Modulation of the tumor microenvironment by natural agents: implications for cancer prevention and therapy

Abstract

The development of cancer is not just the growth and proliferation of a single transformed cell, but its surrounding environment also coevolves with it. Indeed, successful cancer progression depends on the ability of the tumor cells to develop a supportive tumor microenvironment consisting of various types of stromal cells. The interactions between the tumor and stromal cells are bidirectional and mediated through a variety of growth factors, cytokines, metabolites, and other biomolecules secreted by these cells. Tumor-stromal crosstalk creates optimal conditions for the tumor growth, metastasis, evasion of immune surveillance, and therapy resistance, and its targeting is being explored for clinical management of cancer. Natural agents from plants and marine life have been at the forefront of traditional medicine. Numerous epidemiological studies have reported the health benefits imparted on the consumption of certain fruits, vegetables, and their derived products. Indeed, a significant majority of anti-cancer drugs in clinical use are either naturally occurring compounds or their derivatives. In this review, we describe fundamental cellular and non-cellular components of the tumor microenvironment and discuss the significance of natural compounds in their targeting. Existing literature provides hope that novel prevention and therapeutic approaches will emerge from ongoing scientific efforts leading to the reduced tumor burden and improve clinical outcomes in cancer patients.

1. Introduction

Cancer is one of the significant causes of death, affecting millions of people globally. About 1,806,590 new cancer diagnoses and nearly 606,520 cancer-related deaths are expected in 2020 in the United States alone [1]. To address this significant clinical problem, we have made extensive efforts on several fronts, including the basic biology of cancer. Over the past several years, we have witnessed significant progress in our understanding of cancer genetics and relevant signaling pathways affecting tumor phenotypes. We have also learned that tumors are not just a mass of transformed cells but are comprised of other non-malignant cells as well, ranging from fibroblasts, endothelial cells, and immune cells to even microbes [2–8]. As tumor cells progress to become highly malignant, their microenvironment also co-evolves. Indeed, tumor cells continue to corrupt the non-malignant stromal cells as they grow and use them to support their growth, metastasis, therapy resistance, and to evade the immune defense [9–15]. Tumor-stromal interactions are driven by growth factors, cytokines, metabolites, and other biomolecules secreted by the cells within the tumor microenvironment (TME). These interactions are often bidirectional and essential for optimal tumor growth, and thus provide targeting opportunities for effective clinical management.

Natural products are generated in abundance by plants and marine organisms and have been utilized for centuries in traditional medicines [16, 17]. Most of the bioactive natural products are secondary metabolites that aid in survival by often serving as mediators of innate defense mechanisms [18]. These compounds gained significant attention after different epidemiological studies reported the health benefits of the consumption of certain fruits, vegetables, and their derived products [19–22]. It also renewed the interest in ancient literature from Middle Eastern and South Asian countries that reports healing powers of natural compounds and associated practices. Over the years, bioactive constituents from natural sources have been isolated and experimentally validated for their anti-viral, anti-bacterial, anti-inflammatory, anti-fungal, and anti-cancer properties [21, 23–25]. Indeed, about forty-seven percent of all anti-cancer drugs in clinical use are derived from the naturally-occurring compounds [26]. These natural compounds exhibit significant diversity in structure, function, and biosynthesis, and therefore it is hard to classify them under strict categories. However, they are broadly put under four structural classes that include terpenoids and steroids, alkaloids, fatty acid derivatives and polyketides, and phenylpropanoids [27]. While the majority of the earlier studies investigated the effect of natural compounds on the tumor cells [28, 29], the focus later expanded to include TME components as well. These efforts resulted in the demonstration of TME-modulating effects of natural agents in multiple cancer types, as reviewed in [30–35]. Recent years have witnessed significantly increased efforts towards understanding the roles of TME in cancer pathogenesis after the reported success of immunotherapy in some cancer types [36–38]. Driven with this impetus, more and more natural compounds are now being evaluated for their TME-modulating properties in parallel investigations [39–41]. In light of the new data emerging from these efforts, it has become imperative that we revisit natural compounds to understand their broader significance in the modulation of TME as well as associated mechanisms. This review provides a comprehensive summary of the current literature on TME, especially highlighting its fundamental cellular and acellular components, and discusses their functions as well as the factors regulating the behavior and functional properties of the stromal cells. After that, we discuss the natural products of plant and marine origin that target TME via different mechanisms to confer their anti-cancer activity. The expectation is that the presented discussion would encourage cancer biologists and cancer chemoprevention and therapy experts to engage in cross-disciplinary collaborations to allow future translational advancements.

2. The cellular component of the tumor microenvironment

Principal non-malignant cells within the TME are fibroblasts, endothelial cells, and immune cells (macrophages,myeloid-derived suppressor cells (MDSCs), and lymphocytes. It has also recently been recognized that microbial cells are present within the TME and play an active role in pathobiology and therapy-resistance (Figure 1). Below we discuss in more detail about these cells, their functions, and their characteristics.

Figure 1: Components of the tumor microenvironment.

The tumor microenvironment is a complex network of stromal cells (fibroblasts, endothelial cells, lymphocytes, macrophages, and myeloid-derived suppressor cells), and other acellular entities surrounding the tumor cells. Tumor and stromal cells actively interact and influence each other to support tumor growth by promoting desmoplasia, angiogenesis, and immune suppression.

2.1. Cancer-associated fibroblasts

Fibroblasts are the quiescent mesenchymal cells that are present within the extracellular matrix (ECM) and play important roles in tissue repair. Upon injury, these cells are activated and differentiate into myofibroblasts through paracrine signaling [42, 43]. Their excessive proliferation can result in organ fibrosis and may have unwanted consequences [44, 45]. Within the context of the tumor, these myofibroblasts are referred to as cancer-associated fibroblasts (CAFs) [3, 6]. In some cases, CAFs can also be derived from other precursor cells, such as endothelial cells, smooth muscle cells, and myoepithelial cells [2, 46]. Differentiation into the CAF phenotype is facilitated by factors derived from tumor cells or other cells within the TME, such as IL-6, transforming growth factor-beta (TGF-β), sonic hedgehog (SHH), and platelet-derived growth factor (PDGF) [12, 47, 48]. Microenvironmental stress (extracellular pH, hypoxia, radiation, and chemotherapy) can further aggravate this process likely to protect and support the tumor cells [49–51]. Recently, the role of tumor-derived exosomes in CAFs generation has also been reported [52].

Morphologically, CAFs have a large spindle shape and express a cytoskeletal protein, α-smooth muscle actin (α-SMA). Some other markers, such as fibroblast activation protein (FAP), platelet-derived growth factor receptor-β, and S100 calcium-binding protein A4, are also used to identify CAFs [53, 54]. None of these markers are expressed exclusively by the CAFs because of their heterogeneous nature within tumor tissues [54, 55]. CAFs abundantly produce collagens, fibronectins, and hyaluronan, and these proteins, along with other ECM components, form a highly dense fibrotic covering around the cancer cells [55, 56]. Studies have suggested that CAFs can regulate tumor progression and therapy resistance by inducing EMT, metabolic reprogramming, activation of survival pathways, modulating anti-oxidant system, or stemness in tumor cells [38, 55, 57]. CAFs also help in the recruitment of monocytes to the TME and promote their differentiation into immunosuppressive MDSCs [58].

2.2. Tumor-associated endothelial cells

To meet their high demand for oxygen and nutrients, actively dividing cancer cells recruit and activate the endothelial cells into the TME to facilitate the formation of new blood vessels from pre-existing blood capillaries [4]. Endothelial cells form the inner lining of the blood and lymphatic vessels [59, 60]. Single-cell RNA sequencing data has suggested that tumor-associated endothelial cells (TECs) are distinct from the normal ECs [61]. Normal ECs form uniform monolayer with few cytoplasmic projections, whereas TECs are irregular in shape and size with long fragile, or thin cytoplasmic projections extending across the vessel lumen [4]. TECs are CD31⁺ and CD105⁺ and appear in a mosaic pattern within the tumor tissues [62]. TME- derived cytokines and growth factors such as interleukin-8 (IL-8), vascular endothelial growth factor A (VEGF-A), and basic fibroblast growth factor (bFGF) aberrantly activate the TECs, which makes resultant blood vessels fragile, leaky and of irregular shape [59, 63]. Tumor cell-derived VEGFA, IL-10, and prostaglandin E2 also induce Fas ligand (FasL) expression in TECs, which causes FasL- mediated apoptosis of CD8⁺T cells. However, regulatory T cells (Tregs) are not affected much since they express a high level of anti-apoptotic protein, cellular FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein [64].

2.3. Tumor-associated macrophages

Macrophages are part of the innate immune system and play active and diverse roles due to their highly flexible nature. Based on the environmental stimuli, they can differentiate either into M1 (classical-activated macrophages, anti-tumor) or M2 (alternative-activated macrophages, pro-tumor) phenotypes; a process often referred as macrophage polarization [65, 66]. Innate or adaptive immune cell-derived interferon-γ (IFN-γ) or bacterial cell-derived lipopolysaccharide (LPS) induces M1 polarization, while Th2-type cytokines (IL-4, IL-10, and IL-13) polarize macrophages into M2 phenotype [67]. In the context of cancer, M2 macrophages are often referred to as tumor-associated macrophages (TAMs), and they are the significant component of tumor-infiltrating immune cells within the TME [67, 68]. Unlike M1 macrophages that rely mostly on glycolytic metabolism and produce pro-inflammatory signals [69, 70], M2 macrophages display oxidative phosphorylation and secrete anti-inflammatory cytokines [71, 72]. Moreover, it is possible to convert M2 macrophages into the M1 subtype by altering mitochondrial respiration [70, 72]. Both M1 and M2 macrophages express different markers. M1 subtype expresses inducible nitric oxide synthase (iNOS), suppressor of cytokine signaling 3 (SOCS3), CD68, CD80, CD86, IL-1, IL-16, IL-12, IL-23, TNF-α, IFN-γ, and CXCL-10 whereas M2 macrophages express arginase 1 (Arg-1), CD163, CD200R, IL-10 and TGF-β [73].

2.4. Lymphocytes

Lymphocytes are white blood cells that include T cells, B cells, and natural killer (NK) cells. T-and B- cells are the part of adaptive immune responses, whereas NK cells are part of the innate arm of the immune system [74, 75]. T lymphocytes play a critical role in cancer immunity and are divided into four subtypes, CD4⁺ T helper cell, CD8⁺ cytotoxic T cell, memory T cells, and natural killer T cells (NKT) [76–78]. CD4⁺ T cells help in B cell maturation and can also activate macrophages and cytotoxic T cells [79]. They become functionally active after recognizing the antigen presented by major histocompatibility complex (MHC) class II molecules [79, 80]. On the other hand, CD8⁺ T cells respond through MHC class I molecules and involve in the killing of infected or malignant tumor cells [80, 81]. Tumor cells and TME factors desensitize tumor-infiltrating lymphocytes (TILs), and they, in turn, build a tumor supportive immune microenvironment [5, 82]. The direct anti-tumor activity of CD4⁺ T cells is less explored because a majority of the solid tumor cells do not express MHCII [79, 80]. However, these cells confer anti-tumor activity indirectly either by secreting type I cytokines or activating cytotoxic CD8⁺ T cells [79, 83]. More importantly, a subset of tumor supportive CD4⁺ T cells, Tregsarefound within the TME. The normal biological function of Treg cells is to distinguish between self and non-self-antigens that helps in the prevention of autoimmunity. Treg cells express the IL-2 receptor (CD25) and Forkhead Box P3 (FoxP3) transcription factor, which is a master regulator of Tregs functions [84]. Treg cells cause immune suppression in several ways. Notable among these are; i) secretion of immunosuppressive cytokines, IL-10, TGF- β and IL-35; ii) consumption of IL-2 from cellular milieu; and iii) cytotoxic T lymphocyte-associated protein-4 (CTLA-4)-mediated suppression of antigen-presenting cells or by induction of T cell exhaustion [84]. Recently, it was demonstrated that Treg cells indirectly induce the M2 population of macrophages in TME by inhibiting IFN-γ secretion [85]. B cells infiltrated into the TME induce memory CD4⁺ T cell formation, promote proliferation and survival of activated CD8⁺ T cells, and can also directly induce apoptosis in tumor cells by secreting granzyme B [86]. Indeed, a high density of tumor-infiltrating B cell subtypes correlates with increased survival [87]. NK cells are innate immune cells, express CD56, and are involved in boosting the host immune defense against pathogens [88]. Overall, these cells mediate anti-tumor and anti-viral immune response by executing cytotoxic action through perforin and granzyme [89].

2.5. Myeloid-derived suppressor cells

Myeloid cells are derived from bone marrow and can be either mononuclear (MN) comprising of monocytes, macrophages, and dendritic cells, or polymorphonuclear (PMN) consisting of neutrophils, basophils, eosinophils and mast cells [90, 91]. During chronic inflammation, pro-inflammatory cytokines activate myeloid progenitor cells to proliferate and differentiate into MDSCs [92]. Therefore, the accumulation of MDSCs is usually seen when a pathological condition exists, and they are mostly absent in a healthy body [91]. MDSCs induce immune suppression and help cancer progression in several ways. These cells either enhance Treg cell recruitment into the TME or differentiate into TAMs [92–94]. MDSCs also promote the differentiation of fibroblasts into CAFs. Monocytic-MDSCs (M-MDSCs) are recruited to TME by CCL-5 and CSF-1, while the TME infiltration of PMN-MDSCs is stimulated by a different set of cytokines including chemokine (C-X-C motif) ligand 12 (CXCL-12), CXCL-1, CXCL5, CXCL-6 and CXCL-8 [90, 91, 95]. Interestingly, cytokine CCL-2 has the ability to attract both M-MDSCs and PMN-MDSCs [96]. Relatively larger extent of M-MDSCs is present in TME as compared to PMN-MDSCs. In solid tumors, hypoxia is shown to induce the differentiation of MMDSCs into TAMs in order to sustain immune suppression [94]. Studies havealso correlated the increased chance of cancer-associated death in patients who had greater abundance of circulating MDSCs [97–99].

2.6. Microbiome

A microbiome is the community of microorganisms or microbes (bacteria, fungi, protozoa, and viruses) residing in a specific niche such as the human gut. The microbiome can modulate multiple cellular events such as host physiology, cellular metabolism, and immune function. Disturbance in the host-microbiota is often associated with pathological conditions such as inflammatory bowel diseases and cancer [7]. Microbiota regulates mucosal immunity and tissue remodeling, which collectively modulate TME to promote tumor growth [8, 100]. Microbes are associated with approximately 10–20% of human malignancies [101]. The effect of the intratumoral microbiome in cancer progression is not yet well explored. However, a study suggests that Fusobacterium nucleatum-derived Fap2 protein interacts with the immune receptor, T-cell immunoglobulin, and ITIM domains (TIGIT), and inhibits the activity of TIGIT-expressing NK and T cells [102].

3. Non-cellular components of the tumor microenvironment

In addition to cellular components, TME is also highly abundant in the secreted material consisting of mostly matrix proteins, cytokines, growth factors, nucleic acid, and metabolites [103]. This acellular material is derived from various cell types and supports the growth of the tumor by enabling bi-directional tumor-stromal interactions [103, 104]. ECM makes the scaffold of tissues and organs and is a collection of biomolecules secreted by different cell types and provides structural and biochemical support to the microenvironment. Specialized ECM (basement membrane) is enriched with fibronectin, collagen, and laminin, whereas the other ECM (interstitial matrix) mostly contains proteoglycans, glycoproteins, and collagen. Importantly, a proteoglycan such as hyaluronic acid (HA) abundantly present in ECM of some solid cancers has gained significant interest as a drug target due to its direct and indirect roles in tumor growth and chemoresistance [105, 106]. Other ECM proteins such as fibulins, secreted protein acidic and rich in cysteine (SPARC), osteopontin, periostin, tenascin(s), and thrombospondins also have an active involvement in tumor pathobiology besides serving as a scaffold for ECM structural proteins [107–109].

4. Role of the tumor microenvironment in cancer pathobiology and therapy-resistance

Several lines of evidence suggest that TME plays a critical role in tumor growth, metastatic progression, and failure of the therapeutic intervention [13, 110, 111]. Tumor cells develop a bidirectional functional association with the surrounding stromal cells during their malignant progression[10, 14, 112]. For instance, pancreatic tumor cells utilize CXCL12, a chemokine secreted by the stromal cells, to not only promote tumor growth and metastasis but also resist the cytotoxic effects of chemotherapy [10, 111, 113]. Interestingly, activation of the CXCL12/CXCR4 pathway in pancreatic tumor cells also promotes synthesis and secretion of sonic hedgehog (SHH) that preferentially acts on the stellate cells to promote desmoplasia [10]. Desmoplastic TME plays a diverse and significant role in pancreatic cancer pathobiology and chemoresistance [9, 114–116]. In a study on breast cancer, CAFs were shown to induce proliferation of CD44⁺CD24⁻ cancer stem-like cells via the activation of CXCL12/CXCR4 signaling [117]. Tumor cells also utilize CAFs to fulfill their energy requirements in a limited or nutrient deficient TME. CAFs-derived alanine provides an alternate carbon source to the pancreatic tumor cells under a nutrient-deprived environment [118]. In another report, CAFs-derived IL-6 is shown to induce epithelial to mesenchymal transition (EMT) and confers resistance to cisplatin in non-small cell lung carcinoma [119]. CAFs exposed to chemotherapy have significantly increased the release of exosomes that enhance the expression of Snail in the recipient tumor cells and promote gemcitabine resistance [11]. Exosome-mediated transfer of miR-221 from stromal cells to breast cancer cells triggers the generation of hormonal therapy-resistant metastatic breast cancer [120]. Interestingly, it is shown that gemcitabine is preferentially taken and metabolized by CAFs, limiting its availability to the tumor cells [121]. Moreover, CAFs also secrete various pro-inflammatory molecules (IL-6, CCL2, and TGF-β), which help in the recruitment of immunosuppressive cells [122, 123]. In another study, CXCL12 secreted from FAP⁺-CAFs was shown to induce CTLA-4- and PD-L1-mediated immunosuppression in pancreatic cancer [124].

High accumulation of TAMs and other immune-suppressive cells in the TME has been suggested to induce aggressive cancer progression and therapy-resistance. CD163⁺ TAMs induce immune suppression, and their depletion promotes robust infiltration of cytotoxic T cells into the TME, leading to the inhibition of melanoma growth [125]. In multiple myeloma patients, high CD163⁺TAMs in TME correlate with worse clinical parameters [126]. In breast cancer, TAMs enhance aerobic glycolysis in tumor cells by transferring hypoxia-inducible factor-1alpha (HIF-1α) stabilizing lncRNA (HISLA) through extracellular vesicles [127]. In clinical settings, HISLA expression associated with inadequate drug response and reduced survival of breast cancer patients [127]. Likewise, pancreatic tumors exhibit increased infiltration of TAMs and low presence of cytotoxic T cells in their TME [128, 129]. TAMs induce the expression of cytidine deaminase, a gemcitabine catabolizing enzyme, in pancreatic tumor cells leading to decreased chemotherapeutic efficacy [130]. Pancreatic tumor educated-TAMs also produce different pyrimidine species, of which deoxycytidine competes with gemcitabine for uptake and metabolism and compromises the therapeutic outcome [131]. In hepatocellular carcinoma (HC), EMT hotpots are the sites of high TAMs infiltration, and HC cells co-cultured with TAMs exhibit induced expression of EMT related genes [132]. Itis also reported that TAMs induce the expression of SPARC, which is a crucial mediator for tumor cell growth, migration, and chemoresistance [133].

The role of TAMs in angiogenesis has also been elucidated. The depletion of TAMs results in a significant reduction in vessel density [134]. TAMs also promote lymphangiogenesis, which is critical for the dissemination of tumor cells to lymph nodes and metastasis via the VEGF-C/VEGFR-3 signaling axis [135]. TAMs secrete various MMPs and interleukins, VEGF, PDGF, and TGF-β in TME that facilitate the vascularization in the tumor tissues [136]. In some cases, NK cells are shown to secrete tumor supportive cytokines and activate pro-angiogenic response [137]. TME of glioblastoma is heavily infiltrated with MDSCs, which activate B cell-induced immune suppression by blocking CD8⁺ T-cell activation [138]. MDSCs in breast TME promote metastasis by supporting EMT of tumor cells [139]. In osteosarcoma, the majority of the MDSCs are CXCR4 positive, and activation of this signaling axis promotes their survival and hamper with the programmed death ligand-1(PDL-1) targeted immunotherapy [140]. PDL-1 expressing tumor cells interact with its receptor, programmed death-1 (PD-1) present on the T cells leading to their desensitization and a suppressed host immune response. Emerging studies also provide compelling evidence for a significant role of TME microbiome in cancer pathogenesis and therapeutic outcome. The intra-tumoral burden of F.nucleatum correlates with poor response to neoadjuvant chemotherapy in esophageal squamous cell carcinoma patients [141]. Moreover, the comparative analysis of the tumor microbiome associated with the short- and long-term survival of pancreatic cancer patients suggested that the intra-tumoral signature of microorganisms can be used as a prognostic marker [142].

Non-cellular components of the TME also play important roles in cancer progression, aggressiveness, and chemoresistance. ECM stiffness upregulates integrin signaling and promotes tumor cell survival and proliferation [104]. The high amount of osteopontin in the TME of triple-negative breast cancer patients serves as a prognostic marker and promotes growth and metastasis [143, 144]. Hyaluronic acid, abundantly present in TME of various cancers, is a receptor for CD44, and their interaction leads to the activation of several cancer-promoting signaling pathways and upregulation of various non-coding RNA species (miR-10b/miR-302/miR-21 and lncRNAs) [106, 145]. Pancreatic cancer stroma is highly reactive and contains a large amount of HA, which creates extensive interstitial fluid pressures leading to the collapse of vasculature and poor outcome of chemotherapy [146]. Indeed, enzymatic targeting of pancreatic tumors with recombinant hyaluronidase, which degrades HA, is shown to provide therapeutic enhancement by decreasing metastasis and increasing survival [146].

5. Natural anti-cancer agents and their TME targeting properties

Natural products have been widely explored for their multi-targeted mechanisms of actions on the tumor cells and the TME (Table 1). Several lines of evidence from laboratory and preclinical studies demonstrate the anti-tumor potential of natural agents, of which some have also been evaluated in clinical trials (Table 2). The following section discusses some of the most promising plant- and marine-based natural agents that confer anti-cancer properties through their targeting of not only the tumor cells but also the TME (Figure 2).

Table1:

List of plant- and marine-based natural compounds and their TME targeting potential

Natural Agent (chemical category)	Source	Effect on TME	References
Terpenoids
Zerumbone (sesquiterpene)	Zingiber zerumbet Sm. (Ginger)	Downregulation of CD1d Inhibition of angiogenesis via targeting VEGF/VEGFR Inhibition in the colonization of enterotoxigenic Bacteroides fragilis	[252, 253, 255]
Curcumin (curcuminoid)	Curcuma longa (Turmeric plant)	Modulation of immunomodulatory cytokines Modulation of the ECM Disruption of tumor-fibroblast interaction Polarization towards M1 macrophage phenotype Decreased Tregs cells Increased central memory T cell	[221–225, 328, 332–333]
Alkaloids
Berberine (benzylisoquinoline alkaloids)	Berberis plants	Increased M1 macrophage polarization Active in a hypoxic microenvironment Disruption of tumor-fibroblast interaction Decreased Tregs cells Increased NK cell activity	[230, 231, 233, 334–336]
Trabectedin	Ecteinascidia turbinate (Marine ascidian)	Activation of NK cells Inhibition of macrophage polarization Targeting of PD-L1 positive tumor cells and immune cells Induction of CD8+ T cells infiltration	[291,292, 296, 337]
Phenylpropanoids
Honokiol (lignan)	Magnolia species	Activation of immunomodulatory cytokines Inhibition of angiogenesis Inhibition of immunosuppressive Tregs Stimulation of T-lymphocytes Disruption of tumor-fibroblast interaction	[157–160]
Hydroxytyrosol (catechol)	Oleaeuropaea (Olive plant)	Inhibits remodeling of ECM and angiogenesis Modulation of oxidative stress Active in a hypoxic microenvironment Modulation of immunomodulatory cytokines Disruption of tumor-stromal interaction	[167, 168, 343–345]
Resveratrol (stilbenoid)	Pine trees, peanut plants, grapevines, and vaccinium shrubs	Disables immunosuppressive Tregs Active in a hypoxic microenvironment Inhibition of cancer-associated fibroblasts Modulation of oxidative stress Inhibition of M2 polarization of macrophages	[199–203]
Epigallocatechin-3-gallate (EGCG; catechin)	Camellia sine sis (Tea)	Inhibition of metabolic circuitry in fibroblasts Decreased M2 macrophage infiltration Inhibition of tumor angiogenesis Improvement of immunotherapy response via downregulation of PD-L1	[240, 241, 350, 351]
Genistein (isoflavone)	Genistatinctoria	Inhibition of tumor angiogenesis Modulation of the ECM Inhibition of fibroblast growth Disruption of macrophage-tumor cell interaction	[261–263, 266]
Naringenin (flavones)	Grapefruit, bergamot, sour orange, tomatoes, coca and others	Decreased accumulation of ECM AbrogatesTGF-β mediated immunosuppression Inhibitionof VEGF-mediated angiogenesis	[279–281]
Onionin A (flavonoids and phenols)	Allium cepa (Onion)	Inhibition of macophage polarization Inhibition of the MDSCs activity Inhibitionof PDL-1 expression	[268, 270–272]
Others *
Plitidepsin (cyclodepsipeptide)	Aplidiumalbica ns (Mediterranean tunicate)	Inhibition of angiogenesis Activation of immunomodulatory cytokines Induce CD8+T cell infiltraion	[302, 304, 306]
Fucoidan (sulfated polysaccharides )	Brown algae and brown seaweed	Inhibition of M2 macrophage activity Activation of macrophage-induced tumor cell killing Activation of immunomodulatory cytokines	[317, 352, 353]

^*Nonribosomal polypeptides and modified polysaccharides.

Table 2:

List of compounds alone or as a co-treatment with other compounds undergo clinical trials

Compound name	Type of cancer	Clinical trial phase	Current status	Clinicaltrials.gov Identifier
Hydroxytyrosol	Breast cancer	Phase 2 and 3	Active, recruiting	NCT02068092
Resveratrol	Colon cancer Liver cancer Colorectal cancer	Phase 1 Phase 1 and 2 Phase 1	Completed Withdrawn Completed	NCT00256334 NCT02261844 NCT00433576
Curcumin	Prostate cancer Breast cancer Colorectal cancer	Phase 3 Phase 1 Phase 1	Active Recruiting Unkown	NCT03769766 NCT03980509 NCT00973869
Curcumin + Avastin/FOLFIRI	Colorectal cancer	Phase 2	Active, not recruiting	NCT02439385
Curcumin + 5- Fluorouracil	Metastatic colon cancer	Early phase 1	Active, not recruiting	NCT02724202
Berberine Hydrochloride	Colorectal Adenomas	Phase 2	Recruiting	NCT03281096
	Colorectal cancer patients with ulcerative colitis remission	Phase 1	Active, not recruiting	NCT02365480
Berberine + Gefitinib	Lung adenocarcinoma and EGFR mutation	Phase 2	Not yet recruiting	NCT03486496
EGCG	Colorectal cancer	Early phase 1	Recruiting	NCT02891538
	Patients with Lung cancer receiving radical radiotherapy	Phase 2	Recruiting	NCT02577393
Trabectedin (ET 743)	Prostate cancer Pancreatic cancer	Phase 2 Phase 2	Completed Completed	NCT00147212 NCT01339754
Trabectedin+Bevacizumab	Ovarian Epithelial cancer recurrent	Phase 2	Completed	NCT01735071
Plitidepsin + Sorafenib	Advanced solid tumors	Phase 1	Completed	NCT00788099
Plitidepsin + Gemcitabine	Lymphomas
Plitidepsin + Dexamethasone	Relapsed/Refractory Multiple Myeloma	Phase 3	Completed	NCT01102426
Oligo Fucoidan	Advanced Hepatocellular Carcinoma	Phase 2	Active	NCT04066660
Genistein + FOLFOX or FOLFOX- Avastin	Colon cancer Rectal cancer Colorectal cancer	Phase 1 and 2	Completed	NCT01985763
Genistein + Gemcitabine	Breast cancer	Phase 2	Completed	NCT00244933
Genistein	Prostate cancer	Phase 2	Recruiting	NCT02766478
Genistein + Decitabine	Non-Small Cell lung cancer	Phase 1 and Phase 2	Completed	NCT01628471

Figure 2: Targeting of the tumor and stromal cells by natural agents.

Plant- or marine-derived agents can either directly impact the tumor or stromal cells or confer their anti-tumor properties by interfering with the tumor supportive tumor-stromal interactions or both. CAFs:cancer-associated fibroblast cells, NK cells: natural killer cells, TECs: tumor endothelial cells, Treg cells: regulatory T cells, MDSCs: monocytes derived suppressor cells, M1/M2 MØ: M1/M2 macrophages.^#CAFs are shown to have diverse or sometimes opposing functions. While the majority of the data suggest their tumor-supporting roles, there are indications that they could also oppose tumor growth. Solid arrows indicate direct effect, and broken arrows show likely interactions through unknown mechanisms.

5.1. Plant-based products

Several plant-derived molecules possess a broad spectrum of pharmacological properties. The principal components recognized to impart the health benefits are the secondary metabolites. Plants have an almost limitless ability to synthesize secondary metabolites, which mainly serve as a defense mechanism against predation. Some of these that have been shown to alter TME and have undergone extensive preclinical evaluation are discussed below:

5.1.1. Honokiol

Honokiol is a small lignan with two phenolic rings. It is isolated from various parts of the plants of Magnolia species and has been used in traditional medicine to treat anxiety, stroke, and gastrointestinal conditions [147]. The extensive attention to honokiol’s anti-cancer property stems from its desirable spectrum of bioavailability and high potency in preclinical studies [148]. We have demonstrated that honokiol induces cell cycle arrest and apoptosis, and sensitizes the pancreatic cancer cells to chemotherapy by targeting the NF-κB activation [149]. Honokiol enhances the therapeutic efficacy of other anti-cancer drugs as well both in vitro and in vivo [149–151]. Honokiol induces autophagy in KRAS mutant cancer cells through the modulation of the AMPK-mTOR signaling pathway [152]. It inhibits the growth and aggressiveness of breast cancer cells in vitro and in vivo through the activation of the AMPK/LKB axis [153, 154]. An earlier study reports the modulation of TME upon exogenous expression of KRAS in mouse ovarian cancer cells [155]. Enhanced levels of immunomodulatory cytokines (IL-6 and TNF-α) and neutrophils were found in the ascetic fluid of mice injected with KRAS-expressing mouse ovarian cancer cells [155]. Another preclinical study suggested that c-Rel, a subunit of NF-κB, controls the activity of Tregs, which are vital contributors in immunosuppressive TME. In another study, the role of mTOR signaling in modulating immune cell response by regulation of differentiation/activation of TAMs, T cells, and antigen-presenting cells has also been demonstrated [156]. Thus, targeting mTOR signaling by honokiol could be useful in activating the immune response. Honokiol also induces cholangiocarcinoma cell apoptosis and increases the release of damage-associated molecular patterns (DAMPs). These DAMPs have the potential to stimulate T-lymphocyte proliferation and type I cytokine production suggesting yet another mechanism for immune modulation by honokiol [157]. In an orthotopic mouse model of pancreatic cancer, we demonstrated that honokiol inhibited tumor growth and metastasis by targeting the crosstalk of tumor and stromal cells [151]. Pancreatic tumor cells treated with honokiol showed reduced synthesis and secretion of sonic hedgehog (SHH) as well as decreased expression of CXCR4 in the tumor cells, which was associated with decreased desmoplastic reaction in the TME [151]. In other studies, honokiol and its derivative (H2P) are shown to alter the TME by inhibiting angiogenesis [158–161]. These findings indicate that honokiol is an important modulator of TME and should be explored further in various combination therapies to improve the therapeutic outcome.

5.1.2. Hydroxytyrosol

Olive oil, a major constituent of the Mediterranean diet, is considered one of the healthiest dietary supplements with multiple proven health benefits [162]. The major components identified to impart these health benefits potentially are the phenolic alcohols, particularly the 3,4-dihydroxyphenyl ethanol, also referred to as hydroxytyrosol (HT). HT is found in red wines and may also be produced endogenously as well, through dopamine metabolism [163, 164]. Multiple preclinical studies have demonstrated the anticancer properties of HT in various cancers including prostate [165], breast [166–168], pancreas [169], liver [170, 171], leukemia [172, 173], and colon [174–176]. HT possesses significant reactive oxygen species (ROS) scavenging ability, which decreases oxidative DNA damage in mammary cells [177]. In contrast, HT was shown to increase the ROS levels in human colon and prostate cancer cells and inhibited their growth [178]. ROS acts as a double-edged sword. At low levels, it promotes cancer pathogenesis, but at higher levels, it induces cell death by causing DNA damage or other cellular mechanisms [179–181]. All tumors exhibit a variable degree of hypoxia and the anti-oxidant action of HTis particularly effective under the hypoxic TME [182]. Furthermore, it is shown that the TME-dependent action of HT involves differences in mRNA translation by a yet undefined mechanism. Thus, HT not only affects the TME, but its response is modulated by different TME conditions as well. Studies from our group and others have demonstrated that HT inhibits Akt phosphorylation, leading to suppression of NF-κB activation [165, 170]. Anti-cancer activity of HT against prostate tumor cells has also been reported via inhibition of other cancer-relevant signaling pathways, including STAT3 and androgen receptor [165, 183–186]. It is significant as published data also demonstrate that olive oil components could inhibit pro-angiogenic and pro-inflammatory signaling in prostate cancer by modulating the IL-6/STAT3 axis and downregulating the expression of growth factors and cytokines (CXCL8, VEGF, CXCL12) [182]. Tumor-derived CXCL8 augments CCL2 and CXCL12 secretion by stromal cells, which in turn promote the growth and invasion of prostate cancer cells [187]. It is shown that HT interferes with the tumor-stromal interaction by inhibiting CCL5 expression in fibroblasts [188]. Further, the role of CXCL8, CCL2, CCL5, and CXCL12 in immune modulation is also reported in several cancers [189]. Thus, HT can potentially abrogate tumor cell-TME crosstalk to exert its diverse anti-tumor activity.

5.1.3. Resveratrol

Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a phytoalexin isolated from the grapevine, peanuts, and pines, exhibits chemopreventive and therapeutic properties against several cancers [190, 191]. A preclinical study also demonstrated that resveratrol could reduce high-fat diet-induced obesity, improve insulin sensitivity, and lower glucose levels in blood [192]. Multiple mechanisms are proposed for the anti-tumor activities of resveratrol including (i) acting as an anti-oxidant/pro-oxidant and anti-mutagen, (ii) inducing activation of phase II drug-metabolizing enzymes, (iii) mediating anti-inflammatory effects, (iv) inhibiting cyclooxygenase (COX) and hydroperoxidase functions, and (iv) inducing tumor cell differentiation [24, 193–196]. With regard to its effect on TME, resveratrol is shown to counter the upregulation of carbonic anhydrase through the modulation of metastasis-associated lung adenocarcinoma transcript 1expression [197]. CA regulates the intracellular pH in cancer cells under hypoxia or during intense glycolytic dependence to maintain it at physiological levels [197]. Resveratrol also imparts anti-cancer properties through copper-mediated inter-nucleosomal DNA fragmentation at low pH that mimics stringent cancer cell growth conditions [198]. Resveratrol could suppress hypoxia-driven ROS-induced pancreatic cancer progression by inhibiting SHH upregulation, thus supporting its potential role in the interference of the tumor-stromal crosstalk [199]. In addition, resveratrol is shown to induce the activation of transient receptor potential ankyrin 1 in prostate CAFs leading to an increase in the intracellular calcium and secretion of HGF and VEGF [200]. Resveratrol also alters the “senescence-associated secretory phenotype” of senescent stromal fibroblasts associated with melanoma by reducing expression of TGF-β, CXCR4, and others [201]. In another study, resveratrol inhibited the proliferation and aggressiveness of human breast cancer cells treated with CAF-CM through downregulation of several growth- and EMT- promoting genes and signaling pathways [202]. Resveratrol treatment of spleen cells isolated from the tumor-bearing mice exhibited a dramatic decrease in the number of CD4⁺CD25⁺ cells, along with a significant reduction in the ratio of CD4⁺CD25⁺ to CD4⁺ cells. In addition, the FoxP3⁺ cells within the CD4⁺CD25⁺ cell population also decreased in numbers [203]. Treatment of a breast cancer preclinical model with resveratrol analog, HS-1793, significantly inhibited the growth of implanted tumor cells with a substantial increase in IFN-γ-secreting cells in the splenocytes and decreased CD206⁺ macrophage infiltration in the TME [204]. Resveratrol also reduced lung cancer growth by inhibiting macrophage infiltration and its polarization into M2 like phenotype [205]. Thus, resveratrol controls tumor growth through its direct effect on the tumor cells as well as by targeting the pro-tumorigenic TME.

5.1.4. Curcumin

Curcumin (diferuloylmethane) is a polyphenolic compound isolated from Curcuma longa (the turmeric plant). Turmeric powder has been widely used in Ayurvedic therapies for its potent anti-oxidant, anti-inflammatory, anti-viral, anti-bacterial, and anti-fungal activities [23, 206, 207]. Studies suggest that the hydroxyl groups in curcumin are essential for its anti-oxidant activity, while methoxy groups are required for anti-inflammatory and anti-proliferative activities [208]. Curcumin has also been used as a lead molecule to generate semi-synthetic derivatives exhibiting improved anti-cancer efficacy and desired pharmacological properties [209, 210]. In colorectal cancer, curcumin administration caused the apoptosis of tumor cells in a p53-mediated manner and also improved the general health of the patients [211]. On the contrary, in a lymphoma cell line, curcumin-induced the growth arrest via downregulation of p53, c-Myc, and early growth response 1 [212]. This context-dependent action could be cell-specific or attributed to TP53 mutational status. Studies from switchable mouse models of Myc-induced β-cell and skin malignancies suggest that Myc regulates tumorigenesis by impacting the tumor cell as well as by remodeling the tumor stroma and angiogenesis [213, 214]. Activation of Myc in β-cell tumors induces the secretion of chemoattractant cytokines and promotes the infiltration of inflammatory immune cells and mast cells to the islet-associated stroma. Furthermore, Myc-induced β-cells fail to expand in mice lacking mast cell function, thus suggesting an essential role for tumor-stromal crosstalk [215]. Treatment of pancreatic cancer cells with curcumin also decreases the secretion of inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-8, due to the repression of NF-κB [216]. Curcumin also inhibits the expression of matrix metalloproteinases, which are responsible for ECM degradation to facilitate cellular movement and promote tumor angiogenesis [217]. The anti-inflammatory property of curcumin is also attributed to its ability to modulate the expression of COX-2 within the TME cells [218, 219]. Recently, single-cell transcriptome analysis of primary prostate tumor and lung metastases from an orthotopic mouse model was performed to delineate the role of tumor-stromal interaction in tumorigenesis. It was demonstrated that tumor-derived COX-2 product, prostaglandin E2, induced upregulation of prolactin in lung stromal cells, and in return, prolactin promoted proliferative signaling in tumor cells [220].

It has also been demonstrated that the anti-tumor activity of curcumin in metastatic breast cancer involves the infiltration of macrophages at the tumor site, predominantly of the M1 phenotype [221]. Similarly, in glioblastoma, curcumin treatment led to an increase in macrophage polarization towards the Arginase-1^low, iNOS^high M1-type tumoricidal microglia with high activation of microglial NF-κB and STAT1 [222]. Curcumin also improved anti-tumor immunity in lung cancer patients by decreasing Tregs in TME with a concomitant increase in Th1 cells [223]. It was determined ex vivo that potential conversion of Tregs to Th1 cells by curcumin resulted from the repression of FoxP3 and an increase in IFN-γ [223]. Curcumin also increased the accumulation and function of CD8⁺ T cells in the TME by blocking multiple immunosuppressors [224]. In another study, curcumin treatment of tumor-bearing mice prevented the overall loss of T cells with an expansion of the central memory T cell/effector memory T cell populations. Curcumin treatment also inhibited the suppressive activity of Tregs cells by downregulating the production of TGF-β and IL-10 [225].

5.1.5. Berberine

Berberine is a bioactive isoquinoline alkaloid found in many herbal substances. It is a common part of the Indian and Chinese traditional medicine and possesses multiple desirable pharmacological properties [226, 227]. Despite low plasma bioavailability of berberine, its metabolic derivatives are capable of exerting similar biological activities [228]. Berberine’s chemopreventive properties have been reported in colon cancer, where it inhibits the growth of intestinal polyp in animals as well as in patients with familial adenomatous polyposis [229]. Chemopreventive efficacy of berberine is also attributed to its ability to regulate macrophage polarization, as evidenced in the Apc (min/+) mouse model. Treatment of berberine in these mice led to a decrease in the number and size of polyps with a concomitant decrease in the levels of F4/80, mannose receptor (MR), and COX-2 and increase in the level of iNOS [229]. Berberine has been shown to promote chemotherapeutic efficacy in hypoxic breast cancer cells by inducing the downregulation of AMPK and HIF-1α [230]. AMPK is activated in hypoxic TME and maintains energy homeostasis [220]. In a study, berberine was predicted to bind Lys395 and Tyr394/Phe484 of Smo, a downstream effector of hedgehog signaling, through hydrogen bonding and π- π interactions, respectively [231]. In a similar approach, candidates for immunotherapy potentiation were tested for their ability to repress IFNγ-induced indoleamine 2,3-dioxygenase (IDO) promoter activity. IDO regulates the activation of Tregs cells in the TME and IDO⁺plasmacytoid dendritic cells (pDCs) from mouse tumor-draining lymph nodes. It activates resting CD4⁺CD25⁺Foxp3⁺ Tregs and upregulates PD-L1 and PD-L2 expression on DCs[232]. Twenty-five new berberine derivatives tested in silico and structure-activity assays identified a subgroup to have potential anti-cancer activity. These compounds could decrease IDO expression and enhanced NK cell-mediated specific lysis of A549 cells [233].

5.1.6. Epigallocatechin-3-gallate

Next to water, tea is the most served beverage around the globe. Catechins are the bioactive constituents of tea and considered to be one of the most active molecules for cancer chemoprevention and therapy [21]. Among these, epigallocatechin-3-gallate (EGCG) is the most potent apoptosis-inducer in cancer cells. EGCG has both anti-oxidant and pro-oxidant properties and is capable of inducing cell cycle arrest, apoptosis, and autophagic cell death in multiple cancer cell lines [234–237]. EGCG inhibits the proliferation and aggressiveness of thyroid carcinoma cells in vitro and in a mouse xenograft model by downregulating proteins of the EGFR and RAS signaling pathways [238]. Another mechanism of anti-cancer actions of EGCG is their ability to alter the expression of long, non-coding RNAs and mRNAs [239]. In a study with the endometrial cancer xenograft model, EGCG-treatment reduced VEGFA expression through downregulation of the HIF-1α protein. Moreover, in the host stromal cells, EGCG induced a reduction in CXCL12 expression and release, which, in turn, led to a decrease in macrophage infiltration and polarization [240]. miR-16 was found to be abundantly present in exosomes from EGCG-treated cancer cells, which reversed the polarization of TAMs to the M1 phenotype by inhibiting IKKα [241]. In another study, EGCG-treatment improved the immunotherapeutic response in a leukemic xenograft mouse model [242]. Furthermore, EGCG treatment increased the percentage of CD3⁺ T-cells, CD19 B-cells, and Mac-3 macrophages. In a more direct effect on immunotherapy response, EGCG was shown to inhibit the expression of PD-L1 in non-small cell lung cancer cells [243]. These findings suggest that EGCG holds significant potential as a combination therapy drug to improve the clinical outcome.

5.1.7. Zerumbone

Zerumbone, a sesquiterpene isolated from the rhizomes of Zingiber zerumbet, has been shown to possess immunomodulatory and anti-cancer properties [244]. It inhibits the inflammatory response by several means, such as by suppression of MAPK and NF-κB signaling, and reduced ROS production [245–247]. Zerumbone reduces TPA-induced ROS generation in leukemic and Chinese hamster ovary cells and induces the killing of tumor cells. Simultaneously, it also inhibits the expression of pro-inflammatory molecules (COX-2, TNF-α, and iNOS) in LPS- and IFN-γ- induced RAW macrophages [248]. Studies have shown that zerumbonecan block Toll-Like Receptors (TLRs)/MyD88-dependent NF-κB activation leading to the downregulation of pro-inflammatory response in LPS-induced macrophages [249, 250]. Zerumbone inhibits ovarian tumor cell growth and reduces the secretion of IL-6 [251]. Zerumbone also downregulates the expression of CD1d by blocking the antigen presentation pathway, and treatment with anti-CD1d antibody augments the zerumbone-mediated killing of breast cancer cells [252]. An antiangiogenic role of zerumbone has also been reported in liver cancer, where it abrogates the expression of VEGF/VEGFR and matrix metalloproteinase-9 [253]. Similarly, another study demonstrates zerumbone-mediated inhibition of angiogenesis due to reduced expression and secretion of VEGF and IL-8 resulting from NF-Kb downregulation [254]. Recently, it was found that the oral administration of zerumbone in enterotoxigenic Bacteroides fragilis (ETBF)-colonized mice reduced colon carcinogenesis by decreasing ETBF-colonization and intestinal inflammation [255].

5.1.8. Genistein

Genistein is an isoflavone present in soybeans and soy food and confers immunomodulatory role in various pathological conditions [256, 257]. It induces an anti-tumor immune response (NKT cell activation) in ovarian cancer by reducing VEGF-mediated upregulation of ganglioside [258]. In glioblastoma cells, genistein induces the upregulation of C/EBP homologous protein that impedes C/EBPβ interaction with IL-6 promoter leading to the downregulation of IL-6 expression and release tumor growth reduction [259]. Further, genistein can inhibit the release of urokinase-type plasminogen activator (uPA), which is a promoter of ECM remodeling and angiogenesis. As a result, its intraperitoneal administration in mice implanted with melanoma or mammary tumor cells reduces angiogenesis and tumor cell migration [260]. Genistein also influences the expression of multiple matrix metalloproteinases (MMPs) in fibroblast and T-cells suggesting its broader functional impact [261, 262]. Besides, genistein inhibits the expression of multiple essential genes associated with cell cycle, DNA replication, and growth in fibroblasts [263]. Treatment of transgenic mouse prostate adenocarcinoma mice with genistein decreased osteopontin expression in TAMs and prevented the development of advanced-stage prostate cancer [264]. Pancreatic tumor-bearing rats treated with genistein also exhibited a decrease in TAMs and reduced blood vessel density [265]. Genistein also abrogates M2 polarization of TAMs and decreased accumulation of IL-10, IL-8, and nitric oxide in the conditioned media of the macrophages and ovarian cancer cells co-culture with [266]. Another study demonstrated that genistein derivative, GEN-27, decreased LPS-induced secretion of IL-6 and IL-1β from THP-1 cells, which, in turn, reduce the proliferation of colon cancer cells [267].

5.1.9. Onionin A

Onionin A, a sulfur-containing compound isolated from Allium cepa (onion), has been used in the prevention of various chronic diseases [268, 269]. Oral administration of aqueous extract of onion boosts the immune response in the rat by increasing the immune cell count, especially white blood cell and CD4⁺T cells [270]. In other studies, onionin A is shown to inhibit macrophage polarization into the M2 phenotype by reducing the expression of CD163 as well as reduced the immunosuppressive functions of MDSCs by enhancing T cell activity [268, 271]. Onionin A also blocks the macrophage-induced ovarian cancer cell proliferation by inhibiting M2 polarization of human monocyte-derived macrophages (HMDMs) and reduces LPS-induced PDL-1 expression in HMDMs [272].

5.1.10. Naringenin

Naringenin is a flavonoid found in various citric fruits such as lemons, oranges, grapefruits, and tangerines [273]. It has emerged as a potent chemopreventive and therapeutic agent due to its anti-inflammatory, anti-cancer, and neuroprotective properties. Like other phytochemicals, it has an anti-oxidant property and can activate anti-oxidant signaling pathways [274, 275]. Naringenin ameliorated experimental autoimmune encephalomyelitis in mice by blocking effector T cell proliferation and secretion of IL-6, IFN-γ, and IL-17 [276]. Naringenin also inhibited the production of pro-inflammatory mediators such as TLR4, iNOS, COX-2, and NADPH oxidase-2 in LPS-treated murine macrophages and protected the mice from endotoxemia [277]. Naringenin prevented breast cancer metastasis by reducing the secretion of TGF-β1 from tumor cells and inhibits TGF-β1-mediated immunosuppression in tumor-bearing mice via increased T cell activation [278]. A similar study demonstrated that naringenin inhibited lung metastases and increased the survival of tumor-bearing mice by attenuating immunosuppressive TME through the downregulation of TGF-β1 and Tregs [279]. Naringenin can also act as an antifibrogenic phytochemical and reduce the secretion of ECM proteins through downregulation of TGF-β1/Smad3 signaling axis in the rat hepatic stellate cells [280]. Moreover, naringenin could inhibit VEGF-mediated angiogenesis by blocking intracellular calcium signaling in the endothelial cells [281]. In another study, naringenin inhibited angiogenesis by upregulating SOCS-3 and inhibiting IL-6/STAT3 signaling in vascular endothelial cells [282].

5.2. Marine-based products

Like the plant world, the biological and chemical diversity of marine life is also immense, and therefore, it also serves as an excellent source for the discovery of anti-cancer agents [283, 284]. In recent years, there has been an increase in the number of anti-cancer compounds from marine life being tested in preclinical studies and clinical trials [285, 286]. This brief section is intended to showcase some of these marine natural products that target the TME as a mechanism of their anti-cancer ability.

5.2.1. Trabectedin

Trabectedin was first isolated from the marine ascidian, Ecteinascidia turbinate, and can now be prepared synthetically. It is the first marine-derived anti-cancer drug currently being used in advanced soft-tissue sarcoma (STS) after the failure of anthracyclines and ifosfamide in STS patients [287, 288]. It is also used to treat patients with relapsed, platinum-sensitiveovarian cancer [289]. The most widely studied mechanisms of its anti-cancer property include the inhibition of active transcription by direct blocking of the transcribing RNA polymerase II. It can also act by causing the displacement of the transcription factors mediated through the structural changes in the DNA that hinder the recognition of transcription factor- specific DNA consensus sequence [290]. Trabectedin has targets the key members of the TME. A recent study showed that trabectedin-treated multiple myeloma cells had upregulation of NK activating receptor ligands leading to the activation of the NK cells [291]. In a study utilizing in vitro assays and four tumor models, trabectedin caused selective depletion of mononuclear phagocytes as well as splenic and tumor-infiltrated macrophages with a simultaneous decrease in angiogenesis [292]. Moreover, sub-cytotoxic concentrations of trabectedin significantly inhibited macrophage differentiation in vitro and reduced the production of inflammatory mediators, IL-6, and CCL2 [292]. Similar to solid tumors, leukemic cells also enhance the frequency of immunosuppressive cells to create a favorable microenvironment [293–295]. Trabectedin conferred cytotoxicity to human primary leukemic cells by inhibiting the myeloid and lymphoid immunosuppressive microenvironment mainly through the upregulation of the TRAIL/TNF pathway. Trabectedin also targeted the PD-L1⁺ CLL cells, PD-L1⁺ monocytes/macrophages, and PD-1⁺T cells [296]. Similar to this observation, trabectedin induced the infiltration of CD8⁺T cells in the osteosarcoma site, and its co-treatment with α-PD-1 mAb led to a stronger anti-tumor immune response [297].

5.2.2. Plitidepsin

Plitidepsin is a cyclodepsipeptide that was first isolated from the Mediterranean tunicate Aplidiumalbicans. It is currently marketed by PharmaMar (Madrid, Spain) under the trade name Aplidin. Plitidepsin demonstrates intense anti-cancer activity against multiple types of human cancer cells in vitro and in preclinical mouse models [298–301]. It inhibits proliferation by inducing cell cycle arrest and mediate apoptosis through Rac1/JNK activation [298]. JNK knockout (Kras; JNK1^−/−) mice develop low weight pancreatic tumors, and tumor-induced JNK activation in stroma reduces infiltration of effector T cells (CD8⁺) cells in the TME through downregulation of CCL20 [302]. Plitidepsin targets the eukaryotic elongation factor 1A2, which is frequently overexpressed in multiple myeloma cells [303]. Additionally, plitidepsin activates p38 and c-Jun signaling- mediated Fas/CD95 translocation to lipid rafts, ultimately causing the caspase activation [299–301]. The therapeutic activity of plitidepsin in chronic lymphocytic leukemia (CLL) is mediated through a direct effect on the leukemic cells as well as an indirect effect on monocyte-derived nurse-like cells [304]. The nurse-like cells protect the CLL cells from undergoing apoptosis by increasing CXCL12-mediated ERK activation [305]. Additionally, plitidepsin inhibits angiogenic signaling by reducing the expression of VEGF and VEGFR [306–308].

5.2.3. Fucoidan

Fucoidan is a natural sulfated polysaccharide that exists in the cell wall matrix of various brown seaweed. It has multiple biological properties such as anti-viral, anti-arthritic, and immunomodulatory potency, and its anti-cancer property has gained much attention in recent years. Fucoidan polymers induce cell cycle arrest and apoptosis in cancer cell lines [309–313]. They also induce the loss of mitochondrial membrane potential, release of cytochrome c, and increase ROS generation and ER stress, leading to DNA damage-induced apoptosis [309–311, 314]. Additionally, oligo-fucoidan has been shown to inhibit the expression of DNA methyltransferases, which leads to the upregulation of p21, a cell cycle inhibitor [315]. Fucoidan also enhances the expression of filamin A (FLNA)-derived circular RNA, which leads to the alteration of multiple genes associated with cell cycle, proliferation, and survival [313]. Similarly, fucoidan has been demonstrated to elevate the expression of miRNA29b that inhibits EMT, and lncRNA-Saf and lncRNA-p21 that target apoptosis and cell cycle pathways in hepatocellular carcinoma cells [316]. Treatment of M2 macrophages with fucoidan resulted in the downregulation of chemokines, CCL22, which mediated the growth and aggressiveness of cancer cells through its interaction with the CCR4 [317]. Elevated expression of CCL2 is associated with poor prognosis in multiple cancers [318–320]. In another study, RAW macrophages stimulated with fucoidan had an increased anti-cancer potential against S-180 cells due to the ability of fucoidan to induce nitric oxide production through upregulation of nitric oxide synthase (NOS) [317]. Fucoidan-mediated decrease of M2 macrophages in the TME is synergistic with the p53 wild type status of the cancer cells [321].

6. Conclusion and future perspective

Clinical management of cancer has been challenging due to the complex genetics and biology of cancer cells. Therefore, significant efforts have shifted towards the cancer microenvironment, which is relatively less diverse and yet plays essential roles in successful cancer progression and its resistance to therapies. In recent years, reactivation of the immune system has met remarkable success for some cancer types and has opened new avenues for cancer management. The studies briefly discussed in the above sections highlight the benefits of natural agents in cancer chemoprevention and therapeutic intervention either as a stand-alone treatment or through their enhancement of the efficacy of currently employed clinical therapies. There is substantial literature to suggest that natural agents target not only the transformed malignant cells but also effectively modulate the tumor-supportive microenvironment. It is, in most cases, achieved by blocking the bi-directional tumor-stromal crosstalk and by empowering the body’s immune system. Clearly, the potential for their future clinical exploitation is immense in terms of the development of novel preventive and therapeutic approaches against cancer. It is, however, important to mention that most of the currently available information discussed here has been generated either in vitro, ex vivo or in preclinical mouse models. There is still a lot to be achieved before we could successfully translate this knowledge from the lab to the bedside.

A major limitation with most natural agents is their low aqueous solubility leading to poor absorption and delivery to the tumor cells. Also, most compounds get quickly metabolized in the systemic circulation [322]. Also, although rare, some natural agents can pose toxicity concerns at pharmacological doses along with instances leading to the development of secondary diseases. For example, chronic consumption of a 2% caffeic acid-containing diet in rats led to the development of forestomach squamous cell papilloma with toxic lesions seen in the kidneys [323]. Similarly, high consumption of green tea catechins (1%) enhanced tumor development in rat colon [324]. Some dietary flavonoids also exhibit structural similarity to thyroid hormones and can interfere with hormone biosynthesis [325]. Epidemiological data also caution that the consumption of flavonoids by pregnant mothers could increase the risk of developing acute myeloid leukemia (MLL⁺) in the offspring [326]. To overcome these limitations, multiple approaches are being employed. One of them is to develop novel semi-synthetic derivatives of natural compounds to enhance their bioavailability and retention. In other cases, novel strategies are being developed to achieve their tumor-targeted, protected delivery using nanosystems. It is also being suggested that the use of natural compounds should also be tailored based on the molecular characteristics of the tumor. Of course, this requires a more in-depth understanding of their mechanisms of action. Despite these hurdles, discoveries are continuously being made to identify novel bioactive natural compounds and to highlight their molecular targets and underlying mechanisms. Furthermore, several natural agents and/or their derivatives have entered and/or continue to enter in clinical trials as new drugs or for therapeutic enhancement of existing drugs in combination therapiesIt is hoped that we will witness novel therapies and prevention approaches emerging from the scientific efforts being made with natural agents in not very distant future.