Cancer Biology

The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods (Ziegler et al., 2021). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures.

Graphic abstract:

Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

Acute myeloid leukaemia (AML) is a highly heterogenous blood cancer, in which the expansion of aberrant myeloid blood cells interferes with the generation and function of normal blood cells. Although key driver mutations and their associated inhibitors have been identified in the last decade, they have not been fully translated into better survival rates for AML patients, which remain dismal. In addition to DNA mutation, studies in mouse models strongly suggest that the cell of origin, where the driver mutation (such as MLL fusions) occurs, emerges as an additional factor that determines the treatment outcome in AML. To investigate its functional relevance in human disease, we have recently reported that AML driven by MLL fusions can transform immunophenotypically and functionally distinctive human hematopoietic stem cells (HSCs) or myeloid progenitors resulting in immunophenotypically indistinguishable human AML. Intriguingly, these cells display differential treatment sensitivities to current treatments, attesting the cell of origin as an important determinant governing treatment outcome for AML. To further facilitate this line of investigation, here we describe a comprehensive disease modelling protocol using human primary haematopoietic cells, which covers all the key steps, from the isolation of immunophenotypically defined human primary haematopoietic stem and progenitor populations, to oncogene transfer via viral transduction, the in vitro liquid culture assay, and finally the xenotransplantation into immunocompromised mice.

With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.