Splenic/Portal-Vein Liver Metastasis Xenograft

Reagents and chemicals

• Sterile cell-suspension vehicle is used to prepare viable tumor cells or tumor organoids for splenic or portal-vein injection, and D-luciferin is used when luciferase-expressing cells or organoids are monitored by bioluminescence imaging^[2][3][5].

• Hemostatic material is used in direct portal-vein injection protocols to control bleeding at the injection site, and small injection volumes with fine needles were used in an optimized breast-cancer liver-metastasis portal-vein protocol^[3].

Antibodies, probes, dyes, or kits

• Firefly luciferase and fluorescent reporters are used to monitor tumor growth kinetics in vivo and to confirm liver tumor burden ex vivo^[1][2][5][7].

• H&E staining and immunohistochemistry are used to confirm hepatic tumor lesions and evaluate tumor histology or stromal features, including the desmoplastic reaction in colorectal cancer organoid portal-vein models^[2][6].

Equipment and instruments

• Required equipment includes cell-culture tools, cell counter or viability-assay equipment, anesthesia and surgical instruments, microsyringes or fine needles for splenic or portal-vein injection, hemostatic materials, and necropsy tools for liver collection^[1][2][3].

• Bioluminescence or fluorescence imaging systems are used for longitudinal monitoring when labeled cells are implanted, while ultrasound-guided injection systems can be used for direct portal-vein injection and tumor surveillance in specialized protocols^[1][4][5][7].

Preparation Steps

• Select immunodeficient mice for human xenograft cells or patient-derived organoids and syngeneic mice for mouse tumor cells; reported examples include human colorectal cancer cells or organoids in immunodeficient mice, CT26 or MC38 murine colorectal cancer cells in syngeneic mice, and mammary tumor cells in BALB/c mice^{[1][2][3][5][6]}.

• Prepare a single-cell or organoid suspension with consistent viable input across groups, confirm reporter expression when optical imaging is planned, and use the same route, cell number, and endpoint across comparison groups because liver tumor burden depends on tumor-cell type, injected cell number, and delivery route^[1][2][3][7].

Operation Steps

• For splenic injection, anesthetize the mouse, expose the spleen, inject tumor cells into the spleen so they drain through the portal circulation, and either leave the spleen in place or remove it depending on the validated model; published splenic models have used splenectomy to reduce splenic tumor growth, while uveal melanoma work found no significant tumor-growth difference between splenic injection with and without splenectomy^[1][5][7].

• For direct portal-vein injection, anesthetize the mouse, surgically expose or ultrasound-visualize the portal vein, inject tumor cells or organoids directly into the portal vein, achieve hemostasis at the injection site, and close the abdomen according to the approved surgical protocol^[2][3][4].

• Monitor liver tumor development by in vivo bioluminescence or fluorescence imaging when labeled cells are used, and collect livers at the predefined endpoint or humane endpoint for gross tumor assessment, ex vivo imaging, histology, immunostaining, or molecular assays^[1][2][5][7].

Data Acquisition and Analysis

• Successful liver metastasis modeling is indicated by liver-localized imaging signal, visible hepatic nodules, increased liver tumor burden, or histologic confirmation of tumor growth in hepatic tissue^[1][2][5][6].

• Interpret splenic and portal-vein injection models as liver-colonization models rather than full spontaneous-metastasis models because they bypass primary tumor formation, local invasion, and intravasation; portal-vein injection can reduce confounding from splenic tumor growth or splenectomy when the question focuses on liver colonization^[2][3][8].

• Use vehicle or sham-surgery controls when procedure-related liver effects are relevant, use a validated liver-colonizing cell line as a positive model-control, randomize mice before treatment studies, and confirm optical imaging results with ex vivo liver imaging or histology^[1][2][3][5].

Problem: Liver metastasis is weak or inconsistent.

• Possible Cause: Tumor-cell type, injected cell number, cell viability, host strain, or delivery route does not support reliable liver colonization.
• Literature-supported Solution: Pilot the injected cell number and endpoint for the selected tumor line and host strain, because liver tumor growth and survival varied with injected cell number in splenic uveal melanoma models and with metastatic potential in portal-vein breast-cancer models^[3][7].

Problem: Splenic injection causes confounding splenic tumor growth or immune alteration from splenectomy.

• Possible Cause: Tumor cells may grow at the injection site, and spleen removal can alter immune context.
• Literature-supported Solution: Use direct portal-vein injection when liver-specific colonization without splenectomy is required, because portal-vein models were developed to deliver cells first and directly to the liver without removal of the spleen^[2][3].

Problem: Imaging signal is uncertain or does not prove liver metastasis.

• Possible Cause: In vivo optical signal provides localization and kinetics but does not by itself define histologic tumor architecture.
• Literature-supported Solution: Confirm liver signal using ex vivo liver imaging, gross liver assessment, and histology, as used in bioluminescence-based colorectal liver metastasis and portal-vein organoid models^[2][5].