ORIGINAL ARTICLE
Tumor-derived inducible heat-shock protein 70(HSP70)is an esntial component of anti-tumor immunity
二月七号是什么星座K Dodd 1,S Nance 1,M Quezada 1,L Janke 2,JB Morrison 3,RT Williams 3,4and HM Beere 1
INTRODUCTION
Heat-shock proteins (HSPs),the functional components of the inducible heat-shock respon,are implicated in the regulation of tumorigenesis by virtue of their ability to promote tumor cell survival.1Ho
wever,the only in vivo genetic evidence of a pro-tumorigenic role for the stress respon is the abrogation of tumor formation in mice de ficient for heat shock factor-1(HSF-1),the transcription factor esntial for the expression of multiple HSPs.2,3Nevertheless,evidence suggests that ‘addiction ’to components of the stress respon,including HSP70,may sustain tumor survival and drive tumor growth.1,4
清蒸毛蟹HSP70also regulates immune function,including antigen cross prentation,5,6dendritic cell maturation 7,8and natural killer (NK)cell,9,10and myeloid-derived suppressor cell 11activities.Extra-cellular HSP70regulates the diver immunoregulatory activities by acting as a cytokine to stimulate the relea of pro-in flammatory factors from immune cells 12or from tumor cells.13HSP70is relead from tumor cells via passive relea from dying cells and active traf ficking via the endolysosomal pathway 14or relea within lipid-bound exosomes.11,15
Discrimination between a need for tolerance and the demand for immunity reprents a fundamental principal of maintaining immunological homeostasis.Tolerance prevents autoimmunity but becau of the extensive overlap of lf-peptides with tumor-associated antigens,also suppress anti-tumor immunity.The immune system can distinguish ‘normal ’from ‘abnormal ’lf to overcome tolerance and instead invoke immunity via mechanisms,such as the relea of immunogenic ‘danger signals ’,
16that include HSPs.17,18Although HSPs may be critical determinants of a need for tolerance or circumstances requiring an immune respon,19,20it remains controversial whether this is
mediated by promoting immunity or by suppressing immune respons to maintain tolerance.18,21
To date,no studies have utilized the Hsp70.1/3−/−murine model to address whether HSP70,like HSF-1,2,3is a critical pro-survival signal for tumor cells in vivo or to evaluate the conquences of HSP70-mediated immune regulation in the context of anti-tumor immunity and tumor growth in situ .Clearly,HSP70can contribute to multiple aspects of immune regulation,but it remains unclear if this manifests in the suppression of tumor growth by activation of anti-tumor immunity 22,23or immunosuppression to exacerbate tumorigenesis.21
油菜霜霉病We utilized the Hsp70.1/.3−/−murine model,in which both alleles of inducible HSP70are deleted,24to ask whether HSP70is esntial for oncogene-induced transformation;whether HSP70has a non-redundant role in tumor growth in vivo ;and whether the immunoregulatory activity of HSP70inhibits or promotes tumor growth in vivo ?We prent data here that challenge an esntial pro-tumorigenic role for tumor-derived HSP70but instead support a model in which it negatively regulates tumor growth in vivo by engaging T-cell dependent immunity.For the first time,using the Hsp70.1.3−/−murin
e model,we demonstrate that HSP70is a non-esntial pro-tumorigenic factor but instead functions as a danger signal to facilitate anti-tumor immunity and suppress tumor growth in vivo .
RESULTS
HSP70is neither required for oncogene-induced transformation in vitro nor tumor growth in vivo
Wild-type (WT)or Hsp70−/−murine embryonic fibroblasts (MEFs)transduced with E1A and Ras ,but not empty vector or E1A or
1Department of Immunology,St Jude Children's Rearch Hospital,Memphis,TN,USA;2Veterinary Pathology Core,St Jude Children's Rearch Hospital,Memphis,TN,USA;3
Department of Oncology,St Jude Children's Rearch Hospital,Memphis,TN,USA and 4PUMA Biotechnology,Los Angeles,CA,USA.Correspondence:Dr HM Beere,Department of Immunology,St Jude Children's Rearch Hospital,262Danny Thomas Place,Memphis,TN 38105,USA.E-mail:helen.beere@stjude
Received 24January 2014;Received 12February 2014;accepted 14February 2014
Oncogene (2014),1–11
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Ras alone,generated drug-resistant colonies of approximately equivalent number (Figure 1a).However,tho colonies lacking Hsp70appeared larger in size,although no difference in the in vitro growth rates of WT and Hsp70−/−MEFs was obrved (not shown).Consistent with obrvations using immortalized HSF-1−/−MEFs,3primary HSF-1−/−MEFs did not transform (Figure 1a).Quantitative PCR of Hsp70expression con firmed the genotype of emergent clones (Figure 1b).WT and Hsp70−/−E1A/Ras transformants generated tumors in immunode ficient mice with no signi ficant difference in tumor size (Figures 1c and d).Similar data were obtained using independently generated WT and Hsp70−/−E1A/Ras transformants.All tumors were classi fied as sarcomas (Supplementary Figures S1A and B),and neither the固定值怎么设置
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frequency of karyomegaly nor mitos revealed any signi ficant difference between WT and Hsp70−/−tumors (Supplementary Figure S1C).
Selection for HSP70expression in WT tumors
WT tumors from CD1-Foxn1nu mice had elevated Hsp70gene expression,compared with the MEFs ud for inoculation (Figure 1e).Hsp70−/−tumors were not completely devoid of Hsp70expression,pr
esumably due to host-derived contamination such as vasculature (Figure 1e),and Hsp90AA1gene expression in WT and Hsp70−/−tumors was comparable and largely unchanged from that in the MEFs ud for inoculation (Figure
1f).
Figure 1.Hsp70is neither required for in vitro transformation nor tumor growth in vivo .WT,HSF-1−/−or Hsp70−/−primary MEFs were transduced with empty vector control,E1A or Ras alone or both E1A and Ras retroviral vectors as indicated,and after re-plating (4×104–2×105/well)and antibiotic lection for approximately 14days,colonies were visualized by methylene blue staining (a ).Quantitative PCR for Hsp70(normalized to L32expression)of emergent colonies (b ).WT or Hsp70−/−E1A/Ras transformants were injected subcutaneously into each of the flanks of CD1-Foxn1nu mice,and tumor growth monitored by ultrasound.Volumetric tumor measurements (mm 3)were derived from 3-D reconstructions of the ultrasound data ts.Data are shown as the average (n =5–10mice/group)±s.d.and is reprentative of six independent experiments (c ).Reprentative images of one tumor (marked with asterisk (*)in panel (c ))in which the left panels show 2-D slices of the tumor and the right panels show the 3-D volumetric reconstruction (d ).Relative expression of Hsp 70(e )and Hsp90AA1(f )normalized to L32.Asterisks denote the expression levels of Hsp70and Hsp90AA1in the MEFs ud for inoculation.
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Neither Bcr-Abl nor E μ-Myc requires HSP70to induce leukemogenesis
We also examined the requirement for HSP70in (i)Bcr-Abl -induced leukemogenesis 25and (ii)E μ-Myc -induced B-cell lymphoma.26WT and Hsp70−/−bone marrow (BM)was trans-duced with a retroviral vector expressing Bcr-Abl-GFP before culture in vitro or transfer into lethally irradiated C57BL/6J WT hosts.WT and Hsp70−/−BM cultures proliferated at similar rates before (Figure 2a)and after (Figure 2b)removal from stromal support.Equivalent green fluorescent protein (GFP)expression was detected in the WT and Hsp70−/−BM transformants (Figure 2c),and evaluation of Hsp70gene expression con firmed
genotype speci ficity (Figure 2d).Similar dia ont was obrved in animals receiving WT or Hsp70−/−BM,with no signi ficant difference in overall survival rates (Figure 2e).No genotype-speci fic differences were obrved in spleen weight (Figure 2f)or percentage of GFP positivity,a surrogate determi-nant of Bcr-Abl +cells (Figure 2g).
E μ-Myc transgenic mice,26WT,heterozygous or null for Hsp70developed dia with a similar time of ont (Figure 2h),spleen weight (Figure 2j)and dia classi fication (Figure 2k).However,although the median survival rate of WT and Hsp70+/−-E μ-Myc mice was similar,Hsp70−/−mice exhibited a signi ficant increa in lifespan (Figures 2h and
i).
Figure 2.Neither Bcr-Abl nor E μ-Myc requires HSP70to induce lymphoma.BM from each of two WT and two Hsp70−/−mice was transduced with a retroviral vector expressing Bcr-Abl-GFP .Cell growth was monitored before (a )and after cells were removed from the stromal layer (b ).After approximately 2weeks,GFP expression was assd via flow cytomtery (c )and Hsp70gene expression via quantitative PCR (d ).Survival of the two cohorts receiving Bcr-Abl-GFP transduced WT or Hsp70−/−BM (e ),spleen weight (f )and percenatge of GFP +cells in the BM,spleen and blood (g ).Lymphoma-free survival of WT,Hsp70+/−and Hsp70−/−E μ-Myc mice (h )and average spleen weight (j ).Statistical evaluation of lymphoma-free survival using a Log Rank Mantel Cox test (Prism software)and median survival (in days)(i ).Relative frequencies (expresd as a percentage)of IgM −pre-B-cell lymphomas and IgM +mature B-cell lymphomas as determined by the percentage of IgM staining of the B220+population in the spleen (o 50%IgM +was assigned a pre-B phenotype)(k ).
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Tumor-derived HSP70only retards tumor growth in an immune-competent host
To speci fically address the role of intra-tumoral HSP70,we further utilized the MEF-derived sarcoma model.WT E1A/Ras cells introduced into immune-competent hosts formed small tumors that subquently regresd,whereas Hsp70−/−transformants generated signi ficantly larger tumors (Figures 3a and b).This contrasts with equivalent WT and Hsp70−/−tumor growth in immunode ficient hosts (Figures 3c and 1c).WT tumors generated in C57BL/6J hosts showed incread Hsp70gene expression compared with the cells ud for inoculation (Figure 3e),although the average fold difference was less than in the CD1-Foxn1nu mice (compare Figures 3e and 1e).Immunohistochemistry con firmed
HSP70expression in WT tumors that was largely abnt from Hsp70−/−tumors (Figure 3d and Supplementary Figures S1F and G).Hsp90AA1expression was incread in Hsp70−/−but not WT tumors (Figure 3f),and constitutive Hsc70remained at or below control levels in WT and Hsp70−/−tumors (Figure 3g).Hsp70−/−tumors display a marked reduction in the in filtration of immune cells
WT tumors from C57BL/6J hosts displayed macrophage in filtra-tion throughout the tumor mass whil
e tho lacking Hsp70showed a reduction in MAC-2staining that was largely restricted to the tumor periphery (Figure 4a and Supplementary Figures
S2C
Figure 3.Growth of WT and Hsp70−/−tumors is dependent on the immune status of the host.WT or Hsp70−/−E1A/Ras MEF transformants were injected subcutaneously into each of the flanks of C57BL/6J (a )or CD1-Foxn1nu (c )mice.Volumetric tumor measurements (mm 3)were derived from 3-D reconstructions of the ultrasound data ts (one reprentative tumor from each of the WT and Hsp70−/−groups is shown panel (b )).A total of 32WT C57BL/6J mice (in three independent experiments)and 55CD1-Foxn1nu mice (in six independent experiments)were ud to asss in vivo tumor growth.One reprentative C57BL/6J experiment is shown in (a ),and for comparison,two independent CD1-Foxn1nu experiments are shown in panel (c )and Figure 1c.HSP70expression in tumors from C57BL/6J mice was determined via immunostaining (d ).The scale bars correspond to 250or 50microns in the ×10and ×40images,respectively.RNA from the tumors was ud to determine the expression of Hsp70(e ),Hsp90AA1(f )and Hsc70(g )via quantitative PCR.Relative gene expression is shown,and all data were normalized to the control gene L32.Left (L)and right (R)flank tumors are numbered quentially,and asterisks denote the expression levels in the transformants ud for inoculation.
Tumor-derived HSP70induces host immunity
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and D).Although Hsp70−/−tumors from CD1-Foxn1nu hosts also showed a reduction in macrophage number compared with WT tumors,they were distributed throughout the tumor mass (Figure 4b and Supplementary Figures 2A and B).
Differential growth of WT and Hsp70−/−tumors in C57BL/6J hosts compared with equivalent growth in CD1-Foxn1nu mice suggests that HSP70may suppress tumor growth in a T-cell-dependent manner.Indeed,WT tumors displayed extensive intra-tumoral lymphocytic in filtration,whereas Hsp70−/−tumors were characterized by a reduced number of CD3+cells,largely restricted to the tumor periphery (Figure 4c and Supplementary Figure S3).Extensive intra-tumoral CD3+/CD4+(Figure 5a)and CD8+/GrB +(Figure 5b)co-staining was obrved in WT tumors that was reduced and largely undetectable in Hsp70−/−tumors.NKT cells were detected in WT and,to a lesr extent,in
Hsp70−/−tumors (Figure 5c)and while WT tumors had extensive granzyme B and perforin expression,Hsp70−/−tumors exhibited a reduction in the expression of both (Figures 5d and e and Supplementary Figures 4D and G).
Knockdown of Hsp70abrogates intra-tumoral immune cell in filtration and promotes tumor growth
WT MEFs stably expressing Hsp70or control shRNAs vectors co-expressing red fluorescent protein (RFP)were evaluated for their ability to generate tumors in vivo .Hsp70shRNAs reduced HSP70protein levels while RFP expression was equivalent in both control and Hsp70shRNA expressing cells (Figure 6a and Supplementary Figures S5A and B).Tumors expressing Hsp70or control shRNAs were equal in size in immunode ficient mice (Figure 6b),but in C57BL/6J hosts,cells expressing Hsp70shRNA generated larger tumors compared with tho expressing control shRNAs (Figure 6d).Furthermore,Hsp70shRNA tumors from immune-competent mice displayed a lective retention of RFP and reduced HSP70proteins (Figure 6e and Supplementary Figures S5I and L).In contrast,RFP protein levels were variable in tumors isolated from CD1-Foxn1nu hosts,with no preferential expression in the Hsp70−/−tumors (Figure 6c and Supplementary Figures S5C and H).Hsp70shRNAs expressing tumors displayed a marked reduction in macrophage and T-cell in filtration
as
Figure 4.Hsp70−/−tumors are associated with a signi ficant reduction in immune cell in filtration.MAC-2staining of tumors from C57BL/6J (a )or CD1-Foxn1nu (b )hosts.Quanti fication of MAC-2staining in tumors collected from CD1-Foxn1nu hosts was evaluated and expresd as ‘percent positivity ’.CD3staining of WT and Hsp70−/−tumors to visualize T-cell distribution (c ).Images shown are reprentative from two WT and two Hsp70−/−tumors at the magni fications are shown.The scale bars correspond to 250or 50microns in the ×10and ×40images,respectively.
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