Hepa1-6 Metastatic Tumor Model Service for Liver Cancer

The Hepa1-6 metastatic tumor model is a murine hepatocarcinoma system in which tumor cells of C57L origin grow in immunocompetent C57BL/6 hosts, preserving full immune competence while supporting hepatic, splenic, and pulmonary metastasis formats. Alfa Cytology delivers Hepa1-6 metastasis programs from authenticated cell preparation through route-specific implantation, scheduled imaging, and immune-informed endpoint analysis for liver cancer drug development.

Overview of Hepa1-6 Metastatic Tumor Model for Liver Cancer

Hepa1-6 is a derivative of the BW7756 hepatoma that arose in a C57/L mouse and was adapted to culture by G.J. Darlington in the early 1980s (ATCC CRL-1830). The cells grow as adherent, epithelial-like monolayers with a population doubling time of about 24–30 hours and continue to produce liver-lineage products—albumin, alpha-fetoprotein, alpha-1-antitrypsin, and amylase—alongside cytochrome P450 enzymes, which gives the line value beyond oncology in drug-metabolism and hepatotoxicity work. Repository testing confirms the cells are negative for ectromelia virus, and their relatively low immunogenicity allows them to engraft in fully immunocompetent animals, where they have been described as capable of forming tumors both subcutaneously and orthotopically.

Fig 1. In vivo bioluminescence and histological characterization of the Hepa1-6 orthotopic metastatic model: longitudinal BLI of primary liver tumors under control, norathyriol, anti-PD-1, and combination treatment; ex vivo fluorescence of liver/lung; gross specimens; and lung H&E at 10×/100×/400× magnification confirming metastatic foci and treatment-induced suppression. (Lan, Tian, et al., 2025)

That host compatibility is the model’s core asset. Because C57L and C57BL/6 share the same MHC background, Hepa1-6 can be implanted into C57BL/6 mice without rejection, leaving T, B, and NK compartments intact—an arrangement that has made the line a routine testbed for hepatocellular carcinoma immunotherapy, including NK-cell and IL-15 axis studies, oncolytic viruses, vaccine platforms, and cellular therapies. Subcutaneous inoculation of about 5 × 10⁶ cells produces measurable flank tumors within roughly two weeks; intrahepatic injection of about 2 × 10⁶ cells establishes an orthotopic liver tumor; intrasplenic delivery routes cells through the portal circulation toward liver colonization; and tail-vein injection seeds the lung. Luciferase-tagged derivatives maintained under puromycin selection allow these patterns to be followed non-invasively by whole-body imaging.

Cell Line Information: Hepa1-6

For routine passage, the cells are grown in DMEM containing 10% serum, incubated at 37 °C under 5% CO₂, and split 1:3 to 1:8 after trypsin-EDTA detachment two or three times weekly; a defined serum-free alternative based on a DMEM/Waymouth blend with selenium at 3 × 10^-8 M has also been described. The line tolerates transfection well and is documented as a suitable transfection host, which simplifies the construction of reporter or engineered variants. Chromosome counts cluster between 59 and 65, consistent with an aneuploid hepatoma karyotype.

Our Services

Because Hepa1-6 grows in hosts with intact immunity, Alfa Cytology treats immune context as part of the experimental design: checkpoint agents, cytokine-armed constructs, cellular therapies, and oncolytic viruses can each be evaluated against the same tumor while standard efficacy readouts remain available. Engagements are structured so that in vitro qualification feeds directly into in vivo efficacy evaluation and survival arms, with flow-cytometric immune profiling and hepatotoxicity panels included as the design requires.

Workflow of Hepa1-6 Metastatic Tumor Model Construction

Hepa1-6 metastasis studies proceed from cell qualification to route selection, implantation, and immune-informed endpoint analysis. The sequence below describes the most common configurations; doses and schedules are tailored to each sponsor’s route and readout requirements.

1. Cell qualification and expansion: Hepa1-6 stocks are verified by STR profiling, confirmed ectromelia-negative and mycoplasma-free, and in vitro expanded in DMEM-based medium with passage history recorded.
2. Route and dose definition: The metastatic format is fixed—subcutaneous flank for routine efficacy, intrahepatic injection for orthotopic growth, intrasplenic delivery for portal-route liver colonization, or tail-vein injection for pulmonary dissemination—with cell doses and any supportive matrix specified in the plan.
3. Implantation: Typical inocula are 5 × 10⁶ cells subcutaneously, about 2 × 10⁶ cells into the liver parenchyma, or 1 × 10⁶ to 2 × 10⁶ cells via spleen or tail vein; luciferase-tagged cells are used where imaging readouts are planned.
4. Establishment monitoring: Tumor take is tracked on a fixed schedule with caliper or IVIS readouts, and cohort enrollment proceeds as tumors cross the predefined size threshold.
5. Randomization and dosing: Cohorts are distributed across vehicle, benchmark, and investigational groups, with immune-modulating agents administered per the sponsor-defined schedule.
6. In-life observations: Tumor burden, body weight, behavior, and clinical chemistry readouts are captured at defined intervals, with humane endpoints enforced throughout.
7. Endpoint and analysis: At study termination, tumors, livers, and lungs are harvested; lesions are scored, histology and IHC are completed, and tumor-infiltrating immune populations are phenotyped before the final report is delivered.

Fig 2. Hepa1-6 Metastatic Tumor Model construction workflow.

Case Study-Hepa1-6 Metastatic Tumor Model Development

Alfa Cytology has conducted Hepa1-6 metastasis programs covering intrahepatic and intrasplenic liver-tumor formats, tail-vein pulmonary dissemination studies, and flank efficacy work supporting immune-modulating constructs such as cytokine-armed therapies and oncolytic agents, with combination activity confirmed in vivo. Readouts have spanned tumor growth, survival, metastatic lesion quantification, and the immune-cell composition of tumor tissue, with hepatotoxicity and clinical chemistry panels added where the design required them; study-specific details can be discussed under confidentiality.

Why Choose Alfa Cytology?

Liver cancer modeling asks for a partner fluent in both tumor biology and immune readouts, since the syngeneic setting is precisely where Hepa1-6 adds value. Alfa Cytology operates the model with that dual focus.

• Immune-competent platform: C57BL/6 hosts preserve T, B, and NK compartments for immuno-oncology questions.
• Route flexibility: subcutaneous, intrahepatic, intrasplenic, and tail-vein formats from one authenticated cell source.
• Imaging-ready: luciferase-tagged derivatives with scheduled IVIS monitoring.
• Hepatic context: liver-lineage marker expression (albumin, AFP) supports HCC-relevant biomarker work.
• Quality and biosafety: STR-verified, ectromelia-negative, mycoplasma-free cells with documented handling.
• Integrated delivery: efficacy, immune profiling, and hepatotoxicity panels consolidated into one report.

Contact Us

Developing a liver cancer candidate that needs an immunocompetent metastatic setting? Contact us to discuss the Hepa1-6 format that fits your program—hepatic, splenic, or pulmonary—and receive a tailored proposal with timelines. Alfa Cytology is ready when you are; reach out to start the conversation.

For research use only. Not intended for any clinical use.