Tumor immune microenvironment is a key factor in cancer development and treatment response. Its dynamics and complexity determine tumor progression, immune escape, and treatment resistance. This article will analyze TIME in detail from three aspects: cell composition, molecular dialogue, and analysis strategy.

What is TIME

The tumor immune microenvironment (TIME) is made up of many different types of cells. These include cancer cells, various immune cells, supportive stromal cells like fibroblasts and endothelial cells, and the extracellular matrix that surrounds the cells. All of these components constantly interact with each other through complex molecular signals, creating a unique environment where the tumor can grow and survive.

As the tumor develops, the TIME is not fixed—it changes over time and can adapt to different conditions. In the early stages, the body's immune system can often recognize and destroy abnormal cancer cells. Cytotoxic T cells and natural killer (NK) cells work together to attack and remove these harmful cells. However, as cancer progresses, it becomes more skilled at avoiding the immune system. The tumor gradually changes its surroundings, disrupting the balance of the microenvironment and making it harder for the immune system to fight back.

Key Cellular and Molecular Constituents of the TIME

Immune cell subset functional map

In the tumor immune microenvironment, different types of immune cells play unique and important roles, working together through complex systems. Among them, T cells are key players in the body's defense. One important type, called CD8+ cytotoxic T cells, helps find and destroy cancer cells. These T cells can recognize special signals (tumor antigens) on the surface of cancer cells and then kill these dangerous cells directly.

On the other hand, another type of T cell, called regulatory T cells, can actually protect the tumor by calming down the immune response. These cells, known by their markers CD4+CD25+Foxp3+, stop other immune cells from attacking. They do this by sending out chemical signals (cytokines) or by directly interacting with other cells. This allows the tumor to grow and escape the immune system's attack.

Macrophages are immune cells that can change their behavior depending on what the body needs. They can become two very different types, each with opposite effects on cancer. One type is called M1 macrophages, which are good at fighting cancer. These cells cause inflammation, help kill cancer cells, and send out signals that support the immune system's attack on tumors. M1 macrophages can be recognized by special markers on their surface, like CD80 and CD86.

The other type is called M2 macrophages, which can actually help cancer grow. M2 macrophages are known by their surface markers CD163 and CD206. They calm down the immune system, help tumors grow new blood vessels (a process called angiogenesis), and support the spread of cancer to other parts of the body. Instead of fighting tumors, M2 macrophages make it easier for tumors to survive and spread.

Dendritic cells are special immune cells that help start the body's defense against cancer. Their main job is to find and capture pieces of the tumor, called antigens, and show them to other immune cells. This process helps "train" naive T cells (T cells that haven't seen a threat before) to recognize and attack the cancer.

Dendritic cells can be identified by special surface markers, like CD11c and MHC class II molecules, which show they are experts at presenting antigens. Inside the tumor, dendritic cells play a key role in waking up the immune system to fight against cancer.

Figure 1. Roles of innate immune cells in metastatic cancers.(Gonzalez, H., et al. 2018)

In addition, NK cells have natural cytotoxicity and can kill tumor cells without prior sensitization, assisting immune surveillance; B cells can produce antibodies, participate in humoral immunity, and assist immune defense. The following is a comparison of the phenotypic markers and functional characteristics of different immune cells:

The supportive role of stromal cells

Stromal cells are important parts of the tumor environment because they provide structure and support for the tumor to grow. One key type of stromal cell is called cancer-associated fibroblasts (CAFs). These fibroblasts play a big role in changing the area around the tumor, called the extracellular matrix.

Cancer-associated fibroblasts make and release a lot of proteins, especially collagen and fibronectin, which help build the framework around the tumor. By changing this framework, they create a space that makes it easier for the tumor to keep growing and spread to other parts of the body.

Besides shaping the structure around the tumor, cancer-associated fibroblasts (CAFs) also release many active substances, like growth factors and inflammatory signals (cytokines). These substances help cancer cells grow faster, survive longer, and spread to other parts of the body.

Cancer-associated fibroblasts are very important in the tumor environment. They not only build the physical space for the tumor but also send out signals that help cancer grow and move. Their actions make them powerful helpers in cancer progression.

Figure 2. Signaling Between CAFs and Immune Cells. (Barrett, RL, et al. 2020)

Vascular endothelial cells are the main cells that build new blood vessels in tumors. As tumors grow and spread, they need a steady supply of oxygen and nutrients to survive. To get this, endothelial cells create new tiny blood vessels through a process called angiogenesis.These new blood vessels not only feed the growing tumor but also provide pathways for cancer cells to enter the bloodstream. Once in the blood, cancer cells can travel to other parts of the body and form new tumors.

Pericyte cells wrap around blood vessels and help keep them strong and stable. They control how easily substances pass through the vessel walls and help regulate blood flow.At the same time, fat tissue cells release special fat-based signals that affect how cancer cells use energy and grow.

Together, these different support cells work as a team to create an environment that helps tumors grow and spread by supporting them in many ways.

Extracellular matrix and secretory factor network

The extracellular matrix (ECM) and the substances it releases create a complex network around tumors. Two important parts of the ECM are collagen and hyaluronic acid.Collagen acts like a strong framework that supports the shape and structure of the tumor. It also forms a physical barrier that helps protect tumor cells by blocking immune cells from reaching and attacking them.Hyaluronic acid holds a lot of water, making the tumor tissue harder and thicker. This also helps shield the tumor from the immune system.

Cytokines and chemokines are important molecules that help cells send signals to each other.For example, Interleukin-6 (IL-6) helps tumor cells grow and survive, while at the same time it can weaken the immune cells that try to fight the tumor.Another molecule, Transforming Growth Factor-β (TGF-β), also slows down the immune system by stopping important immune cells like T cells and natural killer (NK) cells from working properly.The chemokine CXCL12 attracts immune cells that suppress the immune response to the tumor, helping the tumor avoid being attacked by the body's defenses.

Secretory factors have a dynamic change pattern in time and space. In the early stage of tumorigenesis, some pro-inflammatory factors may be activated in an attempt to initiate an immune response. As the tumor develops, immunosuppressive factors gradually become dominant.

Functional Interplay Between Tumors and TIME

Cytokine-mediated bidirectional communication

In the complicated tumor environment, cells communicate using signals called cytokines that go back and forth between cancer cells and immune cells.Many cancer cells produce a lot of a protein called PD-L1. When PD-L1 binds to PD-1 receptors on the surface of T cells, it sends a "stop" signal that makes the T cells weaker and less able to kill cancer cells.Another important "brake" on the immune system is a protein called CTLA-4. It attaches to proteins called B7 on special immune cells, stopping T cells from getting fully activated and multiplying to fight the tumor.

At the same time, there are signals that help activate the immune system inside the tumor environment.One important molecule, Interferon-gamma (IFN-γ), boosts the ability of immune cells by increasing the levels of MHC molecules on antigen-presenting cells. This helps the immune system better recognize and attack tumor cells.Another molecule, Interleukin-12 (IL-12), helps both T cells and natural killer (NK) cells grow and become more active, making them better at fighting cancer.

Cancer cells and immune cells send signals to each other using special molecules called cytokines.Cancer cells can release Interleukin-6, which helps them grow and survive while also weakening the immune cells that try to fight them.On the other hand, immune cells produce molecules like Interferon-gamma (IFN-γ) that tell cancer cells to show more signals (called MHC molecules) on their surface. This makes it easier for the immune system to find and attack the cancer.

Competition and adaptation in metabolic microenvironment

The metabolic characteristics of tumor cells have a significant impact on the function of immune cells. The Warburg effect of tumor cells leads to the production of a large amount of lactic acid, which accumulates in the tumor microenvironment, reduces the extracellular pH, and inhibits the activity and proliferation of T cells. Indoleamine 2,3-dioxygenase 1 (IDO1) can catalyze the decomposition of tryptophan into kynurenine, leading to arginine depletion in the tumor microenvironment, limiting the proliferation and function of T cells.

In addition to lactate and arginine, metabolites such as tryptophan and glucose also participate in the competition in the microenvironment. The high uptake of glucose by tumor cells reduces the amount of glucose available to immune cells, affecting their energy metabolism and function. Lack of tryptophan can lead to T cell dysfunction.

Figure 3. Changes in tumor cell metabolism that shape the inflammatory tumor
microenvironment.(Netea-Maier., et al. 2018)

Hypoxia-driven microenvironmental remodeling

Lack of oxygen is a common feature in tumors and causes many changes in their surroundings. A protein called HIF-1α helps control these changes by increasing the production of VEGF, a signal that tells blood vessel cells to grow and form new blood vessels. These new vessels bring more oxygen and nutrients to the growing tumor, helping it survive and expand.

At the same time, when HIF-1α is active, it also activates cancer-associated fibroblasts (CAFs). These cells make more proteins that build up the support structure around the tumor, helping cancer cells grow and spread. Low oxygen levels also cause macrophages to change into a type called M2, which helps tumors by suppressing the immune system. The lack of oxygen and acidic conditions in the tumor work together to make things worse: acidity boosts HIF-1α activity, leading to more blood vessel growth and even more activation of the fibroblasts that support tumor growth.

Throughout these interconnected processes, HIF-1α functions as the central regulatory hub, coordinating downstream gene expression programs that systematically reconstruct the tumor microenvironment. This transcriptional control mechanism drives multiple pathways supporting neoplastic progression, including enhanced angiogenesis, stromal remodeling, and immune suppression, ultimately facilitating both local tumor expansion and distant metastatic dissemination. The integrated nature of these HIF-1α-mediated responses demonstrates the sophisticated adaptation mechanisms tumors employ to overcome environmental constraints and promote their continued growth.

Technologies for TIME Profiling

Spatial transcriptomics applications

Spatial transcriptomic methodologies offer innovative approaches for investigating tumor immune microenvironment architecture and cellular interactions. These technologies enable researchers to characterize gene expression patterns while preserving critical spatial context within tissue specimens.

The Visium platform employs comprehensive transcriptome profiling of histological sections through a sophisticated technical framework. This methodology integrates tissue specimens with specialized slides containing spatially-barcoded oligonucleotide capture probes, facilitating mRNA hybridization to designated locations. Following reverse transcription and high-throughput sequencing procedures, researchers obtain spatially-resolved gene expression data across entire tissue sections. While this approach provides broad transcriptomic coverage with excellent throughput capabilities, spatial resolution remains somewhat limited compared to alternative technologies.

In contrast, the GeoMx Digital Spatial Profiler represents a region-specific targeted sequencing platform utilizing distinct technical principles. This system employs barcoded antibodies or oligonucleotide probes to mark RNA molecules within precisely defined tissue regions of interest. Laser-based capture mechanisms subsequently isolate these labeled areas for downstream sequencing analysis. Although GeoMx DSP delivers superior spatial resolution for focused transcriptomic characterization, experimental throughput is correspondingly reduced relative to whole-section approaches.

These spatial transcriptomic platforms demonstrate particular utility in tumor-immune neighborhood characterization studies. By maintaining spatial context during gene expression analysis, these technologies reveal critical information regarding the geographic distribution patterns of malignant and immune cell populations within tissue architecture. Furthermore, spatial transcriptomics enables investigation of intercellular communication networks and molecular interactions between tumor cells and their surrounding immune microenvironment, providing insights that traditional bulk sequencing approaches cannot achieve.

Multiplex immunohistochemistry practice

Multiplex immunohistochemistry (mIHC) is an important TIME analysis technology, which mainly includes two methods: fluorescent labeling and metal labeling. Fluorescent labeling, such as Opal technology, has a multi-target detection process: first, the tissue section is antigen repaired, and then different fluorescent labeled antibodies are incubated in sequence, each antibody labels a specific antigen. Through multiple staining and imaging, multiple targets are identified using a combination of different fluorescent signals.

Metal labeling, such as CyTOF technology, combines metal isotope-labeled antibodies with tissue sections and then detects metal signals using a mass spectrometer, thereby achieving multi-target detection.

In terms of quantitative analysis of immune cell spatial localization, mIHC can count and locate differently labeled immune cells through image analysis software. For example, when calculating the density of T cell infiltration, the number of T cells per unit area can be counted. The principle of selecting markers is to be specific and representative, and to accurately reflect the type and function of immune cells.

Single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-seq) is a powerful method to study different types of immune cells in detail.One popular technology, 10x Genomics, uses tiny droplets to capture individual cells and then reads their gene activity. It can process many cells at once and captures most of the cells, but it doesn't read as many genes in each cell.Another method, Smart-seq2, is more sensitive and can detect more genes in each cell by reading the full length of RNA molecules. However, it captures fewer cells at a time.

Conclusion

To understand the tumor immune microenvironment (TIME) well, scientists use several advanced methods together. These include spatial transcriptomics, single-cell analysis, and metabolomic profiling.Spatial transcriptomics shows where different cells are located in the tissue and how they communicate with each other.Single-cell RNA sequencing looks closely at the gene activity inside individual cells.Metabolomic profiling studies the chemicals and energy processes inside cells to learn how they are functioning.By combining these methods, researchers can get a complete picture of the tumor environment.