Rat somatic genome editing enables ER+ breast cancer modeling

Authors

Wen Bu

Tobie Lee

Meenakshi Anurag

Yunfeng Ding

Ruixin Xu

Lillian He

Alexandria Z. Bu

Chandandeep Nagi

Carolina Gutierrez

Susan Hilsenbeck

Chonghui Cheng

Bing Zhang

Shixia Huang

Jianming Xu

C. Kent Osborne

Arun Sreekumar

Eric Chang

Chad J. Creighton

Xiang Zhang

Yi Li

Doi

10.1101/2025.09.15.675961

PMID: 26437033 · DOI: 10.1101/2025.09.15.675961 · Journal: bioRxiv (2025)

TL;DR

Bu et al. develop an AAV-CRISPR somatic genome editing platform in [[../genes/TP53.md|Cas9-transgenic rats]] that delivers Indel and HDR-based substitution mutations to mammary epithelium via intraductal injection. Editing of [[../genes/TP53.md|Tp53]], [[../genes/NF1.md|Nf1]], and [[../genes/PIK3CA.md|Pik3ca]] (alone and in combinations) produced ER+/PR+ ductal carcinomas in rats that mirror the histology, hormone dependence, and transcriptome of human ER+ breast cancer, whereas identical edits in mice yielded ER-/PR- metaplastic tumors with squamous differentiation. Nf1Indel tumors regressed completely under ovariectomy or fulvestrant, while Nf1Indel/Tp53Indel tumors mimicked endocrine-resistant disease. The work closes a long-standing species gap in modeling the ~70% of human breast cancers that are ER+ (PMID:26437033).

Cohort & data

  • Animal models — Cas9-transgenic rats generated by breeding LE-ROSA26tm1(LSL-Cas9)Ottc with Wistar-Tg(CAG-Ncre)81Jmsk (RRRC #00833 and #301); Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh mice (Jackson Laboratory). Female animals only.
  • Tumor cohorts — Six rat genotype groups (Tp53Indel n≈5; Nf1Indel n=13; Nf1Indel/Tp53Indel n=8; Pik3caH1047R n=10; Pik3caH1047R/Tp53Indel n=5; Pik3caH1047R/Tp53Indel/Nf1Indel n=7) plus rat KrasG12D and mouse comparators.
  • Cancer types — Models of [[../cancer_types/BRCA.md|breast carcinoma]], including [[../cancer_types/IDC.md|invasive ductal carcinoma]], [[../cancer_types/DCIS.md|ductal carcinoma in situ]], and contrast with mouse [[../cancer_types/MBC.md|metaplastic breast carcinoma]].
  • Assays — Bulk [[../methods/rna-seq.md|RNA-Seq]] (35 rat tumors), [[../methods/scrna-seq.md|scRNA-Seq]] (10x Chromium Single Cell Gene Expression 3’v3.1), amplicon deep sequencing of edited loci, [[../methods/immunohistochemistry.md|IHC]] for ER/PR/Ki67, [[../methods/multiparameter-flow-cytometry.md|multiparameter flow cytometry]].
  • External comparisons — [[../datasets/brca_metabric.md|METABRIC]] (10.5% TP53/PIK3CA co-mutation), [[../datasets/brca_tcga_pub.md|TCGA breast cancer]] (TP53+PIK3CA vs PIK3CA-only WT-TP53), and patient neoadjuvant endocrine therapy datasets GSE5462 (letrozole) and GSE71791 (fulvestrant) for cross-species transcriptome comparison (PMID:26437033).

Key findings

  • Efficient somatic Indel editing in rat mammary gland. Intraductal AAV2/9 carrying a Tp53 gRNA produced 4.2% Indel editing of whole-gland alleles at day 4 (95% frameshift), translating to an estimated 10.5–21% editing rate in luminal epithelial cells (~25–30% of total mammary cells); Tp53Indel tumors arose with 50% incidence by 10 months (PMID:26437033).
  • Single-gene Nf1 editing is potent. Intraductal AAV-N (Nf1 gRNA, 2.8×10^11 gc) yielded palpable tumors at median latency 4 weeks with ~80% allelic editing, substantially faster than germline Nf1 heterozygous deletion (18 days vs 70–90 days), arguing that biallelic somatic Nf1 loss bypasses LOH requirements (PMID:26437033).
  • Oncogenic cooperation reduces latency stepwise. AAV-NT (Nf1+Tp53 dual gRNA) produced tumors in 100% of rats within one month (p<0.01 vs single editing). AAV-PT (Pik3caH1047R + Tp53Indel) shortened median latency to 42 days. AAV-PNT (triple-edit Pik3ca + Nf1 + Tp53) reached an exceedingly short median latency of 8 days (PMID:26437033).
  • HDR-based substitution editing of Pik3ca. AAV-P with sgRNA + HDR donor introduced [[../genes/PIK3CA.md|PIK3CA]] H1047R in ~40% of tumor alleles; tumors developed at median 4.5 months and were predominantly benign fibroadenomas (8/10) rather than carcinomas (PMID:26437033).
  • Histology mirrors human ER+ ductal carcinoma. All six rat genotype groups produced ductal lesions with robust hormone receptor expression and no squamous differentiation. Nf1Indel tumors were 12/13 DCIS; Nf1Indel/Tp53Indel tumors were uniformly invasive ductal carcinoma with moderate differentiation; Pik3caH1047R/Tp53Indel tumors were invasive ductal carcinoma with papillary features; the triple-edit tumors showed high grade, nuclear pleomorphism, and necrosis (PMID:26437033).
  • Transcriptome subtypes. Among 35 rat tumors, ANOVA (FDR<1%) yielded 1,579 differentially expressed genes; intrinsic subtype assignment using the Hoadley centroids classified most tumors as Luminal A or Luminal B, with sporadic Basal-like and HER2 assignments (PMID:26437033).
  • Cross-species transcriptome concordance. Of 1,469 genes up in rat Pik3caH1047R/Tp53Indel vs Pik3caH1047R-only tumors, 297 were also enriched in [[../datasets/brca_tcga_pub.md|TCGA]] PIK3CA+TP53 vs PIK3CA-only/WT-TP53 breast cancers (one-sided Fisher’s exact p<1×10^-14); of 1,156 down-regulated genes, 211 overlapped (p<1×10^-6) (PMID:26437033).
  • Endocrine sensitivity dichotomy. Nf1Indel tumors regressed completely with ovariectomy (3/3 rats) and with 5-week fulvestrant (4/4 rats); 3/4 fulvestrant-treated animals remained in remission for 8 months after cessation. Nf1Indel/Tp53Indel tumors showed only modest, transient shrinkage by week 3 (p<0.01 ovariectomy; p<0.05 fulvestrant) and resumed growth by weeks 4–5, modeling endocrine-refractory disease (PMID:26437033).
  • Patient transcriptome alignment under endocrine therapy. GSEA of 486 fulvestrant-responsive rat DEGs showed significant enrichment of genes induced by neoadjuvant letrozole (GSE5462) and fulvestrant (GSE71791) in patients; 58 genes including PGR, AREG, SGK3, STC2, DHFR, KLF10/TIEG1, TSKU, PFKFB3, PHB, AGPAT5, TIPARP, WNT5A, and CXCL3 overlapped (PMID:26437033).
  • Species divergence is reproducible across drivers. Identical KrasG12D, Pik3caH1047R, and Pik3caH1047R+Tp53Indel edits delivered intraductally produced ER+/PR+ ductal carcinomas in rats but ER-/PR- metaplastic tumors with squamous differentiation in mice; the lenti-HrasQ61L approach reproduced the same divergence in both species (PMID:26437033).
  • Heterogeneous immune microenvironment. UMAP of scRNA-Seq across models showed adaptive immune cell enrichment in Pik3caH1047R-only and Tp53Indel-only tumors and depletion in multi-edit tumors. A macrophage/plasmacytoid dendritic cell vs neutrophil dichotomy segregated by genotype: macrophages enriched in Nf1Indel and Nf1Indel/Tp53Indel; neutrophils enriched in Tp53Indel/Pik3caH1047R and triple-edit tumors (PMID:26437033).

Genes & alterations

  • [[../genes/TP53.md|TP53]] — Somatic Indel editing of rat Tp53 exon 1 produced ductal carcinomas at ~10-month median latency (5/10 rats); 60% allelic editing in tumors. In combination with Nf1Indel or Pik3caH1047R, Tp53 loss drove progression to invasive carcinoma, suppressed metaplastic transdifferentiation, and conferred endocrine therapy resistance. The authors cite clinical evidence that TP53 mutation associates with endocrine resistance in patients (PMID:26437033).
  • NF1 — Indel editing of rat Nf1 alone produced ER+/PR+/Ki67+ ductal carcinoma in situ at 4-week median latency in 12/13 cases; ~80% allelic editing. The authors invoke NF1’s dual role as a RAS GAP and ER-α transcriptional co-repressor, citing 4% mutation and 20% copy-number loss rates in early ER+ breast cancer; tumors were highly endocrine-sensitive (full regression on ovariectomy and fulvestrant) (PMID:26437033).
  • [[../genes/PIK3CA.md|PIK3CA]] — HDR-based introduction of H1047R (the most frequent PIK3CA hotspot) into rat Pik3ca yielded predominantly benign fibroadenomas (8/10), with the remainder being a lactational adenoma and a ductal carcinoma NOS. ER expression averaged ~27%, similar to normal rat terminal duct lobular units. Combined with Tp53Indel, the same edit produced moderately differentiated invasive ductal carcinoma with papillary features at 42-day median latency (PMID:26437033).
  • KRAS — KrasG12D introduced by AAV-CRISPR HDR into rats yielded ER+/PR+ ductal carcinoma with papillary features at ~70-day median latency; the same editing in mice produced ER-/PR- metaplastic tumors with widespread squamous differentiation, illustrating species-specific lineage plasticity. The authors note KRAS is not commonly mutated in human breast cancer (PMID:26437033).
  • HRAS — Cited as the prior lenti-HrasQ61L mammary intraductal model, which produces ER-/metaplastic tumors in mice but ER+/PR+ ductal carcinoma in rats — historical evidence motivating the CRISPR-based species comparison (PMID:26437033).
  • ESR1 — Rat tumors broadly express ER protein; Esr1 mRNA correlates with Pgr expression and with “group 3” cytoplasmic-ribosomal / mammary-gland gene cluster activity, especially in Nf1Indel models. ESR1 ligand-binding-domain mutations are flagged as a future expansion of the platform (PMID:26437033).
  • [[../genes/PGR.md|PGR]] — PR expression correlates with ER expression across tumor models; PR/PGR transcript appears among the 58-gene set responsive to fulvestrant in both rat tumors and human GSE5462/GSE71791 datasets (PMID:26437033).

Clinical implications

  • Preclinical platform for ER+ breast cancer therapeutics. The Nf1Indel and Nf1Indel/Tp53Indel rat models bracket endocrine-sensitive and endocrine-resistant disease in an immunocompetent host, providing tractable systems to test SERDs (e.g., [[../drugs/fulvestrant.md|fulvestrant]]), aromatase inhibitors (cited [[../drugs/letrozole.md|letrozole]] benchmark dataset GSE5462), CDK4/6 inhibitors, and rational combinations (PMID:26437033).
  • NF1 as an endocrine-response biomarker. Tumors driven by Nf1 loss retain strong ER dependence; the authors cite Zheng et al. (2020) showing NF1 acts as an ER-α transcriptional co-repressor in human ER+ breast cancer and that NF1 loss promotes endocrine resistance, but the model here suggests Nf1-loss alone yields endocrine-sensitive disease — resistance emerges only when Tp53 is co-disrupted (PMID:26437033).
  • TP53 status as a resistance determinant. Combined Nf1/Tp53 editing converts endocrine-sensitive to endocrine-refractory phenotype, consistent with clinical reports that TP53 mutation associates with endocrine resistance (PMID:26437033).
  • Modeling DCIS progression. The Nf1Indel tumors recapitulate DCIS histology and offer an in vivo model for studying the DCIS-to-invasive-cancer transition, which is poorly captured by existing GEM mouse models (PMID:26437033).
  • Immune microenvironment as a therapeutic axis. scRNA-Seq reveals genotype-driven enrichment of suppressive myeloid populations (macrophages vs neutrophils) in immunologically “cold” ER+ tumors, suggesting that immunosuppression in ER+ disease may reflect active myeloid recruitment rather than low mutational burden — a hypothesis that opens immunotherapy reprogramming strategies (PMID:26437033).

Limitations & open questions

  • Preprint, not peer-reviewed. This is a bioRxiv preprint (doi 10.1101/2025.09.15.675961, posted 2025-09-18); findings have not yet been independently certified (PMID:26437033).
  • Limited driver coverage. Models focus on TP53, NF1, PIK3CA, and KRAS; broader human ER+ disease alterations (ESR1 LBD mutations, MAP3K1, GATA3, chromatin regulators) are not yet covered (PMID:26437033).
  • Secondary spontaneous mutations. Long tumor latencies in some single-edit groups (Tp53Indel ~10 months, Pik3caH1047R ~4.5 months) imply accrual of secondary events; whole-genome interrogation and lineage tracing are needed to parse the minimal cancer-permissive changes the authors call out (PMID:26437033).
  • Species mechanism unresolved. The molecular determinants of the rat-versus-mouse ER+/PR+ vs metaplastic divergence remain unknown; head-to-head immune and stromal benchmarking across rat, mouse, and human tissues is proposed for follow-up (PMID:26437033).
  • Metastasis modeling pending. Distant metastases of these rat tumors have not yet been characterized; ER+ breast cancer metastasis remains poorly modeled across rodents (PMID:26437033).
  • PMID provenance. The raw frontmatter lists pmid 26437033 but the manuscript content is the September 2025 bioRxiv preprint by Bu et al.; the PMID may not yet be assigned to this preprint at the time of compilation.

Citations from this paper used in the wiki

  • “Approximately 70% of ductal carcinomas express ER, often along with PR” — motivation for ER+ rat models.
  • “By 10 months, 50% of rats developed palpable tumors” — Tp53Indel single-edit tumor incidence.
  • “Tumors initiated by Nf1 Indel-editing were predominantly ductal carcinoma in situ (12/13 cases)” — DCIS phenotype.
  • “Intraductal delivery of AAV-NT (2x10^10 gc/gland) into 10 Cas9 rats led to tumors within one month for all injected rats” — Nf1+Tp53 cooperation.
  • “Intraductal injection of AAV-PNT into Cas9 rats led to tumors with an exceedingly short median latency of only 8 days” — triple-edit cooperation.
  • “Among the top 1,469 genes higher in Tp53Indel/Pik3caH1047R tumors over Pik3caH1047R only tumors, 297 genes are also enriched in genes higher in TCGA breast cancer cases mutated for both TP53 … and PIK3CA … (enrichment p value <1E-14, one-sided Fisher’s exact test)” — TCGA concordance.
  • “A 5-week course of fulvestrant treatment caused rapid and full regression of all four tumors” — Nf1Indel endocrine sensitivity.
  • “These rat tumors are all ER+/PR+ ductal carcinoma with papillary features but no metaplasia” — KrasG12D species divergence.

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