Cancer Biology

Immunohistochemistry (IHC) is a highly specific and widely used laboratory technique for assessing protein localization and expression in tissue samples. Interpretation of 3,3’ diamino benzidine (DAB)-based IHC is often based on observer-dependent manual scoring or traditional imaging software, which may show variability in DAB staining quantification. Furthermore, conventional image analysis tools often face limitations in precisely defining cell boundaries and quantifying membrane-specific signals. In this study, we present a standardized image analysis workflow using CellProfiler, an open-source software for image analysis for the quantification of membrane staining intensity in IHC images captured from slides prepared using formalin-fixed paraffin-embedded (FFPE) human cervical cancer tissue sections. The image analysis workflow was demonstrated using ASCT2 (SLC1A5), a membrane-localized amino acid transporter, as a representative biomarker for membrane-associated protein expression. This protocol involves image preprocessing, object identification, segmentation, and intensity measurement modules to distinguish cell membranes from cytoplasmic regions, enabling automated quantification of membrane intensity signals. The CellProfiler pipeline demonstrated improved accuracy in cell boundary identification and quantification of membrane-specific staining intensity. This is a rapid quantification process, since processing of each image only takes a few seconds; therefore, the analysis for 100 images can be performed within 10–15 min. This segmentation and quantification strategy is applicable to other membrane-based biomarkers after appropriate optimization of segmentation parameters. Following further minor modifications to the object identification modules, this pipeline can be used to detect and quantify cytoplasm- or nuclei-localized DAB-IHC markers across different tissue types. Overall, this protocol provides a standardized, user-friendly, and reproducible workflow for quantitative IHC image analysis that can be broadly applied to the study of protein biomarkers of different localizations, such as nuclei, cytoplasm, and cell membranes from different tissue types.

ADP ribosylation (ADPr) is a crucial post-translational modification that plays a vital role in DNA damage repair. Catalyzed by ADP ribose polymerases using NAD⁺ as a substrate, ADPr activates DNA repair pathways rapidly, thereby maintaining genomic integrity. The involvement of ADP ribose hydrolases in this process is significant, as they hydrolyze PAR chains, facilitating the release of ADPr-modified proteins from DNA or other proteins, which is essential for subsequent DNA repair steps. This protocol outlines a high-throughput screening method for identifying inhibitors of ADP ribose hydrolases, utilizing His-Tb-conjugated and ADPr-modified His-ADP ribose polymerase as the signal donor, and GST-d2-conjugated GST-XRCC1 as the signal receptor. The detection of time-resolved fluorescence signals enables efficient evaluation of compounds with potential therapeutic activity against cancer.

Gastric cancer remains a major global health challenge, and reliable prognostic biomarkers are urgently needed to guide treatment decisions. Here, we present a simple and efficient protocol for a novel liquid biopsy approach based on quantifying gastric fluid DNA (gfDNA) collected during routine esophagogastroduodenoscopy (EGD). We have previously shown that gfDNA carries gastric cancer–derived mutations; moreover, its concentration increases with tumor progression and varies according to cancer prognosis. This empirically observed increase in gfDNA may mechanistically stem from enhanced cellular turnover, tissue disorganization, dysbiosis of the local microbiota, and/or fluctuations in immune cell infiltrates. Surprisingly, however, in patients diagnosed with gastric cancer, elevated gfDNA levels were also associated with improved survival. This paradoxical finding may be reconciled by an increased anti-tumor immune cell response in treatment-responsive gastric cancers, as well as by the contribution of non-tumoral DNA from inflammatory processes within the microenvironment of the stomach. Here, we detail a standardized protocol for gastric fluid collection and processing, designed to support downstream gfDNA quantification among other potential molecular applications.

Breast cancer is the most frequently diagnosed cancer in women, representing approximately 25% of all cancers in women worldwide. Both breast cancer research and histopathological diagnostics mainly show a two-dimensional planar view of the three-dimensional breast cancerous architecture. Recently, the application of optical tissue clearing, together with 3D microscopy, has been applied to visualize the complexity of whole tumor samples. Preliminary studies on whole-organ mouse mammary glands and tissues from human breast cancer patients subjected to optical tissue clearing and volumetric imaging have enabled the detection of previously unrecognized spatial cellular interactions and structural features within intact breast tissue. There is currently no standardized clearing workflow for breast and lymph node tissues. In this protocol, we optimized and validated the MASH (multiscale architectonic staining of human cortex) immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO)-like clearing and labeling pipeline for the investigation of formalin-fixed and paraffin-embedded (FFPE) breast tissue and lymph nodes obtained from breast cancer patients. This illustrates the application of the protocol in a new biological and clinical context, as human breast and lymph node tissues differ substantially from brain tissues in their composition, architecture, and optical properties. Whole FFPE tissue blocks are deparaffinized in liquid paraffin and xylene, bleached through methanol dehydration and a subsequent hydrogen peroxide incubation, and stained with a diverse set of small molecule dyes. As a next step, the tissues are delipidated and subjected to refractive index matching with ethyl cinnamate to reach optimal tissue transparency. Importantly, the applied dehydration and delipidation nicely preserve the morphology of the tissue, and the shrinkage is minimal. This allows reliable 3D imaging of large tissue samples within a timeframe of 10 days, providing clinicians and biomedical researchers with a more holistic view of the FFPE tissue sample and its spatial organization.

Tumor mechanical microenvironment, particularly extracellular matrix stiffness, plays a critical role in regulating cancer cell behavior, including proliferation, quiescence, and drug resistance. Conventional 2D culture or stiff 3D scaffolds fail to recapitulate the physiological soft (normal) or pathologically stiff (tumoral) mechanical niches. Here, we present a detailed protocol for establishing a tunable 3D tumor spheroid culture system using sodium alginate–based hydrogels crosslinked with calcium ions at different concentrations to achieve soft or stiff conditions that mimic normal colon and colorectal cancer tissues, respectively. We describe the step-by-step procedures for fabricating stiffness-tunable hydrogels, culturing colorectal cancer spheroids, releasing spheroids for downstream analysis, and performing immunohistochemical staining on intact spheroids. This protocol enables the reproducible investigation of mechanosensitive pathways and drug resistance mechanisms in a physiologically relevant 3D context.

Cell therapy holds great promise for cancer immunotherapy, but its clinical efficacy is severely hindered by poor post-transplant cell survival, low homing efficiency, and host immune clearance. To address these challenges, this study develops a novel light-controlled immunotherapy strategy that integrates a red/far-red light genetic switch with single-cell encapsulation engineering. The red/far-red light (660/730 nm) reversible regulatory system enables precise spatiotemporal control over the expression of therapeutic proteins in engineered cells (e.g., CAR-T or engineered HEK 293T cells), allowing on-demand activation of anti-tumor immune responses. On this basis, a mild enzyme-mediated single-cell encapsulation technique is further employed to rapidly form a protective hydrogel coating in situ on the cell surface, thereby enhancing the survival of transplanted cells under hostile in vivo microenvironments. This strategy combines precise gene expression regulation with physical protection, improving therapeutic outcomes without the need for genomic modification of the cells. It provides a new paradigm for developing safe, controllable, and efficient cancer immunotherapy.

Organoids as a drug discovery platform represent an emerging field that continues to refine its tools. NanoBRET (bioluminescence resonance energy transfer) has emerged as a proximity-based and highly sensitive assay to measure protein–protein and protein–ligand interactions. NanoBRET assays were developed and are currently used for 2D cell line experiments. Here, we present the development of the first organoid-compatible Nanoluciferase (Nluc) for 3D model systems. We utilise the Nluc for NanoBRET assays to test drug–target engagement. We describe steps for seeding, transfecting, and replating of mouse colorectal cancer organoids. In addition, we provide detailed procedures for the NanoBRET assay. Various lines of evidence have shown significant difference in drug response between 2D human cell lines and 3D model systems, including patient-derived organoids. Our protocol provides a template for measuring this difference in the context of drug–target engagement.

Anoikis resistance, or the ability of cancer cells to evade cell death triggered by immediate detachment from the extracellular matrix, is a critical established hallmark of metastatic cancer. While suspension culture models have been used to study anoikis, most focus on defined single time points or prolonged suspension that may not recapitulate the effects of repeated stress that tumor cells experience during metastatic dissemination. Here, we describe a detailed protocol for generating anoikis-resistant (AnR) cancer cells that have adapted to such stress through exposure to repeated cycles of suspension stress on poly-HEMA-coated plates, followed by recovery under standard attached conditions. The protocol includes methods for determining baseline anoikis sensitivity, generating AnR cells over 7–9 attachment-detachment cycles, assessing the stability and reversion of the anoikis-resistant phenotype, and characterizing AnR cells using Live/Dead staining of spheroids, flow cytometry–based apoptosis assays, and immunofluorescence for proliferation markers. This approach produces a non-genetic, reversible anoikis-resistant state that models the adaptive transcriptional reprogramming underlying metastatic progression, providing a reproducible and physiologically relevant in vitro system for studying anoikis resistance mechanisms and evaluating therapeutic strategies for prevention and reversal of such adaptations.

Cyclic peptides are emerging as a promising class of recognition modules for chimeric antigen receptor (CAR) engineering. Compared with single-chain variable fragment (scFv)-based CARs, disulfide-directed multicyclic peptides (DDMPs) represent a novel alternative, offering a markedly smaller molecular size (<5 kDa), enhanced structural stability through disulfide-directed cyclization, and broad tolerance to sequence diversification that supports systematic affinity and specificity optimization. DDMP-based CAR T cells leverage these properties to mediate antigen-dependent cytotoxicity while exhibiting an attenuated cytokine secretion profile, supporting the development of potentially safer immunotherapies for solid tumors. Here, we present a comprehensive workflow spanning CAR construct design and generation through in vitro and in vivo functional evaluation. While DDMPs are used as the exemplar recognition module, sections A and C–L of the protocol are directly applicable to any CAR format, including scFv- and nanobody-based designs with minimal modifications, making the workflow accessible to the broader CAR T-cell research community. The protocol includes the generation of Jurkat NFAT reporter cell lines and luciferase-expressing tumor target lines, which are widely used in different assays. Together, these standardized readouts enable rigorous, objective comparison of CAR T-cell efficacy and safety across tumor models.

Epigenetic modifications play essential roles in regulating gene expression and maintaining cellular identity. Accumulating evidence suggests that chemical agents can contribute to carcinogenesis through epigenetic alterations, such as changes in DNA methylation and histone modifications, even in the absence of direct DNA damage. Here, we have developed a simple, cost-effective, and quantitative reporter assay, termed the epi-TK assay, to evaluate chemically induced epigenetic alterations. The assay is built upon the thymidine kinase (TK) gene mutation assay, a standardized and widely used in vitro genotoxicity assay for chemical safety evaluation. This system is based on an engineered human lymphoblastoid cell line (mTK6), in which the promoter region of the endogenous housekeeping TK gene is site-specifically methylated using epigenome-editing technology, resulting in stable transcriptional repression. Following chemical exposure, epigenetic perturbations at the TK locus are detected by culturing cells under hypoxanthine–aminopterin–thymidine selection and quantifying the frequency of TK revertant colonies, which reflects restoration of TK gene expression. Using the DNA methyltransferase 1 inhibitor GSK3484862 as a model compound, this protocol demonstrates that the epi-TK assay enables sensitive and quantitative detection of epigenetic state transitions. Importantly, this assay allows bi-directional detection of epigenetic changes, including DNA demethylation events and broader alterations in histone modification landscapes. Together, the epi-TK assay provides a practical and quantitative platform for evaluating epigenetic toxicity, with potential applications in chemical safety assessment frameworks.