AZ1366

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Flow cytometric DNA-content cell-cycle staining measures the fluorescence intensity of DNA-bound fluorochromes in single cells or nuclei to estimate DNA content distributions, allowing assignment of populations to G0/G1, S, and G2/M phases by DNA histogram deconvolution. Propidium iodide (PI) intercalates into DNA, and PI fluorescence is proportional to cellular DNA content when staining is performed under conditions that make DNA accessible and minimize non-DNA signal. Cells with G2/M DNA content are expected to show approximately twice the fluorescence intensity of G0/G1 cells, while S-phase cells occupy intermediate fluorescence values. PI-based DNA-content analysis can also detect cells with fractional DNA content, often reported as sub-G1, when DNA fragmentation and extraction during staining reduce retained DNA signal in apoptotic cells. DAPI is an alternative DNA fluorochrome for univariate DNA-content analysis, while bivariate approaches combining DNA content with proliferation

Patient-derived xenograft (PDX) models are generated by engrafting primary human tumor tissue directly into immunodeficient mice, allowing in vivo propagation of patient tumor biology without initial in vitro adaptation. These models are used to preserve key histopathological and molecular characteristics of the original tumor and enable assessment of tumor growth dynamics and therapeutic response in a living organism. The biological readout is tumor engraftment and subsequent growth in the murine host, which reflects the ability of human tumor cells to survive, vascularize, and expand in an immunocompromised microenvironment.

Patient-derived orthotopic xenograft (PDOX) modeling implants fresh patient tumor tissue or patient-derived tumor cells into the anatomically corresponding organ or tissue site of immunodeficient mice, usually by surgical orthotopic implantation, to preserve patient tumor histology, local microenvironmental context, invasion, metastatic behavior, and treatment-response features better than subcutaneous implantation. PDOX readouts include tumor engraftment, orthotopic tumor growth, local invasion, metastasis, recurrence after resection, histologic similarity to the donor tumor, biomarker retention, molecular concordance, survival, and response or resistance to therapy. PDOX models are used for preclinical drug testing and individualized therapy evaluation, but engraftment success varies by tumor type and specimen quality.

Cell-cycle analysis by flow cytometry measures DNA content in single cells to estimate the fraction of cells in G0/G1, S, and G2/M phases. Propidium iodide intercalates into DNA, and after RNA removal with RNase, fluorescence intensity reflects cellular DNA content: 2N cells are assigned to G0/G1, cells between 2N and 4N to S phase, and 4N cells to G2/M. DNA-content analysis alone cannot reliably separate G0 from G1 or G2 from M. Ki-67 can distinguish quiescent G0 cells from cycling cells, EdU or BrdU incorporation marks active DNA synthesis in S phase, and phospho-histone H3 staining identifies mitotic cells within the 4N population.

Splenic and portal-vein liver metastasis xenograft models deliver tumor cells into the portal circulation so that cells reach the liver first and form hepatic metastatic lesions; splenic injection uses the spleen as an access route to the portal system, while direct portal-vein injection introduces cells into the portal vein without requiring splenectomy. The assay detects liver colonization, intrahepatic tumor growth, tumor distribution, treatment response, survival, and liver-metastasis microenvironment changes; readouts include bioluminescence or fluorescence imaging, gross liver nodule counts, liver weight or tumor burden, histology, and survival.

Subcutaneous cell-line-derived xenograft (CDX) models are established by implanting cultured human cancer cell lines into immunodeficient mice, where the injected cells form localized tumors that can be monitored in vivo as a measure of tumorigenic potential, growth kinetics, and treatment response. These models are widely used in oncology research because they allow reproducible tumor formation and enable comparative assessment of tumor growth between different cell lines or genetic manipulations in a controlled in vivo microenvironment. Subcutaneous implantation of cancer cells in immunodeficient mice is a standard approach for evaluating tumor growth behavior and therapeutic response across multiple cancer types, including prostate, esophageal, pancreatic, and colon cancer models.

Orthotopic cell-line xenograft models involve implantation of human cancer cell lines into the anatomically corresponding organ of immunodeficient mice to reproduce tumor growth within a native microenvironment, enabling more clinically relevant tumor behavior compared with subcutaneous models. These models are widely used because orthotopic placement better recapitulates tumor progression, including invasion and metastatic spread, which are often underrepresented in heterotopic implantation systems. Compared with conventional xenografts, orthotopic implantation is described as more technically complex but provides improved simulation of tumor-microenvironment interactions and metastatic behavior, making it particularly valuable for translational oncology research. Surgical orthotopic implantation approaches have been emphasized as enabling faithful reproduction of clinical cancer features, including metastasis and disease progression patterns that align with the tumor’s organ of origi

Intraperitoneal (IP) or peritoneal dissemination xenograft models are based on the introduction of human cancer cells into the peritoneal cavity of immunodeficient mice, where they attach to peritoneal surfaces, form multicellular aggregates or spheroids, and progressively generate disseminated tumor nodules that mimic advanced peritoneal metastatic disease. These models are widely used to study ovarian cancer progression, tumor-microenvironment interactions, and intraperitoneal therapeutic responses, often incorporating bioluminescence or fluorescence imaging to longitudinally monitor tumor burden in vivo. The biological principle relies on the capacity of tumor cells such as SKOV3 or related ovarian carcinoma lines to survive in suspension, aggregate within ascites-like fluid, adhere to mesothelial surfaces, and invade peritoneal organs, thereby recapitulating human peritoneal carcinomatosis patterns observed in advanced disease.

The following protocol uses the MTT colorimetric assay as a classic literature-established method for assessing cell viability/metabolic activity in cultured mammalian cells. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by metabolically active cells to a colored formazan product; the amount of formazan is quantified spectrophotometrically and provides an indirect measure of metabolically active viable cells. Importantly, MTT reduction reflects cellular oxidoreductase/metabolic activity rather than an absolute direct count of living cells, so changes in cellular metabolism can alter the signal independently of cell number.