I ett samlingsprojekt har ett helt gäng forskare från Australien,Taiwan,Kina och USA genomfört studier om en riktigt eländig och svårbehandlad bröstcancer..Det handlar om trippelnegativ bröstcancer som drabbar ca 10% av alla bröstcancerpatienter.
Trippelnegativ bröstcancer betyder att cancertumören saknar tre receptorer.Avsaknad av receptorer för de kvinnliga könshormonerna, östrogen och progesteron, liksom proteinet HER2 gör att behandlingsalternativen är färre för trippelnegativ bröstcancer jämfört med andra typer av bröstcancer.
Forskarna (med b.l.a HoloMonitorkände Derek J. Richards) har genomfört extremt komplicerade studier undertecknad har svårt att tolka. Det givet såklart att få andra forskare lyckats kommit denna cancer närmare "in på livet". Som jag förstår det har man i denna aktuella studie använt sig av en inte så vanlig metod/teknik,Cold atmospheric plasma (
CAP),att göra sina observationer.Och att denna metod har gett nya insikter som kan vara början på att förstå cancern och med det kunna lista ut hur bota.
Studien är betitlad :
Ur den klipper jag valda delar.
Abstract
Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills basal-like breast cancer cells. We used CAP-activated media (PAM) to capture the multi-modal chemical species of CAP. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in upregulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells, such that a strategy based on the synergism of CAP and EGF therapy may provide new opportunities to improve the clinical management of TNBC.
INTRODUCTION
Breast cancer (BC) is one of the most prevalent and malignant cancers in women, and affects men at a lower rate (1/100th). Among various breast cancer subtypes, triple negative breast cancers (TNBCs) are known for their poor clinical outcome and dearth of effective targeted therapies with acceptable toxicity profiles, due to the absence of druggable targets seen in other breast cancer subtypes 1,2. There is a need to explore novel therapeutic modalities that, in combination with current therapies, can help improve clinical outcomes and reduce side effects, especially in TNBC. One such potential therapy is cold atmospheric plasma (CAP), which has proven selectivity against many types of cancer cells 3 and represents an emerging oncotherapeutic approach offering a new opportunity to effectively manage a wide range of cancers.
CAP, the plasma generated at room temperature and atmospheric pressure, has shown great potential for biomedical applications 4–8, due to a favorable combination of reactive physical and chemical species, such as UV radiation, electrons, free radicals, ions and excited molecules 9. More recently, plasma activated medium (PAM) produced by exposure of aqueous medium to CAP10, was also found to inhibit cancer cells, sometimes as effectively as the direct CAP treatment 11, increasing the scope and flexibility of plasma-based therapeutics 12. We have previously reported the remarkable anti-cancer capacity of PAM against TNBCs 13,14, and here identify a critical role for epidermal growth factor receptor (EGFR), which is highly expressed in TNBC 15–17, and its response to PAM. Increasing evidence has associated high EGFR with immune resistance 18 and with high PD-L1 expression 19. Synergies between PAM and EGF were identified in this project, which explored the underlying biochemical mechanisms triggered in TNBCs upon treatment with PAM or PAM and EGF together. Combined treatment of TNBC cells with EGF and PAM caused remarkably potent and selective cell killing that is contradictive of conventional anti-cancer approaches using EGFR inhibitors, through cooperative increases in intracellular levels of reactive oxygen and nitrogen species (RONS).
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| Figure 3.EGF enhances the selectivity of PAM against TNBC cells. a Effects of treatment with 50% 10PAM, 10 ng mL−1 EGF or both for 12h on viability (% of control (C); mean +/− SEM; N=3) of breast cancer cell lines as indicated (pink shading of TBNC lines: HCC70, MDA-MB-468, SUM-149PT, SUM159-PT, MDA-MB-231). b Effects of EGF, 50%PAM or EGF+50%PAM treatments for 12h as in (a), for different time durations as indicated, on cell viability (% of control (C); mean +/− SEM; N=3). c Representative HoloMonitor images (taken every 5 min, 3 days in total) of MDA-MB-231 cell examples treated with EGF, 50%PAM, or EGF+50%PAM. The time points of cell division (Control and EGF group) and apoptosis (PAM and PAM+EGF group) have been selected for display. d Cell area (µm2) and optical thickness avg (µm) of MDA-MB-468 and MDA-MB-231 cells measured by the Holomonitor Hstudio after EGF, PAM or EGF+PAM treatments as in (a) above, for 3 days, versus Control in MDA-MB-468 and MDA-MB-231 cultures. e ATP production rates by MCF-10A, MCF-7, T47D, MDA-MB-468 and MDA-MB-231 (from left to right) upon Control, EGF, PAM or EGF+PAM treatments, for 30 min. OCR and ECAR were measured by Seahorse XF Analyzer to determine rates of glycolysis (glycol) or OXPHOS (mito). f Cell apoptosis determined by PI and Annexin V analysis of MDA-MB-231 cells after 50%PAM, EGF or 50%PAM+EGF treatments (as per (a) above) for 1 h. g Viabilities (% of control (DMEM); mean +/− SEM; N=3) of MCF-7, MDA-MB-468 and MDA-MB-231 cells plated for 24h and pre-treated with 50 ng mL−1 PmAb, 1 nM erlotinib and 17 nM gefitinib for 1 h before being treated with 50%PAM, 50%PAM+EGF, or 50%PAM+EGF plus EGFR inhibitors as above for 12 h. h Viabilities (% of Control; mean +/− SEM; N=3) of MDA-MB-231 variants (SC, KO2 EGFR, KO TGFβR) treated with 50% PAM, 10 ng mL−1 EGF+ 50% PAM, 10 ng mL−1 TGFα+ 50% PAM, 10 ng mL1 TGFβ+ 50% PAM for 12 h. |
Morphology of MDA-MB-231 cells as viewed in the Holomonitor changed from a spindle-like shape to a small and roundish shape after the combined treatment of 50%PAM and EGF for 3 h (Figure 3c). While MDA-MB-231 cell division in the control group was observed at 12h, PAM-induced apoptosis was evident at 8 h and cell apoptosis was initiated at 3 h when cells were exposed to combined EGF and 50% 10PAM treatment. Under the control conditions, the correlation between cell area (µm2) and optical thickness avg (µm) as measured by Holomonitor Hstudio was evident in video imaging (Figure 3d and Figure S3a-b, S4a-b).
The proportion of apoptotic and necrotic cells after 50%PAM and/or EGF treatment for one-hour was determined by Annexin V and PI double staining. Quadrants Q1, Q2, Q3 and Q4 denote necrotic cell (Annexin V−/PI+), late apoptotic cell (Annexin V+/PI+), early apoptotic cell (Annexin V+/PI−) and viable cell (Annexin V−/PI−) regions, respectively (Figure 3f). EGF treatment did not cause any significant change as compared with the control. 50%PAM treatment increased the percentages of both necrosis and apoptosis, while combined 50%PAM + EGF treatment boosted the apoptotic proportion. Among cells receiving combined PAM and EGF exposure, 74% (32%+42%) underwent apoptosis and only 2% underwent necrosis (p<0.0001), which was consistent with Holomonitor observations.
Material and Methods
HoloMonitor imager measurements
SUM159 and MDA-MB-231 cells were plated in 96-well plates at 5,000 cells per well for 24h before real-time monitoring. When the cell density was around 30%, the digital holograms of cells were set up using the HoloMonitor M4 Digital Holography Cytometer (Phase Holographic Imaging PHI AB, Lund, Sweden). The results were calculated using Hstudio M4 software (Phase Holographic Imaging PHI AB, Lund, Sweden)
Acknowledgements
The authors would like to thank Tony Blick for data analysis, Laura Croft and Jaimie Mulders for Holomonitor data collection and analysis,
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Forskningsrapport 2 berör den för män bekanta cancern, prostatacancer. Men hur effektivt, om möjligt ens, bota en redan behandlad prostatacancer som ändå återkommer och dessutom sprider sig. Den frågeställningen hade 5 engelska forskare inför denna studie. Resultatet av sina studier de levererar är hoppingivande för de olycksaliga som återfått denna jävla cancer. Pardon my french !
Men till studien betitlad :
Abstract
Prostate cancer (PCa) is one of the leading causes of cancer-related deaths in men. Localised PCa can be treated effectively, but most patients relapse/progress to more aggressive disease. One possible mechanism underlying this progression is alternative splicing of the androgen receptor, with AR variant 7(ARV7) considered to play a major role. Using viability assays, we confirmed that ARV7-positive PCa cells were less sensitive to treatment with cabazitaxel and an anti-androgen-enzalutamide. Also, using live-holographic imaging, we showed that PCa cells with ARV7 exhibited an increased rate of cell division, proliferation, and motility, which could potentially contribute to a more aggressive phenotype. Furthermore, protein analysis demonstrated that ARV7 knock-down was associated with a decrease in insulin-like growth factor-2 (IGFBP-2) and forkhead box protein A1(FOXA1). This correlation was confirmed in-vivo using PCa tissue samples. Spearman rank correlation analysis showed significant positive associations between ARV7 and IGFBP-2 or FOXA1 in tissue from patients with PCa. This association was not present with the AR. These data suggest an interplay of FOXA1 and IGFBP-2 with ARV7-mediated acquisition of an aggressive prostate cancer phenotype.
Introduction
Worldwide, prostate cancer (PCa) is the most commonly diagnosed cancer in males, contributing to 6.6% of all deaths from cancer. Localised PCa can be treated effectively by radical prostatectomy together with radio- and hormone therapy. Advanced and metastatic PCa is managed using androgen deprivation therapy (ADT), androgen receptor (AR) agonists/antagonists or inhibitors like enzalutamide (MDV-3100) that block translocation of AR to the nucleus therefore preventing ligand binding or with chemotherapy using taxanes, like cabazitaxel. A recent systematic review showed that the treatment sequence for metastatic castration resistant PCa (mCRPC) patients of abiraterone followed by enzalutamide improved progression free survival (PFS) that was quicker and less costly [6,7]. After an initial response, most patients develop resistance and relapse mainly due to alternative mechanisms of AR signalling (AR amplification, alternative splicing, intra-tumoral androgen production and others) and progress to CRPC that accounts for the majority of PCa-related deaths. Alternative splicing of the AR results in generation of AR variants, including AR variant7 (ARV7)(Fig 1Ai). These variants lose their AR binding domains, are constitutively activated, and maintain their nuclear localisation sequence and DNA-binding domain. ARV7 is generated by splicing inclusion of CE3, a cryptic exon found in intron 3 that results in active nuclear ARV7 protein regardless of androgen presence. The expression of ARV7 correlates with androgen-independent cell proliferation and progression to CRPC that has been further reviewed recently.
In addition to the established role of AR in PCa, other key molecules are recognised to play a part. For example, circulating insulin-like growth factor binding protein-2(IGFBP-2) is increased in patients with PCa and positively correlates with stage/grade of the tumour. We also previously reported that IGFBP-2 induces PCa cell growth and contributes to chemoresistance. More recently, we reported a novel correlation between IGFBP-2 and a pioneer transcription factor, FOXA1 in prostate cancer cells, that opens the chromatin for binding of additional transcription factors, such as the AR and its variants.
FOXA1 is considered an oncogene in PCa, involved for example in promoting proliferation and migration of PCa cells. We reported that FOXA1 interacted with the IGFBP-2 gene in normal prostate epithelial cells that negatively regulated IGFBP-2. However, in cancer cells FOXA1 associating with the IGFBP-2 gene was minimal, which suggested a loss of the negative regulation. This study aimed to further understand the role of ARV7 in prostate cancer and to interrogate potential links with and regulation of FOXA1 and IGFBP-2 with a view to identifying novel ways of optimising sensitivity to current treatments.
Materials and methods (urval)
Live cell imaging using phase holographic imaging
Cells were plated on 24-well Lumox plates (Sarstedt, 94.6000.014) and imaged using a Holomonitor M4 live cell imaging system (Phase Holographic Imaging, PHI) inside an incubator (set to 37°C and 5% CO2). Analysis of data was performed using HoloMonitor App Suite 3.5.1 (PHI AB, Lund, Sweden). In depth analysis based on selected position per treatment was performed using HStudio (PHI AB, Lund, Sweden).
Min kommentar
Cancerforskningen fortskrider utan avbrott. Tyvärr får man säga. Det då denna best är svår att besegra.
Men med assistans av det excellenta instrumentet HoloMonitor knappar forskarna in i jakten på denna förbannade amöba. Världens cancerdrabbade ska vara tacksamma för att ett litet företag från Lund, Sverige kan hjälpa forskarna på vägen till en lösning.
Mvh the99
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