Oncogenic nature of cancer, cancer-associated cells and immune cells at TME

The process of transformation of cells acquiring cancerous nature is favored with specific conditions at TME including acidic pH, hypoxia and elevated ROS. Its impact on diverse cell population such as cancer/tumor cells, tumor specific adipocytes (TSAs, energy supply), tumor specific fibroblasts (TSFs), tumor specific endothelial cells (TSECs, nutritional supply), tumor specific pericytes (TSPs, remodeling at TME) enables them to participate in tumorigenesis [10]. TSAs act as tumor supporter through the recruitment and differentiation of immune cells with protumor fate under the influence of mediator molecules like [IL-8/VEGF/TGF-β/TNF-α/hepatocyte growth factor (HGF)/plasminogen activator inhibitor-1/monocyte chemoattractant protein-1 (MCP1)] [11]. Similarly, TSECs were associated with tumor evasion due to dysfunctional surface expression of TRAIL/Fas ligand (FasL)/PD-L1/PD-L2, which might also have deleterious consequences relating to antitumor responses such as reduced infiltrating immune cells within TME [10, 12, 13]. TSPs in synchronization with TSECs promote PD-L1 expressions that will correspond to its protumor nature. TSFs are known to have reciprocal relation with cancer cells, various secretions from cancer cells like growth factors: platelet derived growth factor, fibroblast growth factor (PDGF, FGF), IL-1, cancer vesicles or exosomes interact with TSFs and introduce differential changes in it. TSFs further allow prolonged survival of tumor cells along with oncogenic invasion via specific matrix metalloproteinase [MMPs (MMP-2/MMP-9)], oncogenic proliferation [stromal cell-derived factor-1 (SDF-1)/FGF/IL-6/TGF-β/osteopontin] and oncogenic angiogenesis (VEGF/FGF2/FGF7) etc. [10, 14].

Resident immune cells at TME include protumor cohort and antitumor cohort, both performing distinctive functional roles [15, 16]. Resident tumor associated neutrophils (TANs) at TME are represented as antitumor neutrophils (TAN1) and protumor neutrophils (TAN2). Presence of TAN1 type neutrophils instruct antitumor immune cells such as cytotoxic T lymphocytes cells (T cells) to eliminate tumor through cell death initiation and ROS generation eventually leading to tumor regression. Also, TAN1 via antibody dependent cellular cytotoxicity (ADCC) initiate their accumulation at tumor site for antitumor response. TAN2 type neutrophils are involved in immunosuppression and they also restrain functional response of antitumor CD8⁺ T cells [16, 17]. Resident tumor associated macrophages (TAMs) at TME are classified as tumor suppressive (TAM1) and tumor supportive (TAM2) macrophages. They undergo differentiation to acquire a regulatory function by various pro/anti-inflammatory cytokines, chemokines, growth factors, ROS, hypoxia etc. Immunosuppression is regulated by targeting programmed cell death 1 (PD-1)/PD-L1 immune checkpoint signaling pathway that can be critical for tumor regression [16, 18, 19]. Similarly, active myeloid derived suppressor cells (MDSCs) amplify the immunosuppressive expression of TAMs with the release of arginase (ARG1)/inducible nitric oxide synthase (iNOS)/ROS, like protumor factors. They also overexpress indoleamine 2, 3-dioxygenase (IDO) thereby targeting antitumor potential of active natural killer cells (NK cells), T cells, B lymphocytes cells (B cells). MDSCs stimulate CD4⁺ T cell tolerance in an MHC dependent manner. Correlation in MDSCs and T regulatory cells (Tregs) is such that MDSCs stimulate Tregs for their functional response and this encourages Tregs to enhance the differentiation of MDSCs by releasing TGF-β [16, 20]. Tregs are cancer promoting in direct and indirect ways with the release of various chemokines [C-C motif chemokine ligands (CCLs)] and cytokines (TGF-β/IL-10/IL-35). Indirectly, they induce tolerance in dendritic cells (DCs) by removing peptide MHC complexes, therefore blocking their antigen presentation and processing to T cells and directly by increasing levels of IL-10, TGF-β and CD25 where TGF-β further inhibits functional response for NK cells. Further, Tregs promote various inhibitory signal mediators such as PD-1, lymphocyte-activation gene 3 (LAG-3), and transmembrane immunoglobulin and mucin domain 3 (TIM-3). These mediators are further involved in lysis of antitumor expression of T cells along with other antigen presenting cells (APCs) at TME [16, 21].

The inappropriate DCs stimulation at TME encourages tolerogenic DCs (tDCs) to represent the tumor antigen, which will further ensure tolerant response generation at T cells [16, 22, 23]. Recruitment of tumor infiltrating lymphocytes (TILs) at TME confronts tumor growth with substantial support from other antitumor immune cells CD3⁺/CD4⁺/CD8⁺ TILs along with NK cells, but multiple targeting of antitumor immune cells blocks the cytotoxic/chemokine response from them, responsible for immunosuppressive conditions at TME. Cytotoxic actions of CD8⁺ TILs against growing tumor are mediated through cell death pathways involving dysregulated receptor ligand interaction (death receptors, FasL and TRAIL) or by release of perforin and granzyme B. Tumor cells show shielded response against killer cytotoxic T lymphocytes (CTLs) by downregulated expression of antigen recognition, its processing and presenting ability. CTLs also undergo regulatory changes where they represent upregulated expression of checkpoint receptors like PD-1, cytotoxic T lymphocyte associated antigen 4 (CTLA-4), LAG-3, and TIM-3, making them more vulnerable to inhibitory signals responsible for CTLs undergoing anergy. Role of T helper type 1 (Th1) cells is to assist CTLs killer response by releasing IL-2 or interferon-γ (IFN-γ), but CD4⁺ Th2 cells might encourage tumor growth by obstructing Th1 mediated antitumor immunity [16, 23]. Similarly, NK cells are also killer immune cells and tumor cells manage to flee from NK cells surveillance at TME with the help of mediators such as TGF-β, IFN-γ, signal transducer and activator of transcription 3 (STAT3) and hypoxia like factors by downregulating NK cells specific activating expression of CD16, natural killer group 2D (NKG2D), and DNAX accessory molecule-1 (DNAM-1) [24] (Table 1).

Molecular signaling pathways and its regulation at TME

Principle management of cancer related adversity necessitates identification of pathways that are involved in suppressive or resistant immune response at TME [25, 26]. Various cancer studies have reported the involvement of β-catenin in immune suppressive activities at TME [27]. In vitro and in vivo studies in melanoma mice models, in an induced or effector phase response reveal the involvement of CTLs and DCs like immune cells for effective antitumor immune response. But overexpressed levels of β-catenin obstruct the production of IFN-γ from CTLs. This indicates that partial regulation from IL-10 introduces more immune suppression and resistance in residing immune cells at TME [27]. Melanoma derived Wnt5a also promote IDO stimulated immunotolerance in DCs [28]. Constitutively active STAT3 and dominant negative STAT3 signaling represents elevated expression of proinflammatory chemokines such as CCL5 and C-X-C motif chemokine ligand 10 (CXCL10), indicating their involvement in immune-resistance at TME [29]. Role of phosphoinositide-3-kinase/phosphatase and tensin homolog/protein kinase B/mammalian target of rapamycin (PI3K/PTEN/AKT/mTOR) signaling pathway disturbs antitumor immune regulation by downmodulating the functional immune cells activity. Studies from triple negative breast cancer (TNBC) [30], human lung squamous cell carcinoma and gliomas [31] showed inverse relation between active tumor suppressor (PTEN) and PD-L1 expression. Their association with PI3K pathway in infiltrating T cell at TME is well described in TNBC [30]. In sarcoma [32] and prostate cancer [33], recruitment of MDSCs, TAMs, and TANs at tumor site are also guided by PI3K expression. These findings indicate that activation of the PI3K/AKT signaling pathway represents a new mechanism of immune escape that has important implications for the development of a novel cancer immunotherapeutic strategy against immune resistant tumors. p53 has a tumor suppressive role, but its mutant variant is known to be associated with oncogenic events at TME therefore, targeting such signaling pathway can be an immunological strategy for modulating immune response at TME [34, 35]. p53 related targeting of melanoma both in vitro and in vivo reveals that, antitumor suppression was reinstated with subsequent administration of nutlin-3a (p53 activator). The process involves two different ways of targeting: p53-dependent restoration of immunostimulatory response at TME and p53-dependent tumor targeting and augmented antitumor immunity. Collectively, these findings represent steady-state role of p53 signaling pathway in cancer immune regulations [36]. Nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) signaling pathway with constitutive expression of NF-κB also regulates oncogenic events and emphasizes significant role of intrinsic NF-κB expression in immune response regulation at TME [37]. Involvement of (sarcoma virus, rat sarcoma virus) RAS/rapidly accelerated fibrosarcoma (RAF)/mitogen activated protein kinase (MAPK) signaling pathway initiates signaling cascade that activates genes imperative for oncogenesis and it further upregulates the expression of PD-1 by stimulating extracellular signal-regulated kinase (p-ERK) signaling [38, 39]. Its role in recruitment of various immunosuppressive cells like Tregs, TAMs, TANs, and MDSCs, at tumor specific site is depicted in non-small cell lung cancer (NSCLC) along with other cancer studies. These studies reveal the involvement of serine/tyrosine/threonine kinase-extracellular signal-regulated kinase-activator protein 1 (MEK-ERK-AP1) pathway with MYC co-activation [40–42]. Literature survey regarding lung adenocarcinomas reveals that targeting IFN-I/stimulator of interferon gene (STING) signaling pathway at glycogen branching enzyme (GBE1), inhibits the PD-L1 expression, and promotes release of chemokines (CCL5 and CXCL10) for recruitment of antitumor CD8⁺ T lymphocytes at TME. Kirsten rat sarcoma (KRAS)-mutation along with co-activated expression of MYC is known for introducing more deleterious immune suppressive response by excluding the recruitment of many antitumor adaptive immune cells such as B cells, T cells, and NK cells [43], through amplified expression of IL-23, chemokine (CCL9). The epidermal growth factor receptor (EGFR) was regulated or involved with other signaling pathways in different cancers. NSCLC, it is regulated through IL-6/JAK/STAT3 [44], in head and neck cancers JAK2/STAT1 pathway is involved [45], and in esophageal squamous cell carcinoma EGFR/PI3K/AKT [46], EGFR/RAS/RAF/ERK, and early growth response/phospholipase C-gamma (EGR/PLC-γ) signaling pathways are involved. The EGFR signaling pathways are responsible for regulating PD-L1 expression and immune suppression too [47]. Further involvement of Hippo signaling pathway is also evident and targeting it at large tumor suppressor kinase 1/2 (LATS1/2) can be promising for immunotherapeutic efficacy against cancer [48, 49]. The studies from thyroid carcinoma have showed the direct association between IDO1 expression and the oncogenic stimulation of rearranged during transfection (RET) and therefore, illustrate the involved signal transduction pathway [50, 51]. While mutant isocitrate dehydrogenase in glioma is associated with possible mechanism of immune suppression, as NK cells acquire resistance via epigenetic regulation [52]. The cancer immune signaling pathways will enable researchers and clinicians to come up with new therapeutic strategies for effective targeting of cancer immune regulation with advancement in existing immunotherapies (Table 2).

Nano-assisted cytokine therapies

Nanocarriers encapsulating cytokines can help cytokine therapies to be included as mainstream therapies, as they can assist in overpowering the challenges of the intricate TME. With an in-depth understanding of the multifaceted antitumor response mediated by cytokines, nano-assisted cytokine therapies can emerge as frontrunner in nano-immunotherapies [78, 79]. Shen and coworkers [80] worked on effective delivery of trap genes IL-10 trap and CXCL12 trap, these were encapsulated into lipid protamine DNA (LPD) nanoparticles and was delivered to TME in 4T1 breast cancer mouse model. This nano-DDSs boosted infiltrating response of CTLs along with enhanced antitumor activity of other immune cells such as tDCs, NK cells, and macrophages to promote tumor regression. Further, Curnis along with coworkers [81] has demonstrated effectiveness of surface functionalized Au nanoparticles (AuNPs) for tumor targeting in fibrosarcoma mouse (BALB/c) model. Functionalized AuNPs were tagged with peptide comprising asparagine-glycine-arginine (NGR) motif and TNF in a well decorated form. The former peptide acts as a driving agent for TNF to reach at target tumor site, where it initiates its antitumor immune response with minimized toxicity to nearby surrounding tissue. Wang et al. [82] have focused on targeting strategies for solid tumors and have synthesized poly (β-amino ester) copolymer nanoparticles (P1) loaded with interleukin IL-12 (IL-12⊂P1). Delivery of this nanoformulation through intravenous route initiates antitumor response, and it also reprograms the TAM at TME with macrophage polarization achieved through immune modulation in solid tumors. Li and colleagues [83] demonstrated poly (d,l-lactide-co-glycolide) nanoparticles surface functionalized with CD8 and glypican-3 antibodies and encapsulated with IL-12 was significant enough in regulating functional antitumor T cell response against hepatocellular carcinoma G2 (HepG2) liver cancer cells with rapid identification of surface marker and immediate release of encapsulated IL-12 cytokine at site of action.

Nano-assisted antibody therapies

Presence of nanocarriers can lend a hand to antibody therapies to overcome unusual responses, since its tendency to modify immune microenvironment and related heterogeneity can be redirected to desired target site at TME [84]. Wei and coworkers [85] demonstrated efficacy of chemo-immunotherapy for cancer targeting in well-defined nanocarriers system. For this, chemotherapeutic drug doxorubicin and immune adjuvant imiquimod (IMQ), a toll-like receptor 7 (TLR7) agonist were loaded in low molecular weight heparin (LMWH)-d-α-tocopheryl succinate (TOS) micelles (LT) along with anti-PD-L1. Targeting of immune checkpoints through PD-L1/PD-1 axis in immune cells triggers the activation of DCs, with an increased ratio of CD8⁺ CTLs to CD4⁺ T effector cells. Further, Wang and colleagues [86] proved the potential role of PTT along with nano-immunotherapy. Here, surface functionalized CuS NPs were initially modified with maleimide polyethylene glycol, and the presence of anti-PD-L1 as checkpoint blocker in nanocarrier system resulted in noticeable increase in the levels of inflammatory cytokines. This strategic targeting achieved by active CD8⁺ T cells was remarkable against the primary and distant tumor in 4T1 mouse model. Duan and coworkers [87] designed Zn pyrophosphate (ZnP) nanoparticles loaded with photosensitizer (pyrolipid) and anti-PD-L1 blocker to potentiate immune therapeutic responses by actively targeting innate and adaptive immune cells at TME. Wang and colleagues [88] reported synergistic antitumor immune response generation in metastasizing cancer when treated with carbon nanotubes (CNTs) loaded with anti-CTLA-4 antibodies along with photothermal therapy. Further, Mi and coworkers [89] employed combinational immunotherapy approach for cancer targeting, where NPs were conjugated with antagonistic antibodies αPD1 and agonistic antibodies antitumor necrosis factor receptor superfamily member 4 (αOX40), and the two were co-administered with spatiotemporal precision for enhanced efficacy in tumor models. Doing so initiates optimal activation of T cell via immunomodulatory mechanism when compared to respective controls (free antibody as immunotherapy). Wang and coworkers [90] designed pH responsive dextran NPs integrated with hyaluronic acid, loaded with anti-PD-1 (aPD1) and glucose oxidase (GOx). The low pH of TME promotes self-dissociation of intratumoral injected dextran NPs causing the release of aPD1 that subsequently induces robust immune response as compared to plain aPD1. This administration strategy integrated with immunomodulators can be used as combinatorial immunomodulation approach for improved efficacy against tumors.

Nano-assisted adoptive cell transfer therapies

Adoptive T cell transfer has been exploited to eliminate solid tumors, but the clinical results have been disappointing owing to limited T cell expansion within the immunosuppressive TME. Nanocarriers can target specific subsets of immune cells within the TME. T cells are found within the TME, and higher cytotoxic T cell infiltration is frequently correlated with improved survival, while increased numbers of Tregs may be related with the worst prognosis.

Nanocarriers offer protection to T cells from immunosuppressive signals that can improve the T cell infiltrations within TME [91, 92]. Zheng and coworkers [93] have designed nanocarriers for effective adoptive cell transfer therapy. Here, IL-2 Fc-PEGylated liposomes and anti-Thy1.1-PEGylated liposomes performed dual action by stimulating and tracking adoptive cell therapy (ACT) T cells in tumor bearing mouse model. These outcomes exhibit the possibility for purposeful and quick reprogramming of targeting lymphocytes through engineered nanocarrier system. Smith and coworkers [94] demonstrated that DNA within synthesized polymeric nanocarriers can target and reprogram CAR genes in T cells extracted from leukemia cancer patient. Further reinfusing them will build a strong antitumor immune response in T cells to combat against progressive tumor growth. Generally, the synthesized nanocarriers were capable of producing activation, proliferation, and memory response in T cells. Such nanocarriers can provide broad realistic applications and purposeful clinical approach for cancer management. Fadel and colleagues [95] reveal strategic expansion of T cells with nanocarriers working as artificial APCs for adoptive therapies. Here, nanocomposites of CNT and polymers were ornamented with antigens and IL-2 specific for T cell activation. Such presentation of nanocarrier has encouraged as a promising approach for tumor regression in melanoma murine model. The surface engineered CAR T cells designed to deliver adenosine antagonist encapsulated in liposomal nanoparticles within TME of solid tumors and obstruct immunosuppressive response in antitumor immune cells [96]. Chen with coworkers [97] proved that the presence of indocyanine green (ICG) in poly(lactic-co-glycolic) acid (PLGA) nanoparticles can promote enhanced recruitment of CAR T cell for superior antitumor immune activity at TME in melanoma tumors model with photothermal therapy. However, further research investigation in this direction by Nie and colleagues [98] demonstrated that magnetic nanoclusters can also be employed for similar work with technical assistance as applied neodymium magnet. This represents improved enrollment of nanocluster T cells to tumor specific site where they undergo disassembling of nanocluster, thus encouraging antitumor effects of T cells at TME.

Nano-assisted cancer vaccines

Nanocarriers will enable loading or encapsulation of the cancer vaccines that are specially designed based on tumor specific antigen. Apart from the physical and chemical attributes of nanocarriers, other potential features that focus on multiple deliveries wherein multiple attributes can be targeted through an administered route required for effective nano-immunotherapy. This includes immunomodulatory or adjuvant molecules coupled with tumor antigens, raising the levels of nano-immunotherapy [99–101]. Studies in in vivo melanoma mouse model demonstrate antitumor and immunoregulatory nature of melittin (specialized bee venom from Europe). But its encapsulation in lipid nanoparticles enables effective targeting of cancer in primary and metastatic melanoma via modulated antitumor immune response in lymph nodes, where activated TAAs are presented by macrophages and dendritic cells to CD8⁺ T cell generating effector immune response [102, 103]. Thus, it can be considered as a potent nano-vaccine for treatment of breast and other cancers [104]. Nguyen et al. [105] have synthesized nano-vaccine composed of silica nanorods and silica nanoparticles clustered together, loaded with ovalbumin (OVA) protein antigen, immune adjuvant granulocyte macrophage colony-stimulating factor (chemokine), TLR9 agonist (CpG oligo-deoxynucleotides, CpG-ODNs) for effective recruitment of activated DC in myeloma mouse model. Further, its synergistic nature upon treatment with immune checkpoint anti-CTLA-4 (a-CTLA-4) also reveals its efficacy in tumor mouse model. Zamani et al. [106] have studied human epidermal growth factor 2 (HER2/neu) and a Pan HLA-DR (PADRE) tumor’s specific long epitope (peptide) on conjugation with nanoliposomes. These nanoformulations were administered subcutaneously to HER2⁺ TUBO breast cancer mice model and their potential role as an effective nano-vaccine was reported as they enhanced helper T cell and CTL immune responses via activated DC expression against metastatic growth. Masjedi and coworkers [107] demonstrated antitumor immune responses in 4T1 breast tumor mice model (Balb/C) where, siRNA for adenosine type 2 receptors signaling were loaded into polymeric nanoparticles [polyethylene glycol (PEG)-chitosan-lactate (PCL)]. This signaling was known to interfere with the function [down regulation of adenosine A_2A receptor (A2AR)] and differentiation (silencing of naive T cell CD4⁺ CD25⁻) response generated by T cells. Successive immune stimulation, post treatment with polymeric nanoformulation was a resultant of immune signaling event. Where downregulation of protein kinase A/cAMP-response element binding protein (PKA/CREB) occurs and upregulation of NF-κB enhances antitumor performance of T cells at TME. Another study led by Jadidi-Niaragh and coworkers [108] demonstrated CD73 as a leading immune suppressive agent related to adenosine levels. The intravenous administration and targeting of adenosine levels by CD73 siRNA-loaded chitosan-lactate nanoparticles and tumor lysate pulse DCs vaccine has illustrated immense efficacy against tumor growth by reducing the immune suppressive activities of protumor immune cells (Treg, MDSCs, TAMs etc.) at TME. Liu et al. [109] worked on synergistic administration of curcumin loaded poly(ε-caprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone)-poly(ethyleneglycol)-poly(εcaprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone (PECT) polymeric nanoparticles and nano-vaccine loaded with CpG and antigenic peptides that triggered immunogenic cancer cell death (ICD) in TME through immune stimulatory and cytotoxic immune response initiation in DCs and CD8⁺ T cells in 4T1 breast cancer model. This greatly signifies the efficacy of nano-assisted chemo-immunotherapeutics for treatment of breast and other cancers.

Nano-assisted oncolytic virus therapies

Recent reports for potential role of nanotechnologies in providing platform as oncolytic virus therapies have initiated neo-trends in cancer nano-immunotherapies where these nano-assisted oncolytic virus therapies can prevail over the limitations thus making virus nanocarriers safer, efficient and potent for cancer patients [110, 111]. Work led by Lizotte and coworkers [112] demonstrated that cowpea mosaic virus (CPMV) used as nanoparticles which was administered via inhalation in different cancer models (B16F10 lung melanoma, ovarian, colon, and breast tumor models), where its efficacy was attained through systemic activation of the immune responses. Efficiency of the antitumor immune responses of CPMV nanoparticles was primarily due to its stable and nontoxic nature. Flexibility in the desired cargo through drug/antigen loading or nano fabrication has attracted researchers and clinicians as novel approach to target immune responses against metastatic cancer. Further, Arab and coworkers [113, 114] have demonstrated the role of phage nanoparticles in breast cancer where coat protein gpD of lambda phage (λF7) nanoparticles was linked to HER2 protein (E75 and AE37 peptide). In vitro studies along with effective delivery in HER⁺ TUBO breast cancer Balb/c mouse model were also evident.