TY - JOUR
AU - Mao,, Haiting
AB - Abstract Interleukin 35 (IL-35) is a potent immunosuppressive cytokine, consisting of an Epstein–Barr virus-induced gene 3 (EBI3) subunit and a p35 subunit. IL-35 is mainly produced by regulatory T and regulatory B cells, and plays a crucial role in the development and prevention of infectious and autoimmune diseases. However, the effect of IL-35 in malignant disease is not well understood. In this study, we demonstrated that breast cancer cells (BCCs) also expressed and secreted IL-35 and higher level of IL-35 in BCCs was closely associated with poor prognosis of patients and was an independent unfavorable prognostic factor for breast cancer. Subsequent study revealed that BCC-derived IL-35 inhibited conventional T (Tconv) cell proliferation and further induced suppressed Tconv cells into IL-35-producing induced regulatory T (iTr35) cells. Furthermore, BCC-derived IL-35 promoted the secretion of inhibitory cytokine IL-10 and obviously decreased the secretion of Th1-type cytokine IFN-γ and Th17-type cytokine IL-17 in Tconv cells. Meanwhile, the expression of inhibitory receptor CD73 was also elevated on the surface of Tconv cells following the BCCs’ supernatant treatment. Mechanistically, BCC-derived IL-35 exhausted Tconv cells and induced iTr35 by activating transcription factor STAT1/STAT3. Hence, our results indicate functions of BCC-derived IL-35 in promoting tumor progression through proliferation inhibition of tumor-infiltrating Tconv cells and induction of iTr35 cells in tumor microenvironment. This study highlights that IL-35 produced by BCCs are a potential therapeutic target for breast cancer. Introduction Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer-related deaths among Chinese women (1). In spite of improvements in surgical and pharmaceutical strategies, there remains high rates of recurrence and metastases of breast cancer and a major cause of tumor progression is immune suppression in cancer (2,3). Regulatory T cells (Tregs), a unique subset of CD4+ T cells, have been found to play key roles in maintaining suppressive tumor microenvironment and thus contribute to cancer progression. Although Tregs normally comprise only ~4% of CD4+ T cells in adult peripheral blood, they can make up as much as 20–30% of the total CD4+ T cell population in tumor microenvironment (4,5). Increased numbers of Tregs are correlated with poor prognosis in various types of cancers including breast cancer (6). Two subsets of Tregs have been confirmed: naturally occurring Tregs (nTregs) that occur and develop in the thymus and induced Tregs (iTregs) that generate from natural conventional T (Tconv) cells in the periphery. According to the cytokines that induced them, three types of iTregs have been described: iTr-TGF-β, iTr-IL-10 and iTr-IL-35 (iTr35). Immunosuppressive cytokine interleukin-35 (IL-35) induces the conversion of Tconv cells into IL-35-producing regulatory T (iTr35) cells. IL-35, a novel member of IL-12 family, exists as a heterodimer of EBI3 (IL-27β) and p35 (IL-12a) (7). IL-35 is mainly produced by Tregs and regulatory B cells and contributes to the immune suppression function of these cells (8,9). Recent studies have found that IL-35 is also expressed in some types of cancer cells, such as pancreatic cancer, colon cancer and hepatocellular carcinoma (10–12). In vivo studies have shown that IL-35 neutralization limits tumor growth in multiple mouse models of human cancer (13). Therefore, IL-35 has emerged as a new biomarker and a potential therapeutic target for cancer. In this study, we demonstrated that breast cancer cells (BCCs) expressed and secreted IL-35, and BCC-derived IL-35 was capable of inhibiting the proliferation of Tconv cells and converting them into iTr35 cells, which contributed to the progression of breast cancer and poor prognosis of patients. Materials and methods Cell lines and cell culture Human breast cancer cell lines MDA-MB-231 (231), MCF-7, SKBR-3 and T47D were obtained from the American Type Culture Collection. HEK293T cell line was obtained from Chinese Academy of Sciences Cell Bank. All cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA,) medium containing 10% fetal bovine serum (Gibco, Auckland, New Zealand) in a humidified atmosphere of 5% CO2 at 37°C. All cell lines were cytogenetically tested and authenticated before the cells were frozen. Each vial of frozen cells was thawed and maintained for a maximum of 12 weeks. Breast cancer patients Eighty-six unrelated Chinese women with breast cancer who underwent lumpectomy or mastectomy were from the Second Hospital of Shandong University and Dongying People’s Hospital of Shandong Province, excluding patients receiving neoadjuvant chemotherapy or neoadjuvant radiotherapy. Clinicopathological characteristics included age of onset, histological grade, pathological tumor, node, metastases (TNM) stage, the status of estrogen receptor, progesterone receptor (PR), human epithelial growth factor receptor 2, lymph node involvement, progression-free survival and overall survival. Human study was approved by the Human Investigation Committee of the Second Hospital of Shandong University and Dongying People’s Hospital of Shandong Province, and informed consent was obtained from each study participant. Isolation of Tconv cells Human peripheral blood mononuclear cells from leukocyte-enriched buffy coats were separated on a Ficoll (Sigma) gradient and Tconv cells (CD4+CD25−CD45RO−) were purified by negative selection using a Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec, Germany). The purity was >97% as confirmed by flow cytometry. Immunohistochemistry The immunohistochemistry (IHC) was used to detect Epstein–Barr virus-induced gene 3 (EBI3) and p35 expression in breast cancer tissues. In brief, the paraffin-embedded tissue sections were deparaffinized followed by microwave treatment in ethylenediaminetetraacetic acid solution for 15 min. After being treated with 3% hydrogen peroxide and blocked with 10% goat serum, the consecutive tissue sections were probed with primary antibodies, including anti-EBI3 (5 μg/ml; Novus Biologicals) and anti-p35 (5 μg/ml; R&D Systems, Minneapolis, MN), at 4ºC overnight. Then, tissue sections were incubated with the biotinylated secondary antibody, followed by performing the chromogenic reaction using a DAB Substrate Kit. The images were captured using an Olympus microscope. All the slides were independently analyzed by two pathologists. The intensity of the staining was evaluated using the following criteria: 0, negative; 1, low; 2, medium and 3, high. The extent of staining was scored by using the following criteria: 0, 0% stained; 1, 1–25% stained; 2, 26–50% stained and 3, 51–100% stained. Five random fields (×20 magnification) were evaluated under a light microscope. The final scores were calculated by multiplying intensity and extent scores, and the samples were divided into four grades: 0, negative (−); 1–2, low staining (+); 3–5, medium staining (++) and 6–9, high staining (+++). The following criteria were used to quantify the expression levels of IL-35 in breast cancer tissues: high expression, both EBI3 and P35 were scored as ++/+++; low expression, other than high expression. Induction of iTr35 cells Tconv cells were activated with CD3/CD28 T cell activator (12 μl/ml; STEMCELL Technologies, Canada) and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/ml rhIL-2 (PeproTech). For induction of iTr35 cells, rhIL-35 (50 or 100 ng/ml; PeproTech) or culture supernatants (SN) of 231 or MCF-7 at a volume 30% of the total culture volume were added to T cell culture system for 5 days. Cell Counting Kit-8 assay The direct effect of BCC-derived IL-35 on Tconv cells growth was measured by Cell Counting Kit-8 (CCK-8) assay. Briefly, Tconv cells were seeded in triplicate into 96-well plates at 1 × 105 cells per well with rhIL-35, or BCCs’ SN in the presence of a neutralizing anti-IL35 monoclonal antibody (mAb) (10 μg/ml, clone 27537; R&D Systems) or not for 5 days and the fresh conditional medium was added every 2 days (14). After treatment, 10% of CCK-8 buffer (Dojindo, Kumamoto, Japan) was added to culture medium for the final 2 h at 37°C until visible color conversion occurred. The absorbance value was detected at 450 nm wavelength by a Microplate Reader (Bio-Rad, Hercules, CA). Results were representative of three independent experiments. RNA extraction and real-time quantitative reverse transcription PCR Total RNA was extracted from the BCCs or T cells with TRIzol reagent (Invitrogen) in accordance with the manufacturer’s protocol. Equal amounts of total RNA from each sample were then reverse-transcribed into cDNA using a Revert Ace kit (Toyobo, 538100). The following sequence-specific primers were used for PCR amplification: (i) the internal control GAPDH gene: forward, 5′-GGT GGT CTC CTC TGA CTT CAA CAG-3′, reverse, 5′-GTT GTT GTA GCC AAA TTC GTT GT-3′; (ii) ebi3 gene: forward, 5′-GCA GCA GAC GCC AAC GT-3′, reverse, 5′-CCA TGG AGA ACA GCT GGA CAT-3′; (iii) p35 gene: forward, 5′-CCT TCA CCA CTC CCA AAA C-3′, reverse, 5′-TGT CTG GCC TTC TGG AGC AT-3′. The primers used in Figure 1I and J were as follows: (i) ebi3 gene: forward, 5′-TTC ATT GCC ACG TAC AGG CT-3′, reverse, 5′-GGA TGA GGA CGT GGC TTC AA-3′; (ii) p35 gene: forward, 5′-TTC CCA TGC CTT CAC CAC TC-3′, reverse, 5′-ACA GGG CCA TCA TAA AAG AGG T-3′. Amplification cycle conditions were set up as follows: 95°C for 60 s, followed by 40 cycles of 94°C for 5 s, 60°C for 10 s and 72°C for 15 s, with final extension of 45 s at 72°C. Data were normalized to the internal control and relative expression levels were evaluated using the 2−ΔΔCt method. All experiments were conducted in triplicate. Figure 1. View largeDownload slide IL-35 expression and clinical significance in breast cancer. The expression levels of two subunits of IL-35: EBI3 and p35 were detected in consecutive sections of breast cancer tissues. (A) Representative images were shown as negative(−), low(+), medium(++) and high(+++) expressions of EBI3 and p35 protein in IHC staining of breast cancer. Red arrows indicates TILs and BCCs. (B) Staining extent correlation and (C) IHC score correlation analysis of EBI3 and p35 in 86 breast cancer patients (Spearman’s correlation analysis). (D, E) Kaplan–Meier analysis of overall survival (OS) (D) and progression-free survival (PFS) (E) of 86 breast cancer patients (log-rank test) according to different IL-35 levels. (F–H) Detection of IL-35 level in four representative breast cancer cell lines (MDA-MB-231, MCF-7, T47D, SKBR3). (F, G) The mRNA level of ebi3 and p35 in breast cancer cell lines. (H) After culturing for 48 h, the cell-free supernatants of breast cancer cell lines were collected and analyzed for the IL-35 production by ELISA. (I–K) After generation of stable MDA-MB-231 cells using CRISPR/Cas9 system, mRNA level of ebi3 and p35 (I, J) and secretion of IL-35 (K) in MDA-MB-231 cells were detected. Data are representative of three independent experiments and presented as mean ± SEM of three replicates (F–K). TILs, tumor-infiltrating lymphocytes; BCCs, breast cancer cells. Scale bars, 100 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Figure 1. View largeDownload slide IL-35 expression and clinical significance in breast cancer. The expression levels of two subunits of IL-35: EBI3 and p35 were detected in consecutive sections of breast cancer tissues. (A) Representative images were shown as negative(−), low(+), medium(++) and high(+++) expressions of EBI3 and p35 protein in IHC staining of breast cancer. Red arrows indicates TILs and BCCs. (B) Staining extent correlation and (C) IHC score correlation analysis of EBI3 and p35 in 86 breast cancer patients (Spearman’s correlation analysis). (D, E) Kaplan–Meier analysis of overall survival (OS) (D) and progression-free survival (PFS) (E) of 86 breast cancer patients (log-rank test) according to different IL-35 levels. (F–H) Detection of IL-35 level in four representative breast cancer cell lines (MDA-MB-231, MCF-7, T47D, SKBR3). (F, G) The mRNA level of ebi3 and p35 in breast cancer cell lines. (H) After culturing for 48 h, the cell-free supernatants of breast cancer cell lines were collected and analyzed for the IL-35 production by ELISA. (I–K) After generation of stable MDA-MB-231 cells using CRISPR/Cas9 system, mRNA level of ebi3 and p35 (I, J) and secretion of IL-35 (K) in MDA-MB-231 cells were detected. Data are representative of three independent experiments and presented as mean ± SEM of three replicates (F–K). TILs, tumor-infiltrating lymphocytes; BCCs, breast cancer cells. Scale bars, 100 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Flow cytometry and intracellular staining For cell-surface markers, T cells were harvested and incubated with anti-human CD4-FITC, CD73-PE, LAG3-PE, CTLA-4-PE or appropriate isotype controls (eBioscience, San Diego, CA) for 30 min at 4°C. For intracellular cytokine detection, cells were stimulated with 1× cell stimulation cocktail (eBioscience) for 10–12 h and were fixed and permeabilized using Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (eBiosciences). The harvested cells were stained with FITC-labeled anti-CD4 and then with PE-labeled anti-EBI3, or isotype-matched control antibody (eBioscience). Finally, the stained cells were analyzed by flow cytometry and data analysis was done using FCS Express, V3 (De Novo). Cytokine measurement For IL-35 level measurement, SN from BCCs were harvested and determined using human IL-35 enzyme-linked immunosorbent assay (ELISA) kits (sensitivity: 15.6 pg/ml; Cusabio, China). For cytokine analysis of Tconv culture system, Tconv cells were cultured for 5 d after stimulation with rhIL-35 or BCCs’ SN. Cytokine production of IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17a, TNF-α and IFN-γ was simultaneously determined by the commercially available Human High Sensitivity Panel (eBioscience). The assay was performed according to the manufacturer’s instructions and each sample was run in duplicate. Western blotting The harvested cells were washed with phosphate-buffered saline and suspended in radioimmunoprecipitation lysis buffer. The protein concentration was evaluated using the BCA Protein Assay (Beyotime). An aliquot of total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and was further electrotransferred onto polyvinylidene difluoride membranes. After being blocked using 5% non-fat dried milk for 1 h at room temperature, the membranes were probed with anti-EBI3 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p35(1 μg/ml; R&D Systems), anti-phospho-STAT1(Tyr701) (1:1000), anti-STAT1 (1:1000), anti-phospho-STAT3 (1:1000), anti-STAT3 (1:1000), anti-phospho-STAT4(Tyr693) (1:1000), anti-STAT4 (1:1000) and anti-GAPDH (1:1000) (Cell Signal Technology, Beverly, MA) antibodies overnight at 4°C, followed by treatment with horseradish-peroxidase-conjugated secondary antibodies. The signals of labeled proteins were detected by enhanced chemiluminescence (Life Technologies, Carlsbad, CA). CRISPR/Cas9 targeting and generation of lentiviral constructs Single guide RNAs (sgRNA) targeted to ebi3 and p35, respectively, were designed and then cloned into pLenti CRISPR V2 (Hu6-sgRNA-EF1a-CAS9-puro) vectors (LncBio Shanghai, China). All plasmids were sequenced to confirm successful ligation (Supplementary Figure 2, available at Carcinogenesis Online). The following sgRNA sequences were utilized: (i) sg ebi3#1: 5′-GGCGGTGACATTGAGCACGTAGG-3′, (ii) sg ebi3#2: 5′-GAACAGCTGGACATCCGTGATGG-3′, (iii) sg ebi3#3: 5′-TGGACGTTGGCG TCTGCTGCAGG-3′, (iv) sg p35#1: 5′-GGTTAATTCCAATGGTAAACAGG-3′, (v) sg p35#2: 5′-TAAACAGGCCTCCACTGTGCTGG-3′, (vi) sg p35#3: 5′-GTAAAA TTCTAGAGTTTGTCTGG-3′. The HEK293T cells were maintained as recommended by the manufacturer in 100 mm dishes. For each transfection, 4 µg CRISPR/Cas9 vector carrying the construct of interest, 4 µg psPAX2 envelope plasmid and 2 µg pMD2.G packaging plasmids were used. The transfection was carried out using jetPRIME (Polyplus, New York) following the manufacturer’s recommendations. Virus was harvested at 48 h post-transfection. Generation of recombinant stable cell lines To knock out gene expression of ebi3 or p35, MDA-MB-231 cells were transducted with CRISPR/Cas9 virus for 24 h using 8 μg/ml polybrene (LncBio). Two days after transduction, selection of recombinant cells was performed in the presence of 2.0 µg/ml puromycin (Solarbio, Beijing, China). Statistical analysis IHC scores of different subgroups were compared by chi-square test. Kaplan–Meier curves were analyzed for relevant variables. The log-rank test was used to analyze the differences in survival time among the patient subgroups. The Cox’s proportional hazard regression model was used to evaluate risk factors associated with the prognoses. Pearson’s correlation coefficient (r value) was calculated assuming linear relationship between variables. The data represent the mean values ± the standard error of mean. Comparison between groups was performed using unpaired Student’s t-test with GraphPad Prism5 software. A P ≤ 0.05 (two-sided) was considered statistically significant. Results IL-35 was expressed in breast cancer tissues and was positively associated with poor prognosis We first examined the expression of IL-35 in breast cancer tissues. Since IL-35 is a heterodimeric protein, which is composed of EBI3 and p35 subunits, we detected the expression of EBI3 and p35 using serial sections by IHC. As shown in Figure 1A, EBI3 and p35 were co-expressed in the cytoplasm of most cancer cells and part of tumor-infiltrating lymphocytes. Furthermore, the expression of EBI3 and p35 was correlated strongly (r = 0.77, P < 0.0001) (Figure 1B and C). These results demonstrated that BCCs expressed IL-35. Subsequently, we performed the analysis of association between the expression of IL-35 and clinicopathological information in 86 patients with breast cancer. According to the IHC scores of EBI3 and p35, we stratified breast cancer samples into two groups: ‘high IL-35’ and ‘low IL-35’. As shown in Table 1, higher level of IL-35 was significantly associated with poor differentiation, negative estrogen receptor status, negative PR status and positive Her-2 status. The survival data from 86 patients with breast cancer were also assessed in this study. The results showed that IL-35 level was significantly correlated with the survival rate of patients. Both the overall survival rate and progression-free survival rate of patients with low IL-35 expression were better than those with high IL-35 expression (P = 0.0009 and 0.0008) (Figure 1D and E). The multivariate Cox’s regression analysis revealed that IL-35 expression was an independent unfavorable prognostic factor for breast cancer (hazards ratio, 3.779; 95% confidence interval, 1.046–13.653; P = 0.043; Table 2). Table 1. Association between IL-35 expression levels and clinicopathological features of breast cancer patients Features Cases (n) IL-35 expression χ2 P-value Negative/ Low (n) High (n) Age (y) 0.154 0.695  ＜60 73 38 35  ≥ 60 13 6 7 Pathological TNM 0.178 0.673  І 41 20 21  Ⅱ–Ⅲ 45 24 21 Differentiation 4.620 0.032*  Well/moderate 45 28 17  Poor 41 16 25 PR status 5.587 0.018*  Negative 40 15 25  Positive 46 29 17 ER status 7.878 0.005**  Negative 36 12 24  Positive 50 32 18 Her-2 status 4.654 0.031*  Negative 43 27 16  Positive 43 17 26 Lymph node involvement 0.745 0.388  Negative 43 24 19  Positive 43 20 23 Features Cases (n) IL-35 expression χ2 P-value Negative/ Low (n) High (n) Age (y) 0.154 0.695  ＜60 73 38 35  ≥ 60 13 6 7 Pathological TNM 0.178 0.673  І 41 20 21  Ⅱ–Ⅲ 45 24 21 Differentiation 4.620 0.032*  Well/moderate 45 28 17  Poor 41 16 25 PR status 5.587 0.018*  Negative 40 15 25  Positive 46 29 17 ER status 7.878 0.005**  Negative 36 12 24  Positive 50 32 18 Her-2 status 4.654 0.031*  Negative 43 27 16  Positive 43 17 26 Lymph node involvement 0.745 0.388  Negative 43 24 19  Positive 43 20 23 According to 7th edition of UICC TNM classification of malignant tumors. Bold values in italics are statistically significant. *P ≤ 0.05, **P ≤ 0.01. View Large Table 1. Association between IL-35 expression levels and clinicopathological features of breast cancer patients Features Cases (n) IL-35 expression χ2 P-value Negative/ Low (n) High (n) Age (y) 0.154 0.695  ＜60 73 38 35  ≥ 60 13 6 7 Pathological TNM 0.178 0.673  І 41 20 21  Ⅱ–Ⅲ 45 24 21 Differentiation 4.620 0.032*  Well/moderate 45 28 17  Poor 41 16 25 PR status 5.587 0.018*  Negative 40 15 25  Positive 46 29 17 ER status 7.878 0.005**  Negative 36 12 24  Positive 50 32 18 Her-2 status 4.654 0.031*  Negative 43 27 16  Positive 43 17 26 Lymph node involvement 0.745 0.388  Negative 43 24 19  Positive 43 20 23 Features Cases (n) IL-35 expression χ2 P-value Negative/ Low (n) High (n) Age (y) 0.154 0.695  ＜60 73 38 35  ≥ 60 13 6 7 Pathological TNM 0.178 0.673  І 41 20 21  Ⅱ–Ⅲ 45 24 21 Differentiation 4.620 0.032*  Well/moderate 45 28 17  Poor 41 16 25 PR status 5.587 0.018*  Negative 40 15 25  Positive 46 29 17 ER status 7.878 0.005**  Negative 36 12 24  Positive 50 32 18 Her-2 status 4.654 0.031*  Negative 43 27 16  Positive 43 17 26 Lymph node involvement 0.745 0.388  Negative 43 24 19  Positive 43 20 23 According to 7th edition of UICC TNM classification of malignant tumors. Bold values in italics are statistically significant. *P ≤ 0.05, **P ≤ 0.01. View Large Table 2. Multivariate analysis of prognostic factors associated with overall survival of breast cancer patients Variables Unfavorable versus favorable Overall survival Hazards ratio (95% CI) P-value IL-35 Negative/low versus high 3.779(1.046–13.653) 0.043* Age (y) ≥ 60 versus ＜60 2.843(1.035–7.811) 0.043* Differentiation Well/moderate versus poor 5.055(1.361–18.774) 0.015* Lymph node involvement Negative versus positive 4.893(1.344–17.811) 0.016* Variables Unfavorable versus favorable Overall survival Hazards ratio (95% CI) P-value IL-35 Negative/low versus high 3.779(1.046–13.653) 0.043* Age (y) ≥ 60 versus ＜60 2.843(1.035–7.811) 0.043* Differentiation Well/moderate versus poor 5.055(1.361–18.774) 0.015* Lymph node involvement Negative versus positive 4.893(1.344–17.811) 0.016* Bold values in italics are statistically significant. *P ≤ 0.05. View Large Table 2. Multivariate analysis of prognostic factors associated with overall survival of breast cancer patients Variables Unfavorable versus favorable Overall survival Hazards ratio (95% CI) P-value IL-35 Negative/low versus high 3.779(1.046–13.653) 0.043* Age (y) ≥ 60 versus ＜60 2.843(1.035–7.811) 0.043* Differentiation Well/moderate versus poor 5.055(1.361–18.774) 0.015* Lymph node involvement Negative versus positive 4.893(1.344–17.811) 0.016* Variables Unfavorable versus favorable Overall survival Hazards ratio (95% CI) P-value IL-35 Negative/low versus high 3.779(1.046–13.653) 0.043* Age (y) ≥ 60 versus ＜60 2.843(1.035–7.811) 0.043* Differentiation Well/moderate versus poor 5.055(1.361–18.774) 0.015* Lymph node involvement Negative versus positive 4.893(1.344–17.811) 0.016* Bold values in italics are statistically significant. *P ≤ 0.05. View Large BCCs expressed and secreted IL-35 To screen appropriate breast cancer cell lines for in vitro study, we investigated the expression and secretion of IL-35 in four breast cancer cell lines. The two subunits of IL-35 were detectable in all of four cell lines and the higher IL-35 level was observed in 231 and MCF-7 cells, which were verified by real-time reverse-transcription PCR (Figure 1F and G) and ELISA (Figure 1H). The specificity of the IL-35 ELISA was demonstrated using 231 cells in which the ebi3 or p35 gene was inactivated using the CRISPR/CAS9 system (Figure 1I–K). On the basis of these results, 231 and MCF-7 cell lines were chosen for subsequent experiments. BCCs’ SN inhibited the proliferation of Tconv cells in IL-35-dependent manner IL-35 secreted by Tregs suppressed the proliferation of Tconv cells (14). To evaluate whether IL-35 produced by BCCs also had similar function, we tested the inhibitory effect of BCCs’ SN on Tconv cell proliferation using neutralizing IL-35 mAb and CCK-8 assay. As shown in Figure 2A, both SN from 231 and MCF-7 cells markedly suppressed the proliferation of Tconv cells and IL-35-blocking could partially reverse these effects. Therefore, BCCs’ SN inhibited Tconv cell proliferation in an IL-35-dependent manner. Figure 2. View largeDownload slide BCC-derived IL-35 inhibited proliferation of Tconv cells and induced iTr35. Purified CD4+CD25−CD45RO−Tconv cells were cultured with IL-35 or BCCs’ SN, and anti-CD3/CD28 for 5 days. (A) CCK-8 assays showed the effects of BCCs’ SN on proliferation of Tconv cells. (B–D) All cells were collected for analysis of IL-35 level by real-time RT-PCR (B, C) and western blot (D), respectively. (E) Flow cytometry quantification of IL-35 in cells after activation for 10–12 h with the cell-stimulation cocktail, then cells were stained with anti-EBI3 or isotype control antibody. Data are representative of at least three independent experiments and presented as mean ± SEM of three replicates (A–C). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Figure 2. View largeDownload slide BCC-derived IL-35 inhibited proliferation of Tconv cells and induced iTr35. Purified CD4+CD25−CD45RO−Tconv cells were cultured with IL-35 or BCCs’ SN, and anti-CD3/CD28 for 5 days. (A) CCK-8 assays showed the effects of BCCs’ SN on proliferation of Tconv cells. (B–D) All cells were collected for analysis of IL-35 level by real-time RT-PCR (B, C) and western blot (D), respectively. (E) Flow cytometry quantification of IL-35 in cells after activation for 10–12 h with the cell-stimulation cocktail, then cells were stained with anti-EBI3 or isotype control antibody. Data are representative of at least three independent experiments and presented as mean ± SEM of three replicates (A–C). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. BCC-derived IL-35 induced iTr35 cells Given our finding that IL-35 secreted by BCCs suppressed the proliferation of Tconv cells strongly, we next sought to determine whether BCC-derived IL-35 could convert Tconv cells to iTr35 cells. We monitored that, compared with control, Tconv cells activated with anti-CD3/CD28 in the presence of BCCs’ SN simultaneously upregulated the expression of IL-35 at both mRNA and protein levels (Figure 2B–D). Single-cell analysis by intracellular cytokine staining also suggested that treatment with BCCs’ SN or rhIL-35 induced the expression of IL-35 in Tconv cells (Figure 2E). These data confirmed that BCC-derived IL-35 converted Tconv cells into suppressive iTr35 cells. Cytokine secretion profiles of Tconv cells treated by BCCs’ SN Next, we analyzed the cytokine secretion profiles of Tconv cells treated with BCCs’ SN, including IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17a, TNF-α and IFN-γ (Figure 3AH). Secretion of IL-10 and IL-12p70 was obviously upregulated, whereas the secretion of Th1-type cytokine IFN-γ and Th17-type cytokine IL-17 was markedly depressed in both IL-35 groups and BCCs’ SN-treated groups (Figure 3A–D). Furthermore, compared with BCCs’ SN-treated Tconv cells, the addition of IL-35 functional mAb reversed these effects. In addition, we also found that the levels of IL-1β and IL-4 were significantly increased in BCCs’ SN-treated groups compared with those in the control group, but these changes were uncorrelated with IL-35 treatment (Figure 3E and F). Figure 3. View largeDownload slide Cytokine secretion profiles and IRs expression of Tconv cells treated by BCCs’ SN. Tconv cells were cultured with IL-35, or BCCs’ SN at volume of 30% or IL-35Ab for 5 days. (A–H) Then, cell-free supernatants were collected for analysis of the IL-10 (A), IL-12p70 (B), IL-17a (C), IFN-γ (D), IL-1β (E), IL-4 (F), IL-6 (G) and TNF-α (H) production by the Bio-Plex Protein Array system. The secretion levels of cytokines in BCCs’ SN and BCCs’ SN+IL-35Ab groups (231, 231+IL-35Ab, MCF-7, MCF-7+IL-35Ab) were corrected by excluding the basic cytokines level in BCCs’ SN. (I) The expression levels of cell surface CD73, LAG3 and CTLA-4 were detected by flow cytometry. Data are representative of at least three independent experiments and presented as mean ± SEM of three replicates. *P ≤ 0.05, **P ≤ 0.01. Figure 3. View largeDownload slide Cytokine secretion profiles and IRs expression of Tconv cells treated by BCCs’ SN. Tconv cells were cultured with IL-35, or BCCs’ SN at volume of 30% or IL-35Ab for 5 days. (A–H) Then, cell-free supernatants were collected for analysis of the IL-10 (A), IL-12p70 (B), IL-17a (C), IFN-γ (D), IL-1β (E), IL-4 (F), IL-6 (G) and TNF-α (H) production by the Bio-Plex Protein Array system. The secretion levels of cytokines in BCCs’ SN and BCCs’ SN+IL-35Ab groups (231, 231+IL-35Ab, MCF-7, MCF-7+IL-35Ab) were corrected by excluding the basic cytokines level in BCCs’ SN. (I) The expression levels of cell surface CD73, LAG3 and CTLA-4 were detected by flow cytometry. Data are representative of at least three independent experiments and presented as mean ± SEM of three replicates. *P ≤ 0.05, **P ≤ 0.01. Inhibitory receptor expression on Tconv cells treated by BCCs’ SN It has been reported that nTregs express surface inhibitory receptors (IRs), such as CD73, LAG3 and CTLA-4, which contribute to the suppressive function of Tregs themselves (15–17). In this study, we also detected the surface expression of IRs on Tconv cells treated by BCCs’ SN. As shown in Figure 3I, the percentage of CD73+ Tconv cells is upregulated in BCCs’ SN-treated groups. However, BCCs’ SN treatment had no effect in the induction of LAG3 and CTLA-4, which had been described as mediators of nTregs suppression. BCC-derived IL-35 signaled through STAT1 and STAT3 On the basis of the earlier results, we further explored the molecular mechanisms involved in Tconv cell proliferation inhibition and iTr35 cell induction by BCC-derived IL-35. The STAT protein family played a pivotal role during cellular differentiation and immunoregulation (18). Published reports showed that both IL-27 and IL-12, which shared subunits with IL-35, induced the phosphorylation of STAT1, STAT3 and STAT4 (19). The treatment of mouse Tconv cells with IL-35 resulted in intracellular phosphorylation of STAT1 and STAT4 (20). In this study, we examined the phosphorylation status of STAT1, STAT3 and STAT4 in Tconv cells treated by BCC-derived IL-35 by western blot. As shown in Figure 4A, increased p-STAT1 and p-STAT3 expressions were observed in IL-35-treated groups and BCCs’ SN-treated groups compared with control Tconv cells, and the addition of IL-35-blocking mAb in BCCs’ SN-treated groups could partially reverse these effects. Moreover, the effect of BCCs’ SN groups was more pronounced than that of rhIL-35 groups (Figure 4A). However, the expression of p-STAT4 was not detectable in all groups (Figure 4A). These indicated that BCC-derived IL-35 inhibited Tconv cell proliferation and converted Tconv cells into iTr35 cells through activation of STAT1/STAT3 pathway. Figure 4. View largeDownload slide BCC-derived IL-35 signals through STAT1/STAT3. (A) Tconv cells cultured with anti-CD3/CD28 and IL-35 or BCCs’ SN in the presence of IL-35 mAb or not for 1 h, the protein levels of phosphorylated (p) and total STAT1, STAT3 and STAT4 were determined by western blot. The figures are representative of at least three independent experiments. (B) The schematic diagram of BCC-derived IL-35-mediated breast cancer progression. BCC-derived IL-35 inhibits the proliferation of tumor-infiltrated Tconv cells and induces suppressed Tconv cells into iTr35 cells via STAT1/STAT3 signal pathway. Reciprocally, induced iTr35 cells produce high level of IL-35, resulting in accumulation of iTr35 cells in tumor microenvironment, thus forming a positive feedback loop. Figure 4. View largeDownload slide BCC-derived IL-35 signals through STAT1/STAT3. (A) Tconv cells cultured with anti-CD3/CD28 and IL-35 or BCCs’ SN in the presence of IL-35 mAb or not for 1 h, the protein levels of phosphorylated (p) and total STAT1, STAT3 and STAT4 were determined by western blot. The figures are representative of at least three independent experiments. (B) The schematic diagram of BCC-derived IL-35-mediated breast cancer progression. BCC-derived IL-35 inhibits the proliferation of tumor-infiltrated Tconv cells and induces suppressed Tconv cells into iTr35 cells via STAT1/STAT3 signal pathway. Reciprocally, induced iTr35 cells produce high level of IL-35, resulting in accumulation of iTr35 cells in tumor microenvironment, thus forming a positive feedback loop. Discussion Tumor progression is a multistep process depending on both tumor behavior and the immune function of the host. Immune function is generally compromised in most patients with cancer (21–23). It is reported that there are increased numbers of functionally suppressive Tregs in the peripheral blood and tumor microenvironment of patients with cancer, including breast cancer, colorectal cancer and lung cancer (24–26). Moreover, elevated Tregs number in tumor microenvironment is coincident with an increase of intratumoral IL-35+Tregs level and these IL-35+Tregs are significantly associated with cancer development and progression (4,5). In this study, we found that BCCs also expressed and secreted IL-35 in addition to nTregs and iTr35 cells. Furthermore, elevated IL-35 expression in BCCs was significantly correlated with more aggressive tumor phenotypes, including poor differentiation, PR-negative expression and estrogen receptor-negative expression. We also assessed the prognostic value of IL-35 by a long-term follow-up investigation and found that BCC-derived IL-35 was an independent unfavorable prognostic factor for breast cancer. As an inhibitory cytokine, IL-35 has three known biological functions: suppression of T-cell proliferation, the conversion of Tconv cells into iTr35 cells and downregulation of Th17 cell development and differentiation (14,27). To determine whether BCC-derived IL-35 also have these effects, we first examined the proliferation inhibition of Tconv cells. The results showed that BCC-derived IL-35 obviously suppressed the proliferation of Tconv cells. Meanwhile, BCC-derived IL-35 also induced the conversion of these suppressed Tconv cells into iTr35 cells in an IL-35-dependent manner. The induction of iTr35 cells further raised the possibility of more IL-35 accumulation and infectious tolerance in tumor environment, which orchestrated a positive feedback loop contributing to maximal immunosuppressive effect. Moreover, the inhibitory effect of BCC-derived IL-35 on Tconv cells could be blocked by IL-35 mAb treatment, which further underlined the role of BCC-derived IL-35 in the inhibition of antitumor immunity and provided possibility for using of IL-35 mAb in breast cancer therapy. Our results suggest that BCC-derived IL-35 may promote tumor progression by exhausting tumor infiltratory Tconv cells and enhance iTr35 cells population in breast cancer microenvironment. Another important finding of this study was that BCC-derived IL-35 treatment led to the change of cytokine production in Tconv cells. A previous study showed that both Tconv cells and IL-35-treated Tconv cells had similar production of cytokines in a mouse model (14). However, in contrast to the previous report, our study demonstrated that BCC-derived IL-35 induced higher levels of IL-10 and IL-12 (IL-12p70) secretion and lower levels of IFN-γ and IL-17a secretion in Tconv cells, compared with control group in humans. It was known that IL-12 shared p35 subunit with IL-35; therefore, we speculated that evaluated IL-12p70 level might be attributed to high level of IL-35 that was secreted by BCCs and iTr35 cells induced by BCC-derived IL-35. On the other hand, increased secretion of IL-10 could promote Tconv cells to differentiate into inhibitory iTr-IL-10 cells and suppress the function of Th1 and Th17 cells (28). And it was further confirmed by our results that Th1-type cytokine IFN-γ and Th17 cytokine IL-17a were dramatically decreased in both rhIL-35 treatment group and BCC-derived IL-35 treatment group. IFN-γ and IL-17a played important antitumor roles by inducing the differentiation of Th1 and Th17 cells, respectively, and inhibiting Th2 formation (29,30). The earlier results proved that BCC-derived IL-35 not only directly suppressed the proliferation of Th1 and Th17 cells, but also indirectly inhibited their functions by promoting the secretion of IL-10 in T cells, which further clearly pointed out the role of BCC-derived IL-35 in maintaining suppressive tumor microenvironment. However, IL-35 mAb treatment rebounded IFN-γ and IL-17a production of Tconv cells and brought about the recovery of BCC-derived IL-35-induced proliferation inhibition of Tconv cells. It has been reported that surface IRs LAG3, CD73 and CTLA-4 mediated the immunosuppression function of nTregs (13,31,32). In this study, we also tested whether these membrane IRs changed in rhIL-35-treated and BCCs’ SN-treated Tconv cells. The results showed that BCCs’ SN facilitated CD73 expression of Tconv cells. In general, Tregs preferentially express CD73, which converts adenosine monophosphate to highly immunosuppressive adenosine and enhances suppressive function of Tregs (33). Therefore, elevated CD73 may promote inhibitory function of BCCs’ SN-induced Tconv cells. Nevertheless, no difference on the surface expression of LAG3 and CTLA-4 was observed between rhIL-35-treated or BCCs’ SN-treated Tconv cells and control group. This result was supported by the work of Collison LW that multi-inhibitory receptors were at same level in both Tconv cells and iTr35 cells (14). Lastly, we explored the molecular mechanisms involved in the induction of iTr35 cells by BCC-derived IL-35. As IL-35 receptor uses IL-12Rβ2 and gp130, it is possible that signaling is mediated via the STAT family of transcription factors (20). In fact, previous studies from mouse models revealed that IL-35 induced the transcription of STAT1 and STAT4 in Tconv cells and STAT1 and STAT3 in B cells (8,20). In humans, only STAT1 and STAT3 were activated by IL-35 expressed by tumor-infiltrating Tregs in colorectal cancer during the induction of iTr35 cells (24). Our previous results confirmed that IL-35 treatment of human monocyte-derived dendritic cells resulted in phosphorylation of STAT1 and STAT3 (34). These data suggested the possibility that IL-35 might use different signaling components in different species or different types of cells, or different results derived from different sources of IL-35. In this study, we demonstrated that STAT1 and STAT3 but not STAT4 were involved in the induction of iTr35 cells by BCC-derived IL-35. STAT1 is a well-known transcription factor, which plays a critical role in the generation of immunosuppressive function of Tregs and regulatory B cells (8,35,36). On the other hand, activation of STAT3 is important for enhancing human iTreg phenotype and function (37). Therefore, the activation of both STAT1 and STAT3 led to the phenotypic differentiation and functional performance of iTr35 cells induced by BCC-derived IL-35. In conclusion, BCCs expressed and secreted IL-35. BCC-derived IL-35 inhibited Tconv cell proliferation and further converted suppressed Tconv cells into iTr35 cells via activation of STAT1/STAT3 signaling pathway. This might constitute a positive feedback loop: BCC-derived IL-35 inhibited the proliferation of tumor-infiltrating lymphocytes and further induced them into iTr35 cells, which subsequently produced more IL-35 and iTr35 cells in tumor microenvironment (Figure 4B). By this means, BCCs promoted their own development and progression and led to the poor prognosis of breast cancer patients. Our study provided evidence for a novel tumor-evading mechanism in breast cancer and suggested that IL-35 might be a potential therapeutic target for the treatment of breast cancer. Funding Natural Science Foundation of China (31570919, 31270970 and 81602476); Taishan Scholar Foundation; the Science and Technology Project of Shandong, China (2008GG10002035 and 2012G0021821); Science and Technology Project of Jinan of China (201202197). Conflict of Interest Statement None declared. Abbreviations BCC breast cancer cell EBI3 Epstein–Barr virus-induced gene 3 IL interleukin IR inhibitory receptor iTr35 induced regulatory T cells mAb monoclonal antibody nTregs naturally occurring Tregs SN supernatants Tconv conventional T cells References 1. Chen , W. , et al. ( 2016 ) Cancer statistics in China, 2015 . CA. Cancer J. Clin ., 66 , 115 – 132 . Google Scholar Crossref Search ADS PubMed 2. Siegel , R. , et al. ( 2012 ) Cancer statistics, 2012 . CA. Cancer J. Clin ., 62 , 10 – 29 . Google Scholar Crossref Search ADS PubMed 3. Ma , R. , et al. ( 2015 ) Mechanisms involved in breast cancer liver metastasis . J. Transl. Med ., 13 , 64 . 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TI - Breast cancer cell–derived IL-35 promotes tumor progression via induction of IL-35-producing induced regulatory T cells
JO - Carcinogenesis
DO - 10.1093/carcin/bgy136
DA - 2018-12-31
UR - https://www.deepdyve.com/lp/oxford-university-press/breast-cancer-cell-derived-il-35-promotes-tumor-progression-via-FU0XFTPndb
SP - 1488
VL - 39
IS - 12
DP - DeepDyve
ER -