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Immunotherapy: Newer Therapeutic Armamentarium against Cancer Stem Cells

Immunotherapy: Newer Therapeutic Armamentarium against Cancer Stem Cells Hindawi Journal of Oncology Volume 2020, Article ID 3963561, 15 pages https://doi.org/10.1155/2020/3963561 Review Article Immunotherapy: Newer Therapeutic Armamentarium against Cancer Stem Cells 1 2 3 1 Saurabh Pratap Singh , Richa Singh, Om Prakash Gupta, Shalini Gupta , and Madan Lal Brahma Bhatt Department of Oral Pathology & Microbiology, King George’s Medical University, Lucknow, India Centre for Integrated and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow, India Depatment of Surgery, Prasad Institute of Medical Sciences, Lucknow, India Vice Chancellor, King George’s Medical University, Lucknow, India Correspondence should be addressed to Shalini Gupta; sgmds2002@yahoo.co.in Received 14 March 2019; Accepted 4 February 2020; Published 9 March 2020 Academic Editor: P. Neven Copyright © 2020 Saurabh Pratap Singh et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mounting evidence from the literature suggests the existence of a subpopulation of cancer stem cells (CSCs) in almost all types of human cancers. (ese CSCs possessing a self-renewal capacity inhabit primary tumors and are more defiant to standard an- timitotic and molecularly targeted therapies which are used for eliminating actively proliferating and differentiated cancer cells. Clinical relevance of CSCs emerges from the fact that they are the root cause of therapy resistance, relapse, and metastasis. Earlier, surgery, chemotherapy, and radiotherapy were established as cancer treatment modalities, but recently, immunotherapy is also gaining importance in the management of various cancer patients, mostly those of the advanced stage. (is review abridges potential off-target effects of inhibiting CSC self-renewal pathways on immune cells and some recent immunological studies specifically targeting CSCs on the basis of their antigen expression profile, even though molecular markers or antigens that have been described till date as expressed by cancer stem cells are not specifically expressed by these cells which is a major limitation to target CSCs. We propose that owing to CSC stemness property to mediate immunotherapy response, we can apply a combination therapy approach by targeting CSCs and tumor microenvironment (TME) along with conventional treatment strategies as an effective means to eradicate cancer cells. seen in parental tumors [3]. Accumulated evidences advo- 1. Introduction cate that from the time when CSCs were initially identified in Cancer is a diverse heterogeneous disease which is char- human acute myeloid leukemia (AML), they have been acterized by phenotypically and functionally discrete subsets isolated from many divergent malignancies, including of cells. Data amassed from the literature suggests the cancers of the breast, prostate, colon, brain, pancreas, lung, presence of a small population of cancer cells with stemlike liver, bladder, and ovary [4–8]. properties in a wide continuum of human cancers. Char- CSCs also appear to have resistance to anticancer acterized by self-renewal and differentiation, these cells have therapies leading to relapse. (is deleterious feature of CSC been termed as cancer stem cells or tumor-initiating cells, causes a dire impact on cancer management and hence and we have used CSCs to denote these cells throughout the makes CSCs promising targets for elimination. (ere are a review. CSCs are biologically similar to normal stem cells few ongoing trials that involve immunotherapy strategies (SCs) [1, 2]. CSCs are characterized functionally by the against CSCs [9]. However, in order to design newer intrinsic ability to initiate and long-term repopulate tumors therapeutic approaches, we need a clearer understanding of with a recapitulation of the lineage/cellular heterogeneity the biology of these cells. (e present review aims to 2 Journal of Oncology carcinogenesis due to this pathway [17, 23, 24]. (e focus of determine the feasibility of immune targeting CSCs in solid tumors and also highlights that some of the biological treatment research in cancer is to inhibit the adverse pathway in CSCs [25]. However, the Wnt/β-catenin pathway targetings of CSCs may be ambivalent by also affecting immune responses. is a double-edged sword as it functions as a major player in the regulation of T-cell development and activation, Wnt/ β-catenin signalling is crucial for CD8 memory T-cell 2. RoleofDevelopmentalSignallingPathwaysin development, and on the other hand, the efficacy of im- the Regulation of CSCs munotherapy responses is improved by agonists [26, 27]. Tumorigenesis bears resemblance to abnormal organogen- (e Hedgehog (Hh) signalling pathway plays a key role esis. CSCs exhibit three cell-intrinsic fundamental proper- in tissue wear and tear and homeostasis and epithelial to ties: self-renewal, quiescence, and differentiation. (erefore, mesenchymal transition (EMT) in normal tissues. Bellig- any genetic or epigenetic program that can regulate one or erent manifestations like cancer may be prompted by tu- more of these three properties could theoretically have an morigenesis via aberrant Hh signalling defined by the impact on CSC biology [3]. CSC phenotypes change due to overexpression or loss of function of its ligands or receptor altered genetics via various mechanisms. CSC biology is and dysregulation of transcription factors. Hh signalling can majorly governed by developmental pathways, stem cell be triggered by an array of factors in the tumor microen- factors, cell cycle regulation and apoptosis, epithelial-mes- vironment, such as transforming growth factor- (TGF-) β, enchymal transition (EMT), and epigenetics apart from tumor necrosis factor- (TNF-) α, and interleukin (IL)-6. physiological metabolism. Owing to complex interactions Attenuation of Hh signalling is also attracting focus of re- and overlap between mechanistic programs driving CSC searchers worldwide and undergoing intense exploration for regulation, defining five mechanisms is rather arbitrary, as cancer treatment [28]. Hh signalling is relevant in immune they could all ultimately converge on transcriptional regu- cell development and function [29–32]. Also, Hh inhibitors lation driven by myriad transcription factors [1]. may deliver additional benefits as they are also involved in An array of signalling pathways, namely, Myc, Notch, myeloid-derived suppressor cell (MDSC) function. Singly Hedgehog (Hh), Wnt, FGF/FGFR, EGF/EGFR, NF-κB, targeting any pathway may not yield a physiologically rel- MAPK, PTEN/PI3K, HER2, and JAK/STAT operative in evant level of inhibition as there is a considerable overlap normal SCs during development and homeostasis are fre- between these pathways. Since it is responsible for normal quently found to be deregulated in CSCs [10–14]. Devel- tissue homeostasis and development, including immune cell opmental pathways such as Notch, Wnt/β-catenin, and behaviour and peripheral effector function, targeting them is Hedgehog participate instrumentally in cancers and are a challenging job [33]. frequently altered and are implicated in CSCs regulation [15–18]. 3. Identification and Isolation of CSCs Notch ligands, residing in plasma membrane of neighbouring cells, are transactivated, eliciting the tran- 3.1. Identification Based on Immunophenotypic Marker. scription of Notch target genes, such as c-myc, p21 and p27, Coherent immune targeting of CSCs depends on the PI3K, the hairy and enhancer of split- (HES-) related family, categorization of (i) stem cell-like CSC markers used for protein kinase B (AKT), peroxisome proliferator-activated its isolation and (ii) Antigens expressed on CSCs which receptor (PPAR), NF-κB, and cyclin D1. Depending on the are not preferably express on non-CSC/normal cells. (e particular signalling milieu, these downstream targets get SC biology of the tissues from which tumor originates activated and in turn regulate cell fate leading to differen- forms the basis of identification of CSC markers since the tiation, cell-cycle progression, and survival. Notch may lineage relationship between CSCs and tissue SCs remains decelerate differentiation and endorse cell survival in stem- vague in most tumor systems. For detection and segre- like cells [19, 20]. (ere are numerous ongoing Phase I and II gation of alleged CSC populations, not only are functional clinical trials in cancer with a range of targets and mech- assays like ALDEFLUOR, mammosphere, and neuro- anisms investigating the efficacy of Notch targeting, singly or sphere employed, but also side population (SP) and cell in amalgamation with other therapeutic strategies [19]. surface marker coupled with a fluorescence-activated cell Notch signalling has, nevertheless, also been correlated to sorter (FACS) are put to use. (e immunophenotypic peripheral T-cell maturation into effector cells, such as markers for potential CSCs have been reported in many developing cytotoxic T-cell function, or cytokine production human malignancies [1, 3, 6–8] (Table 1). A range of CSC [21]. Similarly, T-cell activity has been weakened by Notch- markers (CD44, CD133, and HER2) represent promising inhibition using c-secretase inhibitor [22]. targets for CSC immunotherapy, such as antibody ther- Two signalling pathways have been recognized which are apy. However, probable safety issues should be addressed the β-catenin-dependent pathway that is involved in cell fate when utilizing these markers as CSCs since normal SCs determination (canonical), and the β-catenin independent share similar phenotypic marker profiles. Also, a limited pathway that is involved in cell polarity and movement number of CSC markers in solid malignancies are cur- (noncanonical). Commencement of Wnt signalling is fos- rently accessible and their biological function is less tered by the release of soluble ligands by the cells in the near categorized. vicinity. (e gain of function, regulation by methylation, and It is a well-established notion that CSCs in different histone modification have all been implicated in tumor types demonstrate distinctive transcriptomic profiles Journal of Oncology 3 Table 1: CSC Markers in different cancers. CSC markers reviewed in [1, 3, 6–8] Cancer type Positive markers Negative markers Leukemia CD34, CD96, ALDH, CD47, CD44, CD123, TIM-3, CD32, CD25, CLL-1 CD38 Prostate ABCG2, ALDH, CD44, α2β1, CXCR4, CD133, NANOG, TRA-1-60, CD151, CD 166 PSA, CK18, CK20, EMA, Bladder CD44, CK5, CD44v6, 67LR, CK17, ALDH, SOX2 CD66c, Breast CD44, PKH26, CD49f, ALDH, CD133, CD90 CD24 Lung CD133, ALDH, CD117/c-kit, OCT4, NANOG, CD44, TPBG/5 T4, CD166, CD44, EpCAM Pancreatic CD44v6, α6β4, TSPAN8, CXCR4, CD44, CD24, ESA, c-Met, ALDH, CD133, CD133, ABCG2, SSEA-1, SOX2, BMI1, MUSASHI1, NESTIN, OLIG2, CD49f, A2B5, L1CAM, EGFR, Glioblastoma CD44, ID1, MYC, ALDH Ovarian ALDH, CD44, CD117/c-kit, CD44, MyD88, CD24, CD133, CXCR4 Colon CD133, EpCAM, CD44, CD166, ALDH, LGR5, ABCB5 Liver CD133, ALDH, EpCAM, CD90, CD44, CD13, SALL4 CD45 and thereby express discrete antigens. (ey express a downregulation or masking, or expression of splice variants. continuum of tumor-associated antigens (TAAs) that have (e ALDEFLUOR assay uses N,N-dieth- the capacity to be identified by the immune system of the ylaminobenzaldehyde (DEAB) to measure the activity of the host [3]. TAAs may be categorically illustrated in CSCs as ALDH1 family for which it acts as a specific inhibitor [35]. four subgroups [3, 8, 31], namely, (i) nominal expression by Nevertheless, it can be inferred that the occurrence of high normal tissues but constitutively highly expressed by tu- ALDH cells is underrated in tissues in which the chief mors as part of being their malignant phenotype like EGFR ALDH isozyme is not a member of the ALDH1 family. and surviving, (ii) differentiation antigens that are tissue- Studies on a variety of cancers such as prostate cancer, specific and expressed by both benign and cancer cells, (iii) ovarian cancer, breast cancer, lung cancer-derived primary CT cancer/testis antigens inadvertently activated in tumors culture, and nonmetastatic/metastatic cell lines of these like MAGE-A3 and MAGE-A4 that are normally confined cancers suggest variation in ALDH expression [36–47]. (is to placenta and testicular germ cells, and (iv) neoantigens inconsistency could refute potentially applicable CSC in cancer genomes derived from mutations leading to new markers and also brings into limelight the significance of epitopes identifiable by immune system like MUM-1 and testing in multiple cell lines/primary tissues. (e clinical CDK4 in melanoma. Innumerable lineage-specific markers outcome in a variety of cancers has been attributed to ALDH are categorised as differentiation antigens, namely, PSA in expression [48–50]. ALDH has been implicated in resistance PCa and MART-1 in melanoma. CSC markers can mechanisms to radiotherapy and chemotherapy; thus, a henceforth be thought to act as overexpressed antigens more specific, diverse therapeutic approach must be although not all are expressed minimally in normal cells. embarked upon. Some research groups have been attracted CT antigens and neoantigens have been considered best for towards investigating inhibitors and involved in developing CSC-targeting immunotherapy although lineage differen- more specific inhibitors through drug discovery approaches, tiation antigens are expressed in low amounts in CSCs as the common expression of ALDH in healthy stem cells [3, 8, 31]. which can cause an off-target activity [35]. It is possible that functional redundancy within the isozyme family could high recompense the inhibition for one ALDH. ALDH CSC- 3.2. Identification Based on Metabolism. (ere is a meta- loaded dendritic cells (DCs) have been used fruitfully in two bolism-based identification which is based on the activity of in vivo melanoma models in the immunotherapy setting certain enzymes such as aldehyde dehydrogenase (ALDH) [51, 52]. In transplantation setting, ALDH activity has been and mitochondrial glycolytic activity. demonstrated in the T regulatory cell (T ) immune subset; reg (e ALDH family is encompassed by a gamut of 19 genes thus, targeting of ALDH may also show an antitumor impact (in humans) that articulate enzymes for catalysis of aldehyde through modulation of T subset [53]. reg oxidation. Metabolism of vitamins, amino acids, and lipids CSCs possess a lesser number of mitochondria and requires aldehyde oxidation [34]. Elevated ALDH activity is consequently show more glycolytic activity than other tumor linked with both normal SCs and CSCs. (ese enzymes also cells, as evident in liver, breast, melanoma, and lung cancer exert a shielding detoxifying effect by catabolizing aldehydes [54–57]. It can be perceived by attenuated mitochondrial derived from pharmacological substrates. Identification of activity, diminished intracellular concentrations of reactive CSCs in the liver, breast, colon, and head and neck is done oxygen species (ROS) and ATP, perinuclear mitochondrial measuring ALDH activity employing flow cytometry-based distribution, and a smaller concentration of mitochondrial ALDEFLUOR assay. (is method is more instantaneous and DNA in CSCs [58]. CSC inhibitors have promising off-target is the direct readout of a fluorescent signal, based on enzyme effects on other cell types focussing on either self-renewal activity as compared to other phenotypical detection pathways, surface markers, or enzymes. Table 2 illustrates methods. (is method is less prone to discrepancies en- the potential off-target activities of some of these inhibitors countered by antibody-based staining, namely, epitope on immune cells [59–61]. 4 Journal of Oncology Table 2: Off-target effect of CSC-targeting inhibitors on immune cells. Inhibition of CSC pathways, Off targeting effect on immune cells Reference function and markers β-catenin Impairs polarization, differentiation, and maturation of T cells [23, 24] Notch receptor Blocks cytotoxic T cells’ function and cytotoxin production [19, 20] WnT receptor Inhibits CD8 positive T cell’s development [23, 24] Hedgehog receptor Inhibits myeloid-derived suppressor cell function [30] ABCB complex Inhibits transporter associated with antigen processing (TAP) in antigen-presenting cells [69] ALDH Inhibits regulatory T cells’ function [52] IL-6 receptor Impairs central and na¨ıve memory T cells’ proliferation, survival, and effector function [59] CD44 Impairs (1 cell survival, memory, effector function, and IFNc production [60, 61] 3.3. Identification on the Basis of Functional Alteration. ROS and may selectively kill the relatively radiosensitive CSCs can be functionally distinguished from SCs by the fact tumor cell populations leaving the therapy-resistant CSCs alive, thus leading to temporary or permanent cell cycle that they exhibit a sluggish rate of cell division, amplified drug, and radiotherapy resistance and display an activation arrest via the selective repopulation from the surviving CSCs through DNA damage repair (DDR). DDR arbi- of detoxification pathways which forms the basis for their identification as well. trates senescence or cell death if the damage is irreparable Characteristic staining of retaining dyes like PKH, [72]. (e cell may die if the antioxidant defence mech- carboxyfluorescein succinimidyl ester (CFSE), or bromo- anism is not able to bring out the cell from the state of deoxyuridine (BrdU) that mostly become dilute during the oxidative stress caused by ROS. Intrinsic radioresistance proliferation phase of the cell cycle can be delineated by the in CSCs can be majorly attributed to both the two highly poor rate of cell division of CSCs especially in growth efficient pathways, namely, DDR and ROS operating in preparatory phase or G0. (ese dye retaining cells give rise to them. Extrinsic or acquired radioresistance may be as- xenotransplants in a number of cancers of the breast, cribed to the localization of CSCs in hypoxic zones inside the tumor. Survival of CSCs is fostered to hypoxia-in- melanoma, pancreas, and glioma [62–66]. (e incremented intensity of drug resistance has been ducible factors (HIF) 1α and HIF2α which have been shown to activate Sonic Hedgehog, Wnt, and Notch found in CSCs due to detoxifying pathways. ABCB1, ABCB5, ABCG2, and ABCC1 which are the members of pathways for stem cell renewal and multipotency. Also, ATP binding cassette transporter family of proteins are an accumulating body of evidence demonstrates that CSC active in CSCs and inactive during differentiation [67]. rich cancers respond negatively or less efficaciously to (ey function to pump out complex molecules from the radiotherapy [73, 74]. Nevertheless, the impact of hyp- cell cytoplasm, thereby, shielding the cells from exoge- oxia is not inhibitory on immune cell function. HIF1α nous toxins like various drugs utilized for chemotherapy. regulates long-term survival of activated DC. HIF2α turns Peptides, lipids, proteins, polysaccharides, and a number on the cytolytic machinery of effector T cells that are of diverse hydrophobic drugs act as their substrates [68]. hypoxia resistant. Effector T cells displaying glycolytic characteristics exert their effector function even in the Targeting them with highly selective and specific inhibitor molecules remains a research niche that attracts the in- areas with poor vasculature, may be where CSCs reside. Low-dose radiations harm na¨ıve T cells unlike the terest of cancer researchers all over the world [69]. Hy- drophobic Hoechst dyes are also excluded/expelled by memory T cells and effector T cells [75–77]. Blending CSCs owing to this mechanism; a side population based radiotherapy with T-cell targeting of stem cells can be on low dye levels is formed by CSCs which aids in their more fruitful stratagem which would impart promising identification [70]. Certain ABCB proteins such as synergistic effects. transporter associated with antigen processing (TAP CSCs may, however, be abolished by high-dose radia- protein) play an instrumental part in intracellular traf- tions as witnessed in prostate and lung cancers of the early ficking of peptides across the membranes with major stage. Partly, it may occur due to immunogenic cell death, association with dendritic cells (DCs) and major histo- which commences by uptake of tumor antigens and antigen cross-presentation by DC [78]. It can be contemplated if compatibility complex (MHC) class I function [71]. As a consequence, the off-target effect of tumor ABCB tar- CSC death due to radiation is immunogenic or if radiation gives rise to specific effector and memory T cells leading to geting may be lethal for generating proficient antitumor T-cell responses. abscopal effect and ultimate failure to recur. Lots of scientific (ere is strong experimental and clinical evidence minds are engrossed to address this predicament of para- that CSCs are intimately involved in both intrinsic and mount clinical significance. acquired tumor resistance to anticancer treatments in- cluding radiotherapy. Unraveling the underpinning 4. Immunomodulating and Immunoresistance mechanisms that govern the maintenance of CSCs and Properties of CSCs their resistance to therapy continues to remain a daunting task. Radiation damages the DNA by causing single- or 4.1. Immunomodulation. CSCs show immunomodulation double-strand breaks or opting an indirect route through mainly through immunosuppression of immune cells. In vitro Journal of Oncology 5 bioassays displayed an exemplary instance for the inhibition to bedside. Here, we are discussing some possible strategies of proliferative T-cell responses and IL-2 production by CSCs to immunologically target CSCs. in melanoma and glioblastoma. Treg frequencies were not increased in glioblastoma and increased in melanoma CSC-T- cell cocultures [79, 80]. Immunosuppression of antigen- 5.1. Innate Immune Response to CSCs. An appropriate presenting cells, Natural Killer (NK) cells, and T cells is candidate for immunotherapy of hematologic and solid exerted by tumorigenic growth factor- (TGF-) β, Interleukin- types of tumors is NK cells that are major effector cells for (IL-) 4, IL-13, and IL-10 secreted by CSCs [80–82]. innate immunity [91, 92]. Nonetheless, the exact partici- Immune responses are weakened by cell surface molecules pation of NK cells in anti-CSC immune surveillance remains on CSCs like CD200. Experiments on breast cancer in vivo discordant. Wu and his coworkers scrutinized the immu- model have revealed that minimised T helper (()1 re- nogenicity of CD133 brain tumor stem cells (BTSCs) and sponses, elevated IL10 production, and attenuated neutrophil showed that MHC I or NK cell-activating ligands fail to be infiltration are credited to CD200 overexpression [83, 84]. expressed by a large proportion of CD133 cells, thereby Overexpression of programmed death ligand 1 (PD-L1) in making them defiant to innate and adaptive immune sur- cancers is associated with increased glycolytic behaviour. (e veillance [93]. Wang et al. verified that the immune CSCs in high expression on CSCs triggers PD-L1 upregulation that is breast cancer evade NK cell killing owing to the marginal plausibly localization dependent or tumor type [85]. Mela- expression of MICA and MICB (MHC class I-related chains nomas do not express high eminent levels of PD-L1 but A and B), two ligands for the stimulatory NK cell receptor overexpression is a conspicuous feature in gastric, head, and NKG2D due to aberrant expression of oncogenic micro- neck and CD133 colorectal cancers [61, 79, 86, 87]. RNA miR-20a [94]. On the other hand, a research group led by Castriconi accounted that a range of ligands of NK cell activation receptors are expressed by glioma stem cells 4.2. Immunoresistance. In contrast to non-CSCs, glioblas- (GSCs) which evokes optimal NK cell cytotoxicity. (ey toma CSCs display downregulation of MHC class I. On the deduced that GSCs fall prey to lysis mediated by both al- other hand, autologous T-cell responses were induced in logeneic and autologous IL-2 (or IL-15)-activated NK cells vitro by these CSCs. Moreover, the vulnerability of CSCs to [95]. T-cell-mediated immune responses is boosted by IFN- In another investigation by Tseng et al., it was inferred c-treatment [80]. Even though the feasibility of therapeutic that NK cell-mediated cytotoxicity may harm primary oral vaccines in clinical settings lies in infancy, cancer vaccines squamous carcinoma stem cells (OSCSCs) more signifi- that generate T-cell and antibody responses against tumor- cantly as compared to their differentiated counterpart [96]. associated antigens (TAAs) yield promising inferences in Similarly, allogenic NK cell recognition of colorectal ade- preclinical models. Vaccine specificity for CSCs antigens is nocarcinoma CSCs was analyzed by Tallerico et al. and also majorly lacking which could also contribute to increased showed that these CSCs are more predisposed to NK cells in resistance to T-cell attack. Development of stem-like fea- contrast to their non-CSC counterpart. Elevated levels of tures like Nanog in surviving cancer cells subsequently after ligands for natural cytotoxicity receptors in colorectal CSCs vaccination has been reported [88]. Silencing Nanog could make them more vulnerable to NK cell killing as compared aid in getting rid of T-cell resistance. (is sheds light on the to non-CSCs [97]. fact that stem-like features may build up in a cell population Exceptional cδ T cells comprise 1–5% of as a consequence of immunological stress; however, it does circulating lymphocytes and are of Vc9Vδ2 phenotype and not show inherent SC resistance. are part of innate effector group family. (ey are charac- (e evidence that purified CSCs are employed as vaccines terized by potent non-HLA restricted cytotoxicity against brings an end to the entire dispute that cancer stem-like cells tumor units and identify and present antigens to αβ T cells; may not be inherently resistant to immune attack. T cells hence, immunotherapy targeting cδ T cells is grabbing the generated through this treatment provide better protection in attention of the scientists worldwide [98, 99]. cδ Tcells target the D5 melanoma model against pulmonary metastasis in isopentenyl pyrophosphate (IPP) which is crucial for iso- compression to the vaccination using unseparated cells. (is prenoid biosynthesis in eukaryotes [100, 101]. Cytolysis of study along with others proves T-cell susceptibility and in- sphere forming neuroblastoma cells sensitized to zoledro- dicates the feasibility of CSCs targeting by T cells [51, 89]. nate is mediated by Vc9Vδ2 T cells as demonstrated by Nishio and his group [102]. Similarly, zoledronate-sensitized human colon CSCs cause Vc9Vδ2 T cells to exhibit an 5. Immunological Targeting of CSCs increased rate of proliferation, tend to secrete TNF-α and Immunotherapy targeting tactics chiefly focus to hamper the IFN-c, and produce the apoptotic and cytotoxic molecules immunosuppressive TME, disrupt immune-repressive reg- granzymes and TRAIL [103]. Increased CD69 expression by ulatory networks, and activate cytolytic lymphocytes [90]. A activated Vc9Vδ2 T cells after zoledronate exposure was gamut of strategies targeting immune responses in cancer shown in a clinical study. Decreased expression of the cells and CSCs, in general, has been investigated. However, lymphoid-homing receptors CCR7 and CXCR5 was ob- such immunotherapy aiming strategies in CSCs are in the served in Vc9Vδ2 T cells. On the contrary, upregulated budding phase in the preventive preclinical stage and need to peripheral tissue-homing chemokine receptors CCR5 and be investigated further so that they can be taken from bench CXCR3 were also displayed by Vc9Vδ2 T cells [102]. In vitro 6 Journal of Oncology these Vc9Vδ2 T cells were found cytotoxic. (ese trans- engineered to target virtually any TAAs to recognize cell ferred Vc9Vδ2 T cells homed predominantly to the spleen, surface protein in an MHC-unrestricted manner [108]. In the preclinical experimental setting of solid tumors, lung, and liver and to the metastatic sites outside these organs [104]. (is signifies that in vitro expansion of the CAR T cells have been perceived to target CSC-associated autologous cδT cells together with different antineoplastic antigens, such as CSPG4 in many discrete types, EpCAM in drugs may be for the betterment of cancer treatment via prostatic carcinoma (PCa), CD133 in glioblastoma, and CSC obliteration. Further investigations to substantiate EGFRvIII and IL13Rα2 in gliomas [109–113]. Antitumor direct targeting of CSCs by cδ T cells are warranted. (e impacts of CAR T cells mediated by CSCs are highlighted dearth of clinical data relating to the use of nonspecific through these studies although there is a paucity of studies in killer cells in the adoptive immunotherapy of cancers this area. Significantly, an elevated rate of toxicities has been mediated by CSCs is the major lacuna in this area and needs observed for CAR T therapies targeting TAAs including to be addressed at the earliest. ERBB2 in metastatic colon cancer and carbonic anhydrase IX (CAIX) in renal cell carcinoma apparently accredited to the common expression of targeted antigens by both cancer 5.2. Antigen-Specific Targeting by T-Cell Immunotherapy. and normal cells [114, 115]. Isolation and expansion of (e T-cell based immunotherapy needs to generate effector T cells restricted to specific TAAs are a daunting task due to T cells followed by adoptive transfer of CD8 T cells back technical cumbersomeness. A meticulous approach towards into patients. Here, we are describing the routes to generate recognition of CSC-specific antigens is expected to be vital in CSC-specific T cells that include CSC-primed T cells and this approach for targeting CSCs. Propitious candidacy for genetic engineering of T cells with chimeric antigen re- focussing on CAR T therapy is of neoantigens that can be ceptors (CARs). acknowledged skillfully by T cells without self-tolerance Regarding CSC-primed T cells, CD8 T-cell response mechanisms. could be elicited by employing CSCs derived from breast, Although the existing knowledge on CSC genomics is in pancreatic, and head and neck cell lines [105]. For instance, its nascent phase, studies suggest the use of in vitro generated an in vitro stimulus of human CD8 T cells secluded from and expanded CSC-specific T cells for adoptive transfer into peripheral blood of normal HLA-A2 donors with tumor-bearing hosts in vivo to target CSCs for eliminating ALDH1A1 peptide-pulsed autologous DCs could trigger the tumor. (eoretically, in vitro CSC-primed T cells may ALDH-specific CD8 T cells. Remarkably, animal survival represent a newer and pragmatic immunotherapy to pre- after adoptive transfer in preclinical bioassays can be ex- cisely focus on CSC [116] but practically this approach is still tended by these ALDH-specific CD8 T cells that can under the developmental stage. hi identify and abolish ALDH CSCs (decreased by 60%–89%) in vitro and restrain metastasis and xenograft growth in vivo [105]. 5.3. CSCs Vaccines. (e immunotherapeutic strategies are hi Likewise, lung CSCs were isolated as ALDH population based on the activation of T-cell responses endogenously to target malignant cells via transferring TAAs to patients, and exploited for lysate-pulsed DCs to stimulate CD8 T cells by cocultivation as demonstrated by Luo et al. [106]. which can be successfully achieved by vaccines such as whole cell, genetic, DC, and peptide. At present, the most effica- Subsequently, noteworthy anticancer effects, ensuing in retarded tumor growth and increased survival, were revealed cious and most interrogated strategy to prevent diseases is high + DC vaccination. Ex vivo induction of DCs, the professional by these ALDH -CD8 T cells [106]. Moreover, HLA-I molecules and autologous cytotoxic T lymphocytes (CTLs) antigen-presenting cells (APCs) from peripheral blood were expressed by CSCs that were purified as SP from bone monocytes or marrow cells, pulsed with tumor antigens, malignant fibrous histiocytoma (MFH) in vitro coculture maturated, and eventually administered to the patient. with SP cells [107]. Also, an important indication that the Comprehensive data from different experiments have CTL clone specifically recognizes MFH CSC-specific anti- thrown light on the use of DCs to initiate tumor-specific gens came from the observation that CTL clones are biased T-cell responses which epitomises a highly potential cancer vaccination approach [116–118]. In particular, DC-mediated towards the recognition of SP cells rather than non-SP cells [107]. tumor-specific immune responses have been fostered by CSCs that function as antigen sources [119, 120]. In prin- Nevertheless, it is imperative to note that CSC-primed T cells distinguished antigens are predominantly unidentified. ciple, the employment of CSC lysates as an antigen source could facilitate synchronized targeting of diverse antigens Otherwise, CAR T cells symbolize a unique and a potential cancer immunotherapy. (e past few decades have wit- and thereby be less prone to antigen loss due to tumor escape nessed a furious attention of the scientific community to- [116]. Also, non-CSCs are less effective in inducing anti- wards applying this therapy in solid tumors which is driven tumor immunity against CSC epitopes as compared to by the profound success and advances in the treatment of enriched CSCs that are immunogenic and better as an hematological malignancies using the adoptive transfer of antigen source. For example, the fact that can be considered CAR T cells. T cells can be genetically crafted employing ex is that, in various syngeneic tumor models of immuno- competent mice, the CSC-DC vaccination notably cir- vivo gene transfer for specifically recognizing a TAA or for expressing the novel T-cell receptor, hence, arbitrating cumvents lung metastasis and slows down the growth of squamous carcinoma compared to immunization with bulk neoplastic cells. Consequently, CAR T cells can be Journal of Oncology 7 tumor cells [51]. (e vaccine comprised of DCs pulsed with immunotherapy. (e endeavours targeting CSCs with irradiated PCSC (prostate cancer stem cells) lines derived specific antibodies have been highly fruitful in terms of therapy response. For instance, tumor progression is de- from the TRAMP (transgenic adenocarcinoma of the mouse prostate) tumors induces a more robust tumor-specific celerated and apoptosis is triggered in leukemic stem cells by immune response than the response induced by DCs pulsed an anti-CD44 mAb [131, 132]. Similarly, human melanoma with differentiated prostate tumor cells. It has also been metastasis is lessened, animal survival in SCID mouse is observed that the CSC-DC vaccine retards tumor growth enhanced, tumor growth reduced, and apoptosis com- [121]. menced in murine breast tumors due to anti-CD44 anti- In a study, cytotoxic T lymphocytes (CTL) antitumor bodies [133, 134]. + + response and activation of CD8 and CD45 T cells were Certainly, targeting CSCs with antibodies has proved to also credited to the breast CSC-DC vaccine [122]. Fasci- be beneficial in improving treatment responses. Also in vivo tumor growth and in vitro proliferation of CD133 natingly, it has been observed that, in the postsurgery gastric phase, CSC-DC vaccines are extremely efficacious when and hepatocellular CSCs can be subdued by drug-conjugated deployed in an adjuvant setting to eradicate microscopic anti-CD133 antibody [135]. A bispecific antibody consisting CSCs, or as combinatorial therapy with radiation and/or of CD133 and CD3 antibodies which were asymmetric in chemotherapy in treating macroscopic tumors. (is was nature displayed a strong antitumor efficacy in a variety of observed in established murine melanoma D5 and squa- tumors [136, 137]. Studies have revealed that antitumor mous cancer SCC7 tumor models where CSC-DC was more capability in solid tumors can be made better with a CSC- efficient when compared to DCs pulsed with non-CSCs in specific antibody-incorporated liposomal nanoparticle de- curing microscopic tumors in [123]. Furthermore, livery system loaded with drugs or a suicide gene [138, 139]. high ALDH CSC-DC vaccines in the adjuvant setting were In yet another classic example, trastuzumab (HER2-tar- more promising than traditional DC vaccines as they re- geting antibody) dramatically lessens the chances of breast duced tumor relapse and lung metastasis coupled with cancer recurrence by targeting HER2, an important regu- enhanced host survival further and obstructing PD-L1 lator of breast CSC self-renewal [140]. Similarly, other [124]. DNA vaccination is gaining hype as compared to HER2-targeting agents, like monoclonal antibody pertu- conventional cell-based vaccines, as they involve injecting zumab and the immunotoxin conjugate ado-trastuzumab emtansine (TDM-1), have further upgraded the efficacy of plasmids for direct antigen production culminating in a protective immunological response, thus preventing cancer human epidermal growth factor receptor (HER2) targeting [125]. Acceptability of DNA immunization and their ap- in the clinical settings [141, 142]. titude to skilfully trigger antigen-specific T cells has been Outstandingly, HER2 expression can also be targeted in efficiently established by clinical trials. PAP and PSA have GBM CSCs [143]. As the “CSC markers” may not necessarily been acclaimed as DNA-based vaccines in human castra- be unique to CSCs, single-agent mAb therapy may adversely tion-resistant prostate cancer (CRPC) [126, 127]. In a affect normal cells [130]. Combination therapy using an study, Nishizawa et al. observed that, in comparison to assortment of different antibodies targeting multiple CSC immunization with the TAA survivin, immunization with markers could potentially reduce doses of individual anti- CSC-specific DNAJB8 expression plasmids shows better bodies to accomplish the efficient abolishment of CSCs while antitumor immune response [128]. reducing side effects due to huge concentrations of single Recently, an experimental DNA vaccine has been de- anti-CSC mAbs. veloped against sternness-specific marker Sox2 and an an- titumor effect also has been seen after immunization against 5.5. Tumor Microenvironment Targeting Immunotherapy. murine lung TC-1/B7 cancer cells expressing oncogenic Sox2 [129]. In a nutshell, it can be contemplated that given (ere are few important factors present in the surrounding tumor microenvironment of CSCs such as hypoxia, chronic the ease and theoretical applicability to target antigens, CSC- specific DNA vaccination holds promise to serve as a tool for inflammation, inflammatory cytokines, and perivascular niches (role in the regulation of proliferation and differ- immunotherapy. entiation) [144–146]. Stat3/NF-κB pathways in tumor and stromal cells are activated by inflammatory cytokines such as IL-1β, IL-6, and IL-8 to further secrete cytokines in a positive 5.4. Antibody-Based Immunotherapy. (e past two decades have brought into limelight the significance and feasibility of feedback loop that elicits angiogenesis, CSC self-renewal, monoclonal antibodies- (mAb-) based treatments as ther- and metastasis [144, 147]. In addition, the CSC population apies against cancer treatment. Antibody-drug conjugates which coevolved in the tumor microenvironment are ad- are powerful newer discovered weapons for the fight against jacent blood vasculature that forms a niche defined by severe cancer [130], and immunomodulatory antibodies, illustrated enhanced angiogenesis and hypoxia [145, 146]. (ese facets by anti-PD-L1, anti-PD-1, and immune checkpoints tar- of the tumor microenvironment have been deciphered as geting anti-CTLA-4 antibodies, have also recently attained probable pharmaceutical targets of CSCs. outstanding clinical success (discussed in immune check- Recent studies have verified that reduced tumor growth points). (e expression levels of some markers (Table 1) in is achieved via IL-6 and/or IL-8 cytokine signalling blockade CSCs considerably dissimilar to the other tumor cells, [148, 149]. Repertaxin, a pharmaceutical commodity, a which provides promising targets for antibody-based noncompetitive inhibitor of IL-8 and CXCR1 signalling, 8 Journal of Oncology could decrease tumor size and enhance chemotherapy ef- 6. Limitation and Challenges ficacy in breast cancer model [150]. However, it has been It has been found that owing to stemlike features like low seen that blocking single cytokines induces limited effects as both IL-6 and IL-8 are crucial for tumor growth and the immunogenicity and intrinsic conventional therapy resis- expression of these genes combined with dismal prognosis in tance, CSCs are demonstrated to be participants in the breast cancer patients. Hence, simultaneously inhibiting IL- processes of tumor progression, maintenance, metastasis, 6 and IL-8 expression was a more lucrative line of action to and relapse. Consequently, CSCs form a crucial target to induce appreciable changes in tumor growth [151]. treat residual disease and circumvent the process of re- Tumor hypoxia is another fascinating means to hit the currence. Immunotherapy is a pragmatic promising tool as CSC niches. Hypoxia causes chemo- and radioresistance by evidenced by the results of a number of clinical trials on activating the HIF pathway and upregulating HIF-1α, by cancer patients. However, in the clinical trials, it has been shown that the objective response rate varies significantly mediating multiple biological effects of hypoxia in tissues. Albeit HIF pathway inhibitor molecules have been underway and a durable response is often confined to a small patient population. It is important to note that the results of the in clinical trials, they seldom become successful enough to cross the clinical trial stage [152]. Also, the CSC niche can be current mono-immunotherapies in solid malignancies are dislocated by targeting tumor vasculature. Limited success generally unsatisfactory. One of the fundamental reasons for has been achieved from the clinical trials targeting angio- this inadequacy might be due to the presence of CSCs that genesis by blocking the vascular endothelial growth factor are not efficiently targeted by the available regimens for pathway [145]. immunotherapy. (e concept of CSC-specific immuno- therapy remains in its budding phase, although it has established utility in a few preclinical and clinical trials 5.6. Immune Checkpoints. Immune checkpoints are known (Table 3); however, challenges still exist. In order to fabricate for declining autoimmunity by intervening coinhibitory more promising strategies and novel therapeutics, genomic, signalling pathways [153]. In cancer, these inhibitory immunological, and biological characterization of CSCs pathways are believed to be implicated in tumor immune along with immune cell interactions in the TME is war- resistance [154]. PD-1/PD-L1 axis and the cytotoxic ranted. Despite the fact that considerable knowledge has T lymphocyte antigen 4 (CTLA-4)/B7 axis are the two in- been accumulated on human CSC properties, most of our strumental pathways of immunoinhibition that have been current understanding is attributed to derived from xeno- identified to date. (ese negative mechanisms contribute to graft studies in immune-compromised nude mice. In the the development of a suppressive microenvironment ren- future, employing humanized mice, immunodeficient dering cell resistance to immune therapy [155, 156]. CSCs strains with engrafted human immune systems, may help to might secrete paracrine factors or direct cell-cell contact and throw light in this area [166]. Yet, another hurdle is the jointly transform the immune cells in the CSC niche because heterogeneity in the population of CSCs and the property of physiologic stem cells have immunoprivilege and active plasticity of cancerous cells. CSCs are acknowledged as immunoregulatory functions [3, 157–160]. Schatton et al. heterogeneous [2] revealed by the fact that different sub- accounted that T-cell activation is downregulated by CSCs populations of CSCs sometimes express different phenotypic [79, 161]. (ey identified malignant melanoma initiating markers in a single cancer type. For example, a previous cells (MMICs), a novel type of CSCs, on the basis of the research group has confirmed that prostate CSCs are mainly expression of ABCB5 (chemoresistance determinant) [161]. PSA-/lo but this is rather heterogeneous inhabiting a vast (e human ABCB5 MMICs express PD-1 and B7.2 and low array of tumorigenic cell subsets that can be prospectively expression of PD-L1 in comparison to ABCD5 cells. (e purified out using distinctive markers [167]. clinical benefit of PD1 and PD-L1 was witnessed in Data amassed from different laboratories worldwide Hodgkin’s disease, melanoma, and lung cancers [162–164]. ascertains that the association between various CSC subsets A better-prolonged response was observed in a patient within a similar cancer type remains largely unidentified, subset that was considerably more durable compared to and if CSC subpopulations share common immunological targeted or cytotoxic therapies. features is also an area to be explored. Hence, there may be a It is proposed that activated T-cell responses may be possibility of a situation that a CSC-specific immunological downregulated by tumor expression of PD-L1 through the treatment leads to the eradication of a particular subset of PD-L1/PD-1 axis and its blocking results. Even though CSCs not all. Furthermore, CSCs and non-CSCs can be Schatton et al. suggested reduced expression of PD-L1 in thought to exhibit diverse plasticity (not discussed in this MMICs [79], in another study, a better expression of PD-L1 review but reviewed in [2]). (is tumor cell plasticity is a was observed in CSCs of head and neck carcinoma [165]. It major impediment in the establishment of long-lasting and can be inferred from this that CSC subsets may reduce T-cell more effective targeted cancer therapies, because therapeutic response through the PD-1/PD-L1 axis. However, future annihilation of present CSC populations might be then clinical trials on immune checkpoint blockade are manda- followed by their regeneration from non-CSC origin within tory for the establishment of these results. Also, the syn- the tumor due to treatment pressures [2]. ergistic effects of immune checkpoint therapies with CSC- It is gripping to see that various agents aim at targeting targeting immunotherapies are suspected to magnify the TME components that demonstrate clinical relevance in clinical applicability of each approach. the treatment of cancers, as our present knowledge about Journal of Oncology 9 Table 3: CSC-targeting strategies and their effect in different types of cancer. Type Cancer types Effects of the CSC-targeting strategies Reference ALDH1A1-specific CD8 T cells distinguish and eradicate ALDHhi CSCs in in Head and neck vitro bioassays, retard xenograft growth and metastases in in vivo bioassays, and [105] CSC-primed prolong survival T cells high + ALDH -CD8 T cells resulted in the inhibition of tumor growth and Lung [106] prolonged survival, hence, bestowing more considerable antitumor effects CSC-DC vaccine that was administered in the adjuvant setting after localized radiation therapy of established tumors resulted in a reduction of tumor growth, and vaccination significantly inhibited tumor growth, abridged [123] high ALDH CSC frequency in primary tumors, and metastases through Squamous cell stimulation of humoral immune responses against CSCs Carcinoma/Melanoma SCC growth was regressed compared to immunization with bulk tumor cells [51] and lung metastasis of melanoma cells was appreciably curtailed In the adjuvant setting, simultaneous PD-L1 blockade further enhanced local CSC-lysate DC tumor recurrence and spontaneous pulmonary metastasis and also increased [125] vaccine survival of the host Tumor regression was witnessed in TRAMP mice, tumor growth was delayed in Prostate mice challenged with prostate CSCs, and tumor-specific immune response was [121] induced that was stronger than differentiated tumor cells Antigen-specific T-cell responses against CSCs were elicited and survival in Glioblastomas [120] animals was improved + + Migration of DCs to the spleen activated CD8 and CD45 T cells; in turn, CTL Breast [122] antitumor responses were induced CSC-mRNA-DC Seven patients vaccinated with an mRNA-DC vaccine exhibited a common Glioblastomas [172] vaccine immune response Stronger antitumor effects were observed in immunization with DNAJB8 DNA vaccine Renal cell carcinoma expression plasmids in contrast with immunization with the tumor-associated [128] antigen survivin, which was expressed in both CSCs and non-CSCs Neural stem cells derived from tumor specimens were prone to attack by lysis Glioblastomas mediated by both autologous IL-2 (or IL-15) activated NK cells but resisted [95] freshly isolated NK cells NK cells CSCs isolated from an array of human cancer cell lines in vitro and dissociated Pancreatic/Breast/ primary cancer specimens ex vivo were preferentially targeted by allogenic [173] Glioblastomas activated human NK cells (e growth of hepatic and pancreatic cancer cells was inhibited in vitro and in Liver/Pancreatic vivo and CD133 high CSCs were targeted by CIK cells bound with anti-CD133/ [137] anti-CD3 bispecific antibodies mAb Human melanoma metastasis was inhibited and the survival of tumor-bearing Melanoma [133] animals was prolonged by anti-CD44 antibodies Murine breast tumor growth was inhibited and apoptosis was induced by anti- Breast [134] CD44 antibodies Patient-derived GBM CSCs were annihilated in an orthotopic tumor model and Glioblastomas [112] in vitro by anti-CD133 CAR T cells CSC-CAR T Significant antitumor efficacy was exhibited by EpCAM-specific CAR T cells in Prostate [113] vitro and in vivo systems the TME is limited. Notably, current research studies have cancer. While therapies that efficiently and selectively get advocated that conventional therapies of cancer are more rid of CSCs do not have clinical applicability, researches on prone to augment CSCs and restructure the TME which various immunotherapeutic strategies to target CSCs may modify the immunotherapy responding ability of (Table 3) are in progress and many of them have displayed CSCs [3]. For instance, chemotherapy enhanced the fre- efficacy in diminishing tumor growth and metastasis in quency of the CSCs in the tumors and downregulated the preclinical and clinical settings. expression of HLA1 molecules in HNSCC and PCa Like all the monotherapies, mono-immunotherapy is improbable to treat cancer, and stratagem that merges both [168, 169] that may cause immunoresistance. Collectively, a CSC epitomises a continuously reshaping target, as it keeps conventional therapies and CSC-specific immunotherapies on developing along with tumor development and pro- would be attractive and promising tactics to combat the gression, particularly, under the influence of treatment. It deadly disease cancer. Combinatorial approaches can reduce can be concluded that the area of immune targeting of drug resistance and cancer cell plasticity and help attain CSCs holds noteworthy promise in curing patients with efficacious treatment outcomes as compared to the 10 Journal of Oncology monotherapies. (eoretically speaking, the immunogenicity References of CSCs can be incremented by the inhibition of negative [1] D. G. 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Immunotherapy: Newer Therapeutic Armamentarium against Cancer Stem Cells

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Copyright © 2020 Saurabh Pratap Singh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2020, Article ID 3963561, 15 pages https://doi.org/10.1155/2020/3963561 Review Article Immunotherapy: Newer Therapeutic Armamentarium against Cancer Stem Cells 1 2 3 1 Saurabh Pratap Singh , Richa Singh, Om Prakash Gupta, Shalini Gupta , and Madan Lal Brahma Bhatt Department of Oral Pathology & Microbiology, King George’s Medical University, Lucknow, India Centre for Integrated and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow, India Depatment of Surgery, Prasad Institute of Medical Sciences, Lucknow, India Vice Chancellor, King George’s Medical University, Lucknow, India Correspondence should be addressed to Shalini Gupta; sgmds2002@yahoo.co.in Received 14 March 2019; Accepted 4 February 2020; Published 9 March 2020 Academic Editor: P. Neven Copyright © 2020 Saurabh Pratap Singh et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mounting evidence from the literature suggests the existence of a subpopulation of cancer stem cells (CSCs) in almost all types of human cancers. (ese CSCs possessing a self-renewal capacity inhabit primary tumors and are more defiant to standard an- timitotic and molecularly targeted therapies which are used for eliminating actively proliferating and differentiated cancer cells. Clinical relevance of CSCs emerges from the fact that they are the root cause of therapy resistance, relapse, and metastasis. Earlier, surgery, chemotherapy, and radiotherapy were established as cancer treatment modalities, but recently, immunotherapy is also gaining importance in the management of various cancer patients, mostly those of the advanced stage. (is review abridges potential off-target effects of inhibiting CSC self-renewal pathways on immune cells and some recent immunological studies specifically targeting CSCs on the basis of their antigen expression profile, even though molecular markers or antigens that have been described till date as expressed by cancer stem cells are not specifically expressed by these cells which is a major limitation to target CSCs. We propose that owing to CSC stemness property to mediate immunotherapy response, we can apply a combination therapy approach by targeting CSCs and tumor microenvironment (TME) along with conventional treatment strategies as an effective means to eradicate cancer cells. seen in parental tumors [3]. Accumulated evidences advo- 1. Introduction cate that from the time when CSCs were initially identified in Cancer is a diverse heterogeneous disease which is char- human acute myeloid leukemia (AML), they have been acterized by phenotypically and functionally discrete subsets isolated from many divergent malignancies, including of cells. Data amassed from the literature suggests the cancers of the breast, prostate, colon, brain, pancreas, lung, presence of a small population of cancer cells with stemlike liver, bladder, and ovary [4–8]. properties in a wide continuum of human cancers. Char- CSCs also appear to have resistance to anticancer acterized by self-renewal and differentiation, these cells have therapies leading to relapse. (is deleterious feature of CSC been termed as cancer stem cells or tumor-initiating cells, causes a dire impact on cancer management and hence and we have used CSCs to denote these cells throughout the makes CSCs promising targets for elimination. (ere are a review. CSCs are biologically similar to normal stem cells few ongoing trials that involve immunotherapy strategies (SCs) [1, 2]. CSCs are characterized functionally by the against CSCs [9]. However, in order to design newer intrinsic ability to initiate and long-term repopulate tumors therapeutic approaches, we need a clearer understanding of with a recapitulation of the lineage/cellular heterogeneity the biology of these cells. (e present review aims to 2 Journal of Oncology carcinogenesis due to this pathway [17, 23, 24]. (e focus of determine the feasibility of immune targeting CSCs in solid tumors and also highlights that some of the biological treatment research in cancer is to inhibit the adverse pathway in CSCs [25]. However, the Wnt/β-catenin pathway targetings of CSCs may be ambivalent by also affecting immune responses. is a double-edged sword as it functions as a major player in the regulation of T-cell development and activation, Wnt/ β-catenin signalling is crucial for CD8 memory T-cell 2. RoleofDevelopmentalSignallingPathwaysin development, and on the other hand, the efficacy of im- the Regulation of CSCs munotherapy responses is improved by agonists [26, 27]. Tumorigenesis bears resemblance to abnormal organogen- (e Hedgehog (Hh) signalling pathway plays a key role esis. CSCs exhibit three cell-intrinsic fundamental proper- in tissue wear and tear and homeostasis and epithelial to ties: self-renewal, quiescence, and differentiation. (erefore, mesenchymal transition (EMT) in normal tissues. Bellig- any genetic or epigenetic program that can regulate one or erent manifestations like cancer may be prompted by tu- more of these three properties could theoretically have an morigenesis via aberrant Hh signalling defined by the impact on CSC biology [3]. CSC phenotypes change due to overexpression or loss of function of its ligands or receptor altered genetics via various mechanisms. CSC biology is and dysregulation of transcription factors. Hh signalling can majorly governed by developmental pathways, stem cell be triggered by an array of factors in the tumor microen- factors, cell cycle regulation and apoptosis, epithelial-mes- vironment, such as transforming growth factor- (TGF-) β, enchymal transition (EMT), and epigenetics apart from tumor necrosis factor- (TNF-) α, and interleukin (IL)-6. physiological metabolism. Owing to complex interactions Attenuation of Hh signalling is also attracting focus of re- and overlap between mechanistic programs driving CSC searchers worldwide and undergoing intense exploration for regulation, defining five mechanisms is rather arbitrary, as cancer treatment [28]. Hh signalling is relevant in immune they could all ultimately converge on transcriptional regu- cell development and function [29–32]. Also, Hh inhibitors lation driven by myriad transcription factors [1]. may deliver additional benefits as they are also involved in An array of signalling pathways, namely, Myc, Notch, myeloid-derived suppressor cell (MDSC) function. Singly Hedgehog (Hh), Wnt, FGF/FGFR, EGF/EGFR, NF-κB, targeting any pathway may not yield a physiologically rel- MAPK, PTEN/PI3K, HER2, and JAK/STAT operative in evant level of inhibition as there is a considerable overlap normal SCs during development and homeostasis are fre- between these pathways. Since it is responsible for normal quently found to be deregulated in CSCs [10–14]. Devel- tissue homeostasis and development, including immune cell opmental pathways such as Notch, Wnt/β-catenin, and behaviour and peripheral effector function, targeting them is Hedgehog participate instrumentally in cancers and are a challenging job [33]. frequently altered and are implicated in CSCs regulation [15–18]. 3. Identification and Isolation of CSCs Notch ligands, residing in plasma membrane of neighbouring cells, are transactivated, eliciting the tran- 3.1. Identification Based on Immunophenotypic Marker. scription of Notch target genes, such as c-myc, p21 and p27, Coherent immune targeting of CSCs depends on the PI3K, the hairy and enhancer of split- (HES-) related family, categorization of (i) stem cell-like CSC markers used for protein kinase B (AKT), peroxisome proliferator-activated its isolation and (ii) Antigens expressed on CSCs which receptor (PPAR), NF-κB, and cyclin D1. Depending on the are not preferably express on non-CSC/normal cells. (e particular signalling milieu, these downstream targets get SC biology of the tissues from which tumor originates activated and in turn regulate cell fate leading to differen- forms the basis of identification of CSC markers since the tiation, cell-cycle progression, and survival. Notch may lineage relationship between CSCs and tissue SCs remains decelerate differentiation and endorse cell survival in stem- vague in most tumor systems. For detection and segre- like cells [19, 20]. (ere are numerous ongoing Phase I and II gation of alleged CSC populations, not only are functional clinical trials in cancer with a range of targets and mech- assays like ALDEFLUOR, mammosphere, and neuro- anisms investigating the efficacy of Notch targeting, singly or sphere employed, but also side population (SP) and cell in amalgamation with other therapeutic strategies [19]. surface marker coupled with a fluorescence-activated cell Notch signalling has, nevertheless, also been correlated to sorter (FACS) are put to use. (e immunophenotypic peripheral T-cell maturation into effector cells, such as markers for potential CSCs have been reported in many developing cytotoxic T-cell function, or cytokine production human malignancies [1, 3, 6–8] (Table 1). A range of CSC [21]. Similarly, T-cell activity has been weakened by Notch- markers (CD44, CD133, and HER2) represent promising inhibition using c-secretase inhibitor [22]. targets for CSC immunotherapy, such as antibody ther- Two signalling pathways have been recognized which are apy. However, probable safety issues should be addressed the β-catenin-dependent pathway that is involved in cell fate when utilizing these markers as CSCs since normal SCs determination (canonical), and the β-catenin independent share similar phenotypic marker profiles. Also, a limited pathway that is involved in cell polarity and movement number of CSC markers in solid malignancies are cur- (noncanonical). Commencement of Wnt signalling is fos- rently accessible and their biological function is less tered by the release of soluble ligands by the cells in the near categorized. vicinity. (e gain of function, regulation by methylation, and It is a well-established notion that CSCs in different histone modification have all been implicated in tumor types demonstrate distinctive transcriptomic profiles Journal of Oncology 3 Table 1: CSC Markers in different cancers. CSC markers reviewed in [1, 3, 6–8] Cancer type Positive markers Negative markers Leukemia CD34, CD96, ALDH, CD47, CD44, CD123, TIM-3, CD32, CD25, CLL-1 CD38 Prostate ABCG2, ALDH, CD44, α2β1, CXCR4, CD133, NANOG, TRA-1-60, CD151, CD 166 PSA, CK18, CK20, EMA, Bladder CD44, CK5, CD44v6, 67LR, CK17, ALDH, SOX2 CD66c, Breast CD44, PKH26, CD49f, ALDH, CD133, CD90 CD24 Lung CD133, ALDH, CD117/c-kit, OCT4, NANOG, CD44, TPBG/5 T4, CD166, CD44, EpCAM Pancreatic CD44v6, α6β4, TSPAN8, CXCR4, CD44, CD24, ESA, c-Met, ALDH, CD133, CD133, ABCG2, SSEA-1, SOX2, BMI1, MUSASHI1, NESTIN, OLIG2, CD49f, A2B5, L1CAM, EGFR, Glioblastoma CD44, ID1, MYC, ALDH Ovarian ALDH, CD44, CD117/c-kit, CD44, MyD88, CD24, CD133, CXCR4 Colon CD133, EpCAM, CD44, CD166, ALDH, LGR5, ABCB5 Liver CD133, ALDH, EpCAM, CD90, CD44, CD13, SALL4 CD45 and thereby express discrete antigens. (ey express a downregulation or masking, or expression of splice variants. continuum of tumor-associated antigens (TAAs) that have (e ALDEFLUOR assay uses N,N-dieth- the capacity to be identified by the immune system of the ylaminobenzaldehyde (DEAB) to measure the activity of the host [3]. TAAs may be categorically illustrated in CSCs as ALDH1 family for which it acts as a specific inhibitor [35]. four subgroups [3, 8, 31], namely, (i) nominal expression by Nevertheless, it can be inferred that the occurrence of high normal tissues but constitutively highly expressed by tu- ALDH cells is underrated in tissues in which the chief mors as part of being their malignant phenotype like EGFR ALDH isozyme is not a member of the ALDH1 family. and surviving, (ii) differentiation antigens that are tissue- Studies on a variety of cancers such as prostate cancer, specific and expressed by both benign and cancer cells, (iii) ovarian cancer, breast cancer, lung cancer-derived primary CT cancer/testis antigens inadvertently activated in tumors culture, and nonmetastatic/metastatic cell lines of these like MAGE-A3 and MAGE-A4 that are normally confined cancers suggest variation in ALDH expression [36–47]. (is to placenta and testicular germ cells, and (iv) neoantigens inconsistency could refute potentially applicable CSC in cancer genomes derived from mutations leading to new markers and also brings into limelight the significance of epitopes identifiable by immune system like MUM-1 and testing in multiple cell lines/primary tissues. (e clinical CDK4 in melanoma. Innumerable lineage-specific markers outcome in a variety of cancers has been attributed to ALDH are categorised as differentiation antigens, namely, PSA in expression [48–50]. ALDH has been implicated in resistance PCa and MART-1 in melanoma. CSC markers can mechanisms to radiotherapy and chemotherapy; thus, a henceforth be thought to act as overexpressed antigens more specific, diverse therapeutic approach must be although not all are expressed minimally in normal cells. embarked upon. Some research groups have been attracted CT antigens and neoantigens have been considered best for towards investigating inhibitors and involved in developing CSC-targeting immunotherapy although lineage differen- more specific inhibitors through drug discovery approaches, tiation antigens are expressed in low amounts in CSCs as the common expression of ALDH in healthy stem cells [3, 8, 31]. which can cause an off-target activity [35]. It is possible that functional redundancy within the isozyme family could high recompense the inhibition for one ALDH. ALDH CSC- 3.2. Identification Based on Metabolism. (ere is a meta- loaded dendritic cells (DCs) have been used fruitfully in two bolism-based identification which is based on the activity of in vivo melanoma models in the immunotherapy setting certain enzymes such as aldehyde dehydrogenase (ALDH) [51, 52]. In transplantation setting, ALDH activity has been and mitochondrial glycolytic activity. demonstrated in the T regulatory cell (T ) immune subset; reg (e ALDH family is encompassed by a gamut of 19 genes thus, targeting of ALDH may also show an antitumor impact (in humans) that articulate enzymes for catalysis of aldehyde through modulation of T subset [53]. reg oxidation. Metabolism of vitamins, amino acids, and lipids CSCs possess a lesser number of mitochondria and requires aldehyde oxidation [34]. Elevated ALDH activity is consequently show more glycolytic activity than other tumor linked with both normal SCs and CSCs. (ese enzymes also cells, as evident in liver, breast, melanoma, and lung cancer exert a shielding detoxifying effect by catabolizing aldehydes [54–57]. It can be perceived by attenuated mitochondrial derived from pharmacological substrates. Identification of activity, diminished intracellular concentrations of reactive CSCs in the liver, breast, colon, and head and neck is done oxygen species (ROS) and ATP, perinuclear mitochondrial measuring ALDH activity employing flow cytometry-based distribution, and a smaller concentration of mitochondrial ALDEFLUOR assay. (is method is more instantaneous and DNA in CSCs [58]. CSC inhibitors have promising off-target is the direct readout of a fluorescent signal, based on enzyme effects on other cell types focussing on either self-renewal activity as compared to other phenotypical detection pathways, surface markers, or enzymes. Table 2 illustrates methods. (is method is less prone to discrepancies en- the potential off-target activities of some of these inhibitors countered by antibody-based staining, namely, epitope on immune cells [59–61]. 4 Journal of Oncology Table 2: Off-target effect of CSC-targeting inhibitors on immune cells. Inhibition of CSC pathways, Off targeting effect on immune cells Reference function and markers β-catenin Impairs polarization, differentiation, and maturation of T cells [23, 24] Notch receptor Blocks cytotoxic T cells’ function and cytotoxin production [19, 20] WnT receptor Inhibits CD8 positive T cell’s development [23, 24] Hedgehog receptor Inhibits myeloid-derived suppressor cell function [30] ABCB complex Inhibits transporter associated with antigen processing (TAP) in antigen-presenting cells [69] ALDH Inhibits regulatory T cells’ function [52] IL-6 receptor Impairs central and na¨ıve memory T cells’ proliferation, survival, and effector function [59] CD44 Impairs (1 cell survival, memory, effector function, and IFNc production [60, 61] 3.3. Identification on the Basis of Functional Alteration. ROS and may selectively kill the relatively radiosensitive CSCs can be functionally distinguished from SCs by the fact tumor cell populations leaving the therapy-resistant CSCs alive, thus leading to temporary or permanent cell cycle that they exhibit a sluggish rate of cell division, amplified drug, and radiotherapy resistance and display an activation arrest via the selective repopulation from the surviving CSCs through DNA damage repair (DDR). DDR arbi- of detoxification pathways which forms the basis for their identification as well. trates senescence or cell death if the damage is irreparable Characteristic staining of retaining dyes like PKH, [72]. (e cell may die if the antioxidant defence mech- carboxyfluorescein succinimidyl ester (CFSE), or bromo- anism is not able to bring out the cell from the state of deoxyuridine (BrdU) that mostly become dilute during the oxidative stress caused by ROS. Intrinsic radioresistance proliferation phase of the cell cycle can be delineated by the in CSCs can be majorly attributed to both the two highly poor rate of cell division of CSCs especially in growth efficient pathways, namely, DDR and ROS operating in preparatory phase or G0. (ese dye retaining cells give rise to them. Extrinsic or acquired radioresistance may be as- xenotransplants in a number of cancers of the breast, cribed to the localization of CSCs in hypoxic zones inside the tumor. Survival of CSCs is fostered to hypoxia-in- melanoma, pancreas, and glioma [62–66]. (e incremented intensity of drug resistance has been ducible factors (HIF) 1α and HIF2α which have been shown to activate Sonic Hedgehog, Wnt, and Notch found in CSCs due to detoxifying pathways. ABCB1, ABCB5, ABCG2, and ABCC1 which are the members of pathways for stem cell renewal and multipotency. Also, ATP binding cassette transporter family of proteins are an accumulating body of evidence demonstrates that CSC active in CSCs and inactive during differentiation [67]. rich cancers respond negatively or less efficaciously to (ey function to pump out complex molecules from the radiotherapy [73, 74]. Nevertheless, the impact of hyp- cell cytoplasm, thereby, shielding the cells from exoge- oxia is not inhibitory on immune cell function. HIF1α nous toxins like various drugs utilized for chemotherapy. regulates long-term survival of activated DC. HIF2α turns Peptides, lipids, proteins, polysaccharides, and a number on the cytolytic machinery of effector T cells that are of diverse hydrophobic drugs act as their substrates [68]. hypoxia resistant. Effector T cells displaying glycolytic characteristics exert their effector function even in the Targeting them with highly selective and specific inhibitor molecules remains a research niche that attracts the in- areas with poor vasculature, may be where CSCs reside. Low-dose radiations harm na¨ıve T cells unlike the terest of cancer researchers all over the world [69]. Hy- drophobic Hoechst dyes are also excluded/expelled by memory T cells and effector T cells [75–77]. Blending CSCs owing to this mechanism; a side population based radiotherapy with T-cell targeting of stem cells can be on low dye levels is formed by CSCs which aids in their more fruitful stratagem which would impart promising identification [70]. Certain ABCB proteins such as synergistic effects. transporter associated with antigen processing (TAP CSCs may, however, be abolished by high-dose radia- protein) play an instrumental part in intracellular traf- tions as witnessed in prostate and lung cancers of the early ficking of peptides across the membranes with major stage. Partly, it may occur due to immunogenic cell death, association with dendritic cells (DCs) and major histo- which commences by uptake of tumor antigens and antigen cross-presentation by DC [78]. It can be contemplated if compatibility complex (MHC) class I function [71]. As a consequence, the off-target effect of tumor ABCB tar- CSC death due to radiation is immunogenic or if radiation gives rise to specific effector and memory T cells leading to geting may be lethal for generating proficient antitumor T-cell responses. abscopal effect and ultimate failure to recur. Lots of scientific (ere is strong experimental and clinical evidence minds are engrossed to address this predicament of para- that CSCs are intimately involved in both intrinsic and mount clinical significance. acquired tumor resistance to anticancer treatments in- cluding radiotherapy. Unraveling the underpinning 4. Immunomodulating and Immunoresistance mechanisms that govern the maintenance of CSCs and Properties of CSCs their resistance to therapy continues to remain a daunting task. Radiation damages the DNA by causing single- or 4.1. Immunomodulation. CSCs show immunomodulation double-strand breaks or opting an indirect route through mainly through immunosuppression of immune cells. In vitro Journal of Oncology 5 bioassays displayed an exemplary instance for the inhibition to bedside. Here, we are discussing some possible strategies of proliferative T-cell responses and IL-2 production by CSCs to immunologically target CSCs. in melanoma and glioblastoma. Treg frequencies were not increased in glioblastoma and increased in melanoma CSC-T- cell cocultures [79, 80]. Immunosuppression of antigen- 5.1. Innate Immune Response to CSCs. An appropriate presenting cells, Natural Killer (NK) cells, and T cells is candidate for immunotherapy of hematologic and solid exerted by tumorigenic growth factor- (TGF-) β, Interleukin- types of tumors is NK cells that are major effector cells for (IL-) 4, IL-13, and IL-10 secreted by CSCs [80–82]. innate immunity [91, 92]. Nonetheless, the exact partici- Immune responses are weakened by cell surface molecules pation of NK cells in anti-CSC immune surveillance remains on CSCs like CD200. Experiments on breast cancer in vivo discordant. Wu and his coworkers scrutinized the immu- model have revealed that minimised T helper (()1 re- nogenicity of CD133 brain tumor stem cells (BTSCs) and sponses, elevated IL10 production, and attenuated neutrophil showed that MHC I or NK cell-activating ligands fail to be infiltration are credited to CD200 overexpression [83, 84]. expressed by a large proportion of CD133 cells, thereby Overexpression of programmed death ligand 1 (PD-L1) in making them defiant to innate and adaptive immune sur- cancers is associated with increased glycolytic behaviour. (e veillance [93]. Wang et al. verified that the immune CSCs in high expression on CSCs triggers PD-L1 upregulation that is breast cancer evade NK cell killing owing to the marginal plausibly localization dependent or tumor type [85]. Mela- expression of MICA and MICB (MHC class I-related chains nomas do not express high eminent levels of PD-L1 but A and B), two ligands for the stimulatory NK cell receptor overexpression is a conspicuous feature in gastric, head, and NKG2D due to aberrant expression of oncogenic micro- neck and CD133 colorectal cancers [61, 79, 86, 87]. RNA miR-20a [94]. On the other hand, a research group led by Castriconi accounted that a range of ligands of NK cell activation receptors are expressed by glioma stem cells 4.2. Immunoresistance. In contrast to non-CSCs, glioblas- (GSCs) which evokes optimal NK cell cytotoxicity. (ey toma CSCs display downregulation of MHC class I. On the deduced that GSCs fall prey to lysis mediated by both al- other hand, autologous T-cell responses were induced in logeneic and autologous IL-2 (or IL-15)-activated NK cells vitro by these CSCs. Moreover, the vulnerability of CSCs to [95]. T-cell-mediated immune responses is boosted by IFN- In another investigation by Tseng et al., it was inferred c-treatment [80]. Even though the feasibility of therapeutic that NK cell-mediated cytotoxicity may harm primary oral vaccines in clinical settings lies in infancy, cancer vaccines squamous carcinoma stem cells (OSCSCs) more signifi- that generate T-cell and antibody responses against tumor- cantly as compared to their differentiated counterpart [96]. associated antigens (TAAs) yield promising inferences in Similarly, allogenic NK cell recognition of colorectal ade- preclinical models. Vaccine specificity for CSCs antigens is nocarcinoma CSCs was analyzed by Tallerico et al. and also majorly lacking which could also contribute to increased showed that these CSCs are more predisposed to NK cells in resistance to T-cell attack. Development of stem-like fea- contrast to their non-CSC counterpart. Elevated levels of tures like Nanog in surviving cancer cells subsequently after ligands for natural cytotoxicity receptors in colorectal CSCs vaccination has been reported [88]. Silencing Nanog could make them more vulnerable to NK cell killing as compared aid in getting rid of T-cell resistance. (is sheds light on the to non-CSCs [97]. fact that stem-like features may build up in a cell population Exceptional cδ T cells comprise 1–5% of as a consequence of immunological stress; however, it does circulating lymphocytes and are of Vc9Vδ2 phenotype and not show inherent SC resistance. are part of innate effector group family. (ey are charac- (e evidence that purified CSCs are employed as vaccines terized by potent non-HLA restricted cytotoxicity against brings an end to the entire dispute that cancer stem-like cells tumor units and identify and present antigens to αβ T cells; may not be inherently resistant to immune attack. T cells hence, immunotherapy targeting cδ T cells is grabbing the generated through this treatment provide better protection in attention of the scientists worldwide [98, 99]. cδ Tcells target the D5 melanoma model against pulmonary metastasis in isopentenyl pyrophosphate (IPP) which is crucial for iso- compression to the vaccination using unseparated cells. (is prenoid biosynthesis in eukaryotes [100, 101]. Cytolysis of study along with others proves T-cell susceptibility and in- sphere forming neuroblastoma cells sensitized to zoledro- dicates the feasibility of CSCs targeting by T cells [51, 89]. nate is mediated by Vc9Vδ2 T cells as demonstrated by Nishio and his group [102]. Similarly, zoledronate-sensitized human colon CSCs cause Vc9Vδ2 T cells to exhibit an 5. Immunological Targeting of CSCs increased rate of proliferation, tend to secrete TNF-α and Immunotherapy targeting tactics chiefly focus to hamper the IFN-c, and produce the apoptotic and cytotoxic molecules immunosuppressive TME, disrupt immune-repressive reg- granzymes and TRAIL [103]. Increased CD69 expression by ulatory networks, and activate cytolytic lymphocytes [90]. A activated Vc9Vδ2 T cells after zoledronate exposure was gamut of strategies targeting immune responses in cancer shown in a clinical study. Decreased expression of the cells and CSCs, in general, has been investigated. However, lymphoid-homing receptors CCR7 and CXCR5 was ob- such immunotherapy aiming strategies in CSCs are in the served in Vc9Vδ2 T cells. On the contrary, upregulated budding phase in the preventive preclinical stage and need to peripheral tissue-homing chemokine receptors CCR5 and be investigated further so that they can be taken from bench CXCR3 were also displayed by Vc9Vδ2 T cells [102]. In vitro 6 Journal of Oncology these Vc9Vδ2 T cells were found cytotoxic. (ese trans- engineered to target virtually any TAAs to recognize cell ferred Vc9Vδ2 T cells homed predominantly to the spleen, surface protein in an MHC-unrestricted manner [108]. In the preclinical experimental setting of solid tumors, lung, and liver and to the metastatic sites outside these organs [104]. (is signifies that in vitro expansion of the CAR T cells have been perceived to target CSC-associated autologous cδT cells together with different antineoplastic antigens, such as CSPG4 in many discrete types, EpCAM in drugs may be for the betterment of cancer treatment via prostatic carcinoma (PCa), CD133 in glioblastoma, and CSC obliteration. Further investigations to substantiate EGFRvIII and IL13Rα2 in gliomas [109–113]. Antitumor direct targeting of CSCs by cδ T cells are warranted. (e impacts of CAR T cells mediated by CSCs are highlighted dearth of clinical data relating to the use of nonspecific through these studies although there is a paucity of studies in killer cells in the adoptive immunotherapy of cancers this area. Significantly, an elevated rate of toxicities has been mediated by CSCs is the major lacuna in this area and needs observed for CAR T therapies targeting TAAs including to be addressed at the earliest. ERBB2 in metastatic colon cancer and carbonic anhydrase IX (CAIX) in renal cell carcinoma apparently accredited to the common expression of targeted antigens by both cancer 5.2. Antigen-Specific Targeting by T-Cell Immunotherapy. and normal cells [114, 115]. Isolation and expansion of (e T-cell based immunotherapy needs to generate effector T cells restricted to specific TAAs are a daunting task due to T cells followed by adoptive transfer of CD8 T cells back technical cumbersomeness. A meticulous approach towards into patients. Here, we are describing the routes to generate recognition of CSC-specific antigens is expected to be vital in CSC-specific T cells that include CSC-primed T cells and this approach for targeting CSCs. Propitious candidacy for genetic engineering of T cells with chimeric antigen re- focussing on CAR T therapy is of neoantigens that can be ceptors (CARs). acknowledged skillfully by T cells without self-tolerance Regarding CSC-primed T cells, CD8 T-cell response mechanisms. could be elicited by employing CSCs derived from breast, Although the existing knowledge on CSC genomics is in pancreatic, and head and neck cell lines [105]. For instance, its nascent phase, studies suggest the use of in vitro generated an in vitro stimulus of human CD8 T cells secluded from and expanded CSC-specific T cells for adoptive transfer into peripheral blood of normal HLA-A2 donors with tumor-bearing hosts in vivo to target CSCs for eliminating ALDH1A1 peptide-pulsed autologous DCs could trigger the tumor. (eoretically, in vitro CSC-primed T cells may ALDH-specific CD8 T cells. Remarkably, animal survival represent a newer and pragmatic immunotherapy to pre- after adoptive transfer in preclinical bioassays can be ex- cisely focus on CSC [116] but practically this approach is still tended by these ALDH-specific CD8 T cells that can under the developmental stage. hi identify and abolish ALDH CSCs (decreased by 60%–89%) in vitro and restrain metastasis and xenograft growth in vivo [105]. 5.3. CSCs Vaccines. (e immunotherapeutic strategies are hi Likewise, lung CSCs were isolated as ALDH population based on the activation of T-cell responses endogenously to target malignant cells via transferring TAAs to patients, and exploited for lysate-pulsed DCs to stimulate CD8 T cells by cocultivation as demonstrated by Luo et al. [106]. which can be successfully achieved by vaccines such as whole cell, genetic, DC, and peptide. At present, the most effica- Subsequently, noteworthy anticancer effects, ensuing in retarded tumor growth and increased survival, were revealed cious and most interrogated strategy to prevent diseases is high + DC vaccination. Ex vivo induction of DCs, the professional by these ALDH -CD8 T cells [106]. Moreover, HLA-I molecules and autologous cytotoxic T lymphocytes (CTLs) antigen-presenting cells (APCs) from peripheral blood were expressed by CSCs that were purified as SP from bone monocytes or marrow cells, pulsed with tumor antigens, malignant fibrous histiocytoma (MFH) in vitro coculture maturated, and eventually administered to the patient. with SP cells [107]. Also, an important indication that the Comprehensive data from different experiments have CTL clone specifically recognizes MFH CSC-specific anti- thrown light on the use of DCs to initiate tumor-specific gens came from the observation that CTL clones are biased T-cell responses which epitomises a highly potential cancer vaccination approach [116–118]. In particular, DC-mediated towards the recognition of SP cells rather than non-SP cells [107]. tumor-specific immune responses have been fostered by CSCs that function as antigen sources [119, 120]. In prin- Nevertheless, it is imperative to note that CSC-primed T cells distinguished antigens are predominantly unidentified. ciple, the employment of CSC lysates as an antigen source could facilitate synchronized targeting of diverse antigens Otherwise, CAR T cells symbolize a unique and a potential cancer immunotherapy. (e past few decades have wit- and thereby be less prone to antigen loss due to tumor escape nessed a furious attention of the scientific community to- [116]. Also, non-CSCs are less effective in inducing anti- wards applying this therapy in solid tumors which is driven tumor immunity against CSC epitopes as compared to by the profound success and advances in the treatment of enriched CSCs that are immunogenic and better as an hematological malignancies using the adoptive transfer of antigen source. For example, the fact that can be considered CAR T cells. T cells can be genetically crafted employing ex is that, in various syngeneic tumor models of immuno- competent mice, the CSC-DC vaccination notably cir- vivo gene transfer for specifically recognizing a TAA or for expressing the novel T-cell receptor, hence, arbitrating cumvents lung metastasis and slows down the growth of squamous carcinoma compared to immunization with bulk neoplastic cells. Consequently, CAR T cells can be Journal of Oncology 7 tumor cells [51]. (e vaccine comprised of DCs pulsed with immunotherapy. (e endeavours targeting CSCs with irradiated PCSC (prostate cancer stem cells) lines derived specific antibodies have been highly fruitful in terms of therapy response. For instance, tumor progression is de- from the TRAMP (transgenic adenocarcinoma of the mouse prostate) tumors induces a more robust tumor-specific celerated and apoptosis is triggered in leukemic stem cells by immune response than the response induced by DCs pulsed an anti-CD44 mAb [131, 132]. Similarly, human melanoma with differentiated prostate tumor cells. It has also been metastasis is lessened, animal survival in SCID mouse is observed that the CSC-DC vaccine retards tumor growth enhanced, tumor growth reduced, and apoptosis com- [121]. menced in murine breast tumors due to anti-CD44 anti- In a study, cytotoxic T lymphocytes (CTL) antitumor bodies [133, 134]. + + response and activation of CD8 and CD45 T cells were Certainly, targeting CSCs with antibodies has proved to also credited to the breast CSC-DC vaccine [122]. Fasci- be beneficial in improving treatment responses. Also in vivo tumor growth and in vitro proliferation of CD133 natingly, it has been observed that, in the postsurgery gastric phase, CSC-DC vaccines are extremely efficacious when and hepatocellular CSCs can be subdued by drug-conjugated deployed in an adjuvant setting to eradicate microscopic anti-CD133 antibody [135]. A bispecific antibody consisting CSCs, or as combinatorial therapy with radiation and/or of CD133 and CD3 antibodies which were asymmetric in chemotherapy in treating macroscopic tumors. (is was nature displayed a strong antitumor efficacy in a variety of observed in established murine melanoma D5 and squa- tumors [136, 137]. Studies have revealed that antitumor mous cancer SCC7 tumor models where CSC-DC was more capability in solid tumors can be made better with a CSC- efficient when compared to DCs pulsed with non-CSCs in specific antibody-incorporated liposomal nanoparticle de- curing microscopic tumors in [123]. Furthermore, livery system loaded with drugs or a suicide gene [138, 139]. high ALDH CSC-DC vaccines in the adjuvant setting were In yet another classic example, trastuzumab (HER2-tar- more promising than traditional DC vaccines as they re- geting antibody) dramatically lessens the chances of breast duced tumor relapse and lung metastasis coupled with cancer recurrence by targeting HER2, an important regu- enhanced host survival further and obstructing PD-L1 lator of breast CSC self-renewal [140]. Similarly, other [124]. DNA vaccination is gaining hype as compared to HER2-targeting agents, like monoclonal antibody pertu- conventional cell-based vaccines, as they involve injecting zumab and the immunotoxin conjugate ado-trastuzumab emtansine (TDM-1), have further upgraded the efficacy of plasmids for direct antigen production culminating in a protective immunological response, thus preventing cancer human epidermal growth factor receptor (HER2) targeting [125]. Acceptability of DNA immunization and their ap- in the clinical settings [141, 142]. titude to skilfully trigger antigen-specific T cells has been Outstandingly, HER2 expression can also be targeted in efficiently established by clinical trials. PAP and PSA have GBM CSCs [143]. As the “CSC markers” may not necessarily been acclaimed as DNA-based vaccines in human castra- be unique to CSCs, single-agent mAb therapy may adversely tion-resistant prostate cancer (CRPC) [126, 127]. In a affect normal cells [130]. Combination therapy using an study, Nishizawa et al. observed that, in comparison to assortment of different antibodies targeting multiple CSC immunization with the TAA survivin, immunization with markers could potentially reduce doses of individual anti- CSC-specific DNAJB8 expression plasmids shows better bodies to accomplish the efficient abolishment of CSCs while antitumor immune response [128]. reducing side effects due to huge concentrations of single Recently, an experimental DNA vaccine has been de- anti-CSC mAbs. veloped against sternness-specific marker Sox2 and an an- titumor effect also has been seen after immunization against 5.5. Tumor Microenvironment Targeting Immunotherapy. murine lung TC-1/B7 cancer cells expressing oncogenic Sox2 [129]. In a nutshell, it can be contemplated that given (ere are few important factors present in the surrounding tumor microenvironment of CSCs such as hypoxia, chronic the ease and theoretical applicability to target antigens, CSC- specific DNA vaccination holds promise to serve as a tool for inflammation, inflammatory cytokines, and perivascular niches (role in the regulation of proliferation and differ- immunotherapy. entiation) [144–146]. Stat3/NF-κB pathways in tumor and stromal cells are activated by inflammatory cytokines such as IL-1β, IL-6, and IL-8 to further secrete cytokines in a positive 5.4. Antibody-Based Immunotherapy. (e past two decades have brought into limelight the significance and feasibility of feedback loop that elicits angiogenesis, CSC self-renewal, monoclonal antibodies- (mAb-) based treatments as ther- and metastasis [144, 147]. In addition, the CSC population apies against cancer treatment. Antibody-drug conjugates which coevolved in the tumor microenvironment are ad- are powerful newer discovered weapons for the fight against jacent blood vasculature that forms a niche defined by severe cancer [130], and immunomodulatory antibodies, illustrated enhanced angiogenesis and hypoxia [145, 146]. (ese facets by anti-PD-L1, anti-PD-1, and immune checkpoints tar- of the tumor microenvironment have been deciphered as geting anti-CTLA-4 antibodies, have also recently attained probable pharmaceutical targets of CSCs. outstanding clinical success (discussed in immune check- Recent studies have verified that reduced tumor growth points). (e expression levels of some markers (Table 1) in is achieved via IL-6 and/or IL-8 cytokine signalling blockade CSCs considerably dissimilar to the other tumor cells, [148, 149]. Repertaxin, a pharmaceutical commodity, a which provides promising targets for antibody-based noncompetitive inhibitor of IL-8 and CXCR1 signalling, 8 Journal of Oncology could decrease tumor size and enhance chemotherapy ef- 6. Limitation and Challenges ficacy in breast cancer model [150]. However, it has been It has been found that owing to stemlike features like low seen that blocking single cytokines induces limited effects as both IL-6 and IL-8 are crucial for tumor growth and the immunogenicity and intrinsic conventional therapy resis- expression of these genes combined with dismal prognosis in tance, CSCs are demonstrated to be participants in the breast cancer patients. Hence, simultaneously inhibiting IL- processes of tumor progression, maintenance, metastasis, 6 and IL-8 expression was a more lucrative line of action to and relapse. Consequently, CSCs form a crucial target to induce appreciable changes in tumor growth [151]. treat residual disease and circumvent the process of re- Tumor hypoxia is another fascinating means to hit the currence. Immunotherapy is a pragmatic promising tool as CSC niches. Hypoxia causes chemo- and radioresistance by evidenced by the results of a number of clinical trials on activating the HIF pathway and upregulating HIF-1α, by cancer patients. However, in the clinical trials, it has been shown that the objective response rate varies significantly mediating multiple biological effects of hypoxia in tissues. Albeit HIF pathway inhibitor molecules have been underway and a durable response is often confined to a small patient population. It is important to note that the results of the in clinical trials, they seldom become successful enough to cross the clinical trial stage [152]. Also, the CSC niche can be current mono-immunotherapies in solid malignancies are dislocated by targeting tumor vasculature. Limited success generally unsatisfactory. One of the fundamental reasons for has been achieved from the clinical trials targeting angio- this inadequacy might be due to the presence of CSCs that genesis by blocking the vascular endothelial growth factor are not efficiently targeted by the available regimens for pathway [145]. immunotherapy. (e concept of CSC-specific immuno- therapy remains in its budding phase, although it has established utility in a few preclinical and clinical trials 5.6. Immune Checkpoints. Immune checkpoints are known (Table 3); however, challenges still exist. In order to fabricate for declining autoimmunity by intervening coinhibitory more promising strategies and novel therapeutics, genomic, signalling pathways [153]. In cancer, these inhibitory immunological, and biological characterization of CSCs pathways are believed to be implicated in tumor immune along with immune cell interactions in the TME is war- resistance [154]. PD-1/PD-L1 axis and the cytotoxic ranted. Despite the fact that considerable knowledge has T lymphocyte antigen 4 (CTLA-4)/B7 axis are the two in- been accumulated on human CSC properties, most of our strumental pathways of immunoinhibition that have been current understanding is attributed to derived from xeno- identified to date. (ese negative mechanisms contribute to graft studies in immune-compromised nude mice. In the the development of a suppressive microenvironment ren- future, employing humanized mice, immunodeficient dering cell resistance to immune therapy [155, 156]. CSCs strains with engrafted human immune systems, may help to might secrete paracrine factors or direct cell-cell contact and throw light in this area [166]. Yet, another hurdle is the jointly transform the immune cells in the CSC niche because heterogeneity in the population of CSCs and the property of physiologic stem cells have immunoprivilege and active plasticity of cancerous cells. CSCs are acknowledged as immunoregulatory functions [3, 157–160]. Schatton et al. heterogeneous [2] revealed by the fact that different sub- accounted that T-cell activation is downregulated by CSCs populations of CSCs sometimes express different phenotypic [79, 161]. (ey identified malignant melanoma initiating markers in a single cancer type. For example, a previous cells (MMICs), a novel type of CSCs, on the basis of the research group has confirmed that prostate CSCs are mainly expression of ABCB5 (chemoresistance determinant) [161]. PSA-/lo but this is rather heterogeneous inhabiting a vast (e human ABCB5 MMICs express PD-1 and B7.2 and low array of tumorigenic cell subsets that can be prospectively expression of PD-L1 in comparison to ABCD5 cells. (e purified out using distinctive markers [167]. clinical benefit of PD1 and PD-L1 was witnessed in Data amassed from different laboratories worldwide Hodgkin’s disease, melanoma, and lung cancers [162–164]. ascertains that the association between various CSC subsets A better-prolonged response was observed in a patient within a similar cancer type remains largely unidentified, subset that was considerably more durable compared to and if CSC subpopulations share common immunological targeted or cytotoxic therapies. features is also an area to be explored. Hence, there may be a It is proposed that activated T-cell responses may be possibility of a situation that a CSC-specific immunological downregulated by tumor expression of PD-L1 through the treatment leads to the eradication of a particular subset of PD-L1/PD-1 axis and its blocking results. Even though CSCs not all. Furthermore, CSCs and non-CSCs can be Schatton et al. suggested reduced expression of PD-L1 in thought to exhibit diverse plasticity (not discussed in this MMICs [79], in another study, a better expression of PD-L1 review but reviewed in [2]). (is tumor cell plasticity is a was observed in CSCs of head and neck carcinoma [165]. It major impediment in the establishment of long-lasting and can be inferred from this that CSC subsets may reduce T-cell more effective targeted cancer therapies, because therapeutic response through the PD-1/PD-L1 axis. However, future annihilation of present CSC populations might be then clinical trials on immune checkpoint blockade are manda- followed by their regeneration from non-CSC origin within tory for the establishment of these results. Also, the syn- the tumor due to treatment pressures [2]. ergistic effects of immune checkpoint therapies with CSC- It is gripping to see that various agents aim at targeting targeting immunotherapies are suspected to magnify the TME components that demonstrate clinical relevance in clinical applicability of each approach. the treatment of cancers, as our present knowledge about Journal of Oncology 9 Table 3: CSC-targeting strategies and their effect in different types of cancer. Type Cancer types Effects of the CSC-targeting strategies Reference ALDH1A1-specific CD8 T cells distinguish and eradicate ALDHhi CSCs in in Head and neck vitro bioassays, retard xenograft growth and metastases in in vivo bioassays, and [105] CSC-primed prolong survival T cells high + ALDH -CD8 T cells resulted in the inhibition of tumor growth and Lung [106] prolonged survival, hence, bestowing more considerable antitumor effects CSC-DC vaccine that was administered in the adjuvant setting after localized radiation therapy of established tumors resulted in a reduction of tumor growth, and vaccination significantly inhibited tumor growth, abridged [123] high ALDH CSC frequency in primary tumors, and metastases through Squamous cell stimulation of humoral immune responses against CSCs Carcinoma/Melanoma SCC growth was regressed compared to immunization with bulk tumor cells [51] and lung metastasis of melanoma cells was appreciably curtailed In the adjuvant setting, simultaneous PD-L1 blockade further enhanced local CSC-lysate DC tumor recurrence and spontaneous pulmonary metastasis and also increased [125] vaccine survival of the host Tumor regression was witnessed in TRAMP mice, tumor growth was delayed in Prostate mice challenged with prostate CSCs, and tumor-specific immune response was [121] induced that was stronger than differentiated tumor cells Antigen-specific T-cell responses against CSCs were elicited and survival in Glioblastomas [120] animals was improved + + Migration of DCs to the spleen activated CD8 and CD45 T cells; in turn, CTL Breast [122] antitumor responses were induced CSC-mRNA-DC Seven patients vaccinated with an mRNA-DC vaccine exhibited a common Glioblastomas [172] vaccine immune response Stronger antitumor effects were observed in immunization with DNAJB8 DNA vaccine Renal cell carcinoma expression plasmids in contrast with immunization with the tumor-associated [128] antigen survivin, which was expressed in both CSCs and non-CSCs Neural stem cells derived from tumor specimens were prone to attack by lysis Glioblastomas mediated by both autologous IL-2 (or IL-15) activated NK cells but resisted [95] freshly isolated NK cells NK cells CSCs isolated from an array of human cancer cell lines in vitro and dissociated Pancreatic/Breast/ primary cancer specimens ex vivo were preferentially targeted by allogenic [173] Glioblastomas activated human NK cells (e growth of hepatic and pancreatic cancer cells was inhibited in vitro and in Liver/Pancreatic vivo and CD133 high CSCs were targeted by CIK cells bound with anti-CD133/ [137] anti-CD3 bispecific antibodies mAb Human melanoma metastasis was inhibited and the survival of tumor-bearing Melanoma [133] animals was prolonged by anti-CD44 antibodies Murine breast tumor growth was inhibited and apoptosis was induced by anti- Breast [134] CD44 antibodies Patient-derived GBM CSCs were annihilated in an orthotopic tumor model and Glioblastomas [112] in vitro by anti-CD133 CAR T cells CSC-CAR T Significant antitumor efficacy was exhibited by EpCAM-specific CAR T cells in Prostate [113] vitro and in vivo systems the TME is limited. Notably, current research studies have cancer. While therapies that efficiently and selectively get advocated that conventional therapies of cancer are more rid of CSCs do not have clinical applicability, researches on prone to augment CSCs and restructure the TME which various immunotherapeutic strategies to target CSCs may modify the immunotherapy responding ability of (Table 3) are in progress and many of them have displayed CSCs [3]. For instance, chemotherapy enhanced the fre- efficacy in diminishing tumor growth and metastasis in quency of the CSCs in the tumors and downregulated the preclinical and clinical settings. expression of HLA1 molecules in HNSCC and PCa Like all the monotherapies, mono-immunotherapy is improbable to treat cancer, and stratagem that merges both [168, 169] that may cause immunoresistance. Collectively, a CSC epitomises a continuously reshaping target, as it keeps conventional therapies and CSC-specific immunotherapies on developing along with tumor development and pro- would be attractive and promising tactics to combat the gression, particularly, under the influence of treatment. It deadly disease cancer. Combinatorial approaches can reduce can be concluded that the area of immune targeting of drug resistance and cancer cell plasticity and help attain CSCs holds noteworthy promise in curing patients with efficacious treatment outcomes as compared to the 10 Journal of Oncology monotherapies. (eoretically speaking, the immunogenicity References of CSCs can be incremented by the inhibition of negative [1] D. G. 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