Cancer Biology

This protocol describes the preparation, administration, and analysis of a nanoparticle-based therapeutic strategy (nanoPDLIM2) in combination with PD-1 immune checkpoint blockade immunotherapy and chemotherapy for the treatment of lung cancer in mouse preclinical studies. NanoPDLIM2 uses a polyethyleneimine (PEI)-based delivery system that encapsulates PDLIM2 expression plasmids for reconstituting PDLIM2 that is repressed in tumors. This approach induces tumor immunogenicity, suppresses drug resistance, and improves treatment efficacy when used in combination with carboplatin, paclitaxel, and anti-PD-1 antibodies. The protocol describes steps for mouse lung tumor induction, nanoPDLIM2 and other therapeutic reagents’ preparation and administration, and subsequent analysis of tumor burden, immune response, and toxicity, providing a reproducible approach for investigators.

Quantification of DNA double-strand breaks (DSBs) is critical for assessing genomic damage and cellular response to stress. γH2AX is a well-established marker for DNA double-strand breaks, but its quantification is often performed manually or semi-quantitatively, lacking standardization and reproducibility. Here, we present a standardized and automated workflow for γH2AX foci quantification in irradiated cells using immunofluorescence and a custom Fiji macro. The protocol includes steps for cell irradiation, immunostaining, image acquisition, and automated foci counting. The protocol is also adaptable to colony-like formations in multi-well plates, extending its utility to clonogenic assays. This protocol enables high-throughput, reproducible quantification of DNA damage with minimal user bias and can be readily implemented in routine laboratory settings.

This protocol describes an ex vivo co-culture method to assess CD8⁺ T-cell activation, proliferation, and cytotoxic potential using bulk splenocytes isolated from immunocompetent mice. Mouse splenocytes are stimulated with anti-CD3 and anti-CD28 antibodies to activate CD8⁺ T cells, which are then co-incubated with either cancer cells or cancer cell–derived conditioned media (CM) to evaluate tumor-driven modulation of immune cell functions. The use of unfractionated splenocytes preserves physiological cell–cell interactions, eliminating the need for exogenous interleukin (IL-2) and bypassing flow sorting, which simplifies the workflow and reduces experimental variability. CD8⁺ T-cell responses are measured via flow cytometry, using markers of proliferation (CFSE dilution), activation (CD69), and effector function (Granzyme B and IFNγ). Additionally, immune-mediated tumor cell death is evaluated by Annexin-V/7-AAD staining. Together, this experimental platform supports the investigation of both cell contact-dependent and contact-independent mechanisms of immune cell modulation in a cost-effective and reproducible setting.

Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.

Cancer-associated mesenchymal stem cells (Ca-MSCs), an integral part of the tumor microenvironment, play a major role in modulating tumor progression; they have been reported to progress as well as inhibit various cancers, including cervical cancer. To understand the exact role of Ca-MSCs in tumor modulation, it is necessary to have an optimized protocol for Ca-MSCs isolation. This work demonstrates the isolation and expansion of a primary culture of cervical cancer–associated MSCs (CCa-MSCs) from the biopsy sample of cervical cancer patients using the explant culture technique. The isolated cells were characterized according to International Society for Cellular Therapy (ISCT) guidelines. Morphological analysis revealed that cells were adherent to the plastic surface and possessed spindle-shaped morphology. Flow cytometry analysis of the cells showed high expression (~98%) for MSC-specific cell surface markers (CD90, CD73, and CD105), negative expression (<0.5%) for endothelial cell marker (CD34) and hematopoietic cell marker (CD45), and negligible expression for HLA-DR, as recommended by ISCT. Further, trilineage differentiation potential analysis of the cells showed their osteogenic and chondrogenic potential and adipogenic differentiation. This standardized protocol will assist in the cultivation of CCa-MSCs and the study of their interactions with tumor cells and other components of the tumor microenvironment. This protocol may be utilized in the establishment of Ca-MSCs from other types of cancers as well.

Stable isotopes have frequently been used to study metabolic processes in live cells both in vitro and in vivo. Glutamine, the most abundant amino acid in human blood, plays multiple roles in cellular metabolism by contributing to the production of nucleotides, lipids, glutathione, and other amino acids. It also supports energy production via anaplerosis of tricarboxylic acid cycle intermediates. While ¹³C-glutamine has been extensively employed to study glutamine metabolism in various cell types, detailed analyses of specific lipids derived from ¹³C-glutamine via the reductive carboxylation pathway are limited. In this protocol, we present a detailed procedure to investigate glutamine metabolism in human glioblastoma (GBM) cells by conducting ¹³C-glutamine tracing coupled with untargeted metabolomics analysis using liquid chromatography–mass spectrometry (LC–MS/MS). The method includes step-by-step instructions for the extraction and detection of polar metabolites and long-chain fatty acids (LCFAs) derived from ¹³C-glutamine in GBM cells. Notably, this approach enables the distinction between isomers of two monounsaturated FAs with identical masses: palmitoleic acid (16:1n-7) (cis-9-hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-hexadecenoic acid) derived from ¹³C-glutamine through the reductive carboxylation process. In addition, using this protocol, we also unveil previously unknown metabolic alterations in GBM cells following lysosome inhibition by the antipsychotic drug pimozide.

Pericytes are essential for tissue homeostasis, functioning to regulate capillary blood flow. Dysfunctional pericytes are implicated in various pathologies, including cancer progression. Despite their important function in both health and disease, pericytes remain understudied due to a lack of robust model systems that accurately reflect their in vivo biology. Here, we present a comprehensive protocol for isolating and culturing primary pericytes from murine lung, brain, bone, and liver tissues, based on NG2 expression using an antibody-conjugated magnetic bead approach. Our protocol emphasizes the importance of physiological oxygen tension during ex vivo culture (10% O₂ for lung pericytes and 5% O₂ for brain, bone, and liver pericytes). These conditions stabilize the expression of characteristic pericyte markers at both the transcriptional and protein levels. Importantly, we optimized growth conditions to limit the expression of the plasticity factor Klf4 in order to prevent spontaneous phenotypic switching in vitro. This protocol provides a reliable and reproducible method for obtaining pericytes suitable for high-throughput analyses in order to explore pericyte biology in both physiological and pathological contexts.

Laser-assisted microdissection (LAM) coupled with next-generation sequencing technologies offers a powerful approach to dissecting the complex cellular heterogeneity within lung adenocarcinoma (LUAD) tumors. This protocol outlines the method for isolating specific high-risk LUAD tissues containing micropapillary/solid (MIP/SOL) patterns, which is linked to poor prognosis. We detail the process of LAM, which involves tissue fixation, microtome sectioning, and the precise dissection and collection of cells of interest under microscopic guidance. The isolated cells are then subjected to RNA extraction, library preparation, and sequencing to profile transfer RNA–derived fragments (tRFs) and tRNA-derived stress-induced RNAs (tiRNAs), which are emerging as key regulators in cancer. This protocol enables researchers to obtain high-quality transcriptomic data from specific LUAD cell populations, aiming to uncover tRF-Val-CAC-024 and tiRNA-Gly-CCC as potential biomarkers for early diagnosis and therapeutic targets for LUAD treatment.

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks. Hereditary breast and ovarian cancer syndrome (HBOC) is caused by germline pathogenic variants of HR-related genes, such as BRCA1 and BRCA2 (BRCA1/2). Cancer cells with HR deficiency are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, accurate evaluation of HR activity is helpful to diagnose HBOC and predict the effects of PARP inhibitors. The direct-repeat GFP (DR-GFP) assay has been utilized to evaluate cellular HR activity. However, evaluation by the DR-GFP assay tends to be qualitative and requires the establishment of stable cell lines. Therefore, we developed an assay to quantitatively measure HR activity called Assay for Site-Specific HR Activity (ASHRA), which can be performed by transiently transfecting two plasmids. In ASHRA, we use Cas9 endonuclease to create DNA double-strand breaks at specific sites in the genome, enabling the targeting of any endogenous loci. Quantification of HR products by real-time PCR using genomic DNA allows HR activity evaluated at the DNA level. Thus, ASHRA is an easy and quantitative method to evaluate HR activity at any genomic locus in various samples. Here, we present the protocols for adherent cells, suspension cells, and tumor tissues.

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.