Thymosin β10 promotes tumor-associated macrophages M2 conversion and proliferation via the PI3K/Akt pathway in lung adenocarcinoma

Abstract

Background: Thymosin β10 (TMSB10) has been reported to play a protumorigenic role in a majority of solid cancers. However, the function of TMSB10 in immune microenvironment may contribute to the pathobiology of lung adenocarcinoma has not been previously explored.

Method: TAMs-associated TMSB10 expression was evaluated by immunohistochemistry (IHC) of 184 lung adenocarcinomas. Xenograft mice model was established to investigate the effect of TMSB10 shRNA on TAMs. The macrophages phenotype associated cytokines IL-6, IL-8 and TNF-α were detected by ELISA. Furthermore, the target proteins were detected by immunoblot.

Results: We found that high TAMs-associated TMSB10 expression was significantly correlated with the advanced TNM stage and T3/T4 tumor size. And high TAMs-associated TMSB10 expression was significantly correlated with poor overall and progression-free survival of lung adenocarcinoma, acted as an independent prognostic factor for lung adenocarcinoma. Furthermore, we investigated the biological functions of TMSB10 in macrophages in vivo and in vitro. TMSB10 knockdown dramatically reduced TAMs, THP-1 and RAW264.7 cell proliferation, and promoted macrophages conversion of M2 to M1, and TMSB10 knockdown reduced the levels of p-Akt (Sec473), p-mTOR (Sec2448) and p-p70S6K (Thr389) without effect on Akt, mTOR and p70S6K expression.

Conclusions: These results demonstrate that TAMs-associated TMSB10 promotes tumor growth through increasing TAMs M2 conversion and proliferation via PI3K/Akt signaling pathway, providing a promising tumor biomarker for predicting prognosis and a potential therapeutic target for lung adenocarcinoma.

Background

Lung cancer accounts for a quarter of all cancer deaths worldwide. Fortunately, lung cancer morbidity shows a continuous tendency to decline in recent years (1). Although great progress has been achieved in early detection and treatment of lung cancer by surgery, chemotherapy, radiotherapy and target therapy, most patients yet suffer from recurrence or death. For early lung adenocarcinoma, the first-line treatment is surgical removal, tumor recurrence is still an inevitable obstacle to the operative effect (2). Postoperative recurrence rate reach to over 30% in patients with early stage lung adenocarcinoma (3). And the number of postoperative patients that could benefit from the adjuvant chemotherapy treatment is limited (4, 5). Therefore, it is urgent to find a new strategy for those recurrent patients. In recent years, the great value of immunotherapy in anticancer treatment has been confirmed, which accelerate the exploration of diverse effective immunotherapeutic strategies for various malignancies. The immune checkpoints such as cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death 1 (PD-1) and programmed death ligand-1 (PD-L1) were the most promising targets, meanwhile their immunosuppressant have been clinically demonstrated effective in early and advanced NSCLC, melanoma and bladder cancer et al (6–11). However, acquired resistance to immunotherapies was unavoidable after receiving immune checkpoints blockade (12, 13).

Tumor-associated macrophages (TAMs) are the major component of the tumor immune microenvironment in solid tumors, which help tumor cells to escape immune surveillance by recruiting immunosuppressors like regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (14). TAMs are mainly consisted by antitumorigenic M1 phenotype macrophages and protumorigenic M2 phenotype macrophages. Therefore, studies try to restore the antitumorigenic talents of macrophages and repress protumorigenic TAMs are prevailing in recent years (14). Manipulating these arsenals of the innate immunity combined with known curative immunotherapies that activate the adaptive immunity looks more promising than either therapy alone.

Thymosin β10 (TMSB10) belongs to the family of β-thymosins which contain highly conserved amino acid sequence (15). TMSB10 mainly function as actin sequestering proteins to inhibit the formation of F-actin. TMSB10 has been proven to be overexpressed in most human solid tumors, and regulate cancer cell proliferation and metastasis (16–20). Remarkably,Miriam Merad et al. reported that TMSB10 mRNA was unregulated in TAMs of early lung adenocarcinoma compared with those in mononuclear macrophages of adjacent normal lung and peripheral blood (21).

However, the relation between TMSB10 and TAMs remains unclear, which needs to be furtherly clarified. Our studies firstly explore the role of TMSB10 in early lung adenocarcinoma through analyzing the association between its clinicopathological characteristics and TAMs-associated TMSB10. Moreover, we conducted the in vitro and in vivo assay to investigate the role of TMSB10 in TAMs phenotype conversion.

Methods

Patients. The clinicopathological and survival data were obtained from patients with lung adenocarcinoma who had undergone surgery between January 2013 and December 2016. Tumors were classified according to the 8th edition of the AJCC TNM system for lung cancer. The inclusion criteria were as follows: (1) primary lung adenocarcinoma diagnosed by pathological diagnosis; (2) cases had received no previous treatment before operation. The age, gender, smoking history, tumor site, tumor size, lymphatic metastasis, TNM stage, and survival information were acquired from medical records. This retrospective study was approved by the ethics committee of our hospital, and the informed consent was exempted.

Animal experiments. The mouse adenocarcinoma cell line (LLC) was purchased from ATCC (Manassas, USA). LLC were cultured in DMEM supplemented with 10% FBS. All C57BL/6 mice were obtained from the vital river company. For tumor growth experiments, 2 × 10⁶ LLC cells were suspended in 100 ul of DMEM and injected subcutaneously into the right back of 6 weeks old male mice. Mice were randomly divided into experimental or control (scramble) group matched for the tumor volumes on day 4 post tumor inoculation. The experimental group mice were injected intravenously with TMSB10 shRNA lentivirus (GENE, Shanghai) every week, and scramble cohorts were injected intravenously with scramble shRNA lentivirus. Tumor sizes were measured with calipers each 5 days throughout the in vivo experiment.

Isolation of tumor-infiltrating immune cells. The excised tumors were finely minced and suspended in HBSS (Thermo Fisher, 14025134) supplemented with 2%FBS (Gibco, 1921005PJ), 1% hyaluronidase (Sigma, 37326-33-3), Collagenase 4 at 1 mg/mL (Sigma, SCR103) and 0.25% DNase I (Roche, 11284932001) at 37℃ on a shaker at 80 rpm for 2 hours. The mixture was then mixed with 10% PBS (suspended in HBSS) in a 1:1 ratio and filtered through a 50um mesh. Percoll (Sigma, P1644) was used to purify tumor-infiltrating leukocytes (TILs) via density gradient centrifugation. Briefly, 60% Percoll was firstly added to the bottom of a glass tube and then 30% Percoll was carefully added by a Pasteur pipette. Finally, the filtered cells were gently resuspended in the 30% Percoll. The suspension was centrifuged at 400 g for 25 min at room temperature. After centrifugation, the pellet of erythrocytes and excess buffer/percoll were removed, leaving a purified population of leukocytes at the interface. The purified tumor-infiltrating leukocytes were washed twice with FACS buffer prior to flow cytometry analysis.

Flow cytometry. Cell staining was operated on ice and away from light. For surface staining, the purified cells from tumor tissues were incubated with mouse Fc block (1:200, BD Biosciences, BD553141) in 4℃ for 30 min before staining with relevant conjugated antibodies. The relevant fluorescent-labeled antibodies were diluted in FACS buffer and applied to incubate for 30 min. For intracellular staining, cells were subsequently washed by FACS buffer and fixed (Fixation and Permeabilization Solution, BD Biosciences, BD554722), then the relevant fluorescent-labeled antibodies, diluted in Perm/Wash Buffer (BD Biosciences, BD557885), were used to incubate for 30 min. Upon staining, cells were resuspended in FACS buffer (25 mM HEPES, 2% FBS, 10 mM EDTA, 0.1% sodium azide in PBS) and passed through 50um mesh filter before flow cytometry analysis. The stained cells were run on a BD LSRFortessa X-20 Flow Cytometer equipped with FACSDiva software (BD Biosciences). FlowJo software (Treestar) was applied for analyzing flow cytometry data. Forward scatter (FSC) and side scatter (SSC) were used to gate on single nucleated cells and to eliminate cell debris and doublets.

Immunoblotting and ELISA assay. THP-1 and RAW264.7 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS. After treatment with indicated shRNA lentivirus or pcDNA3.1 plasmid vectors, THP-1 and RAW264.7 cells were harvested in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitors and phosphatase inhibitors (Roche). Next, equal amounts of total protein lysates (40 µg) were separated by SDS-PAGE before transferred to a PVDF microporous membrane, and stained with antibodies against TMSB10 (1:1000, Sant cruz, sc-514309), AKT (1:1000, Abcam, ab8805), p-AKT(Ser473) (a1:3000, Abcam, ab81283), mTOR (1:2000, Abcam, ab2732) p-mTOR(sec2448) (1:1000, Abcam, ab109268), p70S6K (1:2000, CST, 9202S), p-p70S6K(Thr389) (1:1000, CST, 9234T) and GAPDH (1:5000,CST, 5174S) as previously described (18). Meanwhile, the extracellular IL-6, IL-10 and TNF-α level in serum-free conditioned media from THP-1 and RAW264.7 cells treated with indicated lentivirus (1 µg) or LPS (100 ng/ml) for 72 hr were measured using the human ELISA kit (Elabscience, Wuhan).

Immunohistochemistry. Immunohistochemical assay was performed on the 5 mm thick slides, using mouse anti-human TMSB10 (1:500, Sant cruz, sc-514309) and CD68 (1:1000, CST, 76437) primary antibody, as previously (22). Two experienced investigator independently scored the TAMs-associated TMSB10 without knew the clinical data. The proportion of positive macrophage was scored as 0 (0–9% positive macrophages), 1 (10–25% positive macrophages), 2 (26–50% positive macrophages) and 3 (> 50% positive macrophages) respectively. Staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The final scores ranged from 0 to 3, ≥ 2 score was defined as high TAMs-associated TMSB10 expression and < 2 score was defined as low TAMs-associated TMSB10 expression according to previous method (17).

Cell counting Kit-8 (CCK8)

The THP-1 and RAW264.7 cell proliferation were measured by a CCK-8 kit (Beyotime, Shanghai, China). Briefly, cells were seeded into 96-well plates. After transfection of indicated shRNA lentivirus for 72 hr, the WST-8 reagent was added to each well for 1 hr incubation. The optical density (OD) value (450 nm) was measured using a microplate reader.

Statistical analysis. Statistical analyses were performed by SPSS 22.0 software (IBM Corporation, NY, USA). The correlation between TMSB10 expression and clinical pathological variables were investigated by Chi-square test. The Kaplan-Meier method and Cox proportional hazard model were used to investigate the prognostic factors for progression free survival (PFS) and overall survival (OS). Unpaired two-tailed Student’s t tests and/or analysis of variance (ANOVA) were used to calculate the significance. P < 0.05 was considered significant unless otherwise noted.

Results

High TAMs-associated TMSB10 is correlated with clinicopathologic characteristics of lung adenocarcinoma.

257 cases with lung adenocarcinoma were preliminary enrolled in our study. 73 cases of them were initially excluded: (1) 65 patients had received systemic therapy before enrollment and (2) 8 cases had other malignancies or multiple primary tumors. Finally, 184 cases were enrolled in this study. The clinicopathologic features of the enrolled 184 cases are presented in Table I. The average age of diagnosis was 63 years (age from 35 to 86 years) with mean follow-up of 37 months (range from 4 to 83 months). Of the whole patients, 44.6% were female, 49.5% were non-smokers. 64.1% were at stage 1 + II and 35.9% at stage III. 114 patients (62.0%) showed no lymphatic metastasis.

The CD 68 protein was mainly expressed in TAMs,and TMSB10 protein was expressed in both tumor cells and TAMs (Fig. 1A-1C). High TAMs-associated TMSB10 expression prefers to be detected in older patients (P < 0.001). High TAMs-associated TMSB10 expression was significantly associated with advanced TNM stage (P = 0.036) and T3/T4 tumor size (P = 0.007) (Table I), indicating TAMs-associated TMSB10 may be related to tumor proliferation or metastasis. There are no statistically differences in the relation between TAMs-associated TMSB10 expression and sex, smoking, tumor location, N stage.

High TAMs-associated TMSB10 is correlated the poor prognosis of lung adenocarcinoma.

To investigate the prognostic value of TAMs-associated TMSB10 in patients with lung adenocarcinoma, we had collected follow-up data and performed survival analysis. Kaplan-Meier analysis showed that patients with low TAMs-associated TMSB10 expression had significantly better PFS (5-year survival rate: 53.0% versus 30.1%, P = 0.002) as well as better OS (5-year survival rate: 57.0% versus 25.0%, P < 0.001) compared with those with high TAMs-associated TMSB10 expression (Fig. 1D).

The Cox regression analysis of PFS showed that high TAMs-associated TMSB10 expression (HR: 1.726, 95%CI: 1.061–2.808, P = 0.028) might be independent risk factors of PFS. Meanwhile, The Cox regression analysis of OS showed that high TAMs-associated TMSB10 expression (HR: 1.94, 95%CI: 1.189–3.167, P = 0.008) might be independent risk factors of PFS.

Discussion

TMSB10, a 43-amino acid residues β-thymosin, is found to be overexpressed in human lung cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, pancreatic cancer, thyroid cancer and renal cell carcinoma et al (17, 20, 23–27). And TMSB10 mRNA is found to be overexpressed in TAMs of early lung adenocarcinoma compared with those in adjacent normal lung macrophages and peripheral blood monocytes (21). However, the clinicopathological significance of TAMs-associated TMSB10 in lung cancer remains largely unknown. We firstly carry out the investigation on the relationship between TAMs-associated TMSB10 and the clinicopathological features of lung adenocarcinoma. First, we found that high TAMs-associated TMSB10 expression significantly associated with advanced TNM stage and bigger tumor size. Moreover, High TAMs-associated TMSB10 was significantly correlated with poor PFS and OS. Next, we conducted exnograft model to investigate the role of TMSB10 in tumors. We found that TMSB10 knockdown significantly attenuated the exnograft tumor growth, and the infiltrating TAMs and TAMs-associated TMSB10 expression was reduced by TMSB10 knockdown. The biological role of TMSB10 in lung adenocarcinoma TAMs was still unknown.

Most studies showed that knockdown of TMSB10 reduced cancer cell proliferation or migration (17, 18, 28–30). However, Lee et al. reported that overexpression of TMSB10 caused F-actin stress fibers disruption, notably reduced cell growth and increased apoptosis (23). Zhang et al. showed that TMSB10 played a contradictory role in cell proliferation in different kind of cell (31). Therefore, for investigating the role of TMSB10 in TAMs of lung adenocarcinoma, we isolated the tumor-infiltrating immune cells in exnograft tumors, and found that CSF1R⁺ TAMs and Foxp3⁺ Tregs were reduced in TMSB10 knockdown group. TMSB10 knockdown could significantly promote the M2 to M1 phenotype conversion of TAMs in vivo. Meanwhile, the proliferation of macrophages was repressed by TMSB10 knockdown. And the level of macrophage M1 markers IL-6, TNF-α were increased by TMSB10 knockdown, yet the level of macrophage M1 marker IL-10 was repressed by TMSB10 knockdown in vitro. Further studies are needed to elucidate the underling mechanism for its biological role.

PI3K/Akt is an important pathway for cellular growth, survival and protein synthesis. Recent studies have reported that resident macrophages can proliferate via different signaling so as to maintain cell number in type 2 immunity (32, 33). Meanwhile, the anti-inflammatory M2 macrophage could been activated by PI3K/Akt signaling (34). We further demonstrated that THP-1 and RAW264.7 could undergo rapid proliferation and M2 phenotype conversion by activating TMSB10-dependent PI3K/Akt signaling pathway.

Conclusions

In summary, these results demonstrate that TMSB10 is remarkably overexpressed in TAMs and negatively associated with the prognosis of lung adenocarcinoma, and TMSB10 knockdown dramatically represses exnograft tumor growth in vivo. TMSB10 knockdown promotes antitumorgenic M1 conversion and represses the proliferation of macrophages via PI3K/Akt signaling in vivo and in vitro. Taken together, our findings suggest that TAMs-associated TMSB10 promotes tumor through increasing TAMs proliferation and M2 phenotype conversion via PI3K/Akt signaling, which might be an indicator for prognosis, seems as a promising novel therapeutic target for human lung adenocarcinoma.

Abbreviations

TAMs: Tumor-associated macrophages; Tregs: Regulatory T cells; NSCLC: Non-small cell lung cancer; LLC: Lewis lung cancer. MDSC: myeloid-derived suppressor cells; FSC: Forward scatter; SSC: Side scatter.

Declarations

Consent for publication

Not applicable.

Availability of data and materials

The software, databases and all relevant raw data are freely available to scientists.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding: This work was supported by the Foundation for High-level Talents of the First Affiliated Hospital of Chengdu Medical College (CYFY-GQ03), the Youth Innovation Project of Sichuan Medical Association (Q17025), and the Key Project of the Education Department of Sichuan Province (18ZA0157) and the Natural Science Foundation of China (81600388).

Author contributions: JZ and YXZ designed and performed experiments, YXZ analyzed data, YXZ and LY analyzed histology, and ZJ wrote the manuscript.

Acknowledgements：

None.

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Table

Table I. The Correlation between TAMs-associated TMSB10 expression and clinicopathological characteristics of lung adenocarcinoma.