10x Genomics and Single Cell RNA Sequencing: Interview with Philip Bischoff and Angela Churchill
Single cell sequencing holds the key to an unprecedented understanding of the tumour microenvironment. However, its potential has historically been constrained by the limited range of available sample types for analysis and the challenge of capturing high-quality data. The prospect of acquiring reliable data through single cell analysis of formalin-fixed paraffin-embedded (FFPE) samples is particularly captivating, especially for retrospective studies involving archived clinical samples.
Oxford Global recently had the pleasure of conversing with collaborators from 10x Genomics and Charité University Hospital. Dr. Angela Churchill, the Product Marketing Manager of Single Cell Applications at 10x Genomics, is dedicated to strategizing and executing the potential of 10x's single cell platforms. Dr. Philip Bischoff, a resident pathologist and clinical scientist at Charité University Hospital in Berlin, played a pivotal role in a project that employed 10x's technology for single cell analysis of FFPE samples.
Dr. Bischoff's educational journey took him through medical studies at Charité University Hospital in Berlin, followed by residency at the Institute of Pathology at Charité, which he started in 2018. Since 2020, he has been a fellow of the local Clinician Scientist Program, allowing him to dedicate significant time to research. Dr. Bischoff's research centres on using single cell RNA sequencing (scRNAseq) to delve into the intricacies of lung tumours, with a specific focus on the tumour microenvironment.
We had the privilege of taking a moment from the schedules of Drs Bischoff and Churchill to pose a few questions about single cell analysis in clinical research.
So Philip, could you please describe your current research projects?
PB: Our recent study was based on the first protocol - published last year - that showed how to isolate single nuclei from FFPE tissue for single nucleus RNA sequencing. We were very excited when we found out about these protocols because this has been a major limitation in single cell analysis so far.
But before we could start bigger projects using FFPE tissue for single cell analysis, we first wondered, are the results of FFPE sample data comparable to fresh sample data? Or, is there a risk of losing important information when only looking at the FFPE sample data?
So in our study, we systematically compared different tissue sample types. We collected fresh and snap frozen tissues of three non-small cell lung cancers. Then we took tissue samples of the corresponding paraffin blocks after these have gone through all the routine procedures at our Institute of Pathology. We compared the fresh and the FFPE sample data and looked into the quantity of different cell types, gene expression patterns, patient specific characteristics, and altogether, got very encouraging results.
How did you first become interested in studying this field and using single cell?
Well, as a pathologist, you see every day that tumours are very complex tissues. You have tumour cells which exhibit different morphologies, but you also see infiltrating immune cells and stromal cells. If you're doing it the conventional way, all this complexity of the tissue is more or less lost when you move on to molecular methods like gene expression profiling. So I've always found it very interesting to use methods that preserve this complexity. This has only been possible for a couple of years now: gene expression profiling via scRNAseq.
What, in your opinion, are the benefits of using FFPE rather than a fresh tissue sample?
Well, when you use fresh tissue in single cell studies, you can only prospectively collect your tissue samples. That means you start collecting the tissue when you start conducting your study. For this, you need a collaboration partner in the clinic who talks to the patients and obtains the informed patient consent. Then you have to ensure the quick transport of the tissue samples from the surgery to your lab, and the tissue should be processed on the same day.
So altogether, this not only requires a lot of time and effort, but also limits the number of samples you are able to analyse in your study. This is very different when you're using FFPE tissue which gives you a lot more flexibility in your research.
FFPE tissue has been formalin fixed and paraffin embedded, and this is the standard method to preserve tissue in pathology. This way, it can be stored for a long time and under very simple conditions. You just have to keep the FFPE blocks dry and at room temperature, and they can be archived in pathology for many, many years.
So, when you use FFPE tissue for your single cell study, you can use retrospective samples which have been collected before you start your project. You can multiplex your samples, analyse a lot of samples together in batches. And this makes single cell analysis much more time and cost effective.
What was the technology that you use to analyse the FFPE? And why did you choose that technology in particular?
For dissociation of the tissues, we used a former version of the FFPE tissue dissociation protocol published by 10x Genomics. We also adapted this protocol a little bit and included some steps from the snPATHO-Seq protocol, published by Luciano Martelotto, at the same time.
I don't think there's a one-size-fits-all solution for tissue dissociation, you may have to adapt your protocol according to the type of tissue you're analysing. That being said, in general, your tissue will be dissociated using an enzyme mix and we used a GentleMACS dissociator for mechanical dissociation.
In addition to the 10x Genomics protocol, we also added a FACS step to remove debris from the nuclei suspension. And then we subjected the single nuclei suspension to the Chromium Single Cell Gene Expression Flex, loaded the single cell suspension on the Chromium X, prepped the library, and sequenced on an Illumina sequencer.
My next question is for Angela: could you tell us a bit about the Chromium Single Cell Gene Expression Flex, when was it launched, and what's been the motivation behind its development?
AC: Gene Expression Flex was launched in 2022. One of our drivers for its development is to increase the access of single cell sequencing to researchers across different applications.
So with Flex, we had the opportunity to address a number of barriers that have limited the accessibility of single cell analysis to researchers. This has included sample acquisition and logistical constraints, difficulties to process and generate high quality data, and also the ability to easily scale at a lower cost.
Gene Expression Flex enables the fixation of cells which allows for these optional stopping points. And it provides the ability to store or ship your samples, batch them, and then you can proceed with them at a later time. This is advantageous for prospective translational studies where researchers don't always have control over when the samples are available to be processed.
Philip, what are some considerations when conducting a translational research study that involves prospective collection of fresh clinical tissue samples?
PB: When you prospectively collect fresh tissue samples for a study, you first need a collaboration partner in the clinics who identifies and talks to the patients. Here, you also have to plan how many samples you want to analyse. At the same time, you need to know how many of the patients you're studying will be actually undergoing surgery over a certain period of time at the hospital you're collaborating with.
Another very important point is that at the time of surgery, when you want to collect the tissue, you often don't have all the information required for identification of suitable samples. For example, when studying lung cancer with EGFR mutations, you very quickly find out that, at the time of surgery, the mutational status has very often not yet been determined. You just don't know at the time of surgery if this is an EGFR mutant lung cancer or not, and this is one of the limitations which you do not have when you're using FFPE samples.
When you're using FFPE tissue, you rather collaborate with the department of pathology, and you can screen the pathology archive for suitable tissue samples. There, you can specifically select tissue samples according to tumour characteristics, such as the mutational status of the tumour. You can also select tissue samples according to clinical characteristics, like patient survival or therapy response, this is information which just isn't available at the time of surgery, but only months or even years afterwards.
And how comparable was the single cell analysis of the FFPE versus the fresh sample data?
So in general, we were very surprised how adequately comparable fresh and FFPE sample data was. In both fresh and FFPE sample data, we obtained all the cell types we expected; we didn't systematically lose any. But we still observed some shifts. For example, we obtained more stromal cells, and in particular, more fibroblasts in the FFPE samples, which is possibly due to different tissue dissociation approaches.
When we looked into gene expression, we found a very high correlation of cell type marker genes and gene signatures of relevant oncogenic pathways. But when looking at individual genes, you have to be a bit more careful because we observed some variability when looking at individual genes which are expressed at lower levels. So, when you stick to looking at gene signatures or highly expressed individual genes, then you can observe very robust, patient-specific differences to a similar extent in both fresh and FFPE samples.
When you were doing this comparison, was data quality or reliability impacted between the two different methods?
So, we observed a slightly lower number of genes and lower number of UMIs (unique molecular identifiers) in the FFPE samples, and I guess this is due to different kit chemistries and the lower RNA quality in FFPE tissue compared to fresh tissue. But we did not observe that this had any negative effect on data integration or any subsequent analysis.
Another point that you have to keep in mind is that you have different artefacts in different types of tissue samples. The FFPE tissue rests at room temperature for a couple of hours until the formalin fixation is completed, so this can introduce some tissue artefacts. But on the other hand, the fresh tissue is mechanically and enzymatically dissociated when the cells are still viable. So this procedure also can induce stress signalling pathways. In fact, in our study, we found that in the fresh sample data we observed marginally higher scores of dissociation stress-related gene signatures compared to the FFPE samples.
Does processing and storage have an impact on the results as well? Do you think that preexisting samples that have been stored in pathology archives are accessible for scRNAseq?
This is a very good question. In principle, I would say yes, they are accessible because the FFPE tissue blocks we used in our study underwent the regular procedure at our Institute of Pathology. This involves being processed in the pathology lab and being stored at room temperature, in our case, for four to five months. But, if you have tissue blocks that have been stored for longer, then I can imagine that there would be a higher degree of RNA degradation.
And also, you have to consider the different types of surgery specimens. When you put a surgery specimen in formalin for fixation, then the formalin diffuses into the tissue over time. Therefore, in the centre of your surgery specimen you will have lower tissue quality and consequently, lower RNA quality. In the end, the faster the tissue has been formalin fixed, the better I also expect the RNA quality to be.
What applications or research questions do you see single cell on FFPE opening up for in the future?
First of all, since many thousands of FFPE tissue samples are stored in pathology archives, single cell analysis of FFPE tissue unlocks a very large resource of new samples. For me as a pathologist, it's very important to be able to have a look at the tissue first - which is possible when you're using FFPE blocks - and this allows you to specifically analyse distinct tumour regions, for example, the invasive front of the tumour. This can also, in a very nice way, be combined with methods with a higher spatial resolution, for example, the Xenium platform by 10x Genomics.
From a clinical perspective, I think single cell analysis of FFPE tissue really helps to move the single cell field forward to more translational studies, because it now allows us to address very important clinical questions. For example, now you can specifically select patients who received specific targeted therapy where some of these patients might have responded very well to the therapy and others did not. And now you can study the differences between responding and non-responding tumours on the single cell level. This enables us to identify novel biomarkers to help clinicians in the future predict therapy outcomes and further personalise tumour therapies.
Angela, please could you tell us a bit more about the Chromium Single Cell Gene Expression Flex protocol and what the future developments of it might be?
AC: At 10x Genomics, we continually strive to innovate and improve our assays and also expand on their capabilities. So Flex, from a technological view, presents an enormous opportunity for us to extend and create new product capabilities, to hopefully enable researchers to further their biological discoveries. For Gene Expression Flex overall, we recently launched a new capability to profile both gene expression and protein expression from the same cell at any scale. This strengthens our leadership position in single cell multiomics and emphasises the capabilities of the Flex assay.
Our final question is for both of you: how do you think this technology will shape your work - and the greater field - going forward?
AC: I think that thanks to the expanded sample access with FFPE, it will be really interesting to see how single cell sequencing will be applied to these retrospective studies. It'll be great to see the discoveries that can be made and the new research questions that can be asked as the technology unlocks these different sample types.
PB: Yes, I totally agree. And I think that single cell analysis is now much more easily accessible to a lot of translational and clinical researchers, and scRNAseq of FFPE tissue will be applied in many more translational studies.
More Resources:
- Preprint: Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue
- FFPE Sample Analysis - Access the archives: Single cell, spatial, and in situ for FFPE
- Webinar: FFPE success: single cell insights from tumor biopsies
- Blog: Answering your questions about single cell analysis in clinical FFPE samples
- Blog: Behind the preprint: Combining single cell, spatial, and in situ for deeper biological understanding
