Cell death can be divided into two modes: regulated cell death (RCD) and accidental cell death (ACD). The RCD represents the apoptosis pathways, which refers to the autonomous and orderly cell death controlled by different genes in order to maintain a stable internal environment. In contrast, non-physiological stimuli, such as passive and non-programmed necrosis induced by external factors such as physical, mechanical, and chemical stress, are representatives of ACD.

Necroptosis combines the characteristics of necrosis and apoptosis. During necroptosis, morphological changes such as swelling of organelle, cell membrane rupture, and cytoplasmic as well as nuclear disassembly can be observed.

Studies suggest that necrosis of tumor cells is a self-sacrificed strategy to create a favorable environment for its proliferation and metastasis, but necroptosis plays a tumor suppressive role in most cases. In 2016, Aaes et al. first identified necroptosis in tumors as an immunogenic cell death. In the article titled "Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity", Aaes et al. made preventive vaccines from necroptotic tumor cells and injected them into mice in advance. They found that pretreatment with the prophylactic vaccine inhibited tumor growth in mice, suggesting that necroptotic tumor cells are themselves immunogenic and stimulate the adaptive immune system. Aaes et al. also pointed out that tumor cells undergoing necroptosis can induce the maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ^[2].

On August 21, 2021, You-Sun Kim's team from South Korea published a research paper entitled "RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment" on Molecular Cancer. Using a TAP-MS approach, coupled with analysis of RNA sequencing (RNA-Seq) datasets, the researchers identified TRIM28 as a co-repressor that regulates transcriptional activity during necroptosis. Activated RIPK3 phosphorylates TRIM28, inhibiting the chromatin-binding activity of TRIM28, thereby promoting the transactivation of NF-κB and other transcription factors such as SOX9. These ultimately lead to increased expression of cytokines, which then enhance immune regulatory pathways such as dendritic cell maturation, among others. In conclusion, RIPK3 expression was significantly and positively correlated with tumor-infiltrating immune cell populations across various tumor types, thereby activating antitumor immune responses (Figure 2)^[3].

On May 25, 2022, the field of tumor immunity has made new discoveries about necroptosis. Nature published a paper titled "ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis". Studies have pointed out that the right-handed state of DNA (B-DNA) is the most common state existing in organisms, and under special circumstances, B-DNA flips to form a "freaky" left-handed state, Z-DNA. Both Adenosine deaminase RNA specific 1 (ADAR1) and Z-form nucleic acid binding protein 1 (ZBP1) can specifically recognize and bind to Z-DNA. ADAR1 blocks the action of immune checkpoint blockade (ICB) by inhibiting immunogenic double-stranded RNAs (dsRNAs), while ZBP1 drives tumor cell necroptosis.

CBL0137 triggers the formation of left-handed Z-DNA in cells and activates the ZBP1-dependent necroptosis pathway in cancer-associated fibroblasts (Figure 3). CBL0137 induces ZBP1-dependent necroptosis in cancer-associated fibroblasts. Regardless of the cancer-causing mutation, CBL0137 kills fibroblasts that support tumor growth. ICB unresponsiveness was also reversed in a mouse model of melanoma^[4].

In the detection of necroptosis, the key challenge is to distinguish it from apoptosis and necrosis. In many cases, different modes of death, such as apoptosis, necrosis and necroptosis, will simultaneously exist in the cell population. Therefore, detection of the cell death should be supplemented by real-time morphological analysis and inhibition or knockdown of death pathways. At the same time, it is also crucial to find the right time point for sampling.

Changes in morphology

The morphological change of cell death is a dynamic process. Therefore, it is necessary to "monitor" the whole process in real time to confirm the occurrence of necroptosis. Time-lapse video microscopy can correlate morphological changes of individual cells with live ongoing molecular, subcellular, and biochemical events to detect necroptosis (although this approach is expensive and labor-intensive) (Figure 4).

Inhibition and knockdown

Necroptosis inhibitors, RIPK1 inhibitor Necrostatin-1 and MLKL inhibitor Necrosulfonamide, are usually used to block necroptosis, and the survival rate of cells is measured to reverse verify the occurrence of necroptosis. Some inhibitors can induce apoptosis in specific cells and are not precise enough to distinguish necroptosis from apoptosis. Thus, evidence from inhibition experiments needs to be complemented by knockdown of key proteins, such as RIPK3 or MLKL, to block necroptosis (Figure 4).

Detection of key proteins

The changes of key proteins in the necroptosis pathway, such as RIPK3, RIPK1 and MLKL, has been detected by western blot, immunohistochemistry and flow cytometry. Phosphorylated MLKL (Ser358 and Thr357) is the most commonly detected protein which can confirm the necroptotic pathway via RIPK3-mediated activation of MLKL. In addition, the high ratio of RIPK1/pro-caspase-8 also illustrates the favorable intracellular environment towards necroptosis (Figure 4).

Detection of key proteins

In cancer, necroptosis occurs mainly in the center of advanced tumors, where necrotic cells are scattered. It is difficult to detect key protein markers without enrichment by microdissection. In the article "Examining MLKL phosphorylation to detect necroptosis in murine mammary tumors", Baik et al. has provided a detailed immunohistochemistry-directed approach. The approach includes tumor isolation from a mouse mammary tumor model, identification of necrotic areas by H&E staining, and detection of phosphorylated MLKL to detect necroptosis (Figure 5). This method can also be applied to other types of solid tumors.