Sunitinib inhibits PD-L1 expression in osteosarcoma by targeting STAT3 and remodels the immune system in tumor-bearing mice

Xian Liang Duan1, Jian Ping Guo1, Fan Li1, Chao Xiu2 & Hua Wang *,3
1 The Second Department of Orthopedic, The Affiliated Hospital of Beihua University, Jilin 132011, PR China
2 Medical Imaging Center, The Affiliated Hospital of Beihua University, Jilin 132011, PR China.
3 The Department of Orthopedic, The Affiliated Hospital of Beihua University, Jilin 132011, PR China.
*Author for correspondence: Fax: 0432 6216 6003; [email protected]

Aim: Exploring the mechanisms of the combination therapy using VEGFR-TKI and immune checkpoint in- hibitors might be useful to control the development of osteosarcoma. Materials & methods: The expres- sion of PD-L1 and STAT3 in osteosarcoma were determined with western blot. Proliferation, migration and invasion were determined with CCK-8 and Transwell assays. Lung metastases, tumor growth, survival and immune cell populations were performed in tumor-bearing mice. Results: Sunitinib reduced the ex- pression of PD-L1 by inhibiting the activation of STAT3 and suppressed the migration and invasion in osteosarcoma cells. Combination therapy reduced lung metastases, tumor growth, improved survival and reverse tumor microenvironment in tumor-bearing mice. Conclusion: Sunitinib inhibits PD-L1 expression by targeting STAT3 and remodels the immune system in tumor-bearing mice.

First draft submitted: 8 November 2019; Accepted for publication: 15 May 2020; Published online: 8 June 2020
Keywords: immunotherapy • metastasis • migration and invasion • osteosarcoma • PD-L1 • sunitinib
Osteosarcoma is the most common primary mesenchymal tumor in adolescents. It is also one of the strict malignant bone tumors that endanger the health of adolescents [1,2]. Because of the poor outcomes of the metastatic osteosarcoma, its 5-year survival rate is less than 20% [3,4]. The main treatment of osteosarcoma is surgery combined with chemotherapy; however, there has been no significant improvement in the survival rate of osteosarcoma patients in the past decade. Therefore, it is particularly urgent to explore the mechanisms underlying osteosarcoma development and find new approaches to treat osteosarcoma.

Immunotherapy is a promising therapeutic strategy against osteosarcoma. The relationship between immune checkpoints and the tumor immune system is a hot topic in recent research [5]. Immune checkpoints are necessary to maintain self-tolerance and limit immune responses to prevent autoimmune diseases. But it also leads to the escape of local antitumor immune responses [6]. For the past few years, checkpoint inhibitor-based immunotherapy has attracted much attention. The expression of PD-1 and PD-L1 in osteosarcoma cells was negatively correlated with prognosis. Moreover, the expression level in metastatic osteosarcoma was significantly higher than that of primary osteosarcoma [7]. Therefore, we hypothesize that using antibody blockade of such inhibitory proteins may be an effective option for the treatment of metastatic osteosarcoma.
ImageImageImageImageImageImageMore recently, vascular endothelial growth factor receptor tyrosine kinase inhibitor, VEGFR-TKI, has been shown to enhance the immunogenicity of tumor cells and reverse immunosuppressive tumor microenvironment [8]. According to previous studies, sunitinib, a multitargeted tyrosine kinase, exerts promising antitumor effect in various tumors [9,10]. However, the effects of sunitinib on osteosarcoma is little known. Combination of immunotherapy with antiangiogenic agents has achieved significant improvements in various carcinomas [11,12]. It is hypothesized that combination strategies are necessary to optimize immunotherapy for osteosarcoma. The combination of an anti-PD-L1 antibody and TKI may theoretically enhance the antitumor effects; however, the principle and mechanism are still unknown.

In this study, we investigated whether sunitinib suppressed the PD-L1 expression in osteosarcoma cells and inhibited the migration and invasion of osteosarcoma cell by inactivating STAT3. In addition, we explored that sunitinib remodeled the immune system in tumor-bearing mice. We also demonstrated that antiangiogenic agents, in combination with a-PD-L1 antibody, displayed significant effects in promoting immune response and significantly prolonged the survival.

Materials & methods
Cell lines & primary cultures
Osteosarcoma cell lines KRIB, U2OS, 143B, MG63.2, LM-7 and SAOS-2 cells were purchased from American Type Culture Collection (MD, USA). All cells were cultured in complete Dulbecco’s modified Eagle’s medium (Gibco, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, CA, USA), 100 U/ml penicillin,
and 100 lg/ml streptomycin (Sangon Biotech, Shanghai, China) at 37◦C with 5% CO2 and 95% air humidified
incubator. All cells were tested for mycoplasma using MycoAlert™ Mycoplasma Kit (Lonza, NJ, USA).

Western blot
Proteins were collected from various cell lysed using RIPA. A 10% SDS/PAGE was run after protein quantification
(Bio-Rad, CA) and transferred onto NC membranes. After blocking in nonfat milk for 1 h, the membranes were incubated with primary antibodies (1:1000): PD-L1, p-STAT3, STAT3 overnight at 4◦C. The next day, after incubated with secondary antibodies, the bands were probed with the western blot detection system (BioRad, CA,
USA), and quantification was performed using Image J.

Cell viability assays

Cells were seeded in a 96-well plate (1 104cells/well) and cultured overnight. Then, the cells were treated with sunitinib at different concentrations, followed by incubation at 37◦C for 24 h. At the end of incubation, cell viability was analyzed using Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) according to the protocol.

Transwell assay
Cells (6 × 104) were seeded into the noncoated upper chamber of 24-well plates (Corning, Inc., NY, USA) with an 8 μm pore size for a migration assay. For an invasion assay, the upper chamber was coated with Matrigel (BD
Bioscience, 354234). Cells in upper chamber were cultured in medium with 1% FBS treated with or without 2 mM sunitinib, and 500 μl medium with 10% FBS was added to the lower chamber. After culturing for 24 h, cells on
the bottom of the upper chamber were washed with phosphate-buffered saline (PBS) and fixed with methanol for 30 min at 4◦C. And then, the cells were stained with 0.1% crystal violet. Migrated cell populations were evaluated in five fields per well under a microscope. Experiments were performed in triplicate.

Animal model
Female BALB/c mice, 6–8 weeks old, body weight 20 g, were purchased from Charles River Laboratories (Beijing, China) and raised in an animal room under specific pathogen-free conditions. All procedures in the animal experiments were carried out in accordance with the current regulations and standards of the Animal Ethics Study Committee of Beihua University (Jilin, China).
To establish primary osteosarcoma tumors in mice, 5 × 106 KHOS cells were injected subcutaneously into
the right flanks of six-week-old female BALB/c nude mice (Vital river, Beijing, China). To establish metastatic osteosarcoma tumors in mice, 5 × 106 K7M2 cells were injected via the lateral tail vein in 100 μl of PBS. All treatments were started on day 5 after the injection. The mice were randomly divided into four groups (n = 6/7
per group) and administered sunitinib 40 mg/kg or 10 mg/kg PD-L1 antibody or combination therapy or PBS control intraperitoneally every 3 days for 4 weeks. The volume of the tumor was measured in every 5 days (tumor
volume = [length × width2]/2) until sacrifice at end point. Metastatic osteosarcoma lung tissues were counted and
stained to determine the percentage of osteosarcoma cells.

Flow cytometry staining
Single cell tumors were obtained using a 100 μm syringe filter (Corning Inc., NY, USA) and then suspended in RPMI1640 complete medium supplemented with 5% FBS. For the regulatory T cells (Tregs) analysis, staining was performed with CD5 (clone#53-7.3, eBioscience), CD4 (clone#GK1.5, eBioscience), CD25 (clone#PC61.5,

eBioscience) and Foxp3 (clone#NRRF-30, eBioscience). For the CD8+ T cell analysis, staining was performed with CD8 (clone#53-6.7, eBioscience). For cell surface markers, cells were stained with antibodies on ice for 30 min. For intracellular antigen detection, an intracellular staining kit containing fixation/perm reagents from eBioscience
was used. Flow cytometry analyses were performed on BD LSR Fortessa (BD Biosciences). Flowjo 10 was used to analyze the data.

Statistical analysis
Survival curves were illustrated using Kaplan–Meyer method. Statistical analysis was performed by one-way ANOVA with Bonferroni’s multiple comparison. All the analysis was performed on GraphPad Prism 6. Data are expressed
as mean ± SD. In all statistical analyses, a p < 0.05 was considered statistically significant.

Results
Sunitinib suppresses the PD-L1 expression in osteosarcoma cells by targeting STAT3
PD-L1 expression in the tumor microenvironment has been associated with poor outcomes in cancer patients. To investigate whether sunitinib suppresses PD-L1 in osteosarcoma, we first examined the expression of PD-L1 in several osteosarcoma cell lines. The PD-L1 expression in human osteosarcoma cells was examined by western blot. The results showed that the high levels PD- L1 protein were seen in KRIB, U2OS, 143B and LM7 cells, whereas very low levels were seen in MG63.2 and SAOS-2 cells (Figure 1A & B). Next, we examined that whether sunitinib affected PD-L1 expression in KRIB and LM7 cells by western blot. As results showed (Figure 1C, D & E), the expressions of PD-L1 were downregulated after sunitinib treatment. Sunitinib is a known tyrosine kinase inhibitor, and previous studies suggested that STAT3 could regulate PD-L1 expression [13]. We examined the downstream signaling molecules of STAT3 in osteosarcoma cells. As results showed (Figure 1C–E), sunitinib decreased the activation of STAT3 in KRIB and LM7 cells. Together, these findings strongly indicate that sunitinib inhibits PD-L1 expression by the inactivation of STAT3.

Sunitinib reduces the migration & invasion of osteosarcoma cells
We first found that sunitinib did not appreciably affect the vitality of KRIB and LM7 cells at concentrations of 0.5, 1.0, 2 or 1.5 μM (Figure 2A). Then, the concentration of 2 μM was selected as the maximum for further investigations. Furthermore, sunitinib decreased the migratory (Figure 2B & C) and invasive capacities of KRIB and LM7 cells by trans-well assays (Figure 2D & E). These results indicate that sunitinib could effectively reduce the migration and invasion of osteosarcoma cells.

Sunitinib significantly reduces osteosarcoma lung metastases, controls tumor growth & improves survival in osteosarcoma tumor-bearing mice
As osteosarcoma is a metastatic disease, we monitored two osteosarcoma mice models that underwent different treatment regimens in both in situ and metastasis models and then treated them with sunitinib and/or a-PD-L1 antibody. As shown in (Figure 3A& B), in the in situ model, sunitinib and a-PD-L1 antibody treatment suppressed inhibited tumor growth and prolonged mouse survival. Moreover, combination treatment revealed even stronger benefit. As the same in the metastasis model (Figure 3C & D), both sunitinib or a-PD-L1 antibody treatment caused a significant decrease in the mean number of LM7 OS metastases and prolonged mouse survival than the mice receiving PBS. Moreover, combination treatment revealed even stronger benefit.

Sunitinib changes the immune cell populations in osteosarcoma tumor-bearing mice
According to a previous study, increased PD-L1 expression in the tumor microenvironment can lead to a subsequent decrease in tumor-specific T-cell activity, which promotes the development of immune tolerance [14]. Therefore, we evaluated the potential immunomodulatory effect of sunitinib and the anti-PD-L1 antibody in osteosarcoma tumor-bearing mice. Single-cell suspensions from tumor tissue samples collected from the above-mentioned three
groups were examined for the proportions of CD8+ T lymphocytes and Treg cells. As the results showed (Figure 4A & B), the proportions of CD8+T cells in the sunitinib or anti-PD-L1 antibody treatment groups were higher
compared with those in the control group. By contrast, the proportions of Treg cells in the sunitinib or anti-PD-L1 antibody treatment groups were lower than that in the control group (Figure 4C & D). These results suggest that sunitinib increased the number of tumor-infiltrating lymphocytes and alleviated the immunosuppressive microenvironment.

Protein quantification
Figure 1. Sunitinib suppresses PD-L1 expression in osteosarcoma cells. (A) PD-L1 expression in osteosarcoma cells was determined by western blot. (B) ImageJ analysis was used for PD-L1 (relative to GAPDH) quantification. (C) The protein levels of PD-L1, STAT3 and
p- ±
p STAT3 on KRIB and LM7 cells treated with Sunitinib or DMSO were determined by western blot. (D & E) ImageJ analysis was used for Protein (relative to GAPDH) quantification. All experiments were repeated three-times. Data are presented as mean SD.
***p < 0.001.

Discussion
The treatment of osteosarcoma has not markedly progressed for several decades, and the most effective strategy is still neoadjuvant chemotherapy combined with surgical resection. However, in up to 40% of patients, chemotherapy cannot prevent metastasis and treatment failure. Therefore, it is urgent to find a new way to improve the prognosis of patients with osteosarcoma. In recent years, immunotherapy has achieved remarkable progress and attracted much attention in cancer treatment [15]. The most interesting immune therapeutic drug is directed against the programmed death receptor 1, PD-1, and its ligand PD-L1 antibody. Such immunological checkpoint inhibitors have achieved significant efficacy in the treatment of malignant tumors such as melanoma, non-small-cell lung cancer, and renal cancer [16–18]. Owing to the pathogenic mechanism and immune characteristics, osteosarcoma could be the perfect candidate for immunotherapy.
Anti-angiogenic targeted therapy can change the immune microenvironment of tumors by reducing the vascular density of tumors and normalizing tumor blood vessels [19]. In a Phase III randomized clinical trial, one of the 28

Sunitinib significantly reduces osteosarcoma lung metastases, controls tumor growth and improves survival in osteosarcoma tumor-bearing mice. (A) Sunitinib or anti-PD-L1 therapy substantially inhibited tumor growth when compared with the control. (B) Survival rates among various groups of mice bearing in situ osteosarcoma. (C) Sunitinib or anti-PD-L1 therapy substantially inhibited tumor lung metastases when compared with the control. (D) Survival rates among various groups of mice bearing in situ metastatic osteosarcoma. n = 7/group.
**p < 0.01; ***p < 0.001patients with maxillary osteosarcoma had partial remission after treatment with nivolumab plus pazopanib [20]. In our study, osteosarcoma cells are exposed to 2 μM sunitinib, and sunitinib attenuates the migration and invasion of osteosarcoma cells. Meanwhile, there was no detectable cytotoxicity of 2 μM sunitinib in osteosarcoma cells, suggesting that sunitinib exhibits a positive effect in inhibiting metastasis in osteosarcoma. However, the underlying mechanism about the inhibition of metastasis by sunitinib is veiled in osteosarcoma. Signal transducer and activator of transcription STAT3 are important components of the common tumor signaling pathway JAK/STAT involved in tumor development [21]. STAT3 is responsible for the transmission of extracellular signals to the nucleus. STAT3 induces the proliferation and differentiation of anti-apoptotic genes, such as Bcl-2, Bcl-xl and Survivin [22]. Blocking STAT3 can increase the expression of cytokines and chemokines in tumor tissues and activate innate immune activated dendritic cells, which leads to tumor T-cell response [23]. In our study, in vitro tumor cell experiments confirmed that sunitinib inhibits PD-L1 expression by targeting STAT3.

Recently, studies have reported that sunitinib not only solely targets tumor cells but also enhances antitumor immunity [24]. Sunitinib creates a favorable microenvironment depleted of myeloid-derived suppressor cells and acts synergistically with a cancer vaccine resulting in enhanced levels of active tumor-antigen specific cytotoxic T
lymphocyte (CTLs), thus changing the balance in favor of antitumor immunity [25]. In this study, we demonstrate that immunization with sunitinib increases the proportion of CD8+ T cells among tumor-infiltrating lymphocytes. We conjectured that it might be associated with decreased expression of PD-L1 in tumor tissues and we will confirm
it in the future. Because CTLs kill cancer cells by directly contacting them and releasing perforin and granzyme B [26], it is critical to develop optimal combination immunotherapies, which can increase CTL infiltration in osteosarcoma

Sunitinib changes the immune cell populations in the osteosarcoma tumor-bearing mice. (A & B) Sunitinib therapy increased the proportion of CD8+ T cells in the tumor tissue of mice bearing in situ osteosarcoma. (C & D) Sunitinib therapy decreased the proportion of Treg cells in the tumor tissue of mice bearing in osteosarcoma. The results are displayed as mean. n = 6/group.
**p < 0.01; ***p < 0.001. Treg: Regulatory T celltumors. Tregs inhibit the migration of T lymphocytes to the tumor and promoting T cell exhaustion [27]. In our work, we found that sunitinib decreased the proportion of tumor-infiltrating Tregs in osteosarcoma. This finding suggests that sunitinib can remodel the tumor microenvironment. Combining PD-L1 blockade with CTLA-4 blockade has been shown to enhance T-cell cytokine production and result in overall increases in survival in osteosarcoma [28]. The effect of VEGFR- TKI combined with an anti-PD-L1 antibody as an osteosarcoma therapy is still unknown. In the present study, we confirmed that the combination of sunitinib and an anti-PD-L1 antibody is an effective strategy for osteosarcoma treatment using a subcutaneous osteosarcoma model. Our results have proven that combined therapy with sunitinib and an anti-PD-L1 antibody is a better choice for osteosarcoma treatment than sunitinib alone. We can expect that cocktail immunotherapy, sunitinib, PD-1/PD-L1 blockade and CTLA-4 blockade may achieve most powerful anticancer effects.
Our study demonstrates that sunitinib reduces PD-L1 expression in osteosarcoma cells. These results provide direct evidence that sunitinib enhances antitumor activity not only through antiangiogenic effect but also via suppressing immune escape. In summary, sunitinib inhibits PD-L1 expression in osteosarcoma cells by targeting

STAT3 and attenuates metastatic capacities. Additionally, sunitinib increases the proportion of CD8+ T cells and
reduces the proportion of Tregs in osteosarcoma-bearing mice, which suggests that sunitinib can remodel the tumor
microenvironment.
⦁ Previous studies have reported that sunitinib not only solely targets tumor cells but also enhances antitumor immunity. However, the regulatory mechanisms still need to be clarified.
⦁ PD-L1 was upregulated and associated with the progression of osteosarcoma.
⦁ Sunitinib inhibited PD-L1 expression by targeting STAT3 in osteosarcoma cells.
⦁ Sunitinib also inhibited the migration and invasion of osteosarcoma cells.
⦁ The combination therapy using vascular endothelial growth factor receptor and immune checkpoint inhibitors significantly reduced osteosarcoma lung metastases, controlled tumor growth and improved survival in osteosarcoma tumor-bearing mice.
⦁ We demonstrated that sunitinib enhanced the activation of CD8+ T cells and decreased the regulatory T cells.
⦁ Our study suggested that sunitinib suppressed immune escape and reversed immunosuppressive tumor
microenvironment.
⦁ The combination therapy using VEGFR-TKI and immune checkpoint inhibitors might be a potential therapeutic target for metastatic osteosarcoma.

Summary points
Financial & competing interests disclosure
This work was funded by the Science and technology research project of the Jilin Provincial Department of Education (2020; JJKH20200070KJ), Health Technology Innovation Project of Jilin Province (2019J045). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
The paper was edited by Elsevier Language Editing Services.

Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.

References
Papers of special note have been highlighted as: • of interest; •• of considerable interest
1. Heare T, Hensley MA, Dell’Orfano S. Bone tumors: osteosarcoma and Ewing’s sarcoma. Curr. Opin. Pediatr. 21, 365–372 (2009).
2. Jeon DG, Song WS. How can survival be improved in localized osteosarcoma? Expert Rev. Anticancer Ther. 10, 1313–1325 (2010).
⦁ A review of the most effective therapies that have been used for the treatment of osteosarcoma.
3. Luetke A, Meyers PA, Lewis I et al. Osteosarcoma treatment – where do we stand? Cancer Treat. Rev. 40, 523–532 (2014).
4. Harrison DJ, Geller DS, Gill JD et al. Current and future therapeutic approaches for osteosarcoma. Expert Rev. Anticancer Ther. 18(1), 39–50 (2018).
5. Hegde PS, Karanikas V, Evers S. The where the when, and the how of immune monitoring for cancer immunotherapies in the era of checkpoint inhibition. Clin. Cancer Res. 22, 1865–1874 (2016).

6. Miwa S, Shirai T, Yamamoto N et al. Current and emerging targets in immunotherapy for osteosarcoma. J. Oncol. 2019, 7035045 (2019).
7. Lussier DM, Johnson JL, Hingorani P et al. Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J. Immunother. Cancer 3, 21 (2015).
•• Suggests that the combination therapy with immune check-point inhibitors anti-PD-1 and anti-CTLA-4 may serve as a new candidate against tumor growth of metastatic osteosarcoma.
8. Ozao-Choy J, Ma G, Kao J et al. The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies. Cancer Res. 69, 2514–2522 (2009).
•• Reports that the novel role of tyrosine kinase inhibitor can remodel the tumor microenvironment and could serve as a likely therapeutic intervention in cancer therapies.
9. Castillo-Avila W, Piulats JM, Garcia Del Muro X et al. Sunitinib inhibits tumor growth and synergizes with cisplatin in orthotopic models of cisplatin-sensitive and cisplatin-resistant human testicular germ cell tumors. Clin. Cancer Res. 15, 3384–3395 (2009).
10. Figlin RA, Hutson TE, Tomczak P et al. Overall survival with sunitinib versus interferon (IFN)-alfa as first-line treatment of metastatic renal cell carcinoma (mRCC). Am. Soc. Clin. Oncol. 26, 15 (2008).
11. Jaini R, Rayman P, Cohen PA et al. Combination of sunitinib with anti-tumor vaccination inhibits T cell priming and requires careful scheduling to achieve productive immunotherapy. Int. J. Cancer 134, 1695–1705 (2014).
⦁ Presents the evidences for the key role of sunitinib in cancer progression.
12. Farsaci B, Higgins JP, Hodge JW et al. Consequence of dose scheduling of sunitinib on host immune response elements and vaccine combination therapy. Int. J. Cancer 130, 1948–1959 (2012).
13. Seki N, Kan-O K, Matsumoto K et al. Interleukin-22 attenuates double-stranded RNA induced upregulation of PD-L1 in airway epithelial cells via a STAT3-dependent mechanism. Biochem. Biophys. Res. Commun. 494, 242e248 (2017).
⦁ Establishes the molecular basis of PD-L1 blockade-dependent immunotherapy and provides a rationale for immune checkpoint blockade as a promising approach in cancer treatment.
14. Gao Q, Wang XY, Qiu SJ et al. Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma. Clin. Cancer Res. 15, 971–979 (2009).
15. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).
16. Rizvi NA, Hellmann MD, Snyder A et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124–128 (2015).
17. Tumeh PC, Harview CL, Yearley JH et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).
18. Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 27, 450–461 (2015).
⦁ A review of immune checkpoint blockade may be the most effective therapy for the treatment of cancer.
19. Cao M, Liu C. Kinase inhibitor sorafenib modulates immunosuppressive cell populations in a murine liver cancer model. Lab. Invest. 91 (4), 598–608 (2011).
20. Hu X, Cao J, Hu W et al. Multicenter Phase II study of apatinib in nontriplenegative metastatic breast cancer. BMC Cancer 2014(14), 820 (2009).
21. Yu H, Kortylewski M, Pardoll D et al. Crosstalk between cancer and immune cells: role of STAT3 in the tumor microenvironment. Nat. Rev. Immunol. 7, 41–51 (2007).
•• Presents the important role of STAT3 in the tumor microenvironment.
22. Yu H, Pardoll D, Jove R et al. STATS in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9, 798–809 (2009).
23. Germain D, Frank DA. Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin. Cancer Res. 3, 5665–5669 (2007).
24. Potapova O, Laird AD, Nannini MA et al. Contribution of individual targets to the SU11248 antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248. Mol. Cancer Ther. 5, 1280–1289 (2006).
25. Draghiciu O, Nijman HW, Hoogeboom BN et al. Sunitinib depletes myeloid-derived suppressor cells and synergizes with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication. Oncoimmunology 4(3), e989764 (2015).
26. Trapani JA, Smyth MJ et al. Functional significance of the perforin/granzyme cell death pathway. Nat. Rev. Immunol. 2 (10), 735–747 (2001).
27. Preston CC, Maurer MJ, Oberg AL et al. The ratios of CD8+T cells to CD4+ CD25+ Foxp3+ and Foxp3− T cells correlate with poor clinical outcome in human serous ovarian cancer. PloS ONE 8, e80063 (2013).
28. Lussier DM, Zamarin D, Munn DH et al. Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J. Immunother. Cancer 3, 21 (2015).
•• Suggests the crosstalk between poor clinical outcome of the cancer and immune cells and provides a novel therapeutic approach for the treatment of cancer.