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Oxygenation and (Tumour) Biology Cell Models & 3D culture - - PDF document

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11/15/2017 Biological Roles of O2 and principles of quenched-phosphorescence O 2 sensing Demonstration in in vivo studies Demonstration in ex-vivo models 3D tissue models and physiological studies in Dmitri B. Papkovsky vitro

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Dmitri B. Papkovsky

School of Biochemistry and Cell Biology, University College Cork, Ireland  Biological Roles of O2 and principles of

quenched-phosphorescence O2 sensing

 Demonstration in in vivo studies  Demonstration in ex-vivo models  3D tissue models and physiological studies in

vitro

Tumour Metabolism

Oxygenation and (Tumour) Biology

McKeown SR. Defining normoxia, physoxia and hypoxia in tumours—implicationsfor treatmentresponse. Br J Radiol 2014;87:20130676

Therapeutic Efficacy

~7% O2 ~21% O2 ~1% O2

  • Therapeutic Efficacy
  • Delineating hypoxia signalling (HIF stabilisation)
  • Ischemia Reperfusion Modelling

0.E+00 2.E-08 4.E-08 6.E-08 8.E-08 1.E-07 iO2 2 = 17% 7% iO2 2 = 0.5% Glycolytic Flux ([H+] /h)

Oxygenation and Physiological Relevance

5 10 15 20

Inspired air Kidney Liver Muscle

Cell Models & 3D culture

‘Normoxia’

Hyperoxic Oxygen

Q) Is culturing cells at 18.6% O2 physiologically relevant ? A) No. It is Hyperoxic w.r.t. to most tissues. Cell Culture ~18.6% ROS / Metabolism / Signalling?

‘Hypoxia’ ‘Hypoxia’ ‘Normoxia’

O2%

Anoxia ‘Physoxia’

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In situ [O [O2] determines s cell physi siology !!

Zhdanov et al - Integr. Biol. 2010, 2:443

S* S* T* T* hn

fluorescence phosphorescence

O2

ns ms

  • Non-chemical , reversible
  • Quantitative, real-time, stable
  • Optical, minimally invasive

Relationship : [O [O2] = (t

(to/t -1)/ Kq

Analyt ytical Appr proach Features

In vivo imaging with intravascular O2 probes Systemic administration in live animals (IV):

  • High doses, rapid clearance

(~3h), once-off

  • Poorly suitable for in vitro use
  • Bulk tissue is dark
  • Complex synthesis, costs

Intracellular O2 probes & micro- imaging Local administration in tissue:

  • low doses, controlled location
  • long retention time (many days)
  • Low toxicity
  • Micro & macro imaging,

Miscellaneous: O2 probes Imaging systems, Various technologies, often 2D or point measurements, semi- quantitative

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  • Small molecules
  • Bioconjugates, e.g. peptides
  • Polymeric NPs (by inclusion)
  • Polymeric NPs (by conjugation)
  • Multi-functional NP composites

Probe name, composition lexc lem to (ms) KSV

Ru(bpy)2(pic)2+ - CPP conjugate 458 nm 610 nm 775 ns ND PtCP - CPP conjugates 390 nm 650 nm 50-70 ms 0.006 mM-1 Ir-BTP coumarin C343 conjugate 405 nm 620, 680 nm 480 nm (ref) 5.6 ms 0.064 mmHg-1 IrOEP – CPP complexes 386 654 58-69 ms 0.074 mM-1 PtTFPP Pt-Glc conjugate 395 650 57 ms 0.03 mM-1 PdTCBP-HiLyte680 dendrimer in PAAG NPs modified with TAT peptide (30-50 nm) 442, 632 nm 678 nm (ref) 790 nm (O2) 699 nm (ref) Not reported (250 ms for G2) 0.034 mM-1 [Ru(dpp(SO3Na)2)3]Cl2 in PAA NPs (45 nm) 454 nm 608 nm 3.88-4.06 ms ND PtTFPP in RL100 polymer (35 nm NPs) 395 nm 650 nm 69.1 ms 0.04 mM-1 PtTFPP-naphtalimide dye in PS NPs (410-430 nm) 395 nm 650 nm 490 nm (ref) ND ND PtTFPP and PFO in RL100 NPs (70 nm) 405 nm - 1P 760 nm – 2P 650 nm 430 nm (ref) 66 ms 0.041 mM-1 [Ru(dpp)3](TMSPS)2 in amino modified PS NPs (121 nm) 488 – 1P 830 nm – 2P 630 nm ND ~0.8? PtTBP in RL100 NPs 440, 614 nm 760 nm 57 ms ~0.02 mM-1 PtTFPP in PS NPs (50 nm) 395 650 61 ms ND PtTFPP and PFO in acrylic polymer NPs (95 nm). 405 – 1P 760 – 2P 650 nm 430 nm (ref) 68 ms 0.086 mM-1 WPF-Ir4 and WPF-Ir8 NPs (19 nm). 405 nm 630 nm 450 nm (ref) 0.6 ms 0.006 mmHg ? [Ru(dpp)3]2+Cl2 and NaYF4:Yb/Tm@NaYF4 in mesoporous silica NP (50 nm) 980 nm – UC 613 nm 477 nm (ref) ns ND PtTFPP in PS-silane hybrid NPs (77 nm) 395 605 nm Ms ND

Detection Modes:

Fercher er A. et al – ACS Nano,

  • , 2011
  • Biocompatible polymer
  • Average size 35-50 nm
  • Z potential +45mV
  • Stable, bright, low toxicity

Prof.

  • f. Sergey Borisov

sov, , Graz, , Austria

100 200 300 400 300 350 400 450 500 550 600 650 700 Wavelength, nm

MM2 MM2

100 200 300 400 500 600 300 350 400 450 500 550 600 650 700 Intensity, a.u. Wavelength, nm

NanO2

Dmitriev ev RI et al . – Adv . . Funct . Mater er 2012

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400 500 600 700 0.0 2.0x10

5

4.0x10

5

6.0x10

5

8.0x10

5

1.0x10

6

1.2x10

6

500 600 700 800 1x10

5

2x10

5

3x10

5

4x10

5

5 10 15 20 1 2 3 4

Luminescence Intensity, a.u. Luminescence Wavelength, nm

pO2, kPa 0.98 1.96 3.92 7.82 11.74 19.56

(a)

Intensity, a.u. Wavelength, nm

pO2, kPa 0.98 1.96 3.92 7.82 11.74 19.56

(b)

SII-0.1

+

SI-0.15

+/0.05

  • R0/R

pO2, kPa

(c)

Conjugated Polymer NPs

Advantages:

  • Enhanced brightness - up to 10-fold, 1P, 2P
  • Tunable spectra, surface charge, cell

specificity

  • Improved stability, tissue staining and

penetration

Dmitriev ev RI et al . – ACS Nano,

  • , 2015

Pt-Glc structure

Dmitriev ev RI et al . – Biom

  • mater
  • er. Sci., 2014
  • Hydrophilic, water-soluble, neutral
  • Efficient cell and tissue penetration
  • Stable calibration
  • High photostability, low toxicity
  • Moderate brightness

 Procedure:

  • Anaesthesia, surgery
  • Probe/Sensor application
  • Mounting cranial window
  • Commercial Intensity based imager
  • Sacrificing animal
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 VSD - Cell depolarisation

  • fast, localised response - ~50 ms

 O2 Probe - tissue oxygenation, metabolism, hemodynamics

  • Delayed, bi-phasic response
  • Affects larger area
  • Resembles BOLD-MRI, fNIRS signals

Tsytsarev ev et al – J. Neuros

  • sci Meth, 2013,

Ex-Vivo Tissue: Experimental procedure

Euthanasia Tissue excision & mounting Pins Pins Pt-Glc Pins Pins Pt-Glc

1-3 h at 37oC

Pins Pins Stimulations

  • Distal colon
  • Urinary bladder
  • Carotid artery

Axio Examiner microscope (Zeiss) DCS-120 Confocal FLIM (B&H) supercontinuum ps laser Fianium, 400-650 nm, 4W

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180 iO2 [mM] 6 weeks after ligation Control carotid artery

  • Bright staining of the tunica intima layer
  • Dramatic effect of ligation on tissue oxygenation

Occluded Carotid Artery Oxygenation

Zhdanov

  • v AV et al . – CMLS , 2016

10 20 30 40 50 60 70 80 10 20 30 40 50 60 Control DSS O2 levels in the samples [mM]

G

90 95 100 105 Normalised OCR [%] 0.7 0.8 0.9 1 1.1 1.2 1.3 Control DSS OCR [nM/min × mg protein]

H

p = 0.116

I

1.4

135 iO2 [mM] WT KO

WT KO S 1 S 2

0.5 1 1.5 2 2.5 3 3.5 4 50 100

O2

TOP MIDDLE BOTTOM

Prominent diff fference ces in ROS S generation, , but marginal diff fference ces s in tiss ssue O2 levels !

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25 50 75 100 125 10 20 30 40 50

Chart Title

aver aver

m m +/ +/- SEM values for 25%, 50% and d 75% quartiles show the diffe ference in actual O2 levels

Detailed Analysis - Group comparison Non-parametric Mann-Whitney U-test

Pircalabior

  • ru G et al . – Cell Host Microb
  • be. 2016,19:651.

Line 1 Line 2 5 O2 [microM] 65 Pt-Glc intensity iO2 100 200 300 Distance [micrometres] 20 40 60 20 40 60 iO2 [microM]

Cell border

Line 1 Line 2

40 80 25 50 75 iO2 [mM] Cell cross section [mm] 120 FCCP / EGTA Resting AntA

FCCP / EGTA Resting AntA (80 min)

O2 [mM] 100 0 20 40 60 80 40 80 Average iO2 [mM] 120 AntA treatment [min] 160 160 1 2 3 4 5 6 7 8 9 25 30 35 40 45 50 Relative LT frequency [ *105] LT [ms]

30 min 5 m i n 8 m i n

* *

9 mM 6 mM

Zhdanov

  • v AV – Am . J. Cell Physiol
  • l.. 2015

5-10 2-3.8 5 O2 [mM] 85 10-15 3.8-5.7 15-20 5.7-7.4 20-30 7.4-10.7 30-40 10.7-13.8 40-50 13.8-16.7 Putative PHD2 activity, v [%Vmax] iO2 [mM] v [%Vmax] 5 O2 [mM] 100 12-15 mM O2 4.6-5.7 %Vmax 40 mM O2 13.8 %Vmax 35 mM O2 12.3 %Vmax 10-13 mM O2 3.8-4.9 %Vmax

Calculated local PHD2 activity : Km (O2) = 250 mM; v = Vmax[S]/(Km+[S])

Calculated HIF prolyl hydroxylase 2 activities

Zhdanov

  • v A et al, AJP-CP, 2015
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  • Multicellular spheroids
  • Cell co-cultures
  • Organoids
  • Engineered tissue scaffolds
  • Vascularised tissue

Environmental control and standardization remain bottlenecks FLIM platforms can address these

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Jenki kins J. et al . – Biochem em . J. 2016

  • Neurospheres cultured

at 21% and 4% O2 with NanO2 probe.

  • Imaged at 21% O2.

O2 gradient between basal (blue) and apical (violet) membranes (n=10): 500μm

 Intestinal Organoid (SIO) Models:

  • Establish cultures
  • Characterise O2 and respiration activity
  • Standardize, reduce heterogeneity
  • Conduct physiological studies
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 T – probe (NanO2 analog) –

Anal Chem, m, 2016, 88: 10566

 pH probe - J. Mater. Chem.

  • m. B, 2014, 2: 6792

 Cell Cycle assay (Hoechst 2334 and dBrU) –

PLOS One, 2016, 11: e0167385

 K+-probe - Adv Funct Mater (in press)  More in development 

Biophysics Lab (University College Cork):

  • Dr. Alex Zhdanov, Dr. Ruslan Dmitriev
  • Dr. Irina Okkelman
  • Dr. James Jenkins
  • All former lab members

  • Profs. Sergei Borisov (Graz University of Technology, Austria)

  • Dr. H. Dussmann (Royal College of Surgeons Ireland)

  • Dr. V.P. Baklaushev (Pirogov Medical University, Russia)

  • Dr. M. Tangney, (CCRC, University College Cork)

Prof John Cryan, Dr Anna Golubeva

Dr Silvia Melgar, Dr Niall Hyland

Prof Ulla Knaus, Dr Gabriella Aviello Funding: Science Foundation Ireland, Enterprise Ireland Follow us at: http://photobiolabcork.blogspot.ie