Hide-and-seek: the interplay between cancer stem cells and the immune system
Hide-and-seek: the interplay between cancer stem cells and the immune system
Mohammad Sultan 1
Krysta Mila Coyle 1
Dejan Vidovic 1
Margaret Lois Thomas 1
Shashi Gujar 0 1
Paola Marcato 0 1
0 Department of Microbiology and Immunology, Dalhousie University , 5850 College Street, Halifax, Nova Scotia B3H 4R2 , Canada
1 Department of Pathology
The enhanced ability of cancer stem cells (CSCs) to give rise to new tumors suggests that these cells may also have an advantage in evading immune detection and elimination. This tumor-forming ability, combined with the known plasticity of the immune system, which can play both protumorigenic and antitumorigenic roles, has motivated investigations into the interaction between CSCs and the immune system. Herein, we review the interplay between host immunity and CSCs by examining the immune-related mechanisms that favor CSCs and the CSC-mediated expansion of protumorigenic immune cells. Furthermore, we discuss immune cells, such as natural killer cells, that preferentially target CSCs and the strategies used by CSCs to evade immune detection and destruction. An increased understanding of these interactions and the pathways that regulate them may allow us to harness immune system components to create new adjuvant therapies that eradicate CSCs and improve patient survival.
Our defense against invading pathogens is due to innate and
adaptive immune cells and the extensive network of cytokines
that facilitate their interaction ensure the eradication of foreign
antigens. Beyond ridding the body of invading pathogens, the
defensive role of immune cells extends to eliminating
abnormal cells, including mutated transformed cells, which can
prevent tumorigenesis. In the early 1900s, Paul Ehrlich introduced
his magic bullet theory hypothesizing that cancer would occur
at high rates if it was not for the surveillance exerted by the
immune system (
). However, the theory faced strong
opposition. Evidence of tumor formation at sites of inflammation
caused the rise of opposing opinions against the hypothesized
antitumorigenesis role of the immune system (
) and stalled
cancer immunology research for almost 50 years.
In the late 1950s, enhanced understanding of the immune
system and tumorigenesis led Frank Macfarlane Burnet and
Lewis Thomas to individually reinvestigate the possible role
of the immune system in cancer. Although Thomas
postulated that under normal conditions the immune system
prevents tumorigenesis in a similar fashion to graft rejection 3(),
Macfarlane Burnet hypothesized that cancer cells contain
neospecific antigens that can provoke an immune response
leading to the elimination of neoplastic cells (
). Together, their
work laid the foundation for the immune surveillance
hypothesis—that the immune system eliminates nascent cancer cells
and prevents tumor formation (6). Other groups disagreed with
the immune surveillance hypothesis and suggested that the
immune system would be unable to differentiate tumor cells
from normal cells due to their similarities (
). The next wave
of research provided evidence for the protumorigenic nature of
innate immunity after enhanced tumor growth was observed
in proinflammatory settings (
). In the 1990s, the use of
various immune-deficient mouse models revealed that the immune
system may have both supportive and inhibitory roles in
tumorigenesis and metastasis.
More recent studies into the opposing role of the immune
system in cancer progression led to the development of
several new concepts, which define our current understanding of
cancer immunology. The pressure of immune surveillance, in
addition to the highly adaptive and mutagenic nature of tumor
cells, leads to the emergence of specific cancer phenotypes
that are capable of forming tumors in the presence of a
functional immune system (i.e. immunoediting) (
). As discussed in
detail later in this review, immunoediting mechanisms, such as
downregulation of antigen presentation to avoid detection by
immune cells and recruitment of
protumorigenic/anti-inflammatory immune cells to promote cancer progression, are well
utilized by a subset of highly tumorigenic cancer cells, cancer
stem cells (CSCs).
CSCs [also commonly referred to as tumor-initiating cells
(TICs)], cancer-initiating cells or stem-like cancer cells are
believed to initiate cancer, mediate metastasis and contribute
to therapeutic resistance and recurrence (
). CSCs were first
defined in the 1990s by Dr John Dick’s laboratory, which isolated
a population of acute myeloid leukemia (AML) cells with distinct
cell surface marker expression (i.e. CD34+CD38−) that had
significantly enhanced leukemogenesis properties in
immunocompromised mice (
). Shortly after, the same group was able to
identify CSCs, or leukemia-initiating cells, in three other forms
of leukemia (
). Investigation for the presence of cell
populations with tumor-initiating and self-renewal properties led to
the discovery of the first CSC population in solid tumors by Dr
Michael Clark’s group in breast cancer 1(0). They illustrated that
breast cancer cells with CD44+CD24− surface marker expression
had increased tumorigenicity and yielded tumors with
heterogeneous cell surface marker expression. Subsequently, CSCs
were isolated and described in other forms of solid tumors.
Many of these CSC populations were identified by CD133+, such
as in glioblastoma (
), liver (
), colon (
) and pancreatic
), or by CD44+CD24−, such as in pancreatic cancer (
prostate cancer (
). Additional CSC cell surface markers have
since been discovered, such as CD90 for glioma, liver and breast
cancer; CD166 for colon and prostate cancer; CD117 for lung and
ovarian cancer; CD166 for colon and prostate cancer; and CD20
and CD271 for melanoma (
). In addition to identification by
cell surface marker expression, the CSCs of many cancers can
be identified by vital dye exclusion due to the increased
expression of efflux pumps (23). In fact, transporter ABCG2 identifies
pancreatic CSCs, and transporters ABCB5 and ABCB1 (MDR1) are
used to identify melanoma CSCs (
). Additionally, increased
aldehyde dehydrogenase (ALDH) activity as measured by the
Aldefluor assay is used to identify the CSCs of a number of
cancers including breast, colon, lung, melanoma, pancreatic and
prostate cancer (
). It is important to note that the
identification of cells that express these markers do not necessarily
define them as CSCs, but rather identifies a population of tumor
cells that is enriched for the cells with the CSC phenotype. The
identification of tumor cells based on a combination of these
markers (e.g. ALDH+CD44+CD24−) identifies tumor cells of greater
tumorigenicity than those identified based on single markers
(11). This suggests that the most robust CSC studies should use
multiple markers and assays to confirm identification of the
In addition to increased tumorigenicity, further
investigation of CSCs revealed that they have increased resistance to
conventional treatments, which could contribute to
incomplete response to chemotherapy and radiation, and therefore
cause relapse and decreased patient survival. Several
mechanisms have been described that cause decreased sensitivity to
these conventional treatments including increased expression
of detoxification enzymes such as ALDHs (
), quiescence (
hyperactivation of the DNA damage response (
) and increased
drug efflux pump activity (
The interaction between CSCs and the immune system is
of much interest, as their increased tumorigenicity suggests
they may have enhanced immunoediting mechanisms. In this
review, we will discuss the role of major immune cells in
promoting CSC expansion and therefore promoting
tumorigenesis. Furthermore, we will review the evidence for CSC-specific
avoidance of immune cell detection and destruction, as well as
studies illustrating that CSCs can elicit protumorigenic immune
cell activities. Finally, we will discuss the therapeutic potential
of targeting these immune cell interactions to eliminate CSCs.
Role of immune cells in regulating CSC maintenance and differentiation
The role of the immune system in promoting and maintaining
tumorigenesis has been largely attributed to innate immune
cells [i.e. macrophages, myeloid-derived suppressor cells
(MDSCs), dendritic cells (DCs) and T regulatory (Treg) cells] (
The mechanisms for this have been largely explored outside of
the context of CSCs. Below, we summarize the role of distinct
innate and adaptive immune cells in tumorigenesis followed by
the evidence for their role in CSC-specific expansion and
maintenance (Figure 1, Table 1).
Macrophages are important innate immune effector cells. Along
with neutrophils, they constitute the first line of defense against
invading pathogens. Normally, macrophages bind and ingest
invading pathogens, act as antigen-presenting cells (APCs)
and produce proinflammatory cytokines (
). In cancer,
tumorassociated macrophages (TAMs) are an essential component of
the tumor microenvironment. Macrophages/TAMs can either
target cancer cells and mediate their destruction or promote
tumorigenesis and metastasis, depending on their
predominant phenotype: M1 (proinflammatory) or M2
(anti-inflammatory), respectively (
). The predominant TAM phenotype
is governed by the cytokine profile and the interaction with
other immune cells. Adding to the complexity, the distinction
between the M1 and M2 phenotypes is not always made in the
The interaction between TAMs and CSCs has been exten
sively studied. It can be direct or indirect and can have a range
of consequences on CSCs including effects on chemoresistance
properties, maintenance and differentiation. TAMs produce
many cytokines including milk fat globule epidermal growth
factor 8 (MFG-E8) and interleukin 6 (IL6), which activate signal
transducer and activator of transcription 3 (STAT3) and the
Hedgehog signaling pathway in CSCs, promoting their drug
resistance (Figure 1A) (
). In hepatocellular carcinoma, IL6
produced by TAMs activates STAT3, which causes the expansion of
CD44+ CSCs, and ultimately enhances tumorigenesis. This
TAMmediated expansion of hepatocellular CSCs was halted in vitro
and in vivo by inhibiting IL6 signaling with anti-IL6-R antibody
). TAMs have also been found to induce
transforming growth factor β1 (TGFβ1)-driven
epithelial-to-mesenchymal transition (EMT) in a hepatoma cell line, promoting CSC
properties; this effect was blocked by depleting TGFβ1 (Figure 1B)
). Staining of a 96-pancreatic ductal adenocarcinoma (PDAC)
patient tumor microarray for CSC markers CD4+4CD133+ and
TAM marker CD204+ revealed a potential association between
CSC and TAM prevalence in the tumors, which was associated
with shorter overall and disease-free survival 4(0). Additionally,
a recent study showed that breast CSCs interact directly with
TAMs and tumor-associated monocytes via CD90/CD11b
anchoring (Figure 1C) (
). This physical interaction leads to the
activation of the EphA4 receptor, which activates nuclear
factor-kappaB (NF-κB) as well as Src signaling pathways in CSCs.
The juxtracrine signaling cascade culminates with the
secretion of cytokines [IL6, IL8 and granulocyte macrophage
colonystimulating factor (GM-CSF)], by the CSCs, which maintain the
CSC state. CSC and TAM interactions can be mediated by other
components of the extracellular matrix. Breast CSCs isolated
from cell lines via cell surface markers upregulate hyaluronan
synthase 2 (HAS2), which produces the extracellular matrix
component hyaluronan (Figure 1D) (
). Pericellular hyaluronan
mediates the interaction between CSCs and TAMs and promotes
the secretion of platelet-derived growth factor BB (PDGF-BB) by
TAMs. PDGF-BB activates stromal cells (e.g. fibroblasts) in the
tumor microenvironment, which enhance CSC self-renewal
ability by secreting fibroblast growth factor 7 (FGF7) and FGF9.
Importantly, inhibiting HAS2 in CSCs with
4-methylumbelliferone halted tumor growth and resulted in fewer incidences of
metastasis. This suggests that the inhibition of HAS2 and the
interaction between CSCs and TAMs may be a potential novel
therapeutic strategy. Finally, TAMs may indirectly drive CSC
differentiation by promoting the production of interferon gamma
(IFN-γ) by other immune cells such as natural killer (NK) cells
). Importantly, the direct and indirect effects of TAMs and
other immune cells such as NK cells on CSC maintenance and
differentiation may also have the eventual consequence of
altering the tumor’s ability to evade the immune system (e.g.
expression of major histocompatibility complex class I; MHC-I) (44).
Myeloid-derived suppressor cells
MDSCs are a heterogeneous population of immature myeloid
cells derived from myeloid progenitor cells, and they
regulate immune responses to infection, inflammation and cancer
). MDSCs affect immune responses by inhibiting NK cells,
DCs, natural killer T (NKT) cells and T cells (
). Within the
tumor microenvironment, MDSCs induce immune suppression
), and there is some evidence of CSC-specific interactions
with MDSCs. MDSCs were reported to induce microRNA-101
Immunotherapy and potential avenues for targeting cancer stem cells
Targeting PD1/PDL1 on T cells and CSCs, respectively
Targeting STAT3 signaling
(mir-101) expression in ovarian cancer cells 4(
of mir-101 inhibits C-terminal binding protein-2 (CTBP2),
resulting in increased expression of stemness genes and
CSCassociated properties (Figure 1E). Furthermore, the pancreatic
cancer tumor microenvironment can induce monocyte
transformation to monocytic-MDSCs by activating STAT3 signaling
in the cell (Figure 1F) (
). This coincided with increased EMT
of the tumor cells and frequency of ALDH+ tumor cells. This
suggests that targeting STAT3 might inhibit MDSC-mediated
expansion of CSCs.
A well-orchestrated immune response against an invading
pathogen is composed of complementing innate and adaptive
responses, which ensure removal of the pathogen. DCs link
innate and adaptive immunity and facilitate interaction between
different immune cells (
). They are the most important APC
and are critical in T and B lymphocyte activation and
expansion. The key role of DCs in antigen presentation made them a
prime candidate in investigation and development of
immunotherapies and vaccines. However, there is also evidence that DCs
can have adverse effects in cancer by promoting CSC enhanced
tumorigenicity. In follicular lymphoma, follicular DCs promoted
CSC chemoresistance and tumorigenesis via the CXCL12/CXCR4
signaling axis (Figure 1G) (
). Importantly, tumor formation was
abolished on treatment with CXCL12/CXCR4-specific inhibitor,
AMD3100, suggesting potential avenues of therapeutic
intervention. Moreover, tumor-infiltrating DCs can play an important role
in inducing immune tolerance and halt antitumor immunity (
DC-induced tolerization has been attributed to hampered
endocytic activity (
) and low expression of costimulatory molecules
leading to inefficient antigen presentation (
). Although the
role of CSCs in promoting DC tolerization is yet to be explored, it
could provide important insight on the process of tumor
initiation under immune surveillance.
As their name indicates, Tregs regulate the function and
activation of other immune cells. Importantly, they prevent
autoimmunity by inducing tolerance to self-antigens and dampening
immune cell activation (e.g. anergize T cells) (
). Tregs also
play an essential role in the ability of cancer cells to evade
antitumor immunity by promoting anergy of effector immune
cells, rendering these cells unable to recognize and destroy
cancer cells (
). Although not much is known about the
interaction between CSCs and Tregs specifically, a recent
colorectal cancer study showed Foxp3+IL17+-identified Tregs promote
the development TICs by secretion of hypoxia-induced IL17
(Figure 1H) (
Immune evasion mechanisms of CSCs
Antigen presentation by the cells in our bodies is a constant
process by which self-tolerance is established and abnormal or
stressed cells are eliminated by the immune system. Therefore,
a key initial step in adaptive immune recognition and targeting
of tumor cells is antigen processing and presentation of
neoantigens or altered self-antigens. Antigen processing requires
proteosomal degradation of cytosolic proteins to form peptides.
These peptides are then transported to the endoplasmic
reticulum (ER) by the transporter associated with antigen processing
(TAP) heterodimer, which is composed of TAP1 and TAP2. From
the ER, peptides that originated in the cytosol can be loaded
onto MHC-I molecules, which are expressed on all nucleated
cells. Alternatively, antigens which have been internalized by
phagocytosis, macropinocytosis, endocytosis or similar
mechanisms are typically presented on MHC-II molecules by
professional APCs (DCs, macrophages, B cells) (
Upon antigen presentation, naive CD4+ T helper cells and
CD8+ cytotoxic T lymphocytes can be activated to recognize and
destroy cells presenting foreign antigen (Figure 2A). Tumors cells
commonly evade immune detection by downregulating several
important components of the antigen processing and
presentation pathways, and thereby prevent the exposure of
neo-antigens. This phenomenon appears to be enhanced in the CSCs of
several cancer types (Figure 2, Table 1). For example, CD44+
putative CSCs from head and neck squamous cell carcinoma have
decreased MHC-I and TAP2 expression in comparison with the
CD44− putative non-CSCs (Figure 2B and C) (
). Similarly, MHC
and/or TAP molecules expression was lower in the putative
CSCs of melanoma, glioblastoma and colorectal cancer (
However, it should be noted that at least one study reported no
difference in MHC-I expression between the CSCs and non-CSCs
of colon cancer (
). The decreased efficiency in antigen
processing and downregulation of antigen presentation makes CSCs
poor targets for the cell-mediated immune response of T cells.
This contributes to the immune privileged status of CSCs within
the tumor microenvironment. Downregulation of antigen
processing and presentation is also evident in stem cells. Embryonic
stem cells and the undifferentiated NTera2 teratocarcinoma cell
line (pluripotent human embryonal carcinoma cell line) have
reduced levels of restricted antigen processing machinery (e.g.
TAP1) and absent expression of β2-microglobulin, which
limits expression of MHC-I on the cell surface of these cells (
Differentiation of the cells increased expression of the antigen
presentation genes, connecting the differentiation state of cells
with expression of MHC-I. The lack or low expression of the
antigen presentation machinery in undifferentiated cells is in part
Figure 2. Cancer stem cells employ several mechanisms to evade detection. (A) Several signals are required to activate T cells: MHC-I/II antigen presentation to TCR
of TAP1/2-processed antigen, costimulatory signals via CD80/86 and activating cytokines. (B–E) CSCs interfere with antigen presentation and costimulatory signals to
prevent cytotoxic T cells from targeting them (
). (B) Head and neck squamous cell carcinoma CSCs can downregulate MHC-I/II molecules to prevent antigen
presentation to T cells and therefore limit their activation (60). (C) CSCs from various cancers downregulate TAP1/2 resulting in lack of antigen presentation to T cells,
limiting their activation (
). (D) CSCs downregulate CD80 and (E) upregulate PDL1 to induce T-cell anergy (
due to epigenetic regulation, including repression by histone
modifications and DNA methylation. Similarly, hematopoietic
stem cells also demonstrate low levels of MHC molecules ( 67),
suggesting that immune privilege may be a feature of stemness
maintained in CSCs. Treatment with a DNA demethylation agent
increased expression of antigen presentation machinery in
glioblastoma CSCs, although to a lesser extent than in differentiated
tumor cells, suggesting that several mechanisms contribute to
the altered expression of the genes (
). Furthermore, as
discussed in a later section of this review, decreased expression of
MHC-I expression also makes CSCs targets of other immune cells
(e.g. NK cells).Therefore, it is often the balance of multiple signals
that determines the fate of CSCs and tumor cells—eradication by
the immune system versus evasion of the immune system.
Antigen presentation on MHC molecules is rarely sufficient
to activate T cells. In the absence of a costimulatory signal, T
cells become apoptotic or anergic—they cannot be activated.
This costimulatory signal can be provided by CD80 and CD86
on the APC, which interact with CD28 on T cells. Alternatively,
inhibitory molecules like PDL1 (B7-H1), which can be found on
APCs and tumor cells, bind to PD1, causing T cells to become
anergic or apoptotic (Figure 2D and E). Upregulation of inhibitory
molecule PDL1 is a known mechanism utilized by tumor cells to
inhibit T-cell activity (
). Similarly, putative CSCs from
glioblastoma and head and neck squamous cell carcinoma expressed
very low levels of the costimulatory molecule CD80 and high
levels of the inhibitory molecule PDL1 (
). This suggests that
CSC interactions with T cells may be especially inhibitory and
promote T-cell anergy (
). As discussed in the final section
of this review, elevated levels of PDL1 on CSCs could provide a
potential avenue for CSC-targeting immunotherapy.
CSCs promote the expansion of protumorigenic immune phenotypes
Although certain immune cells (TAMs, MDSCs, DCs andTregs)
contribute to the maintenance of CSCs, the inverse is also true—CSCs
induce the expansion of protumorigenic immune cell activities
and eliminate antitumorigenic cell activities (Figure 3, Table 1).
This CSC-induced activity on components of the immune system
has the same end result of promoting tumorigenesis.
CSCs can promote macrophage polarization toward the
immunosuppressive M2 phenotype, away from the proinflammatory
M1 phenotype (Figure 3A) (
). Glioblastoma CSCs produce
soluble colony-stimulating factor (sCSF), TGFβ1 and macrophage
inhibitory cytokine (MIC-1) promoting macrophage recruitment,
polarization toward the M2 phenotype and inhibition of
macrophage phagocytic activity. This helps shield the tumor from
the immune system. Furthermore, incubating macrophages in
glioblastoma CSC-conditioned medium promoted the
secretion of immunosuppressive cytokines including IL10 and TGFβ1
and enhanced their ability to inhibit proliferation of T cells. The
capacity of CSCs to induce immunosuppressive macrophages
could be reversed by inhibiting STAT3 signaling in the CSCs (
In addition, CSCs can inhibit the expansion of
antitumorigenic immune cells. A xenotransplant model of AML demon
strated the ability of leukemia stem cells to inhibit the activity
of antitumor macrophages through SIRPα signaling via
engagement with the CD47 receptor (Figure 3B). Treatment with
SIRPα-Fc fusion protein disturbed the SIRPα-CD47 engagement
and enhanced the ability of macrophages to phagocytose AML
stem cells without affecting normal hematopoietic cells (
CSC-induced expansion of protumorigenic immune cells goes
beyond macrophages and includes other innate effector immune
cells, such as neutrophils. Neutrophils are the most common
leukocytes in the blood. They have a well-characterized role in
the elimination of pathogens and a more recently discovered
role in the regulatory circuits of adaptive immunity. The role of
neutrophils in cancer progression and metastasis has been
generally associated with their capacity as inflammatory immune
). A recent study has also linked neutrophils with glioma
stem-like cells (GSLCs) by examining glioma patient tumor
samples (Figure 3C) (
). They identified niches where GSLCs reside
in close proximity to endothelial cells. These niches were
positive for stromal derived factor-1α (SDF1α; also known as CXCL12),
CXCR4, osteopontin and protease cathepsin K. SDF1α/CXCL12
binds to CXCR4 on GSLCs and retain them in the niche, whereas
osteopontin promotes neutrophil and macrophage infiltration in
the niche. In turn, these infiltrating leukocytes produce neutrophil
elastase and matrix metalloproteinase 9 (MMP-9), which together
with cathepsin K, may degrade SDF1α/CXCL12. The action of the
proteases on SDF1α/CXCL12 may release GSLCs from their niche,
facilitating glioma invasion.
CSCs can alter the adaptive immune landscape by targeting
effector T cells and inducing the expansion of
immunosuppressive, cancer-promoting Tregs. Two recent studies showed
that the CSCs from glioblastoma and head and neck squamous
cell carcinoma impair the proliferation of effector T cells 6(
and induce the expansion of Tregs (Figure 3D) (
cytokines produced by ABCB5+ identified malignant
melanomainitiating cells inhibit effector T cells and instead induce
recruitment of CD4+CD25+FoxP3+-identified Tregs to the tumor site (
NK and NKT cells may play a key role in targeting CSCs
In contrast with most of the evidence discussed thus far, there
are indications that certain elements of the immune system
may be effective at eliminating CSCs, specifically NK and NKT
cells (Figure 4, Table 1).
A subset of granular innate lymphocytes, NK cells are
characterized by the presence of specialized lytic organelles containing
preformed cytotoxic and proteolytic enzymes such as perforin,
granzymes and granulysin (
). NK cells recognize and destroy
virus-infected cells, cells undergoing stress response and
abnormal cells in an antigen-independent fashion
(circumventing the adaptive immunity requirement of prior sensitization).
Moreover, as is often in the case of tumor cells, NK cells target
cells that have low expression of, or lack, MHC-I molecules (
This targeted response can be initiated via binding of the NK
cell’s activating receptor NKG2D with its ligands
(stress-inducible MHC-like molecules MICA/B and ULBPs), the binding of
natural cytotoxicity receptors (NCRs) such as NKp30 and NKp44
with their respective ligands, or by antibody-dependent
cellular cytotoxicity (ADCC) where the CD16 Fc receptor on NK cells
binds to cell-bound antibodies. Subsequently, NK cell killing is
mediated by the release of cytotoxic and proteolytic enzymes
and/or the binding of death receptors Fas and DR5. Importantly,
NK activity is necessarily highly regulated to prevent the killing
of healthy normal cells (Figure 4A). NK cells also express
inhibitory receptors such as killer-cell immunoglobulin-like receptors
(KIRs), which bind to inhibitory MHC-I molecules expressed on
cells, and prevent NK cell lysis. A balance between the activating
and inhibitory receptors, as well as activation by cytokines such
as IL2, is required for NK cells to attain their full lytic potential
). NK cells may also play a key role in regulating stem cell
maintenance and differentiation, potentially killing extraneous
non-differentiated stem cells (which have low or lack MHC
) and driving differentiation of cells required for
the regeneration of damaged tissue (
). This dual function
of NK cells is dependent on the predominant phenotype of NK
cells (cytotoxic toward stem cells or anergic and cytokine
producing, which drives the differentiation of the cells) (
NK cells play an especially important role in the
elimination of nascent tumor cells due to their recognition of
abnormal or stressed cells (
). Furthermore, the selective pressure
on cancer cells to downregulate MHC-I to avoid cytotoxic T-cell
destruction makes them susceptible to killing by NK cells. The
innate role of NK cells in cancer surveillance makes them
attractive candidates for immunotherapy; especially in the context of
expanded and activated allogeneic NK cells obtained from the
peripheral blood of healthy donors, since autologous NK cells
harvested from cancer patients often exhibit anergy (83). As
discussed below, in terms of CSC-specific targeting, the majority
of evidence suggests that NK cells exhibit increased targeting
of CSCs; however, there is also some of evidence of enhanced
evasion of NK cell-mediated attack by CSCs (
A number of studies have illustrated that although CSCs are
generally resistant to killing by resting/non-activated NK cells,
activated NK cells have increased anti-CSC activity (Figure 4B)
). In 2009, Castriconi et al. reported that both
allogenic and autologous NK cells, upon activation by IL2 and IL5,
exhibited heightened activity toward patient-derived
glioblastoma cells with CSC-associated characteristics (i.e. enhanced
tumorigenicity, capable of multilineage differentiation) (
Analysis of the CSC population revealed that these cells had
increased expression of NK cell-activating ligands and low MHC-I
expression. Avril et al. later reported that glioblastoma-derived
CSCs were highly susceptible to allogeneic NK cells that were
activated by lectin or IL2 (
). More recently, Ames et al. reported
that activated allogeneic and autologous NK cells preferentially
target CSCs derived from patient tumors of liposarcoma, Ewing’s
sarcoma and PDAC, as well as putative CSCs identified in cell
lines from pancreatic cancer, breast cancer, glioma and sarcoma.
Their analyses revealed that CSC killing by NK cells was
dependent on NKG2D binding to upregulated activating ligands MICA/B
and death receptors FAS and DR5 (
). Similarly, IL2-activated
allogeneic NK cells eliminated primary and metastatic
osteosarcoma cells, including the TIC cell population (
). NK cell killing
was dependent on NKG2D activation with its ligand (NKG2DL) on
tumor cells and TICs. The addition of NKG2DL-inducer
spironolactone enhanced NK cell killing and reduced the osteosarcoma
TIC population (
). Although activated allogeneic NK cells exhib
ited greater cytotoxic activity, both allogeneic and autologous
activated NK cells preferentially killed the cancer-initiating cells
of colorectal adenocarcinomas (
). The cancer-initiating cells
had reduced expression of NK cell inhibitory MHC-I molecules
but elevated expression of ligands to NK cell-activating receptors
NKp30 and NKp44. Finally, a body of evidence suggests that NK
cells can control the CSC population of tumors by two
mechanisms; the killing of undifferentiated CSCs by cytotoxic NK cells
and the differentiation of CSCs by cytokine production of anergic
NK cells (
). Tseng et al. observed that oral squamous CSCs
are more sensitive to activated allogeneic NK cell-mediated
cytotoxicity compared with their differentiated counterparts and the
cytotoxicity of NK cells was dependent on the differentiation state
of the cells (78). The same group found that glioblastoma CSCs
are highly susceptible to activated allogeneic NK cell-mediated
killing by direct lysis or ADCC, in comparison with their
serumdifferentiated counterparts (
). However, glioblastoma CSCs can
become insensitive to NK cell-mediated killing either due to NK
cells anergy or as result of their differentiation, thus, limiting the
ability of NK cells to curtail glioblastoma progression. The
inclusion of a secondary strategy for targeting NK-induced
differentiated tumor cells may improve tumor eradication. Notably, NK
cell-mediated differentiation of CSCs increased their expression
of MHC-I, which may facilitate their targeting by cytotoxic T cells
). Taken together, these findings suggest that CSCs from
multiple cancer types are especially susceptible to lysis by activated
cytotoxic NK cells, that NK cells affect the differentiation state of
the heterogenous tumor and that if the inactivation of NK cells
induced by the tumor microenvironment is overcome, the use
of activated allogenic NK cells as adjuvants in
immunotherapybased approaches targeting CSCs is a viable strategy.
Despite the wealth of evidence suggesting that activated NK
cells have enhanced CSC-targeting ability, there is some evidence
suggesting that glioblastoma, AML and breast CSCs may be
resistant to NK cells (Figure 4C) (
). First, Di Tomaso et al.
characterized CSCs isolated from glioblastomas and revealed that the
cells had decreased expression of inhibitory MHC-I, which would
make them sensitive to NK cell killing; however, the CSCs also
had decreased expression of activating ligands, MICA and UPLBs,
which suggests a potential resistance to NK cells (
unlike the study by Tseng et al. that measured NK cell killing of
glioblastoma CSCs (
), the effect of NK cells on the glioblastoma
CSCs was not assessed in the study by Di Tomaso et al. (
it remains uncertain if the decreased expression of activating
ligands in the CSC population observed by Di Tomaso et al. is
consequential. For AML, She et al. reported that cell line KG1a bore
leukemia stem cells that were resistant to lysis by allogeneic NK
cells activated by IL15 and CpG oligodeoxynucleotide and had low
expression of NKG2D ligands MICA/B and ULBPs (
). Wang et al.
reported that CSCs isolated from breast cancer patient tumors
had low levels of MICA/B and were resistant to lysis by both
allogeneic and autologous IL2-activated NK cells, although in general
the autologous NK cells were less cytolytic than the allogeneic NK
). The downregulation of MICA/B in the breast CSCs was
mediated by increased expression of the oncogenic microRNA
miR20-a. Hindrance of immune surveillance by tumor-infiltrating
NK cells culminated in increased lung metastasis (
contrasting results between these studies (
could be due to differences in how studies were performed, such
as if the CSCs were isolated from patient tumor tissues versus
cell lines, discrepancy in the markers used to identify the putative
CSCs, differences in the tumor microenvironment, which affect
expression of the activating ligands, and the protocols employed
to activate the NK cells. In these studies, activated allogeneic NK
cells were utilized; however, strategies for activation were not
consistent, which may affect the response of the immune cells
to CSCs of certain cancers. Additionally, many of the studies
only addressed the cytotoxic effect of NK cells on CSCs, without
addressing potential effects on differentiation. Future studies are
required to determine how generally applicable NK cell
targeting of CSCs is across all cancers, and whether a combination of
markers that better identify the discrete CSC population should
Expressing both NK cell markers (CD161 receptor; NK1.1) and
the T-cell receptor (TCR), NKT cells link innate and adaptive
immunity and are restricted to CD1d-presented lipid and
glycolipid antigens rather than peptide antigens presented by
). A recent study of colorectal CSCs (identified
by CD44+CD24+CD133+ cell surface marker expression) with
upregulated CD1d was highly sensitive to killing by NKT cells
(Figure 4D) (
). The effect was amplified by cotreating CSCs with
thymosin-α1 (an immune adjuvant and modulator of
). This could potentially lead to the development of NKT
cell adjuvant-based vaccines that target the heterogeneity of
solid tumors by effectively killing CSCs and other tumor cells.
Immunotherapy and potential avenues for targeting CSCs
In addition to the possibility of harnessing NK and NKT cells
to target CSCs (described above), the interactions between CSCs
and immune cells provide other potential avenues of
immunotherapy-based intervention. Below we describe some of the
more clinically advanced immunotherapy approaches being
studied in the context of CSCs ( Table 1).
Higher expression of PDL1 (B7H1) expressed by tumor cells
and activated immune cells of the tumor microenvironment
is associated with more aggressive and advanced disease and
poorer prognosis (
). Cytokines produced in the tumor
microenvironment such as IFN-γ contribute to the increased
expression of PDL1 in the tumor. PDL1 binds to PD1 on CD8+ T cells,
blocking their activation and leading to an immunosuppressive
tumor microenvironment. This is known as immune checkpoint.
Several immunotherapeutic strategies inhibit this interaction,
which prevents T-cell anergy and facilitates T-cell activity against
cancer cells (i.e. checkpoint blockade). For example, nivolumab,
an anti-PD1 monoclonal antibody, has been approved for
melanoma and non-small cell lung cancer treatment (
as mentioned previously, several studies have shown that the
CSCs of some tumors (glioblastoma and head and neck cancer)
express higher levels of PDL1 than other tumor cells (
Similarly, Zhi et al. found that CD133+ colorectal CSCs have
elevated levels of PDL1 and this increase was associated with EMT,
highlighting a possible immune evasion mechanism employed
by CSCs during metastasis (
). Interestingly, PDL1 binding to
PD1 in gastric cancer TICs enhanced their proliferation,
suggesting a potential growth-stimulating role for PDL1 in CSCs (
Furthermore, the CSCs of head and neck squamous cell
carcinoma had elevated PDL1 expression compared with non-CSCs
due to constitutive activation of STAT3 signaling pathway (
IFN-γ treatment further increased PDL1 expression in the CSCs.
The increase in PDL1 correlated with decreased immunogenicity
which can be reverted by STAT3 inhibition or antibody blockade
of PD1 (
). Most recently, Gupta et al. reported that PDL1
knockdown in B16 murine melanoma cells significantly reduced the
CSC population (
). Together these studies suggest that immu
nosuppression conferred by PDL1 expression contributes to
the maintenance and differentiation of CSCs within the tumor.
Importantly, the elevated PDL1 levels in CSCs suggest that using
checkpoint blockade agents such as nivolumab may be an effec
tive way of targeting CSCs via immunotherapy.
Inhibiting STAT3 signaling may also be a potential strategy
for targeting CSCs and their effects on the immune system.
In response to growth factors and cytokines, STAT3 is
phosphorylated and translocates to the nucleus where it induces
transcription of genes. This mediates cellular responses such
as cell proliferation, differentiation and apoptosis, which in
transformed cells contributes to tumorigenesis and metastasis
). Specific to its role in the tumor microenvironment,
activation of STAT3 leads to production of cytokines such as
IL6, which causes an increase in immunosuppressive cells such
as MDSCs and Tregs, and a decrease in the maturation of DCs.
Therefore, STAT3 is a key inducer of an immunosuppressive
tumor microenvironment. As mentioned throughout this review,
STAT3 plays an important role in CSC/immune cell interaction,
with evidence of inhibition of STAT3 as a mechanism of
blocking CSC-associated tumor immune evasion activity (
Several studies illustrated the elevated levels of STAT3 signaling
in the CSC population and its role in promoting the
immunosuppressive properties of CSCs (
immunosuppressive immune cells such as macrophages can activate
STAT3 signaling and promote the drug resistance of CSCs (
In these studies, inhibiting STAT3 blocked the immunosuppres
sive effects of CSCs. Together these finding highlight the
importance of STAT3 signaling in the immunosuppressive network
of CSCs. Although at this time it is unclear what the clinical
impact of STAT3 inhibition is on the immunosuppressive
properties of CSCs, several STAT3 inhibitors are undergoing clinical
trials to assess their effectiveness on different types of cancers.
For example, inhibitor OPB-51602 is showing promising effects
DCs pulsed with the lysate of
DCs pulsed with the lysate of
DCs pulsed with the lysate of
DCs pulsed with the lysate of
DCs pulsed with the lysate of
DCs pulsed with the lysate of
DC transfected with hTERT and
survivin mRNA + CSC mRNA
DCs pulsed with the lysate
of allogeneic glioblastoma
stem-like cell line cultured
DCs pulsed with the lysate of
DC transfected with CSC mRNA
CD105/Yb-1/SOX2/CDH3/MDM2polyepitope plasmid DNA
Stages I and II clinical trials investigating the effect of DC- and DNA-based vaccine in targeting CSCs and eliminating tumors of different types of cancer are
completed or currently recruiting (summarized from information available aCtlinicalTrials.gov).
against non-small cell lung carcinoma (
). Despite this poten
tial success, the ubiquitous presence and general importance of
STAT3 signaling suggests that targeting this pathway may
present some toxicity.
DC-based vaccines are being developed with the goal of
circumventing the downregulation of immune responses by tumor
cells and facilitate tumor cell recognition and killing by
adaptive immune cells. Bone marrow-derived DCs loaded with tumor
antigens to induce antitumor immune responses can either be
specific to a tumor antigen or generated from whole-cell lysates
of tumor cells. For example, DCs presenting mutated RAS
peptides (neo-antigens) or tumor-associated antigens (TAAs) such
as mucin 1, HER2/Neu and melanoma-associated antigen 3
(MAGE-A3) are in clinical trials and show promising results 1(05).
Some studies suggest that this approach will also be able to
target CSCs (
). The elimination of CSCs by DC-based
therapeutic vaccines requires either the identification of CSC-associated
tumor antigens or the isolation of CSC populations from patient
tumors for the generation of whole-cell lysates. Potential
CSCassociated antigens being investigated for DC-based vaccines
include CD133, CD44 and octamer-binding transcription factor
4 (OCT-4), survivin and telomerase reverse transcriptase (TERT)
). Animal models and clinical trials have shown high
efficacy in establishing antitumor immunity and selectively
targeting CSCs by inducing anti-CSC antibodies and cytotoxic T cells
). Together, these findings led to the development of sev
eral clinical trials to assess the effect of DC vaccines loaded with
CSC antigens on several types of tumors (Table 2). However, due
to the potential immunosuppressive nature of CSCs,
combination treatment strategies where checkpoint blockade therapies
are administered concomitantly with DC-based vaccines may be
required to evoke a potent anti-CSC immune response.
The immune system can play opposing roles in the progression
of cancer, having both protumorigenic and antitumorigenic
consequences. Innate and adaptive immune cells contribute
to tumor establishment, growth and metastasis by creating an
anti-inflammatory, protumorigenic environment and
inducing tumor cell migration. In contrast, immune cells also inhibit
tumor development and promote destruction of established
tumors by direct lysis of tumor cells by cytotoxic immune cells.
The immune surveillance hypothesis suggests that when this
balance is shifted toward the cancer cell by immunoediting
mechanisms tumors develop. Studies reviewed here suggest
that CSCs play a pivotal role in shifting this balance, having
profound effects on regulating immune responses. Compounded
by the impact that CSCs have on tumor development,
progression and chemotherapy resistance, understanding the specific
interaction between CSCs and the immune system has become
increasingly important. However, it is apparent that delineating
CSC-specific immune interactions is complex and only partially
deciphered. Reports are at times conflicting, with evidence of
both CSC targeting and evasion with the same immune cell
(e.g. NK cells). Regardless, these finding have triggered
interest into targeting CSCs via immunotherapy-based approaches.
Delineating the molecular components of CSC-specific
immunity will aid in developing immunotherapy approaches that
eliminate the entire tumor without leaving behind
chemotherapy-resistant CSCs. In ongoing and future
immunotherapybased clinical trials, it will be important not only to determine
the effect on tumor regression and patient survival but also to
monitor the effect on CSCs and the immune system.
Canadian Institutes of Health Research (CIHR; MOP-130304 to
P.M.); studentship or trainee awards from the Beatrice Hunter
Cancer Research Institute (to M.S., K.M.C. and M.L.T.); Canadian
Breast Cancer Foundation (to M.S. and M.L.T.); CIBC Graduate
Scholarship in Medical Research (to M.S.); CGS-D award from
CIHR (to K.M.C.); Nova Scotia Health Research Foundation (to
K.M.C., M.S. and M.L.T.); NS graduate scholarship (to M.S., M.L.T.
and D.V.); Killam Laureate scholarship (to K.M.C., M.L.T. and
Conflict of Interest Statement: None declared.
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