5 nya studier med HoloMonitor

Det börjar komma in fler och fler forskningsrapporter där användande av HoloMonitor är central. Med den ökade kunskapen av hur använda instrumentet effektivt till att få fram nya fakta annars svåra att komma till insikt om,är det kanske inte så förvånande att antalet forskningsrapporter förmodligen kommer att öka snabbt i det korta perspektivet. Bloggen berättar här om de senast uppgrävda. 
Då i förkortad version mtp det utrymme som krävs annars.

För 2 dagar sen,16/11, publicerades dessa 2 forskningsrapporter. 

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest
Published: November 16, 2023

Summary

CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.

Time-lapse imaging

 Holographic imaging movies were capture using a Holomonitor M4 microscope to quantify single-cell mitotic volumes, which were calculated using the Holomonitor App Suite version 3.4.0.158. All holomonitor analysis was carried out with a Holomonitor M4. A total of 15,000 cells were plated into either a Sarstedt multiwell, or an Ibidi μ-plate glass-bottomed 24 well plate. After 24hrs cells were treated and imaging began immediately. Images were scheduled every 20 minutes for a total of 96 hours.

Länk till Molecular Cell där studien hittas i sin helhet.

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence
Published: November 16, 2023

Summary

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2− breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Method details (urval)

Cell volumes were measured using a HoloMonitor M4 in either Sarstedt Lumox, or Ibidi μ-plate glass-bottomed 24 well plates and the associated hololids. 
Optical volumes were determined using the holomonitor app suite.

Länk till Molecular Cell där studien hittas i sin helhet.

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För 4 dagar sen,14/11, publicerades följande forskningsrapport: 

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration
Published: 14 November 2023

Abstract

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Materials and Methods (urval)

Cell migration was monitored in time under a holographic microscope Holomonitor M4 live cell imaging system (Phase Holographic Imaging, PHI AB, Lund, Sweden). Data analysis was performed using HStudio (PHI AB, Lund, Sweden). Pictures were taken after 24 h. Data are reported as increase in cell-covered areas after 24 h with respect to the control condition (DMSO).

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Chalmers universitet i Göteborg har publicerat denna Masters Thesis av David Perhed. Notera användande av Microfluidics.

Using open-volume microfluidics to develop gap closure assay in a confluent monolayer of cells

Abstract 
Understanding the basic mechanisms of wound healing in living organisms is important to enhance healing as well as to avoid infections and scarring. Today, cell-free areas (gaps) in a confluent monolayer of cells in vitro, are commonly formed to mimic wounds in vivo. Migration and proliferation of cells are essential mechanisms for gap closure, in vitro. In many studies, measuring cell migration is of interest, thus suppressing cell proliferation is desired. Standard gap closure assays provide simple ways of monitoring cell migration in vitro. Commonly, these assays focus on formation of gaps with simple geometries, such as lines circles, squares and triangles. Other methods for gap formation have therefore been developed, however these methods have limited flexibility of forming gaps with arbitrary size and geometry. This study aims to use open-volume microfluidics to develop an in vitro gap-closure assay, allowing for time-dependent gap closure to be monitored and analysed for gaps with different initial geometry. Gaps of different initial geometries and sizes were successfully formed in a confluent monolayer of HaCaT cells. Gap closure of square, circle and crescent moon geometries (300x300 µm) was monitored under conditions where cells were allowed to proliferate and under conditions of no proliferation. Additionally, gap closure of larger square gaps (1000x1000 µm) was monitored under conditions of proliferation. This study developed an assay serving as a foundation for future studies of HaCaT gap closure in vitro. Furthermore, the study provides insights about printing fibronectin with the Biopixlar technology, important for modulating cellular migration and adhesion during gap closure. These conclusions, further add to the current research field of gap closure assays and provides principal knowledge needed for future studies aiming towards formation of gaps with more complex geometries.

Figure 3.5: Phase contrast microscopy image of small crescent moon gap in a confluent monolayer of HaCaT cells immediately after gap formation. Imaging was performed using a Holomonitor M4 (Phase holographic imaging, PHI AB). Scale bar (upper left) = 500 µm. The obtained height profile is observered in the bottom right of the image and is based on the blue line over the gap.

Methodology (urval) 
Quantitative holographic phase microscopy was performing using a Holomonitor M4 (Phase holographic imaging, PHI AB). Data was acquired and processed using the Hstudio suite (Phase holographic imaging, PHI AB). The technology allowed for the comparison of the height profile for gaps.

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Det 5e och sista bidraget kommer från USA och PHI`s partner Northeastern university

TAAR1 Functions as a Cancer-Inhibiting Gene in Breast Cancer

Background and Research Goals 
The G-protein coupled-receptor Trace Amine-Associated Receptor 1 (TAAR1) is expressed in the brain and immune system and its upregulation is inducible by cellular activation via immunological challenge, suggesting a possible role in the immune response. TAAR1 binds a variety of ligands including its most potent endogenous ligand 3-iodothryonamine (T1AM). T1AM is a derivative of the T4 thyroid hormone and has been shown to reduce cancer cell proliferation, growth, and survival in cancer cell lines. However, it is not known whether these actions are mediated through TAAR1. TAAR1 protein expression is positively correlated to overall survival in breast cancer, and TAAR1 is over-expressed in breast cancer, leading me to hypothesize that T1AM signals through TAAR1 to negatively modulate tumor cell function to reduce cancer growth and survival, and that upregulation of TAAR1 may be a protective anti-tumor response mechanism of the immune system.

Methods (urval)
Live-cell imaging of cell migration 

A small silicone insert (Ibidi) with two chambers separated by a 500µM silicone divider will be placed in a 24-well plate and cells will be seeded into both wells in media containing T1AM or vehicle and allowed to attach for 24 hours. The silicone insert will then be removed to reveal a cell-free gap, and cells will be replenished with media with or without T1AM. Cells are then placed in an incubator equipped with a HoloMonitor live-cell imaging camera and images taken automatically every 5 minutes over the next 20 hours. Data was then collected and analyzed using the accompanying HStudio software. Visualization of wound closure over time is captured in playable video files, and individual frames will also be compiled into 3-D surface plots to allow for observation of cell migration over time by stacking the image frames chronologically using a base function of ImageJ.

Min kommentar
Behöver jag kommentera hur användbar HoloMonitor är för cancerforskningen? Känslan är att PHI nästan helt kunde inrikta sig på världens alla cancerforskare och få merparten av dem att börja använda instrumentet.

Nu låter det sig inte göras då bearbetning och övrig marknadsföring till alla dessa 10,100 tusentals forskare kräver enorma resurser enbart de absolut största aktörerna förfogar över. I ljuset av detta förstår man hur högt PHI skattar den potential Reg Med vibbar om,det då man kör nästan All-In på sagda område. Tanken gnager ändå,hur värdefull HoloMonitor är för cancerforskningen och i slutändan världens alla cancerdrabbade. Men ok, Reg Med utesluter ingalunda cancerforskningen så man kan se det som att PHI sitter med 2 starka kort bevisligen centrala för respektive område.

Annat.

Sajten WordWideScience.org jag prenumererar på har idag fått fnatt? och mejlar över 16 st forskningsrapporter med HoloMonitor. "Gamla" sådana ska sägas.Men ovanligt då sajten allt som oftast orkar leverera max 2-3 forskningsrapporter i månaden. Anledning till utskicket med just dessa 16 studier kan man fundera och spekulera över.Hittar dock ingen gemensam nämnare på dessa så tillsvidare hänför jag det till en intern algoritm som fått fnatt.

Sen får jag mejl med frågor om CAR och deras aktiesäljerier.Motiv,volym,tidslängd..osv...

Svaret på dessa frågor kan enbart CAR själva svara på. Vi övriga får ägna oss åt spekulation och gissa anledning. Min syn är som jag skrivit tidigare att CAR "rensar bordet (innehavet)" av annat än kursrelaterad anledning.Har man likvida medel och tror på en rejäl kursstegring inom det hyfsat korta men även det längre perspektivet avhjälper man dem såklart med att bygga på sitt eget innehav. 

Så gör jag,men det är ingen rekommendation till andra naturligtvis. Ok? Comprende?

Avslutningsvis,en del undrar min syn på aktiens utveckling under 2024.Det är en omöjlig fråga att svara på egentligen.Allt kan hända inom aktieområdet,det vet de flesta. Men med senaste händelserna,GlycoImaging,Reg Med,Altiums inträde och Gorans högst offensiva uttalanden siktar jag på 20-30 kr under vårkanten.Det utan nåt revolutionerande PM. Sen utesluter jag inte en återgång till tidigare AllTimeHigh runt 65-70 kr under året. 

Så pass bra är PHI positionerat enligt min högst subjektiva åsikt. OBS! Subjektiva åsikt.

Jag ser samma mönster som i "mitt" gamla bolag,C-rad,där de harvade med emissioner och misstroende som gav börsvärde under och runt 100 Msek en längre tid vill jag minnas. Det fast "produkten" var och är sanslöst innovativ och användbar för cancerbehandling.Idag när tekniken fått fäste och behandlingsaktörer ser fördelarna har Bolaget förärats en värdering runt 1300 Msek.Nu är ju detta ingen garanti för att PHI kommer gå i samma spår,men som jag ser det har PHI större potential om allt går Bolagets väg. 

OBS! Min högst subjektiva åsikt och absolut ingen utfästelse. Ok? Comprende?

                                             Mvh the99

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