Ny forskningsrapport från Huntsman

PHI`s partner Huntsman Cancer Institute fick i lördags (27/6) sin forskningsrapport om programmerad celldöd publicerad på Applied Sciences.

Det är Robert L. Judson-Torres m kollegor som ligger bakom denna studie. Med det kan vi lista ut att det rör hudcancer vilket i dessa dagar får mer uppmärksamhet än vanligtvis.Läs den här hjärtknipande berättelsen :CANCER TRAGEDY

Mum-of-two, 26, died of skin cancer after GP ‘told her mole was nothing to worry about’

Men till Roberts studier :

Label-Free Classification of Apoptosis, Ferroptosis and Necroptosis Using Digital Holographic Cytometry

Received: 26 May 2020 / Revised: 23 June 2020 / Accepted: 25 June 2020 / Published: 27 June 2020

Abstract

Apoptosis, ferroptosis and necroptosis are three distinct forms of programmed cell death. Each of these pathways can be exploited to terminate cancer cells. One promising therapeutic strategy is to activate alternative programmed cell death pathways subsequent to cancer cells evolving mechanisms to evade apoptosis. However, the interplay between distinct programmed cell death pathways and cancer progression is complex and can paradoxically promote the disease. There is a need for high-throughput assays for real-time classification of programmed cell death, both to further investigate these important biologic processes and to assess the case-by-case efficacy of targeting each pathway in patient-derived tumor cells. Here, we sought to develop a label-free, live-imaging-based assay for classifying forms of programmed cell death with single cell resolution. We used digital holographic cytometry (DHC) to monitor human melanoma cells undergoing apoptosis, ferroptosis, and necroptosis. We developed and validated models that used DHC-derived features to classify each form of cell death with 91–93% accuracy in the test sets. We conclude that high-accuracy, high-throughput, label-free classification of apoptosis, ferroptosis and necroptosis can be achieved with DHC.

Keywords: digital holographic cytometry; quantitative phase imaging; apoptosis; ferroptosis; necroptosis

1. Introduction

The ability to evade programmed cell death is one of the hallmarks of cancer, leading to therapeutic resistance or failure. Although apoptosis is perhaps the most famous, other forms of programed cell death have been well-characterized. Previous studies have demonstrated that cancer cells that are resistant to one type of cell death may be more vulnerable to another. Ferroptosis and necroptosis, for example, can be exploited to induce death in cancer cells that have successfully evaded apoptosis . However, the interplay between each of these programed cell death pathways and their involvement in cancer progression is complex. For example, pan-caspase inhibitors have been shown to inhibit apoptosis while simultaneously inducing necroptosis. Induction of necroptosis can be antitumorigenic in some settings or promote tumor progression in others. In another example, vulnerability of melanoma cells to induction of ferroptosis is dependent on the phenotypic subtype. Such observations highlight the need for further investigation and suggest that the therapeutic potential of targeting distinct programmed cell death pathways might require case-by-case assessment. Methods that efficiently and simultaneously quantify apoptosis, ferroptosis, and necroptosis would be valuable for further exploring the clinical utility of agents that regulate cell death pathways.

Apoptosis, ferroptosis and necroptosis are all active forms of cell death, meaning they comprise defined molecular pathways that drive organized termination. The key regulatory molecules that activate, inhibit, or orchestrate each process permit molecular classification of cell death pathways using a variety of methods. For example, changes in expression, post-translational processing, or subcellular localization of key proteins can be monitored using immunological methods such as fluorescence microscopy, immunoblotting, flow analysis, or enzyme-linked immunoassays (ELISA). Other useful methodologies detect outer membrane permeabilization or mitochondria function and health—both of which change during the process of apoptosis . While each of these approaches has distinct advantages and limitations, none permit both high-throughput classification of all three forms of cell death in real time and with single cell resolution.

Digital Holographic Cytometry (DHC) is a label-free technique that allows for live cell imaging and real time morphologic assessment. DHC uses a laser that is split into two beams—a reference beam and an object beam. The reference beam is undisturbed, while the object beam is directed through the specimen. When the two beams are merged, an image can be generated through interpretation of the resulting interference pattern, with pixel intensity representing optical thickness—a function of both physical thickness and optical density. Cells are identified using standard image segmentation techniques, and individual cell features (optical volume, optical thickness, perimeter length, etc.) are derived. Quantitative phase images can be captured over a period of time, permitting tracking of DHC-derived features as cells undergo change, for example, due to exposure to a therapeutic agent. DHC has previously been used to distinguish different cell states based upon morphology, including identification of pre-apoptotic and dying cells.

In addition to molecular changes, a distinct set of morphological changes accompanies each form of programmed cell death. Apoptosis is associated with an increase in cell thickness, plasma membrane blebbing, decrease in cell volume, and chromatin condensation. Ferroptosis is associated with iron-dependent membrane lipid oxidation and damage to the plasma membrane. Necroptosis is associated with cell swelling, an increase in cell thickness, decrease in cell area, and eventual rupture of the plasma membrane. We therefore reasoned that analysis of cellular morphologic change with DHC might permit label-free, live, and high-throughput classification of apoptosis, ferroptosis and necroptosis with single cell resolution. The goal of this study was to train and test a classifier to separate three types of cell death using DHC-derived cell features.

2. Materials and Methods (urval av mig)

 The standard plate lid was replaced with an optically clear Hololid (Phase Holographic Imaging, Lund, Sweden). After addition of the Hololid, plates were incubated for 45 min to eliminate condensation. The dose–response application in AppSuite (v2.1.2.0, Phase Holographic Imaging, Lund, Sweden) was then used to determine the IC50.

For classification experiments, cells were cultured and treated as above, except that each plate contained a single well of each compound at the calculated IC50. The 6-well plate was then loaded onto the DHC platform and imaged via AppSuite. The parameters were set to 48 h, with images taken every 1 h from 20 random fields. The training and test sets were generated using independent passages, plates of cells, and freshly generated working solutions of each compound, and were conducted at different times. In two test set experiments, the erastin-treated well was lost due to technical reasons resulting in a sample size of two instead of four for this condition.

 

2.2. Digital Holographic Imaging and Cytometry

The M4 HoloMonitor (Phase Holographic Imaging, Lund, Sweden) uses a form of quantitative phase imaging called digital holographic microscopy to create a digitally reconstructed image of cells. Images were segmented and analyzed using the Hstudio software (v2.7.5, Phase Holographic Imaging, Lund, Sweden). HStudio was used to derive 32 quantitative cell features for individual cells. For each condition, image stacks were monitored for cell death and time points selected at which cells were undergoing morphological changes prior to cell death. In control conditions, M-phase cells were excluded from the analyses.

Figure 3. Digital holographic cytometry (DHC) images of indicated conditions. Expected form of programmed cell death indicated in parentheses.

Min kommentar

Det är fasiken för varmt för att orka gå igenom hela studien ord för ord (översätta) så ni får läsa igenom den själva och lista ut vad Robert har på gång med bas av dessa 3 typer av programmerad celldöd. Ett Nobelt steg vidare mot...

                                             Mvh the99

Tillägg (viktig info)

Hudcancer ökar kraftigt i Sverige

Upprepad hudcancer av typen malignt melanom har ökat kraftigt och blivit tio gånger så vanligt i Sverige, jämfört med siffror från 1960-talet, visar ny forskning.
Hudcancer är den cancerform som ökar mest i Sverige. Den nya forskningen visar också att den allra farligaste varianten ökar mest, nämligen upprepade fall av malignt melanom.
Trots ökad kunskap om att solbad kan orsaka hudcancer har attityderna bland svenskarna inte förändrats, säger Hilda Helgadottir på Karolinska institutet.