Biotechnol Lett DOI 10.1007/s10529-014-1746-4
ORIGINAL RESEARCH PAPER
CXCL10/XCL1 fusokine elicits in vitro and in vivo chemotaxis Yessica E. Sanchez-Lugo • Jose J. Perez-Trujillo • Yolanda Gutierrez-Puente • Aracely Garcia-Garcia • Humberto Rodriguez-Rocha • Oralia Barboza-Quintana • Gerardo E. Mun˜oz-Maldonado • Odila Saucedo-Cardenas • Roberto Montes de Oca-Luna Maria J. Loera-Arias
•
Received: 13 August 2014 / Accepted: 10 December 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Fusokines are proteins formed by the fusion of two cytokines. They have greater bioavailability and therapeutic potential than individual cytokines or a combination of different cytokines. Interferon-gamma-inducible protein 10 (CXCL10) and lymphotactin (XCL1) are members of the chemotactic family of cytokines, which induce tumor regression by eliciting immune-system cell chemotaxis. We engineered a replication-deficient adenoviral system expressing CXCL10/XCL1 fusokine (Ad FIL) and assessed its chemotactic response in vitro and in vivo. The CXCL10/XCL1 fusokine elicited a greater chemotactic effect in IL-2 stimulated lymphocytes than individual or combined cytokines in vitro.
CXCL10/XCL1 fusokine biological activity was demonstrated in vivo by intratumoral chemoattraction of CXCR3? cells. Thus, this novel CXCL10/XCL1 fusokine may represent a potential tool for gene therapy treatment of cancer and other illnesses that require triggering immune-system cell recruitment.
Y. E. Sanchez-Lugo J. J. Perez-Trujillo A. Garcia-Garcia H. Rodriguez-Rocha O. Saucedo-Cardenas R. M. de Oca-Luna M. J. Loera-Arias (&) Departamento de Histologia, Facultad de Medicina, Universidad Autonoma de Nuevo Leon (UANL), Madero y Aguirre Pequen˜o s/n Mitras Centro, CP: 64665 Monterrey, NL, Mexico e-mail: [email protected]
R. M. de Oca-Luna e-mail: [email protected]
J. J. Perez-Trujillo e-mail: [email protected] A. Garcia-Garcia e-mail: [email protected] H. Rodriguez-Rocha e-mail: [email protected]
Keywords Adenovirus Cancer therapy CXCL10 Fusokine Gene therapy Immunotherapy Interferon-gamma-inducible protein 10 Lymphotactin
Y. Gutierrez-Puente Departamento de Bioquimica, Facultad de Ciencias Biologicas, Universidad Autonoma de Nuevo Leon (UANL), CP: 66450 Monterrey, NL, Mexico e-mail: [email protected] O. Barboza-Quintana Servicio de Anatomia Patologica y Citopatologia, Hospital Universitario ‘‘Dr. Jose Eleuterio Gonzalez’’ de la Facultad de Medicina, Universidad Autonoma de Nuevo Leon (UANL), CP: 64665 Monterrey, NL, Mexico e-mail: [email protected]
O. Saucedo-Cardenas e-mail: [email protected]
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Introduction Cytokines are intercellular messengers that are secreted by several cell types. Chemokines belong to a cytokine subfamily that is primarily responsible for chemoattraction (chemotaxis) of immune effector cells (Proudfoot 2002) and its function is recognized as crucial for cancer progression (Raman et al. 2007). Interferon-gamma-inducible protein 10 (IP-10 or CXCL10) is a small chemokine peptide (10 kDa) that binds to the CXCR3 receptor and induces chemoattraction of lymphocytes (CD4? and CD8?), macrophages, NK cells, and dendritic cells (Dufour et al. 2002). CXCL10 can generate antitumor activity through the chemoattraction of tumor-specific T cells into the local tissue, and through the functional activation, proliferation and survival of infiltrating T lymphocytes (Yang et al. 2006; Wennerberg et al. 2014). Lymphotactin (XCL1) is a small chemokine peptide (12 kDa) expressed by activated CD8? T cells, CD4? T cells, NK cells, and NKT cells. It binds the XCL1 receptor in CD8? dendritic cells (Lei and Takahama 2012). The therapeutic effect of XCL1 against cancer has been proven, and it can be used in combination with different therapies to induce cancer regression (Russell et al. 2007; Zhang et al. 2011). The synergetic effect of combining adenovirus (Ad) CXCL10 and Ad XCL1 on tumor progression was demonstrated previously in mice with wellestablished tumor masses(Huang and Xiang 2004). Considering the strongly positive effect of this combined therapy, these two chemokines are good candidates to study the effects of their combined activity as a fusion protein. Fusion proteins of different cytokines are known as fusokines. Previous studies report that fusokines may synergistically enhance the therapeutic potential of individual proteins (Williams and Galipeau 2011). In
this study, a CXCL10 and XCL1 fusokine (FIL) recombinant adenovirus was developed, and its biological activity was investigated in vitro and in vivo.
Materials and methods Cell lines, animals, and antibodies The cell lines HEK293, HeLa, and TC-1 were obtained from ATCC. HEK293 and HeLa were kept in medium Advanced DMEM supplemented with 4 % (v/v) fetal bovine serum, L-glutamine and antibiotics. TC-1 is an epithelial tumoral cell line derived from C57BL6 mice strain co-transformed by HPV-16 E6 and E7 and the oncogene c-Ha-ras. These were maintained in RPMI 1640 medium supplemented with 10 % fetal bovine serum, sodium pyruvate and antibiotics. All cells were maintained at 37 °C in a 5 % CO2 atmosphere. Female C57BL6 mice (6–8 weeks old) were purchased from Harlan, Me´xico, housed with 12 h of light and dark cycles and ad libitum access to food and water. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The protocol was approved by the Ethics Committee of the School of Medicine, Universidad Autonoma de Nuevo Leon (Protocol HT13-001). Antibodies for goat anti-human CXCL10, goat antihuman XCL1, HRP anti-goat IgG, and recombinant IL2 were purchased from R&D Systems, Inc.Adenovirus hexon protein monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. The monoclonal antibody anti-CXCR3 was purchased from United States Biological. The monoclonal antibody anti-mouse CD16/CD32 was purchased from BD Pharmingen. Adenoviral engineering
G. E. Mun˜oz-Maldonado Servicio de Cirugia General, Hospital Universitario ‘‘Dr. Jose Eleuterio Gonza´lez’’ de la Facultad de Medicina, Universidad Autonoma de Nuevo Leon (UANL), CP: 64665 Monterrey, NL, Mexico e-mail: [email protected] O. Saucedo-Cardenas Division de Gene´tica, Centro de Investigacion Biomedica del Noreste, Instituto Mexicano del Seguro Social (IMSS), CP: 64665 Monterrey, NL, Mexico
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The fusokine construct and the individual chemokines (to be used as controls) were cloned and optimized for expression in mammalian cells by GeneArt. Sequences for these constructs were deposited in GenBank under accession numbers KM222438, KM222439, and KM222440, for fusokine FIL, CXCL10 and XCL1, respectively. A three-dimensional (3D) model of FIL was generated by Raptor X
Biotechnol Lett
simulation (Ka¨llberg et al. 2012). Recombinant adenoviral constructs Ad FIL, Ad CXCL10, Ad XCL1, and Ad Empty were generated in our laboratory using the AdEasy Adenoviral Vector System (Agilent Technologies, Inc.) according to the manufacturer instructions. The ViraBind Adenovirus Purification Kit and the QuickTiter Adenovirus Quantitation Kit (Cell Biolabs, Inc.) were used for adenovirus purification and quantification. Western blot The recombinant proteins were expressed by infecting 3 9 105 HeLa cells with 1 9 106 adenovirus viral particles (VP). At 24 h post-infection, 10 ll of supernatant mixed with 29 loading buffer were loaded onto 15 % acrylamide gels. Proteins were transferred to PVDF membranes (Thermo Fisher Scientific, Inc.), blocked, incubated overnight with anti-CXCL10 and anti-XCL1 primary antibodies (0.05 lg/ml), and then incubated for 2 h with the secondary antibody HRPanti-goat IgG (0.005 lg/ml). The SuperSignal West Dura kit (Thermo Fisher Scientific, Inc.) was used to detect immunoreactive protein bands. In vitro chemotaxis assay To demonstrate that the recombinant proteins secreted by cells infected with the engineered adenoviruses are biologically active in vitro, we performed a chemotaxis assay. Human lymphocytes were isolated by FicollPaque Plus (GE Healthcare Life Sciences) and cultured for 21 days (37 °C, 5 % CO2) in the presence of 10U interleukin 2 (IL-2). 3 9 105 HeLa cells were seeded in 6 well plates and infected with the adenovirus previously constructed (MOI 3). Supernatants were collected 48 hafter infection, stored at -80 °C, and used as chemoattractants. Chemoattractants (300 ll) were placed in the bottom well of a ChemoTx cell migration system (Neuroprobe, Inc.) and 5 9 104 IL2stimulated lymphocytes were seeded above the membrane. The chamber was incubated for 3 h at 37 °C with 5 % CO2. Zymozan-activated fetal bovine serum was used as positive control. A control with 5 9 104 IL2-stimulated lymphocytes were seeded in the bottom of three wells to be used as the 100 % reference value. Migrating cells were quantified by adding 10 ll of WST-1 (Roche) to the bottom of the wells. The reaction was incubated for 20 h (37 °C, 5 % CO2), and
sample absorbance was measured at 450 nm using a microplate reader. Results were plotted as the % of chemoattracted cells. This experiment was independently repeated twice with similar results. In vivo chemotaxis assay Twenty female C57BL/6 mice were inoculated subcutaneously in the left flank with 104 TC-1 cells. When tumors reached 6–7 mm diam. The mice were vaccinated with the plasmid pSh:CRT/E7 (Calreticulina/ E7) to induce lymphocyte activation. The plasmid was introduced via particle bombardment into the shaved abdominal region using a Bio-Rad Helios gene gun (1 lg gold particles, 1 lg DNA, and 300 psi). The plasmid pSh:CRT/E7 was a gift from Dr. GomezGutierrez at University of Louisville, KY (GomezGutierrez et al. 2007). Seven days later, the mice were randomly assigned to five groups (four mice per group), and 106 VP of each adenoviral construct was administrated by intratumoral injection. Mice were sacrificed by sodium pentobarbital overdose 48 h after adenoviral administration, and the tumors were collected and fixed in 4 % (v/v) paraformaldehyde. Paraffin-embedded tumor sections were incubated with anti-CXCR3, anti-CD16/CD32, and anti-hexon antibodies at 1 lg/ml in PBS and incubated overnight. Finally, slides were incubated with the Vectastain ABC System and the DAB Peroxidase Substrate Kit (Vector Laboratories) to label immunoreactive moieties, and 15 regions were analyzed for each slide. A qualitative criteria from 0 to 4 was used to measure the amount of positive cells expressing the different markers in the slides: 0 no positive cells; 1 rare; 2 few; 3 moderate; and 4 many positive cells. The score was normalized with hexon expression. Statistical analysis ANOVA was performed using Prism (GraphPad Software). P values \ 0.05 were considered significant.
Results Design and characterization of the fusokine FIL A fusokine fusion construct was generated using an open reading frame for both cytokines. To create this
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Fig. 1 Design and 3D protein simulation of fusokine FIL. a Expression cassettes for fusokine FIL, CXCL10, and XCL1. b 3D simulation of fusokine FIL using the RaptorX web server.
SP, signal peptide; DUAG, deletion of stop codon; DSP, deletion of signal peptide; Linker, (G4S)3
fusokine fusion, the CXCL10 stop codon was deleted, a flexible peptide [(G4S)3] sequence was added, and the mature XCL1 coding sequence was added in-frame. This new fusokine fusion encoding CXCL10-(G4S)3XCL1 was designated as FIL (Fig. 1a). We performed a 3D simulation of the fusokine FIL using the RaptorX web server. This model indicated that the chimeric protein conformation consisted of two domains, with each domain preserving the respective chemokine structure (Fig. 1b). This structural preservation is important for the biological activity of the fusokine.
Cells infected with Ad CXCL10, Ad XCL1, or fusokine FIL secrete recombinant chemokines To assess chemokine expression and secretion, HeLa cells were infected with the indicated adenovirus. At 24 h post-infection, medium was collected and subjected to immunoblotting with anti-CXCL10 (Fig. 2a) and anti-XCL1(Fig. 2b). The theoretical weights calculated for recombinant proteins Ad FIL, AdCXCL10, and Ad XCL1 were 22, 10, and 12 kDa, respectively. Western blots confirmed that all proteins were detected at the calculated molecular masses. Supernatants of cells infected with the Ad Empty vector showed no expression. These results indicate that the engineered adenoviral vectors are competent to infect cells and are able to secrete the recombinant proteins. Fusokine FIL elicits in vitro chemotactic activity
Fig. 2 Adenovirus-directed expression of fusokine FIL. Western blot analysis of supernatants from HeLa cells infected with different recombinant adenovirus (MOI 3). Blots were probed using anti-CXCL10 (a) or anti-XCL1 (b)
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To determine the biological activity of fusokine FIL, we performed a chemotaxis assay. Conditioned medium from HeLa cells infected with the adenovirus was used as chemoattractant for IL-2 stimulated lymphocytes. The Ad Empty control induced chemotaxis in 20 % of cells, whereas the individual or combined chemokines (Ad CXCL10, Ad XCL1, and Ad CXCL10? Ad XCL1) induced chemotaxis in30–40 % of cells. By contrast, the fusokine FIL
Biotechnol Lett
with Ad FIL (Fig. 4b). These results demonstrate that the fusokine recombinant adenovirus elicit an intratumoral chemoattraction in vivo.
Discussion
Fig. 3 In vitro chemotactic activity of fusokine FIL. Lymphocytes obtained from human peripheral blood were stimulated with human IL-2 for 21 days. Chemotaxis was assayed using supernatants of HeLa cells infected with the adenoviral constructs (assays were performed in 96-well microchambers); cells in the bottom wells were counted and plotted as the % of migratory cells. ***P values \ 0.05 were considered significant
induced chemotaxis in 60 % of cells. This result indicates that fusion of the chemokines CXCL10 and XCL1 enhanced their chemoattractant activity compared with that of the individual or combined chemokines (Fig. 3). Similar results were obtained using murine splenocytes (data not shown). Fusokine FIL elicits in vivo chemotactic activity We assessed the in vivo chemotaxis induced by fusokine FIL in mice bearing tumors induced by TC-1 cells.T lymphocytes were activated by DNA vaccination using the CRT/E7 antigen two weeks after the recombinant adenovirus was injected intratumorally. Immunization with a DNA vaccine, which encodes this fusion protein, activates E7-specific T lymphocytes (Cheng et al. 2001). Immunostaining for hexon protein on paraffin sections of tumors was positive for all adenovirus treatments, as expected (Fig. 4a). Next, we assessed whether the adenoviruses elicited chemotaxis by immunostaining for the CXCL10 (antiCXCR3) and FcR (anti-CD16/32) receptors. These receptors are present inT, B, monocyte/macrophage, NK, dendritic, neutrophil, eosinophil, mast, and basophil cells. A cell migration response to the adenoviral constructs was observed (but not the empty adenovirus), which was most pronounced for tumors injected
A key event for successful immunotherapy is the generation of antigen-specific T cells that infiltrate into the tumor to destroy malignant cells. The presence of tumor infiltrating lymphocytes (TILs) has been associated with a better outcome in patients with HPV16-associated oropharynx cancer (Wansom et al. 2012) and other cancer types (Gooden et al. 2011). Thus, the generation of new therapeutic tools that enhance the presence of tumor-localized TILs may improve the efficacy of immunotherapy in patients with tumors (Gattinoni et al. 2006). Chemokines are small cytokines that guide the migration of responsive cells. CXCL10 and XCL1 are both chemokines with antitumor activity (Cairns et al. 2001; Wennerberg et al. 2014). The effect of these chemokines is reported to be synergistically enhanced when used in combination (Huang and Xiang 2004; Zhang et al. 2011). CXCL10 has been used in fusion protein constructs with other molecules. A chimeric construct, engineered by substituting the N-terminal and N-loop region of CXCL10 with those of CXCL11,is reported to induce stronger antitumor effects than individual or combined parental chemokines by eliciting CXCR3? chemotaxis and antiangiogenesis (Wang et al. 2010). Another fusion protein composed of CXCL10 and mutant EGFR in combination with activated CD8? lymphocytes synergistically inhibited glioma growth and induced an increased number of CXCR3?CD8? T cells in a mouse glioma model (Wang et al. 2013). In this study, we report the fusion of CXCL10 and XCL1 as a fusokine for the first time, and demonstrate its biological activity by performing in vitro chemotaxis assays and in vivo chemotaxis of CXCR3? cells. The advantage of this construction over previous designs is the inclusion of chemokine CXCL10, which chemoattracts activated TCD8? lymphocytes induces the functional activation, proliferation, and survival of infiltrating T lymphocytes, and has antiangiogenic effects (Yang et al. 2006). Our strategy also utilizes chemokine XCL1, which chemoattracts XCR1-expressing dendritic cells (DCs) and may facilitate antigen
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Fig. 4 In vivo chemotactic activity of fusokine FIL. Female C57BL/6 mice were inoculated subcutaneously in the left flank with 1 9 104 TC-1 cells. When the tumors reached 100 mm3, mice were biolistically vaccinated with the plasmid CRT/E7 (1 lg gold particles, 1 lg DNA, and 300 psi). Seven days after vaccination, 1 9 106 viral particles from each adenovirus were
administrated by an intratumoral-injection. Mice were sacrificed 48 h after infection. Tumor sections were incubated with antihexon, anti-CXCR3and anti-CD16/CD32 antibodies. a Representative field of each treatment. Magnification is 209. b The relative amount of positive cells in each group was scored as rare = 1, few = 2, moderate = 3, and many = 4. Mean ± SE
presentation of DCs to CD8? T cells. These results provide an experimental foundation that supports further studies of fusokines as cancer immunotherapies.
calreticulin-human papillomavirus 16 E7 fusion protein eradicates E7 expressing established tumors in mice. Cancer Immunol Immunother CII 56:997–1007 Gooden MJM, de Bock GH, Leffers N et al (2011) The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis. Br J Cancer 105:93–103 Huang H, Xiang J (2004) Synergistic effect of lymphotactin and interferon gamma-inducible protein-10 transgene expression in T-cell localization and adoptive T-cell therapy of tumors. Int J Cancer J Int Cancer 109:817–825 Ka¨llberg M, Wang H, Wang S et al (2012) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7:1511–1522 Lei Y, Takahama Y (2012) XCL1 and XCR1 in the immune system. Microbes Infect Inst Pasteur 14:262–267 Proudfoot AEI (2002) Chemokine receptors: multifaceted therapeutic targets. Nat Rev Immunol 2:106–115 Raman D, Baugher PJ, Thu YM, Richmond A (2007) Role of chemokines in tumor growth. Cancer Lett 256:137–165 Russell HV, Strother D, Mei Z et al (2007) Phase I trial of vaccination with autologous neuroblastoma tumor cells genetically modified to secrete IL-2 and lymphotactin. J Immunother Hagerstown Md 1997 30:227–233 Wang P, Yang X, Xu W et al (2010) Integrating individual functional moieties of CXCL10 and CXCL11 into a novel chimeric chemokine leads to synergistic antitumor effects: a strategy for chemokine-based multi-target-directed cancer therapy. Cancer Immunol Immunother CII 59:1715–1726 Wang X, Lu X-L, Zhao H-Y et al (2013) A novel recombinant protein of IP10-EGFRvIIIscFv and CD8(?) cytotoxic T lymphocytes synergistically inhibits the growth of implanted glioma in mice. Cancer Immunol Immunother CII 62:1261–1272 Wansom D, Light E, Thomas D et al (2012) Infiltrating lymphocytes and human papillomavirus-16 associated oropharynx cancer. Laryngoscope 122:121–127
Acknowledgments This work was supported by Programa de Apoyo a la Investigacio´n en Ciencia y Tecnologı´a (PAICYT) No. SA381-10 from the UANL, and by ConsejoNacional de Ciencia y Tecnologı´a (CONACYT) Grant No. CB-10-158509. YESL and JJPT were recipients of scholarships from CONACYT. Conflict of interest conflict of interest.
The authors declare that they have no
References Cairns CM, Gordon JR, Li F et al (2001) Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4? and CD8? T cells and neutrophils expressing XCR1 receptor. J Immunol Baltim Md 1950 167:57–65 Cheng W-F, Hung C-F, Chai C-Y et al (2001) Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen. J Clin Invest 108:669–678 Dufour JH, Dziejman M, Liu MT et al (2002) IFN-c-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 168:3195–3204. doi:10.4049/jimmunol.168.7. 3195 Gattinoni L, Powell DJ, Rosenberg SA, Restifo NP (2006) Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol 6:383–393 Gomez-Gutierrez JG, Elpek KG, de Oca-Luna RM et al (2007) Vaccination with an adenoviral vector expressing
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Biotechnol Lett Wennerberg E, Kremer V, Childs R, Lundqvist A (2014) CXCL10-induced migration of adoptively transferred human natural killer cells toward solid tumors causes regression of tumor growth in vivo. Cancer Immunol Immunother CII. doi:10.1007/s00262-014-1629-5 Williams P, Galipeau J (2011) GM-CSF–based fusion Cytokines as ligands for immune modulation. J Immunol 186:5527–5532
Yang X, Chu Y, Wang Y et al (2006) Targeted in vivo expression of IFN-gamma-inducible protein 10 induces specific antitumor activity. J Leukoc Biol 80:1434–1444 Zhang J, Zhou Z, Wang C et al (2011) Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism. Cancer Immunol Immunother CII 60:559–573
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ORIGINAL RESEARCH PAPER
CXCL10/XCL1 fusokine elicits in vitro and in vivo chemotaxis Yessica E. Sanchez-Lugo • Jose J. Perez-Trujillo • Yolanda Gutierrez-Puente • Aracely Garcia-Garcia • Humberto Rodriguez-Rocha • Oralia Barboza-Quintana • Gerardo E. Mun˜oz-Maldonado • Odila Saucedo-Cardenas • Roberto Montes de Oca-Luna Maria J. Loera-Arias
•
Received: 13 August 2014 / Accepted: 10 December 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Fusokines are proteins formed by the fusion of two cytokines. They have greater bioavailability and therapeutic potential than individual cytokines or a combination of different cytokines. Interferon-gamma-inducible protein 10 (CXCL10) and lymphotactin (XCL1) are members of the chemotactic family of cytokines, which induce tumor regression by eliciting immune-system cell chemotaxis. We engineered a replication-deficient adenoviral system expressing CXCL10/XCL1 fusokine (Ad FIL) and assessed its chemotactic response in vitro and in vivo. The CXCL10/XCL1 fusokine elicited a greater chemotactic effect in IL-2 stimulated lymphocytes than individual or combined cytokines in vitro.
CXCL10/XCL1 fusokine biological activity was demonstrated in vivo by intratumoral chemoattraction of CXCR3? cells. Thus, this novel CXCL10/XCL1 fusokine may represent a potential tool for gene therapy treatment of cancer and other illnesses that require triggering immune-system cell recruitment.
Y. E. Sanchez-Lugo J. J. Perez-Trujillo A. Garcia-Garcia H. Rodriguez-Rocha O. Saucedo-Cardenas R. M. de Oca-Luna M. J. Loera-Arias (&) Departamento de Histologia, Facultad de Medicina, Universidad Autonoma de Nuevo Leon (UANL), Madero y Aguirre Pequen˜o s/n Mitras Centro, CP: 64665 Monterrey, NL, Mexico e-mail: [email protected]
R. M. de Oca-Luna e-mail: [email protected]
J. J. Perez-Trujillo e-mail: [email protected] A. Garcia-Garcia e-mail: [email protected] H. Rodriguez-Rocha e-mail: [email protected]
Keywords Adenovirus Cancer therapy CXCL10 Fusokine Gene therapy Immunotherapy Interferon-gamma-inducible protein 10 Lymphotactin
Y. Gutierrez-Puente Departamento de Bioquimica, Facultad de Ciencias Biologicas, Universidad Autonoma de Nuevo Leon (UANL), CP: 66450 Monterrey, NL, Mexico e-mail: [email protected] O. Barboza-Quintana Servicio de Anatomia Patologica y Citopatologia, Hospital Universitario ‘‘Dr. Jose Eleuterio Gonzalez’’ de la Facultad de Medicina, Universidad Autonoma de Nuevo Leon (UANL), CP: 64665 Monterrey, NL, Mexico e-mail: [email protected]
O. Saucedo-Cardenas e-mail: [email protected]
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Introduction Cytokines are intercellular messengers that are secreted by several cell types. Chemokines belong to a cytokine subfamily that is primarily responsible for chemoattraction (chemotaxis) of immune effector cells (Proudfoot 2002) and its function is recognized as crucial for cancer progression (Raman et al. 2007). Interferon-gamma-inducible protein 10 (IP-10 or CXCL10) is a small chemokine peptide (10 kDa) that binds to the CXCR3 receptor and induces chemoattraction of lymphocytes (CD4? and CD8?), macrophages, NK cells, and dendritic cells (Dufour et al. 2002). CXCL10 can generate antitumor activity through the chemoattraction of tumor-specific T cells into the local tissue, and through the functional activation, proliferation and survival of infiltrating T lymphocytes (Yang et al. 2006; Wennerberg et al. 2014). Lymphotactin (XCL1) is a small chemokine peptide (12 kDa) expressed by activated CD8? T cells, CD4? T cells, NK cells, and NKT cells. It binds the XCL1 receptor in CD8? dendritic cells (Lei and Takahama 2012). The therapeutic effect of XCL1 against cancer has been proven, and it can be used in combination with different therapies to induce cancer regression (Russell et al. 2007; Zhang et al. 2011). The synergetic effect of combining adenovirus (Ad) CXCL10 and Ad XCL1 on tumor progression was demonstrated previously in mice with wellestablished tumor masses(Huang and Xiang 2004). Considering the strongly positive effect of this combined therapy, these two chemokines are good candidates to study the effects of their combined activity as a fusion protein. Fusion proteins of different cytokines are known as fusokines. Previous studies report that fusokines may synergistically enhance the therapeutic potential of individual proteins (Williams and Galipeau 2011). In
this study, a CXCL10 and XCL1 fusokine (FIL) recombinant adenovirus was developed, and its biological activity was investigated in vitro and in vivo.
Materials and methods Cell lines, animals, and antibodies The cell lines HEK293, HeLa, and TC-1 were obtained from ATCC. HEK293 and HeLa were kept in medium Advanced DMEM supplemented with 4 % (v/v) fetal bovine serum, L-glutamine and antibiotics. TC-1 is an epithelial tumoral cell line derived from C57BL6 mice strain co-transformed by HPV-16 E6 and E7 and the oncogene c-Ha-ras. These were maintained in RPMI 1640 medium supplemented with 10 % fetal bovine serum, sodium pyruvate and antibiotics. All cells were maintained at 37 °C in a 5 % CO2 atmosphere. Female C57BL6 mice (6–8 weeks old) were purchased from Harlan, Me´xico, housed with 12 h of light and dark cycles and ad libitum access to food and water. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The protocol was approved by the Ethics Committee of the School of Medicine, Universidad Autonoma de Nuevo Leon (Protocol HT13-001). Antibodies for goat anti-human CXCL10, goat antihuman XCL1, HRP anti-goat IgG, and recombinant IL2 were purchased from R&D Systems, Inc.Adenovirus hexon protein monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. The monoclonal antibody anti-CXCR3 was purchased from United States Biological. The monoclonal antibody anti-mouse CD16/CD32 was purchased from BD Pharmingen. Adenoviral engineering
G. E. Mun˜oz-Maldonado Servicio de Cirugia General, Hospital Universitario ‘‘Dr. Jose Eleuterio Gonza´lez’’ de la Facultad de Medicina, Universidad Autonoma de Nuevo Leon (UANL), CP: 64665 Monterrey, NL, Mexico e-mail: [email protected] O. Saucedo-Cardenas Division de Gene´tica, Centro de Investigacion Biomedica del Noreste, Instituto Mexicano del Seguro Social (IMSS), CP: 64665 Monterrey, NL, Mexico
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The fusokine construct and the individual chemokines (to be used as controls) were cloned and optimized for expression in mammalian cells by GeneArt. Sequences for these constructs were deposited in GenBank under accession numbers KM222438, KM222439, and KM222440, for fusokine FIL, CXCL10 and XCL1, respectively. A three-dimensional (3D) model of FIL was generated by Raptor X
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simulation (Ka¨llberg et al. 2012). Recombinant adenoviral constructs Ad FIL, Ad CXCL10, Ad XCL1, and Ad Empty were generated in our laboratory using the AdEasy Adenoviral Vector System (Agilent Technologies, Inc.) according to the manufacturer instructions. The ViraBind Adenovirus Purification Kit and the QuickTiter Adenovirus Quantitation Kit (Cell Biolabs, Inc.) were used for adenovirus purification and quantification. Western blot The recombinant proteins were expressed by infecting 3 9 105 HeLa cells with 1 9 106 adenovirus viral particles (VP). At 24 h post-infection, 10 ll of supernatant mixed with 29 loading buffer were loaded onto 15 % acrylamide gels. Proteins were transferred to PVDF membranes (Thermo Fisher Scientific, Inc.), blocked, incubated overnight with anti-CXCL10 and anti-XCL1 primary antibodies (0.05 lg/ml), and then incubated for 2 h with the secondary antibody HRPanti-goat IgG (0.005 lg/ml). The SuperSignal West Dura kit (Thermo Fisher Scientific, Inc.) was used to detect immunoreactive protein bands. In vitro chemotaxis assay To demonstrate that the recombinant proteins secreted by cells infected with the engineered adenoviruses are biologically active in vitro, we performed a chemotaxis assay. Human lymphocytes were isolated by FicollPaque Plus (GE Healthcare Life Sciences) and cultured for 21 days (37 °C, 5 % CO2) in the presence of 10U interleukin 2 (IL-2). 3 9 105 HeLa cells were seeded in 6 well plates and infected with the adenovirus previously constructed (MOI 3). Supernatants were collected 48 hafter infection, stored at -80 °C, and used as chemoattractants. Chemoattractants (300 ll) were placed in the bottom well of a ChemoTx cell migration system (Neuroprobe, Inc.) and 5 9 104 IL2stimulated lymphocytes were seeded above the membrane. The chamber was incubated for 3 h at 37 °C with 5 % CO2. Zymozan-activated fetal bovine serum was used as positive control. A control with 5 9 104 IL2-stimulated lymphocytes were seeded in the bottom of three wells to be used as the 100 % reference value. Migrating cells were quantified by adding 10 ll of WST-1 (Roche) to the bottom of the wells. The reaction was incubated for 20 h (37 °C, 5 % CO2), and
sample absorbance was measured at 450 nm using a microplate reader. Results were plotted as the % of chemoattracted cells. This experiment was independently repeated twice with similar results. In vivo chemotaxis assay Twenty female C57BL/6 mice were inoculated subcutaneously in the left flank with 104 TC-1 cells. When tumors reached 6–7 mm diam. The mice were vaccinated with the plasmid pSh:CRT/E7 (Calreticulina/ E7) to induce lymphocyte activation. The plasmid was introduced via particle bombardment into the shaved abdominal region using a Bio-Rad Helios gene gun (1 lg gold particles, 1 lg DNA, and 300 psi). The plasmid pSh:CRT/E7 was a gift from Dr. GomezGutierrez at University of Louisville, KY (GomezGutierrez et al. 2007). Seven days later, the mice were randomly assigned to five groups (four mice per group), and 106 VP of each adenoviral construct was administrated by intratumoral injection. Mice were sacrificed by sodium pentobarbital overdose 48 h after adenoviral administration, and the tumors were collected and fixed in 4 % (v/v) paraformaldehyde. Paraffin-embedded tumor sections were incubated with anti-CXCR3, anti-CD16/CD32, and anti-hexon antibodies at 1 lg/ml in PBS and incubated overnight. Finally, slides were incubated with the Vectastain ABC System and the DAB Peroxidase Substrate Kit (Vector Laboratories) to label immunoreactive moieties, and 15 regions were analyzed for each slide. A qualitative criteria from 0 to 4 was used to measure the amount of positive cells expressing the different markers in the slides: 0 no positive cells; 1 rare; 2 few; 3 moderate; and 4 many positive cells. The score was normalized with hexon expression. Statistical analysis ANOVA was performed using Prism (GraphPad Software). P values \ 0.05 were considered significant.
Results Design and characterization of the fusokine FIL A fusokine fusion construct was generated using an open reading frame for both cytokines. To create this
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Fig. 1 Design and 3D protein simulation of fusokine FIL. a Expression cassettes for fusokine FIL, CXCL10, and XCL1. b 3D simulation of fusokine FIL using the RaptorX web server.
SP, signal peptide; DUAG, deletion of stop codon; DSP, deletion of signal peptide; Linker, (G4S)3
fusokine fusion, the CXCL10 stop codon was deleted, a flexible peptide [(G4S)3] sequence was added, and the mature XCL1 coding sequence was added in-frame. This new fusokine fusion encoding CXCL10-(G4S)3XCL1 was designated as FIL (Fig. 1a). We performed a 3D simulation of the fusokine FIL using the RaptorX web server. This model indicated that the chimeric protein conformation consisted of two domains, with each domain preserving the respective chemokine structure (Fig. 1b). This structural preservation is important for the biological activity of the fusokine.
Cells infected with Ad CXCL10, Ad XCL1, or fusokine FIL secrete recombinant chemokines To assess chemokine expression and secretion, HeLa cells were infected with the indicated adenovirus. At 24 h post-infection, medium was collected and subjected to immunoblotting with anti-CXCL10 (Fig. 2a) and anti-XCL1(Fig. 2b). The theoretical weights calculated for recombinant proteins Ad FIL, AdCXCL10, and Ad XCL1 were 22, 10, and 12 kDa, respectively. Western blots confirmed that all proteins were detected at the calculated molecular masses. Supernatants of cells infected with the Ad Empty vector showed no expression. These results indicate that the engineered adenoviral vectors are competent to infect cells and are able to secrete the recombinant proteins. Fusokine FIL elicits in vitro chemotactic activity
Fig. 2 Adenovirus-directed expression of fusokine FIL. Western blot analysis of supernatants from HeLa cells infected with different recombinant adenovirus (MOI 3). Blots were probed using anti-CXCL10 (a) or anti-XCL1 (b)
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To determine the biological activity of fusokine FIL, we performed a chemotaxis assay. Conditioned medium from HeLa cells infected with the adenovirus was used as chemoattractant for IL-2 stimulated lymphocytes. The Ad Empty control induced chemotaxis in 20 % of cells, whereas the individual or combined chemokines (Ad CXCL10, Ad XCL1, and Ad CXCL10? Ad XCL1) induced chemotaxis in30–40 % of cells. By contrast, the fusokine FIL
Biotechnol Lett
with Ad FIL (Fig. 4b). These results demonstrate that the fusokine recombinant adenovirus elicit an intratumoral chemoattraction in vivo.
Discussion
Fig. 3 In vitro chemotactic activity of fusokine FIL. Lymphocytes obtained from human peripheral blood were stimulated with human IL-2 for 21 days. Chemotaxis was assayed using supernatants of HeLa cells infected with the adenoviral constructs (assays were performed in 96-well microchambers); cells in the bottom wells were counted and plotted as the % of migratory cells. ***P values \ 0.05 were considered significant
induced chemotaxis in 60 % of cells. This result indicates that fusion of the chemokines CXCL10 and XCL1 enhanced their chemoattractant activity compared with that of the individual or combined chemokines (Fig. 3). Similar results were obtained using murine splenocytes (data not shown). Fusokine FIL elicits in vivo chemotactic activity We assessed the in vivo chemotaxis induced by fusokine FIL in mice bearing tumors induced by TC-1 cells.T lymphocytes were activated by DNA vaccination using the CRT/E7 antigen two weeks after the recombinant adenovirus was injected intratumorally. Immunization with a DNA vaccine, which encodes this fusion protein, activates E7-specific T lymphocytes (Cheng et al. 2001). Immunostaining for hexon protein on paraffin sections of tumors was positive for all adenovirus treatments, as expected (Fig. 4a). Next, we assessed whether the adenoviruses elicited chemotaxis by immunostaining for the CXCL10 (antiCXCR3) and FcR (anti-CD16/32) receptors. These receptors are present inT, B, monocyte/macrophage, NK, dendritic, neutrophil, eosinophil, mast, and basophil cells. A cell migration response to the adenoviral constructs was observed (but not the empty adenovirus), which was most pronounced for tumors injected
A key event for successful immunotherapy is the generation of antigen-specific T cells that infiltrate into the tumor to destroy malignant cells. The presence of tumor infiltrating lymphocytes (TILs) has been associated with a better outcome in patients with HPV16-associated oropharynx cancer (Wansom et al. 2012) and other cancer types (Gooden et al. 2011). Thus, the generation of new therapeutic tools that enhance the presence of tumor-localized TILs may improve the efficacy of immunotherapy in patients with tumors (Gattinoni et al. 2006). Chemokines are small cytokines that guide the migration of responsive cells. CXCL10 and XCL1 are both chemokines with antitumor activity (Cairns et al. 2001; Wennerberg et al. 2014). The effect of these chemokines is reported to be synergistically enhanced when used in combination (Huang and Xiang 2004; Zhang et al. 2011). CXCL10 has been used in fusion protein constructs with other molecules. A chimeric construct, engineered by substituting the N-terminal and N-loop region of CXCL10 with those of CXCL11,is reported to induce stronger antitumor effects than individual or combined parental chemokines by eliciting CXCR3? chemotaxis and antiangiogenesis (Wang et al. 2010). Another fusion protein composed of CXCL10 and mutant EGFR in combination with activated CD8? lymphocytes synergistically inhibited glioma growth and induced an increased number of CXCR3?CD8? T cells in a mouse glioma model (Wang et al. 2013). In this study, we report the fusion of CXCL10 and XCL1 as a fusokine for the first time, and demonstrate its biological activity by performing in vitro chemotaxis assays and in vivo chemotaxis of CXCR3? cells. The advantage of this construction over previous designs is the inclusion of chemokine CXCL10, which chemoattracts activated TCD8? lymphocytes induces the functional activation, proliferation, and survival of infiltrating T lymphocytes, and has antiangiogenic effects (Yang et al. 2006). Our strategy also utilizes chemokine XCL1, which chemoattracts XCR1-expressing dendritic cells (DCs) and may facilitate antigen
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Fig. 4 In vivo chemotactic activity of fusokine FIL. Female C57BL/6 mice were inoculated subcutaneously in the left flank with 1 9 104 TC-1 cells. When the tumors reached 100 mm3, mice were biolistically vaccinated with the plasmid CRT/E7 (1 lg gold particles, 1 lg DNA, and 300 psi). Seven days after vaccination, 1 9 106 viral particles from each adenovirus were
administrated by an intratumoral-injection. Mice were sacrificed 48 h after infection. Tumor sections were incubated with antihexon, anti-CXCR3and anti-CD16/CD32 antibodies. a Representative field of each treatment. Magnification is 209. b The relative amount of positive cells in each group was scored as rare = 1, few = 2, moderate = 3, and many = 4. Mean ± SE
presentation of DCs to CD8? T cells. These results provide an experimental foundation that supports further studies of fusokines as cancer immunotherapies.
calreticulin-human papillomavirus 16 E7 fusion protein eradicates E7 expressing established tumors in mice. Cancer Immunol Immunother CII 56:997–1007 Gooden MJM, de Bock GH, Leffers N et al (2011) The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis. Br J Cancer 105:93–103 Huang H, Xiang J (2004) Synergistic effect of lymphotactin and interferon gamma-inducible protein-10 transgene expression in T-cell localization and adoptive T-cell therapy of tumors. Int J Cancer J Int Cancer 109:817–825 Ka¨llberg M, Wang H, Wang S et al (2012) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7:1511–1522 Lei Y, Takahama Y (2012) XCL1 and XCR1 in the immune system. Microbes Infect Inst Pasteur 14:262–267 Proudfoot AEI (2002) Chemokine receptors: multifaceted therapeutic targets. Nat Rev Immunol 2:106–115 Raman D, Baugher PJ, Thu YM, Richmond A (2007) Role of chemokines in tumor growth. Cancer Lett 256:137–165 Russell HV, Strother D, Mei Z et al (2007) Phase I trial of vaccination with autologous neuroblastoma tumor cells genetically modified to secrete IL-2 and lymphotactin. J Immunother Hagerstown Md 1997 30:227–233 Wang P, Yang X, Xu W et al (2010) Integrating individual functional moieties of CXCL10 and CXCL11 into a novel chimeric chemokine leads to synergistic antitumor effects: a strategy for chemokine-based multi-target-directed cancer therapy. Cancer Immunol Immunother CII 59:1715–1726 Wang X, Lu X-L, Zhao H-Y et al (2013) A novel recombinant protein of IP10-EGFRvIIIscFv and CD8(?) cytotoxic T lymphocytes synergistically inhibits the growth of implanted glioma in mice. Cancer Immunol Immunother CII 62:1261–1272 Wansom D, Light E, Thomas D et al (2012) Infiltrating lymphocytes and human papillomavirus-16 associated oropharynx cancer. Laryngoscope 122:121–127
Acknowledgments This work was supported by Programa de Apoyo a la Investigacio´n en Ciencia y Tecnologı´a (PAICYT) No. SA381-10 from the UANL, and by ConsejoNacional de Ciencia y Tecnologı´a (CONACYT) Grant No. CB-10-158509. YESL and JJPT were recipients of scholarships from CONACYT. Conflict of interest conflict of interest.
The authors declare that they have no
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