Jeffery James Rodvold, PhD
Postdoctoral Researcher, UCSD, San Diego, CA, USA
Jeffrey is an expert in tumor microenvironment biology. Currently a postdoctoral researcher, he completed his PhD in Biomedical Sciences at UC San Diego focused on the unfolded protein response (UPR) within the tumor microenvironment. His research involved elucidating how the tumor microenvironment becomes dysregulated within both the cancer cell and immune infiltrate compartments through the production of signaling molecules cancers cells producing during the UPR.
He earned his BS in Bioengineering: (Biotechnology) from UC San Diego in 2010 where he performed research in the fields of biomaterials and stem cell mechanotransduction.
Learn about Jeffery's research
Title: Cancer cells send signals boosting survival and drug resistance in other cancer cells
- Understand how to design an in vitro system to model phenomenon within the tumor microenvironment
- Understand how ER stress in cancer cells may facilitate immune dysregulation and chemoresistance in vivo
Successful tumor outgrowth requires the coordination of a variety of cell intrinsic and cell extrinsic signaling events. These events include those establishing a tumor microenvironment (TME) that both enables tumor cell survival and disables anti-tumor immunity. Recent reports demonstrate that these events require molecules produced by resident tumor cells. Tumor-borne signals within the tumor microenvironment propagate tumor cell fitness and immune hijacking. The endoplasmic reticulum (ER) stress is an adaptive response to a variety of TME insults, including hypoxia and nutrient deprivation, raising the possibility that ER stress could serve as a potential source of tumor cell fitness and immune dysregulation. To that end, we induced cancer cells of various origin to undergo ER stress and harvested the resulting conditioned medium (CM) to explore its effects on both recipient cancer cells and immune cells. Cell culture medium became an invaluable tool to our studies as it allowed us to recreate stimuli existent within the tumor microenvironment under controlled conditions. Our results revealed that the CM of cancer cells undergoing ER stress transmits ER stress to recipient cells. On the one hand, myeloid cells (macrophages and dendritic cells) treated with the CM of ER stressed cancer cells acquire a mixed pro-inflammatory and immune suppressive phenotype, which restrained T cell anti-tumor immunity and facilitated tumor growth in vivo. On the other hand, cancer cells treated with the CM of ER stressed cancer cells acquire cellular fitness to a variety of challenges including nutrient deprivation and chemotherapies. When implanted into immune competent hosts, ER stress experienced cancer cells grew at markedly faster rates than inexperienced ones. These findings support the existence of a novel mechanism used by tumor cells to restrain anti-tumor immunity while enhancing cellular fitness with the TME.
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Presenter: Dr. Jeffrey Rodvold, Postdoctoral Researcher at UC San Diego
0:00:00 – Slide 1
Moderator: Hello everyone, and welcome to today's life webinar, Cancer Cells Send Signals Boosting Survival and Drug Resistance in other cancer cells presented by Dr. Jeffrey Rodvold, a Postdoctoral Researcher at UC San Diego. I'm Susie Valdez and I will be your moderator for this educational webinar presented by LabRoots and sponsored by Thermo Fisher Scientific. Before we begin, I'd like to remind everyone that this event is interactive. We encourage you to participate by submitting as many questions as you want at any time you want during the presentation. (0:00:38 )Just click on that green Q&A button located at the lower left of your presentation window. Type your questions into the box that appear on the screen and we'll answer as many questions as we have time for at the end of the presentation. Also notice you'll be viewing this presentation in a slide window. (0:00:54)To enlarge that window just click on the screen icon located on the lower right. If you have any trouble seeing or hearing this presentation, click on the support button at the top right of your presentation window or use that Q&A button and let us know you're having a problem. (0:01:09)This presentation is educational and thus offers continuing education credits. Click on the button in the bottom left-hand corner and follow the process to obtain your credits.
I'd now like to introduce today's speaker, Dr. Jeffrey Rodvold. Jeff is an expert in tumor microenvironment biology. (0:01:28 ) Currently a postdoctoral researcher he completed his Ph.D. in biomedical sciences at UC San Diego focusing on the unfolded protein response within the tumor microenvironment. His research involved elucidating how the tumor microenvironment becomes dysregulated within both the cancer cell and the immune infiltrate compartments through the production of signaling molecule cancer cells producing during the UPR. (0:01:57) He earned his BS in bioengineering from UC San Diego in 2010 where he performed research in the field of biomaterials and stem cell mechanotransduction. Please join me in welcoming Dr. Jeffrey Rodvold. I will now turn the presentation over to him. Welcome, Jeff. (0:02:27)
Dr. Jeffrey Rodvold: Great, thank you very much, Susie, for that introduction and thank you to Thermo Fisher Scientific for the opportunity today to talk to you about the cell extrinsic consequences of the tumor UPR through a title called cancer cells send signals boosting survival and drug resistance in other cancer cells. My name is Jeff Rodvold. I'm a postdoctoral scientist at UC San Diego and the Zanetti Lab where the data of today's resource has been found.
0:03:03 – Slide 2
In today's talk what I would like to do is first provide an introduction to the endoplasmic reticulum, ER stress and the unfolded protein response. I will then introduce how the UPR is involved in the tumor microenvironment as a cell intrinsic driver towards tumor growth. I will then extrapolate these findings by providing evidence that there are cell extrinsic consequences of the tumor UPR through a phenomenon called transmissible ER stress or TERS. (0:03:35). I'll introduce the TERS hypothesis and review how we designed the culture technique in vitro to generate the condition media revealing this phenomenon. In some detail I'll review our studies concerning myeloid cells and the affects of TERS on myeloid cells, specifically macrophages and dendritic cells and how they're polarized to an inflammatory and immune-suppressive phenotype and provide evidence that this phenotype can be recovered in vivo. (0:04:05).
Finally with more detail I'll be reviewing how TERS affects cancer cells, specifically how it endows cytoprotection, Wnt signaling, the mechanism behind both of these phenomenon and demonstrate that clonal fitness can occur both in vitro and in vivo through the effects of TERS. And without further ado let's jump right into it.
0:04:27 – Slide 3
The endoplasmic reticulum is a massive and sprawling organelle found in all cells. It's responsible for the proper folding, maturation and secretion of nascent polypeptide chains as they become shallowed from within the ER to the specific organelle of interest. In this way the ER is a massive and sprawling organelle necessary to traffic protein throughout all compartments of the cell. You can appreciate in the right-hand figure that this network looks awfully like a highway system and there's plenty of an analogy between the two.
0:05:10 – Slide 4
Specifically, the ER can accommodate a massive amount of cargo at any one time. A massive amount of proteins can be properly folded, matured and trafficked out of the ER without there being any issues. But as a Southern Californian as many others are very familiar traffic is never homeostatic and in fact...
0:05:29 – Slide 5
...often traffic jams can occur that look often like this. The biologic significance of traffic jams is an accumulation of miss or unfolded protein that sit in the ER that can be a consequence of a variety of insults.
0:05:47 – Slide 6
To cope with ER stress cells initiate the unfolded protein response, the UPR. (inaudible) carry outs this evolutionary (inaudible) phenomenon is mediated by three transmembrane proteins, PERK, IRE1a, and ATF6. They're held in an inactive state throughout their association with a master chaperone protein called GRP78. However, when there's an accumulation of miss or unfolded protein, GRP78 preferentially disassociates from these transmembrane proteins and to aid in a chaperone function of folding polypeptide chains. (0:06:20) In so doing it derepresses each of these three arms to allow for downstream effects. PERK dimerizes an auto-phosphorylates ultimately promoting the activation of the activating transcription factor 4 or ATF4 seen here which provides generally speaking a pro-apoptotic signaling through its induction of a pro-apoptotic protein CHOP. IRE1 alpha oligomerizes and auto-phosphorylates. Its primary function is an endonuclease function where it splices the XPP1 MRNA leading to a spliced isotherm of this MRNA leading to the production of the transcription factor XPP1 which serves to restore ER homeostasis as well as preferentially activate a variety of genes. ATF6 translocates to the Golgi where it is modified to an active form where it then serves as a transcription factor itself. (0:07:27) Again aiding in ER in balancing homeostasis within the ER. One of the compelling of the UPR is it is neither strictly pro-apoptotic nor strictly pro-survival but depending on the duration and the amount of ER stress that occur, these three arms will decide whether a cell should go through apoptosis or persevere and survive. For this reason the URP has been implicated in a variety of pathologies including neurodegeneration, diabetes and in fact cancer. (0:08:05) One of the prime locations for this to occur is within the tumor microenvironment. The tumor microenvironment is an awful mess of cells, both of transformed and non-transformed nature as represented in the left cartoon. The self-interested cancer cells have no interest in creating a balanced vasculature. This in turn leads to exogenous stressors such as hypoxia, nutrient deprivation and acidic pH all of which create ER stress. Indeed there are also endogenous stressors within cancer cells that can cause ER stress such as aneuploidy. (0:08:47) There's numerous signs of evidence to suggest that the UPR is involved in cell intrinsic features to lead to tumorigenicity of various solid malignancies including XBP1, GRP78 and PERK in a variety of different tissues. Most of these studies have been focused on how the UPR provides cell intrinsic benefits predominantly through signaling prosurvival pathways as well as lead to metastatic function. (0:09:17 ) We sought to demonstrate that there could also be cell extrinsic consequences of the tumor UPR, specifically as an initial hypothesis that there is an affect on the immune infiltrate of the tumor microenvironment through cell extrinsic features of the tumor UPR.
0:09:44 – Slide 7
To that end strictly our hypothesis became that there exist tumor-cell borne cues produced by UPR signaling that extrinsically affect infiltrating cells or cells within the tumor microenvironment. To that end it was important for us to establish an in vitro setting where we could very easily control all of the factors and model some of these stimuli in a very controlled manner. The conclusion of our studies as I will review today demonstrate that a phenomenon known as transmissible ER stress affects both immunity as well as resident cancer cells to facilitate tumor outgrowth.
0:10:27 – Slide 8
I will be reviewing in some brief detail our key findings on an innate immunity as it relates to TERS which comes from two main publications, the first concerned with the roles of TERS on macrophages, the second concerned with the effects of TERS on dendritic cells and thereby adaptive immunity. And then finally in greater detail we'll be discussing how transmissible ER stress affects cancer cells.
0:10:59 – Slide 9
In order to establish an experimental model based off of our hypothesis, we had to consider some requirements. The first and foremost is we wanted to demonstrate that cancer cells of various origin undergoing ER stress must product some molecule. In order to do that it was important for us that we could control for the stress of a transmitting cell is producing a molecule that is then carried over to a recipient cell as opposed to just loading cells with a drug, taking the resulting condition media still containing the drug and loading it onto recipient cells which becomes far more precarious when you're trying to de-convolute what contributions are made by the transmitting cell and what contributions are made simply because of drug carryover. (0:11:50) Of course this system also had to be affordable, rapid and reproducible.
0:11:55 – Slide 10
The model that we designed was the following. We took tumor cells of various origin and seated them in about 80% confluence and created ER stress through the treatment of the drug Thapsigargin. Thapsigargin is a serif inhibitor that effectively defeats the ER out of calcium leading to creating ER stress. Because of this it allows us to treat cells for an acute length of time in our hands of 300 nanomolar concentration for two hours which depletes the ER of calcium. (0:12:31) In so doing we then wash twice to remove any residual or un-metabolized drug of Thapsigargin and then load onto these treated cells with standard growth media as you'll find in subsequent slides in addition to the Tg treated cells we use vehicle condition media as a control for any effects that are just carried over simply by the cancer cells in a homeostatic environment. In both cases we harvest 16 hours later. We harvest the condition media by filtering it through a .22 micron filter and then load it onto naive cells. (0:13:09 ) In our initial experiments we loaded this onto unstressed J774 mirroring macrophages. And what we were surprised with finding from foremost was in fact that ER stress, that the macrophages treated with this condition media underwent ER stress which is to say ER stress was being transmitted from a tumor cell to a macrophage cell and thus the term transmissible ER stress or TERS. In reviewing a large collection of data...
0:13:41 – Slide 11
...what I'd like to draw your attention to is the following synoptic review of a bunch of experiments whereby we took cancer cells of various origin. Here you're looking at TC1 which are a mirroring prostate cancer cell line and we treated bone marrow derived macrophages with the vehicle or TERS condition media of TC1 cells for 24 hours. You'll notice the first three panels include ER stress related genes, GRP78, XPP1S and CHOP where they all had an increased transcriptional up regulation. (0:14:15 ) In addition these cells became pro-inflammatory through the increases of inflammatory genes IL6, IL23 and CNFa. Our studies also showed that these molecules could be produced and secreted as well and are not just affected at the transcriptional level. Perhaps most striking, however, is that cells also acquired a suppressive feature through the up regulation of Arginase, an immune suppressive molecule that restrains T cell proliferation. (0:14:47) We used various cell lines including mirroring lung cell carcinoma, LLC's, or mirroring melanoma cell lines, all of which had similar effects, though there was some biologic variability. In terms of recipient cells, we've also used immortalized J774 cells and bone marrow derived macrophages where again we find a similar phenomenon to be true.
0:15:17 – Slide 12
We then were interested in demonstrating that the phenomenon that we describe is not exclusively due to Thapsigargin mediated ER stress. To that end we took different cancer cell lines including TC1, B16 and LLC and starved them of glucose, both in the growth media as well as using dialyzed FPS which had a molecular cutoff that was able to remove glucose. (0:15:42) And we pretreated these cancer cells for 24 hours in the absence of glucose effectively creating ER stress. We then harvested this condition media and treated macrophages with either this condition media or either standard growth media denoted as RPMI + or glucose starved media itself, RPMI -. As you'll appreciate in the transcriptional data on the right, you'll find that these cells, macrophages, had increases in UPR related genes in the top row, GRP78, GADD34 and CHOP both when they themselves underwent nutrient depravation in the second panel, but they had substantially increased ER stress when they were treated with the nutrient deprived media preconditioned by the various cancer cell types. (0:16:36) Concordantly they were also polarized to a pro-inflammatory state meaning that there was seen to be some other factor to enhance and potentiate the effects of the glucose depravation. To this end we concluded that transmissible ER stress could occur through the very relevant tumor microenvironmental insults glucose depravation.
0:16:59 – Slide 13
Indeed the profile that I've described to you is neither classically M1 or M2 but in fact TERS treated myeloid cells acquired this mixed phenotype that is both pro-inflammatory and immune suppressive. Of equal interest is the fact that a separate group at University of Washington in 2012 showed that VEGF was also produced using similar techniques to what we described today. (0:17:25) Meaning in fact that these cells are both pro-inflammatory, immune suppressive and angiogenic. We then wanted to re-see if this profile, this TERS profile as I described which is both ER stress inflammatory and immune suppressive exists within the tumor microenvironment.
0:17:43 – Slide 14
To that end we used two different models, an orthotopic model which involved mirroring melanoma B16 cells subcutaneously injected into an immune competent host and the second model, a spontaneous model in which we used APC mice which developed small intestinal adenomas at about three months of age. In both models we harvested the myeloid infiltrate from both the tumor as well as the spleen and bone marrow where we would predict ER stress is not nearly as commonly occurring as it is within the tumor microenvironment. We then performed qPCR of the CD11 diapositive, the myeloid positive infiltrate.
0:18:23 – Slide 15
What we found was that in both models the CD11 diapositive cells that infiltrated the tumor microenvironment had transcriptional increases in ER stress genes, inflammatory genes and immune suppressive genes relative to those of the splenic or bone marrow drive controls confirming that the profile that we were able to demonstrate so cleanly in vitro existed in vivo.
0:18:46 – Slide 16
To that end a working hypothesis of how transmissible ER stress may affect immune infiltrate is the following: In the absence of ER stress cancer cells are properly able to coordinate—or pre-cancer cells are able to properly coordinate with infiltrating antigen presenting cells including macrophages and dendritic cell (inaudible) coordinate this with T-lymphocytes to lead to clinical expansion of the adaptive arm of immunity ultimately cleaning any pre-malignant lesion. (0:19:20)
However, in the presence of ER stress, cancer cells are per se able to transmit factors or a factor that affects infiltrating immune infiltrate macrophage or dendritic cell which in turn suppresses any adaptive arm through the production of Arginase as well as the surface expression of PD-L1 which I didn't have time to review today, as well as the production of pro-inflammatory cytokines including IL-6, IL-23 and of course the angiogenic factor VEGF. All of these converge in which to enable the tumor to grow and escape from immune surveillance.
0:20:05 – Slide 17
That left us wondering through due to cell extrinsic effects of the UPR affect unstressed cancer cells. It's well and good to describe the phenomenon that I've introduced to you but in fact were overlooking the majority population of the tumor, which is to say other cancer cells. (0:20:20) And so we were left wondering what effects TERS might have on neighboring cancer cells which is to say if one cell is undergoing ER stress because of either a lack of nutrients or a lack of oxygen or defects that lead to exogenous—or indigenous effects such as the aneuploidy how would that affect a neighboring cancer cell that is otherwise homeostatic and is (inaudible) folding.
0:20:46 – Slide 18
We drew inspiration from a provocative paper published in 2014 from the Poliac Group in the Journal of Nature who challenged this classical idea that clonal dynamics within the tumor microenvironment are driven in cell autonomous manners. The paradigm that once existed that a cell acquires a driver and mutation and then becomes the predominant clone.
0:21:16 – Slides 19
The Poliac Group challenged this by showing that in fact in a cell not in a non-cell autonomous manner when you control for any changes in genetic lineage there still is a dynamic influence of different subclones within the tumor microenvironment which is to say during times of tumor development one clone, like Clone A, maybe the predominant clone but during times of chemotherapy or if there is any temporal imbalance Clones B, C, P or Z would be the predominant clone. (0:21:53) The conclusion of this paper was in fact that tumor growth could be sustained by minor cell populations and acted in a cell non-autonomous manner. But leaving us to hypothesize that TERS could drive tumor clonal heterogeneity and provides tumor cell fitness.
0:22:13 – Slide 20
To that end we used human prostate cancer cells as a model just so we could focus on one specific tissue. The research being done today was predominantly using PC3 cells (inaudible) cells and DU145 cells. In the first experiment that we performed, we produced vehicle or TERS condition media using our classical Thapsigargin method in PC3 cells, harvested the condition media and loaded it onto unstressed PC3 cells. (0:22:48) What we find in panels A and B is in fact these cells underwent substantial ER stress throughout the five-day treatment of the condition media. We did re-supplement every other day for these conditions to occur. And we found that in fact these cells that did survive had substantial ER stress. (0:23:11) Concordantly however this was not restricted to alike cells but heterogenous cells as well which is to say we could generate TERS using a PC3 line and load it onto DE145 cells as we did in Panel C where again you find a similar trending TERS, the conclusion of which is transmissible ER stress is certainly not restricted to just affecting innate immunity but in fact neighboring cancer cells and it's not restricted of course, therefore, to alike clones.
0:23:44 – Slide 21
Additionally these cells acquire pro-inflammatory features such as the increase of IL6 and as I described they had an increase in the master chaperone protein GRP78, both intracellularly as visualized in Panel E, but also at the cell surface as visualized in Panel F by flow cytometry. (0:24:06) This is particularly striking because the surface relocation of GRP78 which still remains to be a controversial topic in the field of UPR has been implicated in tumor genicity in a variety of solid malignancies such that it's become a drugable target in various modules and it's suggested to provide a new perhaps monoclonal intervention against it. (0:24:34)
In terms of our kinetics we notice that surface GRP78 didn't occur—the relocation of surface GRP78 didn't occur on Day 1 but it took a protracted period of time on Day 3 and even substantially on Day 5 suggesting that this is not an acute experience that the cells must undergo but something that must be a little bit more protracted. This let us theorize that in fact that TERS could operate within a iterative manner within the tumor microenvironment. And so to better illustrate these effects what we decided to do was...
0:25:11 – Slide 22
...treat cells for two days on either vehicle or TERS condition media represented here. We then would remove the TERS or vehicle condition media washing twice and then re-supplement with standard growth media for two days off. (0:25:28 ) During this time the treated cells could restore their ER and restore homeostatic function. Again, the belief being if a cancer cell is producing these factors it likely is not a faucet fully on all the time but is turned on and off. This population we refer to as TERS primed or vehicle primed. Interestingly TERS primed cells had a surface relocation of GRP78 that was quite profound relative to vehicle primed cells. (0:26:02 ) Because of this and the role of surface GRP78 and enabling tumor cell survival we then wanted to look at how this might affect cells during one of the most common insults of the tumor microenvironment which again is glucose depravation. So we starved vehicle primed or TERS primed cells for 48 hours. (0:26:25 ) In the first two panels you will appreciate we have—is our negative control using just complete rough media while in the latter two we have starved the cells of glucose and FBS. GRP78 was increased at both the unstimulated and stimulated conditions. Conversely the PERK arm which we consider to be the pro-apoptotic arm was repressed or had the less expression in the TERS primed population than the vehicle primed population drawing your attention to the fact that above ATF4 and CHOP had markedly reduced expression in the TERS primed cells during neutron depravation. These two observations, increases in GRP78 and altered PERK signaling would suggest that the cells might be surviving better within the glucose starvation.
0:27:19 – Slide 23
To that end we started cells for 48 hours a previous described and then probed them for viability by an Annexin V apoptosis assay by flow cytometry. As you can appreciate for those who may not be familiar with how to read these assays it's presented in the lower right-hand-corner where you have your live cells in the lower left. In the lower right quadrant are your dying cells and in your upper right quadrant are your dead cells. TERS primed cells survived about three times as much as the vehicle primed cohorts as you can appreciate 27% versus 66% survival after 48 hours. If we carried the starvation out for another day it could be as compelling as 40% relative to 2%. We were really excited by this data but we wondered if the cells also were protected against exogenous insult, not just those that are contained within the tumor microenvironment.
0:28:16 – Slide 24
To that end we used the stress inducing chemotherapy Bortezomib which is a proteasome inhibitor that creates massive amounts of ER stress to ultimately drive ER tumor cell death. Again, across the titration of the drug we find that GRP78 is increased whereas PERK signaling again remains altered.
0:28:39 – Slide 25
Concordantly we observed vehicle primed cells had poor survival relative to TERS primed cells across the titration of the drug. For instance at 100 nanomolar we have 75% survival of TERS primed cells relative to 38% in vehicle primed cells. We were satisfied with these results but we wanted to know if this was a durable cytoprotection or simply lost after two days. And so using GRP78 abundant as a proxy for...
0:29:10 – Slide 26
...cytoprotection we detected its changes in expression between vehicle and TERS primed cells two days post priming and up to five days post priming in which the cells had not experienced any transmissible ER stress condition media for one week. Again, you can appreciate that we had increases in fact in GRP78 and the TERS primed cells. This concordantly was found to be cytoprotective where we could again find increases in TERS primed cells treated with Bortezomib over vehicle primed cells. However, proteasome inhibitions are not always done with solid malignancy and so we wondered if non-stress inducing chemotherapy also was less effective in TERS primed cells.
0:29:58 – Slide 27
We use the microtubule inhibitor Paclitaxel which in our hands causes no ER stress in cells to see if we could find a similar phenomenon. I'll be quick to point out at this point that others have reported that Paclitaxel can cause ER stress, particularly in ovarian cancer, but in our hands here is shown in Panels A, B and C we found that no ER stress could be produced at the transcriptional level in A or at the protein level in the Western blots in B and C.
0:30:28 – Slide 28
However, here again we find that TERS primed cells survive better than their vehicle primed cohort. I'll draw your attention to the fact that we have 23% death in vehicle primed cells relative to 6% death in TERS primed cells.
0:30:45 – Slide 29
Because Paclitaxel is mediating cytotoxicity through creating double stranded DNA breaks by preventing the proper microtubule assembly we wondered if there was a change in the cell cycle and in fact we find that there is a change in the G2/M phase where DNA repair occurs whereby TERS primed cells were enriched somewhere between two to three fold relative to vehicle primed cells.
0:31:12 – Slide 30
Concordantly if we probed for double stranded DNA breaks in Paclitaxel vehicle primed or TERS primed cells, we find this is in IHC standing for Gamma H2AX which stains double trended DNA breaks. You again find a marked difference in pattern between the two suggestive of the fact that TERS prime cells are not undergoing double stranded DNA breaks induced by Paclitaxel. We couldn't quite explain this phenomenon, enrichment in the G2/M phase due to ER stress. However, one pathway that's incriminated in tumor genicity and in the enrichment in the G2/M phase...
0:31:50 – Slide 31
...is Wnt signaling. So we did PCR for the abundance of two genes associated with Wnt signaling, both beta CTNN and it's negative regular AXIN2 and we found that the protracted treatment of TERS primed condition media I should say and these PC3 cells analyzed here was increased relative to vehicle condition media. To better visualize and appreciate the kinetics of this phenomenon we took advantage of a reporter system called the TCF optimal promotor or top promotor assay in which when beta CTNN is stabilized ready leading to when signaling, it associates with a complex including TCF leading to downstream signaling and activation of Wnt associated genes. (0:32:38) The top reporter takes advantage of this by putting a GFP transcript downstream of this assembly so we could detect GFP expression by flow cytometry in top transduced cells to associate how TERS condition media affects when signaling. In Panel C as you can see on the two days off of TERS condition media we had robust Wnt signaling relative to vehicle condition media concluding that TERS promotes Wnt signaling.
0:33:11 – Slide 32
To understand the mechanism behind this, we treated cells in the absence or presence of vehicle or TERS condition media with the addition of in Panel C and IRE1 inhibit chemical inhibitor 4u8C or in the case of Panel D a PERK inhibitor GSK2656157. Whereas PERK inhibition had no effect on top expression visualized in Panel D, the IRE1 inhibitor did suggesting that IRE1 inhibition mediated by TERS effects has an effect on Wnt signaling. (0:33:44) We wanted to know if this phenomenon was restricted to simply ER stress related effects or TERS related effects. We then treated cells with Tunicamycin and probed for top expression where we could find no changes in top GFP product despite the fact that the cells underwent ER stress suggestive of the fact that the effects of TERS on Wnt signaling are privileged and not generalizable to ER stress.
0:34:15 – Slide 33
Importantly a recent paper in Oncogene from a separate group reported that IRE1 seemed to be involved in Wnt signaling.
0:34:24 – Slide 34
We then wanted to de-convolute what mechanism is responsible for its cytoprotective effects we've observed. Predicting that the UPR was centrally involved, we decided to go to a mouse embryonic fibroblasts or MEF model. In Panel A we first demonstrated that the priming model of TERS could exist in MEF where you find increases in protection in both Bortezomib, Taxol and nutrient depravation. However, abandoning wild type MEFs for PERK knockout MEFs we find that we can no longer recover this cytoprotection. (0:35:03) I'll point out that we've also done these studies using IRE1 alpha and AGF6 knockout MEFs where the pattern is less compelling, but they do have equally contributing roles to this phenomenon. To better demonstrate however that PERK was involved, we decided to use leverage CRISPR technology. In Panel C you can see our guide against Exon 2 of ATF4 one of the central targets of PERK signaling. (0:35:31) In Panel D you see successful deletion of alteration of the ATF4 construct and in Panel E using 293T cells you see again using wild type cells we have primed cells that have cytoprotection during TERS endowed features. However, using ATF4 CRISPR 293T cells the cytoprotective effects are lost suggestive again that PERK signaling and perhaps particularly ATF4 are central in the cytoprotective effects that we've observed.
0:36:07 – Slide 35
Bringing it back to our opening hypothesis which is that clonal heterogeneity and clonal fitness could be endowed by TERS, we took advantage of a mirroring tagging model whereby we transduced TC1 cells with RFP that would consistently express red fluorescent protein and we primed them with TC1 vehicle condition media. Separately we TERS primed untagged TC1 cells. We then co-cultured these two populations together and challenged them for 24 hours with a variety of insults and by flow cytometry determined the relative abundance of RFP positive and RFP negative cells. (0:36:45) What we find is that across a variety of challenges including Thapsigargin to DOX ED glucose which serves as a model for nutrient starvation or glucose starvation, Bortezomib and Taxol the TERS primed clone always emerged as the fit clone. If we reverse the conditions which is to say TC1 RFP cells are TERS primed and untagged TC1 cells are vehicle primed we find the same results.
0:37:20 – Slide 36
Concordantly, if we took TC1 cells and vehicle primed them or TERS primed them and contralaterally injected them into an immune competent host we were curious as to how this might affect tumor growth which we observed over one-month's time.
What we find is that TERS primed tumors grew at much faster rates than their vehicle primed cohort. After one month's time in Panel D you can see there's a substantial change in tumor weight and you'll have to forgive in Panel E the gross morphology of these tumors at the time. I was quite inexperienced at removing tumors from tumor bearing mice.
0:38:00 – Slide 37
The conclusion of our studies, however, is the following: That in the absence of ER stress there could be or of any challenge there could be these subpopulations in blue that are TERS primed. However, the introduction of a chemotherapy or a tumor microenvironmental pressure would force the clonal dynamics to change whereby a naive cancer cell may die from these challenges whereas a TERS experienced cancer cell through its increases in GRP78 both intracellularly and at the cell surface increases in Wnt signaling and altered PERK signaling all enable it to cope with these insults to ultimately emerge as the predominant clone within the tumor microenvironment.
0:38:43 – Slide 38
The conclusion of these studies are the following therefore: Using in vitro systems we were able to provide a precise tool to recreate some of the stimuli that may be made up to the tumor microenvironment. I'll point out that our observations in vitro and in vivo were only enabled—or rather I should say, our in vivo observations were predicated off of the observations in vitro. So again these using condition media allowed us to de-convolute a very challenging problem that would be very difficult to solve using mouse models or more advanced models.
And then finally I hope today I've convinced you that transmissible ER stress dually affects cancer cells as well as innate immunity, all of which these features seem to converge to enable tumor cell growth.
0:39:35 – Slide 39
I'll conclude by revisiting the Hanahan and Weinberg hallmarks of cancer in which they outline that there are ten features that tumor cells must acquire for the successful outgrowth. In today's talk and through some features I haven't had time to review with you today...
0:39:52 – Slide 40
...transmissible ER stress seems to affect at least six of these programs. Today I've put particular emphasis on resisting cell death, deregulating cells that are energetics through the survival of glucose depravation. I've introduced observations that affect angiogenesis, tumor promoting inflammation and indeed avoiding immune destruction. And data I didn't have time to review today, there also seems to be effects on replicative immortality by affecting telomerase reverse transcriptase or TERTS. With that I'd like to conclude...
0:40:33 – Slide 41
...by first thanking the members of the Zanetti Lab and predominantly Navijn Mahadevan who was the original mind that laid out a lot of these experiments. In the Lin Lab Jonathan H. Lin's Lab at UC San Diego, Nobu Hiramatsu was a tremendous help to understand how transmissible ER stress is affecting cancer cells. And finally I would like to again reiterate my appreciation to Thermo Fisher Scientific for this great opportunity to talk to you today about the effects of transmissible ER stress. And with that I'm looking forward to fielding your questions and look forward to the dialog. Thank you very much.
Moderator: Thank you, Dr. Rodvold for that informative presentation. We will now start the live Q&A portion of the webinar. If you have any questions you'd like to ask please do so now. Just click on the green Q&A button at the lower left of the presentation window, type your questions into the box that appear on the screen and click the send button. We'll answer as many questions as we have time for today.
So let's take a look at our audience member's questions that are already coming in.
(0:41:55) Question: Our first question, Dr. Rodvold is do all cancer cell lines experience the same level of TERS provided cytoprotection. And a second part of that is, what about production of TERS?
Answer: Sure, okay, yes. So that's a fair question. So to answer the first one, do all cancer cell lines experience the same level of cytoprotection. The answer is not all at the same level, I would say. (0:42:27) Most of the data we saw today was from PC3 cells and in the paper that we described we also looked at LNCaP cells in DU145 cells where again we do find the same phenomenon is true, that they're protected. Obviously it was most pronounced in the PC3 cells and we do think that this must be some sort of generalizable phenomenon as we've been able to do it with breast cancer cell lines, mirroring TC cell line. (0:42:57) You saw some of the data on using MEFs cells and 293T cells. So we don't think it's quite restricted to just one particular line but of course like anything else some lines are better at it than others. And I think the same could be said for TERS production. If you recall, I think it was one of the earlier slides that I introduced where we treat these cells with a variety—here we are—where we use TC1 cells, B16 cells and LLC cells. (0:43:31) You can appreciate that and each has the same production effects. That is to say they all transmit ER stress; however, the levels of ER stress seem to be a little variable from one line to the next. So we do think that this is a programming that most cells if not all cells can do, but some might do it better than others.
Moderator: Thank you. And here's our next question.
(0:44:03) Question: Does TERS treatment cause any cytotoxic effect on tested cell lines?
Answer: Yes, that is a fair point. It definitely can. When you're protract for the length of time that we have done, even for the two days on or the iterative every other day for five days, you do can have a certain percent of cell death. (0:44:30) We have observed this to be anywhere between 20-40%. At the time of these in our in vitro models we had some cells that we were provided that were unhealthy that these effects were then pronounced even further to 80% to 90% death. Obviously we didn't use those because they were contaminated, but yes, there are cytotoxic effects that could be observed. (0:44:54) And again while that may seem artificial, it's not too surprising given the fact that this likely would occur in the tumor microenvironment and if you see TERS almost as a Darwinian effect, that is to say that there are— some are going to survive and some will thrive. The ones that do thrive seem to thrive very well. So, yes, I think that there could be cytotoxic effects, but the ones that persevere are really the concerning clones.
Moderator: Thank you. And I think you two are audience members. What a wonderful live participation we have today. Here's our next question.
(0:45:35) Question: Can you further discuss how you see TERS effects tumor immunity?
Answer: Sure, yes. If I understand the question correctly, the question is kind of how we see this affect TERS immun—or how immunity, and I think they probably are talking about adaptive immunity here and how TERS can affect it. Through a paper that I didn't have time to discuss today, but I'll jump onto this slide whereby, let's see, here we are, so whereby we kind of see that there are a variety of effects that can occur on this T cell on the far right. (0:46:19) Through my predecessor, Navijn Mahadevan, he did a lot of this work. He's really capable of demonstrating that these T cells become anergic when experienced with myeloid cells that had been treated with TERS which is to say that the T cells are able to clonally expand and aren't able to clear any tumor. The effects of TERS directly on T cells is something that we're really interested in that we haven't had time to go into any great length but we do think that there could be effects on promoting either Treg function or something to that effect. (0:47:00) So we do think it could affect adaptive immunity as well but currently our understanding is that the kind of the fulcrum of this between is tumor cell to myeloid cell and then the myeloid cell pivots that to the adaptive arm. So, yes, hopefully that answers the question, but we do think that there are effects on adaptive immunity certainly.
Moderator: Thank you, Dr. Rodvold.
(0:47:26) Question: And what percent of oxygen was used during in vitro culture normoxic or hypoxic?
Answer: So we've used normoxic conditions. For those studies that I presented today we just did normoxic cultures and the reason is because we want to control for any other bystander effects like hypoxia that certainly would occur within the tumor microenvironment but we didn't want to make it assignment to this. (0:47:58) I will say, however, we have been able to generate TERS like effects with hypoxia where we pre-incubated these lines, the B16 TC1 and LC lines in a hypoxic environment in which we used 0.5% oxygen for 48 hours and then normalized oxygen levels and then loaded it onto receiver macrophages where we find similar effects. (0:48:26) I really want to do, if I understand the question correctly, I would love to have done an experiment where we show how this affects hypoxic conditions as well; however, we didn't have access to a incubator to demonstrate that. But I would be willing to bet that some of the cytoprotective effects that I talked about with nutrient depravation are also experienced in a hypoxic setting as well.
Moderator: Thank you.
(0:48:55) Question: And what can you tell about the identity of the phenomenon?
Answer: Yes. So there's scant information in today's talk about that because in truth we haven't been able to fully identify it. That's something near and dear to our heart and something that every day we're working towards. What I can say right now is the molecule is not a protein. It seems to be a small molecule. We think it's a lipid of some sort. (0:49:31) Without saying exactly what it is obviously because we don't know truly its identity, I would say it's in really low abundance and that's made it particularly difficult for identity efforts. We've paired up with a organic chemist at Scripps Institute of Oceanography and they're kind of helping us out with these isolation techniques using HPLC chromatographic techniques and NMR and mass spec; however, any ambition we had of it being just a transmitted protein does not seem to be the case. (0:50:12) And I'd also like to point out there in some sense it may not even be one factor. And in fact I would emphasize the Wnt story as kind of evidence for that where we don't think—at least personally I don't think that while we know that Wnt protein is not responsible for the Wnt signaling effects, it's certainly likely that there could be other transmitted molecules that in one sense endow ER stress or pro-inflammation and other produced factors that either stimulate Wnt signaling or angiogenic or immune suppressive effects. So obviously each of those are separate questions, but in terms of the factor that seems to transmit ER stress right now we think it's a small signaling lipid.
Moderator: Thank you.
(0:51:04) Question: And why do you think this phenomenon you describe occurs in MEF.
Answer: Yes, that's also a fine question to ask which is something that, you know, at the time we used MEFs just as one of the more clean and elegant models to address the question of what effects are—or what arm of the UPR seems to mediate the cytoprotection we observe. In terms of the why MEFs would be able to transmit ER stress and have gains in cytoprotection, that's obviously a larger question. (0:51:47) But I think it speaks to potentially the fact that this transmissible event was being that we're describing may be evolutionally conserved. I know that's kind of a bold statement but there was a series of reports that came from Andy Dillon's group, currently now at UC Berkeley where he used c elegans and was able to demonstrate that if he were to create ER stress in neuronal lines or neuronal tissue it affected other organ systems as well, meaning that there seemed to be some effect that there seemed to be some effect that was being transmitted from one cell line to another in c elegans that ultimate promoted longevity in those organisms. (0:52:37) So that's kind of a stretch to go from c elegans to MEFs and to say that the same phenomenon could be true. But if it were to be true it would suggest that this is a conserved event that probably was once used for good purposes as demonstrated in what it can do to c elegans and like a lot of other programming the cancer cells hijack it for their own benefit for survival. And it's really only when you can produce these stimuli which we think would only really occur in chaotic environments like the tumor microenvironment of during times of severe stress that these signals would occur that make it relevant. (0:53:22) So I kind of got off on a tangent there. But I think the reason why we ultimate use MEFs is because they are a clean model and they likely just have the programming innate in them as any other cell line does.
Moderator: Thank you, Dr. Rodvold. And we have time for one final question, because I want to give you some time for final comments. I want to thank our audience again and remind them that any questions not answered today will be answered via e-mail following the presentation. So here's our final question for today.
(0:53:51) Question: What do you think the implications of TERS are in relation to different lines of intervention like for example radiation or chemotherapy or even surgery for that matter?
Answer: Sure. So I think the point of our study, of the latter study that we talked about here is to provide a new line of consideration of how cells might adapt to standard lines of chemotherapy. Obvious chemo resistance is a topic that a lot of people are familiar with and we're trying to provide evidence that this cytoprotective effects might be through cell non-autonomous manners. (0:53:22) And so I suppose for chemotherapy the first point I would make is that if you're to intervene using standard chemotherapy you might also want to consider intervening against the UPR or ER stress or, you know, when the time does come that we identify that factor to intervene against that factor that seems to be endowing these features. (0:55:04) In terms of how it should affect radiation, that's a great question and it's something we've never really broached. So I would be really interested in those studies. And then in terms of surgery, I recall a few papers that have been produced in various groups. (0:55:29) I think one at M.D. Anderson whose name is escaping me to show that surgery obviously can cause a massive amount of stress as well on a patient and that stress in turn can actually do more harm than good. So it would be interesting to see if the stress associated with someone cutting into you and removing a tumor may in fact have affects in producing this molecule which kind of re-invigorate the transmissible ER stress phenomenon and in fact put a little bit of gasoline on the effects. (0:56:04) So I would say that that could definitely be an area of future topic as we kind of transition from our basic in vitro models to more clinically relevant in vivo models. See, I suppose that would be my final point on that.
Moderator: Thank you, Dr. Rodvold. Did you want to provide our audience with any final comments before we close today?
Comment: Sure, I'd love to. So I would obviously conclude by first of all reiterating this opportunity from Thermo Fisher Scientific to give today's talk. I do hope that if anything the point would be to say that we've tried to provide a new line of mechanism that might be occurring with the tumor microenvironment needed by ER stress and we certainly don't think this is the end all be all of how the tumor microenvironment operates but I hope it provides the audience with a new line of consideration. (0:57:04) And then of course I would love if anyone had any further questions on today's talk that as it comes to them I would love to field those, so feel free to e-mail me for any further curiosities that might strike you. I think my e-mail is attached but if not I'll just reiterate it as firstname.lastname@example.org. And again I'd love to hear your guy's thoughts or hopefully this will provide as a template for further investigation. So, yes, that would be my concluding thought.
Moderator: Thank you, Dr. Rodvold. And I'd like to also take the opportunity to thank Labroots and our sponsor, Thermo Fisher Scientific for making today's educational webcast possible. Before we go, I want to let everyone know that Jay's webcast will be available for on demand viewing through May of 2018. So receive an e-mail from Labroots letting us know that the webcast will be available for replay. Please share that announcement with your colleague who may have missed today's live event. That's all for now and thanks for joining us. We hope to see you again soon. Goodbye.
End Presentation: 58:22
Get to know Jeffery
Why did you choose cancer research?
Cancer is fascinating to me because of the unique challenges it poses: often acting like a foreign agent but originating from self, recruiting healthy cells to aid in counterproductive functions, adapting to the therapies thrown at it, and seldom being the exact same from one patient to the next. The enormity of these complexities make it such a challenge, it is hard not to be captivated by it.
What are your top three things to do outside of the lab?
Experimenting in the kitchen, travelling, watching/playing sports
If you could choose any other career what would it be? Why?
Conservation biologist; it is awesome seeing state-of-the-art biomedical techniques being leveraged to attempt to restore endangered or extinct species. What a tremendous impact that could begin to undue so many ecological missteps?
What role have the mentors had in your passion for basic research?
Significant. I’ve been fortunate enough to have mentors that have kept me in the game when I could have easily exited. It is all too easy to get dissuaded in science with bad results. To have mentors that not only contextualize setbacks but realign those setbacks as progress have buoyed me in more times than one. Their passion for science nurtured mine.
What is your favorite phrase?
“There are none so blind as those who will not see”
Which scientist current or past would you most like to meet and why?
Norman Borlaug. He is one of the unique upper echelon of scientists that through his innovation had enormous scientific impact on the world but through his compassion equally became a humanitarian, ala Salk. I would love to learn his thoughts on everything from CRISPR to where science should focus next to where it has so far missed the mark.
What are some small things that make your day better?
Coffee. Easily coffee.
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