2.1.1. T-Lymphocyte

T cells play a central role in the immune microenvironment of CRC and are key factors in regulating tumor immune responses. The level of tumor-infiltrating lymphocytes (TILs) is closely related to tumor antigen load and patient prognosis. CD8⁺ T cells are cytotoxic and can recognize and kill tumor cells [12]. Studies have shown that the level of CD8⁺ T-cell infiltration contributes to tumor antigenicity, facilitating the transformation of “cold tumors” into “hot tumors,” thereby enhancing the benefits of immunotherapy [12, 13]. Conversely, cluster of differentiation 4 (CD4)⁺Foxp3⁺ regulatory T cells (Tregs) act as immunosuppressors in antitumor immunity, typically accumulating in the TME of CRC patients and entering peripheral blood as the tumor progresses, inducing immune tolerance and promoting immune evasion [14]. The ratio and functional status of T-cell subsets influence tumor development and treatment responses.

Cytotoxic T lymphocytes (CTLs) contain granules that are released upon specific interaction with target cells. These granules primarily consist of perforin, granzymes, and proteoglycans [15]. Killing of target cells occurs at a specialized contact site called the immunological synapse (IS), where regulated exocytosis of cytotoxic granules (CGs) takes place. CGs contain perforin-1 (Prf1), granzyme B (GzmB), and Fas ligand (FasL), which induce target cell death via apoptosis and necrosis. CGs are lysosome-related organelles that sharing similarities with endosomes and lysosomes [16]. These cytotoxic vesicles are transported along the cytoskeleton to the IS and fuse with the plasma membrane to release their contents. Due to the hazardous nature of CGs, their fusion is tightly regulated both spatially and temporally [17].

Research indicates that positive expression of programed death-ligand 1 (PD-L1) in tumor cells is closely associated with CD8⁺ T-cell infiltration, meaning that extensive cytotoxic T-cell infiltration is a driving factor for enhancing the antigenicity of CRC cells [14, 18, 19]. Activated effector T cells can not only secrete cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) to enhance immune responses and recruit other cytotoxic cells like dendritic cells (DCs) and natural killer cells (NKCs) to collaborate in antitumor activities, but they can also differentiate into memory T cells under strong tumor antigen stimulation, allowing for rapid response and control of tumor recurrence and metastasis upon re-exposure to tumor antigens [20–24]. However, as the tumor progresses, CD8⁺ T cells may experience exhaustion, showing functional decline and decreased cytokine secretion, in which Tregs play a significant role [25, 26]. The proportion of Tregs is higher in CRC patients, and their infiltration is negatively correlated with tumor malignancy and prognosis [27]. Additionally, the increased surface expression of inhibitory receptors such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programed cell death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin and mucin domain 3 (TIM-3), and T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) indicates T-cell exhaustion—a state of functional impairment characterized by gradual loss of effector function and changes in transcription and metabolism [28, 29]. PD-1 and CTLA-4 inhibitory molecules play a significant role in T-cell immune suppression and have shown strong prognostic predictive performance in CRC, representing a breakthrough point for immunotherapy.

The expression of LAG-3 on TILs in certain CRC tissues has been linked to advanced tumor stages, microsatellite instability-high (MSI-H), and poor prognosis, suggesting that LAG-3 could serve as a potential prognostic marker for CRC [30].  A recent study found that CD8⁺ T cells deficient in both PD-1 and LAG-3 exhibited enhanced tumor clearance and improved long-term survival compared to those lacking either PD-1 or LAG-3 alone. LAG-3 and PD-1 work together to promote T-cell exhaustion and are key regulators of TOX expression [31]. Additionally, the negative immune regulator TIM3 plays a role in tumor evasion. Elevated TIM3 expression reduces Th1 cell activity, diminishes the secretion of IFN-γ, and weakens other antitumor responses, thereby impairing the immune system’s ability to fight the tumor [32].

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades tryptophan, an essential amino acid and is overexpressed in tissues such as the colon, intestines, and lungs. In CRC, tumor-driven regulation of IDO is linked to metastasis and is negatively correlated with T-cell infiltration [33]. IDO1 expression can be induced by inflammatory signals like IFN-γ, lipopolysaccharide, and TNF. Under pathophysiological conditions, IDO1 is highly upregulated in response to these inflammatory stimuli, and its overexpression is associated with improved detection across various cancer types, including CRC [34]. TIGIT is primarily expressed on activated T cells, Tregs, memory T cells, and NK cells. It exerts its immunosuppressive effect by binding to CD155 on DCs, thereby modulating cytokine production by DCs and indirectly influencing T-cell function. TIGIT also promotes T-cell exhaustion by competing with the costimulatory receptor CD226 for binding to CD155. In microsatellite unstable CRC (MSI-CRC), immune checkpoint molecules such as IDO1, LAG-3, and TIGIT are expressed at higher levels compared to microsatellite stable (MSS) CRC. Combining checkpoint inhibitors has shown to be more effective in MSI-CRC, enhancing immune cell cytotoxicity and targeting the immune evasion mechanisms associated with microsatellite instability [35].

Recently, Liu et al. [36] highlighted the pivotal role of RUNX proteins in regulating CD8⁺ T and CD103⁺CD8⁺ T cell–mediated antitumor responses within the TME of human CRC. Their study showed significant differential expression of RUNX genes between CRC and normal tissues. Patients with a higher proportion of infiltrating CD8⁺RUNX1⁺, CD103⁺CD8⁺RUNX1⁺, CD8⁺RUNX2⁺, CD103⁺CD8⁺RUNX2⁺, CD8⁺RUNX3⁺, or CD103⁺CD8⁺RUNX3⁺ T cells had better outcomes compared to those with lower proportions. The RUNX family—comprising RUNX1, RUNX2, and RUNX3—has long been recognized as essential regulators in developmental processes, and dysregulation of these genes has been linked to tumorigenesis and cancer progression.

The immune costimulatory molecule OX40 (CD134) is a promising novel target for CRC immunotherapy [37].

It is now ascertained that immune checkpoints include both inhibitors and promoters of T-cell activation. Inhibitory checkpoints, like CTLA-4, PD-1, and PD-L1, dampen T-cell activity while activating checkpoints such as LAG-3, OX40, and glucocorticoid-induced TNF receptor family-related proteins promote T-cell responses. Additionally, checkpoints-like IDO are involved in regulating T-cell metabolism. Blocking suppressive checkpoints, particularly PD-1, PD-L1, and CTLA-4, has demonstrated clear clinical benefits in patients with MSI-H or deficient mismatch repair (dMMR) metastatic CRC (mCRC).

Currently, significant progress has been made in the development of immune checkpoint inhibitors (ICIs) for solid tumors, and their combination with radiotherapy/chemotherapy has further improved clinical outcomes [38, 39]. However, their approval is limited to tumors with mismatch repair deficiencies or microsatellite instability, which is a major constraint [40, 41]. To address the deficiency of secondary TILs in immunotherapy that blocks T-cell inhibitory receptors, adoptive cell therapy (ACT) has emerged strongly [42–44]. As a highly personalized cancer treatment method, ACT involves genetically reprograming and expanding tumor-specific TILs, which are then reinfused into the patient to induce an autologous immune response to kill tumor cells. This approach has the advantages of eliminating certain T-cell suppressions and maintaining prolonged proliferation and cytotoxicity. However, the lack of universality of patient-specific TILs, along with the inefficiency of transporting TILs to the tumor center and the associated high toxicity, pose both economic and technical challenges for its clinical promotion in solid tumors. Therefore, a detailed classification and functional understanding of these T-cell populations can help guide the development of personalized immunotherapy strategies, improving treatment efficacy and patient survival rates.

2.1.2. B-Lymphocyte

For a long time, T cells and NKCs have been considered the main mediators of antitumor immunity, while B-cell populations were thought to provide negligible antitumor effects merely as antibody producers [45]. Therefore, research targeting B cells for antitumor purposes has been quite limited. However, with the deepening understanding of the TME and advancements in single-cell sequencing technology, the role of B cells in the TME of CRC has gradually gained attention [46–48]. The infiltration composition of B-cell subpopulations in CRC tissues has been continuously clarified, and the infiltration levels of tumor-infiltrating B cells (TIL-Bs) vary at different stages of tumor development [49, 50]. In early primary CRC tissues, there is a rich presence of activated or terminally differentiated memory B cells and plasma cells compared to the peripheral blood of patients and nontumor patients. These cells work together to produce high-affinity antibodies that directly target tumor cell surface antigens or indirectly induce other immune cells to recognize and eliminate tumor cells, thereby enhancing tumor-specific immune responses and providing sustained and effective immune protection. In contrast, in late-stage tumors and metastatic sites, chronic inflammation caused by persistent tumor antigen immune responses leads to significant infiltration of regulatory B cells (Bregs), which promote immune tolerance and tumor escape by secreting regulatory factors such as interleukin 10 (IL-10) and transforming growth factor-beta (TGF-β), affecting tumor development and treatment [48, 51]. The mature B-cell subpopulations adjacent to tumor tissues are closely related to lymph node metastasis in stages I–III and low survival rates in CRC patients. TIL-Bs can also promote antitumor immunity in T cells through their antigen-presenting function, maintaining an immune “hot” TME [52, 53]. This indicates that these B-cell populations can combat immune editing and tumor heterogeneity by relaxing self-tolerance mechanisms, providing new therapeutic opportunities for cancer immunotherapy, and showing potential as biomarkers for prognosis prediction and immune targets in CRC progression.

Although the specific role of B cells in CRC tissues still requires further research, a large number of B cell–targeted drugs currently on the market or in development primarily focus on the treatment of autoimmune diseases and B-cell malignancies [54, 55]. Nonetheless, investigating their existence and functions offers fresh insights into the development of immune therapeutic strategies targeting B cells, with some progress already achieved. Researchers have utilized multidimensional omics data to map the genetic regulatory landscape of immune infiltration in CRC within the Chinese population, identifying a total of 568 immuno-QTLs [56]. These loci are significantly enriched in susceptibility regions for CRC and have been validated in the cancer genome atlas TCGA. Additionally, researchers have constructed a polygenic risk score (PRS) model, which effectively enhances the risk stratification efficiency for CRC and the identification of high-risk populations. In summary, B lymphocyte populations play an important role in the TME of CRC, and current research advancements provide new strategies and targets for immunotherapy in CRC. Therefore, gaining a more detailed understanding of tumor-associated B-cell subpopulations and their impact on tumor growth will facilitate the timely transition to antitumor clinical interventions.

2.2.1. TAMs

TAMs are the predominant myeloid cells infiltrating solid tumors, playing a critical role in regulating the TME and driving tumor progression and metastasis. Their involvement in the clearance of apoptotic cells (cytophagy) aids in suppressing inflammation and contributes to immune evasion in CRC. Earlier studies have found poorer progression-free survival and chemotherapy response rates in the high TAMs group, which may be associated with inefficient drug distribution and chemoresistance due to TAMs [57]. Additionally, a high density of M2-type TAMs at the tumor center and infiltration margins is associated with poor differentiation and shorter disease-free survival. However, the same study suggested that a high density of pan-macrophages in these regions correlates with a more favorable prognosis [58, 59]. This apparent contradiction has sparked significant interest among researchers. With advances in technology and more refined studies, it has been observed that TAMs are typically induced by macrophage colony-stimulating factor (M-CSF), which differentiates monocytes into M0 macrophages. Subsequently, under the influence of specific cytokines, M1-type TAMs exhibit proinflammatory and antitumor effects. They can eliminate pathogens and stimulate antitumor immune responses by secreting proinflammatory cytokines such as TNF-α, IL-6, and IL-12. When appropriately activated, M1-type TAMs are capable of mediating phagocytosis and exerting cytotoxic effects to kill cancer cells [60]. However, in malignant tumors, TAMs are regulated by a variety of factors and mostly exhibit M2-type features, which promote tumor angiogenesis, alter CRC drug resistance, induce immune escape, and promote tumor proliferation and metastasis by inhibiting acute intense inflammation, secreting immunosuppressive factors, and regulating metastasis-related proteins [61, 62]. Furthermore, the recruitment of M2-type TAMs (marked by CD163) is significantly higher in metastatic CRC tissues compared to nonmetastatic ones. CD163 levels are also markedly elevated in mid-to-late-stage (stages III–IV) CRC tissues compared to early-to-mid-stage (stages I–II) [63–65]. In contrast, no significant difference was observed in the levels of M1-type TAMs (marked by CD68). These findings suggest that a high density of M1-type TAMs in the CRC stroma is associated with improved cancer-specific survival, whereas a high density of M2-type TAMs correlates with poorer survival outcomes in CRC patients [66, 67]. However, TAMs contribute far beyond their role in CRC progression. Acting as a bridge between inflammation and cancer, they induce early-onset CRC through metabolic reprograming, acidification of the TME, reduction of oxygen levels, and creating conditions favorable for tumor growth. Moreover, under the influence of various regulatory factors and chemokines, TAMs actively polarize toward the M2 phenotype. This polarization can even drive the secondary conversion of M1-type TAMs into M2-type TAMs, facilitating the aggregation of Tregs and increasing PD-1 expression, ultimately promoting immune evasion [60, 68].

As our understanding of the functions and mechanisms of TAMs deepens, distinct functional subtypes of M1 and M2 TAMs have been identified. These subtypes, when combined with other marker molecules, offer valuable potential for effectively predicting clinical outcomes. Targeted drugs based on different stages of CRC and subtypes of TAMs, such as probiotics that maintain microbial homeostasis, show strong potential for development, with the ultimate goal of inhibiting the polarization of M2-type TAMs. A study at Fudan University found that activation of the NOD1 receptor on the surface of TAMs could shift TAMs from the immunosuppressive M2 type to the CD8⁺ T-cell-activated M1 type and enhance the immune response, which provides a new strategy to enhance immunotherapy by regulating the polarization status of TAMs [69, 70]. On the other hand, metabolic remodeling can also be addressed. Studies have shown that M1-type TAMs predominantly rely on glycolytic metabolism for energy, while M2-type TAMs derive energy primarily through oxidative phosphorylation and fatty acid oxidation. This metabolic distinction suggests that reducing lipid uptake in TAMs by downregulating the scavenger receptor CD36 could decrease lipid accumulation within TAMs. Consequently, this reduction would impair fatty acid oxidation and oxidative phosphorylation, thereby inhibiting the polarization of TAMs toward the M2 state [71, 72]. The emergence of nanomaterials offers promising strategies for inhibiting tumor progression. For example, cationic polymers acting as TLR-4 ligands can activate the TLR-4 signaling pathway, stimulate the specific secretion of IL-12, and reverse the polarization of M2-type TAMs to the M1 phenotype [73]. These advancements provide novel perspectives for CRC immunotherapy, prognostic assessment, and the identification of potential therapeutic targets.

2.2.2. DCs

DCs are a highly heterogeneous population of innate immune cells, and the recognition, capture, and processing of antigens by immature DCs in the peripheral blood and their presentation to naïve T lymphocytes are the most unique features of this subset [74–76]. A low level of antigenic stimulation can prompt DCs to transition from an immature to a mature state. This maturation reduces their antigen-capturing capacity while enhancing the expression of major histocompatibility (MHC) class I and II antigens, costimulatory molecules, and effector cytokines. These changes initiate T-cell immune responses and activate TILs and NKCs to exert cytotoxic effects. As a result, DCs play a pivotal role in inhibiting local tumor growth, proliferation, and metastasis [77, 78]. Compared with the normal population, the number and function of peripheral blood DCs subpopulations are impaired in CRC patients, limiting effector T-cell activity, and this effect correlates with patient prognosis. The infiltration of mature DCs is significantly higher in early-stage CRC primary tissues compared to metastatic tissues, with even higher levels observed in patients with microsatellite-unstable CRC. This suggests that DCs could represent a promising new target for cancer immunotherapy [79, 80]. However, tumor cells can suppress DC function by secreting immunosuppressive factors such as TGF-β and IL-10, which alter cytokine secretion patterns. This induces the production of immunosuppressive tumor-associated DCs (TADCs) and modifies metabolism to adjust the microenvironmental pH. These changes directly or indirectly impair DC functions, including antigen presentation, T-cell activation, and maturation, contributing to tumor cell evasion of immune surveillance and resistance to immune clearance. Therefore, understanding the inhibitory mechanisms of DCs by tumor cells is important for developing more effective immunotherapy strategies [81, 82].

DCs-based immunotherapy is in full swing: it has been found that vascular endothelial growth factor (VEGF) can impair the migratory ability and immune function of mature DCs through the VEGFR-2-mediated RhoA-cofilin1 signaling pathway, suggesting that blockade of this signaling pathway should be considered when administering DCs-based antitumor immunotherapy in the clinic in order to improve the clinical efficiency of treatment [83]. DCs vaccines have shown significant efficacy in clinical trials for a variety of cancers, especially in enhancing immune response and improving patient survival [84–86]. These vaccines activate the TILs response by activating the patient’s immune system and prompting the presentation of potent tumor antigens and have been shown to have promising applications in a variety of types of cancers, including CRC, breast cancer, prostate cancer, and other types of cancers [87]. Additionally, researchers have developed a dual-response nanomedicine designed for programed antitumor immune activation. Delivered via the lymphatic system, this nanomedicine releases STING agonists in acidic lymph nodes, effectively activating DCs and T cells. It then accumulates in tumors to release aPD-1 for immune checkpoint blockade (ICB) therapy while promoting DC maturation and initiating robust antitumor immune responses [88]. These advancements not only offer valuable insights into the safety and efficacy of DC-targeted immunotherapy but also provide a crucial foundation for designing novel vaccines and enhancing existing therapeutic strategies. By conducting in-depth analyses of response variations among different patient groups and optimizing vaccine formulation and administration methods, the clinical efficacy of such treatments is expected to improve significantly in the future, offering new hope for advancing cancer therapy.

2.2.3. NKCs

NKCs in bone marrow are one of the innate immune cell populations, accounting for less than other cells, about 5% or less, and varying with individual differences, age, health status, and immune status [89–91]. As cytotoxic members of the innate lymphoid cell (ILC) family, these cells play a crucial role in immunosurveillance and tumor clearance despite their low abundance. Circulating through blood, lymphatic, and nonlymphatic tissues, they are capable of mounting a rapid and robust response against cancer cells [92].

NKCs play a critical role in controlling cancer progression by killing mutant cells via death receptors and CGs. However, within the TME, NKCs often become exhausted, impairing their ability to surveil and respond to tumors, thus contributing to immune evasion. NKCs release CGs, such as perforin and GzmB, to induce tumor cell lysis and apoptosis [93]. Additionally, the Fcγ receptor III (CD16) on NKC recognizes antibodies bound to cancer cells, triggering NKC to release more CGs in a process known as antibody-dependent cell-mediated cytotoxicity (ADCC) [94]. Unlike CTLs, which require activation, NK cells constitutively express these granules [95, 96].

A study investigating the relationship between NKC activity and CRC risk found that NKC activity levels were lowest in the CRC group, intermediate in the colorectal adenoma group, and highest in the tumor-free group [82]. These findings suggest that NKC activity declines as the risk of CRC increases and that reduced NKC activity is associated with a higher incidence of CRC and colorectal adenomas. However, CRC tissues have high levels of chemokines such as CXCL10, CXCL8, CXCL5, and CXCL1, which are more likely to recruit T cells for aggregation than NKCs, which contributes to the general scarcity of NKCs in CRC tumor tissues and this phenomenon shows no correlation with the expression of the human leukocyte antigen MHC class I [97]. NKCs operate independently of MHC complex class I-mediated antigen presentation, highlighting their ability to function without relying on traditional MHC-based mechanisms [98]. This finding raises an intriguing question: could NKCs offer immunotherapeutic advantages over TILs that translate into clinical benefits? With ongoing research, it has been observed that activated NKCs in the TME exhibit potent cytotoxicity and killing activity. They can directly and independently destroy tumor cells by secreting cytotoxic and proinflammatory factors such as perforin, granzyme, and IFN-γ. Additionally, they enhance the antitumor immune response by recruiting other antitumor cell aggregates. Similar to T cells, a subset of NKCs can develop a memory-like effect, enabling them to quickly recognize and attack previously encountered tumor cells, further boosting the efficiency of immune clearance. Furthermore, epithelial-to-mesenchymal transition (EMT), a key driver of metastasis, can alter the immunogenicity of CRC cells, increasing their sensitivity to NKC-mediated cytotoxic cleavage and facilitating their elimination [99, 100]. However, the killing activity of NKCs is tightly regulated by various inhibitory signals, which can weaken their function or even turn them into “accomplices” of tumor cells. In the suppressive TME, functionally exhausted NKCs exhibit increased expression of PD-L1. This crosstalk between immune cells and tumor cells promotes EMT and suppresses the antitumor immune response [101]. As a result, the development of truly adaptive NKC-based therapies faces significant challenges. Nevertheless, achieving success in this area would represent a major milestone in the field of immunotherapy. Research on NKC-based tumor immunotherapies has advanced rapidly, encompassing a wide range of approaches such as chimeric antigen receptor (CAR) NKC therapy, cytokine mobilization, ICB, bi- or tri-specific killer cell engagers, and NKC-derived extracellular vesicles (NKEVs). For instance, in a human CRC NSG mouse model, NKG2D CAR-modified NKCs (NKG2D-DAP12CAR-NKCs) demonstrated enhanced IFN-γ secretion and a robust cytotoxic immune response, resulting in significant tumor suppression. Furthermore, local infusion therapy in patients with metastatic CRC has been shown to effectively prolong median survival times while demonstrating safety and feasibility [102]. In addition, the knockdown of TIPE2 using CRISPR-Cas9 technology can reverse the depletion state of NKCs, significantly enhancing their antitumor activity, suggesting that targeting TIPE2 represents a promising immunotherapy strategy [103]. Finally, NKEVs not only retain functional proteins from NKCs and exhibit antitumor activity both in vitro and in vivo but also effectively target tumor cells, inhibiting CRC growth and immune evasion through multiple mechanisms [104–106]. These advancements underscore the potential of NKCs in improving immunotherapeutic efficacy, driving the development of novel strategies, and leveraging NKC derivatives, paving the way for more effective cancer treatment options.

2.2.4. BMDCs

Another significant population of innate immune cells with a high proportion in BMDCs is the granulocyte population. As key players in inflammation, granulocytes initiate, promote, and sustain inflammatory responses through chemotaxis, phagocytosis of pathogens, and the release of inflammatory factors, thereby protecting damaged tissues from infection. In addition, granulocytes facilitate the maturation and antigen presentation of DCs and activate NKCs and T cells, synergistically enhancing the immune response. Granulocytes are classified based on their specific cytoplasmic granule content into neutrophils (N), eosinophils (E), and basophils (B) [107].

Neutrophils release both cytoplasmic cytokines and factors stored in their granules. They contain three types of granules and secretory vesicles [108]. Azurophil granules hold MPO, which aids ROS production, and several serine proteases like neutrophil elastase, cathepsin G, and proteinase-3, which promote tumor invasion and proliferation [109]. Specific (secondary) granules contain lactoferrin and neutrophil collagenase (matrix metalloproteinase-8 [MMP8]), while gelatinase (tertiary) granules are rich in gelatinase B (matrix metalloproteinase-9 [MMP9]). Secretory vesicles release proteins integrated into the plasma membrane upon stimulation [108].

The neutrophil immune function relies on both the biogenesis of granules, which store antimicrobial proteins and their ability to generate an oxidative burst. Defects in either process can result in severe immunodeficiencies, such as neutropenia, neutrophil-specific granule deficiencies, or chronic granulomatous disease when oxidative burst is absent [110]. Granulocyte CSF (G-CSF) is the primary cytokine regulating granulocytopoiesis, and its absence impairs granulocyte production, leading to neutropenia and immune deficiency [111, 112]. Neutrophilic recruitment and activation can have broad effects on tumor cells and the microenvironment, including direct cellular injury from oxidant release, remodeling of the ECM, proangiogenic product release, and cross-signaling to other inflammatory cells and stromal constituents [113]. Neutrophils interact at these sites via cell contact or mediators such as reactive oxidants, granular proteins, and cytokines, which can prime or dampen antigen-dependent immune responses.

Eosinophils, traditionally known for combating parasites and contributing to allergic inflammation [114], produce a range of toxic granule proteins and proinflammatory mediators that can lead to tissue damage. They exert potent cytotoxic functions through the release of cationic proteins like major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPX), and eosinophil-derived neurotoxin (EDN). Studies suggest that cytokines can influence eosinophil phenotype, determining their polarization and tumor immune responses. For example, Reichman and colleagues reported that in experimental CRC, intratumoral eosinophils exhibited an IFN-γ-related signature, which helped prevent the development of CRC in mice [115].

Although basophils make up a small proportion of circulating blood leukocytes, they are powerful immune effector cells. In CRC, low levels of circulating basophils have been associated with more advanced tumor stages (both T and N stages), venous and perineural invasion, and poorer patient survival [116]. One study suggested that basophils might be resistant to the immunosuppressive effects of Tregs (CD4⁺CD25⁺FoxP3⁺) and may even be activated by these cells instead [117]. Basophils are also capable of producing extracellular DNA traps containing mitochondrial DNA and granule proteins [118].

2.2.5. Mesenchymal Stem Cells (MSCs)

The most specialized cell population among BMDCs is the multipotent stem cell, known as MSCs. These cells possess the unique abilities of self-renewal, multilineage differentiation, and tissue repopulation. MSCs can be isolated from various tissues and are extensively utilized in tissue repair, graft-versus-host disease treatment, autoimmune disease management, and oncology research [144, 145]. The classification and functional mechanisms of MSCs in CRC remain a topic of debate. Insights from single-cell transcriptome analyses have identified five distinct MSC subpopulations: (a) Stem cell-like subpopulations, which are involved in self-renewal and differentiation, providing essential support for sustained tumor growth. (b) MSC subpopulations, characterized by their ability to differentiate into various cell types, contribute to the TME and influence tumor invasiveness and metastatic potential. (c) Monopotent adipose precursor cells, associated with fat metabolism, potentially impacting CRC progression through energy metabolism regulation. (d) Bipotent chondrogenic-osteogenic precursors linked to bone metastasis and tumor-associated bone disorders. (e) Chondrogenic precursor cells, which possess immunomodulatory properties and may play a role in immune system regulation [146].

The identified MSC subpopulations highlight their dual role in CRC progression. On the one hand, MSCs can synergistically promote tumorigenesis by recruiting protumor inflammatory factors that induce the differentiation of CAFs [147, 148]. This contributes to tumor growth, invasion, migration, and metastasis, driven by their proangiogenic potential. Furthermore, MSCs subtly reshape the peritumoral cellular and stromal landscape, fostering the formation of an inhibitory TME, increasing drug resistance, tumor recurrence, and suppressing antitumor immunity, ultimately worsening patient prognosis [149, 150]. On the other hand, MSCs exhibit antitumor effects, particularly in the early stages of tumor development. During the initial phases of CRC, MSCs can migrate to the colorectal region, mitigate chronic inflammation, and regulate imbalances in the intestinal flora. By recruiting and activating Tregs, they can help inhibit tumor progression and contribute to antitumor responses [131, 132, 151]. MSCs have demonstrated remarkable tumor-homing properties, enabling precise migration to tumor sites and the targeted release of therapeutic agents. Their derived exosomes are effective drug carriers, positioning MSCs as potential nanomaterial delivery systems. This capability significantly enhances the efficacy of chemotherapeutic drugs while reducing their adverse effects, improving both the safety and effectiveness of CRC treatments. MSCs’ unique tumor tropism has already led to their inclusion in clinical trials, with over 50 drugs currently registered for CRC treatment [133]. However, it is important to note that the role of MSCs can vary across different stages and subtypes of CRC. This duality underscores the need for continued research into their mechanisms and interactions within the TME. A deeper understanding of MSCs roles will be crucial for refining therapeutic strategies and improving the accuracy of patient prognosis.

2.4.1. Extra Cellular Matrix

The ECM serves as a crucial structural support for CRC cell growth and migration within the TME [174, 175]. Its composition and regulation, influenced by CAFs and their secreted factors, have garnered significant interest in CRC research [176–178]. The ECM is primarily composed of the following components: (a) Collagen: as the most abundant ECM protein, collagen provides tissue strength and reduces deformation. In CRC, it indirectly promotes tumor progression by activating the PI3K/AKT pathway through integrin α2β1 signaling. (b) Fibronectin (FN): a protumorigenic, noncollagenous protein found in the ECM and basement membrane, predominantly produced by liver and vascular endothelial cells. FN induces EMT and promotes CRC progression via the PI3K/AKT pathway. Its levels can also serve as a prognostic marker. (c) Laminin (LN): an essential component of the basement membrane, in conjunction with type IV collagen. LN derivatives enhance CRC liver metastasis, promote branching angiogenesis, and inhibit the Notch signaling pathway. (d) Hyaluronic acid (HA): this glycosaminoglycan binds to collagen, aiding in hydration and tissue compressive resistance. Interestingly, HA overexpression suppresses CRC liver metastases, underscoring its nuanced role. (e) Periostin (POSTN): a protein secreted by fibroblasts that plays a key role in regulating intercellular adhesion. POSTN is heavily involved in metastatic invasion and is a critical ECM component. (f) Follistatin-like 3 (FSTL3): overexpressed in CRC tissues, FSTL3 is associated with lymph node metastasis and resistance to chemotherapy. However, it exhibits sensitivity to immunotherapy, offering a potential therapeutic target.

These ECM components not only provide structural integrity but also actively influence tumor progression, metastasis, and therapeutic responses. Understanding their roles could unlock novel strategies for CRC treatment and prognosis. Abnormal deposition and cross-linking of the ECM result in increased matrix stiffness within the TME [179, 180]. This alteration not only provides structural support to the tumor but also creates a multifaceted barrier with significant implications: (a) Physical barrier formation: increased ECM stiffness physically compresses microvessels, leading to reduced blood flow and contributing to the development of a hypoxic environment. (b) Impact on therapeutic efficacy: hypoxia and physical barriers hinder neovascularization, impair the delivery of chemotherapeutic agents and reduce the penetration effectiveness of radiation therapy. (c) Immune modulation: the altered ECM stiffness influences the infiltration, activation, and function of immune cells within the TME. This regulation significantly impacts the success of immunotherapeutic approaches.

These factors highlight ECM stiffness as a critical determinant in CRC progression and treatment outcomes, underscoring its role as a potential therapeutic target to improve drug delivery, radiation efficiency, and immune cell function in the TME.

In CRC, the abundance and composition of the ECM are strongly associated with patient survival, with a negative correlation observed between ECM levels and prognosis. ECM proteins contribute to tumor progression and metastasis by promoting tumor cell proliferation and survival [181, 182]. However, studies have primarily established a link between elevated ECM proteins derived from tumor cells (parenchyma) and poor patient survival, while the relationship between ECM proteins from mesenchymal cells and survival remains uncertain and lacks a clear explanation.

It is well-established that increased ECM stiffness exacerbates the immunosuppressive environment within the TME, reducing the effectiveness of immunotherapy. One mechanism involves the activation of the ROCK-myosin II signaling pathway, which weakens CRC cell immunogenicity and induces the conversion of TAMs to the anti-inflammatory M2 phenotype, thereby impairing immune responses [183]. Additionally, studies have shown that DCs cultured on softer substrates demonstrate higher differentiation rates, whereas DCs grown on stiffer substrates show impaired differentiation and antigen presentation, affecting their ability to activate TILs. The remodeling of ECM also forms a physical barrier that hinders the migration of TILs, induces hypoxia, and weakens the function of NK cells [184].

Current therapeutic strategies targeting ECM hardness, such as matrix modulators, inhibitors of mechano-transduction pathways, and biomaterials designed to mimic the ECM, aim to enhance tumor immunotherapy. These strategies seek to improve drug and immune cell infiltration, modulate immune cell function, and reduce the immunosuppressive TME. However, inconsistent efficacy and a lack of conclusive evidence present significant challenges, requiring further exploration to optimize these approaches.

2.4.2. Complementary System

The complement system is a crucial component of the body’s immune defense, consisting of approximately 30 different plasma proteins typically found in an inactive state in the peripheral blood. The system can be activated through three distinct pathways: the classical pathway, which is antibody-dependent and involves the binding of pathogens to antibodies; the alternative pathway, which is antibody-independent and directly activates on the surface of pathogens; and the lectin pathway, which depends on specific glycans. Once activated, complement components perform vital functions, including marking pathogens for destruction, attracting immune cells, and directly killing infected cells. The complement system plays a central role in immune responses, such as anti-infective defense, inflammation, and tumor immune surveillance.

Both tumor cells and stromal cells in the TME of CRC produce abnormal complement proteins, leading to the aberrant activation of the complement system. Evidence suggests that these complement components may have an immunosuppressive role in the TME, acting as a bridge between tumor progression and immune responses that suppress tumor activity. For instance, C1q, an initiator protein in the classical complement pathway, has been linked to poor prognosis in CRC patients, with significant deposition in the TME. Studies have shown that C1q induces a tolerogenic or immunosuppressive phenotype in TAMs, favoring the polarization of M2-type TAMs and promoting their chemotaxis toward CRC tissues. This process correlates with the upregulation of PD-L1 and PD-L2, as well as reduced T-cell proliferation.

In contrast, other complement proteins, such as C3a and C5a, act as allergenic toxins that promote tumor metastasis and tumor-associated angiogenesis by activating CD4⁺ T cells. Specifically, C5a has been shown to facilitate CRC metastasis by stimulating the secretion of leukotriene B4 from epithelial and endothelial cells, as well as promoting the recruitment of TANs. Furthermore, C5aR1 can inhibit Th1 production and enhance the M2 polarization of TAMs [185, 186]. These findings emphasize the complex roles of the complement system in CRC, highlighting its involvement in tumor progression and immune modulation while also offering new insights and potential targets for CRC diagnosis and therapy.

Inhibiting the activation of the downstream complement system through complement inhibitors has been shown to modify the natural progression of diseases, improving conditions such as renal impairment, thrombosis, and pulmonary hypertension. This approach is considered an important strategy for the treatment of CRC [187]. Targeting specific complement-regulating proteins offers another potential therapeutic strategy. For example, a current Phase I clinical trial has demonstrated that combining C5aR1 antagonists with PD-1 inhibitors in the treatment of advanced solid tumors produces promising synergistic effects [188]. However, while this combination shows potential, there is currently insufficient evidence in CRC, making it a promising avenue for future research.