Our study's successful application of the RIP3-Caspase3-assay in heterogeneous organoids highlights its methodological strengths. By employing directly conjugated monoclonal antibodies, our assay provides a nuanced analysis of cell death pathways, revealing the complexity of these processes in greater detail. This approach aligns with Lee et al.’s findings, which distinguished between apoptosis, necroptosis, and RIP1-dependent apoptosis in both viable and non-viable cell populations [9]. Our extension of these advancements to spheroid cultures marks a first in the field, showcasing the assay's reliability and its capability to discern between cell death mechanisms. In our study, the treatment of isolated intestinal epithelial cells from spheroids with TNFα accelerated the cell death rate, as indicated by Zombie-stained cells. The study observed an initial lower response to TNFα that increased with higher concentrations. In this context, this pattern suggests a possible adaptive or threshold response of the cells to TNFα-induced stress. TNFα is known to activate multiple signaling pathways, including NF-κB, MAPKs, and the apoptotic pathway [10]. The activation of NF-κB, for instance, can lead to the expression of survival genes, which might help cells adapt and survive under increasing TNFα concentrations [11]. However, in this study we had a linear response to the TNFα concentration with relevant damage of the organoids at 100ng/ml after 48h. This finding was in line with observed morphological changes of treated organoids (Fig. 1).
Upon TNFα stimulation, we observed an increase in Caspase3 expression and a decrease in RIP3 expression across all samples. FACS analysis further revealed that TNFα-treated cells showed a significant increase in apoptosis, while simultaneously shifting away from RIP1-dependent apoptotic mechanisms, without involving necroptosis (Fig. 2C-E). This finding suggests a preference for RIP1-independent cell death in both living cells undergoing cell death and those that had already died. The shift towards apoptosis in the presence of TNFα is a key finding. Apoptosis is a programmed and orderly cell death process, often considered immunologically silent, while necroptosis is a form of programmed necrosis that is inflammatory in nature. The decision between apoptosis and necroptosis is often determined by the availability and activity of key regulatory proteins like RIP1, RIP3, and Caspase8 [11]. This shift in the type of cell death can also be attributed to the inflammatory context, where the co-sensing of multiple cytokines and the activation of various pathways, such as the non-canonical NF-κB pathway, lead to different forms of cell death. For instance, the combination of TNFα with other cytokines can result in TNFR1-induced RIP1 kinase activity-dependent apoptosis [11]. Also, the balance between apoptosis and necroptosis is depended on the concentration of TNFα [12]. In contrast, TNFα treatment did not impact the proportion of "other" cell death types substantially. However, cells sorted into this category showed a higher proportion in the dead cell section than in the living cells, which may be attributable to the disruption of cell membrane integrity and subsequent cell death during assay preparation.
Another important finding is the differential response in living versus dead cells (Fig. 2D-E). The significant changes in the dead cell population, as opposed to the living cells, suggest that TNFα effects are more pronounced in cells already compromised or stressed. This could imply that TNFα is a more potent inducer of cell death pathways in cells that are already damaged. This is because stressed or damaged cells might have compromised defense mechanisms (for example reduced expression of anti-apoptotic proteins), making them more prone to TNFα cytotoxic effects [11]. Also, the absence of necroptotic cells in both living and dead cells suggests that, under these conditions, TNFα does not induce necroptosis and underscores TNFα role in promoting apoptosis under these experimental conditions in colonic epithelial organoids.
The assay's ability to detect both major and minor cell populations undergoing various forms of cell death is particularly valuable in the context of spheroid cultures, where cell heterogeneity and complexity are inherent [7]. Notably, unlike the original technique described by Lee et al., our approach did not use CalTag fixative, known to upregulate RIP3. This modification is noteworthy because the detection of RIP3 in our results is not influenced by the fixative. Since we were still able to detect RIP3 as an individual cell death marker in all organoids, suggests that RIP3 is inherently present in the organoids and is not an artifact of the fixation process. Interestingly, necroptotic RIP3+/Caspase3− cells were not identified in downstream analysis, regardless of stimulation or Zombie status. Since RIP3 was present in RIP1-dependent apoptosis, as indicated by RIP3+/Caspase3+ status, the reasons why the assay did not determine necroptosis remain unclear. This could be due to several factors, such as the specific cellular context, the sensitivity of the assay, or other regulatory mechanisms in the cells that may inhibit necroptosis. To further explore this, our ongoing investigations are utilizing a cytokine cocktail containing IL-6 and IL-1ꞵ to simulate intestinal inflammation in a more holistic manner.
This study, while showcasing several strengths, also acknowledges certain limitations. Colonic organoid cultures, composed of different cell types like the colonic epithelium, provide insights into the composition and interaction of diverse cell populations in organoids. However, our analysis did not include cell type determination, which may mask specific dysregulations in signaling pathways. Additionally, for FACS analysis, organoids must be dissociated into single cells, a process that can inevitably cause cell damage and produce debris. The amount of “other” cell death observed may partly result from mechanical damage during this process. To mitigate these challenges, we employed lower centrifugal accelerations and carefully separated cell clusters using a dissociation reagent, which positively influenced cell death rates. Moreover, cell damage and death, accompanied by an increase in debris, were noted after differentiation periods exceeding 96 hours. To ensure sufficient differentiation of the organoids while minimizing these effects, we precisely matched the supplement addition and time points of differentiation. Considering the varied growth rates of organoid cultures derived from primary patient material, ensuring comparability of our results required measuring the same number of single cells in each tube, leading to a smaller number of analyzed cells. The challenges in reaching the necessary cell count during assay preparation led to the exclusion of samples with lower cell numbers, with minimal cell loss achieved by transferring organoids twice during preparation. Despite these challenges, the robustness of our results across a highly heterogeneous set of organoids highlights the substantial broader implications of this methodological innovation. The assay’s adaptability to spheroid cultures significantly enhances our understanding of diseases marked by cell death dysregulation. By providing detailed insights into the mechanisms of cell death in these physiologically relevant 3D models, our approach paves the way for more accurate disease modeling and the potential development of targeted therapies [7].
In conclusion, the RIP3-Caspase3-assay represents a significant advancement in organoid research. The application of this assay to spheroid cultures, a first in this field, has demonstrated its robustness and reliability in discerning between various cell death mechanisms, such as apoptosis and necroptosis.