AACR Annual Meeting 2026

Gamma delta (γδ) T cells are a unique subset of lymphocytes that possess innate and adaptive immune functions. Unlike conventional αβ T cells, γδ T cells recognize antigens in an MHC-independent manner. This allows them to detect a wide range of stress-induced ligands that are commonly expressed on tumor cells. This property makes γδ T cells particularly attractive for cancer immunotherapy, especially in cases where tumors evade immune surveillance by downregulating MHC molecules. Recent advances in cellular engineering have made it possible to generate γδ T cells that express chimeric antigen receptors (CARs), which combines their natural tumor-recognition capabilities with the targeted specificity of CARs. γδ CAR T cells offer several advantages over αβ CAR T cells, including lower graft-versus-host disease risk, suitability for allogeneic applications (“out-of-the-shelf”), and enhanced solid tumor infiltration due to tissue-homing properties. These properties provide a strong rationale for investigating γδ CAR T cells as a novel cancer therapy approach and underscore the importance of continued optimization to realize their full therapeutic potential.

This study explores strategies to improve the intracellular signaling domain of CAR constructs with a focus on γδ T cells. The CD3ζ domain has traditionally been used as the primary activation motif in CAR design. However, emerging evidence suggests that it may not be optimal for all T cell subsets. Therefore, we investigated alternative CD3 subunits, specifically, CD3δ, CD3ε, and CD3γ, as intracellular signaling domains in CAR constructs. Our in vitro data show that CARs with these alternative CD3 cytoplasmic tails are more effective than conventional CD3ζ-based CARs in terms of activation and cytotoxicity. In vivo validation of αβ CAR T cells incorporating alternative CD3 subunits has demonstrated enhanced antitumor efficacy. To extend these findings, in vivo experiments are currently underway using the NALM-6_luc (luciferase expressing) xenograft model, a well-established system for evaluating next-generation cellular therapies. These experiments aim to explore whether similar benefits apply to γδ CAR T cells. If confirmed, these results would underscore the potential of leveraging alternative CD3 signaling domains to optimize CAR T cell performance, particularly in γδ T cell-based therapies.

Our findings provide a compelling rationale for reevaluating CAR construct design, suggesting that alternative CD3 subunits could significantly enhance the therapeutic potential of γδ CAR T cells in cancer treatment.

Chimeric antigen receptor (CAR) T cells have emerged as a powerful tool in cancer immunotherapy, particularly in hematologic malignancies such as B-cell leukemia and lymphoma. CAR T cell therapy involves genetically engineering patient-derived T cells to express synthetic receptors that target tumor-associated antigens, enabling precise and potent immune responses against cancer cells. Despite remarkable clinical successes, especially with CD19-targeted CAR T cells, the therapy has limitations that hinder broader application and long-term efficacy. A significant challenge is T cell exhaustion, a dysfunctional state involving reduced proliferation, diminished cytokine production, and impaired cytotoxicity. Exhaustion often arises in the tumor microenvironment, where persistent antigen stimulation, immunosuppressive signals, and metabolic stress collectively impair CAR T cell function. This limits durability and contributes to relapse in many patients. Overcoming CAR T cell exhaustion is essential to improving efficacy and durability of cellular therapies. Strategies under investigation include optimizing CAR architecture by using alternative co-stimulatory domains, transiently modulating inhibitory pathways, and refining manufacturing protocols to preserve T cell fitness.

An in vitro assay that reliably mimics exhaustion is essential for identifying and validating improvements. In this study, we present several approaches to inducing exhaustion-like phenotypes in CAR T cells. Chronic, antigen-agnostic stimulation and repeated co-culture with antigen-expressing target cells are used to model persistent activation, and to compare various functional readouts. We discuss the advantages and limitations of different assay setups to help identify the most suitable approach depending on the specific experimental needs. A robust and reproducible assay system is key to reliably screening novel CAR T cell designs and therapeutic interventions aimed at mitigating T cell exhaustion. We compare T cell functionality using multiple readout technologies, including impedance-based measurements using xCelligence for adherent and selected suspension cells and luminescence assays with luciferase-expressing target cells as well as flow cytometry. This allows us to highlight the strengths and constraints of each method in capturing functional decline and exhaustion phenotypes.

Our findings support the use of standardized in vitro exhaustion models as valuable tools for the preclinical evaluation of next-generation CAR T cell therapies and combination strategies. Furthermore, our antigen-agnostic model enables evaluation of the impact of any T cell modulation approach on exhaustion dynamics. These models enable more predictive and efficient development of strategies to enhance therapeutic performance.

Background: G protein-coupled receptors (GPCRs) are increasingly recognized as important targets in cancer therapy due to their roles in tumor growth, angiogenesis, and metastasis. Linklight technology is a protein-protein interaction (PPI) detection platform that monitors β-arrestin recruitment to activated G protein-coupled receptors (GPCRs}, complementing second messenger-based assays for ligand activity characterization. Here, we use Linklight stable cell lines coexpressing TEV-protease-tagged GPCRs and permuted luciferase (pluc)-tagged β-arrestin to study β-arrestin recruitment, cAMP signal, and calcium-influx within the same cellular context. Methods: Cell-based functional assays were conducted in both agonist and antagonist modes across three GPCR Linklight stable cell lines representing distinct G-protein couplings (Gs, Gi, and Gq). β-Arrestin-1/2 recruitment was quantified using the Linklight PPI luminescence readout, while G-protein-mediated signaling was measured via cAMP or calcium-influx fluorescence, depending on receptor class.

Results: Ligands produced consistent, mechanism-appropriate responses across β-arrestin and second-messenger pathways. For Gq-coupled ADRA 1A, the agonist Cirazoline induced robust β-arrestin-1/2 recruitment (EC50 = 2.51 x10-8 M) and calcium influx (EC50 = 8.2x10-9 M), while the antagonist Prazosin inhibited both (β-arrestin EC50 = 2.51 x10-8 M; calcium EC50 = 2.06x10-7 M). For Gi-coupled ADRA2A, the agonist Brimonidine stimulated β-arrestin-1/2 recruitment (EC50 = 2.64x 10-8 M) and suppressed cAMP (EC50 = 8.88x 10-10 M), whereas the antagonist Yohimbine blocked β-arrestin recruitment (EC50 = 1.78x10-8 M) and reversed cAMP inhibition (EC50 = 2.69x10-7 M). For Gs-coupled ADRB2, the agonist Fenoterol induced β-arrestin-2 recruitment (EC50 = 8.83x10-10 M) and cAMP signaling (EC50 = 1.52x10-9 M), while the antagonist Yohimbine (S)Propranolol inhibited both (β-arrestin EC50 = 3.98x10-8 M; cAMP EC50 = 3.6x10-8 M). Across all receptors, β-arrestin recruitment aligned with G-protein-specific signaling in response to the same ligand, demonstrating the robustness of the comprehensive GPCR portfolio.

Summary: By enabling simultaneous assessment of β-arrestin recruitment and G-protein signaling in a unified cellular context, Linklight technology offers a comprehensive, mechanistically informative platform for GPCR pharmacology. Integrated with G-protein activation data, Linklight technology also supports high throughput profiling of ligand efficacy, bias, and receptor
regulation for GPCR drug discovery against cancer.

The serine/threonine phosphatase WIP1 (PPM1D) is a key negative regulator of the DNA damage response (DDR) and a recognized oncogene, frequently amplified or truncated in various cancers including breast, ovarian, neuroblastoma, and glioblastoma. Its inhibition restores DDR signaling, offering a promising therapeutic strategy. Despite initial efforts, including the development of the allosteric inhibitor GSK2830371, clinical translation has been limited. We report the establishment of a comprehensive assay platform to support drug development targeting WIP1. Biochemical assays were optimized using fluorescein diphosphate (FDP), malachite green with phosphopeptides (p53, H2AX, p38 MAPK), and TR-FRET formats, enabling robust activity profiling. Cellular mechanism-of-action assays were developed to monitor phosphorylation changes in WIP1 substrates, with pS15-p53 ELISA in U2OS cells selected for medium-throughput iterative compound profiling. Phenotypic
assays demonstrated compound efficacy in 2D and 3D proliferation models, with leukemic cell lines showing pronounced sensitivity. Combination studies revealed strong synergy between WIP1 inhibitors and MDM2 antagonists (e.g., Nutlin3a), supporting a dual-targeting approach for p53 pathway reactivation. In vivo efficacy was confirmed in MV4-11 xenograft models, with dose-dependent tumor growth inhibition and favorable tolerability profiles. Our integrated assay suite enables iterative screening and lead optimization, culminating in the identification of a potent allosteric WIP1 inhibitor with nanomolar activity and promising preclinical efficacy. These findings support WIP1 as a viable target in oncology and provide a foundation for future clinical development.

Antibody-drug conjugates (ADCs) are a rapidly evolving class of targeted cancer therapeutics that combine the specificity of monoclonal antibodies with the potency of small-molecule cytotoxic drugs. It is critical for ADCs to be stable in the systemic circulation in order to prevent premature release of the payload, which can lead to off-target toxicity and reduced therapeutic efficacy. The pharmacokinetic (PK) behavior of ADCs is influenced by several factors, such as antibody structural heterogeneity, linker type, and payload physicochemical properties. Stability is a key quality attribute that directly affects dosing strategies, the therapeutic window, and clinical outcomes. Therefore, robust and sensitive analytical platforms are essential for monitoring ADC integrity and payload release over time. While traditional ELISA-based methods are widely used, they often lack the sensitivity and dynamic range required to detect subtle changes in ADC composition during circulation. In  this study, we use the Meso Scale Discovery® (MSD) platform, which uses plate-based electrochemiluminescence (ECL) technology, to evaluate the in vivo PK and stability of ADCs. By applying this technology to preclinical in vivo models, we aim to generate high-resolution PK profiles and stability data that support the rational design and optimization of ADCs for clinical development.

Standard 1-Spot SECTOR plates were used to detect the antibody backbone by coating them with a specific capture antibody. Small Spot Streptavidin SECTOR plates were used to detect the intact ADC, capturing the molecule via anti-payload antibodies. In both assays, detection was performed using a sulfo-tagged anti-human IgG antibody. After optimizing the setup, it demonstrated a broad dynamic range of 4-5 orders of magnitude and a lower limit of detection of less than 100 pg/mL for both the antibody and the intact ADC. Subsequent in vivo PK studies involved administering ADCs via a single intravenous injection to SCID beige mice. Serum samples were diluted 1:1000 in PBS containing 1% BSA, and 25 µL per well was applied to the MSD plates. This high dilution factor permits minimal blood volume collection per time point, thereby reducing the number of animals required. The assay demonstrated excellent reproducibility with minimal variability between replicates. To further increase the relevance of the results, future studies may use mFcRn⁻/⁻ hFcRn transgenic mice or HSA/hFcRn/hFcγR triple transgenic models. These models more accurately reflect the serum half-life of human IgG and correlate well with data from cynomolgus monkeys and humans.

In summary, this MSD-based assay platform offers a highly sensitive and reproducible approach for in vivo PK analysis of ADCs that spares animals. There is potential to further refine this approach using humanized mouse models to improve clinical predictability.

Cyclin-dependent kinases (CDKs) and their associated cyclins are central regulators of cell cycle progression and transcriptional control. Dysregulation of CDK/Cyclin complexes is a hallmark of many cancers, driving uncontrolled proliferation and tumor development. Consequently, these complexes have become critical targets in oncology, with CDK4/6 inhibitors already established as a cornerstone in the treatment of e.g. hormone receptor-positive breast cancer and under investigation for other malignancies. Early preclinical development of CDK inhibitors relies on biochemical screening assays to identify small molecules capable of inhibiting CDK/Cyclin activity. These assays, frequently based on kinase activity measurements, enable high-throughput evaluation of compound libraries and provide essential insights into inhibitor potency and selectivity. These approaches are mandatory for rational drug design, guiding the optimization of lead compounds toward improved efficacy and reduced off-target effects. Despite their obvious limitations in predicting cellular and in vivo responses, these early screening strategies are instrumental in shaping the current generation of CDK- and other kinase-targeted therapeutics. In subsequent development stages, cellular and in vivo model systems are employed to validate inhibitor activity and assess pharmacodynamics and toxicity. Many of these models are based on non-human mammalian species, such as murine or primate systems. These studies are essential for bridging the gap between biochemical screening and clinical application, ensuring that candidate molecules demonstrate efficacy and safety before entering human trials. However, despite the importance of such data, it is rarely assessed in early biochemical screening whether results from human and non-human assays are consistent. Such early biochemical evaluation of potential differences in the effects of drug candidates on the kinase target from different species could generate valuable insights to select the most relevant cellular or in-vivo model systems for advanced drug-development. Here, we present comparative biochemical data for late-stage development or already approved CDK inhibitors tested against CDK/Cyclin complexes from human, rat, mouse, dog and primate origin, focusing on CDK4/CycD1. Notably, differential inhibitory potency was observed for several compounds, including palbociclib, which showed an approximately ten-fold difference in CDK4/CycD1 inhibition between human and murine enzymes.

Brain metastases remain exceptionally challenging to treat, in part because many candidate therapies fail to demonstrate the clinical benefit observed in preclinical studies. A major contributor to this gap is the limited ability of traditional animal models to accurately replicate the functional blood–brain barrier (BBB). Conventional intracranial orthotopic tumor models are commonly used but inherently compromise BBB integrity, reducing their utility for evaluating drug penetration and therapeutic performance. Other implantation routes, such as intravenous or intracardiac delivery, better preserve the BBB but often produce extensive extra-cranial metastases, shortening study duration and diminishing relevance for brain-specific disease.

To overcome these limitations, we established an intra-carotid tumor cell delivery method that selectively seeds the brain while minimizing systemic spread. This technique enables localized tumor growth in disease relevant brain regions without disrupting BBB architecture. Tumor progression and spatial distribution were followed longitudinally using magnetic resonance imaging (MRI) together with bioluminescence imaging (BLI). These modalities consistently demonstrated reliable brain colonization and supported high-resolution tracking of tumor growth dynamics.

Our intra-carotid model provides a more physiologically faithful system for studying metastatic brain disease. By retaining BBB functionality and supporting reproducible, long-term monitoring, this platform offers a powerful tool for assessing BBB-permeant therapeutics and may enhance the translational value of preclinical studies in for brain targeted therapies, particularly in the context of metastatic disease. 

Macrophages play a central role in the tumor microenvironment. Depending on their activation state and local signals, they can exert both pro- and anti-tumorigenic effects. Tumor-associated macrophages (TAMs) often exhibit an immunosuppressive, pro-tumoral phenotype that promotes tumor growth, angiogenesis, metastasis, and therapy resistance. Due to their plasticity and abundance in solid tumors, TAMs are attractive therapeutic targets: either by reprogramming them toward an anti-tumoral state or depleting their tumor-supportive functions. Understanding macrophage behavior in cancer is critical for developing effective immunotherapies, given their dual roles. This requires reliable, physiologically relevant in vitro assays modeling key functions, such as phagocytosis, efferocytosis, cytokine secretion, and tumor cell interaction.

Macrophage phagocytosis and efferocytosis assays assess the impact of test compounds on the uptake activity of human M0, M1, and M2 macrophages. CD14+ monocytes are isolated from cryopreserved PBMCs and differentiated into M0 macrophages using M-CSF for six days, then polarized into M1 (IFN-γ + LPS), M2 (IL-4 + IL-13) or TAM-like (IL-4 + IL-10 + TGF-β) phenotypes. After polarization, cells are treated with test compounds at defined concentrations and time points, followed by incubation with either pHrodo™ bioparticles or apoptotic pHrodo™ labelled tumor cells (camptothecin-treated Raji cells) to assess phagocytosis or efferocytosis, respectively. Quantification is performed by measuring red fluorescence over 12 - 24 hours using a Cytation 5 reader.

Our experiments indicate that M0, M2 and TAM-like macrophages have a strong capacity for phagocytosis and efferocytosis, while M1 macrophages have minimal particle or apoptotic tumor cell uptake. Time-resolved analysis reveals distinct uptake kinetics. M0 and M2 macrophages rapidly internalize targets within 2-4 hours. However, M0 cells subsequently decline in activity, while M2 macrophages maintain a sustained plateau for up to 12 hours with an overall higher uptake. Repolarizing M2 macrophages to an M1 phenotype using LPS and IFN-γ markedly reduces their phagocytic capacity. Blocking the "don't eat me" signal with an anti-CD47 antibody significantly enhances the efferocytosis of Raji cells. We anticipate that cytokine profiling will reveal mechanistic differences between phagocytosis mediated by pattern recognition receptors and efferocytosis, which is critical for tissue homeostasis.

These findings suggest that the phenotype of macrophages strongly influences their phagocytic and efferocytic behavior, with M2 cells exhibiting the highest capacity. Strategies that modulate macrophage polarization or relieve inhibitory signals could enhance these functions and support further investigation into their therapeutic potential.

Kinase inhibitors have been on spotlight for cancer drug discovery ever since the approval of Imatinib and recently the 100th small molecule kinase inhibitor was approved by the FDA showing the potential of kinase inhibitors for cancer therapeutics and beyond. HER2 kinase is an established drug target in breast cancer therapeutics with multiple inhibitors developed against this target over time. However, cancer developing drug resistance to these inhibitors over time remain as a prognostic challenge for aggressive breast cancer treatment. Recent studies investigating the HER2 resistance mechanism of breast cancers identify HUNK (hormonally upregulated neu-associated Kinase) playing a crucial role in promoting HER2+ breast cancer cell survival and inhibitors resistance via phosphorylation of Rubicon. Recently a first in class HUNK targeted inhibitor HSL119 has been identified and characterized to demonstrate potential of targeting HUNK as a treatment strategy for HER2 resistant breast cancers, but more potent inhibitors for this target are yet to be identified. To address this, we screened 145 FDA approved compounds in HUNK biochemical kinase assay in Reaction Biology’s HotSpot assay platform and identified Momelotinib, an orally available JAK1/JAK2/ACVR1 inhibitor used to treat myelofibrosis, inhibiting HUNK with 7.9 nM IC50. NanoBRET target engagement assay confirmed Momelotinib binds cellular HUNK in live HEK293 cells. Furthermore, CellTiter-Glo assays demonstrated Momelotinib inhibits proliferation of HER2+ JIMT1 cells and triple-negative 4T1 breast cancer cells, while caspase-3/7 activation assays indicated Momelotinib induces apoptotic cell death in both lines. These findings highlight Momelotinib as a potential therapeutic candidate for overcoming HER2 inhibitor resistance in breast cancer.

Drug development costs exceed $172m and increase to over $500m when clinical trial failures are considered (Sertkaya 2024, DOI: 10.1001/jamanetworkopen.2024.15445). It can take 20 years to bring a drug to market so it is critical to identify safety liabilities early in the discovery process to ensure valuable time and money are not invested in preclinical and clinical trials. In vitro safety panels, such as InVEST44™, can identify potential toxicities of drugs cost-effectively during early-stage drug development.

This is particularly relevant as, between 2006 and 2023, the proportion of venture capital investments in early-stage drug discovery has decreased from 50% to 16% while increasing for later stage from 20 to 54% (The Biopharma Industry Investment Report 2015. Biotechnology Innovation Organization; Larka 2024, Trends in Venture Capital Funding for Biopharma Startups. GlobalData). This highlights a trend towards expensive, high-risk but potentially lucrative, clinical trials and away from early drug discovery. The goal is to demonstrate that early-stage in vitro safety panels are fiscally and scientifically critical steps in drug development to identify off-target effects before advancing drugs to animal testing and clinical trials.

InVEST44™ is an in vitro safety panel designed to identify off-target effects of lead compounds for oncology and other therapeutic areas in early-stage drug development, ensuring a more efficient path from discovery to clinical trials. It comprises 44 industry standard, high risk targets based on Bowes et al. (2012; DOI: 10.1038/nrd3845), including GPCRs, transporters, ion channels, nuclear receptors and enzymes. Various assay types are utilized, including radioligand binding, fluorescence polarization, enzymatic activity, cell reporter, calcium mobilization and electrophysiology assays.

We identified 20 drug candidates from 17 failed clinical oncology trials between 2001 and 2025, all terminated because of reported severe adverse effects (clinicaltrials.org). These were screened at 10 µM in InVEST44™: 17 drugs elicited >50% effect on at least 1 target, including 8 with 4+ target interactions and one interacting with 15 safety targets.

This study demonstrates the importance of in vitro safety panels, such as InVEST44™, by identifying off-targets effect of lead compounds before pursuing time-consuming and expensive animal testing and potentially failed clinical trials. Data from such panels are increasingly being provided to enhance IND applications (Scott, 2022, DOI: 10.1016/j.vascn.2022.107205). In summary, by providing cost-efficient, early-stage identification of off-target effects by lead compounds, in vitro safety panels, such as InVEST44™, have become integral components of drug discovery and warrant increased early-stage consideration and investment in the future.

Protein arginine methyltransferase 5 (PRMT5) is a type II methyltransferase that symmetrically dimethylates arginine residues on histone and non-histone proteins, an essential epigenetic modification that shapes chromatin structure and regulates gene expression. Through these activities, PRMT5 controls key cellular processes, including transcription, RNA splicing, DNA repair, and signal transduction. PRMT5 operates in complex with MEP50, which is required for efficient catalytic activity. Dysregulated PRMT5 is strongly associated with diseases, particularly cancer. PRMT5 is frequently overexpressed in breast, lung, prostate, and hematologic malignancies, where it promotes tumor growth, metastasis, and therapy resistance. Small-molecule PRMT5 inhibitors have emerged as promising agents that block methyltransferase activity, reverse aberrant epigenetic marks, and disrupt oncogenic pathways. Here, we established comprehensive assay platforms for PRMT5-targeted drug discovery and validated them using five known inhibitors (LLY-283, JNJ-64619178, GSK591, EPZ015666, and GSK33326595). Our biochemical FlashPlate assay demonstrated potent inhibition of PRMT5/MEP50 activity, with IC₅₀ values in the low nanomolar range (0.6-17 nM). Surface Plasmon Resonance (SPR) revealed distinct binding profiles: substrate-competitive inhibitors (EPZ015666, GSK33326595, GSK591) showed weak affinity for apo PRMT5, enhanced by cofactors (MTA, SAH, SAM), whereas cofactor-competitive LLY-283 bound tightly to apo PRMT5 but exhibited >50-fold reduced affinity with cofactors. JNJ-64619178 displayed reduced signal upon cofactor binding, driven by a slow off-rate consistent with pseudo-irreversible inhibition. NanoBRET target engagement intracellular assay results indicated that the PRMT5 inhibitors engaged with the PRMT5/MEP50 complex within one hour of incubation in live HEK293 cells, and Western blot confirmed inhibition of histone H4R3me2s methylation, a key substrate of PRMT5, in MV4-11, Jeko-1, 22RV1, and PC3 cancer cell lines. Collectively, these platforms enable identification, optimization, and mechanistic characterization of PRMT5 inhibitors, accelerating development of selective and potent therapeutic candidates.

 Cancer cachexia remains a major unmet need in oncology, characterized by progressive loss of skeletal muscle and adipose tissue that negatively impacts treatment tolerance and survival. Anamorelin (AML), a ghrelin receptor agonist, is currently the only approved therapeutic for cancer cachexia; however, its clinical benefits remain modest, highlighting the need for improved preclinical models and more informative physiological endpoints. Here, we established and characterized a C26 colorectal carcinoma cachexia model using both traditional metrics and non-invasive body composition imaging performed on a Bruker MiniSpec. This platform enabled longitudinal quantification of lean and fat mass changes with high sensitivity, providing a refined assessment of cachexia progression and therapeutic response.In vitro and in vivo studies confirmed the robust cachexia-inducing phenotype of the C26 model. Consistent with published literature, AML treatment increased food intake in tumor-bearing mice but did not reduce circulating pro-inflammatory cytokines. Combination therapy, evaluated based on the mechanistic rationale that AML-mediated appetite stimulation may complement the anti-inflammatory activity of CYA, demonstrated enhanced preservation of body mass and adipose tissue compared with either monotherapy. Our findings support the utility of integrating body composition imaging with standard physiological and biochemical measures to generate a comprehensive efficacy profile. These results highlight the value of this refined platform for evaluating emerging cachexia therapeutics and suggest that multi-modal combinations such as CYA+AML may provide superior benefit over current single-agent approaches.  

Brain metastases are one of the most common and serious complications of solid tumors, affecting 20-40% of patients. They are common in lung, breast, and melanoma cancers and are associated with a poor prognosis and limited treatment options. Key challenges include the blood-brain barrier (BBB), which restricts penetration of many therapeutic agents, and the brain's historically immune-privileged status, limiting immune responses against tumor cells. Establishing in vivo models that accurately replicate the natural metastatic process to the brain is essential for understanding disease mechanisms and advancing therapeutic research. Most current preclinical brain metastasis models rely on intracranial implantation of tumor cells directly into the brain or systemic delivery via intracardiac injection. Although intracranial implantation enables rapid local tumor formation, this approach disrupts the BBB integrity and limits physiological relevance for studying natural metastatic progression and evaluating compounds with specific BBB-crossing properties. In contrast, intracardiac injection allows systemic dissemination of tumor cells and can lead to brain metastases in ~50% of cases using MDA-MB-231 cells and 70%-80% using JIMT-1 cells while likely preserving BBB integrity. However, these models are limited by suboptimal brain metastasis rates or early euthanasia due to aggressive extracranial tumor growth, hindering study of brain-specific
disease progression over time.

In this study, we present a more physiologically relevant brain metastasis model based on intracarotid injection. This approach enhances the targeted delivery of tumor cells to the brain while preserving BBB integrity, increasing translational relevance. We report take rate and intracranial tumor growth kinetics of human breast cancer cell lines following intracarotid injection and compare these outcomes with those from conventional intracardiac injection. The intracarotid technique yields a high rate of brain metastasis and confines tumor growth to the brain region. To further validate the model, we evaluate efficacy of standard-of-care (SOC) therapies within this system, providing insights into therapeutic responsiveness under conditions closely mimicking clinical scenarios. Additionally, we present data on the growth of 4T1 syngeneic tumor cells in the brain, enabling the study of immune-tumor interactions in an immunocompetent host. This syngeneic model adds another dimension by allowing exploration of immunological mechanisms and testing of immunotherapies in a controlled, biologically relevant setting.

In conclusion, these findings suggest that the intracarotid injection model is a valuable tool for preclinical evaluation of novel therapies targeting brain metastases.

 Accurate characterization of microglia and infiltrating immune cell populations is essential for understanding the immunological landscape of brain tumors and for evaluating therapeutic responses. Here, we report the development and optimization of a multicolor flow cytometry panel tailored to discriminate resident microglia from peripheral myeloid and lymphoid subsets within an experimental brain tumor model. Marker selection was guided by tissue-specific expression profiles, leveraging differential levels of CD45, CX3CR1, and P2RY12 to resolve microglia, while incorporating canonical markers for infiltrating macrophages, dendritic cells, neutrophils, and T cells. Enzymatic dissociation conditions, viability dye selection, and gating hierarchy were systematically optimized to preserve epitope integrity and maximize signal resolution. Validation using tumor-bearing and control brain tissues demonstrated robust separation of microglial and infiltrating populations with reproducible quantification across biological replicates. This workflow provides a reliable and scalable approach for immune profiling in brain tumor research, enabling deeper insight into neuroimmune interactions and immunotherapy-driven changes within the tumor microenvironment.  

In the preclinical space of cancer drug discovery, multi-omics characterization of cancer models is essential for model selection, understanding mechanisms of action, and early biomarker discovery. We launched a panel of 160 cell lines (CL Proliferation Panel) to study drug responses across a large variety of cancer types and validated their molecular characteristics at both the transcriptomic and genomic levels. Recently, we characterized this set of models at the proteomic level using mass spectrometry, identifying a total of more than 15.000 proteins. In the present work, we aim to investigate the relevance of this new dataset in the daily practice of cancer model utilization. First, we demonstrated the robustness of the established proteomic profiles, showing high concordance among duplicate samples. Using a multiomics comparison and dimensionality reduction approaches, we curated the proteomic dataset to remove outlier and irrelevant protein profiles, ensuring high data quality. Next, unsupervised hierarchical clustering of the proteomic profiles revealed accurate classification of models according to their tumor type of origin, confirming the relevance of the data. Given the growing importance of proteomic information in the context of antibody-drug conjugate (ADC) development, we further assessed the relevance of our dataset for evaluating the expression of top 20 ADC targets such as ERBB2 and TACSTD2, among others. We also established a multi-omics strategy integrating transcriptomic and proteomic data to enhance target characterization. Finally, we explored the relationship between protein expression of the targets and drug sensitivity by analyzing ADC targets in the context of responses to clinically approved ADCs, including trastuzumab emtansine (Kadcyla), and sacituzumab govitecan.

 Diffuse intrinsic pontine glioma (DIPG) and meningioma are central nervous system tumors where anatomical location critically influences disease behavior and therapeutic response. To investigate this impact, we have developed and compared preclinical models using SF8628-GFP⁺/Luc⁺ (DIPG) and Ben-Men-1-Luc (meningioma) across three inoculation sites: (1) clinically relevant locations (pons for DIPG; skull base for meningioma), (2) general intracranial sites, and (3) subcutaneous sites. Tumor progression was monitored longitudinally using bioluminescence imaging and confirmed by histopathology. This study sought to determine how site-specific microenvironments affect growth kinetics, infiltration patterns, and imaging characteristics. Understanding these differences is essential for drug development, as orthotopic models better replicate blood-brain barrier constraints, tumor vascularization, and local tissue interactions that influence therapeutic delivery and efficacy.