Triple negative breast cancer (TNBC) lacks targeted treatment resulting in poor prognosis. Targeting overexpressing mesothelin (MSLN) using MSLN-specific T cells is an attractive treatment approach and the aim of the present study. The expression of MSLN in human TNBC paraffin sections was analyzed by immunohistochemistry. Lentiviral vector harbored granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and MSLN cDNAs was constructed to generate self-differentiated myeloid-derived antigen-presenting-cells reactive against tumor expressing MSLN dendritic cell (MSLN-SmartDC) for MSLN-specific T cell activation. The results showed high MSLN in 32.8% of all breast cancer subtypes and 57% in TNBC. High MSLN was significantly associated with TNBC subtype and the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. MSLN-SmartDC exhibited comparable phenotype to DC generated by exogenous cytokine treatment and an addition of 40s ribosomal protein subunit 3 (RPS3), a toll-like receptor 4 ligand, enhanced DC maturation and function by upregulation of CD40, CD80 and CD83 expressions and IL-12p70 secretion. MSLN-specific CD8+CD69+ IFN-γ+ T cells were detected in T cells activated by both MSLN-SmartDC and RPS3-MSLN-SmartDC. MSLN-specific T cells activated by these DCs showed more specific killing capability against naturally expressed MSLN-HCC70 and artificially MSLN-overexpressing MDA-MB-231 compared with parental MDA-MB-231 in both two dimensional (2D)- and 3D-culture systems. In conclusion, the results demonstrated the efficacy of MSLN-SmartDC to promote MSLN-specific T cells response against TNBC and RPS3 can enhance the cytolytic activity of these T cells providing an alternative treatment approach for patients with TNBC.
Triple negative breast cancer (TNBC) is a subtype of breast cancer accounting for 15–20% of all breast cancer and characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) (
Dendritic cells (DCs)-based immunotherapy aims to induce an antigen-specific immune response through DCs presenting antigens to stimulate cancer-specific T cells returned to the patients to eliminate cancer cells (
Mesothelin (MSLN) is a glycophosphatidylinositol-linked glycoprotein limitedly expressed in mesothelial cells but reported to be aberrantly expressed in various types of cancer, such as ovarian cancer, pancreatic cancer, mesothelioma and TNBC (
The efficacy of T cells activated by DCs is greatly affected by the maturation status of DCs including the human leukocyte antigens (HLAs), co-stimulatory molecules and cytokines expressions (
A total of 351 cases of paraffin-embedded breast cancer tissues and clinicopathological data (
MDA-MB-231, HCC70 and T2 cell lines were from American Type Culture Collection. Lenti-X™ 293T cells were from Takara Bio (Takara Bio, San Jose, CA). The Lenti-X™ 293T cells, MDA-MB-231 and MSLN-MDA-MB-231, produced by lentiviral transduction, were maintained in DMEM with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.). HCC70 cells were cultured in RPMI-1640 with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). The T2 cell line was cultured in RPMI-1640 with 10% FBS and 2 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.). All cell lines were cultured at 37°C with 5% CO2.
The construction of lentiviral vector was performed as previously described (
The peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of HLA-A2+ healthy donor blood with written consent under Siriraj Institutional Review Board ethical approval (COA no. Si 580/2018) by density centrifugation at 800 × g for 20 min at RT in lymphocyte separating medium (Corning Life Sciences). The monocytes were isolated and incubated for 1 h at 37°C. The non-adherence cells were collected and cryopreserved in human AB serum (MilliporeSigma) containing 10% dimethyl sulfoxide until use. The monocytes were transduced with IRFP-SmartDC or MSLN-SmartDC at 75 multiplicity of infection (MOI) together with 10 µg/ml protamine sulfate in AIM-V medium (Invitrogen; Thermo Fisher Scientific, Inc.). On day 5 post-transduction, 1 µg/ml of recombinant human RPS3 (cat. no. NBP2-90977; Novus Biologicals, LLC) was added. Monocytes were cultured in 100 ng/ml of rhGM-CSF (cat. no. 11343125; ImmunoTools GmbH) and 50 ng/ml of rhIL-4 (cat. no. 11340045; ImmunoTools GmbH) for five days and treated with 100 ng/ml of rhIFN-γ (cat. no. 11343536; ImmunoTools GmbH) and rhTNF-α (cat. no. 11343015; ImmunoTools GmbH) for additional 48 h served as positive control or conventional DC (conv. DC). All DCs were harvested on day 7 to check the activated characters.
The cryopreserved T cells were thawed and co-cultured with DCs for 48 h at a 10:1 ratio to support T cell activation and proliferation (
The immunophenotype of monocytes and DCs were assessed by anti-CD14 (cat. no. 21620143, ImmunoTools GmbH), anti-CD40 (cat. no. 21270403, ImmunoTools GmbH) and anti-human leukocyte antigen (HLA)-DR (cat. no. 21278993, ImmunoTools GmbH), anti-CD80 (cat. no. 12-0809-42, Thermo Fisher Scientific, Rockford, IL), anti-CD83 (cat. no. 12-0839-42, Thermo Fisher Scientific) and anti-CD86 antibodies (cat. no. 12-0869-42, Thermo Fisher Scientific). All antibodies were used at 1:50 dilution for 30 min at 4°C. Isotype antibodies were used as negative controls.
The memory T cell subsets were stained by anti-CD3 (cat. no. 21620033, ImmunoTools GmbH), anti-CD45RA (cat. no. 21819456, ImmunoTools GmbH) and anti-CD62L (cat. no. 21819624, ImmunoTools GmbH), anti-CD4 (cat. no. 56-0049-42, Thermo Fisher Scientific) and anti-CD8 (cat. no. A15448, Thermo Fisher Scientific). For intracellular cytokines, the activated T cells were re-stimulated with 10 µg of MSLN antigenic peptides (SLLFLLFSL and VLPLTVAEV) (GenScript, Jiangsu, China) (
The flow cytometry data of DCs and T cell immunophenotypes were acquired by CytoFLEX (Beckman Coulter, Inc.); the intracellular cytokine staining was acquired by BD LSRFortessa (BD Biosciences). The data were analyzed by FlowJo version 10.7 (FlowJo LLC) and shown as geometric mean fluorescence intensity (MFI) of each marker normalized by isotype control.
GM-CSF, IL-4 and IL-12p70 were measured using Human GM-CSF (cat. no. DGM00), IL-4 (cat. no. D4050) and IL-12p70 (cat. no. D1200) Quantikine ELISA kits (R&D systems, Inc.). IFN-γ was measured in medium collected from activated T cells co-cultured with target cancer cells using Human IFN-γ Quantikine ELISA kit (cat. no. DIF50, R&D systems, Inc.).
The IFN-γ ELISpot assay was performed using a Human IFN-γ ELISpotBASIC kit (Mabtech AB). Briefly, 15 µg/ml of IFN-γ capture antibody was coated for overnight at 4°C. The 2×105 activated T cells were then re-stimulated with 1×104 MSLN peptides-pulsed HLA-A2+ T2 cells. T cells treated with 20 ng/ml of phorbol 12-myristate 13-acetate and 1 µg/ml of ionomycin (MilliporeSigma) served as positive controls. After removing these T cells, 1 µg/ml of biotinylated-IFN-γ antibody was incubated for 2 h and ALP-conjugated streptavidin for 1 h at RT. The IFN-γ spots were evaluated by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium plus substrate (Mabtech AB) and captured by ELISpot plate reader (BIOREADER® 5000 Fγ, BIOSyS). The numbers of spots were counted by CellCounter software (
Lenti-X™ 293T and cancer cell lysates were prepared in RIPA Lysis Buffer System (Santa Cruz Biotechnology). The protein concentration was determined by Bradford assay and 30 µg of protein lysates were separated in 12% SDS-PAGE before transferred to PVDF membrane (GE Healthcare, Buckinghamshire, UK). The membrane was blocked in 5% skimmed milk (MilliporeSigma) for 30 min at RT. The membrane was incubated with 1:500 anti-MSLN and 1:5,000 anti-β-actin antibodies (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The 1:1,000 HRP-conjugated goat anti-mouse antibody (cat. no. 7076; Cell Signaling Technology, Inc.) was added and incubated for 1 h at RT. The signal was detected by Clarity™ western ECL substrate (Bio-Rad Laboratories, Inc.) using a Gel Doc instrument (G:Box Chemi XR5; Syngene Europe). The band intensity was analyzed by ImageJ software version 1.53 (NIH).
T cells were co-cultured with 5,000 cells of luciferase expressing HCC70, MDA-MB-231 and MSLN-MDA-MB-231 cells at Effector: Target ratio of 1:1, 5:1 and 10:1 for 24 h at 37°C with 5% CO2. The luciferase activity was determined using Pierce Firefly Luciferase Glow Assay kit (Pierce; Thermo Fisher Scientific, Inc.) and Lumat LB 9507 Ultra-sensitive Luminometer (Berthold Technologies GmbH & Co. KG). After normalizing the luciferase activity of every condition with target cell alone condition, the percentage of cancer cell lysis was calculated. The luciferase activity of target cell alone was served as an internal control.
The 1×103 target mWasabi-transduced cancer cells were formed into spheroid in 96-well ultra-low attachment plates (Corning Life Sciences) in 200 µl culture medium containing 2.5% cold Matrigel (BD Biosciences) by centrifugation at 300 × g for 3 min at 4°C. The spheroid was incubated for 4 days at 37°C with 5% CO2 with CellTracker™ Orange CMRA Dye (Invitrogen; Thermo Fisher Scientific, Inc.)-labelled activated T cells at Effector: Target ratios of 1:1, 5:1, 10:1 and 20:1 for 48 h at 37°C with 5% CO2. The mWasabi and CMRA fluorescence signals were detected by inverted fluorescence microscope and cellSens standard program version 1.15 (Olympus Corporation).
The association between MSLN score and clinicopathological data were accessed by Fisher's exact test. One-way ANOVA and Tukey's post-hoc test was used for all experiments except in the intracellular cytokine staining for comparison of control- and peptide-challenged T cells from the same condition in which Student's t-test was used. The Fisher's exact test was performed in SPSS 17.0 (SPSS, Inc.), whereas GraphPad prism V (GraphPad Software, Inc.) was used for one-way ANOVA and Student's t-test. All results were shown as mean ± standard deviation from at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.
All patient cases were female with mean age of 54±11.5 years diagnosed as luminal, HER2-positive and TNBC subtypes for 52 (15%), 154 (38%) and 165 (47%) cases, respectively (
MSLN-SmartDC was generated by transducing the lentiviral vector containing
The results showed slight changes in CD4+ and CD8+ T cells frequencies and memory T cells subsets compared with those in PBMCs at day 0 (
The MSLN showed the highest level in MSLN-MDA-MB-231, followed by HCC70, whereas MDA-MB-231 had no MSLN (
The results exhibited no difference in MDA-MB-231 cell lysis co-cultured with both MSLN-activated T cells and RPS3-MSLN-activated T cells at Effector: Target ratios of 1:1, 5:1 and 10:1 (
MDA-MB-231 spheroids co-cultured with activated T cells or unactivated T cells for 48 h revealed no differences in mWasabi green fluorescence signals representing viable cancer cells (
The lack of ER/PR and HER2 in TNBC limits the available treatment to systemic chemotherapy and surgical resection (
Several studies have reported the utility of DCs generated by lentiviral transduction of cytokine genes for DCs differentiation and by tumor-associated antigen gene to induce antigen-specific immune response leading to the tumor growth inhibition (
The T cells characteristics following co-culturing with MSLN-SmartDC with or without RPS3 showed slightly changed in frequencies of CD4+, CD8+ and the memory T cells subsets. This may be due to the effect of cytokines used during T cells expansion process which can non-specifically promote T cell proliferation (
The cytolytic activity of T cells activated by MSLN-SmartDC in 3D-cancer spheroid was consistent with that observed in 2D culture system. To minimize the effect of non-specific T cells killing mediated by HLA-mismatched between the target cells and T cells, healthy donors with HLA-A2 partially matched with MDA-MB-231, but not HCC70 (HLA-A3), were selected. Using MSLN-MDA-MB-231 in comparison with parental MDA-MB-231 could eliminate the intrinsic factors of target cells that may interfere with the T cells cytolytic activity, except the presence of MSLN. Future study using the
The MSLN-specific T cells in cancer patients have been reported (
In conclusion, the efficacy of MSLN-SmartDC promoting MSLN-specific immune response killing MSLN-expressing TNBC cells was successfully developed. The MSLN-SmartDC maturation enhancement by RPS3 treatment can improve the cytolytic activity of T cells against high MSLN TNBC cells. Though the clinical use of MSLN-SmartDC and RPS3-MSLN-SmartDC needs more convincing data in the
The authors gratefully thank Miss Surat Phumphuang (Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand) for her effort in clinicopathological data collection. The authors would also like to thank Emeritus Professor James A. Will (senior editor for the Faculty of Medicine, Khon Kaen University) for the English version of the present manuscript.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
CT and PY conceived the study. NJ, TC, PT, PY and CT designed the experiments. NJ, ST and WC performed the experiments. DS and MW performed data resource and analysis. NJ, ST and CT analyzed and interpreted the data. CT supervised the overall research. NJ and CT confirm the authenticity of all raw data. NJ, ST and CT wrote the manuscript. CT reviewed and/or edited the manuscript. All authors reviewed and approved the final manuscript.
All experiments in this study were approved by Siriraj Institutional Review Board (COA no. Si580/2018) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All blood donors in this study provided written informed consent for the use of blood samples for research.
Not applicable.
The authors declare that they have no competing interests.
Expression of MSLN in human breast cancer tissue samples. Representative images of MSLN expression levels in breast cancer tissues ranging from (A) negative, (B) low, (C) moderate and (D) strong staining. (E) Normal lobules (black arrow) showed no MSLN expression. Negative staining of MSLN in (F) stromal cells (yellow asterisk) and (G) immune cells (white asterisk). (H) Proportion of MSLN expressing samples in full cohort and stratified subtypes. (I) Mean MSLN score stratified by subtypes of breast cancer. Original magnification, 200×; scale bars=200 µm. ***P<0.001. MSLN, mesothelin.
The lentiviral vector schematic maps and immunophenotype of MSLN-SmartDC and RPS3-MSLN-SmartDC. (A) Construction of MSLN-SmartDC lentiviral vector. (B) Expression of MSLN protein in Lenti-X™ 293T transfected with IRFP-SmartDC and MSLN-SmartDC lentiviral vectors. (C) Representative images of monocytes at day 0 and DCs at day 7 of the experiment. Production of (D) GM-CSF, (E) IL-4 and (F) IL-12p70 by monocytes and different DCs. The geometric mean fluorescence intensity of surface markers of DCs in different treatment conditions including (G) CD14, (H) CD40, (I) HLA-DR, (J) CD80, (K) CD83 and (L) CD86 in monocytes and DCs. Original magnification, ×100 and scale bar=50 µm. The results were collected from four independent experiments. ND, not detected. #P<0.05, ##P<0.01 and ###P<0.001 vs. monocytes. **P<0.01 and ***P<0.001. MSLN, mesothelin; MSLN-SmartDC, MSLN dendritic cells; RPS3, ribosomal protein subunit 3; GM-CSF, granulocyte-macrophage colony stimulating factor; DCs, dendritic cells; HLA, human leukocyte antigen.
Presence of MSLN-specific T cells after SmartDCs activation. (A) Representative ELISpot well images and the quantification of IFN-γ secreting cells after restimulation with MSLN antigenic peptides. Frequency of (B) IFN-γ+ and (C) MSLN-specific T cells (CD69+ IFN-γ+) gated from CD8+ T cells after restimulation with MSLN antigenic peptides. The results were collected from thee independent experiments. *P<0.05. MSLN, mesothelin.
MSLN expression in TNBC cells and 2D killing assay by T cells activated by MSLN-SmartDC and RPS3-MSLN-SmartDC. (A) Western blot analysis of MSLN in TNBC cell lines. (B) IFN-γ concentration in culture medium after 24 h of co-culturing with MDA-MB-231, MSLN-MDA-MB-231 and HCC70 at E:T ratio of 10:1. Percentage of cell lysis of (C) MDA-MB-231, (D) MSLN-overexpressing MDA-MB-231 (MSLN-MDA-MB-231) and (E) HCC70 co-cultured with activated T cells at various ratios of E:T. The results were collected from four independent experiments. *P<0.05, **P<0.01 and ***P<0.001. MSLN, mesothelin; TNBC, triple negative breast cancer; 2D, two dimensional; MSLN-SmartDC, MSLN dendritic cells; RPS3, ribosomal protein subunit 3; E:T, Effector cell: Target cell.
Cytotoxicity of cancer cells mediated by MSLN-SmartDC- and RPS3-MSLN-activated T cells in 3D cancer spheroid assay. Representative cancer spheroid (green) co-cultured with T cells (orange) for 48 h and the changes in MFI of mWasabi signal in (A) MDA-MB-231, (B) MSLN-MDA-MB-231 and (C) HCC70 representing the remaining viable cancer cells after exposure to T cells. The results were collected from four independent experiments. Original magnification, ×100 and scale bar=50 µm. *P<0.05, **P<0.01 and ***P<0.001. MSLN, mesothelin; MSLN-SmartDC, MSLN dendritic cells; RPS3, ribosomal protein subunit 3; 3D, three dimensional; MFI, mean fluorescence intensity.
Relationship between MSLN expression and clinicopathological factors analyzed by Fisher's exact test. (Some clinical data were not available for some patient samples).
Full cohort (n=351) | HER2 cohort (n=134) | TNBC cohort (n=165) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Characteristic | Low | High | P-value | Low | High | P-value | Low | High | P-value | |
Age (years) (n=351) | ≤50 | 83 | 46 | 0.410 | 28 | 5 | 1.000 | 24 | 41 | 0.260 |
>50 | 153 | 69 | 86 | 15 | 47 | 53 | ||||
pT (n=351) | 1-2 | 210 | 107 | 0.255 | 103 | 19 | 0.693 | 63 | 87 | 0.424 |
3-4 | 26 | 8 | 11 | 1 | 8 | 7 | ||||
pN (n=350) | 0 | 121 | 71 | 0.086 | 56 | 9 | 0.810 | 41 | 62 | 0.331 |
1-3 | 114 | 44 | 57 | 11 | 30 | 32 | ||||
pM (n=348) | 0 | 226 | 114 | 0.175 | 112 | 20 | 1.000 | 71 | 94 | NA |
1 | 9 | 1 | 1 | 0 | 0 | 0 | ||||
Clinical staging (n=348) | 1-2 | 168 | 92 | 0.087 | 80 | 14 | 1.000 | 53 | 78 | 0.244 |
3-4 | 66 | 22 | 32 | 5 | 18 | 16 | ||||
Subtype (n=351) | TNBC | 71 | 94 | <0.001 | - | - | NA | - | - | NA |
HER2 | 114 | 20 | - | - | - | - | ||||
Luminal | 51 | 1 | - | - | - | - | ||||
ER (n=351) | Neg | 185 | 114 | <0.001 | - | - | NA | - | - | NA |
Pos | 51 | 1 | - | - | - | - | ||||
PR (n=351) | Neg | 190 | 114 | <0.001 | - | - | NA | - | - | NA |
Pos | 46 | 1 | - | - | - | - | ||||
HER2 (n=342) | Neg | 69 | 63 | <0.001 | - | - | NA | - | - | NA |
Pos | 158 | 52 | - | - | - | - | ||||
LN metastasis (n=350) | Neg | 129 | 73 | 0.136 | 55 | 10 | 1.000 | 44 | 63 | 0.515 |
Pos | 106 | 42 | 58 | 10 | 27 | 31 | ||||
Perineural metastasis (n=351) | Neg | 185 | 99 | 0.111 | 83 | 15 | 1.000 | 59 | 83 | 0.370 |
Pos | 51 | 16 | 31 | 5 | 12 | 11 |
MSLN, mesothelin; HER2, human epidermal growth factor receptor 2; TNBC, triple negative breast cancer; p, pathological stage; ER, estrogen receptor; PR, progesterone receptor; LN, lymph node; NA, Not available.