Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is ...a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (Unk
) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in Unk
mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that Unk
mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.
Prion diseases typically involve brain deposition of abnormally folded prion protein, which is associated with activated glia and increased cytokine production. Cyclophilin A (CypA) is a ubiquitous ...protein with peptidyl prolyl cis-trans isomerase activity, which regulates protein folding, and can be secreted by cells in response to inflammatory stimuli. On the basis of in vitro studies, CypA was proposed to mediate glial activation during prion infection. To investigate the role of CypA in vivo, we inoculated CypA+/+, CypA+/− and CypA−/− mice with the RML prion strain, and recorded the time to onset of neurological signs and to terminal disease, and the astrocyte and microglia response at presymptomatic and symptomatic stages. Time to onset of disease and survival were significantly shorter in CypA-deficient mice than CypA-expressing controls. CypA-deficient mice had significantly greater microglial activation in the presymptomatic stage, and analysis of anti- and pro-inflammatory microglial markers indicated a shift towards a pro-inflammatory phenotype. There was no difference in astrocyte activation. This suggests that CypA contributes to dampening the pro-inflammatory microglial response during the early stage of RML-induced prion disease.
•Prion disease is hastened in Cyclophillin A (CypA)-deficient mice•CypA deficiency does not influence PrPScaccumulation•CypA deficiency does not exacerbate neurodegeneration•CypA deficiency promotes proinflammatory microglia activation
Fatal familial insomnia (FFI) is a dominantly inherited prion disease linked to the D178N mutation in the gene encoding the prion protein (PrP). Symptoms, including insomnia, memory loss and motor ...abnormalities, appear around 50 years of age, leading to death within two years. No treatment is available. A ten-year clinical trial of doxycycline (doxy) is under way in healthy individuals at risk of FFI to test whether presymptomatic doxy prevents or delays the onset of disease.
To assess the drug's effect in a tractable disease model, we used Tg(FFI-26) mice, which accumulate aggregated and protease-resistant PrP in their brains and develop a fatal neurological illness highly reminiscent of FFI. Mice were treated daily with 10 mg/kg doxy starting from a presymptomatic stage for twenty weeks. Doxy rescued memory deficits and restored circadian motor rhythmicity in Tg(FFI-26) mice. However, it did not prevent the onset and progression of motor dysfunction, clinical signs and progression to terminal disease. Doxy did not change the amount of aggregated and protease-resistant PrP, but reduced microglial activation in the hippocampus. Presymptomatic doxy treatment rescues cognitive impairment and the motor correlates of sleep dysfunction in Tg(FFI-26) mice but does not prevent fatal disease.
•Early doxycycline treatment improves memory in fatal familial insomnia mice.•Doxycycline restores normal circadian motor rhythmicity.•Doxycycline reduces microglial activation in the hippocampus.•Doxycycline does not reduce mutant prion protein aggregation or protease resistance.•Doxycycline does not delay disease onset or prolong survival of the mice.
Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype ...is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ductal carcinoma in-situ (DCIS) accounts for 20-25% of all new breast cancer diagnoses. DCIS has an uncertain risk of progression to invasive breast cancer and a lack of predictive biomarkers may ...result in relatively high levels (~ 75%) of overtreatment. To identify unique prognostic biomarkers of invasive progression, crystallographic and chemical features of DCIS microcalcifications have been explored. Samples from patients with at least 5-years of follow up and no known recurrence (174 calcifications in 67 patients) or ipsilateral invasive breast cancer recurrence (179 microcalcifications in 57 patients) were studied. Significant differences were noted between the two groups including whitlockite relative mass, hydroxyapatite and whitlockite crystal maturity and, elementally, sodium to calcium ion ratio. A preliminary predictive model for DCIS to invasive cancer progression was developed from these parameters with an AUC of 0.797. These results provide insights into the differing DCIS tissue microenvironments, and how these impact microcalcification formation.
The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies ...have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described. These TAMs can recruit and prolong the lifespan of neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.
The prognostic value of cytonuclear grade in ductal carcinoma in situ (DCIS) is debated, partly due to high interobserver variability and the use of multiple guidelines. The aim of this study was to ...evaluate interobserver agreement in grading DCIS between Dutch, British, and American pathologists. Haematoxylin and eosin‐stained slides of 425 women with primary DCIS were independently reviewed by nine breast pathologists based in the Netherlands, the UK, and the USA. Chance‐corrected kappa (κma) for association between pathologists was calculated based on a generalised linear mixed model using the ordinal package in R. Overall κma for grade of DCIS (low, intermediate, or high) was estimated to be 0.50 (95% confidence interval CI 0.44–0.56), indicating a moderate association between pathologists. When the model was adjusted for national guidelines, the association for grade did not change (κma = 0.53; 95% CI 0.48–0.57); subgroup analysis for pathologists using the UK pathology guidelines only had significantly higher association (κma = 0.58; 95% CI 0.56–0.61). To assess if concordance of grading relates to the expression of the oestrogen receptor (ER) and HER2, archived immunohistochemistry was analysed on a subgroup (n = 106). This showed that non‐high grade according to the majority opinion was associated with ER positivity and HER2 negativity (100 and 89% of non‐high grade cases, respectively). In conclusion, DCIS grade showed only moderate association using whole slide images scored by nine breast pathologists. As therapeutic decisions and inclusion in ongoing clinical trials are guided by DCIS grade, there is a pressing need to reduce interobserver variability in grading. ER and HER2 might be supportive to prevent the accidental and unwanted inclusion of high‐grade DCIS in such trials.
Abstract
Introduction: Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer. The uncertain trajectory of DCIS, to progress to invasive disease or to remain in situ, ...currently drives treatment, despite lack of proven benefit. Therefore, understanding the molecular features of the DCIS trajectory may prevent overtreatment of this disease. Breast calcifications are a common feature in DCIS and are seen mammographically in over 80% of cases. Calcifications have been largely characterised based only on x-ray morphology; their chemical composition, their association with the surrounding soft tissue and role in DCIS and invasive breast cancer biology is largely unexplored. In this regard, a bio-photonic approach, based on infrared (IR) and Raman spectroscopy in combination with machine learning, was used to study DCIS by probing the chemical composition of calcifications and the surrounding soft tissue in breast lesions. The main aim of the work is to identify molecular compositional changes in calcifications and in soft tissue that potentially accompany or drive the progression of DCIS to invasive breast cancer, or indicates a stable DCIS phenotype. Methods: Serial tissue sections from 303 patients with (i) ‘pure DCIS’ (DCIS without recurrence) (n=158), (ii) ‘DCIS with invasive recurrence’ (DCIS from a patient who subsequently was known to develop invasive disease) (n=123) and (iii) ‘DCIS plus invasive cancer contemporaneously’ (n=22), were measured using mid-IR imaging and Raman mapping. The same calcifications and soft tissue regions from specific DCIS ducts were targeted across the techniques on the serial sections. Spectral images were analysed using cluster analysis followed by unsupervised and supervised machine learning classification models to identify spectral features associated with the progression of DCIS to invasive breast cancer. Results: Segmentation of IR and Raman spectral images based on cluster analysis identified important histopathological features including calcifications, epithelium, necrotic areas, connective tissue and stroma based on the spectral heterogeneity. Based on analysis of Raman calcification data from 145 patients with (i) ‘pure DCIS’ (n=90) and (ii) ‘DCIS with invasive recurrence’ (n=55), an area under the receiver operating characteristic (AUROC) mean value of 85% was obtained in distinguish pure DCIS from DCIS that later recurred as invasive cancer. The calcification features appeared to indicate pathology specific changes in phosphate and carbonate content and appearance of magnesium whitlockite. Similar analysis of the surrounding soft tissue spectral features showed an AUROC mean value of 76%, which showed changes in protein secondary structure and content, particularly in the necrotic regions surrounding calcifications. In addition, classification models are being developed and refined from the IR spectral data, the initial results of which have shown an AUROC value of only 54% from the same patients’ data. Perspectives: It is anticipated that the current novel approaches allowing label-free measurement of calcifications and soft tissue will provide important cues in understanding DCIS prognosis and could be a promising way forward in determining DCIS management. Current and future efforts include identification of specific discriminatory spectral features for molecular and pathological correlation. Acknowledgments: This work was supported by Cancer Research UK and by KWF Kankerbestrijding (ref. C38317/A24043).
Citation Format: Jayakrupakar Nallala, Doriana Calabrese, Sarah Gosling, Allison Hall, Sarah Pinder, Ihssane Bouybayoune, Lorraine King, Jeffrey Marks, Esther Lips, Thomas Lynch, Donna Pinto, Jelle Wesseling, Shelley Hwang, Keith Rogers, Nick Stone, on behalf of the Grand Challenge PRECISION consortium. Predicting DCIS prognosis using infrared and raman spectroscopy of breast calcifications and soft-tissue microstructure abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-07.
Abstract
Despite the many pro-tumoral functions that have been described for the various stromal populations in cancer, it is becoming apparent that these cells are not working alone, but instead in ...concert with one another as part of a wider network of cross-communication to facilitate disease progression. Identifying non-redundant signaling pathways within the stromal network is desirable, as therapeutically targeting these signals could result in an unraveling of the stromal support network upon which the tumor cells rely. Tumor associated macrophages (TAMs) have been implicated in a variety of pro-tumoral processes and are polarized by signals in the tumor microenvironment (TME) to adopt a range of functionally distinct subsets to perform these tasks. Single-cell RNA-sequencing of the F4/80+ TAMs from the MMTV-PyMT murine model of mammary adenocarcinoma identified one subset selectively expressing Lyve-1 and adopting a CD206hiMHCIIlo phenotype. This population was identified to spatially reside in the niche proximal to blood vasculature within the tumor. We demonstrate that selective depletion of the Lyve-1+ TAM population using liposome-based approaches controlled tumor growth and was associated with a loss of perivascular alpha smooth muscle actin (αSMA)-expressing stromal cells. The αSMA+ stromal cells were characterized ex vivo and expressed a surface phenotype associated with pericytes (CD90+CD34-NG2+), highlighting a possible role for Lyve-1+ TAMs in maintaining the population. To exclude a role of Lyve-1+ TAMs in recruiting a pericyte-progenitor cell to the tumor, we selectively photo-labelled the TME using MMTV-PyMT mice ubiquitously expressing the photo-convertible green/red Kaede protein. Labelling the TME Kaede-red through exposure to ultraviolet light, demonstrated that pericytes and CD90+ mesenchymal cells more broadly, were not recruited from a peripheral site but instead suggested these populations must rely on local proliferation to maintain their prevalence in the TME. Indeed, we demonstrate through Edu incorporation, that loss of Lyve-1+ TAMs from the perivascular niche renders pericytes senescent. Using computational approaches, we characterized the signaling cross-talk in the perivascular niche between Lyve-1+ TAMs, pericytes and endothelial cells and identified a specific interaction involving platelet derived growth factor-CC (PDGF-CC) expression by Lyve-1+ TAMs and PDGF-receptor alpha (PDGFRα) on the pericytes. Using blocking antibodies to PDGF-CC in tumor bearing mice we demonstrate that blocking this axis of stromal crosstalk can render pericytes senescent. These data highlight that local expansion of pericytes in cancer is not an autonomous event, nor regulated by the endothelium alone, but tightly regulated by the perivascular Lyve-1+ TAM population, which dictates the success of angiogenesis in cancer.
Citation Format: James W. Opzoomer, Joanne E. Anstee, Isaac Dean, Emily J. Hill, Ihssane Bouybayoune, Jonathan Caron, Tamara Muliaditan, Peter Gordon, Rosamond Nuamah, Sarah E. Pinder, Tony Ng, Francesco Dazzi, Shahram Kordasti, David R. Withers, Toby Lawrence, James N. Arnold. Lyve-1 expressing macrophages direct the expansion of pericytes within the perivascular niche to support angiogenesis in cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 113.
Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their ...morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 μm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.
We aim to develop a protocol for efficiently analysing breast microcalcifications and their microenvironment in detail by combining O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 μm).
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