The syngeneic murine Ff-iCCA model is introduced, providing a platform for functional investigation of cancer cell-TME crosstalk in this molecular subtype, and overcoming the key limitation of prior models relying on human transgenes in immunodeficient hosts.
Abstract
Background
&
Aims
Fibroblast growth factor receptor 2 gene (FGFR2) rearrangements are among the most common oncogenic drivers in intrahepatic cholangiocarcinoma (iCCA). While FGFR inhibitors are clinically approved, primary and secondary resistance remain major limitations. Preclinical investigation of resistance mechanisms, including cancer cell-extrinsic crosstalk, is hampered by current models that rely on human FGFR2-fusion transgenes in immunodeficient hosts. We therefore aimed to generate an entirely murine FGFR2-fusion driven iCCA (Ff-iCCA) model to study immunomodulatory mechanisms in the tumor microenvironment (TME).
Methods
A syngeneic cholangiocyte organoid-based iCCA mouse model was engineered via endogenous chromosomal rearrangement of the Fgfr2 gene combined with Trp53 deletion (PFf) and other co-occurring genetic alterations. KrasG12D-mutated lines (PK) served as comparison. TME characterization was performed using 30-plex spatial proteomics on ∼250,000 cells. Bulk RNA-sequencing was conducted on FGFR inhibitor-treated PFf and PK organoids. Pharmacodynamics of FGFR inhibition on Ff-iCCA were assessed by immunostaining and quantitative RT-PCR.
Results
Intrahepatic implantation produced well-differentiated Ff-iCCA with morphologic features resembling human small duct type iCCA. In immunocompetent hosts, additional co-alterations were required for tumor penetrance, with Pten deletion being most robust with 75%. Compared to KRAS-driven iCCA, Ff-iCCA showed a significantly increased infiltration by Ly-6C/G+ neutrophils (19-fold, p=0.004) and CD8+ T cells (8-fold, p<0.001). Transcriptome analysis revealed increased chemokine expression in PFf versus PK organoids, which was not reversed by FGFR inhibition. Ff-iCCA responded to FGFR inhibition with a 6.5-fold reduced proliferation in vivo (p=0.029), without observation of significant TME remodeling.
Conclusions
Our syngeneic murine Ff-iCCA model recapitulates hallmarks of human FGFR2-fusion iCCA, providing a platform for functional investigation of cancer cell-TME crosstalk in this molecular subtype.
IMPACT AND IMPLICATIONS
This study introduces the first fully syngeneic, endogenously engineered murine model of FGFR2-fusion driven iCCA, overcoming the key limitation of prior models relying on human transgenes in immunodeficient hosts. The model faithfully recapitulates hallmarks of human FGFR2-fusion iCCA, including a small duct type morphology and a distinct neutrophil-enriched tumor immune microenvironment, validating its translational relevance. The finding that an upregulated chemotaxis signature in FGFR2-fusion persists despite FGFR inhibition, alongside upregulation of interferon-stimulated genes upon treatment, points to compensatory immunomodulatory mechanisms that remain to be mechanistically resolved. Overall, this work provides a physiologically relevant platform to interrogate cancer cell-tumor microenvironment crosstalk in FGFR2-fusion driven iCCA.
FGFR2 fusion is a common alteration in malignancies, including intrahepatic cholangiocarcinoma (iCCA). While FGFR2 fusion has oncogenic properties, iCCA patients harboring this alteration demonstrate favorable prognosis, remaining a paradox. Here, we delineated the transcriptomic landscape of FGFR2 fusion-positive iCCA. We observed transcriptional features related to PI3K-AKT signaling in tumor cells and reduced neutrophil infiltration, particularly of the PD-L1+ neutrophil subtype. Mechanistically, FGFR2 fusion was associated with reduced H3K27ac enrichment at the CXCL3 promoter and decreased CXCL3 expression in tumor cells, which may contribute to impaired neutrophil recruitment to tumor tissues. In preclinical models, pharmacological FGFR inhibition increased CXCL3 levels and neutrophil infiltration. Combining neutrophil blockade with clinically available FGFR inhibitors enhanced antitumor activity. In conclusion, this study provides mechanistic insights into the paradox between the oncogenic properties of FGFR2 fusion and favorable clinical outcomes in FGFR2 fusion-positive iCCA, and suggests a potential strategy to optimize FGFR2-targeted therapies.
Tao Zhu, Jia-Cheng Lu, Yan-Zi Pei et al.· Cancer Letters· 0 citations
Fibroblast growth factor receptor 3 (FGFR3) is one of the most frequently altered genes in bladder cancer, primarily through activating mutations that drive oncogenesis and are enriched in luminal tumors. However, the underlying gene regulatory network (GRN) remains poorly characterized. Here, we constructed an FGFR3-mutated GRN using a bottom-up bioinformatics approach, integrating transcriptomic data from bladder cancer cell lines, FGFR3-mutated tumors, and FGFR3 perturbation experiments in human and mouse models. Using publicly available CRISPR/Cas9 screening data, we identified transcription factors from this GRN that regulate the viability of FGFR3-mutated cells, with a focus on p63 (TP63). We showed that FGFR3 activation upregulates p63 in patient-derived xenografts and cell lines, while single-cell RNA sequencing revealed heterogeneous p63 activation associated with basal differentiation. Functional studies, including TP63 knockdown in FGFR3-dependent in vitro and in vivo models and RNA-seq along with p63 ChIP-seq, demonstrated that p63 directly promotes cell proliferation and migration and uncovered a positive feedback loop between FGFR3 and p63. Together, these findings support p63 as a protumorigenic regulator in FGFR3-mutated tumors despite their luminal differentiation and provide a detailed FGFR3-driven GRN, offering insights into FGFR3-induced oncogenic dependency and potential strategies to circumvent resistance to FGFR inhibitors.
A. Moreno-Vega, M. Zambrano, Lilia Estrada-Virrueta et al.· Journal of Clinical Investig...· 0 citations
The in vitro proliferation of AKP-M4 cells, but not KRAS wild-type SNU-1079 and SSP-25 cells, was reduced by the KRAS G12D mutation inhibitor MRTX-1133, which supports the development of KRAS mutation inhibitor for CCA treatment.
J. Hung, Tsai-Hsien Hung, Chun-Nan Yeh et al.· Journal of Immunology· 0 citations
PURPOSE
Intrahepatic cholangiocarcinoma (iCCA) is rising in incidence and is associated with limited treatment options and poor prognosis, particularly in advanced disease. Recently, biologic strategies such as antibody-drug conjugates (ADCs) have expanded therapeutic opportunities across many tumor types, including HER2/neu-altered iCCA. However, validated and prevalent cell surface targets suitable for biologic therapies remain uncommon in iCCA, representing a critical bottleneck to the development of novel targeted treatments. In particular, gene-level analyses overlook tumor-enriched protein isoforms produced through alternative splicing.
EXPERIMENTAL DESIGN
To address this gap, we applied a novel isoform-resolved proteogenomic approach comprising transcriptomics, in silico translation, and cell surfaceomics to identify prevalent cell surface targets in iCCA, and further assessed their specificity through analysis of expression data from the TCGA and GTEx datasets.
RESULTS
This approach identified a unique peptide corresponding to FGFR2b, a splicing isoform of FGFR2, that demonstrated markedly higher tumor enrichment than the alternative FGFR2 isoform, FGFR2c. FGFR2b was the predominant FGFR2 isoform in BTC, including iCCA, in both our institutional cohort (88.6%) and TCGA (88.2%). FGFR2 fusions, in particular, were associated with high FGFR2b expression. High FGFR2b expression was associated with improved surgical outcomes, epithelial differentiation, and reduced CD8+ T-cell infiltration. Orthogonal validation using immunohistochemistry in a subset of 20 patients with available tissue samples confirmed membrane-bound FGFR2b expression in 31.6% of iCCA cases, including all patients with FGFR2 fusions.
CONCLUSIONS
Our findings identify FGFR2b as a prevalent and compelling therapeutic target in iCCA and provide strong rationale for ongoing and future FGFR2b-targeted clinical trials.
N. Shah, Beatriz Alvarado-Hernandez, Xinyue Chen et al.· Clinical Cancer Research· 0 citations
Oncogenic
KRAS
mutations rank among the most prevalent driver alterations in human malignancies, reaching near-universal frequency (~98%) in pancreatic ductal adenocarcinoma (PDAC) and high prevalence in colorectal cancer (CRC, ~52%) and lung adenocarcinoma (LAC, ~32%). Beyond their canonical roles in promoting cell-intrinsic proliferation and survival through the MAPK/ERK and PI3K/AKT cascades, KRAS mutations actively sculpt a profoundly immunosuppressive tumor microenvironment (TME), which constitutes a major barrier to both targeted therapy and immunotherapy. Through coordinated programs encompassing inflammatory cytokine secretion, downregulation of antigen presentation machinery, tumor-associated macrophage (TAM) reprogramming, myeloid-derived suppressor cell (MDSC) expansion, and PD-L1 upregulation, KRAS-mutant tumors establish robust immune exclusion. These programs are further stratified by co-mutations in
STK11
,
KEAP1
, and
TP53
, which define distinct immune phenotypes ranging from inflamed to profoundly immune-excluded “cold” tumors. The recent approval of covalent KRAS G12C inhibitors, sotorasib and adagrasib, has revealed that targeted KRAS blockade can remodel the TME toward an immunostimulatory state, providing a mechanistic rationale for combining KRAS-directed agents with immune checkpoint blockade, STING agonists, and neoantigen vaccines. This mini-review synthesizes the current knowledge of KRAS-immune crosstalk, highlights existing controversies and research gaps, and evaluates emerging combination strategies designed to convert immune exclusion into durable anti-tumor immunity.
Vasudevan Ramachandran, H. Koyou, Siddarth Raajasekar et al.· Frontiers in Oncology· 0 citations
Acquired resistance significantly limits the long-term clinical benefit of osimertinib in EGFR-mutant lung adenocarcinoma (LUAD). However, the early non-genetic adaptations and microenvironmental crosstalk that enable initial tumor survival remain poorly defined. This study aimed to elucidate the epigenetic-immune mechanisms driving osimertinib resistance and to identify novel actionable therapeutic targets.
We integrated transcriptomic profiling and clinical cohort analyses to identify therapy-induced secreted factors during the evolution of EGFR-TKI resistance. The upstream regulatory mechanisms were characterized using ChIP, Co-IP, and dual-luciferase reporter assays. The dual functional roles of the identified factor, serum amyloid A1 (SAA1), in tumor-intrinsic signaling and macrophage polarization were investigated in vitro. Finally, the therapeutic efficacy of combining SAA1 neutralization with osimertinib was evaluated across patient-derived organoids, xenografts, and immunocompetent spontaneous lung tumor models.
We identified SAA1 as a critical therapy-induced secreted factor that progressively increases during the transition from drug tolerance to established resistance, which negatively correlates with clinical outcomes in patients receiving osimertinib. Mechanistically, osimertinib treatment suppressed the ERK-dependent phosphorylation of the transcriptional repressor GATAD2A at Ser114. This event destabilized the NuRD complex, thereby relieving the epigenetic transcriptional repression of SAA1. Secreted SAA1 promoted therapeutic resistance through dual mechanisms: activating the autocrine TLR4/NF-κB survival signaling in tumor cells and inducing the paracrine polarization of pro-tumorigenic SPP1⁺ macrophages. Therapeutically, targeted neutralization of SAA1 synergized robustly with osimertinib to reverse immunosuppressive remodeling and induce profound tumor regression in multiple preclinical models.
Our findings define a novel therapy-induced ERK/GATAD2A/SAA1 epigenetic-immune axis that drives acquired resistance to osimertinib. SAA1 acts as a pivotal bridge between tumor-intrinsic survival adaptations and microenvironmental immune suppression, highlighting SAA1 neutralization as a tractable combinatorial strategy to enhance EGFR-TKI efficacy in LUAD.
Junkan Zhu, Shencheng Ren, Tao Cheng et al.· Journal of Experimental &...· 0 citations
