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