scholarly journals Generation of in vivo labelling tumour cell lines

2019 ◽  
Author(s):  
Luigi Ombrato ◽  
Ilaria Malanchi
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Breast Tumour ◽  
Tumour Microenvironment ◽  
Tumour Cells ◽  
In Vivo Labelling ◽  
4T1 Cells ◽  
Novel Strategy

Abstract The characterization of the tumour microenvironment is highly desirable in order to get a better understating on how the tumour cells exploit their neighbours to support their own growth. In Ombrato et al., Nature 2019 we describe a novel strategy that allows 4T1 breast tumour cells to label their lung metastatic niche in vivo. The labelled cells surrounding the tumour cells can be freshly isolated within the all tissue and used for ex vivo assays. Here, we describe how we generated Labelling-4T1 cells. The same procedure can be used to generate labelling cells from other cell lines, but it might require optimization. This protocol is a suggested guide.

Characterization of stem cell–like cancer cells in immune-competent mice

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3906-3912 ◽  
Author(s):  
Jorg A. Kruger ◽  
Charles D. Kaplan ◽  
Yunping Luo ◽  
He Zhou ◽  
Dorothy Markowitz ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Side Population ◽  
Carcinoma Cell Lines ◽  
Tumorigenic Potential ◽  
4T1 Cells

AbstractRecently, the cancer stem cell hypothesis has gained significant recognition as the descriptor of tumorigenesis. Although previous studies relied on transplanting human or rat tumor cells into immunecompromised mice, our study used the Hoechst 33342 dye–based side population (SP) technique to isolate and transplant stem cell–like cancer cells (SCLCCs) from the 4T1 and NXS2 murine carcinoma cell lines into the immune-competent microenvironment of syngeneic mice. 4T1 cells displayed an SP of 2% with a Sca-1highc-Kit–CD45– phenotype, whereas NXS2 cells contained an SP of 0.2% with a Sca-1highCD24highc-Kit–CD45–GD high2 phenotype. Reverse transcription–polymerase chain reaction (RT-PCR) further revealed up-regulation in SP cells of ABCG2, Sca-1, Wnt-1, and TGF-β2. Additionally, 4T1 and NXS2 SP cells exhibited increased resistance to chemotherapy, and 4T1 SP cells also showed an increased ability to efflux doxorubicin, which correlated with a selective increase in the percentage of SP cells found in the tumors of doxorubicin-treated mice. Most importantly, SP cells showed a markedly higher repopulation and tumorigenic potential in vivo, which correlated with an increased number of cells in the SP compartment of SP-derived tumors. Taken together, these results show that we successfully characterized SCLCCs from 2 murine carcinoma cell lines in the immune-competent microenvironment of syngeneic mice.

scholarly journals Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1791
Author(s):  
Rosa Scala ◽  
Fatima Maqoud ◽  
Nicola Zizzo ◽  
Giuseppe Passantino ◽  
Antonietta Mele ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Katp Channel ◽  
Ex Vivo ◽  
Channel Modulation ◽  
Flexor Digitorum Brevis ◽  
Inflammatory Edema ◽  
Flexor Digitorum ◽  
Channel Currents

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10−5 M in SUR2wt/AV and 8.6 ± 0.4 × 10−6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] “knock-in” mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

scholarly journals Characterization of Intrinsically Radiolabeled Poly(lactic-co-glycolic acid) Nanoparticles for ex Vivo Autologous Cell Labeling and in Vivo Tracking

2021 ◽  
Author(s):  
Massis Krekorian ◽  
Gerwin G. W. Sandker ◽  
Kimberley R. G. Cortenbach ◽  
Oya Tagit ◽  
N. Koen van Riessen ◽  
...  
Keyword(s):  
Glycolic Acid ◽  
Ex Vivo ◽  
Cell Labeling ◽  
Autologous Cell

About Puncture Testing Applied for Mechanical Characterization of Fetal Membranes

2014 ◽  
Vol 136 (11) ◽  
Author(s):  
Wilfried Bürzle ◽  
Edoardo Mazza ◽  
John J. Moore
Keyword(s):  
Deformation Behavior ◽  
Mechanical Characterization ◽  
Ex Vivo ◽  
Fetal Membrane ◽  
Membrane Rupture ◽  
Resistance To Deformation ◽  
Ex Vivo Testing ◽  
Insight Into

Puncture testing has been applied in several studies for the mechanical characterization of human fetal membrane (FM) tissue, and significant knowledge has been gained from these investigations. When comparing results of mechanical testing (puncture, inflation, and uniaxial tension), we have observed discrepancies in the rupture sequence of FM tissue and significant differences in the deformation behavior. This study was undertaken to clarify these discrepancies. Puncture experiments on FM samples were performed to reproduce previous findings, and numerical simulations were carried out to rationalize particular aspects of membrane failure. The results demonstrate that both rupture sequence and resistance to deformation depend on the samples' fixation. Soft fixation leads to slippage in the clamping, which reduces mechanical loading of the amnion layer and results in chorion rupturing first. Conversely, the stiffer, stronger, and less extensible amnion layer fails first if tight fixation is used. The results provide a novel insight into the interpretation of ex vivo testing as well as in vivo membrane rupture.

In vivo characterization of the transitional bronchioles by aerosol-derived airway morphometry

1999 ◽  
Vol 87 (3) ◽  
pp. 920-927 ◽  
Author(s):  
Kirby L. Zeman ◽  
Gerhard Scheuch ◽  
Knut Sommerer ◽  
James S. Brown ◽  
William D. Bennett
Keyword(s):  
Ex Vivo ◽  
Normal Subjects ◽  
Characteristic Line ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Lung Capacity ◽  
Single Breath ◽  
In Vivo Characterization

Effective airway dimensions (EADs) were determined in vivo by aerosol-derived airway morphometry as a function of volumetric lung depth (VLD) to identify and characterize, noninvasively, the caliber of the transitional bronchiole region of the human lung and to compare the EADs by age, gender, and disease. By logarithmically plotting EAD vs. VLD, two distinct regions of the lung emerged that were identified by characteristic line slopes. The intersection of proximal and distal segments was defined as VLDtransand associated EADtrans. In our normal subjects ( n = 20), VLDtrans [345 ± 83 (SD) ml] correlated significantly with anatomic dead space (224 ± 34 ml) and end of phase II of single-breath nitrogen washout (360 ± 53 ml). The corresponding EADtranswas 0.42 ± 0.07 mm, in agreement with other ex vivo measurements of the transitional bronchioles. VLDtrans was smaller (216 ± 64 ml) and EADtrans was larger (0.83 ± 0.04 mm) in our patients with chronic obstructive pulmonary disease ( n = 13). VLDtrans increased with age for children (age 8–18 yr; P = 0.006, n = 26) and with total lung capacity for age 8–81 yr ( P < 0.001, n = 61). This study extends the usefulness of aerosol-derived airway morphometry to in vivo measurements of the transitional bronchioles.

scholarly journals Ex Vivo Isolation and Characterization of Cd4+Cd25+ T Cells with Regulatory Properties from Human Blood

2001 ◽  
Vol 193 (11) ◽  
pp. 1303-1310 ◽  
Author(s):  
Detlef Dieckmann ◽  
Heidi Plottner ◽  
Susanne Berchtold ◽  
Thomas Berger ◽  
Gerold Schuler
Keyword(s):  
T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Regulatory Cells ◽  
Suppressor T Cells ◽  
Isolation And Characterization ◽  
Regulatory Properties

It has been known for years that rodents harbor a unique population of CD4+CD25+ “professional” regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4+CD25+CD45RO+ T cells (mean 6% of CD4+ T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4+CD25+ T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte–associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4+CD25+ T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4+CD25+ T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4+ and CD8+ T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4+CD25+ T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.

scholarly journals SCIDOT-05. DEVELOPING VARIABLE LYMPHOCYTE RECEPTORS THAT TARGET PATHOLOGICALLY EXPOSED NEURAL ECM TO TREAT GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi273-vi273
Author(s):  
Benjamin J Umlauf ◽  
Paul A Clark ◽  
Jason M Lajoie ◽  
Julia V Georgieva ◽  
Samantha Bremner ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
High Specificity ◽  
Recognition System ◽  
Therapeutic Benefit ◽  
Immunogenic Peptide ◽  
Immunogenic Peptides ◽  
Novel Strategy ◽  
Lymphocyte Receptors

Abstract INTRODUCTION The median survival of gliobastoma (GBM) patients remains less than two years despite aggressive treatments. Current targeted GBM therapies demonstrate initial therapeutic benefit; however, patients relapse due to therapeutic resistance and failure to eliminate GBM cells at the invasive margin. Therefore, we propose a two-prong approach: first, target pathologic disruption of the blood brain barrier (BBB) via exposure of neural ECM rather than disease markers to overcome therapy-resistant GBM; and second, designing therapeutic payloads that extracellularly spread throughout the tumor volume. METHODS Variable Lymphocyte Receptors (VLRs, a lamprey-derived antigen recognition system) were identified with high specificity for neural ECM. Candidate VLRs underwent further refinement using ex vivo tissue staining. Utilizing pathologic disruption of BBB as an approach for targeting GBM was confirmed in vivo with intracranial murine glioblastoma models. Finally, an immunogenic peptide was attached via a cleavable linker to the neural ECM binding VLRs for conditional release extracellularly to spread throughout the tumor. RESULTS The lead neural ECM-binding VLR candidate, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to other tissues. P1C10 demonstrates nanomolar affinity for neural ECM, and preferentially accumulates in intracranial GL261 and U87 murine GBM models. Finally, P1C10-targeted doxorubicin-loaded liposomes significant increased survival of mice with intracranial GBM. In additional studies, treating murine GBM models with a P1C10 VLR linked to an immunogenic peptide reduced GBM proliferation and increased infiltration of cytotoxic T cells. CONCLUSIONS We present proof-of-concept demonstration for targeting intracranial GBM via neural ECM exposed at pathological BBB disrupted sites. Additionally, P1C10 neural ECM-targeting VLR delivers chemotherapy-loaded nanoparticles and immunogenic peptides designed to spread extracellularly throughout the tumor. Thus, this novel strategy links a physiological ECM targeting scheme with extracellular-released therapeutics to treat primary GBM, and has potential for delivering therapies to other CNS diseases with pathological BBB.

Ex vivo and in vivo characterization of cold preserved cartilage for cell transplantation

2016 ◽  
Vol 17 (4) ◽  
pp. 721-734 ◽  
Author(s):  
Wichaya Sriuttha ◽  
Nantawat Uttamo ◽  
Apisek Kongkaew ◽  
Jongkolnee Settakorn ◽  
Suchanan Rattanasalee ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Ex Vivo ◽  
In Vivo Characterization

scholarly journals A novel strategy for the characterization of glaucocalyxin A metabolites in vivo and in vitro by UHPLC-Q-TOF-MS based on DDA and DIA data acquisitions

RSC Advances ◽  
2020 ◽  
Vol 10 (18) ◽  
pp. 10431-10446
Author(s):  
Wenjing Sun ◽  
Yiran Jin ◽  
Shuai Guan ◽  
Mengxin Yang ◽  
Miaoting Zhang ◽  
...  
Keyword(s):  
Process Flow ◽  
Experimental Process ◽  
Novel Strategy ◽  
Tof Ms

The experimental process flow.

scholarly journals P01.16 Effects of the STAT3 inhibitors on senescent tumour cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A16.1-A16
Author(s):  
O Sapega ◽  
R Mikyskova ◽  
K Musilek ◽  
J Bieblova ◽  
Z Hodny ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Czech Republic ◽  
Cell Lines ◽  
Cellular Senescence ◽  
Tumour Cells ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway ◽  
Stat3 Phosphorylation

BackgroundCellular senescence is the process of cell proliferation arrest. Premature cellular senescence can be induced by chemotherapy, irradiation and, under certain circumstances, by cytokines. Senescent cells produce a number of secreted proteins and growth factors that may either stimulate or inhibit cell proliferation. One of the major cytokines that play role in regulation of cellular senescence is IL-6. IL-6/STAT3 signaling pathway represent decisive regulatory factors in cellular senescence. The objective of this study was to compare the effects of the STAT3 inhibitors on senescent and proliferative tumour cells. Further, the therapeutic potential of the STAT3 inhibitors was evaluated using murine tumour models.Materials and MethodsIn vitro, as well as in vivo experiments were performed using TC-1 (model for HPV16-associated tumours) TRAMP-C2 (prostate cancer) cell lines. C57Bl/6NCrl mice, 7–8 weeks old, were obtained from Velaz (Prague, Czech Republic). Experimental protocols were approved by the Institutional Animal Care Committee of the Institute of Molecular Genetics (Prague, Czech Republic). STAT3 inhibitors, namely STATTIC, BP-102 (synthesised at the University of Hradec Kralove) and their derivatives were tested for their effects on tumour cells, such as cytotoxicity, ability to inhibit STAT3 phosphorylation, cell proliferation and tumour growth in syngeneic mice.ResultsWe have previously demonstrated that docetaxel-induced senescence in the TC-1 and TRAMP-C2 murine tumour cell lines, which was proved by in vitro (detection of increased p21 expression, positive beta-galactosidase staining, and the typical SASP capable to induce ‘bystander’ senescence), and in vivo experiments, using C57BL/6 mice [1]. Both TC-1 and TRAMP-C2 cells displayed elevated IL-6 secretion and activated STAT3 signaling pathway. Therefore, we tested efficacy of the STAT3 inhibitors on these cell lines. Cytotoxic and STAT3 phosphorylation inhibitory effects of the inhibitors were observed in both proliferating and senescent cells. Antitumor effects of selected inhibitors were evaluated.ConclusionsCollectively, STAT3 is an attractive target for therapeutic approaches in cancer treatment and we can assume that inhibition of the STAT3 pathway can be used for elimination of the pernicious effects of the senescent cells.ReferenceSimova J, Sapega O, Imrichova T, Stepanek I, Kyjacova L, Mikyskova R, Indrova M, Bieblova J, Bubenik J, Bartek J, et al: Tumor growth accelerated by chemotherapy-induced senescent cells is suppressed by treatment with IL-12 producing cellular vaccines. Oncotarget7: 54952–54964, 2016. This work was supported by the research grant No. NV18-05-00562 provided by the Grant Agency of the Ministry of Health of the Czech Republic.Disclosure InformationO. Sapega: None. R. Mikyskova: None. K. Musilek: None. J. Bieblova: None. Z. Hodny: None. M. Reinis: None.

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