Perspectives on reprograming cancer-associated dendritic cells for anti-tumor therapies
- 1Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, Athens, OH, USA
- 2Diabetes Institute, Ohio University, Athens, OH, USA
- 3Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA
- 4Molecular and Cell Biology Program, Ohio University, Athens, OH, USA
- 5Department of Biological Sciences, Ohio University, Athens, OH, USA
In recent years, the relevance of the tumor microenvironment (TME) in the progression of cancer has gained considerable attention. It has been shown that the TME is capable of inactivating various components of the immune system responsible for tumor clearance, thus favoring cancer cell growth and tumor metastasis. In particular, effects of the TME on antigen-presenting cells, such as dendritic cells (DCs) include rendering these cells unable to promote specific immune responses or transform them into suppressive cells capable of inducing regulatory T cells. In addition, under the influence of the TME, DCs can produce growth factors that induce neovascularization, therefore further contributing to tumor development. Interestingly, cancer-associated DCs harbor tumor antigens and thus have the potential to become anti-tumor vaccines in situ if properly reactivated. This perspective article provides an overview of the scientific background and experimental basis for reprograming cancer-associated DCs in situ to generate anti-tumor immune responses.
Tumors are composed of cancerous cells and non-cancerous cells such as fibroblasts, endothelial cells, and infiltrating leukocytes. Together with non-cellular components (extracellular matrix proteins), this constitutes the tumor microenvironment (TME). The non-cellular components often support the growth and survival of cancer cells. Moreover, cancer cell growth and survival are influenced by the activation state and responses of infiltrating leukocytes. In particular, leukocytes such as macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and dendritic cells (DCs) have all been shown to participate in tumor development in various settings. For instance, on one hand, chronic inflammation, either induced by infection (e.g., H. pylori, Hepatitis virus) or irritants (tobacco smoke, asbestos) constitutes an important risk factor for the development of cancer (1–4). On the contrary, tumor-infiltrating leukocytes, such as cytotoxic T cells can mediate an immune response against the tumor by recognizing tumor antigens and attacking tumor cells in a specific manner (5, 6). Indeed, this is the basis of cancer immunotherapies. Thus, immunosuppression is also able to support tumor growth. Furthermore, existing evidence supports that adaptive immune response influences the behavior of human tumors. In situ analysis of tumor-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of colorectal cancer and possibly other malignancies (7).
There are two main ways in which leukocytes can collaborate with tumor development (i.e., pro-tumorigenic processes): suppression of the anti-tumor immune response and production of growth factors. In particular, cancer-associated immune cells such as regulatory T cells (Treg) or MDSCs have been shown to directly inhibit the activity of specific anti-tumor cytotoxic T cell responses (8, 9). In addition, infiltrating inflammatory cells secrete a diverse repertoire of growth factors that can enhance cancer cell proliferation and survival directly [e.g., interleukin (IL)-6 and TNF-α] or by stimulating angiogenesis (10–17). In this context, DCs are very interesting players, especially taking into account their ability to participate in both pro-tumorigenic and anti-tumor processes. For more detailed reviews on DCs in cancer biology and immunotherapy, please refer to Ref. (18–21).
Immune Properties of Dendritic Cells
Dendritic cells scan peripheral tissues where they recognize, take up, and process antigens and then migrate to lymphoid organs to present antigenic peptides to naive T lymphocytes in the context of major histocompatibility molecules (MHC) (13, 22–24). During this process, DCs become activated, upregulating MHC class II molecules and co-stimulatory molecules such as CD40, CD80, CD86, or OX40L. Upon activation, DCs typically show a decrease in their phagocytic capability, an augment in their efficacy to present processed antigens in the context of MHC molecules, and consequently an improved capability to activate T cells. Through the expression of both MHC class I and II molecules, DCs are able to activate antigen-specific CD8+ T cytotoxic and CD4+ T helper lymphocytes respectively (25–27). By means of various signals, DCs do not only activate specific T cells, but also drive their differentiation into distinct subsets and even can imprint a migration pattern on these cells toward particular organs or tissues (28). Depending on the stimulus and tissue microenvironment, activated DCs produce an array of cytokines including IL-6, IL-12, IFN-γ, and TNF-α, in addition to several chemokines such as CCL2, CCL3, and CCL5 (29), and thus can play a critical role in shaping the cytokine milieu and leukocyte recruitment and activation.
Dendritic cells are a diverse group of professional antigen-presenting cells that link innate and adaptive immune systems. Several distinct subsets of DCs have been identified and broadly subcategorized into conventional (cDCs) and plasmacytoid (pDCs) (30). Each subset is considered functionally unique, with different TLR expression profiles, response, and outcomes leading to activation of alternate branches of the immune system. For instance, mDC express TLR-2, -4, and -5 whose activation induces IL-12 and IL-6 production. In contrast, pDCs express TLR-7 and -9 ligation resulting in a strong type-I interferon namely IFN-α and are critical players in the innate anti-viral response (31). Such subset differences may have critical implications in success or failure of reprograming cancer-associated DCs in situ to generate anti-tumor immune responses.
Characteristics of Cancer-Associated DCs
The presence of DCs in the stroma of various types of cancer has been well-established (11, 32–35). Interestingly, often these cells do not exert a positive immune influence but act as co-conspirators of tumor growth by inducing regulatory T cell expansion, or directly suppressing T cell responses. These cancer-associated DCs, albeit carrying tumor antigen as we have previously shown (36), express low levels of co-stimulatory molecules (37). Thus, upon encounter with antigen-specific naïve T cells, they can induce an anergic state in these cells favoring tumor immune-escape. This DC phenotype could be caused by products generated by cancer cells or non-cancer cells present in the microenvironment of the tumor. For example, tumor-associated cytokines such as vascular endothelial growth factor (VEGF), IL-10, prostaglandin E-2 (PGE2), and transforming growth factor (TGF)-β can profoundly affect the nature of DCs (38, 39). Indeed, we have previously shown that DCs that were co-opted by the mouse tumors upon injection, acquired angiogenic properties (10). As we have recently reported, the particular characteristics of the extracellular matrix components can also shape the immune properties of these cells (40). Importantly, tumor factors usually exert a systemic effect as previously described (41, 42). For example, it has been demonstrated that VEGF induces a potent systemic effect on both primary and secondary immune organs (41). Therefore, DCs at lymphoid organs can be influenced by tumor factors and/or immunosuppressive leukocytes that can affect their properties (43).
Cancer-associated DCs can also contribute to tumor development by producing factors that promote angiogenesis (44). In the mouse model, we have recently shown that myeloid DCs are able to produce an array of angiogenic molecules in vitro, including matrix metalloproteases, VEGF, angiogenin, heparanase, and basic fibroblast growth factors among others (40). We have also previously shown that DC precursors participate in tumor progression and angiogenesis in a mouse model of ovarian cancer (10). Moreover, depletion of cancer-associated DCs in vivo was found to reduce tumor growth and decrease angiogenesis in a mouse model of ovarian cancer (45, 46). Not surprisingly, in the same way DCs contributed to angiogenesis in the Lewis lung carcinoma model (47). In humans, cancer-associated DCs have also been shown to produce angiogenic factors and promote neovascularization in the TME (11, 35, 48).
Collectively, these studies provide ample evidence in support of tumors’ capability to reprogram the biology of DCs, inducing them to exert immunosuppressive or angiogenic effects, favoring tumor growth and survival.
Reprograming Cancer-Associated DC to Induce Anti-Tumor Immunity
The “immune paralysis” of cancer-associated DCs can be overcome in an experimental setting by blocking IL-10R while simultaneously activating specific pattern recognition receptors (PRRs). Upon treatment, the cells regain their ability to activate antigen-specific T cells (10, 49, 50). Considering that cancer-associated DCs can harbor tumor antigen, a compelling strategy would be to reprogram them in vivo. Thus, these cells will be transformed into effective antigen-presenting cells capable of promoting anti-tumor immunity and combating tumor growth.
In the mouse model, targeted delivery of antigens to DCs via specific molecules expressed on the DC surface has been investigated. For example, antibodies specific to these surface molecules have been fused with antigens in order to induce an immune response mediated by specific DC populations. Targeting ovalbumin to CD205 and 33D1 molecules on the surface of DCs in vivo helped to markedly enhance and qualitatively direct the antigen-presenting properties of CD8+ and CD8− DC subpopulations of splenic DCs. This difference in antigen processing is suggested to be intrinsic to the DC subsets in association with increased expression of proteins involved in MHC processing (51). Likewise, immunization strategies have been designed using antibody–tumor antigen fusion proteins targeting DCs via CD205 (52) or CD11c (53). In addition, antibodies specific to DC surface molecules have been used to coat liposomes or nanoparticles to deliver antigens and inflammatory compounds to DCs in situ in a mouse model (54) or to target human DCs (55). Other strategies involve the design of antigen-carrying lentiviral vectors capable of selectively binding to DCs (56).
Evidence that phenotype of cancer-associated DCs can be altered in vivo is found in human clinical trials. Anti-tumor therapies using anti-VEFG antibodies, alone or in combination with other drugs, have been evaluated in preclinical and clinical studies (57–60). Interestingly, tumor patients treated with anti-VEGF antibody showed decreased levels of immunosuppressive DCs (61). Similarly, it has been demonstrated that the endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces DC maturation (62). On the other hand, further highlighting the complexity of DC modulation by the TME, cancer patients treated with VEGF-trap [a fusion protein of extracellular domains of VEGF receptor(R)-1 and -2, which can capture all VEGF isoforms] did not show a significant improvement in their immune response, despite a significant increase in the proportion of activated DCs (63). Thus, therapies directly focused on targeting DC in vivo must be designed to enhance this effect.
Pioneering research has been performed by the Conejo-Garcia group aimed at reprograming cancer-associated DCs in order to generate a vaccine in situ (64). For these studies, a mouse model of ovarian cancer was used. Ovarian cancer characteristically exhibits metastasis within the peritoneal cavity, and is thus an excellent target for localized immunotherapies (65). In a mouse model of ovarian cancer ascites, the group showed that intraperitoneal co-delivery of TLR3 ligands and CD40-activating antibodies induced up-regulation of co-stimulatory molecules in cancer-associated DCs together with increased antigen presentation and anti-tumor T cell response (66). A more focused strategy involved directly targeting cancer-associated DCs with nanoparticles carrying pre-miRNA oligonucleotides that were able to reprogram these immunosuppressive cells into promoters of anti-tumor immune response by increasing miR-155 activity in the targeted cells (67). In addition, similar results were obtained when cancer-associated DCs were targeted by linear polyethylenimine nanoparticles encapsulating non-viral siRNA. These particles were avidly engulfed by the cells, activating them through TLR5 and inducing a potent anti-tumor immune response (64). Lastly, an alternative procedure to activate cancer-associated DCs in situ was recently reported. As described by Baird et al. (68), intratumoral administration of an avirulent strain of Toxoplasma gondii in a model of ovarian cancer specifically infected cancer-associated DCs (68). These cells reversed their immunosuppressive status and were able to activate a robust anti-tumor T cell response. Finally, future studies will also need to focus on enhancing the migratory capability of reprogramed DCs toward lymph nodes in order to generate a robust T cell response.
Dendritic cells comprise a population of leukocytes with the capability of activating specific immune responses to promote immunity or induce tolerance. They capture, process, and present antigens thereby activating T cells that carry cognate receptors for these presented antigens. Consequently, DCs serve vital function in initiating adaptive immunity and orchestrating the immune response outcome. The TME can exert undesirable effects on DCs by either rendering them unable to promote specific immune responses, or transforming them into suppressive cells capable of inducing regulatory T cells collectively creating significant obstacles and challenges in cancer immunotherapy. However, ample evidence supports the feasibility to overcome the immune paralysis of cancer-associated DCs. Herein, we summarized our perspective overview of cancer-associated DCs reprograming in situ to generate anti-tumor immune responses that will orchestrate a desirable outcome by halting tumor growth and survival. Knowledge of TME, DC biology, and DC response to specific signals will promote the discovery of new strategies for the reprograming of cancer-associated DCs. The fact that cancer-associated DCs harbor tumor antigens also opens up the tantalizing possibility of reprograming these cells in vivo, thus inducing a de facto patient personalized vaccine. Using innovative approaches to target DCs is vital, and these types of studies will be important in revealing the most effective strategies to overcome setbacks that troubled the field for so long, subsequently helping advance anti-tumor immunotherapy.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported in part by the NIH under Grant R15 CA137499-01 (Fabian Benencia), the RSAC grant (RP1206) from the Heritage College of Osteopathic Medicine, OU (Fabian Benencia) and a SEA grant from Ohio University (Maria Muccioli). Maria Muccioli was supported by the MCB program (OU).
5. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med (2003) 348:203–13. doi:10.1056/NEJMoa020177
7. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science (2006) 313:1960–4. doi:10.1126/science.1129139
10. Conejo-Garcia JR, Benencia F, Courreges MC, Kang E, Mohamed-Hadley A, Buckanovich RJ, et al. Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A. Nat Med (2004) 10:950–8. doi:10.1038/nm1097
11. Curiel TJ, Cheng P, Mottram P, Alvarez X, Moons L, Evdemon-Hogan M, et al. Dendritic cell subsets differentially regulate angiogenesis in human ovarian cancer. Cancer Res (2004) 64:5535–8. doi:10.1158/0008-5472.CAN-04-1272
14. Conejo-Garcia JR, Buckanovich RJ, Benencia F, Courreges MC, Rubin SC, Carroll RG, et al. Vascular leukocytes contribute to tumor vascularization. Blood (2005) 105:679–81. doi:10.1182/blood-2004-05-1906
16. Luo JL, Maeda S, Hsu LC, Yagita H, Karin M. Inhibition of NF-kappaB in cancer cells converts inflammation-induced tumor growth mediated by TNFalpha to TRAIL-mediated tumor regression. Cancer Cell (2004) 6:297–305. doi:10.1016/j.ccr.2004.08.012
17. Yang H, Bocchetta M, Kroczynska B, Elmishad AG, Chen Y, Liu Z, et al. TNF-alpha inhibits asbestos-induced cytotoxicity via a NF-kappaB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis. Proc Natl Acad Sci U S A (2006) 103:10397–402. doi:10.1073/pnas.0604008103
29. Morelli AE, Zahorchak AF, Larregina AT, Colvin BL, Logar AJ, Takayama T, et al. Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation. Blood (2001) 98:1512–23. doi:10.1182/blood.V98.5.1512
32. Baleeiro RB, Anselmo LB, Soares FA, Pinto CA, Ramos O, Gross JL, et al. High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer. Cancer Immunol Immunother (2008) 57:1335–45. doi:10.1007/s00262-008-0468-7
33. Shurin MR, Shurin GV, Lokshin A, Yurkovetsky ZR, Gutkin DW, Chatta G, et al. Intratumoral cytokines/chemokines/growth factors and tumor infiltrating dendritic cells: friends or enemies? Cancer Metastasis Rev (2006) 25:333–56. doi:10.1007/s10555-006-9010-6
35. Mantovani A, Sozzani S, Locati M, Schioppa T, Saccani A, Allavena P, et al. Infiltration of tumours by macrophages and dendritic cells: tumour-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Novartis Found Symp (2004) 256:137–45; discussion 146–138, 259–169. doi:10.1002/0470856734.ch10
36. Benencia F, Courreges MC, Fraser NW, Coukos G. Herpes virus oncolytic therapy reverses tumor immune dysfunction and facilitates tumor antigen presentation. Cancer Biol Ther (2008) 7:1194–205. doi:10.4161/cbt.7.8.6216
37. Gabrilovich DI, Ishida T, Nadaf S, Ohm JE, Carbone DP. Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function. Clin Cancer Res (1999) 5:2963–70.
40. Sprague L, Muccioli M, Pate M, Meles E, McGinty J, Nandigam H, et al. The interplay between surfaces and soluble factors define the immunologic and angiogenic properties of myeloid dendritic cells. BMC Immunol (2011) 12:35. doi:10.1186/1471-2172-12-35
41. Gabrilovich DI, Chen HL, Girgis KR, Cunningham HT, Meny GM, Nadaf S, et al. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med (1996) 2:1096–103. doi:10.1038/nm1096-1096
42. McAllister SS, Gifford AM, Greiner AL, Kelleher SP, Saelzler MP, Ince TA, et al. Systemic endocrine instigation of indolent tumor growth requires osteopontin. Cell (2008) 133:994–1005. doi:10.1016/j.cell.2008.04.045
43. Sharma MD, Baban B, Chandler P, Hou DY, Singh N, Yagita H, et al. Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase. J Clin Invest (2007) 117:2570–82. doi:10.1172/JCI31911
45. Bak SP, Walters JJ, Takeya M, Conejo-Garcia JR, Berwin BL. Scavenger receptor-A-targeted leukocyte depletion inhibits peritoneal ovarian tumor progression. Cancer Res (2007) 67:4783–9. doi:10.1158/0008-5472.CAN-06-4410
46. Huarte E, Cubillos-Ruiz JR, Nesbeth YC, Scarlett UK, Martinez DG, Buckanovich RJ, et al. Depletion of dendritic cells delays ovarian cancer progression by boosting antitumor immunity. Cancer Res (2008) 68:7684–91. doi:10.1158/0008-5472.CAN-08-1167
47. Fainaru O, Adini A, Benny O, Adini I, Short S, Bazinet L, et al. Dendritic cells support angiogenesis and promote lesion growth in a murine model of endometriosis. FASEB J (2008) 22:522–9. doi:10.1096/fj.07-9034com
49. Vicari AP, Chiodoni C, Vaure C, Ait-Yahia S, Dercamp C, Matsos F, et al. Reversal of tumor-induced dendritic cell paralysis by CpG immunostimulatory oligonucleotide and anti-interleukin 10 receptor antibody. J Exp Med (2002) 196:541–9. doi:10.1084/jem.20020732
50. Osterbur J, Sprague L, Muccioli M, Pate M, Mansfield K, McGinty J, et al. Adhesion to substrates induces dendritic cell endothelization and decreases immunological response. Immunobiology (2013) 218:64–75. doi:10.1016/j.imbio.2012.02.003
51. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, Yamazaki S, et al. Differential antigen processing by dendritic cell subsets in vivo. Science (2007) 315:107–11. doi:10.1126/science.1136080
52. Wang B, Kuroiwa JM, He LZ, Charalambous A, Keler T, Steinman RM. The human cancer antigen mesothelin is more efficiently presented to the mouse immune system when targeted to the DEC-205/CD205 receptor on dendritic cells. Ann N Y Acad Sci (2009) 1174:6–17. doi:10.1111/j.1749-6632.2009.04933.x
53. Wei H, Wang S, Zhang D, Hou S, Qian W, Li B, et al. Targeted delivery of tumor antigens to activated dendritic cells via CD11c molecules induces potent antitumor immunity in mice. Clin Cancer Res (2009) 15:4612–21. doi:10.1158/1078-0432.CCR-08-3321
54. Faham A, Altin JG. Antigen-containing liposomes engrafted with flagellin-related peptides are effective vaccines that can induce potent antitumor immunity and immunotherapeutic effect. J Immunol (2010) 185:1744–54. doi:10.4049/jimmunol.1000027
55. Cruz LJ, Tacken PJ, Fokkink R, Joosten B, Stuart MC, Albericio F, et al. Targeted PLGA nano but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro. J Control Release (2010) 144:118–26. doi:10.1016/j.jconrel.2010.02.013
56. Hu B, Dai B, Wang P. Vaccines delivered by integration-deficient lentiviral vectors targeting dendritic cells induces strong antigen-specific immunity. Vaccine (2010) 28:6675–83. doi:10.1016/j.vaccine.2010.08.012
61. Osada T, Chong G, Tansik R, Hong T, Spector N, Kumar R, et al. The effect of anti-VEGF therapy on immature myeloid cell and dendritic cells in cancer patients. Cancer Immunol Immunother (2008) 57:1115–24. doi:10.1007/s00262-007-0441-x
62. Tian F, Grimaldo S, Fujita M, Cutts J, Vujanovic NL, Li LY. The endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces dendritic cell maturation. J Immunol (2007) 179:3742–51.
63. Fricke I, Mirza N, Dupont J, Lockhart C, Jackson A, Lee JH, et al. Vascular endothelial growth factor-trap overcomes defects in dendritic cell differentiation but does not improve antigen-specific immune responses. Clin Cancer Res (2007) 13:4840–8. doi:10.1158/1078-0432.CCR-07-0409
64. Cubillos-Ruiz JR, Engle X, Scarlett UK, Martinez D, Barber A, Elgueta R, et al. Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity. J Clin Invest (2009) 119:2231–44. doi:10.1172/JCI37716
66. Scarlett UK, Cubillos-Ruiz JR, Nesbeth YC, Martinez DG, Engle X, Gewirtz AT, et al. In situ stimulation of CD40 and Toll-like receptor 3 transforms ovarian cancer-infiltrating dendritic cells from immunosuppressive to immunostimulatory cells. Cancer Res (2009) 69:7329–37. doi:10.1158/0008-5472.CAN-09-0835
67. Cubillos-Ruiz JR, Baird JR, Tesone AJ, Rutkowski MR, Scarlett UK, Camposeco-Jacobs AL, et al. Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer. Cancer Res (2012) 72:1683–93. doi:10.1158/0008-5472.CAN-11-3160
68. Baird JR, Fox BA, Sanders KL, Lizotte PH, Cubillos-Ruiz JR, Scarlett UK, et al. Avirulent Toxoplasma gondii generates therapeutic antitumor immunity by reversing immunosuppression in the ovarian cancer microenvironment. Cancer Res (2013) 73:3842–51. doi:10.1158/0008-5472.CAN-12-1974
Keywords: tumor microenvironment, dendritic cells, vaccines, angiogenesis, targeted delivery
Citation: Benencia F, Muccioli M and Alnaeeli M (2014) Perspectives on reprograming cancer-associated dendritic cells for anti-tumor therapies. Front. Oncol. 4:72. doi: 10.3389/fonc.2014.00072
Received: 10 January 2014; Paper pending published: 26 January 2014;
Accepted: 21 March 2014; Published online: 07 April 2014.
Edited by:Jozsef Dudas, Innsbruck Medical University, Austria
Reviewed by:Daniel Benitez-Ribas, CIBERehd, Spain
Nikolaus Romani, Innsbruck Medical University, Austria
Copyright: © 2014 Benencia, Muccioli and Alnaeeli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Fabian Benencia, Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, ARC 202c, Athens, OH 45701, USA e-mail: email@example.com