Aveneu Park, Starling, Australia

In progressing and several antiangiogenic drugs has

In the present study, for the
first time we reported that an engineered antiangiogenic peptide derived from
endostatin, ES-SS, has no immunogenic effect on healthy mice and is completely
safe. Surprisingly, this peptide could activate cellular and humoral immunity
in mammary carcinoma-bearing mice through increasing TNF-?, IL-10, IFN-?, IL-4,
and IL-17 production. Consequently, it can be proposed as a promising adjuvant
in cancer immunotherapy.

In the last three decades,
development of angiogenesis inhibitors for treatment of cancer has been
progressing and several antiangiogenic drugs has been approved by F.D.A (Samant
and Shevde 2011). Recently, we demonstrated that ES-SS
comprises potent antiangiogenic and antitumor activities in vivo (Chamani
et al. 2015). No immunologic data is available about this
fragment of endostatin. Therefore, the present study was focused on
investigating the effect of ES-SS on the cellular and humoral immune system of
both healthy and mammary carcinoma-bearing mice. Treatment of healthy mice with
1 and 5 mg/kg/day ES-SS demonstrated no significant increase in the level of TNF-?,
IL-10, IFN-?, IL-4, and IL-17 in comparison to the control group after 25 days (p<0.05). Accordingly, this peptide is safe and has no adverse and immunological effects on the normal cells and tissues. The comparison of tumor volume in animal received 1 and 5 mg/kg/day ES-SS with control demonstrated 62% and 55% inhibition of tumor growth, respectively. This result is in accordance with our previous study that ES-SS could remarkably inhibit the growth of breast adenocarcinoma, derived from MC4-L2 cell line, in mice (Chamani et al. 2015). We measured five key cytokines that are important in cellular and humoral immunity. TNF-? is secreted primarily by macrophages in response to inflammatory stimuli and is capable of applying significant tumor necrosis in certain animal models (Roberts et al. 2011). IL-10 is an anti-inflammatory cytokine that can directly regulate innate and adaptive Th1 and Th2 responses by restraining T cell activation and differentiation in the lymph nodes and tissues (Couper, Blount, and Riley 2008). In contrast with its inhibitory effects on T cells, IL-10 promotes survival, proliferation, and differentiation of human B cells and increases the production of IgG4 and/or IgA (Meiler et al. 2008). IFN-? plays a role in the development of Th1 response and the B-cell isotype switching to IgG2a (Akdis et al. 2011). IL-17 is expressed by activated CD4+ Th17 cells and also by CD8+ T cells, NK cells, and neutrophils. IL-17 acts on a variety of cells, which respond by upregulating expression of proinflammatory cytokines, chemokines, and metalloproteases (Korn et al. 2009). IL-4 induces IgE class switching in B cells, as well as increasing the B-cell receptors and expression of class II MHC molecules in B cells (Akdis et al. 2011), so is a key regulator of humoral immunity. When secretion of above mentioned cytokines from splenocytes of tumor-bearing mice was evaluated 14 and 25 days after tumor implantation, an increase in the level of cytokines was detected after treating with 1 and 5 mg/kg/day ES-SS compared to the control, and IL-10 and IL-4 secretion were significantly higher than others, particularly in 1 mg/kg/day ES-SS. Furthermore, calculation of IFN-?/IL-10 ratio for analysis of Th1/Th2 response indicated potential capacity of this peptide in induction of higher and longer Th2 response in immunized mice, as well (Fig. 4). The reason why 1 mg/kg/day ES-SS could increase the secretion of IL-10, IL-4 and TNF-? more than 5 mg/kg/day is not clear and needs to be investigated, but we observed this behavior of ES-SS previously, where 50 µg/kg concentration (twice a day) inhibited the growth of breast tumor in mice more than 0.5 and 2.5 mg/kg (twice a day) (Chamani et al. 2015). In addition, a U-shape antitumor response was observed for endostatin and certain angiogenesis inhibitors, so that a narrow range of concentration is able to prevent tumor growth in mice and below and above this concentration, antitumor activity reduces (Javaherian et al. 2011). This immunostimulatory effect was also explained by other researchers, previously. For instance, Chaves et al. (2012) demonstrated that endostatin gene therapy induced a significant increase in both CD4+ and CD8+ T cells in the lymph nodes and the spleen, as well as an increased percentage of the CD4-IFN-?, CD8-IFN-? and CD49b-TNF-? cells in the metastatic lung tumor-infiltrating lymphocytes of mice. Therefore, endostatin has an antitumor inflammatory effect in an metastatic renal cell carcinoma model, and an important role in the amplification of the immune response in the lymph nodes (Chaves et al. 2012). In addition, Rocha et al. (2010) showed endostatin treatment produced a significant increase in regulatory CD4 and non-specific tumor-killing CD49b cells in a mouse model of metastatic renal cell carcinoma (Rocha et al. 2010). Endostatin gene therapy was shown to lead to a tumor volume reduction of ~50%, associated with tumor infiltration of leukocytes (Coutinho et al. 2007). It has been previously shown that endostatin may interfere with endothelial cell anergy, leading to increased leukocyte endothelium interactions that promote leukocyte infiltration within tumors (Dirkx 2006). Besides endostatin, other antiangiogenic agents like sunitinib and bevacizumab induce immune response in tumor models. Sunitinib treatment enhances the functional capacity of tumor infiltrating T cells by stimulating IFN-? production and cytolytic activity against the tumor (Ozao-Choy et al. 2009) and improves Th1 response after stimulation of PBMC by anti-CD3/CD28 antibodies in metastatic renal cell carcinoma patients 23, 24. Bevacizumab treatment, enhances absolute number of CD4, CD8, and CD3 lymphocytes in metastatic colorectal cancer patients (Manzoni M, Rovati B, Ronzoni M, Loupakis F, Mariucci S, Ricci V, Gattoni E, Salvatore L, Tinelli C, Villa E 2010) and IL-2 and IFN-? production after restimulation of PBMC by anti-CD3 antibody (Tsavaris N, Voutsas IF, Kosmas C, Gritzapis AD 2012). In addition to the stimulation of immune response, some anti-angiogenic molecules modulate immunosuppression developed by the tumor to escape the immune system, enhance Th1 response after mitogenic restimulation, and increase T-cell infiltration into the tumors (Terme et al. 2012). For instance, Sunitinib treatment results in a decrease of Treg percentage in spleens and tumors of different mouse tumor models as well as in the peripheral blood and tumors of metastatic renal cell carcinoma patients 23, 24. Consequently, inhibition of tumor-induced immunosuppressive conditions makes suitable environment to induce an efficient anti-tumor immune response after vaccination. Hence, combination therapies of different vaccines with antiangiogenic molecules have been tested in mouse tumor models. For example, administration of IL-2 combined with endostatin gene therapy was able to produced an increase in CD4+ T, CD8+ T, and CD49b cells and demonstrated additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma (Rocha et al. 2010). Liang et al. (2016) used tumor antigen-pulsed dendridic cells (DC)-T cells in combination with endostatin to treat tumor bearing mice with Lewis lung carcinoma. Compared with control and DC-T cells alone, adoptively transferred tumor antigen specific DC-T cells combined with endostatin reduced immunosuppressive myeloid derived suppressor cells (MDSC) and M2 tumor-associated macrophages (TAM), down-regulated immunoinhibitory cytokines (IL-6, IL-10, TGF-? and IL-17), increased M1 TAM and the expression of IFN-? involved in oncolysis and significantly elevated tumor-infiltrated myeloid DC and CD8+ T cells, so as to reverse immunosuppression in tumor microenvironment and enhance antitumor effect (Liang et al. 2016). In addition, antitumor effect of combined gene therapy of endostatin and interleukin 12 (IL-12) with polyvinylpyrrolidone (PVP) was studied on mouse transplanted hepatoma. Results demonstrated that angiogenesis in hepatoma was inhibited synergistically, lymphocytes activated to infiltrate, and tumor cells are induced to apoptosis. Hepatoma highly inhibited or eradiated (P. Y. Li et al. 2004). Immunotherapy of  mouse colorectal cancer and melanoma with adenoviral vectors expressing soluble VEGFR-1 and -2 associated with a GM-CSF-secreting tumor cell enhanced anti-tumor immune response in these tumor models (B. Li et al. 2006). Furthermore,  in a model of hepatitis virus-induced hepatocellular carcinoma in eastern woodchuck, administration of two adenoviral vectors, one encoding GM-CSF and IL-12 and the other expressing endostatin and pigment epithelium-derived factor (with anti-angiogenic properties), enhanced natural killer (NK) cell infiltration and decreased immunoregulatory molecules CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and PD-1 (Programmed cell death protein 1) and tumor volume (K.-W. Huang et al. 2010). In conclusion, ES-SS for some reason may serve as a potent and promising adjuvant to immunotherapy of cancer and deserves further studies. First of all, angiogenesis inhibitors could normalize tumor vascularization (Jain 2005). This vascular normalization could help CD8+ T cell influx in the tumor after vaccination (Y. Huang et al. 2012). Moreover, the abnormal tumor vasculature creates a hypoxic microenvironment that polarizes inflammatory cells toward immune suppression. So, the suppression of hypoxia could also be a mechanism of modulation of tumor-induced immunosuppression (Yuhui Huang et al. 2013). Secondly, antiangiogenic molecules could modulate immunosuppressive microenvironment of tumors leading to effective vaccination strategy. Finally, ES-SS could augment cellular and humoral immunity to tumor-bearing but not normal mice. Our data strongly support the immunostimulatory effect of endostatin and thereby highlighting the use of ES-SS as a promising possible therapeutic tool. This potential benefit of ES-SS in combination with an immunotherapeutic strategy will be tested by our team and hopefully synergistic responses would be expected from this combination.

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