2.1. Mechanism of Action of Soluble PD-1

Programmed cell death-1 (PD-1, CD279) is a 55-kDa transmembrane protein containing 288 amino acids, extracellular N-terminal, membrane-permeating domain and a cytoplasmic tail at N and C ends with two tyrosine bases [16]. PD-1 is expressed in activated T cells, natural killer (NK) cells, monocytes, macrophages, dendritic cells (DCs) and B lymphocytes. Mechanistically, it is an inhibitor of both adaptive and innate immune responses, and is known to be highly expressed on tumor-specific T cells [17]. With regards to its role in the immune microenvironment, it is described to have both protective and harmful roles. On one hand, it plays a significant role in the regulation/maintenance of immune tolerance to prevent host tissue damage while, on the other hand, it facilitates tumor immune escape via its overexpression [18].

With respect to soluble PD-1 (sPD-1), it is generated via proteolytic cleavage of membrane-bound PD-1. Soluble PD-1 primarily exists in two forms: one form is completely homologous with membrane-bound PD-1, while others are the spliced-out parts of membrane-bound form [19]. With regards to its role in immune microenvironment, studies have shown that sPD-1 functions as a blocker of PD-1 ligands and as such suppresses the interaction of membrane-bound PD-1 with immune checkpoint ligands and receptors, PD-L1, PD-L2 and CD80 respectively [20]. This interaction leads to several immune modulatory effects. On one hand, it can lead to enhancement of the anti-tumor response via early activation of CD8+ lymphocytes, boost lytic activity of macrophages, up regulate TNF-α, IFN-γ, inducible nitric oxide synthase (iNOS) production, reduce OX40 and IL-10 expression and block PD-L1expression on tumor cells [21]. On the other hand, it confers immune inhibitory effects such as reverse signaling effect on dendritic cells via the PD-L1/PD-L2 pathway, subsequently leading to a reduction in DC maturation, and inhibition of IL-2 production and T cell activation [22].

2.2. Role of Soluble PD-1 as a Biomarker in NSCLC Patients Treated with Immunotherapy

Several studies have reported on the role of soluble PD-1 as a dynamic biomarker in immunotherapy treated NSCLC patients. Soluble forms of PD-L1 (sPD-L1), such as secreted splice variants (secPD-L1) and exosomal PD-L1 (exo-PD-L1), have recently been found in peripheral blood and have been shown to suppress T cell functions, allow tumor evasion, and accelerate tumor growth [23]. A study including 189 NSCLC patients monitored levels of soluble PD-L1 (sPD-L1) and soluble PD-1 (sPD-1) before and after starting ICI therapy. The results showed improved overall survival (OS) (hazard ratio 0.24, p value: 0.037) and high levels of sPD-1 for patients after receiving anti-PD-1 monotherapy.

On the other hand, patients receiving combination of ICI and chemotherapy, no significant change in expression levels were seen, suggesting that sPD-1 can be a potential predictive response biomarker on ICI monotherapy [24]. Similarly, another study on 13 NSCLC patients, that measured change in levels of sPD-1 before and after anti-PD-1 treatment showed that increased expression of sPD-1 is significantly correlated to disease progression (p = 0.024) [14]. Another study, measuring the change in the expression of sPD-1 before anti-PD-1 (Nivolumab) treatment and after two cycles (post 28 days) in 51 NSCLC patients reported that patients with >30% increase in sPD-1 level, after two cycles of nivolumab, had a better progression-free and overall survival as compared to those with decreased levels of sPD-1 [25]. Similarly, a study on 22 NSCLC patients, investigating the role of soluble PD-1 on treatment dynamics showed comparable results to the above-mentioned studies. The level of soluble PD-1 before anti-PD-1 treatment and at the time of first clinical evaluation (after 3 months of treatment) showed dynamic modulation. A significant decrease during Nivolumab treatment was found to be associated with clinical response while increase was associated with non-response to treatment [26]. A study examined the predictive significance of baseline and dynamic changes in soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) levels concerning treatment outcomes. The positivity of either sPD-L1 or sPD-1 was used to calculate a composite score known as sCombo. The baseline positive of this score was substantially linked with shorter progression-free survival (PFS) of 78 days (95% CI: 55–109) compared to 658 days (95% CI: 222-not achieved) in score-negative individuals (p-value = 0.0002). Overall survival (OS) was also worse for individuals with a positive score, with a hazard ratio (HR) of 3.99 (95% CI: 1.63–9.80, p = 0.003). In multivariate analysis, sCombo score positive continued to be an independent predictor of shorter PFS, with an HR of 2.66 (95% CI: 1.17–6.08, p = 0.02) [27]. Though, limited studies have been performed on sPD-1 profiling in immunotherapy-treated NSCLC patients, results from published literature indicate a trend and scope of sPD-1 kinetics with anti-PD-1 treatment dynamics, indicating its significance as a prognostic and predictive biomarker.

3.1. Mechanism of Action of Soluble PD-L1

Programmed Death Ligand-1 (PD-L1, CD274) is a 40 kDa type I transmembrane glycoprotein that can exist as multiple splice variants, PD-L1 lncRNA splice isoforms and truncated PD-L1 [28]. Two possible alternative splicing events-either at the mid-exon or alternative polyadenylation led to the removal of the exon encoding the PD-L1 transmembrane domain in the CD274 transcript, thus, leading to the generation of soluble PD-L1 [28,29]. Studies have documented that sPD-L1 can only be generated by tumor cells and activated mature DCs [28]. Though the exact mechanism of action of soluble PD-L1 in the immune microenvironment is still unclear, studies have documented various roles of this mediator in immune regulatory processes. For instance, sPD-L1 has been reported to induce negative regulation of T-cell function by binding to PD-1. In addition to this, it has been documented that specific interaction between sPD-1/sPD-L1 may alter the activity of immune cells, leading to immune suppression via the reduction in TH1 cytokines and increased TH2 cytokines expression [30,31]. Moreover, it has been reported that a unique variant of sPD-L1, consisting of a unique18 aa tail can homodimerize and induce immunosuppressive activity via T-cell apoptosis, inhibition of lymphocyte function, T-cell proliferation, and IFN-gamma production [32]. However, studies have reported that sPD-L1 lacks measurable T-cell inhibitory activity but rather functions as a receptor antagonist by blocking the inhibitory activity of membrane-bound PD-L1 [28].

3.2. Role of Soluble PD-L1 as a Biomarker in NSCLC Patients Treated with Immunotherapy

Studies investigating the role of PD-L1 as a prognostic and predictive biomarker have shown interesting results. For instance, a study investigating the role of pre-treatment sPD-L1 levels in 233 NSCLC patients treated with anti-PD-1 (Nivolumab) found that high pre-treatment sPD-L1 levels were significantly associated with a lower disease control rate and poorer progression-free, and overall survival [33]. Similarly, a study on the impact of pre-treatment sPD-L1 levels on anti-PD-1 treatment dynamics/clinical outcomes was assessed in 39 NSCLC patients. The study reported that increased pre-treatment sPD-L1 levels confer poor response and survival in ICI-treated patients, indicating a prognostic role of this biomarker in NSCLC [34].

On the other hand, several studies have reported the kinetics of this biomarker during immunotherapy indicating its role as a dynamic marker. A study of 189 NSCLC patients determined the change in the expression level of soluble PD-L1 (sPD-L1) prior to and after immune checkpoint inhibitor (ICI) therapy. Higher baseline sPD-L1 was associated with poor progression-free survival (PFS) and OS in patients receiving ICI monotherapy, with HR of 15.4 (p = 0.009) and 11.4 (p = 0.007), respectively. Baseline sPD-L1 levels were linked with other soluble components produced by zinc-binding proteases ADAM10/17 [24]. According to a study, individuals with advanced NSCLC who exhibit elevated levels of soluble PD-L1 (sPD-L1) are more likely to have unfavorable outcomes with immunotherapy. Researchers analyzed 120 patients and discovered that those with disease control had longer PFS and OS compared to those with high sPD-L1 levels, as well as significantly lower sPD-L1 concentrations (p = 0.0006). The median PFS was 5.8 months vs. 2.5 months (HR = 0.6021, p = 0.0156), and the median OS was 16.5 months vs. 7 months (HR = 0.5354, p = 0.0071). The levels of sPD-L1 and tumor PD-L1 expression did not significantly correlate. Further, the study’s conclusion emphasizes sPD-L1’s potential for treatment guidance by showing that it is a useful marker for predicting resistance to anti-PD-1 or anti-PD-L1 therapy [35].

Another study by Costantini et al. investigated sPD-L1 levels in the plasma at three time points; at the time of diagnosis, at the initiation of nivolumab, and after 2 months (first tumor evaluation). The study found that an increase in sPD-L1 levels from baseline to the first tumor evaluation was associated with poorer progression-free survival (PFS) and overall survival (OS) [36]. Interestingly, the authors did not see any correlation between sPD-L1 concentration with tissue PD-L1, indicating that sPD-L1 can serve as an additional biomarker (in combination with tissue PD-L1) for treatment monitoring. Another study on sPD-L1 levels in 22 NSCLC patients before nivolumab treatment and after the first clinical evaluation (after 3 months of treatment) reported dynamic modulation of sPD-L1 levels with significantly higher levels observed in non-responding patients [26]. The pharmacodynamics of anti-PD-1 treatment and sPD-L1 were further observed in a large case control study. In 51 nivolumab-treated patients, plasma PD-L1 was assessed at the time of initiation of treatment and after 28 days of treatment. The results showed that the reduction in sPD-L1 levels after 28 days was associated with poor prognosis [25].

In addition to anti-PD-1 monotherapy, the role of sPD-L1 in combination therapy (chemotherapy in combination with immunotherapy) has also been investigated. A clinical trial by Bonomi et al. investigated the role of sPD-L1 in two randomized arms of patients: one arm with 10 patients on pembrolizumab monotherapy, and another arm with 10 patients on pembrolizumab combination with carboplatin and paclitaxel. Immune cell phenotyping to assess sPD-L1 levels in patients prior to treatment and at weeks 4 and 7. In both arms, increased baseline levels of sPD-L1 were associated with disease progression [37]. Another study conducted an open-label, randomized phase II trial encompassing three arms. Pembrolizumab was given to 7 patients for four cycles in Arm A, then four cycles of nab-paclitaxel in a sequential treatment regimen. Another 7 patients in Arm B followed a comparable sequential regimen, starting with four cycles of nab-paclitaxel followed by four cycles of pembrolizumab. Pembrolizumab and nab-paclitaxel were given concurrently to 6 patients in Arm C for four cycles of therapy. PFS was 10.1, 8.4, and 10.2 months for Arm A, B and C, respectively. Due to the approval of PD-1 blocking as the preferred first-line therapy, the trial was prematurely discontinued. However, it was shown that the administration of chemoimmunotherapy was successfully endured and capable of establishing long-term disease management in a particular subset of patients, in line with subsequent findings [38]. However, this is the only study in such a cohort, and larger studies on this aspect will be able to provide a better understanding of the kinetics of sPD-L1 in combination immunotherapy and its role as a biomarker of response.

Though limited studies have been performed on soluble PD-L1, the results of the studies are comparable with clear attribution of sPD-L1 in poor prognosis, indicating its role as a prognostic and predictive biomarker in immunotherapy-treated NSCLC patients.

4.1. Mechanism of Action of Soluble PD-L2

PD-L2 (CD273) is a 273-amino acid long-cell surface receptor, consisting of an immunoglobulin-like variable domain (Ig-V) and an immunoglobulin-like constant domain (Ig-C) in the extracellular region, a transmembrane domain, and a cytoplasmic domain [39]. Its expression is mainly found on dendritic cells, macrophages, and sometimes in T helper cell subsets and cytotoxic T cells [40]. PD-L2 serves as the second ligand for PD-1 and is therefore involved in regulation of T cell activity. Studies report contrasting role of PD-L2 with respect to immune regulation. On one hand, PD-L2 has been documented to function as an inhibitory receptor with its interaction with PD-1, leading to decreased cytokine production by CD4+T cells and inhibition of T cell receptor (TCR)-mediated proliferation [39]. Furthermore, some studies have reported its role in the activation of T cells via IL-12 production and reverse signaling in dendritic cells [41].

Though both PD-L1 and PD-L2 serve as ligands for PD-1 with similar influence on immune regulation, structural differences exist between both which affect their binding affinity. Studies have documented structural differences of amino acids homologous to their binding sites (in PD-L2, alanine exists at position 110 while tryptophan exists at position 121). In addition to this, PD-L2 contains N64PD-L2 glycan, which is absent in PD-L1. It is postulated that this structural change affects the “snug-fit” interaction of PD-L2 with PD-1, thus, weakening its binding affinity. However, PD-L2 carries a C–D latch region (which is missing in PD-L1), that facilitates the extension of PD-L2 contact area with PD-1. Therefore, the presence of this latch helps to counterbalance the weakness brought on by alanine and N64 glycan and instead increases its binding affinity for PD-1, approximately 3-4-fold higher than PD-L1 [42,43,44,45].

Studies have reported that soluble PD-L2 is generated due to a splicing event involving a frameshift of exon 3 to an alternative acceptor site 5 base pairs downstream of the canonical acceptor site. As a result, the generated soluble PD-L2 lacks the transmembrane domain but retains the IgV-like domain. It should be noted that since no deletion of the exon encoding transmembrane domain occurs during the splicing event, it is postulated that soluble PD-L2 can play a role in immune regulation due to its Ig-V domain and the existing exon for transmembrane region thus facilitating its binding with PD-1 [46].

4.2. Role of Soluble PD-L2 as a Biomarker in NSCLC Patients Treated with Immunotherapy

The role of soluble PD-L2 as a biomarker in immunotherapy-treated NSCLC patients has been reported in limited studies. A study on 22 NSCLC patients treated with Nivolumab assessed the role of soluble mediators in treatment response. Changes in the kinetics of sPD-L2 before initiation of treatment and after 6 cycles (3 months’ post-treatment) of Nivolumab showed interesting results. Dynamic modulation of sPD-L2 was observed, with levels of sPD-L2 significantly decreasing during nivolumab treatment in responding patients (though non-significantly). However, a combination of decreased levels of sPD-L2 with soluble mediators PD-L1, CD137, Tim-3, and BTLA-4 during treatment was significantly associated with a favorable clinical response. This suggests that these soluble markers may work synergistically to modulate the tumor microenvironment. Interestingly, the authors also found a prognostic value of sL2. Low levels of sPD-L2 before treatment initiation were significantly associated with longer clinical response indicating the prognostic value of pre-treatment sPD-L2 as a biomarker of response [26]. Another research investigated the potential relevance of sPD-L2, sPD-L1, sIL-2, sIFN-γ, granzyme B and microRNAs as indicators of ICI responsiveness among 43 NSCLC patients treated with Nivolumab, the results of sPD-L2 demonstrated no association with clinical outcomes. Additionally, grade 3–4 toxicities were linked to low sPD-L2, low sIL-2, and high sIFN-γ [47]. Another study on 43 patients in Nivolumab-treated NSCLC patients aimed to assess the role of sPD-L2 as a predictive biomarker. Plasma sPD-L2 levels were assessed at diagnosis, at the start of treatment and after 2 months (first tumor evaluation). It was observed that, though sPD-L2, was not associated with progression-free or overall survival, lower levels of sPD-L2 at diagnosis and start of treatment were significantly associated with grade 3–4 adverse events. This suggests a dynamic role for this biomarker in predicting immune-related adverse events [36].

5.1. Mechanism of Action of Soluble CTLA-4

CTLA-4, binds to CD80 and CD86 ligands present on APCs and regulates the immune system. The second signal needed for complete T cell activation and proliferation is blocked by this interaction between the ligands and the CD28 receptor on T cells. Through efficient inhibition of T cell activation caused by the blockage of the CD28-CD80/CD86 interaction, CTLA-4 produces an immunosuppressive effect [48]. This suppression can be harmful in the setting of cancer by impeding the body’s capacity to generate an efficient anti-tumor response, but it is advantageous in avoiding hyperactive immune responses and autoimmunity [49]. The method by which CTLA-4 controls immunological responses demonstrates its dual function in immune regulation. On the one hand, it promotes immunological homeostasis and the prevention of autoimmune disorders by suppressing T cell activation [50]. However, in the case of cancer, this same technique can be detrimental since it reduces the immune system’s capacity to efficiently fight tumor cells. This complicated role of sCTLA-4 emphasizes the need for more study to better understand its activities and create ways to modulate its levels, thereby improving treatment outcomes in cancer patients [51].

In addition to being expressed on the cell surface, the CTLA-4 protein is also found in soluble form as soluble CTLA-4 (sCTLA-4), which was initially discovered by Magistrelli et al. Non-activated T cells release the fully functional 137-amino-acid polypeptide sCTLA-4, which is also seen in normal human serum. Extracellular, transmembrane, and cytoplasmic sections make up mature CTLA-4 molecules. Although having no transmembrane domain, sCTLA-4 secretes from cells while maintaining its amino acid residues that bind to B7 [52]. Although sCTLA-4 lacks the transmembrane domain, which allows cell secretion, mature CTLA-4 has extracellular, transmembrane, and cytoplasmic sections. Additionally, in sCTLA-4, the amino acid residues that bind to B7 remain unchanged [53]. In vitro research on human T cells has demonstrated that sCTLA-4 release peaks throughout immunological responses and has a potent inhibitory effect. In particular, when sCTLA-4 is blocked, antigen-driven proliferation and cytokine release are markedly enhanced [54]. Recent studies indicate that solublesCTLA-4 has a significant down-regulatory influence on the immune response by preferentially binding to B7 molecules on antigen-presenting cells (APCs), as opposed to CD28 on T lymphocytes [52].

In cancer patients, notably individuals with NSCLC, increased levels of sCTLA-4 have been linked to poorer clinical results when treated with ICIs [55]. According to studies, high plasma levels of sCTLA-4 are associated with a lack of reactivity to therapy such as anti-PD-1 medications [56]. This shows that sCTLA-4 might serve as a predictive biomarker, assisting in identifying individuals who are less likely to benefit from ICI therapy and possibly driving more tailored treatment methods [57].

5.2. Role of Soluble CTLA-4 as a Biomarker in NSCLC Patients Treated with Immunotherapy

sCTLA-4 is a potential biomarker for determining the effectiveness of immunotherapy in patients with NSCLC. Recent research has investigated its ability to predict treatment response and correlate with clinical outcomes [57]. For example, a study conducted by Hayashi et al. [51] investigated the role of sCTLA-4 as a biomarker in 50 individuals with NSCLC who received nivolumab. Higher baseline levels of sCTLA-4 were related to worse progression-free survival, indicating a negative relationship between sCTLA-4 levels and the therapeutic response. Patients with high sCTLA-4 levels had a poorer response to nivolumab. The study found a significant p-value (<0.05), highlighting the strength of the correlation [51]. A study of 536 patients with primary resected stage I–IIIA NSCLC evaluated the immunohistochemistry expression of CTLA-4 in the tumor and stromal tissues of the main tumors, as well as the locoregional metastatic lymph nodes. The study discovered that CTLA-4 expression in primary tumors’ tumor epithelial cells (T-CTLA-4) and stromal cells (S-CTLA-4) were not correlated with each patient’s disease-specific survival (DSS). In the squamous cell carcinoma subgroup, however, increased sCTLA-4 expression predicted significantly better DSS (HR 0.62, 95% CI 0.41–0.93, p = 0.021). On the other hand, there was a negative predictive effect associated with T-CTLA-4 expression in metastatic lymph nodes (HR 1.65, 95% CI 1.03–2.65, p = 0.039). These results reveal significant variations in respective tumor microenvironments and imply that CTLA-4 expression has different prognostic consequences in original tumors vs. metastatic NSCLC lymph nodes [58].

6.1. Mechanism of Action of Soluble LAG-3

Lymphocyte-activation gene 3 (LAG-3) or CD223 is an immunological checkpoint receptor that regulates immune responses, namely self-tolerance and autoimmunity. LAG-3 is expressed on a variety of cell types, including CD4+ and CD8+ T cells, as well as Tregs, and is necessary for proper T cell regulation and homeostasis [59]. While LAG-3 is often investigated in its membrane-bound variant on the surface of T cells, sLAG-3 may also be found in the extracellular environment. The mechanism of action for soluble LAG-3 differs from that of its membrane-bound counterpart and includes numerous critical roles [60]. LAG-3 is thought to regulate the immune response by attaching MHC-II molecules to APCs. This connection can alter the activation and function of many immune cells, including APCs, dendritic cells, and T-cells, and hence affect the overall immune response [59]. LAG-3 suppresses the proliferation and activation of T cells by binding to MHC-II on APCs, and this helps to prevent the immune system from becoming overactive, which can result in autoimmunity or chronic inflammation [61].

LAG-3 plays a major role in stimulating the development of regulatory T cells (Tregs), which helps reduce exaggerated immune responses and promotes immunological tolerance and homeostasis [62]. Additionally, LAG-3 interacts with immune checkpoint molecules such as CTLA-4 and PD-1 to form a complex regulatory network that modulates the immune response. These interactions can strengthen the inhibitory signals produced by these checkpoints, further suppressing the immune response. The interaction of several checkpoints demonstrates the complexity of immune regulation and the potential for addressing these pathways in therapeutic procedures [63].

sLAG3, a soluble form of LAG3, is formed by alternative splicing and proteolytic cleavage of the extracellular domain and is mostly secreted by dendritic cells (DCs), but also by B and T lymphocytes, and its function is largely unknown. Some studies suggest its involvement as a costimulatory molecule by binding to MHC-II and encouraging DC maturation; however, these are largely connected to synthetic sLAG3-Ig proteins employed in therapeutics, which have a high MHC-II affinity compared to human sLAG3 [64]. However, human sLAG3 has been postulated to function as a co-inhibitor of immune responses by decreasing monocyte development and antigen presentation capabilities, inhibiting their activation of T-cell proliferation [65].

6.2. Role of Soluble LAG-3 as a Biomarker in NSCLC Patients Treated with Immunotherapy

sLAG-3 has emerged as a promising biomarker in NSCLC, providing insights into tumor immune suppression and treatment response by reflecting the dynamic interaction of immunological checkpoints and tumor growth [63]. A study examined soluble lymphocyte activation gene-3 (sLAG-3) and CD4/CD8 ratio dynamics as biomarkers for predicting outcomes in advanced solid cancer patients following immune checkpoint inhibitor (ICI) treatment. Higher pre-treatment sLAG-3 levels were linked with poor progression-free (HR: 1.005, p = 0.039) and overall survival (HR: 1.006, p = 0.015) outcomes, but a lowering CD4/CD8 ratio following therapy was associated with improved overall survival (p = 0.012, HR: 3.32). A combined immunological score that included sLAG-3 levels and CD4/CD8 ratio dynamics had the highest predictive potential for survival (HR: 10.3), indicating that they might be used as robust biomarkers to guide ICI treatment [64]. Another study looked at the role of soluble LAG-3 (sLAG-3) as a biomarker in non-small cell lung cancer (NSCLC) and how it is associated with the disease stage. Serum sLAG-3 levels were evaluated using enzyme-linked immunosorbent assay (ELISA) in 247 individuals, comprising 71 with benign illnesses and 176 with NSCLC. The study discovered that sLAG-3 levels were significantly higher in early-stage NSCLC (stage I-II) compared to later stages (stage III-IV, p < 0.001). This suggests that lower sLAG-3 expression in advanced NSCLC may indicate a weaker immune response. The findings emphasize the possibility of boosting sLAG-3 levels as a treatment option for advanced NSCLC [66].

7.1. Mechanism of Action of Exosomes

The endosomal compartment of most eukaryotic cells produces membrane extracellular vesicles, or exosomes, typically ranging in size from 30 to 150 nanometers. A diverse range of biological elements, including proteins, short RNAs, lipids, deoxyribonucleic acids (DNAs), messenger RNAs (mRNAs), and more, are found within the lipid bilayer structure that makes up exosomes. By transmitting these useful proteins, these extracellular vehicles (EVs) fulfill their role as mediators of intercellular communication [67,68]. Specifically, exosomes are actively released by cancer cells into the circulation, where they travel throughout the body and become a component of the intricate communication network that is supported by the tumor microenvironment. These exosomes can promote cellular communication and function as non-invasive cancer indicators. By interacting with the immune cell protein PD-1, this substance can aggressively inhibit CD8+ cells. Exosomes can also prevent monocyte differentiation, decrease NK cell function, and transform myeloid precursor cells into myeloid suppressor cells. In vitro studies have demonstrated that elevated levels of exosomal PD-L1 can inhibit T cell activation, even though the role of exosomal PD-L1 in vivo is still unknown. They are often found in a variety of physiological fluids. Exosomal-PD-L1 is noticeably detectable in human plasma and has the potential to become a crucial biomarker [69,70].

7.2. Role of Exosomal PD-L1 as a Biomarker in NSCLC Patients Treated with Immunotherapy

Studies have investigated the relationship between the expression of PD-L1 mRNA in exosomes obtained from plasma and the responsiveness of anti-PD-1 antibodies in melanoma and NSCLC. For example, a study was conducted by Del Re et al. (2018) [71] on 8 patients with non-small-cell lung cancer (NSCLC) where 2 individuals had a partial response (PR), 2 had stable disease (SD), and 4 had progressive disease (PD). The study demonstrated that the response to anti-PD-1 antibodies in people with melanoma and non-small cell lung cancer (NSCLC) was strongly correlated with the presence of PD-L1 mRNA within exosomes extracted from plasma. The tumor response was correlated with the number of copies of PD-L1 mRNA per milliliter. number of copies of PD-L1 mRNA per milliliter was related to tumor response. Patients with PD showed a considerable increase (mean: 295 to 550 copies per mL, from baseline to 2 months), but those who achieved PR showed a decrease (mean: 1015 to 300 copies per mL). Patients with SD showed a moderate change (mean: 455 to 285 copies per mL). 231 ± 55.7, p = 0.012). There was a noticeable variation in the number of PD-L1 mRNA copies per milliliter in the entire research cohort. This difference was statistically significant in with progressing disease (204 vs. 416, baseline vs. 2 months later, p = 0.001) and patients exhibiting complete or partial responses (830 at baseline compared to 242 after 2 months, p = 0.016). The difference between patients with stable disease (298 at baseline vs. 247 after 2 months, p = 0.586) was not as noticeable as expected. It must be noted that, at first, people who had complete and partial responses had much more PD-L1 mRNA copies per milliliter than people who had stable disease or who were progressing from their disease (830 vs. 231 p = 0.012) [71]. In a trial of 17 patients with advanced or metastatic NSCLC, researchers investigated the possibility of serum-derived exosomal PD-L1 (exoPD-L1), exoPD-L2, and exoPD-1 as biomarkers for monitoring immunotherapy responses. The study discovered that exoPD-L1 levels in patients were substantially greater than in their soluble forms and healthy controls. Importantly, a drop in exoPD-L1 levels was related to better treatment outcomes, whereas an increase was connected to tumor growth. The study indicated that exosomal biomarkers may be more successful than standard tissue biopsies in predicting and monitoring treatment results in NSCLC patients undergoing immunotherapy [72].

In another study, the clinical significance of Exo-PD-L1, sPDL1, and the overall PD-L1 profile was investigated in 65 stage I-IIIA NSCLC patients, who had not previously received therapeutic procedures such as chemotherapy, targeted therapy, surgical removal, or radiation. The results showed a concentration of 1.84 pg/mg exosomal PD-L1 protein concentration in healthy controls, whereas in patients with stage I/II and III/IV the levels were 3.02–1.67 pg/mg and 5.17–3.16 pg/mg respectively. In healthy controls the level of Exo-PD-L1 was low when compared to affected individuals. Furthermore, large tumor size and advanced disease with profound metastasis was associated with higher exo-PD-L1 indicating its potential to be used as a biomarker in NSCLC [73].

8.1. Mechanism of Action of Cytokines

Cytokines, such as tumor necrosis factors (TNF), interleukins, interferons, and chemokines, as well as growth factors including TGF-β, VEGF, and EGF, can promote or inhibit tumor growth depending on external factors [74]. Cytokines, being important immune response regulators, promote inflammation and modulate several intracellular signaling pathways that govern the growth, proliferation, and migration of cells, contributing to the development as well as the advancement of cancer [75] (Table 2). Recent improvements in targeting these molecules have resulted in the creation of innovative treatment approaches, including monoclonal antibodies, bispecific antibodies, receptor inhibitors, fusion proteins, and synthetic cytokine variations [76]. These techniques seek to control the immune system, slow tumor growth, and overcome resistance to standard treatments. Combining these tailored medicines with additional therapeutic options shows promise for improving patient outcomes. Ongoing research and clinical trials are crucial for improving our understanding and implementation of cytokine- and chemokine-targeted treatments in cancer [77].

8.2. Mechanism of Action of Pro-Inflammatory Cytokines

8.3. Mechanism of Action of Anti-Inflammatory Cytokines

8.4. Transforming Growth Factor-Beta (TGF-β)

TGF-β is a multifunctional cytokine that is essential for controlling tissue healing, cell division, and immunological responses. TGF-β binds to its receptor complex, which consists of TGF-β receptor types I and II (TGFBR1), on target cells to produce its effects [104]. SMAD proteins, especially SMAD2 and SMAD3, are phosphorylated and activated through the activation of receptor serine/threonine kinases resulting from this interaction. The phosphorylated SMAD2/3 forms a complex with SMAD4 that translocates into the nucleus and affects the transcription of genes involved in immunological modulation, cell proliferation, and differentiation [105]. By stimulating the formation of regulatory T cells (Tregs) and reducing the generation of pro-inflammatory cytokines, TGF-β prevents the activation and proliferation of immune cells, including T cells and B cells. Subsequently, it also affects macrophage and dendritic cell function, diminishing their capacity to trigger T-cell responses and inflammatory cytokine production [106]. TGF-β plays a role in immune regulation, fibrosis, and cancer progression. It promotes EMT, increases extracellular matrix production, and creates an immunosuppressive microenvironment, facilitating tumor progression. TGF-β has a critical part in both normal and pathological processes, as evidenced by its various roles [107].

8.5. Role of Pro and Anti-Inflammatory Cytokines as Biomarkers in NSCLC Patients Treated with Immunotherapy

In a study, serum-derived exosomes from 17 NSCLC receiving ICI treatment were assessed for TNF-alpha levels. According to the research, TNF-alpha was increased in responding patients, and this was linked to a considerable rise in exosomal PD-1 (p = 0.0156) and IFN-γ (p = 0.0156). TNF-alpha may be a sign of immune system activation and treatment response, while non-responding individuals showed a distinct cytokine profile. These findings highlight the potential of TNF-alpha as a useful biomarker for evaluating and forecasting the effectiveness of ICI treatment in patients with NSCLC [72]. A study of 29 stage IV NSCLC patients receiving single-agent PD-1 inhibitors showed high baseline levels of inflammatory markers. The results were associated with worse PFS, with p-values < 0.001. Increased IFN-γ levels were linked to improved PFS (p < 0.001). Long-term responders (PFS > 30 weeks) showed decreased IL-6, NLR, and SII levels, but greater IFN-γ levels (all p-values < 0.001). Inflammatory indicators increased throughout time, indicating that the condition was progressing. There was no significant correlation between these indicators and irAEs [108]. According to another study, cytokine levels in untreated advanced NSCLC patients were higher than in healthy controls. Chemotherapy was shown to reduce these levels, whereas immunotherapy alone did not. With hazard ratios of 0.28–0.34 for OS and 0.42–0.54 for PFS lower baseline TNF-RI levels were linked to improved survival outcomes. P-values ranged from 0.014 to 0.001. irAEs were associated with higher baseline levels of IL-1β and angiogenin, with fold increases ranging from 7–9 and p-values from 0.009 to 0.0002. Nonetheless, neither the course of the disease nor its clinical prognosis was predicted by alterations in cytokine levels after therapy. irAEs and treatment benefits may be predicted with baseline cytokine levels, but long-term illness monitoring is not possible with these measurements [109].

In a study of 88 participants (53 NSCLC patients, 17 with non-malignant lung diseases, and 18 healthy volunteers), researchers discovered that NSCLC patients had a higher number of CD4(+) CD25(+) FoxP3(+) regulatory T (Treg) cells with increased expressions of immunosuppressive molecules such as CTLA-4, LAG-3, and PD-1. Intratumorally Treg cells had the greatest quantities of these co-inhibitory compounds, indicating a potent immunosuppressive action. Additionally, the number of Treg cells and their functional molecules increased as cancer progressed. NSCLC patients had greater plasma levels of TGF-β, and IL-10 compared to healthy controls, indicating the importance of Treg cells in tumor microenvironments [110]. Another study of 60 NSCLC stage III/IV patients treated with anti-PD-1-based chemotherapy found that greater on-treatment IL-1β and IFN-γ levels correlated with better outcomes, including enhanced response and extended PFS (p = 0.013 and p = 0.007, respectively). In contrast, higher on-treatment IL-6 levels were associated with a worse response, shorter PFS, and OS (p = 0.012, p = 0.020, and p = 0.037, respectively). High pretreatment IL-2 levels indicated longer PFS (p = 0.049), but high IL-8 levels predicted shorter OS (p = 0.006) [111]. In addition, a trial of 102 NSCLC patients undergoing immunotherapy showed how increased CXCL12 levels were linked with significantly poorer outcomes, including shorter OS (12.20 vs. 44.84 months, p = 0.008) and PFS (3.76 vs. 14.40 months, p < 0.001). Patients with PD-L1 positive had superior OS (44.84 vs. 20.42 months, p = 0.087) and ORR (70.0% vs. 28.8%, p < 0.001). PD-L1 positive patients exhibited a significantly longer median PFS (25.35 vs. 4.64 months, p = 0.003). Patients with high CXCL12 levels and low PD-L1 expression had the worst results, with a lower ORR (27.3% vs. 73.7%, p < 0.001) and shorter PFS (2.44 vs. 25.35 months, p < 0.001). Combining PD-L1 and CXCL12 levels has a higher predictive value for clinical outcomes.

By enabling customized therapies based on individual biomarker profiles, the use of cytokines, chemokines, and new interleukins as cancer biomarkers has the potential to transform personalized medicine by increasing therapeutic efficacy and reducing adverse effects. Strict clinical verification and standardized testing procedures are necessary to guarantee these biomarkers’ dependability [112]. Enhancing treatment methods and providing a full knowledge of cancer biology can be achieved by integrating data on cytokines and chemokines with genomes, proteomics, and metabolomics. Technological developments like liquid biopsies and high-throughput tests will enhance biomarker sensitivity and non-invasive monitoring, enabling more accurate tracking of cancer growth and treatment outcomes [113]. Management of patients and customized therapy planning will be improved by incorporating cytokine and chemokine data into clinical decision support systems. For novel biomarkers to be clinically adopted, it is essential that they satisfy regulatory requirements and that ethical issues be addressed. Lastly, global collaboration in research and the involvement of varied populations will speed the creation and validation of novel biomarkers, ensuring widespread use and efficacy [112].