HoloMonitor i Nature

Bloggen har fått flera mejl från uppmärksamma phi,are om en helt ny forskningsrapport som publiceradades i en edition till Nature igår.Jag förstår varför man vill att jag ska återge den här på bloggen,den är jäkligt bra.Ämnesområdet och resultaten vill säga. Det är den HoloMonitorfrälste forskaren från Ungern Robert Horvath med team som via sitt HoloMonitorinstrument studerat en cancercells utveckling och kommit fram till ganska häpnadsväckande resultat.

Det ungerska teamet studerade cancerceller som övergick till att meastasera och kunde via sitt instrument bedöma 2 faktorer. 

1. Hur stor risk för patient att dottertumören är dödlig. 

2. Hur tillsatt cancerläkemedel påverkar cancerutvecklingen.

Men till studien som är betitlad :

Simple and automatic monitoring of cancer cell invasion into an epithelial monolayer using label-free holographic microscopy

• Published: 16 June 2022

 

Abstract

The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner.

En googleöversättning blir :

Invasiviteten hos cancerceller beskriver metastaserande förmåga hos en primär tumör. Den enkla upptäckten och kvantifieringen av cancercellsinvasion är viktig för att förutsäga överlevnadsgraden för en cancerpatient och för att testa hur anti-cancerföreningar påverkar cancerutvecklingen. 

Digital holografisk mikroskopi baserad M4 Holomonitor (HM) är en teknik som möjliggör märkningsfri övervakning av cellmorfologiska och kinetiska parametrar i realtid. Här etablerades ett helt sammanflytande epitelialt monolager härlett från den afrikanska gröna apnjuren (Vero) på en gelatinbelagd yta, sedan såddes HeLa-celler ovanpå monoskiktet och deras beteende övervakades under 24 timmar med användning av HM. 

Flera cancerceller som visade invasivitet upptäcktes under denna period, medan andra HeLa-celler inte visade några tecken på aggressivitet. Det visades att invasionen av enstaka cancerceller är väl observerbar och även kvantifierbar genom övervakningsparametrar som fasförskjutning, optisk volym, area och rörlighet, vilka parametrar lätt kan erhållas och bearbetas automatiskt. 

Baserat på experimentella data kan invasionshastigheten för cancerceller som kommer in i epitelskiktet definieras som krympningen av detekterad encellsvolym per tidsenhet. Invasionshastigheten och dess korrelation med cellmigreringsparametrar analyserades på djupet. Ett tydligt linjärt samband mellan migration och invasionshastighet hittades, cancerceller med starkare migration har lägre invasionshastighet. 

Dessa resultat beskriver inte bara effekten av hur cancerceller invaderar det underliggande monoskiktet i motsats till icke-invasiva HeLa-celler, utan skulle kunna hjälpa till i framtida forskning för att optimera läkemedel som påverkar cellinvasibilitet på ett helt automatiserat, etikettfritt och högeffektivt sätt.

Sen måste ni läsa nästa stycke noggrannt.Speciellt sista 2 delarna som mer eller mindre är en hyllning till HoloMonitor och instrumentets möjligheter att studera cancerceller utan infärgning.Teamet har även använt HoloMonitor vid tekniken att studera cellerna i en microfluidic miljö. Alltså på ett litet chip. Dvs Lab-On-a-Chip (LOC).

Introduction

Malignant cancer cells can reproduce themselves without control and penetrate distant tissues via the chain of metastatic events¹, which is the primary cause of cancer-related deaths². The first step of metastasis is the detachment of individual cancer cells from the primary tumor and invasion into the epithelial cell layer of the host tissue. A successful invasion is followed by the intravasation into the circulation and then exiting it with extravasation, after which processes the invading cancer cells can form new colonies in the attacked tissue. The dissemination of cancer cells can occur during different stages of tumor progression, and one cellular key-program that initiates this process is the Epithelial-Mesenchymal Transition (EMT). During EMT, epithelial cells receive mesenchymal phenotype, losing the physical connection with the basement membrane, become detached, migrate, degrade the components of the extracellular matrix (ECM) and invade the tissue. The transition from collective to single-cell-based migration events can also be accounted for the EMT. Cultured cancer cells (e.g., HeLa) are selected from high-stage cancers that have already become genotypically and phenotypically distinct from the tissue of origin⁸. Therefore, HeLa cells, which are widely used in cancer research and drug development, are an ideal candidate to study the metastatic cascade since these cells have already been transformed by EMT and are used worldwide in numerous laboratories. Classic in vitro assays studying cancer cell behavior and drug response include wound healing, gap closure, transwell, microfluidic, 3D platform assays combined with immunofluorescent labeling, and microscopy techniques. 

However, new in vitro, label-free, high-throughput techniques are emerging that provide real-time visualization of cancer cell movement, adhesion, division, and pharmaceutical affinity.

Such an effective, label-free, real-time, and high-throughput instrument is the digital holographic microscopy based M4 Holomonitor(HM). The HM technique uses a diode laser, and a semi-transparent mirror splits its beam to a reference and a sample beam. The sample beam is directed with a mirror through the observed cell culture, and so when the laser light meets translucent objects (e.g., cells and layers) with a specific refractive index, its phase will be shifted compared to the reference light. The reference beam and the sample beam will create interfering wave patterns, which an image sensor camera will then record. Therefore, the primary detected physical quantity is the phase shift of the laser beam caused by cultured cells reconstructing the image of the sample. Since the phase shift of the laser light is affected by the thickness and refractive index of the cells, it is possible to create 3D topographies of the objects in the laser’s path. The constructed images are visualized in the HM software, and parameters related to the kinetics and morphologies of the cells can be exported.

In comparison with other techniques, HM also allows observation in a microfluidic environment, where cells are seeded into a well and chemokines into a different well connected with a microfluidic channel. Samples containing cells are placed on the stage of the HM instrument. The microscope is then focusing on the channel and captures cellular motion in 3D. Holomonitor M4 is an incubator-proof digital holography technique-based instrument. The main advantage of Holomonitor M4 is the real-time visualization, which does not require the labeling or staining with cytofluorescent molecules, fixation, or any other substance, which could influence the microenvironment of the cells, therefore, it is label-free and cost-effective. The Holomonitor M4 is a precise instrument that monitors cells in a humidified, temperature and gas-controlled environment in real-time, and no post-visualization of the cells is required since the software already calculates cellular features related to dynamics and morphology. These instrumental abilities potentially open up the possibility for the automatic and straightforward detection of cancer cell invasion into compact monolayers, but such investigations were not conducted before.

Google får översätta halva ovanstående stycke från However.

Men nya in vitro, etikettfria tekniker med hög genomströmning dyker upp som ger realtidsvisualisering av cancercellers rörelse, vidhäftning, delning och farmaceutisk affinitet.

Ett sådant effektivt, etikettfritt instrument i realtid och hög genomströmning är den digitala holografiska mikroskopibaserade M4 Holomonitor (HM). HM-tekniken använder en diodlaser, och en halvtransparent spegel delar sin stråle till en referens och en provstråle. Provstrålen riktas med en spegel genom den observerade cellkulturen, och så när laserljuset möter genomskinliga föremål (t.ex. celler och skikt) med ett specifikt brytningsindex, kommer dess fas att förskjutas jämfört med referensljuset. Referensstrålen och provstrålen kommer att skapa störande vågmönster, som en bildsensorkamera sedan registrerar. Därför är den primära detekterade fysiska kvantiteten fasförskjutningen av laserstrålen orsakad av odlade celler som rekonstruerar bilden av provet. Eftersom fasförskjutningen av laserljuset påverkas av cellernas tjocklek och brytningsindex, är det möjligt att skapa 3D-topografier av objekten i laserns väg. De konstruerade bilderna visualiseras i HM-mjukvaran, och parametrar relaterade till cellernas kinetik och morfologi kan exporteras. I jämförelse med andra tekniker tillåter HM också observation i en mikrofluidisk miljö, där celler sås in i en brunn och kemokiner i en annan brunn ansluten till en mikrofluidisk kanal. Prover som innehåller celler placeras på HM-instrumentets scen. Mikroskopet fokuserar sedan på kanalen och fångar cellulära rörelser i 3D. Holomonitor M4 är ett inkubatorsäkert digitalt holografiteknikbaserat instrument. Den största fördelen med Holomonitor M4 är realtidsvisualiseringen, som inte kräver märkning eller färgning med cytofluorescerande molekyler, fixering eller någon annan substans, som kan påverka cellernas mikromiljö, därför är den etikettfri och kostnadseffektiv.

Holomonitor M4 är ett exakt instrument som övervakar celler i en fuktad, temperatur- och gaskontrollerad miljö i realtid, och ingen eftervisualisering av cellerna krävs eftersom programvaran redan beräknar cellulära funktioner relaterade till dynamik och morfologi. Dessa instrumentella förmågor öppnar potentiellt möjligheten för automatisk och okomplicerad upptäckt av cancercellsinvasion i kompakta monolager, men sådana undersökningar har inte utförts tidigare.

Materials and methods

Instrumentation

For the detection of Vero monolayer assembly and HeLa invasion, the digital holographic cytometer Holomonitor M4 (Phase Holographic Imaging PHI AB, Lund, Sweden) was used. During the experiments, the Holomonitor is placed inside a humidified incubator with 37 °C and 5% CO₂. For imaging purposes, a 35 mm glass bottom dish (VWR) was used, which was covered with a HoloLid (Phase Holographic Imaging PHI AB, Lund, Sweden). The set-up of the Holomonitor and the HoloLid are determined by the manufacturer, and all guidelines were followed according to the manual.

Schematics of experimental design, execution, and results. (A) Seeding of Vero cells (green) on gelatin-coated dish. (B) Seeding of HeLa cells (red) on top of the self-assembled 100% confluent Vero monolayer (ML). (C) Holomonitor M4 was used to image monolayer assembly and invasion for 24 h. (D) Illustration of the expected result, the HeLa cells seeded on top of the Vero monolayer infiltrate by searching for optimal invasion positions. 

HeLa cells were also picked up from a confluent sustained cell culture with the same procedure as Vero cells. To visualize invasion, after rinsing the confluent Vero monolayer with completed cell culture media and refilling the dish, 100 µl HeLa cell suspension (~ 1.2 × 10⁵ cells) was added on top of the fully confluent Vero monolayer (Fig. 1), for which step the recording on the Holomonitor M4 was paused and the HoloLid covered dish was removed from the stage. After the injection of HeLa cells onto the monolayer, the dish was covered again and placed back to the stage of the instrument, and recording was immediately continued. The recording was proceeded for 24 h to determine the invasive nature of HeLa cells (Fig. 2).

Data acquisition and evaluation

The software HStudio M4 (Phase Holographic Imaging PHI AB, Lund, Sweden) is used by the Holomonitor M4 instrument to visualize, detect, track and determine cell morphology and movement. HStudio can export obtained data for further processing and data analysis, which was done in OriginPro 9.5.

Discussion

The investigation of the invasive nature of HeLa cells on a confluent monolayer of epithelial Vero cells has proved to be a valuable model for future research and development, and could demonstrate the most important features present during cellular invasion processes. It was shown that it is possible to determine and quantify invasion with the digital holography technique using Holomonitor M4. Factors on which an invasion can be quantified are cell morphology features such as area and volume, and behavioral parameters, including motility, invasion speed, and change in the movement and lock-in phases of the cell. These parameters can be used for the automatic detection of cancer cell invasion into monolayers under environmentally controlled conditions.

We found that the motility of invasive HeLa cells falls within the average and error range of non-invasive HeLa cells. The discovered loss in the holomonitor signal due to cancer cell invasion could greatly help in the development of automated detection methods in the future. For example, when investigating large samples, the linearly rising motility curve would be broken at some point due to the signal loss and identification of invasive cancer cells could be done based on this unique feature. Moreover, when using the evaluation software of Holomonitor M4 the loss of signal can also be considered as easily detectable in all parameters because the software tries to continue the tracking even when the original cell has already vanished from the sight.

The approach presented in our manuscript is mainly a technical demonstration that the Holomonitor M4 can be used to effectively track and measure cancer cell invasion into cell sheets in a simple label-free manner. 

Summarized, the results show a new opportunity to study cancer cell invasion into cellular monolayers in depth. Most of the prior studies (Table 1) investigated the relative number of migrating cells into gels, since they did not investigate motility or morphology parameters further, due to the fact that most of the methods are not capable of extracting real-time motility and morphological parameters, compared to the parameters calculated by the software of the Holomonitor M4⁴⁷. However, investigations on the movement speed of cancer cells in gels have similar results to ours¹³. Also, methods capable of monitoring the speed of invasion in an incubator-proof and label-free manner are relatively rare²². The Holomonitor system can be extended with high-throughput applications with a motorized xy-stage that allows monitoring of up to 96-wells. Also, combination of the instrument with fluorescent mode could capture fluorescent images in real-time with short pulses of laser illumination, which would presumably not interfere with a cell’s normal homeostasis. With the help of holomonitor also the establishment of 3D spheroids could be monitored⁴⁸. However, some drawbacks can only be eliminated by the aforementioned combined techniques, since Holomonitor does not allow specific, or molecular level single-cell analysis. Also, the instrument is incubator proof and so requires a high-precision maintenance and handling. 

HM has the potential for automatic detection of cancer cell penetration of confluent monolayers, the possibility to extend the measurements to multiple wells and cell types gives the ability to test new substances and analyze the motility and morphological parameters of invading cells in a high-throughput manner. 

I publikationen finns 6 filmer tagna med HoloMonitor.Scrolla ner till Supplementary Information och kolla.

Min kommentar

Man förstår att forskaren Robert Horvath m team valt Nature för publicering av denna studie.

Genomslagskraften ut till forskarvärlden blir stort.Deras resultat är i mitt tycke rent sprängstoff.

Att kunna bedöma en tumörs dödlighet för patient och sen ge svar på hur häva den risken är i min bok stort.

Forskarvärlden kommer med all rätt applådera teamets resultat,för att inte nämna världens alla cancerpatienter.

Slutklämmen i studien : HM (HoloMonitor) has the potential for automatic detection of cancer cell penetration....är ovärderlig för PHI. Denna studie hoppas jag Bolaget skickar ut till världens alla cancerforskare,så bra är den.

Tack till er uppmärksamma phi,are som mejlade över denna studie.

Fredagsbonus.Klicka och se nåt spektakulärt.

                                          Mvh the99

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