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. 2015 Sep 24:14:174.
doi: 10.1186/s12943-015-0435-9.

Shikonin-enhanced cell immunogenicity of tumor vaccine is mediated by the differential effects of DAMP components

Tien-Jen Lin  1   2   3   4   5 , Hsin-Ting Lin  2   3 , Wei-Ting Chang  3 , Pradeep Mitapalli S  3   4   5 , Pei-Wen Hsiao  3   4   5 , Shu-Yi Yin  6 , Ning-Sun Yang  7   8   9   10
Affiliations

Shikonin-enhanced cell immunogenicity of tumor vaccine is mediated by the differential effects of DAMP components

Tien-Jen Lin et al. Mol Cancer. .

Abstract

Background: The tumor cell lysate-pulsed, dendritic cell (DC)-based cancer vaccine approaches are being actively evaluated for application to cancer immunotherapy, hopefully at a personalized medicine base. There is apparently an emerging technical problem however, the lack of highly efficacious potency in activation of patient's DCs for T-cell priming and the associated process for presenting tumor immunogenicity.

Methods: One strategy to address this is to consider the manipulation of the tumor immunogenic cells death (ICD) complex ex-vivo for maximal activation of DC efficacy. In our previous study we showed that phytochemical shikonin (SK) can drastically enhance ICD activity in mouse tumor cells treated ex-vivo, and the resultant tumor cell lysate (TCL) can effectively augment such SK-TCL pulsed DC vaccine activity in vivo in anti-tumor activities. In this study, we investigated the specifics and the multi-functional effects of various damaged associated molecular pattern (DAMP) components of the ICD complex for their participation, roles and potential cross talks in activating DCs, as measured by five different functional assays.

Results: Among three DAMPs tested, HSP70 and CRT mediate a key role in SK-TCL-induced DC immunity for both CD4(+) and CD8(+) T cell proliferations in vitro. HSP70 is the most important component, followed by CRT, then HMGB1 in facilitating DC immunity on suppressing metastasis of mouse 4 T1 mammary tumors and prolonging survival in test mice. Only HSP70, but not CRT or HMGB1, is effective for the suppression of both granulocytic and monocytic MDSC populations in vivo. Both HSP70 and HMGB1, but not CRT, are essential in activating the expression of three key ICD molecules-associated receptors on test DCs. Each of the three test ICD proteins can exhibit a distinguishable pattern in stimulating the expression of four key chemokines in test DCs.

Conclusion: Our findings on the differential roles or effect of various ICD components in activating vaccinated DCs may help formulate new strategies for future cancer vaccine designs.

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Figures

Fig. 1
Fig. 1
Shikonin-treated 4 T1 tumor cell lysate effectively activates DCs that in turn induce T-cell proliferation in vitro. SK-TCL or Dox-TCL samples were prepared from transgenic Luc2-labelled 4 T1 cells that were treated with shikinon or doxorubicin at 5 μM for 24 h. Mouse bone marrow-derived DCs were then treated with Naive-, SK- or Dox-TCL samples and used as stimulator cells. Splenic CD4+ and CD8+ T cells were collected from syngeneic mice and employed as responder cells. Proliferation activities of a CD4+ and b CD8+ T cells were analyzed by mixed lymphocyte reaction (MLR) assay. Ratios of stimulator to responder cells were set between 1:1000 and 1:5. T-cell proliferation activity is represented as fold change over the control (T cells only). Data represent the mean ± SD of three biological replicates, and an independent experiment showed similar results
Fig. 2
Fig. 2
Adjuvant effect of SK-TCL (formulation) used with a DC vaccine, in 4 T1 tumor metastasis in mice. Test mice were injected subcutaneously with 4 T1-Luc2 cells (5 × 105 cells/100 μl PBS/mouse) into the mammary fat pad under isoflurane anesthesia. At 15 days post tumor cell implantation, primary tumors were surgically resected. Specifically formulated DC vaccines (1 × 106 DCs/200 μl PBS/mouse) were delivered via tail vain injection to mice at 0, 7 and 14 days post tumor resection. a Representative in vivo bioluminescent images of test mice (8 mice/group) treated with PBS, mDCs, mDCs + Naive-TCL, mDCs + SK-TCL and mDCs + Dox-TCL vaccines after resection of the 4 T1 orthotopic primary tumors. The red signals represent the highest level on the colorimetric scale. b Percentage of whole body (all organs) free from metastasis in mice (8 mice/group). The metastasis levels of tumors in test mice were scored within the indicated time course as revealed by bioluminescence imaging. c Survival rate of test mice after resection of 4 T1 tumors and treatment with the indicated DC vaccine regimes. Similar trends of results were obtained from four independent experiments
Fig. 3
Fig. 3
Western blot analyses for expression of HSP70, CRT and HMGB1 in 4 T1 cells. Test 4 T1 cells dispensed in 6-well plates (3 × 105 cells/well) were incubated with vehicle, SK or Dox at 5 μg/ml for 24 h. Beta-actin was used as a loading control. Three independent experiments showed similar patterns as the data shown here
Fig. 4
Fig. 4
HSP70 and CRT may play an important role in shikonin-induced ICD-derived proteins in treated tumor cells, that may differentially activate DCs and mediate T-cell proliferation. The shikonin-treated 4 T1 cells and the resultant-ICD derived TCLs (i.e., SK-TCL) and the specific protein-deleted SK-TCL [(i.e., SK-TCL(−HSP70), SK-TCL(−CRT) or SK-TCL(−HMGB1)] were used to stimulate DCs and for subsequent activation of T-cell proliferation. (a) Western blot analysis of ICD component proteins in SK-TCL(−HSP70), SK-TCL(−CRT) and SK-TCL(−HMGB1) samples, representing specific depletion of HSP70, CRT or HMGB1 proteins in the correspondent SK-TCL sample preparations, respectively. These DC vaccine samples were then used as stimulator cells for T-cell proliferation assays. Splenic CD4+ and CD8+ T cells were collected from syngeneic mice as responder cells. Proliferation activities of (b) CD4+ and (c) CD8+ T cells were analyzed by MLR assay. Ratios of stimulator to responder cells were set between at 1:1000 and 1:5. T-cell proliferation activity is represented as the fold change over the control (T cells only). Data represent the mean ± SD of three replicates. Similar results were obtained from three independent experiments
Fig. 5
Fig. 5
HSP70 may play a key role in the shikonin-induced ICD for enhancing anti-metastasis activity of DC-based cancer vaccines. Specific ICD protein components (i.e., HSP70, CRT or HMGB1) were depleted from SK-TCL samples before they were used as an adjuvant for preparation of a DC-based cancer vaccine. Test mice (8 mice/group) were injected with different vaccine formulations [i.e., iDCs, mDCs + Naive-TCL, mDCs + SK-TCL, mDCs + SK-TCL(−HSP70), mDCs + SK-TCL(−CRT), mDCs + SK-TCL(−HMGB1)] three times (see Materials and Methods), and mice were monitored for metastasis status and survival rate was scored for three months. a Representative in vivo bioluminescent images of test mice treated with different vaccine regimes after the resection of the 4 T1 orthotopic primary tumors. The red signals represent the highest level on the colorimetric scale. b Percentage of whole body organ free from metastasis in mice. The metastasis levels of tumors in test mice were scored by bioluminescence imaging within the indicated time period. c Survival rate of test mice treated with the indicated DC vaccine formulations, after tumor resection. A P value of less than 0.05 was considered significant (*, P < 0.05). d Ten days after the last vaccination, the percentages of monocytic MDSCs (CD11b+Ly6C+) and granulocytic MDSCs (CD11b+Ly6G+) in the blood of test mice were analyzed using flow cytometry. Similar trends of results were obtained from three independent experiments shown in (a), (b) and (c), and from two independent experiments shown in (d)
Fig. 6
Fig. 6
Cytokine profiling array analysis of conditioned culture media from various 4 T1 TCL-treated DCs. a Cytokine array membranes were incubated with cultured media from immature DCs or DCs that were treated with different TCL samples, including Naive-TCL, Dox-TCL, SK-TCL, SK-TCL(−Hsp70) , SK-TCL(−CRT) and SK-TCL(−HMGB1) for 2 h, and then stimulated with LPS for another 22 h. Squares mark the chemokines secreted from DCs that were increased in the SK-TCL-loaded group, as compared with those from Naive-TCL-loaded groups. Green square: G-CSF, blue square: SDF-1/CXCL12, red square: MIP-1α/CCL3 and yellow square: MCP-1/CCL2. b Stimulation (in fold change) of ICD-responsive chemokines in DCs loaded with Naive-TCL, Dox-TCL, SK-TCL, SK-TCL(−Hsp70), SK-TCL(−CRT) or SK-TCL(−HMGB1). Secretion level of each chemokine was normalized by value of Naive-TCL-loaded group (Fold change = Each TCL-loaded group/Naive-TCL-loaded group). Color of cone for each chemokine is correspondent to the indication in (a)
Fig. 7
Fig. 7
Western blot analyses on expression of CD91, TLR2 and TLR4 proteins on various TCL-activated DCs. Test 4 T1 cells were dispensed in 6-well plates (3 × 105cells/well) and incubated with vehicle, SK or Dox at 5 μg/ml for 24 h. Some sets as replicates collected from SK-TCL samples were depleted for specific ICD molecules, i.e., HSP70, CRT or HMGB1, using an antibody-mediated pull-down procedure (see Materials and Methods). Different ICD molecule-depleted SK-TCL samples were then compared for their stimulatory activity on expression of CD91, TLR2 and TLR4 on TCL-pulsed DCs. Beta-actin was used as a loading control. The results represent the similar trend of data obtained from three independent experiments
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