Exploring the Tumor Microenvironment: Macrophages and PD-1 Checkpoint Inhibitors

This review provides an in-depth examination of the tumor microenvironment, macrophages, and PD-1 checkpoint inhibitors. It delves into the fundamental aspects of the tumor microenvironment and macrophages, discussing the evolution of key concepts, the involvement of macrophages in tumor progression, and their interactions with tumor cells. The historical development of PD-1 checkpoint inhibitors is explored, detailing their discovery, early clinical trials, and initial challenges. Current insights into macrophages within the tumor microenvironment are presented, including their polarization, mechanisms of immunosuppression, and diagnostic techniques. The clinical application of PD-1 inhibitors is reviewed, encompassing therapeutic strategies, comparative efficacy across various cancers, and management of side effects. Additionally, the paper addresses technological advancements in tumor microenvironment research, future directions, and ongoing controversies, aiming to provide a comprehensive overview to inform further research and clinical applications.

Evolution of tumor microenvironment concepts

The understanding of the Tumor Microenvironment (TME) has undergone substantial evolution over time. Initially perceived merely as the surrounding milieu of tumor cells, the TME is now acknowledged as a complex and dynamic ecosystem [1]. For instance, gliomas arise in a distinct Tumor Microenvironment (TME) that includes a variety of cells such as those residing in the brain, cells of the immune system and blood vessels. These cells engage in reciprocal interactions that influence the development of the TME. Mouse models play a pivotal role in elucidating the processes and mechanisms underlying TME evolution [2].

Cellular communication within the Tumor Microenvironment (TME) has become recognized as a critical area of study. Innovative mechanisms such as vesicle transfer and cell fusion are increasingly recognized for their roles in cellular reprogramming within the TME [3]. Specifically, the information exchange between cells and the matrix, among tumor cells, and between tumor cells and the host immune system is mediated through exosomes and microvesicles. This interaction significantly impacts our comprehension of how tumors start and develop. Furthermore, the TME is characterized by spatial heterogeneity, with conditions that differ significantly from those in healthy tissues. Aneuploidy and polyploidy in cancer cells engage in a bidirectional interaction with the TME, wherein abnormal cells alter the TME, which in turn selects for the most adaptable cells under specific conditions [4].

Role of macrophages in tumor progression

Macrophages are pivotal in tumor progression, affecting various processes such as immune response modulation, angiogenesis, and tissue remodelling. Their interactions with tumor cells can facilitate tumor growth and metastasis, underscoring their importance in cancer development.

Macrophages are crucial and have multiple roles in the advancement of tumors. The primary stromal cells present in the Tumor Microenvironment (TME) are Tumor-Associated Macrophages (TAMs), can differentiate into either anti-tumorigenic (M1) or pro-tumorigenic (M2) phenotypes [5]. In advanced tumors, M2-like TAMs primarily modulate the TME. The release of multiple cytokines, chemokines, and growth factors by them encourages tumor growth. The tumor microenvironment, macrophages, immune cell surface PD-1/PD-L1, and the interaction between tumor cells (Figure1).

Figure 1: Macrophages play a crucial role in tumor progression. Tumor-associated macrophages (TAMs) are the main stromal cells in the tumor microenvironment and can differentiate into anti-tumor (M1) or pro-tumor (M2) phenotypes. M2 TAMs mainly regulate the Tumor Microenvironment (TME), while M1 TAMs exert direct antitumor effects. PD-1/PD-L1 can directly act on tumor cells.

In the context of breast cancer, TAMs within the adipose microenvironment contribute to tumor progression. A portion of these macrophages expresses CD163, CCL2, and CCL5, which points to an immunosuppressive phenotype [6]. Macrophages associated with tumors are crucial in enhancing the development of new blood vessels, facilitating tumor cell metastasis, and contributing to multidrug resistance [7], thereby significantly advancing tumor progression in hepatocellular carcinoma. Macrophages can differentiate into M2-like TAMs, which possess immunoregulatory functions, contribute to the polarization of Th2 responses, and support tumor progression [8]. The emerging understanding of the phagocytic function of TAMs introduces a novel mechanism for cancer treatment. Boosting the phagocytic function of TAMs could strengthen natural cancer-fighting immunity and encourage T-cell-driven responses of the adaptive immune system [9].

Interaction between macrophages and tumor cells

The interplay between macrophages and tumor cells represents a complex and reciprocal process. Within the framework of Small Cell Lung Cancer (SCLC), Tumor-Associated Macrophages (TAMs) are implicated in tumor progression via the activation of STAT3 [10]. Immunohistochemically analyses indicate that the activation of STAT3 in tumor cells mainly occurs in the peripheral regions of tumor nests, adjacent to TAMs within the stromal compartment. Studies using In vitro co-culture have shown that when SCLC cells are indirectly co-cultured with macrophages, STAT3 is activated in both types of cells. The activation of STAT3 in SCLC cells is markedly enhanced by the culture supernatant derived from macrophages, thereby facilitating tumor cell proliferation, invasion, chemo resistance, and sphere formation.

Similarly, glioblastoma is characterized by intricate Interactions among cancerous cells and TAMs [11]. Cancer cells emit chemokines, cytokines, and growth factors to recruit TAMs, which in turn secrete factors that establish an immunosuppressive and tumor-promoting microenvironment. In papillary thyroid cancer, CD36+ pro-inflammatory macrophages interact with ZCCHC12+ tumor cells. These CD36+ macrophages secrete SPP1, which activates the PI3K-AKT signalling pathway in cancer cells, thereby augmenting their proliferation [12].

Macrophage polarization in tumor context

Within the tumor microenvironment, macrophages can differentiate into distinct phenotypes, predominantly the pro-inflammatory M1 and the anti-inflammatory M2 types. In this context, macrophages often polarize towards the M2 phenotype, which facilitates tumor progression [ 13]. Metabolic reprogramming plays a critical role in macrophage polarization, enabling both tumor cells and macrophages to meet their energy demands. A comprehensive understanding of the metabolic transitions between pro-tumor and anti-tumor states in Tumor-Associated Macrophages (TAMs) is essential for elucidating immune escape mechanisms [14].

Glutamine is recognized as an essential metabolite for both M1 and M2 macrophage types. Studies using monoculture and coculture cell models indicate that M1 polarization relies more heavily on the availability of glutamine in the culture environment compared to M2 polarization. A deficiency in glutamine impairs M1 polarization, whereas its impact on M2 polarization is comparatively milder [15]. Furthermore, apoptotic tumor cells can modulate macrophages to adopt a phenotype that facilitates tumor growth. Soluble mediators, such as Sphingosine-1-Phosphate (S1P) derived from apoptotic cells, can induce the expression of arginase 2 in macrophages, thereby reducing citrulline/nitric oxide synthesis while enhancing ornithine production [16].

Mechanisms of macrophage - mediated immunosuppression

Macrophages play a pivotal role in mediating immunosuppression through diverse mechanisms. In the context of sepsis, mitochondrial dysfunction within macrophages is closely linked to immunosuppressive outcomes. Specifically, the S1PR2 receptor on macrophages exacerbates sepsis-induced immunosuppression by promoting mitochondrial fragmentation [17]. This is evidenced by the elevated expression of S1PR2 in peripheral blood monocytes of septic patients, which is concomitant with mitochondrial fragmentation and dysfunction. The activation of S1PR2 triggers mitochondrial fragmentation in macrophages via the activation of ROCK I, which subsequently phosphorylates Drp1, thereby playing a crucial role in septic immunosuppression.

Furthermore, inside the microenvironment of the tumor, the interaction between tumor cells and macrophages mediated by CD147-K148me2 induces immunosuppression in Non-Small Cell Lung Cancer (NSCLC) through the CCL5/CCR5 pathway [18]. The interaction between CD147 and cyclophilin A is strengthened by the demethylation of CD147 at lysine 148, which in turn activates the p38–ZBTB32 signalling pathway to increase CCL5 gene transcription. This process increases CCL5 secretion from NSCLC cells, thereby promoting the infiltration of M2-like Tumor-Associated Macrophages (TAMs) in NSCLC tissues through CCL5/CCR5 axis-dependent communication between macrophages and cancer cells.

Diagnostic techniques for tumor - associated macrophages

Precisely identifying Tumor-Associated Macrophages (TAMs) is essential for comprehending tumor development and creating effective treatments. One promising method involves the application of nanoparticle-based Magnetic Resonance Imaging (MRI). Specifically, iron oxide nanoparticles with Superparamagnetic Properties (SPIONs) are used to non-invasively evaluate TAMs in animal studies and clinical research [19]. Tumors and metastases exhibiting macrophage infiltration tend to accumulate these iron oxide nanoparticles, resulting in a decreased T2-relaxation time and generating negative (dark) contrast in quantitative T2-weighted MRI (qT2wMRI).

One different method is to generate monocyte-derived Tumor-Associated Macrophages (TAMs) In vitro using media conditioned by tumors [15]. The co-expression of CD163/CD206 is more pronounced in these TAMs than in M2-like macrophages, with expression rates of 87% and 36%, respectively. They also exhibit elevated levels of transcripts for functional markers such as IL-6, IL-10, CCL2, c-Myc, iNOS, and arginase. Functionally, In vitro-generated TAMs suppress T cell proliferation by 47% relative to M1-like macrophages and reduce IFN-γ production by natural killer cells by 44%.

The exploration and advancement of the PD-1 pathway

The development and identification of the PD-1 pathway constitute a key advancement in the field of cancer immunotherapy. In 1992, Honjo and his team at Kyoto University identified PD-1, marking the beginning of extensive research into its functions [20]. Over the past two decades, research on human PD-1 can be delineated into three distinct phases. Initially, the elucidation of the PD-1 gene's structure and genomic organization provided a foundational understanding. Subsequently, it became vital to grasp the mechanism by which PD-1, along with its ligands PD-L1 and PD-L2, manages immune checkpoints. PD-1 plays a critical role in modulating immune responses and enhancing self-tolerance by inhibiting T cell activity and facilitating the differentiation of regulatory T cells [21].

Preclinical animal studies indicate that PD-1 blockade effectively inhibits tumorigenesis and metastasis while exhibiting fewer side effects compared to CTLA-4 blockade. This finding has led to the creation of monoclonal antibodies tailored for human use, such as nivolumab, which targets human PD-1. Notably, PD-1 blockade exerts its therapeutic effects by targeting lymphocytes rather than tumor cells, allowing its efficacy to persist despite mutations that may occur during tumorigenesis. Additionally, its therapeutic applicability spans multiple tumor types, as it does not depend on specific tumor antigens. Nonetheless, challenges persist, particularly concerning the wide variability in response rates to PD-1 immune-checkpoint inhibition therapy across different cancer types, which range from 18% to 87% [22].

Initial clinical evaluations of agents that inhibit PD-1

Initial clinical trials of PD-1 inhibitors have played a pivotal role in evaluating their efficacy and safety. A meta-analysis encompassing 51 pairs of early-phase and phase III clinical trials for PD-1/PD-L1 inhibitors demonstrated that early-phase trials consistently overestimated the outcomes observed in phase III trials, with an Odds Ratio (OR) of 1.66 for the Overall Response Rate (ORR) (95% CI 1.43 to 1.92, p < 0.05) [23]. This overestimation was observed consistently in trials evaluating both PD-1/PD-L1 monotherapies and combination therapies.

Challenges in the application of PD-1 inhibitors

One of the primary challenges in the application of PD-1 inhibitors is the heterogeneous response rate observed across various cancer types. In lymphoid malignancies, although immunochemotherapy can achieve remission in some patients, a significant number experience relapse or disease progression despite undergoing intensive treatment. Further research is required to elucidate the role of PD-1 activation and its potential as a target for therapy in these malignancies [24]. The PD-1/PD-L1 pathway is crucial for tumors to evade the immune system, which is pivotal in the pathogenesis and progression of leukaemia [25].

Furthermore, a significant issue is the absence of dependable biomarkers to predict how individual patients will respond to PD-1 inhibitory immunotherapy. Patients may exhibit primary, adaptive, or acquired resistance to PD-1 immune checkpoint inhibitors, and some may develop hyper progressive disease after treatment. These issues highlight the urgent need for further investigation to understand the mechanisms underlying responses to PD-1 inhibitors and to develop strategies to overcome resistance.

Current approaches to treatment with PD-1 inhibitors

Combination therapies incorporating PD-1 inhibitors are increasingly recognized as significant therapeutic strategies. A meta-analysis examining the combination of PD-1/PD-L1 inhibitors, radiotherapy, and anti-angiogenesis agents in solid tumors demonstrated a pooled overall response rate of 59% (95% CI: 48-70%), a disease control rate of 92% (95% CI: 81-103%), and a complete remission rate of 48% (95% CI: 35-61%) [26]. In contrast, neither monotherapy nor dual-combination treatments improved overall survival (HR = 0.499, 95% CI: 0.399-0.734) or progression-free survival (HR = 0.522, 95% CI: 0.352-0.774) when compared to the triple regimen.

Checkpoint inhibitors that target PD-L1 and PD-1 have revolutionized the therapeutic strategy for advanced urothelial bladder cancer. Currently, five approved agents are available for the management of platinum-refractory bladder cancer. In a randomized Phase III clinical trial, pembrolizumab exhibited superior efficacy compared to standard chemotherapy. Both pembrolizumab and atezolizumab have received FDA approval and are well-tolerated as first-line treatments for patients who are ineligible for cisplatin-based therapy [27].

Evaluating the relative effectiveness of PD-1 inhibitors across various cancer types

The effectiveness of PD-1 inhibitors exhibits variability across different cancer types. In a comparative study of PD-L1 and PD-1 inhibitors for Small Cell Lung Cancer (SCLC), the Overall Response Rate (ORR) was observed to be 50.0% in the PD-L1 cohort, compared to 36.8% in the PD-1 cohort, with no statistically significant difference (p = 0.308). The median survival durations were 21.0 months for the PD-L1 group and 17.0 months for the PD-1 group, also showing no statistically significant difference (p = 0.180) [28]. Additionally, the PD-L1 group experienced significantly milder bone marrow suppression and gastrointestinal adverse effects.

A network meta-analysis encompassing 37 randomized controlled trials and involving a total of 31,779 patients revealed that, in the context of Non-Small Cell Lung Cancer (NSCLC), the agents tislelizumab, pembrolizumab, and nivolumab significantly improved overall survival in comparison to chemotherapy. Among these, tislelizumab exhibited the maximum probability of being the most efficacious treatment in terms of enhancing overall survival and disease control rate. While cemiplimab and tislelizumab were associated with the greatest likelihood of improving progression-free survival, no statistically notable distinctions were observed among all PD-1/PD-L1 inhibitors [29].

Side effects and management of PD - 1 inhibitor therapy

Programmed Death-1 (PD-1) inhibitor therapy is associated with a range of adverse effects. In a study comprising 496 patients with metastatic melanoma who were administered nivolumab or pembrolizumab, 138 individuals experienced a total of 242 adverse events. These events predominantly affected the integumentary, gastrointestinal, hepatic, endocrine, and renal systems. Notably, the rare adverse reactions comprised the emergence of lichen planus, diabetes mellitus, and pancreatic insufficiency as a result of pancreatitis [30].

immune-related Adverse Events (irAEs) involving the ocular system may also occur. An analysis of 70 case reports indicated that the most prevalent malignancies were melanoma (n = 41; 58.6%) and lung cancer (n = 13; 18.6%). The primary PD-1 inhibitors administered were pembrolizumab (n = 38; 54.3%) and nivolumab (n = 28; 40%). The most frequently observed ocular complications were uveitis (n = 35; 50%) and myasthenia gravis (n = 13; 18.57%). The majority of these toxicities were responsive to treatment with topical and systemic steroids. However, severe cases may necessitate the temporary or permanent discontinuation of PD-1 inhibitor therapy [31].

Novel imaging techniques for tumor microenvironment

Advanced imaging techniques are offering novel insights into the tumor microenvironment. Uncommon side effects included the development of lichen planus, diabetes mellitus, and pancreatic deficiency due to pancreatitis [32]. These techniques allow for the study of cells within the complete tumor microenvironment in a mouse, facilitating prolonged observation of cellular interactions and the analysis of specific cell types，Interactions occur between T cells, neutrophils, monocytes, and the vasculature of tumors as well as cancer cells.

Molecular imaging modalities, including Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and fluorescence imaging, offer real-time, non-invasive, and longitudinal approaches for investigating the Tumor Microenvironment (TME) [33]. The Targeted Molecular Localization (TML) technique utilizes a singular imaging sequence and reconstruction process to co-localize super-resolved vasculature with molecular imaging signatures, thereby providing simultaneous anatomical and biological insights that may facilitate multiscale disease assessment [34] (The TML method employs a single imaging sequence and reconstruction technique to simultaneously map super-resolved blood vessels and molecular imaging markers. Thus offering concurrent anatomical and biological insights that could aid in multiscale disease evaluation). In a murine hind limb tumor model, the TML method achieved microvasculature resolution as fine as 28.8 μm [34].

Genomic and proteomic approaches to study macrophages

Genomic and proteomic methodologies are advancing our comprehension of macrophage biology. Genetic and genomic strategies have been employed to elucidate macrophage identity and functionality. Recent studies propose a model in which lineage-determining and signal-dependent transcription factors interact in a collaborative and hierarchical manner to delineate macrophage identity and function [36]. Additionally, natural genetic variation serves as a valuable tool for elucidating the context-dependent acquisition of specific macrophage phenotypes.

Proteomic analysis has been utilized to investigate the macrophage response to various stimuli. Employing a gel-free proteomic approach, a study identified 504 differentially expressed proteins with strong assurance (defined as ≥ 5 peptide spectral matches) in macrophages exposed to low-dose ethanol and lipopolysaccharide [37]. Among these items are, 319 proteins were consistently present under every treatment condition, whereas 69 proteins were uniquely identified in cells treated with ethanol or stimulated with lipopolysaccharide. Bioinformatics tools were employed to assess the combined impact of ethanol and lipopolysaccharide on the macrophage proteome, assisting in pinpointing proteins that respond differently, networks of protein interactions, and established pathways.

Novel biomarkers for predicting response to PD-1 inhibitors

The identification of biomarkers predictive of response to PD-1 inhibitors is crucial for the advancement of personalized therapy. In a study involving cancer patients treated with PD-1 inhibitors, early treatment levels of anti-thyroglobulin antibody (TgAb) (OR = 2.831, 95% CI 1.077-7.443, p = 0.035) and anti-Thyroperoxidase Antibody (TPOAb) (OR = 9.565, 95% CI 3.399-26.921, p < 0.001) were found to be independent predictors of immune-related Thyroid Dysfunction (irTD) [38]. These early-stage biomarkers exhibited a superior predictive capability for irTD, as evidenced by a larger Area Under the Curve (AUC) compared to pre-treatment biomarkers (0.655 vs. 0.571).

Through proteomic profiling, machine learning algorithms identified ten protein biomarkers associated with gastric cancer. The model demonstrated exceptional performance in predicting responses to PD-1 inhibitor immunotherapy in an independent validation cohort (n = 14; AUC = 0.959) [39]. These biomarkers were associated with pathways such as the complement system and blood clotting processes, and the presence of activated CD8 T cells was correlated with a positive response to immunotherapy.

Exploring combination therapies with PD-1 inhibitors offers significant potential

PD-1 inhibitor combination therapies demonstrate considerable potential. A retrospective cohort analysis of metastatic melanoma indicated that combination Immune Checkpoint Inhibitor (ICI) therapy (anti-PD-1 ± anti-CTLA-4) significantly enhanced clinical Progression-Free Survival (cPFS) (HR 0.57, 95% CI 0.38 to 0.86, p = 0.007) and Overall Survival (OS) (HR 0.42, 95% CI 0.28 to 0.65, p < 0.001) in comparison to anti-PD-1 monotherapy [40]. Nonetheless, the incidence of grade 3 or 4 toxicities was higher with the combination therapy (31%) relative to PD-1 monotherapy (7%).

The use of PD-1/PD-L1 inhibitors alongside radiotherapy and anti-angiogenesis agents has demonstrated success in treating solid tumors. A meta-analysis reported a pooled overall response rate of 59% (95% CI: 48-70%) for solid tumors managed with this combination [26]. This therapeutic strategy may enhance survival outcomes more effectively than monotherapy or dual-combination therapies, highlighting the potential of multi-modal approaches in advancing cancer treatment outcomes.

Controversies in macrophage targeting strategies

One of the primary controversies in macrophage-targeting strategies pertains to the intricate nature of macrophage phenotypes and functions. Macrophages demonstrate considerable plasticity and heterogeneity, impacting the tumor microenvironment through both pro-tumor and tumor-suppressing activities, depending on various influencing factors [41]. The transformation of macrophages from the M2 phenotype, which promotes tumor growth, to the M1 phenotype, which suppresses tumors, emerges as a promising strategy. However, achieving precise control over this polarization process remains a significant challenge. Additionally, the design and development of Chimeric Antigen Receptor (CAR) macrophages encounter obstacles, including the need to ensure their specificity, efficacy, and safety.

The targeting of phagocytosis checkpoints on macrophages represents a contentious area of research. Although augmenting macrophage phagocytosis holds promise for enhancing anticancer immunity, the precise mechanisms and optimal targets for intervention remain incompletely elucidated. Furthermore, a thorough assessment of potential side effects and off-target effects associated with macrophage-targeting therapies is essential to ascertain their clinical feasibility and effectiveness.

Future prospects for personalized medicine in tumor microenvironment management

The management of the tumor microenvironment through personalized medicine offers significant potential. The application of radionuclide-based imaging and therapeutic techniques to target the tumor microenvironment represents a personalized medicine approach that is applicable across various cancer types [42]. Radio ligands designed to target modified procedures, the extracellular matrix, and cellular components within the Tumor Microenvironment (TME) have been designed and tested in both before clinical trials and during clinical trials research. These radio ligands have the potential to be employed for therapy and imaging with targeted radionuclides, facilitating more personalized interventions within the field of nuclear medicine.

The application of 3-Dimensional (3D) tumor models, including spheroids and organoids, is gaining recognition as a promising strategy in contemporary research [43]. These models, which are derived from patient tissues, employ high-throughput approaches in individualized medicine techniques to ascertain suitable therapies for individual patients. They significantly enhance the comprehension of tumor biology and its surrounding environment, support the advancement of innovative In vitro drug testing platforms, and facilitate the development of personalized therapeutic strategies.

This work was supported by Science and Technology Plan of the Health Commission of Jiangxi Province (202311840).

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