Analy?cal strategy. Tissue imaging. Prof. Detlef Günther Prof. Renato Zenobi. Sahar Ghiasikhou Kaspar Andreas Kuster Beatrice

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1 Analy?cal strategy Tissue imaging Prof. Detlef Günther Prof. Renato Zenobi Sahar Ghiasikhou Kaspar Andreas Kuster Beatrice

2 Introduc?on Cellular imaging Mul?disciplinary techniques that detects and analyzes cellular organelles and macromolecules. Important tool to perform diagnosis and treatment of diseases. Cancer Major cause of human suffering accoun?ng for almost 25% of mortality in Europe. Breast cancer is the most widespread cancer among women, 28% of the total in the European Region 1. Successful treatment requires à determina?on of breast cancer subtype and state of tumor via. different methods hsp:// topics/noncommunicable 2. Virchows Arch., 2012 Jan; 460(1):3-8. PMID: Tissue imaging

3 Classifica?on of breast cancer subtypes Main subtypes: Luminal HER2-, Luminal HER2+, HER2+, triple nega?ve. 1. Histology Microscopic examina?on of?ssue (surgical specimen) by a pathologist such as Hematoxylin and Eosin (H&E) test. More diagnos?c informa?on than prognos?c 2. Molecular pathology Used for Immunochemical quan?fica?on and quan?ta?on. Microscopy, Immunochemical methods and Fluorescence in situ hybridiza?on (FISH), Immunohistochemistry (IHC). 3. Gene;c Compara?ve genomic hybridiza?on (CGH) to demonstrate gene?c differences between tumor molecular subtypes (such as defec?on in chromosome). 4. Gene- expression Such as measurement of the expression level of HER2 genes. Nat. Clin. Pract. Oncol., 2007 Sep; 4(9): PMID: Tissue imaging

4 Marker Marker A kind of indicator that serves to iden?fy, predict, or quan?fies the interested object. Bio marker Defini;on : Biomarker is a biological substance which exists in body fluids or?ssue and can be indica?ve of an abnormal process such as cancer 1. Visualizing or measurement of the biomarker expression level can give informa?on on cancer diagnosis, anatomical loca?on of malignant cells, type of cancer and stage of the spreading. Bio markers AR ( androgen receptor), BCL2 ( B- cell CLL/lymphoma 2), CK ( cytokera7n ), EGFR ( epidermal growth factor receptor ), ER ( estrogen receptor), ERBB2 (HER2) (erythroblas7c leukemia viral oncogene homolog 2 ), PARP ( poly (ADP- ribose) polymerase ), PR ( progesterone receptor) 2. Targe?ng an individual biomarker depends on the purpose of the study. 1. Comprehensive Cancer Informa?on, Na?onal Cancer Ins?tute. [Online]. Available: hsp:// [Accessed: 22- Jul- 2014]. 2. Virchows Arch., 2012 Jan; 460(1):3-8. PMID: Tissue imaging

5 An?body An?gen reac?on M hsp://en.wikipedia.org/wiki/an?body 2. hsp:// Tissue imaging

6 Elemental markers Reporters: Stable isotopes of nonbiological rare earth metals. Tissue imaging hsp://

7 Formalyin fixed parafin embadedd sample (FFPE) 1) Fresh tumor - Aqer resec?on surgery 2) Formalin solu;on (Formaldehyde in Phosphate buffered saline PBS) - Deac?vate epitops - Decrease cellular process and?ssue degrada?on 3) Tissue embedded in paraffin - For longterm preserva?on and help tumoractumy to be cut 4) A microtome crea;ng thin slices - Tickness 4-5 micrometer Tissue imaging hsp:// introduc?on- to- rou?ne- and- special- staining/ 7

8 Analy;cal methods: 1) Mass cytometry Induc;vely couples plasma MS 2) Secondary ion mass spectroscopy (SIMS) 3) Fluorescence microscopy (MxIF) Tissue imaging

9 Analy;cal methods: 1) Mass cytometry Induc;vely couples plasma MS 2) Secondary ion mass spectroscopy (SIMS) 3) Fluorescence microscopy (MxIF) Tissue imaging

10 Mass Cytometry Is the adapta?on of Induc?vely Coupled Plasma Mass Spectroscopy (ICP- MS) to single- cell analysis based on rare earth metal labeled an?body and an?gen reac?on. Metal abundance à marker expression Tissue imaging hsp://

11 Validity of the approach a) To check the influence of metal labeling on the An?body performance. b) To check the performance of Mass cytometry in compare with IFM. Samples) Formalin fixed paraffin embedded (FFPE) breast cancer?ssues from tumor of the same type. Tissue imaging hsp://

12 Validity of the approach a) To check the influence of metal labeling on the An?body performance. b) To check the performance of Mass cytometry in compare with IFM. Samples) Formalin fixed paraffin embedded (FFPE) breast cancer?ssues from tumor of the same type. Tissue imaging hsp://

13 a) Check the influence of metal labeling on the An?body performance IFM on serial breast cancer sec?on of the luminal HER2+ subtype using unlabeled and metal labeled an?bodies recognizing the indicated markers. Observa;on No apparent change in the AB specifity due to metal labeling. Quan;ta;ve analysis Differences of the mean single cell fluorescence intensity of each marker between unlabeled and metal labeled AB were small. Note: Tissue sec?ons are similar but not iden?cal. Tissue imaging Nat. Methods., 2014 Apr; 11(4): PMID:

14 Validity of the approach a) To check the influence of metal labeling on the An?body performance. b) To check the performance of Mass cytometry in compare with IFM. Samples) Formalin fixed paraffin embedded (FFPE) breast cancer?ssues from tumor of the same type. Tissue imaging hsp://

15 b)to check the performance of Mass cytometry in compare with IFM Inves?ga?on inves?ga?on of 1) reproducibility of staining pasern by Mass Cytometer 2) percentage of cells expressing a given marker IFM and CyTOF imaging mass cytometry on breast cancer?ssue sec?ons of the luminal HER2+ subtype using unlabeled and metal labeled an?bodies recognizing the indicated markers. Result Both methods gave AB staining pasern expected for this subtype The percentage of tumor cells expressing the analyzed markers were almost similar in both methods Tissue imaging Nat. Methods., 2014 Apr; 11(4): PMID:

16 Tissue features as future biomarkers Spa?al rela?onships of complex cell states : Layers of 'omics informa?on Cell- cell interac?ons and communica?on Transcellular networks Mathema?cal models of celular assemblies Tissue imaging

17 Improvements - Op?mizing the laser spot size, clock rate and the laser energy - Proper?es of the Element (Ionisa?on poten?al, natural abundance) - Sample introduc?on area is the achilles heel (1-2 % finds its way to plasma) - Improve transport rate (studies suggest % ) - Instead of TOF design a machine with SEM (secondary electron mul?plier) or CEM (channel electron mul?plier), increases the dynamic range by 10³ or more decrease in mass resolu?on increases the ion transi?on (sensi?vity) - More ion- markers per biomarker - An?bodies binding to different places - An?bodies binding to other an?bod Tissue imaging

18 Advantageous of mass cytometry Simultaneous, rapid and spa?ally resolved analysis of 32 proteins and their modifica?ons ( over 100 markers can be detected simultaneously). No sample auto florescence. Low background effect. NO need for amplifica?on system such as applied in IHC Tissue is completely sampled Approach has wide dynamic range of 10^5, high resolu?on and high throughput. Appropriate standard will enable the absolute quan?fica?on of cellular markers => determina?on of accurate thresholds to define the pa?ent s state. Tissue imaging

19 Limita?ons of mass cytometry Some?mes Abs are not available for a given target or in the format needed for mass cytometry, those that work well in single- plex assays behave differently in mul?plex assays. Analysis an area of 0.5 mm * 0.5 mm at 1- Micrometer resolu?on takes almost 3.5 hours. Price Spot size limit (diffrac?on defined) - > limited lateral resolu?on Ar?ficial high values at edges, cleqs and holes in the sample because of too low pixel values due to par?al volume effects Polyatomic isobaric interferences Tissue imaging

20 Analy;cal methods: 1) Mass cytometry Induc;vely couples plasma MS 2) Secondary ion mass spectroscopy (SIMS) 3) Fluorescence microscopy (MxIF) Tissue imaging

21 Secondary ion mass spectroscopy (SIMS) Imaging an?bodies tagged with isotopically pure elemental metal reporters, capable of analyzing up to 100 metal- isotopes simultaneously. hsp:// Tissue imaging

22 Mul?plex ion beam imaging (MIBI) analysis Nat. Med., 2014 Apr; 20(4): PMID: Tissue imaging

23 Validity of the Approach : MIBI Vs. Mass cytometry Sample Peripheral blood mononuclear cells (PBMC), stained with seven metal isotopes labeled an?body. MIBI cell were immobilize on poly- l- lysine coated silicon wafer, dried under vacuum, analyzed by Nano SIMS- MS. Results MIBI yielded both qualita?vely and quan?ta?vely equivalent results as conven?onal analy?cal plazorm. Nat. Med., 2014 Apr; 20(4): PMID: Tissue imaging

24 Discussion on MIBI MIBI is capable of analyzing up to 100 metal- isotopes simultaneously, over 10^5 dynamic range. Can achieve as low as parts per billion sensi?vity. Can be used on vacuum compa?ble specimen, including FFPE?ssues. Absence of auto fluorescence signal. No spectral overlap between mass adjacent elemental reporters. Assay is more linear in compare with IHC (Fluorescent and chromogenic method), due to no need to secondary labeling and amplified detec?on. Less throughput : 500 micrometer field of view requires 2h to be imaged. Tissue imaging

25 Analy;cal methods: 1) Mass cytometry Induc;vely couples plasma MS 2) Secondary ion mass spectroscopy (SIMS) 3) Fluorescence microscopy (MxIF) Tissue imaging

26 Interobserver agreement and reproducability in classifica?on of invasive breast cancer carcinoma Kappa sta?s?cs: κ = Pr (α) Pr (e)/1 Pr (e) Pr(α) = rela?ve observed agreement between raters Pr(e) = hypothe?cal probability of chance agreement κ = 0 only chance agreement; κ < 0.4 rela?vely poor agreement κ = moderate agreement κ = good agreement κ > 0.8 excellent agreement κ = 1 complete agreement Tissue imaging

27 Interobserver agreement and reproducability in classifica?on of invasive breast cancer carcinoma Kappa sta?s?cs: κ = Pr (α) Pr (e)/1 Pr (e) Pr(α) = rela?ve observed agreement between raters Pr(e) = hypothe?cal probability of chance agreement κ = 0 only chance agreement; κ < 0.4 rela?vely poor agreement κ = moderate agreement κ = good agreement κ > 0.8 excellent agreement κ = 1 complete agreement

28 Interobserver agreement and reproducability in classifica?on of invasive breast cancer carcinoma Modern Pathology, 2006 Feb; 19(2): Tissue imaging

29 Fluorescence microscopy Staining of?ssue samples with an?bodies with covalently bound fluorescence dyes. Illumina?on of sample with light of a specific wavelentght to induce fluorescence. Fluorescence is separated form illumina?on light by spectral emission filters hsp:// 2000x- infinity- plan- fluorescent- trinocular- microscope- 5mp- fluorescent- camera.html Tissue imaging hsps://

30 Mul?plexed fluorescence microscopy MxIF allows mul?plexed fluorescence microscopy by irreversible chemical deac?va?on of the fluorescent dyes between measurements. Tested with up to 32 rounds of dye deac?va?on. Tissue imaging Proc. Natl. Acad. Sci. U S A., 2013 Jul; 110(29): PMID:

31 Fluorescence microscopy + established method subcellular resolu?on nm (diffrac?on limit) fast measurement?me rela?vely cheap - autofluorescence photobleaching spectral overlap limits number of markers (normally around 2 up to 7 for special cases) measurement?me for many marker (MxIF) dye inac?va?on chemistry affec?ng target molecule (MxIF) Tissue imaging

32 MALDI- IMS In matrix assisted laser desorp?on/ ionisa?on a suitable matrix material is applied to the?ssue sample surface. Sample is irradiated by a pulsed laser which leads to abla?on and desorp?on of matrix and brings sample into the gas phase. Detec?on of ions by mass spectrometry or by tandem mass spectrometry if small molecules should be detected. Nat. Methods., 2007 Act; 4(10): Tissue imaging

33 MALDI- IMS + visualisa?on of thousands of analytes (proteins, lipids, metabolites drugs) iden??es of proteins observed do not need to be known in advance m/z range can be well over qualita?ve matrix applica?on difficult matrix effects resolu?on μm not many photocleavable mass tags available for targeted approach Tissue imaging

34 Thank you for your asen?on Tissue imaging

35 Importance of exact determina?on of cancer subtypes 0 / + Nega;ve ++ Ambiguous +++ Posi;ve A. T. Ciqlik, H.- A. Lehr, and M. A. M. Gijs, Microfluidic processor allows rapid HER2 immunohistochemistry of breast carcinomas and Tissue imaging significantly reduces ambiguous (2+) read- outs, Proc. Natl. Acad. Sci., vol. 110, no. 14, pp , Apr c