Cancer Biology


Protocols in Current Issue
0 Q&A 75 Views Jun 20, 2024

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector–target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality.

Protocols in Past Issues
0 Q&A 269 Views Jun 5, 2024

Neutrophils, constituting 50%–70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies.

0 Q&A 2543 Views Apr 20, 2024

The CTC1-STN1-TEN1 (CST) complex is a single-strand DNA-binding protein complex that plays an important role in genome maintenance in various model eukaryotes. Dysfunction of CST is the underlying cause of the rare genetic disorder known as Coats plus disease. In addition, down regulation of STN1 promotes colorectal cancer development in mice. While prior studies have utilized RNAi to knock down CST components in mammalian cells, this approach is associated with off-target effects. Attempts to employ CRISPR/Cas9-based knockout of CST components in somatic cell lines have been unsuccessful due to CST's indispensable role in DNA replication and cell proliferation. To address these challenges, we outline a novel approach utilizing a Cre-loxP-based conditional knockout in mouse embryonic fibroblasts (MEFs). This method offers an alternative means to investigate the function and characteristics of the CST complex in mammalian systems, potentially shedding new light on its roles in genome maintenance.

0 Q&A 668 Views Jan 20, 2024

The blood–brain barrier (BBB) is a major obstacle to the diagnostics and treatment of many central nervous system (CNS) diseases. A prime example of this challenge is seen in glioblastoma (GBM), the most aggressive and malignant primary brain tumor. The BBB in brain tumors, or the blood–brain–tumor barrier (BBTB), prevents the efficient delivery of most therapeutics to brain tumors. Current strategies to overcome the BBB for therapeutic delivery, such as using hyperosmotic agents (mannitol), have impeded progress in clinical translation limited by the lack of spatial resolution, high incidences of complications, and potential for toxicity. Focused ultrasound combined with intravenously administered microbubbles enables the transient disruption of the BBB and has progressed to early-phase clinical trials. However, the poor survival with currently approved treatments for GBM highlights the compelling need to develop and validate treatment strategies as well as the screening for more potent anticancer drugs. In this protocol, we introduce an optical method to open the BBTB (OptoBBTB) for therapeutic delivery via ultrashort pulse laser stimulation of vascular targeting plasmonic gold nanoparticles (AuNPs). Specifically, the protocol includes the synthesis and characterization of vascular-targeting AuNPs and a detailed procedure of optoBBTB. We also report the downstream characterization of the drug delivery and tumor treatment efficacy after BBB modulation. Compared with other barrier modulation methods, our optical approach has advantages in high spatial resolution and minimally invasive access to tissues. Overall, optoBBTB allows for the delivery of a variety of therapeutics into the brain and will accelerate drug delivery and screening for CNS disease treatment.

Key features

• Pulsed laser excitation of vascular-targeting gold nanoparticles non-invasively and reversibly modulates the blood–brain barrier permeability.

• OptoBBTB enhances drug delivery in clinically relevant glioblastoma models.

• OptoBBTB has the potential for drug screening and evaluation for superficial brain tumor treatment.

Graphical overview

0 Q&A 1427 Views Nov 20, 2023

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes.

Key features

• This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I.

• Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

0 Q&A 449 Views Nov 5, 2023

Cell signaling is highly integrated for the process of various cell activities. Although previous studies have shown how individual genes contribute to cell migration, it remains unclear how the integration of these signaling pathways is involved in the modulation of cell migration. In our two-hit migration screen, we revealed that serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK) worked synergistically, and the suppression of both genes could further lead to suppression in cell migration. Furthermore, based on our analysis of cellular focal adhesion (FA) parameters using MATLAB analysis, we are able to find out the synergistic reduction of STK40 and MAPK that further abolished the increased FA by shSTK40. While FA identification in previous studies includes image analysis using manual selection, our protocol provides a semi-automatic manual selection of FAs using MATLAB. Here, we provide a method that can shorten the amount of time required for manual identification of FAs and increase the precision for discerning individual FAs for various analyses, such as FA numbers, area, and mean signals.

0 Q&A 347 Views Nov 5, 2023

Cell migration is an essential biological process for organisms, in processes including embryonic development, immune response, and cancer metastasis. To elucidate the regulatory machinery of this vital process, methods that mimic in vivo migration, including in vitro wound healing assay and random migration assay, are widely used for cell behavior investigation. However, several concerns are raised with traditional cell migration experiment analysis. First, a manually scratched wound often presents irregular edges, causing the speed analysis difficult. Second, only the migration speed of leading cells is considered in the wound healing assay. Here, we provide a reliable analysis method to trace each cell in the time-lapse images, eliminating the concern about wound shape and creating a more comprehensive understanding of cell migration—not only of collective migration speed but also single-cell directionality and coordination between cells.

0 Q&A 956 Views Oct 20, 2023

An efficient and precise genome-editing approach is in high demand in any molecular biology or cell biology laboratory worldwide. However, despite a recent rapid progress in the toolbox tailored for precise genome-editing, including the base editors and prime editors, there is still a need for a cost-effective knock-in (KI) approach amenable for long donor DNA cargos with high efficiency. By harnessing the high-efficient double-strand break (DSB) repair pathway of microhomology-mediated end joining, we previously showed that a specially designed 3′-overhang double-strand DNA (odsDNA) donor harboring 50-nt homology arm (HA) allows high-efficient exogenous DNA KI when combined with CRISPR-Cas9 technology. The lengths of the 3′-overhangs of odsDNA donors could be manipulated by the five consecutive phosphorothioate (PT) modifications. In this protocol, we detail the stepwise procedures to conduct the LOCK (Long dsDNA with 3′-Overhangs mediated CRISPR Knock-in) method for gene-sized (~1–3 kb) KI in mammalian cells.

Graphical overview

Improvement of large DNA fragment knock-in rates by attaching odsDNA donors to Cas9-PCV2 fusion protein

1 Q&A 784 Views Oct 20, 2023

Platelets and their activation status play an essential role in cancer metastasis. Therefore, the anti-metastatic potential of antiplatelet drugs has been investigated for many years. However, the initial screening of these antiplatelet drugs to determine which agents can inhibit the interactions of platelets and tumor cells is very limited due to reliance upon expensive, time-consuming, and low-throughput animal experiments for screening. In vitro models of the platelet–tumor cell interaction can be a useful tool to rapidly screen multiple antiplatelet drugs and compare their ability to disrupt platelet–tumor cell interactions, while also identifying optimal concentrations to move forward for in vivo validation. Hence, we adopted methods used in platelet activation research to isolate and label platelets before mixing them with tumor cells (MDA-MB-231-RFP cells) in vitro in a static co-culture model. Platelets were isolated from other blood components by centrifugation, followed by fluorescent labeling using the dye CMFDA (CellTrackerTM Green). Labeling platelets allows microscopic observation of the introduced platelets with tumor cells grown in cell culture dishes. These methods have facilitated the study of platelet–tumor cell interactions in tissue culture. Here, we provide details of the methods we have used for platelet isolation from humans and mice and their staining for further interaction with tumor cells by microscopy and plate reader–based quantification. Moreover, we show the utility of this assay by demonstrating decreased platelet–tumor cell interactions in the presence of the T-Prostanoid receptor (TPr) inhibitor ifetroban. The methods described here will aid in the rapid discovery of antiplatelet agents, which have potential as anti-metastatic agents as well.

Key features

• Analysis of platelet–tumor cell binding dynamics.

• In vitro methods developed for measuring platelet–tumor cell binding to enable rapid testing of antiplatelet and other compounds.

• Complementary analysis of platelet–tumor cell binding by imaging and fluorimetry-based readings.

• Representative results screening the effect of the antiplatelet drug, ifetroban, on platelet–tumor cell binding using the protocol.

• Validation results were presented with both a TPr agonist and ifetroban (antagonist).

Graphical overview

Representative overview of the process to isolate and label platelets, incubate platelets and tumor cells in the presence of antiplatelet agents, and image and/or quantify platelet–tumor cell interactions.

0 Q&A 997 Views Aug 20, 2023

Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.

Graphical overview

0 Q&A 976 Views Aug 5, 2023

Resistance of acute lymphoblastic leukemia (ALL) cells to chemotherapy, whether present at diagnosis or acquired during treatment, is a major cause of treatment failure. Primary ALL cells are accessible for drug sensitivity testing at the time of new diagnosis or at relapse, but there are major limitations with current methods for determining drug sensitivity ex vivo. Here, we describe a functional precision medicine method using a fluorescence imaging platform to test drug sensitivity profiles of primary ALL cells. Leukemia cells are co-cultured with mesenchymal stromal cells and tested with a panel of 40 anti-leukemia drugs to determine individual patterns of drug resistance and sensitivity (“pharmacotype”). This imaging-based pharmacotyping assay addresses the limitations of prior ex vivo drug sensitivity methods by automating data analysis to produce high-throughput data while requiring fewer cells and significantly decreasing the labor-intensive time required to conduct the assay. The integration of drug sensitivity data with genomic profiling provides a basis for rational genomics-guided precision medicine.

Key features

• Analysis of primary acute lymphoblastic leukemia (ALL) blasts obtained at diagnosis from bone marrow aspirate or peripheral blood.

• Experiments are performed ex vivo with mesenchymal stromal cell co-culture and require four days to complete.

• This fluorescence imaging–based protocol enhances previous ex vivo drug sensitivity assays and improves efficiency by requiring fewer primary cells while increasing the number of drugs tested to 40.

• It takes approximately 2–3 h for sample preparation and processing and a 1.5-hour imaging time.

Graphical overview

BM: bone marrow; PB: peripheral blood; ALL: acute lymphoblastic leukemia; MNCs: mononuclear cells, which include leukemia cells when present; MSCs: mesenchymal stromal cells; LC50: drug concentration that kills 50% of the leukemia cells

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