Publications by authors named "Maria Alieva"

14 Publications

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Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D.

Nat Biotechnol 2021 Jun 3. Epub 2021 Jun 3.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.
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http://dx.doi.org/10.1038/s41587-021-00926-3DOI Listing
June 2021

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.

J Vis Exp 2019 05 3(147). Epub 2019 May 3.

Cancer Genomics Netherlands, Prinses Máxima Center for Pediatric Oncology.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.
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http://dx.doi.org/10.3791/59278DOI Listing
May 2019

High-resolution 3D imaging of fixed and cleared organoids.

Nat Protoc 2019 06 3;14(6):1756-1771. Epub 2019 May 3.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
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http://dx.doi.org/10.1038/s41596-019-0160-8DOI Listing
June 2019

Intravital imaging of glioma border morphology reveals distinctive cellular dynamics and contribution to tumor cell invasion.

Sci Rep 2019 02 14;9(1):2054. Epub 2019 Feb 14.

Hubrecht Institute-KNAW & University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.

The pathogenesis of glioblastoma (GBM) is characterized by highly invasive behavior allowing dissemination and progression. A conclusive image of the invasive process is not available. The aim of this work was to study invasion dynamics in GBM using an innovative in vivo imaging approach. Primary brain tumor initiating cell lines from IDH-wild type GBM stably expressing H2B-Dendra2 were implanted orthotopically in the brains of SCID mice. Using high-resolution time-lapse intravital imaging, tumor cell migration in the tumor core, border and invasive front was recorded. Tumor cell dynamics at different border configurations were analyzed and multivariate linear modelling of tumor cell spreading was performed. We found tumor border configurations, recapitulating human tumor border morphologies. Not only tumor borders but also the tumor core was composed of highly dynamic cells, with no clear correlation to the ability to spread into the brain. Two types of border configurations contributed to tumor cell spreading through distinct invasion patterns: an invasive margin that executes slow but directed invasion, and a diffuse infiltration margin with fast but less directed movement. By providing a more detailed view on glioma invasion patterns, our study may improve accuracy of prognosis and serve as a basis for personalized therapeutic approaches.
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http://dx.doi.org/10.1038/s41598-019-38625-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375955PMC
February 2019

Potential impact of invasive surgical procedures on primary tumor growth and metastasis.

Clin Exp Metastasis 2018 04 4;35(4):319-331. Epub 2018 May 4.

Department of Neurology & Neurosurgery, Brain Center Rudolf Magnus, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

Surgical procedures such as tumor resection and biopsy are still the gold standard for diagnosis and (determination of) treatment of solid tumors, and are prognostically beneficial for patients. However, growing evidence suggests that even a minor surgical trauma can influence several (patho) physiological processes that might promote postoperative metastatic spread and tumor recurrence. Local effects include tumor seeding and a wound healing response that can promote tumor cell migration, proliferation, differentiation, extracellular matrix remodeling, angiogenesis and extravasation. In addition, local and systemic immunosuppression impairs antitumor immunity and contributes to tumor cell survival. Surgical manipulation of the tumor can result in cancer cell release into the circulation, thus increasing the chance of tumor cell dissemination. To prevent these undesired effects of surgical interventions, therapeutic strategies targeting immune response exacerbation or alteration have been proposed. This review summarizes the current literature regarding these local, systemic and secondary site effects of surgical interventions on tumor progression and dissemination, and discusses studies that aimed to identify potential therapeutic approaches to prevent these effects in order to further increase the clinical benefit from surgical procedures.
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http://dx.doi.org/10.1007/s10585-018-9896-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063335PMC
April 2018

Preventing inflammation inhibits biopsy-mediated changes in tumor cell behavior.

Sci Rep 2017 08 8;7(1):7529. Epub 2017 Aug 8.

Cancer Genomics Netherlands, Hubrecht Institute-KNAW & University Medical Center Utrecht, Uppsalalaan 8, 3584CT, Utrecht, The Netherlands.

Although biopsies and tumor resection are prognostically beneficial for glioblastomas (GBM), potential negative effects have also been suggested. Here, using retrospective study of patients and intravital imaging of mice, we identify some of these negative aspects, including stimulation of proliferation and migration of non-resected tumor cells, and provide a strategy to prevent these adverse effects. By repeated high-resolution intravital microscopy, we show that biopsy-like injury in GBM induces migration and proliferation of tumor cells through chemokine (C-C motif) ligand 2 (CCL-2)-dependent recruitment of macrophages. Blocking macrophage recruitment or administrating dexamethasone, a commonly used glucocorticoid to prevent brain edema in GBM patients, suppressed the observed inflammatory response and subsequent tumor growth upon biopsy both in mice and in multifocal GBM patients. Taken together, our study suggests that inhibiting CCL-2-dependent recruitment of macrophages may further increase the clinical benefits from surgical and biopsy procedures.
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http://dx.doi.org/10.1038/s41598-017-07660-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548904PMC
August 2017

Imaging windows for long-term intravital imaging: General overview and technical insights.

Intravital 2014 11;3(2):e29917. Epub 2014 Aug 11.

Cancer Genomics Netherlands; Hubrecht Institute-KNAW and University Medical Centre Utrecht; CT Utrecht, The Netherlands.

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.
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http://dx.doi.org/10.4161/intv.29917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312719PMC
August 2014

Fast-proliferating adipose tissue mesenchymal-stromal-like cells for therapy.

Stem Cells Dev 2014 Dec 13;23(23):2908-20. Epub 2014 Aug 13.

1 Human DNA Variability Department, GENYO-Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government) , PTS Granada, Granada, Spain .

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization.
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http://dx.doi.org/10.1089/scd.2014.0231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235596PMC
December 2014

Bioluminescence imaging of cardiomyogenic and vascular differentiation of cardiac and subcutaneous adipose tissue-derived progenitor cells in fibrin patches in a myocardium infarct model.

Int J Cardiol 2013 Nov 6;169(4):288-95. Epub 2013 Oct 6.

Institute for Advanced Chemistry of Catalonia, Barcelona 08034, Spain; Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain.

Background: Adipose tissue-derived progenitor cells (ATDPCs) isolated from human cardiac adipose tissue are useful for cardiac regeneration in rodent models. These cells do not express cardiac troponin I (cTnI) and only express low levels of PECAM-1 when cultured under standard conditions. The purpose of the present study was to evaluate changes in cTnI and PECAM-1 gene expression in cardiac ATDPCs following their delivery through a fibrin patch to a murine model of myocardial infarction using a non-invasive bioluminescence imaging procedure.

Methods And Results: Cardiac and subcutaneous ATDPCs were doubly transduced with lentiviral vectors for the expression of chimerical bioluminescent-fluorescent reporters driven by constitutively active and tissue-specific promoters (cardiac and endothelial for cTnI and PECAM-1, respectively). Labeled cells mixed with fibrin were applied as a 3-D fibrin patch over the infarcted tissue. Both cell types exhibited de novo expression of cTnI, though the levels were remarkably higher in cardiac ATDPCs. Endothelial differentiation was similar in both ATDPCs, though cardiac cells induced vascularization more effectively. The imaging results were corroborated by standard techniques, validating the use of bioluminescence imaging for in vivo analysis of tissue repair strategies. Accordingly, ATDPC treatment translated into detectable functional and morphological improvements in heart function.

Conclusions: Both ATDPCs differentiate to the endothelial lineage at a similar level, cardiac ATDPCs differentiated more readily to the cardiomyogenic lineage than subcutaneous ATDPCs. Non-invasive bioluminescence imaging was a useful tool for real time monitoring of gene expression changes in implanted ATDPCs that could facilitate the development of procedures for tissue repair.
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http://dx.doi.org/10.1016/j.ijcard.2013.09.013DOI Listing
November 2013

Endothelial differentiation of adipose tissue-derived mesenchymal stromal cells in glioma tumors: implications for cell-based therapy.

Mol Ther 2013 Sep 13;21(9):1758-66. Epub 2013 Jun 13.

Instituto de Química Avanzada de Cataluña CSIC, CIBER-BBN, Barcelona, Spain.

Multipotent human adipose tissue mesenchymal stromal cells (hAMSCs) are promising therapy vehicles with tumor-homing capacity that can be easily modified to deliver cytotoxicity activating systems in the proximity of tumors. In a previous work, we observed that hAMSCs are very effective delivering cytotoxicity to glioma tumors. However, these results were difficult to reconcile with the relatively few hAMSCs surviving implantation. We use a bioluminescence imaging (BLI) platform to analyze the behavior of bioluminescent hAMSCs expressing HSV-tTK in a U87 glioma model and gain insight into the therapeutic mechanisms. Tumor-implanted hAMSCs express the endothelial marker PECAM1(CD31), integrate in tumor vessels and associate with CD133-expressing glioma stem cells (GSC). Inhibition of endothelial lineage differentiation in hAMSCs by Notch1 shRNA had no effect on their tumor homing and growth-promoting capacity but abolished the association of hAMSCs with tumor vessels and CD133+ tumor cells and significantly reduced their tumor-killing capacity. The current strategy allowed the study of tumor/stroma interactions, showed that tumor promotion and tumor-killing capacities of hAMSCs are based on different mechanisms. Our data strongly suggest that the therapeutic effectiveness of hAMSCs results from their association with special tumor vascular structures that also contain GSCs.
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http://dx.doi.org/10.1038/mt.2013.145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776633PMC
September 2013

In vivo bioluminescence imaging of cell differentiation in biomaterials: a platform for scaffold development.

Tissue Eng Part A 2013 Mar 21;19(5-6):593-603. Epub 2012 Dec 21.

Cardiovascular Research Center CSIC-ICCC, CIBER-BBN, Barcelona, Spain.

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.
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http://dx.doi.org/10.1089/ten.TEA.2012.0073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568970PMC
March 2013

Calcium phosphate glass improves angiogenesis capacity of poly(lactic acid) scaffolds and stimulates differentiation of adipose tissue-derived mesenchymal stromal cells to the endothelial lineage.

J Biomed Mater Res A 2013 Apr 8;101(4):932-41. Epub 2012 Sep 8.

Cardiovascular Research Center (CSIC-ICCC-UAB), Barcelona, Spain.

The angiogenic capacity of a new biomaterial composite of poly(lactic acid) and calcium phosphate glass (PLA/CaP) was analyzed by noninvasive bioluminescence imaging (BLI) and histological procedures. Human adipose tissue-derived mesenchymal stromal cells expressing cytomegalovirus (CMV) promoter regulated Photinus pyralis luciferase (hAMSC-PLuc) grew up to 30 times the initial cell load, in vitro, when seeded in PLA/CaP scaffolds, but suffered an initial growth crisis followed by recovery when the scaffolds were subcutaneously implanted in SCID mice. To analyze changes in gene expression, hAMSC-PLuc cells were double labeled with a CMV promoter regulated Renilla reniformis luciferase and a Photinus pyralis luciferase reporter regulated by either the PECAM promoter or a hypoxia response element (HRE) artificial promoter and seeded in PLA/CaP and PLA scaffolds implanted in SCID mice. Analysis by BLI showed that hAMSCs in scaffolds were induced to differentiate to the endothelial lineage and did this faster in PLA/CaP than in PLA scaffolds. Endothelial differentiation correlated with a decrease in the activity of HRE regulated luciferase expression, indicative of a reduction of hypoxia. Histological analysis showed that PLA/CaP scaffolds were colonized by a functional host vascular system. Moreover, colonization by isolectin B(4) positive host cells was more effective in PLA/CaP than in PLA scaffolds, corroborating BLI results.
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http://dx.doi.org/10.1002/jbm.a.34391DOI Listing
April 2013

Glioblastoma therapy with cytotoxic mesenchymal stromal cells optimized by bioluminescence imaging of tumor and therapeutic cell response.

PLoS One 2012 17;7(4):e35148. Epub 2012 Apr 17.

Cardiovascular Research Center, CSIC-ICCC, CIBER-BBN, Barcelona, Spain.

Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 10(4) relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035148PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328467PMC
November 2012

Adult-onset hypothyroidism enhances fear memory and upregulates mineralocorticoid and glucocorticoid receptors in the amygdala.

PLoS One 2011 24;6(10):e26582. Epub 2011 Oct 24.

Department of Nervous System and Endocrine Pathophysiology, Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain.

Hypothyroidism is the most common hormonal disease in adults, which is frequently accompanied by learning and memory impairments and emotional disorders. However, the deleterious effects of thyroid hormones deficiency on emotional memory are poorly understood and often underestimated. To evaluate the consequences of hypothyroidism on emotional learning and memory, we have performed a classical Pavlovian fear conditioning paradigm in euthyroid and adult-thyroidectomized Wistar rats. In this experimental model, learning acquisition was not impaired, fear memory was enhanced, memory extinction was delayed and spontaneous recovery of fear memory was exacerbated in hypothyroid rats. The potentiation of emotional memory under hypothyroidism was associated with an increase of corticosterone release after fear conditioning and with higher expression of glucocorticoid and mineralocorticoid receptors in the lateral and basolateral nuclei of the amygdala, nuclei that are critically involved in the circuitry of fear memory. Our results demonstrate for the first time that adult-onset hypothyroidism potentiates fear memory and also increases vulnerability to develop emotional memories. Furthermore, our findings suggest that enhanced corticosterone signaling in the amygdala is involved in the pathophysiological mechanisms of fear memory potentiation. Therefore, we recommend evaluating whether inappropriate regulation of fear in patients with post-traumatic stress and other mental disorders is associated with abnormal levels of thyroid hormones, especially those patients refractory to treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026582PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3200331PMC
February 2012
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