Pathology Grand Rounds: March 2, 2023 - Robert J. Coffey, Jr., MD

March 02, 2023

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Update on Extracellular Vesicles and Nanoparticles in Colorectal Cancer – By Robert J. Coffey Jr., MD

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00:00Was a grand drones today.
00:02It is my honor and great pleasure to
00:05introduce today's speaker, Bob Coffey.
00:08Doctor Coffey wears many hats.
00:11Professor of Medicine and settle
00:13in developmental biology at
00:15Vanderbilt University Medical Center.
00:17And he's Co director of the
00:20Epithelial Biology Center and Ingram
00:22Professor of Cancer Research.
00:24And he's also the principal investigator
00:27of the GI GI Spore at Vanderbilt.
00:30He went to Princeton University
00:33for majoring in politics,
00:35not in biology or chemistry.
00:38And then.
00:38Went to law school at at Georgetown
00:42after graduating from Princeton
00:44and but they dropped off on three
00:47weeks after entering law school.
00:50Then prepare to enter the medical
00:52school and enter the Georgetown
00:55Medical School and then politics
00:58is internal medicine residency at
01:01Emory and then Medical Oncology
01:04Fellowship at Georgetown and then
01:07gastroenterology fellowship double.
01:09Fellowship and at Mayo Clinic,
01:11and stayed there as an assistant
01:15professor for a foreign year before
01:18he moved to Vanderbilt in 1986,
01:22and he has stayed at Vanderbilt since then.
01:27He. Is the most hardworking person
01:33I've I've ever met, actually.
01:35So he's a really successful,
01:38exemplary physician and scientist.
01:41I really admire and.
01:44So his, you know, work day and
01:48during the week is like Monday,
01:49Tuesday, Wednesday, Thursday,
01:51Friday, Friday, Friday.
01:54That's done. And when I try to
01:59join his laboratory as a postdoc.
02:02That he set up our first meeting
02:05on Thursday at 8:00 in the
02:07morning and he always comes in,
02:11you know during the weekend holidays
02:13and then comes in the oldest among
02:16the all the land members and leave the
02:20last yeah in the laboratory I and.
02:23This is Eugene Cliffy.
02:25So after he moved to Vanderbilt
02:27within a year, I just looked at.
02:29I didn't know.
02:30I just realized that he published 5
02:33first author papers within a year,
02:35including Nature, Cancer Research and ACI.
02:41Clearly shows how you know the he's
02:44a really successful physician,
02:46physician, scientist.
02:47And he has also one thing I also admire.
02:53Respect him as though he always
02:56tried to learn new things.
02:58He tried to keep learning at
03:01least one new thing every day,
03:03so he may take notes in every
03:07seminars and conferences and on.
03:10Read the papers every day has
03:14practicing that over 30 years so.
03:19And he has published more than 300
03:23papers so far and I had this many
03:27seminal discoveries including the,
03:29you know, TGF alpha is the,
03:32you know,
03:33pathogenesis for the mandatory disease.
03:35Also performed the clinical trial
03:40treating the military disease patient
03:42with the cetuximab treatment and
03:44also found the Elic wine is a,
03:46you know,
03:47quiescent stem cell marker in intestine.
03:50And also showed that you know,
03:52long non coding RNA's near 100 HD is
03:57the reason why the colorectal cancer.
04:00Shows the resistance to the setup
04:02symmetry kment via the wind better
04:05containing signaling pathways.
04:07And recently he also showed that EGFR
04:11is secreted in within the oxygen from
04:14the colorectal cancer and also showed that.
04:19In the contrary to the fields belief,
04:23RNA's are not included in the EXOGEN,
04:26but it's mainly secreted from the,
04:29you know,
04:29smaller compartment secreted from the cells,
04:33different from the exogen
04:35and recently discovered.
04:38Smaller nano nanoparticles,
04:39smaller than the exosome.
04:41And he named it Super Mere and
04:44showed that it's functionally
04:46important in biology.
04:48So.
04:51Ohh, sorry again.
04:53Maybe that was too much.
04:54OK, so without further ado,
04:56his title on the talk of
04:57his title is the update on
05:00extracellular vesicles and
05:01nanoparticles in colorectal cancer.
05:03Please join me in welcoming
05:05Doctor Patrick. Thank you so much.
05:10So it reminding me that I started
05:14in 1986 at Vanderbilt and I remember
05:17I trained in as an MD and I I met
05:21Stanley Cohen at that time who I
05:23hadn't known before and you know I
05:26said to him can somebody like myself
05:29trained and in medicine do anything
05:32worthwhile and and research and
05:34Stanley used to walk around the 6th
05:37floor the biochemistry with a corncob.
05:40Type and just thinking of the
05:42simplest experiment that would
05:44be the most informative.
05:45And he said to me, yes, it can do two things.
05:48He and he would usually cut his hands over
05:50his eyes when he was going to make a point.
05:53And he said if you pay careful
05:55attention to your data and you're lucky.
05:58And I thought that was some
06:00of the best advice I ever got.
06:02So it's great to be here and
06:06it's nice to see how well.
06:09That one Jay is settling in and how
06:12welcoming everyone has been and that he
06:16has such superb mentors and Katie and Fred.
06:21And So what I'm going to try to do is,
06:25is sort of give you an overview
06:27about some of the things that
06:29we've been doing more recently.
06:31I'd like to keep it informal,
06:33so if you have questions,
06:34don't hesitate to stop and
06:38ask me and I'm also.
06:40I want to present a fair
06:42amount of unpublished data.
06:44So.
06:52Was told if I press that.
06:56It would work. But maybe not.
07:02This one.
07:06OK, let's see it. Yep.
07:09OK, so I wanted to give first.
07:14A little bit of background and
07:16and I wanna tell you about this
07:19overarching 3 pronged approach.
07:21We start taken to study
07:24colorectal cancer at Vanderbilt.
07:26And I think this could be
07:28applied to any solid tumor.
07:30And so we start with polarized
07:33epithelial cells and we're interested in,
07:36in various acts, aspects that I'll,
07:38I'll tell you about in just a moment.
07:40And then we moved from in vitro.
07:44To mouse models of colon cancer and then to
07:48human colorectal cancer and each of these.
07:52These approaches are are going by
07:55directionally and we can in an iterative
07:59way to hopefully make significant advances.
08:02And so as Juan J mentioned,
08:05in 2010 I started the epithelial
08:07Biology Center and that's something
08:09that Jim Golden Ring and I Co direct.
08:12Now we have over 40 members.
08:14Areas of interest include
08:17epithelial polarity,
08:18vesicle trafficking,
08:19stem cells and extracellular vesicles.
08:23And and the center tries to bring
08:25forward new tools that can be used
08:28throughout their the university we
08:30have a pipeline for single cell RNA
08:32seek Multiplex immunofluorescence
08:34that David Rim gave us some good
08:38advice about a few years ago.
08:40And.
08:41Isolation of the ebbs and nanoparticles,
08:45which I'll be telling you about today
08:47and we have a symposium that alternates
08:50with an epithelial pathobiology class.
08:53This year is the symposium
08:55in and on April 3rd.
08:57The theme this year is a basic
09:00biology that therapeutic intervention
09:02and we have Carl Sawyers and Health
09:05Chapman as as our keynote speakers.
09:08And then as far as mouse modeling,
09:11we've been working on trying to determine
09:14the cell of origin and colonic neoplasia
09:17use this elry one pre jot driver.
09:20This panel will be negative regulator
09:22that won Jay mentioned and then
09:25we've made a useful reporter
09:27mouse that I think monitors EGFR
09:29visually and and Juan Jay I think
09:33is going to take really effective
09:36use of that model and then.
09:38We've had our GI Sport since 2002 and.
09:44Presently we have the three projects that
09:48are are listed here and we're facing as
09:52I was telling Katie a little while ago,
09:54we're facing our competitive renewal in
09:57September and one of the projects and I'm
10:00going to be talking about this as I go
10:03through the talk is to try to overcome
10:06immune exclusion and microsatellite stable,
10:09chromosomally unstable colorectal cancer.
10:16So about. Couple years ago now.
10:22We have been involved in human
10:26tumor Atlas network and our project
10:30which was headed by myself,
10:33Ken Lau and Martha Shrubsole was
10:36to do a single cell Atlas of the
10:38two most common pre malignant
10:40tumors of the of the colon and
10:43those are the conventional adenoma.
10:45And and sessile serrated lesions
10:50this is going to be about 85% of pre
10:54malignant tumors these are 15% and
10:57these were really pretty well uncertain
11:01in terms of their origin and what
11:06we found perhaps not unexpectedly
11:09was that the conventional abnormal
11:11was a wind driven expansion of stem
11:13progenitor cells that grip base but.
11:16Very unexpectedly,
11:17the sessile serrated lesions which
11:20are occurring in in a background
11:24of inflammation predominantly
11:25on the right side of the colon,
11:29we're due to gastric metaplasia and and
11:32we think it's driven by a loss of CD,
11:35A CD X2 which is a fine gut fate determinant.
11:40And when that happens you revert to a
11:43more rostral fate and in this case.
11:46Have histologic elements that
11:49are seen in the stomach.
11:52So we were able to provide now
11:54a tool for pathologists to make
11:57this diagnosis because it's a
12:00challenging diagnosis to make.
12:02But now you have a number of markers
12:05that you can do that you can use
12:07to to help make that diagnosis.
12:10And.
12:12What we've done more recently and we've
12:16just submitted a paper is to now advance.
12:19So conventional adenomas are going to be
12:23moving towards microsatellite stable,
12:25chromosomal unstable cancer whereas
12:29the microsatellite unstable.
12:31Or the gastric metaplasia tend to
12:35evolve to a microsatellite unstable
12:38hyper mutated tumor and and these
12:42tumors we see a lot of immune cells,
12:45CDA T cells but but by and large.
12:51CDA T cells are not there in the
12:55microsatellite stable colon cancers.
12:58There are nuances that I don't
12:59have a chance to go in today,
13:01but as a generalization that
13:04appears to be the case.
13:06And So what we've just submitted
13:09now is a four gene Abune exclusion
13:13signature that we've identified in
13:16these cancers and I'll be telling
13:19you more about these proteins as
13:22I go along and that's.
13:24Dipeptidase one TGF beta induced,
13:28not to be confused, which it always is,
13:31with TGF beta 1DR1 and then pack four.
13:36So these are all membrane or
13:39secreted proteins.
13:40This is a cytosolic protein that
13:43in microsatellite stable colon
13:45cancer appears to be associated
13:48with immune exclusion.
13:50So that what I am hoping to
13:52cover today is to tell you.
13:54About work and we've in our isolation
13:58of extracellular vesicles and
14:00examiners are discovery of super
14:02meres and then identification of
14:04these ECM related clinically relevant
14:07cargo and colorectal cancer that
14:09may contribute to immune exclusion
14:13as I've indicated.
14:15So how I got involved in extracellular
14:18vesicles was really an outgrowth
14:20of the basic work in the lab which
14:23is really to understand.
14:25The trafficking of the EGF receptor
14:27ligands and the context of a polarized
14:30epithelial cell and by and large we've
14:33used polarized MDCK cells which we
14:36overexpressed the different ligands
14:38and they have 20 to 40,000 basolateral
14:41egbdf receptors as what we think is a
14:45complement of of a normal epithelial cells.
14:48And So what we've systematically
14:50done over the years is to look at the
14:53trafficking of the ligands in that setting.
14:56And it's really been what I would say,
14:58a mother lode of good cell biology
15:00in terms of each ligand having
15:02nuances in terms of where it goes.
15:07Which surface, who cleaves it and then
15:11how actively it engages the receptor
15:15with different signaling consequences.
15:18And so when we were studying HB EGF,
15:22we were surprised to see that there
15:25was full length HB EGF in the apical
15:27media but but not at the cell surface.
15:30So one formal possibility was that it was
15:33being released and an exosome and so in fact.
15:38By combining sequential ultracentrifugation
15:40and and a technique that we developed
15:43in the lab called fluorescence
15:46activated vesicle sorting,
15:48which I'll mention in a minute,
15:50we were able to show that in fact
15:54these different live bands were present
15:56in individual exosomes from breast
15:58and colorectal cancer cell lines,
16:00but they differ and a rag exosomes
16:03enhanced invasiveness of recipient
16:05cancer cells more than TDF.
16:08Often HEEF exosomes and this was in
16:11the setting of overexpressing these
16:13different ligands in the MDCK cells.
16:17And we were able to.
16:21Identified that there were 24
16:23molecules of amperage on that were
16:25packaged in individual axes on.
16:27So these are like signaling payloads.
16:31And we coined the term that a
16:35Reg was in part working through
16:38EGFR receptor to introduce the
16:41idea of extra print signaling,
16:43which has not become a household
16:46term by any means.
16:48And we were also able to show that EGF
16:52receptor itself was packaged in EB's
16:55and and this is an example of a line
16:58that we frequently use in the lab.
17:01So this is.
17:0350, which is a polar rectal cancer
17:06cell line that has 5,000,000
17:08EGF receptors per cell.
17:10So it's sort of the granddaddy
17:13of an EGF receptor overexpressing
17:16cancer cell lines.
17:18And so Jim Higginbotham in the lab
17:21was able to flow sort with directly
17:26labeled antibodies to either.
17:30Cetuximab,
17:31or a tetraspanin that's commonly
17:34used to mark.
17:36Exosomes and was able to flow
17:40short double positive,
17:42double negative further enriching
17:46for those EB's and then we could
17:50do Western blotting and show in
17:52fact in the double positive we
17:55could see EGFR and Centennial and
17:58other exosome marker and you hear,
18:00see,
18:01hear that CD 81 was present in
18:04both although enriched in the
18:06EGFR double positive.
18:08And then Jeff Franklin in the lab
18:10was able to take a drop and and
18:13put that on a cover slip and then
18:15use storm with antibodies to EGFR
18:18and CD9 and showing that they were
18:21single particle that were of the
18:25right size for an exosome that
18:27were positive for EGFR and CD9.
18:33And.
18:35How we've taken this forward clinically is
18:39in Leo Blastoma where we know in some cases
18:44there's more overexpression of EGF receptor,
18:47there's going to be amplification.
18:50And in this particular case,
18:52we were looking at at patients
18:54that had the V3 mutation,
18:56so they have a chunk of the actual
18:59domain removed and. In this.
19:02We were able to show that we're now using.
19:08Not only she talks about,
19:09but we're also using monoclonal antibody
19:128O6 which was generated to a by a group
19:16in Australia to the Conformationally
19:19active Ectodomain conformationally
19:21active form of EGF receptor.
19:24And so there we could see in the normal
19:27control we we didn't see a signal,
19:30whereas these four patients were
19:34positive although at differing
19:36percentages for double positivity.
19:394806 and setup samap and so.
19:43We could then perform Westerns
19:45and here the lower band the faster
19:49migrating ban is is spurious.
19:53But we can see that the receptor is
19:56present in the normal at the right
20:00size and these B3 individuals are
20:03cancers were were had a a smaller
20:08band appropriate loading control
20:10and then we can see that.
20:13All three of the GBM patients appear
20:16to have active ETF receptor in their
20:19circulating EV's that have clearly
20:22crossed the blood brain barrier.
20:27So I've been fortunate to be
20:30involved in two rounds now of the
20:33extracellular RNA Community consortium,
20:36and this now will just give you a
20:42sense of the complexity of what
20:45one can detect in the circulation.
20:49So here's a cell for or size,
20:52here's some viruses and what
20:55I'm going to be talking about.
20:58Is that there are large EB's
21:00that are thought to.
21:02That are often called micro vesicles.
21:04They're budding from the cell surface
21:06and then they're small eddies,
21:09some of which are exosomes,
21:12and that is if they have the Tetra spaniens.
21:18And uh.
21:21The exosome is rather it's also
21:25starting at the plasma membrane but
21:28then it's endocytosed and during
21:31its late endosomes they pinch off
21:34and forward inward vaccinations
21:36within a multi vesicular body.
21:39So which is really a bag of intraluminal
21:42vesicles in which the topology is changed.
21:46So the transmembrane protein
21:48now has its ectodomain facing.
21:51Outward and the cytoplasmic tail inward.
21:54So when these multi vasectomy
21:57particular bodies choose to budget
21:59the plasma membrane rather than going
22:02to the lysosome that they will.
22:06Release their signaling competent material.
22:11And I'm going to tell you a little bit
22:14more about these a membranous nanoparticles,
22:18which are examiners and super meres.
22:23And and so we've been able to publish
22:26a number of papers in this space most
22:31recently for those of us that are interested,
22:33we have a comprehensive protocol for
22:37isolating extracellular vesicles and
22:40nanoparticles from the same starting
22:43material and then two recent reviews
22:46that for those that are interested.
22:49So the first important paper
22:53we published was by Dennis,
22:56Yep,
22:56Person in the lab who published
23:01this paper and sell and we got a
23:05lot of nice PR in terms of that.
23:08We provided a much needed reappraisal
23:11of what constitutes a bona fide
23:14exosome through a highly stringent
23:17and novel methodology.
23:19And So what Dennis did was he used
23:22the conventional way of of a series
23:25of low speed spans passing through
23:28a filter and then a high speed
23:30spin to get his SB pellet.
23:34Now until somewhat recently that was
23:38considered enough to call it an exosome,
23:42but clearly there's a lot more
23:46there in this evening pellet than.
23:49Just an exosome.
23:50So what Dennis did was he then took
23:53that pellet and then bottom loaded it,
23:56which is very important and then
24:00spun that over this discontinuous
24:05gradient at 120,000 G in this
24:10case overnight and what he was
24:12and and we did this not only in
24:15colorectal cancer cells and breast.
24:19BM primary human renal epithelial
24:21cells in human even human plasma.
24:25And and So what Dennis was able
24:28to show was that when he looked
24:32at at the proteins now both in a
24:35colorectal cancer cell line and a
24:38glioblastoma he could see that in
24:42the lighter fractions was where
24:44he was able to to detect what we.
24:50We consider.
24:51Bicycle exosome markers whereas
24:53in the non vesicular there were a
24:56number of proteins that have been
24:59outed to be in in exosomes but clearly
25:03aren't in in both of these cell lines.
25:06And but he went one step further,
25:09so there's been concern that maybe damaged.
25:12These EB's with a high speed spin.
25:15So what he did was he then within
25:20individual immunity Immunoaffinity
25:21captured with the different Tetris.
25:24Spaniens was able to then prior to the
25:29high speed ultracentrifugation place beads
25:32with these antibodies and then pull down
25:35and then was able to validate that much
25:39of the material that he identified in the.
25:42High speed span was was was shown
25:45to be the same and so at the end of
25:49the day Dennis could say OK,
25:51what's in classical exosomes.
25:54What's weakly associated with
25:57classical exosome?
25:59Absent from classical exosomes and then
26:03completely absent from any type of small EV.
26:08So I think this was an
26:10important advance in the field.
26:12A few of the other highlights of
26:16of that work was is depicted here.
26:21And. About that same time.
26:26A little before,
26:28David Lyden's group had identified examiners,
26:31and he did that by using asymmetric
26:34flow field flow fractionation.
26:37So this required about a $300,000
26:41instrument and it's low yield.
26:45And at Vanderbilt,
26:47we couldn't afford that piece of equipment.
26:50So Kinzang and the lab had the idea, OK,
26:53well, let's just take the supernatant.
26:56From that EV pellet and let's spend
26:59that harder and and then let's
27:01see what we we find.
27:03And so she did that and she was
27:06able to show that I'm using this
27:10simplified method that we were able to
27:13identify pretty much the same cargo,
27:17many of the same cargo that David
27:20had had seen,
27:21although we were using different
27:23cell lines in this case,
27:24but an awful lot of overlap.
27:26And we were able to identify 2 functional
27:30properties of these examiners and
27:33that there was ST6 gal one and examiners.
27:37And it was able to simulate recipient
27:40cell surface targets including beta 1
27:43integrins and EGFR not shown here and
27:46increase the activity of those proteins.
27:49And then we could show that a Reg and
27:52examiners were able to modulate EGFR,
27:54separate tracking, trafficking and.
27:57Increase.
27:58Whom organoid an order of magnitude more
28:02equivalent amounts of recombinant camparada.
28:06So then that set the stage for the
28:09paper I'm about to tell you about,
28:12which was published.
28:14In December of 2021 and that's
28:18identifying super mirrors.
28:20But we did more than just show the
28:23discovery of superiors in this paper.
28:25We did a comprehensive classification
28:28or analysis both at the M RNA
28:31and protein level of of cargo
28:33within these different fractions.
28:36And so that I'm going to tell you
28:38about that work that was carried
28:40out by Dennis and Chin.
28:42So Chen figured out if this trick
28:45worked once, maybe it would work again.
28:48So all she did was take the
28:50supernatant from the examiner palette.
28:53And now she's spun that even harder than
28:57we had spawned things before for 16 hours.
29:01And so we coined the term
29:04super mere because it's
29:05the supernatant of examiners and it also
29:09has really super interesting cargo.
29:12Which. I'm going to tell you about.
29:15So this is just a fluid phase atomic
29:19force microscopy showing that there
29:22were some differences we could detect
29:25between examiners and super meres.
29:28We're in the process of doing
29:30prior OEM of of these a membranous
29:34nanoparticles and that work is underway,
29:37but we were able to show that the
29:40Super mirrors were shorter and
29:42smaller than the examiners and.
29:45Interesting when we took the different
29:48fractions and labeled them IR 800 labeled
29:53and then injected them IP200 micrograms.
29:58IP. And then look 24 hour
30:02later at the biodistribution,
30:04we were able to show that the Super meres
30:09were more enriched in the different
30:14organs and perhaps most notably in
30:18the brain than the other fractions.
30:21And examiners are about 35 nanometers
30:24and super meres are about 25 nanometers.
30:27So I don't think.
30:28That small size is enough to.
30:34To allow for this marked difference in
30:37ability to cross the blood brain barrier
30:40and we've now shown these particles are
30:43taken up in different cells in the brain.
30:47And this is just a quantifying those results.
30:51The other thing that was
30:53really interesting to us?
30:55Was that, you know,
30:56a lot of people are studying
30:58micro RNA's in EB's and there's
31:01a lot of people working on that.
31:04Some people including ourselves
31:06don't think there's all that much
31:09RNA and EB's and access zones.
31:12And what we were able to show in this
31:15study was when we look at total RNA.
31:18We were able to see that most of the
31:21total RNA that was being released
31:23from the cell was in super meres
31:26rather than these two other fraction.
31:29And this just goes to show you that
31:32the small nuclear RNA seemed to be
31:35particularly enriched in the Super meres.
31:38And then in this case,
31:39we did the comprehensive small RNA analysis
31:43and mere 1246 was the most upregulated.
31:49Micro RNA in the Super Myers I should
31:52say that we are 1246 is not a micro RNA.
31:56It's actually processed from
32:00splicing factors R&amp;U 2.1,
32:03but it doesn't mean it
32:05couldn't be a useful biomarker.
32:08And so there was a very nice
32:10editorial in that issue,
32:12nature cell biology trying to now
32:16classify extracellular vesicles with
32:19the lipid bilayer and extracellular
32:22and nanoparticles that include
32:25now super mirrors and examiners
32:28and their associated Carta.
32:30So now I want to delve into some of
32:33the more interesting cargo that we
32:36found in these different fractions.
32:38And remember I tried to set the
32:41stage to tell you that we've
32:44identified in immune exclusion
32:47signature that included deep one,
32:50TGF, beta I and and Dr. one.
32:53And so we could see by principal component
32:57analysis that the and in this case.
33:01We're using Diffie cells again,
33:03but we've done this in other
33:05cell lines as well.
33:06We can see that the small
33:10extracellular vesicles live here.
33:14Not surprising,
33:15the examiners and the non vesicular
33:19material are clustered here
33:22and the Super meres are here.
33:25And what we found was that the
33:31most abundant protein in the small
33:35extracellular vesicles was deep one.
33:38And the work I'm going to tell you
33:41about now is the work of a graduate student,
33:45Sarah Glass.
33:49And this is just to remind you,
33:52this is a vocal logogram and we're
33:55talking now about the sequence of events
33:59in microsatellite stable and positive.
34:04Colon cancer. As contrasted to
34:09microsatellite unstable colon cancer.
34:12And and it was very interesting
34:15to us that when Bert Vogelstein,
34:18some 11 years after that first paper,
34:22he now had all this sage data and he said OK,
34:25how do we decide what genes
34:27are we going to go after?
34:30And so they sat down and said,
34:32OK, gene has to encode either
34:35a membrane or secreted protein.
34:38So it's got to be a target or a
34:41biomarker and it's got to be 20 fold.
34:43Greater in both adenomas and cancers.
34:48And when he did that there were
34:50only 6 genes that they identified.
34:53One was dipeptidase one and
34:56one was a TDF beta induce,
34:59which I'm going to tell you a little
35:01bit more about now, so deep one.
35:04For a long time had thought of being to
35:09be merely an extracellular dipeptidase,
35:13and it's GPI linked.
35:17So it's at the April membrane
35:19of a polarized epithelial cell.
35:23More recently has been found
35:26to have non enzymatic activity
35:28as well as enzymatic activity.
35:30It is expressed in normal kidney,
35:32pancreas and small intestine,
35:34but it's overexpressed
35:35in a number of cancers.
35:37And I'm just going to summarize a
35:39body of work that we've carried out
35:42but haven't published yet and which
35:44we could show that there's increased
35:46standing for deep one and 2527% of
35:51adenomas and that increases to 70.
35:541% in in colorectal cancer and diffuse
35:58staining and colorectal cancer
36:00correlates with a worse performance,
36:03progression free and overall survival.
36:07And importantly it's overexpressed
36:09in microsatellite stable but not
36:12as contrasted to microsatellite
36:15unstable colorectal cancer.
36:17And using fabs we can show that EB's
36:22isolated from the blood of colorectal cancer.
36:24Patients have increased deep one CEA Cam 5.
36:30Compared to healthy individuals and
36:34and this just depicts the enzymatic
36:38activity of of deep one which is
36:41so it's acting extracellularly
36:44and breakdown of glutathione.
36:47So glutathione then is converted
36:50to cysteinyl lysine dipeptide.
36:52Pep one will convert it to cysteine
36:55and glycine which is then thought
36:56to be able to taken up by the
36:59cells and replenish intracellular.
37:01Do the style and it converts Ltd
37:04or to LTE four and this increases
37:09vascular permeability.
37:11And then several years ago it
37:13was shown that in mice,
37:15if they were given LPSS to
37:18activate endothelial cells in
37:21liver and lung endothelial cells,
37:24there is now an increase in deep one and
37:27it was serving as a receptor for neutrophils.
37:32And that really caught our attention
37:36because of a link now between neutrophils
37:40and possibly tumor progression and so.
37:46There is evidence that neutrophils
37:50can also result in immune evasion,
37:55and this is 1 paper where that's
37:58shown where neutrophils are producing
38:01MP nine that's going to activate
38:05latent TGF beta and TGF beta will
38:08impair the activity of CDA T cells
38:12and increase the activity of T Rex.
38:16And and they're actually in the
38:19clinic now drugs that will block
38:21both the enzymatic and non enzymatic
38:23activity of dpep one.
38:25So it's if you have beta lactam
38:29antibodies it turns out deep one
38:32will cleave them and they're half
38:34life is very very short.
38:36So what's done is you give cellar
38:39statin which is a deep one enzymatic
38:42activity inhibitor and then that will
38:45prolong the half life of the antibody.
38:48So it's been used just you know
38:50week 10 days not longer but it's
38:52used and then El Sol peptide which
38:55is actually there's,
38:56it's a 16 amino acid peptide that's
39:00been grown to be able to block the
39:04ability of neutrophils to bind to deep.
39:07And I should say that we don't
39:10know the Libyan in neutrophils
39:13bind that binds to deep one,
39:16but we know that neutrophils bind.
39:19And so this now is used in the
39:21clinic and COVID patients that are
39:24at high risk for either having
39:26lung or kidney problems because
39:29it's going to block the ability
39:32of neutrophils to get there and
39:35it appears to have some efficacy.
39:38So this is just showing now by single
39:40cell RNA seek that we can see that deep
39:45one is relatively up in microsatellite
39:49stable colon cancer and are.
39:55Are adenoma stem cell like cells?
40:00Compared to SSL and
40:03microsatellite instability.
40:05And this is just showing staining now.
40:08So we can see in the normal colon that
40:10there is staining more towards the base
40:13of the **** down in the progenitor zone
40:16and you can see nice staining as you
40:19might expect for this GPI linked protein.
40:23We think it may be a wink targeting,
40:26but we haven't shown that conclusively.
40:28We can see that in adenomas
40:31we see increased standing.
40:33It seems to be restricted.
40:35To the room,
40:37to the apical domain.
40:39And then when we moved to cancer,
40:41we can see either further increase and
40:45then in some cancers we see this I
40:49think what appears to be more diffuse
40:53staining and that's the staining
40:55that when we have K Washington,
40:58our GI pathologist of clinically
41:01well and annotated TMA can show
41:04that that's associated with.
41:06A worse survival, and that's just shown here.
41:14Now we're in a position where
41:16we take abnormal organoids.
41:17And we can show that in this particular case,
41:23we're seeing an adenoma that has deep
41:261 restricted to the apical domain
41:29where it's in this particular organoid.
41:34It looks like there's more diffuse
41:37stain even though this is an abnormal
41:40and and now we have the ability of
41:43placing these organoids on trans
41:46wells and now we'll have a bully.
41:49Polarized. Ordinarily that allows
41:52us the opportunity to now place,
41:57in this case, neutrophils at at the
42:01at the bottom of the transplant.
42:03And now we can look at neutrophil
42:08adenoma interactions and see if
42:10they are any change in the behavior.
42:13These have to be fractionally
42:17isolated neutrophils.
42:18Have a half life of about.
42:216 to 10 hours and they're all
42:25varieties of neutrophils.
42:27As I'm learning.
42:29There's net posis.
42:31They make their own set of
42:34extracellular vesicles.
42:35And then we have our adenomas
42:38with or without deep one,
42:40and we can begin and their vesicles and
42:43we can begin to look at combinations
42:46of of different articles and and look
42:48at some of the genes and biology that.
42:52That are unearthed.
42:54So that's where we stand with the pep
42:58one work in in the extracellular vesicles.
43:02The most abundant protein that we found
43:05in the Super meres was TGF beta I.
43:09And so obviously now we've got two of
43:12the three proteins we're interested
43:15in and that was a interest to us.
43:18So TGF beta I,
43:19it was actually it goes by a number
43:23of different names and Greg Plowman
43:26about 20 years ago added TGF beta
43:29to a 549 lung cancer cells cloned.
43:33Has Gene which he called TGF beta I,
43:36but it probably has little if anything
43:38to do with TGF beta signaling,
43:40at least as far as we can understand
43:43thus far.
43:43It's expressed by epithelial cells
43:46and macrophages and loss of function,
43:48germline loss of function mutations are
43:52associated with corneal dystrophies.
43:54And it's been implicated in
43:57glycolysis past thesis.
43:59Migration and angiogenesis.
44:02We do staining.
44:04We can see that it really it,
44:07it's got a signal peptide and we can
44:11see that it really is enriched in the
44:14stroma of these colorectal cancer cells.
44:18In in vivo and.
44:21When once again Kay store scores the
44:26colorectal cancer TMI I TTF beta I
44:31is associated with worse survival
44:34and we actually now have an ELISA
44:38for TGF beta I and taking plasma and
44:42and this was our first attempts at this.
44:44So I think we can do a better
44:46job of separating the different
44:49fractions but you can see that.
44:53There is a mark enrichment of TGF beta
44:56I in these three colorectal cancer
45:00patients compared to three healthy controls.
45:06So. Now we've accounted for two
45:10of them. And so now we're very
45:13interested in and DDR1 and.
45:19DDR1 is actually a tyrosine kinase.
45:22But rather than having a growth
45:24factor bind, it's activated by column.
45:28So collagen will activate.
45:34DDR1 it's got a PDZ binding motif.
45:39And it's been studied quite a bit
45:42by Ambra Posey at Vanderbilt,
45:44who's found that it plays a
45:47role in kidney fibrosis. Show.
45:54There were two recent nature papers
45:58which found different roles by which
46:02they thought DDR one was working.
46:06The first is from Ron Lee,
46:09an investigator at GW who found that
46:15the shed ectodomain of DDR1 is able
46:20to alter the alignment collagen.
46:24In a way that he's arguing
46:27he's cells we don't know.
46:29Other immune cells are affected as well,
46:32don't get to the action.
46:34And this is in the setting of
46:37triple negative breast cancer.
46:38So that's his model.
46:40And then Michael Karen Year later
46:44comes up with another story.
46:47Which I think is experimentally
46:49flawed but I don't really have
46:51time to go into the reasons.
46:53But he and pancreatic cancer
46:56said that the ectodomain of Dr.
47:00One is not checked in pancreatic cancer.
47:03So now what we're left with OK
47:06is there release of of soluble
47:09ectodomain of of DR1 and so this
47:13is very preliminary data but
47:14we now took our our fractions.
47:17Once again from Diffie cells and
47:20this is overexpression of DDR1 and
47:23Heck 293 cells and then this is
47:26Super Myers isolated from our Diffie
47:28cells and I think you're going to
47:31appreciate there's a large band Dr.
47:35One is glycosylated so but it's
47:39a large band for DR1 in in super.
47:44So that's really what I wanted
47:48to tell you about.
47:50I'm going forward like many in the
47:55field were interested in overcoming
47:57immune exclusion in this case and
48:01microsatellite stable colorectal cancer.
48:05And so we think that DDR1,
48:08TGF data,
48:09ID PEP one are all therapeutic targets.
48:14And our first approach is going to
48:17be to conduct a clinical trial with
48:20a company Parthenon Therapeutics
48:22that is in Boston.
48:25The founders were trained at Vanderbilt,
48:28hence the named Parthenon for
48:30those not interested we have.
48:32A replica of the Parthenon in Nashville,
48:35across the street from Vanderbilt,
48:37so they were favorably disposed from
48:40their experience in at Vanderbilt and
48:42have called the company Parthenon.
48:45And the medical oncologist overlap
48:47with me during my medical oncology
48:50training at Vanderbilt.
48:52So everything's kind of fitting
48:55together here and we're going to try
48:58this neutralizing antibody to see if
49:00it permits return of cytotoxic T cells.
49:03The plan is to do the phase
49:05one and then introduce Contrada
49:08to see if in fact we can get.
49:11T cells to get back to where they need to be.
49:16All three. We have biomarkers 4.
49:21And we can monitor plasma DR1 and TGF beta
49:26and Super mares and deep pep one and EB.
49:30So even though this is an
49:32investigator initiated trial,
49:33we have correlated biomarkers which
49:36will meet the standard of of what
49:39you need for reading or reaching a
49:42translational goal in a sport trial.
49:45And So what I've tried to tell
49:49you about and and very rapid
49:52fashion today is our isolation of
49:55extracellular vesicles and examiners
49:58are discovery of super meres and
50:01and then identification all of these
50:04three proteins that are part of this
50:08gene exclusion signature that that
50:11paper has been submitted to cell.
50:14And we think that we've identified
50:19some tractable targets and
50:22and correlative biomarker.
50:25So obviously this this work.
50:29Couldn't have taken place with with
50:32help from a lot of people I've tried
50:35to highlight Sarah Dennis Chin,
50:38Jim Higginbotham,
50:39Jeff Franklin's a senior member of the
50:43lab Oleg 2 Tonov joined from Siberia.
50:46He left Russia the day after
50:50the exercise in Ukraine.
50:54You know, he's happy to be he
50:57and his wife and in Nashville.
51:00We have both this human tumor
51:03Atlas network and we recently were
51:05awarded a translational and basic
51:08science research in early lesions
51:11and and our project within that is
51:14related to D PEP one and both in the
51:19H10 and T valve can allow Martha
51:22Shrubsole and and Cindy Sears who's
51:25an expert on the microbiome and
51:29colorectal neoplasia at Hopkins.
51:31Are all part of our team and we
51:33have you know tremendous support at
51:36Vanderbilt and and as well elsewhere
51:39and I've been fortunate for the well
51:43funded for the for the time being.
51:46So I'll be happy to answer any questions.
51:57David.
52:19No, really the that that's been done
52:23at Parthenon Therapeutics and it
52:26was only when we recently acquired
52:28this gene exclusion signature
52:32that we were thinking about OK,
52:34which of these proteins do we
52:36want to target and then realized
52:39that actually over a drink with a
52:45colleague that Parthenon Therapeutics
52:47had this neutralizing antibody.
52:49To DDR1 and decided to then
52:52focus on that to start with.
52:58Pardon me? So, so they have data. Right.
53:04That they have data that they think the
53:08effects that they get are independent
53:11of the tyrosine kinase activity,
53:15but that you know, that's their data.
53:17We we haven't repeated that with kinase
53:20dead constructs and and that work is
53:23just really beginning to be carried out
53:25by a graduate student and all that.
53:32How do you get here? These years.
53:38Great question.
53:40Clueless. So these are.
53:44A membranous nanoparticles
53:46they've got a lot of.
53:49Ribosomal components,
53:50they've got a lot of RNA,
53:53they have a lot of RNA binding proteins.
53:58And. I had a a talk today with
54:04somebody who's still awake.
54:10Who just submitted his first R1 after
54:13being up for four days and he gave
54:17me some really great suggestions
54:19for proceeding in that area.
54:22So I'm hoping that we'll be able to
54:25collaborate on on that going forward,
54:27whether it's related to stress,
54:30stress, granules.
54:33Either base separation components
54:35of this with all the RNA there and
54:39some of the positively charged
54:41Eno one is one of the most.
54:44Upregulated proteins we find in
54:46in super mirrors and and that has
54:50a positive charge for him just
54:52hand waving explanations but so
54:54we we really don't know the the
54:57Biogenesis but as some people in
55:00the field have said you know.
55:02One persons, one cells track.
55:06Maybe another sells treasure,
55:09so you know whether this is just junk
55:14being thrown out or whether it has some.
55:20Impact on on recipient sales, we don't.
55:24But we try to proceed, you know,
55:27cautiously and rigorously as we
55:31go forward. Gave. Patrick. So.
55:54Got it.
55:57No, no, that's exactly right.
55:59So and and at the level of light
56:02microscopy we can't see these,
56:04you know that X the Super mirrors
56:07are 25 nanometer examiners are 35
56:10that LED's are you know 80 to 120.
56:17Yeah, distracted.
56:20I don't know, but we're, we're,
56:23we're we're unable to detect
56:25them what with our immuno stain.
56:28Clearly if you have cultured cells
56:30and you have high enough resolution.
56:33You can see these.
56:35And and there are tricks,
56:37you know that you can pH,
56:39Lauren, so it'll light up.
56:43Certain pH. You can just
56:45see things being released.
56:50Yeah.
56:55Right. So that's what we've been doing.
56:56We have very good antibodies.
57:00For. To work carefully with self
57:04taking 94 not one antibody.
57:08And actually. There anybody that
57:10can work very well overtime another.
57:13Yeah and you know less than 12 says
57:17that antibodies PSA I think you're OK
57:20and ER one well once again that cells.
57:26OK, great.
57:30Question about. Yeah. In those.
57:40Yeah. We get. Would go in.
57:45Target Fairmount go now is
57:47what's really interesting.
57:48So we can now we flow sort. You've got.
57:54Now we're close sort, EGFR and TECHSPAN.
57:58And DDR1 by itself,
58:00but a number of other. April.
58:05GPI links like those proteins.
58:08And including CD 73.
58:10So we think that's interesting.
58:13I mean, this is very speculative,
58:16but you might have a dual warhead.
58:18That in that they're being released by a
58:21cancer cell and that local environment.
58:24So you've got.
58:25Now keep one that's gonna.
58:28It's a team and it can be a chemoattractant
58:31and all this after for neutrophils and
58:34mild mild cycle now more recently.
58:37OK, going to bring those in,
58:39but that doesn't exclude detail.
58:42We've got CD73 making Dennis,
58:46which was going to begin suppressive.
58:48So you know churning out studying.
58:51Now we're looking to see if
58:53they can see that predicted.
58:57Presence of neutrophils in the
59:00absence of of CDA sets and there
59:03is a correlation we think,
59:05between deep 1 staining and
59:08we use neutrophil elastase.
59:11Now,
59:11probably HNE probably is is
59:14probably even better than that,
59:17because mutual elastase can
59:18be produced by others.
59:22Yeah, V6. Yeah. But even better,
59:28maybe just. With the ballot.
59:35Yeah.
59:38Question.
59:43Other than.
59:48What I'm thinking?
59:52Face. Face. Here.
59:59So I showed the data for.
01:00:04So we we can exercise.
01:00:08Cancel pensions. You don't like.
01:00:13Detective and all of it. It it is.
01:00:21Thank you. We need to refine our plasma.
01:00:28So yeah, you can measure a cargo in superior.
01:00:43Good question, you mentioned.
01:00:50This.
01:00:55You know, people are starting
01:00:59to use these for drug delivery.
01:01:05And but we and there's a lot of
01:01:08activity in that space right
01:01:10now we really haven't undertaken
01:01:13those experiments we're we're
01:01:16we're more inclined towards you
01:01:18know looking them as biomarkers
01:01:21and therapeutic targets and.
01:01:26Not so much that therapeutic
01:01:28opportunities I think it's it would
01:01:30be it's still a real challenge.
01:01:32You know what cell is gonna be your producer
01:01:36cell and what other cargo might be there.
01:01:41And you know, this field has been tarnished,
01:01:43I think, by a lot of.
01:01:46Extravagant claims that then
01:01:48haven't been able to be reproduced
01:01:51and and we're also as a field,
01:01:54have been giving.
01:01:56Pharmacological industrial doses
01:01:59of of these EB's into into mice
01:02:04and claiming we're seeing a real
01:02:07biological effect so you know it's
01:02:11there's a note of caution here.
01:02:14As the field moves forward.
01:02:22On red cells and. It doesn't.
01:02:29I mean you know platelets are huge
01:02:32producer of abuse and so we're
01:02:35very careful in all of our studies.
01:02:38We're dumbing it down you know,
01:02:40so I can hope to understand what's
01:02:43going on but we our extraction
01:02:47process excludes platelets,
01:02:49but that's that's a another area
01:02:53everybody makes every cell it's making EB.
01:02:57Grapefruit, there are people in the
01:03:00consortium that are studying the the
01:03:03release of EV's from grapefruit and the
01:03:07biological effects that that that has.
01:03:10So in the next time you know you're
01:03:13eating a grapefruit or an orange.
01:03:16Think about all those EV's that are
01:03:20being released and thinking about,
01:03:22you know, what is the consequence of that.