Impact of Proton Irradiation Depending on Breast Cancer Subtype in Patient-Derived Cell Lines

Abstract

Research on different types of ionizing radiation's effects has been ongoing for years, revealing its efficacy in damaging cancer cells. Solid tumors comprise diverse cell types, each being able to respond differently to radiation. This study evaluated the radiobiological response of established (MDA-MB-231 (Triple negative breast cancer, TNBC), MCF-7 (Luminal A)) and patient-derived malignant cell lines, cancer-associated fibroblasts, and skin fibroblasts following proton IRR. All cell line types were irradiated with the proton dose of 2, 4, and 6 Gy. The radiobiological response was assessed using clonogenic assay, γH2AX, and p53 staining. It was noticeable that breast cancer lines of different molecular subtypes displayed no significant variations in their response to proton IRR. In terms of cancer-associated fibroblasts extracted from the tumor tissue, the line derived from a TNBC subtype tumor demonstrated higher resistance to ionizing radiation compared to lines isolated from luminal A tumors. Fibroblasts extracted from patients' skin responded identically to all doses of proton radiation. This study emphasizes that tumor response is not exclusively determined by the elimination of breast cancer cells, but also takes into account tumor microenvironmental variables and skin reactions.

Keywords: breast cancer; oncology; proton therapy; radiobiology; tumor microenvironment.

Figure 1

Phase-contrast images determine the morphology of isolated cell lines ((A) CAF 202, (B) NF202, (C) CAF211, (D) NF211, (E) CAF213, (F) NF213, (G) NF235). They represent an elongated, spindle-like shape morphology corresponding to a fibroblast-like phenotype. Pictures were taken at 100× magnification.

Figure 2

Flow cytometric analysis of the phenotype of isolated CAF and NF lines. The biomarker expression indicates specific cell line characteristics, confirming its phenotype and molecular subtype. For cell phenotyping, CD90, CD44, CD24, CD29, CD140a, and CD140b biomarkers were used. (A) Plot of each cell line’s relative mean fluorescence intensity of selected biomarkers. (B) Representative histograms of CAF and NF isolated from the same patient (no. 202) show the fluorescence intensity level. The gray histogram determines unstained cells (negative group), while the red one marks labeled cells with a specific antigen. The presented results are based on a single experiment.

Figure 3

Characterization of the BC239 cell line. (A): The BC239 line confirmation of BC origin using flow cytometry. The lack of CD90, CD31, and CD45 expression demonstrated exclusion of fibroblast, leukocyte, and endothelial features. The presence of CD44, CD326, and VIM and the lack of E-CAD and CD24 show the increased mesenchymal-like population with a high content of CSC originating from the epithelium. Grey histograms represent isotype control and red histograms correspond to the staining antigen of interest. (B): The relative gene expressions of the BC239 line. The genes characteristic for BC cells and TME were chosen. (C): The microscopic evaluation of BC239 cells showing spindle-like morphology. The pictures were taken using an inverted microscope Axio Vert.A1 (Carl Zeiss, Germany). Magnification ×100. (D): The evaluation of receptor status and p53 expression of the isolated primary breast cancer cell line using the Western blot technique. The blots represent the status of ERα, PRA/PRB, HER2 receptors, and p53 in commercially available breast cancer cell lines (MDA-MB-231, SKBR3, T47D, MCF-7), normal epithelial breast cell line (MCF12A), and the primary breast cancer cell line (BC239). GAPDH was used as a loading control. Below the blots, the signal intensity values normalized to GAPDH are shown.

Figure 4

Cell survival fraction (SF) post proton IRR. All cell lines were irradiated with 2, 4, and 6 Gy doses. The column graphs (left side) show the statistically significant differences between the cell lines for each dose. The curve plots (right side) were determined to illustrate the exponential dependency between the SF and dose. The various cell lines were: BC (A) (red), CAF (B) (orange), and NF (C) (green). The p-value was adjusted to * p < 0.05, ** p < 0.01, and *** p < 0.001. The values presented in the graph are the mean of the obtained results and the deviation is the standard deviation. The experiment was performed in triplicate.

Figure 5

The mean fluorescence intensity (MFI) of γH2AX 1 h post proton IRR. After IRR, the γH2AX was labeled to indicate DNA (DSB). Cells were irradiated using doses of 2, 4, and 6 Gy. The results were compared between different cell lines, and each dose was analyzed. Various cell lines were: BC (red), CAF (orange), and NF (green). The p-value was adjusted to * p < 0.05, ** p < 0.01, and *** p < 0.001. The values presented in the graph are the mean of the obtained results and the deviation is the standard deviation. The experiment was performed in triplicate.

Figure 6

p53 expression 1 h (A) and 24 h (B) post IRR. All cells were irradiated with a proton beam using 2, 4, and 6 Gy doses. The results were compared between different cell lines analyzing each dose. A p53 value for NF202, 24 h post-IRR with a dose of 4 Gy, was missing because of the cell lack. Various cell lines were irradiated with a proton beam: BC (red), CAF (orange), and NF (green). The p-value was adjusted to * p < 0.05, ** p < 0.01, and *** p < 0.001. The values presented in the graph are the mean of the obtained results and the deviation is the standard deviation. The experiment was performed in triplicate.

Figure 7

An example of an experiment that was set-up for cell IRR using a proton beam. This Solid Water^® High Equivalency phantom model enables IRR of cells cultured in 12- and 6-well plates. (A): Side view of positioned measurement set-up, (B): Top view of positioned measurement set-up.

Figure 8

A representative scheme from planning 2 Gy irradiation of a 12-well plate using a modulated scanning technique. (A): transversal view, (B): dose-volume histogram, (C): coronal view, (D): sagittal view.