Adhesion and Migration Response to Radiation Therapy of Mammary Epithelial and Adenocarcinoma Cells Interacting with Different Stiffness Substrates

Abstract

The structural and mechanical properties of the microenvironmental context have a profound impact on cancer cell motility, tumor invasion, and metastasis formation. In fact, cells react to their mechanical environment modulating their adhesion, cytoskeleton organization, changes of shape, and, consequently, the dynamics of their motility. In order to elucidate the role of extracellular matrix stiffness as a driving force in cancer cell motility/invasion and the effects of ionizing radiations on these processes, we evaluated adhesion and migration as biophysical properties of two different mammary cell lines, over a range of pathophysiological stiffness (1–13 kPa) in a control condition and after the exposure to two different X-ray doses (2 and 10 Gy, photon beams). We concluded that the microenvironment mimicking the normal mechanics of healthy tissue has a radioprotective role on both cell lines, preventing cell motility and invasion. Supraphysiological extracellular matrix stiffness promoted tumor cell motility instead, but also had a normalizing effect on the response to radiation of tumor cells, lowering their migratory capability. This work lays the foundation for exploiting the extracellular matrix-mediated mechanism underlying the response of healthy and tumor cells to radiation treatments and opens new frontiers in the diagnostic and therapeutic use of radiotherapy.

Investigation of Biophysical Migration Parameters for Normal Tissue and Metastatic Cancer Cells After Radiotherapy Treatment

“…On stiff substrate, TER reduced only when cells were irradiation with the lower dose (reduction of 12%), whereas the principal displacement diminished by 42 and 46% and AT by 69 and 66% after irradiation with 2 and 10 Gy, respectively (Figures 2B-F, Table 1). In order to interpret our data in relation with previous results concerning cell velocity and reported in [29], we performed also a correlation analysis between TER and velocity data. In the case of normal tissue cells, TER and velocity resulted to be not or negatively correlated in control condition (correlations are equal to −0.11 and close to 0 on soft and stiff substrates, respectively), whereas we observed a weak positive correlation for cells irradiated with the dose of 2 Gy (0.11 on soft substrate and 0.15 on the stiff one) and a moderate positive correlation after irradiation with the dose of 10 Gy (0.26 on soft substrate and 0.24 on the stiff one).…”

“…24 h after cell culture, MCF10A and MDA-MB-231 cells were exposed to two different doses of X-rays, 2 and 10 Gy, with a dose rate equal to 5 Gy/min, using a 6MeV energy beam, at the National Cancer Institute "PASCALE" of Naples. Additional information can be found in [29].…”

“…MCF10A and MDA-MB-231 cells were cultured on PAAm substrates at a final density of 1,000 cells/cm 2 , to guarantee a sparse-cell condition also 72 h after irradiation (time of analysis), necessary to avoid the formation of cell-cell contacts that could override the effect of substrate stiffness. Cell migration experiment were performed as previously reported [29]. Briefly, images of cells were acquired every 10 min for a total duration of 24 h. Single cell trajectories were determined using ImageJ and Manual Tracking plugin (http://rsweb.nih.gov/ij/).…”

“…Actin cytoskeleton and nuclei were stained with Alexa 488 phalloidin and Hoechst 33342, respectively. More detailed information on staining protocol can be found in [29]. Images of stained cells were acquired with Olympus IX81 inverted microscope equipped with a 10× objective.…”

“…The clinical significance of these findings is still largely unknown and new cell biophysical parameters have to be identified in order to assess how RT treatments can promote increased cell migration [26,27] and enhanced growth of distant metastases [25,28] as well as reduction of cell migration [29,30] and inhibition of distant tumor growth, also known as the abscopal effect [31]. Along this path, we proposed here a continuation of our previous work [29] by the introduction of new biophysical parameters that can be used by different researcher interested into basic and clinical translation of mechanobiology investigation. In particular, in this study we focused our attention on breast cancer, a consolidated model used to understand cancer progression and metastasis and generally treated with RT from stages I to III to reduce the risk of recurrence after surgery.…”