Immune modulation by a cellular network of mesenchymal stem cells and breast cancer cell subsets: Implication for cancer therapy

Cellular Immunology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm

Research paper

Immune modulation by a cellular network of mesenchymal stem cells and breast cancer cell subsets: Implication for cancer therapy ⁎

Hussam S. Eltoukhy, Garima Sinha, Caitlyn A. Moore, Oleta A. Sandiford, Pranela Rameshwar Rutgers, New Jersey Medical School, Department of Medicine-Hematology-Oncology, Newark, NJ 07103, USA

A R T I C L E I N F O

A B S T R A C T

Keywords: Cancer stem cell Mesenchymal stem cell Immunosuppression Chimeric antigen receptor Drug delivery Dormancy

The immune modulatory properties of mesenchymal stem cells (MSCs) are mostly controlled by the particular microenvironment. Cancer stem cells (CSCs), which can initiate a clinical tumor, have been the subject of intense research. This review article discusses investigative studies of the roles of MSCs on cancer biology including on CSCs, and the potential as drug delivery to tumors. An understanding of how MSCs behave in the tumor microenvironment to facilitate the survival of tumor cells would be crucial to identify drug targets. More importantly, since CSCs survive for decades in dormancy for later resurgence, studies are presented to show how MSCs could be involved in maintaining dormancy. Although the mechanism by which CSCs survive is complex, this article focus on the cellular involvement of MSCs with regard to immune responses. We discuss the immunomodulatory mechanisms of MSC-CSC interaction in the context of therapeutic outcomes in oncology. We also discuss immunotherapy as a potential to circumventing this immune modulation.

1. Introduction In the mid-20th century, scientists noted that culturing bone marrow and spleen cells grew colonies of fibroblastic phenotype, leading to the concept that precursors of fibroblasts exist and were termed colony forming unit-fibroblasts (CFU-F) [1,2]. These cells were later designated as mesenchymal stroma/stem cells (MSCs). MSCs have undergone extensive studies, which provide in-depth insight into the growth and maintenance of organ-specific tissues, along with insight into how they regulate immune functions within an inflammatory milieu. The information gained in studying MSCs has led to therapeutic potential for these cells, including involvement in cellular delivery of drugs and as immune modulators. As multipotent cells that can selfrenew and differentiate along distinct lineages, MSCs can also regulate immune responses [3]. The immune biology of MSCs is not mutually exclusive of the growing literature on cancer stem cells (CSCs), which are a subgroup of cancer cells with stem cell-like and tumor initiating properties. A common function between MSCs and CSCs is their ability to interact with immune cells to modulate the immune system. Through an interplay between surrounding cells and factors in the microenvironment, stem cells can in many cases suppress the immune system, while in others enhance [4]. These interactions are important when discussing tumorigenesis as this is comprised of a complex system of

⁎

signaling influenced by these cells. By discussing the immune effects of each type of stem cell individually as well as their interrelationship, we can begin to develop a discussion on navigating this interplay of immune modulation to enhance cancer therapies. 2. Mesenchymal stem cells and immune modulation Since their discovery, MSCs have demonstrated the potential to differentiate into cells of a differentiate milieu, an affinity for migration to site of injury and tumor, easy maintenance in vitro, and a crucial role in tissue regeneration and immunomodulatory function [3,5]. These properties of MSCs have allowed for their safe use in clinical applications and the subject of a plethora of clinical trials. MSCs are one of the many stem cells that reside in the bone marrow. These cells play a fundamental role in monitoring afferent and efferent blood flow in the bone marrow and are thus often referred to as ‘the gate keepers’ of the bone marrow. Studies have shown that MSCs possess potent immunosuppressive capacity via soluble factors, insoluble factors, and cell-to-cell interaction [5–7]. In addition, studies have shown that MSCs can reduce inflammation in vivo [7]. Further proof of the immunosuppressive effects of MSCs has been shown when in vivo depletion of a specific tumor stromal cell population restored native antitumor activity [8] MSCs not only reside in the bone marrow but on or in many other vascular sites in the body which allows for easy metastasis to site of injury [5,9].

Corresponding author at: Department of Medicine – Division of Hematology/Oncology, Rutgers, New Jersey Medical School, Newark, NJ 07103 USA. E-mail address: [email protected] (P. Rameshwar).

http://dx.doi.org/10.1016/j.cellimm.2017.07.011 Received 2 February 2017; Received in revised form 28 July 2017; Accepted 29 July 2017 0008-8749/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Eltoukhy, H.S., Cellular Immunology (2017), http://dx.doi.org/10.1016/j.cellimm.2017.07.011

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

Fig. 1. Multiple roles of MSCs. Shown at left are the mechanisms by which MSCs facilitate tumor survival and dormancy. The right section of the diagram shows MSCs as cellular drug delivery and their ability to modulate the immune system.

delivery efficacy. Currently, the method by which MSCs can deliver the drugs is under experimental investigation. There are several strategies currently being investigated to deliver anti-tumor cargo. The methods capitalize on the plasticity of MSCs such as survival in foreign hosts, the ability to form gap junctional intercellular interaction with other cells, expression of cytokine receptors, and response to the microenvironment. The secretome of MSCs has been implicated in the therapeutic effects of MSCs. The loaded cargo can easily be excreted by the cell and directly delivered to the target tumor tissue [25–35]. Based on the discussion in this section, MSCs exert properties that can allow them to deliver anticancer agents directly to tumors. Besides delivering drugs, MSCs can also be used as a delivery system to shuttle small non-coding RNA to tumors. Researchers have demonstrated a non-toxic approach with the use of MSCs to deliver antagomiR-222/223 to dormant breast cancer cell [35]. Similarly, other studies used MSCs to deliver anti-cancer agents via adeno/retroviral vectors, oncolytic viruses or drug loaded polymeric nanoparticles expressing tumor suppressing agents to tumor sites [10,36]. Constructs utilizing gene-directed enzyme prodrug therapy, delivery of pro-apoptotic proteins, and exosome-based cargo delivery will be discussed in more detail in the following sections.

MSCs can either be immune-suppressive or immune-enhancive to cancer cells (Fig. 1) [10]. Upon encountering an immune insult such as cancer activated MSCs have demonstrated the ability to sense microenvironment changes via paracrine molecules which allow docking at the site of injury and the release of therapeutic molecules. Studies indicate that upon homing to the tumor site MSCs release mediators that are detrimental to tumor progression [11]. The source of MSCs can be indicative of the role MSCs play on the tumor. MSCs can be found in many other tissues besides the bone marrow such as umbilical cord blood adipose tissue, amniotic fluid and the placenta. One study demonstrated that adipose-derived stem cells (ADSC) show homing capacity potential to glioma [12]. Thus the difference in phenotype based on the source of MSC can dictate metastatic and homing potential.

3. MSCs and drug delivery This article focuses mostly on the immune suppressive properties of MSCs. However, it is worth noting that MSCs are also multipotent cells with functions similar to stem cells. MSCs show functional plasticity and can respond to tissue insult [13]. These functions provide the MSCs with the ability to navigate organs, including the lymphoid system. Despite MSCs being non-hematological immune cells, they can be given as off-the-shelf cells [14]. This property of MSCs makes these cells attractive for drug delivery [15]. As off-the-shelf method of delivery, this implies that MSCs are able to cross allogeneic barriers. Due to the expression of receptors for chemokines, MSCs can home to sites of tissue injury such as inflammatory sites where the mediators can license them to become immune suppressor cells [13,16–23]. Thus, the MSCs will not be rejected or cleared by the immune system. This will provide the MSCs with a window of opportunity to deliver the desired drug. Also, the prolongation of MSCs in a foreign host might not be an advantage since the MSCs are from an allogeneic host. Since they are multipotent cells, if the environment is permissive, the MSCs could expand at the target site through self-renewal [24]. This will allow the MSCs to deliver the drug for a prolonged period. However, this might not be a desirable outcome since the drugs might not move to the daughter cells. Also, this ability enables improved survivability once at the target site, increasing drug

3.1. Gene-directed enzyme prodrug therapy MSC-based gene-directed enzyme prodrug therapy (GDEPT) or “suicide gene therapy” is designed around the use of MSCs as a pharmacologic pump. A viral vector that encodes a gene for a prodrug-activating enzyme transgene is incorporated into MSCs where the vector is transcribed and translated into the desired enzyme within the target cell. Transduced MSCs are then reinjected and allowed to home to the tumor site at which point the prodrug is administered. The cytotoxic metabolites that are produced within transduced MSCs are then pumped out into the local microenvironment killing neighboring tumor cells by a ‘bystander effect’ [15,37–39]. Herpes simplex virus thymidine kinase (HSV-TK) with Ganciclovir (GCV) and E. coli cytosine deaminase (CD) with 5-fluorocytosine (5-FC) are commonly used suicide gene constructs. Incorporation of a suicide gene enables efficient elimination of transduced MSCs lowering the risk of malignant transformation. Also 2

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

[76–79]. The formation of CSCs is either due to mutations in normal stem cells or due to dedifferentiation of more differentiated cells that have oncogenic changes, resulting in cancer within progenitor cells [80–84]. CSCs can self-renew and similar to other normal cells, differentiate into progenitors but never to fully differentiated cells since the initiating CSCs are malignant [79]. The population of CSCs can adapt dormancy within tissues such as the bone marrow, maintain low numbers, and demonstrate resistance to most available therapy [85,86]. The similarity between CSCs and native stem cells increases the complexity in designing drugs against CSCs. Thus, it is important to understand how CSCs interact with the microenvironment since this could lead to the development of targets. In breast cancer, CSCs migrate to different organs of the body where they maintain cycling quiescence. Clinical evidence supports the bone marrow as the source organ during tertiary metastasis of breast cancer, suggesting that the CSCs may survive within the cavity [66,84,87–91]. Indeed, our group has reported on the survival of dormant breast cancer cells close to the endosteum and with evidence that the transition into dormancy begin at the perivascular region when the MSCs provide immune protection as well as release of miRNA-containing exosomes [35,90,92,93]. It should be noted that MSCs are not the only cells within the bone marrow microenvironment that support cancer dormancy. The bone marrow microenvironment includes non-hematopoietic cells such as fibroblasts, macrophages, endothelial cells, MSCs, and adipocytes, hematopoietic cells such as stem cells (HSCs), progenitors, and differentiated immune cells [66,90,91]. The region of dormancy preferred by the CSCs has been identified as the hypoxic region close to the endosteum, the area of the cavity that also supports the HSCs [94]. Although MSCs can support cancer dormancy, the roles of the other cells within the bone marrow have not been studied. This is in part due to the structure of the bone marrow, making this organ difficult to be reproduced as an organoid structure to mimic the natural organ. In order to recreate the marrow and its microenvironment, 3D modelling with the primary cells to recapitulate the marrow. The model would be able to study how the cellular and soluble factors interact to closely replicate the mechanical features of the bone marrow. Our laboratory are in the process of developing a 3D model of the bone marrow microenvironment. Once this 3D modelling method has been developed, we will study selective cell types to determine how the different regions within the bone marrow supporting dormancy as well as other behavior of different cancer cell subsets. The stromal cells and the MSCs interact with the incoming CSCs either directly or indirectly and signal them to undergo dormancy in the bone marrow (Fig. 2) [79,92,95,96]. Indirect interactions include the release of exosomes which carry non-coding RNA such as miRNA [35]. The exosomes enter the cancer cells and signal them to transition into cycling quiescence. Direct interaction can occur through gap junctional formation between the bone marrow cells and the CSCs for the delivery of miRNA, as well as interaction through the CXCR4-CXCL12 axis [79,92,95,96]. MSCs constitute a major cell niche for HSCs such as supporting their proliferation [97,98]. In fact it has been shown that MSCs when coadministered with HSCs in bone marrow transplants showed improvement with respect to engraftment and graft function [98]. It is plausible to propose the same effect of MSCs on HSC maintenance may be extended to CSCs. As discussed above CSCs share similar properties with other stem cells including HSCs by gene expressions and function. Recent studies have also shown the involvement of autophagy in CSC dormancy [99,100]. Autophagy is a process by which cells survive under stress by forming a membrane around defective proteins. For tumor suppression, autophagy is an important method for cells to destroy any damaged proteins to prevent oncogenic transformation by mutated proteins [101,102]. Autophagy has also been linked to CSC survival and maintenance [99,100]. It is well-established that CSCs survive in hypoxic microenvironments and remain unaffected even

tissue-specific promoters and prodrug activators have been utilized to further mitigate potential side effects of these genes in non-tumor environments by facilitating more direct action [40] 3.2. Pro-apoptotic proteins MSCs can also be used to deliver pro-apoptotic proteins that selectively induce apoptosis of cancer cells. Studies have shown that administration of MSCs engineered to express TRAIL (tumor necrosis factor-related apoptosis induced ligand) can induce apoptosis and reduce tumor cell viability in several cancer models [41–45]. However TRAIL is a membrane protein that requires additional cleavage from its anchoring site to be released into the microenvironment. To address this engineered recombinant secretable TRAIL (S-TRAIL) was developed and shown to exhibit efficient secretion higher cytotoxicity than native TRAIL and significant anti-tumor effects in glioblastoma models [46–48]. Supplementary treatments have also been used to augment the anti-tumor effect by incorporating XIAP inhibitors PI3K inhibitors and antago-miRNA with systemic MSC-based delivery of TRAIL [49] 3.3. Exosomes Exosomes are cup-shaped membrane vesicles with diameters between 20 and 100 nm. These exosomes are present in a wide array of biological fluids and mediate intercellular transfer of proteins and RNA [34,50]. Generally, exosomes exhibit features such as tolerability, ability to cross plasma membranes, intrinsically home to target tissues, and amenability to membrane modifications that make them ideal drug delivery vehicles [51,52]. Notably, MSCs produce a relatively high concentration of non-immunogenic exosomes, making exosomes prime candidates for drug delivery strategies through MSCs [52]. Exosomes are easily isolated from cell culture and physiological fluids, indicating that their delivery through MSCs will not lead to degradation, giving the exosomes time to enter the target cancer cells [34,53–58]. Recently, it has been reported that the anti-inflammatory compound curcumin can form complexes with exosomes. This complex enhances the anti-inflammatory effects of curcumin, inhibiting IL-6 and TNF-α secretion in activated myeloid cells and in a lipopolysaccharide (LPS)induced septic shock mouse model [59]. In a model of LPS-induced brain inflammation, curcumin-exosome complexes have also been shown to cross the blood-brain barrier into microglial cells to reduce inflammation [60]. Limitations of exosome-based drug delivery strategies mainly involve the issue of loading exosomes without compromising self-renewal and potency of the MSCs [61]. However, since MSCs produce large amounts of exosomes, the MSCs can be loaded with the desired drugs for release through the endogenous formation of exosomes. The issue with this approach is that only small non-coding RNA and very small molecules could be delivered through exosomes due to the small size of these vesicles. Also, scalability and reliability of exosome-based MSC drug delivery will be important for clinical application. 3.4. Pitfalls and potential risks There are inherent risks involved with using MSC-based treatments. Administration of MSCs as cargo delivery vehicles can potentially lead to tumorigenesis through enhanced angiogenesis and their ability to support cancer growth [62–71]. Furthermore, drugs administered alongside cargo-loaded MSCs have the potential to affect MSC behavior and cargo release. 4. Immune system to cancer stem cells (CSCs) The stochastic model of tumorigenesis has long been replaced by the CSC model. There is ample clinical evidence that supports the existence of CSCs [72–75]. The small subset of CSCs can initiate tumor formation 3

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

Fig. 2. MSC-CSC interaction in cancer dormancy. Shown at the center is cellular interaction between MSCs and CSCs. This interaction can lead to chemoresistance and cycling quiescence (lower left). The lower right shows the similarities between MSCs and CSCs with respect to shared gene expressions.

almost non-detection, resulting in these cells being unable to act as antigen presenting cells [119]. Interestingly, the non-CSCs could be induced by interferon γ to upregulate the MHC molecules more efficiently than the glioma CSCs, which mounted immune suppression. The induction of T-cell response should, in theory, require that CSCs express co-stimulatory proteins such CD80 and CD86. However, instead they express the inhibitory molecule programmed death ligand-1, thus inhibiting any effector T-cell activation [119,120]. In CD44+ squamous cell carcinoma of the head and neck, blocking the PD-1 receptor chemosensitized resistant tumor-initiating cells [121]. Contrary to tumorigenic cells, healthy cells do not express the stress receptor NKG2D for NK cells, thereby providing the immune system with selective targeting of cancer cells [122]. If CSCs express the stress receptor despite the expression of MHC-1, these cells will be targeted by NK cells [123]. However, as discussed above, the CSCs seem to be able to adapt to stress such as by utilizing the autophagocytic pathways. The CSC microenvironment is often composed of tumor activating macrophages (TAMs) which suppress the immune system and promote cellular dormancy [124,125]. M2 macrophages have been shown to support mammosphere formation by CSCs and also maintain the stemness of the cancer cells [125]. In renal CSCs, TNF-α produced by TAM was shown to maintain its stemness via the NFκB signaling pathway [124]. CD47, an integrin-associated protein, has been shown to be involved in macrophage-associated phagocytosis [126]. CD47, when bound to signal-regulatory protein α (SIRPα), releases an inhibitory signal for phagocytosis. In xenograft models, blocking of CD47 resulted in the CSCs differentiating, making the breast cancer into drugsensitive cells [126,127]. Although the immunosuppressive effect of MSCs has been well studies, their role in the bone marrow is still to be determined. The ongoing studies in our laboratory suggest an axis between MSCs and macrophage in determining how CSCs behave, dormancy versus recurrence into metastatic cancer cells. Chemoresistance of CSCs has been linked to several drug resistance genes. One such gene encodes ATP-binding cassette (ABC) transporters that use ATP-derived energy to pump small molecules/compounds out of the cell [85]. There are different categories of ABC genes expressed on different CSCs. ABCG2 is expressed in both liver and breast CSCs, whereas ABCB1 is upregulated in thyroid and osteosarcoma CSCs [128–131]. There are several factors that play key roles in eliminating CSCs from being targeted. In the future, it is important to include

after aggressive chemo/radiation therapy. Recent studies have shown that induction of stress leads to the CSCs undergoing autophagy to facilitate a dormant phenotype [103,104]. In an experimental study with pancreatic CSCs, blocking autophagy proteins ATG5, ATG7, and BECN1 led to significant reduction of the CSC population [105]. In a mouse model of breast cancer, it was shown that downregulation of FIP200, an autophagy gene negatively affected tumor formation [106]. In a separate study, it was shown the autophagic protein Beclin1 was robustly upregulated in the tumorospheres generated from a breast cancer cell line, under normal conditions as well as under stress [100]. A separate study performed with glioblastoma stem cells showed upregulation of the autophagic genes DRAM1 and SQSTM1, resulting in the regulation of p62, a nucleoprotein that scavenges toxic proteins, and regulated invasion and migration through ATP [107]. Together, these studies suggest that autophagy can regulate the CSC organelles, resulting in drug resistance and transition of cancer cells into dormancy. The signaling pathways utilized by CSCs and native stem cells are similar and include Hedgehog, Notch, Wnt, IL-6/JAK/STAT3, and NFκB, leading to expression of similar types of markers [108–113]. The similarity between CSCs and native stem cells makes it difficult to identify target markers to treat CSCs. The other important reasons for therapeutic failure in targeting CSCs are heterogeneity within the population, their ability to avoid immune targeting, and the high probability of these cells to be drug resistant [80,114]. At times, oncogenic mutations can be immunogenic. In these cases, the cancer cells can be detected by the immune system either by activation of the innate immune system or by activation of MHC-mediated immunity [114,115]. The same does not hold true for CSCs. Recent studies have shown pre-leukemic HSCs to be responsible for AML formation [116]. The pre-leukemic HSCs were not only chemoresistant but were the source for relapse, indicating their ability to evade the immune system [116]. CSCs have the ability to alter the immunogenic microenvironment to an immunosuppressive milieu by the release of immunosuppressive factors and the recruitment of immunosuppressive cells [92,115,117,118]. One such factor is TGFβ, which was reported by our group. The interaction between MSCs and CSCs could lead to the secretion of TGFβ by MSCs, resulting in increase in immune suppressor regulatory T-cells (Tregs) and concomitant decrease in Th17 [92]. In glioblastoma CSCs, MHC-1 and MHC-II were significantly decreased to 4

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

in efficacy of the CAR-Ts [134,135]. Second generation CAR-T cells were thus developed which added a costimulatory domain. One of the costimulatory domains tested was CD28 which showed a more sustained and efficacious response by the T-cells than that of the first generation T-cells [136–138]. Since the advent of CAR-T cells, many various clinical trials have been conducted. The majority of clinical applications have been performed in B-cell malignancies [139–148]. This is due to the specificity of CD19, which is found almost exclusively on B-cells. There are many other studies underway examining the effect of targeting various other surface markers [35,149–156]. This can also be applied to targeting CSC markers, thus potentially eliminating the tumor-initiating cells. There are many potential obstacles to the use of CAR-T cells in augmenting the immune response. The problem of a lack of specificity of surface receptors is one of the major key setbacks. By attacking cells that express a certain surface marker, many ‘innocent bystanders’ are also targeted. In the setting of CD19-specific CAR-T cells, native nonmalignant B-cells become targeted and thus B-cell aplasia may occur. This side effect has been demonstrated as there have been studies to overcome the limitation of B-cell aplasia by blocking the CAR-T cells, which have shown reversal of the B-cell aplasia [154]. In using CAR-T cells to target cancer cells, targeting the cancer cells alone without ensuring that the CSC population is targeted will leave behind the CSC population and thus risk regrowth of the tumor. Regarding targeting of CSCs, the problem arises in that the property of CSCs that differentiates them from the other cancer cells is their stemness. It is well established that many surface markers on CSCs include shared markers with healthy stem cells. If CSCs and thus native stem cells are depleted, this will lead to a significant immunocompromised state. Epithelial cell adhesion molecule (EpCAM), a stem cell marker, has been shown to be expressed in CSCs [157–161]. EpCAM-targeting CART cells were developed in a human derived prostate cancer cell line PC3 and its metastatic form PC3M [162]. Both in vitro and in vivo studies showed killing of PC3M and inhibition of tumor growth of the PC3 clone, therefore providing a new direction towards targeting CSCs using CAR-T cells [162]. In another study using glioblastoma, the AC133 epitope on CD133 was used to isolate the CSC population from the patients [163]. The CAR-T cells designed against them were able to significantly kill the CSC population in glioblastoma cells [163]. The challenge is how to target CSCs, which by themselves could include a heterogeneous population of cells. More importantly, the

treatment regimens that can target the heterogeneous CSC population in order to achieve disease cure. 5. Circumventing immune suppression with immune therapy As discussed above, one of the main effects that MSCs have on the immune system is to exert immune suppressor effects. The influence of MSCs on immunosuppression involves both the innate and adaptive immune systems. In the innate immune system, MSCs have been shown to suppress dendritic cell, NK cell, neutrophil, and macrophage activation [6]. In the adaptive immune system, MSCs prohibit through various mechanisms both B- and T-cell activation. Regarding T-cells, MSCs can increase Tregs and inhibit the activation of cytotoxic T-cells that are needed to eliminate tumor cells as well as infected cells [90]. While the overall immunosuppression by MSCs may be beneficial in some regard, such as in wound healing when MSCs migrate to the site of injury to suppress the inflammatory response in order to facilitate proliferation of stromal cells and allow reconstruction of the extracellular matrix, in many instances such as cancer, this may not be a desirable response [67]. Thus, when one considers past failures of immune therapy, it is not surprising, since the immunology of MSCs have just taken center. Thus, in order to discuss methods of circumventing this immune modulation, one must discuss chimeric antigen receptor T (CAR-T) cells. CAR-T cells are an application in which engineered autologous Tcells are given to tumor patients to target the tumor and to circumvent the suppressor systems such as MSCs and Tregs (Fig. 3). CAR-T-cells are in the clinic mostly for hematological disorder, although the method is gaining ground towards solid tumors. CAR-T cells are generated by engineering T-cells to target a specific receptor. One of the early experiments conducted demonstrated the principle used in CAR T-cells [132]. In this experiment, the T-cell receptor (TcR) constant domain was fused to an antibody’s variable domain resulting in an antigenspecific T-cell, which was activated in a non-MHC-restricted manner. Since then, CAR-T cells have been improved with several generations of the original CAR-T. One of the main modifications over time has been that of the signaling for the T-cell activation. First generation CAR-T cells had the chimeric molecule consisting of single chain Fv domain linked to CD3ζ, the triggering receptor allowing T-cell activation without any co-stimulation [133,134]. This T-cell activation without a costimulatory domain results in T-cell anergy with a resultant decrease

Fig. 3. CAR-T cells as a method for circumventing the immune suppression by MSCs. The immune suppressive property of MSCs are shown to be circumvented by CAR-T cells.

5

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

827–830. [9] A.I. Caplan, MSCs: The sentinel and safe-guards of injury, J. Cell. Physiol. 231 (2016) 1413–1416. [10] D. Gjorgieva, N. Zaidman, D. Bosnakovski, Mesenchymal stem cells for anti-cancer drug delivery, Rec. Pat Anticancer Drug Discov. 8 (2013) 310–318. [11] N. D'Souza, J.S. Burns, G. Grisendi, O. Candini, E. Veronesi, S. Piccinno, E.M. Horwitz, P. Paolucci, P. Conte, M. Dominici, MSC and tumors: homing differentiation, and secretion influence therapeutic potential, Adv. Biochem. Eng. Biotechnol. 130 (2013) 209–266. [12] M. Lamfers, S. Idema, F. van Milligen, T. Schouten, P. van der Valk, P. Vandertop, C. Dirven, D. Noske, Homing properties of adipose-derived stem cells to intracerebral glioma and the effects of adenovirus infection, Cancer Lett. 274 (2009) 78–87. [13] L.S. Sherman, A. Conde-Green, O.A. Sandiford, P. Rameshwar, A discussion on adult mesenchymal stem cells for drug delivery: pros and cons, Ther. Deliv. 6 (2015) 1335–1346. [14] C. Lechanteur, A. Briquet, O. Giet, O. Delloye, E. Baudoux, Y. Beguin, Clinicalscale expansion of mesenchymal stromal cells: a large banking experience, J. Transl. Med. 14 (2016) 145. [15] L.S. Sherman, M. Shaker, V. Mariotti, P. Rameshwar, Mesenchymal stromal/stem cells in drug therapy: new perspective, Cytotherapy 19 (2017) 19–27. [16] A.K. Teo, L. Vallier, Emerging use of stem cells in regenerative medicine, Biochem. J. 428 (2010) 11–23. [17] M.F. Corsten, K. Shah, Therapeutic stem-cells for cancer treatment: hopes and hurdles in tactical warfare, Lancet Oncol. 9 (2008) 376–384. [18] A. Aleynik, K.M. Gernavage, Y. Mourad, L.S. Sherman, K. Liu, Y.A. Gubenko, Stem cell delivery of therapies for brain disorders, Clin. Transl. Med. 3 (2014) 24. [19] W.Y. Jung, J.H. Kang, K.G. Kim, H.S. Kim, B.I. Jang, Y.H. Park, Human adiposederived stem cells attenuate inflammatory bowel disease in IL-10 knockout mice, Tissue Cell 47 (2015) 86–93. [20] K. Le Blanc, C. Tammik, K. Rosendahl, E. Zetterberg, O. Ringden, HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells, Exp. Hematol. 31 (2003) 890–896. [21] J.A. Potian, H. Aviv, N.M. Ponzio, J.S. Harrison, P. Rameshwar, Veto-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens, J. Immunol. 171 (2003) 3423–3434. [22] L.S. Sherman, J.L. Munoz, S.A. Patel, M.A. Dave, I. Paige, P. Rameshwar, Moving from the laboratory bench to patients' bedside: considerations for effective therapy with stem cells, Clin. Transl. Sci. 4 (2011) 380–386. [23] Y.S. Kim, J.Y. Kim, D.M. Shin, J.W. Huh, S.W. Lee, Y.M. Oh, Tracking intravenous adipose-derived mesenchymal stem cells in a model of elastase induced emphysema, Tuberc. Respir. Dis. 77 (2014) 116–123. [24] T.J. Kean, P. Lin, A.I. Caplan, J.E. Dennis, MSCs: delivery routes and engraftment, cell-targeting strategies, and immune modulation, Stem Cells Int. 2013 (2013) 732–742. [25] C.T. Stabler, S. Lecht, P. Lazarovici, P.I. Lelkes, Mesenchymal stem cells for therapeutic applications in pulmonary medicine, Br. Med. Bull. 115 (2015) 45–56. [26] A.I. Caplan, J.E. Dennis, Mesenchymal stem cells as trophic mediators, J. Cell. Biochem. 98 (2006) 1076–1084. [27] T.S. Chen, E.E. Tredget, P.Y. Wu, Y. Wu, Paracrinefactors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing, PloS One 3 (2008). [28] S.C. Hung, R.R. Pochampally, S.C. Chen, S.C. Hsu, D.J. Prockop, Angiogenic effects of human multipotent stromal cell conditioned medium activate the PI3K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase survival, and stimulate angiogenesis, Stem Cells 25 (2007) 2363–2370. [29] T. Kinnaird, E. Stabile, M.S. Burnett, M. Shou, C.W. Lee, S. Barr, Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms, Circulation 109 (2004) 1543–1549. [30] L. Li, S. Zhang, Y. Zhang, B. Yu, Y. Xu, Z. Guan, Paracrine action mediate the antifibrotic effect of transplanted mesenchymal stem cells in a rat model of global heart failure, Mol. Biol. Rep. 36 (2009) 725–731. [31] Y.C. Lin, T.L. Ko, Y.H. Shih, M.Y. Lin, T.W. Fu, H.S. Hsiao, Human umbilical mesenchymal stem cells promote recovery after ischemic stroke, Stroke 42 (2011) 2045–2053. [32] F. Togel, K. Weiss, Y. Yang, Z. Hu, P. Zhang, C. Westenfelder, Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury, Am. J. Physiol. Renal. Physiol. 292 (2007) F1626–1635. [33] D. van Poll, B. Parekkadan, C.H. Cho, F. Berthiaume, Y. Nahmias, A.W. Tilles, Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo, Hepatology 47 (2008) 1634–1643. [34] R.C. Lai, F. Arslan, M.M. Lee, N.S. Sze, A. Choo, T.S. Chen, Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury, Stem Cell Res. 4 (2010) 214–222. [35] S.A. Bliss, G. Sinha, O.A. Sandiford, L. Williams, D.J. Engelberth, K. Guiro, L.L. Isenalumhe, S.J. Greco, S. Ayer, M. Bryan, R. Kumar, N.M. Ponzio, P. Rameshwar, Mesenchymal stem cell-derived exosomes stimulates cycling quiescence and early breast cancer dormancy in bone marrow, Cancer Res. 76 (2016) 1–13. [36] M. Lovkova, Biosynthesis of the piperidine ring in alkaloids (review), Izv Akad Nauk SSSR Biol 3 (1970) 451–455. [37] G.U. Dachs, M.A. Hunt, S. Syddall, D.C. Singleton, A.V. Patterson, Bystander or no bystander for gene directed enzyme prodrug therapy, Molecules 14 (2009) 4517–4545. [38] I. Amara, W. Touati, P. Beaune, I. de Waziers, Mesenchymal stem cells as cellular vehicles for prodrug gene therapy against tumors, Biochimie 105 (2014) 4–11.

heterogeneity of the CSCs may change with the microenvironment and with patient diversity. The current literature has not dissected these differences. Therefore, despite these promising results, questions of safety remain unanswered. The targeting of CD133 and EpCAM may be able to target a subpopulation of CSCs through principle, but this cannot be clinically transitioned as not only does it leave behind nonCD133 and non-EpCAM-expressing CSCs, but it has the potential to target normal stem cells as well because of the similarity to the latter. The reason for concern of the non-CSC population remaining is that these cells, with the appropriate microenvironment, may dedifferentiate into CSCs. One cannot forget that these are malignant cells. 6. Conclusion This review article discussed how CSCs can modulate the immune system and the role of MSCs. Since MSCs are ubiquitous, we assume similarities regardless of the source. Until robust studies are done to compare the different sources of MSCs, it will be difficult to determine if MSCs coming from the adipose tissue will respond to the tumors in a manner similar to those from the bone marrow. Regarding tumor cells, MSCs can enhance and suppress tumor growth depending on the microenvironmental factors [4]. Understanding the immunomodulatory effects of MSCs within various tumor microenvironment is crucial to gain insights into mechanistic studies. This will allow for targeted immune treatment, alone or in combination with current anti-cancer drugs. By understanding of the mechanisms by which MSCs navigate the immune system, going forward, scientists, through research, can develop therapeutic agents. One such therapeutic option using CAR-T cells has been discussed. We believe mechanisms such as adoptive T-cell transfer by which CSCs can be directly targeted to eliminate tumor initiation could provide a reliable and effective method of treating cancer. We propose that the future of oncology must encourage research studies to include MSCs to understand how the immune system evade the tumor cells. This will result in efficient development of targeted immunotherapy. A major arm of MSC-tumor biology is the exciting development of MSCs for drug delivery. This use of MSCs could be developed along with nanotechnology to determine the most efficient use of MSCs. Such development must keep in mind that MSCs are immunosuppressive cells and possess the ability to develop stromal cells within the tumor. We propose that MSCs should be center in future cancer research and stem cell biologists need to be involved within a team of bioengineers, physicians, and cancer biologists. Conflicts of interest The authors declare that they have not conflicts of interest. References [1] A.J. Friedenstein, R.K. Chailakhjan, K.S. Lalykina, The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells, Cell Tissue Kinet. 3 (1970) 393–403. [2] A.J. Friedenstein, J.F. Gorskaja, N.N. Kulagina, Fibroblast precursors in normal and irradiated mouse hematopoietic organs, Exp. Hematol. 4 (1976) 267–274. [3] S. Ramdasi, S. Sarang, C. Viswanathan, Potential of mesenchymal stem cell based application in cancer, Int. J. Hematol-Oncol. Stem Cell Res. 9 (2015) 95–103. [4] A. Torsvik, R. Bjerkvig, Mesenchymal stem cell signaling in cancer progression, Cancer Treat. Rev. 39 (2013) 180–188. [5] A. Uccelli, L. Moretta, V. Pistoia, Immunoregulatory function of mesenchymal stem cells, Eur. J. Immunol. 36 (2006) 2566–2573. [6] M.J. Hoogduijn, F. Popp, R. Verbeek, M. Masoodi, A. Nicolaou, C. Baan, M.H. Dahlke, The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy, Int. Immunopharmacol. 10 (2010) 1496–1500. [7] Y. Moodley, V. Vaghjiani, J. Chan, S. Baltic, M. Ryan, J. Tchongue, C.S. Samuel, P. Murthi, O. Parolini, U. Manuelpillai, Anti-inflammatory effects of adult stem cells in sustained lung injury: a comparative study, PloS One 8 (2013) e69299. [8] M. Kraman, P.J. Bambrough, J.N. Arnold, E.W. Roberts, L. Magiera, J.O. Jones, A. Gopinathan, D.A. Tuveson, D.T. Fearon, Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha, Science 330 (2010)

6

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

[69] M. Kampera, S. Glennie, J. Dyson, D. Scott, R. Laylor, E. Simpson, Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide, Blood 101 (2003) 3722–3729. [70] I. Rasmusson, O. Ringden, B. Sundberg, K. Le Blanc, Mesenchymal stem cells inhibit lymphocyte prolideration by mitogens and alloantigens by different mechanisms, Exp. Cell Res. 305 (2005) 33–41. [71] J. Stagg, S. Pommey, N. Eliopoulos, J. Galipeau, Interferon-gamma-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell, Blood 107 (2006) 2570–2571. [72] J.W. Eng, T.A. Mace, R. Sharma, D.Y. Twum, P. Peng, J.F. Gibbs, R. Pitoniak, C.B. Reed, S.I. Abrams, E.A. Repasky, B.L. Hylander, Pancreatic cancer stem cells in patient pancreatic xenografts are sensitive to drozitumab, an agonistic antibody against DR5, J. Immunother. Cancer 4 (2016) 33. [73] S.V. Chekhun, T.V. Zadvorny, Y.O. Tymovska, M.F. Anikusko, O.E. Novak, L.Z. Polishchuk, CyrillicD44+/CD24-markers of cancer stem cells in patients with breast cancer of different molecular subtypes, Exp. Oncol. 37 (2015) 58–63. [74] F. Grillet, E. Bayet, O. Villeronce, L. Zappia, E.L. Lagerqvist, S. Lunke, E. CharafeJauffret, K. Pham, C. Molck, N. Rolland, J.F. Bourgaux, M. Prudhomme, C. Philippe, S. Bravo, J.C. Boyer, L. Canterel-Thouennon, G.R. Taylor, A. Hsu, J.M. Pascussi, F. Hollande, J. Pannequin, Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture, Gut (2016) in press. [75] B.B. Green, S.H. Taplin, Breast cancer screening controversies, J. Am. Board Family Practice 16 (2003) 233–241. [76] B. Naume, X. Zhao, M. Synnestvedt, E. Borgen, H.G. Russnes, O.C. Lingjaerde, M. Stromberg, G. Wiedswang, G. Kvalheim, R. Karesen, J.M. Nesland, A.L. Borresen-Dale, T. Sorlie, Presence of bone marrow micrometastasis is associated with different recurrence risk within molecular subtypes of breast cancer, Mol. Oncol. 1 (2007) 160–171. [77] J.L. Mansi, U. Berger, T. McDonnell, A. Pople, Z. Rayter, J.C. Gazet, R.C. Coombes, The fate of bone marrow micrometastases in patients with primary breast cancer, J. Clin. Oncol. 7 (1989) 445–449. [78] I. Martin-Padura, P. Marighetti, A. Agliano, F. Colombo, L. Larzabal, M. Redrado, A.M. Bleau, C. Prior, F. Bertolini, A. Calvo, Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts, Lab. Invest. 92 (2012) 952–966. [79] S.A. Patel, S.H. Ramkissoon, M. Bryan, L.F. Pliner, G. Dontu, P.S. Patel, S. Amiri, S.R. Pine, P. Rameshwar, Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy, Sci. Rep. 2 (2012) 906. [80] C. Ginestier, M.H. Hur, E. Charafe-Jauffret, F. Monville, J. Dutcher, M. Brown, J. Jacquemier, P. Viens, C.G. Kleer, S. Liu, A. Schott, D. Hayes, D. Birnbaum, M.S. Wicha, G. Dontu, ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome, Cell Stem Cell 1 (2007) 555–567. [81] M.S. Wicha, S. Liu, G. Dontu, Cancer stem cells: an old idea–a paradigm shift, Cancer Res. 66 (2006) 1883–1890. [82] S. Sell, Stem cell origin of cancer and differentiation therapy, Critical Rev. Oncol/ Hematol. 51 (2004) 1–28. [83] C. Scheel, R.A. Weinberg, Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links, Sem. Cancer Biol. 22 (2012) 396–403. [84] S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, C. Brisken, J. Yang, R.A. Weinberg, The epithelial-mesenchymal transition generates cells with properties of stem cells, Cell 133 (2008) 704–715. [85] L.N. Abdullah, E.K. Chow, Mechanisms of chemoresistance in cancer stem cells, Clin. Transl. Med. 2 (2013) 3. [86] T. Lapidot, C. Sirard, J. Vormoor, B. Murdoch, T. Hoang, J. Caceres-Cortes, M. Minden, B. Paterson, M.A. Caligiuri, J.E. Dick, A cell initiating human acute myeloid leukaemia after transplantation into SCID mice, Nature 367 (1994) 645–648. [87] D. Paez, M.J. Labonte, P. Bohanes, W. Zhang, L. Benhanim, Y. Ning, T. Wakatsuki, F. Loupakis, H.J. Lenz, Cancer dormancy: a model of early dissemination and late cancer recurrence, Clin. Cancer Res. 18 (2012) 645–653. [88] D. Samanta, D.M. Gilkes, P. Chaturvedi, L. Xiang, G.L. Semenza, Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells, Proc. Natl. Acad. Sci. 111 (2014) E5429–E5438. [89] F. Rahim, S. Hajizamani, E. Mortaz, A. Ahmadzadeh, M. Shahjahani, S. Shahrabi, N. Saki, Molecular regulation of bone marrow metastasis in prostate and breast cancer, Bone Marrow Res. 2014 (2014) 405920. [90] S.A. Patel, J.R. Meyer, S.J. Greco, K.E. Corcoran, M. Bryan, P. Rameshwar, Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta, J. Immunol. 184 (2010) 5885–5894. [91] B.Y. Reddy, P.K. Lim, K. Silverio, S.A. Patel, B.W. Won, P. Rameshwar, The microenvironmental effect in the progression metastasis, and dormancy of breast cancer: a model system within bone marrow, Int. J. Breast Cancer 2012 (2012) 721659. [92] S.A. Patel, M.A. Dave, S.A. Bliss, A.B. Giec-Ujda, M. Bryan, L.F. Pliner, P. Rameshwar, T/Th17 polarization by distinct subsets of breast cancer cells is dictated by the interaction with mesenchymal stem cells, J. Cancer Stem Cell Res. 29 (2014) 2014. [93] S.J. Greco, S.A. Patel, M. Bryan, L.F. Pliner, D. Banerjee, P. Rameshwar, AMD3100-mediated production of interleukin-1 from mesenchymal stem cells is key to chemosensitivity of breast cancer cells, Am. J. Cancer Res. 1 (2011) 701–715. [94] H. Clevers, K.M. Loh, R. Nusse, Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control, Science 346 (2014)

[39] O. Greco, G.U. Dachs, Gene directed enzyme/prodrug therapy of cancer: historical appraisal and future prospectives, J. Cell. Physiol. 187 (2001) 22–36. [40] O. Levy, W.N. Brennen, E. Han, D.M. Rosen, J. Musabeyezu, H. Safaee, S. Ranganath, J. Ngai, M. Heinelt, Y. Milton, H. Wang, S.H. Bhagchandani, N. Joshi, N. Bhowmick, S.R. Denmeade, J.T. Isaacs, J.M. Karp, A prodrug-doped cellular Trojan Horse for the potential treatment of prostate cancer, Biomaterials 91 (2016) 140–150. [41] H. Walczak, P.H. Krammer, The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems, Exp. Cell Res. 256 (2000) 58–66. [42] L.P. Mueller, TRAIL-transduced multipotent mesenchymal stromal cells (TRAILMSC) overcome TRAIL resistance in selected CRC cell lines in vitro and in vivo, Cancer Gene Ther. 18 (2011) 229–239. [43] S.K. Kim, PEX-producing human neural stem cells inhibit tumor growth in a mouse glioma model, Clin. Cancer Res. 11 (2005) 5965–5970. [44] M. Ehtesham, Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand, Cancer Res. 62 (2002) 7170–7174. [45] M.R. Loebinger, Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer, Cancer Res. 69 (2009) 4142–4143. [46] L.S. Sasportas, Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy, Proc. Natl. Acad. Sci. 106 (2009) 4822–4827. [47] K. Shah, Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression, Ann. Neurol. 57 (2005) 34–41. [48] K. Shah, Inducible release of TRAIL fusion proteins form a proapoptotic form for tumor therapy, Cancer Res. 64 (2004) 3236–3242. [49] M. Khorashadizadeh, M. Soleimani, H. Khanahmad, A. Fallah, M. Naderi, M. Khorramizadeh, Bypassing the need for pre-sensitization of cancer cells for anticancer TRAIL therapy with secretion of novel cell penetrable form of Smac from hA-MSCs as cellular delivery vehicle, Tumour Biol. 36 (2015) 4213–4221. [50] T.S. Chen, R.C. Lai, M.M. Lee, A.B. Choo, C.N. Lee, S.K. Lim, Mesenchymal stem cel secretes microparticles enriched in pre-microRNAs, Nucleic Acids Res. 38 (2010) 215–224. [51] R.C. Lai, T.S. Chen, S.K. Lim, Mesenchymal stem cell exosome: a novel stem cellbased therapy for cardiovascular disease, Regen. Med. 6 (2011) 481–492. [52] R.C. Lai, R.W. Yeo, K.H. Tan, S.K. Lim, Exosomes for drug delivery - a novel application for the mesenchymal stem cell, Biotechnol. Adv. 31 (2013) 543–551. [53] K. Denzer, M.J. Kleijmeer, H.F. Heijnen, W. Stoorvogel, H.J. Gueze, Exosome: from internal vesicle to the multivesicular body to intercellular signaling device, J. Cell Sci. 113 (2000) 3365–3374. [54] H.G. Lamparski, A. Metha-Damani, J.-Y. Yao, S. Patel, D.-H. Hsu, C. Ruegg, Production and characterization of clinical grade exosomes derived from dendritic cells, J. Immunol. Methods 270 (2002) 211–226. [55] L. Zitvogel, A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell derived exosomes, Nat. Med. 4 (1998) 595–600. [56] R.J. Simpson, S. Mathivanan, Extracellular microvesicles: the need for internationally recognised nomenclature and stringent purification criteria, J. Proteomics Bioinform. 5 (2012). [57] A. Clayton, J. Court, H. Navabi, M. Adams, M.D. Mason, J.A. Hobot, Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry, J. Immunol. Methods 247 (2001) 163–174. [58] S. Mathivanan, J.W.E. Lim, B.J. Tauro, H. Ji, R.L. Moritz, R.J. Simpson, Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature, Mol. Cell. Proteomics 9 (2010) 197–208. [59] D. Sun, X. Zhuang, X. Xiang, Y. Liu, S. Zhang, C. Liu, A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes, Mol. Ther. 18 (2010) 1606–1614. [60] X. Zhuang, X. Xiang, W. Grizzle, D. Sun, S. Zhang, R.C. Axtell, Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region of the brain, Mol. Ther. 19 (2011) 1769–1779. [61] C. Théry, A. Clayton, S. Amigorena, G. Raposo, Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids, Inc Curr Protoc Cell Biol, John Wiley and Sons, 2006. [62] T. Kinnaird, E. Stabile, M.S. Burnett, C.W. Lee, S. Barr, S. Fuchs, Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms, Circ. Res. 94 (2004) 678–685. [63] K.E. Corcoran, K.A. Trzaska, H. Fernandes, M. Bryan, M. Taborga, V. Srinivas, Mesenchymal stem cells in early entry of breast cancer into bone marrow, PloS One 3 (2008) e2563. [64] G. Kasper, N. Dankert, J. Tuischer, M. Hoeft, T. Gaber, J.D. Glaeser, Mesenchymal stem cells regulate angiogenesis accroding to their mechanical environment, Stem Cells 25 (2007) 903–910. [65] M. Studeny, F.C. Marini, R.E. Champlin, C. Zompetta, I.J. Fidler, M. Andreeff, Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors, Cancer Res. 62 (2002) 3603–3608. [66] A.E. Karnoub, A.B. Dash, A.P. Vo, A. Sullivan, M.W. Brooks, G.W. Bell, Mesenchymal stem cells within tumour stroma promote breast cancer metastasis, Nature 449 (2007) 557–563. [67] S. Aggarwal, M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses, Blood 105 (2005) 1815–1822. [68] M. Di Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli, Blood 99 (2002) 3838–3842.

7

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al. 1248012. [95] M. Ono, N. Kosaka, N. Tominaga, Y. Yoshioka, F. Takeshita, R.U. Takahashi, M. Yoshida, H. Tsuda, K. Tamura, T. Ochiya, Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells, Sci. Signaling 7 (2014) ra63. [96] P.K. Lim, S.A. Bliss, S.A. Patel, M. Taborga, M.A. Dave, L.A. Gregory, S.J. Greco, M. Bryan, P.S. Patel, P. Rameshwar, Gap junction-mediated import of microRNA from bone marrow stromal cells can elicit cell cycle quiescence in breast cancer cells, Cancer Res. 71 (2011) 1550–1560. [97] G.R. Fajardo-Orduna, H. Mayani, J.J. Montesinos, Hematopoietic support capacity of mesenchymal stem cells: biology and clinical potential, Arch. Med. Res. 46 (2015) 589–596. [98] L. De Luca, S. Trino, I. Laurenzana, D. Lamorte, A. Caivano, L. Del Vecchio, P. Musto, Mesenchymal stem cell derived extracellular vesicles: a role in hematopoietic transplantation? Int. J. Mol. Sci. 18 (2017). [99] M. Chaterjee, K.L. van Golen, Breast cancer stem cells survive periods of farnesyltransferase inhibitor-induced dormancy by undergoing autophagy, Bone Marrow Res. 2011 (2011) 362938. [100] C. Gong, C. Bauvy, G. Tonelli, W. Yue, C. Delomenie, V. Nicolas, Y. Zhu, V. Domergue, V. Marin-Esteban, H. Tharinger, L. Delbos, H. Gary-Gouy, A.P. Morel, S. Ghavami, E. Song, P. Codogno, M. Mehrpour, Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells, Oncogene 32 (2013) 2261-2272, 2272e 2261-2211. [101] W. Bursch, A. Ellinger, H. Kienzl, L. Torok, S. Pandey, M. Sikorska, R. Walker, R.S. Hermann, Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy, Carcinogenesis 17 (1996) 1595–1607. [102] J. Korah, L. Canaff, J.J. Lebrun, The retinoblastoma tumor suppressor protein (pRb)/E2 promoter binding factor 1 (E2F1) pathway as a novel mediator of TGFbeta-induced autophagy, J. Biol. Chem. 291 (2016) 2043–2054. [103] V. Rausch, L. Liu, A. Apel, T. Rettig, J. Gladkich, S. Labsch, G. Kallifatidis, A. Kaczorowski, A. Groth, W. Gross, M.M. Gebhard, P. Schemmer, J. Werner, A.V. Salnikov, H. Zentgraf, M.W. Buchler, I. Herr, Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment, J. Pathol. 227 (2012) 325–335. [104] P. Maycotte, K.L. Jones, M.L. Goodall, J. Thorburn, A. Thorburn, Autophagy supports breast cancer stem cell maintenance by regulating IL6 secretion, Mol. Cancer Res. 13 (2015) 651–658. [105] M.C. Yang, H.C. Wang, Y.C. Hou, H.L. Tung, T.J. Chiu, Y.S. Shan, Blockade of autophagy reduces pancreatic cancer stem cell activity and potentiates the tumoricidal effect of gemcitabine, Mol. Cancer 14 (2015) 179. [106] H. Wei, S. Wei, B. Gan, X. Peng, W. Zou, J.L. Guan, Suppression of autophagy by FIP200 deletion inhibits mammary tumorigenesis, Genes Dev. 25 (2011) 1510–1527. [107] S. Galavotti, S. Bartesaghi, D. Faccenda, M. Shaked-Rabi, S. Sanzone, A. McEvoy, D. Dinsdale, F. Condorelli, S. Brandner, M. Campanella, R. Grose, C. Jones, P. Salomoni, The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells, Oncogene 32 (2013) 699–712. [108] L. Vermeulen, E.M.F. De Sousa, M. van der Heijden, K. Cameron, J.H. de Jong, T. Borovski, J.B. Tuynman, M. Todaro, C. Merz, H. Rodermond, M.R. Sprick, K. Kemper, D.J. Richel, G. Stassi, J.P. Medema, Wnt activity defines colon cancer stem cells and is regulated by the microenvironment, Nat. Cell Biol. 12 (2010) 468–476. [109] R.C. D'Angelo, M. Ouzounova, A. Davis, D. Choi, S.M. Tchuenkam, G. Kim, T. Luther, A.A. Quraishi, Y. Senbabaoglu, S.J. Conley, S.G. Clouthier, K.A. Hassan, M.S. Wicha, H. Korkaya, Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity, Mol. Cancer Ther. (2015). [110] P.R. Prasetyanti, C.D. Zimberlin, M. Bots, L. Vermeulen, S. Melo Fde, J.P. Medema, Regulation of stem cell self-renewal and differentiation by Wnt and Notch are conserved throughout the adenoma-carcinoma sequence in the colon, Mol Cancer 12 (2013) 126. [111] X. Fan, L. Khaki, T.S. Zhu, M.E. Soules, C.E. Talsma, N. Gul, C. Koh, J. Zhang, Y.M. Li, J. Maciaczyk, G. Nikkhah, F. Dimeco, S. Piccirillo, A.L. Vescovi, C.G. Eberhart, NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts, Stem Cells 28 (2010) 5–16. [112] S. Liu, G. Dontu, I.D. Mantle, S. Patel, N.S. Ahn, K.W. Jackson, P. Suri, M.S. Wicha, Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells, Cancer Res. 66 (2006) 6063–6071. [113] J. Zhou, H. Zhang, P. Gu, J. Bai, J.B. Margolick, Y. Zhang, NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells, Breast Cancer Res. Treatment 111 (2008) 419–427. [114] H. Enderling, L. Hlatky, P. Hahnfeldt, Cancer stem cells: a minor cancer subpopulation that redefines global cancer features, Front. Oncol. 3 (2013) 76. [115] D.H. Munn, V. Bronte, Immune suppressive mechanisms in the tumor microenvironment, Curr. Opin. Immunol. 39 (2016) 1–6. [116] L.I. Shlush, S. Zandi, A. Mitchell, W.C. Chen, J.M. Brandwein, V. Gupta, J.A. Kennedy, A.D. Schimmer, A.C. Schuh, K.W. Yee, J.L. McLeod, M. Doedens, J.J. Medeiros, R. Marke, H.J. Kim, K. Lee, J.D. McPherson, T.J. Hudson, H.P.L.G.P. Consortium, A.M. Brown, F. Yousif, Q.M. Trinh, L.D. Stein, M.D. Minden, J.C. Wang, J.E. Dick, Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia, Nature 506 (2014) 328–333. [117] L. Zitvogel, A. Tesniere, G. Kroemer, Cancer despite immunosurveillance: immunoselection and immunosubversion, Nat. Rev. Immunol. 6 (2006) 715–727. [118] E. Jachetti, S. Caputo, S. Mazzoleni, C.S. Brambillasca, S.M. Parigi, M. Grioni, I.S. Piras, U. Restuccia, A. Calcinotto, M. Freschi, A. Bachi, R. Galli, M. Bellone,

[119]

[120]

[121]

[122] [123] [124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135] [136]

[137]

[138]

[139]

[140]

[141]

[142]

8

Tenascin-C protects cancer stem-like cells from immune surveillance by arresting T-cell activation, Cancer Res. 75 (2015) 2095–2108. T. Di Tomaso, S. Mazzoleni, E. Wang, G. Sovena, D. Clavenna, A. Franzin, P. Mortini, S. Ferrone, C. Doglioni, F.M. Marincola, R. Galli, G. Parmiani, C. Maccalli, Immunobiological characterization of cancer stem cells isolated from glioblastoma patients, Clin. Cancer Res. 16 (2010) 800–813. Y. Yao, X. Wang, K. Jin, J. Zhu, Y. Wang, S. Xiong, Y. Mao, L. Zhou, B7–H4 is preferentially expressed in non-dividing brain tumor cells and in a subset of brain tumor stem-like cells, J. Neurooncol. 89 (2008) 121–129. Y. Lee, J.H. Shin, M. Longmire, H. Wang, H.E. Kohrt, H.Y. Chang, J.B. Sunwoo, CD44+cells in head and neck squamous cell carcinoma suppress T-cell-mediated immunity by selective constitutive and inducible expression of PD-L1, Clin. Cancer Res. 22 (2016) 3571–3581. E.O. Long, S. Rajagopalan, Stress signals activate natural killer cells, J. Exp. Med. 196 (2002) 1399–1402. D.H. Raulet, N. Guerra, Oncogenic stress sensed by the immune system: role of natural killer cell receptors, Nat. Rev. Immunol. 9 (2009) 568–580. B. Sainz Jr., E. Carron, M. Vallespinos, H.L. Machado, Cancer stem cells and macrophages: implications in tumor biology and therapeutic strategies, Mediators Inflamm. 2016 (2016) 9012369. R. Ward, A.H. Sims, A. Lee, C. Lo, L. Wynne, H. Yusuf, H. Gregson, M.P. Lisanti, F. Sotgia, G. Landberg, R. Lamb, Monocytes and macrophages, implications for breast cancer migration and stem cell-like activity and treatment, Oncotarget 6 (2015) 14687–14699. X. Liu, Y. Pu, K. Cron, L. Deng, J. Kline, W.A. Frazier, H. Xu, H. Peng, Y.X. Fu, M.M. Xu, CD47 blockade triggers T cell-mediated destruction of immunogenic tumors, Nat. Med. 21 (2015) 1209–1215. S. Kaur, A.G. Elkahloun, S.P. Singh, Q.R. Chen, D.M. Meerzaman, T. Song, N. Manu, W. Wu, P. Mannan, S.H. Garfield, D.D. Roberts, A function-blocking CD47 antibody suppresses stem cell and EGF signaling in triple-negative breast cancer, Oncotarget 7 (2016) 10133–10152. Y. Wang, J.S. Teng, Increased multi-drug resistance and reduced apoptosis in osteosarcoma side population cells are crucial factors for tumor recurrence, Exp. Ther. Med. 12 (2016) 81–86. X. Zheng, D. Cui, S. Xu, G. Brabant, M. Derwahl, Doxorubicin fails to eradicate cancer stem cells derived from anaplastic thyroid carcinoma cells: characterization of resistant cells, Int. J. Oncol. 37 (2010) 307–315. G. Zhang, Z. Wang, W. Luo, H. Jiao, J. Wu, C. Jiang, Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma, Gastroenterol. Res. Pract. 2013 (2013) 782581. K.M. Britton, R. Eyre, I.J. Harvey, K. Stemke-Hale, D. Browell, T.W. Lennard, A.P. Meeson, Breast cancer, side population cells and ABCG2 expression, Cancer Lett. 323 (2012) 97–105. G. Gross, T. Waks, Z. Eshhar, Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity, Proc. Natl. Acad. Sci. 86 (1989) 10024–10028. Z. Eshhar, T. Waks, G. Gross, D.G. Schindler, Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibodybinding domains and the gamma or zeta subunits of the immunoglobulin and Tcell receptors, Proc. Natl. Acad. Sci. 90 (1993) 720–724. K.J. Curran, H.J. Pegram, R.J. Brentjens, Chimeric antigen receptors for T cell immunotherapy: current understanding and future directions, J. Gene Med. 14 (2012) 405–415. D.J. Lenschow, T.L. Walunas, J.A. Bluestone, CD28/B7 system of T cell costimulation, Annu. Rev. Immunol. 14 (1996) 233–258. H. Koehler, D. Kofler, A. Hombach, H. Abken, CD28 costimulation overcomes transforming growth factor-beta-mediated repression of proliferation of redirected human CD4+and CD8+T cells in an antitumor cell attack, Cancer Res. 67 (2007) 2265–2273. C.M. Kowolik, M.S. Topp, S. Gonzalez, T. Pfeiffer, S. Olivares, N. Gonzalez, D.D. Smith, S.J. Forman, M.C. Jensen, L.J. Cooper, CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells, Cancer Res. 66 (2006) 10995–11004. A. Loskog, V. Giandomenico, C. Rossig, M. Pule, G. Dotti, M.K. Brenner, Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric Tcell resistance to T regulatory cells, Leukemia 20 (2006) 1819–1828. R.J. Brentjens, M.L. Davila, I. Riviere, J. Park, X. Wang, L.G. Cowell, S. Bartido, J. Stefanski, C. Taylor, M. Olszewska, O. Borquez-Ojeda, J. Qu, T. Wasielewska, Q. He, Y. Bernal, I.V. Rijo, C. Hedvat, R. Kobos, K. Curran, P. Steinherz, J. Jurcic, T. Rosenblat, P. Maslak, M. Frattini, M. Sadelain, CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia, Sci. Transl. Med. 5 (2013). C.R. Cruz, K.P. Micklethwaite, B. Savoldo, C.A. Ramos, S. Lam, S. Ku, O. Diouf, E. Liu, A.J. Barrett, S. Ito, E.J. Shpall, R.A. Krance, R.T. Kamble, G. Carrum, C.M. Hosing, A.P. Gee, Z. Mei, B.J. Grilley, H.E. Heslop, C.M. Rooney, M.K. Brenner, C.M. Bollard, G. Dotti, Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study, Blood 122 (2013) 2965–2973. E. Jacoby, Y. Yang, H. Qin, C.D. Chien, J.N. Kochenderfer, T.J. Fry, Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD, Blood 127 (2016) 1361–1370. J.N. Kochenderfer, M.E. Dudley, R.O. Carpenter, S.H. Kassim, J.J. Rose, W.G. Telford, F.T. Hakim, D.C. Halverson, D.H. Fowler, N.M. Hardy, A.R. Mato, D.D. Hickstein, J.C. Gea-Banacloche, S.Z. Pavletic, C. Sportes, I. Maric, S.A. Feldman, B.G. Hansen, J.S. Wilder, B. Blacklock-Schuver, B. Jena,

Cellular Immunology xxx (xxxx) xxx–xxx

H.S. Eltoukhy et al.

[143]

[144]

[145] [146]

[147]

[148] [149]

[150]

[151]

[152]

acute myeloid leukemia, Leukemia 29 (2015) 1637–1647. [153] A. Mardiros, C. Dos Santos, T. McDonald, C.E. Brown, X. Wang, L.E. Budde, L. Hoffman, B. Aguilar, W.C. Chang, W. Bretzlaff, B. Chang, M. Jonnalagadda, R. Starr, J.R. Ostberg, M.C. Jensen, R. Bhatia, S.J. Forman, T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia, Blood 122 (2013) 3138–3148. [154] P.J. Paszkiewicz, S.P. Frassle, S. Srivastava, D. Sommermeyer, M. Hudecek, I. Drexler, M. Sadelain, L. Liu, M.C. Jensen, S.R. Riddell, D.H. Busch, Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia, J. Clin. Invest. 126 (2016) 4262–4272. [155] J.B. Smith, E. Lanitis, D. Dangaj, E. Buza, M. Poussin, C. Stashwick, N. Scholler, D.J. Powell Jr., Tumor regression and delayed onset toxicity following B7–H4 CAR T cell therapy, Mol. Ther. 24 (2016) 1987–1999. [156] L. Wang, N. Ma, S. Okamoto, Y. Amaishi, E. Sato, N. Seo, J. Mineno, K. Takesako, T. Kato, H. Shiku, Efficient tumor regression by adoptively transferred CEA-specific CAR-T cells associated with symptoms of mild cytokine release syndrome, Oncoimmunology 5 (2016) e1211218. [157] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci. 100 (2003) 3983–3988. [158] O. Gires, C.A. Klein, P.A. Baeuerle, On the abundance of EpCAM on cancer stem cells, Nat. Rev. Cancer 9 (2009) 143. [159] R. Adikrisna, S. Tanaka, S. Muramatsu, A. Aihara, D. Ban, T. Ochiai, T. Irie, A. Kudo, N. Nakamura, S. Yamaoka, S. Arii, Identification of pancreatic cancer stem cells and selective toxicity of chemotherapeutic agents, Gastroenterology 143 (2012) 234-245 e237. [160] C.A. O'Brien, A. Pollett, S. Gallinger, J.E. Dick, A human colon cancer cell capable of initiating tumour growth in immunodeficient mice, Nature 445 (2007) 106–110. [161] L. Ricci-Vitiani, D.G. Lombardi, E. Pilozzi, M. Biffoni, M. Todaro, C. Peschle, R. De Maria, Identification and expansion of human colon-cancer-initiating cells, Nature 445 (2007) 111–115. [162] Z. Deng, Y. Wu, W. Ma, S. Zhang, Y.Q. Zhang, Adoptive T-cell therapy of prostate cancer targeting the cancer stem cell antigen EpCAM, BMC Immunol. 16 (2015) 1. [163] X. Zhu, S. Prasad, S. Gaedicke, M. Hettich, E. Firat, G. Niedermann, Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57, Oncotarget 6 (2015) 171–184.

M.R. Bishop, R.E. Gress, S.A. Rosenberg, Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation, Blood 122 (2013) 4129–4139. A.S. Onea, A.R. Jazirehi, CD19 chimeric antigen receptor (CD19 CAR)-redirected adoptive T-cell immunotherapy for the treatment of relapsed or refractory B-cell Non-Hodgkin's Lymphomas, Am. J. Cancer Res. 6 (2016) 403–424. M.L. Schubert, A. Huckelhoven, J.M. Hoffmann, A. Schmitt, P. Wuchter, L. Sellner, S. Hofmann, A.D. Ho, P. Dreger, M. Schmitt, Chimeric antigen receptor T cell therapy targeting CD19-positive leukemia and lymphoma in the context of stem cell transplantation, Hum. Gene. Ther. (In press). N. Singh, Recent advances in engineered T cell therapies targeting B cell malignancies, Discov. Med. 22 (2016) 215–220. C.J. Turtle, L.A. Hanafi, C. Berger, M. Hudecek, B. Pender, E. Robinson, R. Hawkins, C. Chaney, S. Cherian, X. Chen, L. Soma, B. Wood, D. Li, S. Heimfeld, S.R. Riddell, D.G. Maloney, Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+CD19-specific chimeric antigen receptor-modified T cells, Sci Transl Med 8 (2016). M.P. Velasquez, D. Torres, K. Iwahori, S. Kakarla, C. Arber, T. Rodriguez-Cruz, A. Szoor, C.L. Bonifant, C. Gerken, L.J. Cooper, X.T. Song, S. Gottschalk, T cells expressing CD19-specific Engager Molecules for the Immunotherapy of CD19positive Malignancies, Sci Rep 6 (2016) 27130. T. Venkei, J. Sugar, Keratoacanthoma of precarcinomatous character keratoacanthoma, Dermatol. Wochenschr. 138 (1958) 957–965. B.D. Choi, C.M. Suryadevara, P.C. Gedeon, J.E. Herndon 2nd, L. Sanchez-Perez, D.D. Bigner, J.H. Sampson, Intracerebral delivery of a third generation EGFRvIIIspecific chimeric antigen receptor is efficacious against human glioma, J. Clin. Neurosci. 21 (2014) 189–190. T. Gargett, W. Yu, G. Dotti, E.S. Yvon, S.N. Christo, J.D. Hayball, I.D. Lewis, M.K. Brenner, M.P. Brown, GD2-specific CAR T cells undergo potent activation and deletion following antigen encounter but can be protected from activationinduced cell death by PD-1 blockade, Mol. Ther. 24 (2016) 1135–1149. W. Haso, D.W. Lee, N.N. Shah, M. Stetler-Stevenson, C.M. Yuan, I.H. Pastan, D.S. Dimitrov, R.A. Morgan, D.J. FitzGerald, D.M. Barrett, A.S. Wayne, C.L. Mackall, R.J. Orentas, Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia, Blood 121 (2013) 1165–1174. S.S. Kenderian, M. Ruella, O. Shestova, M. Klichinsky, V. Aikawa, J.J. Morrissette, J. Scholler, D. Song, D.L. Porter, M. Carroll, C.H. June, S. Gill, CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human

9