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When immunology meets oncology

Oncology is the most prevalent research area worldwide and has grown to encompass immune tolerance, innate immunity and metabolic regulation in the larger field of Immuno-Oncology.
From an immunity standpoint, tumor management by the organism goes through 7 stages as described by Mellman & Chen in 2013, and is termed the “cancer immunity cycle”. It starts with unaddressed tumor cells releasing tumor antigens that can be captured, digested and presented by dendritic cells. Antigen capture initiates the usual unfolding of an adaptive response through T-cells priming, activation and release in blood. At this point, active T-cells circulate the organism looking for the tumor, which they then infiltrate. Once in the microenvironment of the tumor, T-cells proceed to specifically recognize tumor cells and kill them following immune checkpoint approval. In some cases, a few tumor cells manage to develop strategies or mutations that allow them to evade death, which starts the cycle all over again with a new strain of cells the immune system fail to address.

Depending on their nature, cancers are split in 3 categories that oppose different challenges to cancer immunity cycle 7 stages.

  • tumor in immune deserts are tissues empty of immune cells which cause them to avoid the immune response from the very start
  • immune excluded tumor are characterized by an immune response but remain out of reach for T-cells, which fail to penetrate their microenvironment
  • inflamed tumors are actively patrolled by immune cells that secrete inflammatory messenger in their microenvironment, their usual strategies to evade cellular death involves immune checkpoint alterations that prevent T-cells from killing them.

All 7 stages of the cycle can be addressed with a range of therapeutic strategies to enhance the immune strategies already in place and support the immunity-driven elimination of tumor cells. Depending on the type of cancer those strategies vary and adapt. To support that research, Cisbio grew a dedicated portfolio which covers various aspects of immunity and immune cells as well as solutions and content to support IO studies. We recommend reading our guides to Immunology and Immuno-Oncology therapies to review the basics of the field and update your knowledge on the current state of research.

Take a tour of innate immunity’s core strategies

Innate immunity gathers the non-specific strategies and defenses susceptible of immediate or short-term intervention upon antigen presence in the organism. At the cellular level, these strategies involve a large array of receptors (TLRs) and sensors (STING axis and inflammasomes) that induce inflammatory signals and antigen clearance.
Those receptors and sensors are shared among most unspecified immune cells including dendritic cells, macrophages, neutrophils, natural killer or mastocytes.

Toll-Like Receptors

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that usually bind ligands specific to micro-organisms such as lipids and proteins from bacterial walls or double-stranded RNA. Humans express ten types of them, either at the cell surface (Types 1, 2, 4, 5, 6 and 10) or on intracellular vesicles (3, 7, 8, and 9).
They are used and studied as active immuno-oncology therapies, as well as therapies for infectious and inflammatory diseases. In such therapies, small molecules are designed to activate or block different TLRs and alter the immune response.
Cisbio has offers a range of ready-to-use assays to monitor transcription factors, phosphoproteins and kinases involved in TCR signaling.

Activation of TLRs through ligand binding triggers a signaling cascade involving a variety of intracellular signaling adaptors, including MyD88, IRAKs, and TRAF6. TLR signaling leads to the activation of the MAP kinase, IKKs, TBK1, and IRF signaling pathways, which promotes transcription factors including NF-κB, IRF3, IRF7, and Jun. These mediate inflammation through the production of inflammatory cytokines, type I IFN, chemokines, and antimicrobial peptides.

STING Axis

The STimulator of INterferon Genes (STING), is a cytoplasmic protein that teams up with the cytoplasmic DNA sensor cGAS to detect floating dsDNA from pathogens or shrinking mitochondria and initiate a signal transduction via TBK1, IRF3 and NF-kB to promote the expression of pro-inflammatory mediators and type 1 IFNs (TNF-a, IFN- a, IFN- b).
In immuno-oncology, tumor derived DNA fragments have been shown to drive anti-tumor immune response through the STING pathway and activating it has shown promising anti-tumor effects in pre-clinical models. It has also been deeply investigated in the context of autoimmune inflammation, as diseases such as lupus and psoriasis are suspected to arise from nucleic acid self-antigens.
Cisbio has developed a panel of high-quality assays to pinpoint those pro-inflammatory signal transduction steps as well as the resulting transcription factors and products of STING activity.

cGAS binds cytosolic floating dsDNA, and starts the production of 2’-3’cGAMP, a cyclic dinucleotide, which then binds to STING proteins. In turn, phosphorylated STING interacts with TBK1 leading to the recruitment and activation of active IRF3 dimer. Those translocate to the nucleus and promotes the expression of IFN-α/β. In addition, the STING pathway controls NF-κB dependent inflammatory cytokine expression. Negative feedback is ensured though STING protein degradation via p62 SQSTM1 associated autophagy, which switches off IFNβ production.

Inflammasomes

Inflammasomes are cytosolic complexes that enable the rapid activation of cytokines IL-1b and IL-18 from their precursors pro-IL1b and pro-IL18 and promote the extracellular secretion of the pro-inflammatory alarm protein HMGB1.
They consist of identical ASC specks made up of an upstream sensor protein (NLRP, NLRC or AIM2) linked to a downstream effector pro-caspase-1 by an adaptor protein ASC. This structure allows their assembly into wheel-like complexes upon binding of their sensor proteins.
To better monitor and understand those complexes, Cisbio developed a collection of assays targeting different steps of inflammasome activity and end results.

TLRs signaling promotes the NF-kB pathway and expression of pro-IL1β, pro-IL18, inflammasome sensor proteins and pro-caspase-1. Upon binding of the sensor proteins by pathogens, ASC specks assemble into the larger wheel-shaped inflammasome active complex, which cleaves pro-caspase-1 into caspase-1. Active caspases then proceed to cleave stored pro-IL1β and pro-IL18 into their active forms, promoting inflammation. Inflammasome assembly and caspase-1 activation also regulate and promote the secretion of the alarm protein HMGB1.

Explore the frontier of cancer killing cells

Natural Killer cells (NK) are a subset of lymphoid cells known for their ability to target and lysate a wide spectrum of tumorous, infected, or damaged cells, without prior activation and in a non-specific fashion. They also act as sentinels ready to detect degenerated cells early on and stimulate adaptive immunity with inflammatory cytokines. These abilities make them critical early responders to tumor development.
They are understood to target and kill cells thanks to a collection of activating and inhibitory receptors that guide their decision-making process by respectively binding molecules expressed on the surface of tumorous or infected cells and MHC I or inhibitory ligands. Due to their non-specific lytic properties, they are a promising research axis that calls for a better understanding of their receptor and signaling pathways.

The decision to kill or spare is based on the ratio of activating and inhibitory signals they receive from their interlocutor. The simultaneous binding of multiple activating receptors can override the signal of inhibitory receptors binding MHC I, and trigger NK cell cytotoxic activity. The same override phenomenon has been observed when a potent enough activating receptor is bound. The case of self and healthy cells expressing low or no MHC I, such as red blood cells and neural tissues, is not fully understood within this theory. They could either lack activating ligands or express inhibitory ligands>/figcaption>

Dive into the professional adaptive cells of immunity

Adaptive immunity gathers the antigen-specific strategies and defenses the immune system mobilizes on mid to long-term to handle threats that would override innate mechanisms alone. It mostly consist of T-cells that mediate inflammatory signals and specialized cellular toxicity, and B-cells that are responsible for humoral responses through antibodies secretion.

T-cells & CAR-T therapies

T-cells are star players of the adaptive immunity and contribute to inflammation, recruitment and management of other cells, direct elimination of pathogens and overall surveillance and monitoring of the immune response. They are dependent on their TCR receptors and corresponding signaling pathway to initiate their activity following antigen binding. Consequently, TCR signaling study is a central piece of immuno-oncology and inflammatory research as a readout of T-cells activity.
CAR-T cell therapies are a patient-specific T-cell based therapeutic approach where T-cells are directly collected from patients’ blood and genetically modified to express Chimeric Antigen Receptors (CAR) that allow them to target and kill cancer cells specific to the patient they were taken from. CAR-T cells are then amplified and reinjected to the patient. This approach is both highly technical and long but is expected to yield revolutionary cancer therapies. Due to its technicity, CAR-T cells call for specific detection assays that assess both their functionality and persistence during development.

TCR engagement promotes the phosphorylation of ITAM bearing segments on the cytosolic side of the TCR/CD3 complex by Lck. Zap-70 is recruited to the TCR/CD3 complex where it becomes phosphorylated and activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins. To result in a full activation and expression of T-cell phenotype, TCR engagement needs to be associated with CD4/8 activation and CD28 binding, which respectfully promote Lck activity and cytokine expression. Key immune checkpoints CTLA-4 and PD1 also contribute to the regulation of the overall system via phosphorylation-dephosphorylation of SHP1 & SHP2.

B-cells

B-cells are responsible for the humoral part of the adaptive response by secreting antibodies against pathogens. As such they are critical to all therapies pertaining to tumor detection by dendritic cells and tumor infiltration by T-cells. B-cells rely on their BCR receptors to identify antigens and evolve into antibody-secreting plants. As a results BCR signaling is a pathway of interest for monitoring B-cell activation.

BTK is a Cytoplasmic tyrosine kinase involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Initiation of BCR signaling involves Lyn and Syk, part of the Src family. Lyn phosphorylates the intracellular domain of the BCR, leading to the recruitment and phosphorylation of Syk, and eventually, SLP65 phosphorylation. This leads to the recruitment and phosphorylation of BTK and the promotion of multiple mediators and transcription factors.

An assay platform to advance Immune Checkpoints studies

The blockade of immune checkpoints is a promising approach to activate therapeutic anti-tumor immunity. It is now clear that tumors co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. Because many of the immune checkpoints are initiated by ligand–receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors. To date, Cisbio offers 5 assays to monitor inhibitory checkpoints and investigate strategies that prevent tumor cells from evading cellular death.

Explore and monitor immunosuppressive behaviors

Immunosuppression refers to the mechanisms susceptible of decreasing and stopping the immune response. It is especially important in inhibiting inflammatory signals and killer cells behavior that would otherwise grow dangerous for the organism once an antigenic threat has been dealt with. Regulatory T-cells usually oversee immunosuppressive strategies, but other cell types such as type 2 macrophages also fulfill immunosuppressive roles through TGF-b secretion.
In Immuno-Oncology research, finding ways to monitor and decrease immunosuppression could yield therapies that boost the immune response and empower the immune system against tumors by removing or adjusting the brakes that keep it in check. Cisbio has developed a portfolio to map the main immunosuppressive strategies that regulatory T-cells exhibit, including secretion of anti-inflammatory IL-10, synthesis of immunosuppressive adenosine, immune checkpoint-driven inhibition or TGF-b expression.

IL-10 is the usual anti-inflammatory cytokine and a characteristic product of regulatory T-cells immunosuppressive activity. TGF- is also a regulatory T-cell secretion but is expressed in significant amount by type II (alternatively activated) macrophages, as a part of an overall anti-inflammatory process that both tones down immune cells and promotes tissue healing. The other immunosuppressive strategies illustrated here include immune checkpoint de-activation of T-cells by immune checkpoint couples and adenosine-mediated signaling through the adenosine family receptors.

Do not miss on ubiquitous pathways of inflammation

Proliferation & inflammation pathways

Along with the usual main cell types and signaling pathways that characterize Immuno-Oncology, other inflammatory pathways are relevant to IO research as they provide an insight into cell-to-cell signaling, inflammation and proliferation. Some of those pathways were addressed by Cisbio to provide more complete and polyvalent solutions. These include the P13K/AKT/mTOR axis and SAPK pathway, both in charge of cell survival and proliferation and commonly mis regulated in cancer, as well as the NF-kB and JAK/STAT axis, which play roles in cellular inflammation.