Role of Immune Cells and Metabolism in Cancer

#### **Chapter 4**

## Perspective Chapter: Dendritic Cells in the Tumor Microenvironment

*Dan Jin, Laura Falceto Font and Catherine T. Flores*

#### **Abstract**

Tumor infiltrating dendritic cells (DCs) play a critical role in initiating the process of anti-tumor immune responses. They can uptake tumor antigens either directly at the tumor site or from circulating antigens, and elicit T cell activation and adaptive immunity in secondary lymphoid organs. Subtypes of dendritic cells have various roles in immunity and tumor rejection. In this chapter, we will summarize the role of dendritic cell populations on mounting anti-tumor immunity. Conversely, we will discuss tumormediated dysfunction of dendritic cells that aid immune evasion including prevention of recruitment, impairment in antigen presenting and mediation of tolerance. At last, we briefly introduced the progress in DC vaccine applications in clinic.

**Keywords:** dendritic cell, tumor microenvironment, antigen presenting, T cell activation, DC tolerance, DC vaccine

#### **1. Introduction**

Dendritic cells (DC) are responsible for activating effector responses and mediating adaptive immunity. Immune responses are dependent on multiple factors including the DC type, maturation status, and immunogenicity of antigens. DCs have the capacity of inducing protective immunity as well as generating a tolerogenic immune environment. The complexity of how cancer impacts the spectrum of response varies depending on the cancer type, largely on the cancer immunophenotype. Here we discuss how different DC subtypes interact between cancer and adaptive immunity. We also touch on various cancer-mediated immune evasion strategies that alter DC function. Lastly we evaluate immunotherapeutic strategies that employ DCs to elicit anti-tumor T cell responses.

#### **2. Anti-tumor roles of different dendritic cell sub-populations**

DCs are composed of heterogenous sub-populations with each subtype possessing unique functions to compensate for each other. They cooperate to elicit both innate and adaptive immunity. In this section, we will generally review the roles of different DC sub-populations in anti-tumor immunity (**Figure 1**).

#### **Figure 1.**

 *Roles of DC subpopulations in anti-tumor response. Immature DCs in peripheral tissue can be recruited into tumor site through CCR5/CCL3/4 chemoaxis. Both cDC1 and cDC2 uptake tumor antigens in situ and migrate to tdLN in a CCR7-dependent way, trafficking tumor antigens into tdLN. In tdLN, antigen-loaded cDC1 primes both naïve CD8 T cells and CD4 T cells. Primed CD4 T cells further boost CD8 T cell activation through licensing cDC1 in a CD40/CD40L dependent way. Antigen-loaded cDC2 predominantly primes naïve CD4 T cells. It can alternatively activate CD8 T cells through transferring antigens to LN resident cDC1(rcDC1), which primes naïve T cells in LN. Activated T cells migrate to tumor site in a CXCR3 dependent chemokine recruitment way. Effector T cells can be further stimulated by antigen activated cDCs and moDCs in tumor and exert cytotoxic tumor killing. moDCs exert CD4 and CD8 T priming function to compensate for cDCs when they are dysfunctional or depleted. Activated pDCs secrete CCL chemokines to recruit and activate NK cells through type I IFN and OX40- OX40L interaction. Activated NK cells promote cytotoxic CD8 T priming through activation of cDC in an IFNγdependent way, meanwhile, activated cDCs secrete cytokines like IL12, IL18 that enhance NK cell activation.* 

#### **2.1 Conventional DCs**

 Conventional DCs (cDCs) are derived from common DC precursors (CDP) in the bone marrow, which are comprised of two main subsets: cDC1 and cDC2. The infiltration of cDCs in the tumor has a positive correlation with patient survival in some solid cancers [ 1 ]. cDC1 development is specified by transcriptional factors BATF3, IRF8, and ID2, while cDC2 development depends on transcriptional factors RELB, IRF4, ZEB2 and KLF4 [ 2 , 3 ]. BATF3 is required for maintaining IRF8 expression during cDC1 commitment in specified cDC1 progenitor [ 4 ]. BATF3 is also required for cDC1 cross-presentation function and cross-presentation independent anti-tumor immunity functions [ 5 , 6 ]. BATF-dependent cDC1 is specified by its unique role to initiate naïve CD8 + T cell activation in tumor-draining lymph nodes(tdLN), as well as enhance both T cell accumulation and local CD8 T cell cytotoxicity. The abundance of cDC1 in the tumor microenvironment positively correlates with cancer patient survival and response to immunotherapy across different cancer types [ 7 , 8 ]. CD103 + cDC1s sample tumor antigen in tumor mass and migrate to tumor-draining lymph nodes via CCR7, where they prime T cell responses [ 9 ]. Tumor-resident BATF3 + cDC1s *DOI: http://dx.doi.org/10.5772/intechopen.108586 Perspective Chapter: Dendritic Cells in the Tumor Microenvironment*

secret CXCL9 and CXCL10 to recruit CXCR3 expressing effector T cells and NK cells [10, 11]. In turn, IFNγ produced by tumor effector T cells and NK cells induce CXCL9/10/11 production by myeloid cells, creating a feedback loop in this response [12]. cDCs also secrete IL-12, IL-18 and IL-2 provoking NK cells to produce IFNγ, TNFα, or GM-CSF, which further promotes DC activation [13].

Unlike cDC1s, cDC2s have limited capacity to cross-present tumor antigens to CD8 T cells. The function of cDC2s are largely restricted to priming of CD4 T cells in tdLN or in tumor [14–16]. cDC2s mediate cross-presentation of soluble antigens and is enhanced by TLR7 agonist [17, 18]. cDC2s complement the function of cDC1s by also activating CD8 T cells. Migratory cDC2 capture antigens in tumor and transfer antigens to LN resident cDC1s through antigen vesicles and synaptic transfer, which is capable of activating CD8 T cells [19]. In the absence of cDC1s, activating cDC2s by type I IFN can stimulate CD8 T activation in tumor [20]. In preclinical models where cDC1 function is impaired, deletion of cDC1 population improves cDC2 migration into tdLN and CD4 T activation [1].

#### **2.2 Plasmacytoid dendritic cells**

Plasmacytoid dendritic cells(pDC) are largely regarded as immunomodulating cells through secretion of massive amounts of type-I interferon during anti-virus immune responses. The role of pDC in anti-tumor immunity is controversial. pDC in tumors have been found to have impaired response to Toll-like receptor activation and decreased type-I IFN production. They recruit and expand immune regulatory T cells in the tumor microenvironment (TME) and are associated with poor prognosis [15, 21]. As an escape mechanism, tumor cells attract pDCs to induce an immunosuppressive environment through secreting chemokine CXCL12 [22].

On the contrary, in some solid tumors, pharmacological agents can be used to overcome immunosuppression. For example, imiquimod stimulation can induce pDC mediated tumor killing via secretion of TRAIL and granzyme B independent of adaptive immunity [23]. pDCs can also drive anti-tumor response by activating adaptive T cell immunity mediated by cDC activation dependent on type-I IFN [24]. Direct injection of TLR9 activated pDC into B16 melanoma tumor bearing mice induces robust cytotoxic T lymphocytes (CTL) cross-priming against tumor, leading to tumor regression. TLR9 activated pDCs produce large amounts of chemokines CCL3, CCL4, and CCL5 within the tumor, which recruits CCR5+ NK cells. Recruited NK cells are activated by pDC through cell-to-cell interaction via OX40/OX40L and type I IFN secreted by pDC. Tumor cells lysated by NK cells cause tumor antigen release into cDCs and IFNg secreted by activated NK cells also help activate CTL in dLN [25]. Such an activated subset of pDC with higher levels of OX40 is also found in head and neck squamous cell carcinoma (HNSCC) tumor with distinct immunostimulatory and cytolytic function and can synergize with cDCs in generating tumor antigenspecific CD8+ T cell responses [26].

#### **2.3 Monocyte-derived dendritic cells**

Monocyte-derived dendritic cells (moDCs) are differentiated from monocytes under inflammatory conditions. Activation of p53 in MDSCs and monocytic progenitors can induce moDC-like population differentiation in tumor, which potentiates the anti-tumor response [27]. Increase of moDC in tdLN can be a measurable indication of immune activation, particularly after treatment with pharmacological agents

such as TLR agonists [28]. moDCs in tumor are essential for CD8 T activation and antitumor response after local immunostimulatory agent treatment [29]. In mice with Zbtb46-DTR bone marrow chimeras, which are deficient in cDC production after diphtheria toxin (DT) treatment, moDC compensate for the loss of cDCs and account for intratumoral CTL expansion and function [30]. Compared with cDC, moDCs are less efficient at inducing CD4 T cell proliferation but more efficient at inducing Th1 and Th17 differentiation [31]. However, moDCs are also able to cross-present antigens through the vacuolar pathway and activate naïve CD4 T and CD8 T cells [32]. *In vitro* differentiated moDCs have been used in clinical trials as vaccines for cancer patients and encouraging responses have been shown when combined with other cancer therapies [33–37], which will be further discussed in the last section of this chapter.

#### **3. Tumor-mediated immune evasion: impact on dendritic cells**

#### **3.1 DC tumor infiltration and migration to LN**

cDCs in tumor are found to be sparse among tumor infiltrated immune populations [8, 38]. Increased cDC amount within tumor is associated with improved prognosis and response to check-point inhibitor immunotherapy [8, 39]. Tumor cells secret soluble factors that suppress DCs infiltrating to tumor site and migrating to LN (**Figure 2**).

Tumor cells suppress chemokine CCL4 production through activating betacatenin signaling, and beta-catenin activation induces ATF3 expression. ATF3 binds the promoter of CCL4 gene and suppresses CCL4 expression. Decreased CCL4 leads to decreased intratumoral cDC recruitment by CCL4/CCR5 axis [40]. beta-catenin signaling suppresses CCL5 level in tumor loci. CCL5/CCR5 chemoaxis recruits cDC1 into tumor. Increased CCL5 expression in tumor recruits cDC tumoral infiltration and promotes anti-tumor immune response and promotes efficacy when combined with anti-PD1 [41]. Prostaglandin E2 (PGE2), a prostanoid lipid catalyzed by enzyme cyclooxygenase (COX), is highly produced in tumor [42–44]. cDC1s are absent from

#### **Figure 2.**

*Impact of DC recruitment and migration by tumor. Tumor cells suppress CCL4/5 expression by inducing β-catenin signaling pathway, which inhibits cDC recruitment via CCL4/5/CCR5 chemoaxis. PGE2 produced from tumor directly acts on intratumoral NK cells through EP2/4 receptors. PGE2 inhibits CCL5 and XCL1 secretion by NK cells, which results in decreased cDC recruitment. PGE2 can also inhibit CCR5 and CXR1 expression on cDC to impair cDC recruitment. High concentration of PGE2 inhibits moDC migration into dLN. TGFβ from tumor inhibits LC migration into LN.*

*DOI: http://dx.doi.org/10.5772/intechopen.108586 Perspective Chapter: Dendritic Cells in the Tumor Microenvironment*

PGE2 producing tumor. PGE2 suppresses NK cells mediated cDC1 recruitment in tumor. Intratumoral NK cells secret cDC1 chemoattractant CCL5, XCL1, which are inhibited by tumor derived PGE2 through PGE2 receptor EP2, EP4 on NK cells. However, expressing CCL5, or XCL1 in tumor is insufficient to reverse intratumoral DC exclusion in PGE2 producing tumor. Further study shows that PGE2 can also downregulate CXR1 and CCR5 expression in DC, which leads to impairment of response to chemokine even in the existence of chemoattractant [21, 44, 45].

DC cells uptake antigens in tumor site and then migrate to dLN for priming T cells. TGFβ secreted from tumor inhibits DC migration to dLN in both autocrine and paracrine way. Langerhans cells (LCs), skin-resident DCs, play critical role in eliciting immune response in skin disease. Besides of tumor cells, LCs are also active TGFβ producer. Knock-out of TGFβ or its receptor in LCs induces mass migration of LCs to regional LN in both steady and inflammation states [46]. In skin tumor model, TGFβ inhibits tumor infiltration and migration to skin-dLN by LCs [47]. Role of PGE2 in regulating DC migration relies on its concentration. High concentration of PGE2 suppresses DC migration while it has also been shown as a positive regulator of CCR7 expression and migration of moDCs [48, 49].

#### **3.2 Antigen presentation**

Tumor cells develop mechanisms to impair antigen capture and presenting by DCs (**Figure 3**). Molecules released or exposed from dying cancer cells can act as danger signals to circulating DCs. DCs recognize dying/dead tumor cells or tumor derived debris through danger-associated molecular patterns (DAMPs) mediated by pattern recognition receptors (PRRs) like TLRs, and phagocyze dying-tumor cells or tumor derived debris [50]. DAMPs include ATP, heat shock proteins (HSPs), HMGB1, calreticulin, annexin A1, dsDNA, but are not limited to these [50]. Antigen uptake will stimulate DC maturation and migration to dLN. Internalized antigens will be processed and presented on the DC surface by MHC-I and MHC-II molecules. MHC-I molecules used to be thought for intracellular peptide presenting, while MHC-II is for exogenous peptide. However, this is not always the case. Cross-presenting is termed for presenting exogenous antigens by MHC-I, which plays a critical role in eliciting anti-tumor immune response by DCs [51]. Proteins internalized by DCs are degraded in phagosomes into peptides [52]. Peptides are then translocated into endoplasmic reticulum (ER) by transporter associated with antigen presentation (TAP). The MHC-I heterodimer is assembled in ER from a polymorphic heavy chain and a light chain β2-microglobulin (β2m) and stabilized by chaperone proteins like calreticulin and tapasin when peptide is not loaded. Chaperones will be exchanged when peptide is loaded. Peptides fit into the MHC-I peptide binding groove which stabilizes the peptide-MHC-I complex. MHC-II comprises transmembrane α- and β-chains and an invariant chain. MHC-II will be transported to a MHC-II compartment, an endosomal compartment, for invariant chain digestion, resulting a class II-associated lipid peptide (CLIP). With the help of H2-DM/HLA-DM, CLIP is exchanged with antigen peptide [53]. In LN, DCs present tumor antigens to CD8 T cells and CD4 T cells dependent on MHC-I and MHC-II respectively. Tumor proteins will be processed into immunogenic peptides and loaded on MHC-I or MHC-II molecules on cell surface.

Tumor cells evolved multiple immune escape strategies to prevent recognition by DCs. For example, tumor-derived stanniocalcin 1(STC1) interacts with DAMP signal, calreticulin (CRT), to prevent CRT membrane from exposing to APCs, thereby abrogating membrane CRT-directed phagocytosis by DCs. High expression of STC1

#### **Figure 3.**

*Tumor induced impairment of DC on tumor antigen capture and presentation. Tumor derived factors mediate lipid body accumulation in DC by inducing MSR1 expression. ROS, induced by tumor microenvironment, leads to increase lipid peroxidation in DC. Byproduct of lipid peroxidation, 4-HNE, induces ER stress, which activates XBP1 transcription factor. XBP1 then increases genes synthesizing triglyceride. Increased triglyceride leads to increased oxidized lipid bodies. The lipid bodies competitively bind HSP70 with pMHC, preventing pMHC been translocated onto cell surface. TIM3 competitively binds HMGB1 with dying tumor derive DNA, inhibiting DNA stimulated immune response via preventing DNA been internalized into DC. Tumor inhibits TIM4 expression on DC, thus inhibiting TIM4 mediated tumor associated antigen capture.*

in tumor is significantly correlated with poor responses to immunotherapy in patients [54]. In another immune escape mechanism, the mevalonate (MVA) pathway, which is highly activated in tumor cells, reduces F-actin exposure, a DAMP signal, on tumor cells, evading recognition mediated by Clec9A on cDC1. The MVA increases protein geranylgeranylation on Rac1, a small GTPase controlling actin cytoskeleton, resulting reduced F-actin in tumor [55–57]. The immune modulator TIM-3 is also highly expressed by DCs and has been shown to play an inhibitory role on DC activation. TIM-3 inhibits tumor derived DNA uptake by cDC1 through inhibiting endocytosis. HMGB1, a ligand of TIM-3, also acts as a DAMPS signal and binds tumor-derived DNA and is taken up by DCs. TIM-3 inhibits this process through sequestering HMGB1 bound DNA on cell surface [58]. TIM4, another T- cell immunoglobulin and mucin domain gene as TIM3, is also expressed on APCs like macrophages and dendritic cells. DAMP signal induces TIM4 expression on intratumoral macrophages and DCs [59]. Though TIM4 on tumor associated macrophage (TAM) has been shown impedes tumor antigen presentation through activating autophagy in TAM upon tumor antigen uptake in mouse melanoma model [59], TIM4 on lung resident cDCs in lung adenocarcinoma model shows a positive role in promoting anti-tumor immune activation [60]. TIM4 expression is downregulated in cDC1 from advanced lung tumor. Blocking TIM4 or knocking out of TIM4 abolishes tumor antigen uptake by lung resident cDC1 and impairs antigen presenting to CD8 T cells in vitro and in vivo.

#### *DOI: http://dx.doi.org/10.5772/intechopen.108586 Perspective Chapter: Dendritic Cells in the Tumor Microenvironment*

DCs from tumor-bearing host have been found with accumulated lipids. Tumor derived factors induce oxidized lipid accumulation in cDCs from tumor bearing host. DCs have high oxidized lipids shows impaired cross-presentation while not affecting presenting endogenous antigens, and nor affecting the level of MHC-I. Scavenger receptor, MSR1, induced by tumor derived factors, accounts for the lipid accumulation in DC [61, 62]. ER stress signaling also involved in oxidized lipid accumulation in DC from tumor bearing host. 4-HNE is a byproduct from lipid peroxidation mediated by ROS and triggers ER stress and XBP1 activation. XBP1, a multitask transcription factor in response to ER stress, induces triglyceride biosynthesis. Elevated triglyceride biosynthesis leads to accumulated abnormal lipids and suppresses DC function [63]. HSP70 is a chaperon protein that binds with pMHC, and facilitates pMHC trafficking onto cell surface. Oxidative lipid bodies, not non-oxidized lipid bodies, competitively bind HSP70 covalently, preclude HSP70 interaction with pMHC, thus affect pMHC trafficking to cell surface [64].

#### **3.3 Tolerance**

Tumor cells have evolved different mechanisms to promote DC tolerance to facilitate immune escape (**Figure 4**). Tolerized DCs experience higher co-inhibitory markers, including PD-L1, PD-L2 and higher arginase activity, and lower MHC-II and co-stimulation markers, including CD80, CD86, CD40 [39, 65]. Tolerized DCs are not capable of activating T cells, while promote immune suppression through mechanisms like, for example, Treg upregulation.

#### *3.3.1 Secreted factors from tumor environment*

Secreted tumor-derived factors is one of the major ways of driving DC tolerance, these include PGE2 and TGFβ which lead to subsequent induction of other immune modifiers. Tumor derived PGE2 suppresses cDCs activation by suppressing costimulation, IL-12 production, and increasing PD-L1 and Arg1 [44, 66]. PGE2 is the main inducer of arginase-1 during tumor induced DC tolerization [67]. TGFβ from

#### **Figure 4.**

*DC tolerance mechanism. Secreted factors or metabolites derived from tumor environment induce dendritic cell tolerance through activating or inhibiting cell-intrinsic signaling pathways in DC. DC tolerance leads to induced inhibitory molecular expression, Arg and IDO1 upregulation, anti-inflammation cytokine production and suppress co-stimulation and pro-inflammation cytokine production. And also result into dysregulated chemokine secretion. DC tolerance abolishes anti-tumor immune response through inhibiting T cell and NK recruitment and mediated tumor killing, and promoting immunosuppressive Treg differentiation and recruitment.*

tumor also induces DC tolerization [67, 68]. TGFβ induces IDO expression in pDC and enhances expression of CCL22 by myeloid DCs in tumor. IDO suppresses effector T cell activity and promotes Treg differentiation and activation. DC-derived CCL22 chemokine promotes CCR4-dependent recruitment of Tregs to the tumor microenvironment [69]. pDCs in tumor environments are associated with poor survival. Co-culture with TGFβ containing medium inhibits pDC activation and type I IFN secretion. Tolerized pDCs promote tumor growth through inhibiting NK cell infiltration and recruitment of Treg cells [70]. DCs with high TGFβ expression are poor at eliciting the activation of naive CD4 T cells and sustaining their proliferation and differentiation into Th1 effectors. Vascular endothelial growth factor (VEGF) inhibits LPS induced DC maturation via Nrp-1 receptor on DC. NRP1 interacted with LPS receptor TLR4 and suppressed downstream ERK and NF-κβ signaling, resulting in increased expression of MHC-II and costimulatory molecules (CD40, CD86) as well as proinflammatory cytokine production inhibition [71]. Infiltrating macrophages were the primary source of IL-10 within tumors, blocking IL-10 signaling increases intratumoral dendritic cell expression of IL-12 during chemotherapy in breast cancer [65].

LPA is a bioactive lipid produced by tumor cells. Blocking LPA-generating enzyme autotaxin in ovarian cancer cells elicits anti-tumor immune response driven by type-I IFN. LPA induces PGE2 synthesis by DCs, which suppressed type-I IFN production and response in DC via autocrine EP4 engagement [72]. Lactate is an oncometabolite resulted from metabolic adaption in cancer cells via Warburg effect. Lactate in tumor attenuates pDC activation in response to TLR9 ligand and consequent type I IFN induction. pDC tolerization by lactate is partially through activating GPR81, a cell surface G-protein coupled receptor of lactate. GPR81 activation induces intracellular Ca2<sup>+</sup> mobilization and activates calcineurin phosphatase (CALN) expression. Inhibition of CALN reverses the inhibitory effect by lactate. Extracellular lactate can also influence pDCs through intracellular import via the monocarboxylate transporters (MCT). Inhibition of MCT genes resulte in significant reversal of the lactate-mediated inhibition of IFNα. Thus, both GPR81 triggering and cytosolic import via the MCT transporters mechanism are involved in lactate induced pDC tolerization. Lactate treated pDCs have enhanced tryptonphan metabolism, leading to excessive production of kynurenines which in turn induces Treg cell differentiation [73].

#### *3.3.2 Cell-intrinsic mechanism*

Tumor-derived Wnt5a induces β-catenin signaling activation-dependent IDO expression in DCs. DCs conditioned by wnt5a promote Treg development and suppresses effector T cell activation [74, 75]. β-Catenin complexed with PPAR-γ upon wnt5a stimulation and transcriptionally activates fatty acid oxidation (FAO) synthesis gene, CPT1a, inducing the synthesis of heme prosthetic group, protoporphyrin IX, which is required for IDO enzymatic activity [75]. Wnt1/β-catenin signaling in DC suppresses chemokine production, leading to T cell exclusion in tumor and decreased T cell activation [76]. β-catenin signaling in DC also impairs CD8 T priming through inducing IL-10 secretion via mTOR activation. Even though the negative regulation of initial CD8 T priming by β-catenin/mTOR/IL10 in DC, β-catenin–regulated IL-10 also shown has an opposite anti-tumor immunity role through maintenance of primed CD8 T cells after clonal expansion [77, 78]. β-catenin can also interact with TCF4 and activates gene expression of Aldh1, an enzyme to produce retinoic

acid (RA) from vitamin A, resulting increased RA in DC [79]. Aldh1 expression in mature DC significantly correlated with immunoregulatory module including genes like PD-L1, PD-L2, CD83, and CCL22 [80]. RA induces Treg generation in vitro and in vivo [81–83]. DC maturation suppression could be mediated by E-cadherin based DC-DC adhesion. Disrupting this contact activates DC maturation through activating β-catenin/TCF, leading to increase of co-stimulatory molecules, MHC-II and chemokine receptors. However, such DC maturation is not coupled with proinflammation cytokine secretion and failed to prime CD4 T cells, coupled with a distinct transcriptional profile from those induced by TLR activation. DC matured by E-cadherin disruption also leads to Treg production. The data suggests a DC function regulatory role of E-cadherin/β-catenin/TCF axis [84].

Nuclear factor-κB (NF-κB) is an important transcription factor that participating in cancer inflammation. There are two general types of NF-κB signaling pathways: canonical and non-canonical pathways [85]. Canonical and non-canonical NF-κB pathways play different roles in DC functional regulation. Lung cancer patient derived tumor sera induce canonical NF-κB pathway inhibition, while activates noncanonical NF-kB pathway in human mo-DC [86]. IFNγ has been shown important for myeloid activation [87]. Canonical NF-κb/IRF1 mediated IFNγ response pathway is required for intra-tumoral cDC1 activation. IFNγ knock out or IFNGR1 knock out in cDC1 abolished IL12 production [88]. Impaired NF-κB or IRF1 loses control of tumor growth and expression of maturation markers and chemokines (CXCL9/10) for recruiting T cells [89]. Inhibiting NF-κB in BMDC has no effect on MHC-II or co-stimulation molecules, while promotes Treg differentiation in vitro [80]. VEGF mediated inhibition on LPS stimulated BMDC activation is dependent on the inhibition of canonical NF-κB signaling pathway [66]. Noncanonical NF-kB signaling in dendritic cells is required for IDO induction in the late stage of DC activation by CD40 ligation [90].

Inhibitory molecular expression on DC suppresses T cell activation and induces Treg differentiation. PD-L1 upregulation in tolerized DC is not dependent on the presence of type I and type II IFN signaling, nor is dependent on inflammasome or TRIF/MyD88 signaling. Instead, PD-L1 upregulation is dependent on phagocytic cell-surface receptor AXL activation upon antigen uptake. IL-4 signaling negatively regulates IL-12 production on DC. Blocking IL-4 signaling can increase IL-12 production without upregulating PD-L1 [88]. IRF4 plays a dural role of upregulating antigen presenting capability and tolerization of BMDC. Depletion of IRF4 reduces Aldh1 and PD-L2 expression, coupled with elevated cytokine IL-12 an TNF expression. IRF4 deficient DC is impaired for Treg generation in vivo. TIM-3 is predominantly found expressed in cDC cells in tumor. TIM-3 expression on DC can be induced by IL-10 or VEGF [91]. Blocking TIM-3 improve survival when combined with chemotherapy. The regulatory effect by TIM-3 blocking is neither through affecting cDC infiltration nor through regulating cDC activation. However, TIM-3 blocking increases CXCL9 secretion by cDC1, which is a ligand for CXCR3. CXCL9/CXCR3 chemoaxis attracts T cells into tumor [92]. TIM-3 on DC impaires DC recognition and response to tumor derived nucleic acids. TIM-3 serves as a receptor for DNA sensor, HMGB1, completing with nucleic acids for binding to th A-box domain of HMGB1. The binding of TIM-3 on HMGB1 inhibits nucleic acids to be internalized into endosomes [87].

DC activation is accompanied by an increased glycolysis metabolic process, which is required by both survival and effector function of activated DC. Bioactive gas nitric oxide (NO) is synthesized and secreted by activated DC, playing an

immunomodulating role of DC. Cellular production of NO is catalyzed by NOS enzymes, which converts substrates L-arginine, NADPH and O2 to L-citrulline, NADP+, and NO [93]. Inducible NOS (iNOS) is the primary NO-synthesizing enzyme expressed by DC. iNOS expression in CD103<sup>−</sup> CD11b+ intratumoral DC is required for tumor suppressive Th17 T cell differentiation in PDA model [94]. Glucose could inhibit DC function through mTOR/HIF1a/iNOS signaling axis, inhibiting costimulation molecular expression and IL12 secretion and restricting T cell activation. When T cells encounter DCs, they compete for glucose availability, which suppress the glucose sensitive pathway resulting T cell activation [95]. Monocyte-derived tumor associated DCs are prominent in tumor antigen uptake, but lack of strong T-cell stimulatory capacity due to NO-mediated immunosuppression [96].

#### **4. Application of DC vaccine in tumor immunotherapy**

#### **4.1 DC vaccination**

DCs are the most efficient professional antigen-presenting cells that can initiate an adaptive immune response by presenting antigens to T cells [97, 98]. In the past 25 years, many groups have exploited this characteristic to create dendritic cell vaccines to direct the immune system to fight cancer. DC cell-based vaccine approaches have been proved safe for their minimal toxicity, and their low association rates with autoimmunity [99, 100]. The general process of DC vaccine preparation including DC generation, antigen loading and DC maturation. To date, different strategies have been developed to generate DC vaccine for clinical applications.

The most commonly used approach to generate DCs is through ex-vivo differentiation from peripheral blood. The advantage of this method is the easy generation of sufficient autologous DCs for vaccination. However, therapeutic outcomes still have a lot of room for improvement, with less than 15% the patients showing objective response [101]. Due to the artificial *in vitro* differentiation process, moDCs have compromised functionality compared with naturally-occurring DCs with different transcriptional profiles. The limitation of using naturally-occurring DCs is the low frequency of DCs in peripheral blood, resulting in a highly labor-intensive process in DC isolation for clinical use. To overcome this, a growing effort in the field has been exerted to facilitate the developing a feasible protocol, for example, an automatic system that can prepare DCs [102]. For DC vaccine production, DCs are then be loaded with total tumor lysate or RNAs and tumor associated antigens. The loading methods include pulsing by co-culturing, electroporation, viral transduction or DC-tumor fusion [103]. Maturation cocktails used in the clinic consist of TLR agonists and cytokines, often in combination with co-stimulatory proteins like CD40L. Introducing mRNAs coding constitutively-active TLR4, CD40L and CD70 via electroporation has shown clinical success [33, 104].

#### **4.2 DC vaccination clinical trial in glioblastoma**

DC vaccination in the context of glioblastoma has shown both positive and negative results in clinical trials. Even though a phase III clinical trial aiming to assess DC vaccine targeting the EGFR deletion mutation EGFRvIII in newly diagnosed EGFRvIII-expressing GBM patients failed [105], some other clinical trials have shown promising results. Another phase III clinical trial utilizing an autologous

#### *DOI: http://dx.doi.org/10.5772/intechopen.108586 Perspective Chapter: Dendritic Cells in the Tumor Microenvironment*

tumor lysate-pulsed dendritic cell vaccine combined with standard therapy showed significant overall survival benefit from 15 to 17 months to 23.1 months [36]. In another phase II clinical trial, ICT-107 (autologous dendritic cells (DC) pulsed with six synthetic peptide epitopes targeting GBM tumor/stem cell-associated antigens MAGE-1, HER-2, AIM-2, TRP-2, gp100, and IL13Rα2 was given to newly diagnosed glioblastoma patients in addition to standard therapy. Results showed progression free survival (PFS) increased 2.2 months in ICT-107 cohort compared with matched DC control cohort. HLA-A2 subgroup patients achieved a meaningful therapeutic benefit with ICT-107, in both the MGMT methylated and unmethylated prespecified subgroups, whereas only HLA-A1 methylated patients had an OS benefit [106, 107]. Combination with other intervention methods could help increase DC vaccine efficacy. Pre-conditioning at vaccinated site can improve DC vaccination efficacy. Mitchell et al. (2015) showed that glioblastoma patients pre-exposed to tetanus/diphtheria (Td) toxoid in the vaccine site before vaccination with pp65 RNA-pulsed DCs had improved tumor-antigen-specific DC migration and improved survival compared to the ones that were not pre-exposed to the toxoid through increasing DC migration to dLN [108]. Three phase II clinical trials (ATTAC; ELEVATE; NCT00639639, NCT00639639, NCT02366728) aim to test pp65 DC with Td vaccine in newly diagnosed GBM patients. Results to date have shown that despite a small cohort, three successive trials demonstrate consistent survival outcomes, supporting the efficacy of *cytomegalovirus* DC vaccine therapy in GBM [109].

#### **5. Conclusions**

Dendritic cells, as the most professional APCs, play key roles in mediating the bridge between innate and adaptive immunity in anti-tumor immunity. DC subpopulations, through use of different action mechanisms in activating adaptive immunity, collaborate with each other to elicit anti-tumor immunity. In the battle with tumor, DC functions become regulated by tumor cells or other components in the tumor microenvironment, leading to DC dysfunction. These include impairments on antigen uptake, antigen presentation, migration to LN, and DC tolerance. Secreted factors from the tumor environment play a key role in mediating DC regulation. These suppressive signals act on DCs inducing DC dysfunction through different cellular intrinsic pathways. DC vaccine development for tumor treatment has made significant progress in the last decades, but still faces challenges in achieving a wide and significant therapeutic success. Deepening our understanding on DC function and regulation in the tumor environment will help the field in developing new and more powerful therapeutic intervention approaches.

#### **Acknowledgements**

This work was funded by NIH NINDS 5R01NS111033-03.

#### **Conflict of interest**

CF is a founder of iOncologi.

*Tumor Microenvironment – New Insights*

### **Author details**

Dan Jin, Laura Falceto Font and Catherine T. Flores\* University of Florida, Gainesville, United States

\*Address all correspondence to: catherine.flores@ufl.neurosurgery.edu

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Perspective Chapter: Impact of Tumor Metabolism on Immune Cells in the Tumor Microenvironment

*Adith Kotha, Chikezie Madu and Yi Lu*

#### **Abstract**

Metabolism is essential for a cell to obtain energy for its growth and development. In tumors, the rapid rate of cell proliferation leads to an increased demand for energy. Because nutrients in the tumor microenvironment are scarce, there is great competition between tumor cells and healthy cells to obtain them. Because of this, tumor cells undergo adaptations to outcompete healthy cells for nutrients. These adaptations cause characteristic changes to the tumor microenvironment, which in turn, causes changes to immune cells in the tumor tissue. These changes help the tumor evade immune detection and cause tumor growth and metastasis. This review will analyze the changes that take place in the tumor microenvironment, the impact they have on immune cells, and how this contributes to cancer progression.

**Keywords:** metabolism, nutrients, tumor microenvironment, immune cells, immune detection, cancer progression

#### **1. Introduction**

Metabolic reactions are chemical reactions that take place within cells or organisms and are essential for their survival. Metabolic processes include the breakdown of compounds for energy, the synthesis of necessary biomolecules, etc. Changes to the metabolic processes of cancer cells are a key characteristic of tumorigenesis. In order to supply their rapid rates of cell proliferation, tumor cells are in constant need of nutrients from the tumor microenvironment (TME), which are very scarce. This puts tumor cells in fierce competition with neighboring cells for these resources. Tumor cells undergo various adaptations, such as utilizing anaerobic glycolysis in favor of aerobic respiration, a process that allows them to synthesize ATP at higher rates. Such adaptations allow tumor cells to outcompete neighboring cells and allow the tumor to grow. The adaptations that the tumor cells undergo have an influence on the TME. For example, the aforementioned use of anaerobic respiration causes the TME to become more hypoxic and acidic.

These changes to the characteristics of the TME cause phenotypic alterations of immune cells within the TME. The TME includes cells of both the adaptive and innate immune systems, and they undergo notable changes to their metabolic pathways in response to the conditions of the TME or other signals within it. The former includes T cells and B cells, while the latter consists of tumor-associated macrophages (TAMs), natural killer (NK) cells, dendritic cells, and neutrophils.

These alterations of immune cells in the TME provide numerous benefits to the tumor. Namely, various altered pathways allow for the tumor to evade detection by the immune system, which contributes to the growth of tumors and the progression of cancer. This paper will discuss how the metabolic reprogramming of tumor cells contributes to changes in the conditions of the TME, the impact these changes have on the functionality of immune cells, and how they relate to the spread of cancer.

#### **2. Changes to conditions of the TME**

Tumor growth relies on the rapid proliferation of cells, which is an energetically demanding process. However, nutrients within the TME are often very scarce, and as a result, tumor cells are in fierce competition with healthy cells in the TME for these nutrients. Tumor cells adapt to these increased energy demands by shifting their metabolic pathways [1]. One such adaptation that tumor cells undergo is reprogramming of their glucose metabolism to utilize anaerobic glycolysis in preference to the tricarboxylic (TCA) and oxidative phosphorylation (OXPHOS) pathways ([2], **Figure 1**). This pathway, known as the Warburg effect, is active even in the presence of abundant oxygen, and it is key to a tumor cell's ability to outcompete neighboring cells.

Though the process of anaerobic glycolysis generates lower quantities of net ATP from glucose than the OXPHOS pathway, it allows for the metabolism of glucose to occur much more rapidly in tumor cells, thus leading to tumor cells outcompeting neighboring ones for nutrients. Additionally, other adaptive mechanisms of tumor

#### **Figure 1.**

*(Warburg effect): The Warburg effect is a major metabolic reprogramming that cancer cells undergo. Normal cells exhibit a usage of both glycolytic and OXPHOS pathways, while cancer cells rely on glycolysis and produce excess lactate as a by-product. This reliance on glycolysis and production of molecules, such as lactate, cause major changes to the conditions of the TME [3].*

#### *Perspective Chapter: Impact of Tumor Metabolism on Immune Cells in the Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108830*

cells allow them to overcome this inefficient method of obtaining energy. For example, many tumor cells can carry out autophagy, which allows them to recycle nutrients and prevents nutrient depletion [4]. Additionally, tumor cells can synthesize ATP using two ADP molecules, forming one ATP and one AMP [5, 6]. These adaptations make the Warburg effect a useful mechanism through which tumor cells can outcompete other cells within the TME for nutrients and proliferate. However, the process also causes drastic changes to the conditions of the TME.

The primary change caused by the Warburg effect is the acidification of the TME. These conditions are caused by the higher rates of anaerobic glycolysis and the production of lactic acid [7]. The acidic state of the TME confers numerous advantages for tumor growth, as it promotes the formation of new blood vessels, drug resistance, and suppression of the anticancer immune system [8]. The lactic acid that is produced can also act as a signaling molecule that regulates the migration of tumor cells: areas with a lower pH promote tumor cell invasion and metastasis [8].

Another important characteristic of the TME is its state of hypoxia. The delivery of oxygen and other nutrients to tissue occurs through blood vessels. Because tumors are undergoing constant growth, their receiving of blood flow is often irregular. In order to combat this, tumor tissues can form new blood vessels in a process referred to as angiogenesis [9]. This process allows tumors to continually receive the nutrients required to meet their metabolic demands. However, if angiogenesis fails, the aforementioned conditions of hypoxia and resource scarcity will arise in the TME. The tumor can still thrive under these conditions due to its reliance on anaerobic glycolysis [7]. Additionally, the hypoxic state acts as an additional stressor on the immune system and allows the tumor to evade immune attack.

The conditions in the TME also cause changes to the functionality of the immune system. For example, the hypoxic environment can negatively impact the immune detection of cancer cells and contributes to tumor immunity [10]. Signaling factors, called hypoxia-inducible factors (HIFs), are a key part of the regulation of tumor immunity genes. These factors can also inactivate lymphocytes in the TME, namely NK cells and CD8 T lymphocytes, thus preventing them from combating tumor growth. In this pathway, proinflammatory signals produced in hypoxic regions of the TME attract regulatory T cells (Tregs), which in turn suppress cytotoxic T cells from producing an immune response, thus promoting cancer growth [11]. The hypoxic conditions also act as a stressor on neutrophils and block them from attacking tumors. Finally, HIFs have negative impacts on the maturation of B cells, which they accomplish by increasing their rate of glycolysis. This metabolic change to B cells causes them to divide less rapidly (thus decreasing their immune response), prevents them from altering antibody production, and can even trigger cell death [10].

Additionally, the aerobic glycolysis pathway causes irregularities in the metabolite balance within tumor cells, a factor that causes changes to cell signaling and cell–cell interactions within the TME [12]. For example, the aforementioned acidic conditions of the TME created by the excessive lactate produced through glycolytic pathways interfere with the immune response of cytotoxic T cells. The lactic acid also interferes with the production of IFN-γ by NK cells, which inhibits phagocytic cells from attacking the tumor [13].

Amino acids, namely glutamine, arginine, and tryptophan, are also important metabolites that influence the function of immune cells within the TME. Glutamine is produced as a by-product of the catabolism of proteins in nutrient-scarce environments [13]. It is essential to the function of immune cells because it regulates immune cell activation and determination, namely that of T cells. When its availability is

limited, T-cell functionality is suppressed [13]. Similar to glutamine, arginine plays a role in the activation of T cells and NK cells. Additionally, it regulates the secretion of cytokines [13]. Tumor cells consume a significant amount of the exogenous arginine in the TME, thus inhibiting the effect it has on immune cells [13]. Tryptophan also plays a role in the regulation of T cells, namely its cell cycle. When tryptophan is unavailable, the rate of T-cell apoptosis increases drastically [13].

Finally, lipids play an important role in the regulation of immune cell signaling within the TME. Fatty acids are needed for macrophage maturation and proliferation [13]. Additionally, they are necessary for the synthesis of membranes for effector immune cells. However, the accumulation of fatty acids within the TME can cause metabolic alterations to immune cells and make them anti-inflammatory. [13]. Similar effects can be induced by the accumulation of cholesterol within the TME, which causes T cells to lose their antitumor functionality. This occurs because high cholesterol levels can cause the disruption of T-cell membranes, thus impeding their ability to attack tumors [13].

#### **3. Immune cell subtypes in TME**

The tumor microenvironment is comprised of tumor cells, resident host cells, extracellular matrix, cancer-associated fibroblasts, vascular cells, and tumorinfiltrating immune cells [14]. Although tumor-infiltrating immune cells of both innate and adaptive arms of the immune system are often present in the TME, specific subtypes of immune cells, their number, and function can vary significantly depending on the tumor type and on the different stages of progression [14]. Functionally, tumor-infiltrating immune cells have been shown to be responsible for both tumorinhibitory (antitumor) and tumor-promoting properties [15]. Recruitment of immune cells into the TME is tightly regulated by chemotactic factors and the expression of chemokine receptors on immune cells which together define the recruitment of activator or suppressor type of immune cells into the TME [16]. Based on the extent of immune cell infiltration into tumor tissue, the TME can be classified as immune-infiltrated, immune-excluded, and immune-silent.

Immune cells of the adaptive response in the TME include T- and B- subsets of lymphocytes. Both subtypes of CD3+ T lymphocytes (CD4+ helper T cells and CD8+ cytotoxic T cells) can be observed within the TME, where CD8+ T cells are predominantly responsible for cytotoxicity response against the tumor cells and CD4+ T cells either support CD8+ cell cytotoxic activity or act as regulatory T cells (Tregs) that suppress the antitumor immune responses. The types of chemotactic factors in the TME and expression cytokine receptors therefore collectively determine which subtype of T cells predominate in the TME. For example, chemokines CXCR3, and CXCR4 aid in directing the migration of cytotoxic T cells and NK cells into the tumor, whereas CCR4 expression is linked to the recruitment of suppressor Tregs into the TME [16]. B cells, which are primarily responsible for antibody-mediated immune response, are also observed in the TME but in relatively small numbers when compared with T cells. Tumor-infiltrating B cells appear to mediate the formation of lymphoid-like structures within the TME where their interaction with T cells regulates tumor progression [16].

Immune cells of innate response that are constituents of the TME include NK cells, macrophages, neutrophils, and dendritic cells [14, 16]. Natural killer (NK cells) mediate antitumor activity either via direct cell-mediated killing of tumor cells or by

*Perspective Chapter: Impact of Tumor Metabolism on Immune Cells in the Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108830*

secretion of specific cytokines that indirectly contribute to the antitumor response. NK cells, although present in the TME, are less efficient at killing tumor cells within the tumor microenvironment, are highly effective against circulating tumor cells, and therefore more effective in preventing tumor metastasis [17]. Macrophages by far are the most common type of innate immune cells in TME and macrophage infiltration has been associated with poor prognosis of several solid tumors. Two distinct phenotypes of macrophages that mediate a pro-inflammatory response (M1 macrophages) and wound healing response (M2 macrophages) are commonly present in the tumor tissue [18]. However, the hypoxic state and presence of certain cytokines within the TME favor the M2 phenotype that supports tumor progression [18]. Neutrophils are the next variety of innate immune cells seen in the TME. Neutrophils are recruited into tumor tissue where they initially promote a local inflammatory response thereby promoting tumor cell apoptosis. As the tumor progresses, neutrophils can functionally support tumor growth through the modification of the extracellular matrix, and the release of growth factors that promote angiogenesis [19]. Dendritic cells (DCs), the most potent type of antigen-presenting cells; play an important role in cancer immunosurveillance and infiltration of DC into tumor tissue is associated with delayed tumor progression and metastasis [19].

#### **4. Metabolism in lymphocytes: t cells**

Metabolic pathways in T-lymphocyte vary depending on their differentiation status in their life cycle [20]. Naïve T lymphocytes mainly depend on TCA and OXPHOS to support basal metabolism. Continued signaling from cytokines, such as IL-7, is required to maintain glucose uptake by naïve T cells for sustaining the metabolism [20]. Following antigen recognition and activation, T cells undergo a metabolic change that is dependent on both glucose and amino acids as energy sources to support cell proliferation and to function as effector T cells [21]. Similar to the tumor cells, the effector T cells use Warburg metabolism to support energy demands associated with the secretion of cytotoxic cytokines and enzymes required for the removal of the tumor and virally infected cells. Therefore, within the TME, malignant cells compete with the effector T cells for energy sources and relatively nutrient deficiency in the TME can impair T-cell survival and proliferation [22]. The mechanisms underlying the regulation of T-cell effector functions by metabolic pathways also vary in different subsets of T cells. For example, in CD4 T cells, enzymes of the glycolytic pathway, such as GAPDH, can interact with mRNA of key cytokines, thereby preventing their translation [23]. Additionally, acetyl-CoA produced from citrate in cytosol due to the action of ATP citrate lyase (ACL) in both CD4 and CD8 T cells can directly modify histone acetylation status at the promoter regions of key cytokine genes involved in mediating effector functions [24]. Changes in mitochondrial structure and function are also implicated in the regulation of effector T-cell function as well as memory T-cell formation. Effector T cells, where mitochondria exhibit fragmentation, are poor in supporting electron transport machinery that leads to upregulation of anaerobic glycolysis, whereas in memory T cells, the mitochondrial fusion process allows proper function of ETC and facilitates lipid metabolism via fatty acid oxidation [25].

Due to similarities in the metabolic pathways utilized, within the TME, competition for nutrients exists between tumor cells and the effector T cells [26]. Tumor cells with functional mutations that confer survival advantage can therefore outcompete effector T cells leading to the reduced number and/or function of cytotoxic CD8

cells. Furthermore, lactate produced by tumor cells in the hypoxic regions creates an acidic environment that can inhibit T-cell activation by preventing glycolysis [27]. In contrast with cytotoxic T cells, Tregs, upon activation, induce fatty acid biosynthesis and oxidative phosphorylation, conferring them with a metabolic advantage to thrive within the TME [28]. Tumor cells evade the immune response by upregulation of inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte association protein 4 (CTLA4). These inhibitory receptors, known as immune checkpoints, are widely used as targets in cancer therapy as they also play a role in the metabolic regulation of T cells [29]. PD-1 expression downregulates glycolysis and increases fatty acid oxidation, which reduces their cytotoxic potential. PD-L1 expressed on tumor cells enhances glucose uptake and therefore blockade of PD-1/PD-L1 interaction can collectively potentiate antitumor activity of T cells [30]. CTLA-4 is a receptor expressed transiently on T cells following activation and plays an important role in regulating their activity. One of the mechanisms by which CTLA-4 suppresses T-cell activity is by down-regulating critical amino acid and nutrient transporters and inhibition of CTLA4 can restore the bioenergetic balance that favors the survival of T cells in the TME [29].

Another key area where understanding T-cell metabolism is critical is in cell-based therapies that utilize chimeric antigen receptor (CAR)-T and tumor-infiltrating lymphocytes (TIL). CAR-T treatment is an immunotherapeutic strategy in which samples of T cells taken from a patient's blood are genetically modified to produce receptors that target tumor cells [31]. In TIL therapy, T lymphocytes are taken from the tumor microenvironment and cultured *ex vivo*. The amplified TILs are then infused with the tumor in order to promote the targeting of cancer cells. These cells undergo metabolic reprogramming to inhibit glycolysis *in vivo*, which increases the

#### **Figure 2.**

*(cytokine release syndrome): CRS is an acute immune inflammatory response caused by the activation of the immune system, particularly T cells. This triggers the release of cytokines, which are molecules involved in directing immune function. These excess cytokines pose serious health risks, such as organ failure and potential death [34].*

*Perspective Chapter: Impact of Tumor Metabolism on Immune Cells in the Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108830*

proliferation of T cells and thus increases antitumor efficacy [32]. These methods have proven beneficial as alternative therapies when conventional therapies fail due to the acquisition of tumor resistance or where checkpoint inhibitors therapies are not a viable option due to lack of expression of those receptors as targets [33]. Both CAR-T and TIL therapies require isolation and *ex vivo* expansion of tumor-specific lymphocytes prior to administering to the patients. Despite having tumor-specific activity, engineered CAR-T-cell therapies are prone to adverse events in the form of cytokine release syndrome ([32], **Figure 2**). It is beginning to be understood that some of the mechanisms underlying CAR-T-cell properties, therapeutic efficacy, and potential adverse events are linked to metabolic pathways in the engineered cells. Conditions used for *ex vivo* expansion of TIL and CAR-T cells also may alter the metabolic state of these cells, thus impacting therapeutic effectiveness [35]. It is possible that redirecting the metabolic pathways during their expansion may result in cells with beneficial properties targeting tumors [35].

#### **5. Metabolism in lymphocytes: nk cells**

Resting NK cells predominately use glucose as fuel to carry out glycolysis and oxidative phosphorylation. Activation of NK cells via cytokine stimulation increases glucose uptake and the rate of glycolysis and oxidative phosphorylation, which support biosynthesis and secretion of IFN-Ƴ and other key enzymes, such as granzyme, that are required for NK cell effector function [35]. In contrast with other lymphocytes, pyruvate generated from glycolysis in NK cells is preferentially converted to citrate via citrate-malate shuttle (CMS) rather than metabolism via the TCA cycle [36]. Two subsets of NK cells are recognized based on the expression level of phenotypic marker CD56 (CD56 dim and CD56 bright) that appear to be metabolically distinct. For example, CD56 bright NK cells involved in cytokine production express higher levels of glucose transporter proteins, thus rapidly taking up glucose upon activation [37]. In addition to glucose, glutamine is also important as a fuel source for the metabolism of activated NK cells. Glutamine can regulate the uptake of critical amino acids and the breakdown products of glutamine enter the TCA cycle for generating ATP [37]. Metabolic pathways in NK cells are tightly regulated both during development and activation. Specific signal transduction pathways and transcription factors are involved in regulating the metabolic pathways in NK cells. Transcription factor steroid regulatory element binding protein (SREBP) regulates the expression of the components of the CMS pathway and the mammalian target of the rapamycin (mTOR) pathway regulates NK cell proliferation and metabolism [36]. Consequently, reduced mTOR activity of mature NK cells is associated with diminished metabolic activity that results in impaired effector functions of NK cells. The multifunctional transcription factor c-Myc plays an important role by upregulating glucose transporters and critical enzymes of glycolysis in NK cells [36].

Although NK cells are highly effective in the targeted removal of tumor cells, the tumor microenvironment poses a challenge to the appropriate function of the NK cells. Firstly, changes in the metabolic properties of tumor cells create an environment that is low in critical nutrients (glucose and glutamine) and oxygen (hypoxic state) that are essential for the normal metabolism of NK cells [38]. Secondly, anaerobic glycolysis of tumor cells produces lactic acid that creates an unfavorable acidic environment, leading to reactive oxygen species (ROS) production in NK cells and induction of apoptosis [38]. Furthermore, transforming growth factor β

#### **Figure 3.**

*(targeting of NK-cell metabolic pathways): The targeting of major receptors and metabolites, namely FBP-1 in NK cells, holds great promise in restoring the antitumor efficacy of NK cells in the TME [40].*

(TGF-β), a cytokine that is commonly upregulated in several cancers can inhibit NK cell metabolism, presumably via the inhibition of mTor activity [39]. Metabolic adaptation of NK cells within the TME involves the activation of enzymes in the gluconeogenetic pathways, such as FBP-1, to generate glucose needed for NK cell metabolism. Therefore, dysregulated FBP-1 expression in NK cells further reduces their ability to survive in the TME and thus reduces immune function [40]. The hypoxic state of TME is associated with mitochondrial fragmentation in certain tumors, which perturb the survival and cytotoxic properties of NK cells [41]. In addition to the aforementioned factors, certain other metabolites that are elevated in the TME (adenosine, prostaglandin E2 (PGE2), and kynurenine) may also be responsible for reduced NK cell function via mechanisms that are yet to be understood [36]. Restoring normal metabolic function and survival of NK cells in the TME is one of the bases for pharmacological approaches to treat cancer where infiltrated NK cells have potent antitumor activity. Targeting TGF-β or its downstream signaling pathways and/or restoration of c-Myc protein levels via inhibition of enzymes (GSK3) are potential therapeutic approaches [39]. Additionally, culturing autologous NK cells *ex vivo* and inhibiting FBP-1 has proven to restore immune function, namely cytotoxicity ([40], **Figure 3**). Other cells in the TME (cytotoxic and Tregs, stromal fibroblasts, etc.) have also been shown to modulate the expression of various activating and inhibitory receptors on NK cells that in turn regulate the metabolic and antitumor properties of NK cells [38]. Therefore, targeting inhibitory NK cell receptors, such as NKG2A, is one of the strategies being evaluated as NK cell-mediated antitumor immunotherapy ([38], **Figure 3**).

*Perspective Chapter: Impact of Tumor Metabolism on Immune Cells in the Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108830*

#### **6. Metabolism in the innate immune system: tumor-associated macrophages**

Macrophages are specialized immune cells that develop from myeloid progenitor cells and are highly efficient in phagocytosis and the removal of pathogens [42]. Tumor-associated macrophages (TAMs) are macrophages that are specifically recruited into tumor tissue due to cytokines and growth factors secreted by cells within the tumor microenvironment [43]. TAMs are one of the most abundant leukocytes within the TME and have been implicated in tumor progression and metastasis [44]. Macrophages were further classified as inactive (M0), pro-inflammatory (M1), and anti-inflammatory (M2) subtypes based on specific immune responses elicited by these cells. Inactive macrophages (M0) are undifferentiated cells and can reprogram themselves into polarized M1 and M2 cells after exposure to stimuli [45]. These distinct subtypes of macrophages utilize different metabolic pathways to exert their functional effects and TAMs are further induced to undergo a metabolic switch to survive in the tumor microenvironment. The key features of M1 and M2 macrophages in the utilization of various metabolic pathways are as follows. Although both M1 and M2 macrophages metabolize glucose via glycolytic pathways, in M1 macrophages it is essential for pro-inflammatory properties, such as cytokine production, and in mediating phagocytosis [46]. Similarly, the pentose phosphate pathway, which produces NADPH, is also critical in M1 macrophages, where NADPH-oxidase-dependent generation of reactive oxygen species (ROS) and regeneration of glutathione [47]. Arginine is also metabolized differently in M1 and M2 macrophages by virtue of the

#### **Figure 4.**

*(TAM polarization): TAMs undergo metabolic changes that trigger polarization to the M2 phenotype, which has pro-tumorigenic properties and contributes to cancer progression [51].*

expression of distinct enzymes that break down arginine. Notably, M1 cells express inducible nitric oxide Synthase or iNOs that produces NO from arginine [46] and M2 macrophages express the enzyme arginase that metabolizes arginine to produce ornithine. NO has an important function in mediating pro-inflammatory response and ornithine serves as a precursor for polyamine synthesis that is critical in wound healing and repair processes that are mediated by M2 macrophages [48]. The TCA cycle in M2 macrophages is coupled to mitochondrial oxidative phosphorylation, whereas in M1 macrophages, intermediate metabolites of the TCA cycle, citrate and succinate, accumulate and are redirected toward the processes that lead to the production of inflammatory mediators, such as prostaglandin E2 (PGE2) [49].

The subtypes of macrophages within the TME vary with the progression of tumors. During the early stages of tumors, M1 macrophage polarization is favored, thus leading to the recruitment of cytotoxic CD8 cells and NK cells ([50], **Figure 4**) and the antitumor property of TAMs. However, as tumors progress, polarization to M2 macrophages is favored due to progressive changes in the TME ([50], **Figure 4**). Due to aerobic glycolysis of tumor cells, lactic acid in the TME induces M2-like TAM polarization of TAMs [46]. Additionally, TAMs have also been implicated in regulating tumor metastasis and angiogenesis further supporting the survival and spread of tumors. Metabolically, TAMs utilize glucose as the primary energy source with oxidative phosphorylation favoring their differentiation into pro-tumorogenic M2 macrophages [46]. As TAMs constitute the predominant cell population in the TME, potential therapies for cancer are based on metabolic targeting either to inhibit TAM polarization to an M2 phenotype or to selectively deplete M2 cells within the TME [52]. However, considering the complexity of concurrent metabolic processes occurring in other cells in the TME, these approaches have some limitations. Nevertheless, inhibition of OXPHOS pathways in TAMs has been shown to decrease tumor progression [53]. Future therapies directing metabolic processes via targeted drug delivery to TAMs may prove useful to overcome limitations associated with current strategies [52].

#### **7. Conclusion**

Cancer cells undergo key changes to their metabolic processes as an adaptation to outcompete other cells in the TME. This metabolic reprogramming causes the chemical conditions of the TME to change. The most notable of these changes is the development of hypoxic and acidic conditions due to a reliance on anaerobic glycolysis rather than OXPHOS pathways to produce ATP (Warburg effect), as well as the limited availability of nutrients. Additionally, the unique metabolism of cancer cells causes irregularities in the metabolite balance within the TME. Such changes have significant impacts on immune cells within the TME and their antitumor efficacy. All immune cell types in the TME of both the adaptive and innate immune systems undergo metabolic alterations in response to changes in the TME. These alterations greatly reduce immune function and contribute to tumor progression. The limited availability of nutrients in the TME downregulates the function of effector T cells and cytotoxic T cells and prevent their proliferation, and also prevents the formation of memory T cells. The antitumor efficacy of NK cells is reduced by the acidic and nutrient-scarce TME, which both triggers apoptosis, as well as the hypoxic state, which triggers mitochondrial fragmentation and reduces cytotoxic capabilities. The conditions of the TME cause TAMs to undergo polarization to the M2 subtype, which has pro-tumorigenic properties and can contribute to angiogenesis. Metabolism in

*DOI: http://dx.doi.org/10.5772/intechopen.108830 Perspective Chapter: Impact of Tumor Metabolism on Immune Cells in the Tumor Microenvironment*

the TME has become a focus of cancer treatment. Common treatments are based on culturing autologous immune cell types *ex vivo* and modifying their metabolic properties. These immune cells are amplified in order to improve immune function and are then infused with the tumor. These treatments must be further explored, but the targeting of immune cell metabolism in the TME proves to be a promising strategy in the treatment of cancer.

#### **Acknowledgements**

Funding for the publication of this paper was made possible by a grant from the Assisi Foundation of Memphis. Brown, Chester, PhD (PI). We also thank Yirui Tang for drawing the figures used in this paper.

#### **Conflict of interest**

The authors have declared that no conflict of interest exists.

### **Author details**

Adith Kotha1 , Chikezie Madu1,2 and Yi Lu3 \*

1 Department of Biology, White Station High School, Memphis, USA

2 Department of Biological Sciences, University of Memphis, Memphis, USA

3 Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, USA

\*Address all correspondence to: ylu@utshc.edu

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
