Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture

Abstract

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24 population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs

“…Their results demonstrated that 94.12% of U87 spheroids and 83.91% of U373 spheroids were positive for CD133, whereas the monolayer culture resulted only in 63.56% and 67.6% positive cells for U87 and U373 cell lines, respectively (G et al, 2015). Other studies showed that the spheroids produced with CSC obtained from cancer cell lines (e.g., BT-474, GBM6, H1650, and MDA-MB-231) demonstrated a higher resistance toward different therapeutics (e.g., 5-FU, camptothecin, carmustine, cisplatin, gemcitabine, lomustine, paclitaxel, temozolomide, and trastuzumab) than 2D cell cultures (Godugu et al, 2013;Reynolds et al, 2017;Rodríguez et al, 2018; K. Wang et al, 2016).…”

“…For instance,Todaro et al (2007) produced spheroids with cells obtained from human colon adenocarcinoma tissue fragments and confirmed the expression of a CSC marker, that is, CD133. Other studies showed that the spheroids produced with CSC obtained from cancer cell lines (e.g., BT-474, GBM6, H1650, and MDA-MB-231) demonstrated a higher resistance toward different therapeutics (e.g., 5-FU, camptothecin, carmustine, cisplatin, gemcitabine, lomustine, paclitaxel, temozolomide, and trastuzumab) than 2D cell cultures(Godugu et al, 2013;Reynolds et al, 2017;Rodríguez et al, 2018;K. For example, the 100 µM of oxaliplatin reduced the CD133+ cell viability in only almost equal to 10%, whereas the same treatment reduced the CD133− cell viability to almost equal to 70%(Todaro et al, 2007).Optionally, spheroids can be produced with CSC obtained from cancer cell lines (Figure 3).…”

EPHA5 mediates trastuzumab resistance in HER2‐positive breast cancers through regulating cancer stem cell‐like properties

“…Evidence is mounting that CSCs are relatively quiescent, which contributes to treatment resistance and relapse (9, 10). Therefore, various studies have indicated that relapses observed in HER2‐positive breast cancer patients receiving adjuvant trastuzumab are due to the presence of CSCs that escape the effect of these therapeutic agents (11). There is increasing evidence that the subpopulation of CD44 + /CD24 −/low cells, which is enriched with breast cancer stem cell (BCSC)‐like properties, could explain clinical resistance to trastuzumab therapies.…”

Anti-tumor activity of plant extracts against human breast cancer cells are different in monolayer and three-dimensional cell culture screening models: A comparison on 34 extracts

“…Furthermore, in their study, Imamura and colleagues also showed that expression of Ki-67 was less in 3D breast cancer cell mass than in 2D, suggesting that the greater G0-dormant subpopulation was responsible for drug resistance in 3D culture. Many studies have show that the breast cancer cell population with phenotype CD44 + CD24 − possesses higher tolerability to chemotherapy, hormone therapy, and radiotherapy [16][17][18][19][20][21] . Thus, for the drug screening in our study, these 4 breast cancer cell lines were suitable for our evaluations:…”