Date Published: May 1, 2019
Publisher: Public Library of Science
Author(s): Maria K. Tveitarås, Frode Selheim, Kristina Sortland, Rolf K. Reed, Linda Stuhr, Aamir Ahmad.
The main objective of this study was to identify single proteins or protein networks that might be used as diagnostic biomarkers or for therapeutic purposes by evaluating the protein expression profiling of plasma and lungs at different stages of metastatic development in a human triple negative MDA-MB-231 breast cancer xenograft model. MDA-MB-231 tumour cells were injected into the mammary fat pads on one side of the groin area. The mice were sacrificed day 19 (pre-metastases) and day 54 (metastases). Non-injected mice served as controls. Plasma was collected and lungs harvested for both immunohistochemistry and protein analysis. The most striking observation in plasma was the initial reduction in haptoglobin level at the pre-metastatic stage, to a following significant increase in haptoglobin level at the metastatic stage, with a more than 4000-fold increase from the pre-metastatic to the metastatic phase. A corresponding increase in haptoglobin level was also found in lung tissue after metastasis. Fibrinogen beta chain also had a similar change in expression level in plasma as haptoglobin, however not as prominent. There were also changes in plasma thrombospondin-4 and transferrin receptor protein 1 levels, from an increase at the pre-metastatic stage, to a significant fall when metastases were established. This suggests that especially changes in haptoglobin, but also fibrinogen beta chain, thrombospondin-4 and transferrin receptor protein 1 is indicative of metastasis, at least in this breast cancer model, and should be further evaluated as general breast cancer biomarkers.
Breast cancer is the most common cancer among females worldwide. Each year, approximately 1.8 million patients are diagnosed with breast cancer, and more than 500 000 die from the disease . Metastatic spread of breast cancer is responsible for 90% of breast cancer-related deaths and thus remains the most important negative prognostic predictor . Therefore, recognition and understanding of the metastatic process is very important.
In this study we investigated the proteomic profiles of lungs and plasma at different stages of breast cancer development in mice with MDA-MB-231 xenografts. The establishment of metastasis was confirmed histologically. Five plasma and 5 lung samples from each group (control, pre-metastatic and metastatic) were subjected to identical protein extraction and LC-MC and MS/MS procedures, however one control and two pre-metastatic plasma samples had to be discarded due to haemolysis of red blood cells (erythrocytes). We identified 389 proteins for the plasma proteome, of these 339 proteins had valid label-free quantification values after removal of potential contaminants and reverse hits.
The present study identified a number of proteins and biological processes that showed altered levels in plasma and in lung tissue, both before and after spontaneous metastasis developed from primary breast cancer (MDA-MB-231) in the mammary fat pad in mice.
The procedure of collecting a blood sample from a patient is a simple procedure, and a plasma sample is thus easily obtainable from patients. It is therefore highly favourable to be able to identify proteins in plasma that could aid the identification of breast cancer at an early stage. In plasma the most prominent changes in protein expression were haptoglobin and fibrinogen beta chain, from a marked down-regulation before development of metastasis to significant up-regulation after the establishment of metastasis, with haptoglobin being the most striking observation. This is also reflected in lung tissue after metastasis. There were also changes in plasma thrombospondin-4 and transferring receptor protein 1 levels, from an increase at the pre-metastatic stage, to a significant fall when metastases were established. Taken together this suggests that especially changes in haptoglobin levels, but also fibrinogen beta chain, thrombospondin-4 and transferrin receptor protein 1 levels are indicative of metastasis in at least this breast cancer model, and should be further evaluated as general breast cancer biomarkers.