Evaluation of chemotherapy and P2Et extract combination in ex-vivo derived tumor mammospheres from breast cancer patients

Abstract

The main cause of death by cancer is metastasis rather than local complications of primary tumors. Recent studies suggest that breast cancer stem cells (BCSCs), retains the ability to self-renew and differentiate to repopulate the entire tumor, also, they have been associated with resistance to chemotherapy and tumor recurrence, even after tumor resection. Chemotherapy has been implicated in the induction of resistant phenotypes with highly metastatic potential. Naturally occurring compounds, especially phytochemicals such as P2Et, can target different populations of cancer cells as well as BCSC, favoring the activation of immune response via immunogenic tumor death. Here, we evaluated the presence of BCSC as well as markers related to drug resistance in tumors obtained from 78 patients who had received (or not) chemotherapy before surgery. We evaluated the ex vivo response of patient tumor-derived organoids (or mammospheres) to chemotherapy alone or in combination with P2Et. A xenotransplant model engrafted with MDA-MB-468 was used to evaluate in vivo the activity of P2Et, in this model P2Et delay tumor growth. We show that patients with luminal and TNBC, and those who received neoadjuvant therapy before surgery have a higher frequency of BCSC. Further, the treatment with P2Et in mammospheres and human breast cancer cell lines improve the in vitro tumor death and decrease its viability and proliferation together with the release of immunogenic signals. P2Et could be a good co-adjuvant in antitumor therapy in patients, retarding the tumor growth by enabling the activation of the immune response.

Conflict of interest statement

SF, CU, TS and PL are inventors of a granted patent related to P2Et. The other authors declare no competing interests.

Figures

Figure 1

Cancer stem cell features in breast cancer patients. The patients were classified in Healthy Donor (HD), Luminal A (LA), Luminal B (LB), Triple negative (TN) and Her-2+. (a) Sample processing workflow of breast cancer tumors. (b) Multidimensional reduction analysis (tSNE) of flow cytometry data by breast cancer sub-type, rainbow scale represents relative intensity by channel; bulk population (gray) and CD44 + CD24-EPCAM + CD49f + (light blue). (c) CD45 + tumor-infiltration lymphocytes. (d) Frequency of Aldehyde dehydrogenase 1 (ALDH1) positive cells. (e) Frequency of Lin-CD44 + CD24-CD49f + EPCAM + cells. (f) Frequency of BCRP+. (g) Frequency of Pgp+. (h) Frequency of MRP1 + cells by flow cytometry. Data are presented as violin plots and each point represents independent samples; dotted lines indicate quartiles. Multiple comparisons were calculated by one-way ANOVA with Dunnet T3 correction, and significant exact p-values are shown. *p < 0.05; **p < 0.01; ***p < 0.001, **** p < 0.0001.

Figure 2

Neoadjuvant chemotherapy (NAT) before surgery increase the frequency of BCSC. (a) Frequency of BCSC (Lin-/CD44 + CD24-CD49f + EPCAM+) in healthy donor (HD), breast cancer patients with or without NAT before surgery. (b) Frequency of BCSC in LB patients with or without NAT before surgery. (c) Frequency of BCSC (ALDH +) in HD, breast cancer patients with or without NAT before surgery. (d) Frequency of BCSC (ALDH +) in TN breast cancer patients who received or not NAT before surgery. (AC (Anthracyclines + Ciclophosphamide), AC + TX (Anthracyclines/Ciclophosphamide + Taxanes), TX (Taxanes). Data are presented as violin plots and each point represents an independent sample; dotted lines indicate quartiles. Multiple comparisons were calculated by one-way ANOVA with Tukey post-test analysis and unpaired t-test, significant exact p-value are shown. *p < 0.05; **p < 0.01.

Figure 3

Breast cancer stem cells markers (ALDH+) correlated with BCRP expression in triple negative patients. Correlation of BCRP protein and ABCG2 gene expression with BCSC markers (ALDH+ and CD24-CD44 + CD49f + EPCAM +) in breast cancer patients who received or not NAT before surgery. (a) Correlation of BCRP protein with ALDH + and CD24-CD44 + CD49f+ EPCAM + expression in all samples. (b) Correlation of ABCG2 gene expression with ALDH + and CD24-CD44 + CD49f + EPCAM + in all samples. (c) Correlation of BCRP with ALDH + and CD24-CD44 + CD49f + EPCAM + expression in breast cancer patients who received NAT before surgery. Correlation of BCRP protein with BCSC markers (ALDH+ and CD24-CD44+ CD49f + EPCAM+) expression in (d) LA only, (e) LB only and (f) TN patients who received NAT before surgery. Correlations were assessed using nonparametric Spearman correlation, determination coefficient r and p-value are shown. *p < 0.05.

Figure 4

P2Et extract decrease the viability of mammospheres derived from breast cancer patients after in vitro treatment. (a) Representative confocal microscopy image of the effect of P2Et extract, DX or DX + P2Et on the mammospheres viability of breast cancer patients. Luminal A (LA), Luminal B (LB), Triple negative (TN) and Her2. Calcein AM (green), Ethidium homodimer-1 (Red). (b) Cell death percentage in mammospheres by molecular subgroups of breast cancer patients after treatment. (c) Cell death percentage in all mammospheres from breast cancer patients after treatment. Data are presented as violin plots and each point represents an independent sample; dotted lines indicate quartiles. Exact p-values were calculated using One-way ANOVA with Tukey post-test analysis. (d) Spearman nonparametric correlation between percentage of cell death with ABCB1, ABCG2, ABCC1 gene in mammospheres from breast cancer patients after P2Et, DX or Ethanol (negative control) treatment, coefficient of determination r and exact p-value are shown. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

P2Et extract decrease the viability and proliferation of human triple negative breast cancer cells. (a) Frequency of BCSC ALDH1 + and Lin-CD44 + CD24-CD49f + EPCAM + cells in MCF-7, BT-549 and MDA-MB-468 breast cancer cells measured by flow cytometry. Data are presented as the mean ± SEM. (b) IC50 values of P2Et extract or Doxorubicin (DX) in MCF-7, BT-549 and MDA-MB-468 using MTT Assay. (c) Left side: MDA-MB-468 cell count by trypan blue after treatment with vehicle (Ethanol), P2Et IC50, DX IC50, or P2Et + DX IC50 for 0, 48, 72 and 96 h. Right side: MDA-MB-468 cells were stained with CFSE and treated with P2Et IC50, DX IC50, or P2Et + DX IC50 for 0, 48, 72 and 96 h. For each time the proliferation was evaluated by flow cytometry. (d) The combined inhibitory effects of P2Et (0 to 500 μg/ml) and DX (0 to 1.16 μg/ml) were tested over a range of combinations against MDA-MB-468 cells using MTT Assay (Left side); a dose–response matrix was generated and analyzed for zero-interaction potency method (ZIP), using SynergyFinder pipeline (Right side). Data are presented as the mean of three independent experiments. (e) Frequency of death (Annexin V+ , propidium iodide [PI]−) and Annexin V+ , PI+) MDA-MB-468 cells after 24 and 48 h of treatment with Etanol, P2Et (IC50 and IC50*2) or DX (IC50 and IC50*2). MDA-MB-468 cells were stained with Annexin V-Alexa Fluor 488 and PI. Data are presented as the mean value ± SEM of 4 independent experiments. (f) MDA-MB-468 cells were treated with vehicle (ethanol or DMSO), P2Et (IC50 and IC50*2) or DX (IC50 and IC50*2) for 24 h. Surface exposure of Calreticuline (CRT) was determined by flow cytometry among viable cells (Aqua negative). Data are presented as violin plots and each point represents independent samples; dotted lines indicate quartiles. (g) MDA-MB-468 cells were treated vehicle (ethanol or DMSO), P2Et (IC50 and IC50*2) or DX (IC50 and IC50*2) for 48 h. After treatment cells were stained with quinacrine (1 μM) and PI. Quinacrine low cells were determined among viable cells (PI negative cells). Data are presented as violin plots and each point represents independent samples; dotted lines indicate quartiles. Exact significant p-values are shown. *p < 0.05; **p < 0.01; ***p < 0.001, **** p < 0.0001.

Figure 6

P2Et extract retard initially primary tumor growth but increase survival in triple negative human breast cancer. (a) Experimental scheme of treatment. MDA-MB-468 cells were inoculated into the mouse mammary gland, 8 days later, the mice were treated with PBS (negative control) or 18.7 mg/Kg of P2Et extract twice a week until endpoint. (b) Tumor growth (mm3) of MDA-MB-468 control (PBS) mice or mice treated with P2Et extract. Multiple t-test with Holm-Sidak method correction was performed assuming significance of α = 0.05. *p < 0.05.