Doktorsavhandling från USA

En doktorand från PHI`s Center of Excellence i Boston USA, Northeastern University, har nyligen fått sina studier beträffande bukspotts respektive bröstcancer och hur effektivt behandla dessa cancertyper offentliggjorda.
Doktorranden har varit inne på GlycoImagings område med att studera hur nanoteknik kan användas för att målsöka en specifik cancertumör och via tekniken applicera det mest effektiva läkemedlet på identifierad tumör.
Doktoranden ifråga har haft "vår" forskare Ed Luther från universitet som handledare för att förstå sig på och sen använda universitets HoloMonitor för sina studier.

                    Targeted Nanopreparations For Cancer Therapy 

                                                     STATEMENT OF HYPOTHESIS

Considering the advantages of targeted drug delivery in cancer therapy, I hypothesize that the
development of the tumor-targeted nanomedicines such  as phage  protein-targeted micellar
formulation for pancreatic cancer and the antibody-targeted liposomal formulation for breast 
cancer will facilitate specific drug delivery to the tumor site and produce optimal therapeutic effects
by minimizing the adverse effects associated with the drug and maximizing the therapeutic benefits.

Abstract:
Cancer therapy in the recent years has evolved with the development of novel targeted drug delivery
systems. The conventional chemotherapy approach is plagued by side effects and numerous
disadvantages such as low bioavailability, poor solubility of drugs, toxicity, non-specific drug action
and so forth. With the main goal of producing a drug delivery vehicle that is optimally designed to
address the many limitations associated with the conventional chemotherapy, the work has been
designed as follows: Different lipid-based targeted formulations were prepared, such as the tumor-
targeting micelles and liposomes, and the in vitro effects of the formulations on pancreatic and
breast cancer cells have been studied to determine their optimal therapeutic effects and their ability
to minimize adverse off-target effects. Keeping in mind the need for an ideal therapy requirement
for life threatening illnesses, hard to treat pancreatic cancer and the very commonly diagnosed
cancer among women, breast cancer, were chosen as the targets for testing designed nano-
preparations.


The first approach was to develop polymeric micelles self-assembled with a single drug, paclitaxel
and a targeting agent, a peptide sequence derived from phage coat protein. The drug is hydrophobic
and poorly soluble in aqueous solvents that limit its pharmaceutical applications despite high
efficacy. Cell viability studies on PANC-1 pancreatic cancer cells using the targeted phage micelle
preparation produced significantly higher cell death of ~45 % while the non-targeted preparation
caused only about ~30% cell death at 750ng/ml concentration of paclitaxel. Significantly higher cell
death was observed at 1.5 and 2.75 mg/ml concentrations of paclitaxel-loaded in the formulation, that was dose and time dependent. More than a 30% increase in caspase3/7 activity was observed
in vitro in a monolayer of PANC-1 cells while the in vivo data on lactate dehydrogenase release
indicated a significantly enhanced apoptosis in the PANC-1 spheroid model upon treatment with the
targeted micelle preparation.


The second approach utilized liposomes modified with monoclonal antibody 2C5 (mAb 2C5) as
targeting ligand for breast cancer therapy. The study is important in that a combination of 2 drugs,
paclitaxel, a microtubule inhibitor, and salinomycin, an anti-cancer stem cell targeting agent, has
been used. The main goal with the use of combination chemotherapeutics was to eliminate not just
the bulk cancer cells as with conventional chemotherapy, but also the cancer stem cells, or the
tumor-initiating cells, in the core of the tumor to eliminate possibilities of cancer metastasis and
recurrence. The study produced promising results for cellular interaction, uptake and cytotoxicity in
vitro as was observed both quantitively and qualitatively. Interaction of the mAb 2C5-targeted
liposomes was greater than ~3.25-fold with SK-BR-3 breast cancer cells, while the same formulation
showed over a ~1.3-fold increase in interaction with the MDA-MB-231 breast cancer cells in
comparison to the other controls used. A significantly enhanced fluorescence signal upon treatment
of both the breast cancer cells with rhodamine-labeled mAb 2C5-targeted liposomes was observed
in comparison to the unmodified liposomes that supported cellular specificity and enhanced cellular
uptake of the antibody-targeted formulation. The average of the corrected total cell fluorescence
(CTCF) plotted for the mAb 2C5-modified liposome-treated cells from three different locations on the fluorescence image was statistically significant (p  0.05) in comparison to that of the unmodified liposome-treated cells in both the cell lines. There was a ~4.5-fold and a ~3.0-fold increase in average CTCF with the mAb 2C5-modified liposomes in comparison to the unmodified liposomes in SK-BR-3 and MDA-MB-231 cells respectively. The quantitative data was a further confirmation for the enhanced cellular uptake of the formulations in both the cell lines.
Holographic monitoring confirmed improved cellular killing by showing visible differences in cellular morphology, cell division and proliferation over time (48 hours) that validated the efficacy of the mAb-targeted liposomal formulation in MDA-MB-231 cancer cells. The study also confirmed the specificity of mAb 2C5 for two different breast cancer cell types viz, the triple negative MDA-MB-231 cells and Her2-positive SK-BR-3 cells. Also, preliminary data on hemolysis (%) in female athymic nude mice confirmed lower toxicity for the antibody-targeted combination drug-loaded liposomal formulation in vivo in comparison to the single drug-loaded targeted formulations.


Transitioning from the studies using targeted polymeric micelles for pancreatic cancer therapy to
mAb-targeted liposomes for breast cancer combination drug therapy, additional studies to
demonstrate the stem cell-like properties of the cancer cells were performed. In order to also
confirm the potential of the combination therapeutics for breast cancer, in vitro studies were
performed using free drugs on a monolayer of breast cancer cells. A cell viability study using free
drugs and the study of effects of free drugs (both single drugs and in combination) on cellular
morphology and metastasis were performed to demonstrate efficacy of the combination
therapeutics on breast cancer cells. Lipodox in combination with salinomycin and paclitaxel in
combination with salinomycin were used to treat breast cancer cells in vitro.
In comparison to Lipodox alone, the combination therapy showed significantly improved cellular killing over time and with different doses and produced about 50% more cellular killing at 72 hours in MDA-MB-231 cells.
In comparison to salinomycin alone, the combination killed about 70% cells in 72 hours. MCF-7 cells were more sensitive to the therapy with lower dose of Lipodox giving enhanced cell killing in combination with salinomycin and achieving significantly higher cell killing percentages at 48 hours and 72 hours after treatment.
Significantly improved cell killing percentages were observed with both the cell lines in presence of the combination of drugs as opposed to single agents in a dose and time dependent manner. Characterization of the breast cancer cell lines for presence of cancer stem cell specific markers CD44+/CD24- showed enriched populations of cells expressing these biomarkers over several generations in both the cell lines. Also, unique properties of stem cells such as their differentiation potential and self-renewal properties were demonstrated using the breast cancer cells to successfully confirm the presence of stem cells and to validate the mammospheres generated for their stem cell-like properties. Although the regular expression of CD44+/CD24- marker is variable in different types of breast cancer, the mammospheres generated in MCF-7 cells showed high levels of CD44+/CD24- expression (that is different from the usual expression profile of CD44, CD24 in such basal/ epithelial breast cancer cells) validating the enrichment of cells with stem cell-like properties over generations using the 3D mammosphere culture system. The MDA-MB-231 cancer cells with intrinsically higher expression of CD44+/CD24- marker (as expected with the basal/mesenchymal cancer cells) showed consistent high expression of the marker in the various generations of the cancer cells in the mammospheres. The results also highlighted the biological heterogeneity of different types of breast cancers.
Additional holographic studies on wound healing of a monolayer of breast cancer cells revealed that the combination of free drugs (salinomycin and paclitaxel) treatment of wounds on a monolayer of MDA-MB-231 cells showed a reduction in gap width at the wound site from ~355um at the beginning of holographic monitoring to only ~219um at the end of the wound healing assay in comparison to a reduction to ~7um with salinomycin-only and to ~104um with paclitaxel-only treated wounds. The ability of the combination chemotherapy to prevent cellular migration at the wound suggested its potential to inhibit cellular proliferation and metastasis in vivo.


Overall, the study below is valuable for the development of targeted drug delivery systems for
pancreatic and breast cancer therapy. We hope that the work translates in the clinic and provides an
optimal outcome in people with this life-threatening malady.

Urval av studiens innehåll.

Additional information on the behavior of breast cancer cells in the presence of the targeted
formulation using holographic imaging was obtained to assess the effectiveness of the targeted
combination chemotherapeutics and their mode of action on the cancer cells. The specific
morphological changes induced by the action of the combination chemotherapeutics in the targeted
liposomal formulation were studied in the triple negative breast cancer cell line, MDA-MB-231, using holographic imaging. The cancer cell line is triple negative since it lacks estrogen receptor (ER) and progesterone receptor (PR) expression, as well as HER2 (human epidermal growth factor receptor 2) amplification. Specific information on cell count, proliferation, motility, and killing mechanism over time was assessed using this advanced imaging technique.

The wound was also imaged holographically over 48 hours (at 10-minute intervals) to secure quantitative data on gap widths and the percent cell-free areas following the drug treatments.




12.3.15. Holographic monitoring of MDA-MB-231 cellular morphology and proliferation

Cells, when ~90% confluent, were split and ~350,000 cells/well were seeded. Cells were incubated
for 24 hours and treated with the targeted formulation, the non-targeted formulation or the free
drugs (at starting concentrations of 2.5ug/ml for the single drug formulations and 1.25ug/ml of each 
drug in combination preparations) before imaging using a holomonitor. The final concentrations of
drugs per well were diluted ~4x with the anti-condensation strips dipped thoroughly inside each well
before the start of imaging. The wells were holographically monitored for ~ 48 hours by recording
images every 10 minutes. After 48 hours, independent frames were collected from the beginning,
middle and the end of the monitoring to assay cellular proliferation, division and any other
morphological changes. Videos recorded changes over time.

13.10. Free drug effects on cellular migration and proliferation at the wound site imaged using
holographic monitoring

The wound generated was also imaged holographically over 48 hours (at intervals of 10 minutes) to
determine the rate of change of gap widths following the drug treatments. From about 355um gap
width at the start of the monitoring, the width reduced steadily to less than half over 24 hours and was ~104um wide at the end of 48 hours in the paclitaxel treated group. Likewise, from a 63% cell-
free area at the wound from start of the monitoring, the area decreased to ~18% in the paclitaxel-
treated wound. The other groups, salinomycin-only and untreated, showed an opposite trend, with
increasing cell numbers at the wound over time (reducing wound gaps and cell-free area
percentages). For the salinomycin, combination treated and the untreated wounds, the gap widths
were reduced (from ~355um to) ~7um, ~219um and ~47um respectively. The combination drug
treated wound had a significantly wider gap and a higher percentage of cell- free area at the wound
in comparison to any of the single drug treatments or the untreated wounds. This observation
supported the potential for anti-metastatic and anti-proliferative effects of combination therapy
over either of the independent drug treatments in vivo.

13.15. Holographic imaging of morphology, proliferation and division of breast cancer cells after treatment with drug-loaded mAb 2C5 modified liposomes 

Holographic imaging of breast cancer cells treated with the mAb 2C5-modified drug containing
paclitaxel, salinomycin and combination-loaded liposomes, unmodified liposomes loaded with the
drugs, free drugs, and the untreated cells was performed for 48 hours. The monitoring helped
visualize live changes in morphology, proliferation and division of cells after the various treatments (Figure 43 a-g). The images gave a clear idea on how the different treatments (free drugs, vs. targeted
or non-targeted formulation loaded with drugs (as single agents and in combination)) affected the
viability of breast cancer cells.  

Supporting data obtained via holographic monitoring on the gap-width closure and cellular morphology at the wound site over a period of 48 hours confirmed the efficacy of the combination drug therapy in preventing metastasis.

Holographic imaging was performed using the antibody-modified liposomal formulation over 48
hours to validate the efficacy of the formulation in MDA-MB-231 cell killing. Detailed imaging of
cellular division, proliferation and morphology over 48 hours showed promising results with the
targeted formulation in cell killing.

15. Conclusion:

The major issue with aggressive forms of breast cancer is that they tend to recur over time and thus
are metastatic. A combination drug therapy approach is a possible effective treatment that kills both
cancer stem cells (CSCs) and the bulk cells ofthe tumor35. We have reported a targeted combination-
drug delivery approach using a nanocarrier (liposome) with antibody (mAb 2C5) as the targeting
ligand. The approach confirmed the targeting effectiveness of mAb 2C5- modified liposomal
preparation and good cytotoxicity against two selected breast cancer cell lines, viz. triple negative
MDA-MB-231 cells and Her2-positive breast cancer SK-BR-3 cells. The cancer cell behavior observed holographically, confirmed that the liposomal paclitaxel/salinomycin formulation with the targeting ligand (mAb 2C5) facilitated cellular killing and had profound cancer cell cytotoxicity in an aggressive breast cancer cell line model in vitro. The advanced imaging technique provided a visual confirmation for the anti-cancer effect of the developed targeted liposomal formulation.
This research should be considered a step further in the development of the combination therapy for breast cancer, even in aggressive cases.

16. Concluding remarks

With the goal of developing targeted drug delivery systems for the conventional
chemotherapeutics in cancer therapy, the above study was designed. mAb 2C5-targeted
liposomes and phage coat protein-derived peptide-targeted micelles developed in the study are
expected to provide promising benefits such as improved solubility, bioavailability,
biodistribution and specificity of action in order to enhance the drug’s therapeutic benefits.
Additional advantage such as the delivery of combination chemotherapeutics to target and kill
both the cancer stem cells and the bulk cells using the liposomal preparation for aggressive
breast cancer therapy is an important highlight of the work. The developed drug delivery systems
tested on the hard-to-treat pancreatic and the very commonly diagnosed breast cancer cells, in
vitro, contribute the initial steps for the translation of such targeted formulations into clinic. We
firmly hope the designed experiments using the targeted formulations and the results recorded
thus far are helpful for additional research and prove useful to several people affected with the
life-threatening illness.
  
I thank Mr. Ed Luther for all help he rendered with Keyence microscopy imaging and holographic
monitoring. 

Min kommentar
Denna väl utförda studie hur använda ny bättre teknik,bättre i den bemärkelsen att tekniken (nano) mer effektivt behandlar cancern som att den ger färre biverkningar (läs stycket om biverkningar) är baserad till stor del på användande av PHI`s HoloMonitor.När man läser igenom den 148-sidiga rapporten framträder hur väl man använt sig av PHI`s teknik och därigenom inser man hur viktig HoloMonitor varit för att nå dessa nya resultat.
Notera mina kursiverade markeringar hur doktoranden i sin avhandling beskriver Holotekniken.
PHI`s trogne partner sen 2014, Ed Luther/Northeastern University, producerar förhoppningsvis en riktigt vass framtida forskare i denna doktorand.Cancerforskningen behöver mer braighta snillen för att få stopp på denna best.Denna nya generation forskare kommer vara mer benägna att använda sig av ny state-of-the-art instrumentering för att nå helt nya insikter till gagn för världens alla cancerdrabbade.
Doktoranden Radhika Narayanaswamy lär vi få höra talas om framgent,förhoppningsvis med än mer ny kunskap baserad på användande av HoloMonitor.
För att ära hennes kommande utnämning till Doktor (PhD) tar jag mig friheten att klistra in nedanstående.

Carbiotix - Ökad ämnesuppmärksamhet

Carbiotix´s bransch uppmärksammas mer och mer. Igår visade SVT Vetenskapens Värld en dokumentär som på ett mycket bra sätt belyser tarmflorans betydelse för människan,det ur många aspekter.
Obesity,diabetes,allergier,cancer är några av de sjukdomar som kopplas till tarmfloran i programmet.

                                 Guldet i dina tarmar
Våra tarmbakterier kan revolutionera medicinen - om inte vår livsstil tar knäcken på dem först.
De senaste årens forskning om det mikrouniversum av bakterier som finns i vår mage har gjort det möjligt att bättre förstå sjukdomar som diabetes, övervikt, vissa typer av cancer och även depression.

Bland innehållet:
- Vår tarmflora står i centrum för en medicinsk revolution,nya forskningsfynd har avslöjat en skattkammare,ett bakteriekapital vi bär med oss och behåller under förutsättning att vi tar hand om det.
Man jämförde tarmflora hos överviktiga och normalviktiga och såg tydliga skillnader som kan förklara överviktigas problematik. Tänk här Gut Test.
Att dessa hemmatester kommer öka explosionsartat när väl den bredare skaran av människor förstår att man enkelt kan få info om obalans/brister om sin egen tarmflora, och därmed ett verktyg i att förändra till det bättre.
Man nämner även i programmet att man i en framtid kanske införlivar test av tarmfloran inför ett läkarbesök.
Att läkare med info om patients tarmstatus lättare kan ställa diagnos.

För den som vill skaffa sig info och kunskap om ett växande ämne/område/bransch, vilket kommer låta höra talas om sig stort, är en titt på SVT`s film ett måste.
Men innan rekommenderar jag att man läser mitt tidigare inlägg Carbiotix - prebiotika som förklarar begrepp och sammanhang.
Carbiotix är väl positionerat inom detta nya kommande stora område med sina 4 "ben".
Alltför få har upptäckt Bolaget vilket avspeglas i aktiekursen som dess handel.
Det är mest några trejders vilka dagligen byter aktier med varandra som jag kan se det.
Alltså inga riktiga investerare med längre placeringshorisont som identifierat den potential Carbiotix besitter.
Lite synd då börsvärdet får anses vara riktigt lågt i nuläget.
Undertecknad hoppas att åtminstone några av bloggens läsare tar sig en funderare på Carbiotix som en utmanare till andra aktier att komplettera PHI i aktieportföljen.
Det medan börsvärdet fortfarande ligger på nuvarande nivåer.
På sikt är jag hyfsat säker på att min investering i Carbiotix kommer bli en av de bättre.
Inom kort startar den Italienska studien (där Barilla ingår) vilket bör få fart på aktien och handeln.
Det ser jag som en av de viktigare händelserna innan årsskiftet.
Positiva besked därifrån lär uppmärksammas även utanför Sveriges gränser.

Edit 22:17
När jag kollar dagens handel ser jag att aktien fått in en ny aktör (långsiktig förhoppningsvis).
Någon på Pareto har hittat aktien och köpte sig idag en post på 10 000 aktier.
Bryter mönstret med att Ava och Non är de som köper/säljer/byter aktier med varandra vilket brukar vara legio när jag kollat in en vanlig dags handel.Sett till volymerna alltså.

Ps. De utlovade inläggen om Carbiotix 3 övriga affärsområden kommer inom sinom tid.

                                        Mvh the99

Forskningsrapport om bröstcancer (diagnosverktyg)

Igår 15/7 publicerades då den 10e studien på kort tid.Denna nya forskningsrapport är nånting utöver det ordinära då den berättar om hur HoloMonitor kan användas som ett detekteringsverktyg för att upptäcka metastaserande bröstcancerceller,som är den vanligaste anledningen till dödsfall i bröstcancer.
I studien beskriver man tekniken som HoloMonitor bygger på kunna vara ett framtida kraftfullt diagnosverktyg.
Läs den meningen igen : framtida kraftfullt diagnosverktyg.

Kikar vi då lite närmare på studien och väljer ut enstaka stycken som förklarar hur de kommit fram till denna slutsats.
De inleder med att berätta att dagens system för att försöka hitta bröstcancerceller med profil/risk att vandra vidare ut i kroppen från huvudtumören i bröst är att använda en markör kallad EpCAM som står för Epithelial cell adhesion molecule.
Cancerceller med profil/risk att metastasera och skapa nya tumörer kallas Cirkulating Tumor Cells,förkortat CTC.
EpCAM-markören fungerar så att den identifierar de vanligaste attributen hos CTC.
Men den är inte 100%-ig då den inte känner igen riktigt aggresiva CTC med annan karaktistik än de vanliga.
Det beskrivet i detta stycke :
- The most common marker for CTCs that is used today is the epithelial cell adhesion molecule (EpCAM), but for some malignancies such as aggressive breast cancer metastases that are often of non-epithelial origin or have transitioned through EMT, the EpCAM detection method is not useful. This highlights the need for more reliable CTC markers. The adhesion receptor CD44 and EpCAM function together in preparing the pre-metastatic niche, while CD45 is a leukocyte marker that is exclusively expressed on all nucleated cells of the hematopoietic system. The absence of CD45 on epithelial cells emphasizes the fact that it is an appropriate cell surface marker for distinguishing between CTCs and hematopoietic cells.

Discrimination between Breast Cancer Cells and White Blood Cells by Non-Invasive Measurements: Implications for a Novel In Vitro-Based Circulating Tumor Cell Model Using Digital Holographic Cytometry

 Published: 15 July 2020

Abstract

Breast cancer is the second most common cancer worldwide. Metastasis is the main reason for death in breast cancer, and today, there is a lack of methods to detect and isolate circulating tumor cells (CTCs), mainly due to their heterogeneity and rarity. There are some systems that are designed to detect rare epithelial cancer cells in whole blood based on the most common marker used today, the epithelial cell adhesion molecule (EpCAM). It has been shown that aggressive breast cancer metastases are of non-epithelial origin and are therefore not always detected using EpCAM as a marker. In the present study, we used an in vitro-based circulating tumor cell model comprising a collection of six breast cancer cell lines and white blood cell lines. We used digital holographic cytometry (DHC) to characterize and distinguish between the different cell types by area, volume and thickness. Here, we present significant differences in cell size-related parameters observed when comparing white blood cells and breast cancer cells by using DHC. 

In conclusion, DHC can be a powerful diagnostic tool for the characterization of CTCs in the blood.

Keywords: breast cancer; cell area; cell thickness; cell volume; circulating tumor cell; CD45; digital holographic cytometry; EpCAM

1. Introduction

Breast cancer is the leading cause of cancer deaths among women worldwide. 

Breast cancer metastasis accounts for the majority of deaths from breast cancer. The detection of metastases at the earliest stage is important for the management and estimation of breast cancer progression. Breast cancer treatments today are based on the absence or presence of the hormone estrogen receptor (ER), the progesterone receptor (PR) and the expression of human epidermal growth factor receptor 2 (HER2). 

A tumor with the absence of all of these three receptors, also called triple negative breast cancer, is an aggressive form of breast cancer with a high risk of relapse and metastasis, and is associated with a poor clinical outcome.

Epithelial cells may undergo epithelial-to-mesenchymal transition (EMT), allowing the cells to gain new abilities to cross the extracellular matrix, migrate and become circulating cells. 

The presence of circulating tumor cells (CTC) in patients with metastatic breast cancer is indicative of poor prognosis. The survival rate of women with breast cancer is highly dependent on the absence of metastatic cells and the tumor grade.

The most common marker for CTCs that is used today is the epithelial cell adhesion molecule (EpCAM), but for some malignancies such as aggressive breast cancer metastases that are often of non-epithelial origin or have transitioned through EMT, the EpCAM detection method is not useful. This highlights the need for more reliable CTC markers. The adhesion receptor CD44 and EpCAM function together in preparing the pre-metastatic niche, while CD45 is a leukocyte marker that is exclusively expressed on all nucleated cells of the hematopoietic system. The absence of CD45 on epithelial cells emphasizes the fact that it is an appropriate cell surface marker for distinguishing between CTCs and hematopoietic cells.

CTCs are known to have morphological properties that distinguish them from other circulating cells, such as a larger size than leukocytes and different nuclear morphology. CTCs have been isolated by size, using different methods such as density gradient centrifugation and micropore filtration. 

A more cell-friendly and speedy method of investigating morphological differences would facilitate CTC research. 

Digital holographic cytometry (DHC) is a powerful tool for label-free cell observations and the evaluation of cell morphological and dynamical parameters in vitro. 

Studies using DHC include different cell types, from protozoa, bacteria and plant cells to mammalian cells such as nerve cells, stem cells and tumor cells. DHC has also become popular in the diagnostic field such as for the screening of malaria-infected red blood cells and cervical cancer.

The aim of the present study was to use DHC to investigate the morphological differences between a collection of breast cancer cell lines and white blood cell (WBC) cancer cell lines. 

The flow cytometry analysis of the cell markers EpCAM and CD45 was used to characterize the cell lines. We show that cell types lacking CD45 expression have significantly larger cell area, volume and thickness than CD45⁺ cells without EpCAM expression. 

In conclusion, DHC is a powerful technique for discriminating cancer cells from WBCs by performing non-invasive measurements of cell area, volume and thickness.

2. Materials and Methods (urval)

2.3. DHC and Computer Software

Cell morphology was detected and analyzed using DHC with the HoloMonitor^TM M4 

(Phase Holographic Imaging AB, PHIAB, Lund, Sweden). 

For each analysis, 1 × 10⁶ cells in suspension were washed twice with PBS and then 1 × 10⁴ cells in 10 µL were added to a Countess^TM cell counting chamber slide (ThermoFisher Scientific, Waltham, MA, USA). The chamber slide was placed on a HoloMonitor^TM M4 inside a cell incubator to ensure stable conditions for the cells, 37 °C with 5% CO₂ and 95% humidity for 10 min, during the analysis. For each cell line, more than 10 images at different positions were acquired automatically all over the chamber. 

The image analysis was performed with the proprietary AppSuite software (Phase Holographic Imaging AB). 

The HoloMonitor^TM M4 was equipped with a 635 nm diode laser, which illuminated the cells at 0.2 mW/cm² to prevent phototoxic effects on the cells.

Discussion

In 2004, the CellSearch system was approved for the detection of CTCs in humans by the Food and Drug Administration (FDA). This system is based on magnetic cell sorting, the expression of EpCAM and cytokeratin (CK). The system is reliable and has radically improved the detection of CTCs. However, one drawback is the inability to detect CTCs with downregulated EpCAM expression. The level of EpCAM varies in different tumor types and is known to be downregulated to increase invasiveness and metastatic potential. The CellSearch system may miss EpCAM-negative tumor cells due to the loss of EpCAM expression.

In this study, we have shown that epithelial breast cancer cells can be discriminated from WBCs by measuring the cellular thickness, volume and area using DHC. The investigated parameters showed significant differences between epithelial cells and blood cells. Future applications will include peripheral blood cells and extended collections of epithelial cancer cells.

Conclusions

Non-invasive DHC is a powerful technique to use for the discrimination of epithelial cancer cells and WBCs.  

Upon determining the cell area, volume and thickness, the investigated cell types with CD45 expression, the WBCs, were shown to have significantly smaller cell areas, volumes and thicknesses than CD45⁻ epithelial cells either with or without EpCAM expression.  

We conclude that DHC is a convenient technique with many possibilities for analyzing either suspension or adherent cells, here used in a model for the in vitro analysis of CTCs.

 

Min kommentar

I denna studie får vi många nya fakta,som ex att man använt HoloMonitor (DHC) för att studera vita blodkroppar (WBC = White Blood Cell).Det vet jag inte om jag läst i tidigare forskningsrapporter.

Studien berättar som i ett förbiseende att Holotekniken nu blivit populär som ett diagnosverktyg för malaria och livmoderhalscancer.

- DHC has also become popular in the diagnostic field such as for the screening of malaria-infected red blood cells and cervical cancer.

Till det konstaterandet kan vi nu lägga till att HoloMonitor enligt forskarna är att anse som ett nytt kraftfullt instrument för diagnos av aggresiv bröstcancer.

Det i sin tur adderar världens alla mammografer (stavning?) till potentiella kunder för PHI,men såklart även andra aktörer med samma grundteknik.Marknaden utvidgas mao.

Om ovanstående inte räcker för att få alla phi,are att hoppa jämfota av excitement lägger jag till att denna forskningsrapport kommer ingå i den specialutgåva jag tidigare berättat om.

Avyttrar man sina aktier efter ha läst detta inlägg kanske man ska söka sig till ett diagnosställe för att utröna om man lider av den inte så kända bokstavskombinationen JHIT (jagharintetålamod) eller/och i kombo JVIHBAPMI (jagvillintehabättreavkastningpåmininvestering). 

                                      😎

                                              Mvh the99

Carbiotix - Bolagsvärdering (1/4) Gut Test

Marknaden åsätter Carbiotix en värdering på ca 35 Msek i dagsläget kan konstateras.
Det baserat på dagens stängningskurs 4,12.(Noteringskursen var 4,50)
Är börsvärdet rimligt och rättvist? Frågar ni mig är svaret ja men även nej.
Och varför är undertecknad av den dubbla åsikten då kanske nån undrar.Jo om jag ska ge mig på att försöka förklara hur jag tänker.
Utgår vi från krass försäljning och lägger till den förmildrande omständigheten att Bolaget är relativt nystartat och inte kommit igång med säljeriet ännu så är värderingen rimlig.
Men nu fungerar ju aktiemarknaden så att det är den framtida potentialen (försäljning) man ska bedöma och värdera ett Bolag efter. Och då är dagens värdering på ca 35 Msek knappast rättvis.
Eftersom Carbiotix innehåller 4 affärsområden är det enklast att värdera dessa enskilt och sen addera.
Jag tänker i 4 inlägg gå igenom respektive affärsområde och se hur en mer rättvis värdering skulle kunna se ut.

Det första området är deras konsumentriktade tjänst Gut Test.
Kortfattat innebär tjänsten att man beställer ett kit från deras hemsida,gör enligt instruktionerna och skickar in proverna till deras labbpartner för att sen någon/några veckor få resultatet.
Det är alltså en analys av tarmfloran som görs.Analysens svar kan sen ge vägledning till om man behöver lägga om sin kost eller om man käkar helt rätt så får man kvitto på det.

I Sverige har denna marknad ännu inte kommit igång då företeelsen är tämligen ny,men i USA och framförallt Australien har den fått fäste.England, Tyskland och Frankrike ser ut att även de mogna.
Kollar man på vad dessa tester kostar ligger de flesta mellan 1200 - 3100 kr.
Högst pris har ett amerikanskt företag som säljer sitt kit för ca 4700 kr.
Kikar man då på den Svenska marknaden och vilka aktörer som fysiskt finns här hittar jag bara 2.
Gutfeeling Labs är enbart inriktat på att sälja dessa kit och förmodligen den konkurrent Carbiotix kommer dela den Svenska marknaden med.
Gutfeeling säljer sina kit för 1199 kr.Det är ett engångstest alltså.
Carbiotix anger på sin hemsida att de säljer sina kit för 1/3 av närmaste konkurrents pris.
Den konkurrenten bör vara svenska Gutfeeling.
Dock är skillnaden att Carbiotix säljer kit på 3 resp 6 tester.
Deras 3 variant kostar 999 kr vilket blir 333 kr per test och alltså en tredjedel av konkurrenten Gutfeelings pris.
Från hemsidan:

PM
2020-02-11
"Forskningsbolaget Carbiotix lanserar nu sin andra generationens diagnostiska plattform för tarmhälsa baserad på andra generationens DNA-sekvensering, NGS. "Bolaget kommer nu att erbjuda ett NGS-test i tre exemplar som standard och till en kostnad som är en tredjedel av kostnaden hos närmaste konkurrenten på marknaden per prov", skriver Carbiotix i ett pressmeddelande. 
Detta test ska säljas direkt till konsumenter via Carbiotix webbplats, via partners som en "white label" och som en klinisk diagnostiktjänst.
Carbiotix kommer att rikta in sig på hela marknaden för diagnostiska tjänster för konsumenter och kliniska mikrobiomanalyser som bedöms vara värd mer än 800 miljoner euro per år med en årlig tillväxt på 19 procent."

Kikar vi på Gutfeelings försäljning visar den att de omsatte 1,6 Msek 2019.
Bör vara baserat på att nån gång under året (2019) satte de igång med sitt säljeri.
Hyfsat imponerande om de kom igång först under andra halvåret,men som sagt det vet vi inte så utgå från helår.

För att exemplifiera med marknadens etablerade aktörer och vad deras engångskit kostar:

Biohm 129,99 $, ca 1200 skr
Sungeomics 147 $, ca 1330 skr
Thryve 149 $, ca 1350 skr.
Biomes 139 €, ca 1400 skr
Viome 179 $, ca 1600 skr.
Atlas Biomed 149 £, ca 1700 skr
EasyDNA 199 $, ca 1800 skr
Ixcela 299 $, ca 2700 skr (blodtest)
Onegevity 349 $, ca 3100 skr
Microba 349 $, ca 3100 skr

De här aktörerna slåss på en marknad som omsätter 8 Miljarder Skr årligen (800 milj €).
Att det är en expansiv och lukrativ bransch kan man vara ganska säker på.
Marginalerna på dessa kit lär knappast understiga 80%.

Kikar vi då på Carbiotix inträde på denna marknad.
De har samma typ av produkt som konkurrenterna,samma förfarande att konsument beställer hemma och skickar proverna i ett förfrankerat brev.
Samma förfarande att på resp företags hemsida logga in på sitt konto och ta del av vad resultaten visar.
En smart affärsidé med låga kostnader och troligtvis riktigt höga marginaler.
Carbiotix bör se den Svenska marknaden som Nr 1 och Gutfeeling som sin närmsta konkurrent.
Dock är de inte främmande för att ta upp konkurrensen med de riktigt stora aktörerna.
Det genom att affärsidén är nätbaserad och enbart kräver tillgång till internet.Från deras hemsida :

Ställer vi Carbiotix pris (Price per sample: 333SEK Available worldwide) mot konkurrenternas framstår det ju som tämligen attraktivt.
Hur ska de då komma in på marknaden och få möjlighet att visa upp sitt erbjudande?
Ja,det är där den stora utmaningen ligger och där Bolaget kommer få visa sin smartness.
Internetmarknadsföring har jag ingen direkt pejl på,kostnader osv...Men det tror jag heller inte är den väg Bolaget väljer att gå då det förmodligen kostar alldeles för mycket flis.
Skulle jag gissa kommer man istället inledningsvis medverka i sammanhang där ämnet tarmflora är det centrala. Typ pusha på att synas i artiklar som riktar sig till konsumenter intresserade av ämnet.
Medverka i symposium där branschens alla aktörer medverkar,symposium med rapportering ut till tidningar,sajter med hälsoinriktning. Alltså bygga varumärkeskännedom på ett kostnadseffektivt sätt.
Ponerar vi då att de lyckas tränga sig in bland de redan etablerade kit-säljarna, under förutsättning att de fortfarande har denna pris-konkurrensfördel, bör de relativt snabbt kunna nå volym.
Med VD,s bakgrund (affärande) och nordamerikanska ursprung (Kanada) tror jag att han redan har en utarbetad affärsplan för att slå sig in bland dessa av majoritet USA-baserade konkurrenter.
Vad är då rimligt att anta att deras kit-försäljning kommer landa på då de är hyfsat kända och etablerade?
En försiktig gissning är att år 1 bör de kunna omsätta minst 25 Msek,det i scenariot att de som nyetablerade fått fäste på marknaden.Sen bör omsättningen accelerera ganska brant de följande åren.
Ok,om vi går tillbaka till inläggets inledning om Bolagsvärdering och applicerar modellen P/S, sätter ps-talet till 10,det motiverat av ett växande företag med hög vinstmarginal.
Bolagsvärderingen blir med denna uträkning 250 Msek vilket per aktie landar i ca 29 kr.
Nu ska man vara medveten om att Carbiotix i dagsläget inte är i närheten av att sälja sina testkit i den omfattningen (25 Msek) och därför bör man inte se denna värdering som vägledning i nuläget.
Men uträkningen visar den potential Bolaget har,det enbart på 1 affärsområde.
Min subjektiva åsikt är dock att med denna potential värderas Carbiotix alltför lågt (ca 35 Msek).
Förmodligen hade marknaden gjort en annan högre värdering än dagens om Bolaget enbart sysslade med dessa testkit.Men nu är ju Carbiotix ganska komplext då de har 3 affärsområden till,samtliga inom sina egna branscher/marknader.Efterföljande inlägg kommer beta av dessa områden vart och ett.

                                                 Mvh the99

Rekord i forskningsrapporter

Idag publicerades ytterligare forskningsrapporter,nämligen bestämt 2 st.Med dessa 2 kan vi på 1 månad (33 dagar) räkna in 9 studier som offentliggjorts,samtliga såklart baserade på PHI`s excellenta HoloMonitor.
Detta är ett rekord och borde uppmärksammas på fler ställen än här på bloggen.
Med detta tempo på 1 forskningsrapport nästan var 3e dag (9/33) kan man ju inte annat säga än att vi nu ser den beryktade ketchupeffekten.Frågan är om även branschens större aktörer,dvs bjässarna,noterar detta formliga crescendo i HoloMonitorbaserade studier.Nån slags reaktion tror jag vi kan förvänta oss.Men när och i vilken form återstår att se.Vi är de facto inne i semestertider vilket kan påverka.Men när den perioden är över..då ....
Men till dagens skörd av forskningsrapporter.
Först har vi en studie där PHI`s Svenska ambassadör Professor Stina Oredsson medverkar.
Förutom Stina ser vi idel kända namn som Sofia Kamlund, Birgit Janicke och Kersti Alm.
Denna studie kan vara en förlaga till det som komma skall : Kombon Holo/Fluo-instrumentet.
Det eftersom forskarna medvetet samkört bägge teknikerna.
Som de skriver i inledningen : The aim of the present study is to use longitudinal tracking of cells in images acquired using digital holographic microscopy in a unique combination with fluorescence microscopy to identify the expression of CD24 and E-cadherin on the tracked cells.
 

Salinomycin Treatment Specifically Inhibits Cell Proliferation of Cancer Stem Cells Revealed by Longitudinal Single Cell Tracking in Combination with Fluorescence Microscopy

Published: 9 July 2020 

Abstract

A cell line derived from a tumor is a heterogeneous mixture of phenotypically different cells. Such cancer cell lines are used extensively in the search for new anticancer drugs and for investigating their mechanisms of action. Most studies today are population-based, implying that small subpopulations of cells, reacting differently to the potential drug go undetected. This is a problem specifically related to the most aggressive single cancer cells in a tumor as they appear to be insensitive to the drugs used today. These cells are not detected in population-based studies when developing new anticancer drugs. Thus, to get a deeper understanding of how all individual cancer cells react to chemotherapeutic drugs, longitudinal tracking of individual cells is needed. Here we have used digital holography for long time imaging and longitudinal tracking of individual JIMT-1 breast cancer cells. To gain further knowledge about the tracked cells, we combined digital holography with fluorescence microscopy. We grouped the JIMT-1 cells into different subpopulations based on expression of CD24 and E-cadherin and analyzed cell proliferation and cell migration for 72 h. We investigated how the cancer stem cell (CSC) targeting drug salinomycin affected the different subpopulations. By uniquely combining digital holography with fluorescence microscopy we show that salinomycin specifically targeted the CD24⁻ subpopulation, i.e., the CSCs, by inhibiting cell proliferation, which was evident already after 24 h of drug treatment. We further found that after salinomycin treatment, the surviving cells were more epithelial-like due to the selection of the CD24⁺ cells.

Keywords: digital holographic microscopy; fluorescence microscopy; single cell tracking; cancer stem cells; salinomycin; JIMT-1 breast cancer cells

1. Introduction

The importance of cancer stem cells (CSCs) in the development and recurrence of cancer has gained increased attention in cancer research during the last decade. A tumor contains a mixture of phenotypically heterogeneous cancer cells. Only a few cells in a tumor belong to the CSC population, but it appears that for many tumors of different origin it is mainly the CSCs that can give rise to new tumors with similar phenotypic composition as the original tumor. Since the CSCs are drug resistant and favor cancer metastasis there is an increased need for new drugs and therapeutic strategies that reduce the CSCs specifically. In breast cancer, a high expression of the cell surface marker CD44 in combination with a low expression of the cell surface marker CD24 have been used to identify CSCs.

The cationic ionophore salinomycin was found to target CSCs, in a high throughput screening study aimed to find drugs that target this subpopulation in breast cancer. Subsequently, salinomycin has been found to target CSCs in many other human cancer types including leukemia, gastric cancer , colorectal cancer , osteosarcoma , pancreatic cancer , prostate cancer , head and neck squamous cell carcinoma, and lung cancer . Many different mechanisms of action have been ascribed to salinomycin, such as disruption of actin stress fibers , downregulation of mRNAs and proteins related to stemness , impaired mitochondrial function, induction of autophagy, decreased ATP levels and increased reactive oxygen species production, sequestration of iron in lysosomes , and induction of reactive oxygen species , with outcomes like decreased cell viability, proliferation, and migration, as well as stimulation of mesenchymal-to-epithelial-transition . Salinomycin has also been shown to increase the expression of E-cadherin , a calcium-dependent cell adhesion glycoprotein molecule found on the surface of epithelial cells . The expression level of E-cadherin was inversely correlated with the tumorigenicity of different cell lines, where a high expression of E-cadherin correlated to low tumorigenicity and vice versa . However, for clinical outcomes the role and correlation of E-cadherin is unclear  and the level of expression has been found to vary between tumors of different classification .

We have previously found evidence for the molecular initiating event that explains most down-stream effects observed after salinomycin treatment . Salinomycin was shown to almost immediately after addition to the cell culture medium localize to the endoplasmic reticulum (ER) of breast cancer cells, leading to increased cytosolic Ca²⁺ levels followed by ER stress. This effect was down-stream linked to inhibition of the Wnt signaling pathway, which has previously been reported as an effect of salinomycin treatment. These findings are deduced from population-based studies, but to increase our understanding of CSCs as well as how they can be targeted, more studies of single cell sensitivity are required.

Digital holography is a quantitative phase imaging technique, which can be used to generate large amounts of information about individual cells based on the phase shift of light. The method is label free, thus eliminating possible chemical toxicity, and no phototoxicity has been found. Therefore, the technique allows for long time imaging and when images are acquired with high time resolution they can be used for longitudinal tracking of individual cells, which is a powerful tool to investigate how individual cells in a population react to treatment over time. It can also be used to detect and over time trace subpopulations that react differently than the bulk of cells to drugs, e.g., that are drug insensitive and may therefore be the cause of metastasis. Using digital holography, we have previously shown that JIMT-1 breast cancer cells contain a subpopulation of cells with a decreased response to salinomycin compared to the other subpopulations , an effect that was hidden among the total population data.

Much work is ongoing trying to find holography-derived parameters, such as morphological for cell behavior features, or combinations thereof that can be used to characterize subpopulations among the bulk of cells . It has been shown that a number of empirically-derived parameters obtained by digital holographic microscopy, actually can be applied to computational machine learning to identify effects of drug treatment on individual cells . However, still more work is needed to elucidate if digital holography alone can be used to truly identify different cell populations and subpopulations.

The aim of the present study is to use longitudinal tracking of cells in images acquired using digital holographic microscopy in a unique combination with fluorescence microscopy to identify the expression of CD24 and E-cadherin on the tracked cells. This was used to investigate differences in cell cycle length and migratory behavior between subpopulations of JIMT-1 cells, as well as the effect of salinomycin on those parameters in the different subpopulations. We have used the JIMT-1 breast cancer cell line in studies of the effect of different compounds including salinomycin and salinomycin analogues on CSCs as well in studies using digital holographic microscopy . 

The cell line contains a high proportion of CSCs that are sensitive to different treatments. Here we deepen our insight into dynamics of how the salinomycin treatment decreases the CSC subpopulation of JIMT-1 cells. Altogether the data show that the main difference between subpopulations of JIMT-1 cells is related to cell proliferation and that the initial effect of salinomycin treatment was a decrease in the proliferation of CD24⁻ cells.

2. Materials and Methods (urval)

2.4. Digital Holographic Time-Lapse Imaging in Combination with Fluorescence and Tracking

For digital holographic microscopy (DHM), the HoloMonitor^® M4 (Phase Holographic Imaging AB (PHI), Lund, Sweden), with a motorized stage was used for time-lapse imaging. Images were acquired using the software Hstudio™ (PHI). To increase image quality, the standard lid of the Petri dish was replaced with HoloLid™ 71 110 (PHI) prior to the start of imaging.

The imaging was done using two different time setups. First, the method for combining digital holographic microscopy with fluorescence was evaluated using 24 h-time-lapses as illustrated in the upper part of Figure 1A. Cells were seeded in a number of Petri dishes, of which some were used in time-lapse imaging 24–48 h after seeding (i.e., time-lapse/treatment time 0–24 h) and some in time-lapse imaging 48–72 h after seeding (i.e., time-lapse/treatment time 24–48 h). Thus, a 48-h time-span was divided into two consecutive 24-h time-lapses where parallel samples were imaged for the different time-spans. For the other set-up, samples were imaged uninterrupted for 48 h, i.e., from 24–72 h after seeding (i.e., time-lapse/treatment time 0–48), as illustrated in the lower part of Figure 1A.

Supplementary Materials

Figure S1. Representative images from DHM time-lapses and fluorescence. (A) Images from timepoint 0, 24 and 48 h of a representative DHM time-lapse of JIMT-1 cells in the absence (control) or presence of 0.5 µM salinomycin. The salinomycin was added immediately before the start of the timelapse. The scale bar on top represents 300 µm for control and 400 µm in salinomysin-treated samples, and the scalebar to the left represents the optical thickness 0-46 µm in control and 0-48 µm in salinomycin-treated samples. (B) Fluorescence images of samples fixed after DHM imaging. The cells were labelled with anti-CD24-PE and anti-CD44-FITC or anti-CD24-PE and anti-E-cadherin-Alexa Flour 488 after 24 or 48 h of treatment.

4. Discussion

Most of our current knowledge of how cells react to different kinds of perturbations is based on analysis of the response of an entire cell population. Individual cells may be analyzed e.g., by using flow cytometry as an end response assay, which may display heterogeneity of the studied entity. However, a challenge in biology is to understand the kinetics in the processes of each individual cell that causes the heterogeneity in the end response. Microscopic techniques like confocal microscopy, immunofluorescence microscopy, phase contrast microscopy, and DHM are today used to follow the behavior of live individual cells through time-lapse imaging. All methods have their advantages and disadvantages. Here we have used DHM because of the advantage of low photo toxicity and because it is label-free. The disadvantage of label free microscopy is that there are questions around cell identification. Here we address such questions by analyzing different subpopulations of JIMT-1 breast cancer cells identified by combining DHM with fluorescence microscopy.

5. Conclusions

In conclusion, the combination of DHM and fluorescence microscopy is powerful. 

It allowed us to show for the first time that the decrease in the CD24⁻ CSC subpopulation already after 24 h of salinomycin treatment is caused by specific inhibition of proliferation of the CD24⁻ population while the CD24⁺ population is not affected. This implies that the phenotypic shift towards less stemness caused by salinomycin treatment is due to positive selection of the CD24⁺ cells.

Min kommentar

Överlåter jag till granskaren av denna studie.

"The manuscript by Kamlund et al is interesting, as it shows an original perspective allowing to see how individual cells in a population can be affected by a drug. Something, that would be difficult to observe with classical methods looking at averages in bulk populations."

Värt att ytterligare notera är vilka som finansierat denna studie.
"This work was supported by funding from the Swedish Research Council (VR), Forska Utan Djurförsök, by a donation from Carolina LePrince with the “Kalenderflickorna” and associated sponsors, and by donations to Stina Oredsson’s research group at Lund University (http://biology.lu.se/cancer-stem-cells)."

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Studie nr 2 som offentliggjorts idag ser ut att vara en fortsättning på Robert L. Judsons arbete med att konstruera ett "verktyg" i att känna igen karakteristika för olika typer av cancer. Robert medverkar så jag kan ha rätt i det antagandet,men som vanligt : jag är bara 99% säker:-D

Men till studien benämnd 

Quantifying the Rate, Degree, and Heterogeneity of Morphological Change during an Epithelial to Mesenchymal Transition Using Digital Holographic Cytometry

 Published: 9 July 2020

Abstract

Cells in complex organisms can transition between epithelial and mesenchymal phenotypes during both normal and malignant physiological events. These two phenotypes are not binary, but rather describe a spectrum of cell states along an axis. Mammalian cells can undergo dynamic and heterogenous bidirectional interconversions along the epithelial–mesenchymal phenotypic (EMP) spectrum, and such transitions are marked by morphological change. Here, we exploit digital holographic cytometry (DHC) to develop a tractable method for monitoring the degree, kinetics, and heterogeneity of epithelial and mesenchymal phenotypes in adherent mammalian cell populations. First, we demonstrate that the epithelial and mesenchymal states of the same cell line present distinct DHC-derived morphological features. Second, we identify quantitative changes in these features that occur hours after induction of the epithelial to mesenchymal transition (EMT). We apply this approach to achieve label-free tracking of the degree and the rate of EMP transitions. We conclude that DHC is an efficient method to investigate morphological changes during transitions between epithelial and mesenchymal states. 

Keywords: digital holographic cytometry; quantitative phase imaging; epithelial to mesenchymal transition

1. Introduction

During tumour progression, cancer cells interconvert between epithelial and mesenchymal states with a high degree of plasticity based upon environmental signals. These bidirectional transitions are known as epithelial to mesenchymal transitions (EMT) and mesenchymal to epithelial transitions (MET). Both transitions are required for the complex series of processes that result in metastatic dissemination. For example, EMT is involved in both tumour cell migration and the transformation of cancer cells into a cancer stem cell (CSC) phenotype, each of which promotes growth of new tumours at distant locations. However, the repression of EMT and induction of MET is also essential for metastatic colonization.  

Since metastases cause most cancer-related deaths  it is of vital importance to understand the EMT/MET processes. Efforts to identify candidate therapeutics that interfere with the process are underway. However, dependent on the stage and identity of each cancer cell within a tumour or a patient, inhibition of either EMT or MET could be beneficial or harmful. 

Given the complexity of the transitions and their relationship to metastatic dissemination, more comprehensive characterization of the effect of candidate therapeutics on EMT and MET are needed.

Characterization of EMT and MET is challenging due to several types of substantial heterogeneity. First, the processes of EMT/MET do not describe a binary toggling between two discrete states, but rather transitions along an “epithelial-mesenchymal phenotypic (EMP) spectrum” from more epithelial-like states to more mesenchymal-like states. Second, cells are heterogenous in the ease with which they can transverse this spectrum, often referred to as plasticity. Each cell line and individual cells within a cell line population can occupy distinct locations along the EMP spectrum and possess varying degrees of plasticity dependent on contextual signals. Third, the intracellular gene circuits and morphological changes that define the EMP spectrum differ from cell line to cell line. Finally, the EMP spectrum is only one of many transcriptional programs that define a cell. Some cancers, such as melanoma, are not derived from epithelial cells but, nevertheless, undergo an “EMT-like” transition during metastasis. Thus, simply defining the EMP spectrum for any specific cellular context is non-trivial and it cannot be assumed that parameters that accurately define EMP in one cell line apply to another.

To further develop the candidate therapeutics for clinical application, it is first essential to develop assays capable of capturing and quantifying the degree, kinetics, heterogeneity and cell-line specificities of transitions along the EMP axis. Defined changes in morphology, specifically the acquisition of a spindle-like shape, constitute one hallmark of EMT that could serve as an informative feature. EMT has been monitored in real time by fluorescence imaging of vimentin by Maier et al. Both EMT and MET have been monitored by time-lapse imaging giving insights into the ongoing process. Digital holographic cytometry (DHC) has emerged as a complementary technique to obtain time-lapsed imaging for label free long-term cell analysis. The quantitative 3D images acquired can be used to follow morphological changes or changes in cell movement induced by therapeutic treatment. It has also been presented as a technique suitable for classifying distinct cell phenotypes . We have previously suggested that DHC can be used as a tool for identifying and monitoring EMT or MET  by characterizing the change in cell movements. Recently, it has been suggested that the application of machine learning techniques to DHC-derived features can detect epithelial and mesenchymal characteristics in cell lines of unknown EMP status. However, due to substantial morphological heterogeneity between cell lines, cell types, and species, it is not obvious whether a single model that classifies EMP status based solely on DHC-derived morphological features can be universally applied.

In this study, we evaluated the use of DHC to track the rate, degree, and heterogeneity of transitions within the EMP spectrum based on morphological features. Importantly, we aimed to create an accessible approach for assessing EMP status that could be easily adjusted for different cell systems and was not at risk of over-training as introduced by machine learning approaches. First, we monitored mouse mammary epithelial cells undergoing EMT. Next, we trained a model to classify the degree of EMT using DHC-derived features and applied our model to live-imaged cultures undergoing EMT. Finally, we applied the model to five human and mouse cell lines of known EMP state and observed substantial line-to-line variability. Our data show that DHC-derived morphological parameters can be used to monitor the degree, rate, and heterogeneity of EMP transitions, while highlighting the necessity for developing cell-line specific classifiers.

2. Materials and Methods (urval)

2.3. Digital Holographic Imaging and Analysis

The cells were allowed to attach for 24 h prior to start of the experiment. For NMuMG cells, compounds were added as described above immediately before imaging. The standard lid of the Petri dish was replaced with a HoloLid™ 71,110 for Petri dishes with 35 mm diameter (Phase Holographic Imaging AB (PHI), Lund, Sweden) or HoloLid™ 71,120 for 6-well plates (PHI). The cells were then imaged using the HoloMonitor^® M4 with a motorized stage (PHI). 

The HoloMonitor^® M4 is a quantitative imaging system based on digital holographic microscopy. For imaging, the software Hstudio™ (PHI) was used. Images were acquired at three to five locations (image frames) per well, at time-intervals ranging from every hour to every 24 h, for a total time of 48–72 h.

The images were analyzed using Hstudio™ as follows. First, each image was segmented using thresholding that identified each individual cell in that image. Second, based upon this segmentation, morphological parameters for each cell in each image frame were computationally calculated. 

In the Hstudio™ analysis tool, values for the 27 morphological parameters considered here are presented for each individual cell. To develop the EMP score, we used the average values for each image frame at each timepoint. Data were analyzed for correlations using the free software R (R Core Team, 2015). To generate the score, we first selected four features that positively correlated with mesenchymal conditions in the dataset obtained from a 72-hour time-lapse imaging of NMuMG cells with and without exposure to 0.5 ng/mL TGFβ: average cell eccentricity, average cell hull convexity, average cell roughness skewness, and average cell optical thickness max. We centered each feature set by dividing each value within that set by the mean feature value in control conditions. We then performed standard min max feature scaling (Equation (1)) to restrict all values to the range 0–1 that were then summed to generate a score in the range 0–4 with increasing values correlating with a more mesenchymal phenotype. 

5. Conclusions

We conclude that DHC can be applied to the study of EMP transitions. Specifically, consideration of DHC-derived morphological features permits efficient monitoring of the rate, degree, and heterogeneity of the transition in a label-free manner. We further provide evidence that the morphological changes associated with EMP status are not universal across lines, emphasizing the need to optimize and validate DHC-derived classifiers for each cell system. 

Min kommentar

Stämmer mitt antagande (99% säker) att studien är en utveckling av Roberts tidigare arbete med att "katalogisera" kännetecken på olika typer av cancrar med tekniken HoloMonitor medger är detta en veritabel infobomb.Det under förutsättning att forskarvärlden tar till sig studien och ger det sin acceptans.Konkret skulle det i så fall innebära 2 saker. Det första är att forskare får ett verktyg i att snabbt identifiera vilken cancertyp det handlar om när de startar sina cellstudier.De hoppar alltså över en massa steg i att behöva identifiera och kan istället gå direkt till "andra halvan" i studierna.

För det andra är det oerhört betydelsefullt för forskare som bedriver studier kring läkemedel som är verkningsfulla mot specifik typ av cancer. Med verktyget behöver de inte köra uteslutningsmetoden för att till slut hitta vilken cancer det handlar om,och sen fortsätta forska vilka substanser som är effektiva som bot.Med ett verktyg som ger möjligheten att utgå direkt från cancertyp sparas tid och resurser.Det borde attrahera de större läkemedelstillverkarna (med egna forskningsavdelningar) skulle jag tippa.

Summasummarum.

Dessa 2 forskningsrapporter är nånting utöver det vanliga.

Skulle jag ha 100% rätt i mina antaganden bör de var för sig ha stor betydelse för PHI.

Kombon holo-fluo kommer när den är färdigutvecklad av NIH redan ha fakta ute som bekräftar dess unika funktionalitet vilket borde rendera i förhandsbokningar i den bästa av världar.

Ett verktyg som definierar cancertyp vid forskningsstadiets början kommer mottas med stor tacksamhet,även det i den bästa av världar.

Att verktyget är framtaget med HoloMonitor bör innebära att det fungerar bäst i vidare forskning med just HoloMonitor.

Som extra grädde på moset kommer dessa 2 studier finnas med i den specialutgåva jag berättade om igår.
Gissa om den kommer läsas av världens forskare! Späckad med helt nya fakta som kommer vara högst användbara för framförallt cancerforskare,men troligtvis även andra.

Min spontana tanke med detta sanslösa flöde av forskningsrapporter och en specialutgåva i vardande är att PHI har ett finger med i detta och med det en klar strategi för det som utvecklar sig vid samtalen med sina friare.
Man har väntat in denna "ammunition" och kommer använda den vid de fortsatta förhandlingarna.
Spekulation från bloggens sida? Absolut,och till 100%.Men som pusselgillare är senaste tidens händelser för bra pusselbitar i det stora pusslet för att ignoreras.

                                                 Mvh (en superexcited) the99

 

Strax kommer info ni vill läsa

Undertecknad sitter och färdigställer ett nytt inlägg jag tror alla phi,are vill läsa.
Absolutely worth waiting for.

En excited the99

Svensk forskningsrapport med bravur

Här kommer en riktigt bra forskningsrapport utförd av 4 Svenska forskare + 1 överläkare.
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.

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