Patient-derived Xenograft Models Research Articles

The roles of patient-derived xenograft models and artificial intelligence toward precision medicine.

Patient-derived xenografts (PDX) involve transplanting patient cells or tissues into immunodeficient mice, offering superior disease models compared with cell line xenografts and genetically engineered mice. In contrast to traditional cell-line xenografts and genetically engineered mice, PDX models harbor the molecular and biologic features from the original patient tumor and are generationally stable. This high fidelity makes PDX models particularly suitable for preclinical and coclinical drug testing, therefore better predicting therapeutic efficacy. Although PDX models are becoming more useful, the several factors influencing their reliability and predictive power are not well understood. Several existing studies have looked into the possibility that PDX models could be important in enhancing our knowledge with regard to tumor genetics, biomarker discovery, and personalized medicine; however, a number of problems still need to be addressed, such as the high cost and time-consuming processes involved, together with the variability in tumor take rates. This review addresses these gaps by detailing the methodologies to generate PDX models, their application in cancer research, and their advantages over other models. Further, it elaborates on how artificial intelligence and machine learning were incorporated into PDX studies to fast-track therapeutic evaluation. This review is an overview of the progress that has been done so far in using PDX models for cancer research and shows their potential to be further improved in improving our understanding of oncogenesis.

Unmasking Neuroendocrine Prostate Cancer with a Machine Learning-Driven 7-Gene Stemness Signature that Predicts Progression.

Prostate cancer (PCa) poses a significant global health challenge, particularly due to its progression into aggressive forms like neuroendocrine prostate cancer (NEPC). This study developed and validated a stemness-associated gene signature using advanced machine learning techniques, including Random Forest and Lasso regression, applied to large-scale transcriptomic datasets. The resulting 7-gene signature (KMT5C, MEN1, TYMS, IRF5, DNMT3B, CDC25B and DPP4) was validated across independent cohorts and patient-derived xenograft (PDX) models. The signature demonstrated strong prognostic value for progression-free, disease-free, relapse-free, metastasis-free, and overall survival. Importantly, the signature not only identified specific NEPC subtypes, such as large-cell neuroendocrine carcinoma, which is associated with very poor outcomes, but also predicted a poor prognosis for PCa cases that exhibit this molecular signature, even when they were not histopathologically classified as NEPC. This dual prognostic and classifier capability makes the 7-gene signature a robust tool for personalized medicine, providing a valuable resource for predicting disease progression and guiding treatment strategies in PCa management.

Preclinical Evaluation of AZD6422, an Armored Chimeric Antigen Receptor T Cell Targeting CLDN18.2 in Gastric, Pancreatic, and Esophageal Cancer.

CLDN18.2 is a surface membrane protein crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers. Thus, CLDN18.2 is suited for exploration as a clinical target for chimeric antigen receptor T-cell (CAR-T) therapy in these indications. Although CAR-T therapies show promise, a challenge faced in their development for solid tumors is the immunosuppressive tumor microenvironment, often characterized by the presence of immune and stromal cells secreting high levels of transforming growth factor beta (TGF-β). Addition of TGF-β armoring can potentially expand CAR-T activity in solid tumors. We report on the preclinical development of a CLDN18.2-targeting CAR-T showing effectiveness in CLDN18.2-positive gastric, esophageal, and pancreatic tumor models. The lead lentivirus product contains a unique single-chain variable fragment, CD28 and CD3z costimulatory and signaling domains, and dominant negative TGF-β receptor armoring, enhancing targeting and safety and counteracting suppression. We developed a shortened cell manufacturing process to enhance the potency of the final product, AZD6422. AZD6422 exhibited significant antitumor activity and tolerability in multiple patient-derived tumor xenograft models with various CLDN18.2 and TGF-β levels, as determined by immunohistochemistry. Efficacy of armored CAR-Ts in tumor models with elevated TGF-β was increased in vitro and in vivo. In vitro restimulation assays established greater persistence and cytolytic function of AZD6422 compared with a traditionally manufactured CAR-T. AZD6422 was safe and efficacious in patient-derived, CLDN18.2-positive murine models of gastrointestinal cancers. Our data support further clinical development of AZD6422 for patients with these cancers.

Anti-tissue factor antibody conjugated with monomethyl auristatin E or deruxtecan in pancreatic cancer models.

Antibody-drug conjugates (ADCs) have been recognized as a promising class of cancer therapeutics. Tissue factor (TF), an initiator of the blood coagulation pathway, has been investigated regarding its relationship with cancer, and several preclinical and clinical studies have presented data on anti-TF ADCs, including tisotumab vedotin, which was approved in 2021. However, the feasibility of other payloads in the design of anti-TF ADCs is still unclear because no reports have compared payloads with different cytotoxic mechanisms. For ADCs targeting other antigens, such as Her2, optimizing the payload is also an important issue in order to improve invivo efficacy. In this study, we prepared humanized anti-TF Ab (clone.1084) conjugated with monomethyl auristatin E (MMAE) or deruxtecan (DXd), and evaluated the efficacy in several cell line- and patient-derived xenograft models of pancreatic cancer. As a result, optimizing the drug / Ab ratio was necessary for each payload in order to prevent pharmacokinetic deterioration and maximize delivery efficiency. In addition, MMAE-conjugated anti-TF ADC showed higher antitumor effects in tumors with strong and homogeneous TF expression, while DXd-conjugated anti-TF ADC was more effective in tumors with weak and heterogeneous TF expression. Analysis of a pancreatic cancer tissue array showed weak and heterogeneous TF expression in most TF-positive specimens, indicating that the response rate to pancreatic cancer might be higher for DXd- than MMAE-conjugated anti-TF ADC. Nevertheless, our findings indicated that optimizing the ADC payloads individually in each patient could maximize the potential of ADC therapeutics.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eye’s uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases.

Uveal melanoma (UM) is a rare melanoma originating in the eye's uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Targeting SHCBP1 Inhibits Tumor Progression by Restoring Ciliogenesis in Ductal Carcinoma

Abstract Primary cilia detect and transmit environmental signals into cells. Primary cilia are absent in a subset of ductal carcinomas characterized by distinctive biological activities, and recovery of cilia with normal functionality has been shown to have therapeutic potential in some cancer types. Therefore, elucidation of the underlying mechanism and clinical significance of ciliary loss in ductal carcinomas could help develop effective treatment strategies. Here, we identified a link between SHCBP1 and cilia in ductal carcinomas. Shcbp1 knockout in transgenic mice profoundly impeded tumor progression and metastasis, prolonging survival. Single-cell transcriptome analysis revealed a functional connection between SHCBP1 deficiency and increased tumor ciliogenesis. SHCBP1 ablation restored ciliogenesis in unciliated ductal carcinoma by promoting the proximity between the midbody remnant (MBR) and centrosome through enhanced Rab8 GTPase activity and Rab8GTP positioning within the MBR. Inhibition of tumor progression by SHCBP1 loss relied on the recovery of ciliogenesis. Analysis of a large cohort of patients with ductal carcinoma revealed a negative correlation between SHCBP1-induced ciliary loss and patient prognosis. Restoring ciliogenesis via SHCBP1 ablation elicited therapeutic effects in patient-derived xenograft models. Together, this study delineates that induction of MBR-centrosome proximity through SHCBP1-deficiency reactivates ciliogenesis, offering unique opportunities for the treatment of unciliated ductal carcinomas.

Expression and Clinical Significance of Nuclear Phosphoglucomutase-1 in Hepatocellular Carcinoma.

This study aimed to evaluate the predictive values of phosphoglucomutase-1 (PGM1) expression for prognosis in patients with hepatocellular carcinoma (HCC). PGM1 expression was assessed by immunohistochemistry in tissue microarrays. The relationship of PGM1 expression level with pathologic parameters and prognosis values was respectively analyzed by χ2 test and Cox regression. The accuracy of independent risk factors in predicting prognosis was calculated by receiver operating characteristic curve. HCC patient-derived xenograft models were performed to evaluate the nuclear PGM1 antitumor effect. The results showed that PGM1 expression was low in HCC tissues. Nuclear PGM1 was an independent prognostic factor for overall survival and time to recurrence. Cox regression showed that nuclear PGM1, serum α-fetoprotein, liver cirrhosis, and TNM staging stage were independent risk predictors for HCC. Receiver operating characteristic curve demonstrated that combination of independent predictors had better prognostic value than TNM staging alone. Moreover, patient-derived xenograft models showed antitumor effect of nuclear PGM1. We found that low expression of nuclear PGM1 was detected in HCC tissues and associated with poor prognostic. Nuclear PGM1 was an independent prognostic factor in patients with HCC. Furthermore, nuclear PGM1 combining other independent risk factors showed a better prognostic value. Nuclear PGM1 was a useful prognostic biomarker for patients with HCC.

FABP4-mediated lipid metabolism promotes TNBC progression and breast cancer stem cell activity

Metabolic remodeling is a pivotal feature of cancer, with cancer stem cells frequently showcasing distinctive metabolic behaviors. Nonetheless, understanding the metabolic intricacies of triple-negative breast cancer (TNBC) and breast cancer stem cells (BCSCs) has remained elusive. In this study, we meticulously characterized the metabolic profiles of TNBC and BCSCs and delved into their potential implications for TNBC treatment. Our findings illuminated the robust lipid metabolism activity within TNBC tumors, especially in BCSCs. Furthermore, we discovered that Fabp4, through its mediation of fatty acid uptake, plays a crucial role in regulating TNBC lipid metabolism. Knocking down Fabp4 or inhibiting its activity significantly suppressed TNBC tumor progression in both the MMTV-Wnt1 spontaneous TNBC model and the TNBC patient-derived xenograft model. Mechanistically, Fabp4's influence on TNBC tumor progression was linked to its regulation of mitochondrial stability, the CPT1-mediated fatty acid oxidation process, and ROS production. Notably, in a high-fat diet model, Fabp4 deficiency proved to be a substantial inhibitor of obesity-accelerated TNBC progression. Collectively, these findings shed light on the unique metabolic patterns of TNBC and BCSCs, underscore the biological significance of Fabp4-mediated fatty acid metabolism in governing TNBC progression, and offer a solid theoretical foundation for considering metabolic interventions in breast cancer treatment. SignificanceTriple-negative breast cancer progression and breast cancer stem cell activity can be restricted by targeting a critical regulator of lipid responses, FABP4.

LncRNA-HMG incites colorectal cancer cells to chemoresistance via repressing p53-mediated ferroptosis

Upon chemotherapy, excessive reactive oxygen species (ROS) often lead to the production of massive lipid peroxides in cancer cells and induce cell death, namely ferroptosis. The elimination of ROS is pivotal for tumor cells to escape from ferroptosis and acquire drug resistance. Nevertheless, the precise functions of long non-coding RNAs (lncRNAs) in ROS metabolism and tumor drug-resistance remain elusive. In this study, we identify LncRNA-HMG as a chemoresistance-related lncRNA in colorectal cancer (CRC) by high-throughput screening. Abnormally high expression of LncRNA-HMG predicts poorer prognosis in CRC patients. Concurrently, we found that LncRNA-HMG protects CRC cells from ferroptosis upon chemotherapy, thus enhancing drug resistance of CRC cells. LncRNA-HMG binds to p53 and facilitates MDM2-mediated degradation of p53. Decreased p53 induces upregulation of SLC7A11 and VKORC1L1, which contribute to increase the supply of reducing agents and eliminate excessive ROS. Consequently, CRC cells escape from ferroptosis and acquire chemoresistance. Importantly, inhibition of LncRNA-HMG by anti-sense oligo (ASO) dramatically sensitizes CRC cells to chemotherapy in patient-derived xenograft (PDX) model. LncRNA-HMG is also a transcriptional target of β-catenin/TCF and activated Wnt signals trigger the marked upregulation of LncRNA-HMG. Collectively, these findings demonstrate that LncRNA-HMG promotes CRC chemoresistance and might be a prognostic or therapeutic target for CRC.

Highly Efficient Synergistic Chemotherapy and Magnetic Resonance Imaging for Targeted Ovarian Cancer Therapy Using Hyaluronic Acid-Coated Coordination Polymer Nanoparticles.

The diagnosis and treatment of ovarian cancer (OC) are still a grand challenge, more than 70% of patients are diagnosed at an advanced stage with a dismal prognosis. Magnetic resonance imaging (MRI) has shown superior results to other examinations in preoperative assessment, while cisplatin-based chemotherapy is the first-line treatment for OC. However, few previous studies have brought together the two rapidly expanding fields. Here a technique is presented using cisplatin prodrug (Pt-COOH), Fe3+, and natural polyphenols (Gossypol) to construct the nanoparticles (HA@PFG NPs) that have a stable structure, controllable drug release behavior, and high drug loading capacity. The acidic pH values in tumor sites facilitate the release of Fe3+, Pt-COOH, and Gossypol from HA@PFG NPs. Pt-COOH with GSH consumption and cisplatin-basedchemotherapy plus Gossypol with pro-apoptotic effects displays asynergisticeffect forkillingtumorcells. Furthermore, the release of Fe3+ at the tumor sites promotes ferroptosis and enables MRI imaging of OC. Inthe patient-derived tumor xenograft (PDX)model, HA@PFG NPs alleviate the tumor activity. RNA sequencing analysis reveals that HA@PFG NPs ameliorate OC symptoms mainly through IL-6 signal pathways. This work combines MRI imaging with cisplatin-based chemotherapy, which holds great promise for OC diagnosis and synergistic therapy.

GDF-15 predicts epithelioid hemangioendothelioma aggressiveness and is down-regulated by sirolimus through ATF4/ATF5 suppression.

Epithelioid hemangioendothelioma (EHE) poses a therapeutic challenge due to limited efficacy of conventional chemotherapy in advanced cases, necessitating exploration of new treatment avenues and identification of novel aggressiveness biomarkers. This study aimed at i) utilizing an EHE patient-derived xenograft (PDX) model and its associated cell line to assess the efficacy of sirolimus and ii) analyzing two distinct patient cohorts to pinpoint circulating biomarkers of EHE aggressiveness. A PDX model and corresponding cell line were established from an advanced EHE patient, demonstrating consistency with the original tumor in terms of histomorphology, WWTR1::CAMTA1 fusion presence, and genomic and transcriptomic profiles. Two independent patient series were employed to investigate the association between Growth/Differentiation Factor 15 (GDF-15) serum levels and EHE aggressiveness. ELISA analyses on EHE cell culture medium and blood from EHE-carrying mice revealed the release of GDF-15 by EHE cells. Sirolimus exhibited markedly higher anti-tumor activity compared to doxorubicin, concurrently reducing GDF-15 expression/release both in vivo and in vitro. This reduction was attributed to the drug-induced inhibition of phosphorylation/activation of 4E-BP1 and subsequent downregulation of the GDF-15 transcription factors ATF4 and ATF5. Blood sample analyses from two independent patient series showed a significant correlation between GDF-15 and EHE aggressiveness. This study identifies GDF-15 as a novel biomarker of EHE aggressiveness and underscores the superior efficacy of sirolimus compared to doxorubicin in our experimental models. The observed inhibition of GDF-15 release by sirolimus suggests its potential as a biomarker for monitoring the drug's activity in patients.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

Abstract C025: A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer

Abstract BACKGROUND: The absence of symptoms often leads to late diagnosis of pancreatic ductal adenocarcinoma (PDAC) limiting treatment options. Tumor cell proliferation and invasion/metastasis are usually driven by KRAS mutations and activation of Wnt/β-catenin pathway components. The recent approval of KRASG12C-specific inhibitors for lung cancer has validated RAS as a druggable target but has had limited application for PDAC given the rarity of this codon in PDAC. In addition, no Wnt/β-catenin inhibitors have been FDA approved. Thus, drugs which inhibit these important targets are urgently needed for PDAC and other cancers. Here, we describe treatment results with ADT-030, a novel dual pan-RAS/β-catenin inhibitor in murine models of PDAC and lung cancer. METHODS: In vitro assays of growth inhibition, colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were performed to assess potency and selectivity of ADT-030. Murine 2838c3-Luc KRASG12D PDAC cells were implanted orthotopically into the pancreas of C57BL/6J mice and cells from two PDAC patient-derived xenograft (PDX) models harboring KRASG12D or KRASG12C mutations were implanted subcutaneously (SC) in NSG mice to evaluate the antitumor activity of ADT-030. ADT-030 was administered once daily by gavage 5x/week for 4 weeks. RESULTS: ADT-030 potently inhibited the growth of PDAC cells in vitro harboring the most prevalent KRASG12D mutation as well as those with KRASG12C. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using propidium iodide staining revealed that ADT-030 arrested cells in the G2/M phase. Other experiments showed that ADT-030 simultaneously suppressed both MAPK/AKT signaling and β-catenin transcriptional by inhibiting PDE10 and activating cGMP/PKG signaling. ADT-030 caused tumor regression in SC PDAC PDX mouse tumor models harboring either KRASG12D or KRASG12C mutations without discernable toxicity. Inhibition of orthotopic PDAC tumor growth by ADT-030 was dose-dependent, causing up to 80% reduction in tumor volume following a 4-week treatment period. In addition, ADT-030 significantly extended survival and yielded a high percentage of cures after stopping treatment in a murine model of KRAS- mutant lung cancer. CONCLUSION: ADT-030, novel dual pan-RAS/β-catenin inhibitor, showed strong and durable antitumor activity in clinically relevant and aggressive murine models of PDAC and lung cancer. ADT-030 is a promising development candidate for treatment of PDAC and other RAS-driven cancers, particularly given the complex KRAS mutation landscape of many cancers. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C025.

Abstract A027: Enhancing pancreatic cancer chemotherapy through photochemical internalisation

Abstract Introduction Pancreatic cancer is the deadliest common cancer. Its poor prognosis is associated with its complex biology and lack of early detection strategies. Although much progress has been made in finding more effective therapeutic strategies, pancreatic tumours remain difficult to treat. A major reason why chemotherapy is relatively ineffective against this type of cancer is inefficient delivery of drugs to their target sites combined with multidrug resistance. Our aim is to use the technique called Photochemical Internalisation (PCI), a light-triggered intracellular drug delivery method which combines low dose Photodynamic Therapy (PDT) with chemotherapy, to induce efficient cytosolic delivery of therapeutic compounds to their specific subcellular targets. Methodology Treatment outcomes using saporin (a type I ribosome-inactivating protein) and gemcitabine as monotherapies, or in combination with a light-activated photosensitizer (the porphyrin TPPS2a), were thoroughly analysed in both established pancreatic cancer cell lines and patient-derived xenograft (PDX) models. The efficacy of these treatments was assessed in both 2D and 3D tumour models through various cell viability assays, including MTT, crystal violet staining, and neutral red staining. Additionally, molecular techniques such as western blotting, protein arrays, and flow cytometry were employed to determine the effects on cell viability and to elucidate changes in signalling pathways leading to the therapeutic response. Results In this study, we observed minimal cytotoxicity induced by saporin, gemcitabine or TPPS2a + light monotherapies. However, PCI synergistically enhanced saporin and gemcitabine cytotoxicity (p&amp;lt;0.001) using very low concentrations in all tumour models. Moreover, we determined the mechanism of cell death triggered by these protocols. PCI induced endosomal disruption following light excitation and apoptosis leading to caspases 3/7 activation, in a time dependent manner. Conclusions Overall, our findings demonstrate the potential of PCI to enhance the efficacy of cancer chemotherapy for pancreatic cancer using lower doses than in monotherapy and open a new window for future translational studies. By using low doses of light therapy, we have found a promising avenue for future studies that could eventually lead to better treatments for this challenging disease. Citation Format: Maria Rosado, Ismahan Mahamed, Andres Garcia-Sampedro, Sandy MacRoberts, Pål Selbo, Stephen Pereira, Pilar Acedo. Enhancing pancreatic cancer chemotherapy through photochemical internalisation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A027.

Overcoming Osimertinib Resistance with AKT Inhibition in EGFRm-Driven Non-Small Cell Lung Cancer with PIK3CA/PTEN Alterations.

Osimertinib is an EGFR tyrosine kinase inhibitor indicated for the treatment of EGFR-mutated (EGFRm)-driven lung adenocarcinomas. Osimertinib significantly improves progression-free survival in first-line-treated patients with EGFRm advanced non-small cell lung cancer (NSCLC). Despite the durable disease control, the majority of patients receiving osimertinib eventually develop disease progression. ctDNA profiling analysis of on-progression plasma samples from patients treated with osimertinib in both first- (phase III, FLAURA trial) and second-line trials (phase III, AURA3 trial) revealed a high prevalence of PIK3CA/AKT/PTEN alterations. In vitro and in vivo evidence using CRISPR-engineered NSCLC cell lines and patient-derived xenograft (PDX) models supports a functional role for PIK3CA and PTEN mutations in the development of osimertinib resistance. These alterations are functionally relevant as EGFRm NSCLC cells with engineered PIK3CA/AKT/PTEN alterations develop resistance to osimertinib and can be resensitized by treatment with the combination of osimertinib and the AKT inhibitor capivasertib. Moreover, xenograft and PDX in vivo models with PIK3CA/AKT/PTEN alterations display limited sensitivity to osimertinib relative to models without alterations, and in these double-mutant models, capivasertib and osimertinib combination elicits an improved antitumor effect versus osimertinib alone. Together, this approach offers a potential treatment strategy for patients with EGFRm-driven NSCLC who have a suboptimal response or develop resistance to osimertinib through PIK3CA/AKT/PTEN alterations. See related commentary by Vokes et al., p. 3968.

Dronedarone inhibits the proliferation of esophageal squamous cell carcinoma through the CDK4/CDK6-RB1 axis in vitro and in vivo.

Treatment options for patients with esophageal squamous cell carcinoma (ESCC) often result in poor prognosis and declining health-related quality of life. Screening FDA-approved drugs for cancer chemoprevention is a promising and cost-efficient strategy. Here, we found that dronedarone, an antiarrhythmic drug, could inhibit the proliferation of ESCC cells. Moreover, we conducted phosphorylomics analysis to investigate the mechanism of dronedarone-treated ESCC cells. Through computational docking models and pull-down assays, we demonstrated that dronedarone could directly bind to CDK4 and CDK6 kinases. We also proved that dronedarone effectively inhibited ESCC proliferation by targeting CDK4/CDK6 and blocking the G0/G1 phase through RB1 phosphorylation inhibition by in vitro kinase assays and cell cycle assays. Subsequently, we found that knocking out CDK4 and CDK6 decreased the susceptibility of ESCC cells to dronedarone. Furthermore, dronedarone suppressed the growth of ESCC in patient-derived tumor xenograft models in vivo. Thus, our study demonstrated that dronedarone could be repurposed as a CDK4/6 inhibitor for ESCC chemoprevention.

C-Myc alone is enough to reprogram fibroblasts into functional macrophages

BackgroundMacrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases.MethodsThe composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac.ResultsHere we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models.ConclusionsOur findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

SNHG14 promotes triple-negative breast cancer cell proliferation, invasion, and chemoresistance by regulating the ERK/MAPK signaling pathway.

The functional role and molecular mechanisms of small-nucleolar RNA host gene 14 (SNHG14) in triple-negative breast cancer (TNBC) progression remain unclear. The expression levels of SNHG14 in breast cancer samples and cell lines were determined using real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion abilities were detected using MTS and transwell assays. By RNA sequencing, differentially expressed genes were identified between the SNHG14 siRNA and the negative control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to predict the targets and pathways regulated by SNHG14. pRAF, pMEK, and pERK expression were measured by western blot. The xenograft model was constructed to access the biological function of SNHG14 in vivo. A minimal patient-derived xenograft model was established to evaluate the sensitivity to chemotherapy drugs. Our data indicated that SNHG14 expression was increased in TNBC tissues and cell lines. SNHG14 knockdown attenuated the proliferation, migration, and invasion abilities of TNBC cells both in vivo and in vitro. High SNHG14 expression was associated with lymph node metastasis and a high Ki67 index. The targets of SNHG14 were mainly enriched in the MAPK signaling pathway. pRAF, pMEK, and pERK expression were downregulated after being transfected with SNHG14 siRNA. Compared with the negative control group, the expression of CACNA1I, DUSP8, FGF17, FGFR4, FOS, PDGFRB, and DDIT3 was increased, and the expression of MKNK1 was decreased in the SNHG14 siRNA group. Minimal patient-derived xenograft model demonstrated that knockdown of SNHG14 enhanced the sensitivity to Docetaxel in vivo. Compared with the DMSO group, the proliferation of Docetaxel-resistant MDA-MB-231 cells was decreased in Dabrafenib, PD184352, and FR180204 treatment groups. SNHG14 knockdown inhibits TNBC progression by regulating the ERK/MAPK signaling pathway, which provides evidence for SNHG14 as a potential target for TNBC therapy.