Cancer Biology


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0 Q&A 690 Views Jan 20, 2023

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient–derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders.


Graphical abstract



Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation

0 Q&A 2449 Views Apr 20, 2022

Three-dimensional culture of human normal colorectal epithelium and cancer tissue as organoids and tumoroids has transformed the study of diseases of the large intestine. A widely used strategy for generating patient-derived colorectal organoids and tumoroids involves embedding cells in domes of extracellular matrix (ECM). Despite its success, dome culture is not ideal for scalable expansion, experimentation, and high-throughput screening applications. Our group has developed a protocol for growing patient-derived colorectal organoids and tumoroids in low-viscosity matrix (LVM) suspension culture. Instead of embedding colonic crypts or tumor fragments in solid ECM, these are grown suspended in medium containing only a low percentage of ECM. Compared with dome cultures, LVM suspension culture reduces the labor and cost of establishing and passaging organoids and tumoroids, enables rapid expansion, and is readily adaptable for high-throughput screening.


Graphical abstract:



Generation of organoids and tumoroids from human large intestine using LVM suspension culture (Created with BioRender.com).


0 Q&A 4008 Views Mar 5, 2021

Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are “primed” by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these “primed” cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.

0 Q&A 3498 Views Jan 20, 2020
Infantile hemangioma (IH) is a vascular tumor noted for its excessive blood vessel formation during infancy, glucose-transporter-1 (GLUT1)-positive staining of the blood vessels, and its slow spontaneous involution over several years in early childhood. For most children, IH poses no serious threat because it will eventually involute, but a subset can destroy facial structures and impair vision, breathing and feeding. To unravel the molecular mechanism(s) driving IH-specific vascular overgrowth, which to date remains elusive, investigators have studied IH histopathology, the cellular constituents and mRNA expression. Hemangioma endothelial cells (HemEC) were first isolated from surgically removed IH specimens in 1982 by Mulliken and colleagues (Mulliken et al., 1982). Hemangioma stem cells (HemSC) were isolated in 2008, hemangioma pericytes in 2013 and GLUT1-positive HemEC in 2015. Indeed, as we describe here, it is possible to isolate HemSC, GLUT1-positive HemEC, GLUT1-negative HemEC and HemPericytes from a single proliferating IH tissue specimen. This is accomplished by sequential selection using antibodies against specific cell surface markers: anti-CD133 to select HemSC, anti-GLUT1 and anti-CD31 to select HemECs and anti-PDGFRβ to select HemPericytes. IH-derived cells proliferate well in culture and can be used for in vitro and in vivo vasculogenesis and angiogenesis assays.
0 Q&A 9410 Views Sep 5, 2018
Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in vitro. We further show how ELDA can be used on cells, before and after forced differentiation to amplify inherent differences in sphere-forming potential between mutant and control NSCs. Ultimately, ELDA exploits a difference in the ability of a single or a few seeded stem cells to self-renew, divide and form neurospheres. Importantly, the assay also allows a comparison between genetically distinct cells or between the same cells under different conditions, where the impact of target-specific drugs or other novel cancer stem cell therapies can be tested.
0 Q&A 9344 Views Jun 5, 2018
Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, spheroids cultured in hanging drops, magnetic levitation systems or 3D printing methods are already being widely used in research and for novel drug screening. In this article, we describe a detailed protocol for testing the effect of shRNA-mediated gene silencing on tumorsphere formation and growth. This approach allows researchers to test the impact of gene knockdown on the growth of tumor initiating cells. As verified by our lab, the protocol can be also used for isolation of 3D cancer cell lines directly from tumor tissues.
0 Q&A 6415 Views Apr 5, 2018
Pituitary adenomas are among the more frequent intracranial tumors usually treated with both surgical and pharmacological–based on somatostatin and dopamine agonists–approaches. Although mostly benign tumors, the occurrence of invasive behaviors is often detected resulting in poorer prognosis. The use of primary cultures from human pituitary adenomas represented a significant advancement in the knowledge of the mechanisms of their development and in the definition of the determinants of their pharmacological sensitivity. Moreover, recent studies identified also in pituitary adenomas putative tumor stem cells representing, according to the current hypothesis, the real cellular targets to eradicate most malignancies. In this protocol, we describe the procedure to establish primary cultures from human pituitary adenomas, and how to select, in vitro expand, and phenotypically characterize putative pituitary adenoma stem cells.
0 Q&A 12203 Views Dec 20, 2017
Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and glioma cells with stem cell properties from fresh postmortem and surgical tissues. The ability to prospectively isolate stem cell populations using native ligand-binding ability opens new doors for understanding both normal and tumor cell biology in uncultured conditions, and is applicable for various downstream molecular sequencing studies at both population and single-cell resolution.
0 Q&A 8636 Views Sep 5, 2017
Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further functionally profile the cancer stem cells (RNA sequencing and epigenetic analysis), grow them in culture or use them directly in transplantation assays.
0 Q&A 13625 Views Apr 20, 2017
Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells (or cancer stem cells) self-renewal potential in vitro. When single cells are plated at low density in stem cell culture medium, only the cells endowed with self-renewal are able to grow in tridimensional clusters usually named spheres. In the recent years, SFA has also been used also to test the effect of several drugs, chemical and natural compounds or microenviromental components on stem cells self-renewal capacity. Here we will illustrate a detailed protocol to assess self-renewal of human melanoma stem cells, growing as melanospheres.



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