A375 / A375-luc Orthotopic Mouse Model Service for Melanoma

The A375 / A375-luc orthotopic mouse model of melanoma engrafts the canonical BRAF V600E human melanoma into the skin site, where intradermal tumors grow rapidly and disseminate to lymph nodes, lungs, and brain, with luciferase bioluminescence quantifying primary and metastatic burden in real time. Alfa Cytology delivers this platform as an integrated preclinical service: authenticated CRL-1619 banking, A375-luc reporter qualification, intradermal or flank engraftment in nude or NRG recipients, scheduled whole-body and ex vivo imaging, BRAF/MEK-pathway dosing, and endpoints spanning tumor metrics, metastasis mapping, resistance-marker analysis, and histopathology.

Overview of A375 / A375-luc Orthotopic Mouse Model for Melanoma

A375 is a human malignant melanoma cell line established in 1973 by D. J. Giard and colleagues (Journal of the National Cancer Institute, 51:1417) from the skin of a 54-year-old female patient, and deposited with ATCC as CRL-1619. It is the most cited human melanoma line in the world, and its genetics explain why: a BRAF V600E mutation drives constitutive MAPK signaling with high phospho-ERK, while NRAS remains wild-type and TP53 is wild-type and functional — an unusually clean oncogene-addicted background. Lesions at the CDKN2A locus (undetectable p16 mRNA and homozygous deletion of ARF) complete the picture, and the cells carry a hypotriploid karyotype with modal chromosome number 62. Notably for a melanoma line, A375 is amelanotic, so its tumors are non-pigmented and grow rapidly in immunodeficient mice — a phenotype that made it the substrate on which the first BRAF inhibitor responses were mapped and on which vemurafenib and dabrafenib benchmarking continues today.

Fig 1. Translational imaging study in which female nude mice received intratracheal A375-Luc cells to establish a lung-localized melanoma model, followed by ¹¹¹In-DOTAGA-zampilimab and longitudinal BLI/SPECT imaging at 48/72/96 hours showing declining A375-Luc pulmonary radiance over time with corresponding SPECT-derived target occupancy quantification. (Bellaye, Pierre-Simon, et al., 2016)

Orthotopically, A375 and its derivatives are implanted intradermally, recreating the cutaneous site of the original tumor. Classic orthotopic work with GFP-tagged A375 showed uniform take (22 of 22 implants) with spontaneous metastatic seeding to lung and brain, and modern luciferase-tagged variants extend the design: an in vivo-selected A375 IV Luc2 line injected intradermally at 5×10⁴ cells forms melanomas in NRG mice that disseminate to lymph nodes within five weeks, with GFP-positive lung deposits detectable by confocal imaging at necropsy. Luciferase-only and dual A375-LUC-GFP derivatives serve complementary roles: the former quantifies whole-body and organ burden by bioluminescence after D-luciferin administration, while the latter enables fluorescence-activated sorting of GFP-positive human tumor cells away from murine stroma for resistance-mechanism studies. Published pharmacology on the platform runs from vemurafenib monotherapy through abemaciclib-plus-vemurafenib intermittent regimens and tubulin-inhibitor combinations, with drug-selected clones such as VR1 sustaining phospho-ERK under BRAF inhibition — making the A375-luc orthotopic model the definitive human-melanoma system for BRAF-pathway drug development, combination-schedule optimization, and resistance research.

Cell Line Information: A375 / A375-luc

A375 is the reference human melanoma cell line for BRAF-pathway research, with fully documented genetics and STR identity. Its luciferase derivatives extend it into imaging-guided orthotopic studies. Core attributes follow.

Our Services

A375’s value as the human melanoma benchmark depends on protecting a simple fact — a clean, BRAF-addicted genotype — from the drift that heavy laboratory use invites. Alfa Cytology works from authenticated CRL-1619 master banks with documented STR identity, caps in vitro passage before animal use, and releases A375-luc lots only after uniform in vitro luminescence is confirmed. Engraftment follows the orthotopic intradermal route with imaging-verified randomization, vemurafenib-class reference arms are available, and terminal workups separate GFP-positive human tumor cells from murine stroma for resistance analysis on dual-labeled designs. From first baseline scan to final histopathology, Alfa Cytology runs the A375 / A375-luc orthotopic workflow as one accountable preclinical program.

Workflow of A375 / A375-luc Orthotopic Mouse Model Construction

The A375-luc orthotopic workflow pairs intradermal surgical technique with imaging-native metastasis tracking and resistance-aware endpoints. The sequence below is the standard construction-to-analysis flow.

1. Authentication and reporter release: A375 / A375-luc stocks are expanded from authenticated master banks at capped passage, mycoplasma-tested, STR-verified, and released only after uniform firefly luciferase expression (and GFP signal for dual-labeled variants) is confirmed in vitro.
2. Inoculum preparation: Log-phase adherent cultures are trypsinized, washed, counted, and resuspended in sterile PBS at the study dose (5×10⁴ cells for the A375 IV Luc2 design; 1×10⁶ to 5×10⁶ cells for standard orthotopic or flank cohorts).
3. Recipient preparation: Nude or NRG mice are acclimatized, body-weight stratified, shaved, and positioned under anesthesia with analgesia for intradermal or flank procedures.
4. Orthotopic intradermal implantation: The suspension is delivered intradermally into the dorsal skin using a fine needle, creating a defined bleb at the injection site; flank cohorts are implanted in parallel where designed.
5. Baseline imaging and randomized allocation: Baseline bioluminescence after D-luciferin documents engraftment; once signals are established, cohorts are randomized with matched photon flux and dosing begins — BRAF inhibitors, MEK inhibitors, combinations, or anti-metastatic agents.
6. Longitudinal primary and metastasis monitoring: Whole-body imaging tracks primary tumor signal on a fixed schedule, with focused imaging of the axillary/brachial regions for lymph node spread; body weight and clinical signs are scored throughout.
7. Endpoint analysis and resistance workup: At termination, primary tumors and lymph nodes are excised and weighed, lungs, brain, liver, and spleen are imaged ex vivo and inspected for deposits, histopathology confirms melanoma involvement, and on dual-labeled designs GFP-positive human tumor cells are sorted from stroma for resistance-mechanism analysis.

Fig 2. A375 / A375-luc orthotopic mouse model construction workflow.

Case Study-A375 / A375-luc Orthotopic Mouse Model Development

Alfa Cytology has implemented the A375 orthotopic platform in preclinical programs spanning BRAF/MEK inhibitor benchmarking, combination-schedule optimization, and anti-metastatic agent evaluation. In these studies, serial bioluminescence tracked primary tumor growth alongside early lymph node signal, randomized arms started from matched photon flux, vemurafenib-class reference arms anchored cross-study comparability, and terminal analyses combined tumor and node weights with ex vivo organ imaging and histopathology. On dual-labeled designs, GFP-positive human tumor cells recovered from relapsed lesions were sorted from murine stroma for downstream resistance-mechanism profiling. Program-specific parameters and outcomes are retained as sponsor-confidential deliverables, and representative outlines can be furnished upon request.

Why Choose Alfa Cytology?

The world’s most cited melanoma line deserves benchmark-grade execution, and Alfa Cytology’s A375-luc service is built to deliver it. Sponsors benefit from:

• Authenticated CRL-1619 stocks with documented STR identity and capped passage before animal use.
• Reporter lines qualified for uniform luciferase (and GFP) expression before release into studies.
• True orthotopic intradermal technique with imaging-verified engraftment and randomization.
• Metastasis-native imaging readouts covering lymph nodes, lungs, brain, liver, and spleen.
• Optional dual-label designs enabling FACS isolation of human tumor cells for resistance studies.
• BRAF-pathway reference-arm capability aligned with published vemurafenib-class regimens.
• Single-point study accountability with per-timepoint reporting from implantation through final analysis.

Contact Us

Developing a BRAF-pathway, combination-schedule, or anti-metastatic program in melanoma? Contact us to discuss engraftment route, imaging cadence, and endpoint design for the A375 / A375-luc orthotopic platform. You can also reach out to Alfa Cytology for a customized proposal and quotation — our team will respond with a study design aligned to your therapeutic strategy.

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