小鼠多亚型热休克蛋白/肽疫苗联合PD-L1免疫检查点抑制剂的抗肿瘤实验研究
|
摘要
目的:应用小鼠肉瘤组织制备混合多亚型热休克蛋白/肽疫苗(mHSP/P),联合程序性死亡配体(PD-L1)抑制剂治疗小鼠肉瘤。方法:免疫组织化学染色和Elisa蛋白定量鉴定肉瘤细胞MCA207中的热休克蛋白(HSP70、HSP90、Grp94)的表达。制备蛋白悬液,通过蛋白层析技术获取mHSP/P和Grp94/肽肉瘤疫苗(Grp94/P),以Western blot(WB)鉴定。流式细胞术测定细胞毒性作用。Elisa实验测定mHSP/P和Grp94/P刺激产生的干扰素(IFN-γ)、肿瘤坏死因子(TNF-α)。小鼠实验探究肉瘤疫苗对肉瘤生长以及小鼠生存状况的影响。免疫荧光染色MCA207肉瘤细胞表面PD-L1的表达,WB测定IFN-γ对PD-L1表达的影响。动物实验探究PD-L1抑制剂联合mHSP/P对肿瘤的影响。结果:肿瘤组织携带多种亚型的HSP(HSP70、HSP90、Grp94);成功制备肉瘤组织来源的mHSP/P和Grp94/P,Western blot对肿瘤疫苗鉴定并且流式细胞学测定未发现细胞毒性;制备的mHSP/P较Grp94/P刺激产生更多的IFN-γ和TNF-α细胞因子(P<0.05)。肉瘤细胞表面PD-L1的表达随着IFN-γ的介入而增高;动物实验显示PD-L1抑制剂联合mHSP/P提高了免疫反应的抗肿瘤作用(P<0.05)。结论:肿瘤来源的mHSP/P和Grp94/P可以作为肿瘤疫苗在动物实验中使用。mHSP/P比Grp94/P能诱发更强的抗肿瘤免疫反应。IFN-γ刺激肉瘤细胞PD-L1的表达而导致免疫逃逸。PD-L1抑制剂联合mHSP/P提高了抗肿瘤作用。
Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide(mHSP/P) vaccine in combination with a programmed death ligand 1(PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and enzyme-linked immunosorbent assay(Elisa) was used to quantitatively identify the expression of heat shock proteins(HSP70, HSP90,Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide(Grp94/P) sarcoma vaccines were isolated using chromatography and were identified by Western blot(WB). Flow cytometry was used to determine their cytotoxic effects. The levels of interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes(HSP70, HSP90, Grp94). We successfully isolated sarcoma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cytotoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation(P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor(P<0.05).Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune evasion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.
Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide(mHSP/P) vaccine in combination with a programmed death ligand 1(PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and enzyme-linked immunosorbent assay(Elisa) was used to quantitatively identify the expression of heat shock proteins(HSP70, HSP90,Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide(Grp94/P) sarcoma vaccines were isolated using chromatography and were identified by Western blot(WB). Flow cytometry was used to determine their cytotoxic effects. The levels of interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes(HSP70, HSP90, Grp94). We successfully isolated sarcoma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cytotoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation(P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor(P<0.05).Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune evasion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.
引文
[1]Baldin AV,Zamyatnin AA,Bazhin AV,et al.Advances in the development of anticancer HSP-based vaccines[J].Curr Med Chem,2018,doi:10.2174/0929867325666180129100015.[Epub ahead of print]
[2]Ferrucci PF,Tosti G,Di Pietro A,et al.Newly identified tumor antigens as promising cancer vaccine targets for malignant melanoma treatment[J].Curr Top Med Chem,2012,12(1):11-31.
[3]Kampinga HH,Hageman J,Vos MJ,et al.Guidelines for the nomenclature of the human heat shock proteins[J].Cell Stress Chaperones,2009,14(1):105-111.
[4]De Maio A.Extracellular heat shock proteins,cellular export vesicles,and the stress observation system:a form of communication during injury,infection,and cell damage.It is never known how far a controversial finding will go!Dedicated to Ferruccio Ritossa[J].Cell Stress Chaperones,2011,16(3):235-249.
[5]Multhoff G,Hightower LE.Distinguishing integral and receptorbound heat shock protein 70(Hsp70)on the cell surface by Hsp70-specific antibodies[J].Cell Stress and Chaperones,2011,16(3):251-255.
[6]Jego G,Hazoume A,Seigneuric R,et al.Targeting heat shock proteins in cancer[J].Cancer Lett,2013,332(2):275-285.
[7]Ji N,Zhang Y,Liu Y,et al.Heat shock protein peptide complex-96vaccination for newly diagnosed glioblastoma:a phase I,singlearm trial[J].JCI Insight,2018,3(10):pii:99145.
[8]Weng D,Calderwood SK,Gong J.A novel heat shock protein 70-based vaccine prepared from dc-tumor fusion cells[J].Methods Mol Biol,2018,1709:359-369.
[9]Ohaegbulam KC,Assal A,Lazar-Molnar E,et al.Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway[J].Trends Mol Med,2015,21(1):24-33.
[10]Topalian SL,Taube JM,Anders RA,et al.Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy[J].Nat Rev Cancer,2016,16(5):275.
[11]Kim AK,Gani F,Layman AJ,et al.Multiple immune suppressive mechanisms in fibrolamellar carcinoma[J].Cancer Immunol Res,2019,pii:canimm.0499.2018.
[12]Stahl M,Goldberg AD.Immune checkpoint inhibitors in acute myeloid leukemia:novel combinations and therapeutic targets[J].Curr Oncol Rep,2019,21(4):37.
[13]Ikwegbue PC,Masamba P,Mbatha LS,et al.Interplay between heat shock proteins,inflammation and cancer:a potential cancer therapeutic target[J].Am J Cancer Res,2019,9(2):242-249.
[14]Calderwood SK.Heat shock proteins and cancer:intracellular chaperones or extracellular signalling ligands[J].Philos Trans R Soc Lond B Biol Sci,2018,373(1738).pii:20160524.
[15]Gonzalez FE,Chernobrovkin A,Pereda C,et al.Proteomic identification of heat shock-induced danger signals in a melanoma cell lysate used in dendritic cell-based cancer immunotherapy[J].J Immunol Res,2018,2018:3982942.
[16]Crouch BT,Gallagher J,Wang R,et al.Exploiting heat shock protein expression to develop a non-invasive diagnostic tool for breast cancer[J].Sci Rep,2019,9(1):3461.
[17]Pawaria S,Binder RJ.CD91-dependent programming of T-helper cell responses following heat shock protein immunization[J].Nat Commun,2011,2:521.
[18]Guo QY,Yuan M,Peng J,et al.Antitumor activity of mixed heat shock protein/peptide vaccine and cyclophosphamide plus interleukin-12 in mice sarcoma[J].J Exp Clin Cancer Res,2011,30(1):24-24.
[19]Ribas A,Flaherty KT.Gauging the Long-Term Benefits of Ipilimumab in Melanoma[J].J Clin Oncol,2015,33(17):1865-1866.
[20]Murshid A,Gong J,Calderwood SK.Purification,preparation,and use of chaperone-peptide complexes for tumor immunotherapy[J].Methods Mol Biol,2013,960:209-217.
[2]Ferrucci PF,Tosti G,Di Pietro A,et al.Newly identified tumor antigens as promising cancer vaccine targets for malignant melanoma treatment[J].Curr Top Med Chem,2012,12(1):11-31.
[3]Kampinga HH,Hageman J,Vos MJ,et al.Guidelines for the nomenclature of the human heat shock proteins[J].Cell Stress Chaperones,2009,14(1):105-111.
[4]De Maio A.Extracellular heat shock proteins,cellular export vesicles,and the stress observation system:a form of communication during injury,infection,and cell damage.It is never known how far a controversial finding will go!Dedicated to Ferruccio Ritossa[J].Cell Stress Chaperones,2011,16(3):235-249.
[5]Multhoff G,Hightower LE.Distinguishing integral and receptorbound heat shock protein 70(Hsp70)on the cell surface by Hsp70-specific antibodies[J].Cell Stress and Chaperones,2011,16(3):251-255.
[6]Jego G,Hazoume A,Seigneuric R,et al.Targeting heat shock proteins in cancer[J].Cancer Lett,2013,332(2):275-285.
[7]Ji N,Zhang Y,Liu Y,et al.Heat shock protein peptide complex-96vaccination for newly diagnosed glioblastoma:a phase I,singlearm trial[J].JCI Insight,2018,3(10):pii:99145.
[8]Weng D,Calderwood SK,Gong J.A novel heat shock protein 70-based vaccine prepared from dc-tumor fusion cells[J].Methods Mol Biol,2018,1709:359-369.
[9]Ohaegbulam KC,Assal A,Lazar-Molnar E,et al.Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway[J].Trends Mol Med,2015,21(1):24-33.
[10]Topalian SL,Taube JM,Anders RA,et al.Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy[J].Nat Rev Cancer,2016,16(5):275.
[11]Kim AK,Gani F,Layman AJ,et al.Multiple immune suppressive mechanisms in fibrolamellar carcinoma[J].Cancer Immunol Res,2019,pii:canimm.0499.2018.
[12]Stahl M,Goldberg AD.Immune checkpoint inhibitors in acute myeloid leukemia:novel combinations and therapeutic targets[J].Curr Oncol Rep,2019,21(4):37.
[13]Ikwegbue PC,Masamba P,Mbatha LS,et al.Interplay between heat shock proteins,inflammation and cancer:a potential cancer therapeutic target[J].Am J Cancer Res,2019,9(2):242-249.
[14]Calderwood SK.Heat shock proteins and cancer:intracellular chaperones or extracellular signalling ligands[J].Philos Trans R Soc Lond B Biol Sci,2018,373(1738).pii:20160524.
[15]Gonzalez FE,Chernobrovkin A,Pereda C,et al.Proteomic identification of heat shock-induced danger signals in a melanoma cell lysate used in dendritic cell-based cancer immunotherapy[J].J Immunol Res,2018,2018:3982942.
[16]Crouch BT,Gallagher J,Wang R,et al.Exploiting heat shock protein expression to develop a non-invasive diagnostic tool for breast cancer[J].Sci Rep,2019,9(1):3461.
[17]Pawaria S,Binder RJ.CD91-dependent programming of T-helper cell responses following heat shock protein immunization[J].Nat Commun,2011,2:521.
[18]Guo QY,Yuan M,Peng J,et al.Antitumor activity of mixed heat shock protein/peptide vaccine and cyclophosphamide plus interleukin-12 in mice sarcoma[J].J Exp Clin Cancer Res,2011,30(1):24-24.
[19]Ribas A,Flaherty KT.Gauging the Long-Term Benefits of Ipilimumab in Melanoma[J].J Clin Oncol,2015,33(17):1865-1866.
[20]Murshid A,Gong J,Calderwood SK.Purification,preparation,and use of chaperone-peptide complexes for tumor immunotherapy[J].Methods Mol Biol,2013,960:209-217.