Det handlar om studier av ännu en j..ig cancerform,nämligen en huvud-hals-cancer.Mer specifikt en cancer som sätter sig i munregionen som ex i svalget,vid tonsillerna, del av tungan och gommen.Man har tagit cancerceller från en 48-årig man,ur hans munhåla där cancern sitter.Studerat dessa med HoloMonitor och jämfört med att studera cellerna på ett traditionellt sätt där infärgning ingår.
Studien handlar alltså om skillnader mellan teknik.Ny teknik mot gammal teknik.
Först berättar man om att denna typ av cancer har över 25% återfall efter behandling.
Varför återfallssiffran är så hög förklarar man med att vid screeningen av de första proverna hittar tekniken inte de variabler som gör att cancern återkommer.
Det någorlunda beskrivet i denna mening : "the organoid-forming ability of different cancer-cell phenotypes".
Som jag förstår det visar det risk för senare (efter behandling) metastasering och ny omgång med denna cancerform. Infärgningstekniken (den gamla tekniken) klarar alltså inte på en tillräckligt detaljerad cellnivå se dessa händelser.Man hävdar att single cell analys ger bättre förutsättningar att förutse risken att cancern kommer metastasera och återkomma i ett senare skede.
För att kunna göra det lyfter man fram HoloMonitors egenskaper som den nya tekniken att klara detta.
Men till själva studien då.
Subpopulations of Organoid-Forming Cells Have Different Motility
Published: 7 July 2020
Abstract
Cancer stem cells from oropharyngeal squamous cell carcinoma (OPSCC)
have the ability to self-renew and differentiate into heterogeneous
three-dimensional structures carrying features of tumor cells. Here, we
describe a simple and label-free method for generating tumor organoids,
and imaging them using live digital holographic microscopy (DHM) on the
basis of the phase shift caused by light passing through the cells. We
show early events of cell aggregation during tumor-organoid formation,
and display their heterogeneity in terms of optical parameters up to an
optical volume of 105 µm3. Lastly, by sorting OPSCC epithelial cells, we demonstrate that CD44+ cells displayed greater motility and tumor-forming capacity than those of CD44−
cells. These results were in line with previous reports highlighting
increased invasive and tumorigenic potential in tumor cells expressing
high levels of CD44. Our method provides insight into the formation of
tumor organoids, and could be used to assess stemness-associated
biomarkers and drug screenings on the basis of tumor organoids.
Keywords:
organoid formation; digital holography; cancer stem cells; oropharyngeal squamous-cell carcinoma
1. Introduction
Oropharyngeal
squamous cell carcinoma (OPSCC) is a head-and-neck cancer affecting the
tonsils, base of the tongue, pharyngeal walls, and soft-palate region.
Despite advances in OPSCC treatment, over 25% of patients relapse, 16%
experience local recurrence, and 7% experience distant recurrence.
It is important to develop culture methods that recapitulate key events
in tumorigenesis, allowing for drug screening in an in vivo-like
setting.
Tissue invasion and metastatic
capacity are fundamental traits of cancer stem cells (CSCs), a
heterogeneous subset of cancer cells with the ability to reproduce tumor
complexity at a cellular level.
When cultured in a hydrogel, CSCs generate 3D structures known as
organoids, self-organized cellular structures that exhibit phenotypes
resembling those seen in tumors.
Basement-membrane matrices provide appropriate mechanical stimuli that
allow CSCs embedded within them to aggregate, grow, and differentiate,
resulting in organoids of different sizes and morphologies with the ability to replicate cellular, mechanical, and physical signals that take place in the tumor microenvironment .In OPSCC, CD44 and nerve growth-factor receptor (NGFR) are two candidate markers for identifying cancer stem cells, as shown by a study where CD44+/NGFR+ cells transferred into in vivo models recovered initial tumor heterogeneity. However, loss of NGFR expression in the tonsillar crypt was reported in tonsil-cancer patients.
Therefore, studying the organoid-forming ability of different
cancer-cell phenotypes may improve our understanding about tumor or
metastasis initiation. In vivo tumor behavior can be monitored using
transgenic cancer cells expressing bioluminescent or fluorescent
proteins. Although useful, these methods generally lack single-cell
resolution, are time-consuming, and require extensive manipulation of
the model.
Here,
we present a quick and inexpensive method for imaging organoid
formation over short time periods. Homogeneous tumor organoids could be
generated by embedding OPSCC-tumor cells in a hydrogel. The motility of
single cells and the optical properties of organoids could be monitored,
and used to determine aggregation events and organoid growth over time.
Using this setup, we confirmed the motility and capability of
CD44-enriched OPSCC cells to form organoids using the HoloMonitor M4
(Phase Holographic Imaging AB (PHI), Lund, Sweden) instrument.
2. Materials and Methods (urval)
2.2. Holographic Phase-Contrast Microscopy
Organoid
formation was monitored with HoloMonitor M4, which is based on digital
holography. Low-intensity laser light passes through the cells,
resulting in a cell-density-dependent phase shift of the light. The
phase shift is translated into cell thickness by HoloStudio software
(PHI, Lund, Sweden).
HoloMonitor M4 was placed in a cell-culture incubator at 37 °C with 5% CO2
and 95% humidity. Time-lapse images of organoid formation were taken
from several positions every 30 min.
Data on accumulated cell motility,
aggregation, and organoid growth were obtained by analysis with
HoloStudio v2.7.4 software. In the current work, accumulated motility
was defined as the movement of cells embedded in the hydrogel over time.
Briefly, single cells were identified by establishing a cutoff diameter
of 20 µm. After that, cells were tracked in time-lapse experiments to
monitor cell motility and aggregation events, followed by analysis of
organoid homogeneity according to the increase in area, optical volume,
and optical thickness over time. The percentage of single cells that did
not form organoids was calculated according to the difference between
the number of single cells identified in the first and last frames of a
given time lapse.
I studiens sista stycke Discussion sammanfattar man det mer tydligt,skillnaden mellan gammal och ny teknik.
4. Discussion
Many different organoid systems enable researchers to study cells in a tumor environment in vitro, but the development of methods for understanding organoid formation is needed to study tumor initiation. Cell motility is an important feature as it is suggestive of invasiveness and, when monitored along organoid formation, arguably informs about the metastatic capacity of tumor cells . Nonetheless, quantitative methods for studying these early events are scarce, as conventional migration assays such as wound-healing and transwell assays do not evaluate growth in 3D.
Live fluorescence-microscopy monitoring is useful since it enables the visualization of cell subsets in a complex mixture, but it causes phototoxicity that could compromise obtained results. In this study, we presented a method for generating organoids from cells in suspension and monitoring their formation by measuring optical properties. Collected data using quantitative phase imaging were based on the phase shift of the light, making the images fully quantifiable since light intensity in HoloMonitor M4 is 0.2 mW/cm2, it did not cause any measurable phototoxicity.
In summary, the present method could be a useful tool for evaluating organoid initiation, and thus be implemented in drug screenings attempting to target early stages of tumor initiation in a label-free manner. To this end, cell density should be carefully examined for each cell type, and the tested substances should be able to diffuse into the hydrogel without causing structural changes in the extracellular matrix.
Min kommentar
Denna studie är lite annorlunda mot de vi annars läser.Man har nämligen tagit med en praktiserande läkare inom området,överläkare Lennart Greiff Head and Neck Surgery, Skåne University Hospital.
Det ger tyngd åt studien och gör den förmodligen mer intressant för andra forskare att läsa.
Kan man visa på konkretisering: från labbmiljö (teori) ---> till konkret användande (praktisk funktion) finns knappast några tvivel om studiens innehåll och slutsatser.
Vär att notera (förutom studiens tydliga budskap : HoloMonitor är överlägset som ny teknik att identifiera risk för canceråterfall) är 2 saker.
Först att EU tillsammans med Svenska Cancerfonden finansierar denna studie.
This research was funded by EU-H2020-MSCA-COFUND-2016-754299 and the Cancera Foundation.
Det ger ytterligare tyngd och vibbar om att slutsatserna i studien är tänkta att spridas till labb, forskare och läkare inom området .
Den andra noteringen är att denna studie kommer ingå i en specialutgåva av organet Applied Sciences.
Special Issue "Applications of Digital Holography in Biomedical Engineering"
En specialutgåva med i princip bara cred till PHI`s HoloMonitor då samtliga forskningsrapporter (4 st) är baserade på just HoloMonitor. Det är PR/marknadsföring i en av de bästa kanalerna tänkas kan.Om gårdagens "karamell" var söt och god att suga på,är den närmast salt i jämförelse med denna.
Mvh the99
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