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The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

Modern cytometry methods allow collecting complex, multi-dimensional data sets from heterogeneous cell populations at single-cell resolution. While methods exist to describe the progression and order of cellular processes from snapshots of such populations, these descriptions are limited to arbitrary pseudotime scales. Here we describe MApit, an universal transformation method that recovers realtime dynamics of cellular processes from pseudotime scales by utilising knowledge of the distributions on the real scales. As use cases, we applied MAPiT to two prominent problems in the flow-cytometric analysis of heterogeneous cell populations: (1) recovering the kinetics of cell cycle progression in unsynchronised and thus unperturbed cell populations, and (2) recovering the spatial arrangement of cells within multi-cellular spheroids prior to spheroid dissociation for cytometric analysis. Since MApit provides a theoretic basis for the relation of pseudotime values to real temporal and spatial scales, it can be used broadly in the analysis of cellular processes with snapshot data from heterogeneous cell populations.

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIPL expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIPL knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.

Cancer is one of the most serious diseases for human beings, especially when metastases come into play. In the present article, the example of lung-cancer metastases in the brain is used to discuss the basic problem of cancer growth and atrophy as a result of both nutrients and medication. As the brain itself is a soft tissue that is saturated by blood and interstitial fluid, the biomechanical description of the problem is based on the Theory of Porous Media enhanced by the results of medication tests carried out in in-vitro experiments on cancer-cell cultures. Based on theoretical and experimental results, the consideration of proliferation, necrosis and apoptosis of metastatic cancer cells is included in the description by so-called mass-production terms added to the mass balances of the brain skeleton and the interstitial fluid. Furthermore, the mass interaction of nutrients and medical drugs between the solid and the interstitial fluid and its influence on proliferation, necrosis and apoptosis of cancer cells are considered. As a result, the overall model is appropriate for the description of brain tumour treatment combined with stress and deformation induced by cancer growth in the skull.

Apoptosis, a form of programmed cell death, can be induced in lung-cancer cells by treatment with death-receptor ligands. In this contribution, the coupled multiphasic process is described using a continuum-mechanical model based on the Theory of Porous Media. Furthermore, the data-enriched model incorporates the crucial apoptosis parameters, which are estimated via the maximum likelihood estimation based on cell-culture experiments.

Extrinsic apoptosis relies on TNF-family receptor activation by immune cells or receptor-activating biologics. Here, we monitored cell cycle progression at minutes resolution to relate apoptosis kinetics and cell-to-cell heterogeneities in death decisions to cell cycle phases. Interestingly, we found that cells in S phase delay TRAIL receptor-induced death in favour for mitosis, thereby passing on an apoptosis-primed state to their offspring. This translates into two distinct fates, apoptosis execution post mitosis or cell survival from inefficient apoptosis. Transmitotic resistance is linked to Mcl-1 upregulation and increased accumulation at mitochondria from mid S phase onwards, which allows cells to pass through mitosis with activated caspase-8, and with cells escaping apoptosis after mitosis sustaining sublethal DNA damage. Antagonizing Mcl-1 suppresses cell cycle-dependent delays in apoptosis, prevents apoptosis-resistant progression through mitosis and averts unwanted survival from apoptosis induction. Cell cycle progression therefore modulates signal transduction during extrinsic apoptosis, with Mcl-1 governing decision making between death, proliferation and survival. Cell cycle progression thus is a crucial process from which cell-to-cell heterogeneities in fates and treatment outcomes emerge in isogenic cell populations during extrinsic apoptosis.