In vitro and In vivo studies of metastasis often focus on the first step ? local invasion ? however, it is clear that the number of cells that successfully metastasize is significantly lower than the migratory fraction at the local tumor. What is the underlying cell biology of cells that successfully disseminate and can those characteristics be predicted from knowledge of the primary tumor?
Created by Sara Gosline sgosline sounds good, just signed up. Signed up for a call Thurs at 3pm - hope others can join I will be enroute to airport but hoping to join the call. Also please check and edit the doc if you can... Just a reminder, meeting tomorrow at 2pm @gabor @cncurtis.... @lmheiser and @jiyang.yu will be joining us. I think there could be overlapping interests with complementary innovative strategies and skills. Looking forward to our discussion.
Josh
Gabor here you go...https://docs.google.com/spreadsheets/d/1lhQFZeqU6jzmKNABSISRsaGFHi34tgk6cKk4w3JPO8M/edit?usp=sharing
Thanks Josh, I marked it on my calendar. Looking forward to it!
On the Teleconferences site the last link I saw was for the week of May 22 - is there another place to find the spreadsheet? ok. I just signed up for the last open time slot at 2pm on Friday. Friday at or after 1 pm works for me. Let us also try to work on the doc before the call if possible. Should we try to set up a call this week? I am traveling but could potentially do Tues or Fri. Otherwise, we can iterate via on the doc online. Here is a link to a google doc https://docs.google.com/document/d/1NJdE8ARjAB_UGVoegdAmN0tEj7tMmS6BK3-s3Y8Xm7g/edit?usp=sharing
It is bare bones but should get us started.
Josh @snyderjc1 -- Your best bet for shared editing is going to be a google doc. However, you can also start a Project Folder if you navigate here:
https://www.synapse.org/#!Synapse:syn8063299. You'll need to become a Certified Synapse user (short quiz) to upload documents, but if you create a folder you can keep some things together and also link the Google Doc from that location so you don't have to keep navigating to the end of this long thread. @gabor @cncurtis1 You can either use your own Google doc and share or create a folder in the [shared document folder](syn8063299) and upload files there. You can set permissions specifically for members of this group. @nzahir @gabor @cncurtis1 Is there a space available for a shared document and editing capabilities so that we can start writing up aims etc... If not I will provide a link to a google document so we can finalize the goal, aims, and key experiments. It would be great to get this done soon and maybe have a phone call at the end of next week?
@gabor, @snyderjc1, @cncurtis1; this sounds interesting, but I have committed to a team on a different topic.
Would love to measure cells though, even if with an informal role.
In the table under notes, click on the link for each respective week:
https://www.synapse.org/#!Synapse:syn9693597
I think it would be great to form a team around the topics we discussed last week, a potential title being along the lines "From cellular heterogeneity to evolutionary dynamics of cancer metastasis". Could someone please help me access the notes taken last week? I still cannot find where they are. Yes indeed 5/18 at noon. Sorry about that! 5/18 at noon :) Looking forward to our discussion.
Booked a teleconference slot on Thursday, 5/17 at 12 noon. Anyone interested, please sign up. @scarc @cncurtis1 @gabor this works for me. @snyderjc1 @scarc @cncurtis1 Thursday at noon? @gabor I would prefer Thursday or Friday. I am unavailable wed. @snyderjc1 @cncurtis1 So should we book Wednesday at 12 noon or 2 pm?
I am available Wednesday (all day) and thursday morning. @snyderjc1 (Josh), @cncurtis1 (Christina) Wed-Fri any time works except for 3 pm on Wednesday. Maybe others could join, @scarc, @jiyang.yu, @subhode, @jungwoo? @gabor we had a great conversation this afternoon. It sounds like the end of next week should be another good time for @cncurtis1. Let's get a time and I will sign us up.
Sounds good. I have signed up for tomorrow as well. @gabor maybe we can talk again Wednesday-Friday next week.
I could do noon on Friday and indicated this on the form. Hopefully, a few others can join and we can follow-up again next week.
Unfortunately, Monday does not work for me, but I could join from Wed on. Unfortunately Friday does not work for me. Could we try early next week? Say, 1 pm on Monday? @cncurtis and @gabor it looks like the 1pm slot for this Friday is booked. The noon slot is still available. Will this work? How about a call at 1pm Friday then? All of these times work for me as well @gabor
For sure. I will be around and will keep my schedule open. I will send an email to you to set up a time. @snyderjc1 On the off chance you are around, I thought I would mention that I will be a Duke on Monday for a meeting and should have some time.
Yes, the models can be applied to animal models and other cancer types (we have applied this to multiple solid tumors).
I could do a call Thurs between 2-4pm EST or Fri 1pm or 3pm.
@snyderjc1 For me 1 pm on Monday, 12 noon on Tuesday, 12 noon or 2 pm on Wednesday would work fine next week.
@cncurtis1 Would any of those times work? Would your models be adaptable to animal models of other cancer types? Anyone available early next week to further refine the question and solutions... This is a very interesting concept and discussion of experimental approaches.
Potentially relevant to this, we have developed a fully spatial computational model of tumor growth (simulating up to 80B cells) and statistical inference framework that enables comparison of 'virtual' tumors with patient epigenomic data within an evolutionary population genetic framework. This approach enables the inference of patient-specific parameters such as the timing of metastatic dissemination and patterns of mono vs polyclonal seeding. We have applied this to a cohort of clinically annotated paired primaries and metastases and have assembled several such cohorts. We previously described the application of our spatial computational model to single gland and multi-region genomic data derived from colorectal adenomas and carcinomas, resulting in the description of a Big Bang model of colorectal tumor growth (PMID: 25665006). We have since extended this approach to model different modes of tumor evolution (Nat Gen in press) with extension to the metastatic setting. We thus have some predictions regarding the drivers and dynamics of metastasis and some experimental systems to test these in. As these approaches also enable us to infer the levels of selection operative in different tumors and to quantify subclone fitness , they could potentially be combined in a powerful way with the platform Josh has developed. It would be interesting to discuss this further.
I am traveling this week, but could do a call Wed-Fri. Many apologies for not being able to attend the meeting...I hope that it went well. I am available most of next week if there is a second call in the works.
@snyderjc1, could you join us at 12? Friday noon is good. PMID: 28102402 and PMID: 26436482 @gabor Friday noon is good. For me, it would be PMID: 25339351 and PMID: 23150542. @jungwoo This week, Thursday 11 am and Friday 12 noon work for me. Maybe we should try Friday 12 noon and send PMIDs for 1 or 2 papers for everyone to glance at before the meeting. We have a paper from @snyderjc1. For me it would be PMID 23385595 and PMID 24395801. So how does Friday 12 noon work for others? Please also send PMIDs if you are available. Hi @gabor, Yes. I think we should start in a teleconference. For this week, Thursday 11am, 12pm, and Friday 12pm, 1pm and 4pm slots are good for me.
@jungwoo @scarc we can monitor biomaterials and bone by multiphoton microscopy in vivo, ex vivo and in vitro @snyderjc1 We have been developing dox-inducible systems for precisely tuning the expression of genes. Could we combine this with the Brainbow system and measure fitness versus expression levels of, say WT or mutant LGR5? We would have to turn on a tunable expression system instead of a constitutively expressed gene. Combine this with @jungwoo 's tunable biomaterials and @scarc 's stiffness measurements and we may have an interesting system for studying the effects of intracellular and extracellular factors on cell fitness and stiffness. Maybe we should discuss in a teleconference? Hi @gabor, @scarc, @snyderjc1, and @jungwoo,
It's great to see everyone's ideas coming together. To move it along further I suggest signing up for a teleconference this week. The signup sheet can be found [online](https://docs.google.com/spreadsheets/d/1hY53jRaqoBMnb9HhuE8dN3k4ejX37gG-4C4pattxkuM/edit#gid=1701101959) and as many of you can sign up for as many topics as you'd like. Just put the 'call topic' in column F. The workshop organizing committee is ready to host and facilitate these calls as need be, all you need to to do is call in the number in the column A. You can use this forum or communicate offline to find a time when all of you can call in to discuss further.
The sooner you circle around a project the more time you will have to prepare for a successful project in June. Please feel free to ask me if you have any questions about the process.
-sara We also have data on three different genes, gene fusions, and can breed to homozygosity as a test for gene dosage (i.e. het vs hom). It would be simple enough to change out our promoter for a dox-inducible promoter if we really wanted to alter the concentration of a protein...also tandem proteins tied together via '2a self-cleavage sites would work as well.
PMID 28275053 was interesting to read. So it is possible to define fitness in vivo for up to 2 mutants + wild-type version of a given gene.
An interesting question to explore is fitness versus expression level (instead of mutation). Basically, asking how does fitness depend on the concentration of a certain protein (say, an oncogene) without any additional mutations? Could we extend or modify the method to address that question? Thanks, I will read PMID 28275053. This is cool! Yes they are...we can build any gene model and induce its expression in any tissue/cell-type...so far we have extensive data for the intestine and breast...including precancerous lesions and tumors. We also have some limited data on the pancreas.
@snyderjc1 Are these techniques adaptable to other cell types besides intestinal cells? Mapping gene expression to cellular fitness is fundamental to understanding metastatic progression. We have validated and optimized a methodology for visualizing the effects of multiple tumor driver genes on cell fitness in vivo and in ex vivo organoids. We can monitor up to three driver genes coincidentally and visualize their effects on the stem cell lineage with single cell resolution. We now have data demonstrating that we can identify key genes that can permit an intestinal stem cell to live outside of its microenvironment (unpublished). We recently utilized the the founding principles of this technique to show that it can be used for quantifying cell fitness within the intestinal stem cell. Our current efforts now make this technique scaleable and robust. (PMID: 28275053 )
Our technological platform would enable us to track this infrequent and yet more fit cells in their native environment and test the drivers of this biology.
good @scarc, maybe you can assemble a team and define questions and approaches.
Thank you @JAguirre-Ghiso; and sorry for the late reply.
The questions you pose indeed capture the essence of what we were discussing. I went through the other attendees' proposals and it seems that these question (especially #2 and #3) could benefit from the work and input of @alexandra.naba, @dgilkes and @aedinc. It may be useful to structure these questions around observations from the patterns of metastasis.** Some ideas:**
**1- **Bone marrow seems to be for the most part suppressive of DTC expansion in some cancers irreversibly (HNSCC, gastric cancer, melanoma) in others growth can occur but after long periods of dormancy (breast, prostate). How do the physical and tension constraints impact this biology? Same probably applies for brain, where metastasis appear late after other interventions have taken place.
**2-** Liver and lung can present with metastasis but one can find single cells in the same organ that are non-proliferative. Are there defined micro-environments even within the same organ that are display different stiffens or cellular/ECM components that dictate DTC fate? Liver may be amenable for 50 um depth imaging Giuliano.
**3-** Does the level of stiffness in the primary tumor pre-program the cells for a specific behavior once they arrive to the target organ? In other words, are these physical constraint and stiffness variations in the primary site determinant of DTC fate after extravasation? This may be measurable with imaging but it may be fun to layer on an epigenome or signaling analysis contributed by other participants.
I am concerned though about the transparency of the implant biomaterials. Did I understand correctly that to modify stiffness you introduce beads that make the sample opaque? our ability to image in non-transparent materials is fairly limited (~50 to 100 microns tops in tissue). Our implantable biomaterials are mechanically tunable. Above described experiment is doable with high power imaging tools. Implant biomaterials can be explanted and the culture can be maintained ex vivo, which gives many experimental flexibilities but compromises keeping in vivo context of the tumor microenvironment. Looks like a good plan! @jungwoo , could your tissue engineering platform replicate conditions and phenotypes described by @jjbravo? If validated against in vivo, they could facilitate our analysis. Thanks Javier. Our technique uses Brillouin light scattering spectroscopy to probe local mechanical properties. We couple it to a confocal microscope to make it high 3D resolution. Here's link to a paper explaining (http://www.nature.com/nmeth/journal/v12/n12/full/nmeth.3616.html), and I can certainly tell you more if you are interested. We have never done cells in vivo inside tissue, we can only penetrate ~50 microns in non-transparent tissue. If interesting things can be learned at shallow depths, we could certainly try; otherwise, it is easier to characterize cells in 3D matrices/systems. Can the phenotypes/conditions observed in vivo be replicated faithfully in vitro? @scarc very good point! One feature of highly motile cells in vivo is their ability to move in an single cell ameboid fashion by forming actin-rich pseudopods. This type of movement allow them to change quickly direction and follow chemotactic cues. Cells in vivo have also other types of movements such as collective migration, multicellular streaming. We have characterize the features of single cells and multicellular streaming in vivo. For example, we know that multicellular streaming with macrophages contributes to the directionality of tumor cells towards the blood vessels. However, we don't fully understood how these phenotypes contributes to determine the final fate of a tumor cell towards a particular niche. That point will be interesting to know because they may facilitate interaction with different niches and tumors with certain type of motility patters may seed in one or other organ. In my lab we have done in vivo intravital imaging of mammary, melanoma and head and neck tumors and we can identify these different phenotypes, so they seems to be conserved among tumors. I agree, these cells have an exquisite ability to adapt to different types of ECM contexts . It will be interesting, as you mention to measure the stiffness of these cells in these different types of movements in vivo. What are the characteristics of your imaging technique? In terms of molecular features we know that ameboid cells for example use the RhoC/cofilin/ pathway among other actin related mechanisms to dynamically reorganize their cytoskeleton during migration but how other pathways such as pathways involved in membrane plasticity and stiffness we don't know.
Happy to discuss more!
Javier Thank you Sara!
When I first read this thread, I was expecting the title to be "only the soft survive", because several studies have shown metastatic cells to be softer than non-metastatic ones. And the reason behind this would be that in many of the metastatic processes, cells need to squeeze through matrices, pores etc, so softness would be an advantage. On the other hand, we have a microscopy technique that can measure cell stiffness without contact and we have observed that metastatic cells actually change their stiffness quite dramatically during extravasation for example. Which is why I wonder if, given the many diverse challenges a cell has to face to successfully metastasize, maybe one of the properties that successful metastatic cells need to have is the ability to physically adapt to varying microenvironments. It would be nice to connect this physical property to genetic or molecular characteristics of the cells.
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