Patient-derived Xenograft Mouse Models Research Articles

P10.22.B COMBINATION OF ARTS MIMETICS WITH AUTOPHAGY INHIBITORS AS A NOVEL THERAPEUTIC STRATEGY TO OVERCOME APOPTOSIS RESISTANCE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common malignant brain tumour in adults. The current standard of care for GBM patients consists of maximal surgical resection followed by chemotherapy and radiation. Despite such an aggressive treatment approach, the average 5-year survival rate for GBM patients is approximately 5%. One of the main reasons for poor therapy response in the GBM is resistance to programmed cell death, otherwise known as apoptosis. One of the mechanisms behind apoptosis resistance relies on the upregulation of inhibitor of apoptosis proteins (IAPs) which are known to hinder pro-apoptotic proteins and allow cell survival under abnormal conditions such as severe DNA damage. However, there are natural IAP antagonists such as ARTS which are capable of stimulating cell death by promoting the degradation of IAPs. To harness the pro-apoptotic function of IAP antagonists, small peptide mimetics of ARTS have been developed. This project investigates the efficacy of utilizing ARTS mimetics as a novel strategy to overcome apoptosis resistance in the GBM treatment. MATERIAL AND METHODS This study utilizes patient-derived cells with exogenous gain-of-function (GOF) overexpression to introduce full-length ARTS protein as well as pharmacological small peptide mimetics of ARTS to evaluate the response of GBM cells following the promotion of ARTS activity. We also employ a patient-derived orthotopic xenograft mouse model to test novel combinatorial strategies in vivo, where we used a nanoparticle drug delivery system to ensure treatment delivery across the blood-brain barrier (BBB). RESULTS We found that patient-derived GBM cells were highly resistant to ARTS-induced apoptosis. Further molecular investigation unveiled a novel role of ARTS in the upregulation of autophagy as a resistance mechanism. A combination of ARTS GOF or mimetic peptides with autophagy inhibitors mitigated resistance to ARTS-mediated apoptosis and drastically decreased GBM cell proliferation. This combination was further tested in the patient-derived xenograft mouse model, which demonstrated the successful delivery of ARTS mimetic peptides and autophagy inhibitors through the BBB via nanoparticle lipid carriers. Animals treated with a combination of the autophagy inhibitor Spautin-1 and ARTS mimetic peptides demonstrated significantly prolonged survival, confirming the therapeutic potential of such combinatorial approach for GBM treatment. CONCLUSION Altogether, these findings uncover a previously unknown role of ARTS in autophagy regulation and provide evidence that the combination of IAP-antagonist mimetics with autophagy inhibitors can be an effective strategy against highly aggressive GBM brain tumours. This work was supported by the Health Sciences Centre Foundation (HSCF).

198. MODELING CANCER CACHEXIA USING PATIENT TUMOR ORGANOID-DERIVED XENOGRAFTS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Cancer cachexia is a metabolic and functional multifactorial body wasting syndrome and is one of the major causes of death in late-stage patients. Esophageal squamous cell carcinoma (ESCC) presents one of the highest morbidity and mortality rates due to cancer cachexia. However, there is limited effective and approved targeted treatment. Cancer cachexia animal models are highly limited, with few cancer types available as a pre-clinical drug testing platform and with none related to esophageal carcinoma. Methods We have developed a panel of ESCC patient-derived organoid xenograft (PDOX) mouse models based on our latest patient-derived organoid cultures from Hong Kong patients. Results Our PDOX mouse model displays cachexic phenotypes that traditional cell line-derived xenografts lack, including body wasting and functional signs. The slow-proliferating characteristic of the ESCC PDOX on mice allows development of cachexic conditions before the humane endpoint. Our comprehensive panel of PDOX lines allowed us to identify full spectrum and differential cachexia severities across mice bearing different PDOXs, indicating a heterogenous nature and allowing faithful mimicking of various patient conditions. Molecular analysis showed that several classic cachexia-related gene expressions were up-regulated in the isolated liver and inguinal adipose tissues of cachexic mice, along with elevated circulating Il-6 levels. Conclusion Our ESCC PDOX model potentially serves as a useful pre-clinical cachexia-targeted drug testing platform, providing more treatment options for ESCC patients for better disease management. Acknowledgments We acknowledge the Research Grants Council (Theme-based Research Scheme T12–701/17-R to MLL) and the Food and Health Bureau (Health and Medical Research Fund 06171566 to VZY) of the Hong Kong Special Administrative Region for funding support. We acknowledge DSMZ (German Collection of Microorganisms and Cell Culture) for the KYSE cell lines. We acknowledge the University of Hong Kong LKS Faculty of Medicine Centre for PanorOmic Sciences for providing imaging facilities.

Bcl-2 inhibition combined with PPARα activation synergistically targets leukemic stem cell-like cells in acute myeloid leukemia

Persistence of leukemic stem cells (LSCs) is one of the determining factors to acute myeloid leukemia (AML) treatment failure and responsible for the poor prognosis of the disease. Hence, novel therapeutic strategies that target LSCs are crucial for treatment success. We investigated if targeting Bcl-2 and peroxisome proliferator activated receptor α (PPARα), two distinct cell survival regulating mechanisms could eliminate LSCs. This study demonstrate that the Bcl-2 inhibitor venetoclax combined with the PPARα agonist chiglitazar resulted in synergistic killing of LSC-like cell lines and CD34+ primary AML cells while sparing their normal counterparts. Furthermore, the combination regimen significantly suppressed AML progression in patient-derived xenograft (PDX) mouse models. Mechanistically, chiglitazar-mediated PPARα activation inhibited the transcriptional activity of the PIK3AP1 gene promoter and down-regulated the PI3K/Akt signaling pathway and anti-apoptotic Bcl-2 proteins, leading to cell proliferation inhibition and apoptosis induction, which was synergized with venetoclax. These findings suggest that combinatorial Bcl-2 inhibition and PPARα activation selectively eliminates AML cells in vivo and vitro, representing an effective therapy for patients with relapsed and refractory AML.

Smoking induces WEE1 expression to promote docetaxel resistance in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) patients have poor clinical outcomes, with an overall 5-year survival rate of 20%. Smoking is a significant risk factor for EAC. The role of WEE1, a nuclear kinase that negatively regulates the cell cycle in normal conditions, in EAC tumorigenesis and drug resistance is not fully understood. Immunohistochemistry staining shows significant WEE1 overexpression in human EAC tissues. Nicotine, nicotine-derived nitrosamine ketone, or 2% cigarette smoke extract treatment induces WEE1 protein expression in EAC, detected by western blot and immunofluorescence staining. qRT-PCR and reporter assay indicates that smoking induces WEE1 expression through miR-195-5p downregulation in EAC. ATP-Glo cell viability and clonogenic assay confirmed that WEE1 inhibition sensitizes EAC cells to docetaxel treatment invitro. A TE-10 smoking machine with EAC patient-derived xenograft mouse model demonstrated that smoking induces WEE1 protein expression and resistance to docetaxel invivo. MK-1775 and docetaxel combined treatment improves EAC patient-derived xenograft mouse survival invivo. Our findings demonstrate, for the first time, that smoking-induced WEE1 overexpression through miRNA dysregulation in EAC plays an essential role in EAC drug resistance. WEE1 inhibition is a promising therapeutic method to overcome drug resistance and target treatment refractory cancer cells.

PI3K/mTOR inhibitor VS-5584 combined with PLK1 inhibitor exhibits synergistic anti-cancer effects on non-small cell lung cancer

Small molecule drugs are of significant importance in the treatment of non-small cell lung cancer (NSCLC). Here, we explored biological effects of the PI3K/mTOR inhibitor VS-5584 on NSCLC. Our findings indicated that VS-5584 administration resulted in a dose-dependent inhibition of NSCLC cell proliferation, as well as the induction of apoptosis and cycle arrest. Additionally, we observed a significant increase in intracellular reactive oxygen species (ROS) levels following VS-5584 treatment. The use of the ROS inhibitor N-acetylcysteine (NAC) effectively reduced ROS levels and decreased the proportion of apoptotic cells. Treatment with VS-5584 led to an upregulation of genes associated with apoptosis and cell cycle, such as c-caspase 3 and P21. Conversely, a downregulation of cyclin-dependent kinase 1 (CDK1) expression was observed. Next, transcriptome analyses revealed that VS-5584 treatment altered the abundance of 1520 genes/transcripts in PC-9 cells, one of which was polo-like kinase 1 (PLK1). These differentially expressed genes were primarily enriched in biological processes such as cell cycle regulation and cell apoptosis, which are closely linked to the P53 and apoptosis pathways. Co-treatment with VS-5584 and PLK1 inhibitor NMS-P937 resulted in enhanced cancer cell death, exhibiting synergistic inhibitory activity. Notably, VS-5584 inhibited the growth of NSCLC in a patient-derived xenograft (PDX) mouse model without observable abnormalities in major organs. Overall, VS-5584 effectively suppressed the growth of NSCLC cells both in vitro and in vivo. VS-5584 combined with NMS-P937 exhibited a synergistic effect in inhibiting NSCLC cell growth. These findings suggest that VS-5584 has potential as a therapeutic strategy for treating NSCLC.

HDS screening with patient-derived primary cells guided individualized therapy for esophageal squamous cell carcinoma-in vivo and vitro.

To analyze and evaluate the role of the High-throughput Drug Sensitivity (HDS) screening strategy in identifying highly sensitive drugs against esophageal squamous cell carcinoma (ESCC). A total of 80 patients with progressive ESCC were randomly divided into the observation (40 cases) and the control groups (40 cases). In the observation group, primary ESCC cells were isolated from the tumor tissues with a gastroscope, and drug sensitivity screening was performed on cells derived from the 40 ESCC cases using the HDS method, followed by verification in a patient-derived tumor xenograft (PDX) mouse model. Finally, the differences in the therapeutic efficacy (levels of CEA, CYFRA21-1, SCCA after chemotherapy and the rates of overall survival, local progression, and distant metastasis at 12 months and 18 months time points after chemotherapy) were compared between the observation group (Screened drug-treated) and the control group (Paclitaxel combined with cisplatin regimen-treated). Forty ESCC patients were screened for nine different high-sensitive chemotherapeutics, with the majority showing sensitivity to Bortezomib. Experiments on animal models revealed that the tumor tissue mass of PDX mice treated with the HDS-screened drug was significantly lower than that of the Paclitaxel-treated mice (p < 0.05), and the therapeutic efficacy of the observation group was better than the control group (p < 0.05). HDS screening technology can be beneficial in screening high-efficacy anticancer drugs for advanced-stage ESCC patients, thereby minimizing adverse drug toxicity in critically ill patients. Moreover, this study provides a new avenue for treating advanced ESCC patients with improved outcomes.

PEGylated Polymeric Nanoparticles Loaded with 2-Methoxyestradiol for the Treatment of Uterine Leiomyoma in a Patient-Derived Xenograft Mouse Model

Leiomyomas, the most common benign neoplasms of the female reproductive tract, currently have limited medical treatment options. Drugs targeting estrogen/progesterone signaling are used, but side effects and limited efficacy in many cases are major limitation of their clinical use. Previous studies from our laboratory and others demonstrated that 2-methoxyestradiol (2-ME) is promising treatment for uterine fibroids. However, its poor bioavailability and rapid degradation hinder its development for clinical use. The objective of this study is to evaluate the in vivo effect of biodegradable and biocompatible 2-ME-loaded polymeric nanoparticles in a patient-derived leiomyoma xenograft mouse model. PEGylated poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles loaded with 2-ME were prepared by nanoprecipitation. Female 6-week age immunodeficient NOG (NOD/Shi-scid/IL-2Rγnull) mice were used. Estrogen-progesterone pellets were implanted subcutaneously. Five days later, patient-derived human fibroid tumors were xenografted bilaterally subcutaneously. Engrafted mice were treated with 2-ME-loaded or blank (control) PEGylated nanoparticles. Nanoparticles were injected intraperitoneally and after 28 days of treatment, tumor volume was measured by caliper following hair removal, and tumors were removed and weighed. Up to 99.1% encapsulation efficiency was achieved, and the in vitro release profile showed minimal burst release, thus confirming the high encapsulation efficiency. In vivo administration of the 2-ME-loaded nanoparticles led to 51% growth inhibition of xenografted tumors compared to controls (P < 0.01). Thus, 2-ME-loaded nanoparticles may represent a novel approach for the treatment of uterine fibroids.

C-Met is a chimeric antigen receptor T-cell target for treating recurrent nasopharyngeal carcinoma

Background aimsRadiation therapy is the standard treatment for patients with nasopharyngeal carcinoma (NPC), but relapse occurs in 10% to 20% of patients. The treatment of recurrent nasopharyngeal carcinoma (rNPC) remains challenging. Chimeric antigen receptors (CAR)-T-cell therapy has achieved good outcomes in the treatment of leukemia and seems to be a promising therapeutic strategy for solid tumors. c-Met has been found to be highly expressed in multiple cancer types, and the activation of c-Met leads to the proliferation and metastasis of cancer cells. However, the expression of c-Met in rNPC tissues and whether it can be used as a target for CAR-T therapy in rNPC remain to be investigated. MethodsWe detected the expression of c-Met in 24 primary human rNPC tissues and three NPC cell lines and constructed two different antibody-derived anti-c-Met CARs, namely, Ab928z and Ab1028z. To estimate the function of these two different c-Met-targeted CAR-T cells, CD69 expression, cytotoxicity and cytokine secretion of CAR-T cells were assessed after coculture with target cells. A cell line-derived xenograft mouse model also was used to evaluate these two anti-c-Met CAR-T cells. Furthermore, we determined whether combination with an anti-EGFR antibody could promote the antitumor effect of CAR-T cells in a patient-derived xenograft mouse model. ResultsHigh c-Met expression was detected in 23 of 24 primary human rNPC tissues by immunohistochemistry staining and in three NPC cell lines by flow cytometry. Ab928z-T cells and Ab1028z-T cells showed significantly upregulated expression of CD69 after coculture with targeted cells. However, Ab1028z-T cells showed superior cytokine secretion and antitumor activity. Furthermore, Ab1028z-T cells effectively suppressed tumor growth compared with control CAR-T cells, and the combination with nimotuzumab further enhanced the tumor-clearing ability of Ab1028z-T cells. ConclusionsWe found that c-Met is highly expressed in rNPC tissues and confirmed its potential as a CAR-T target for rNPC. Our study provides a new idea for the clinical treatment of rNPC.

Small Extracellular Vesicle-Derived vWF Induces a Positive Feedback Loop between Tumor and Endothelial Cells to Promote Angiogenesis and Metastasis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a hypervascular malignancy by which its growth and dissemination are largely driven by the modulation of tumor-derived small extracellular vesicles (sEVs). Proteomic profiling of circulating sEVs of control individuals and HCC patients identifies von Willibrand factor (vWF) to be upregulated progressively along HCC stages. Elevated sEV-vWF levels are found in a larger cohort of HCC-sEV samples and metastatic HCC cell lines compared to their respective normal counterparts. Circulating sEVs of late-stage HCC patients markedly augment angiogenesis, tumor-endothelial adhesion, pulmonary vascular leakiness, and metastasis, which are significantly compromised by anti-vWF antibody. The role of vWF is further corroborated by the enhanced promoting effect of sEVs collected from vWF-overexpressing cells. sEV-vWF modulates endothelial cells through an elevated level of vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Mechanistically, secreted FGF2 elicits a positive feedback response in HCC via the FGFR4/ERK1 signaling pathway. The co-administration of anti-vWF antibody or FGFR inhibitor significantly improves the treatment outcome of sorafenib in a patient-derived xenograft mouse model. This study reveals mutual stimulation between HCC and endothelial cells by tumor-derived sEVs and endothelial angiogenic factors, facilitating angiogenesis and metastasis. It also provides insights into a new therapeutic strategy involving blocking tumor-endothelial intercellular communication.

OTHR-10. COLLABORATIVE EFFORTS FOR HOSPITAL-BASED BIOBANK DEVELOPMENT AND OPTIMIZATION TO ENSURE QUALITY PEDIATRIC SPECIMENS FOR TRANSLATIONAL RESEARCH

Abstract In recent years, drastic improvements in sequencing technologies have led to a better understanding of the specific genetic drivers of cancer. However, one of the greatest barriers to these discoveries is access to primary patient tumor tissue, especially in cancers of the Central Nervous System (CNS). In many cases tissue from surgery is processed and stored in Formalin-Fixed Paraffin-Embedded (FFPE) blocks, which sets limitations on downstream applications. In 2015, our team launched an initiative to collect and efficiently store samples from pediatric CNS tumor patients during surgeries and autopsies. Through extensive collaboration between Neuro-Oncology, Neurosurgery, Pathology, and our Translational Laboratory, we have optimized the collection process to effectively preserve sample integrity and therefore maximize their potential usage. To date, we have collected over 4,509 samples from 293 patients (636 flash-frozen tissue, 1,026 plasma &amp; buffy coat, 748 serum, 996 whole blood, 316 Cerebrospinal Fluid (CSF), and 787 parental blood samples). In addition, we have cultured 31 primary cell lines and collected over 242 dissociated tumor specimens for implantation into patient-derived xenograft (PDX) mouse models. Through our partnership with neurosurgery and pathology, we are permitted to attend surgeries and autopsies to immediately flash-freeze tumor tissue in liquid nitrogen, typically within minutes of the tissue being removed from the body. This immediate flash-freezing process aims to ensure specimen integrity and preserve molecular profiles. From there, the tissue is either stored in our -140oC freezer for future use or sent to partnered consortiums, including the Children’s Brain Tumor Network (CBTN) and Gift from a Child. Since the optimization of the collection process, the focus has now shifted to investigating the efficacy of our storage methods by quantifying DNA/RNA integrity over time. By ensuring the successful preservation of the samples, we can maximize their impact in the pediatric neuro-oncology research field.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells

BackgroundBreast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HypothesisEarlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. MethodsCo-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. ResultsData revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. ConclusionIn silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

XMT-1660: A phase 1b trial of a B7-H4 targeted antibody drug conjugate (ADC) in breast, endometrial, and ovarian cancers.

TPS3154 Background: Breast cancer (BC) is the most commonly diagnosed cancer and one of the leading causes of cancer death in women. Despite significant therapeutic advances, the majority of patients with unresectable or recurrent/metastatic disease eventually develop resistance to available standard of care (SOC) therapies. High B7-H4 expression has been observed in several cancers including breast, endometrial, and ovarian, with limited expression in normal tissue. As a member of the CD28/B7 family of cell surface proteins, it promotes tumorigenesis by suppressing anti-tumor immunity. XMT-1660 is a B7-H4-directed Dolasynthen antibody drug conjugate designed with a precise, optimized drug-to-antibody ratio and a DolaLock microtubule inhibitor payload with controlled bystander effect. In the preclinical setting, XMT-1660 has demonstrated anti-tumor activity in TNBC and ER+/HER2- patient-derived xenograft mouse models, which included tumors from heavily pre-treated patients (Collins et al, AACR 2022). Increased anti-tumor activity tended to be more frequent in preclinical models with higher B7-H4 expression, providing rationale for a Ph1 clinical trial. Methods: The Ph1 trial includes a first-in-human open-label dose escalation (DES) portion followed by dose expansion (EXP) evaluating XMT-1660 in patients with BC, EC, and OC following progression on SOC as applicable (i.e., CDK4/6i + ET; platinum-based chemotherapy). In the DES, Bayesian Optimal Interval (BOIN) design will be used to determine the MTD. Patients will receive XMT-1660 IV Q3 weeks. Primary endpoints in DES are to assess safety and determine a recommended phase 2 dose (RP2D) and assessment of preliminary efficacy as a secondary endpoint. In parallel with DES, optional backfill cohorts will enroll patients into tumor-type specific cohorts (TNBC, HR+/HER2- BC, EC, or OC) at a selected dose level from DES and can be initiated at multiple dose levels simultaneously. In the EXP portion, cohorts enrolling TNBC, ER+/HER2- BC, EC/OC are planned and additional patients may be enrolled based on emerging data. The primary endpoint of EXP is to assess safety and tolerability, overall response rate, disease control rate, and duration of response. Secondary endpoints include pharmacokinetic analysis and assessment of antidrug antibodies. Patients are not selected by B7-H4 status, but baseline tumors samples are collected for retrospective tumor tissue evaluation. The trial is currently enrolling patients. NCT05377996. Clinical trial information: NCT05377996 .

Relationship between efficacy of phase I/II drug ispinesib in medulloblastoma, molecular subtypes, and p53-mutant status.

e14001 Background: KIF11, a mitotic kinesin, is responsible for assembly and maintenance of mitotic spindle during mitosis. Tumor cells can upregulate KIF11 to enhance tumor proliferation. Many cancers overexpress KIF11, including medulloblastoma (MB), the most common malignant brain tumor in children. MB is molecularly classified into 4 molecular subtypes: sonic-hedgehog (SHH)-activated MB, Wingless (WNT)-activated, and less characterized Group 3 and 4 subtypes. Group 3 and 4 MBs are the most aggressive subtypes and Group 3 MBs have the worst survival outcome. p53 mutation defines a high-risk group in MB patients. We evaluated the efficacy of a Phase I/II KIF11 inhibitor, ispinesib on MB. Methods: We employed in-vitro proliferation assays (CCK), flow cytometry (Annexin V, Propidium Iodide), western blot, Hoechst staining, and two patient-derived orthotopic xenograft (PDOX) mouse models for in-vivo drug testing. Results: Ispinesib effectively inhibited proliferation at nanomolar concentrations for all 5 MB cell lines. A comparison of IC50 (day 4) indicated that p53-mutational-status did not influence sensitivity to ispinesib. In all five cell lines, regardless of p53 mutational status, ispinesib led to G2/M arrest. However, different p53-mutational-status led to different modes of cell death. High levels of necrotic cell death were observed in p53-mutant, while apoptosis was the predominant mode of cell death in p53-wildtype, on flow cytometry. Apoptotic nuclei were more significantly observed in p53-wildtype (p = 0.002) while p53-mutant cell line demonstrated more micronuclei (p = 0.0302) and multi-nucleation (p= 0.0161), compared to respective DMSO-control cells on Hoechst staining. Ispinesib has demonstrated effective blood brain barrier penetration in brain xenografts. We compared 2 PDOX models of MB. Ispinesib significantly improved survival outcome of Group 3 MB (Control=6, Treated=7, Log-rank p=0.0003), but worsened the survival outcome of p53-mutant SHH MB (Control=14, Treated=11, Log-rank p=0.169). This differential efficacy of ispinesib was recapitulated in the matched 3D-neurosphere cultures of both PDOX models. Cancer-stem-cell (CSC)-enriched neurospheres derived from aggressive Group 3 subtype demonstrated susceptibility to 7-day in-vitro treatment with ispinesib (AUC=0.7743). Monolayer tumor cells demonstrated similar susceptibility (AUC=0.7757). Area under curve (AUC)&gt;0.7 was the significant cut-off. In contrast, both CSC-enriched neurospheres (AUC=0.379) and monolayer tumor cells (AUC=0.491) derived from p53-mutant SHH MB did not demonstrate susceptibility to ispinesib, consistent with matching PDOX models. Conclusions: Preclinically, our results indicate feasibility of ispinesib treatment in MB, specifically aggressive Group 3 MB. Importantly, p53-mutant SHH MB represents a poor molecular-subtype candidate for ispinesib therapy.

From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Biological evaluation of novel gemcitabine analog in patient-derived xenograft models of pancreatic cancer

Gemcitabine (Gem) has been a standard first-line drug for pancreatic cancer (PCa) treatment; however, Gem's rapid metabolism and systemic instability (short half-life) limit its clinical outcome. The objective of this study was to modify Gem into a more stable form called 4-(N)-stearoyl-gemcitabine (4NSG) and evaluate its therapeutic efficacy in patient-derived xenograft (PDX) models from PCa of Black and White patients.Methods 4NSG was synthesized and characterized using nuclear magnetic resonance (NMR), elemental analysis, and high-performance liquid chromatography (HPLC). 4NSG-loaded solid lipid nanoparticles (4NSG-SLN) were developed using the cold homogenization technique and characterized. Patient-derived pancreatic cancer cell lines labeled Black (PPCL-192, PPCL-135) and White (PPCL-46, PPCL-68) were used to assess the in vitro anticancer activity of 4NSG-SLN. Pharmacokinetics (PK) and tumor efficacy studies were conducted using PDX mouse models bearing tumors from Black and White PCa patients.Results 4NSG was significantly stable in liver microsomal solution. The effective mean particle size (hydrodynamic diameter) of 4NSG-SLN was 82 ± 6.7 nm, and the half maximal inhibitory concentration (IC50) values of 4NSG-SLN treated PPCL-192 cells (9 ± 1.1 µM); PPCL-135 (11 ± 1.3 µM); PPCL-46 (12 ± 2.1) and PPCL-68 equaled to 22 ± 2.6 were found to be significantly lower compared to Gem treated PPCL-192 (57 ± 1.5 µM); PPCL-135 (56 ± 1.5 µM); PPCL-46 (56 ± 1.8 µM) and PPCL-68 (57 ± 2.4 µM) cells. The area under the curve (AUC), half-life, and pharmacokinetic clearance parameters for 4NSG-SLN were 3–fourfold higher than that of GemHCl. For in-vivo studies, 4NSG-SLN exhibited a two-fold decrease in tumor growth compared with GemHCl in PDX mice bearing Black and White PCa tumors.Conclusion 4NSG-SLN significantly improved the Gem's pharmacokinetic profile, enhanced Gem's systemic stability increased its antitumor efficacy in PCa PDX mice bearing Black and White patient tumors.

89Zr]-Atezolizumab-PET Imaging Reveals Longitudinal Alterations in PDL1 during Therapy in TNBC Preclinical Models.

Triple-negative breast cancers (TNBCs) currently have limited treatment options; however, PD-L1 is an indicator of susceptibility to immunotherapy. Currently, assessment of PD-L1 is limited to biopsy samples. These limitations may be overcome with molecular imaging. In this work, we describe chemistry development and optimization, in vitro, in vivo, and dosimetry of [89Zr]-Atezolizumab for PD-L1 imaging. Atezolizumab was conjugated to DFO and radiolabeled with 89Zr. Tumor uptake and heterogeneity in TNBC xenograft and patient-derived xenograft (PDX) mouse models were quantified following [89Zr]-Atezolizumab-PET imaging. PD-L1 expression in TNBC PDX models undergoing therapy and immunohistochemistry (IHC) was used to validate imaging. SUV from PET imaging was quantified and used to identify heterogeneity. PET/CT imaging using [89Zr]-Atezolizumab identified a significant increase in tumor:muscle SUVmean 1 and 4 days after niraparib therapy and revealed an increased trend in PD-L1 expression following other cytotoxic therapies. A preliminary dosimetry study indicated the organs that will receive a higher dose are the spleen, adrenals, kidneys, and liver. [89Zr]-Atezolizumab PET/CT imaging reveals potential for the noninvasive detection of PD-L1-positive TNBC tumors and allows for quantitative and longitudinal assessment. This has potential significance for understanding tumor heterogeneity and monitoring early expression changes in PD-L1 induced by therapy.

Delivery of Engineered Primary Tumor-Derived Exosomes Effectively Suppressed the Colorectal Cancer Chemoresistance and Liver Metastasis.

Liver metastasis is one of the major causes of colorectal cancer (CRC)-related morbidity and mortality. Delivering small interfering RNAs (siRNAs) or noncoding RNAs has been reported as a promising method to target liver metastasis and chemoresistance in CRC. Here, we report a noncoding RNA delivery system using exosomes derived from primary patient cells. Coiled-coil domain-containing protein 80 (CCDC80) was strongly associated with CRC liver metastasis and chemoresistance, a finding validated by bioinformatic analysis and clinical specimens. Silencing CCDC80 significantly increased sensitivity to chemotherapy agents in OXA-resistant cell lines and a mouse model. The primary cell-derived exosome delivery system was designed to simultaneously deliver siRNAs targeting CCDC80 and increase chemotherapy sensitivity in the distant CRC liver metastasis mouse models and patient-derived xenograft mouse models. We further validated the antitumor effect in an ex vivo model of chemoresistant CRC organoids and a patient-derived organoid xenograft model. Tumor-bearing mice treated with the siRNA-delivering exosomes and hepatectomy showed ideal overall survival. Our results provide a therapeutic target and represent a possible therapeutic alternative for patients with CRC and distant metastasis and in cases of chemoresistance.

Irbesartan overcomes gemcitabine resistance in pancreatic cancer by suppressing stemness and iron metabolism via inhibition of the Hippo/YAP1/c-Jun axis

BackgroundChemoresistance is the main reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Thus, there is an urgent need to screen out new targets and compounds to reverse chemotherapeutic resistance.MethodsWe established a bio-bank of human PDAC organoid models, covering a representative range of PDAC tumor subtypes. We screened a library of 1304 FDA-approved compounds to identify candidates efficiently overcoming chemotherapy resistance. The effects of the compounds were evaluated with a CellTiter-Glo-3D assay, organoid apoptosis assay and in vivo patient-derived xenograft (PDX), patient-derived organoid (PDO) and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) genetically engineered mouse models. RNA-sequencing, genome editing, sphere formation assays, iron assays and luciferase assays were conducted to elucidate the mechanism.ResultsHigh-throughput drug screening of chemotherapy-resistant PDOs identified irbesartan, an angiotensin ‖ type 1 (AT1) receptor antagonist, which could synergistically enhance the ability of chemotherapy to kill PDAC cells. In vitro and in vivo validation using PDO, PDX and KPC mouse models showed that irbesartan efficiently sensitized PDAC tumors to chemotherapy. Mechanistically, we found that irbesartan decreased c-Jun expression by inhibiting the Hippo/YAP1 pathway and further overcame chemotherapy resistance in PDAC. We also explored c-Jun, a potential target of irbesartan, which can transcriptionally upregulate the expression of key genes involved in stemness maintenance (SOX9/SOX2/OCT4) and iron metabolism (FTH1/FTL/TFRC). More importantly, we observed that PDAC patients with high levels of c-Jun expression demonstrated poor responses to the current standard chemotherapy regimen (gemcitabine plus nab-paclitaxel). Moreover, patients with PDAC had significant survival benefits from treatment with irbesartan plus a standard chemotherapy regimen in two-center retrospective clinical cohorts and patients with high c-Jun expression exhibited a better response to combination chemotherapy.ConclusionsIrbesartan could be used in combination with chemotherapy to improve the therapeutic efficacy in PDAC patients with high levels of c-Jun expression. Irbesartan effectively inhibited chemotherapy resistance by suppressing the Hippo/YAP1/c-Jun/stemness/iron metabolism axis. Based on our findings, we are designing an investigator-initiated phase II clinical trial on the efficacy and safety of irbesartan plus a standard gemcitabine/nab-paclitaxel regimen in the treatment of patients with advanced III/IV staged PDAC and are hopeful that we will observe patient benefits.

Clinicopathological characteristics associated with the engraftment of patient lymphoma cells in NOG mice.

Patient-derived xenograft (PDX) mouse models are useful for deepening our understanding of the biology of malignant lymphoma; however, factors associated with the success of the PDX lymphoma model are largely unknown. We retrospectively analyzed the characteristics of 66 xenotransplantations from 65 patients. In all, 43 (65%) specimens were obtained from patients aged > 60years, and 42 (64%) specimens were obtained at diagnosis. Specimens were obtained from patients with the following diseases: diffuse large B-cell lymphoma (n = 30), intravascular large B-cell lymphoma (n = 12), follicular lymphoma (n = 8), peripheral T-cell lymphoma (n = 7), mantle cell lymphoma (n = 2), and other (n = 7). The specimens were sourced mainly from bone marrow (n = 31, 47%) and extranodal tumors (n = 13, 20%). Engraftment was successful in 33/66 (50%) xenotransplantations. The median age of patients who provided successful specimens was significantly higher than that for unsuccessful specimens (p = 0.013). Specimens with a high proportion of tumor cells in the graft and those obtained from patients with relapsed/refractory disease showed higher tendencies toward successful engraftment. Taken together, these data suggest that tumor cells with a highly malignant potential might have a high likelihood of engraftment.

The National Cancer Institute's Co-Clinical Quantitative Imaging Research Resources for Precision Medicine in Preclinical and Clinical Settings.

Genetically engineered mouse models (GEMMs) and patient-derived xenograft mouse models (PDXs) can recapitulate important biological features of cancer. They are often part of precision medicine studies in a co-clinical setting, in which therapeutic investigations are conducted in patients and in parallel (or sequentially) in cohorts of GEMMs or PDXs. Employing radiology-based quantitative imaging in these studies allows in vivo assessment of disease response in real time, providing an important opportunity to bridge precision medicine from the bench to the bedside. The Co-Clinical Imaging Research Resource Program (CIRP) of the National Cancer Institute focuses on the optimization of quantitative imaging methods to improve co-clinical trials. The CIRP supports 10 different co-clinical trial projects, spanning diverse tumor types, therapeutic interventions, and imaging modalities. Each CIRP project is tasked to deliver a unique web resource to support the cancer community with the necessary methods and tools to conduct co-clinical quantitative imaging studies. This review provides an update of the CIRP web resources, network consensus, technology advances, and a perspective on the future of the CIRP. The presentations in this special issue of Tomography were contributed by the CIRP working groups, teams, and associate members.