癌症干细胞疫苗

Tumor and Stem Cell Biology Cancer Stem Cell Vaccination Confers Significant Antitumor Immunity Ning Ning1,4, Qin Pan1,5, Fang Zheng1,6, Seagal Teitz-Tennenbaum1, Martin Egenti1, Ji Yet2, Mu Li1, Christophe Ginestier3, Max S. Wicha3, Jeffrey S. Moyer2, Mark E.P. Prince2, Yingxin Xu4, Xiao-Lian Zhang5, Shiang Huang6, Alfred E. Chang1, and Qiao Li1 Abstract Most studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosup- pressed mice, preventing an assessment on the immunologic interactions and effects of CSCs. In this study, we examined the vaccination effects produced by CSC-enriched populations fromhistologically distinctmurine tumors after their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. Immune sera fromCSC-vaccinated hosts contained high levels of IgGwhich bound to CSCs, resulting in CSC lysis in the presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established that CSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity. Together, these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based on the development of CSC vaccines that can specifically target CSCs. Cancer Res; 72(7); 1853–64. �2012 AACR. Introduction Clinical trials to treat patients with cancer using adoptively transferred T cells (1–3) or dendritic cells (DC; refs. 4–6) have shown therapeutic efficacy for patients with advanced dis- eases. However, the clinical responses to such immunothera- peutic approaches have been confined to a limited percentage of treated patients. To date, bulk tumor masses with hetero- geneous populations of cancer cells have been used as a source of antigen either to generate effector T cells or to prime DC vaccines. Human tumors are composed of heterogeneous tumor cell clones that differ with respect to proliferation, differentiation, and ability to initiate daughter tumors. The inability to target cancer stemcells (CSC)with current immune approaches may be a significant factor for treatment failures. The identification of human CSCs (7–17) presents a new paradigm for the development of cancer treatments. These stem cells have been shown to be relatively resistant to conventional chemotherapeutic regimens and radiation (18, 19) and are postulated to be the cells responsible for the relapse and progression of cancers after such therapies. In an analo- gous fashion, the CSC phenomenon may adversely affect the development of effective immunotherapies for cancer. These therapies have involved targeting cells that express differen- tiated tumor antigens. However, such antigens may be selec- tively expressed on differentiated tumor cells. CSCs that do not express these antigens may thus escape these immunologic interventions. While a few studies have evaluated the resistance of CSCs to the cytotoxic effects of chemotherapy (18, 20–23) and low-dose radiation treatment (19), the immunogenicity of CSCs and their susceptibility to immune-based therapy have not been determined. So far, the majority of CSC studies have been conducted using human tumors inoculated into severely immunosuppressed hosts [e.g., severe combined immunode- ficient (SCID) mice]. These hosts represent very useful models for the studies of the biology, tumorigenicity, and signaling pathways of human CSCs as well as for the screening of small molecules which may lead to the development of new drugs that target CSCs. A very recent report described the isolation of cancer-initiating cells (CIC) using ALDEFLUOR/ALDH as a marker from human head and neck, breast, and pancreatic carcinoma cell lines and the generation of ALDH1A1-specific Authors' Affiliations: Departments of 1Surgery, 2Otolaryngology, and 3Internal Medicine, University of Michigan, Ann Arbor, Michigan; 4Depart- ment of General Surgery, Chinese PLA General Hospital, Beijing; 5State Key Laboratory of Virology, Department of Immunology and Hubei Prov- ince Key Laboratory of Allergy and Immune-related Diseases, Wuhan University School of Medicine; and 6Center for Stem Cell Research and Application, Institute of Hematology, Union Hospital, Tongji Medical Col- lege, Huazhong University of Science and Technology, Wuhan, China Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). N. Ning, Q. Pan, and F. Zheng contributed equally to the work. Current address for C. Ginestier: Centre de Recherche en Canc�erologie de Marseille, U1068-Inserm-Institut Paoli-Calmettes-CNRS, 27, Bd Lei Roure, BP 30059, 13273 Marseille Cedex 09, France. Current address for N. Ning: Department of General Surgery, Hainan Branch of Chinese PLA General Hospital, Hainan, China. Corresponding Author: Qiao Li, University of Michigan Comprehensive Cancer Center, 1150 West Medical Center Drive, 1520 MSRB-1, Ann Arbor, MI 48109. Phone: 734-615-1977; Fax734-763-4135; E-mail: qiaoli@umich.edu doi: 10.1158/0008-5472.CAN-11-1400 �2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org 1853 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 CD8T cells in vitro (24). These T cells eliminated CICs in vivo by adoptive transfer to immunodeficient (SCID) mice bearing human tumor xenografts. However, the absence of adaptive immune responses in the SCID mouse precludes the ability to investigate the host immune response to CSCs. Although normal mouse mammary stem cells have been isolated (25), there is a need to develop model systems where CSCs can be isolated in the immunocompetent host to evaluate the immu- nogenicity of CSCs. In this study, we isolated and assessed the tumorigenicity of murine CSCs in 2 histologically different tumors from 2 genet- ically distinct immunocompetent hosts. From there, we eval- uated the immunogenicity induced by purified CSCs used as a source of antigen to prime DCs as a vaccine. We found that CSC-based vaccines conferred effective protective antitumor immunity which was associated with the induction of humoral and cellular responses that directly targeted CSCs via comple- ment-dependent cytotoxicity (CDC) and CTLs, respectively. Materials and Methods Mice Female C57BL/6 (B6) and C3H/HeNCrMTV (C3H)mice were from Charles River Laboratories. All the animals were main- tained in a pathogen-free environment and used at age 8 weeks or older. The University of Michigan Laboratory of Animal Medicine (Ann Arbor, MI) approved all the animal protocols. Murine tumors D5 is a clone which our laboratory produced (26) from the B16-BL6 tumor line that is a poorly immunogenicmelanoma of spontaneous origin syngeneic to B6 mice (27, 28). SCC7 is a spontaneously arising squamous cell cancer syngeneic to C3H mice also described in our previous report (29). ALDEFLUOR assay The ALDEFLUOR Kit (StemCell Technologies) labels the ALDEFLUORþ/ALDHhigh population including the stem/pro- genitor cells (30–33). TheALDEFLUORassay uses afluorescent substrate of the enzyme (BAAA) freely diffusible across cell membranes. Polar fluorescent products (BAA) accumulate when this substrate is oxidized in cells that express aldehyde dehydrogenase (ALDH). Consequently, cells with high levels of ALDH enzymatic activity stain more brightly (ALDEFLUORþ also referred to as ALDHþ or ALDHhigh) than cells with lower ALDH (ALDEFLUOR� also referred to as ALDH� or ALDHlow). The fluorescent product BAA is trapped in the cells, due to its negative charges. In each experiment, a sample of cells was stained under identical conditions with specific ALDH inhib- itor diethylaminobenzaldehyde (DEAB) as negative control. Flow cytometry–based sorting is conducted using a FACStar- PLUS. The sorting gates are established using as negative controls the propidium iodide (PI)-stained cells for viability and the ALDEFLUOR-stained cells treated with DEAB. Test of tumorigenicity of ALDEFLUORþ cells Equal number of ALDEFLUORþ or ALDEFLUOR� tumor cells mixed with Matrigel (BD Biosciences; 1:1) were injected into the opposite side of the syngeneic mice. Tumor size was measured every 3 to 4 days. Vaccination To examine the protective antitumor immunity induced by vaccination with DCs pulsed with the lysate of ALDEFLUORþ cells (CSC-TPDC), ALDEFLUORþ/ALDHhigh and ALDEFLUOR�/ALDHlow cells were isolated as described above either from cultured D5 and SCC7 cells or from freshly harvested growing tumors from initial respective ALDEFLUORþ D5 or SCC cell injection. ALDEFLUORþ, ALDEFLUOR�, and unsorted cells were frozen and thawed 3 times to make cell lysate. Bone marrow–derived DCs were cultured in interleukin (IL)-4 and granulocyte macrophage colony—stimulating factor (GM-CSF) as previously described in our laboratory (5, 27) and were pulsed with tumor lysate to generate tumor lysate–pulsed DCs (TPDC). After 24 hours of coculture, normal animals were vaccinated with CSC-TPDC or DC pulsed with lysate from unsorted heterogeneous tumor cells (H-TPDC) or DCs pulsed with sorted ALDEFLUOR� cell lysate (ALDHlow-TPDC) at the same DC to tumor cell lysate ratio as CSC-TPDC. Tumor challenge After vaccination, the B6 mice were challenged with the heterogeneous D5 tumor cells intravenously and the lungs harvested 20 days later to enumerate lung metastases. In SCC7 model, the C3H mice were challenged with the heterogeneous SCC7 tumor cells subcutaneously on the opposite side of the vaccine and the tumor size was monitored. Antibody production To test systematic immune responses conferred by CSC- based vaccine, spleens were harvested at the end of experi- ments. Spleen B cells were activated with lipopolysaccharide (LPS) plus anti-CD40 (FGK45) monoclonal antibody (mAb) ascites as previously described (28). After activation, super- natants were collected and analyzed for IgG production. CSC binding by immune plasma Plasmawas collected from vaccinated hosts at the end of the experiments. IgG level was tested using ELISA. ALDEFLUORþ cells were washed with fluorescence-activated cell-sorting (FACS) buffer, blocked with anti-CD16/CD32 (BD biosciences), and incubated with the plasma with equal quantity of IgG for 60minutes on ice. Cells were washed again and incubated with fluorescein isothiocyanate (FITC) anti-mouse IgG (0.5 mg/106 cells) for 30 minutes on ice. Cells were then washed and their binding to plasma IgG was detected using flow cytometry. Antibody and complement-mediated cytotoxicity CSC lysis mediated by antibodies in plasma was assessed by incubation of 105 viable ALDHþ CSCs or ALDH� non-CSCs (serving as control) with plasma in test tubes on ice for 1 hour followed by culture in the presence of rabbit complement (Calbiochem) in a 37�C water bath for another 1 hour. Viable cells were then counted after trypan blue staining to calculate CSC lysis: % of viable cells ¼ viable cells counted after plasma Ning et al. Cancer Res; 72(7) April 1, 2012 Cancer Research1854 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 and complement incubation/105. Lower percentage of viable cells at the end of incubation indicates more cell lysis. CTL cytotoxicity CTLs were generated from the peripheral blood mononu- clear cells (PBMC) or splenocytes harvested from vaccinated animals by anti-CD3/CD28 activation and IL-2 expansion, which consistently results in more than 90% of CD3þ T cells (data not shown). CTL-mediated CSC cytotoxicity was tested using the lactate dehydrogenase (LDH)ReleaseAssay (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega) according to the manufacturer's protocol. The following formula was used to calculate cytotoxicity: % Cytotoxicity ¼ Experimental�Effector spontaneous�Target spontaneous Target maximum� Target spontaneous �100 Statistical analysis The significance of difference in tumorigenicity, metastatic nodules, tumor size, the concentration of IgG, and CSC lysis by antibodies or CTLs was determined using unpaired t test. P values<0.05were considered statistically significant between the experiment groups. Results Identification of CSCs in two syngeneic immunocompetent animal models We have previously described the isolation of stem cell– enriched populations using ALDEFLUOR/ALDH as a marker (30, 33, 34). Using this technique, we identified CSC-enriched populations in 2 different immunocompetent murine tumor models. As indicated in Fig. 1, approximately 4% to 6% of the cultured murine melanoma D5 and squamous cancer SCC7 cells are ALDEFLUORþ/ALDHþ/high; with the rest (�95%) being ALDEFLUOR�. The existence of a small percentage of ALDEFLUORþ cells in established murine tumors was con- firmed by analyzing freshly harvested tumor cells. Processed tumor cells from in vivo established D5 and SCC7 murine tumors also revealed approximately 5% of the ALDEFLUORþ cells (Fig. 1). To determine the purity of the sorted cells, the whole ALDEFLUORþ/ALDHþ/high cells and an approximately equal number (5%–15%) of the ALDEFLUOR� cells used for the ALDH+/high 91.8% ALDH+/high 82.2% ALDH-/low 90.3% 5.6% 5.3%5.1%6.7% ALDH+/high 93.1% ALDH-/low 94.3% 4.4%15% ALDH-/low 95.2% ALDH+/high 92.7% Culture cells Freshly harvested tumor cells D5 cells 6.1% 5.7% SCC7 cells With DEAB With DEAB With DEAB With DEAB ALDH-/low 95.6% Figure 1. Existence of ALDEFLUORþ/ALDHþ/high populations in murine D5 melanoma and SCC7 squamous cell tumors. The ALDEFLOUR Kit labels the ALDEFLUOR-positive population including the stem/progenitor cells. The ALDEFLOUR assay isolates the populationwith a high ALDH enzymatic activity. As negative control, an aliquot of each sample of cells was treated with 50 mmol/L DEAB, a specific ALDH inhibitor. To test the purity of the sorted cells, the whole ALDEFLUORþ/ALDHþ/high cells (4%–6%) and an approximately equal number (5%–15%) of the ALDEFLUOR� cells used for the subsequent immunogenicity analyses (ALDH�/low cells) were collected and restained with ALDEFLOUR using the same staining protocol. The percentages of ALDHþ/high and ALDH�/low cells are listed with the purity of 7 of the 8 restains being higher than 90%. Antibody and T-cell Targeting of Cancer Stem Cells www.aacrjournals.org Cancer Res; 72(7) April 1, 2012 1855 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 dell 高亮 subsequent immunogenicity analyses (ALDH�/low cells) were collected and restained with ALDEFLUOR using the same staining protocol. High percentages (>90% in 7 of the 8 restains) of the ALDH�/low cells (in blue) and ALDHþ/high cells (in red) after restaining confirmed the purity of originally sorted cells (Fig. 1). The tumorigenicity of sorted D5 melanoma ALDEFLUORþ cells was evaluated in the syngeneic immunocompetent C57BL/6 host. ALDEFLUORþ D5 cells (5,000 per inoculum) generated large size tumors in 19 days (Fig. 2A), whereas equal numbers of ALDEFLUOR� cells injected into the opposite side of the same mouse failed to generate tumors. Separate mice 0 10 20 30 0 50 100 150 50,000 ALDEFLUOR– cells 50,000 ALDEFLUOR+ cells * P = 0.045 (n = 3) Days after tumor inoculation Tu m or s iz e (m m2 ) ALDEFLUOR– 5,000 cells ALDEFLUOR+ 5,000 cells ALDEFLUOR– 500 cells ALDEFLUOR+ 500 cells ALDH– 20,000 cells ALDH+ 20,000 cells ALDH– 2,000 cells ALDH+ 2,000 cells ALDH– 200,000 cells ALDH+ 200,000 ALDH+ 20,000 ALDH– 200,000 ALDH– 20,000 ALDH+ 2,000 ALDH– 2,000 ALDH+ 200,000 cells Day 40 Day 68Day 54 Days 0 20 40 60 80 A D5 B SCC7 Tu m or s iz e (m m2 ) 400 300 200 100 0 Figure 2. Testing of tumorigenicity of ALDEFLUORþ populations in D5 and SCC7 tumor models. Equal numbers of D5 (A) or SCC7 (B) ALDEFLUORþ and ALDEFLUOR� cells were injected into the opposite side of the same mouse. Tumor growth was then observed. ALDEFLUORþ cells can form tumors more efficiently than ALDEFLUOR� cells. Ning et al. Cancer Res; 72(7) April 1, 2012 Cancer Research1856 were injected with much lower numbers of ALDEFLUORþ D5 cells. In 4 weeks, 500 injected ALDEFLUORþ cells formed tumors (Fig. 2A). In contrast, the curve in Fig. 2A shows that as many as 50,000 ALDEFLUOR� D5 cells did not grow. The tumorigenicity of sorted SCC7 ALDEFLUORþ population was evaluated in the syngeneic immunocompetent C3H host. As was the case for the D5 model, only the ALDEFLUORþ (as few as 2,000) cells grew into tumorswhereas equal numbers or even much greater numbers (as many as 200,000) of ALDEFLUOR� cells failed to generate tumor (Fig. 2B). These results indicate that the ALDEFLUOR� tumor cells are less tumorigenic than ALDEFLUORþ cells. Collectively, these data indicate that ALDEFLUOR/ALDH can serve as a reliable marker for the enrichment of murine D5 and SCC7 CSCs. This has allowed us to characterize CSC- induced antitumor immunity in the immunocompetent murine host in the subsequent experiments. CSC vaccination confers significant protective antitumor immunity DCs pulsed with whole tumor lysate have been reported to be an effective vaccine for cancer both in animal studies (35) and in clinical trials (4) including the findings reported by our own group (5, 27). To examine whether DCs pulsed with the lysate of CSCs are more effective in inducing antitumor immu- nity, we evaluated the protective antitumor immunity induced by vaccination with DCs pulsed with the lysate of ALDEFLUORþ cells (CSC-TPDC) and used DCs pulsed with the lysate of whole unsorted heterogeneous tumor cells (H-TPDC) as a positive control. In the D5melanomamodel, we used D5 CSCs as a source of antigen. D5 subcutaneous tumors were established and used as a source of CSCs by sorting for ALDEFLUORþ cells. DCs were pulsed with tumor lysate to generate TPDC to be used as a vaccine. Normal immunocom- petent B6 mice were immunized with D5 CSC-TPDC or D5 H-TPDC (at the same lysate:DC ratio as D5 CSC-TPDC). Control groups received PBS. The TPDCs were inoculated subcutaneously for 2 doses (106/each) given 1 week apart. Seven days after the last vaccine, themicewere challengedwith the heterogeneous D5 tumor cells intravenously and the lungs harvested 20 days later to enumerate lung metastases. The study scheme and results are illustrated in Fig. 3. Compared with nonvaccinated, PBS-injected animals (PBS), H-TPDC induced protective immunity against tumor growth, which corroborates with previous observations (27, 35). Of note, the pulsing of tumor cell lysate to DC to generate H-TPDCs was suboptimal compared with what has been reported in the past (4, 5, 27, 34), which may partially explain why H-TPDCs did not immunize effectively (Fig. 3). In these experiments, DCs were pulsed with the lysate of ALDEFLUORþ cells to generate CSC- TPDCs at the ratio of DCs to ALDEFLUORþ cells 3:1 (D5) and 10:1 (SCC7), respectively. This ratio is much lower than the DC: unsorted tumor cell ratio (1:3) as previously described by our group (4, 5, 27). We used fewer tumor cells to pulse DCs to generate CSC-TPDC due to the following reasons: (i) the number of ALDEFLUORþ cells obtained was limited and (ii) we wanted to see the antitumor potential of DCs pulsed with this limited number of ALDEFLUORþ cells compared with the DCs pulsed with the same number of unsorted cells. Impor- tantly, mice that received CSC-TPDC prepared at an identical low lysate to DC ratio resulted in significantly fewer lung metastases than the PBS control group as well as the H-TPDC vaccine group. These results suggested that D5 CSCs are immunogenic and can induce an immune response, even under a suboptimal CSC to DC pulsing condition, which led to decreased lung colonization upon tumor c