Keywords

8.1 Introduction

Bioactive lipids include a wide variety of metabolites that regulate essential cellular functions including membrane fluidity, cellular energy storage, lipid signaling, inflammation, and immunity. These mediators have pleiotropic and opposing activities secondary to their metabolism. The biosynthesis and metabolism of fatty acids are dependent on dietary intake and composition of polyunsaturated fatty acids (PUFA). Recent studies into bioactive fatty acids and their precursors in the tumor microenvironment have emphasized the importance of improving our understanding of the cellular lipidome as a regulator of tumor initiation, progression and metastasis, inflammatory and immune responses, and their tumor therapeutic potential as targets.

Chronic inflammation has multiple roles in carcinogenesis, tumor progression, and metastasis. Evidence from preclinical and clinical studies supports a role for chronic inflammation in the initiation and progression of cancer. Further, multiple mechanisms contribute to tumor initiation including the induction of genomic instability, alterations in epigenetic events and subsequent inappropriate expression of genes, enhancing the proliferation of tumor initiated cells, resistance to apoptosis, tumor neovascularization, and tumor invasion and metastasis. Inflammation-associated reactive oxygen and nitrogen species can also result in damage to cellular components including DNA, proteins, and lipids, which may directly or indirectly contribute to malignant transformation. Overexpression, elevated secretion, or abnormal activation of proinflammatory mediators, including cytokines, growth factors (GFs), chemokines, cyclooxygenase-2 (COX-2), prostaglandins (PGs), arginase, lipoxygenases (LOX), pattern recognition receptors (toll and notch), inducible nitric oxide synthase (iNOS) and nitric oxide (NO), and a distinct network of intracellular signaling molecules including upstream kinases and transcription factors, can all contribute to tumor progression. While inflammation supports tumor development, the tumor microenvironment, including tumor and stromal cells, as well as, inflammatory/immune cells, both activate in situ, or mobilized into the tumor microenvironment, can result in an inflammatory state by aberrant expression of proinflammatory mediators. Many of the proinflammatory mediators, especially cytokines, GFs, chemokines, PGs, and leukotrienes (LTs), upregulate angiogenic switches inducing inflammatory angiogenesis and tumor-stroma-cell communication, resulting in tumor angiogenesis, local and systemic immune suppression, and tumor invasion and metastasis.

Rodent and clinical studies have shown that myeloid cell infiltration of the tumor microenvironment is associated with poor clinical outcomes, as well as, neutrophilia and lymphocytopenia. In contrast, an increased lymphocytic infiltration of tumors is associated with improved clinical outcomes. Lifestyle parameters, including obesity and diets with high amounts of saturated fat and/or omega (ω)-6 PUFAs, influence tumor leukocytic infiltration, as well as an increase in extramedullary myelopoiesis (EMM). Tumor secretion of GFs and chemokines can regulate tumor-immune-cell crosstalk; dietary lifestyle choices can also contribute to inflammation, tumor induction and progression, and tumor leukocyte infiltration. A relationship between obesity and high-fat diets (notably saturated fats in Western diets) and the regulation of inflammation, tumor induction, metastasis, and poor clinical outcomes is accepted. Further, mechanisms of dietary promotion of an inflammatory microenvironment are little studied, and few targeted drugs to inhibit the clinical sequelae have neither been identified nor studied. Similarly, adipose tissue, within the tumor microenvironment and its regulation by diet, needs additional study. Thus, modifications of obesity and dietary lipids may inform preventative or therapeutic approaches in the control of tumor-associated inflammation and neoplastic progression.

Tumor-associated adipocyte-derived elements can promote tumor growth, as well as, dedifferentiation into fibroblast-like cells. Free-fatty acids (FFAs) released by adipocytes are used by cancer cells to support proliferation. In addition, these FFAs activate and modulate monocytes, macrophages, and vascular endothelial cells so as to favor a protumorigenic microenvironment [1, 2]. Obesity- and tumor-associated adipose tissues regulate tumor development in multiple ways, by providing energy via FFAs or through adipokines, cytokines, and miRNAs. Tumor-associated adipocytes can recruit macrophages to the tumor microenvironment and stimulate their polarization to the alternative M2 functionality via CCL2, interleukin (IL)-1β, and CXCL12 [3]. In addition, adipocytes produce inflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-6, IL-1β, and CCL2 [3, 4], which results in inflammatory cell recruitment, infiltration, and accumulation, resulting in foci of low-grade chronic inflammation [5, 6]. Visceral adipose tissues from obese individuals are also frequently associated with increased systemic levels of CCL2, TNF-α, IL-1, IL-6, and iNOS [7]. Moreover, the level of CCL2 produced is increased due to recognition of cell-free DNA (cfDNA) from degraded adipocytes. Obese mice also have increased cfDNA release resulting in a higher accumulation of macrophages, which aggravates inflammation [8], increases angiogenesis, and supports tumor growth and progression [3].

8.2 Role of Dietary Lipids in Chronic Inflammation and Tumor Initiation, Progression, and Regression

Dietary fats can increase inflammatory responses, particularly Western diets, predominantly due to the inclusion of fatty acids (FAs) from animal sources, which are mainly saturated fatty acids (SFAs), and FAs from plants that are predominantly ω-6 PUFAs. All are proinflammatory. In contrast, FAs derived from some plant-based oils, and fatty fish, which consist mainly of ω-3 PUFA, inhibit inflammation. Rodent and clinical studies have shown that subjects given diets rich in ω-6 PUFAs have an increased risk of inflammatory mediators and diseases, including asthma, rheumatoid arthritis, and inflammatory bowel disease [9]. In contrast, diets with high levels of long chain (LC)-ω-3 PUFAs are anti-inflammatory, such that consuming individuals have a decreased risk of inflammatory diseases [9]. These clinically relevant activities are associated with the oxidization of PUFAs to either proinflammatory or pro-resolving lipid mediators (Fig. 8.1), both of which can regulate inflammation and immunity [10]. Proinflammatory mediators, notably PGs and LTs, are secreted in response to “foreign” substances and cleared by pro-resolving lipid mediators, restoring cellular and tissue homeostasis [11]. Diets with high levels of the ω-3 PUFAs, shorter chain α-linolenic acid (ALA), and more critically, LC eicosapentaenoic (EPA) and docosahexaenoic (DHA) are associated with a decrease in inflammation [12]. The beneficial effects of dietary LC-ω-3 PUFAs include their metabolism into anti-inflammatory metabolites including LTs, thromboxanes (TX), resolvins and a decrease in inflammatory cytokines. The ω-3 PUFAs differ from the ω-6 PUFAs based on the position of their double bonds in the acyl chain, such as linoleic acid (LA) as compared to the ω-6 PUFA arachidonic acid (AA) (Fig. 8.1). However, the inflammatory activities of PUFAs are not clearly separated, based on the number and placement of double bonds, counting from the methyl end of the FA (i.e., ω-3 vs. ω-6). The dietary addition of the short-chain ω-3 PUFA, ALA, an essential FA, and the main precursor of LC-ω-3 PUFAs, is proinflammatory and can result in enhanced secretion of superoxides from macrophages and neutrophils [13], and adhesion of inflammatory cells to endothelial cells [14]. Further, ALA can limit the proliferation of rodent and human lymphocytes [15,16,17], supporting immunosuppressive functions. Consistent with these in vitro activities, rodents provided a high-fat diet, rich in ALA, have a decreased mitogen-stimulated lymphocyte proliferation and natural killer (NK) cell activity [18].

Fig. 8.1
figure 1

This is an outline of eicosanoid synthesis pathways from arachidonic acid (AA) and resolving-related mediators from α-linolenic acid (ALA) and their inflammatory and anti-inflammatory functions. The abbreviations include COX, cyclooxygenase; CYT p450 cytochrome, p450; CXC chemokine subtype, HETE, hydroxyeicosatetraenoic acid; HDHA, hydroxyldocosahexaenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HPDHA, hydroperoxydocosahexaenoic acid; HPEPE, hydroperoxyeicosapentaenoic acid; IL, interleukin, IFN, interferon; LOX, lipoxygenase; LT, leukotriene; LX, lipoxin; PG, prostaglandin; PMN, polymorphonuclear leukocytes; ROS, reactive oxygen synthetase; TNF, tumor necrosis factor, TX, thromboxane

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In vitro studies using the ω-6 PUFA, AA, have revealed an increase in inflammation, including enhanced superoxide release [13], neutrophil attachment to endothelial cells [14], and increased macrophage secretion of IL-1β [19]. Mice given diets with high ω-6 PUFA levels, in a dose-dependent manner, have an increased level of urinary leukotriene E4 (LTE-4) and prostaglandin E2 (PGE-2) following leukocyte stimulation in vivo [20]. Further, it has been reported that diets high in AA result in increased levels of angiotensinogen, IL-6, and monocyte chemoattractant protein (MCP)-1 and increased expression of the proinflammatory transcription factor, nuclear factor κβ (NFκβ) [21]. In studies with rats fed high AA diets for 8 weeks, a decrease in superoxide production by peritoneal macrophages was observed in response to phorbol esters [22] as well as an increase in TNF secretion by resident macrophages, although no effect on TNF production by inflammatory macrophages was observed [23]. Thus, the effects of the shorter ω-3 PUFA, ALA, on lymphocyte functions appear to be dependent on ALA levels and total PUFA diet content [24]. Studies with ALA and ω-6 PUFA contrast with the bioactivity of LC-ω-3 PUFA with 20 or more carbon atoms such as EPA and DHA, which are anti-inflammatory and immune augmenting [25]. The anti-inflammatory activity of diets incorporating LC-ω-3 PUFAs are due to a lower metabolism of ω-6 PUFA into inflammatory eicosanoids, cytokines, and the stimulation of reactive oxygen species (ROS) and nitric oxide synthase (NOS) mediators [26]. Clinically, dietary supplementation with EPA and DHA can decrease intestinal damage and improve gut histology in patients with inflammatory bowel disease [27], as well as decrease arthritic lesions including joint pain, number of tender and swollen joints, and duration of morning stiffness [28].

Tumor blood vessels are structurally and functionally abnormal, lacking a normal hierarchical structure composed of arterioles, capillaries, and venules [29]. Tumor endothelial cells are generally loosely connected and covered by fewer and more abnormal mural pericytes [29,30,31]. Clinically, a poor coverage of tumor blood vessels by pericytes is related to poor patient prognosis [32,33,34], and pericyte dysfunction has been associated with increased numbers of metastases [35]. Prostaglandin I2 (PGI2) is an important vascular prostanoid that provides an important balance in tumor angiogenesis [36, 37]. PGI2 and agonists of PGI2 have been suggested to reduce tumor metastasis by modifying tumor angiogenesis [38]. Thus, administration of PGI 2 analogs that affect endothelium-pericyte interaction has been shown to target angiogenesis tumor microenvironment and control neoplasia progression and growth [39]. The potential interaction between vascular prostacyclin and diet is supported by the finding of increased prostacyclin production by vessel walls in patients given diets with moderate levels of LC-ω-3 PUFAs [40]. Thus, LC-ω-3 PUFAs regulation of prostanoid synthesis by vascular endothelial cells within the tumor vascular provides another potential mechanism for LC-ω-3 PUFAs control of tumor growth.

Chronic inflammation contributes to the initiation and progression of malignancy [41]. The role of inflammation in carcinogenesis was first proposed in 1863 by Rudolf Virchow, when he noticed the presence of leukocytes in neoplastic tissues [42]. Since Virchow’s early observation linking inflammation and cancer, data supporting tumor initiation at sites of infection and chronic inflammation have been reported [43]. Indeed, approximately 25% of all cancers are associated with chronic infections and inflammation [44]. Although inflammation is an adaptive host defense against infection and is primarily a self-limiting process, inadequate resolution contributes to chronic pathologies including cancer [45, 46]. Evidence from laboratory- and population-based studies has suggested that organ-specific carcinogenesis is at least partly associated with inflammation [47,48,49,50]. Thus, the development of gastric, hepatic, gallbladder, prostate, and pancreatic tumors has been attributed to Helicobacter pylori-induced gastric inflammation, chronic hepatitis, cholecystitis, inflammatory atrophy of the prostate and chronic pancreatitis, respectively [46, 51, 52]. Patients suffering from inflammatory bowel disorders also have an increased risk of colorectal cancer [47, 53, 54], while management with anti-inflammatory drugs (COX-2 inhibitors) reduces this risk [55].

Sustained cellular injury can also induce inflammation and stimulate carcinogenesis. Inflammatory and innate immune cells are recruited to sites of infection or inflammation, such that activated myeloid cells generate ROS and reactive nitrogen species (RNS), which facilitate tumor initiation. Thus, one mechanism by which chronic inflammation supports carcinogenesis is the generation of ROS and/or RNS in inflammatory tissue and subsequent DNA damage leading to oncogene activation and/or inactivation of tumor suppressor genes. Chronic exposure to ultraviolet (UV) B radiation can induce inflammatory tissue damage [56], tumor suppressor T-cells [57], and skin cancer [56]. Mutational changes in ras and p53 have also been observed with many lipid mediators resulting in the regulation of inflammation and cancer initiation and progression [58, 59]. The activation of ras oncogene and loss-of-p53 tumor suppressor gene function have been shown to support UVB-induced mouse skin carcinogenesis [60]. ROS-induced DNA damages, including DNA strand breaks, DNA-based modifications, and DNA cross-links, result in replication errors and genomic instability contributing to tumor initiation and progression [61, 62]. NO, another reactive species, has a role in inflammation-associated carcinogenesis by direct modification of DNA and inactivation of DNA repair enzymes [63]. 8-Oxo-7,8-dihydro-20-deoxyguanosine (8-oxo-dG), which is associated with oxidative and mutagenic DNA damage [64], is produced in association with H. pylori-induced gastric [65] and TNF-α-induced pulmonary carcinogenesis [66]. Peroxynitrite, formed by a reaction between NO radical and superoxide anion, causes DNA damage by forming 8-nitroguanine (8-NG) [67, 68], another biomarker of inflammation-associated cancers [69]. Thus, oxidative and nitrosative DNA damage products are associated with inflammation-driven carcinogenesis [70]. ROS and RNS can also induce lipid peroxidation resulting in other reactive species, such as manoldialdehyde and 4-hydroxynonenal (4-HNE), which are capable of forming DNA adducts [71]. Elevated intracellular ROS (e.g., superoxide anion, H2O2, and hydroxyl radicals) and RNS (e.g., peroxynitrite, NO, and S-nitrosothiols) also induce alterations in protein functions, including a perturbation of DNA-protein cross-links and posttranslational modification critical to maintaining cellular homeostasis. For example, NO has been shown to hyperphosphorylate and inactivate retinoblastoma protein, thereby increasing human colon cancer cell proliferation [72]. Moreover, in a mouse model of colitis, the hyperphosphorylation of retinoblastoma protein (Rb) was found to be reduced in the colons of iNOS-null mice as compared to wild-type littermates, suggesting that NO is involved in Rb hyperphosphorylation [72]. In the colon tissues of patients with ulcerative colitis, a positive correlation between iNOS levels and the phosphorylation of p53 as well as the activation of p53 transcriptional activity has been identified [73]. Thus, nitrosative stress also has a role in inflammation-associated carcinogenesis by activating activator protein-1(AP-1), a representative redox-sensitive transcription factor [74], which is involved in cell transformation and proliferation [75, 76].

Further, metabolic reprogramming from glycolysis to lipid metabolism regulates myeloid cell differentiation. For instance, IL-4-induced M2 macrophages rely on fatty acid oxidation (FAO) to proliferate, which is mediated through signal transducer and activator of transcription 6 (STAT6) and peroxisome proliferator-activated receptor gamma (PPARγ)- co-activator 1β (PGC1β) [77, 78]. Indeed, the uptake of triacylglycerols followed by lipolysis is critical for M2 activation [79] and tumor-infiltrating dendritic cells (DCs) develop high levels of intracellular triglycerides [80]. This increased lipid accumulation in DCs impairs their ability to process and present antigens and results in an inhibition of the induction of antigen-specific T-cells [81, 82]. Decreasing the lipid content by inhibiting fatty acid synthesis restores DC functions and may improve the efficacy of cancer vaccines [81]. Overall, it appears that myeloid cells can use the metabolic programs that support their survival and functional demands within their microenvironment. As such, an improved understanding of these metabolic pathways may support the development of novel therapeutic targets in cancer and other chronic inflammatory diseases [83, 84].

Tumor-infiltrating myeloid derived suppressor cells (MDSCs) use FAO as their primary source of adenosine triphosphate (ATP) [85, 86], such that pharmacologic inhibition of FAO blocks the immunosuppressive functions of MDSCs, delaying tumor growth in a T-cell-dependent fashion and enhancing therapeutic responses to low-dose chemotherapy and adoptive T-cell therapy [86]. The mechanisms by which the tumor microenvironment regulates the uptake of exogenous lipids and enhances the metabolic and functional reprogramming of tumor-associated MDSCs is associated with tumor-derived GFs (granulocyte-colony stimulating factor (G-CSF) and granulocyte/macrophage-colony stimulating factor (GM-CSF)) that can upregulate lipid transport receptors in tumor-infiltrating myeloid cells and increase lipid uptake in the tumor microenvironment [87]. This is associated with an enhanced oxidative metabolism and upregulated immunosuppression. Interestingly, human tumor-infiltrating and peripheral blood (PB) MDSCs also have increased levels of lipid transport proteins, supporting the development of more immunosuppressive MDSCs in vitro. Thus, tumor-derived cytokines facilitate myeloid cell lipid uptake, accumulation, and metabolism resulting in the induction of MDSC immunosuppressive functions. As such, lipids contribute to immunosuppressive myeloid cells (M2 macrophages, dendritic cells, and polymorphonuclear leukocyte (PMN) and monocytic (M)-MDSC) in cancer and chronic inflammatory pathologies [78,79,80,81, 83,84,85,86, 88]. Following infiltration of the tumor microenvironment, myeloid cells undergo metabolic reprogramming from glycolysis to FAO that is paralleled by the upregulation of the T-cell immunosuppressive mediators arginase I and iNOS [85, 86]. As such, metabolites in the tumor microenvironment regulate the immunometabolic induction of MDSCs. Tumor-derived inflammatory GFs including G-CSF and GM-CSF upregulate the expression of lipid transport receptors that facilitate the uptake of lipids in the tumor microenvironment, including FFAs and the triacylglycerol-carrying lipoproteins, very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) [89]. The uptake, accumulation, and oxidation [86] of these lipids activate and prolong the survival of immunosuppressive MDSCs [90]. Importantly, cancer-associated MDSCs also express lipid transporters, and therefore, human peripheral blood stem cells (PBSC), cultured in lipid-rich media, develop into highly inhibitory MDSCs [91]. Consistent with other myeloid cells, substantial lipid accumulation occurs with tumor-derived MDSCs [92, 93]. MDSCs with lipid overload have greater immunosuppressive effects on CD8+ T-cells, compared to MDSCs with normal lipid content. Lipid accumulation in tumor-derived MDSCs is linked to an increase in fatty acid uptake. This observation is supported by the study of Cao et al., which demonstrated an increased expression of fatty acid transport protein 4 (FATP4) in murine tumor-derived MDSCs [93]. The lipids in the MDSCs of tumor-bearing mice and cancer patients are oxidized, potentially by the oxidative activities of ROS and myeloperoxidase (MPO) [92, 94]. Inhibition of ROS and MPO can reduce the oxidation of lipids resulting in MDSCs with decreased immunosuppressive activity [92]. Studies such as these document an increase in lipids in the tumor microenvironment that are assimilated by MDSCs supporting previous reports of increased levels of serum triglycerides, LDL-cholesterol, and VLDL-cholesterol in cancer patients [95,96,97] and patients with chronic inflammatory diseases [95,96,97,98,99,100,101,102,103]. However, the origin of lipids in the tumor microenvironment is unclear, although it appears that lipids released from adipose tissue provide energy that supports tumor growth and invasion [104,105,106]. However, the mechanisms by which MDSCs mobilize lipids to support increasing FAO remain obscure. Adipocytes liberate fatty acids for FAO from lipids stored in lipid droplets by lipolysis, which is regulated by adipose triglyceride lipase, hormone-sensitive lipase, and lysosomal acid lipase (LAL) [107]. Recent studies showed that LAL-mediated lipolysis releases fatty acids supporting FAO in IL-4-induced M2 macrophages [79] and IL-15-driven memory T-cells [108]. Furthermore, lipids act as ligands for PPARs [109] that have a key role in the regulation of FAO [110]. In line with this, PPARγ and PPARδ, which are induced by STAT6, can regulate the alternative activation of macrophages [110].

8.3 Lipid Mediators Linking Inflammation and Cancer

8.3.1 Dietary PUFA Regulation of Myeloid Cell Functions

Diets rich in ω-6 PUFAs are proinflammatory, enhancing the expansion of myeloid cells [111] and MDSCs [112]. The increase in MDSCs is observed with both in vitro cultured murine bone marrow cells and in vivo in mice fed diets enriched in ω-6 PUFAs. In the latter studies, mice were given a linseed oil-based diet containing 45% of the shorter ω-3 PUFA, ALA, or a sunflower oil diet containing 45% of the ω-6 PUFA, LA. These studies suggested that the bioactivity of PUFAs occurred through Janus kinase-signal transducer and activator of transcription (JAK-STAT3) signaling, such that a JAK inhibitor reduced the bioactivity of PUFAs on MDSCs. Based on these and other studies, it was concluded that the inflammatory activity of PUFAs may be mediated, in part, by diet [113]. Thus, dietary fat contributes to tumor-associated inflammation that occurs, in part, through AA metabolism [114]. A Western-style diet increases the risk of tumorigenesis via myeloid recruitment, infiltration of tumors, and subsequent activation of TNF-a, PGE2, NF-kβ, and Wnt inflammatory pathways [115]. AA can make up to 40% of the fatty acid composition of cancer cell membranes [116]. The anti-tumorigenic effects of ω-3 PUFAs may be mediated in part by their anti-inflammatory effects [117].

Recently, lipid accumulation in the adipose tissues of obese hosts have been shown to promote infiltrating macrophages with an M2 polarization shift, while M1 phenotype macrophages are observed in lean adipose tissue [118, 119]. Because dietary fish oil with LC-ω-3 PUFAs decreases PGE-2 production, LC-ω-3 PUFAs are considered anti-inflammatory. Such a diet also results in enhanced secretion of Th1-type cytokines and decreased major histocompatibility complex (MHC) II expression, lymphocyte proliferation, and NK cell activity. Consistent with these observations, the culture of human neutrophils with LC-ω-3 PUFAs has been reported to suppress superoxide production and phagocytosis [120]. Similarly, the incubation of murine peritoneal macrophages with EPA or DHA can inhibit MHC II expression [121]. In one study, human monocytes were cultured with EPA or DHA, resulting in a decreased frequency of human leukocyte antigens-DR or DP (HLA-DR or –DP) positive monocytes following addition of interferon gamma (IFN-γ) [122] and depressed antigen (Ag) presentation [123]. Similarly, adding fish oil to rodent diets can decrease superoxide and H2O2 secretion by macrophages [124]. Experiments comparing diets with safflower oil versus fish oil have been found to decrease peak plasma levels of the inflammatory cytokines TNF-α, IL-1β, and IL-6 following lipopolysaccharide (LPS) injection [125]. However, super-pharmacologic doses were used in this study, contrasting with the majority of rodent studies which use dietary fish oil in which EPA plus DHA comprise up to 30% of the lipid fatty acids and up to 12% of dietary energy. The conclusions from these studies have been extended using lower levels of EPA or DHA (4.4% of total FAs or 1.7% of dietary energy), documenting that these levels have anti-inflammatory activities [126].

8.3.2 T-cell Immunoregulation and PUFA

Clearly, ω-6 PUFAs are proinflammatory [127] as they are metabolized to AA and subsequently by COX-/LOX- to inflammatory lipid mediators that include PGs and LTs [128]. These AA metabolites have tumor-promoting actions such that the COX downstream metabolite PGE-2 can enhance tumor growth by inducing tolerogenic DCs and Tregs. 5-LOX metabolites include the four series LTs that can also stimulate tumor growth and progression [129]. This contrasts with LC-ω-3 fatty acids that have alternative COX/LOX activities forming metabolites with alternative bioactivities, including the three series PGEs and five series LTs. AA metabolism results in the LOX products that can stimulate the expansion and differentiation of myeloid progenitor cells [111] including MDSCs. Similarly, tolerogenic DCs contribute to T-cell regulatory functions by inhibiting their activation. In steady-state conditions, tissue-resident, immature DCs internalize, process, and present tumor Ags. These DCs, identified as DC2s, are poorly immunogenic and do not secrete proinflammatory cytokines due to the expression of low levels of costimulatory molecules. Further, DC2s secrete immunosuppressive cytokines, including IL-10 and transforming growth factor – beta (TGF-β), which are critical to the induction of T-reg cell differentiation. The secretion of indoleamine 2,3-dioxygenase (IDO) secretion by DC2s can also contribute to immune tolerance [130]. Another myeloid cell population with a similar functional profile are alternatively activated macrophages (M2s) that differentiate from monocytes by IL-4 stimulation. M2s facilitate tumor angiogenesis, support tumor progression, invasion, and metastasis, and contribute to T-cell immunosuppression by secreting IL-10, facilitating Th2 cell differentiation. This provides a positive feedback cycle for differentiation of additional M2 macrophages, all of which can express programmed death-ligand 1 (PD-L1), further contributing to activated T-cell apoptosis [131].

Similar to M2 macrophages, MDSCs can infiltrate tumors, as well as circulate in the PB of tumor patients. MDSCs can be either of monocytic, PMN or immature in origin [132, 133]. In the blood of cancer patients, MDSCs lack lineage (LIN) markers for T and B lymphocytes (CD3 and CD19) and NK cells (CD56) and thus express an LIN−HLADR−CD11b+ phenotype [134, 135] that can be further subset based on CD14 expression (monocytic), CD15 expression (PMN), or expression of CD33+CD14−CD15− (immature) cells [132, 136]. A positive correlation between the frequency of MDSCs and tumor stage has been reported for numerous tumor pathologies [133]. The function of MDSC inhibition of T-cell activation occurs via arginase, iNOS, ROS, or RNS, as well as secretion of immunosuppressive cytokines [137]. MDSCs also deplete nutrients necessary for lymphocyte function (arginine), disrupt IL-2 receptor signaling, interfere with lymphocyte trafficking, promote activation of T-regs by CD40-CD40L ligation, and suppress CD3-zeta (ζ) expression and secrete IL-10 or TGF-β [138, 139]. While few studies have examined the response of MDSCs to dietary PUFA [140], myeloid cell and myeloid progenitor cell response to dietary ω-6 PUFA as extramedullary hematopoiesis and myeloplasia has been studied [140]. Chronic inflammation has a key role in the expansion and activation of MDSCs in both tumors and various inflammatory disorders. As discussed in this chapter, chronic inflammation is a hallmark of MDSC expansion and immunosuppression. Thus, ω-6 dietary associated inflammation [140] suggests a potential role for dietary PUFA in the regulation of MDSCs. A few studies have shown that dietary PUFA can promote the expansion of MDSCs via JAK-STAT3 signaling in mice [112]. Waight et al. [141] demonstrated that G-CSF and GM-CSF increased the generation of MDSCs by downregulating the expression of interferon regulatory factor 8 (IRF-8) in myeloid progenitors via signaling via the STAT3 and STAT5 pathways. Interestingly, Yan et al. [112] demonstrated that both the ω-3 and ω-6 PUFA treatments significantly enhanced the expansion of cultured bone marrow MDSCs, PMN-MDSCs. The proliferation of T-cells decreased in a dose-dependent manner in mice given a PUFA-containing diet. Murine diets containing PUFAs increased the percentage of PMN-MDSCs in both the bone marrow and the spleen. The administration of PUFAs also stimulated the immunosuppressive properties of MDSCs isolated from mouse spleens. PUFA diets also induced the activation of JAK-STAT3 signaling and the immunosuppression of T-cells was mediated by ROS. Finally, Yan et al. [112] revealed that a PUFA-enriched diet augmented the growth of CT26 and Lewis lung carcinoma in mice. In agreement with these results, given that PUFAs activate PGE-2 production through COX-2, it is interesting to note that PGE-2 can stimulate STAT3 signaling [142]. PGE-2 is a potent inducer of MDSC functions [143] and thus might stimulate their expansion. Indeed, inhibition of COX-2 with Celebrex has been shown to slow tumor growth and inhibit MDSC expansion and numbers [144].

T-regs, a T-cell-based immunosuppressive cell, are divided into two major populations. This includes a thymic origin cell [145] and one that is induced in the PB [146] by TGF-β. Under homeostatic conditions, T-regs can limit the induction of autoimmunity, inhibit tissue destruction, and ensure the maintenance of tolerance to self-antigens [147]. In the PB of cancer patients, the frequency of T-reg cells is increased compared to normal individuals [148]. In addition, tumor-infiltrating T-regs are increased in tumors of cancer patients [149]. T-regs are phenotypically described as CD4+ T-cells that co-express forkhead box P3 (Foxp3) and CD25 [148]. T-regs can also express checkpoint inhibitory molecules, including, but not limited to PD-1, lymphocyte activating gene-3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), glucocorticoid-induced tumor necrosis factor receptor (GITR), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), which can all suppress T-cell responses [148, 150]. T-regs also indirectly suppress effector T-cells by depleting local IL-2 levels, which are needed for the proliferation and survival of effector T-cells [151]. Indirect suppression is also associated with the secretion of immunosuppressive cytokines, such as IL-10 and TGF-β [152].

Lipid mediators derived mainly from dietary PUFAs can also contribute to the control of inflammation. These mediators, which are collectively called specialized pro-resolving lipid mediators (SPMs), include the families of compounds termed “resolvins,” “lipoxins,” “maresins,” and “protectins” [11]. The resolution of an inflammatory response by SPMs is characterized by lipid mediator “class switching,” in which cells downregulate enzymes responsible for the production of proinflammatory lipids, including prostaglandins and leukotrienes, while upregulating enzymes responsible for the production of SPMs [153]. SPMs exert pro-resolving and anti-inflammatory activities without suppressing immunity [153, 154]. This contrasts with corticosteroid and nonsteroidal anti-inflammatory drug (NSAID) anti-inflammatory therapies. SPM’s therapeutic potential is due to their activity in the nanomolar to picomolar range and secretion by many cells with minimal toxicities. LC-ω-3 PUFAs, which are precursors for SPMs, can inhibit T-cell proliferation and cytokine production [155]. Murine diets supplemented with fish oil or DHA have been reported to increase CD4+ T-cell proliferation in response to a Th2 stimulus and decrease Th2 cytokine production by CD8+ T-cells [156]. T-cells also express known SPM receptors, including ALX/FPR2 [157, 158], GPR32 [158], and BLT1 [159], which support their response to SPMs. Overall, SPMs show promise in regulating adaptive immune responses. Receptors for SPMs are found on DCs, T-cells, and B cells. Furthermore, SPM therapy has shown significant activity, including augmentation of pathogen clearance, resolution of inflammation, and the development of immune memory.

8.3.3 Dynamic Anti-inflammatory Activities of ω-3 PUFAs

One of the challenges with dietary studies using PUFA regulation of inflammation is that obesity is also associated with chronic low-grade inflammation and increased levels of FFA, proinflammatory cytokines, hormones, and circulating myeloid cells [160, 161]. Adipocytes can secrete metabolites that either promote or resolve an inflammatory response [162]. Thus, adipose cell hypertrophy and hyperplasia increases oxygen consumption, which can result in hypoxia [163], activation of cellular stress and inflammation in association with proinflammatory cytokine secretion [163]. Adipocyte hyperplasia also results in myeloid infiltration of adipose tissue, notably surrounding both dead and dying adipocytes, and a phenotypic shift of adipose tissue macrophages, that can release proinflammatory cytokines that induce ROS and activate inflammatory signaling pathways [164]. Obesity can also contribute to changes in the tumor microenvironment by increasing inflammatory cell infiltration and the presence of FFAs [165]. High levels of proinflammatory adipokines have also been shown to contribute to tumor infiltration of inflammatory cells within the microenvironment [166, 167] through autocrine and paracrine activation of signaling pathways including NF-κβ [168], STAT3, and extracellular regulated kinase (ERK)1/2, all of which stimulate tumor cell proliferation and inhibit apoptosis [169].

Further, LC-ω-3 PUFAs are metabolized into anti-inflammatory, bioactive SPMs, which can reduce inflammation [170]. These observations suggest that the resolving phase of inflammation is not passive but is actively downregulated by endogenous anti-inflammatory mediators [171]. This contrasts with ω-6 PUFA metabolites, including PGD-2, LTD-4, LTC-4, and LTE-4, which are inflammatory. Although AA is a precursor to LTs, its metabolite PGE-2 can also regulate macrophage and lymphocyte functions. Thus, dietary consumption of the ω-6 PUFA LA, as the precursor of AA, is causally linked to allergic diseases and provides a potential treatment strategy using LC-ω-3 PUFAs [172].

8.4 Dietary Lipid Regulation of Murine and Human Tumor Initiation, Progression, and Metastasis

8.4.1 Dietary LC-ω-3 PUFA Controls of Murine Tumor Growth

Clinically, associations have been reported between PUFA consumption/composition and inflammation, although co-variable includes genetic susceptibility, tissue microenvironments, stress, obesity, age, caloric intake, and dietary duration. Murine models have also suggested mechanisms whereby PUFA composition can regulate tumor initiation and progression. These studies provide insight into various pathologic conditions including infections, autoimmune, inflammatory, neoplastic, and obesity conditions and a relationship with neutrophilia, splenomegaly, and multifocal, hepatic extramedullary myelopoiesis (i.e., the formation of myeloid tissue outside of the bone marrow) [173, 174]. Such inflammatory pathologies, which are associated with tumor initiation, are controlled by multiple risk factors, including hormones, obesity, diet, and age. However, following tumor initiation, inflammation is also regulated by tumor GF and chemokine secretion, as well as, additional risk factors. Thus, within the tumor microenvironment, crosstalk occurs between the immune response and inflammation, including EMM and tumor-secreted GFs.

Dietary PUFA regulation of tumor progression and metastasis has been studied in transplanted syngeneic and xenograft tumor models. In one xenograft model using MDA-MB-435 tumor cells, athymic nude mice were inoculated with tumor cells following the placing of recipients on either LA, EPA, or DHA diets. These studies revealed a significant delay in tumor growth and reduced metastases in mice fed an EPA or DHA diet, including reduced AA levels in tumor membrane phospholipids [175]. The results from one of our studies in a syngeneic tumor model, with two groups of mice receiving pair-fed isocaloric and isolipidic liquid diets documented the impact of PUFA composition on tumor growth [176]. Ten weeks following initiation of the diets, groups of mice received orthotopic injections of 4T1 mammary tumor cells. The results showed that mice consuming a LC-ω-3 PUFA diet had a significant delay in tumor initiation, slower growing tumors, and prolonged survival compared to mice given an ω-6 PUFA diet [176]. Interestingly, when mice were autopsied 35 days post-orthotopic injection, the hosts consuming the ω-6 based diets had a significantly greater number and frequency of pulmonary, hepatic, renal, cardiac, and bone marrow metastases. These results suggested that dietary PUFA composition is not only critical to tumor initiation but also modulates tumor growth and the extent of metastasis and distribution of localization metastatic sites.

As part of this study, tumors were collected 35 days after tumor injection, frozen in OCT and immunohistochemistry (IHC) performed to evaluate the frequency of proliferating cells using antibodies to KI67 (Fig. 8.2a) infiltration by macrophages, by staining with F4/80 (Fig. 8.2b) and infiltration by granulocytes and MDSCs, by staining with antibodies to neutrophil elastase (Fig. 8.2c). In every instance, the tumors in mice receiving an ω-6 based diet had a significant increase in proliferating tumor cells and infiltrating macrophages and granulocytes, as opposed to tumors from mice on an LC-ω-3 PUFA diet as described above. It is noted that there was a slight but significant difference in tumor sizes at this time, with a median tumor volume of 888.5 ± 115.2 mm3 for the tumors from the ω-6 tumor-bearing mice and 446.3 ± 52.3 mm3 in the ω-3 diets. This documents the effect of an ω-6 PUFA diet on innate inflammatory cell infiltration of 4T1 tumors and on tumor cell proliferation. Further, in murine studies of neoadjuvant therapy where treatment with EPA and DHA preceded surgery, the number of pulmonary metastases was significantly decreased compared to mice on an LA diet [177]. Similar results focused on immune-augmentation and therapeutic activities have been documented in R3230RC and MCF-7 breast adenocarcinoma tumor studies [178, 179], which included a reduced number of MDSCs [180]. In addition to a neoadjuvant activity, LC-ω-3 PUFA diets have therapeutic potential. In a tumor survival study, mice were switched from an 8% corn oil (1% ALA) diet to an 8% canola oil (10% ALA) diet, when the average primary tumor volume became 60 mm3. This ω-3 PUFA canola oil diet therapy significantly reduced tumor growth as compared to an ω-6 PUFA corn oil diet [181]. Based on these and other rodent studies, it appears that LC-ω-3 PUFA dietary intervention may be used with therapeutic intent [182].

Fig. 8.2
figure 2

Mice were fed omega-3 and omega-6 diets for 10 weeks prior to injection of 5000 4T1 cells in an abdominal mammary gland. Tumors were collected after 35 days from tumor injection, and IHC was performed for the evaluation of proliferating cells by Ki67 (a), intra-tumor macrophages by (b) F4/80, and neutrophil infiltration by staining with neutrophil elastase (c). (Images were taken at 400× magnification)

Full size image

Murine studies using LC-ω-3 PUFA and autochthonous chemically induced mammary tumors have confirmed and extended these observations using transplanted tumors. In an autochthonous 7, 12-dimethylbenz (α) anthracene (DMBA)-induced mammary tumor model, mice on a fish oil diet had a significantly reduced tumor incidence, slower tumor growth, and a reduced numbers of metastasis [183, 184]. The LC-ω-3 PUFA diet affected tumor induction and growth, which correlated with reduced AA serum levels, suppressed tumor cell proliferation, protection against DNA single-strand breaks, and an increase in tumor cell apoptosis [184,185,186]. Similarly, a comparison of tumorigenic using a tumor model with N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors and diets with differing fat composition were compared, including an SFA diet, a monounsaturated fat (MUFA) diet, an ω-6 PUFA diet, or diets with different ratios of ω-6: ω-3 PUFA. It was found that the diet incorporating a 1:1 ratio of ω-6:ω-3 PUFA could prevent mammary tumor development. Studies into causal relationships revealed that this diet group had decreased levels of COX-2 and 5-LOX transcription levels in mammary tissues and PPAR-γ levels [187]. Together, these and other studies support a role for LC-ω-3 PUFAs in regulating the metabolic inflammatory tumor microenvironment by upregulating PPAR-γ [186, 187]. Consistent with these studies, when dietary LC-ω-3 PUFA content is increased to an ω-6:ω-3 ratio of 1:14.6, as compared to a control diet of 1:0.7, a 60% decrease in tumor growth was observed [188]. Similar studies, using a therapy model with transplanted, orthotopic 4T1 mammary tumors, in which a 5% fish oil diet was initiated when the hosts had developed primary tumors that were 8–10 mm3 in diameter, resulted in significantly reduced growth and metastases that had a correlation with decreased tumor cell proliferation [189]. A similar therapy study using C3(1) Tag mice revealed that a switch to a fish oil concentrate from a corn oil diet slowed prostate tumorigenesis and progression in association with lower estradiol, testosterone, and androgen receptor levels [190]. The ability of LC-ω-3 PUFAs to downregulate inflammatory mediators and increase tumor cell apoptosis supports the importance of its regulation in the tumor microenvironment. In vivo studies have studied the effect of dietary LC-ω-3 PUFA on inflammatory cells in animal models of both LPS inflammatory disease and tumor-induced inflammation. However, the majority of murine models have used diets that were neither isocaloric nor pair-fed, raising the question of obesity versus a role for dietary composition? Since obesity itself is inflammatory, clarifying the effects of obesity-associated inflammation, as opposed to dietary regulation of inflammation, is critical to determining the regulatory activity of dietary components. Thus, using animal models, with an isocaloric, isolipidic liquid diet that allows pair feeding and controlled dietary caloric intake, is needed to assess the impact on host weight and adipose changes, as well as dissociate effects between obesity and dietary composition.

Epidemiologic studies support the role for NSAIDs, including aspirin in reducing the incidence of cancer and can prolong survival if administered postdiagnosis [114]. Initial studies focused on colorectal cancers; however, low-dose aspirin has also demonstrated antitumor activity for other tumors, including lung, breast, prostate, and metastatic cancers [191, 192]. In addition, low-dose aspirin has been found to improve survival and provide chemopreventive benefits in combination with cytotoxic therapy and/or surgical resection [193, 194]. Many of these studies are derived from patients receiving low-dose aspirin for cardio-prevention, in which 20–30% have been suggested to obtain benefit with a decrease in cancer incidence [195]. However, studies also show that neither non-aspirin NSAIDs nor acetaminophen is associated with a reduced risk of cancer or chemoprevention [196, 197]. While the known anti-inflammatory activity of aspirin offers a rational mechanism of action, the unique antitumor mechanisms of aspirin compared with other NSAIDs is confounding. It is noted that the use of low-dose aspirin in cancer patients is limited by gastrointestinal bleeding and hemorrhagic stroke [198].

Investigations into anti-inflammatory mechanisms in cancer patients have focused on the downregulation of proinflammatory mediators, including cytokines, eicosanoids, and enzymes [114]. COX-1 and COX-2 are key targets of aspirin and are involved in the biosynthesis of proinflammatory lipid autacoids, including prostaglandins. Aspirin’s anticancer activity has been suggested to be associated with the irreversible acetylation of cyclooxygenases that are overexpressed in cancer patients [199, 200]. In contrast to other NSAIDs that reversibly block COX enzymes, aspirin qualitatively alters enzymatic substrate specificity and activity of COX. A unique function of aspirin-acetylated COX is the metabolism of aspirin-triggered (AT) SPMs, including AT-lipoxin A4 (AT-LXA4) and AT-resolvins D1 (AT-RvD1) and D3 (AT-RvD3) [201,202,203]. Other NSAIDs do not trigger endogenous SPM production [204]. Thus, SPMs, such as resolvins and lipoxins, promote the resolution of inflammation by stimulating phagocytosis of cellular debris and counter-regulating proinflammatory cytokines without being immunosuppressive [204, 205].

The aspirin-acetylated COX facilitates the biosynthesis of aspirin-triggered SPMs from ω-3 PUFA substrates, including EPA and DHA [206]. While AT-resolvins exhibit potent anti-inflammatory activity characteristic of native resolvins, the AT forms resist rapid inactivation and have longer half-lives [204]. AT-SPMs are increased in patients who respond to the anti-inflammatory activity of aspirin compared with those that do not respond to aspirin [207]. AT-lipoxins also modulate tumor-associated macrophages and reduce bone cancer pain [208, 209]. Further, SPMs, such as resolvins, enhance cytotoxic cancer therapy by promoting the clearance of therapy-generated apoptotic tumor cells by macrophages [210]. AT-resolvins and AT-lipoxins have been shown to be critical for the anticancer activity of low-dose aspirin by resolving tumor-promoting inflammation in mice [211].

8.4.2 Dietary LC-ω-3 PUFAs and Improved Cancer Patient Outcomes

The tumor microenvironment includes, in addition to tumor cells, extracellular matrix, endothelial cells, stromal cells, fibroblasts, adipocytes, and critically infiltrating inflammatory cells (M2 macrophages and MDSCs) as well as suppressive and effector lymphocytes, all of which have a role in regulating tumor progression and metastasis. The infiltrating lymphocytes, particularly mature CD8 T-cells, serve as mediators of antitumor activities [212, 213]. In cancer patients, the infiltration of lymphocytes provides an independent, positive prognostic factor as assessed by IHC staining [214]. Studies into the type of infiltrating immune cells (e.g., CD3+, CD8+, and FOXP3+ T-lymphocytes) and the density or location of infiltrating T-cells also provide prognostic correlations with positive (or negative) outcomes in patients with colorectal cancer (CRC) [215,216,217,218,219,220,221,222], ovarian cancer, and breast cancer [223,224,225,226]. A meta-analysis of these clinical studies assessed the impact of tumor-infiltrating leukocytes on outcomes, including one incorporating 30 studies with 2988 patients [227]. This analysis examined associations between survival and inflammatory cell (N = 12) and T-lymphocyte subset infiltration (N = 18) studies. Pooled analyses documented that a generalized tumor inflammatory infiltrate was associated with significantly improved cancer-specific survival (CS), overall survival (OS), and disease-free survival (DFS). Stratification by cellular location and T-lymphocyte subset suggested that in the tumor microenvironment, CD3+, CD8+, and FoxP3+ cellular infiltrates were not significant prognostic markers for OS or CS. In contrast, a high frequency of infiltrating CD8+ but not CD3+ or FoxP3+ T-cells was predictive of an increased OS. Furthermore, a high frequency of tumor-infiltrating CD3+ T-cells, at the invasive tumor border, was associated with improved OS and DFS [227].

Consistent with the effects of LC-ω-3 PUFAs on tumor-infiltrating leukocytes is an inverse relationship between dietary consumption of LC-ω-3 PUFAs and the probability of developing CRC, as found in case-control studies by Murff et al. [228] and Habermann et al. [229]. However, the benefits were limited such that, in one study [228], an increased LC-ω-3 PUFA intake was associated with a reduced risk of CRCs in women. In a second trial [230], an inverse relationship was observed between low DHA intake and an increased risk of CRC in patients with genetic variants that resulted in higher proinflammatory mediators. Recently, a relationship between LC-ω-3 PUFA consumption and survival was observed in a retrospective analysis of the CALGB 89803 randomized trial of adjuvant chemotherapy for completely resected stage III CRC (n = 1264) [230]. Patients in the highest quartile of LC-ω-3 PUFA dietary intake had a significantly increased DFS compared with the patients in the lowest quartile. Notably, this relationship appeared to be highest for patients with high CRC COX-2 expression [230]. Further, clinical studies have also examined adjuvant therapy with LC-ω-3 PUFA [231] such that higher consumption of LC-ω-3FA was associated with improved OS in patients with established CRC using two independent cohort studies [230, 232].

The EMT study is the only reported randomized trial of purified LC-ω-3 PUFA treatment in patients with metastatic CRC [233]. This is a phase II double-blind, randomized, placebo-controlled trial of EPA, in the FFA form, 2 g daily before surgery in patients (n = 88) undergoing liver resection for CRC liver metastases. In the first 18 months after resection, EPA-treated individuals obtained an OS and DFS benefit compared to the placebo cohort [233]. This preliminary observation from a “window” trial of limited LC-ω-3 PUFA use, prior to metastasis surgery, resulted in a phase III randomized trial of EPA (4 g daily in the ethyl ester form) in patients undergoing liver resection for CRC liver metastasis (the EMT2 trial). In this trial, subjects are randomized to EPA or placebo at least 2 weeks before surgery and continue the drug long term, with progression-free survival (PFS) as the primary endpoint and OS as the key secondary endpoint (ClinicalTrials.gov; NCT03428477).

The possible beneficial effects of ω-3 PUFAs in CRC incidence was first suggested in 1997 in West Coast fishermen [234]. Two years later, it was pointed out that several of the known risk factors for some cancers, including colon cancer, may be reduced by dietary ω-3 PUFAs supplementation, and implementation of clinical chemoprevention trials was encouraged [235]. Based on these observations and other studies, two randomized trials of LC-ω-3 PUFA supplementation are underway that have secondary CRC endpoints. These include the ASCEND (A Study of Cardiovascular Events in Diabetes) trial (NCT00135226), which is a 2 × 2 factorial study of long-term (median 7.5 years) LC-ω-3 PUFA (840 mg EPA/DHA ethyl ester daily) and aspirin (100 mg daily) treatment for the prevention of cardiovascular and cerebrovascular events in patients with diabetes (n = 15,480). In this study, cancer outcomes were a secondary endpoint, with the ability to continue with posttrial follow-up. In the ASCEND trial which tested ω-3 supplementation (at a dose of 1 g per day) in adults with diabetes in the United Kingdom provided generally null results. In contrast, the VITamin D and OmegA-3 Trial (VITAL) study (NCT01169259), which was a 2 × 2 factorial study of the same dose and formulation of LC-ω-3 PUFA (also 840 mg EPA/DHA ethyl ester) and vitamin D3 in 25,871 US male and female participants had different outcomes. During the overall treatment period of 5.3 years, there was a statistically nonsignificant 17% reduction in cancer death, with a hazard ratio of 0.83 [236, 237]. However, the protocol planned to account for a latency period with some analyses that excluded early follow-up. In an analysis that excluded the first 2 years of follow-up, there was a reduction in cancer deaths that was statistically significant, a 25% reduction, as well as a nonsignificant 6% reduction in cancer incidence with vitamin D. A beneficial association between higher consumption of LC-ω-3FA and a lower incidence of CRC was reported to be restricted to a subset of tumors with microsatellite instability (MSI) [238]. MSI occurs in 15% of CRC patients and is caused by a loss of DNA mismatch repair (MMR) activity [239]. Consistent with the anti-inflammatory activities of LC-ω-3FA, data support the critical role of inflammation and dysregulated anti-tumor immune response in the development of MSI tumors [240]. It should be noted that immune checkpoint inhibitor therapy has been shown to be more effective for treating cancers with MSI [241, 242]. This suggests that increased dietary consumption of LC-ω-3FA after diagnosis may benefit patients with MSI tumors [243].

As supported by our transplantable tumor studies as discussed above and shown in Fig. 8.2, IHC analyses of infiltrating lymphocytes, particularly CD3+ T-lymphocytes in primary tumors, provides a biomarker that predicts improved clinical outcomes [244,245,246]. Furthermore, basic histological quantification of T-lymphocyte density, cytotoxicity, and a memory phenotype, by CD3+, CD8+, and CD45RO+ markers, respectively, demonstrated that an increase in T-lymphocyte infiltration is associated with significant improvements in DFS and OS [217, 245, 247]. In CRC, identifying the location of infiltrating cytotoxic T lymphocytes (CTLs), assessed as CD3+CD8+ T-cells within the center (CT) and invading margin (IM) of the primary tumor, predicts clinical outcomes [217]. The quantification of the density, phenotype, and location (CT or IM) of infiltrating CTL provides an immunoscore [248,249,250]. Indeed, the significance of the CD3+ cell infiltration analysis surpasses a diagnosis of tumor stage, lymph node, and metastatic invasion, sub-setting patients into five categories based on the location in the tumor (CT and IM) of CD3+ and CD8+ T-cells [251, 252].

In association with immunoregulatory properties, a patient’s lifestyle, preceding and following diagnosis and therapeutic interventions, is associated with controlling cancer initiation, progression [253], and responses to therapeutic interventions [254]. Specifically, patients who consume a high-fat diet (saturated fat, or ω-6 PUFAs) frequently exhibit neutrophilia that can facilitate tumor initiation, progression, and result in poor outcomes [255, 256]. Conversely, diets that contain a high LC-ω-3 PUFA content have been associated with decreased inflammation, lower EMM, and improved outcomes [173]. The improved clinical outcomes were initially suggested by epidemiological studies into the incidence and progression of breast cancer in American women of Japanese descent, as compared to Japanese women living in Japan. The results from one study indicated a significantly higher breast cancer incidence in American women of Japanese descent compared to Japanese women in Japan [257]. This observation is supported and extended by studies with female children from Japanese immigrants to America, but not the immigrants themselves, who had breast cancer rates similar to the general American population [258]. In the 1990s, dietary components were found to be implicated in these different incidences [259]. These correlative epidemiologic studies are supported by rodent studies, which demonstrated that LC-ω-3 PUFAs can reduce proinflammatory cytokines, inflammation, and cancer development [260].

Case-control studies have also shown an inverse relationship between dietary ω-6 and LC-ω-3 PUFAs ratio and the incidence of breast cancer, supporting their dietary importance [261]. An epidemiological study of 56,007 French women over 8 years revealed that the risk of breast cancer was unrelated to dietary PUFA consumption. Rather, a significant risk was associated with the ratio of dietary ω-6 versus LC-ω-3 PUFAs, which was inversely related to LC-ω-3 PUFA levels in women with the highest intake of ω-6 PUFAs, indicating interactions with PUFA consumption [262]. Subsequent studies revealed a decreased risk of developing breast cancer with dietary LC-ω-3 PUFA in a case-controlled, population-based study [263] that showed a reduction in all-cause mortality that was reduced 16–34% in women consuming high levels of LC-ω-3 PUFAs [264]. Indeed, during the last 20 years, data has accumulated supporting the observation that high ω-6 PUFA dietary consumption is proinflammatory, likely involving COX-2 secretion and NFκβ activation, resulting in an increased incidence of cancer and all-cause mortality. In contrast, consumption of high levels of LC-ω-3 PUFA were found to be protective against neoplasia, including a decreased incidence of cancer associated, all-cause mortality [265]. Indeed, in a meta-analysis of 11 independent prospective studies, it was observed that a decrease in the dietary ω-6: LC-ω-3 PUFA ratio significantly lowered the risk of breast cancer [266]. However, some studies have shown no association between heightened ω-6: LC-ω-3 PUFA ratios in the diet and breast cancer development.

Recent studies have investigated the underlying mechanisms of this observation and its relationship to innate and acquired immune cell infiltration of the tumor microenvironment. The regulatory activity of LC-ω-3 PUFA on macrophage functions has also been documented with the use of antagonists to G protein-coupled receptor (GPR120), which is expressed by some myeloid cell populations and acts as a PUFA receptor [267]. This is supportive of a role for LC-ω-3 PUFA mediation and anti-inflammatory effects via this receptor. However, PPAR-γ also acts as a receptor for PUFAs and the regulatory mechanisms of LC-ω-3 and ω-6 PUFA on obesity [268], postmenopausal breast cancer [269], and microenvironmental inflammation [270], suggesting a need for additional studies. Further, PUFAs contribute to the regulation of bone marrow (BM) and EMH at sites such as the spleen [271, 272] and may also expand the frequency of MDSCs [112].

Unfortunately, LC-ω-3 PUFA dietary supplements can lead to various toxicities. Despite the therapeutic benefits discussed herein, there are potential risks associated with high doses. The primary adverse effects are altered platelet function. The presence of EPA and DHA leads to the production of TX A3, which is a less potent platelet activator than TX A2. Supplementation of EPA and DHA, therefore, can affect platelet activation because of the different eicosanoids produced, resulting in an antithrombotic effect that can impact blood coagulation and wound healing [273]. The impact depends on the amount and the duration of LC-ω-3 PUFA supplementation. When given in combination with other medications, such as aspirin or warfarin LC-ω-3 PUFA interactions may exacerbate adverse effects that can occur with LC-ω-3 PUFA supplementation alone [274,275,276,277,278]. LC-ω-3 PUFAs supplementation is contraindicated during antiplatelet and anticoagulant treatment because of the additive effect on bleeding times when administered together [279].

8.5 Future Trends or Directions

Dietary PUFA consumption may not only affect inflammation and the incidence and progression of neoplasia, but may also support responses to therapeutic interventions in cancer patients via the regulation of inflammation. In general, increased dietary ω-6 PUFA consumption is associated with a heightened risk of cancer that is suggested to be due to a proinflammatory tumor microenvironment. In contrast, an LC-ω-3 PUFA diet has potential protective effect to suppress ω-6 PUFA-associated inflammation. Nutritional recommendations are that individuals should decrease dietary ω-6 PUFA intake and increase LC-ω-3 PUFA consumption with an intake of at least 500 mg/day of LC-ω-3 PUFA [280]. PPAR-γ and GPR120 agonists also have potential for use as chemopreventive drugs, although their use may, perhaps, be better targeted toward either high-risk individuals or as part of therapeutic interventions. Both of these are receptors for LC-ω-3 PUFA [281, 282]. Regardless, we need to further study both pharmacophores and dietary regulation of PUFAs as protective and therapeutic strategies for cancer and their association with leukocyte infiltration of tumors.

Research on the role of LC-ω-3 PUFAs and SPMs on inflammation and cancer is increasing and suggests a positive role for use as an adjuvant in cancer therapy. Increased efforts are needed using high-quality randomized control trials to establish their mechanisms of action, the optimal timing for supplementation, dosage, product source, method of extraction, preparation, and quantification to obtain efficacy, which will optimize their clinical use for cancer prevention and therapy. These future trials should address these questions as well as the impact on the tumor microenvironment, specifically infiltrating cellular subtypes. We also stress the need for translational/preclinical studies that utilize isocaloric and isolipidic pair-fed diets to segregate the regulation of immunity and inflammation by obesity versus dietary PUFA. Further, care must be taken to differentiate between activity on tumor growth as opposed to metastasis, as these biologic parameters are interrelated such that, larger tumors typically have more metastases. In our experience, ω-6 PUFA diets impact not only primary tumor growth but also the extent and critically sites of metastasis, all of which are typically unstudied but highly relevant since metastasis is frequently the ultimate cause of patient mortality.