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Wickremesekera et al. J Cancer Metastasis Treat 2019;5:62 I http://dx.doi.org/10.20517/2394-4722.2019.09 Page 3 of 9

We here hypothesized expression of the RAS by CSCs in MM to the brain and investigated the expression
and localization of components of the RAS: PRR, ACE, ATIIR1 and ATIIR2, in relation to the putative
[21]
CSC subpopulations we have recently identified , using immunohistochemical (IHC) staining, Western
blotting (WB) and NanoString mRNA analysis.

METHODS
Tissue samples
Samples of MM to the brain from ten patients were sourced from the Gillies McIndoe Research Institute
Tissue Bank and used in this study that was approved by the Central Health and Disabilities Ethics
Committee (Ref. 15CEN28) with written informed consent from all participants.

Histology and IHC staining
Hematoxylin and eosin (H&E) staining was performed on 4 μm thick formalin-fixed paraffin-embedded
[21]
sections of MM to the brain from ten patients included in our previous study , to confirm the presence
of the tumor by an anatomical pathologist (HDB). 3,3-diaminobenzidine (DAB) IHC staining for CD34
(ready-to-use, cat# PA0212, Leica), Melan-A (ready-to-use, cat# PA0233, Leica), ACE (1:100; cat# MCA2054,
AbD Serotec, Kidlington, UK), PRR (1:2000; cat# ab40790, Abcam), ATIIR1 (1:30; cat# ab9391, Abcam),
ATIIR2 (1:2000; cat# NBP1-77368, Novus Biologicals, LLC, Littleton, CO, USA) as well as NANOG (1:100;
cat# ab80892, Abcam, Cambridge, UK), OCT4 (1:1000; cat# ab109183, Abcam) and ERG (1:200; EPII, Cell
Marque, Rocklin, CA, USA), diluted with Bond primary antibody diluent (cat# AR9352, Leica), was
TM
[30]
performed as previously described .
+
+
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To localize the components of the RAS in relation to the putative Melan-A and Melan-A OCT4 /
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SALL4 /SOX2 /NANOG CSC subpopulations within the tumor, and the pSTAT3 subpopulation on
+
[21]
the endothelium of microvessels within MM to the brain , immunofluorescence (IF) IHC staining was
performed on two representative samples of MM to the brain from the original cohort of ten patients
included for DAB IHC staining. Components of the RAS were co-stained with ESC markers OCT4 or
+
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NANOG as the surrogate markers for the Melan-A and the Melan-A OCT4 /SALL4 /SOX2 /NANOG
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+
+
+
+
CSC subpopulations, or endothelial markers ERG or CD34 for the pSTAT3 subpopulation on the
endothelium of microvessels within the tumor. Antibodies used for PRR and ATIIR2 detection were
VectaFluor Excel anti-rabbit 594 (ready-to-use; cat# VEDK-1594, Vector Laboratories) and Alexa Fluor
anti-mouse 488 (1:500; cat#A21202, Life Technologies). Antibodies for ACE and ATIIR1 detection were
VectaFluor Excel anti-mouse (ready-to-use; cat# VEDK2488, Vector Laboratories) and Alexa Fluor anti-
rabbit 594 (1:500; cat# A21207, Life Technologies).
Human tissues used for positive controls were placenta for PRR, liver for ATIIR1, and kidney for ATIIR2
and ACE. Negative controls were used in secondary and tertiary antibody staining by omitting the primary
antibodies on a randomly selected sample of MM to the brain.
Image analysis
DAB IHC stained-slides were viewed and images were captured with an Olympus BX53 light microscope
fitted with an Olympus SC100 digital camera (Olympus, Tokyo, Japan), and processed with the CellSens 2.0
software (Olympus). IF IHC-stained images were captured using an Olympus FV1200 biological confocal
laser-scanning microscope and processed with CellSens Dimension 1.11 software using 2D deconvolution
algorithms (Olympus).

WB
Five snap-frozen samples of MM to the brain from the original cohort of ten patients underwent WB as
[28]
described previously , using the primary antibodies: anti-PRR (ATP6IP2, 1:500; cat# ab40790, Abcam),