“Single Cell and Cancer Heterogeneity Analysis”

, PhD) [#17] Wendy responded: “You need somebody that's really, really committed to understanding how new technologies can work. You need really good physician scientists. They need to really get it. Sadly, a lot of physicians don't have time. I am a former patient. I had ovarian cancer. I fired so many doctors you don't even want to know. I told them: ‘I am the patient from hell.

I am going to ask you questions, I am the boss of me. I am a scientist and a cancer biologist, and I am not a dummy.’ I did find somebody who was amazing. She's one of my best friends, and we are collaborating. You need somebody who's really going to engage and get what it is, and a lot of them they just don't want to.

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“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] Meeting Transcript The information and opinions expressed on this website or platform, or during discussions and presentations (both verbal and written) are not intended as health care recommendations or medical advice by Cancer Patient Lab/Prostate Cancer Lab, its principals, presenters, participants, or representatives for any medical treatment, product, or course of action.

You should always consult a doctor about your specific situation before pursuing any health care program, treatment, product or other course of action that might affect your health.

SUMMARY KEYWORDS cell, tumor, antibodies, proteins, ovarian cancer, question, samples, patient, technology, tumor cell, imaging, nk cells, tissue, important, papers, oligo, immune cells, cancer, neighborhood, assays SPEAKERS Wendy Fantl, Anonymous Caregiver, Dr. John Laird, MD, Brian McCloskey, Brad Power, Rick Stanton.

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] My lab is focused on applying single cell proteomic technologies to expedite patient benefit. We're all about translation. The focus areas of my lab are ovarian cancer and kidney cancer. But all this work can be applied to any malignancy.

There are two multiplex single cell proteomic technologies up and running in my lab: mass cytometry or cytometry by time of flight (“CyTOF”), and CODEX multiplex imaging. (Mass cytometry labels antibodies with isotopes of rare-earth metals, resulting in a theoretical capacity to distinguish up to 100 unique characteristics of cells simultaneously.

ing in my lab: mass cytometry or cytometry by time of flight (“CyTOF”), and CODEX multiplex imaging. (Mass cytometry labels antibodies with isotopes of rare-earth metals, resulting in a theoretical capacity to distinguish up to 100 unique characteristics of cells simultaneously.

CODEX is a high- parameter imaging technology that relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently-labeled DNA probes, enabling up to 60 markers to be simultaneously visualized.) I’m also going to talk about the quality of clinical samples.

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] Wendy Fantl 02:04 What is the motivation for developing multiplex single cell technologies? Tumors are a mass of heterogeneous cell types. These multiplex single cell proteomic technologies allow you to deeply phenotype what kind of cell it is. Is it a tumor cell? Is it a blood cell? What is it?

They allow you to unravel tumor heterogeneity. And why is that important? If you took a tumor and you processed it, just mushed it up, you get sort of average values of transcripts and proteins. But because you're analyzing or measuring what's going on in single, intact cells, you can identify minority cell populations and those are the populations that you're interested in.

They have key roles in tumor initiation, maintenance, epithelial-mesenchymal transition (a process by which epithelial cells – cells that come from surfaces of your body, such as your skin, blood vessels, urinary tract, or organs – lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells – multipotent stem cells found in bone marrow that are important for making and repairing skeletal tissues, such as cartilage, bone and the fat found in bone marrow), and drug resistance.

Those are the bad guys. Those are the guys we need to understand. To address this need, about over a decade ago mass cytometry was developed. It characterizes single, intact cells in suspension, and it's based on protein co-expression patterns. I'm not going to talk about transcripts or DNA.

This is all about proteins, which really are the functional convergence point from DNA, to RNA, and to protein.

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] you can look at protein-to-protein interactions. But CyTOF, or mass cytometry, depends on cells in suspension, and so therefore, you lose any structural information. That was the motivation for the development of multiplex imaging technologies, and several are available.

Code detection by indexing, or CODEX, is the technology I have in my lab. There are other technologies, such as Multiplexed Ion Beam Imaging (MIBI) and Cyclic Immunofluorescence (CycIF) imaging, a whole slew of them. But essentially, they're all doing the same thing.

Code detection by indexing, or CODEX, is the technology I have in my lab. There are other technologies, such as Multiplexed Ion Beam Imaging (MIBI) and Cyclic Immunofluorescence (CycIF) imaging, a whole slew of them. But essentially, they're all doing the same thing. They're good at providing information about tissue architecture and allowing you to identify cellular neighborhoods.

Samples of the highest quality are essential. There are many variables to consider when collecting samples, and the devil is in the details. CyTOF cells in suspension means that blood is the perfect medium. People generate peripheral blood mononuclear cells from whole blood.

But what I'm using is a buffer that allows you at the clinic, at the site, not to do any of the peripheral blood mononuclear side preparation, just add this buffer to incubate and freeze it. That's it. And what's really cool about that is that with peripheral blood mononuclear cells, you don't have any granulocytes. Granulocytes are also important in cancer.

Smart2 buffer allows you to preserve granulocytes. For other CyTOF analysis, blood is perfect, but it also works with solid tumors. I think my lab was one of the first to look at solid image ovarian tumors and to work out in excruciating detail all the protocols and details of dissociation into high quality single cells. CODEX doesn't use cells in suspension, but it uses tissue slices.

These can be fresh-frozen or formalin-fixed paraffin embedded (FFPE).

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] And again, the devil is in the details. Everybody is very sort of "hoo ha" about fresh-frozen paraffin embedded samples because they're available worldwide, so it shouldn't be a problem. But I can tell you, there is such variation in the preparation. Here are some of the papers and some of them are from my lab and others.

They go into the details about producing high quality samples. I think this is very important for this group. Maybe we could have a separate meeting about how to request samples and the quality control that we need to have working with the collaborators and the people providing the samples.

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] Rick Stanton 07:43 I mean, I know flow cytometry, but I don't think other people here do. Wendy Fantl 07:48

, I don't think everyone knows how to associate these wavelengths with any problem, or what are these wavelengths?

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] Rick Stanton 07:43 I mean, I know flow cytometry, but I don't think other people here do. Wendy Fantl 07:48 You basically take antibodies that recognize specific proteins on cells. The antibodies are labeled with fluorescence, fluorochrome. The fluorochromes, when they get excited, emit light in these wavelengths.

That's the signal you will measure. What you can see from this graph is tremendous overlap. It's difficult to pull out specific signals because of this overlap. You must apply all these algorithms to do that. Does that clear it up? Rick Stanton 08:30 Fluorophores with these emission spectra, like the red and the green on the right, show there's overlap.

If something was emitting, it would be between 600 and 100. You don't really know whether it was a red or a green, and that red and the green are bound selectively to different antibodies. Without some kind of deconvolution algorithm which is trying to optimize red from green, you don't know.

That's the meaning of this overlap - there's an ambiguity of whether I am looking at a fluorophore that's tagged to this antibody, or that antibody. That is going to make a difference in the cell type in your conclusions because you're querying with these antibodies tagged to these fluorophores. If you don't know where they're coming from, you lose your ability to interpret.

Those metals are rare or absent in biology and they're at the bottom of the periodic table.

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] Here they are - the lanthanides. They're rare or absent in biology and what's cool is they are composed on the planet when they mine stable metal isotopes. There are ways to purify these stable metal isotopes.

Now you can label antibodies with these metals, and you can get rid of the overlap that you've just so beautifully explained. The number of parameters per single cell is just dependent on what Earth provides us, but maybe we'll go to Mars and get more, but I don't know. For example, you see that samarium, or Sm, has 6, a reasonable abundance.

We label our antibodies with an isotopically-enriched lanthanide.

“Single Cell and Cancer Heterogeneity Analysis” (Wendy Fantl, PhD) [#17] Right now, we're up to about 60 parameters per single cell. We label ourselves with antibodies against proteins that delineate surface markers. If it was a T cell, the tumor cell, the cell type,