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
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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
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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
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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