Carcinoma Lung Cancer

now i'd like to introduce our next speakererica jackson and just point out that she and got her phd inpower jacks lab who i think tyler sorta floating in theair around here with all the contributionsthat he's made at two grass biology and and make me take alsoget erica some things and some credit because the mouse that she had developedhours lab is the only reason why i have a career as well and a at the moment that i think that'snot her main purpose i i think i mean term a purpose a

right now as a senior scientist at atgenentech and chill talk to us about the role the at humorheterogeneity in and in response to therapy thank youokay so i am thank you to your eyes fororganizers for inviting me minutes opportunity to speak i'm gonnagive a little disclaimer they don't actually work on wrath signaling per say at this point in mycareer but i do i heavily utilized raskin cancer model fun to do new target a new targetdiscovery as well as to understand how

interesting role heterogeneity contributed therapeutic responses anddisease relapse that the work will be telling you abouttoday so my lad mainly focuses on lung canceralthough we certainly and branch out into other areas when on targets warranted but we mainly focus onlung cancer on which as many you know is the leadingcause of cancer deaths worldwide on non-small cell lung cancer accountsfor the vast majority of lung cancers on with adenocarcinoma being the largestsubtype of non-small cell lung cancer

and here at activating mutations being amajor driver in subtype on life of the lung cancer soonly about 20 percent of non-small cell lung cancer patients present withresectable defeat resectable disease on these patients have a and when theydid it can be excise they have a a pretty good five-year survival rate 43to 50 percent they mean they typically do you gettreated with an adjutant and cisplatin based chemotherapy i'm but i'll evensell up to eighty percent of the patients on will relapse after curative resectionin israel me

me that mean the occurs at distant siteshowever really the vast majority of patients present with numbers thatabilities and these patients are treated withchemotherapy radiation therapy herbal on the standard of care the plan b isdoubly chemotherapy but 80 to 85 percent in these patientswill continue to progress on therapy or relapse with incurable disease onwhat we do have some targeted agents available for treatment of lung cancers particularlyfor treatment the gfr mutant cancers are alpen cancers the vast majority ofpatients really do and get treated with

conventional chemotherapy in thisdisease and so we were wanted to understand what might bemediating this resistance in relapsed following treatment with conventional chemotherapyis and so as many as you may have heard that cancer stem cell theory has beenused to explain human resistance to relapse in a lot of cancers this is the theory that there's a aspecific cell type in the cancer that on is amanda cancer stemcell that is done actually required for the ongoing publication

that you were and that this particularthese cancer stem cells may be refractory kid 3 treatment and then drive relapseas well as on the task with am this is kim the cancer stem cellperiods a bit controversial there are strong supporters there's strongnaysayers on but what we think is injury what wethought was interesting about it is i'm just trying to understand okay is theresome subpopulation of cells within a tumor within heterogeneous tumor method thathas any resistance

on the neck and then going to driverelapse either at the local site on or as metathesis in a distant organan actual pay a little bit my talk today aboutstudies appall tumor propagating capacity is anindividual cell populations as well i'm resistance properties individuals orpopulations within a timber so the first part we decided to do wasortega approach that was like nah stick to stem cell theory and just say okay let's look at the field that don'tget killed by chemotherapy

so you know patients will present withan established sure that the heterogeneous mixture have tumor cells all heterogeneous fishertumor cells as well as stromal cell then took the lead with other than other celltypes and on were undergo some therapeuticintervention which will cause tumor regression button oftentimes tumor cells a leftover eitherat the primary site in circulation is mike metathesis andjustin organs in overtime we refer to these tumor initiating cellsthese residual cells

already initiated humor again either atthe primary site or as a metathesis and in organ so i thought maybe we couldjust find a way to study these residual tumor cells 30 do this wegenerated a variety of different um tumor models that which we hadintroduced a fluorescent labeling to the tumor cells and here we use both in agraft on models as well as geneticallyengineered mouse models and this would allow us to treat theanimals with chemotherapy significantly regressed the tumors and isolate the few remaining tumorcells that were left over time

and need to the cases we developed wecarefully characterized the worst response to chemotherapy and thenthe time to relapse in these models misled them i'llthat either underwent faces a regression and response toconventional chemotherapy and then it's on set time after the endof treatment because patients won't get cute they're people avatar they'll get on you know certainnumber of courses chemotherapy to a certain time aftercessation of treatment the animals will go on to relapse

to use the budget for models and thentell you bout to are astra models that we used on decatur st caltraing-twelve heat-driven genetically engineered mouse model as well as on theage for 41 model which is a carer street well being you and a week collected the residual tumorcells from both these models we saw that there was an enrichment on aregular and one so we found it initially by microarray and then we went on tovalidated on independent samples by qpcr you can see that majoring in paul

the draft in the gym model and here wasshowing you at the protein level did indeed in these regress trees residualtumor cells that are left you can see this big increase in aregular one protein expression as well as an increase in past overthree that was awhile this western meh lot might not look that impressive wereonly working with a couple thousand yourself for being able to see thesignal is an actually quite impressive today we wenton to actually do further characterization up are all themembers at the rbc going to happen in all

get what those are on the next live butsuffice it to say here that we actually didn't just seen aregular which but several ligands and receptors in the derby signaling pathwayin this was consistent across of the models that we looked atsuggesting that upregulation ever be signaling is occurring in these tumors were actually a bit surprised tosee her poor because i'm kinda north's record kinda like that black sheep in thefamily but we consistently song original agent and

and and receptors that would beconsistent with upregulation of her for settling in these residual tumor cells so what is new rail and all or on derby signaling axis absent a regular atthe lake in for derby three energy for receptors nervy three-headed attenuated kindnessto me into in order to transduce in a regular in signal at half the heterodyne rise it typicallydoes this with rb to you or hurt you which is itsfavorite demonization partner

anything that's unique about rb3 it hasa really strong activation of the payette behind it that way it has 6085 a binding site in it i think domain her be four on the otherhand can transduced unregulatingly at signal atthe home or die more or the heterodimer with any other familymembers and here on can activate you're sort ofconventional on grass rat mac or pi week i needdownstream effectors so we also were able to get our hands ona very small number

up neoadjuvant long cancer patientsample so these we don't know what they were treated with me just know thesepatients had received previous therapy now we collected the tumor cells bylaser capture microdissection and look at the rate and then as well ason tumor cells from naive unmatched patient samples and wecould see that there was also an arrangement of irregular and he pay that cells in patients that survivechemotherapy treatment and some other data we had suggestedthat this arm enrichment a regular might actually becoming from and the enrichment a

pre-existing cell population i'm so if we look for instance a so we look at some other conventionalchemotherapy that are used commonly to treat lung cancer van orleaning and gemcitabine and we could see that in the casegemcitabine we got pitching regression and gonna richmond at age 45 a.m. and a regular in however with theeuropean it was a pretty ineffective treatment and we didn't see in richmond have irregularand in those

residual tumor cells suggesting that hewould there be than activating a regular expression that perhaps is enriching forsale that expressed at high and when welooked in the genetically-engineered the gem shimmers on we could actuallyfind small population up in a regular expressing cell pre-existing in the tumor and these arereal this isn't just some scatter on the backs pockets on the sort out these as you can see that their mrna levelthey're highly enriched for a regular expression

but it is a very tiny population itselfcould so we look at what's happeningdownstream unregulated how is what whether the signals that signalingpathway that activating you can see that age 44 oneself this is done in vitrothese don't actually have no detectable regular expression until you regret youknow to you request that you were down to the smallof the population so here we had added dodge's newregulation and it 5 why to be fi three to four to sevenone or anybody 71 it's me like to refer to it as a as an irregularblocking anybody that we generated

engine and hack so if we stimulate with on irregular andwe can see really strong activation for three and astrong activation on the cake anything happily is read outby past week eighty and if we block your and we country withthat irregular blocking anybody and we can attenuate now if we look in the k wrath 53 lung tumors and these arejust four different rumors that we isolatedfrom the animal you can see that

on the tumors express and the for toreceptor they expect a lot of the hermit threeyears after they expressed no detectable her one which is the egfr receptor on and today we generated cell linesfrom these tumors in this is just one other cell lines that we we've generated in my lab and you cansee that when we treat with the antibody we can see wecan a block on her three activation and andreastatement that we can decrease and possibly katie levels as well aspossible 6l

there's very little pasternak actuallyin with that was not modulated by antibody treatment in the self so then we wanted to explore whetherinhibiting regular signaling could enhance the chemotherapy responseor delay the time to relapse after chemotherapy treatment so we used both the we used twodifferent courts in animals one in which there the animals are 5300i get the one in which the animals were 53 homozygote and in this case we use this diable sitewill her for extracellular domain italy

contrast this with operating not allowed by to the south signal we could see that although the her for a td really didn'tyou anything at all provided no benefit the animals when it was given incombination with and says patton that enhance the response to cisplatintreatment and prolong the time to relapse i'm or slowed the relapse the tumorsafter cisplatin i'm not a hugely impressive hacked on i'll at all you can see here than thepre and post-match pre- and

post-treatment skins that we are we r holding the chamber that a muchbetter than this on treatment alone however we think thatperhaps i'm the reason that we don't see suchgreat efficacy here is because we're not really shrinking the tumor that a wholewith line treatment in the them all downthere not that responsive to the spine and it's likely that there are many other cell cancer byaside that'll just dependent on the reagan

in other models again another again herewe're looking at another arrested in tumor model on where we alsosee enrichment up on rb happy components after treatment after to update there between inparticular we see really strong enrichment for four in this on my all we can see that if weuse offer for blocking antibody on in thiscase we can all completely delay relapsingthat decision treatment so that dat but

these also undergone much betterrationed we believe that you know that there's a population upirregular or be i'm a high it yourself and that thecells are wanted the resistant clones are oneother system populations within the tumor and is not genetic it we these tumors are the real at theirre treatment sensitive they will respond just as wellto the original therapies and non-genetic mechanism but through some means there's asubpopulation of tumor cells that we

believe are is expressing high levels ofunregulated or its other or be receptors so what are the cell on what are theseresidual cells are the cancer stem cell this is one of the questions that wewere interested in an asking and so i'm we have a fully andaddress that question fully answered it but weset out the kinda look at this and collaboration with other 100 secreterroneous postdoc unions and stamford and here we're going to try toquery whether these residual cells are

the same felt that are responsible for maintaining arepropagating the tumors i'm for long-term growth so i don't eat introduce you to thesemodels we've heard about him at length when he used to flavor the take a rest room in our long model on we use the k-ras only in which we do get some progression and a carcinomabeginning about sixteen weeks but which the bulk of the tumors in these animalsreally

remain how to know my and then we usedon the here at 53 w and a model in whichnow we can see a hits progression to late-stage diseasewhere the utmost in the animals where where you get a much largerfraction of the chamber's progressing to high-grade and add new carcinoma and the animalsactually development of the seas and acted the regional lymph nodeoccasionally to distant organs and so we also added in a fluorescentreporter a wheel to this strain we used a bunch of different i'mversions of fluorescent reporters

including rfp leila wire pleo itv2 made a leo but on using a ride a different color themand strengthen reporter allow us to isolate the tumor cells andactually we and at first we thought that okay justsorting on the 4th reporter will be enough to atlanta up true isolator population of tumorcells but we were fooled by this and actually went off ona wild goose chase for quite a long time because we found out that it was reallyimportant to also include on

lineage us elections are negativeselection for lineage markers because we can see in the fluorescent population there alotta macrophages in there so we thought it was very important bothto the positive selection for the fluorescent marker and the negativeselection for the lineage on and then we do this we developed anorthotopic crafting system where we interest rate really into baby animalsand introduced a cells into their lungs and you can see that the fluorescent

tumor cells wind a single cells indifferent parts of the long and then give right the co-lead arrived riversand tell on so you can see that when we look do thati'm i a cheaper the ds red transgene that all the team is derivedfrom the crafted self so we found that with both the care andand the camera 53 to ensure models only a small fraction of the cells are tumor tumorigenic on but this is strongly the this fraction orhow big this faction is strongly

influenced by 53 so you can see that when we transplanton tumor cells from the care is only tumors we get a very small number ofchambers for animal on and and this number is greatlyincreased with me have the p53 mentally ill in there andthat that tumor sizes also remains really small onlytransplant came as only tumors and a much larger much more aggressive whenthey have a lot of p53 and in fact we look at thesetransplanted to return to you we initiate the tumors

we let the mites from the cameras onlysituation with that they might go 16 to 20 weeks the donor might then we i think the tumors redraft themlet them go another 12 to 16 weeks and that you were stayingat no the whole time so on the really need to have secondevent that looks like to be able to progress to carcinoma which you know we knew but it was verysurprising i thought you could transplanted use but that it with theyhad no mental where the chaos 53 w down to mouth thetumors were very very aggressive

i'm and you can see just order the tumorinitiating cell frequency in these animals is i won in 2000 with the p53 mutation onein forty thousand with the key with only so this is a rare cell populationand we wanted to also i just mention it i before i moveon that it's a long long care med we're talkingfifteen weeks and then another 16 weeks though we wanted the kinda faster systemto be able to try to keep the part where the two are propagating cells here so we developed the spear dayon

that we hope can be useful as asurrogate for tumor initiating a pass at your trip up getting passe the agency citizens using a 3dsystem with this tumor cells are embedded in majorgeneral this fears are clearly derived his only to a mixing experiments thereeither all quiet the positive for all our peoplethat we never see any next self and its clones and they this year the girl at actually hollowthey form a polarized epithelium as you can see by that

polarized localization e-cadherin ca1 and the express on lung epithelialmarkers such as ittf one which is also known as and hicks 2.1 and surfactant proteins same and when wepassage these fears over time you can see that up so you get more spears from here53 just like you get more tumors from here at 53 and as you passage them over time the p53 null years increase in number wall

that a that he was only thirty twothings suggesting that the self-renewal capacity with in the absence of p53 isgreatly enhanced and then we took the same ourself ampleand grafted them in vitro versus pleaded them in tears invivo at i'm sorry crafted their in vivo versusthing to them if they are in vitro we could see that is a good correlationbetween spear formation and your information sowe believe that this is really a good surrogate for on in devel that lethal get thestarting point to trade with that

different markers after public ok and so we started with just a candidateapproach st 24 has been used to identify to republic eating cells from fromseveral other genetically engineer now 12 cancer including several breastcancer models so we just used c24 the candidate marker and lead early so that indeed c24positive cells were highly-enriched first-year warmingcapacity varsity 24 negative cells in it's a with held up by the york a dress only orkey west 53 double mutant

on but you not really in reaching muchwith the 24 because most to the tumor cells actually 8024positive did so we thought and this the numberc24 positive cells is way higher dead the number spearforming cells in in a chamber so we thought okay maybe wecan look at the 24 positive self do some expression profiling up then ifyou come up with other candidate markers that we might be able to use to it in which further and so i ok before but by four sharia is want tomention that we do see in terms of this

relationship between sure propagatingcell and injury initiating cells we do seethat cd 24 is highly i'm is interesting is a highly but isenriched following first-line treatment in those residual tumor cells and if you look at naive t 24 positivetumor cells their highly enriched for a regular expression which means that isalso you know a calmer up these residualtumor cells that are remaining after chemotherapy though this is correlated have datasuggesting that on

the that you are propagating self i maybe the tumor initiating cells getting a movingon to the candidate markers so following that gene expressionprofiling we came up with for candidate markers 8133 which has been widely described asa cancer stem cell marker idea 1r notched three and injuring datafor then we could see that these rollingbridge dinner 24 positive self so just though we try to look at whether any addiesmight have other on

evidence to suggest they may be onheterogeneous rb represent a subset of the cells in thetumor we could see that if we look at has one which is a downstream target ofnotch we could see that all that sure that welooked at were heterogeneous perhaps one and the proportion up to your cell thatwere positive buried between jim is to begin with a fraction of cells that had activatednotch signaling in the tumors so then we went on and looked at thisyear for me capacity i felt that were sorted for these individual markers sowas not

3 that came up on in the microarrayexperiment but we decided to extend the analysis to allnot receptors given the hath staining pattern we had them and so wecould the in japan beat up for out came up asbeing as enriching first year from capacity eachother noches enriched first year from a capacity but city 133 did not in wichita didn't in that case was notworking at the stem cell marker so the experiments here we're done withthe key rats mice on we also sorry and then going back to thestore to chemotherapy i'm connection

thing out again when we look at those residualtumor cells that remain after because the evidence that they have onnot pathway activation in those cells as well so again providing them correlatedevidence linking potentially that propagating cells tothe initiating cells so moving on to the care at 53 system wedecided to look at you to these markers i'll e when we start compiling them in andout in a stepwise fashion so if we store for on you do that

24 positive cells on our make up about50 percent in a tumor and a in richford your permit capacitywhen you add on that and you can be 24 with the c24 now you get down to about20 percent of their tumor cells and you further enrich first performing capacity and when you're out on the not marker inhere we sorted for panda much on we can further image for spare fromincapacity and further decreased at a small thatthe tumor cells minister foreign numbers are shown herelead and so that's all fine and good that'spure formation in vitro but

what'd had it hold up when we graphedthousand people today we did orthotopic graftingcomparing different i sell doses up the triple positive cells that c24positive integrating data for positive not positive to non-tribal positive cellsand and we could see that both the tumornumber and the tumor size was greatly in creased with the on-scene the triplepositive cells relative to the non-tribal positive cells

and we go for mature initiatingcell-free here on $1,200 with the triple positives to onein 2000 with an triple posited was not perfect yet myhead but these health are definitely highlyenriched further tumor propagating capacity so then we wanted to see okay does thishold up like now is this is this am is this a stable pheno type but these people positivecells are there back and forth in am conversion between shovel positivesand knowledgeable positive

and jim propagating self-knowledgepropagating felt so this is kinda confusing but i'll try to walk you 30 death so we did was we took ourprimary donor mice we separated into triple positive thenon-tribal positives crafted them got a lot a tear in the triple positivenot so many in the non-tribal positive took the dow fractionated them again indigital positive and non-tribal positive channel positive knowledgeable positiveand redrafted and look at your information so what we saw was a kinda but weexpected but it was a nice result in her

left with that when you look and the triple positivecell from a triple positive tumor they make the most generous thenon-tribal positive self from originally triple positive drivechandler make the second most tumors and the non-tribal positive ensure apositive felt that came from a non-jew a positive chandler are thehave the least your public any capacity looks sorry at at the least surepropagating capacity and also give just very very smalltumors that if they're their their growth potential isdecreasing with time

petering out it so now we wanted to say okay so now we knowthat not just marker on he felt but is it a functionally important driver and them that you are propagatingcell phenotype so we started just using again issueprotease inhibitor dappy and we saw the tapti treatment upeither care at only work here spad three i'm double mutantsfears hours decrease your number on but with that on and though side and

it could this decrease could be rescuedif we overexpressed in dodge sony to try this contacting get ticketsat a lot of different target that we wanted look at whetherit's really not dependent it can be rescued with the night cd whati think is even more striking is that effect on self-renewal so if wegrowth fears intact but them passage them to look atsecondary there not inductee anymore %ah we see an evenbigger reduction in the number of secondary years that form suggested that perhapsfor differentiating themselves

on or poor blocking their ability tosell for you with it that the treatment and you seethe opposite when the overexpressed in a ginger say me actually hands cell and so then we looked at i'm each of theindividual not proteins on we isolated the tumor cells and this was really a tour de forceworked up by all the 100th post at union but she ate ladies individual tumors i'm sorry i played at the chamber'sdissociated single-cell

that an acute infection with lentivirusis expressing here and each individual noches multiple hereand on and either plated them its fears over orwe did orthotopic transplantation so all that these in the cell never saw if they were cultured and that in factit was they were affected for a couple hours immediately after they came at theanimal and why they put immediately into itspeers or on the next but i'll show you put immediately back into the animal's so we could see that only knock down anotch three but not the other

three notches had any effect on yourpermission and it with it an effect on primary here as well as ourself and all thesehere pathogen and we crafted these notched threeknockdown cells in vivo we saw really dramatic reduction inthere to reforming capacity you can see that burdened with that hairenergy lp is massive relative to the burden with that on national knocked down and we look atthe individual tumor that did grow they were much smaller

and overall were much smaller than thegym with that group edge p.m. suggesting that notch three isplaying an important role for that publication of the cell that public meeting about 30 at thethoughtful in the show an interview about on and so that we wanted to look atwhether this might be on important in patient samples whetherthis was just them quick the model on and so we were ableto get several different freshly isolatedpatient tumors although we don't know

the wrath status full disclosure a these tumorsbut we were able to get several freshly isolated patient samplesthat we could grow its peers they loved it system it definitelyoptimized for mouth cell fear growth but we did get them onthe growing this your system and we could see each casethat treatment with the game a secret ace inhibitor reduced hear from a capacity of thesetumor cells and in the cases where we were able to pass is

spears we could see that there is areally say there was a reduction in secondaryinformation on with the dow teaching and then with one humor we were able togenerate a pdx as the patient raising a graft an and a and we could show that knockeddown a notch 3 with three different hair pins dramaticallyinhibited the growth that this tumor email an which is shown adjust the volume isshown here

and sorry and then you can see a images that these here and i think that's it so out with etihadon a cell with that i'll just wrap up and acknowledge the people that did thework on does the field in the cruising up atthe head to my lab did them of both the work on than a regularand anarchy story that i showed you for the first part the talk on there were several other people thatwere involved in those at after the genetic as well and then allthe second part talk showed you on that

you are propagating cell capacity work was done is that closecollaboration with alejandra secret a row and as per stocky and it was also veryinvolved in work that as really lovely story so in yoursecondary and tertiary transplantation experiments with that no one with a triple posit ameliorate thechapel positive cells if you look at the charm as they grow tothe recapitulate the heterogeneity of

the original such that the triple positive cells at aminority population they are so would so when we they are aminority population on and that and diffraction even tho soif you look at the triple positive cells may triple positive drive tumor it looks similar to you know your youroriginal unsorted donor if you look at the howmany positive felt there are from a non picture of apositive derived him it's gone down actually they're not asgood with to non-tribal positive l

can make some trouble positives but itdoesn't make a lot and the thing to be petering out overtime so what are the things we wantto do now is we would like to look at this allergy wehaven't really looked extensively at the knology up all those real transplantedtumors and but we have been quick follow-upquestion so obviously there's been a previous claim for the existence of a prank you all theor stem cell that may also be a picture forpropagating celso

do you know if there's any relationshipbetween your triple positive cells and sells previously claimed to be spcpositive cc a positive from college him now we don't know aboutit whether there's a relationship there one thing i will say though is that you know who had published i thatintegral beta 4 is a marker of are normal lung epithelial themselvesand certainly in turn be there for is i want the markers of ourselves aswell but what relationship felt half to death p n double positivecells we don't know but we did look at taiwan which is oneof the market's the car uses for us

sorting itself and we do see enrich ministerfrom a capacity with scott in got one fell haven't looked exactly howthat column overlaps with that with a triple positive more and so in the the first mouse that youtake the primary tumors before you craft them to take all those tumors and pull themtogether and then sort yeah we pull so do you do you know what like thepercentage for each tumor the triple positive

know and to be honest with you we willprobably get like truth the whole because the fact is really inefficient ithink particularly for the night marker the fact that poor it's not a great any body which is partof the reason or we actually cool notch anybody and ithink that we we ended up having to do that becausethey just art accurately i'd the leading a notchpositive as my feeling you don't know if it's whether whetherit's from one tumor or all the tumor well it any what thespirit we did our from all that shimmers

however if you look for instance at thehess stating that i showed you in all tumorswe can see a sub that felt that our hats one am i was curious in in new york for yourshit for story with sydney regular sorry to show if you added exogenously did youget this sem your force for her three signature so iviewed if you just looked at the resistantcells and not edit exhaustion is leniency okay what's the percentage of cells that really go over her threesignal over

her three oh yes oh i only ship that onethere was only one law where we are today added exogenously on there so most thework we were looking at the possible for three-level in the residual felt themselves theywere greatly elevated yeah and so have you damn try to at like agdc inhibitor you know given that there's that link tothe ps3 kinda we haven't done that experiment so on so the combination withchemotherapy

a lot of things are not tolerated i wehaven't done it other thing we haven't done it and i don't know you know i with have a little bit ofconcerned about the combinations at we're heading hard chemo but ok had mystery erica her i noticed it enda dooo positives fears they seem to be no longer hollow seemedto be field just wondering whether you have looked at any emt martyrs becausethis is usually what happens

in a related question is whether your are orthotopic model you can score fromits and see whether de sales have highermetastatic yeah that's a great question so i think thaton okay so this year's from the mouse tumorsactually are always hollow and so the pictures up the human spearsthey seem to be filled so we never cease your dealing with it the one city showedwith down a secret a's inhibitors the two pictures there doword field stickers renault holes so if you haven't looks atit carefully it's just because sir

the one that you showed just the imagesthe phase images these are field spears but take a look at it but that humans arethe humans are usually feel they seem to be filled i mean that's not having section themand look carefully by just looking at them to the maker cisco in the field and when you look at thisyear's and makers but actually quite obvious that there holly you could the density ring and that's what aboutalike come to the center but

but it's ok so for the human spiritthere clearly not hollow then is the system dc at it change in metastatic potential between think we dobut but we haven't carefully analyzed it sowe need it will be very helpful be compared to a lot like i'll like a luciferase reporter and so wecould monitor ii a metathesis more easily and and it's something that we can do wehave enough we just haven't done it yet

okay i wonder emissions there is a lotof such research it macrophages some wonder if you guys have looked at and is there anything who thing interestingthere any have have we look at any role thatyou're associated macrophages know we havent really done much withthat it's certainly an area interests bat wedon't now we do know there's a huge infiltrate wedo know their eyes some kinda strange event going on with those macrophages inthe tumor cells because if you look at those fluorescent macrophages

you can see some evidence and therecombined here essentially all in them so we don't think we think but it not it's not like 121 it's not like in alate not like when you do the pcr the tumor cells themselves on their on the main negative still we thinkthere may be some cell fusion events or something that's going on with themacrophages in the tumor cells but we don't know we're not sure but there's somethinginteresting and strange going on there but we don't know it

her