Cancer Biology

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

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).

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.

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.

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.

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.

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.

HER2 is a tyrosine kinase receptor, which is overexpressed in about 30% of breast cancer patients. Its overexpression leads to mammary tumorigenesis and increased invasion and metastasis (Slamon et al., 1987). HER2 transgenic mouse (FVB/N-MMTVneu mouse) is a well-established model of mammary tumor in human (Fantozzi and Christofori, 2006). Although in vivo models are excellent for assessing the influence of various factors, especially microenvironment, on development of breast cancer, a convenient and less costly way to study the underlying molecular events is utilizing cells derived from the model under evaluation. In order to explore the molecular mechanism by which HOXB7 inhibits initiation, but promotes metastasis of breast tumors, we generated mouse breast cancer cell line from HER2 transgenic mouse (Liu et al., 2015). This protocol may be useful for the generation of breast cancer cell line from mice with other genetic backgrounds.

The intestinal epithelial layer forms tubular invaginations into the underlying connective tissue of the lamina propria. These structures, termed crypts, are the basic functional unit of the intestine. Colon crypts and the surrounding lamina propria house different cell types, including epithelial cells, stem cells, enterocytes, goblet cells, as well as cells of the innate and adaptive immune systems (Clevers, 2013; Mowat and Agace, 2014). Here we describe a technique for the isolation of mouse intestinal crypt cells as well as their characterization by flow cytometry analysis (FACS) (Del Reino et al., 2012).

Glioma Associated Stem Cells (GASCs) represent a population of non-tumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma (Bourkoula et al., 2014).