3.1 Tumor infiltrating lymphocytes and their prognostic value in clinical outcome
A particularly important role in the anti-cancer immune response is played by lymphocytes whose effector functions allow reduction in tumor invasion. On the other hand, during carcinogenesis, the secretory activity of tumor cells can lead to suppression of the host's cell-mediated immunity. The molecular basis of these relationships has a significant impact on the clinical course of the disease because disturbed T cell function contributes to the progression of some cancers [
34]. The exact characterization of tumor-infiltrating lymphocytes (TILs) can even have therapeutic relevance. Neoantigen-specific immune reactivation of TILs, unique for each patient, is currently considered a method of personalized anticancer treatment [
35].
In breast cancer, TILs represent an immunological parameter whose morphological characteristics are of clinical significance. This parameter is related to the histopathological-molecular type of the tumor and may be an independent prognostic biomarker of e.g. the response to adjuvant chemotherapy [
36]. Cancer-induced changes in gene expression and T cell receptor (TCR) activity in the CD8
+ cell population modify TIL density, which determines the response to anticancer treatment and the prognosis of patients, and may be a promising starting point for therapy [
37]. Evaluation of inflammatory immune infiltration in cancer may also reflect the defense capabilities of the immune system. The density of immune cells in cancer illustrates the severity of the host antitumor immune response and usually refers to clinical aspects of the disease.
In CRC, the assessment of CD8
+ T cell density in randomly chosen invasive tumor margin infiltrations has shown a significant relationship with survival [
38]. In CRC, TILs are a favorable prognostic parameter – a high number of immune cells was associated with statistically better overall survival (OS
) values than was a low tendency toward lymphocytic infiltrates [
39]. The distribution of immune cells in tumor tissue may also have prognostic significance. In esophageal squamous cell carcinoma, TILs showed redistribution of the location in favor of the stroma rather than the intraepithelial location, and the higher intensity of infiltration in the stroma showed prognostic advantage [
40]. Nevertheless, the clinical significance of inflammatory immune infiltration can have a different outcome depending on the type of cancer. More pronounced lymphocytic infiltrates are characteristic for lobular breast cancer (LBC) rather than for invasive ductal cancer (IDC), and high levels of TILs have been associated with tumor cell proliferation, with more frequent lymph node infiltration and a worse prognosis [
41]. In breast cancer, the prognostic value of TILs also appears to depend on the molecular specifications of tumor cells. An increased TIL concentration is a beneficial prognostic marker for human epidermal growth factor receptor 2 (HER2)-positive breast cancer and TNBC (triple-negative breast cancer), but is unfavorable for luminal HER2-negative breast cancer [
42]. A significant prognostic role is played by the immunophenotype of tumor-infiltrating immune cells. The prognostic value of TILs depends on the specification of the lymphocyte immunophenotype – the predominance of T cells promoting cellular cytotoxicity and APCs is usually a favorable parameter [
43]. In the case of extrahepatic cholangiocarcinoma, a beneficial prognosis is associated with the presence of memory cells with a CD8
+CD45RO
+ phenotype in the lymphocytic infiltrate, but not with CD8
+ only cells [
44]. The predominance of CD4
+ T cells in tumor-infiltrating immune cells of CRC metastases to the lungs has been found to be associated with a better outcome after surgical resection [
45].
3.2 Tumor associated macrophages
Tumor associated macrophages (TAMs) residing in neoplastic tissue constitute a fairly heterogeneous population of cells. Some cells are derived from monocytes circulating in the blood, the reservoir of which is the bone marrow. The process of migration occurs through chemotaxis and is conditioned by tumor-released ligands, such as chemokine (C-C motif) ligand 2 (CCL2), CCL5 and colony stimulating factor 1 (CSF-1). A distinct subgroup is a population of TAMs derived from embryonic precursors that colonize the tissue at early stages of ontogenesis [
46]. Various subpopulations of macrophages with different regulatory effects on the immune response can be distinguished in the population of monocytes. The subpopulation of M1 macrophages has proinflammatory properties, evokes a Th1-type response, and has strong cytotoxic activity as part of the antitumor response. Subpopulations of M2 macrophages usually have immunoregulatory properties, are responsible for a Th2-type response and produce small amounts of pro-inflammatory cytokines. M2 TAMs secrete immunosuppressive factors, such as IL-10 and TGF-β. In the M2 subpopulation, as many as 3 major subtypes of macrophages with different functions and secretory profiles can be distinguished: M2a, M2b and M2c macrophages [
47]. There are also M2d macrophages, characterized by high levels of vascular endothelial growth factor (VEGF), IL-10 and cytokine-inducible nitric oxide synthase (iNOS) expression [
48].
The subpopulation of M2 macrophages is a common element of the TME and is usually an unfavorable prognostic factor in various types of cancer. Their activity has been associated with greater invasiveness, shorter disease-free survival and induction of epithelial-mesenchymal transition (EMT) [
49]. Neoplastic cells induce polarization of M0 macrophages towards the M2 phenotype. On the other hand, physical properties of the cellular matrix may increase tumor invasiveness by regulating the transcription of EMT-related genes in the population of M2c macrophages [
50]. TNF-α-releasing M2 macrophages promote the progression of HCC and induce EMT, which is associated with regulation of the Wnt/β-catenin pathway by cytokines [
51]. In HCC, upregulation of Wnt/β-catenin signaling depends on Notch signaling. In Kupffer cell-like TAMs (kclTAMs), the process increases cell proliferation, downregulates interleukin-12 (IL-12) synthesis and increases IL-10 release. This in turn leads to the progression of HCC and promotes the potential of some solid cancers to form metastases in the liver - however, this potential is dependent on the type of cancer [
52]. Other immunosuppressive cytokines released by TAMs may also lead to the progression of solid tumors. A higher number of TAMs in CRC is associated with a tendency to form distant metastases. Greater invasiveness and migration of cancer cells results from the regulation of EMT through TGF-β secreted by TAMs via the Smad2,3-4/Snail/E-cadherin pathway [
53]. Overexpression of NOR1 observed in HCC has been associated with an unfavorable prognosis, which is most likely due to acceleration of the immunosuppressive activity of NOR1
+ TAMs by upregulation of arginase 1 (Arg1) expression and preferred polarization of macrophages towards the M2 subpopulation [
54]. Human prostate cancer xenografts showed a clear polarization of the macrophage population towards the M2 phenotype - the higher TAM density stimulated the growth of neoplastic lesions. Moreover, CD206 macrophages were more likely to settle in strongly vascularized regions characterized by co-expression of the CD31 antigen, which may suggest the importance of TAMs in the regulation of angiogenesis [
55]. Hyperactivity of M2 macrophages is also typical of some subtypes of inflammatory cancers. An inflammatory infiltrate formed by M2 macrophages is relatively common in inflammatory breast cancer (IBC). In this type of cancer, TAMs clearly overexpress interleukin-8 (IL-8) and growth-regulated oncogene (GRO) chemokines, which are associated with epithelial-to-mesenchymal transition CSC-like phenotypes through the activation of JAK2/STAT3 signaling [
56]. Overexpression of some inflammatory factors may paradoxically increase promotion of the immunosuppressive phenotype. In breast cancer (basal-like BCC), the S100 calcium-binding protein A4 (S100A4) promotes monocyte differentiation and polarization towards M2 phenotype macrophages as well as induces increased expression of interleukin-6 (IL6) and IL8 in macrophages [
57].
TAMs have an adverse effect on cellular responses, especially on the action of T cells. Tumor cells capable of releasing CXCL2 ligand activate CXCR2
+ TAMs, enhancing their suppressive effect on T cells and pro-angiogenic activity, with consequent tumor growth [
58]. TAMs also impede the migration of CD8
+ T cells into the tumor nests as well as restricted inflammatory infiltration by these cells. This significantly determines the effectiveness of cancer immunotherapy [
59]. An interesting observation is also the relationship between the molecular profile of TAMs and TILs. In pancreatic ductal adenocarcinoma (PDA), a significant relationship has been observed between TAMs and TILs, where different epigenetic profiles of macrophages influenced the regulation of the molecular parameters of T cells infiltrating the tumor. A reduced number of CD11b
+ and CD115
+ cells resulted in an increased number of T cells infiltrating the tumor, a lower number of IL-10-releasing CD4
+ T cells and a smaller Treg population [
60]. The regulation of the migration of TAMs into the tumor nest is also dependent on other factors. In non-small-cell lung carcinoma (NSCLC), the recruitment of macrophages relates to the expression of the VEGF-C cytokine, whose overexpression potentiates tissue infiltration by this population of cells. The ligand secreted by tumor cells activates the VEGFR-2 and VEGFR-3 receptors located on macrophages, leading to their increased migration through Src/p38 signaling [
61].
Although most reports revealed adverse effects of TAMs on the process of oncogenesis, particularly related to the M2 subpopulation, there are also data suggesting a beneficial effect of these cells on prognosis. CD68
+ cells are most numerous in the inflammatory infiltrate of CRC. Their prognostic value seems to depend on the microanatomical location of the infiltrate. Long-term follow-up showed a tendency of CD68
+ macrophages to infiltrate the anterior regions of the invasive tumor as a favorable prognostic parameter [
62]. In CRC, a tendency to induce expression of proinflammatory cytokines has also been reported. Exposure of peripheral blood mononuclear cells to conditioned medium from CaCo-2 cells changed the secretory activity of monocytes by promoting the release of proinflammatory cytokines, such as IL-6, IL-12 and interferon gamma (IFN-γ), with downregulation of the expression of some immunosuppressive interleukins, such as interleukin-4 (IL-4) and IL-10. The group of proinflammatory cytokines also revealed a reduced expression of TNF-α, which may result from an increased pro-angiogenic activity of tumor cells caused by negative regulation by VEGF [
63].
M1 macrophages, whose secretory profile differs from the properties of the M2 population, play a different role in the pathogenesis of tumors. A tendency for an increased expression of TNF-α is typical of M1 macrophages, for which this cytokine remains a marker protein additionally to blocking polarization towards the M2 subtype [
64,
65]. The favorability of macrophage repolarization to M1 cells has been associated with elevated levels of reactive oxygen species (ROS), a higher concentration of proinflammatory agents and a simultaneous decrease in the expression of CD206 and Arg1 [
64]. Additionally, in this case, the M1 subtype is not always a favorable prognostic factor. In breast cancer, M1 macrophages are usually an unfavorable predictor. In vitro studies have shown that the pro-inflammatory secretory activity of M1 macrophages may increase invasiveness by promoting the EMT phenotype. The regulation of this phenomenon is complex and strictly dependent on other molecular parameters of the cancer cells. Bednarczyk et al. noted a positive effect of increased matrix metallopeptidase 9 (MMP-9) expression on the migrative and invasive potential of adenocarcinoma cells induced by the M1 subpopulation [
66].
3.3 Tumor associated neutrophils
The heterogeneity of the tumor-associated neutrophil (TAN) population has not been sufficiently defined and, therefore, requires more detailed studies. Nevertheless, morphologically their population is divided according to differences in density gradient purification, where low-density neutrophils (LDNs) can be distinguished, which are most often associated with cancer progression, as well as a population of so-called high-density neutrophils (HDNs), which comprises highly diverse and mature inflammatory cells. Alternatively, N1 and N2 neutrophils may be distinguished, characterized by different gene expression profiles and secretion parameters. TAN polarization characterized by the N2 phenotype, which is identified as promoting oncogenesis, takes place under the influence of TGF-β. In contrast, the N1 phenotype is characterized by lower Arg1 levels, distinctive anticancer activity and a tendency toward cytokine and chemokine expression, which stimulate an inflammatory response [
67,
68]. Within the cell response range, the N2 phenotype regulates CD4
+ T cell recruitment, whereas N1 TANs seem to be responsible for cytotoxic functions. In the division that concerns cancer-related circulating neutrophils, the LDN phenotype limits the ability to perform phagocytosis and exhibits antiproliferative activity toward CD8
+ T cells, whereas HDN cells have antagonistic properties [
69].
The neutrophil-to-lymphocyte ratio (NLR) is a key prognostic factor in various cancer types and is relatively frequently correlated with recurrence risk [
70‐
72]. A high NLR in bladder cancer positively correlates with determinable concentrations of IL-6 and IL-8 as well as with their Treg expression in peripheral blood, which emphasizes a systemic dependence between neutrophil infiltration in cancer and the secretion profile of cytokines. Furthermore, higher concentrations of both cytokines, of prevailing proinflammatory activity, have shown a positive relation to Treg induction. In contrast, high NLR values have shown a positive correlation with tumor stage, tumor growth and the level of common inflammatory markers such as C-reactive protein (CRP), the plate-to-lymphocyte ratio (PLR), and the monocyte-to-lymphocyte ratio (MLR) [
73].
The presence of TANs in inflammatory infiltrations most often disturbs the accumulation and activity of T cells, which is related to clinical and pathological data. In patients with extrahepatic cholangiocarcinoma, TANs showed an inverse correlation to CD8
+ T cells and a strong association with a poor overall survival (OS) [
74]. Myeloperoxidase (MPO)
+ neutrophils releasing interleukin-17 (IL-17) are a favorable prognostic factor in esophageal squamous cell carcinoma (ESCC). It was confirmed in in vitro models that a high percentage of immune cells releasing IL-17 correlated with the release of CXCL2 and CXCL3 ligands, which resulted in cells having a greater tendency toward migration [
75]. On the other hand, it has been shown that T cells releasing IL-17 may be linked to cancer progression, and that TANs may show properties that enhance anticancer responses via γδ T cell suppression due to oxidative stress intensification and ROS production [
76]. However, under restricted glucose conditions, ROS production in the mitochondria of neutrophils may lead to T cell suppression, which links oxidative stress to increased immunosuppression [
77].
3.4 Myeloid-derived suppressor cells
Myeloid-derived suppressor cells (MDSCs) represent a group of heterogeneous subpopulations of immature myeloid cells with predominant immunosuppressive properties that are widespread in various types of human cancers, as well as in mouse tumor models. In tumor-bearing mice, the expression of relevant molecules enables the classification of MDSCs into two main subtypes: monocytic lineage cells (mo-MDSCs) and granulocytic (polymorphonuclear) lineage cells (PMN-MDSCs). Three subgroups of these cells are distinguished in humans depending on their immunophenotype and immunosuppressive properties: PMN-MDSCs, M-MDSCs and Lin
-HLA
-DR
-CD33
+ cells [
78]. Cancer cells and immune cells have the potential to induce the formation of MDSC subtypes. Human T cells are of pivotal importance in inducing a population of PMN-MDSCs. Through a direct cell-cell mechanism with the use of the transmembrane form of TNF-α (tmTNF-α) activation, CD4
+ T cells promote the development of a PMN-MDSC population using CD33
+ myeloid cell reserves, while the CD3
+ subpopulation shows a correlation with anti-apoptotic activity toward PM-MDSCs [
79]. Cancer cells release CXCL1 and CXCL2 chemokines, which in turn induce the generation of mo-MDSCs as a bone marrow cell subpopulation [
80].
In patients diagnosed with renal clear cell carcinoma (RCC) an estimatable level of total MDSCs, granulocytic MDSCs (G-MDSCs), and immature MDSCs (I-MDSCs) clearly correlates with the degree of histological malignancy and stage of the disease, whereas stromal MDSCs positively correlate with IL-17 and IL-18 co-expression, both at the protein and mRNA level. An increased level of the two interleukins in relation to MDSCs was also noted in peripheral blood, i.e., PBMCs. The MDSCs and cytokine concentrations were significantly higher in RCCs than in controls [
81]. The preoperative number of PMN-MDSCs and mo-MDSCs tend to be different depending on TNM breast cancer stage. The lowest percentages of mo-MDSCs were noted in stage Tis (in situ), whereas a higher number of these cells was observed in the 3rd stage of the disease. In the case of PMN-MDSCs, however, the dependency was reversed, with stage Tis being characterized by the highest percentage of this MDSC subpopulation [
82]. The number of MDSCs in an ovarian cancer mouse model increased with neoplastic process duration and was significantly higher in late stages of the disease. The significant immunosuppressive activity of MDSCs directed against T cells leads to disturbed systemic immunity in animals [
83].
The epithelial-to-mesenchymal transition phenomenon in ovarian cancer is related to the Snail transcription factor, whose expression level is significantly correlated with a lower percentage of survival. Snail also shows a correlation with a higher percentage of intratumor MDSCs, whose number reduces the CD8
+ population in TILs. As demonstrated in a mouse model, MDSC migration toward tumor tissue takes place by chemotaxis and depends on chemokines such as CXCL1 and CXCL2. High levels of these chemokines were noted in cancers with a positive Snail co-expression. The use of a CXCR2 ligand antagonist limited MDSC migration, and the chemokine receptor alone was associated with the promotion of tumor growth and an unfavorable prognosis. Nuclear factor kappa B (NF-κB) has been reported to participate in Snail-dependent regulation of CXCL1 and CXCL2 expression [
84]. Chemotaxis dependent on relevant chemokine expression appears to be important, not only in MDSC migration toward the primary tumor, but also in the promotion of distant metastases and upward regulation of growth factors. Breast cancer cells that secrete CXCL17 increase lung CD11b
+Gr
-1
+ MDSC accumulation and increase the level of platelet-derived growth factor-BB (PDGF-BB) expression in these cells, which is also associated with proangiogenic activity that facilitates lung metastatic niche formation [
85]. The chemokine expression level supporting intratumor MDSC recruitment has been found to be regulated by transcription factors. A high level of ΔNp63 in triple-negative breast cancer (TNBC) is positively correlated with the size of the MDSC population, which represents a direct ΔNp63-dependent activation of chemokines such as CXCL2 and CCL22 [
86]. CXCR2
+ PMN-MDSCs enhance tumor growth, whereas their migration from the periphery to the tumor appears to be dependent on CXCR2. PMN-MDSCs are characterized by pronounced TIL suppression, which may be accompanied by programmed death (PD)-axis signaling. Due to the capability of regulating tumor T cell infiltration, the PMN-MDSC subpopulation may have considerable significance in cancer immunotherapy [
87].
In the regulation of carcinogenesis, MDSCs are crucial not only at advanced stages, but also in early precursor lesions of malignant cancers. In premalignant lesions, the amounts of MDSCs have been found to be considerably greater than those in controls. Its amounts were, however, lower than those in overt cancers, whereas the immunosuppressive activity of these cells was comparable in precancerous and cancer stages [
88]. The transcription profile of peripheral blood MDSCs (PB-MDSCs) reflects molecular changes appearing in different stages of tumor development. Upregulated genes such as those encoding Arg1 and nitric oxide synthase 2 (NOS2) seem to confirm an immunosuppressive tendency of tumor progression. Mammalian target of rapamycin kinase (mTOR) pathway activation and Toll-like receptor (TLR), IL-4, IL-6 and IL-10 signaling take place in early stages. In patients with gastric cancer (GC), IL-6 and IL-8 activate CD45
+CD33
lowCD11b
dim MDSCs, thereby inducing Arg1 release accompanied by activation of the PI3K-AKT signaling pathway. Activation of this pathway suppresses CD8
+ T cells and positively correlates with disease progression and patient survival rates in general. Increased levels of IL-6 and IL-8 in patients with GC have been found to positively correlate with Arg1 and MDSCs [
89]. There are reports stating that Arg1 is not consecutively released by MDSCs and that its synthesis depends on the activity of certain interleukins, i.e., IL-6, IL-4, GM-CSF and IL-10 are known to regulate the secretion of Arg1 by MDSCs.
The induction of Arg1 synthesis seems to be indirectly regulated, with IL-6 regulating IL-4R receptor expression on MDSCs, which subsequently enhances Arg1 synthesis by binding to the ligand. In the second option, GM-CSF induces IL-10R expression, and the binding of the cytokine to the receptor results in Arg1 release [
90]. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in prostate cancer together with Arg1, NOS2 and STAT3 expression, exhibit a suppressive effect on T cell activity. PMN-MDSC suppressive activity seems to be regulated through interactions of these cells with CD40L
+ mast cells resulting from CD40 ligand co-stimulation. This correlates with the induction of oncogenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) and affects cell immunity [
91]. In epithelial ovarian cancer (EOC) a positive correlation has been observed between TGF-β, estimable in plasma, and Arg1
+ MDSCs found in the peritoneal fluid. The plasma level of Arg1 was also found to be positively correlated with TGF-β
+/IDO
+/IL-10
+ PMN-MDSCs [
92]. The immunosuppressive tumor microenvironment is an important regulator of the response to radiotherapy. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibition within the range of IDO1-expressing myeloid-derived suppressor cells in a mouse model of lung cancer decreased the size of the population of these cells, increased the immunosuppressive effect and sensitized the tumor to radiation [
93]. Expression of programmed death 1 (PD-1) on the MDSC surface is regulated by the NK-κB signaling pathway and is increased by MDSC tumor infiltration. The presence of PD-1
+ MDSCs in the TME is associated with acceleration of their proliferation in relation to PD-1
- MDSCs, which suggests that TME immunosuppressive properties may contribute to the promotion of carcinogenesis [
94].
The use of STAT3 inhibitors in prostate cancer limited tumor-associated MDSCs and inhibited interleukin-1β (IL-1β), IL-10 and IL-6 synthesis by monocyte cultures [
95]. STAT3 inhibition in liver-associated MDSCs (L-MDSCs) has been found to exhibit antitumor activity, whereas a decreased level of STAT3 phosphorylation also reduced the size of the L-MDSC population, which in turn led to an increased anticancer activity of chimeric antigen receptor T cells (CAR-T) [
96]. MDSCs generated from induced pluripotent stem cells (iPSCs) in an autoimmune hepatitis murine model disturbed the cellular response while limiting lymphocyte proliferation and CD8
+ T cell inflammatory infiltration in portal tract regions, which was accompanied by significantly decreased alanine aminotransferase levels (ALT) in plasma [
97]. In the case of CRC, the number of MDSCs showed a discernible disturbing effect on the cellular response associated with lymphocyte redistribution toward the monocyte line resulting in a higher level of circulating MDSCs, which was correlated with a lower lymphocyte to monocyte ratio (LMR). A significantly decreased LMR was also found to be associated with a reduced recurrence-free survival [
98]. Abundant MDSC infiltration in osteosarcoma also resulted in increased T cell cytotoxic activity, while its accumulation in cancer tissue appeared to be dependent on expression of the CXCR4 receptor, which enabled the migration of these cells by stromal cell-derived factor 1 (SDF-1). Moreover, binding of this chemokine with CXCR4 inhibited MDSC apoptosis and was inversely correlated with infiltration of the cancer by CD8
+ T cells. CXCR4 blockade, on the other hand, in combination with anti-PD-1 therapy exhibited a synergistic effect [
99]. In CRC, MDSC function is regulated via RIPK3-PGE2. MDSC accumulation has been found to decrease the level of RIPK3, which enhanced oncogenic potential via the promotion of MDSC accumulation and increased immunosuppressive activity resulting from NF-κB upregulation, which regulated COX2 transcription and inhibited prostaglandin E2 (PGE2) release, thereby stimulating cancer cell proliferation while negatively influencing CD8
+ T cells [
100].
The number of MDSCs in patients with breast cancer has been found to be higher than that in the control group and to correlate with both the tumor size and the stage of the disease. The IL-17 level, on the other hand, was found to be lower in the patients than in the controls [
101]. Proinflammatory IL-17 showed an anti-proliferative effect on MDSCs and induced their differentiation, which was accompanied by an altered expression profile of specific cytokines. Under the influence of IL-17, MDSC release lowered the TGF-β and IL-10 levels, whereas the IL-1, IL-1β, IL-6 and TNF-α levels were upregulated. Ma et al. observed a correlation between MDSC accumulation inhibition by IL-17 and STAT3 activation [
101]. In lung cancer, the TLR1/TLR2 expression levels served as a favorable prognostic factor. In an animal model, TLR1/TLR2 activation was found to be linked to a reduction in tumor growth and a selective downregulation of the M-MDSC subpopulation. The use of a TLR2 agonist exhibited a positive effect on M-MDSC redistribution toward M1 macrophages [
102].