Cancer Biology

P18F3-based bi-modular fusion proteins (BMFPs), designed to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, demonstrated efficient biological activity in a mouse tumor model and could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing scFv_2H7-P18F3, a BMFP targeting human CD20, in Escherichia coli (SHuffle^®), and for purifying soluble proteins using a two-step process, namely immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography. This protocol can also be used for expression and purification of other BMFPs with alternative binding specificities.

Mitochondria play decisive roles in bioenergetics and intracellular communication. These organelles contain a circular mitochondrial DNA (mtDNA) genome that is duplicated within one to two hours by a mitochondrial replisome, independently from the nuclear replisome. mtDNA stability is regulated in part at the level of mtDNA replication. Consequently, mutations in mitochondrial replisome components result in mtDNA instability and are associated with diverse disease phenotypes, including premature aging, aberrant cellular energetics, and developmental defects. The mechanisms ensuring mtDNA replication stability are not completely understood. Thus, there remains a need to develop tools to specifically and quantifiably examine mtDNA replication. To date, methods for labeling mtDNA have relied on prolonged exposures of 5′-bromo-2′-deoxyuridine (BrdU) or 5′-ethynyl-2′-deoxyuridine (EdU). However, labeling with these nucleoside analogs for a sufficiently short time in order to monitor nascent mtDNA replication, such as under two hours, does not produce signals suited for efficient or accurate quantitative analysis. The assay system described here, termed Mitochondrial Replication Assay (MIRA), utilizes proximity ligation assay (PLA) combined with EdU-coupled Click-IT chemistry to address this limitation, thereby enabling sensitive and quantitative analysis of nascent in situ mtDNA replication with single-cell resolution. This method can be further paired with conventional immunofluorescence (IF) for multi-parameter cell analysis. By enabling monitoring nascent mtDNA prior to the complete replication of the entire mtDNA genome, this new assay system allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection. Moreover, a modification in primary antibodies application allows the adaptation of our previously described in situ protein Interactions with nascent DNA Replication Forks (SIRF) for the detection of proteins of interest to nascent mtDNA replication forks on a single molecule level (mitoSIRF).

Graphical overview

Schematic overview of Mitochondrial Replication Assay (MIRA). 5′-ethynyl-2′-deoxyuridine (EdU; green) incorporated in DNA is tagged with biotin (blue) using Click-IT chemistry. Subsequent proximity ligation assay (PLA, pink circles) using antibodies against biotin allows the fluorescent tagging of the nascent EdU and amplification of the signal sufficient for visualization by standard immunofluorescence. PLA signals outside the nucleus denote mitochondrial DNA (mtDNA) signals. Ab, antibody. In in situ protein interactions with nascent DNA replication forks (mitoSIRF), one of the primary antibodies is directed against a protein of interest, while the other detects nascent biotinylated EdU, thus enabling in situ protein interactions with nascent mtDNA.

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ER^T2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ER^T2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs.

Graphical overview

Timing

Day 0: Zebrafish spawning

Day 8: Primary tumor induction

Day 11: Chemical treatment

Day 11.5: Metastatic dissemination induction in the presence of a test chemical

Day 16: Data analysis

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5– and serine 2–phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription.

Graphical overview

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-β-galactosidase (SA-β-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-β-D-galactopyranoside (C₁₂FDG), a fluorigenic substrate of β-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents.

Graphical abstract

Over the past decades, the main techniques used to visualize bacteria in tissue have improved but are still mainly based on indirect recognition of bacteria. Both microscopy and molecular recognition are being improved, but most procedures for bacteria detection in tissue involve extensive damage. Here, we describe a method to visualize bacteria in tissue slices from an in vivo model of breast cancer. This method allows examining trafficking and colonization of fluorescein-5-isothiocyanate (FITC)-stained bacteria in various tissues. The protocol provides direct visualization of fusobacterial colonization in breast cancer tissue. Rather than processing the tissue or confirming bacterial colonization by PCR or culture, the tissue is directly imaged using multiphoton microscopy. This direct visualization protocol causes no damage to the tissue; therefore, all structures can be identified. This method can be combined with others to co-visualize bacteria, types of cells, or protein expression in cells.

A rigorous determination of effector contributions of tumor-infiltrating immune cells is critical for identifying targetable molecular mechanisms for the development of novel cancer immunotherapies. A tumor/immune cell–admixture model is an advantageous strategy to study tumor immunology as the fundamental methodology is relatively straightforward, while also being adaptable to scale to address increasingly complex research queries. Ultimately, this method can provide robust experimental information to complement more traditional murine models of tumor immunology. Here, we describe a tumor/macrophage-admixture model using bone marrow–derived macrophages to investigate macrophage-dependent tumorigenesis. Additionally, we provide commentary on potential branch points for optimization with other immune cells, experimental techniques, and cancer types.

Telomeres are structures that cap the ends of linear chromosomes and play critical roles in maintaining genome integrity and establishing the replicative lifespan of cells. In stem and cancer cells, telomeres are actively elongated by either telomerase or the alternative lengthening of telomeres (ALT) pathway. This pathway is characterized by several hallmark features, including extrachromosomal C-rich circular DNAs that can be probed to assess ALT activity. These so-called C-circles are the product of ALT-associated DNA damage repair processes and simultaneously serve as potential templates for iterative telomere extension. This bifunctional nature makes C-circles highly sensitive and specific markers of ALT. Here, we describe a C-circle assay, adapted from previous reports, that enables the quantitation of C-circle abundance in mammalian cells subjected to a wide range of experimental perturbations. This protocol combines the Quick C-circle Preparation (QCP) method for DNA isolation with fluorometry-based DNA quantification, rolling circle amplification (RCA), and detection of C-circles using quantitative PCR. Moreover, the inclusion of internal standards with well-characterized telomere maintenance mechanisms (TMMs) allows for the reliable benchmarking of cells with unknown TMM status. Overall, our work builds upon existing protocols to create a generalizable workflow for in vitro C-circle quantitation and ascertainment of TMM identity.

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

The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell–ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell–ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes.