Flow cytometry technologies and cancer associated fibroblasts

Flow cytometry

Dr Amber Miller, Flow Cytometry Scientist, Fortis Life Sciences, says flow cytometry techniques can help our understanding of cancer associated fibroblasts but there is still work to be done.

Inside any solid tumour there is high heterogeneity, and the types of tumour cells, immune cells, and stromal cells present and their functional states dictate disease severity and progression. Identification of markers specific to tumours and their composite cells can lead to the development of highly specialised therapies, and flow cytometry-based techniques are at the forefront of these advances.

Fibroblasts in the tumour microenvironment

Cancer associated fibroblasts (CAFs) have recently emerged as key players in the tumour microenvironment. Since CAFs are a type of fibroblast, the most common cell type in connective tissue, they play critical roles in the production of extracellular matrix and stroma. This is of particular importance in dense tissues like breast and pancreas. Fibroblasts also play a role in modulating cell growth, tissue vascularisation, and immune cell infiltration1. With so many functions, it is easy to see how these cells can have a dramatic effect on the tumour microenvironment and implications on the effectiveness of cancer therapy.

There have been many limitations in studying CAFs and understanding their role in cancer progression, but new technologies are advancing our understanding of this cell type. There are limited unique cell surface markers for fibroblasts. Instead, fibroblasts are frequently identified using a combination of positive and negative markers along with cell morphology. In addition, CAFs have a high level of plasticity that is influenced by the tumour microenvironment and disease progression, leading to the development of specialised subsets of CAFs within the tumour microenvironment1. These factors make studying CAFs challenging, but advances in flow cytometry technology are providing useful strategies to further our understanding.

Flow cytometry technologies

Conventional flow cytometry

Flow cytometry is a high- throughput, cell-based assay used to examine properties of single cells. It has been used to investigate rare cell populations, screen potential drug therapeutics, and examine sample phenotypes for diagnostic applications2. Flow cytometry revolutionised immuno-oncology by providing detailed, multiparameter analysis of specific cells and cell populations within the heterogeneous tumour microenvironment. Now, flow cytometry is more advanced by integrating it with other powerful tools such as mass spectrometry and spectral technologies.

A limitation of conventional flow cytometry in the era of single cell biology is how many distinct pieces of information can be collected about each cell. Due to the properties of existing fluorophores and the laser composition of traditional flow cytometers, this technique is limited to detecting 10-20 different markers simultaneously. Newer techniques, which will be discussed below, can detect upwards of 50 markers per cell. Even though the number of markers that can be assessed is low, conventional flow cytometry is still a valuable tool in examining CAF populations and functions. Pancreatic ductal adenocarcinoma has a tumour environment that is dominated by fibrous tissue and stromal cells. CAFs are critical regulators of desmoplasia characteristic of this cancer, yet they are poorly characterised. Elyada et al. used conventional flow cytometry analysis and FACS (fluorescence-activated cell sorting) to validate a pan-CAF marker along with CAF subpopulation-specific markers identified using single-cell RNA sequencing3. This strategy identified a novel class of antigen presenting CAFs that were able to interact with CD4+ T cells3. Additional flow cytometry studies of this antigen presenting CAF population found that these CAFs induced the transition to and proliferation of regulatory T cells capable of inhibiting CD8+ T cells4. An increased presence of immunosuppressive regulatory T cells and inhibition of CD8 T cell function has a dramatic effect on the current strategies used to treat cancer. These data provide one example of how conventional flow cytometry can play a critical role in our understanding of CAF populations, functions, and implications on therapeutic strategies.

Spectral flow cytometry

Spectral flow cytometry differs from traditional flow cytometry in that it takes the entire spectral profile for a single fluorophore into account rather than specific peak emission wavelengths. This allows fluorophores with similar peak emission wavelengths but different full spectral profiles to be used in combination.

This increases the number of fluorophores that can be used in a single experiment, increasing the number of parameters that can be investigated for each sample. Spectral flow cytometry is regularly being used to investigate 20-30 different markers simultaneously and advances in fluorophore technology will only increase this number5. The ability to examine a large number of markers at the same time provides multiple advantages when studying the poorly characterised CAFs. This technique can be used to ensure CAF rather than epithelial cells are studied and can examine multiple subtypes of CAFs concurrently.

In colorectal cancer, CAF abundance is inversely proportional to patient survival. A recent study found that CAFs can process and present extracellular antigens to CD8+ T cells via cross-presentation. Spectral flow cytometry was used to examine this cross-presentation on the characteristics of the CD8+ T cells using a panel assessing 28 different activating and inhibitory molecules. This experiment showed that after cross- presentation, CD8+ T cells had an increase in the expression of the inhibitory checkpoint molecules LAG3, CD39, and TIM36. The link of CAFs to CD8 T cell dysfunction could provide a mechanistic link to patient survival. The information provided from this spectral flow cytometry assay not only increases our understanding of what is happening inside the tumour microenvironment, but also provides evidence that modulating CAF function could be an effective therapeutic strategy.

Imaging mass cytometry

Immunohistochemistry is a valuable tool that provides detailed images of the spatial relationships between cells in tissues and proteins in cells, in their most native, non- denatured states. Multiplexing increases the detail of these images through phenotypic and functional marker localisation. Immunohistochemistry, like conventional flow cytometry, is limited in the number of parameters that can be evaluated simultaneously, typically maxing out around eight markers7. Imaging mass cytometry, which combines the high parameter capabilities of mass cytometry with the spatial organisation and localisation data of immunohistochemistry, increases the parameters that can be detected to forty or more.

Mass cytometry combines the techniques of mass spectrometry (measuring mass to charge ratios) with flow cytometry through the use of heavy metal conjugated antibodies. Imaging mass cytometry takes this one step further by incorporating sequential laser ablation of regions on a slide that are then processed by the cytometer. For imaging mass cytometry, heavy metal-conjugated antibodies are added simultaneously to a tissue section adhered to a slide. A laser is used to precisely ablate the tissue one micrometer region at a time, and the vapourised tissue sections are processed using mass cytometry. The identification and differentiation of the heavy metals are then correlated back to the specific region on the slide, providing detailed spatial information for the sample8. Large amounts of data can be generated using imaging mass cytometry; however, as with all techniques, there are limitations.

Since imaging mass cytometry relies on sequential laser ablation of regions on a slide, the time of data collection dramatically increases compared to traditional immunohistochemistry and flow cytometry. The size of the laser directly correlates with the resolution of the markers examined, and while decreasing the laser size increases spatial resolution, it not only increases the processing time, but also affects sensitivity as the number of particles processed during each ablation is reduced. To combat increased time due to the ablation, pathologists often assist in selecting representative regions to process rather than the entire sample. Processing only select regions of a sample can inadvertently introduce sample bias if representation of the entire sample is not ensured8. However, the amount and types of data generated by imaging mass cytometry is incredibly useful in situations like investigating CAFs.

Histological examination of biopsies is a common procedure to diagnose and assess cancer progression. As more information is gained about the tumour microenvironment and factors that impact patient survival, the desired number of parameters examined for each patient will increase. Elaldi et al. created a panel comprised of 39 markers to evaluate immune cells (B cells, T cells, macrophages, neutrophils, dendritic cells, natural killer cells), tumour cells, blood and lymphatic vessels, and CAFs, along with proliferation, activation, maturation, and immune checkpoint markers in cutaneous squamous cell carcinomas9. This panel can be applied to other cancers or modified to better fit researchers’ or clinicians’ needs. Panels like these will be the future of personalised medicine as we will be able to examine the immune environment inside a tumour, response to checkpoint inhibitors, CAF population heterogeneity, and cell proliferation.

Comparing technologies

There are advantages and disadvantages to each of the flow cytometry technologies discussed. Since conventional flow cytometry has been around for decades, there are numerous materials, devices, and resources available. The major limitation is the number of parameters that can be assessed simultaneously. Spectral flow cytometry addresses this limitation by incorporating complete spectral profiles for each fluorophore used rather than peak emission wavelengths; this increases the parameters to 20-40. The challenge for spectral flow cytometry is having to carefully construct panels that take into account the spectral profiles of available fluorophores. Additionally, the increased number of parameters increases the complexity of the data analysis. Streamlined and automated processes are being developed, but they are not as common as the tools available for conventional flow cytometry.

Mass cytometry can combat some of the challenges observed with conventional and spectral flow cytometry. By using heavy metal conjugated antibodies and detection of differences in mass, parameters are more easily and specifically differentiated, increasing the number that can be examined. Additionally, since isotypes are being used instead of fluorophores, many challenges of complex panel design based on identifying complementary fluorophores are not present in mass cytometry10. However, since mass cytometry and imaging mass cytometry are newer techniques, the materials, reagents and machines are not as readily available as those for spectral and conventional flow cytometry. Data analysis is more complex for mass cytometry and imaging mass cytometry because of the number of parameters assessed in each sample. Advances will continue to be made with cytometry technologies that will increase the accessibility and usability of newer techniques while continuing to develop new technologies.

Flow cytometry techniques are increasing our understanding of CAFs, but there is still work to be done. Since CAFs are often embedded into dense extracellular matrix, their isolation using traditional approaches can be challenging, which limits researchers’ ability to study these cells using classical transcriptional and translational approaches. Additionally, the plasticity of CAF populations provides hurdles in ex vivo experiments. Imaging mass cytometry is a useful tool to combat these challenges in studying CAFs because laser ablation can easily gather information from dense extracellular matrix in a more native context.

Currently, the dual role of CAFs to either inhibit anti-tumour therapies and promote tumour growth versus promote immune cell infiltration and suppress cancer progression make the development of therapies targeting these cells challenging. However, flow cytometry technologies are helping increase our understanding of CAFs. As more data is generated about CAFs, so will our ability to target them and modulate their activity.

Volume 23 – Issue 4, Fall 2022

References:

  1. Biffi G, Tuveson DA. Diversity and Biology of Cancer-Associated Fibroblasts. Physiological Reviews. 2021;101(1):147-176. doi:10.1152/ physrev.00048.2019
  2. Flow Cytometry: Principles, Best Practices, and Considerations for Experimental Design. https://www. fortislife.com/products/documents/ flow-cytometry-principles-best- practices-and-considerations-for- experimental-design-/flow-ebook.
  3. Elyada E, Bolisetty M, Laise P, et al. Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer- Associated Fibroblasts. Cancer Discovery. 2019;9(8):1102-1123. doi:10.1158/2159-8290.CD-19-0094
  4. Huang H, Wang Z, Zhang Y, et al. Mesothelial cell-derived antigen- presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer. Cancer Cell. 2022;40(6):656-673.e7. doi:10.1016/j.ccell.2022.04.011
  5. Nolan JP, Condello D. Spectral Flow Cytometry. Current Protocols in Cytometry. 2013;63(1). doi:10.1002/0471142956.cy0127s63
  6. Harryvan TJ, Visser M, de Bruin L, et al. Enhanced antigen cross-presentation in human colorectal cancer-associated fibroblasts through upregulation of the lysosomal protease cathepsin S. Journal for ImmunoTherapy of Cancer. 2022;10(3):e003591. doi:10.1136/jitc- 2021-003591
  7. Immunohistochemistry: A Guide to Investigating Cancer Hallmarks. https:// www.selectscience.net/application- articles/immunohistochemistry- a-guide-to-investigating-cancer- hallmarks?artID=55173.
  8. Chang Q, Ornatsky OI, Siddiqui I, Loboda A, Baranov VI, Hedley DW. Imaging Mass Cytometry. Cytometry Part A. 2017;91(2):160-169. doi:10.1002/ cyto.a.23053
  9. Elaldi R, Hemon P, Petti L, et al. High Dimensional Imaging Mass Cytometry Panel to Visualize the Tumor Immune Microenvironment Contexture. Frontiers in Immunology. 2021;12. doi:10.3389/ fimmu.2021.666233
  10. Spitzer MH, Nolan GP. Mass Cytometry: Single Cells, Many Features. Cell. 2016;165(4):780-791. doi:10.1016/j. cell.2016.04.019

Amber MillerAbout the author

Dr Amber Miller works for Fortis Life Sciences as a Flow Cytometry Scientist at Bethyl Laboratories validating newly developed antibodies for use in flow cytometry. She completed her PhD and post-doctoral training at Baylor College of Medicine. She has a passion for STEM and education which she shares through the podcast, WISEcast.

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