Brain metastases occur in up to 40% of patients with stage IV breast cancer, and the cerebellum is a common site for metastases in HER2-positive breast cancer. We developed a syngeneic, immunocompetent mouse model of breast cancer brain metastases (BCBM) by stereotactically injecting murine breast cancer organoids into the cerebellum. Spatial transcriptomics on these brain metastases revealed that breast cancer cells produce interleukin-34 (IL34), which induces ARG1+ macrophages at the invading edge of metastases. IL34 expression in human brain metastasis samples was measured in two independent datasets, and IL34 was expressed widely in both HER2+ and HER2- human BCBM. Remarkably, IL34-deficient breast cancer cells failed to establish tumors in the mouse cerebellum. Furthermore, treatment with a blocking antibody against the IL34 receptor, CSF1R, led to tumor shrinkage, highlighting the translational potential of these findings, as a CSF1R-blocking antibody is FDA-approved for another indication, graft-versus-host disease. SIGNIFICANCE:Targeting IL34-CSF1R is a potential new approach to treat BCBM and could be combined with existing therapies. IL34 expression is widely found in human BCBM.

Spatial gene expression in mouse BCBM.

Herceptin optical imaging of brain metastases. A, Contrast-enhanced T1- and T2-weighted MRIs of stereotaxic injection at 3 weeks after transplantation of HP organoids in C57BL/6 syngeneic mice. B, Representative BLI images of the cerebellum tumor at 3 weeks after organoid cell transplantation of HER2+ tumor organoids. C, Live animal in vivo imaging of NIR fluorophore–labeled trastuzumab (NIR-trastuzumab). Mice were injected with 100 μg of NIR-trastuzumab via the tail vein 24 hours prior to this imaging. D, Left, Brightfield images of dissected cerebellum tumor at 3 weeks after transplantation. Right, Imaging of NIR-trastuzumab after dissection. E, NIR-trastuzumab imaging was performed after 24 hours of formalin fixation of mouse brain tissues. Left, Brightfield images of formalin-fixed cerebellum tumor slices. Right, Imaging of NIR-trastuzumab after formalin fixation in the same cerebellum tumor slices.

Abstract Background. Advancing therapeutic approaches to brain metastases (BrMets) is an area of critical need. Preclinical models of BrMets are a rare but much-needed tool to investigate novel therapeutic approaches. We developed a biobank of BrMet patient-derived xenograft (PDX) models established from resected BrMets originating from various solid tumor types. Methods: Resected BrMet tissues were collected. PDX models were established, and ex vivo drug screening was performed using previously published methods (Morikawa et al. Can Res Comm 2023). Molecular profiles of PDXs and matched source tumors were compared, and their correlations with drug response were examined. Results: From Nov 2016 to Sept 2023, 142 surgical cases were collected, of which 126 PDX models were established and maintained growth. Tumor types included common (lung, breast, melanoma) and rare (sarcoma, ovarian, cervical, prostate, renal, and gastrointestinal) tumors. Common mutations across these models included BRCA, ATR, ALK, KMT2C, FAT1, ZFXHX3, MAP3K1, COL6A3, FLT3, MLH1, EGFR, IGFN1, and TP53, though many were not predicted to be pathogenic. In addition, there were less prevalent but potentially targetable alterations, such as PI3K mutations. Copy number variation (CNV) analysis demonstrated a predominance of amplifications over deletions. The observed pathways included those associated with neuronal and structural features such as axonal transport (DNA KEGG database) and extracellular matrix (DNA REACTOME database). The RNA seq analysis revealed clustering mostly based on the primary tumor type. Compared to publicly available metastatic PDX models (NCI database) stratified by primary tumor type, these BrMet models demonstrated differences in the molecular pathway enrichment. PDX models generally exhibited high concordance based on Jaccard Index (JI). The majority of the samples showed JI in the range of 0.4-0.6, with melanoma samples demonstrating JI in the lower 0.2 range. We evaluated the PDX and matched pairs for the selected variants predicted to be pathogenic or possibly functionally impactful. Again, melanoma subtypes showed more divergence, but overall, the majority of the variants were retained in the matched PDX models. Drug sensitivity testing was performed on 13 PDXs using a panel of molecularly targeted agents and chemotherapies. Of 13, nine PDXs had potentially actionable molecular targets. Highly active drugs were identified in these models; however, most drug sensitivities were not predictable based solely on genomic profiles, emphasizing the utility of paired functional characterization. Conclusion: We present a novel biobank of BrMet PDX models. These models provide a valuable resource for probing the biology of BrMet and informing therapeutic strategies. Prospective collection is ongoing to expand the biobank, alongside further studies investigating tumor-microenvironment interactions using immune-competent and organ-on-chip microfluidic blood-brain niche models. AI disclosure: AI was used for language editing only; content was verified by the authors Citation Format: Aki Morikawa, Tusharika Rastogi, Noreen Khan, Peter Ulintz, Derek Nancarrow, Habib Serhan, Xu Cheng, Liwei Bao, Aaron Udager, Matthew Soellner, Jason Heth, Nathan Merrill, Sofia D. Merajver. Developing diverse patient-derived xenograft models of common and rare brain metastases to elucidate molecular landscapes and reveal therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B062.

Spatial transcriptomics reveals the clusters of cells and genes in HER2+ BCBM. A, Sample joint clustering in the duplicate samples of spatial transcriptomics in the HER2+ breast cancer cerebellum metastasis model. The average expression of the whole genome in each cluster is provided in Supplementary Table S1. B, Tissue plot with spots colored by UMI count in duplicate samples. C, Hematoxylin and eosin staining of biological duplicate samples for spatial genome-wide sequencing. D, Volcano plot illustrating genes meeting cutoffs for differential expression [log-fold change (logFC2) >1, Padj. < 0.05] between tumor vs. normal cerebellum, tumor vs. invasion clusters, invasion clusters vs. normal cerebellum, and invasion clusters near the normal area vs. invasion near the tumor. A list of the significantly altered genes in replicate two is provided in Supplementary Table S2.

IL34 induced ARG1 expression in BV2 cells.

Enrichment of pathways and cell types in the spatial landscape of tumor, invading, and normal cerebellum clusters. A, Gene sets significantly enriched in tumors compared with normal cerebellum as identified via GSEA (P < 0.05). ES (enrichment score) and −log10 (P values) of pathways are shown. GSEA was performed using the hallmark gene sets in the Molecular Signatures Database (version 7.5.1). A list of the hallmark genes in replicate one is provided in Supplementary Table S3. B, Gene sets significantly enriched in tumors compared with invasion clusters as identified via GSEA (P < 0.05). ES (enrichment score) and −log10 (P values) of pathways are shown from GSEA performed using hallmark gene sets in the Molecular Signatures Database (version 7.5.1). A list of the hallmark genes in replicate one is provided in Supplementary Table S3. C, Integrated spatial distribution panel of cell types of prediction in duplicate samples. Cell types are identified by a combination of snRNA and spatial transcriptomics technology. D, Spatial distribution of macrophages as indicated in duplicate samples. E, Spatial distribution of Arg1 as indicated in duplicate samples.

IL34-induced Arg1 expression at the invading edge in cerebellum metastasis of HER2+ breast cancer. A, Spatial feature plot of IL34 in a representative murine tissue sample. B, ELISA test result of IL34 protein level from astrocytes and microglial cells, HP breast cancer organoid cells. C, Mouse BMDM was cultured with conditioned media supernatant from BO1 cells or HP tumor organoid cells for 24 hours, and the IHC staining of ARG1 was performed on cytospin slides of the cocultured cells. D, Percent change of tumor volume in the control and anti-CSF1R mAb mouse groups. E, The representative contrast-enhanced T1W and T2W images of MRI showed the cerebellar tumor volume change at 0 and 12 days after treatment in the control and anti-CSF1R mAb groups. Briefly, 5,000 cells from HP breast cancer organoids were injected into the mouse cerebellum as described in the methods. F, Change of tumor volume in the control and IL34 knockout mouse groups.

Characterization of cerebellum metastasis mouse model. A, Hematoxylin and eosin (H&E) and IHC stained with GFAP, HER2, and ERα antibodies in the cerebellum metastasis model of HP tumor organoid cells. Scale bars of low-power images are 5 mm. Scale bars of high-power images are 500 μm. B, H&E and IHC stained with GFAP, ERα, and HER2 antibodies in the cerebellum metastasis model of H53 tumor organoid cells. Scale bars of low-power images are 5 mm. Scale bars of high-power images are 500 μm. C, IF stained with IBA1 and TMEM119 antibodies in the cerebellum metastasis model of HP organoid cells. Scale bars of low-power images are 5 mm. Scale bars of high-power images are 500 μm. D, IF stained with IBA1 and TMEM119 antibodies in the cerebellum metastasis model of H53 tumor organoid cells. Scale bars of low-power images are 5 mm. Scale bars of high-power images are 500 μm.

IL34 expression and CD163+ macrophages in human BCBM samples. A, IHC staining of human IL34 on human BCBM samples (N = 19). B, IF costaining of CD206 and IL34 on human BCBM samples (N = 8). C, CD163 IHC on human BCBM samples (N = 19). D, Summary of the IHC results (N = 19). E,IL34 mRNA expression level among the University of Michigan PDX samples (N = 137). GI, gastrointestinal; GU, genitourinary; Gyn, gynecological; HN, head and neck cancers.

Effect of CSF1R blocking antibody and IL34 CRISPR knockout.

IL34-induced ARG1 expression in macrophages at the invading edge of BCBM.

Characterization of murine breast cancer organoids.

Abstract Introduction: Heterogeneous mutation profiles are common in anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK+ NSCLC), especially upon progression, supporting that robust longitudinal monitoring to detect these changes early can play a role in disease management. Circulating tumor cells (CTCs) provide a minimally invasive modality for real-time monitoring. We longitudinally analyze CTC counts and sc-transcriptomes to assess their reflection of clinical responses and correlation with progression risk in ALK+ patients. Methods: Following informed consent, we collected 61 samples from 12 ALK patients (2-10 timepoints/patient; 11 Stage IV, 1 Stage III). Serial peripheral blood samples were processed using the in house developed microfluidic Labyrinth device for CTC enrichment. CTC subgroups were identified by immunofluorescence staining for Cytokeratin (CK), EpCAM, and Vimentin. At each sampling point, clinical response was assessed by a treating physician. Sc-RNA sequencing was performed on enriched CTCs, characterizing differentially expressed gene (DEG), survival, trajectory states, immune interactions, and inferred CNV profiles. Results: Patients receiving a single TKI treatment (Alectinib) with clinically stable disease showed decreasing CTC burden. Patients with prior exposure to multiple TKIs and progressive disease showed fluctuating dynamics. Kaplan-Meier analysis showed a decrease in total CTCs (77%, p=0.016) and a decrease in CK+ CTCs (6.05%, p=0.049) significantly correlated with lower progression risk. scRNA-seq on three Stage IV patient’s CTC collected at multiple time points revealed dynamic immune populations changed between the visits. DEGs in CTC compared to the rest of immune cells in all 3 visits displayed EMT-associated upregulation (TUBB1, PPBP, ITGA2B); and downregulation of GATA2, consistent with its reported reduction in lung cancer. A composite of 45 consistently downregulated genes was correlated with reduced survival, using a TCGA cohort (n=500; p=0.039). Notably, downregulated genes obtained after the patient started progressing showed increasingly more significant associations with poor survival (p=0.21, p=0.019, p=0.0067). Trajectory analysis identified two CTC clusters, with later visits mapping to higher pseudotime states. CNV analysis revealed a CDK4 copy-gain specifically at progression. Intercellular communication analysis showed decreased CTC-B-cell and increased CTC-monocyte interactions at progression, which may suggest reduced anti-tumor surveillance and enhanced myeloid support. Conclusion: Longitudinal monitoring integrating CTC burden and scRNA analysis provides insights into tumor evolution and immune interactions in ALK+ NSCLC, potentially signaling imminent progression and may help guide personalized treatment strategies. Citation Format: Yuru Chen, Shamileh Fouladdel, Leah Kidder, Yuehang Tang, Harrison Ball, Habib Serhan, Zhaoping Qin, Albert Liu, Xu Cheng, Liwei Bao, Varun Kathawate, Larua Goo, Mary Horn, Stacy Fry, Aaron N. Hata, Justin Gainor, Jessica J. Lin, Stuart Hinton, Chao H. Huang, Nathan Merrill, Aaron M. Udager, Peter J. Ulintz, Angel Qin, Sofia Merajver, Sunitha Nagrath. Longitudinal monitoring of circulating tumor cells (CTC) reveals dynamic CTC behaviors associated with disease progression and survival in ALK-positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1062.

Abstract Introductory Sentence: This study investigates the therapeutic potential of dual alectinib and everolimus inhibition to prolong the clinical benefit of alectinib in ALK-positive non-small cell lung cancer. Pertinent experimental procedures: ALK-rearranged NSCLC cell lines (CUTO8, CUTO9, CUTO29.1, CUTO39, CUTO41, CUTO43, CUTO46) obtained from the University of Colorado; DFCI032 from Dana-Farber; NCI-H2228 and NCI-H3122 from ATCC; and SNU2292 and SNU2535 from Seoul National University were screened using a high-throughput drug-combination platform to identify synergistic interactions between alectinib and a curated library of 1,600 approved and experimental compounds. Functional validation in NCI-H3122 included colony-formation and Glo-Caspase 3/7 apoptosis assays with therapeutically relevant concentrations of everolimus (Cmax and Caverage) plus 100 nM alectinib, ∼ten-fold lower than reported Cmax/Caverage. Apoptotic activation was further confirmed by Western blot for cleaved PARP, cleaved caspase-3, and the pro-survival protein MCL-1. In parallel, ex vivo screening was conducted on ALK167-T-01, an ALK-positive PDX model harboring the p.Leu1196Met ALK mutation. Summary of new unpublished data: High-throughput screening identified a consistent synergistic response to alectinib combined with multiple mTOR inhibitors across all the ALK-rearranged models tested. Synergy was assessed by the Chou-Talalay method, with Cl <1 in most ALK-positive cell lines tested. In NCI-H3122, the combination significantly reduced clonogenic potential (alectinib alone VS combo Caverage; p<0.01 and alectinib alone VS combo Cmax; p<0.001) and induced apoptosis, evidenced by increased Caspase 3/7 activity (alectinib alone VS combo Caverage; p<0.01 and alectinib alone VS combo Cmax; p<0.001) and significant PARP and caspase-3 cleavage. Ex vivo PDX screening using ALK167-T-01 with Glo-Caspase 3/7 assays at therapeutically relevant everolimus concentrations plus 100 nM alectinib showed higher apoptosis vs single agents (alectinib alone VS combo Caverage; p<0.001 and alectinib alone VS combo Cmax; p<0.0001), confirming synergy is not exclusive to sensitive lines. Statement of the conclusions: Dual ALK and mTOR targeting with alectinib and everolimus produces synergistic antitumor activity in vitro and ex vivo, reflected by significantly increased apoptosis vs single agents. These findings support further investigation in in vivo models, including alectinib-sensitive and -resistant tumors. AI disclosure: AI was used for language editing only; content was verified by the authors. Citation Format: Hamadi Madhi, Habib Serhan, Rachel Mercer, Benjamin Levy, Anna Rottinghaus, Liwei Bao, Xu Cheng, Sharon R. Pine, Ross Camidge, Angel Qin, Nathan M. Merrill, Sofia D. Merajver, Matthew B. Soellner. Preclinical evidence for synergistic activity of alectinib and everolimus in ALK-positive non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6501.

Abstract Patient-derived organoids from breast cancer brain metastases enable real-time drug sensitivity testing integrated with genomic profiling. Drug response varied by subtype and molecular alterations. PI3K inhibitors showed activity regardless of PIK3CA mutation status. Pronounced tumor heterogeneity highlighted the urgent need for effective therapies personalized for each patient. Functional assays and molecular matching can help tailor therapy for patients who need the most effective next treatment quickly and warrant further translational evaluation to address this unmet need.

Abstract We developed a syngeneic, immunocompetent mouse model of breast cancer brain metastases by stereotactically injecting murine breast cancer organoids into the cerebellum. The cerebellum was chosen as the site of injection because it is a frequent site for metastasis from HER2+ breast cancer. Spatial transcriptomics on these brain metastases revealed that breast cancer cells produce interleukin-34 (IL34), which recruits or polarizes ARG1+ macrophages to the invading edge of the metastases. IL34 expression in human brain metastasis samples was measured in two independent patient datasets, and IL34 was expressed widely in both HER2+ and HER2- human breast cancer brain metastasis. Remarkably, CRISPR knock-out of IL34 in the breast cancer cells prevented growth of metastases in the mouse cerebellum. As a control, these IL34 KO breast cancer cells showed equal growth in vitro and equal invasion in Boyden chamber assay. Finally, in vivo treatment of mice with a blocking antibody against the IL34 receptor, CSF1R, led to breast cancer brain metastasis shrinkage, highlighting the translational potential of these findings, as a CSF1R-blocking antibody is FDA-approved for another indication, graft-versus-host disease.

Abstract Background: Non-Small Cell Lung Carcinoma (NSCLC) harboring the EML4-ALK fusion gene (Echinoderm Microtubule-Associated Protein-Like 4-Anaplastic Lymphoma Kinase) comprises about 5% of NSCLC cases. Tumors with this genetic alteration are initially responsive to ALK Tyrosine Kinase Inhibitors (TKIs), which constitute first- and second-line therapy. However, nearly all ALK-positive (ALK+) lung cancers ultimately develop resistance to ALK TKIs, highlighting the urgent need for alternative treatment options. Methods and Results: Tumor Suppressor Candidate 2 (TUSC2) is a tumor suppressor gene with low endogenous expression in NSCLC. Quaratusugene ozeplasmid (QO), developed by Genprex, is a novel gene therapy that encapsulates the TUSC2 plasmid in non-viral lipid nanoparticles, effectively upregulating TUSC2 in cancer cells. We evaluated TUSC2 expression in a range of ALK+ cell lines and patient-derived organoids (PDOs), both prior to and following exposure to QO. Our findings show that QO-driven TUSC2 overexpression initiates a robust pro-apoptotic response in ALK+ models, not only in cells that are sensitive but also with acquired resistance (generated in the lab) to the ALK inhibitor, alectinib. This is evidenced by increased pro-apoptotic markers and lower cell viability when QO is used in combination with alectinib. To further assess the QO and alectinib combination, we tested it in two in vivo models: (1) an alectinib-sensitive model using subcutaneous injection of NCI-H2228 ALK+ cells into nude mice, and (2) an alectinib-resistant model using ALK167 PDX implants in NSG mice. Once tumors reached ∼ 100 mm3, mice were randomized into four groups: vehicle control; QO alone (25 µg/mouse, IV, every three days); alectinib alone (0.5 mg/kg for sensitive or 15 mg/kg for resistant, oral, daily); and QO plus alectinib at the same doses. In the sensitive model, tumors in the alectinib-treated group shrank by 60%. Notably, treatment with QO alone, and particularly QO combined with alectinib, resulted in 79% tumor shrinkage (p value 0.0135 versus control), demonstrating a 23% improved outcome than alectinib alone. This suggests that QO might serve as a valuable adjunct therapy, especially for patients who have advanced disease and/or experience resistance to TKIs. Major new unpublished results: In the resistant model, the QO and alectinib combination produced a synergistic effect, achieving the greatest tumor reduction and improved overall survival (p value 0.0001 versus control), further supporting the clinical potential of this therapeutic strategy in ALK+ NSCLC. Altogether, our in vitro and in vivo studies indicate that QO-mediated TUSC2 overexpression in ALK+ NSCLC effectively curtails tumor growth and proliferation via activation of apoptotic pathways, providing a compelling rationale for progressing towards clinical trial. Citation Format: Ananya Banerjee, Neeke Busette, Xu Cheng, Kerslee Kohagen, Liwei Bao, Lluis Lopez-Barcons, Mark S. Berger, Matthew B. Soellner, Angel Qin, Sofia Merajver, Nathan Merrill. Quaratusugene ozeplasmid mediated TUSC2 upregulation in EML4-ALK bearing non-small cell lung carcinoma induces apoptosis and is highly effective in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 469.

Spatial feature plot of macrophage markers and related chemokines and cytokines.

Organoid transplant model for HER2-positive BCBMs. A, MRI of a patient with HER2+ breast cancer shows two cerebellar metastases in the brain. B, Schema for establishing BCBM in mice. C, Dorsal and coronal brain slice views of the mouse cerebellum. D, Brightfield images of dissected and postfixation cerebellar metastasis. E, Contrast-enhanced T1-weighted and T2-weighted MRI of stereotaxic injection at 3 and 4 weeks after transplantation of HER2+ organoids in C57BL/6 syngeneic mice. F, Top, Hematoxylin and eosin (H&E) staining distinguishes the highly cellular breast cancer cells from the normal brain parenchyma, which is more eosinophilic and has sparse cell nuclei. Along the border of the lesion, areas of invasion and edema with microvacuolization can be seen. Bottom, IHC stained with ErbB2 antibodies confirms the overexpression of HER2 in the intracranial lesion, as well as HER2 expression in the invading cells. Scale bars of lower power images are 1 mm. Scale bars of high-power images are 100 μm. G, IHC staining of the BCBM with GATA3, GFAP, and IBA1 antibodies. Scale bars of lower power images are 5 mm. Scale bars of high-power images are 500 μm.