10x Flex brings single-cell sequencing to paraformaldehyde-fixed human and mouse samples. Read on to learn how we have advanced that protocol to work on patient-derived mouse xenografts.
Patient-derived xenografts (PDx) are widely adopted in preclinical drug testing. They are created by implanting tumor tissue from a patient into immunodeficient mice or other animal models. This allows the tumor to grow, develop, and undergo treatment in a living organism. They are frequent model systems in preclinical research for studying tumor biology and predicting drug response, safety, and resistance mechanisms.
A highly valued trait of PDx is that the tissue retains the original tumor’s heterogeneity. Because single-cell sequencing is the most accurate assay for heterogeneous tissues, many researchers have also shown interest in applying the technology on PDx tissue.
Until recently, however, studying PDx tissue at single-cell resolution was mainly feasible for researchers who had their PDx and single-cell sequencing facility side-to-side. To preserve PDx quality during transport or storage, researchers often perform tissue fixation with formaldehyde. Yet, formaldehyde fixation decreases RNA quality too much for single-cell sequencing.
As an answer to a widespread need, 10x Genomics has established a protocol titled 10x Flex for paraformaldehyde-fixed tissues based on RNA probes. These probes are organism-specific, either for mouse or human tissue. Unfortunately, the protocol did not support tissues of mixed origin, such as xenografts, because of the high amount of similarities between human and mouse transcripts.
Now, Single Cell Discoveries has developed a technique to reliably mix these probe sets and perform single-cell sequencing on human-mouse xenograft tissue. Read on to learn more.
Key Takeaways
Single Cell Discoveries has successfully developed a protocol that enables mixing human and mouse probes and deconvoluting the data afterward with minimal information loss. This enables full, probe-based, single-cell sequencing of formaldehyde-fixed PDx tissues with the 10x Genomic Gene Expression Flex (10x Flex) kit.
Applications include PDx, cell line–derived tumor xenografts (CDx), and humanized mice.
A manuscript with the results will be published shortly.
Jump to a section:
- Background: Why study xenograft tissue at single-cell resolution?
- What is 10x Flex: Fixed RNA Profiling?
- Adapting 10x Flex for Human-Mouse Models
- Frequently Asked Questions
10x Flex: Why study xenograft samples at single-cell resolution?
The creation of PDx mice was a significant breakthrough in cancer research in the 1980s. It provided a model system for studying human tumors in a more biologically relevant context, allowing researchers to explore patient-specific tumor biology, treatment responses, and therapeutic strategies inside a complex organism.
This tissue retains the characteristics of the patient’s tumor, including heterogeneity and molecular features. They are valuable for testing drug responses that are more reflective of patient outcomes. As a result, PDx models quickly became essential in the study of cancer resistance mechanisms and personalized therapy options. In line with this, PDx models also proved helpful for studying tumor-immune interactions, examining the tumor microenvironment, and for testing CAR T-cells or other immunotherapies.
Single-cell sequencing of PDx models improves drug development
Researchers have demonstrated a strong interest in pursuing single-cell sequencing on PDx tissue. The translational value of this application becomes clear when you look at some of the existing research.
Finding rare, pre-existing resistant tumor cells
Firstly, with single-cell sequencing, you can detect rare cell populations, including cancer stem cells, immune cells, and other subpopulations with specific roles in tumor progression and drug response.
Moghal et al. (2022) used 10x Genomics Gene Expression in a study of drug-tolerant, persistent lung adenocarcinoma cells. Specifically, they examined cells tolerant to EGFR inhibitor erlotinib. In PDx models that they had given erlotinib, the researchers identified a rare population of tumor cells that transcriptionally resembled the persistent adenocarcinoma cells of patients. Comprising only 4% of all cells, it is likely that this rare population would have been overlooked had the PDx tissue been studied in bulk.
With an in-depth gene expression analysis of the rare persistent cells, the team identified several biomarkers and potential drug targets to improve lung adenocarcinoma treatment.
Studying mechanism of action in vivo
Secondly, single-cell sequencing in PDx models can help researchers better understand the gene expression changes in tumors as responses to treatment. This knowledge can expedite drug discovery and lead to more effective therapies.
For instance, Jermakowicz et al. (2021) studied the effects of a new drug for glioblastoma, a type of brain tumor. The drug candidate, called UM-002, showed success in vitro and in human brain organoids. To study the mechanism of action in vivo, they tested the drug on PDx models.
They found that UM-002 induced changes in cell cycle–related gene expression at single-cell resolution in vivo. After closer inspection, they observed that the treatment reduced the proportion of the most proliferative tumor cell clusters. They found it likely that UM-002 alters the transcriptional state of glioblastoma cells. Moreover, compared to similar compounds, UM-002 was the most effective drug doing so.
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Identifying drivers of disease progression
Another example shows how scRNA-seq can elucidate signaling pathways and cellular communication networks within the tumor. This information can help researchers identify potential drug targets.
Bondoc et al. (2021) used 10x Genomics Gene Expression to study the mechanisms of disease of hepatoblastoma, a precancerous liver disease in children. By comparing the gene expression patterns in tumor, background liver, and PDx tissue, the team could characterize several cell clusters.
In addition, after close examination of the gene expression patterns, they assigned functional roles to each cell cluster. One cluster showed signatures of a potential driver cell population, while other clusters seemed to support tumor growth and stability. The gene expression profile moreover highlighted the importance of several gene pathways that the researchers suggested as possible drug targets.
Assessing PDx model validity
Finally, to safeguard its validity in preclinical research, it is crucial to understand the tissue dynamics of a PDx model. One observation is that the tumor microenvironment partially formed by human stromal cells is slowly replaced by the murine host cells. How this phenomenon influences the cancer cells is important for understanding how well the models reflect what happens in patients.
Such a study benefits from high-resolution assessment of the interactions between human cancer cells and the surrounding mouse microenvironment.
What is 10x Flex: Fixed RNA Profiling?
In 2022, 10x Genomics presented a new technology dubbed Gene Expression Flex, or 10x Flex, which enables single-cell RNA sequencing of formaldehyde-fixed, FFPE, and FPO tissues.
In the technology, 100s of designated probes bind transcripts to generate a comprehensive RNA profile. These RNA probe sets target predetermined regions in the transcriptome designated for high-quality single-cell sequencing results. Currently, there’s a human and a mouse probe set.
- The human transcriptome probe set consists of 53,957 probes and delivers information on a maximum of 18,082 different genes.
- The mouse transcriptome probe set consists of 55,479 probes and delivers information on a maximum of 19,059 different genes.
The probe sets are barcoded so that they may be processed individually (singleplex workflow) or pooled with up to 16 samples in a single lane of a Chromium X chip (multiplex workflow).
Learn about 10x Flex in more detail in our blog “Fixed RNA Profiling: Benefits and Limitations”
10x Flex for Human-Mouse PDx Models
The need for single-cell profiling fixed PDx tissue is widespread: any situation where it’s impractical to load freshly dissected PDx material directly on a 10x Chromium chip benefits from some type of preservation. This includes the following scenarios:
- Researchers who do not have a 10x Chromium controller on site;
- Time-course experiments in which it is impractical to load samples from each timepoint on 10x Chromium chips immediately;
- Large-scale experiments with too many samples per condition for a single 10x run;
- Consortia in which the PDx facility needs to ship samples to a collaborator;
An example of the last scenario is PERSIST-seq, a consortium focused on therapeutic resistance in cancer, to which Single Cell Discoveries contributes their single-cell expertise. PDx models play a significant role in the study of therapeutic resistance in cancer for reasons mentioned in the previous chapter. By the collaborative nature of the consortium, PDx tissues had to be preserved for transport and storage between the research site and the single-cell sequencing facility. It was this situation that prompted Single Cell Discoveries to adapt 10x Flex to human-mouse cell mixtures.
Defining the problem
Human-mouse cell mixtures exposed to mouse and human probes, in essence, result in an RNA profile in which some signals double. Namely, an individual cell’s transcripts can be captured by human and mouse probes. The result would be that one cell’s RNA profile is determined by a mixture of signals stemming from transcripts binding to 10x Flex human and mouse probes.
It is thus crucial that cells can be confidently labeled by their species. Then, for example, a mouse cell can be viewed as a mouse cell, and signals stemming from human probe-binding can be ignored.
Developing a new assay
An important success was that the single-cell sequencing data of a cell-line mixture of human and mouse cells, exposed to human RNA probes, could be separated into two data groups. Based on a combination of properties of each cell, a cell could be classified as either human or mouse with a newly developed data analysis pipeline.
This could be repeated in a mixed cell line exposed to a mixture of mouse and human probes. The data analysis pipeline proved successful in deconvoluting the doubling signals in the data and was optimized further.
Crucially, the new method also proved successful in PDx samples, validating the method’s efficacy. Each time, the proportion of cells classified as mouse or human was as expected. Moreover, the cell types classified as mouse cells made sense, as they were cells natural to the microenvironment, like endothelial cells, monocytes, and fibroblasts.
Gene expression analysis of cells classified as human still enables clustering and differential gene expression analysis. So, for example, treated cells still validly clustered differently from untreated cells despite the classification step. Importantly, very little to no data quality was lost.
Summary
In summary, we can conclude that:
- The 10x Genomics Gene Expression Flex can be used for mixed populations of human and mouse cells like PDx and CDx tissue;
- Mixing both human and mouse probes allows the correct classification of each cell species;
- Data can be pulled apart to perform downstream, species-specific analysis.
relevance of 10x Flex for Fixed Xenograft tissue
The advanced 10x Flex protocol has prevailed over the challenge of performing single-cell sequencing on preserved, high-quality PDx tissue. It has lifted the logistical hurdles. As a result, we have already processed over 500,000 cells with 10x Flex in the PERSIST-seq consortium.
Importantly, other researchers working with PDx, CDx, humanized mice or other mixed mouse-human tissue can now also consider single-cell sequencing. Connect with our PhD-level scientists to discuss your specific needs, and we’ll develop a tailored strategic plan and determine the next steps to help you achieve your goals.
PDx with 10x Flex – FAQs
Is this technology compatible with cell-line-derived tumor xenografts?
Yes, this technology is compatible with cell-line-derived tumor xenografts. In addition to PDx, many researchers perform initial studies in cell-line-derived tumor xenografts (CDx). Although they lack the full spectrum of genetic and phenotypic diversity of PDx models, CDx models can provide a faster and more standardized method of drug testing. Hence, they are often used in large screens or early-stage research.
Would you use the same strategy to study humanized mice?
If you have an interest in how the microenvironment interacts with the patient-derived tumor cells in humanized mice, this is possible with this protocol.
How many samples can you multiplex when you mix the probes?
We can use 8 unique human probe sets and 8 unique mouse probe sets together. Hence, we can multiplex up to 8 samples.
For which species of xenograft does this method apply?
Xenograft analysis of paraformaldehyde-fixed tissues applies to all species supported by the 10x Flex probes. Currently, that is mouse and human.
Find out more
Find out more information on fixed RNA profiling, our other 10x Genomics services, resources, and much more in our information guide:
