Breaking Barriers in Spatial Proteomics with Single-Round Imaging

TRANSCRIPT

Welcome everybody and thank you for joining RareCyte for today's webinar, raking Barriers in Spatial Proteomics with Single Round Imaging. I'd like to introduce Tad George. Tad is the Senior VP of Biology at RareCyte and has been with RareCyte for about eight years. He has a long history of introducing breakthrough technology into research, clinical, and translational environments is part of numerous startup instrumentation companies in the past. He has worked closely with the engineering team at RareCyte to develop and introduce the Orion technology for translational spatial biology, which is the subject of today's webinar.

Tad George

Hello everyone. Thank you for joining our webinar about breaking barriers and spatial proteomics using the Orion system with single round imaging. So before we get going, I want to kind of introduce RareCyte. So we're a precision biology company that really creates breakthrough technologies focused on areas related to biopsies like liquid and tissue biopsies. And we design cell and cell instruments, reagents, consumables and services both for ultra rare cell analysis and retrieval, but also for highly multiplexed tissue analysis. And that's going to be the focus of what we talk about during this webinar with the Orion system. As most of you know, that study kind of tissue and spatial biology tissue really contains lots of different cell types. They have different functions, they're also in different functional states. And their spatial arrangement really dramatically can impact patient health status.

Unlocking Spatial Biology with Orion^™

So if you're going to be studying spatial biology, you're going to be using scanning and imaging technologies to get that spatial relationships resolved. But traditionally in this space, if you're working with fluorescence, resolving this complexity has been challenged by fluorescence overlap, also autofluorescence, which leads to techniques that really challenge throughput. So Orion technology is really focused on breaking that throughput barrier by doing really ultra-fast, highly multiplex multiplex tissue analysis in a single round. So really Orion is what we like to call spatial biology at light speed and primarily the main thing about the Orion is that we're able in a single round of staining and imaging to do 20 channels of data. But by doing that it means the technology is ultra-fast, which in academic core facility setting you can get 10 times the users per week if you're doing small studies. But if you're in the translational clinical space, it's really important to get through large numbers of samples to get statistical prognostic predictive data or mechanistic data. We like to say Kaplan-Meier curves for clinical studies. So ultra-fast if you do this in a single round, it's also really efficient in terms of running costs and also by doing everything a single round, the data quality is high because the tissue is preserved. There are several other examples we'll talk about as I go through this webinar that are listed on this slide, we'll come to in a bit. But the other thing that's I think really important to think about is the flexibility of the panel design, which we'll cover extensively during this webinar.

Any Tissue, Any Indication, 20 Channels in a Single Round

So upfront, really any tissue, any indication, 20 channels in a single round, we've done over probably 250 panels. There really is not any kind of limitation of the type of tissue or sample type that can be addressed. It doesn't even have to be tissue, it can be spheroids, organoids, anything, but mostly usually people are doing FFPE tissue. I do encourage you to look at our interactive page on our website. It gives you some nice examples. So these are just some interactive data viewing examples that span various tissue types and applications. For example, if you look at mouse ileum here, so this is, I think it's 16 plex mouse ileum. You can zoom in to see the high data quality that we get, but also it's whole slide and I kind of glossed over it. We can also do same section HE. So you see this little monocle here, we see these little goblet cells with that morphology aligned to the molecular information for the, if that's pretty easy to do in this case I don't think we used SMA, but you can definitely see the smooth muscle. So that's another nice feature of doing things in a single round is you can really get pathologists involved quite easily with the same section H&E.

It's not only whole slide, it's definitely whole study. So one of our customers actually did a 74 patient cohort colorectal cancer cohort. All of those examples are here. You can click on any of those and just like I showed you before with the ileum, you can zoom in and pan and see super high quality but also whole slide. I think on average these tissues were about five square centimeters and of course it's not just one sample, it's hundreds of samples, right? So that's kind of from a big picture.

Quantitative data for spatial biomarker analysis

The breakthrough for Orion in the field in terms and of course the data's fully quantitative so you can segment the images into cells which allow you to get molecular biomarkers and mean intensity for all the different biomarkers along with the centroid positions, which of course allows you to classify cells into different populations and leads forward into spatial biomarkers.

Case Study 1

So fully quantitative data, and I'll give a couple of case studies here. So the first one was a drug company looking to Orion to try to uncover what the mechanism of action was for their dual immunotherapy trial where they had found actually that treating patients with two of their drugs empirically reduced colorectal cancer tumor size prior to surgical resection, but they didn't really know what the mechanism was. So they enrolled 12 patients into this study, got pre-treatment biopsy as well as post-treatment surgical samples, processed them with this 13 plex IO-based Orion panel and then sort of measures sort of density of different cell types within the tumor lesion. So this is one of the patient's surgical resections. This is normal colonic mucosa. In yellow is the Pan-CK, white is T-cell. This is pretty normal architecture, but what's not normal is this swarm of T cells here to the right.

So if we pan further to the right, you can see this is where the aggressive tumor is. So lots of highly dividing tumor polyps that are being massively invaded by T cells and it looks like the T cells are doing a nice job of killing the tumor off. And as I mentioned, this is quantitative so we can look at this particular patient pre-treatment biopsy in blue post-treatment surgical resection in gold, and you can see that the treatment really caused recruitment of all the major immune cell types into the tumor lesion associated with the regression of the tumor prior to resection.

Case Study 2

Another case study, I kind of alluded to this, our customer at Harvard that did the 74 patient colorectal cancer sample. In this particular study because they had clinical outcome data, they could derive actually prognostic spatial biomarkers. This paper is published in nature but it was essentially 74 patients blocks where processed with this 17 plex Orion panel.

And really we simulated the Immunoscore®, which is quite is a very nice prognostic test for colorectal cancer, but they actually derived several hundred potential spatial biomarkers that theoretically outperformed the Immunoscore. So this is a nice example of using the system and the throughput to derive potential prognostic tests in colorectal cancer in this case.

Translational Spatial Biology

So from a big picture, Orion really unleashes translational spatial biology by kind of including all of these things which are necessary. First of all, the high quality is the foundation for trustworthy quantitative data. That's true of any platform. But Orion is ultra-sensitive, high dynamic range, good resolution image quality, but really that single round sample integrity helps quite a bit to give nice foundation for your quantitative results. Complete spatial context is really important for deriving a lot of those spatial biomarkers. You can see these are the cores drilled out of the middle here. We wouldn't have gotten or Harvard wouldn't have gotten those spatial biomarkers. They were just looking at the TMA. So that sort of whole slide ability was really important. You need the sufficient plex for the biomarker discovery and MOA work and fundamentally that throughput necessary to image those large cohorts for statistical power. So that's kind of the foundations that we have.

Panel Building Blocks

In terms of building panels, the building blocks that we provide are really centered around the Orion reagent portfolio. So we sell ArgoFluor conjugated antibodies, all of them are IHC validated and known to work on the Orion system. There's well over a hundred biomarkers available. We also sell off the shelf panels, 16 for human, six for mouse. And as we recognize, a lot of times there'll be biomarkers that are not in our, so we enable customers to make their own reagents with conjugation kits and services to customize biomarkers for their panels and there's a nice panel design tool to help configure custom panels for any application.

The other thing is because all of our reagents are sold as direct conjugates and individual vials, even our panels that way, so for example, if you were to start with ordering this type one diabetes panel, Q138. If I click on this button, it will show the 16 markers that come in that panel kit. But you don't have to take all of those reagents. You can say I don't really want CD20. You can remove that, put in say DC-Lamp and order that. So very flexible, easy to mix and match and build panels with the system.

Orion Panel Development Workflow

In terms of the panel development workflow, it's super simple. You use the panel design tool to select your biomarkers. If there are no custom antibodies required, really the panel designer will assign those channels for you. Then you order those reagents and you verify the panel performance at your site in a simple titration on your intended tissue. The reason this is important is in production we validate all of these reagents against control tissues that we know work. Obviously we don't validate against every single tissue type. Sometimes the abundance or the titration needs to be adjusted a little bit before begining your study. But this is a very simple experiment to do just to verify the performance on your tissue type of interests. In cases where you have custom biomarkers, it's really simple. You basically just validate that reagent versus IHC. Really the main job there is just to validate the clone that you ordered from the vendor and the control tissue block that you're using actually gives you the results you want by IHC.

Once you've established that you conjugate your clone to an Argo Fluor to an epitope tag. One thing that's really nice about the ArgoFluor conjugation is either small molecule organic dyes, very similar to what's done in flow cytometry and we really haven't had a case where we could not reproduce an IF with the Argo Fluor conjugated antibody, the results we got in IHC. So this is super high rate of success. Then you just validate that conjugate in single plex titration versus IHC. This takes probably a lab about two week process to validate up to four custom conjugates in parallel. So really easy to even customize your panels.

Orion Sample Testing Workflow

Once you have your panel running and you want to do your study. Super straightforward, you can stain, we often will stain up to 24 slides per day off the instrument. There's no staining process done on the scanner, so that's done separately from the scanners that's done in your scanning and parallel to staining. There's not really much of a limitation on what you can put on the slide, so large and or multiple specimens per slide. Another nice thing is that because the fluors are very photo stable, once you've done the staining, if you want you can go straight to scanning or you can bank them, which is really convenient for a multi-user site because multiple labs can be preparing their samples and then the scanning can be scheduled anytime that the scanner's available. And of course we scan 20 channels per round. So very simple, straightforward workflow and the output of course is fully quantitative.

ArgoFluor Dyes, Lasers, and Narrow Emission Bands

In terms of the technology and validation. So I didn't really tell you how we did what we do, And really the Orion was an adventure in sort of finding the best set of dyes and instrumentation attributes that allowed us to image at high plex with direct conjugates with sensitivity that enables you to see even low abundance biomarkers, which is critical. So that was really made possible by several things. One, normally with fluorescent scanners, sensitivity is a major barrier, so there's usually wide excitation and emission bands to get as much excitation and light as possible. We're trying to do 20 channels at once. You typically want narrow band excitation and narrow emission bands. So of course we went to lasers. So we have on the system nine high powered lasers in order to give you lots of sensitivity and very specific excitation.

The narrow band filters, what's nice about them is in the factory we're able to tune them with one nanometer resolution between 425 and 900 nanometers. This really allowed us to screen. We actually screened over 300 small molecule organic fluors for brightness, photo stability and spectral spacing. And we found the 17 that worked well together in that space and went to a laser manufacturer, custom built the laser to match the pallet of ArgoFluors. As I mentioned before, these dyes are very small molecules attached to antibodies with simple amine conjugation chemistry. All it requires is knowledge of how to use a pipette and spin columns. They're very light touch on the reagents. So as I mentioned, we typically always are able to reproduce our IFC results. The other thing, they're stable for many years after conjugation enabling single lot multi-year study. So the technology is really quite a bit of work went into achieving the throughput that we're able to with single rounds.

Spectral Extraction

Of course we have a spectral overlap, so spectral extraction is done. The other major advance we had is a nice algorithm that automatically will detect the overlap and remove it from the crosstalk channels. So this is an example of say CD3 and CD8. These are spatially colocalized and spectrally overlapping in this particular panel. So CD3 is on Argofluor 572, CD8 is on Argofluor 602. These are 30 nanometers apart, so there's lots of overlap if we do not extract. But the system has an automated algorithm that will extract the crosstalk and essentially get, essentially flow like plots where you can see here CD3 versus CD8. We see that the CTLs are here and the non CTLs are down here. You can see before extraction, there's a few cells over here, CD3 cross selecting to CD8 after extraction completely removed. So that was another major advance to enable the single round.

Single-Plex IF vs IHC

I mentioned validation, super easy. This an example from SOX10. We essentially established the pattern in IHC with clone SB267. Labeled that with an ArgoFluor stained an IF on the Orion and then in serial section the pattern match. That's kind of how we validate our reagents. Also, the nice thing of course the IF you can look at specificity as well in this case IL-4I1 with IHC, nice punctate stains. It's typically expected to be in macrophages, but of course single plex IHC, we don't actually know for sure if those are within macrophages. So when we label the IL-4I1 with the ArgoFluor, we could also do a mini panel with CD68 and CD163 and verify that punctate yellow was residing within macrophages. So these are the types of things you do to validate your reagents.

Accuracy and Precision

System is super accurate and precise.So biomarker detection usually matches or outperforms IHC for challenging biomarkers. In this case we had a pathologist score immune IC for PD-1 in colon. This is really dim PD-1 staining. We had no problem seeing it with directly conjugated reagent PD-1 here and verified that we have more cells that were PD-1+ and we verified that they were T-cells by co-staining with CD3. PD-L1 here also in the colon, medium sort of expression by IHC, very robust in the IF. One of our CROs actually validated a 16 plex Orion panel that we developed and transferred to them against the Tyramide based system and got basically similar sensitivity for MFIs and percent positives. Again, Tyramide is a very powerful technology that sometimes gives a hundredfold amplification. It's necessary for regular scanners, not for the Orion. It's very repeatable and reproducible. So very suitable for running large studies.

Onboarding Overview

And another key point is that because it's so easy for us to develop panels, one of the key features of adopting the Orion system is part of our onboarding program is that we actually endeavor to design and promote success with a customer panel shortly after training. So the key components of onboarding is once an instrument is ordered, our customer success team will work with your site to prepare the site to ensure the instrument's network and the laboratory is set up to support installation and training. We do provide training with a standardized 7-plex training panel, but we typically also transfer a customer specified panel within two weeks of training completion. So this is, we try to shorten that sort of Orion order to first experiment by providing that kind of onboarding success.

Orion

And with that, that's an overall summary of Orion. As I mentioned, we like to call it spatial biology at light speed by having that sort of single round approach. And yeah, let me know if you have any questions. Thank you.

Q&A

Alright, thank you all for your attention. I did get at least five or six questions so I will go ahead and answer those. Let me also share my screen in case we need to look at some of the slides as we answer these questions. A couple of these are related to sort of developing custom panels.

So one question was do we offer customer antibody conjugation kits or services? So a couple things here. So yes we do. We sell the dyes, the conjugation of antibodies to our ArgoFluor dyes are very simple because they're small molecule dyes, you just need pipettes and spin columns. So we do sell those kits for you to label them in your own lab, but we also do perform or provide custom biomarker services if you want us to develop those for you. One of the nice things about the conjugation services is we actually will not only do the validation against the tissue type that you tell us to, but we'll also transfer some of that reagent to make sure you can reproduce it in your lab. But we do also train you how to do it as well and it's very easy to do.

The other question is sort of how long does it take to develop a custom panel? Really I think the main fork here is whether or not you have to develop your own customs. If you don't, as I mentioned before, it's super easy. It's basically order the reagents and titrate it because one of the nice things about having all of the reagents validated on Orion, we already know they work on the Orion. That cuts out a lot of the trial and error that you might have if you were looking for new reagents. If you're doing customs. The main work here really is that validation of that clone and control tissue by IHC. So we really highly recommend not skipping that step because a lot of times you'll see nice IHC data from the vendor. But it may not be done on your tissue or with the same management retrieval, et cetera. So really once you've established a clone and a controlled tissue IHC, the rest of this is actually quite easy.

Another question is a little bit related to the signal processing here. So it's kind of when do we actually do this signal extraction, right? So as we mentioned here, we did nice spectral spacing, but you do have spectral overlap. The kind of image processing pipeline is once the scanner scans your slide, it generates a raw image file onto the hard drive. That is then processed. There's some image corrections that are done upstream of extracting out the spectral overlap. So that's done post-acquisition but done kind of in parallel to scanning. So it's definitely done after scanning and you do have the raw image which is unextracted if you should need to go back and review any of the extractions. So it's done post scanning but definitely kicked off in parallel while you scan your first slide. That processing happens while you're scanning your second one.

There was another question related to this sort of maximum plex. So we get asked this a lot, will we ever go beyond this 20 channels space? I think I kind of glossed over it here, but if you notice this classification scheme, this was actually done on a 51 plex Orion cyclic experiment. So really I think if going beyond 20 channels you can definitely cycle on the Orion and a lot of people are starting to do this where they kind of have these discovery level panels that might be 50 plex that inform a single round panel somewhere, 12 to 16, for the translational work. So that's kind of how we think people will sort sort of go beyond 20 with the Orion is just by doing maybe two rounds or three rounds to get that high level plex. And because otherwise I think this relatively crowded space here, we could certainly try to add more fluors, but I think you start to impact a little bit of data quality.

And I think one other question was related. We do, I didn't mention this, but we have a dedicated autofluorescence channel here. So we actually deal with autofluorescence in two ways. One is that when you're preparing your sample prior to staining, the system is so sensitive that we actually do quench autofluorescence, which knocks autofluorescence down probably about 90- 95%. We do still observe it on the Orion, but it's collected into its own channel. So we have a dedicated 445 laser, this is an autofluorescence trace here. We collected into this channel and because we collect autofluorescence, we could use that as a basis to subtract it from overlap into all the other channels to give really nice low background in the ArgoFluor channels. The system does also have a secondary auto fluorescence channel. Some tissues will have more than one distinct signature, so you can do more complex auto fluorescent subtraction as well.

And I think that was all the questions that were typed in.

I have another, actually several questions just came in. So one of them, is it possible to do intracellular staining? If you want to see cells that are secreting certain cytokines, you can certainly do intracellular staining typically in tissue with cytokines that because they're usually sort of secreted, it's not so easy to stain the protein because it's not really concentrated in the Golgi. One of the ways that's done kind of in live cells is you'll block the expression of cytokines that'll build up in the Golgi and stain. That's not really practical to do in tissue. We do have people sort of doing same section RNA and protein. There's some sort of endeavors to do that on the system. That's probably the better way to look at cytokines, I would say by RNA.

And then there was another question related to what are my options if I'm not ready to purchase an instrument? Do we offer services? Yes we do. We certainly offer both instrument sales and support, but definitely services. So certainly reach out if you have any questions along that route.

And another question, do we have any examples of brain tissue staining or pre conjugated seen as antigens of interest? So yes, brain works very well with the system. We have a couple customers doing glioblastoma and normal brain works quite well. It is true that I would say our biomarker list is more IO bias. It's mostly because the decision that we make about putting reagents into the catalog is dictated entirely about what our customers are doing. We have a couple of customers where we're transferring neuropathology or CNS based panels, so there'll be quite a few brain oriented and CNS oriented biomarkers available in the catalog soon. But even if they're not, it's actually quite easy to develop your own reagents.

Okay, I'm checking Rob to see if there are any other questions.

No, it looks like at the moment that's all the questions. Did anybody else like to submit a question before we wrap this up today? Okay, Tad, it does not look like there are. Certainly everybody, thank you for joining us today. If you have any questions, feel free to email us at info@rarecyte.com or visit our website. And if you want to come back and see this webinar again, we will have it on demand later today for you to view again. Again, thank you very much everybody for joining us today. Have a great day.