Development And Mechanism Of A Novel Personalized Lung Cancer Preventive Vaccine

Background and aimLung cancer has the highest mortality rate among malignant tumors.Despite various treatment measures,such as targeted drugs,the 5-year survival rate is still less than 20%.Compared to a large number of treatment regimens,the outcome of preventive methods is very limited.The incidence of lung cancer is related to smoking and family history,and air pollution caused by rapid economic development is also a potential factor.As an important cancer prevention method,the prophylactic vaccine has a broad application prospect because of its application before the formation of tumor-associated immune suppression.Some preventive vaccines target tumors caused by viruses,such as Gardasil and Cervarix,which are used to prevent cervical cancer caused by human papillomavirus infection.However,there are few preventive vaccines for non-viral malignancies.For example,lung cancer vaccines are almost therapeutic vaccines,including whole tumor cell vaccines,single tumor-related peptides such as EGF,multiple tumor-related peptides,dendritic cell vaccines,etc.These therapeutic vaccines are effective against tumor-specific antigens(TSAs)and tumor-associated antigens(TAAs)that evolved from tumor-specific mutations.It is reasonable to assume that prophylactic vaccines with minimal tumor burden would be more effective.This study started to consider one of the key issues of prophylactic cancer vaccines,namely the acquisition of early-stage tumor cells.Due to the rapid development of high-throughput sequencing technology,the driver mutations of tumors can be known at the early stage of tumor progression.Personalized neoantigen vaccines are effective in the treatment of advanced melanoma.However,the application of personalized neoantigen vaccines for driver mutations in tumor prevention has not been reported.We explored the preventive effects of a whole lung cancer cell vaccine containing individualized neoantigens on lung cancer.We edited the iPS cells derived from healthy mouse somatic cells using inactive mutant driver genes of tumor,and finally induced to lung cancer cells.The protective effect of somatic cell derived lung cancer cells on the subcutaneous lung cancer model and spontaneous lung cancer model was tested by immunizing mice with this prophylactic vaccine.Based on the curative efficacy,the immunological mechanism of the vaccine was further investigated.Based on the efficacy of autologous lung cancer cell vaccine derived from iPS cells,combined with the advantages of higher operability and lower cost of allogeneic vaccine in practice,we continued to establish two lung cancer cell lines cultured from KRas ^LSL-G12D/+;p53^LSL-R172H/+mice inhalated with Ad-Cre and explore the key antigens of tumor cells affecting prophylactic vaccines.These two lung cancer cell lines preinfected with two different oncolytic viruses were used to immunise this kind of lung cancer mice model,respectively.Interestingly,although the driver mutations of the two lung cancer cell lines and the derived lung cancer mice model were the same,the efficacy was significantly different.The lung cancer cell line derived from early staged lung cancer mice model could prolong the lifetime of lung cancer mice,but the lung cancer cell line derived from the advanced lung cancer mice model had no protective effect.Therefore,identification of key differences between the two cell lines would be important for improving the preventive vaccine for lung cancer.Afterwards,we analyzed the differential neoantigens between these two cell lines and explored the key neoantigens which influenced the efficacy of vaccines in vitro and in vivo.This study aimed to provide a safe and effective method for the prevention of lung cancer.Part?Establishment and characterisation of lung cancer cell line derived from inactive KRas ^LSL-G12D/+;p53^LSL-R172H/+modified induced pluripotent stem cells1 ObjectiveTo differentiate iPS cells edited by KRas ^LSL-G12D/+;p53^LSL-R172H/+to lung cancer cell line,and compare its similarity with the lung cancer cell lines derived from the primary tumor cells from transgenic mice.2 Methods2.1 Pluripotency identification of KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cellsiPS cells edited by inactive KRas ^LSL-G12D/+;p53^LSL-R172H/+were inoculated to nude mice subcutaneously.The H&E staining of the teratoma sections was used to determine the differentiation and development of iPS cells,to verify their pluripotency.2.2 Reprogramme KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells to lung cancer cell linesKRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells were induced to endoderm,anterior foregut endoderm,lung progenitors by small molecular compounds.Afterwards,cells were infected with Ad-Cre to activate mutant KRas ^G12Dand p53^R172Hto transform the differentiated lung progenitor cells into lung cancer cells.2.3 Verification of cell type markers at various stages of differentiation of iPS cellsThe markers of KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells in various stages of iPSC,endoderm,anterior foregut endoderm and lung progenitors were verified by immunofluorescence and q PCR.2.4 Characterisation of iPSCs-derived lung cancer cell line KPLCThe phenotype and biological features of the lung cancer cell line KPLC derived from KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells,KPL 160302S and KPL 160424S obtained from KP transgenic mice were compared,including LSL cassette removal,karyotype analysis,growth curve in vitro and in vivo,histopathology,wound healing assay,soft agar clone formation assay and plate clone formation assay.2.5 Cytotoxicity and replication of replicating oncolytic viruses Ad5 and VVL15-RFP in KPLCCytotoxicity and replication of Ad5 and VVL15-RFP were tested in KPLC,KPL160302S and KPL 160424S,respectively,with or without treatment of mitomycin C.2.6 Transcriptome analysis of similarity and gene differential expressionThree lung cancer cell lines KPLC,KPL160302S,KPL 160424S,two iPS cell lines WT iPSCs,WT-KP iPSCs,and three pancreatic cancer cell lines TB11381,KP-AC,KPC,were subjected to transcriptome sequencing.Three repeated samples were sequenced for each cell line.Bioinformatic methods were then used to measure the transcriptome similarity of these cell lines,and analyse the genes expressed higher in KPLC than WT iPSCs.3 Results3.1 Pluripotency identification of KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cellsThe KRas ^LSL-G12D/+;p53^LSL-R172H/+genome modified iPS cells were implanted to nude mice to form teratoma.H&E staining showed that the iPS cells were differentiated into different tissues of endoderm,mesoderm,and ectoderm,thus proving that the inactive mutant KRas and p53 gene-edited iPS cells still had pluripotency.3.2 Reprogramme KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells to lung cancer cell linesKRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells underwent differentiation to endoderm,anterior foregut endoderm,lung progenitors at different culture medium.The differentiated lung progenitor cells were able to be transformed to lung cancer cells by Ad-Cre infection,and their morphology changed gradually.The lung progenitor cells without Ad-Cre infection showed senescence in four passages.3.3 Verification of cell type markers at various stages of differentiation of iPS cellsImmunofluorescence and q PCR results showed the tissue-specific marker expression of KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells in various stages of iPSC,endoderm,anterior foregut endoderm and lung progenitor cells,demonstrating the differentiation process and efficiency were not affected by the transferred inactive mutant KRas and p53.3.4 Characterisation of iPSCs-derived lung cancer cell line KPLCIn lung cancer cell lines KPLC derived from KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells and KPL 160302S,KPL 160424S derived from primary tumor of KP transgenic mice,mutant KRas ^G12Dand p53^R172Hhave been activated,showing obvious chromosome number abnormalities.What's more,these three cell lines could form subcutaneous tumors in KP littermates and C57BL/6 mice,and malignancy could be verified by H&E staining of subcutaneous tumor sections.In the aspects of growth rate in vitro,histopathology,wound healing,soft agar clone formation,and plate clone formation,KPLC had a certain similarity with the primary cultured lung cancer cell lines derived from transgenic mice.3.5 Cytotoxicity and replication of replicating oncolytic viruses Ad5 and VVL15-RFP in KPLCIn the virus cytotoxicity experiment,the EC50 values(viral dose needed to kill50%of tumor cells)of KPLC for Ad5 and VVL15-RFP were between KPL 160302S and KPL 160424S.In the virus replication experiment,Ad5 barely replicated in murine derived cells.While VVL15-RFP replicated well in KPLC,but its replication can be inhibited by mitomycin C.3.6 Transcriptome analysis of similarity and gene differential expressionThe iPSCs-derived lung cancer cell line KPLC had the highest similarity with the early staged lung cancer model derived lung cancer cell line KPL 160302S(0.91),followed by the advanced lung cancer model derived lung cancer cell line KPL160424S(0.84).KPLC had low similarity with the two iPS cell lines WT iPSCs and WT-KP iPSCs,the iPSCs transformed pancreatic cancer cell lines KP-AC and KPC,and the primary cultured pancreatic cancer cell line TB11381.4 ConclusionThe KRas ^LSL-G12D/+;p53^LSL-R172H/+modified iPS cells can be programmed to lung cancer cell line.Its tumor characteristics and transcriptome were similar to primary cultured mice lung cancer cell lines in vitro and in vivo.Part?Preventive efficacy and functional mechanism of the vaccine by the induced pluripotent stem cells derived lung cancer cell line to spontaneous and subcutaneous tumor models of lung cancer1 ObjectiveTo use the lung cancer cell line KPLC derived from KRas ^LSL-G12D/+;p53^LSL-R172H/+-iPS cells as a prophylactic vaccine,and investigate its preventive effect on lung cancer spontaneous model and subcutaneous model and the underlying functional mechanisms.2 Methods2.1 Production of whole tumor cell vaccineAfter infection by oncolytic virus Ad5/VVL15-RFP,KPLC cells were treated with mitomycin C.To verify the safety of the whole tumor cell vaccine,the proliferation of the cells was tested by MTS and plate colony formation assays.The expression of virus protein was examined by Western Blotting.2.2 The preventive effect of KPLC on mice bearing subcutaneous lung cancer model and its functional mechanismsKP littermates mice were immunized by virus-infected KPLC prophylactic vaccine and then rechallenged with KPL 160302S/KPL 160424S to form subcutaneous tumors.The tumors were measured regularly,and were taken for immunohistochemistry to explore its immune mechanism.2.3 The preventive effect of KPLC on mice bearing spontaneous lung cancer model and its functional mechanismsKP mice delivered Ad-Cre intranasally were immunized by virus-infected KPLC prophylactic vaccine.The progression and survival period was observed.The immunological mechanism of this vaccine was explored through immunohistochemistry,IFN?stimulation of spleen cells,flow cytometry,antibody deletion experiment,etc.3 Results3.1 Production of whole tumor cell vaccineAfter infection by oncolytic virus Ad5/VVL15-RFP and followed the treatment with mitomycin C,the proliferation of KPLC was completely inhibited and the ability of plate colony formation was lost.However,viral proteins expression of Ad5 and VVL15-RFP were still present continuously.3.2 The preventive effect of KPLC on mice bearing subcutaneous lung cancer model and its functional mechanismsKPLC vaccine can delay the growth of subcutaneous tumor of lung cancer cell lines KPL 160302S/KPL 160424S,and significantly increase the number of infiltrating CD8⁺T cells and CD4⁺T cells within the tumors.3.3 The preventive effect of KPLC on mice bearing spontaneous lung cancer model and its functional mechanismsVirus-infected KPLC prophylactic vaccine significantly delayed the progression and prolonged the survival time of lung cancer mice.It can cause an increase of CD8⁺T cells infiltrated in lung tissues at 1-3 weeks and CD4⁺T cells infiltrated in lung tissues at 1-2 weeks post booster vaccination.This prophylactic vaccine increased the proportion of effective memory T cells(TEM)in spleen CD8⁺T cells of the immunized mice.And the spleen cells of immunized mice could be stimulated to increase the secretion of IFN?significantly by various lung cancer cell lines and KRas peptide.When the KPLC vaccine has coupled with CD8⁺T cells or CD4⁺T cells depletion antibodies,the survival period was significantly shorter than that without deletion and was of no difference with PBS group.Furthermore,the KPLC vaccine combined with the PD-1 antibody failed to improve the efficacy of the vaccine significantly.4 ConclusionVirus-infected KPLC prophylactic vaccine could significantly delay the growth of lung subcutaneous tumor and prolong the survival period of KP mice,the spontaneous lung cancer model.The efficacy of the vaccine was associated with the increase of infiltrating CD8⁺T cells and CD4⁺T cells.What's more,this vaccine contributed to the amplification of CD8⁺TEM cells.Part?Preventive efficacy and mechanism of primary-cultured lung cancer cell lines1 ObjectiveTo establish lung cancer cell lines from different stages of lung cancer mice model,and explore their preventive efficacy to lung cancer and identify the key neoantigens.2 Methods2.1 Establishment and identification of lung cancer mice modelAfter the genotype of KRas ^LSL-G12D/+;p53^LSL-R172H/+(KP)mice was identified,the mice were administrated with Ad-Cre intranasally at 10-12 weeks old.Then lung tissues were collected at different stages after Ad-Cre infection.The initiation and progression of lung cancer were verified by H&E staining.2.2 Establishment and identification of primary-cultured mice lung cancer cell linesLung tissues were collected to do primary culture at different stages after Ad-Cre delivery,respectively.The obtained cell lines were screened and purified by a round of subcutaneous tumor to acquire mice lung cancer cell lines KPL 160302S and KPL160424S,then their tumor associated characteristics were verified.2.3 The preventive efficacy of KPL 160302S,KPL 160424S on mice bearing spontaneous lung cancer modelThe two lung cancer cell lines derived from different stages of mice with lung cancer were infected with oncolytic viruses to enhance their immunogenicity and treated by mitomycin C to inhibit their proliferation.Then,the KP lung cancer mice were immunised with the virus-infected cancer cells.The efficacy of the vaccine was determined by observing the survival period.2.4 Functional mechanism of KPL 160302S,KPL 160424S vaccine on prevention of mice bearing spontaneous lung cancer modelLung tissues of mice with lung cancer were collected at different periods after prophylactic vaccination,and immunohistochemical experiments were conducted to determine the effects of vaccination on promoting the infiltration of the CD8⁺T cells and CD4⁺T cells into lung tissues.2.5 Analysis and verification of differential neoantigens between KPL 160302S and KPL 160424SWhole exome and transcriptome of the two mice lung cancer cell lines were sequenced.Neoantigen candidates exist in the valid cell line were analyzed by bioinformatics and tested in vitro and in vivo.3 Results3.1 Establishment and identification of lung cancer mice modelH&E staining of lung sections was observed after Ad-Cre was delivered intranasally.Early lesions were not observed three weeks post-infection.Afterwards,tumor cells with enlarged nuclei and prominent nucleoli were observed since 8 weeks post Ad-Cre infection,and tumors progressed gradually.3.2 Establishment and identification of primary-cultured mice lung cancer cell linesLung tissues were collected to do primary culture at 8 and 16 weeks after Ad-Cre infection,respectively.The two cell lines obtained were purified by a round of subcutaneous tumor.The two purified mice lung cancer cell lines KPL 160302S(8weeks)and KPL 160424S(16 weeks)had the tumorigenecity and their malignance were proved by H&E staining.3.3 The preventive efficacy of KPL 160302S,KPL 160424S on mice bearing spontaneous lung cancer modelTwo mice lung cancer cell lines were infected by oncolytic virus and treated by mitomycin C.Then,the cell lines were vaccinated to KP lung cancer mice model as prophylactic vaccines.One cell line prolonged the lifetime of lung cancer mice,the other cell line didn't.3.4 Functional mechanism of KPL 160302S,KPL 160424S vaccine on prevention of mice bearing spontaneous lung cancer modelAfter two mice lung cancer cell lines were vaccinated to KP lung cancer mice,lung tissues were collected at different time and analyzed by immunohistochemical experiments.The valid cell line elicited more CD8⁺T cells infiltration 1-3 weeks post booster vaccination,and elicited more CD4⁺T cells infiltration 1-2 weeks post booster vaccination.While the invalid cell line only elicited more CD8⁺T cells infiltration 1-2 weeks post booster vaccination.3.5 Analysis and verification of differential neoantigens between KPL 160302S and KPL 160424S12 candidate neoantigens only existing in the valid cell line were analyzed by bioinformatics.Among them,two neoantigens,Ddx21MT(SNFPIFCDL)and Zfp760MT(KCFRSYSSL)elicited more IFN?secretion in vitro.Immunized by Ddx21MT peptide,the CD8⁺and CD4⁺TCM cells in spleens of B6 and WT129 mice were both increased.4 ConclusionTwo lung cancer cell lines were obtained from lung cancer mice model.Although their driver mutations were the same,one cell line can prolong the survival period of lung cancer mice,while the other one can't.Ddx21MT(SNFPIFCDL)and Zfp760MT(KCFRSYSSL)neoantigens existed in the valid cells increased IFN?secretion significantly in vitro.What's more,Ddx21MT peptide was conducive to the augmentation of Central Memory T cells in spleens of mice.