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Title: Uncovering the Immunogenicity of Neonatal Nav1.5 by Utilising Serum Samples from 4T1 Orthotopic Mice and Breast Cancer Patients

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Harishini Rajaratinam at Universiti Sains Malaysia
Harishini Rajaratinam
Wan Zainira
Wan Zainira
Wan Zainira
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Wan Zain
Wan Zain
Wan Zain
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Maya Mazuwin Yahya at Universiti Sains Malaysia
Maya Mazuwin Yahya
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Abstract

Neonatal Nav1.5 (nNav1.5) potentiates breast cancer (BCa) metastasis. However, its immunogenicity remains unexplored. The study's objective was to confirm the presence of circulating anti-nNav1.5 antibodies (anti-nNav1.5-Ab) in 4T1 orthotopic mice model and BCa patients. In the pre-clinical phase, 37 female BALB/c mice, were divided into two groups: a) control (n=20) b) 4T1 orthotopic mice (n=17). 4T1 cell injection was administered subcutaneously at the 3rd mammary fat pad of the 4T1 orthotopic mice, whereas PBS was introduced at a similar site of the control mice. After tumour development, both mice groups were sacrificed, followed by the collection of serum, 4T1 tumours and target organs. In-house indirect ELISA was conducted to detect anti-nNav1.5-Ab in the serum. Histopathology of the organs was carried out to validate BCa metastasis within the model. As for the clinical study, healthy females (n=64, mean age= 39.98 ± 1.79) and BCa patients (n=64, mean age= 47.81 ± 1.43) were recruited. Approximately 3 ml of blood was withdrawn, and the serum was separated. Similarly, an in-house indirect anti-nNav1.5-Ab ELISA assay, specifically for human antibodies, was designed. Both pre-clinical and clinical studies portrayed the positive presence of anti-nNav1.5-Ab. There was a significant difference in the absorbances of anti-nNav1.5-Ab between the control and 4T1 orthotopic mice (P<0.0001****). There were signs of metastasis on the heart, lungs, spleen, liver and kidneys of the 4T1 mice. Based on the ROC analysis, all the 4T1 mice exhibited absorbances of anti-nNav1.5-Ab above the cut-off value (>2.759). Similarly, the clinical study also reported a significant difference in the absorbances of anti-nNav1.5-Ab between healthy participants and BCa patients (P<0.0001****). The ROC analysis for the clinical study presented a cut-off value of >0.280. Unlike the pre-clinical study, 9.38% of control samples had a reading greater than the cut-off value. In conclusion, nNav1.5 is an immunogenic protein. Keywords Neonatal; Nav1.5; breast cancer; metastasis; immunogenicity
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Title: Uncovering the Immunogenicity of Neonatal Nav1.5 by Utilising Serum
Samples from 4T1 Orthotopic Mice and Breast Cancer Patients
Harishini Rajaratinam1, Wan Zainira Wan Zain2, Maya Mazuwin Yahya2,3,
Sabreena Safuan1, Nurul Asma-Abdullah1, Noor Fatmawati Mokhtar4, Wan Ezumi Mohd Fuad1*
1School of Health Sciences, Health Campus, Universiti Sains Malaysia (USM), 16150, Kubang Kerian, Kelantan, Malaysia.
2Department of Surgery, School of Medical Sciences, Health Campus, Universiti Sains Malaysia (USM), 16150, Kubang Kerian, Kelantan, Malaysia.
3Breast Cancer Awareness and Research (BestARi) Unit, Hospital Universiti Sains Malaysia (HUSM), 16150, Kubang Kerian, Kelantan, Malaysia.
4Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia (USM), 16150, Kubang Kerian, Kelantan, Malaysia.
Corresponding author: wanezumi@usm.my
Introduction and Objectives:
Neonatal Nav1.5 (nNav1.5) is the alternative
splice variant of Nav1.5. Nav1.5 is one of the nine
members of the voltage-gated sodium channel-
alpha subunit (VGSCα) family [1]. The
overexpression of nNav1.5 potentiates breast cancer
(BCa) metastasis. Its detection has been mainly
conducted using cell lines and breast tissues [2,3].
However, its immunogenicity remains unexplored.
Therefore, the study has aimed to validate the
presence of circulating anti-nNav1.5 antibodies
(anti-nNav1.5-Ab) in serum of 4T1 orthotopic mice
model (pre-clinical) and BCa patients (clinical).
The detection of anti-nNav1.5-Ab reflects the
immunogenicity of nNav1.5.
Methodology:
Preclinical study:37 female Balb/c mice, were
divided into two groups: a) control (n=20)b) 4T1
orthotopic mice (n=17). 4T1 murine mammary
cancerous cell injection was administered
subcutaneously at the 3rd mammary fat pad of the
4T1 orthotopic mice, whereas PBS was introduced at
a similar site of the control mice. After tumour
development, both mice groups were sacrificed,
followed by the collection of serum, 4T1 tumours
and target organs.
Clinical study: Healthy females (n=64, mean age=
39.98 ± 1.79) and BCa patients (n=64, mean age=
47.81 ± 1.43) were recruited based on the inclusion
and exclusion criteria. Approximately 3 ml of blood
was withdrawn, and the serum was separated.
For both preclinical and clinical study, in house
indirect ELISA assays were developed. For preclinical
study, an additional histopathology analysis was
included to validate the presence of BCa metastasis
in the 4T1 orthotopic mice. Heart control Heart 4T1 mice
Kidney control Kidney 4T1 mice
Spleen 4T1 mice
Lungs control
Liver control Liver 4T1 mice
Lungs 4T1 mice
Spleen control
Flow Chart of Methodology
Results (Preclinical and clinical studies)
(a) Development of 4T1 orthotopic
breast cancer mice model
Figure 1: The multi panel reflects the growth of tumour at the
3rd mammary fat pad (from day 40 till post mortem)
(b) Figure 2: Histopathology validated the presence of metastasis to
the heart, kidney, lungs, liver and spleen of 4T1 mice
(c) Figure 3: Significant difference in
the expression of anti-nNav1.5-Ab in
the serum of 4T1 and control mice
(P<0.0001****)
(d) Figure 4: Significant difference in
the expression of anti-nNav1.5-Ab in
the serum of healthy participants
and BCa patients (P<0.0001****)
Discussion and Conclusion
Both pre-clinical and clinical studies portrayed the
positive presence of anti-nNav1.5-Ab. The detection
of anti-nNav1.5-Ab reflects the immunogenicity of
nNav1.5. Even though, none of the control mice
exhibited these novel antibodies, 9.38%of the
healthy participants portrayed otherwise, which is in
agreement with the findings from Yamaci et al. [4].
References
1. Brackenbury WJ 2012 Voltage-gated sodium channels and metastatic disease.
Channels (Austin). 6352-361
2. Brackenbury WJ, Chioni AM, Diss JKJ and Djamgoz MBA 2007 The neonatal splice
variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human
breast cancer cells. Breast Cancer Res. Treat. 101 149-160
3. Fraser SP, Diss JK, Chioni AM, Mycielska ME, Pan H, et al. 2005 Voltage-gated sodium
channel expression and potentiation of human breast cancer metastasis. Clin. Cancer
Res. 11 5381-5389.
4. Yamaci RF, Fraser SP, Battaloglu E, Kaya H, Erguler K, et al. 2017 Neonatal Nav1.5
protein expression in normal adult human tissues and breast cancer. Pathol. Res.
Pract. 213 900-907 Acknowledgement: Funding from Research University Individual (RUI) grant (1001/PPSK/8012275).
ResearchGate has not been able to resolve any citations for this publication.
Voltage-gated sodium channels and metastatic disease
Article
Full-text available
Voltage-gated Na (+) channels (VGSCs) are macromolecular protein complexes containing a pore-forming α subunit and smaller non-pore-forming β subunits. VGSCs are expressed in metastatic cells from a number of cancers. In these cells, Na (+) current carried by α subunits enhances migration, invasion and metastasis in vivo. In contrast, the β subunits mediate cellular adhesion and process extension. The prevailing hypothesis is that VGSCs are upregulated in cancer, in general favoring an invasive/metastatic phenotype, although the mechanisms are still not fully clear. Expression of the Nav 1.5 α subunit associates with poor prognosis in clinical breast cancer specimens, suggesting that VGSCs may have utility as prognostic markers for cancer progression. Furthermore, repurposing existing VGSC-blocking therapeutic drugs may provide a new strategy to improve outcomes in patients suffering from metastatic disease, which is the major cause of cancer-related deaths, and for which there is currently no cure.
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The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells
Article
Full-text available
  • Feb 2007
Upregulation of functional voltage-gated Na+ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the "neonatal" splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by approximately 90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (approximately 30%), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33%, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43%, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42%, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.
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