so um I celebrate and acknowledge the traditional owners and custardians on the land on which we meet today the Warren jury people of the cool Nation I pay my respects to their Elders past and present and all Aboriginal and Torres Strait Islander peoples from other communities who may be here today uh and so I'll introduce Thomas Thomas is a cancer cell biologist working in the field of the extracellular Matrix and ECM remodeling in the progression and metastasis of solid tumors he graduated with a PhD from the University of Durham UK in 2008 followed by postdoctoral research positions at The Institute of cancer research in London UK and Copenhagen University Denmark uh in 2016 Thomas was recruited to the Garden where he currently leads the Matrix and metastas's lab the lab's creative research program integrates Matrix Biology with Precision oncology aligning with the Garvin strategic Vision to make fundamental advances in the understanding treatment and prevention of human disease in this case personalized stormal targeting in solid tumors Thomas takes an active role in training emerging scientists honors and PhD students and their projects and Thomas is the president of the Matrix Biology Society of Australia in New Zealand as well as director of communications and an executive board member of the international metastasis research society and Thomas is also a co-founder of the Australian pancreatic cancer Matrix Atlas so thank you very much for being here Thomas and yeah please take it away great uh thanks Somali for the introduction and um also just thank you to the organizers for giving me this opportunity to come and talk to you today um and and to give you some insight into uh the tumor micro environment so as mentioned I'm based at the Garden Institute here in Sydney and have been now for about five and a half years or so and so my lab is interested in really how the extracellular Matrix which is sort of a key component of that tumor microenvironment really feeds into the initiation development progression of solid tumors and also how it affects your response to therapy and so as my sort of I guess you know remit for today is to really go through with you guys what is the tumor micro environment what are some of the more most important components of the tumor microenvironment um and given the fact that most of my research is based around the extracellular Matrix and what it is and also what role that plays in the tumor micro environment I'm going to talk a little bit about some of the ways which we've been developing to study the extracellular Matrix in tumors and then touch on why the extracellular Matrix is important in cancer research and also treatment I mean if we get time at the end what I'm going to do is run quickly through a research example of one of our recent Publications uh looking at cancer cell Matrix interactions in pancreatic cancer so what is that tumor microenvironment and what is it made up of well I'm sure you're aware that tumors are complex ecosystems they are made up of more than just cancer cells but what's important is it's not just cells that there are acellular components of a tumor as well and collectively all these things together are known as the tumor microenvironment and so what I've just shown you on the right hand side here is sort of a stylized diagram of a tumor now you don't need to worry too much about some of the boxes around but what I'm using this is an example to highlight how you have tumor cells in this case these are pancreatic diplomacarcinoma or pancreatic cancer cells shown in the middle but these are essentially surrounded by fibroblasts immune cells of many different flavors B cells T cells macrophages and so on uh you so these are all depicted here at what's known as extracellular Matrix these are these little gray lines that you'll see around you have presence of blood vessels uh within tumors and an important factor as well is nutrients and other factors such as uh growth factor like tgfbs are shown here um but also things such as hypoxia and collectively that is considered to be the tumor now whilst the malignant part of that tumor is the cancer cells themselves they typically are the ones with the genetic mutations that are driving them to be malignant the whole entity itself should be considered a tumor and so a tumor doesn't just encapsulate these sort of central regions showing here of cancer cells but actually extend much larger than that and that's something I'll touch on a little bit later is that actually in some tumors cancer cells may make up less than 50 percent of the mass of a particular tumor so my lab is important interested in studying The extracellular Matrix component of that tumor microenvironment so what is the extracellular Matrix and for those of you who are aware indulge me a little um whilst I explain to those who perhaps haven't come across so we know that cells are that basic building block of life but how they come together to form tissues and ultimately organs is through interacting with and essentially uh with the extracellular Matrix and this is depicted here in the center a lovely green gfp labeled cell here and you'll see these uh sort of fibers now I'm illustrating on the right hand side here is with this video um uh so this just to orientate you this is a um uh intravital Imaging so this is um second harmonic generation Imaging inside a mouse the purple strands that you can see are collagen one fibers collagen being the major Matrix component within our bodies the green are actually neutrophils that have been labeled with a green fluorescent protein so this is this itself is not a cancer but I'm using this video to emphasize a couple of points the first is that if you watch these neutrophils as they move along these fibers you'll see that their migration is you know essentially controlled by the presence of the fibers they move along the fibers they squeeze between the fibers so we have a direction of communication in which the Matrix is altering cell Behavior but if you continue to watch the movie you'll see that some of these green cells are essentially altering those purple strands those collagen 1 fibers and you this uh here you'll see them moving them aside in some places degrading them and so this speaks to a concept we call Dynamic reciprocity the idea that the extracellular Matrix influences cell Behavior but that cells can also change that Matrix which may go on to have further effects on Cell behavior and that idea of dynamic reciprocity is something I'll touch on a couple of times and also something to keep in mind so we talk about many solid tumors being what's known as highly stromal stromal being another way of saying highly fibrotic desmoplastic or essentially just containing a lot of extracellular Matrix and non-tune apart or non-tumor cells I should say and so what I'm showing on the left hand side here this is a h e section so this is um I say this is a histology section stained with a dye called picker series red this binds to collagens and essentially using a very clever trick of the light uh known as um polarizing light microscopy you can look at the birefringent signal that's generated when this dye binds to collagen and so what this essentially allows you to do is to look at the fibular organization and bundling of collagen now the reason I'm showing you this is to highlight how we have you can see this sort of pseudo green and yellow sort of pseudo fluorescent image to show you um what this extra cellular matrix looks like around this tumor this is a pancreatic tumor and you can see that in this case we've got this very dense fibrotic capsule forming around the outside that also appears to penetrate into the center of the tumor however it's not just um this matrix it's not just form at the edge of tumors and using this example on the right hand side of breast cancer also stained with pick or serous red we can see that actually The Matrix also penetrates deeply within tumors and so we have both this extra or sort of um sort of invasive Edge capsule but we often also see a lot of Matrix Within These tumors and this is important because in some cases and some Cancers and pancreatic cancer is a good example up to 50 of the mass of the tumor can be this extracellular Matrix and so it really just shows that we shouldn't just be thinking about cancer cells in isolation when we study tumors now I'm sure most of you are aware of the different stages of cancer progression and metastasis and I just want to use this slide really to emphasize one point which is that the Matrix and the tumor microenvironment as a whole really plays a key role not just the primary tumors where we might have Invasion into or away from tumors and through interstitial tissues towards blood vessels but also in secondary sites metastatic sites where we know that the Matrix and the tumor microenvironment as a whole is important in the colonization of secondary tissues and also in dormancy which is a very important feature and some solid tumors but at all of these stages it's this sort of cell intrinsic properties of the tumor cell so mutations which these tumor cells have and that interactions with the cell extrinsic properties of the tumor micro environment that really control uh progression so when we think about the stroma the The Matrix the tumor microenvironment in solid tumors uh we I just want to make the point is that every tissue in our body contains a matrix either soluble or insoluble and I say that because if we think about blood even as a liquid it contains a huge amount of soluble Matrix components which the minute you cut yourself begin clotting and that's exactly what it's meant to do and so this Matrix is critical for the correct tissue function but what happens is this it gets dramatically changed in cancer and just on the right hand side you'll be able to see some of the different characteristics of the Matrix that are important composition in terms of biochemistry binding of growth factors hydration cross-linking stiffness which I'll touch on shortly um which obviously in the biomechanical properties things like density the actual Ultra structure even degradation and turnover and the availability of interaction ligands all play critical roles in normal tissues and are typically disrupted in cancer and so again it goes back to this point of this Dynamic reciprocity between cell intrinsic and cell extrinsic um properties and just to highlight this a little bit um what I'm sure going to show you here on the right hand side so this is collagen one this is um imaged by a second harmonic generation Imaging this is a multi-photon microscopy approach that allows you to image collagen one fibers without the need to stain them and what you can see this is normal healthy fat pad uh you see this rather sort of curly um sort of collagen appearance and on the right hand side you'll see the collagen one structure as tumors are growing and so we can immediately see that there is a huge difference in the way that the collagen is organized between healthy tissue and a growing tumor and so we know that these changes really sort of Mark either transitional events in the progression of cancer or play a key role in regulating things such as response to therapy however what we also know is that there's sometimes similarity and so what I'm showing in the bottom two images is this is collagen one in the normal lung it's getting got this sort of curly wavy appearance this is actually um in part due to the ability of the lung to expand and contract during breathing but in a developing lung metastasis we see a reorganization of that collagen one structure and if you look between the top right and bottom right images you'll see that there is a huge similarity and what this really says to us is that there are likely to be underlying remodeling events within the tumor microenvironment at both primary and secondary tumors that may be similar and if we can understand both these similarities and differences can we begin to think about therapies that might co-target these remodeling programs in order to improve outcome and treat both primary and metastatic disease now for those who are interested in this how this architecture and structure and three-dimensional organization changes and I just want to point you to a recent paper which just came out in Matrix Biology plus um by uh Raphael Alejandro and Janine this talks about what we know as the matry texture The Matrix architecture and so it's worth a read for those who are interested in understanding a little bit more about how that architecture is being studied but also important in tumors well something I just wanted to highlight and and was that one of the biggest changes associated with remodeling of the Matrix in the tumor micro environment is changing stiffness of the tumor and we all know or at least anecdotally know that tumors are stiffer than normal tissue and many years ago now and some great work by uh Valerie Weaver's lab and colleagues showed that as um transformation events occur and the Matrix begins to be remodeled around a developing tumor as you can see here going from this curly appearance to these sort of straight linearized fibers is that we see an increase in stiffness and this stiffness is known as from a biomechanical perspective to feed back onto many of the mechano transductive transduction pathways within the cell but also we look at it the other way and think well Matrix might be modeling and has actually been exploited for diagnostics for many many years now as I mentioned one of the first indicators of uh the developing tumor is often Palpatine and the feeling of a hard lump within tissue and so that's really actually what uh elastograms as this is a media letter a big mammogram and on the left is an elastogram both are essentially uh highlighting or picking up changes in density in organization of the extracellular Matrix and as a result of the developing tumors no it's not to say that it's only the Matrix which contributes to these Recaps but the Matrix and the tumor microbiome is a significant indicator so not only is this a way to diagnose tumors but remembering back to the previous slide it also subsequently activates many of the pathways known to make cancer cells more aggressive so how does the tumor Matrix Drive disease progression so we know that it Alters cancer cell Behavior to make them more aggressive I've mentioned mechanic transduction and activation of Pathways but also deposition of new or reorganized metrics combine other cell surface receptors and activate cells uh intracellular signaling Pathways that may normally be switched off during normal tissue homeostasis importantly it doesn't just act on cancer cells and we often see significant reprogramming of what we call stromal cells and examples being fibroblasts um either recruited or local within the tissue immune cells especially there's a lot of work going on at the moment looking at how for example collagen receptors or other Matrix receptors are immune cells might alter either activation or exhaustion and I know I believe you have an immunotherapy time session coming up later in this course we know that it can both directly and indirectly alter cancer cell sensitivity um to therapies and I'll touch on some of our work on that toward the end of this lecture importantly it can also act as a physical barrier to the delivery of our therapies I.E so this capsule or this sort of fibrotic wall almost that you can see that encapsulates tumors okay prevent chemotherapy from being able to enter into the tumors I also mentioned that we see increases in stiffness we see changes in intratumor pressure which can cause blood vessel collapse as a result of Matrix Remodeling and all of these can act to physically stop can add chemotherapy from entering into the tumors and the other thing which has not been touched on yet is that it can provide a physical Highway for cancer cells to spread or metastasize to other parts of the body and so these dense and thick collagen fibers don't always Orient in this way that you can see here this looks like a kind of a circumferential um organization in some tissues such as breast cancer we actually see radially orientated uh Matrix fibers which known as tumor Associated collagen signatures or tax if you're interested in looking that up these are known to also be indicative of poor outcome and metastasis in patients and so not only does it provide as I say biochemical cues but it can also provide physical um routes of dissemination now just want to touch briefly on really what you can see is the key architects of the tumor microenvironment in terms of the extracellular Matrix and that would be the cancer-associated fibroblasts these are the ones that produce significantly produce large amounts of extra cell in a matrix cancer cells I will say cancer cells do produce extracellular Matrix however they tend to be in a lower overall quantity that said they do also tend to secrete let's say more exotic or unusual Matrix components those that you typically wouldn't see within the tumor and that's important because a lot of these cancer secreted Matrix molecules actually act as scaffolding molecules to change the organization of other Matrix molecules and so this sort of combination of large amounts as well as changed organization is really what underpins the importance of The Matrix as I briefly alluded to earlier this sort of Matrix remodeling occurs both the primary tumor and secondary sites and is driven by a number of factors typically secreted by cancer cells but also in some cases immune cells and this as I mentioned before feedback to Dynamic reciprocity there it is again that term uh which really just sums up this intracellular extracellular uh communication and co-regulation and so really the premise of a lot of these approaches is that if we can Target that deposition or remodeling of the tumor Matrix and we find a way to break that cycle and ultimately improve treatment and patient outcome so what I want to do over the next sort of maybe 10 minutes or so is just talk about how we study some uh The extracellular Matrix in some different tumors and then talk about the role that it might play so over the years um there's been a lot of tissue clearing protocols being produced uh this slide is a little bit out of date now and there are certainly a lot more um than just those ones uh showing up here and essentially these have all been designed um to enable you to image whole organs without the need to section in 3D in fact I've seen recent ones where whole animals are on now cleared and made transparent and this has been fantastic it allows image very deep into tissues and really map cells within that three-dimensional context however most of these approaches in fact almost all of them are designed to improve Imaging of the cells and were not designed to image the extra cellular matrix in fact many of them actually involve partial digestion steps where they degrade The Matrix to allow tissues to swell in order to help improve resolution and so a couple of years ago now um whilst actually while I was still in Copenhagen and doing my postdoc um a couple of post-ops got together and said Well there must be a better way to do this for extracellular Matrix and so we came up with a way to decellularize tissues so we remove the cells using a vascular shunting approach and so these images here are really just representative of the different surgical procedures that you can use to use animal vasculature to essentially decellularize tissues from the inside and and so what's more important is showing you these images and so what you can see this is over time is that the shunting of vascular tissue decelerization agents through the lung tissue which is what we're looking at here leads to that clearing of or removal of cells um and so on the right hand side here this is after 18 hours this is decentralized lung tissue and so this paper was published in 2017 for anybody who does want to go and look a little bit more into it um but I will I'll talk you through some of the parts of it um in the next few slides importantly we showed that this our decellularization approach doesn't affect Matrix Integrity but is really good at improving Imaging proteomic cataloging of the Matrix in that three-dimensional structure and spatial organization and so here are some examples of this here um these are dark field microscopy images just for those of you who aren't familiar um what uh so there's no fancy microscopy here as such but what you're looking at is uh tissues so this is my breast cancer models and on the right hand side are pancreatic cancer models and you'll see that we've got various tissues liver lung hopefully you can appreciate is um that the structure of these tissues is still fully intact many of them are still clearly um identifiable but there are no cells in any of these images so this is a lobe of the lung part of the liver again liver over here in fact you can see the fenestrations of the liver Within These tissues here and so this just really emphasizes just how much Matrix you have in tissues and how important that's going to be in controlling and regulating cell Behavior here's just another example um so this is the pancreas this is a spleen and one of our Mouse models um they can be stained for your favorite uh ECM molecule this is an example of collagen four and you hope you can see this sort of grape like or ductal structure of the pancreas here on the right um in green and this can be imaged in hormones so we don't need to section and process these the other thing is because it's hormone and there's no need to section is that we can look at the The extracellular Matrix organization over scales of magnitude so this image itself is a tiled image of the mammary gland um staying for collagen four which is important basement membrane protein and shg which is collagen one some of the same as the images I show in some of the earlier slides the scale on here is 500 microns so these images are about three millimeters by about five millimeters which is for those familiar with microscopy huge field of view and what hopefully you can see is that this this is normal tissue this sort of again grape-like ductile structure these are the mammary gland uh or the thermal end Buds where all your milk gets produced um supported by a collagen one sort of fibrous Matrix and so what we can do is we can begin to zoom in into some of these regions and so what we can do is zoom right in and so what you're looking at here this is collagen 4 still these little great white structures of it as I said the milk duct but what you'll see these sort of um vessels that are snaking over the outside these are the capillaries that sit on the outside of those mammary ducts and so you can see that once we've removed all the cells even deep within tissue we can begin to map that three-dimensional organization now we can do this in our tumor models quite nicely um and so that's one of the key ways we actually begin to understand what's happening in the tumor microenvironment and how it's important and you can see so this is a decellularized uh lung and so there's no cells here present again um this has got developing lung metastases in it you can see uh it really doesn't take an expert to see that there's a region up here in the top left with a big white arrow pointing at it and also down here in the sort of Center where the Matrix looks very very very different it's very dense and opaque in this image and so we can begin to zoom in and we can begin to look at this this is just shg so collagen one uh looking at how the extracellular Matrix within that tumor micro environment appears in relation to that matched next to it right adjacent to that developing moment and if you remember those images from one of the first slides I showed you when I talked about the differences uh this is exactly where they came from from a 3D decellularized lung metastasis but that's not all because one of the key things we're interested is understanding not just what happens inside a tumor and so this is collagen 4 staining um within a developing lung metastasis you can see this sort of very fragmented um organization and also what happens in the normal lungs so we can see this is the normal alveolar structure um of the lung staying for collagen 4. but what we like to call transition zones that region where tumor essentially meets host and you should hopefully be able to see that there's a very big difference As you move from inside the metastasis through this transition zone the collagen four changes organization becomes very dense very disordered and moves out into normal lung tissue what you'll also see is the uh beginning to say hopefully is this white dense collagen Matrix being deposited and if you remember back to one of the earlier images uh showing that you often get this encapsulation and this is important because obviously the tumor micro environment here the Matrix will give the tumor cells inside the metastasis one set of instructions but as they move through this either the primary tumor or in metastatic sites into these transition zones you're likely to be encountering a completely different set of extrinsic excuse and if they break free and or migrate into normal tissue again that may be a third set and so we're interested in understanding how do these different regions and especially if things such as these invasive edges feedback into progression we also like to do make lots of pretty pictures as well and so this is just an example of how we're creating three-dimensional reconstructions of multiple Matrix components and so what you'll begin to appreciate with just four ECM components shown on this right hand image is just how dense that extracellular Matrix is now we don't just do this in Mouse models and we actually got a program in the lab where we're doing this in two human tissues and working with a number of different collaborators um and where we can take tissues he's a breast cancer example take tissue post surgery this is just an example of normal tissue shown here in blue you can see these are the this is the ductile structure of the memory tissue the purple is the collagen one um again inside the tumor of that matched patient you'll see that there's a significant loss for example of collagen for organization so again that Matrix has been heavily remodeled Gmail and we're also doing this actually with in pancreatic cancer models and we launched the Australian pancreatic cancer Matrix Atlas a few years ago now um where we're actually collecting patient samples from those undergoing surgery and again just look at this schematic in the middle looking at the importance of how the ECM changes between the sort of marginal invasive edges of the tumors versus the core tumor region and also a fairly revealing so those are just an example of how we're also able to apply many of these Imaging techniques to Patient material as well as to animal models the other thing we can do which is nice which is um and uh it really allows us to study the interaction uh deep inside tissues is re-cellularization so once we've removed the cells we can then introduce back in cells and this is just a very quick example showing that we can um reseed tumor cells back into uh by the tissues such as the lung or back into decellularized tumors and begin to ask how they interact with the Matrix and image in real time and that's ongoing work actually not by a number of labs around the world are now using these approaches to really unpick what it is the Matrix is doing to the cells one of the last things I want to show you in terms of the Imaging um is that we can also do imaging of the Matrix in real time in intravitally so inside live and Living Mice And this is work has been pioneered by a number of individuals um Paul timson here at the Garden Adrian Hawkins down um obviously we hire which many of you may know and up here at Garden what they use is these titanium window imagings which looks like a small washing machine window I guess which is essentially put into the abdomen of mice that allows you to essentially image the organs underneath this can be done for for weeks uh on end it allows you to go back to the same spot in either an organ or a tissue or a developing tumor and look at what might be happening and eyes are perfectly fine with this hopefully you'll see this video running and there's a little window on the mouse and this is courtesy of Paul timson's lab um and you'll see that this allows to pop this Imaging window on the microscope and can we use it many times and what that allows us to do is to look at how the Matrix is purple again is that collagen one Matrix how that might change over time as a tumor grows or in response to a particular therapy and if you combine that with fluorescently labeled tumor cells or immune cells or whatever cell you're interested in you'll be able to actually image those interactions in real time as well so one of the other things that we like to do um and this actually has been facilitated significantly by decabularization Technologies is to catalog that Matrix and so one of the important things we do is proteomics based Mass spectrometry analysis um and I'm not going to spend too much time on that but what I want to just say is that what it allows us to do by removing the cells is really make in-depth analysis of how that Matrix is changing um within healthy tissues versus tumors metaphatic sites versus primary tumors and that then gives us a number of leads that we can then begin to dissect mechanistically as to the role that they might be playing one of the things that we obviously don't get when we use proteomics is that we sort of blend everything up into a bit of a smoothie for one of a better word and then catalog what's in there what we lose is that spatial information and so recently we've been working on mass spectrometry Imaging as a new way to study the Matrix in cancer and for those of you who aren't familiar Mass Spec Imaging is essentially capable of Imaging a number of different small molecules for example proteins lipids metabolites but it uh it maintained well and maintains the spatial information and so very briefly essentially it involves uh putting a section onto a special slide uh blasting it with a laser to ionize um the various uh analytics you're interested in and then running mass spectrometry at each of those individual coordinates this creates essentially a series of mass spectrums across your tissue and then allows you to create maps of the distribution of those specific analytes As you move across the tumor or any tissue of Interest now what we've been doing recently is to develop this um and through we recently published um a paper on a new technique called hitmap which allows us to begin mapping um tumor microenvironment Matrix within so this is a h e of breast cancer you'll see this H in here in the bottom uh left hand side of the slide here it's fairly homogeneous it's pretty much all purple but once we begin to Overlay the different Matrix spatial maps you can see that they had a very very marked heterogeneity and we can begin to look at how they particular ECM components might you know relate to one another and how they overlap don't forget the Matrix you know individual components never operate in isolation they're always part of larger super molecular structures and so we're often interested in finding out what else is sort of co-regulating co-regulated with particular Matrix components what we're also doing is generating segmentation patterns um and so again this is just another breast tumor this is a h e you'll see maybe you might be able to identify a little bit of histology within this if you've got a good eye but by looking at how Matrix signatures specifically cluster within the tumor we can begin and so that's what this Center image is showing you and this is five clusters where Matrix signatures most closely match one another and so you can begin to look at how heterogeneity within a seemingly otherwise homogeneous tumor might be important and actually we believe that a lot of this microenvironmental heterogeneity underlies and feeds into a lot of the cellular heterogeneity that we're seeing come out at the moment and so one of the most sort of I guess exciting things that we're doing or trying to the moment and this is working actually very closely with Alex swarbrick here at the Garden is to integrate our spatial transcriptomics programs where we can spatially map micro-environmental changes back to transcriptomic programs and look at how cells are changing transcriptional programs and that's actually quite important because this allows us to really ask the question of well what happens in the microenvironment and how is that sensed and responded to within the cells present within that region be it cancer cells immune cells or other so what I want to do really now is sort of try and run through why is the Matrix important and and and really highlight some of the work that we've been doing and so I've probably alluded to already that patients uh often don't respond to current therapies and that's mainly because tumor cells are very good at finding ways to escape but that includes the generation of these protective niches and and the creation of tumor micro environments that really blunt the efficacy of therapy and so we now accept that it's really going to be more powerful to Target multiple elements of that tumor microenvironment either together or sequentially so by essentially taking away or you know removing those support systems of the tumor such as the Matrix we may actually be able to augment if this efficacy has already approved standard care therapy the other thing I just want to highlight is that we can use Matrix signatures to help stage cancer so just as we've seen large amounts of molecular phenotyping coming out we also argue that you can now or we should also use a matrix stratification approaches to complement that and this is just an example we're showing here uh purple on this is that collagen one again we can see we can very easily have patients with high medium and low levels of collagen one within those tissues and actually given the longevity of a lot of Matrix components most of most of the ECM components in your body last for days weeks maybe even months in fact some of the collagens in your body you're born with and you will die with so this is sort of a historical record of what's happened during tumor development and so if we can really tease out both the biological and mechanistic sort of understanding of what this is doing and it'll allow us to begin to sort of add subtype certain tumors the other thing I will just say is that in almost every tumor biopsy varies extracellular Matrix in fact in some tumor biopsies there's only Matrix and no cells and historically Pathologists have been unable to score tumors if there aren't any cells and so we know that there are millions of archived bits of tissue out there that we can go back to and so not only can we image with developing spatial proteomics approaches that we can apply to tissue microarrays as well okay so how are we doing for time what I'm going to do in the last 10 minutes or so is just talk about a research example of some of the work that we published a couple of years ago right um looking at that cancer cell Matrix feedback uh in pancreatic cancer and so this again was mentioned it's published in 2019 in nature Communications um for those of you who find this interesting I would suggest you go and have a look over the paper um really I'm going to give you the the sort of the highlights and the punch line but there's an enormous amount of work that um really advise you to check out the paper um and so this this work started around um the idea of trying to understand um the role that p53 played in pancreatic cancer and so we have two mouse genetically engineered Mouse models um so we know all those p53 is one of the major oncogenes but there are two types of p53 aberration one is a loss of p53 um and our Mouse model uh is the is a flux model this um which is represented here in Blue by the KP FLC and this is loss of p53 the other is a gain of function mutation um in p53 so they both have the same K res mutation they have different p53 mutations or loss and everything else in theory remains identical between the two moles what we're interested in is is that these loss of p53 models are highly tumorgenic but they're very poorly metastatic whereas the p53 gain of function are highly metastatic so highly tumorogenic but also highly metastatic and so most people attribute this to the p53 that either loss of function or gain a function but what we noticed when just looking at the tumors was that in the loss of p53 model we have a much lower stromal Matrix component in that tumor micro environment than we do in the gain a function as you can see here in these pictures red images there's so many reason that we'll maybe there's something in the extracellular Matrix that's feeding back along with that p53 difference that might be important and so to test this what we did is we took mice mice from both these models we are isolated by tumors and we took the counts Associated fibroblasts the cats the known sort of Architects of Matrix remodeling in tumors and matched KPC cancer cells so we have cancer cells and cancer Associated fibrous from each model what we then did and this is kind of a long story short is a number of proteomic characterizations of exactly what it is that these KPC caths are secreting what is it that's pumping out into the micro environment or not and um that might be in some way playing a role and so what we uh did as this is just shown here with this heat map this is um sort of highlighting a number of differences between what we know is to call as the matrixome matrixome here being the proteome of The Matrix um and one of the ones that struck our um was essentially this protein that we saw called headphones like proteoglycan 2 or perlican and for sure um and this is very highly conserved um protein it's incredibly important in binding to not only growth factors but other Matrix molecules and Scaffolding that extracellular Matrix and so it controls organization assembly integrity and cell interaction and so this is created in very very high levels in the p53 gain of function tumors compared to the loss so again coming to the to really the punch line we did a number of experiments uh to manipulate this perlican within those calves and then looking at co-implantation studies and how that might affect the micro environment and the one of the take-home messages is that depletion of perlican in the calves only uh leads to changes in local Invasion uh primary tumor and so this is the The Fox and the mutant model just shown here but knock out our Perler canning calfs only and so this actually made us realize that well actually just by knocking out one Matrix component from the calves in these Tunes was enough to begin affecting invasion and it also leads to a significant decrease in metastasis to the liver now what I didn't say on the previous slide was I just want to iterate here is that actually the calves would have started that life as normal pancreatic fibroblasts or pancreatic is still Aid cells so the only difference between the calves is that the tumor cells that were that grow and develop within the pancreas have a different p53 mutation or loss the calfs don't have this so what's happened is that the cancer cells have re-educated in some way the local fibroblasts during the onset of that tumor to create either mutant p53 or loss of p53 educated caps basically what this is then arguing is that that education process and then the subsequent secretion of the high levels of permacan might be a strong um or a key factor in controlling the metastatic potential of the p53 Newman cast I say cancer cells the other thing we're interested in doing was to just check at how depleting power can might decrease um or might affect chemotherapy um so this is just the time to metastasis graph from the previous uh slide and this overall changes in survivals this is say just knocking out Perla canning the cancer Associated fibers but we find that in by adding chemotherapy to our knockdown compared to the wild type so we're we're looking at purple is the cancer cells co-implanted with normal wild type cats if we knock out uh so if we add chemotherapy so purple to Green we get this small Improvement in survival in the context of a knockout of perlican in the tumor microenvironment chemotherapy appears to be even better and extends survival and I'm not going to go into this but you can certainly read the paper we showed that this was through um the presence of powder can delays the response to Gem cytopene and practicing which is the standards of care chemo and allows cancer cells to essentially escape the genotoxic effects so just to sum that up um as I said there's a very large paper um is that um p53 mutant cancer cells and I didn't talk about this but through intracellular Pathways regarding um NF Kappa B signaling to create high levels of Tina Alpha which is a known activator of fiberglass to educate calfs within that local environment and we call these mutant educated calfs coming from the p53 mutant uh cancer cell tumors this triggers these casts to secrete noise levels of Perler can into the extracellular space into that tumor micro environment and that presence of parallel account is enough to generate both a pro-invasive and chemo-protective Niche therefore blocking parallel can and so we showed this with genetic manipulation and we're now following this up with domain blocking um antibodies um is likely to lead to the loss of this generation of pro-invasive nation and chemo protective niche so just to start summarizing everything and bringing it all to close and what we know that is as tumors grow um if left untreated we see a large deposition and remodeling of the extracellular Matrix recruitment of actuated sternal cells or calves and this leads to progression and ultimately dissemination now a few years ago there actually a couple of papers out of Ragu kalu Ben Steiner's lab in the US actually showed that if you you know I'm arguing the tumor Matrix is bad so they put forward a couple of papers that showed that if you completely remove the tumor Matrix that tumor stroma so you've completely changed the tumor microenvironment and this is blocking the ability of the fibroblasts to essentially create this Matrix what happened was that the tumors grew faster and spread wildly around the body so that actually suggested that well hey whoa hang on a minute maybe there are some elements of that Matrix that are restraining the tumor and that we can't just completely ablate it incidentally what was really interesting was that once if you did do this then immunotherapy immune cell access into the tumors was significantly enhanced and that it's likely that that would increase the efficacy of immunotherapy so the argument now is that the thinking and the field is that if we can normalize or re-engineer the Matrix rather than completely ablate and remove it so return that tumor micro environment back to a more normal looking micro environment in combination with tumatology therapies then we will likely be able to remove those protein reject support systems and ultimately improve efficacy and outcome so last slide I promise um just to summarize all of that uh The Matrix is everywhere it is all around us even now In This Very Room I'd say that as a joke but I also say it seriously you cannot think about and study cells in isolation in any disease you work in you should always be thinking about the three-dimensional context in which cells exist in and in particular that scaffold that you know Matrix which really acts as a Nexus of communication between all cells present within tissues healthy or diseased hopefully I've convinced you the homeostasis and remodeling are really important and not just normal tissue homeostasis but uh also in tumors and they are an instrumental part of the tumor micro environment I've shown you some inches some cool Imaging Technologies that we're using now to try and study the importance of the Matrix and how it changes in diseases such as cancer and also that there's a huge amount of unexplored targets biomarkers prognostic indicators that we're only just beginning to tap into and this can involve repurposing old drugs for example anti-fibratic compounds or Target generating new drugs or even targeting Downstream effectors of cell Matrix interactions blocking the ability of cancer cells calves to see and respond to proteomogenic Matrix cues as I mentioned we might be able to start using Matrix signatures to further stratify patients who might benefit from additional stromal targeting therapies and that by normalizing that Matrix we can hopefully improve the accuracy efficacy of current standards of care Therapies so with that I think I'll finish um and I would love to take some questions thanks very much Thomas that was such an interesting and informative talk um I I know I've never really considered the um ECM components as therapeutic targets and so I'll definitely be reading that nature comes paper after this that was cool to hear about uh and so we'll take questions from the audience please put your hands up um and ask a question here or pop your questions in the chat and I'll read them out um so I know we have students who have volunteered to ask questions today as well and uh while we're doing the Q a I'll just remind everyone to respond to the poll Kerry has shared in the chat as well about your preferred format for week high training workshops uh and while we're waiting for questions I'm happy to kick us off um I was thinking about your uh using the Matrix stratification as a um uh method to Stage cancers I thought that was very interesting um is there a correlation between Matrix structure and patient outcomes can you use that um ECM structure of you know patient biopsies as a prognostic tool as well yeah um that's a really great question and I sort of alluded to it a little bit when I was talking about the tax signature which is um from uh pakily I was the original uh proponent of this um what they had shown was that the invasive edge of tumors you get this radial alignment so fibers that sort of extend away from a primary tumor um and that correlates they generated like a tax one two and three um and it's a little bit subjective but it actually um taxed three highly correlated with poor survival tax one good survival um so that's an example of the action the architecture itself so just that physical alignment but what we're actually beginning to realize is that sometimes just loads of a particular Matrix isn't necessarily the best indicator but the ratio metric amounts so if you get an increase in one or decrease in another can have again that similar effect or you know or be correlated closely with survival and it's just it comes down to the fact that interact with each other and they scaffold one another and changed their you know the organization and so that's a that's actually a new way that we're thinking about it is is rather than well that's just high and that's just low and that correlates is to say well how do these nuances how do these things all come together and you know especially in that scaffolding sense an increase in one with a common commitment decrease in another maybe more powerful than just looking at one alone and that that speaks to architecture although we don't quite necessarily understand how the architecture has been changed yet that's really interesting thanks um I'll take the question from Harry who's got his hand up hi Thomas Thanks for the talk um you were speaking about how kind of in the developmental stage uh the ACM can be quite similar even between different Schumer types um I was wondering based on that as well as calfs if there are any other sort of uh infiltrating cells that aren't usually in the ACM that are common across various cancer types that's a that's a really great question um so we tend to sort of lump them I suppose we have you know fibroblasts and calves um and you're not you I suppose in different tumors you would have the precursors to those so style excels is a good example in the liver or in the pancreas um and then we obviously lump most immune cells to together when we talk about immune infiltration um at the moment I don't really I can't say that we've looked into whether there's additional cell types I mean there will be obviously all of those cells are the normally present a good example would be I suppose in your case here within pancreatic tumors do things like islet cells play a role um are they somehow involved in that tumor microenvironment and remodeling um my simple question I don't know we haven't looked whether there are cells outside of that and it's a great point we should really consider everything rather than just those that we're familiar with Excellence um I'll guard the chat now Sophie has asked is it possible to upregulate parts of the ECM which promote tumor containment while also targeting the ECM to allow immune cell entry yeah I think that would be the million dollar question um so we've got some programs at work where we are essentially uh doing the first part um so we know that some Matrix components are tumor suppressive um they seem to at least um I guess restrain or constrain tumors and some great early work by the likes of mina Bissell um and co uh show and this was many many years ago now um several decades ago was that if you take transformed mammary epithelial cells and put them into normal Matrix then they they actually almost behave like normal cells suggesting that actually a a normal Matrix can for a while just restrain that that sort of malignant phenotype um and more recently there's been some some great work coming out from um some colleagues over in the US where they've actually been looking at aged versus young Matrix um and with aging we see a lot of changes happen in The Matrix the most common one that we're probably all familiar with is we get wrinkles as we get older um as the balance of Matrix molecules changes and so age DCM appears to have um effects on promoting cancer whereas young ecn seems to restrain them so there are definitely work on the way to say what is it that restrains and can we push that to essentially the upregulated of course the question is how is it controlled and how can we how can we up-regulate that um I'm not sure we're at that process where we can sort of you know inject in Matrix components yet to to temperature a great Point that's definitely one of them just speaking to the second part whether you could upregulate one down regulate another that might be pro-tumorogenic I mean that's really where you want to start going and removing or pruning away those that you know might have been proteomogenic and whilst retaining those anti-tumogenic functions but that's a great point it's certainly work that's ongoing all around the world and I would say probably in the next 10 years or so we'll start to see things like that coming through it's really cool thank you um I saw Anna had her hands up Anna if you've still got a question please pop it in the chat or put your hand up um hanari asks how does extracellular matrix remodeling relate to tumor dormancy ah my brilliant question um there's been some uh super exciting work coming out of the labs of people like Cyrus gujar over in Seattle um at the Fred Hutch that are basically looking at um how single cells interact with different Matrix components especially within perivascular niches within the brain within the bone and so on um and so what we find um is that at that single cell level your cells are often in touch or in direct contact with various elements and that they have that as we were just talking about in the previous question that kind of tumor suppressive effect now a matrix component is not going to kill a cancer cell but it may stop it from overtly proliferating and colonizing and so that's where the dormancy angle comes in and it speaks to what is known in the field and I didn't mention but something known as the seed and soil hypothesis that was originally proposed by Stephen Padgett many many years ago now and that's this idea that a a tumor cell requires that might write that correct microenvironment to in order to colonize so the seed requires the right soil and so this is critically important in examination to secondary organs where tumor cell finds itself in a very you know new or other you know it's never experienced before environment and the elements of that restraining over time something happens and it escapes from that and then obviously dormancy uh the dormant cells become reactivated but yeah there's some great work Cyrus kajar is a great person to to look up and I know that uh and and he's been ridden a few really good reviews on it for those who are interested cool thank you uh Anna if you wanted to ask her a question can you hear me yeah I'm sorry thanks some of the questions have been asked that's why I put my hand down but I'll just still ask um so what about the polarization of the collagen like it doesn't have any effects um in the development of the micro environment or that you can work do anything with that in terms of the Imaging that I showed yeah so um so so the Imaging which um of the collagen the polarized light Imaging well that's um it's basically exploiting uh the anisotropic properties of the dye so when when it binds to the dye we um Can essentially measure it's a sorry about how many how many dye molecules are present and how tightly they're packed to one another and so it actually gives us really useful information about bundling density thickness and so that actually by extracting different elements of that can tell us um about tumor it's obviously a static sort of um measure but it gives us more information than just a h e that says Ah there's there's a matrix um and so yeah we do we do extract some of those different bits of information it's a little bit more bulk so obviously that's not on uh you're sort of standard Imaging a standard light microscope um we tend to use the second harmonic generation Imaging the shg Imaging that I showed you all is beautiful purple and white strands that allows us to really get down to almost that single Matrix fiber level and then start asking about the architecture in the role that it might play just answer the question yeah so so far you don't have much input you know okay cool and just a very quick question just out of curiosity you know that in your pediatric tumor model the over expression of perlicon how exactly that creates the chemo protective Niche like is it like a physical barrier or it's some modulation receptors or how does that yeah absolutely I mean I was nervous I totally skipped over that bit um and so read the paper because it'll it'll definitely um there's so much there but what I will say is that um we actually carried out a number of um fret biosensor readings of cell cycle um so using cdk1 threat biosensors and what we see is that we get an altered cell cycle in the presence of perlicam and what that does is that therefore so all of your chemotherapy is essentially disrupt or try to disrupt you know DNA causing cell cycle stalling and so on what we see is that due to sort of a slightly delayed um or altered cell cycle is that you get a delayed response to your chemotherapy and so we didn't necessarily track back to know which receptors were being activated that was leading to the change in cell cycle but we know that that was likely to explain so the chemotherapy partially works but it has a blunted effect and so the problem with pancreatic cancer is it's hyper aggressive so any slight blunting in your efficacy is quickly going to be overwhelmed so so that's why um but this beautiful fret um cell cycle analysis in there that gives you an insight into really how that's affecting the proliferation super I'll read that thank you so much we had a question from Antoine who said your deceleration of tissue technique is incredible uh it says I was wondering if you had ever tried to stain for cell membrane markers anyway to see if fixation might cross-link some membrane glycoproteins with the ECM this way maybe even with decellularization you might get an idea of what cells are around yeah uh that's a great point and that's I probably didn't make it entirely clear in we don't there's no fixation of the tissues um so they are still fresh um we never stay in for cell membrane um primarily because when we did things we were looking for cells and so we we tried a number of things we were looking for fluid in staining to see if there was any sales DNA content um and so on however it is a great Point um in one sense because receptors and fragmented cell membrane may still be present um we haven't looked um at it specifically the D cell approach is it does use detergents so I do wonder if it might be washing out most of they're sort of lipid-based uh things such as cell membranes but no we've never looked and it's a really interesting question uh cool thank you um Chris Neal asked if the decellularization and receding process uh he said it was a really interesting um as a way to study ECM remodeling due to cancer and asks are these protocols also being used to study tissue transplants yes and there's been some amazing work by people and this is partly where some of the the tips and tricks that we got have come from um and I've I've actually been recently been talking with um a a chat down in uh in Melbourne actually about uh who's got a very similar approach and so so this idea of recellularization recreating matrix in order to create humanized material so there's a lot of work trying to take for example Pig tissues decellularize and remove those and then re-cellularize with human tissues it's not quite made it into the clinic yet but it's definitely um going that way mainly because and I know it's getting better but trying to 3D print an entire Matrix structure it's still pretty challenging we're still a way away from that um that would be the ultimate goal um so yeah it's tissue engineering um and and biomedical engineering sort of approaches is where online of this is has come from we're almost in a way using some of our hard work to apply this now to what happens in that diseased setting when things uh go sideways for example in tumors all right thank you uh we've got a couple more questions in the chat I wasn't sure Gemma do we do we run a bit over is that okay if it's okay with Thomas sure yeah yeah there's a there's a couple more coming there so I'm just right um so Raymond asked uh he said thanks for the great talk and asked do you think chemokines binding to hspg play a role in driving slash inhibiting metastasis or immune cell infiltration yeah uh you've again I totally got I said it's a large multi-domain protein it has a number of growth factor binding domains on it um nothing gets past your your students they're they're awesome um yeah absolutely we didn't look into um the that role but we know that there are a lot of um growth factor binding domains as well as Matrix scaffolding domains so that's what's really happening now is that we're actually doing some work following up with the AstraZeneca and antibody Alliance to specifically generate antibodies targeting domains and so the idea is that we can block different functional domains be it a matrix binding a growth factor binding a cell receptor binding um and then start to tease out what's the most crucial what's what's critical is it the fact it binds other Matrix proteins and organizes that structure or is it just sequestering a growth factor that's known to trigger Invasion I suspect that it's probably going to be a combination of all of them as many of these things are and that it'll take us a long time to tease out which is the most important thank you um Jackson says thanks for a great talk Thomas my question is in regards to transplantation animal studies to get some tumors to grow on mice sometimes mitrogel and growth factors are required to allow for tumor initiation how does this affect extrapolating data for determining driver mutations in the cancer thinking in the context of your KP and KPC experiments yep so you're right that's there are some models that do require that um it's possibly down to this idea that you need some kind of supportive Niche just to give them that foothold in the in the paper I discussed the um um we we avoid where we can the cow implantation with maitre gel um some of the models do require especially in grafting into the pancreas the reason major gel is used in the pancreas is because it has a nice tendency to gel when it gets to about you know body temperature so it's liquid on Ice anyone who's worked with it knows you have to keep it on ice otherwise it starts to go nice and thick and upon the implantation into for example the pancreas which is a very diffuse tissue it gels and so that actually holds the pain the tumor cells in place while staying graft um ideally we would love to move away from that but what's important is that in our models is that we have for example the Perler can we had perlican uh knocked out calves with cancer cells Perler Can wild type calves with cancer cells we then have the flux model each of those is obviously co-implanted in the same way so any effect of that nature Shell is having would be applied to all settings well I thought he was going to ask a question about well if I just have a minute more was that can we use I've repopulated to re-implant back in as a way to look at the effects that might have and that's something we're actually really excited about doing at the moment so we can say well we've got a bit of detailed tissue that we can put cancer cells back into let's re-implant that within that 3D structure and then look at what happens does that affect metastasis response to therapy and so on so that's some of the other cool work which maybe I'll be able to talk about in the future at some point that sounds very exciting thanks um our last question is from Jeffrey who asked uh the images of the ECM give impression of it being almost cobweb like mostly open and not occupying much of the volume even though images were decellularized presumably the ECM contains water and is a lot less viscous than intracellular Matrix cytoplasm is that right yep all correct um the one thing I will say is that the beautiful Imaging I was showing you was one of hundreds of extracellular Matrix components and so there was there was that one slide we'd stay in for four and it looked like a very much more dense um so when we look at individual components it does appear to be quite a bit of space however when you start to layer in I mean any time there can be four or five hundred Matrix components present in different amounts within a particular tissue and so that certainly begins to similar to the cytoplasm begins to start crowding in terms of viscosity matrices are often very well hydrated that hydration can change obviously depending on the it's controlled really by the level of proteoglycans we don't see unless it's a specialized tissue um like the vitreous humor in the eye for example we don't see a sort of viscous property in the sense that we might with a very high protein soluble protein which um fluid because a lot of extracellular Matrix is insoluble so it's it tends to be deposited as an insoluble Matrix um the only difference I guess would also be the blood which has a soluble Matrix so I would say that these cells are definitely as a viscous fluid probably more viscous but that there are a lot more than you can definitely see in the Imaging I've shown you present within there so great thank you so much Thomas I think that was the the last of the questions today awesome really appreciate your time that was yeah fascinating stuff thank you bye yep thanks parents that was great and a huge amount of interest from the students