THING IS HERE, SURVIVAL OF BREAST CANCER IS FAMILIESIAL. WHAT THEY SHOW IS THAT IF HAVE YOU MOTHER-DAUGHTER PAIRS WHO BOTH GOT BREAST CANCER, IF THE MOTHER HAD METASTASIS, THE DAUGHTER WAS MORE LIKELY TO HAVE METASTASIS THAN BY CHANCE. DOES THAT PROVE THAT THIS IS--THESE ARE EXISTING THE HUMAN POPULATION, NO, BUT IT IS ENTIRE LIE CONSISTENT WITH OUR HYPOTHESIS THAT THERE IS INHERITED PREDISPOSITION IN HUMANS. THIS STUDY LOOKED NOT ONLY AT BREAST CANCER MANY OTHER CANCERS IF THEY HAD ENOUGH FAMILY PAIRS TO LOOK AT THAT AND THEY SEE THE SAME THING IN THE LUNG AND THE SAME THING IN RENAL CANCER AND SEE THE SAME THING I BELIEVE IN PROSTATE CANCER. SO IT APPEARS THAT ACTUALLY YOU DO SEE THESE SORTS OF METASTATIC ABILITIES THROUGH FAMILY. AND THE INTERESTING THING IF THE MOTHER HAS BREASTS CANCER AND THE DAUGHTER HAS COLON CANCER THERE'S NO CORRELATION, SO IT'S TISSUE SPECIFIC BUT IT DOES RUN WITHIN FAMILY WITH THOSE TISSUES. OKAY? SO MY LABORATORY USING THESE SORTS OF TECHNIQUES HAVE BEEN CLONING A NUMBER OF THESE GENES NOW AND THIS IS ACTUALLY A FAIR AMOUNT OF DATA NOW BUT WE'VE IDENTIFIED A NUMBER OF THESE GENES, EACH OF THESE HAS SOME LEVEL OF EVIDENCE BOTH IN THE--DEFINITELY IN THE MOUSE AND IN MANY CASES AS WELL IN THE HUMAN EPIDEMIOLOGICAL ANALYSIS AND WE'VE BEEN TRYING TO FIT THREES INTO PATHWAYS BECAUSE AS WE ALL KNOW, GENES DO NOT OPERATE IN YOU KNOW ISOLATION, THEY ARE--THEY ARE INTERACTING WITH OTHER PROTEINS, THEY'VE IMPACT OTHER NETWORKS, AND WHAT WE'VE BEEN ABLE TO SHOW IS, AND THEY ARE INTERESTING THEY ARE PROTEINS RIGHT THERE THAT INTERACT AND THIS BINDS BRDFOUR AND THIS COLLECTION RIGHT HERE SEEMS TO BE A VERY IMPORTANT NOTE IN BREAST CASTER METASTASIS AT LEAST IN THE LUMINAL TUMORS THAT OUR LABORATORY IS MODELING THE MOST. BUT AS WE ALL KNOW THIS IS NOT A NETWORK EITHER, THIS IS A NICE STICK DIAGRAM, IT'S GOT MULTIPLE CONNECTIONS, THIS IS NOT WHAT HAPPENS IN THE BIOLOGY AND PHYSIOLOGY, WHAT WE EELLY WANT TO DO IS TO--REALLY WANT TO DO IS USE THIS ANALYSIS TO DEVELOP WHAT I CALL THE CIRCUIT DECEMBER OF METASTASIS SUSEPTIBILITY. WE WANT TO BUILD A HUGE NETWORK TO TELL US WHAT'S ANYTHING ON IN THE CELL, SO CAN YOU IDENTIFY THESE SORTS OF NODES FOR INTERVENTION SO WHAT WE'RE INTERESTED IN TRYING TO DO IS DEVELOP SOMETHING LIKE THIS. SO THIS IS A NETWORK THAT I STOLE FROM THE LITERATURE FROM ALAN WHO WAS LOOKING AT SUSEPTIBILITY IN THIS CASE, THE SKIN CANCER SO THE IMPORTANT THING IS YOU CAN SEE NODES THAT LOOK LIKE THIS, PRESENT IN THESE NETWORKING AND WHAT THESE NODES ARE ARE INTERESTING TARGETS, IF WE CAN CONTROL THOSE NODES RATHER THAN AFFECTING ALL HUNDREDS OF GENES IN HERE THAT'S INVOLVED IN HERE, IF YOU CAN TARGET SPECIFIC NODES YOU MAY ACTUALLY BE ABLE TO INTERVENE, IN A REASONABLE WAY, YOU DON'T HAVE TO TARGET EVERYTHING, JUST PICK OUT THE MOST IMPORTANT THINGS. SO THAT'S WHAT I'VE JUST SAID SO WHAT WE'VE BEEN DOING NOW IS GOING BACK AND TRYING TO BUILD THE SAME SORT OF NETWORKS USING ALL OF THE ARRAY DATA WE GENERATE INDEED OUR MOUSE MODELS AS WELL AS THESE HUMAN DATA SETS AND THE INTERESTING THING HERE IS AGAIN, REMEMBER, ALL OF THIS DATA THAT I'M GOING TO TALK ABOUT FROM THE MOUSE, IS ALL INDUCED BY ONE GENOMIC INSULT AND THAT IS THE EXPRESSION OF POLEYOMA MIDDLE T. UNLIKE THE HUMAN DATA WHICH I'M GOING TO TALK ABOUT, ALL OF THIS DAT IS JUST WHATEVER THEY HAD IN THE TUMOR BANKS AND THERE ARE MANY DIFFERENT DRIVER MUTATIONS PRESENT THERE. SO WHAT WE'VE DONE IS TAKEN ALL THESE DATA SETS AND THESE ARE ALL DATA SETS, TWO HUMAN FROM THE GENE EXPRESSION FROM THE O--METABOLIZED NIBUS, AS WELL AS A SERIES OF MOUSE BACK CROSS OR RECOMBIN ABT INBRED DATA AND STARTED BUILDING OUR OWN NETWORKS AND COMBINING THE MOUSE AND HUMAN DATA TO START LOOKING FOR COMMONNALLITYS THAT ARE ACTUALLY THESE KEY NOTES. WHAT DO YOU GET WHEN DO YOU THIS, YOU GET DIAGRAMS THAT LOOK LIKE THIS. THIS IS ALL GENE EXPRESSION DATA THAT IS ALL IN THE LINE HERE ARE COLLECTIONS OF GENES THAT ARE CO REGULATED. AND WHAT THIS HAS HOPEFULLY TELLING YOU IS THAT THESE ARE ALL CO-REGULATED BECAUSE THEY'RE ALL IN A NETWORK AND YOU CAN GO IN AND PULL THESE NODES OUT. AND WHEN DO YOU THIS, GET PRETTY LITTLE DIAGRAMS LIKE THIS, AND YOU FIGURE OUT WHAT THESE ARE. AND YOU END UP, WHEN WE DID THIS, WE EPPEDDED UP ABOUT 20 OF THESE FOR EACH OF THE NETWORKS. I'M GOING TO FOCUS ON THIS NETWORK, THIS IS THE NETWORK FROM THIS HUMAN DATA SET. IT'S CALLED THE TPX TWO, MODULE BECAUSE THIS GENE RIGHT HERE IN THE CENTER, IS THIS GENE CALLED TPX TWO, IT IS ASSOCIATE WIDE TUBEULAR FORMATION AND CHROMOSOME SEGGREGATION, WHY IS THIS PARTICULAR MODULE INTERESTING, WELL, WHEN YOU LOOK AT THE MULTIPLE DATA SETS HERE'S ONE HUMAN DATA SET, HERE'S ANOTHER DATA SET, THIS IS THE EQUIVALENT MODULE HERE IN THIS OTHER HUMAN DATA SET. UNLIKE OTHER GENE EXPRESSION ANALYSIS WHERE PEOPLE HAVE GONE OUT AND LOOKED FOR GENES ASSOCIATED WITH METASTASIS, THOSE GENE EXPRESSION PROFILES LIKE NAMA PRINT OR ONCA TYPE OR OTHER THINGS THAT ARE USE INDEED CLINICAL TRIAL TO PREDICT THE OUTCOME, THOSE HAVE ALMOST NO OVERLAP. HOWEVER, WHEN YOU DO IT THIS WAY, WHEN YOU LOOK AT NETWORKING RATHER THAN TRYING TO LIMIT THE GENES DOWN TO A COUPLE, WHAT YOU CAN ACTUALLY SEE THERE'S SIGNIFICANT OVERLAP BETWEEN THESE DATA SETS THEN. AS SCION BY--COME ON, HERE. SO, THOSE TWO NETWORKING FOR THE TWO DIFFERENT DATA SETS OVERLAB P-228, SO THESE ARE REPRODUCIBLE NETWORKS THAT YOU CAN BUILD USING GENE EXPRESSION PROFILING PRACTICES HUMAN DATA SETS. WHAT HAPPEN WHEN IS YOU START LAYERING THE MOUSE DATA SETS, WHAT YOU CAN SEE HERE IS NOW EVERYTHING IN RED IS SHARED BETWEEN THOSE TWO HUMAN DATA SETS AS WELL AS ONE OF THE MOUSE DATA SETS. I STARTED PUTTING THE GENE NAMES IN BECAUSE YOU CAN START TO READ THEM. WHEN YOU LAYER ALL FIVE OF THOSE DATA SETS TOGETHER, WHAT YOU FIND IS IT'S REALLY START SEEING COMMONNALLITYS. PARTICULARLY NODES THAT ARE POPPING UP HERE THAT ARE SHARED PLUNGEST ALL OF THOSE NETWORKS, AND THESE INTERESTING THING ABOUT THESE NODES, THEY CONTROL THESE NINE NODES PRETTY MUCH CONTROL OR CAPTURE ALL OF THE VARIABILITY FOR THIS 290 SOME-ODD GENE NETWORK. SO IS THIS RELATED TO METASTASIS? WELL, WELL, IF YOU TAKE THOSE NINE GENES, AND LOOK AT A COHORT OF ABOUT 1300 PATIENTS AND SIMPLY SEPARATE THOSE BASED ON WHETHER THEY LOOK LIKE THEY'RE UPREGULATING OR DOWN REGULATING THOSE NINE GENES WHAT YOU CAN SEE IS, IT DOES A VERY GOOD JOB OF DISTINGUISHING THOSE PATIENT WHO IS ARE GOING TO DEVELOP METASTASIS OR NOT. OKAY, SO THIS IS NOT TERRIBLY DIFFERENT FROM THE GENE EXPRESSION PROFILES THAT ARE IN CLINICAL TRIALS YET, BUT INTERESTINGLY ENOUGH, IF YOU LOOK AT THOSE NODES, FOUR OF THEM ACTUALLY PHYSICALLY INTERACT AND THEY INTERACT AT THE SPINDLE CHECK POINT. SO WHILE YOUR CELLS ARE THERE BUSILY TRYING TO GET READY FOR MIRROR IMAGE TOESIS, THE MITOTIC SPINDLE IS THERE AND NOT ALL OF THEM ALINEDDED AND--ALIGNED AND THESE ARE IN THE WAY TO STOP THE CELL FROM GOING INTO THE THE DIVISION AND SEPARATING THE CHROMOSOMES. AND WHAT IS THIS--THIS IS SUGGESTING UPREGULATION OF THESE GENES PREVENTING CHROMOSOME MISSEGGREGATION MAYBE INVOLVE IN PROMOTING METASTATIC PROGRESSION. OKAY? AND IF YOU LOOK AT JUST THOSE FOUR GENES, YOU STILL GET HIGHLY SIGNIFICANT DISCRIMINATION BETWEEN PATIENT WHO IS ARE GOING TO METASTASIZE OR NOT. YOU CAN ALSO START STRATIFYING THE PATIENTS BECAUSE YOU STARTED OFF WITH 1300 PATIENTS. IF YOU LOOK AT ALL THE THE LOOK THE THE PATIENTS WHO ARE POSITIVE, YOU SEE A VERY GOOD SEPARATION BECAUSE KHI IS GOOD BECAUSE OUR MOWS MODEL WE DERIVED ALL THIS STUFF FROM AS WELL AS HUMAN ARE LOOKING AT LUMINAL TUMORS IN THEORY. IT'S SUPPOSED TO BE A MODEL OF LUMINAL TUMORS WHICH IS AN ER POSITIVE STYLE TUMOR. BUT, IF YOU LOOK AT ER NEGATIVE PATIENTS, THERE'S NO DISCRIMINATION WHATSOEVER. IT SAYS THAT ER POSITIVE AND NEGATIVE TUMORS USE DIFFERENT METASTASIS THAT'S TRUE, WE HAVE VALIDATION BECAUSE WE ALSO HAVE GENE WHEN YOU LOOK AT THE EPIDEMIOLOGY AT SEGGREGATION YOU SEE BASICALLY THE SAME MULTIPLE MECHANISMS YOU DEVELOPED AT--INTERESTINGLY ENOUGH, IN YOU LOOK AT ONE OF THE OTHER HIGHLY CONSERVED NETWORKS OUT THIS, THIS IS THE SAME ANALYSIS, THAT IS DISSOLVED ALMOST ENTIRELY AN IMMUNOLOGICAL RESPONSE, IL10 RECEPTOR A, CD53 AND THIS ALMOST ALL OF THESE GENES ARE NOW IMMUNOLOGICAL CELL SURFACE MARKERS, THIS IS A DIFFERENT OUTCOME, PARTICULARLY IN THE ER NEGATIVE PATIENTS WHICH SUGGESTS THAT IS IMPORTANT IN BUT THE DIFFERENCES IN THE MIGHTOTIC TUMOR MANNER AND IN THIS CASE, AN IMMUNE RESPONSE IS IMPORTANT IF YOU ARE ER NEGAATIVE. WHICH IS SUGGESTING THAT WE NEED TO START THINKING ABOUT BREAKING JUST LIKE TUMOR SUBTYPES AND WE NEED TO START BREAKING METASTASIS INTO DIFFERENT SUBTYPES IN ORDER TO UNDERSTAND IT BECAUSE THEY ARE BEING MODULATED SIGNIFICANTLY BY DIFFERENT MECHANISMS ANY QUESTIONS SO FAR? >> [INDISCERNIBLE] SURE, THAT WOULD HAVE BEEN GREAT. >> THIS IS WHAT WE SEE--I MEAN--I WOULD LOVE TO HAVE IT BE THE OTHER WAY BUT THIS IS--THIS IS NOT WHAT WE'RE SEEING. SO ... SO BASICALLY THIS IS JUST SAYING WHAT I'VE ALREADY SAID, INHERITED PREDISPOSITIONS TO METASTASIS IS TUMOR AUTONOMOUS IF YOU'RE ER POSITIVE BUT IT'S TUMOR STROMAL OR IMMUNOLOGICAL OR NEGAATIVE. WE CAN ACTUALLY TEST SOME OF THAT IN THE MOUSE. WE CAN GO BACK TO OUR MOUSE MODELS. HERE WE HAVE OUR DBA AND AKR MICE, BOTH OF TREES IS A POLYOMA MIDDLE T SITUATION WITH THE TRANSGENE FORM TUMORS THE SAME TIME AND THEY GO THE SAME WAY, THERE'S 20 FOLD DIFFERENCE IN THE CAPACITY OF THESE ANIMALS INVOLVED. DOES THE IMMUNE SYSTEM PLAY A ROLE HERE, CAN WE ACTUALLY USE THIS TO FIGURE THIS OUT. IS THIS A LEGITIMATE INTERPRETATION. WELL, WE CAN DO A VERY SIMPLE EXPERIMENT HERE, INSTEAD OF LOOKING AT THE TRANSGENE POSITIVE ANIMAL FIST YOU TAKE THE LITTER MATES HERE, AND INJECT THEM WITH THE HIGHLY METASTATIC CELL LINE TURNED OUT TO BE IN FEB, SO NEITHER OF THESE--BOTH OF THESE ANIMALS WILL ACCEPT THESE ANIMAL TUMORS SINCE THEY'RE F-ONE AND AKR AND DBA, LET THE TUMORS GROW UP, HARVEST THE TUMORS DO GENE EXPRESSION ANALYSIS AND LOOK AT WHAT'S DIFFERENT BETWEEN THESE TWO, WHATEVER'S DIFFERENT HERE WILL BE DUE TO HOST BACKGROUND Ö%)SE THE TUMOR CELLS ARE IDENTICAL. SO IT'S EITHER DIFFERENT IN THE DIFFERENT STROMA OR COMBINATION OF STROMA IN DIFFERENCES, IF YOU TAKE THAT, AND LOOK AT WHETHER OR NOT THE TPX TWO SIGNATURE IS DIFFERENT BETWEEN THESE ANIMALS, BASICALLY]Fq+ ANIMAL IS NO, THEY'RE NOT DIFFERENT. SO, THIS MITOTIC CHECK POINT IS SAME IN THE PRIMARY TUMOR BECAUSE IT'S IT IS SAME CELL LINE BASICALLY IN BOTH OF THESE ANIMALS. IN CONTRAST, YOU KNOW AT THE LEVEL OF STATISTICAL THRESH HOLDS, WE SET AT THIS ONE-FIFTH OF THE MODULE GENES HERE ARE DIFFERENT SUGGESTING AGAIN THAT YOU'RE LOOKING AT AN IMMUNE INVASION RATHER THAN A RESPONSE THERE TO THE PRIMARY TUMOR. IN THE PRIMARY TUMOR. AND YOU CAN ACTUALLY TAKE THE GENES THAT ARE DIFFERENT HERE, AGAIN FROM THE MOUSE AND PERFORM THE SAME SORT OF ANALYSIS AND YOU SEE AGAIN, IT DOES PRETTY WELL PREDICTING OUTCOME IN ER NEGATIVE PATIENTS. SO AGAIN, IN IS A STROMAL REINTONES THAT APPEARS TO BE PRESENT IN ER NEGAATIVE PATIENTS. SO AGAIN NOW THE QUESTION COMES BACK TO WE'RE INTERESTED IN THESE GENES DO THEY CONTRIBUTE TO THESE GENE EXPRESSION SIGNATURES WE'RE INTERESTED AND THE ANSWER TO THAT IS YES, THE EXPERIMENTS WE DID WERE PRETTY SIMPLE WE HAD OUR CELL LINES THAT HAD HIGHLY--THAT WE INTRODUCED TO OUR GENES OF INTEREST IN, WE DID MICROARRAYS FROM THOSE TISSUES--EXCUSE ME THOSE CELL LINES COMPARED THEM TO CONTROL, PICKED OUT THE GENES THAT DISTINGUISHED THEM AND AGAIN, DID CAP LANMEYER ANALYSIS, BASED ON THE EXPRESSION OF OUR METASTASIS EXPRESSING GENES SUSEPTIBILITY GENES AND WHAT YOU SEE IS THESE GENES THAT ARE POLYMORPHIC BOTH IN THE MOUSE AND HUMAN, WHEN YOU ALTER THE LEVELS IN TUMOR CELLS, ARE ALSO CAPABLE OF GENERATING SIGNATURES THAT PREDICT OUTCOME. SO THESE GENES ARE PROBABLY INVOLVED IN THE MANIPULATION OR THE MODIFICATION OF THESE TRANSCRIPTIONAL PROFILES WITHIN THE TUMOR CELLS AND ALSO WITHIN THE STROMA THAT RESULT IN DIFFERENT SUSEPTIBILITY. OKAY, TO KIND OF GET BACK TO YOUR QUESTION OVER THERE, IF THESE GENES EXIST, CAN WE USE THEM? ARE THEY HELPFUL FOR US? OR IS THIS JUST AN INTERESTING BASIC SCIENCE QUESTION? WELL, YES, OBVIOUSLY AS I MENTIONED WE CAN DO HUMAN EPIDEMIOLOGY. YOU CAN SEE HERE, HERE'S A SNP FROM RPONE B AND THIS IS, THIS IS PARTICULARLY USEFUL IN THE CLINIC AND IT DISCRIMINATES BUT YOU'RE NOT GOING TO CHANGE YOUR OUTCOME BASED ON THIS IF YOU STRATIFY BY WHETHER THEY GO OR NOT IS ASSOCIATED WITH THOSE PATIENT WHO IS DOT DO NOT INVADE THE LYMPHATICS COMPARED TO THOSE THAT DO. AGAIN THEY ARE MULTIPLE MECHANISMS GOING ON HERE THIS, IS NOT A UNIQUE MECHANISM ASSOCIATE WIDE ALL BREAST CANCER. HERE'S AGAIN, THE TWO DIFFERENT SNPS, HERE'S ONE, CAN WE--NEITHER OF THESE WERE PARTICULARLY USEFUL IN THE CLINIC IN MY OPINION, CAN WE ACTUALLY START COMBINING THESE THOUGH TO ACTUALLY GET BETTER DISCRIMINATION, WHEN YOU START DOING THAT, ACTUALLY YES, YOU CAN, YOU GET THE SAME SORT OF LEVEL OF DISCRIP NATION THAT'S SEEN FOR THINGS LIKE MAMMA PRINT AND ONCA TYPE DX. THE DIFFERENCE IS I DON'T NEED TO BI OOMSY TO DO THIS. KIDO THIS FROM YOUR BLOOD. AND WE TAKE BLOOD AND THROW IT OUT FOR PATIENTS ALL THE TIME, NOT ONLY CAN I DO THIS WITH A BLOOD SAMPLE, THEORETICALLY I CAN PREDICT YOUR RISK FROM OUTCOME IF WE GET ENOUGH OF THESE GENES AT BIRTH. SO IN THE DAYS TO COME WHEN WE SEQUENCE EVERYBODY AS PART OF OUR CLINICAL OUTCOME, THE PROGNOSTICS FOR EVERYTHING WE'RE GOING TO DO, THIS IS ONE OF THE THINGS WE'RE GOING TO BE ABLE TO DO TO END UP WITH LUNG CANCER WITH BAD SHAPE AND BREAST CASTER YOU WON'T, THESE ARE THINGS WE CAN DO NOW OR WE COULD SEQUENCE NOW, BUT WE CAN'T PREDICT AT THIS POINT BUT ULTIMATELY YOU SHOULD BE ABLE TO. OKAY CAN WE INTERVENE, CAN WE--ONCE WE KNOW YOUR GENOTYPE, WE KNOW WHETHER YOU'RE AT RISK OR NOT, CAN WE DO SOMETHING ABOUT IT? BECAUSE IF YOU CAN'T INTERVENE, IT'S POINTLESS. IT'S ACTUALLY PROBABLY WORSE BECAUSE ONE OF THE THINGS WE KNOW IS IF YOU STRESS SOMEBODY OUT THEY'RE MORE LIKELY TO DEVELOP METASTASIS, SO IF I COME UP TO SOMEBODY, PATIENTS AND SAY, HAVE YOU BREAST CANCER, WE GENOTYPED YOU, AND YOU HAVE BAD SUSEPTIBILITY, CAN'T DO ANYTHING, SEE YOU. THAT PERSON IS GOING TO BE STRESSED AND MORE LIKELY TO DEVELOP METASTASIS, SO YOU'RE PROBABLY DOING HARM. IF WE CAN'T GENOTYPE IN AND DO SOMETHING TO HELP THESE PEOPLE, WE SHOULDN'T DID IT AT ALL. SO, TO GIVE YOU AN IDEA OF OF THE PROBLEM, REMEMBER, BEGINNING I TOLD YOU THESE CELLS WERE FLOATING AROUND. THIS IS I SLIDE I BOREIED FROM A PATHOLOGIST AT UC SAN DIEGO IS TRY TO ELSTRAIGHT THE PROBLEM, WHAT HE'S DONE HERE IS HE LABELED A TUMOR CELL WITH GFP AND PLANT TODAY INTO THE ORTHOTOPIC SITE, THE MEMORY FAT PATH, LET THE TUMORS GROW UP AND SACRIFICE THE ANIMALS AND LOOK THE LUNGS OUT AND PLACED THEM ON THE GLASS SLIDE. WHAT YOU SAW HERE ARE ALL THE CELLS IN THE LUNG SO THERE ARE A LOT OF THESE TUMOR CELLS AROUND AND THEY'RE THERE AND SOME OF THEM EACH ONE OF THEM HAVE THE POSSIBILITY OF CONVERTING INTO A PROLIFERATIVE AND CLINICALLY RELEVANCE METASTASIS. , IS THIS CELL LINE IS CONSIDERED METASTATIC EMPLOY. , NEVER THE THELESS, YOU HAVE LARGE NUMBERS OF TUMOR CELLS FLOATING AROUND THE BODY THAT YOU HAVE TO SOMEHOW DEAL WITH. SO IN MY OPINION, AS A BASIC SCIENTIST AND I'M NOT INVOLVED IN DRUG DEVELOPMENT SO ANYONE WHO IS CAN FEEL FREE TO CORRECT ME BUT WHAT I THINK WE NEED ARE TWO DIFFERENT TYPES OF NEW AGENTS WE NEED AGENTS THAT ARE CYTOTOXIC TO METALESION AND DISSEMINATED TUMOR SILLS, THAT'S OBVIOUS, WE KNOW THAT UKES THE METASTASIS TEND TO BE MUCH MORE RESISTANT TO CHEMEE THERAPY THAN THE PRIMARY TUMORS AND WE HAVE TO SOMEHOW GET THAT BETTER, AND/OR WE ALSO NEED CYTOSTATIC AGENTS TO PREVENT THESE TUMOR SEMESTERS FROM GREEN CELLING BECAUSE WE KNOW NOW THAT TUMOR CELL K'S METASTASIZE FROM VERY EARLY, CAN SIM SEMESTERINATE TO THE BODY FROM THE TUMORS SO BY THE TIME A WOMAN COMES IN WITH BREAST CANCER, DIAGNOSED BREAST CANCER THERE'S A VERY GOOD CHANCE SHE'S ALREADY GOT TUMOR CELLS SPRET OUT THAT THROUGHOUT HER BODY IF YOU CATCH IT AT A EARLY STAGE. SO WHAT WE WANT TO DO IS SOMEHOW REDUCE THE LIKELIHOOD THAT THOSE TUMOR CELLS WILL CONVERT TO PROLIFERATIVE LESIONS LESIONS LESIONS AND RESULT IN MORBIDITY AND METABOLISM. WE IF WE FIND SOMETHING WE CAN GIVE TO PATIENTS CHRONICALLY, THAT PRESENTS SHE'S FROM DEVELOPING INTO MICROSCOPIC METASTASIS AND THE PATIENTS DIES OF OLD AGE OR YOU KNOW STROKE OR HEART DISEASE OR SOMETHING, THEN AS AN ONCOLOGIST WE'VE WON. EVEN THOUGH WE HAVEN'T ERADICATED THEM IF WE PREVENTED THEM FROM GROWING THEN SOMEHOW WE'VE HELP THD PATIENT. SO ARE THERE ANY SORT OF THESE PATIENTS PATIENTS AROUND THERE, LOOKING AT THIS ALREADY, THE ANSWER IS, CAFFEINE. IN OUR MOUSE MODELS, CAFFEINE IS NOT A BAD METASTATIC SUPPRESSOR. AND BEFORE YOU ASK, THE REASON WE GOT INVOLVED IN CAFFEINE IS ONE OF THE GENETICALLY DEFINED REGIONS THAT HARBORS A METASTASIS GENE FALLS ON TOP OF THE THE ATM GENE, WE SEQUENCE THE ATM GENE IT HAS MULTIPLE SUBSTITUTIONS AND THE STRAINS WE'RE INTERESTED IN AND NONE OF WHICH ARE OBVIOUSLY FUNCTIONALLY DIFFERENT SO, I DID THE LITERATURE RESEARCH AND FOUND OUT THAT CAFFEINE IS AN ATM INHIBITOR AND WHAT IS THE HARM IN GIVING THESE ANIMALS WITH CAFFEINE AND SEE WHAT HAPPENS. SO WE DID IT. WE GAVE OUR ANIMAL CAFFEINE AND TWO DIFFERENT TIME POINTS. AND WE HAD A IS PSEUDOPREVENTAATIVE MEASURE, PREVEBTAATIVE REGIME BECAUSE THEY HAVE HYPER PLACIA AT THIS POINT BUT DO NOT HAVE CARCINOMAS, OR WE GAVE IT TO THEM AT 60 DAYS OF AGE BY WHICH TIME THEY HAD CARCINOMAS SO THIS IS WHAT YOU DO IF SOMEONE CAME INTO THE CLINIC AND YOU DIAGNOSE THEM WITH A TUMOR AND YOU GIVE THEM CAFFEINE TO TROY TO PREVENT ANY MORE TUMOR CELLS FROM ARISING. IN THIS CASE WE SAY WE'RE KNOW YOU'RE AT HIGH RISK, THIS WILL BE A PREVENTAATIVE REGIME, WE HOPEFULLY WILL PREVENT IT FROM METASTASIZING. AT ONE POINT WE EUTHANIZE ANIMALS AND WE SECTION THE LUNGS AND COUNT THE NODULES IN THE LUNG, AND THE AMOUNT OF CAFFEINE WE GAVE THEM IS .4, AND THE LITERATURE SAYS IS THE AVERAGE FOR GREEN TEA. SO WHEN DO YOU THAT, THIS IS WHAT YOU GET, THE CONTROL ANIMAL IS SOMETHING OF THE METASTASIS I WAS HIDING FOR. WHEN YOU GIVE THESE ANIMALS CAFFEINE AT WEANING, IT SUDDENLY STARTS AND IT'S IN THE LIVER OR IF THESE ANIMALS ALREADY HAVE THEM, AND THIS IS REDUCTION AGAIN, DON'T DEVELOP METASTASIS, YEP THEY'RE GIVING KAF NIGHT THE WATER AND THAT'S ALL THEY'RE ALLOWED TO DRINK FOR THE REST OF THEIR LIFE.w+Z) THEY RIP THEIR HAIR HOW OUT, THE CARE TAKERS SUGGESTS--NO I'M NOT SUGGESTING WE GO OUT AND TELL PEOPLE TO DRINK SIX VENTYS A DAY, THAT'S NOT A GOOD IDEA. BUT I THEN IS AN IDEA THAT THERE MIGHT BE SMALL MOLECULES WE COULD USE. >> DID HAVE YOU A QUESTION? >> [INDISCERNIBLE]. >> WE HAVE NOT TRIED INCREASING THE DOSE OF CAFFEINE. THIS SUFFICIENCY--THEY WERE UNHAPPY ENOUGH ABOUT THIS. SO THE NEXT QUESTION IS, IS THERE ANY EVIDENCE OF THE HUMANS. SO IF YOU DIG DEEP ENOUGH INTO THE LITERATURE AND AGAIN, I AM NOT, I AM NOT TRYING TO CONVINCE YOU THIS IS PROOF, YOU CAN FIND A COUPLE OF STUDIES, HERE'S ONE THE COMA COLA STUDY, IF YOU DRINK BETWEEN ONE AND THREE LET LITERS OF COKE A DAY, WHAT YOU SEE IS THE CANCER RELATED DEATH GOES DOWN, IT'S ABOUT A QUARTER. HERE'S THE KICKER, WHAT'S CHD? >> CORONARY HEART DISEASE. >> CORONARY HEART DISEASE SO YOU'RE NOT DYING OF CANCER BI YOU'RE STROKING OUT AND HAVING HEART ATTACKS. THE SAME GROUP STUDIED COFFEE, IF YOU DRINK AND GET THIS, 10 CUPS OF COFFEE A DAY, IT'S REDUCED. OKAY? NOW THE OTHER REASON I LIKE THIS STUDY, BECAUSE THEY ALSO SAID, WELL, YOU KNOW, IS IT JUST BECAUSE THESE PEOPLE ARE ACTIVE, RUNNING AROUND MORE, ARE THEY JUST--IF I DRAIRCHG 10 CUPS OF COFFEE A DAY, I WOULD BE RICOCHETING OFF THE WALLS SO THESE TWO DRUG GROUPS VALIUM OF COURSE EVERYONE KNOWS IF YOU'RE VERY RELAXED AND HAPPY, YOU HAVE LOW CORONARY HEART DISEASE BUT THESE PATIENTS ARE DYING MORE OF CANCER. BEING IF YOU GIVE THEM THIS DRUG WHICH IS AN ANTIDEPRESSANT AND STIMULATE ANT, NOT MUCH IS HAPPENING HERE AND THESE PEOPLE ARE STILL DYING LESS. IS THIS PROOF? ABSOLUTELY NOT. IS IT CONSITTENT? YEAH. SO, I THINK THERE MAY BE MOLECULES OUT THERE. IS IT CAFFEINE, NO. I DO NOT WANT PEOPLE TO GO OUT AND DRINK LOTS AND LOTS OF STARBUCKS, I'M NOT FUNDED BY STARBUCKS, IT'S NOT A GOOD IDEA. THERE MAY BE THINGS OUT THERE CHRONICALLY THAT WILL REDUCE THE PROBABILITY OF METASTASIS WILL DEVELOP THEREBY SPENDING THEIR LIVES AND REDUCING MORTALITY AND MORBIDITY AND ALL THAT GOOD STUFF. THE TRICK IS WE HAVE TO FIND THEM. OKAY. >> SO I JUST WANT TO END WITH SOME ANIMATIONS THEY DID WHEN I HAD WAY TOO MUCH TIME ON MY HANDS A COUPLE OF DAYS AND JUST TO TRY TO DRIVE HOME WHAT I THINK IS HAPPENING WITH GENETIC GROUND. , YOU KNOW IT'S CLEARLY THE DRIVER BECAUSE IF YOU DON'T HAVE THE TUMOR YOU'RE NOT GOING TO HAVE METASTASIS, BUT I WANT YOU TO TRY TO REMEMBER THAT YOU KNOW THE BACKGROUND IS IMPORTANT AND HOW WE THINK THIS IS OPERATING, WHAT DO YOU HAVE TO DO TO BE SUCCESSFUL WITH METASTASIS. JOSH SAID YEARS AGO, THAT YOU CAN'T USE THE ANALOGY THAT A METASTATIC CELL HAS TO DO A LOT, A LOT OF THINGS IN ORDER TO DEVELOP INTO A CLINICAL LESION. WE MODIFY IT A LITTLE BIT FOR OUR ANAILINGNOLOGY LIKE A HURTLER, WHAT DOES IT HAVE TO DO TESCAPES BY THE PRIMARY TUMORS IT HAS TO CONE VEY STROMA AND THEIR BRICK WALLS BECAUSE I CAN'T DRAW A HURDLES INTO THE VASCULAR AREA AND WHERE ELSE, SURVIVE DEPENDENCE AND IT HAPPENS VERY EASILY, AND THE SURVIVAL OF THE MICROENVIRONMENT IS WHERE MOST OF THESE GUYS GO. AND THEN THEY HAVE TO GET TO A SECOND PLACE AND ACTUALLY PROLIFERATE BEFORE THEY BECOME CLINICALLY RELEVANT, OKAY, IF THEY FAIL ANY ONE OF THOSE STEPS, THEY'RE IRRELEVANT FOR METASTASIS. YOU KNOW JUST IF THEY DIE SOMEWHERE, THEY WIPE OUT. S-[ SO HOW DOES GENETIC BACKGROUND, HOW DOES POLYMORPHISM AFFECT THIS, WELL, I THINK IT'S BASICALLY MODULATING THE HEIGHT OF THESE BARRIERS, AND IT'S EITHER GOOD OR BAD, IMMUNOLOGICAL SURVEYANCE IF HAVE YOU ANY NEGAATIVE TUMORS SO IF YOU'RE--YOU'RE UNFORTUNATE AND HAVE A HIGH METASTATIC GENOTYPE, IT'S THE HURDLES ON THE WAY. IF YOU HAVE A LESS PERMISSIVE GENE, IT STILL CAN HAPPEN, AND IT CAN HAPPEN, IT'S JUST MUCH HARDER. SO YOU HAVE A LOWER PROBABILITY OF THIS GUY CREEPING HIS WAY OUT OF THE CELL. BY FINDING THE GENES AND UNDERSTANDING THE MECHANISMS THE TUMOR CELL, AND SINCE SOME OF THESE, THEY MAY BE NORMAL PROCESSES, METASTATIC DISEASE, RATHER THAN TRYING TO RECONFIGURE SOMETHING THAT HAS A PERMANENT MUTATION OR ALTERATION BY CONSITTATIVE ACTIVATION OR LOSS OF TUMOR SUPPRESSOR GENE AND WITH THAT HERE'S A WHOLE BUNCH OF PEOPLE IN MY LABORATORY WHO HELPED US WITH THIS WORK AS WELL AS THE NUMBER OF OUR COLLABORATORS AND I'LL BE HAPPY TO TAKE ANY QUESTIONS YOU HAVE IN IT IS NEXT FEW MINNITES BEFORE THE NEXT TALK. [ APPLAUSE ] ANYTHING ELSE SORE DID WE ANSWER IT--ELSE OR DID WE ANSWER IT ALREADY? >> [INDISCERNIBLE]. >> SO THAT'S A GOOD QUESTION, ALL OF OUR WORK IS DONE IN IMMUNOLOGICALLY COMPETENT ANIMALS AND THAT'S BECAUSE NOT ONLY OUR WORK WHICH IS RECENTLY SHOWN THIS ASSOCIATION WITH THE IMMUNE SYSTEM BUT OTHER PEOPLE'S WORK PARTICULARLY PEOPLE IN ALBERT EINSTEIN HAVE SHOWN THAT MACK WOPHAGES ARE PARTICULARLY IMPORTANT IN A LOT OF THE METASTATIC PROCESS, SO WE ALSO DO ALL OF OUR WORK IN IMMUNO COMPETENT ANIMALS SO WE'RE NOT ELIMINATE WAG'S KNOWN TO BE AN IMPORTANT PART OF THE PROCESS. >> [INDISCERNIBLE] >> THE IMMUNE SYSTEM CAN PROMOTE OR INHIBIT DEPEBBLEDS HOW IT'S OPERATING. >> THE MACROPHAGES PROMOTE IT. DRNCHL ANYTHING ELSE OR CAN I TURNOVER THE MICROPHONE. OKAY.MY >> OKAY, OUR NEXT SPEAKER IS ENRIQUE SUDARI, HE IS WITH THE NCI CARCINOGENESIS, AND HE HAS SOME MOVIES FOR YOU TO WATCH. >> THANK YOU. >> WELL, GOOD AFTERNOON, OR GOOD EVENING, I KNOW IT'S LATE BUT TALKING TO AN EMPTY ROOM IS VERY DISCOURAGING. SO THANK YOU SO MUCH FOR STAYING I'M GOING TO TALK ABOUT ANGIOGENESIS IN CANCER, AND THIS IS INTERVENTION, TO I'M SURE ALL OF YOU ARE FAMILIAR WITH ANGIOGENESIS BUT I WANT TO START MAKING A DIFFERENT--AND AT THE SAME TIME A DIFFERENT ANGIOGENESIS BY COMPARING VASC LO GENESIS AND ANGIOGENESIS, BUT TWO SEMESTERULAR TERMS BUT AT THE SAME TIME VERY DIFFERENT. BOTH OF THEM VASCULAR GENESIS AND ANGIOGENESIS WERE SOLD IN CREATION OF VASCULATURE, OKAY? HOWEVER, VASCULAR GENESIS, WHICH WILL BE REPRESENTED OVER HERE, IS THE GENERATION OF THOSE CELLS FROM--AND DIFFERENTIATED ANGIO BLAST. OKAY, CELLS THAT ARE PRECURIOUSORS AND THE VASCULATURE, AND ANGIOGENESIS ON THE OTHER SIDE IS THE FORMATION OF VASCULAR SPROUTS FROM PREEXISTING VASCULATURE, AND AND IT LOOKS LIKE THIS, SO YOU HAVE THEM FORMING LIKE A TISSUE, AND, AND THIS IS A SMALL TIME LAPSE OF ANGIOGENESIS AND YOU CAN SEE, PROUD, THIS IS A VESSEL OVERHERE AND YOU CAN SEE SPROUTS, AND ANGIOGENESIS ON VASCULAR DIFFERENCES BETWEEN ANGIOGENESIS AND VASC LO GENESIS, AND OCCURS MAINLY DURING EMBRYO DEVELOPMENT NOW THERE IS AN INTERESTING PAPER LIKE LAST YEAR, SHOWING DEMONSTRATING VASCULAR GENESIS IN ADULTS, BUT IT IS RARE. ANGIOGENESIS, IT IS COMMON IN EVERY OTHER DEVELOPMENT BUT ALSO IT IS COMMON IN ADULTHOOD, IN ADULTS MAYBE HAVE YOU HEARD ARMINGIO GENESIS IN A PATHOLOGICAL CONTEST BECAUSE IT'S RELATED TO CANCER AND OTHER DISEASES. HOWEVER, I'M JUST GOING TO SAY THAT OVERAGEIO GENESIS IS NOT ONLY NOT ONLY OCCURRING BUT IT'S NECESSARY FOR A NORMAL HOMEOSTATIC ADULT. WHERE IS IT NECESSARY? FEMALE PRODUCTIONS DO IT, HAIR GROWTH, EVEN IN PATHOLOGICAL CONDITIONS, HAVING ANGIOGENESIS IS A GOOD THING, FOR INSTANCE HEART ATTACK, AFTER A HEART ATTACK WHAT WE WANT TO DO IS WE WANT TO REOX GENERATEDDATE THE HEART SO WE WE NEED ARMINGIO GENESIS TO REOX GENERATED EIGHT THE HEART. >> AND WE'RE GOING TO TALK MORE ABOUT THAT. AND AT THE SAME TIME, ANGIOGENESIS IS A BAD THING SUCCESSIVE ANGIOGENESIS YOU HAVE TOO MUCH OR TOO LITTLE AND IT'S LINKED TO A CERTAIN DISEASE STATE. MY INTEREST IS TUMOR ANGIOGENESIS AND THAT'S WHAT I'M GOING TO TALK ABOUT A LITTLE BIT OVER THE NEXT SLIDE. NOW, I HAVE A TWO YEAR-OLD DAUGHTER AND FROM KNEES DOWN SHE'S RUNNING ALL THE TIME, SHE FALLS ALL THE TIME AND AND AND THE TYPE OF CANCER BY SAYING THAT, WELL, HERE WE HAVE A NORMAL CELL THAT BECOMES TUMOR GENIC AND WE'RE NOT GOING TO TALK ABOUT WHY THAT BECOMES TUMOR GENIC AND OF COURSE, ONE OF THE TUMORS THAT THEY GROW, THEY DUPLICATE AND SO THESE CELLS CAN GROW TO A CERTAINICIZE, AND WE HAVE A TUMOR, AND NOW THESE CELLS WOULDIy JUST NOT GROW, THIS IS WHAT WE CALL DORMANT INSIGHTUE CANCER, THERE'S A LOT OF EVIDENCE IN THESE CANCERS. THIS A SEMINAL PAPER THAT I LIKE TO CITE BUT AGAIN THERE'S A LOT OF LITERATURE IN THIS. IN 90-93 WHERE BLACK AND WELSH THEY LOOK AT TRAUMA YOU KNOW PATIENTS THEY DIE FROM AN AX, WHATEVER, THEY LOOK AT THE--FOR EVIDENCE OF THESE MICROTUMORS IN DIFFERENT ORGANS, NOW WHAT THEY FOUND WAS OUTSTANDING, AGES 40-50, 39% OF WOMEN SHOWED THIS NODULE IN BREAST AND 60-70, 46% OF MEN IN PROSTATE AND 50-7100% EVERY SINGLE PATIENT CONTAINED THESE MICROTUMORS IF YOU WANT TO CALL THEM LIKE THAT. IN THE THYROID. NOW OF COURSE, NONE OF THESE WERE VERY, VERY SMALL PERCENTAGE OF THESE TUMORS WERE DIAGNOSED. SO THAT MEANS THAT THESE TUMORS YOU SEE HERE ARE NOT CLINICALLY RELEVANT, THEY ARE THERE BUT THEY'RE NOT CLINICALLY RELEVANT. NOW, NEW LITERATURE, NEW DATA ON THIS SAME IDEA SHOW THAT THESE AGES ARE LOWER THAN THEY SHOW HERE IN THIS PERCENTAGES ARE HIGHER MEANING THAT PROBABLILY MOST OF US IN HERE CONTAIN ONE OF THESE ONE OR MORE OF THESE, YOU KNOW MICROTUMORS, BUT WE'LL NEVER KNOW ABOUT IT. OKAY BECAUSE IT IS ALSO A FACT THAT MOST OF US WILL NEVER ACTUALLY DEVELOP CANCER. AND WE'LL NEVER BE DIAGNOSED SO THESE--THESE SMALL MICROTUMORS DON'T HAVE THE ABILITY EVENT THOUGH THE CELLS ARE FULLY TUMOR GENIC, THEY DON'T HAVE THE ABILITY TO EXPAND AND TO BECOMEŤ— CLINICALLY IRRELEVANT. THE REASON WHY THEY CAN'T DO THAT, IS THE VASCULATURE, THEY DON'T HAVE VASCULATURE, WELL THEY HAVEN'T BEEN ABLE TO ATTRACT VASCULATURE TOWARDSS THAT MASS. IF YOU THINK OF A TUMOR AS AN ORGAN, JUST THINK OF A TUMOR AS AN ORGAN, AN ORGAN REQUIRES THE VASCULATURE FOR NUTRIENTS, OXYGEN, TO RELEASE, TO ELIMINATE ALL THE BYPRODUCTS OF THE METABOLISM OF THE TUMOR, SO A MASS THAT IS GROWING AND IS NOT VASCULARIZED WILL NOT BE ABLE TO GROW TOO FAR. NOW SOMETHING HAPPENS WITH CERTAIN TUMORS THAT AT A CERTAIN POINT THOSE TUMORS AND WE'LL TALK A BIT ABOUT THIS LATER CHANGE, THERE'S A CHANGE IN THOSE TUMORS AND THOSE TUMORS ACQUIRE THE ABILITY TO THAT VASCULATURE. SOMETHING HAPPENS TO THE CELLS AND THEN THEY BECOME ABLE TO ATTRACT VASCULATURE OR TO CREATE THE--OR TO INDUCE THE FORMATION OF VASCULATURE, THIS IS WHAT WE CALL THE ANGIOGENESIS SUITE AND WE WILL TALK ABOUT THAT MORE IN A SECOND. BUT AT THAT POINT, WHEN THE VASCULATURE REACHES THE TUMOR AND IT STARTS IRRIGATING THAT TUMOR, THE TUMOR BECOMES CLINICALLY RELEVANT. SOME EXAMPLES OF THIS WHERE THE TUMOR CAN BE LIKE THIS FOREVER, FOR YEARS AND SUDDENLY THE ANGIO GENIC SWITCH HAPPENS IN A VERY SHORT PERIOD OF TIME IN A MANNER OF WEEKS THAT TUMOR BECOMES CLINICALLY RELEVANT. OF COURSE, YOU KNOW EYE ADDED METASTASIS AND EVERYTHING YOU HEARD ABOUT METASTASIS IN THE TALK BEFORE, ONE OF THE MAJOR PROBLEMS WITH ANGIOGENESIS IN THE TUMOR IS IT ALLOWS THE CELLS TO GO TO OTHER PLACES. RIGHT? TO METRICS TAFT CISE TO OTHER PLACES. SO, THE ANGIOGENESIS IS NOT ONLY BENEFICIAL FOR THE TUMOR BECAUSE HE'S PROVIDING THEOX GENERATED AND NUTRIENTS AND IS RELEASING THE WASTE PRODUCT IT IS ALSO PROVIDES THE AVENUE FOR THOSE CELLS TO METASTASIZE TEST TEST TEST TEST TEST OTHER SECONDARY LOCATIONS IN THE BODY. OKAY, SO THESE ANGIOGENESIS SWITCH. YOU CAN SEE HOW THESE ANGIOGENESIS SWITCH, IT IS A VERY IMPORTANT STEP. DURING THE PROCESS OF CANCER DEVELOPMENT AGAIN, WE'RE GOING TO REMIND THAT YOU THESE CELLS ARE FUEL FULLY TUMOR GENIC AND IF YOU TAKE THESE CELLS, WHICH WERE NOT IN THE TUMOR IN WHATEVER YOU'RE TALKING ABOUT AND YOU PUT THEM IN THE RIGHT ENVIRONMENT, THEY SUDDENLY BECOME FULLY--THEY DEVELOP INTO A CLINICALLY IRRELEVANT TUMOR, INTO A FRYING TUMOR HOWEVER IN THIS PARTICULAR LOCATION AND BECAUSE THEY HAD NOT UNDERGONE THE ANGIO GENERATED SICK SWITCH, THEY WERE NOT ABLE TO FULLY DEVELOPED. THIS ANGIOGENESIS SWITCH HAPPENS IN THIS PLACE THAT WE CALL THE TUMOR MICROENVIRONMENT. IT'S IT IS MICROENVIRONMENT WHERE THE CELLS ARE IN THE BODY. THERE ARE MANY DIFFERENT TYPES OF TUMOR MICROENVIRONMENT AND EVERY ONE OF THOSE ENVIRONMENTS IS DIFFERENT AND NEEDS TO BE STUDIED SEPARATELY. BUT THERE ARE CERTAIN, YOU KNOW IDEAS GENERAL IDEAS THAT WE CAN DISCUSS ABOUT THESE TUMOR MICROENVIRONMENT. THESE ARE TUMOR CELLS IN HERE AND THIS IS OUR DORM ABT TUMOR, OKAY, TUMOR GENIC CELLS THAT HAVE NOT DEVELOPED INTO A CLINICALLY RELEVANT TUMOR, HE IS OUR PREEXISTING AND OF COURSE BECAUSE OXYGEN DIFFUSES IN TISSUES, AT LEAST TO A CERTAIN DISTANCE, THERE'S A GRADIENT OF OXYGEN FROM THESE PREEXISTING VASCULATURE TO THESE TUMORS. ONE OF THE THINGS THAT HAPPENS TO THIS TUMOR IS THE AMOUNT OF OXYGEN THE TUMOR RECEIVES ARE VERY SMALL. SO THAT MEANS THAT THE TUMOR IS HIPOXIC. AND WHAT WE CALL HIPOXIC AND MOST OF YOU ARE FAMILIAR PROBABLILY WITH THIS TERM HIPOXIA, SO, THE FACT THAT THE TUMOR IS HIPOXIC INDUCES IN THE TUMOR, CERTAIN CHANGES AND MANY OF YOU MIGHT BE FAMILIAR ALSO WITH A--WITH A FACTOR COAL HIPOXIA INDUCIBLE FACTOR, WHICH IS RESPONSIBLE FOR ALL THE--NOT ALL BUT YOU KNOW PART OF THE OXYGEN SENSING WITHIN TUMOR CELLS AND NORMAL CELLS AS WELL. SO THESE TUMOR CELLS THROUGH CERTAIN MECHANISMS WHICH FOR INSTANCE INVOLVE HIF, HIPOXIA INDUCIBLE FACTOR START CHANGING AND START INTRODUCING THE ANGIOGENESIS SWITCH THAT'S A MECHANISM BY WHICH, THEY CAN ATTRACT VASCULAR TOWARDS THE TUMOR NOW THE ANGIOGENESIS SWITCH IS DEPENDENT ON A NUMBER OF FACTORS, SOLUBLE FACTORS THAT ARE RELEASED INTO THE TUMOR MICROENVIRONMENT, THE FACTORS FOR INSTANCE ARE VEG F AND WE'RE GOING TO TALK ABOUT THIS, LATER. MOST OF THE FACTORS ARE GROWTH FACTORS, ANGIOGENESIS FACTORS IS AS WE'RE GOING TO SEE LATER AND THESE FACTORS CREATE A GRADIENT FROM THE TUMOR TO THE VASCULATURE. WHEN THESE FACTORS REACH THE VASCULATURE, SEVERAL THINGS HAPPEN, BUT, I'M JUST GOING TO TALK ABOUT ONE OF THE PROCESSES THAT HAPPENS THAT THE TUMOR TAKES ADVANTAGE OF TO IN. DUCE ANGIO GENESIS, ONE OF THE THINGS THAT HAPPENS HERE IS THAT A LOT OF THESE MOLECULES ARE NOT ONLY GROWTH FACTORS AND ANGIOGENESIS FACTORS BUT ARE A KEY ATTRACT ANT FACTORS FOR OLDER CELLS FOR INSTANCE CELLS THAT BELONG TO THE UMPIRESUNE SYSTEM, OKAY? --IMMUNE SYSTEM, OKAY? AND ONE OF THE PROGENYS WE DID A FEW YEARS AGO IS WE LOOKED AT THE INNUN ENS OF THESE FACTORS AND ONE OF THESE CELL TYPES THAT IS MASS CELLS, I DON'T KNOW IF YOU'RE FAMILIAR WITH MASSLES, THEY'RE VERY FUN CELL TYPE. THEY'RE LIKE--THE CELLS RESPONSIBLE FOR ALLERGIES, RIGHT? THEY RELEASE HISTA MINE AND THEY ARE RESPONSIBLE FOR ALLERGIES. BUT THEY HAVE A LOT OF THEM--THEY ARE IMPLICATE INDEED A LOT OF MANY DIFFERENT PROCESSES, AND ONE OF THEM IS THIS ONE IN HERE. SO A LOT OF THESE FACTORS AND INCLUDING ALL OF THESE FACTORS IN HERE, ARE A CHEMEE ATTRACT APT FOR THESE MASS CELLS, SO THOSE ARE RECRUITED WITHIN THE TUMOR MICROENVIRONMENT, AND NOW MASS CELLS ARE LIKE BAGS THAT ARE FULL OF FACTORS, DIFFERENT FACTORS WHICH HERE I HAVE REPRESENTED WITH ALL THOSE SPOTS IN THERE. AND THESE ARE GROWTH FACTORS AND ANGIOGENESIS FACTORS AND ET CETERA, ET CETERA UKES ET CETERA, SO YOU CAN SEE HOW THIS PROCESS IS UP--BECOMES A POSITIVE LOOP BECAUSE THE TUMOR CELLS ARE RELEASING THESE FACTOR WHICH IS ATTRACTING OTHER CELL INTO'S THE TUMOR MICROENVIRONMENT WHICH AT THE SAME TIME HAVE THE ABILITY TO RELEASE ALL THESE FACTORS WHICH ARE A GROWTH FACTORS AND ANGIOGENESIS FACTORS, ET CETERA, ET CETERA. THE IDEA I WANT TO CONVEY HERE IS THAT THIS PROCESS NOT JUST THE SIMPLE PROCESS CALLING THE VASCULATURE, IF YOU WILL AND THE VASCULATURE OF GROWING. IT INVOLVES MANY DIFFERENT CELL TYPES, IT INVOLVES A LOT OF DIFFERENT GROWTH FACTORS, A LOT OF DIFFERENT COMPONENTS OF THE EXTRA CELLULARa by MATRIXS WE'RE GOING TO SEE NOW, ET CETERA. SO ONE OF OF THESE FACTORS HAVE RELEASED NEW VASCULATURE STARTING GROWING FROM THE PREEXISTING VASCULATURE TOWARDS THE TUMOR. THIS VASCULATURE, WE ARE GOING TO SEE LATER HAS VARIOUS SPECIFIC CHARACTERISTICS AND THOSE CHARACTERISTICS ARE THEN GOING TO REPRESENT OPPORTUNITIES FOR US, AS SURROGATES FOR THERAPEUTIC INTERVENTIONS. ONE OF THE VERY IMPORTANT COMPONENTS WITHIN THE MICROENVIRONMENT IS THE EXTRA CELLULAR MATRIX, IS THE VESSELS AT THE ENDOTHELIAL CELLS WHICH ARE FORMING THESE VESSELS WANT TO REACH THE TUMOR, THEY'RE GOING TO HAVE TO REMODEL THE EXTRA CELLULAR MATRIX, THIS LIKE--YOU CAN PICTURE THE CELLULAR MATRIX, IT'S A CHEESE, RIGHT IN THE ROOM, IMAGINE THIS ROOM FULL OF CHEESE AND THE END O THELLIAL CELLS HAVE TO CARVE THE CHEESE TO GET TO THE OTHER END WHERE THE TUMOR IS AND IT'S AS HARD AS THAT. IT IS A PROCESS THAT IS AN ACTIVE PROCESS, NOT THE CELLS KIND OF LIKE SLIDE THROUGH THE TISSUES, UNTIL THEY GET TO WHERE THE TUMOR IS. THEY HAVE TO ACTIVELY REMODEL THE EXTRA CELLULOID MATE MATRIX AND ALL THESE PROCESSES ARE A CASCADE OF PROCESSES WHICH WILL TALK ABOUT LATER AND ALL OF THEM ARE REQUIRED FOR THE ENDOTHELIAL CELLS TO GET TO THE TUMOR CELLS AND FOR ANGIOGENESIS TO HAPPEN. IF ONE OF THESE PROCESSES IS BLOCKED OR DOESN'T HAPPEN, THERE'S NO ANGIOGENESIS OR WE CAN BLOCK ANGIOGENESIS. AND THIS IS OF COURSE, AS WE DISCUSSEDś/ś]Ť BEFORE, ONE OF THE AVENUES THAT THE TUMOR CELLS CAN UTILIZE FOR METASTASIS. THE VASCULATURE. THE TUMOR VASCULATURE, IS VERY DIFFERENT COMPARED TO THE NORMAL VASCULATURE, THIS IS JUST A REPRESENTATION OF A CHROMAL SIGNIFY TELOMERA WHICH HAS BEEN DISTRACTED HERE. IT'S A CHROMOSY TELOMERA GROW NOTHING A 14 YEAR-OLD BOY WHICH AS YOU CAN SEE IS GROWING ON TOP OF THE KIDNEYS ON TOP OF ONE OF THE KIDNEYS OVERHERE, AND AS YOU CAN SEE, IT HAS ATTRACTED TREMED EPPEDDOUS AMOUNT OF VASCULATURE AND IT IS A LARGE TUMOR ALSO BUT IT HAS ATTRACTAD A TREMENDOUS AMOUNT OF VASCULATURE AND AS THE FIGURE ROTATES YOU CAN APPRECIATE HOW THESE THIS IS VERY DIFFERENT COP PAIRED TO OTHER VASCULATURE THAT CAN YOU SEE IN THE IMAGE FOR INSTANCE HERE IN THE KIDNEY, SO THIS--IF YOU LOOK FIRST AT THE VESSEL COMPARED TO THESE VESSELS OVERHERE, THEY ARE MORPH LOGICALLY VERY DIFFERENT, OKAY? ONE OF THE THINGS THAT YOU KNOW ONE OF THE CHARACTERISTICS THAT YOU CAN OBSERVE RIGHT A WAY IS THAT THAT THAT VASCULATURE SEEMS TO BE VERY STOUTERUOUS, INSTEAD OF BEING A STRAIGHT VESSEL THEY GO FROM ONE PLACE TO ANOTHER, THEY ARE VERY TORTUOUS. IN FACT VASCULATURE, THE TUMOR VASCULATURE AS I SAID IS VERY DIFFERENT TO THE NORMAL VASCULATURE, IN THE NORMAL, APART FROM THE MORPH LOGICAL DIFFERENCES WE OBSERVE, WHICH IS THAT THEm STRAIGHT COMPARED TO THE TUMOR VASCULATURE WHICH IS MUCH MORE TORTUOUS, FOR INSTANCE A NORMAL VASCULATURE, IS COVERED WITH SOME ACCESSORY CELLS, EVERY VESSEL IS WRAPPED WITH ACEASESSORY CELLS ONE OF THOSE CELL TYPES AND CALLED PAIR SIGHTS I'M NOT SURE YOU'RE FAMILIAR WITH PERICITES THEY WRAP AROUND VESSELS AND THEY HAVE MANY FUNCTIONS BUT ONE OF THE FUNCTION SYSTEM TO SUPPORT THE VASCULATURE BASICALLY. THAT HAPPENS IN NORMAL VASCULATURE AND THESE ARE VERY TIGHTLY, WRAPPED VASCULATURE. IN TUMOR VASCULATURE, THERE ARE NOT AS MANY PRAIRIE CITES. OKAY? SO AND THE ENDOTHELIAL CELLS ALTHOUGH THEY ARE FORMING JUNCTIONS TO EACH OTHER, THOSE JUNCTIONS ARE A LOT LESS TIGHTER THAN THE ONE WEES FOUND IN NORMAL VASCULATURE, AS A CONSEQUENCE, THE VASCULATURE IN THE TUMOR IS VERY LEAKY. SO IT'S MUCH MORE LEAKY THAN THE--THE NORMAL VASCULATURE. THERE'S ALSO--THE GENE EXPRESSION BETWEEN THE NORMAL VASCULATURE AND THE TUMOR VASCULATURE IS DIFFERENT. OR AT LEAST, IT IS SOMEWHAT DIFFERENT, A PERSON HERE AT THE NCI THAT HAS DEVOTED A LOT OF TIME TO THE BRIGHT SIDE, HE'S HAPPY IN FREDERICK, AND HE HAS WORKED A LOT IN WHAT--THEY'RE CALLED TUMOR ENDOTHELIAL MARK E-PRESCRIBINGS SO THESE ARE GENES AND PROTEINS THAT ARE EXPRESSED EXCLUSIVELY IN THE TUMOR VASCULATURE. WHY ALL THESE THINGS ARE IMPORTANT, ALL THESE THINGS ARE IMPORTANT BECAUSE THEY GIVE AGAIN--AT A GIVE THIS OPPORTUNITY FOR THERAPEUTIC INTERVENTION, IF WE KNOW THERE IS A TUMOR IN THE ENDOTHELIAL MARKER, IT IS A PROTEIN LIKE A VASCULATURE OR NORMAL VASCULATURE, THE TUMOR VASCULATURE BUT NOT IN THE NORMAL VASCULATURE, WE COULD UTILIZE THAT TO TARGET THE TUMOR VASCULATURE, THE FACT THAT THE TUMOR VASCULATURE, IS LUKEY CAN ALSO BEE ARE BE THOUGHT AS A AN OPPORTUNITY FOR THERAPEUTIC INTERVENTION. IT IS I COMPARISON BETWEEN THE TUMOR VASCULATURE AND THE NORMAL VASCULAR FOR THIS PARTICULAR REASON. AS WE LEARN MORE ABOUT TUMOR VASCULATURE WE ALSO REALIZE THAT DIFFERENT TUMOR VS DIFFERENT VASCULATURES AND TUMORS ARE DIFFERENT FROM A GENOMICS PERSPECTIVE FROM A MORPH LOGICAL PERSPECTIVE, WE ALSO KNOW THAT DIFFERENT ANATOMICAL LOCATION VS DIFFERENT VASCULATURES NOT THE SAME, THE ENDOTHELIAL CELLS IN THE LIVER, AND THE ENDY THELLIAL CELLS IN THE BRAIN OR THE PRAIRIE SIDES IN THE LIVER AND THE BRAIN, ALL THIS MAKES THE WHOLE PIPGHTURE VERY COMPLICATE BUD AT THE SAME TIME IT GIVES OPPORTUNITIES FOR THERAPEUTIC INTERVENTION. THESE ARE JUST VISUAL IMAGES OF NORMAL5a VASCULATURE AND TUMOR VASCULATURE, YOU CAN SEE THE BIG DIFFERENCE WEAN THE TWO, BETWEEN THE TWO VASCULATURES. AND THIS IS AN EXAMPLE OF THIS TUMOR ENDOTHELIAL MARKERS THAT I WAS TALKING B. HERE'S THE TUMOR, OUTSIDE THE TUMOR AND YOU CAN SEE HOW THIS IN THIS PARTICULAR CASE LOOKING AT THIS TUMOR WITH THE IMMUNO CYTOCHEMMISTRY, YOU CAN SEE HOW THE TUMOR LABELSG WITH THIS TUMOR IN THE MARKER BUT NOT THE NORMAL PICTURE. ANGIOGENESIS AS ANY OTHER PROCESS IS REGULATED BY FACTORS. SO FAR, WE KNOW DOZENS OF PROANGIO GENIC FACTORS AND DOZENS OF ANGIO GENIC FACTORS HERE ARE SOME OF THE PROANGIO GENIC FACTORS AND HERE ARE SOME OF THE ANTIANGIO GENIC FACTORS AND THESE ARE ALL ENDOGENOUS FACTORS WITHIN THE BODY THAT ARE PRO OR ANGIO GENIC FACTORS, NOW THERE IS THIS BALANCE HYPOTHESIS FOR THE ANGIOGENESIS SWITCH. ANGIOGENESIS, WHICH IS NECESSARY AS WE SAW BEFORE, OCCURS NORMALLY WHEN THERE IS A BALANCE BETWEEN THE PRO ANGIO GENIC FACTORS AND THE ANTIANGIO GENIC FACTORS, OKAY? SO WHEN MY DAUGHTER FALLS, WHICH IS PROBABLY SHE DID TODAY, A NUMBER OF TIMES, AND OPEN A WOUND THAT'S GOING TO BE ANGIOGENESIS IN THERE AND THAT WILL BE REGULATED. TELL BE THE RIGHT AMOUNT OF FULL ANGIO GENIC FACTORS, THE RIGHT AMOUNT OF FRONT ANGIO GENIC PROCESSES TO GENERATE THE PROCESS FOR THE WOUND TO HEAL, THE PROBLEM IS WHEN WE TILT THIS BALANCE EITHER ON ONE SIDE OR THE OTHER SIDE. WE CAN HAVE EXTRA ANGIOGENESIS FACTORS, A LOT OF ANGIOGENESIS FACTORS AND NOT ENOUGH ANGIO GENIC FACTORS AND THEN WE HAVE AN EXCESS, O THE OPPOSITE, WE CAN HAVE A LOT OF ANTIANGIO GENIC FACTORS AND NOT HAVE SUFFICIENCY FOR THOSE FACTOR SYSTEM THEREFORE HAVE DEFICIENCY IN ANG JOE GENESIS. THOSE BOTH SITUATIONS ARE LINKED TO DISEASE STATE: >> I'M SORRY? >> WHAT IS PSP? >> [INDISCERNIBLE]. >> THROMBOASIUM ONE. ONE OF THE--ONE OF MY FAVORITE ANTIANGIO VEG F AS YOU MIGHT KNOW IS VASCULAR AND ENDOTHELIAL GROWTH FACTOR. WITH MANY HAVE CONSIDERED FOR A LONG TIME THE GOLDEN STANDARD OF THE PRO ANGIO GENIC FACTORS AND I WILL TALK MORE ABOUT VEG F IN A MINUTE. SO THESE BOTH AGAIN, TILTING THE BALANCE IN ONE OR ANOTHER, WILL RESULT IN A DISEASE STATE. I MENTIONED HERE VASCULAR ENDO GROWTH FACTOR. CAN YOU IMAGINE HOW COMPLEX THESE BALANCES WITH ALL THE MOLECULES IN HERE, LET ME CONCENTRATE IN THIS ONE FOR A MINUTE. AND I'M JUST GOING TO TALK ABOUT VEG F BECAUSE WE KNOW A LOT ABOUT VEG F. OF COURSE, ALL THESE FACTORS WHICH MANY OF THEM ARE SOLUBLE AND YOU KNOW THEY'RE RELEASED, FOR INSTANCE BY TUMOR CELLS INTO THE TUMOR MICROENVIRONMENT REACH THE ENDOTHELIAL CELLS AND REACT WITH THE ENDOTHELIAL CELLS. AND THOSE RECEPTORS FOR INSTANCE FOR VEG F, IT'S A VERY COMPLICATED SITUATION. THERE ARE MANY DIFFERENT VEG F RECEPTORS. SO HERE FOR INSTANCE I'M SHOWING, VEG F RECEPTOR ONE, TWO, AND THREE. BUT THE VEG F RECEPTORS ARE MADE OUT OF LIKE TWO DIFFERENT CHAINS WITH FINDING EACH IN THE MEMBRANE AND WE CAN HAVE ACTUALLY MIXED OR HETERODIMER, THESE CHANGE TO FORM VEG F RECEPTOR ONE AND TWO, TWO AND THREE, SO THERE'S A LOT OF DIFFERENT OF THESE THAT COMBINE TO DIFFERENT VEG FAR. AND NOT TO MENTION THERE'S ONE VEG F, THERE ARE MULTIPLE FORMS OF VF OUT THERE AND THESE MULTIPLE FORMS OF VF BIND WITH THESE AFFINITIES TO THESE RECEPTORS HERE AND THEY ACT IN DIFFERENT WAYS. SO THE POINT IN THIS SLIDE IS TO CONVEY THE IDEA THAT THE ANGIO GENESIS IS VERY COMPLEX PROCESS. OKAY, WHICH IS TIGHTLY REGULATED, OR A--IN THIS WE TIGHTLY REGULATE, IN TUMOR GENESIS WE HAVE DEREGULATION IN THIS PROCESS BY AN INCREASE IN THE PROANGIO GENIC FACTORS AND WE TAKE THESE RECEPTORS, THE PATHWAYS RELATED TO THE RECEPTORS ARE ALSO VERY COMPLEX. AND KNOWLEDGE OF THIS PATHWAYS HAVE ACTUALLY ENCOURAGED THE DEVELOPMENT OF THERAPEUTICS, FOR INSTANCE IN THE CASE OF VEG F, LET ME SAY A LOT OF THESE RECEPTORS HAVE KINASE ACTIVITY. THEY HAVE RECEPTOR TYROSEEN KINASE ACTIVITY. AND THEY HAVE A LOT OF STRATEGIES THAT HAVE BEEN PUT INTO MOTION UNTIL NOW ARE FOCUS ON TARGETING THESE KINASE REGULATION OF THIS RECEPTORS. THIS IS ONE OF THE THE--FOR INSTANCE THE GOLDEN STANDARD ANTIANGIO GENIC THERAPY WHICH MANY OF YOU HAVE HEARD AND IF YOU LISTEN TO THIS ALL OF YOU IS AVASTINE. SO AVASTINE IS A MULTIBILLION DOLLAR DRUG WHICH TARGETS, IT'S A VEG F. IT'S A HUMANIZED MONOCLONEIN ANTIBODY AGAINST VEG F. SO THE ONLY THING THIS MONOCLONAL ANTIBODY DOES IT TRAPS VEG FAR AND IT DOESN'T ALLOW IT TO BIND TO THE ENDORECEPTORS IN THE THE CELLS AND PREVEBT AVERAGE YE GENESIS. O SO AT THE END, THEY ARE DOING, IS THEY ARE LOCKING THIS KINASE ACTIVITY OF THESE RECEPTORS. AND THE REASON WHY I'M MENTIONING THESE IS BECAUSE THERE'S GOING TO BE IMPORTANT AND YOU WILL SEE, THAT IN A MINUTE. SO THERE IS THIS WHOLE IDEA, THIS WHOLE IDEA THAT WE CAN LOOK AT THE ANGIOGENESIS SWITCH, TARGET THE ANGIOGENESIS SWITCH AND DEVELOP THERAPEUTICS BASED ON THE FACT THAT WE CAN BLOCK ANGIOGENESIS. NOW THIS IDEA WAS PUT FORWARD BY JUDITH FAULKMAN, HE'S CONSIDERED THE FATHER OF ANGIOGENESIS AND HIS PAPER WAS PUBLISHED IN 1971 SO THAT WAS A WHILE AGO, PROBABLY MANY OF US WERE NOT AROUND BACK THEN. SO IT TOOK WHEN HE PUBLISHED HIS PAPER IN 1971, NOT A LOT OF PEOPLE LISTEN TO THE THE IDEA, IF WE VERY SIMPLE IF WE BLOCK ANGIOGENESIS, WE WILL BLOCK TUMOR GROWTH, MEANING THE TUMOR GROWTH IS DEPENDENT ON ANGIOGENESIS. THAT WAS HIS IDEA. AND THAT IS WHAT HE'S REPRESENTING HERE. THIS IS WHAT YOU WILL SEE, YOU WILL WILL SEE NOT MANY PEOPLE ACTUALLY PAY ATTENTION INSHALLLY TO HIS HYPOTHESIS, NOT REALIZING THAT IT WILL BE KIND OF MULTIBILLION DOLLAR BUSINESS 30 YEARS DOWN THE LINE. NOW, IN 2000--LET SEE, THIS WAS TAKE PHREN A PAPER IN 2000, SO THE IDEA IS, OKAY, THIS IS THE TUMOR, THIS IS THE VASCULAR BLOCK ANGIOGENESIS TOGETHER FOR THE TUMOR, THAT WAS THE INITIAL IDEA, NOW THIS IS PUBLISHED BY TWO WELL KNOWN INVESTIGATORS IN THE ANGIOGENESIS FIELD INCLUDING JANE, BY 2005, THEY PUBLISHED ANOTHER PAPER WHERE THEY RECOGNIZE SOMETHING DIFFERENT. WE CAN'T ACTUALLY KILL ALL THE VASCULATURE IN THE TUMOR. AND THATY A. --A PROBLEM. SO IF WE USE THE ANGIO GENESIS THERAPY AND WE ELIMINATE THE SIGNS IN THE TUMOR, THERE ARE COMPLICATIONS WITH THAT, LET ME GIVE YOU TWO IDEAS TO THINK ABOUT. NUMBER ONE, THE ONLY WAY WE HAVE TO ACCESS THE TUMOR IS THROUGH THE VASCULATURE, SO IF WE ELIMINATE THE VASCULATURE IN THE TUMOR, WE HAVE ELIMINATED ANY POSSIBILITY FOR INSTANCE DELIVER ANY CREAM O THERAPY TO THE TUMOR. OKAY? SO THAT'S NOT A GOOD IDEA. NUMBER TWO, IF WE ELIMINATE ALL THE VASCULATURE IN THE TUMOR, THAT TUMOR IS GOING TO BE WHAT? IS GOING TO BE HIPOXIC AGAIN. OKAY, SO IF WE MAKE THEÖ PYPOXIC, THAT TUMOR IS GOING TO START AGAIN THE WHOLE PROCESS OF OKAY, I'M HYPOXIC, KEY ONE ALPHA, THE WHOLE PROCESS OF ATTRACTING METAGENESIS AND METASTATIC AND AGGRESSIVE IN ORDER TO SURVIVE IN AN ENVIRONMENT THAT IS HARD FOR A TUMOR. SO ALL THIS PROMPTED THE IDEA OF OKAY, LET'S JUST NOT KILL ALL THE VASCULATURE IN THE TUMOR, LET'S NORMALIZE THE VASCULATURE. SO WE TALKED ABOUT HOW THE VASCULATURE IN THE TUMOR IS AN ABNORMAL VASCULATURE. SO THE WHOLE IDEA HERE THAT'S PROPOSED IS, LET'S JUST NORMALIZE THE VASCULATURE IN THE VASCULATURE IN THE TUMOR, LET'S FIND DRUGS, LET'S FIND THERAPEUTICS THAT WHEN THEY DO IS THEY REDUCE FOR INSTANCE THE LEAKINESS IN THE TUMOR. SO THEY ALLOW PERIICIZE TO COME BACK AND GET THE VASCULATURE MORE SIMILAR TO NORMAL VASCULATURE OUTSIDE OF THE TUMOR. SO YOU KNOW THEY PROPOSE THESE DOZE VERSES TIME NOT FOR EFFICIENT TIME THERE'S NO EFFECT. IF WE GIVE A SUPER DOSE OF THE DRUG, IF WILL HAVE TOXIC STATE. IF WE GIVE THE DRUG UP LONG ENOUGH AND IT'S AN EFFECTIVE DRUG OR AND IT'S AFFECTED DOSE, WE WILL HAVE TO HAVE EXCESSIVE PRUNING SO WE HAVE TO LIMIT IT. SO THERE'S A WINDOW OF OPPORTUNITY THAT THEY CAN THE NORMALIZATION WINDOW AND THIS IS WHAT THEY ARGUE BASICALLY IS THAT IF YOU FIND A COMPOUND AND IT'S ELIMINATING VASCULATURE, THAT MIGHT NOT BE THE RIGHT COMPOUND. YOU MIGHT WANT TO FIND SOMETHING NOT AS AGGRESSIVE AS THE COMPOUND D. SO BASED ON THESE IDEAS, THIS HAS BEEN A TREMENDOUS FIELD, OF OF ANGIOGENESIS WE HAVE A LOT OF PRO ANGIO GENIC MOLECULES OR FACTORS, IN OUR BODIES THAT PARTICIPATE IN TUMOR ANGIOGENESIS AND WE HAVE DEVELOPED DRUGS, BASICALLY THAT TARGET THAT TUMOR ANGIOGENESIS, IT'S A MULTIBILLION DOLLAR DRUG AND HAD IS A BIT OUTDATED BUT THESE NEVER WENT DOWN, IT'S GOING OUT, AND A LOT OF THESE THERAPEUTICS THAT WE FOUND, A LOT OF THEM WHERE THEY TARGET ANGIO GENIC ROAD FACTORS, SO AVASTIN WILL BE IN THESE CATEGORY HERE. OKAY? FACTORS THAT ARE ABLE TO BLOCK PROANGIO GENIC COMPOUNDS HOWEVER, THERE ARE OTHERS LIKE ENDOGENOUS INHIBITORS MOLECULE INHIBITORS, INHIBITORS, THERE'S A LOT OF DIFFERENT FLAVORS IF YOU WILL OF ANTIANGIO GENIC THERAPY AND PAPPYS THAT ARE BEING--THERAPIES THAT ARE BEING INVESTIGATED. THIS IS A SUMMARY WHICH ALTHOUGH IT IS A LITTLE OLD IT WAS PUBLISHED BY ROBERT KERRVILLE IN 2001 IT IS A LITTLE OLD BUT THINGS HAVEN'T ACTUALLY CHANGED THAT MUCH UNTIL NOW. THAT'S WHY I'M STILL USING IT. SO THESE ARE THE DIFFERENT PATHWAYS THAT ARE BEING TARGETED BY ANTIANGIOGENESIS THERAPY AND WE HAVE THE GROWTH FACTORS, FIBROFACTORS ANDW#e[ GENETICALLIER LUNG CANCER KINASE AND A LOT OF DIFFERENT PROANGIO GENIC COMPOUNDS, HERE WE HAVE RECEPTOR TYROSEEN KINASE RECEPTORS. LIKE FOR INSTANCE, KDR THAT IS THE VEG F RECEPTOR SO THESE ARE ALL THE RECEPTORS I SHOWED YOU BEFORE THAT HAVE THE TYROSEEN KINASE ACTIVITY SO A LOT OF THE COMPOUNDS RIGHT NOW, IN THE MARKET, HAVE THESE RECEPTOR TYROSEEN KINASE INHIBITOR POTENTIAL AND THEN YOU KNOW THERE IS A COUPLE MORE OVER HERE SO THE RED NAMES IN THIS SLIDE ARE THE CURRENTLY APPROVED FDA ANTIANGIO GENIC THERAPIES AND I THINK THERE ARE 13 OR 14. AS YOU CAN SEE, MOST OF THEM--ONE, TWO, THREE, FOUR, FIVE, SIX, SEVEN, EIGHT, NINE, 10. 10 OF THEM, TARGET EITHER GROWTH FACTORS OR ARE RECEPTOR TYROSEEN KINASE INHIBITORS. OKAY? SO MOST OF THEM, MOST OF THE PHARMA HAS FOCUSED ON THIS PARTICULAR SEGMENT OF THE ANGIOGENESIS PROCESS. A COUPLE OF THEM, YOU KNOW THEY TARGET THE M-TORPATHWAY AND THEY THERE ARE TWO THAT WE DON'T KNOW WHAT THEY DO. WE DO A LOT OF THINGS WIEW BEDON'T KNOW WHAT THEY DO. SO, THERE ARE SOME PROBLEMS WITH ANTIANGIOGENESIS THERAPY. SO THERE'S A LOT OF LITERATURE ON THIS, BUT WHEN YOU INJECT AN ANTIANGIOGENESIS THERAPY OF COURSE BECAUSE OUR BODIES ARE LIKE FULL OF THESE VESSELS EVERYWHERE OUR BODY ISN'T LIKE THAT AND THERE'S A LOT OF CONSEQUENCE AND SECONDARY EFFECT BASICALLY. ONE OF THEM IS HYPERTENSION, BUT THERE ARE MANY, MANY OF THEM. ACTUALLY ANTIANGIO GENIC THERAPY, IS WELL KNOWN FOR CAUSING IT THIS SECONDARY EFFECT. ONE OF THE--THAT'S ONE OF THE PROBLEMS THAT WE HAVE WITH ANGIOGENESIS THERAPY AND HAPPY. THIS IS I ANOTHER PROBLEM THAT I SEE WITH ANGIOGENESIS THERAPY AND THIS IS A NUMBER OF PUBLICATIONS FROM 1960-2010 IN THE ANGIOGENESIS FIELD AND THIS IS JUDEA FAULKMAN'S PAPER IN 1971. EVERYONE SAID YOU'RE CRAZY. WE'RE PHOT GOING TO PAY ATTENTION TO YOU. NO ONE STUDIED ANGIOGENESIS UNTIL MID90S AND THEN THERE IS AN EXPLOSION OF PUBLICATIONS MOSTLY BECAUSE THE PHARMA SAID THERE'S A LOT OF MONEY HERE AND THEN A LOT OF PEOPLE SAID, LET'S STUDY ANGIOGENESIS AND THIS IS IN--THIS IS 2010, 2011, THIS IS GOING UP, SO IT'S NOT SHOWING A SIGN OF GOING DOWN. SO THIS IS A HISTORY OF RESEARCH IN ANGIOGENESIS, NOW, THIS IS--FROM ALL THESE PUBLICATIONS IN HERE, THIS IS HOW THEY SEPARATE IN DIFFERENT CATEGORIES, THE BLUE LINE IN HERE IS VEG F OR PUBLICATIONS THAT ARE RELATED TO VEG F WHICH ARE 50%, EVEN IN 2010. THE REST OF THE FIELDS ARE BARELY 10% AND MAYBE GOING DOWN A LITTLE BIT. SO, ONE OF THE PROBLEMS WITH THE ANGIOGENESIS IS THAT EVERYBODY'S STUDYING VEG F. NOT EVERYBODY. THERE ARE SOME PEOPLE HERE THAT DON'T STUDY VEG F. BUT PRETTY MUCH EVERYBODY IS STUDYING VEG F SO AVASTIN CAME ALONG AND THEY SHOWED THERE A COULD DO A LITTLE BIT IN PATIENTS AND WE'RE GOING TO SHOW NOW WHAT AVASTIN DOES AND THEN YOU KNOW WE WILL FOCUS ON THAT. SO ONE OF THE THE PROBLEMS I SEE IN THE ANGIOGENESIS FIELD IS THAT EVERYBODY'S LOOKING IN THE SAME DIRECTION. SO THE ANGIOGENESIS PROCESS, WE SEE BEFORE IS EXTREMELY COMPLEX, AND AND A LOT OF DIFFERENT FACTORS INVOLVED IN THE PROCESS, MOLECULE, AND THIS IS WHAT I ASKED THEM, AND THIS IS A BIT UPDATED, LOOK AT NUMBERS OVER HERE, 15.6, AND THIS IS THE EXTENSION, THIS IS COLORECTAL CANCER. LUNG CANCER FROM 10-12 MONTHS. BREAST CANCER FROM SIX-12 MONTHS. FROM MY PERSPECTIVE THAT'S NOT A LOT, RIGHT? SO THIS IS WHAT--TREATMENT WITH AVASTIN DOES IN THE PATIENTS, SO YOU MIGHT HAVE HEARD IF YOU LISTEN TO THE NEWS, YOU MIGHT HAVE HEARD THIS ACTUALLY BEING PULLED FROM BREAST CANCER TREATMENT, FOR A NUMBER OF REASONS, THAT WE'RE GOING TO TALK ABOUT NOW, BUT IT'S BEING PULLED FROM BIRTH CANCER TREATMENT, THE REASON IS BECAUSE THE BENEFIT IS NOT THAT MUCH AND THE PROBLEM THAT IS CAUSES ARE A LOT, SO ONE HAS TO BALANCE THAT RISK, WITH THE BENEFIT OF USING THERAPEUTIC LIKE THIS. LAST YEAR THERE WERE TWO PAPERS ONE BY ROBERT KERRVILLE AND THIS ONE BY ROBERT KERRVILLE AND THIS ONE BY A GROUP IN SPAIN CASANOVAS SHOWING SOMETHING DISTURBING AND WHEN YOU TREAT CHRONICALLY IN THIS CASE, CLINICAL MODELS OF ANGIOGENESIS OR TUMOR GENESIS, WITH AVASTIN, WHAT HAPPENS IN THE LONG-TERM IS THAT, WHAT YOU'RE DO SUGGEST DURING THE METASTATIC CAPABILITY OF THE TUMOR AND YOU ARE ENHANCING THE ANGIO GENIC POTENTIAL OF THE TUMOR THIS, IS A LITTLE BIT COUNTER INTUITIVE BUT THIS HAPPENS. WHEN YOU TREAT THESE ANIMALS CHRONICALLY WITH THESE DRUGS WHAT HAPPEN SYSTEM THAT YOU MAKE THINGS WORSE OPPOSE TO CURING THE TUMORS, OKAY? AND THESE PAPERS, I PUT THEM IN HERE BECAUSE THEY'RE COMPLEX PAPERS AND YOU KNOW, YOU CAN GO AND READ THE WHOLE STORY FOR BOTH PAPERS, THEY WERE BOTH PUBLISHED IN THE SAME NUMBER OF CELLS. I THINK IT WAS LAST YEAR OR TWO YEARS AGO, ROBERT GOES ONE STEP BEYOND AND HE SAYS, WELL THIS IS A PROBLEM WITH RECEPTOR TYROSEEN KINASE INHIBITORS, SO HE SAYS THAT RECEPTOR TYROSEEN INHIBITORS ARE NOT THE RIGHT ANTIANGIO GENIC THERAPY BECAUSE THEY CAUSE ALL THESE BAD EFFECTS BASICALLY ON THE PATIENTS. OKAY? SO, LET'S GO BACK TO OUR THERAPIES IN HERE. SO WE HAVE AVASTIN SERAT NIB AND SER AFNIB WERE THE DRUGS WE'RE USING BEFORE. SO THESE THREE DRUGS HAVE THESE PROBLEMATIC SECONDARY EFFECTS ON THE SIDE EFFECTS ON THE PRECLINICAL MODELS, NOW THESE ARE EVENTUALLY, YOU KNOW THEY ALL TARGET EITHER A GROWTH FACTOR, THAT BINDS TO A RECEPTOR TO ROSINEI KINASE, OR THEY DIRECTLY ACT ON OUR RECEPTOR TYROSEEN KINASE, SO, IF YOU LOOK AT THESE OVER HERE, THIS ONE, THIS ONE AND THIS ONE AND THIS ONE THEY ALL TARGET RECEPTOR TYROSEEN KINASE SO WHAT HAPPENS OVER HERE IS THAT MOST OF THE CURRENTLY APPROVED FDA ANTIANGIC DRUGS ARE IN THE SAME GROUP OF DRUGS ARE IN THIS GROUP OF DRUGS THAT CAUSE THESE PROBLEMS IN PATIENTS NOW THOSE TWO PAPERS HAVE BEEN FOLLOWED THERE BY CLINICAL STUDY SYSTEM AND ET CETERA, ET CETERA AND CONFIRMING HUMANS AS WELL IN PATIENTS, SO, THIS--SO WE'RE LEFT WITH THESE TWO OVERHERE AND THESE TWO OVERHERE WHICH I PERSONALLY DON'T KNOW MUCH ABOUT, AS FAR AS REGUARDING COSTS IN THESE POTENTIAL PROBLEM IN A POTENTIAL PROBLEMS IN PATIENT. SO THIS IS A--THIS IS A VERY EXCITING TIME BASICALLY FOR THE ANGIOGENESIS FIELD WHICH IS SEEN OVER THE PAST 30 YEARS THAT ANGIOGENESIS, THE TUMORS ARE DEPENDENT ON ANGIOGENESIS AND WE'VE SEEN HOW ANGIOGENESIS IS REGULATED AND WE'VE BEEN ABLE TO DEVELOP DRUGS THAT BLOCK ANGIOGENESIS AND WE SEE THOSE PROBLEMS WITH THOSE DRUGS AND THERE IS A NEED FOR NEW OR NOVEL ANTIANG GENIC THERAPIES SO THIS IS LIKE THE SECOND WAVE OF ANGIOGENESIS THERAPY, THE QUESTION IS HOW, WHERE DO WE GO FROM HERE. SO THERE IS A LOT OF RESEARCH, NOW, SHOWING THAT THIS IS A Fh: PAPER BY BELTON'S GROUP THAT SHOWS AS A MATTER OF FACT THERE ARE MORE GENES THAT ARE INVOLVED IN ANGIOGENESIS THAT WE DON'T KNOW THAT GENES'RE INVOMPLE INDEED ANGIOGENESIS THAT WE KNOW. OKAY? SO IN THESE EXPERIMENT WHAT THEY DO IS THEY TAKE--A PANEL OF GENES AND THEY SAY, WELL LET'S SEE IF THESE JEENCHS ARE INVOLVED IN ANGIOGENESIS, THERE ARE MORE THAT ARE INVOLVE INDEED ANGIOGENESIS THAT WE DON'T KNOW ABOUT THAT ARE INVOLVED SO AGAIN, THE PROBLEM IS THAT WE'RE LOOKING IN ALL--WE'RE ALL LOOKING IN THE SAME DIRECTION AND WE'RE FORGETTING ABOUT THE GENES THAT ARE NOT--THAT ARE INVOLVED IN ANGIOGENESIS THAT WE DON'T KNOW ABOUT. SO THERE'S A BUNCH OF PEOPLE THAT--A NUMBER OF INVESTIGATORS HAVE DONE SIMILAR STUDIES FINDING THE SAME THING INCLUDING OURSELVES, WE HAVE CONFIRMATION THAT THERE'S A LOT OF GENES, SOME GENES THAT DON'T EVEN HAVE A NAME THAT WE THINK THAT THEY'RE VERY CRITICAL FOR TUMOR ANGIOGENESIS AND THOUGH ONE IS PAYING ATTENTION TO THOSE GENES AND SO ONE OF THE WAYS ONE CAN GO ABOUT FINDING NOVEL THERAPEUTICS FOR ANGIOGENESIS GOING BACK TO THE THE SQUARE ONE, LOOKING AT THE PROCESS OF ANGIOGENESIS AND TARGET THESE TO FIND NEW THERAPEUTICS SO IN THE ANGIO GENICS PROCESS QUICKLY, WE OF COURSE, YOU KNOW WE HAVE HERE, Arn A TUMOR HERE WHICH IS PRODUCING ANGIOGENESIS, FACTORS, THESE FACTORS ARE RELEASED THEY REACH THE ENDOTHELIAL CELLS THEY ACTIVATE THE ENDOTHELIAL CELLS, AND THEY MAKE THE ENDOTHELIAL CELLS PROLIFERATE IF THERE IS NO ENDOTHELIAL CELL PROLIFERATION, AND THERE'S NO ANGIOGENESIS, RIGHT? THERE'S MIGRATION OF THE ENDOTHELIAL CELLS REMEMBER THE CHEESE IN THE ROOM, THEY NEED TO MIGRATE, THEY NEED TO CARVE THE EXTRA CELLULAR MATRIX, THAT'S GOTTA BE EXTRA CELLULAR MATRIX REMODELING. WE NEED TWO FORMATIONS, THOSE CELLS NEED TO ARRANGE THEMSELVES IN TUBES AND FORM A VESSEL. LOOP FORMATION AND VASCULAR STABILIZATION AND THOSE HAVE TO DO WITH THE SIGHTS THAT WE--SITES THAT WE TALKED ABOUT BEFORE. A LOT OF THESE STEPS ARE VERY TUMOR ANGIOGENESIS, NOT SO MUCH THIS ONE IN HERE BECAUSE THE TUMOR VASCULATURE GROWING REALLY FAST AND SOMETIMES IT DOESN'T HAVE SUFFICIENT TIME ACTUALLY FOR--TO BE STABILIZED BUT A LOT OF THESE STEPS ARE IMPORTANT IN THE TUMOR ANGIOGENESIS PROCESS, HOW CAN WE TARGET THIS PROCESS? HOW CAN WE INVESTIGATE THIS PROCESS? THERE ARE A LOT OF WAYS SO WE CAN DO I TYROSIGN COIN ACE TYPE, AND THESE HAVE BEEN DOING FOR THE PAST 10 YEARS OR WHATEVER, THEY DO RECEPTOR TYROSEEN KINASE ASSAYS AND THEY SAY, OKAY, I FOUND THE RESEARCHER TINE ACE INHIBITOR THIS IS AN ANTIANGIO GENERATED SICK FACTOR AND SURENOUGH, SO THIS IS ONE--SURE EENOUGH THIS, IS ONE WAY ONE CAN DO THIS. HOWEVER, BECAUSE WE'RE TARGETING ONLY THESE PARTICULAR STEPS OVER HERE, THESE ASSAYS ARE NOT VERY INFORMATION RICH. WE FOUND A RECEPTOR TYROSEEN KINASE INHIBITOR, DOESN'T MEAN THIS IS GOING TO WORK ON ANGIO GENERATED SICK THERAPY. --ANGIO GENIC THERAPY. WE CAN DO THIS FOR THE ASSAY. THIS INVOLVES CELLS. WE HAVE CELLS AND WE TARPGET A GROWTH ASSAY, AN ENDOTHELIAL GROWTH ASSAY, WITH DIFFERENT COMPOUNDS, AND WE DISCOVER SOME COMPOUNDS THAT BLOCK THE GROWTH OF ENDOTHELIAL CELLS, WELL THAT'S ANOTHER STEP, AT LEAST IN THIS CASE, WE KNOW THAT THE OUR COMPANY IS DOING SOMETHING. OKAY? IT'S BLOCKING THE GROWTH OF ENDOTHELIAL CELLS, STILL WE'RE MISSING A LOT OF THESE STEPS ON THE ANGIOGENESIS PROCESS, WE DO OTHER DIFFERENT ASSAYS ONE OF THEM I'M GOING TO TALK ABOUT TODAY IS THE TWO FORMATION ASSAY. THIS ASSAY ARE MORE INFORMATION RICH, THEY RECAPITULATE MORE OF THE STEPS OF THE ANGIOGENESIS PROCESS. AND THEREFORE, COMPOUNDS THAT BLOCK THESE ASSAYS, CAN BE UTILIZING THESE ASSAYS, ARE HIGH IN POETIC TESHTIAL BASICALLY OR TRANSLATED INTO PATIENTS OR PRECLINICAL MODELS AND OF COURSE WE HAVE THE IN VIVO ASSAYS, IT WOULD BE GREAT IF WE COULD DO A SCREENING IN VIVO, RIGHT? IT WOULD BE GREAT BUTW@ś REALITY IS THAT WE CAN TAKE A MILLION MICE AND TRY A MILLION COMPOUNDS AND YOU KNOW FOR LOOKING FOR ANGIOGENESIS INHIBITORS SO THIS IS REALLY NOT AN OPTION WHEN TALKING ABOUT SCREENING FOR A COMPOUNDS. SO WE'RE LEFT WITH SOMETHING LIKE THIS, YOU KNOW WE HAVE A NUMBER OF ASSAYS THAT YOU CAN UTILIZE TO INVESTIGATE AN OME OF WHICH ARE INFORMATION RICH, LIKE IF WE CAN LOOK AT THE PATIENT AND WE CAN TRY A COMPOUND IN THE PATIENT, IF THAT PARTICULAR ONE DOESN'T WORK, IT'S JUST--IT'S NOT GOING TO WORK SO THIS IS VERY INFORMATIONf HAVE ALL THE WAY ON THE RECOLLECT END OF THE ASSAYS WE HAVE ASSAYS THAT ARE NOT VERY INFORMATION RICH WHICH ARE VERY EASY TO DO, WHICH ARE VERY SUITABLE WITH HIGH THROUGHOUT PUT TECHNIQUES LIKE THE ONES THAT PHARMA COMPANIES ARE USING. AS I SAID, A LOT OF THESE CURRENTLY AVAILABLE ANTIANGIO GENIC DRUGS WERE FOUND LIKE THIS. DOING ASSAYS OF THIS KIND OVER HERE. SO WE NEED TO FIND A BALANCE BETWEEN HOW MUCH INFORMATION WE GET FROM OUR ASSAYS AND THE FACT THAT THOSE ARE COMPATIBLE WITH HIGH THROUGHOUT PUT TECHNIQUES SO WE CAN TEST A LOT OF COMPOUNDS. SO WE TOOK THE APPROACH OF GETTING HERE, SOMEWHERE IN THE MISTED AND TRY TO UTILIZE THESE ASSAYS THAT ARE COMPLEX IASSAYS BUT TO GIVE US A LOT OF INFORMATION OF THE PROCESS OF ANGIOGENESIS. THIS IS A TWO FORMATION ASSAY, NOT SURE HOW MANY OF YOU, HOW MANY OF HAVE YOU HEARD OF THE TWO-FORMATION ASSAY. ONE. WOW. OKAY. A TWO FORMATION ASSAY IS A VERY INTERESTING ASSAY, IT'S A THREE DIMENSIONAL ASSAY. YOU TAKE A MATRIX, AND YOU EMBED ENDOTHELLIAL CELLSIAL CELLS ON THE MATRIX SO INITIAL LOW AT THE BEGINNING OF THE ASSAY, THIS IS HOW THEY LOOK. THEY ARE HOMOGENOUSLY DESCRIEBTED IN THE MATRIX. ONCE YOU INCUBATE THESE CELLS UNDER CERTAIN CONDITIONS, YOU CAN SEE IT HAVE WELL, THE MOVIE IS NOT PLAYING WELL, SO YOU SEE HOW THE CELLS REARRANGE IN A CAPILLARY LIKE STRUCTURE. OKAY? NOW, THIS CAPILLARY LIKE 7 STRUCTURE IS SIMILAR TO WHAT YOU CAN FIND IN AN IN VIVO SYSTEM, BASICALLY, THESE ARE LIKE CAPILLARIES THERE'S BEEN A LOT OF STUDIES ON THESE TWO FORMATION ASSAYS. IF WE TAKE ONE OF THE SEGMENTS, OF ONE OF THOSE TUBES, YOU SEE THAT IN BLUE HERE YOU HAVE THE DAPI, IS THE NUCLEI OF THE CELLS, AND IN GREEN IS THE CYTOPLASM, CAN YOU SEE THEY'RE FORMED BY MANY, MANY CELLS, THERE'S NOT JUST A SINGLE CELL, BUT THERE MANY CELLS, THERE IS ALSO A CLASS OF FORMS FORMED BY MANY DIFFERENT CELLS, THEY'VE BEEN A LOT OF STUDIES SHOWING THAT IF WE DO CROSS SECTIONS, OF THESE TUBES OVER HERE, THESE TUBES ARE ACTUALLY HOLLOW, SO THEY DO ACTUALLY MIMIC THE VASCULATURE IN VIVO, THEY'RE HOLLOW, THEY HAVE THE LUMEN AND THEY HAVE EXTRA CELLULAR MATRIX, EXTRA CELLULAR MATRIX AROUND THEM. THEY DEPOSIT ACTUALLY CERTAIN COMPONENTS OF THE EXTRA CELLULAR MATRIX, AROUND THEM. SO THIS STRIKES ME AS A GOOD MODEL TO, IF WE WERE ABLE ACTUALLY TO UTILIZE IT IN A GOOD SCREEN MANNER, IT STRIKE ME AS A GOOD MODEL THAT RECAPITULATES A LOT OF THE COMPONENTS OF AND THE STEPS OF ANGIOGENESIS PROCESS, REMEMBER THAT THE CELLS ARE IN THE THREE DIMENSIONAL MATRIX SO THEY NEED TO MOVE AROUND? SO THE ACTIVATION OF THE CELLS IS THERE, THE MOTILITY OF THE CELLS IS THERE, THE TUBE FORMATION CAPABILITY OF THE CELLS IS THERE, SO THERE'S A LOT OF THE STEPS OF THE ANGIOGENESIS PROCESS IS PRESENT IN THIS PARTICULAR ASSAY. NOW WE'LL SEE OKAY THIS IS LIKE AN INVITRO CONSTRUCT, THAT DOESN'T REELINGLY WORK SO THAT IT'S NOT FUNCTIONAL? IN SOME OF THESE STUDIES, A NUMBER OF THEM RIGHT THAT HAVE SHOWN OTHER ONES AND THIS IS A STUDY FOR INSTANCE THEY DO ENDOTHELIAL CELLS IN A CULTURE SYSTEM IN A THREE DIMENSIONAL MATRIX, SO THESE ORANGEY COLOR OVER HERE IS THE THREE DIMENSIONAL MATRIX AND WE HAVE THE CARDIO SIGHTS AND THE ENDOTHELIAL CELLS THIS IS HOW IT LOOK WHEN IS WE LOOK AT IT, UNDER THE MICROSCOPE. BUT WHAT THESE PEOPLE ARE DOING IS VERY INTERESTING, ONCE THESE VASCULATURE IS FORMED, WITH THE SIMILAR TO WHAT I SHOW YOU BEFORE, THEY TAKE THIS, AND THEY IMPLANT IT INTO--THEY TRANSPLANT IT INTO A MODEL OF STROKE IN A RAT. OKAY? SO THIS IS THE HEART THAT WENT THROUGH A STROKE, TRYING TO RECOVER. THEY WILL PUT THIS OVER HERE. AND SO THEY OPERATE THEIR RIDE, SIMPLY THEY TAKE THE WHOLE MATRIX WHERE THE TWO FORMATION HAS HAPPENED AND THEY JUST PLACE IT OVER THE HEART. SIMPLE AS THAT. NOW, WHAT I THINK IS FASCINATING ABOUT THIS PARTICULAR STUDY, IS THE FACT THAT THE EPPED O THELLIAL CELLS VERY QUICKLY GET INCORPORATED INTO THE NORMAL VASCULATURE OF THE HEART. SO IN THIS CASE, IN HERE, WHAT THEY'VE DONE ACTUALLY IN THIS PARTICULAR STUDY, IS THEY STAIN WITH RED, THE HOST VASCULATURE, OKAY. AND IN GREEN, AS YOU SAW HERE, BEFORE, WE HAVE THE INVITRO VASCULATURE IF YOU WANT TO CALL IT THAT. SO IN A MANY OF DAYS, AS A MATTER OF FACT, THE INVITRO VASCULATURE, GETS INTEGRATED INTO THE NORMAL VASCULATURE OF THE HOST AND IT'S ACTIVE. IT'S FUNCTIONAL. SO THAT MEANS THAT IN MY OPINION, VALIDATES THESE INVITRO EXPERIMENTS AS FUNCTIONAL. YOU KNOW THESE ARE STRUCTURES THAT ARE FORM TED INVITRO, THEY'RE ACTUALLY FUNCTIONAL, THERE'S A NUMBER OF STUDIES THAT HAVE SHOWN SIMILAR THINGS TO THIS ONE. YOU KNOW PEOPLE THAT USE IT IN MODELS OF THE SPINE INJURY, SPINE INJURY RECOVERY FROM A SPINE INJURY IS VERY MUCH DEPENDENT ON ANGIOGENESIS. SO THEY CREATE THIS INVITRO MODELS OF VASC--CAPILLARY LIKE STRUCTURES INVITRO AND THEY TRANSPLANT THOSE IN THE ANIMALS AND THE AFTERNOON MALS RECOVER MUCH FASTER THAN THE ONES THAT HAVEN'T RECEIVED THE IMPLANTS JUST BECAUSE DATA VASCULATURE IS FUNCTIONAL. WE HAVE USES FOR A NUMBER OF THINGS LIKE IN THIS PARTICULAR CASE, WE STUDIED THE EFFECT OF A TUMOR SUPPRESSOR GENE IN ANGIOGENESIS AND WE FIRST DID THIS INVITRO, WE SAW THAT THE TUMOR SUPPRESSOR GENE THAT WE THOUGHT THAT IT COULD REGULATE ANGIOGENESIS AND WE COO THAT WHEN THE TUMOR SUPPRESSOR GENE IS PRESENT IT BLOCKS ANGIOGENESIS AND THIS TRANSLATES VERY WELL INTO THE THE VIVO SCENARIO. NOW, THIS THIS BEING CASE ISSUES IN THE CELLS AND INDEPENDENTLY OF THE GENE, IT'S ALSO I THINK, ALSO VALIDATION THAT THE INVITRO ASSAY, THAT PARTICULAR INVITRO ASSAY, IS REPRESENTATION FOR--HAS A PREDICTED POWER OF WHAT HAPPENS IN VIVO. ONE OF THE CHALLENGES SO WE DECIDE TO USE THESE AS A HIGH THROUGH PUT TECHNIQUE TO SCREEN FOR THE ANTIGENIC COMPOUNDS AND ONE OF THE PROBLEMS WE FOUND WAS ACTUALLY THE LEAST EXPECTED PROBLEM WAS THAT WE DIDN'T KNOW WHAT TO DO WITH ALL THE IMAGES SO WHAT WE DID WAS THIS HAPPENEDS OF THESE ASSAYS, WITH FLUORESCENT ENDOTHELIAL CELLS AND TESTED COMPOUNDS AND THEN WE HAD THESE THOUSANDS OF MOVIES AND IMAGES AND WE DIDN'T KNOW WHAT TO DO WITH THEM. SO WE WENT TO THE PRIVATE SECTOR AND ASKED THE QUESTION, DO HAVE YOU A SOFTWARE, THAT WILL HELP US ANALYZE THIS, SO $30,000, $20,000, SO AT THE END OF THE DAY, MAKE A LONG STORY SHORT OF NONE OF THOSE WORKS FOR WHAT WE WANT TO DO AND WE DESIGNED OUR OWN SOFTWARE. IT'S CALLED ANGIO APPLICATION AND IT TAKES YOU INTO THESE IMAGES OVER HERE AND IT'S ABLE TO IDENTIFY THIS CLASS OF CELLS AND THE TUBES AND THAT A LOT OF DIFFERENT MEASUREMENTS AND MEASUREMENTS ON THAT IN IT. SO THESE ACTUALLY WAS A CRITICAL STEP IN ORDER TO IDENTIFY SOMEONE IN THE THE COMPOUND. JUST EXAMPLES ON HOW WE'RE ABLE TO DETERMINE THE SAME IC50S FOR COMPOUNDS THAT WE'RE ALREADY KNOWN, FOR INSTANCE SHERMAN IS A COMPOUND THAT'S ALREADY KNOWN IT'S AN AVERAGEIO GENIC COMPOUND AND WE'RE ABLE TO FIND USE IN THIS SYSTEM, FOR THIS COMPOUNDS, AND THIS IS JUST SOME OF THE RESULTS THAT WE GOT TESTING 2000 DIFFERENT COMPOUNDS USING THIS SYSTEM SO HERE WE HAVE YOUR PLASTIC CONTROL, NOT RISE ROPINGS OF--DISRUPTION OF THE VASCULATURE. ONE OF THE COMPOUNDS HERE DISRUPTED THE VASCULATURE TO A CERTAIN EXTENT AND USE THIS SOFTWARE AND THIS TECHNIQUE WE PUT TOGETHER BUT WE'RE ABLE TO IDENTIFY AND THESE ARE NEGATIVE CONTROLS OVER HERE IN RED. SO YOU KNOW THE CLOSEST THESE COMPOUNDS EVERY ONE–r OF--OF THESE DOTS REPRESENTS ONE OF THOSE COMPOUNDS SO THE CLOSEST WE GET TO THE NEGAATIVE CONTROL THE BETTER IN THIS PARTICULAR CASE. SO, SOME OF THE COMPOUNDS THAT WE FOUND USING THESE TECHNIQUES WE TESTED IN VIVO. AND WE COMPARED THEIR ABILITY TO BLOCK ANGIOGENESIS TO THE AVASTIN WHICH IS LIKE THE GOLDEN STANDARD FOR ARMINGIO GENESIS, SO THIS IS OUR CONTROL AND THIS A VERY ANGIO GENIC TUMOR. THIS IS A SARCOMA AND IT'S A VERY ANGIO GENIC TUMOR AND AS CAN YOU SEE THE TUMOR DOESN'T DO MUCH, WHEN YOU INJECT IT, IT DOESN'T DOO MUCH AND THE REASON WHY IS BECAUSE IT'S NOT VASCULARIZED AT LEAST AND FIRST AND THE TUMOR DOESN'T GROW MUCH BUT THEN IT STARTS BECOMES VASCULARIZED AND IT HAS AN EXPLOSION OF EXPONENTIAL GROWTH AND HAD IS OUR CONTROL OR TREATED WITH PBS, AND THIS IS THE ONES TREATED WITH AVASTIN OR SOME OF THE THE COMPOUNDS WE FOUND USING THIS SYSTEM. SO ONE OF THE THINGS THAT IS IMPORTANT ABOUT THIS IS )jRz GOING TO SAY THESE TWO j" COMPOUNDS IN HERE WHERE THE FIGHTER TWO COMPOUNDS THAT WE--WE DISCOVERED SO ONE OF THE THINGS IMPORTANT ABOUT THIS SYSTEM IS THAT IT'S VERY PREDICTIVE FOR WHAT HAPPEN IN VIVO, SO NOW WE HAVE AN INVITRO OSAY THAT WE CAN USE AND WE CAN TELL YOU WHETHER OR NOT THAT PARTICULAR COMPOPPED IS GOING TO BE EFFECTIVE IN VIVO OR IS NOT GOING TO EFFECTIVE. WE DON'T NEED TO DO THE IN VIVO ASSAY, WITH THE INVITRO ASSAY WE CAN DO THAT, WITH A LOT OF THE STUDIES THAT WE'VE GONE DONE TO VALIDATE THIS AND EXTEND THIS AFTER INITIAL STUDIES AND EVERYTHING SEEMS TO BE HOLDING TRUE SO FAR. SO, THE IMPLICATIONS WHAT I THINK IN MY OPINION, THE IMPLICATIONS FOR THE TRACT DISCOVERY PROCESS ARE THIS, THIS SAME TRADITIONAL MODEL IS OUR DISCOVERY, WE SPEND, YOU KNOW 20 YEARS FINDING VASCULAR ENDOTHELIAL GROWTH FACTOR OR WHATEVER ANY OTHER FACTOR SO WE SPEND A NUMBER OF YEARS DOING THAT, THEN HERE COMES THE PHARMA COMPANIES, AND THEY DEVELOP ASSAYS, YOU KNOW RELATIVELY SIMPLE ASSAYS TO TEST FOR THOSE VEG F FGF, ALL THOSE COMPOUNDS,e"Mv SO, THIS PARTICULAR SEGMENT OVERHERE OF EARLY DRUG DISCOVERY IS CALLED THE BLACK BOX AND A LOT OF COMPANIES THEY CALL IT BLACK BOX, THEY CALL AT THIS TIME BLACK BOX BECAUSE WHAT THEY PULL OUT OF THERE THEY DON'T REALLY KNOW WHAT'S GOING TO HAPPEN WITH THOSE WHEN THEY GO TO PRECLINICAL MODELS OR THEY GO TO CLINICAL TRIALS. SO THE MAIN REASON FOR ADRYINGS, OF DRUGS IF CLINICAL TRIALS OCCURS IN PHASE TWO AND THAT'S BECAUSE THEY'RE NOT EFFECTIVE, THOSE DRUGS ARE NOT EFFECT EENOUGH, AND THESE CAN BE TRACED TWO OF THESE BLOCK TED BOXES OVER HERE, SO WHAT WE PROPOSE WITH THESE NEW MODELS WITH THE ANGIO GENERATED SICK DRUG SYSTEM TRY TO SAY, OKAY, WE'RE GOING NOT GOING TO FOCUS ON ANYTHING, WE'RE GOING TO FOCUS ON DRUGS THAT ACTUALLY WORK. THAT'S THE ONLY THING WE'RE GOING TO DO SEE WE ELIMINATE ALL THIS FACING HERE, WE'RE NOT GOING TO LOOK FOR COMPOUNDS THAT I THINK THAT THERE ARE GOING TO BE IN THE FUTURE THEY'RE GOING TO BE FANTASTIC WOO WHEN WE TRANSLATE THESE INTO HUMANS, SO WE ELIMINATE THESE HERE, WE GENERATE ASSAYS THAT ARE VERY PREDICTIVE OF WHAT HAPPENS IN VIVO, AND WE DISCOVER MOLECULES IN THERE THAT WE KNOW THAT THEY'RE GOING TO WORK, THEN WE INVEST THE TIME TO„i CHARACTERIZE THOSE MOLECULES IN THERE. SO ANYWAY, IN SHORT, WE THINK THAT THESE MODELS IN GENERAL CAN SHORTEN THE AMOUNT OF TIME THAT WE NEED TO USE TO DISCOVER NEW DRUGS THIS THIS PARTICULAR CASE NEOANTIGENIC DRUGS AND IT INCREASES A LOT OF THE YIELD OF MOLECULES THAT ARE DEFECTIVE SO WHEN WE GO TO PRECLINICAL MODELS AND EVENTUALLY TO CLINICAL TRIALS WE HAVE A LOT MORE OPTIONS BASICALLY TO CHOOSE FROM, A LOT MORE MOLECULES THAT ARE POTENTIALLY GOING TO BE EFFECTIVE IN CLINICAL TRIALS WHICH IS EVENTUALLY WHAT WE WANT TO DO, RIGHT? SO, BASICALLY, THE WHOLE IDEA THAT I WOULD LIKE TO COON VEY AND THIS IS JUST TO FINISH IS I DON'T KNOW IF YOU KNOW THIS, THIS SENTENCE FROM ICE ZACH NEWTON BUT MY IDEA IS THAT WE KNOW VERY LITTLE ABOUT THE ARMINGIO GENESIS FIELD, THERE IS NOT MUCH THAT HAS BEEN EXPLORE AND WE'RE ALREADY MAKING ASUSMGDZS ABOUT WHETHER OR NOT ANGIO GENIC THERAPY WILL WORK OR NOT WORK, MY PERSONAL FEEL SUGGEST THAT IT CAN BECOME WORK AND IT WILL WORK, THE THING IS WE NEED TO EXPLORE A LOT MORE THAN WHAT WE HAVE EXPLORED SO FAR. SO I THEN IS A VERY EXCITING TIME ACTUALLY FOR THE ANGIOGENESIS FIELD. IF ANYONE IS INTERESTED IN IT, DEVELOPMENTAL ENDOCRINOLOGY FITLY THIS IS A VERY GOOD FIELD TO GET IN. NOT ONLY FOR THE 232 BILLION DOLLARS THAT ARE BEING USED IN THE ANGIOGENESIS OR INVEST INDEED ANGIOGENESIS FOR OTHER MORE ALLOT TRUISTIC RESEARCH. QUESTIONS? YES? >> [INDISCERNIBLE] NTHAT'S A VERY GOOD QUESTION. SO THIS IS WHAT HAPPENED WITH THE COMPOUND THAT I SHOWED YOU IN THERE, WHICH WE'RE IN ACTIVELY. SO IF YOU LOOKING HERE, LET'S LOOK AT THIS APPROACH FOR INSTANCE NOUR APPROACH, THIS MATRIX, OKAY, IS A TUMOR MATRIX, AND THESE CELLS ARE TUMOR CELLS. SO WHAT HAPPENS IS THAT THE MOLECULES YOU'RE FINDING ARE THE MOLECULES THAT ARE TARGETING THE INTERACTION BETWEEN THE ENDOTHELIAL CELLS AND THE TUMOR MATRIX AND THE ENDOTHELIAL CELLS AND THE TUMOR CELLS. SO,--[INDISCERNIBLE]. >> [INDISCERNIBLE] >> WE'VE DONE THAT IN ASSAYS OR ACTUALLY EVEN TUMOR CELLS THAT ARE NOT VERY ANGIO GENIC, AND THEY DON'T GENERATE GENESIS EVEN INVITRO. SO, WHAT HAPPENS IS, TO MY SURPRISE, ACTUALLY BECAUSE I WAS ON YOUR PAGE, I WAS THINKING EXACTLY THE SAME THING, IS WHEN WE INJECTED THESE COMPOUNDS IN THE ANIMALS, FOR INSTANCE WITH THEM, WE'VE DONE SOME MICROSCOPY TO SEE WHAT HAPPENS TO THE VASCULATURE IN VIVO AND IT TURNS OUT THAT EXCLUSIVELY, THE TUMOR IS AFFECTED. YOU LOOK OUTSIDE THE TUMOR AND THEY COMPOUND IS NOT DOG ANYTHING TO THE TUMOR OUTSIDE THE TUMOR. SO IT TURNS OUT THAT THESE COMPOUNDS AT LEAST THIS ONE COMPOUND I'M TALK ABOUT IS VERY SPECIFIC FOR THE TUMOR VASCULATURE AND THE REASON WHY I THINK IT'S SPECIFIC IS BECAUSE OF THE DESIGN OF THE INVITRO EXPERIMENT. YOU'RE MAKING KIND OF LIKE AN INVITRO TUMOR MICROENVIRONMENT SO THE COMPOUNDS THAT YOU ARE GOING TO FIND OUT, USING THIS, YOU'RE GOING TOO FIND USING THESE SYSTEMS ARE GOING TO BE RELATED TO THE MICROENVIRONMENT NOT TO THE NORMAL TISSUE. >> YOU KNOW YOU HAVE THE DIFFERENCE BETWEEN THOSE AND THE NORMAL CELLS, FOR THIS AND USING IT BACK THERE, IT WOULD BE A DIFFERENCE? YES. >> SO ONE OF THE THINGS WE IF FOR INSTANCE WAS TO TEST THESE COMPOUNDS FOR OTHER THINGS. YOU KNOW? DO THESE COMPOUNDS BLOCK TUMOR GROWTH IN PARTICULAR, THIS ONE THAT I'M TALKING ABOUT DIDN'T. DO THESE COMPOUNDS AFFECT OUR CYTOTOXIC, TO NORMAL CELLS, OR TO OTHER CELLS, AND THEY'RE NONSIT O TOXIC TO OTHER CELLS SO THERE'S A LOT OF THINGS CAN YOU DO TO NARROW DOWN THE KIND OF COMPOUNDS BASICALLY THAT YOU GET OUT OF THESE ASSAYS. YES? >> [INDISCERNIBLE]. >> FOR THE METAMORPH, I DID USE IT BUT DIDN'T DO WHAT I WANTED. >> [INDISCERNIBLE]. >> SO WE'LL TALK LATER ABOUT„i YOUR PROBLEM. >> YOUR APPROACH INVITRO, STILL DOES NOT EXCLUDE THE POSSIBILITY TO CAUSE SOME EXTRAINIOUS SIDE EFFECT LIKE HYPERTENSION. >> YOU HAVE TO GO TO THE IN VIVO MODEL X'S THEN TO THE PATIENTS THAT'S ABSOLUTELY CORRECT. WE'RE NOT PRETENDING TO PREDICT A HUNDRED PERCENT WHAT HAPPENS IN VIVO ON A PATIENT, BUT BETTER THE ODDS WHAT YOU FIND IS GOING TO, ON THE PATIENT. >> SO RELATED TO THAT, IN YOUR EXPERIENCE, HOW, HOW, BIG IS THAT OFF? >> WELL, WHAT WE KNOW AND WE HAVE TESTED THEM FOR CLINICAL MODELS THEY ALL WORK A HUNDRED PERCENT. SO TAKEN--THEY'S WHAT WE KNOW SO FAR. >> WITHOUT HYPERTENSION? >> THE ANIMALS ARE NOT LOSING WEIGHT OR DYING SO THE ANIMALS ARE GROWING FINE. >> OKAY,. >> BESIDE THE POINT THEY HAVE THE VARIANT TUMOR OF COURSE. >> THANK YOU. >> MORE QUESTIONS? >> THANK YOU SO MUCH. >> [ APPLAUSE ]2oR#3