Cancer Biology

Recent advancements in high-throughput functional genomics have substantially enhanced our comprehension of the genetic and molecular dimensions of cancer, facilitating the identification of novel therapeutic targets. One of the key methodological innovations in this field is the CRISPR screening strategy, which has proven efficacy in elucidating essential gene functions and pathway alterations critical to cancer cell survival and fitness. The construction of custom CRISPR libraries permits the integration of tailored single-guide RNAs (gRNAs), offering greater flexibility as well as specificity in comparison to the commercially available libraries, and enables more refined secondary screening strategies to attenuate the selection of false positive potential gene candidates. Among various molecular cloning techniques, circular polymerase extension cloning (CPEC) has emerged as a highly efficient and cost-effective approach. CPEC utilizes polymerase overlap extension to assemble overlapping DNA fragments into circular plasmids, eliminating the need for restriction digestion and ligation and thus streamlining the creation of both single and multi-fragment constructs. In this protocol, we present the application of the CPEC method to construct the EpiTransNuc knockout gRNA library, specifically designed to target epigenetic regulators, transcription factors, and nuclear proteins. The custom library, assembled using the lentiGuide-Puro backbone, comprises 40,820 gRNAs, with 10 gRNAs per gene, along with 100 non-targeting control gRNAs. Importantly, the CPEC method can be tailored to meet the specific requirements of other custom gRNA libraries, offering flexibility for diverse research applications.

Recurrent hormone receptor-positive (HR+) breast cancer is a leading cause of cancer mortality in women. Recurrence and resistance to targeted therapies have been difficult to study due to the long clinical course of the disease, the complex nature of resistance, and the lack of clinically relevant model systems. Existing models are limited to a few HR+ cell lines, organoid models, and patient-derived xenograft models, all lacking components of the human tumor microenvironment. Furthermore, the low take rate and loss of estrogen receptor (ER) expression in patient-derived organoids (PDOs) has been challenging. Our protocol allows simultaneous isolation of PDOs and matching cancer-associated fibroblasts (CAFs) from primary and metastatic HR+ breast cancers. Importantly, our protocol has a higher take rate and enables long-term culturing of PDOs that retain ER expression. Our matching PDOs and CAFs will provide researchers with a new resource to study the influence of the tumor microenvironment on various aspects of cancer biology such as cell growth and drug resistance in HR+ breast cancer.

The initiation and progression of prostate cancer (PCa) are associated with aging. In the history of age-related PCa research, mice have become a more popular animal model option than any other species due to their short lifespan and rapid reproduction. However, PCa in mice is usually induced at a relatively young age, while it spontaneously develops in humans at an older age. Thus, it is essential to develop a method by which the PCa initiation and progression timeline can be strictly controlled to mimic human physiological conditions. One milestone in this field was the identification of the prostate-specific transcription factor, Probasin (Pb), which allowed for the prostate-specific expression of genes knocked into the mice's genome. Another milestone is the establishment of the preclinical mouse model with Pten conditionally knocked out in the prostate tissue, which closely mimics the formation and growth of human PCa. Hereby, we present the prostate-specific temporally and spatially controlled Pten knockout PCa mouse model that can be induced using an adenovirus-based Cre-LoxP system. The Cre recombinase (Cre) is inserted into an adenovirus vector. Unlike Pb-Cre knock-in models (which are spatially but not temporally controlled), the expression of Cre is activated to knock out Pten from the mice's prostate epithelial cells once injected. The viral delivery procedures strictly control the location and time of Pten knockout. This novel approach provides a powerful age-related murine model for PCa, emphasizing the effect of aging on prostate carcinogenesis.

Endometrial cancer (EC) is the leading cause of gynecologic cancer morbidity and mortality in the U.S. Despite advancements in cancer research, EC death rates are increasing, particularly high-grade endometrial cancers. The development of three-dimensional (3D) patient-derived organoid (PDO) models for EC is crucial, as they provide a more accurate representation of the biological and genetic complexity of a patient’s tumor compared to traditional 2D cell lines. Here, we describe a protocol for cultivating PDO models from normal endometrium and EC across different EC subtypes. These EC PDO models can be expanded across multiple passages and facilitate the exploration of tumor behavior and drug responses, thereby advancing our understanding of the disease and potentially leading to more effective and individualized novel therapeutic strategies.

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

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

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

Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader.

Graphical overview

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.

CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50–300 gene knockouts on various cellular parameters within eight weeks.

Graphical abstract