Tumor–Immune Cell Interactions and Experimental Detection Methods

This article systematically reviews the mechanisms of action of four core types of immune cells in tumors: tumor-associated macrophages (TAMs), CD8⁺ T cells, natural killer (NK) cells, and neutrophils (along with their reactive oxygen/nitrogen species products), and also introduces the corresponding research methods and detection strategies.

Table of Contents

1. Roles of Immune Cells in the Tumor Microenvironment

2. Experimental Models and Functional Research Methods

3. Molecular and Cellular Detection of Tumor-Infiltrating Immune Cells

4. Omics and Spatial Analysis of the Tumor Immune Microenvironment

01 Roles of Immune Cells in the Tumor Microenvironment

1.1 Tumor-Associated Macrophages and Their Pro-Tumor Functions

TAMs are the most abundant infiltrating leukocytes in solid tumors. Their pro-tumor functions mainly include ^[1]: ① Immunosuppression: TAMs reduce IL-12 production while producing inhibitory factors such as IL-10, TGF-β, and PGE2; they recruit Tregs via CCL22, directly suppress T cells through ARG1-mediated L-arginine depletion and NOS2-derived NO production, and induce T cell apoptosis via surface PD-L1 expression. ② Pro-angiogenesis and metastasis: TAMs secrete VEGF and other factors to promote angiogenesis and hematogenous dissemination. ③ Promotion of tumor proliferation and stemness: TAMs activate the NF-κB/STAT3 pathway through TNF-α and IL-6, driving tumor cell proliferation and maintaining stem cell-like properties.

The phenotypic classification of TAMs has moved beyond the traditional M1/M2 binary model. Lactate and hypoxia reshape TAM functions by stabilizing HIF1α, which upregulates VEGF and ARG1 expression. In most tumors, TAMs are primarily derived from CCR2⁺ monocytes rather than tissue-resident macrophages ^[1].

1.2 CD8⁺ T Cell Activation and Exhaustion

CD8⁺ T cells are the core effector cells in antitumor immunity. Naïve CD8⁺ T cells, upon activation by dendritic cells (DCs) via cross-presentation of tumor antigens, differentiate into cytotoxic T lymphocytes (CTLs) that kill tumor cells. CD4⁺ Th1 cells synergistically enhance their function, while regulatory T cells (Tregs) exert suppressive effects, and the three maintain immune homeostasis ^[2,3]. Under chronic antigen stimulation, CD8⁺ tumor-infiltrating lymphocytes (TILs) differentiate along an exhaustion trajectory, with progressively declining effector function and proliferative capacity, while inhibitory receptors such as PD-1, TIM-3, and LAG-3 are persistently highly expressed; among these, the PD-1⁺TIM-3⁺ subpopulation exhibits the most severe functional impairment ^[2]. Exhausted T cells display heterogeneity, and Tpex (progenitor exhausted T cells) precursor cells are critical for the efficacy of immune checkpoint blockade (ICB) therapy, with their ability to differentiate into effector-like cells determining therapeutic responsiveness ^[3].

1.3 NK Cell Function and Metabolic Regulation

Natural killer (NK) cells serve as the first line of immune surveillance, capable of directly killing tumor cells without prior sensitization ^[4]. The maintenance of their effector function depends on dual metabolic reprogramming driven by SREBP/cMyc, which simultaneously enhances both glycolysis and oxidative phosphorylation (OXPHOS) metabolic pathways ^[4,5]. However, the tumor microenvironment (TME) induces NK cell dysfunction through multiple metabolic suppression mechanisms: glucose deprivation and hypoxia restrict energy supply; lactate accumulation triggers mitochondrial stress and generates reactive oxygen species (ROS), which in turn induce apoptosis; and adenosine accumulation along with CD36-mediated lipid accumulation impairs NK cell activity by inhibiting mTOR signaling ^[4]. The presence of these metabolic obstacles provides a theoretical basis for metabolic optimization strategies in CAR-NK cell therapy.

1.4 Neutrophils, Myeloid Cells and Reactive Oxygen Species (ROS)

Tumor-infiltrating myeloid cells are the primary source of ROS/reactive nitrogen species (RNS) ^[5]. Myeloid-derived suppressor cells (MDSCs) produce reactive oxygen species via Nox1, which react with NO generated by NOS2 to form peroxynitrite (PNT). PNT suppresses immunity through a three-pronged mechanism: nitrating the TCR/CD8 complex to induce T cell tolerance, modifying peptide–MHC I complexes to enable tumor evasion from CTL recognition, and nitrating CCL2 to block T cell recruitment (while myeloid cells remain unaffected). ARG1 and NOS2 compete for arginine metabolism and synergistically drive PNT production, thereby constituting a core mechanism of myeloid-mediated immunosuppression ^[5].

02 Experimental Models and Functional Research Methods 

2.1 In Vivo Tumor Models for Immune Microenvironment Studies

Syngeneic transplantable tumor models are the most commonly used in vivo platform for studying the tumor immune microenvironment. These models are established by subcutaneously implanting MHC-matched tumor cell lines (e.g., B16F10, MC38, CT26, 4T1, etc.) into immunocompetent syngeneic mice ^[6,7]. Taking the 4T1 breast cancer model as an example: prepare a cell suspension at 5 × 10⁶ cells/mL, subcutaneously inoculate 5 × 10⁵ cells into the right dorsal flank of female BALB/c mice, and harvest tumors on day 13 for analysis.

Different models exhibit distinct immune characteristics. RENCA tumors are the most highly infiltrated, B16F10 represents a "cold tumor," CD8⁺ T cell function is suppressed in CT26 tumors, and EMT6 tumors are enriched with PMN-MDSC chemokines. For studies of "hot tumors" and ICB responses, RENCA/CT26 are recommended; for studies of immune evasion, B16F10 is preferred ^[6]. In mechanistic validation, antibody-mediated cell depletion experiments (e.g., anti-CD8, anti-NK1.1) are commonly used to demonstrate dependence on specific lymphocyte subsets ^[7].

2.2 In Vitro Assays for Immune Cell Function

(1) Antigen Presentation and T Cell Activation/Proliferation Assays

To evaluate the activation and proliferative capacity of tumor antigen-specific CD8⁺ T cells, an in vitro antigen presentation co-culture system can be established. First, CD8⁺ T cells are isolated from mouse spleens using immunomagnetic bead sorting or fluorescence-activated cell sorting (FACS) kits. Post-sorting purity is assessed by flow cytometry (typically exceeding 90%; in this study, the sorting purity achieved was 92.30%). Meanwhile, bone marrow-derived dendritic cells (BMDCs) are induced to mature in vitro, and inactivated tumor cells (e.g., Raw264.7 cells subjected to repeated freeze–thaw cycles or ultraviolet irradiation) are used as antigen sources to load the mature DCs. Subsequently, antigen-loaded DCs are co-cultured with CFSE-labeled CD8⁺ T cells at an appropriate ratio (e.g., 1:10) for 72 hours. T cell proliferation is evaluated by flow cytometric detection of CFSE dilution, which can be further validated by Ki-67 staining or EdU incorporation assays. This co-culture system can be used to verify the immunogenicity of tumor antigens, assess the antigen-presenting function of DCs, and analyze the impact of inhibitory factors in the microenvironment on T cell activation.

Fig. 1 Antigen-presenting cell (APC)-mediated T cell activation.

Table 1. Experimental products for antigen presentation, T cell isolation, and T cell proliferation analysis

(2) NK Cell Cytotoxicity Assay

To comprehensively characterize NK cell functional status, this section employs two complementary approaches: degranulation assays to assess immediate effector function, and metabolic profiling to reveal the underlying metabolic basis for functional maintenance.

Degranulation assay (CD107a mobilization assay): When NK cells kill target cells, the lysosome-associated membrane protein CD107a/LAMP-1 is transiently exposed on the cell surface upon exocytosis of granule contents (granzymes and perforin). This can be detected by flow cytometry using anti-CD107a antibodies to quantify the frequency of cytotoxic effector cells ^[8].

NK cell metabolic evaluation: Assessment of glucose uptake (2-NBDG), nutrient transporter expression (GLUT1, CD71, CD98), glycolytic and oxidative phosphorylation (OXPHOS) rates, as well as mitochondrial morphology and membrane potential, can be used to evaluate the metabolic suppression of NK cell function imposed by the tumor microenvironment (TME).

Application:

Fig. 2 NK cells sorted from human PBMCs were co-cultured with Jurkat target cells at E: T ratios of 1:1, 3:1, and 10:1 for 24 h. DAPI staining revealed a significant increase in DAPI-positive Jurkat cells in the co-culture groups, demonstrating potent NK cell-mediated cytotoxicity and growth inhibition of Jurkat cells in vitro.

(3) Assessment of Myeloid Cell Immunosuppressive Function

Evaluation of the immunosuppressive microenvironment mediated by myeloid cells can be conducted from two perspectives: arginine metabolism inhibition and neutrophil extracellular trap (NET) formation.

ARG1 and ROS/RNS: ARG1 activity can be quantified by measuring L-arginine consumption or L-ornithine/urea production; ARG1 inhibitor-mediated restoration of T cell proliferation assays can be used to verify the specificity of this suppression ^[7]. Meanwhile, respiratory burst-derived ROS/RNS can be assessed by dihydrorhodamine (DHR) flow cytometry or nitroblue tetrazolium (NBT) reduction assays ^[9]. Detection of tissue nitrotyrosine (a PNT marker) and Nox1/NOS2 expression can further validate ROS/RNS-mediated immunosuppression.

NETosis assay: NETs are composed of a DNA scaffold embedded with proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (H3Cit), and play important roles in tumor metastasis and immune evasion. Quantification of NE activity using NE-specific substrates directly reflects the extent of NETosis and is suitable for high-throughput screening.

03 Molecular and Cellular Phenotypic Detection

3.1 Flow Cytometric Immunophenotyping

Flow cytometry is the gold standard for quantitative analysis of tumor-infiltrating immune cells. A typical multicolor panel includes: CD45 (leukocytes), CD3/CD4/CD8 (T cells), CD25/FoxP3 (Tregs), CD49b/NKp46 (NK cells), CD11b/Gr1 (MDSCs), CD11b/F4-80 (macrophages), CD11c (DCs), and B220 (B cells), among others ^[6]. Intracellular staining (for FoxP3, cytokines, and transcription factors) requires the use of fixation/permeabilization buffers ^[10].

3.2 TAM Phenotyping and Functional Assessment

Flow cytometry: Murine TAMs are defined as CD45⁺CD11b⁺F4/80⁺; M1-like TAMs highly express CD80, CD86, and MHC-II, while M2-like TAMs highly express CD206 and CD163; intracellular ARG1 staining can identify suppressive myeloid cells ^[7].

Immunohistochemistry (IHC): Double staining with CD68 (pan-macrophage marker) in combination with M1 markers (HLA-DR, iNOS) or M2 markers (CD163, CD206) is commonly used. The M1/M2 ratio has been shown to have greater prognostic value than total TAM density.

Application:

Fig. 3 Phenotypic and functional characterization of mouse TAMs. Female BALB/c mice were subcutaneously inoculated with 4T1 cells (5 × 10⁵ per mouse). Tumors were harvested on day 13 and digested with enzymes to prepare single-cell suspensions. TAMs were characterized by flow cytometry: M1 macrophages were defined as CD45⁺F4/80⁺CD11b⁺CD86⁺MHC-II⁺, and M2 macrophages as CD45⁺F4/80⁺CD11b⁺CD206⁺MHC-II⁻. The results showed that M1 macrophages accounted for 25.65% of the population, while M2 macrophages accounted for 6.55%.

3.3 Assessment of T Cell Exhaustion

Inhibitory receptors: Markers such as PD-1, TIM-3, LAG-3, TIGIT, CTLA-4, 2B4, and CD39. The PD-1⁺TIM-3⁺ phenotype is a hallmark of terminal exhaustion ^[2,10].

Transcription factors: TOX (increases with exhaustion) and TCF1 (a marker of Tpex cells). Analyzing TOX expression separately in PD-1⁺TIM-3⁺ versus PD-1⁺TIM-3⁻ subpopulations can quantitatively assess the severity of exhaustion ^[10].

Progenitor vs. terminal exhaustion: Tpex cells (TCF1⁺, PD-1⁺, TOX⁺, TIM-3ᵈⁱᵐ, CD39⁻) sustain responsiveness to immune checkpoint blockade (ICB); while Ttex cells (TCF1⁻, TIM-3⁺, CD39⁺, granzyme B⁺) indicate severely impaired effector function ^[3].

Functional assays: Following restimulation with PMA/ionomycin, intracellular staining for IFN-γ and TNF-α can be performed; in exhausted cells, the production of these cytokines is reduced ^[2,10]. Additionally, proliferative capacity can be assessed by CFSE dilution or Ki-67 staining. TOX knockdown via siRNA can be used to validate its regulatory function.

Application:

Fig. 4 Detection of inhibitory receptor expression on human exhausted/dysfunctional T cells.

Table 2. Antibodies used for flow cytometric detection of inhibitory receptors on human T cells

Fig. 5 Detection of inhibitory receptor expression on mouse exhausted/dysfunctional T cells.

Table 3. Antibodies used for flow cytometric detection of exhaustion-associated markers on mouse T cells

04 Omics and Spatial Analysis of the Tumor Immune Microenvironment

Omics and spatial analyses can be performed from four perspectives: NanoString immune profiling, single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (SRT) integration, and IHC spatial localization.

● NanoString immune profiling: The PanCancer Immune Profiling panel (>500 genes) can be used to evaluate functional modules including T cells, NK cells, macrophages, complement, and chemokines ^[6].

● Single-cell RNA sequencing (scRNA-seq): This approach resolves immune cell heterogeneity and differentiation trajectories; differential analysis between PDCD1-high and PDCD1-low subpopulations can identify exhaustion-related regulatory factors, and Monocle pseudotime analysis can reconstruct differentiation trajectories ^[10].

● Spatial transcriptomics (SRT) integration: Through mapping and deconvolution strategies, the spatial distribution of TAMs, CD8⁺ T cells, and Tregs, as well as their interactions with tumor structures, can be resolved in situ ^[12].

● IHC spatial localization: Immunohistochemistry can reveal differential distribution of immune cells between the invasive margin and the tumor core.

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