Nalm-6 / Nalm-6-luc Orthotopic Mouse Model Service for Leukemia

The Nalm-6 / Nalm-6-luc orthotopic mouse model disseminates a CD19-positive human B-cell precursor acute lymphoblastic leukemia through the bone marrow, spleen, and blood of immunodeficient mice — and, characteristically, onward to the central nervous system — recreating the systemic and sanctuary-site biology that makes B-ALL so difficult to eradicate. Alfa Cytology delivers this benchmark immunotherapy model end-to-end: authenticated Nalm-6-luc banks with monoclonal luciferase quality control, tail-vein inoculation with flux-matched randomization, scheduled bioluminescence imaging in vivo, and study designs compatible with CAR-T cells, bispecific engagers, and CAR-NK products.

Overview of Nalm-6 / Nalm-6-luc Orthotopic Mouse Model for Leukemia

Nalm-6 was established in 1976 from the peripheral blood of a 19-year-old man with acute lymphoblastic leukemia in relapse; typed "non-T, non-B" at origin, it was soon characterized by Hurwitz and colleagues as a leukemic line of the pre-B phenotype, and it remains the reference model for precursor B-cell ALL. Its immunophenotype is precisely what modern immunotherapy requires: CD19 is expressed at high levels, CD10 (CALLA) and terminal deoxynucleotidyl transferase (TdT) are positive, CD22 is endogenously expressed, CD34 is absent, and CD20 and BCMA are barely detectable, while TP53 remains wild-type. Injected intravenously into NSG or NOD-SCID mice, Nalm-6 engrafts in bone marrow, spleen, liver, and lymph nodes, with histology showing sheets of small round lymphoblasts positive for TdT, CD19, and CD10; notably, the cells also display neurotropism, accumulating in the meninges and brain parenchyma — a faithful mimic of CNS sanctuary disease. The line responds predictably to the classical B-ALL armamentarium, including vincristine, dexamethasone, methotrexate, and L-asparaginase.、

Fig 1. Longitudinal bioluminescence and tissue-based characterization of a NALM6-Luc B-ALL xenograft, demonstrating systemic leukemia progression and leukemic infiltration of bone marrow, spleen, and peripheral blood. (Huang, Yan, et al., 2025)

What has made Nalm-6 — and especially Nalm-6-luc — indispensable is its performance as an immunotherapy testbed. After intravenous challenge with 10⁶ luciferase-tagged cells, bioluminescence is measurable within 3 days at roughly 4–6 × 10⁵ photons per second, rises to 2–4 × 10⁶ p/s by day 7, and reaches the 2–3 × 10⁹ p/s endpoint between days 21 and 26 — a wide, well-characterized therapeutic window that accommodates CAR-T infusions on days 3 to 14, repeated CAR-NK dosing, bispecific antibodies, and antibody–drug conjugates. Because CD19 and CD22 are both natively present, antigen-escape studies can be built by spiking in CD19-negative or CD22-negative subpopulations; PD-L1-overexpressing variants model checkpoint-enriched disease; and tumor rechallenge experiments test CAR durability. The cranial signal that accompanies systemic disease further enables quantification of CNS involvement by comparing cranial to whole-body flux — a capability few solid tumor models can offer. Reporter configuration matters: as established for luciferase-tagged leukemia lines, monoclonal selection of high-signal clones in vitro is recommended for uniform, quantitative bioluminescence in vivo, and dual luciferase–GFP reporters allow both imaging and single-cell flow cytometric tracking.

Cell Line Information: Nalm-6 / Nalm-6-luc

Nalm-6 (DSMZ ACC 128; ATCC CRL-3273) grows in suspension in vitro in RPMI-1640 supplemented with 10% fetal bovine serum, expanding rapidly and tolerating cryopreservation and thaw with consistent recovery. Its defined antigen profile — abundant CD19, endogenous CD22, and negligible CD20 or BCMA — makes it the default target line for CD19-directed CAR-T programs and a natural fit for CD22 bispecifics and CAR-NK products, while its p53-competent, DNA-repair-proficient genome has made it a staple of radiobiology research as well. Genomic resources are extensive: the line belongs to the LL-100 blood cancer cell line panel with publicly available whole-exome and RNA-sequencing data. The Nalm-6-luc derivative carries a firefly luciferase reporter (luciferase–GFP dual reporters are also in common use), with monoclonal, high-signal clones recommended to keep imaging signals uniform across animals and studies.

Our Services

An immunotherapy study lives or dies by the quality of its imaging: inconsistent luciferase expression or unmatched starting burdens can erase real therapeutic differences. Alfa Cytology runs the Nalm-6 platform with monoclonal reporter quality control, IVIS-quantified flux-matched randomization, and calendar-driven imaging from engraftment through rechallenge, then couples the imaging data with peripheral-blood flow cytometry for leukemia load and CAR persistence — one continuous dataset rather than fragments assembled after the fact.

Workflow of Nalm-6 / Nalm-6-luc Orthotopic Mouse Model Construction

The Nalm-6 orthotopic procedure has been standardized across hundreds of published immunotherapy studies, giving it an unusually tight set of operating parameters. The workflow below follows the validated configuration, from reporter-verified cell preparation through survival analysis.

1. Cell Expansion and Quality Control: Nalm-6 or Nalm-6-luc cells are expanded in vitro in RPMI-1640 + 10% FBS to the logarithmic phase; batches are confirmed mycoplasma-negative and STR-authenticated, and luciferase-tagged lots are monoclonal, high-signal populations — with fluorescence-activated sorting applied to dual luciferase–GFP reporters.
2. Host Selection and Conditioning: Female NSG mice, 6–8 weeks of age, are acclimatized under barrier conditions; where study design calls for enhanced marrow engraftment, animals receive low-dose sub-lethal irradiation, and electronic transponders are implanted for individual identification one day before tumor inoculation.
3. Intravenous Inoculation: Cells are washed twice in cold sterile PBS and resuspended immediately before use; 5 × 10⁴–10⁶ Nalm-6-luc cells in 100–200 µL are injected into the tail vein, with day 0 recorded for the treatment calendar (a subcutaneous 10⁶-cell configuration is available for localized disease questions).
4. Engraftment Verification and Baseline Imaging: Whole-body bioluminescence imaging in vivo is performed within the first three days after D-luciferin administration; a flux of 4–6 × 10⁵ photons/s confirms engraftment, and animals are stratified by measured signal.
5. Flux-Matched Randomization and Treatment: Mice are randomized using IVIS-quantified photon flux so all arms begin with equivalent leukemia burdens; effector cells or biologics — for example, 3–10 × 10⁶ CAR-T cells, repeated CAR-NK doses, or bispecific antibody schedules — are administered on the client-defined day, typically between days 3 and 14.
6. Longitudinal Imaging and Special Readouts: Tumor burden is imaged twice weekly in vivo and quantified as total flux; cranial-to-whole-body signal ratios are computed to monitor CNS sanctuary involvement, and tumor rechallenge with fresh Nalm-6 cells is performed at defined time points to assess CAR durability.
7. Endpoint Analysis and Reporting: Peripheral blood is sampled serially and at termination for flow cytometric enumeration of human CD45-positive leukemia cells and CAR-positive T cells (including CD45RA, CD62L, and CCR7 phenotyping); spleen, marrow, liver, and brain are imaged ex vivo, sectioned for TdT/CD19/CD10 immunohistochemistry, and compiled with Kaplan–Meier survival analysis into the final report.

Fig 2. Nalm-6 / Nalm-6-luc orthotopic mouse model construction workflow.

Case Study-Nalm-6 / Nalm-6-luc Orthotopic Mouse Model Development

The Nalm-6 platform is the most widely cited preclinical B-ALL immunotherapy system in the literature, and it has anchored a broad range of Alfa Cytology client programs — CD19 and CD22 CAR-T benchmarking, bispecific engager dosing studies, CAR-NK evaluation, antigen-escape and PD-L1-variant studies, rechallenge durability designs, and CNS-penetration assessment. Across these programs, readouts have combined quantitative bioluminescence kinetics, peripheral-blood flow cytometry for leukemia and effector-cell persistence, and ex vivo organ imaging with survival analysis. De-identified case summaries and representative data packages are available on request.

Why Choose Alfa Cytology?

Nalm-6 is a demanding model to run well — its value lies in quantitative imaging, precise randomization, and effector-cell timing. Alfa Cytology has engineered its service around those exacting requirements.

• The reference B-ALL immunotherapy model, with published, reproducible bioluminescence kinetics that anchor every study against a known benchmark.
• Monoclonal, high-signal Nalm-6-luc banks with fluorescence-sorted dual reporters available for imaging-plus-flow designs.
• IVIS-quantified, flux-matched randomization that eliminates starting-burden bias between treatment arms.
• Native CD19 and CD22 expression, with CD19-negative, CD22-negative, and PD-L1 variant populations available for escape-mechanism studies.
• CNS neurotropism enabling cranial-flux quantification for sanctuary-site disease and BBB-penetration questions.
• Proven compatibility with CAR-T, CAR-NK, bispecific, and ADC dosing schedules, including rechallenge designs.
• Serial peripheral-blood flow cytometry integrated with imaging for leukemia load, CAR persistence, and T-cell phenotype readouts.

Contact Us

If your next B-ALL program involves a CAR construct, a bispecific engager, or a CAR-NK product, the Nalm-6 / Nalm-6-luc orthotopic model is the system your reviewers will expect to see — and Alfa Cytology can have it running to specification. Contact us to discuss antigen-variant options, effector-cell dosing formats, and imaging schedules, and request a tailored study quotation today.

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