Institut za onkologiju i radiologiju Srbije / Institute for Oncology and Radiology of Serbia
Published June 4, 2022 | Version v.1
Journal article Open

Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

Creators

  • 1. Department for Radiobiology and Molecular Genetics, Institute of Nuclear Sciences "Vinča", University of Belgrade, Belgrade, 11001 Serbia
  • 2. Department for Radiobiology and Molecular Genetics, Institute of Nuclear Sciences "Vinča", University of Belgrade, Belgrade, 11001 Serbia; State University of Novi Pazar, Department of Natural Sciences and Mathemathics, Novi Pazar, Serbia
  • 3. Institute of Oncology and Radiology of Serbia, Belgrade, Serbia
  • 4. Oncology Institute of Vojvodina, Sremska Kamenica, Serbia.
  • 5. Department for Radiobiology and Molecular Genetics, Institute of Nuclear Sciences "Vinča", University of Belgrade, Belgrade, 11001, Serbia.

Description

Abstract

Background: Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor β1 (TGFβ1) in BC patients.

Results: To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFβ1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN/nALN results revealed overexpression of both nuclear cFOS and cytosolic TGFβ1 protein levels. However, only the TGFβ1 data exhibited statistically significant overexpression, which was proportional to the respective values of mALN diameter of tumor deposits.

Conclusions: Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

Notes

Funding Financial support for this work, under Research Theme "Molecular alterations as prognostic and predictive markers in human malignant tumors" - 0802103 (VM, Principal Investigator), was provided by the Ministry of Education, Science and Technological Development, Republic of Serbia Registration number: 451-03-9/2021-14/200017). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript, apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Related works

Is documented by
3565-8894 (ISSN)
Is identical to
PMC9166494 (pmcid)
Is part of
1480-9222 (ISSN)