Page 60 - Read Online

Narayanan et al. J Cancer Metastasis Treat 2019;5:36 I http://dx.doi.org/10.20517/2394-4722.2018.77 Page 3 of 11

is physiologically released from the liver into the circulation and is converted to angiotensin I (ATI) by
renin - the active form of the proenzyme, pro-renin. The receptor for both renin and pro-renin is pro-renin
[23]
receptor (PRR) . ATI is then converted to angiotensin II (ATII) by angiotensin converting enzyme (ACE).
The effects of ATII are mediated through its receptors, angiotensin II receptor 1 (ATIIR1) and angiotensin II
[24]
receptor 2 (ATIIR2) .

Recent studies show that the RAS promotes tumor growth at a local tissue level by modulating angiogenesis,
[24]
tumor cell proliferation, immune responses and extracellular matrix formation . Patients who are
administered RAS modulators have a reduced incidence of CRC as well as lower rate of developing distant
metastasis from CRC [25,26] .

[17]
CSCs in a number of cancer types, including OCSCC [16,27,28] and GB have been shown to express
components of the RAS. Hence CSCs may be a novel therapeutic target by modulation of the RAS. A
recent meta-analysis reports an average 20% reduced risk of metastasis and improved survival from cancer
[29]
including CRC, in patients who are administered RAS modulators .

Currently there are few publications showing the presence of the components of the RAS in LMCA.
This study aimed to investigate the expression of the components of the RAS: PRR, ACE, ATIIR1, and
[21]
ATIIR2 within LMCA, in relation to the three CSC subpopulations we have recently identified , using
3,3-diaminobenzidine (DAB) and immunofluorescence (IF) immunohistochemical (IHC) staining, Western
blotting (WB), and NanoStrong mRNA expression analysis.

METHODS
Tissue samples
LMCA tissue samples from 16 male patients aged 50-80 (mean 65) years including those in our previous
[21]
study were sourced from the Gillies McIndoe Research Institute Tissue Bank and used for this study
which was approved by the Central Health and Disabilities Ethics Committee. Informed written consent
was obtained from all participants.

Histochemical and immunohistochemical staining
Hematoxylin and eosin (H&E) staining was carried out on 4 μm-thick formalin-fixed paraffin-embedded
sections of 16 LMCA tissue samples to confirm the presence of the tumor on the slides by an anatomical
pathologist (HDB). DAB IHC staining was then performed on these sections using the Leica Bond Rx
auto-stainer (Leica, Nussloch, Germany) with primary antibodies for PRR (1:2000; cat# AB40790, Abcam,
Cambridge, MA, USA), ACE (1:100; cat# MAB4399, AbD Serotec, Oxford, UK), ATIIR1 (1:300; cat#
AB9391, Abcam), and ATIIR2 (1:2000; cat# NBP1-77368, Novus Biologicals, Littleton, CO, USA), OCT4
(1:30; cat# MRQ-10, Cell Marque), SOX2 (1:200; cat# PA1-094, Thermo Fisher Scientific, Rockford, IL,
USA). Antibodies were diluted in Bond primary antibody diluent (Leica). All DAB IHC-stained slides were
mounted in Surgipath Micromount (Leica, Nussloch, Germany). To confirm co-expression of two proteins,
two representative samples of LMCA from the original cohort of 16 patients used for DAB IHC staining,
underwent IF IHC staining. Vectafluor Excel anti-mouse 488 (ready-to-use; cat# VEDK2488, Vector
Laboratories, Burlingame, CA, USA) and Alexa Fluor anti-rabbit 594 (1:500; cat# A21207, Life Technologies,
Carlsbad, CA, USA) were used to detect the combinations. All IF IHC-stained slides were mounted in Vecta
Shield Hardset mounting medium with 4, 6-diamino-2-phenylindone (Vector Laboratories). All antibodies
were diluted in Bond primary diluent (Leica). All DAB and IF IHC staining was performed using the Leica
Bond Rx auto-stainer (Leica), as previously described [16,28,30] .

Image analysis
The DAB IHC-stained slides were viewed and the images were captured using an Olympus BX53 light
microscope fitted with an Olympus DP21 digital camera (Olympus, Tokyo, Japan). An Olympus FV1200