The tumor immunity cycle provides a useful framework for understanding how immune cells recognize, respond to, and ultimately eliminate malignant cells. From antigen release to T cell priming, tumor infiltration, and cytolytic killing, each phase can be disrupted by the tumor through suppressive cytokines, checkpoint ligand expression, or recruitment of regulatory cells. Because these regulatory signals occur through both surface molecules and secreted factors, measuring soluble immune proteins has become an increasingly important strategy for characterizing functional immune states in cancer.

 

T cell-mediated immunity is central to anti-tumor responses, and immune checkpoint molecules such as PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3 regulate the balance between activation and exhaustion. Although these proteins are best known as membrane bound receptors and ligands, many also exist in soluble forms generated through proteolytic shedding or alternative splicing. These soluble isoforms can modulate signaling and frequently reflect the level of immune activation occurring in the tumor microenvironment or in peripheral blood. Due to immune checkpoint inhibitors (ICI) acting directly on these pathways, soluble checkpoints have emerged as important biomarkers for predicting and monitoring ICI responsiveness.¹

 

Detection of critical soluble immune checkpoint molecules and tumor microenvironment factors with LEGENDplex panels

To assess how inflammatory stimuli regulate checkpoint release, peripheral blood mononuclear cells (PBMCs) were stimulated with LPS or with LPS in combination with IFN-γ and then assayed using the Human Immune Checkpoint Panel 1. This dataset reveals distinct soluble checkpoint signatures associated with innate and interferon driven activation states, underscoring the relationship between inflammatory signaling and checkpoint regulation.

 

 

Human PBMCs (1 x 10⁶ cells/mL) were cultured unstimulated or stimulated with LPS (100 ng/mL) alone or with LPS and IFN-γ (100 ng/mL). Supernatants were collected after 42 hours of culturing and assayed with LEGENDplex™ Human Immune Checkpoint Panel 1. Each bar represents the measured analyte concentration for each condition.

 

Beyond checkpoint profiling, additional multiplex panels expand the ability to monitor T cell function. The Human CD8/NK Panel V02 enables characterization of cytolytic mediators such as perforins, granzymes, and chemokines associated with effector activity, while the Human TNFSF Family Panel 1 captures a range of co-stimulatory and co-inhibitory ligands important for T cell priming and maintenance of memory responses. Together, these panels create an integrated view of T cell activation, suppression, and effector function that supports mechanistic immunology and translational biomarker studies. 

 

For preclinical and translational research, our new mouse-focused solutions, including the Mouse CD8/NK Panel and the Mouse Immune Checkpoint Panel, extend these capabilities to small animal tumor models and cell-based systems.

 

Tumor-associated macrophages in anti-tumor responses

Myeloid-mediated immunity represents a parallel branch of the anti-tumor response, with tumor-associated macrophages (TAMs) playing a particularly influential role.² TAM populations range along a continuum often described as M1-like or M2-like states. M1-like macrophages support anti-tumor immunity through production of IL-12, TNF-α, and CXCL9/10, while M2-like macrophages tend to drive tissue repair, angiogenesis, and immunosuppression through factors such as IL-10, CCL17, CCL22, and TGF-β associated pathways. The relative abundance or activity of these macrophage subtypes has become a compelling biomarker of therapeutic response, especially in contexts where macrophage targeted therapies aim to shift the TME from an M2 dominant to an M1 dominant state.³

 

Markers included in the Human Tumor-Associated Macrophage Panel and the Human Macrophage/Microglia Panel support detailed assessment of macrophage polarization and function. These panels quantify soluble cytokines and chemokines linked to inflammatory and immunoregulatory macrophage states.

 

Diagram highlighting differences between M1 and M2 macrophage function and polarization.

 

To demonstrate how macrophage signatures shift in a tumor-like environment, PBMC-derived macrophages were polarized to M1- or M2-like states and then co-cultured with tumor cells before being analyzed with the Human Macrophage Panel and Human TAM Panel. These data revealed clear distinctions between proinflammatory and pro-tumorigenic cytokine signatures and highlighted how tumor cell signals can reshape macrophage function.

 

 

Cytokine profiling by LEGENDplex: Heatmaps of secreted cytokines
THP1 monocytes (3 × 10⁶ cells in 5 mL) were seeded into T25 flasks and treated with PMA for 48 hours. In parallel, HT29, MDAMB231, and A549 cells were sub-cultured for 48 hours, and the tumor-conditioned media (tCM) were collected. Following differentiation, medium containing PMA was removed, and adherent THP1-derived macrophages were rinsed once with fresh RPMI1640 medium. Macrophages were then co-cultured with tCM from each cancer cell line for 72 hours, and supernatants were collected to evaluate cytokine secretion profiles from the co-culture systems. Heatmaps show the mean z-scores of cytokine levels measured in cell culture-conditioned media (CM) using the LEGENDplex assay (A) Macrophage/Microglia Panel; (B) Human Tumor-Associated Macrophage Panel. Data are visualized using a red–white–blue color scale, where red represents higher expression and blue represents lower expression across the different culture conditions. Cytokine markers with low signal levels were excluded from the dataset.

 

Soluble protein measurements provide a powerful means of dissecting the cellular crosstalk that drives anti-tumor immunity. By integrating checkpoint signaling, T cell effector activity, and macrophage polarization into existing workflows, researchers can better understand cancer immune evasion mechanisms, uncover biomarkers of responsiveness, and design more effective immuno-oncology strategies.

 

Custom solutions for immuno-oncology research

As researchers study the complexities of cancer and the immune systems’ relationship with the tumor network, collaboration is paramount. We provide custom solutions developed with your research in mind, providing scientists with versatile tools for cancer immunotherapy studies, like recombinant bispecific antibodies to target multiple specific antigens, custom conjugates, and immunoassays. 

 

For additional resources, visit our cancer research hub for educational content, webinars, and insights into the latest discoveries.

 

References

1. Chen, L., Chao, Y., Li, W., Wu, Z., & Wang, Q. (2024). Soluble immune checkpoint molecules in cancer risk, outcomes prediction, and therapeutic applications. Biomark Res, 12, 95. https://doi.org/10.1186/s40364-024-00647-0.  
2. Saeed, A. F. (2025). Tumor‑associated macrophages: Polarization, immunoregulation, and immunotherapy. Cells, 14(10), 741. https://doi.org/10.3390/cells14100741. 
3. Shapouri‑Moghaddam, A., Mohammadian, S., Vazini, H., Taghadosi, M., Esmaeili, S., Mardani, F., et al. (2018). Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol, 233, 6425–6440. https://doi.org/10.1002/jcp.26429.