NCI-H23 / NCI-H23-luc Orthotopic Mouse Model Service for Lung Cancer

The NCI-H23 / NCI-H23-luc orthotopic mouse model carries one of the best-characterized genotypes in lung cancer research — homozygous KRAS G12C with homozygous TP53 M246I — into the pulmonary microenvironment, where its moderate drug sensitivity and documented resistance pathways mirror the partial-responder population that defines the clinical reality of KRAS-targeted therapy. Alfa Cytology delivers this NCI-60 panel line with authenticated banking, intercostal orthotopic implantation or validated subcutaneous benchmarking configurations, quantitative luciferase imaging in vivo, and pairing against its acquired-resistant H23AR derivative, so your KRAS G12C inhibitors, combinations, and next-generation resistance strategies are tested against the pharmacology the field already trusts.

Overview of NCI-H23 / NCI-H23-luc Orthotopic Mouse Model for Lung Cancer

NCI-H23 originated in the NCI-Navy Medical Oncology Branch cell line series of the mid-1980s, derived from the pleural effusion of a 51-year-old Caucasian woman with lung adenocarcinoma, and it has since been profiled in the NCI-60 panel for decades — one of the most densely annotated cancer genomes in existence, with public pharmacogenomic datasets stretching back to the earliest molecular-target screens. Its genotype sits at the center of modern NSCLC pharmacology: a homozygous KRAS G12C mutation combined with homozygous TP53 M246I, on an EGFR wild-type background. Importantly, NCI-H23 represents the "partial responder" segment: its sotorasib sensitivity in vitro (IC₅₀ ≈ 3.2 µM) is markedly lower than the hypersensitive NCI-H358 line (≈ 0.13 µM), while adagrasib retains strong potency (IC₅₀ ≈ 200 nM); on-target engagement is cleanly demonstrable through suppression of phospho-ERK and phospho-AKT, caspase-3 and PARP cleavage, topoisomerase IIα degradation, and γ-H2AX DNA-damage foci.

Fig 1. Growth of orthotopic NSCLC of three different cell lines (Upper-NCI-H1703, middle-NCI-H23 and lower-A549) assessed by BLI and CBCT. (Aktar, Rozina, et al., 2019)

In vivo, NCI-H23 xenografts have become the reference testbed of the KRAS G12C era. Subcutaneous tumors respond to sotorasib at 100 mg/kg once daily with clear growth inhibition, while the addition of the pan-RAF–MEK nondegrading molecular glue NST-628 at 2 mg/kg daily converts that modest effect into deep, durable tumor regressions maintained to the end of a 40-day study — one of the cleanest published demonstrations of vertical pathway blockade in this tumor type. Resistance biology is equally well mapped: continuous sotorasib pressure generates the H23AR acquired-resistant subline, in which MET amplification emerges and PI3K-mTOR inhibition (copanlisib) restores sensitivity in NSG xenografts. For central nervous system questions, intracranial NCI-H23-luc brain-tumor xenografts support quantitative bioluminescence evaluation of CNS-penetrant inhibitors against benchmark compounds. Placed orthotopically into the lung by the intercostal technique, NCI-H23-luc adds thoracic bioluminescence kinetics and lung-specific endpoints to this pharmacological pedigree, giving programs a single line that spans benchmark efficacy, combination strategy, resistance, and brain-penetration assessment.

Cell Line Information: NCI-H23 / NCI-H23-luc

NCI-H23 (ATCC CRL-5800; RRID CVCL_1547) is an adherent epithelial line that grows readily in vitro in RPMI-1640 with 10% fetal bovine serum and has been distributed and re-annotated by the community for forty years, making it one of the most reproducible xenograft substrates in oncology. Its homozygous KRAS G12C/TP53 M246I genotype, moderate sotorasib sensitivity, and well-characterized acquired-resistance derivative give it a pharmacological depth that few NSCLC lines match. The NCI-H23-luc variant adds firefly luciferase for quantitative imaging in vivo, with monoclonal selection of high-signal clones in vitro recommended for uniform bioluminescence.

Our Services

KRAS programs succeed or fail on the quality of their reference data: a mismatched genotype or an unverified resistant derivative can quietly invalidate months of work. Alfa Cytology banks NCI-H23 and NCI-H23-luc under continuous authentication, pairs parental and H23AR arms within single studies, executes orthotopic implantation with published intercostal technique, and reports against benchmark dosing regimens — including sotorasib 100 mg/kg once daily and published combination schedules — so your efficacy readouts land on the same scale the field already uses.

Workflow of NCI-H23 / NCI-H23-luc Orthotopic Mouse Model Construction

Orthotopic NCI-H23 construction adapts the validated intercostal microinjection framework to this KRAS G12C reference line, with study arms configured for benchmarking, combination, and resistance questions. The workflow below details construction and execution.

1. Cell Expansion and Quality Control: NCI-H23 or NCI-H23-luc cells are expanded in vitro in RPMI-1640 + 10% FBS to the logarithmic phase; batches are confirmed mycoplasma-negative, STR-authenticated, and KRAS G12C/TP53 M246I genotype-verified, with monoclonal luciferase expression confirmed for tagged lines.
2. Host Selection and Acclimatization: BALB/c nude, NOD-SCID, or NSG mice, typically 5–6 weeks of age, are acclimatized for approximately one week under SPF conditions; animals intended for intracranial configurations receive electronic transponder identification before surgery.
3. Inoculum Preparation: Cells are washed in PBS and resuspended at high concentration (for example, 5 × 10⁷ cells/mL) with 1 mg/mL Matrigel, kept chilled; standard challenges are 10⁶ cells in 20 µL for intercostal implantation or 5 × 10⁶ cells for subcutaneous benchmarking.
4. Orthotopic Implantation: Under isoflurane anesthesia, the left chest is opened through a small incision and a fine needle advanced approximately 3 mm through the intercostal space into the lung parenchyma delivers 20 µL slowly, with the needle held briefly on withdrawal; intracranial configurations use stereotaxic injection of NCI-H23-luc cells into the brain parenchyma at defined coordinates.
5. Closure and Recovery: The skin is closed with wound clips or sutures, and animals recover on a warming platform with analgesia per IACUC protocol; body weight and respiratory status are monitored daily, with weight decline serving as the early sentinel of thoracic tumor growth.
6. Staging and Randomization: NCI-H23-luc tumors are imaged by bioluminescence in vivo within the first week and weekly thereafter; subcutaneous arms are staged by caliper to approximately 200 mm³; animals are randomized to vehicle, benchmark inhibitor (sotorasib 100 mg/kg once daily), and combination or resistant-derivative arms.
7. Treatment and Resistance Monitoring: Test agents are dosed on study-defined schedules — oral KRAS G12C inhibitors, pan-RAF/MEK glue, SHP2 or PI3K-mTOR partners — while imaging or caliper kinetics track response; H23AR arms monitor on-treatment progression to evaluate resistance-overcoming strategies.
8. Endpoint Analysis and Reporting: At termination, thoracic tumors are harvested and weighed, lungs screened for contralateral and metastatic nodules, and brain tissue processed where intracranial arms are included; phospho-ERK, Ki-67, γ-H2AX, and cleaved caspase-3 immunohistochemistry document pathway engagement and mechanism, ex vivo bioluminescence maps signal distribution, and tumor-growth inhibition, regression, and survival data are compiled into the final report.

Fig 2. NCI-H23 / NCI-H23-luc orthotopic mouse model construction workflow.

Case Study-NCI-H23 / NCI-H23-luc Orthotopic Mouse Model Development

NCI-H23 xenografts have served as a cornerstone of the KRAS G12C preclinical literature, and the platform has carried a wide range of Alfa Cytology client programs — benchmark efficacy studies for covalent KRAS inhibitors, vertical pathway blockade combinations producing durable regressions, acquired-resistance evaluations pairing parental and H23AR tumors, and intracranial luciferase configurations for CNS-penetrant agents. Across these programs, readouts have combined imaging or caliper kinetics in vivo, pathway-engagement immunohistochemistry, and survival analysis against published benchmark regimens. Anonymized case summaries and representative deliverable formats are available on request.

Why Choose Alfa Cytology?

Few lines carry as much reference weight as NCI-H23, and extracting that value requires genotype discipline, benchmark-calibrated dosing, and imaging rigor. Alfa Cytology has built its service around those exacting standards.

• A homozygous KRAS G12C / TP53 M246I genotype, verified by sequencing in every study batch, mirroring the partial-responder biology seen with KRAS inhibitors in patients.
• NCI-60 pedigree with decades of public pharmacogenomic data behind every comparison your program will make.
• Documented in vivo benchmarks — sotorasib growth inhibition and deep durable regressions with pan-RAF/MEK glue combinations — available as built-in reference arms.
• A matched acquired-resistance pair (parental NCI-H23 and H23AR with documented MET amplification) for resistance-overcoming strategies.
• Validated intracranial NCI-H23-luc configuration for brain metastasis and CNS-penetration questions.
• Monoclonal, high-signal luciferase banks with quantitative thoracic bioluminescence in vivo from the first week.
• Mechanism-anchored endpoints: phospho-ERK, γ-H2AX, cleaved caspase-3, and Ki-67 immunohistochemistry tying tumor response to pathway engagement.

Contact Us

If your KRAS G12C program needs a reference model with real pharmacological pedigree — and the resistance configurations to outflank it — the NCI-H23 / NCI-H23-luc orthotopic model is the system to build on, and Alfa Cytology is ready to run it to benchmark standard. Contact us to review genotype verification, benchmark and combination arm designs, and intracranial options, and receive a customized study quotation.

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