ANALYTISCHE STRATEGIE Tissue Imaging. Bernd Bodenmiller Institute of Molecular Life Sciences University of Zurich

ANALYTISCHE STRATEGIE Tissue Imaging Bernd Bodenmiller Institute of Molecular Life Sciences University of Zurich

Quantitative Breast single cancer cell analysis Switzerland Brain Breast Lung Colon-rectum Pancreas Ovary New cases Deaths Lung Liver Primary tumor Bones 0 2000 4000 6000 # cases Metastatic breast cancer 2

Quantitative Breast single cancer cell analysis lobe ducts normal lobules non-invasive fat tissue 3

Silencing of kinases Tumor heterogeneity: can induce Quantitative single cell analysis cell state heterogeneity Intra-tumor heterogeneity Cancer stem cell (CSC) - Differential treatment response of clones - Responsible for future events, e.g. metastasis (EMT) Nat Rev Cancer. 2012 Apr 19;12(5):323-34. 4

Silencing of kinases Tumor heterogeneity: can induce Quantitative single cell analysis cell state heterogeneity Inter-tumor heterogeneity Patients of same classification, e.g. HER2 + - Differential treatment response and prognosis between patients - Better classification needed Nat Rev Cancer. 2012 Apr 19;12(5):323-34. 5

Infiltrating normal cells: Quantitative single cell analysis Tumor as an organ - Diverse cells contribute to tumor development - Tumor microenvironment (TME) induces CSCs, EMT,.. Nat Rev Cancer. 2012 Apr 19;12(5):323-34. 6

Why is understanding the tumor Quantitative single cell analysis composition and structure important? Subopulations CSCs TME cell types (TAMs) Cell-cell interactions Clinical data Biomarkers/ classification Targeting all cells & TME Subpopulation biology NEED FOR MULTIPLEXED IMAGING 7

Quantitative Question single cell 1: analysis Classification of most cancer patients currently relies on an experience-based assessment of biomarker distribution and tumor morphology by a pathologist. The marker distribution and morphology is typically visualized by immunohistochemical methods. How many markers are analyzed in breast cancer and how reproducible are the classifications by a pathologist? 8

PR expression on a breast cancer tissue Quantitative single cell analysis microarray 9

PR expression on a breast cancer tissue Quantitative single cell analysis microarray Where do you set the threshold? 10

Quantitative Question single cell 1: analysis Possible answer Subtype ER PR HER2 Therapy normal-like + + + anthracylines, taxanes luminal A + + - endocrine therapy luminal B + + +/- endocrine therapy HER2-positive - - ++ growth factor receptor inhibitors triple negative - - - PARP inhibitors, platinum salts Resistance Relapse Metastasis Agreement among pathologist between 70%-90% depending on type/question. But: no gold standard to test against available! 11

Quantitative Question single cell 1: analysis Possible answer But more subtypes exist, currently prognosis not accurately determined! 12

Quantitative Question single cell 2a: analysis Recently, three novel methods to visualize tumor samples in a highly multiplexed manner were published, including two imaging mass spectrometry based approaches. A) What promise do these methods hold for a personalized patient classification? B) What are the strengths and weaknesses of each of these methods? C) How could imaging mass cytometry, the LA-ICP-MS based imaging method, find its way into clinical practice? For clinical use, the technology must be fast, reliable, and affordable. D) What tissue features imaged by LA-ICP-MS could be used as biomarkers? 13

Quantitative Question single cell 2a: analysis 1. More markers for more accurate classification (akin to genomics). 2. Additional probes for classification, e.g. genomic features (FISH). 3. Biomarkers that guide therapy (e.g. HER2). 4. Biomarkers that reveal the biology and thus treatment plans (see D). 14

Quantitative Question single cell 2b: analysis Recently, three novel methods to visualize tumor samples in a highly multiplexed manner were published, including two imaging mass spectrometry based approaches. A) What promise do these methods hold for a personalized patient classification? B) What are the strengths and weaknesses of each of these methods? C) How could imaging mass cytometry, the LA-ICP-MS based imaging method, find its way into clinical practice? For clinical use, the technology must be fast, reliable, and affordable. D) What tissue features imaged by LA-ICP-MS could be used as biomarkers? 15

Quantitative LA-ICP-MS single cell analysis Koch J. et al., Applied Spectroscopy. 2011. 16

Quantitative The single mass cell cytometer: analysis CyTOF 3. Time-of-flight (TOF) ion separation and detection 2. Ion filtering 1. Sample introduction and ionization Bandura D. et al., Anal. Chem. 2009.

Quantitative Ion single separation cell analysis Push-out plate: ions pushed into TOF chamber at 13 µsec intervals ( pushes )

Quantitative Laser single ablation cell analysis system 20 Hz ablation CyTOF Intensity Time Hao Wang and Detlef Guenther Wang HAO et al., Anal. Chem. 2013. 19

Quantitative Workflow single cell imaging analysis mass cytometry Giesen C., Wang HAO et al. Nat. Methods. 2014. 20

Quantitative Validation single cell of analysis approach Giesen C., Wang HAO et al. Nat. Methods. 2014. 21

Quantitative nano-sims single cell analysis based tissue imaging Angelo M. et al., Nat. Med. 2014. 22

Quantitative Serial single IFM cell analysis Gerdes MJ. et al., PNAS. 2013. 23

Quantitative Question single cell 2b: analysis LA-ICP-MS Nano-SIMS Serial IFM Resolution 1000 nm 100 nm 200 nm-300 nm Multiplexing 120 (44) 7(7)/Serial 61(3)/Serial Repeat scanning No / serial section Quantitative Yes* No Yes* Ease of use Medium Low High Through put Yes Yes ~6 hrs / mm 2 ~6 hrs / mm 2 66 hrs / 500 mm 2 Cost High Very high Low Sensitivity *Need of appropriate standards None is applicable to clinical practice 24

Quantitative Question single cell 2c: analysis Recently, three novel methods to visualize tumor samples in a highly multiplexed manner were published, including two imaging mass spectrometry based approaches. A) What promise do these methods hold for a personalized patient classification? B) What are the strengths and weaknesses of each of these methods? C) How could imaging mass cytometry, the LA-ICP-MS based imaging method, find its way into clinical practice? For clinical use, the technology must be fast, reliable, and affordable. D) What tissue features imaged by LA-ICP-MS could be used as biomarkers? 25

Quantitative Question single cell 2c: analysis 1. Centralized large facilities. 2. Instruments must be developed that are robust and self monitoring. 3. LA-ICP-MS for dummies. 4. Instruments should be cheaper (though this matters less than you think). 5. Increased throughput: not hours, but minutes per tissue. 6. Most informative antibodies must be determined for classification. 7. Antibody panels must be validated and approved. 8. Appropriate standards must be developed. 9. Algorithms need to be developed to analyze this high dimensional data for patient classification. 10. Biomarkers that reveal the biology and thus treatment plans (see c). 11. Pathologist will only look at cases in which algorithms struggled. 12. Clinicians/pathologists must accept such a new approach (this matters more than you think). 26

Quantitative Question single cell 2d: analysis Recently, three novel methods to visualize tumor samples in a highly multiplexed manner were published, including two imaging mass spectrometry based approaches. A) What promise do these methods hold for a personalized patient classification? B) What are the strengths and weaknesses of each of these methods? C) How could imaging mass cytometry, the LA-ICP-MS based imaging method, find its way into clinical practice? For clinical use, the technology must be fast, reliable, and affordable. D) What tissue features imaged by LA-ICP-MS could be used as biomarkers? 27

Luminal B Her2 Quantitative single cell analysis + breast cancer (8/32 markers shown) Besides proteins and p-sites: - Genome mutations (FISH) - Epigenetics a: CK8/18, H3, Vimentin - Transcripts b: CK7, H3, CD44 - Metabolites c: Pan-actin, PR, CD68 - Directly metals - Etc. Giesen C., Wang HAO et al. Nat. Methods. 2014. 28

Cell subpopulations present Quantitative single cell analysis in 21 breast cancer patients Patient classification based on cell states Giesen C., Wang HAO et al. Nat. Methods. 2014. 29

Intra- and inter-patient Quantitative single cell analysis tumor heterogeneity TN Normal Luminal HER2- Luminal HER2+ HER2+ TN HER2+ Luminal HER2+ Giesen C., Wang HAO et al. Nat. Methods. 2014. 30

Quantitative Classification single cell analysis of patients 31

Quantitative Question single cell 2d: analysis Subopulations CSCs TME cell types (TAMs) Cell-cell interactions Clinical data Biomarkers/ classification Targeting all cells & TME Subpopulation biology 32

Quantitative Question single cell 3: analysis Describe analytical strategies to determine the absolute copy number of the analyzed biomarkers per cell in a tissue section using imaging mass cytometry. Of note, a typical tissue section has a thickness between 2-4 micrometers. The thickness variability is up to 50% within a given tissue section. 33

Quantitative Question single cell 3: analysis What do we need First we need to make sure LA-ICP-MS is quantitative: 1. Representative sampling: a representative aerosol composition <200 nm lasers (e.g. ArF eximer) 2. High transport efficiencies of all particles: Ideally homogenous size distribution 3. Representative (complete) decomposition, ionization and sampling of particles that reach the ICP 4. Mass spectrometer must be quantitative over the analyzed dynamic range 34

Quantitative Question single cell 3: analysis What do we need Standards: 1. Internal standard correct for the drift in sensitivity, matrix effects, and the ablated mass, in our case tissue thickness. Requirements: Equal distribution in tissue Same behaviour as analytes 2. Matrix matched external standard 3. (Non-matrix matched external standards) but two problems remain 37

[Antibody] Quantitative Antibodies single cell analysis are NOT quantitative ABs [c] dependent Epitope accessibility Background binding Binding site occupancy Intensity Antibody labeling Polyclonal antibodies Antibody binding (capacity) epitope copy number 38

Need to generate an external calibrant for Quantitative single cell analysis every AB Calibrate still one problem remains 39

Quantitative A single tissue cell section analysis only part of a cell 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 3D reconstruction needed to quantify per cell expression 40

Quantitative Question single cell 4: analysis What advantages does absolute copy number quantification have over relative quantification for patient classification? 41

Quantitative Question single cell 4: analysis Standardization -ABs -labs Copy numbers to diagnose Defines thresholds So far classification is subjective Different datasets cannot be compared 42

Quantitative Question single cell 5: analysis Some clinically relevant proteins are present at less than 100 proteins per single cell. The CyTOF mass cytometer can detect ~1 out of 10,000 ions generated in the plasma; in imaging mass cytometry a single cell contains ~400 measurement points; and an antibody on average is coupled to 100 lanthanide ions. Describe strategies that could be used to detect such low abundance proteins by imaging mass cytometry. 43

Quantitative Question single cell 5: analysis We need to increase the signal (S/N)! 1. Protein localization? 2. Increase tissue thickness to whole cell (problems with that?). 3. Increase laser spot size. 4. Reduce sample loss during transport. 5. Use antibodies that target different epitopes on same protein. 6. Increase number of metals bound per antibody. 7. Improve instruments sensitivity. 8. Your ideas. 9. Etc. 44