Breast cancer (BC) is the most common cancer in women. Although standard treatments are successful in patients with BC diagnosed at an early stage, an alternative treatment is required for patients with advanced-stage disease who do not respond to these treatments. The concept of using chemotherapy to sensitize cancer cells to become susceptible to immunotherapy was recently introduced and may be used as an alternative treatment for BC. The chemotherapeutic drug doxorubicin has been reported to sensitize cancer cells; however, the efficacy to sensitize the solid spheroids, in addition to its underlying mechanism regarding how doxorubicin sensitizes BC, has not previously been explored. In the present study, the effectiveness of a combined treatment of doxorubicin and natural killer-92 (NK-92) cells against BC in either 2D or 3D spheroid models, and its association with Fas receptor (FasR) expression, was demonstrated. The BC (MCF7) cell line expressing a higher level of FasR was more sensitive to NK-92 cell killing than the MDA-MB-231 cell line, which expressed a lower level of FasR. A sublethal dose of doxorubicin caused a significant improvement in NK cytotoxicity. Concordantly, a significant reduction in cell viability was observed in the doxorubicin-treated MCF7 spheroids. Notably, flow cytometric analysis revealed significantly increased FasR expression in the MCF7 cells, suggesting the underlying sensitization mechanism of doxorubicin in BC was related to the FasR upregulation. The present findings supported the use of combined doxorubicin and NK immunotherapy in BC treatment.
Breast cancer (BC) is the most common cancer diagnosed in women worldwide (
Immunotherapy functions by prompting the immune system to fight against cancer, since host immunity is the key element for controlling carcinogenesis, progression and metastasis (
Previously, the combined effects of chemotherapeutic drugs and immunotherapy have been reported to enhance the cytotoxic activity of immunotherapy against cancer cells and promote the therapeutic effect (
To investigate the sensitization mechanism of doxorubicin in BC, the killing activity of NK cells in two different BC cell lines, including MCF7 (ER+/PR+/HER2−) and MDA-MB-231 (ER−/PR−/HER2−), with and without doxorubicin treatment, was compared in the present study. The cytotoxicity of doxorubicin and its ability to sensitize MCF7 and MDA-MB-231 cells to NK-92 cell killing was investigated. It was demonstrated that the treatment with a sublethal dose of doxorubicin caused MCF7, but not MDA-MB-231 cells, to become more sensitive to NK-92 cells and primary NK cells isolated from healthy donors via upregulation of Fas-receptor (FasR) expression. The knowledge obtained from the present study supports the use of a combined chemotherapy-immunotherapy regimen for the treatment of BC, and suggests that FasR could be used as a marker for predicting the response to combined doxorubicin-NK cell therapy.
BC cell lines, MCF7 (ER+/PR+/HER2−), T47D (ER+/PR+/HER2−) and MDA-MB-231 (ER−/PR−/HER2−) were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), and 100 mg/ml of penicillin/streptomycin (MilliporeSigma). The lymphoblast cell line K562 was obtained from the ATCC and maintained in Roswell Park Memorial Institute-1640 (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 100 mg/ml penicillin/streptomycin. An NK cell line, namely NK-92, was purchased from the ATCC and maintained in Minimum Essential Medium Eagle-α Modification supplemented with 12.5% horse serum (Gibco; Thermo Fisher Scientific, Inc.) and 12.5% FBS, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid and 100-200 U/ml recombinant IL-2 (R&D Systems, Inc.). Mycoplasma testing was performed for the cell lines used. The cells were cultured at 37°C in a 5% CO2 humidified incubator.
The lentivirus transfer plasmid (pCDH vector) was a kind gift from Associate Professor Naravat Poungvarin (Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand). The RFP-encoding lentivirus, namely pCDH-RFP, was constructed by cloning the RFP coding sequences to the vector backbone. Using the 2nd generation lentivirus packing system, the construct (500 ng) was used to transfect into the 293T cells (ATCC; 5×105 cells in a 35-mm dish; maintained in DMEM supplemented with 10% FBS and 100 mg/ml penicillin/streptomycin), together with 50 ng of envelope (pMD2.G) and 500 ng of packaging plasmids (psPAX2) to produce the lentivirus using the Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were incubated for 6 h at 37°C, and the 2 ml of fresh media was replaced. The lentivirus was harvested at 48 h after transfection and titrated using qPCR Lentivirus Titer kit (cat. no. LV900; Applied Biological Materials). The virus was used to transduce the MCF7 and MDA-MB-231 cells at a multiplicity of infection of 100 for 24 h. Subsequently, puromycin (Thermo Fisher Scientific, Inc.) was added at the concentration of 2
Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors using Lymphocyte Separation Medium (Corning, Inc.) according to the manufacturer's protocol. The present study was conducted according to the guidelines of the Declaration of Helsinki and was approved (approval no. COA 286/2021) by the Siriraj Institutional Review Board of the Faculty of Medicine Siriraj Hospital (Bangkok, Thailand). Written consent was obtained during April-June 2021 and followed the approved protocol by which all donors agreed to the use of their samples in scientific research. The primary NK cells were sorted using CD56 MicroBeads (Miltenyi Biotec GmbH) according to the manufacturer's protocol. The primary NK cells were maintained in RPMI-1640 medium, 10% FBS, 2 mM GlutaMAX™ (Gibco; Thermo Fisher Scientific, Inc.), 1% non-essential amino acid and 1% penicillin/streptomycin, supplemented with 100 U/ml recombinant IL-2. The CD56+ cells were expanded by co-culturing with irradiated membrane-bound IL-21 expressing K562 cells (artificial antigen-presenting cells) at a 1:1 ratio in the culture medium supplemented with 100 U/ml recombinant IL-2. The NK cells were expanded for 6 days, with the medium being changed on day 3 of co-culture.
The cytotoxicity of doxorubicin (MilliporeSigma) was evaluated in the MCF7 and MDA-MB-231 cells using a cell viability assay. The cells were plated in a 96-well plate at a density of 7,000 cells/well 1 day before the experiment. Various concentrations of doxorubicin (0, 0.0064, 0.032, 0.16, 0.8, 4, 20 and 100
Primary NK cells were propagated
The effect of doxorubicin on surface protein expression was investigated. MCF7 and MDA-MB-231 cells were plated onto a 24-well plate at a density of 30,000 cells/well 1 day before the experiment. Doxorubicin was added to the cells at various concentrations (0, 40, 80 and 160 nM). The cells were cultured for 24 h at 37°C, in a 5% CO2 cell culture incubator, and harvested for flow cytometric analysis. Briefly, MCF7 and MDA-MB-231 cells were harvested via centrifugation (800 × g) at 4°C for 5 min and incubated with monoclonal antibodies (at 1:50 dilution), including anti-TRAIL1-PE (cat. no. 12-6644-42), anti-TRAIL2-PE (cat. no. 12-9908-42), anti-CD95-APC (cat. no. 17-0959-42), anti-MHC class I polypeptide-related sequence (MIC)-A/B-FITC (cat. no. 53-5788-42), anti-human leukocyte antigen (HLA)-ABC-FITC (cat. no. 11-9983-42) (all Thermo Fisher Scientific, Inc.) and anti-programmed death-ligand 1 (PD-L1)-FITC (cat. no. 393606; BioLegend, Inc.), for 15 min at room temperature. The respective isotype controls were also obtained from eBioscience; Thermo Fisher Scientific, Inc. The stained cells were analyzed using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences) using FlowJo software version 10.
The BC cell lines, MCF7 and MDA-MB-231, treated with various concentrations of doxorubicin, were collected for total RNA extraction using TRIzol® reagent (Thermo Fisher Scientific, Inc.). RNA was converted into cDNA using a SensiFAST cDNA Synthesis kit (Bioline; Meridian Bioscience) according to the manufacturer's protocol. The qPCR analysis was performed using a iCycler iQ™ real-time PCR detection system (Bio-Rad Laboratories, Inc.) using the 2X SensiFast™ SYBR® kit (Meridian Bioscience) and the following primers: FasR forward, 5′-ATG CTG GGC ATC TGG ACC CT-3′ and reverse, 5′-CAA CAT CAG ATA AAT TTA TTG CCA-3′; and GAPDH forward, 5′-CGA CCA CTT TGT CAA GCT CA-3′ and reverse, 5′-AGG GGT CTA CAT GGC AAC TG-3′. The PCR conditions were as follows: An initial denaturation step at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec and extension at 72°C for 45 sec. The final extension was performed at 72°C for 5 min. The housekeeping gene
A killing assay was conducted to determine the NK-92 killing activity against BC cells. The MCF7 and MDA-MB-231 cells were genetically engineered to express RFP. The RFP-expressing MCF7 and MDA-MB-231 cells were used to examine the killing efficiency of NK-92 cells. The cells were plated in a 96-well plate at a density of 7,000 cells/well 1 day before the experiment. Co-culture of the effector NK-92 cells (E) and the RFP-expressing MCF7 or MDA-MB-231 target cells (T) was performed using different E:T ratios, including 1:1, 2.5:1 and 5:1. At 24 h after co-culture at 37°C in a 5% CO2 humidified incubator, the NK-92 cells and the dead cells were removed by pipetting and 100
In addition, the killing assay was conducted using a 3D spheroid model. Briefly, the MCF7 cells (2×103 cells) were labelled with CellTracker™ Green 5-chloromethylfluorescein diacetate dye (Thermo Fisher Scientific, Inc.) at 37°C for 10 min before seeding onto an ultra-low attachment 96-well round-bottomed plate containing 2.5% Corning Matrigel matrix (both from Corning, Inc.). The plate was then centrifuged at 1,000 × g at 4°C for 10 min to allow spheroid formation for 24 h at 37°C, in a 5% CO2 cell culture incubator. Doxorubicin (320 nM) was added to the spheroids for 24 h before introducing the NK cells in the culture medium at an E:T ratio of 5:1. The remaining living cells were analyzed by confocal microscopy (Nikon Corporation). Images were acquired by Nikon Eclipse Ti confocal microscope and analyzed using the NIS-Element software, version 4.2. Cell viability was calculated using the following formula: (mean fluorescence intensity (MFI) of spheroid with treatment condition/MFI of spheroid alone) ×100.
All experiments were conducted in triplicate. P<0.05 was used to indicate a statistically significant difference. Data were analyzed using the unpaired Student's t-test for two groups, in addition to a one-way ANOVA with Tukey's post hoc test for multiple comparisons, using GraphPad Prism software version 8 (GraphPad Software, Inc.).
The ability of NK cells to eliminate BC cells was examined in MCF7 cells and the triple-negative BC MDA-MB-231 cells. The viability of cancer cells after 24 h of co-culturing with NK-92 cells was determined at E:T ratios of 0:1, 2.5:1 and 5:1. The NK-92 cells demonstrated their killing ability in a dose-dependent manner (
To explain the distinct sensitivity of MCF7 and MDA-MB-231 cells to NK-92 cytotoxicity, the expression levels of TRAIL receptors, FasR, MIC-A/B, HLA class I and PD-L1, were investigated (
Notably, the death receptors, including TRAIL receptors and FasR, were expressed in both cancer cell lines, but in the opposite manner. The MDA-MB-231 cells expressed higher levels of TRAIL1 and TRAIL2 receptors than those of the MCF7 cells. In more detail, the mean TRAIL1 receptor expression was 24.18% in the MDA-MB-231 cells compared with 13.04% in the MCF7 cells and the mean TRAIL2 receptor expression was 82.98% in the MDA-MB-231 cells compared with 45.17% in the MCF7 cells. By contrast, the MCF7 cells expressed significantly higher levels of FasR than that of the MDA-MB-231 cells (44.47 vs. 19.81%) (
The immune-modulation activity of doxorubicin has previously been reported (
The sublethal dose of doxorubicin (160 nM) was selected and tested for its activity in modulating TRAIL receptor, FasR, MIC-A/B, HLA class I and PD-L1 expression (
Based on the flow cytometric results, it was speculated that the expression profiles of FasR, PD-L1 and HLA class I may play an important role in MCF7 sensitivity to NK cells. Additionally, the treatment with doxorubicin resulted in an increase in FasR expression in the MCF7 cells, but not in the MDA-MB-231 cells. It was then investigated whether this alteration in FasR expression sensitized the MCF7 cells and increased the cytotoxic activity of the NK-92 cells. The percentages of MDA-MB-231 and MCF7 cell death were compared after co-culturing with the NK-92 cells in the absence and presence of 160 nM of doxorubicin. The results demonstrated that the NK-92 cells killed both types of cells in a dose-dependent manner (
The effect of doxorubicin to increase the FasR expression and its relationship to enhance the NK killing activity were further investigated. Another BC cell line, T47D, was used to demonstrate the doxorubicin sensitization in BC cells. Treatment of doxorubicin at 160 nM increased the expression of FasR and the corresponding MFI (
The potential of doxorubicin in combination with the NK-92 cells was further examined in the present study using spheroid 3D culture to reflect its effect on controlling tumor mass. The MCF7 spheroids were generated and treated with doxorubicin at 320 nM prior to co-culturing with the NK-92 cells. The number of living cells (green color) was monitored. The cell viability observed after 24 and 48 h of co-culturing with the NK-92 cells tended to decrease, particularly in the doxorubicin-treated cells (
To confirm the effect of doxorubicin on cancer cell sensitization, the effect of doxorubicin on enhancing primary NK killing activity was investigated. Primary NK cells were isolated from the PBMCs of two healthy donors and were expanded
The use of immunotherapy to enhance immune response is currently the front-line strategy for cancer treatment and control. Among the different types of immune cells used to fight against cancer cells, NK cells are an attractive cellular immunotherapy strategy, as reflected by a large number of ongoing clinical trials (
Variations in tumor subpopulations caused by genetic or non-genetic factors, so-called heterogeneity, are known to exert significant influence on the outcome of cancer treatment (
FasR (CD95; TNF receptor superfamily member 6) is constitutionally expressed in human cells, and functions to mediate the antitumor cytotoxicity of NK cells and lymphocytes (
Apart from FasR/FasL signaling, chemotherapy treatment has been reported to promote sensitization of cancer cells via other mechanisms, i.e., the stress pathway and damage-associated molecular pattern (DAMP) (
BC cell sensitization by doxorubicin could be applied to adoptive T cell therapy, since both perforin and FasL individually enhanced the killing ability of T cells and NK cells (
A broad range of immunotherapeutic approaches has previously been developed. Adoptive immune cell therapy has been reported to treat BC (
In conclusion, the results of the present study demonstrated the role of FasR modulation in the response to doxorubicin treatment to enhance NK cell killing activity in BC cells. This finding supports the development and use of combined chemo-immunotherapy and immuno-modulation for the treatment of BC.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
NS planned the studies, designed and conducted experimental works, analyzed data and edited the manuscript. MW and NT conducted the primary NK isolation and culture, and edited the manuscript. NP conducted the killing assay. CC conducted the RT-qPCR. CT designed experiments. PTY designed experiments and edited the manuscript. AP managed the research team, planned the studies, designed and conducted experiments, analyzed data, and drafted and edited the manuscript. NS and AP confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.
The present study was conducted according to the guidelines of the Declaration of Helsinki and was approved (approval no. COA 286/2021) by the Siriraj Institutional Review Board of the Faculty of Medicine Siriraj Hospital, Mahidol University, (Bangkok, Thailand).
Not applicable.
The authors declare that they have no competing interests.
The authors would like to thank Assistant Professor Kevin P. Jones (Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand) for language editing of the original manuscript.
Sensitivity of breast cancer cells to NK cytotoxicity. (A) The cytotoxic ability of the NK-92 cells for killing red fluorescence protein-expressing MDA-MB-231 and MCF7 cells was measured under a fluorescence microscope at different E:T ratios. (B) Representative merged images of the NK-92 cells (no color) and the target cells (red). (C) The viability of the target cells after co-culturing with the NK92 cells. Data were obtained from three independent experiments. Data are presented as the mean ± SEM. Scale bar, 100
Protein expression profiles of TRAIL receptors, FasR, MIC-A/B, HLA class I and PD-L1 in BC cell lines. The expression profiles of (A) TRAIL1 receptor (A and B) TRAIL2 receptor, (C) FasR, (D) MIC-A/B, (E) HLA class I (ABC) and (F) PD-L1 were compared between two different BC cell lines, MDA-MB-231 (orange bar) and MCF7 (cyan bar), using flow cytometric analysis. Data**) were obtained from three independent experiments. Data are presented as the mean ± SEM. TRAIL, TNF-related apoptosis-inducing ligand; FasR, Fas receptor; HLA, human leukocyte antigen; PD-L1, programmed death-ligand 1; BC, breast cancer.
Cytotoxicity of doxorubicin in two different breast cancer cell lines. The cytotoxicity of doxorubicin at various concentrations (0-100
Effects of doxorubicin on the modulation of TRAIL receptor, FasR, MIC-A/B, HLA class I and PD-L1 expression in breast cancer cell lines. The MDA-MB-231 and MCF7 cells were treated with a sublethal dose of doxorubicin (160 nM). The percentage of positive cells expressing TRAIL receptors, FasR, MIC-A/B, HLA class I and PD-L1 protein was monitored after a 24-h treatment with doxorubicin in (A) the MDA-MB-231 cells and the (B) MCF7 cells as analyzed by flow cytometry. The dose-dependent effects of doxorubicin on the expression of (C) FasR mRNA and the number of (D) FasR-positive cells were also examined. Data were obtained from three independent experiments. Data are presented as the mean ± SEM. TRAIL, TNF-related apoptosis-inducing ligand; FasR, Fas receptor; HLA, human leukocyte antigen; PD-L1, programmed death-ligand 1; ns, not significant.
Doxorubicin sensitization increases NK cytotoxicity in the MCF7 cells. The effect of doxorubicin in sensitizing (A) the MDA-MB-231 cells and (B) the MCF7 cells to NK-92 cell killing was evaluated. The numbers of cancer cells after doxorubicin treatment, NK-92 cell treatment (at 2.5:1 and 5:1 E:T ratios), or treatment with a combination of doxorubicin and NK-92 cells were determined under a fluorescence microscope (left). The percentage of cancer cell viability was determined using crystal violet staining relative to the non-treated control (set as 100%). The data were analyzed from at least three independent experiments. Data are presented as the mean ± SEM. Scale bar, 100
Doxorubicin sensitization increases NK cytotoxicity of the T47D breast cancer cells. (A) The effect of doxorubicin on FasR expression levels in the T47D cells was determined by flow cytometric analysis. The histograms demonstrate mean fluorescence intensities of the isotype control (gray), the untreated cells (pink) and the doxorubicin-treated cells (cyan). (B) Crystal violet staining of the T47D cells after the NK-92 cell treatment (at E:T ratios of 0:1, 1:1 and 2.5:1), or the combination of doxorubicin (160 nM) and NK-92 cell treatment. Data were obtained from three independent experiments. Data are presented as the mean ± SEM. NK, natural killer; E, effector NK-92 cells; T, MCF7 or MDA-MB-231 target cells; APC, antigen presenting cells.
Doxorubicin sensitizes the MCF7 spheroid to be killed by the NK-92 cells. The effect of doxorubicin to sensitize the MCF7 cells to be susceptible to cytotoxicity by the NK-92 cells was evaluated in the spheroid model. The numbers of the MCF7 living cells (green) were monitored by confocal microscope at (A) 24 h and (B) 48 h after co-culturing with the NK-92 cells. The four experimental conditions included the untreated spheroid, the doxorubicin-treated spheroid, spheroid with the NK-92 cells, and the doxorubicin-treated spheroid and the NK-92 cells. (C) The viability of the MCF7 cells at 24 and 48 h after co-culture. The data were obtained from three independent experiments, which were normalized to the untreated control. Data are presented as the mean ± SEM. NK, natural killer.
Combination of primary NK cells and doxorubicin increases MCF7 cell death. (A) Primary NK cells from two healthy donors were expanded and characterized for the expression of CD45, CD56, CD3 and CD16 using flow cytometric analysis. (B) The ability of primary NK cells to kill the MCF7 cells in the presence or absence of doxorubicin. Data are presented as the mean ± SEM. NK, natural killer; ns, not significant.