Patient-Derived Xenograft Models for Leukemias.

Patient-derived xenograft (PDX) modeling is a valuable tool for the study of leukemia pathogenesis, progression, and therapy response. Engraftment of human leukemia cells occurs following injection into the tail vein (or retro-orbital vein) of preconditioned immunocompromised mice. Injected mice are maintained in a sterile and supportive housing environment until leukemia engraftment is observed, at which time studies such as drug treatments or leukemia sampling can occur. Here, we outline a method for generating PDXs from Acute Myeloid Leukemia (AML) patient samples using tail vein injection; however it can also be readily applied to T- and B- Acute Lymphoblastic Leukemia (ALL) samples.

Patient-Derived Xenografts: Historical Evolution, Immunocompromised Host Models, and Translational Significance.

Patient-derived xenografts (PDXs) represent a critical advancement in preclinical cancer research, wherein human tumor samples are implanted into animal models for evaluation of therapeutic responses. PDXs have emerged as indispensable tools in translational cancer research, facilitating investigation into tumor microenvironments and personalized medicine. This chapter elucidates the historical evolution of PDXs, from early attempts in the eighteenth century to contemporary immunocompromised host models that enhance engraftment success.

Generation of Orthotopic and Subcutaneous Patient-Derived Xenograft Models from Diverse Clinical Tissue Samples of Pediatric Extracranial Solid Tumors.

Realistic and renewable laboratory models that accurately reflect the distinct clinical features of childhood cancers have enormous potential to speed research progress. These models help us to understand disease biology, develop new research methods, advance new therapies to clinical trial, and implement personalized medicine. This chapter describes methods to generate patient-derived xenograft models of neuroblastoma and rhabdomyosarcoma, two tumor types for which children with high-risk disease have abysmal survival outcomes and survivors have lifelong-debilitating effects from treatment. Further, this protocol addresses model development from diverse clinical tumor tissue samples, subcutaneous and orthotopic engraftment, and approaches to avoid model loss.

Patient-Derived Xenograft Models in Cancer Research: Methodology, Applications, and Future Prospects.

Patient-derived xenografts (PDXs) have emerged as a pivotal tool in translational cancer research, addressing limitations of traditional methods and facilitating improved therapeutic interventions. These models involve engrafting human primary malignant cells or tissues into immunodeficient mice, allowing for the investigation of cancer mechanobiology, validation of therapeutic targets, and preclinical assessment of treatment strategies. This chapter provides an overview of PDXs methodology and their applications in both basic cancer research and preclinical studies. Despite current limitations, ongoing advancements in humanized xenochimeric models and autologous immune cell engraftment hold promise for enhancing PDX model accuracy and relevance. As PDX models continue to refine and extend their applications, they are poised to play a pivotal role in shaping the future of translational cancer research.

Development of Orthotopic Patient-Derived Xenograft Models of Pediatric Intracranial Tumors.

The development of clinically relevant and reliable models of central nervous system tumors has been instrumental in advancing the field of Neuro-Oncology. The orthotopic intracranial injection is widely used to study the growth, invasion, and spread of tumors in a controlled environment. Orthotopic models are performed to examine tumor cells isolated from a specific region in a patient in the same site or location in an animal model. Orthotopic brain tumor models are also utilized for preclinical testing of therapeutics as they closely recapitulate the behavior of such cancer and the brain environment of patients. Below, we describe our experiences in the development of murine models of pediatric brain tumors including diffuse midline glioma (DMG), glioblastoma (GBM), and medulloblastoma. The method provides an overview of intracranial stereotactic injections in mice.

Development and Expansion of Patient-Derived Xenografts for Endometrial Cancer.

Patient-Derived Xenografts (PDXs) are established by implanting a fragment of a patient tumor into rodents either subcutaneously or orthotopically. PDX models faithfully recapitulate the histologic and molecular profile of the donor patient's cancer and are regarded as authentic preclinical models for drug testing, understanding of tumor biology and biomarker discovery. This Chapter describes the detailed method for establishing robust PDXs for endometrial cancer and provide important notes for users of the protocol to consider during PDXs development.

Patient-Derived Orthotopic Xenograft Models for High-Grade Pediatric Brain Cancers.

Patient-derived orthotopic xenograft (PDOX) mouse models are considered the gold standard for evidence-based preclinical research in pediatric neuro-oncology. This protocol describes the generation of PDOX models by intracranial implantation of human pediatric brain cancer cells into immune-deficient mice, and their continued propagation to establish cohorts of animals for preclinical research.

A Humanized Patient-Derived Xenograft Model for Pancreatic Cancer.

Pancreatic cancer is associated with a high mortality rate, and there are still very few effective treatment options. Patient-derived xenografts have proven to be invaluable preclinical disease models to study cancer biology and facilitate testing of novel therapeutics. However, the severely immune-deficient mice used to generate standard models lack any functional immune system, thereby limiting their utility as a tool to investigate the tumor-immune cell interface. This chapter will outline a method for establishment of "humanized" patient-derived xenografts, which are reconstituted with human immune cells to imitate the immune-rich microenvironment of pancreatic cancer.

Patient-Derived Xenograft Models for Ovarian Cancer.

Patient-derived xenograft (PDX) models play a crucial role for in vivo research. They maintain the original molecular characteristics of the human tumor and provide a more accurate tumor microenvironment, which cannot be replicated by in vitro models. This chapter describes four different transplantation methods, namely, intra-bursal, intrarenal capsule, intraperitoneal, and subcutaneous, to develop PDX models for ovarian cancer research.

Generation of Metastatic Cholangiocarcinoma Patient-Derived Xenograft Models.

Cholangiocarcinoma (CCA) poses a substantial clinical hurdle as it is often detected at advanced metastatic stages with limited therapeutic options. To enhance our understanding of advanced CCA, it is imperative to establish preclinical models that faithfully recapitulate the disease's characteristics. Patient-derived xenograft (PDX) models have emerged as a valuable approach in cancer research, offering an avenue to reproduce and study the genomic, histologic, and molecular features of the original human tumors. By faithfully preserving the heterogeneity, microenvironmental interactions, and drug responses observed in human tumors, PDX models serve as highly relevant and predictive preclinical tools. Here, we present a comprehensive protocol that outlines the step-by-step process of generating and maintaining PDX models using biopsy samples from patients with advanced metastatic CCA. The protocol encompasses crucial aspects such as tissue processing, xenograft transplantation, and subsequent monitoring of the PDX models. By employing this protocol, we aim to establish a robust collection of PDX models that accurately reflect the genomic landscape, histologic diversity, and therapeutic responses observed in advanced CCA, thereby enabling improved translational research, drug development, and personalized treatment strategies for patients facing this challenging disease.

A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies.

Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor panel comprising 30 distinct PDX models covering a range of histotypes and molecular subtypes and cocultured with fibroblasts and PBMCs in planar (flat) extracellular matrix hydrogels to reflect the three compartments of the TME-tumor, stroma, and immune cells. The panel was constructed in a 96-well plate format and assayed tumor size, tumor killing, and T-cell infiltration using high-content image analysis after 4 days of treatment. We screened the panel first against the chemotherapy drug Cisplatin to demonstrate feasibility and robustness, and subsequently assayed immuno-oncology agents Solitomab (CD3/EpCAM bispecific T-cell engager) and the immune checkpoint inhibitors (ICIs) Atezolizumab (anti-PDL1), Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4). Solitomab displayed a strong response across many PDX models in terms of tumor reduction and killing, allowing for its subsequent use as a positive control for ICIs. Interestingly, Atezolizumab and Nivolumab demonstrated a mild response compared to Ipilimumab in a subset of models from the panel. We later determined that PBMC spatial proximity in the assay setup was important for the PD1 inhibitor, hypothesizing that both duration and concentration of antigen exposure may be critical. The described 30-model panel represents a significant advancement toward screening in vitro models of the tumor microenvironment that include tumor, fibroblast, and immune cell populations in an extracellular matrix hydrogel, with robust and standardized high content image analysis in a planar hydrogel. The platform is aimed at rapidly screening various combinations and novel agents and forming a critical conduit to the clinic, thus accelerating drug discovery for the next generation of therapeutics.

Omega-3 fatty acids decrease CRYAB, production of oncogenic prostaglandin E2 and suppress tumor growth in medulloblastoma.

AIMS: Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the ω3-long chain polyunsaturated fatty acids (ω3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model. MAIN METHODS: Effects of ω3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography. KEY FINDINGS: ω3-LCPUFA decreased prostaglandin E2 (PGE2) secretion from MB cells, and impaired MB cell viability and colony forming ability and increased apoptosis in a dose-dependent manner. DHA reduced tumor growth in vivo, and both PGE2 and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All ω3-LCPUFA and dihomo-γ-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among ω3-LCPUFA treated tumors. CRYAB was the most significantly altered gene and the downregulation was confirmed by immunohistochemistry. SIGNIFICANCE: Our findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment.

Ruxolitinib reduces severe CRS response by suspending CAR-T cell function instead of damaging CAR-T cells.

The therapeutic effect of CAR-T is often accompanied by sCRS, which is the main obstacle to the promotion of CAR-T therapy. The JAK1/2 inhibitor ruxolitinib has recently been confirmed as clinically effective in maintaining control over sCRS, however, its mechanism remains unclear. In this study, we firstly revealed that ruxolitinib significantly inhibited the proliferation of CAR-T cells without damaging viability, and induced an efficacy-favored differentiation phenotype. Second, ruxolitinib reduced the level of cytokine release not only from CAR-T cells, but also from other cells in the immune system. Third, the cytolytic activity of CAR-T cells was restored once the ruxolitinib was removed; however, the cytokines released from the CAR-T cells maintained an inhibited state to some degree. Finally, ruxolitinib significantly reduced the proliferation rate of CAR-T cells in vivo without affecting the therapeutic efficacy after withdrawal at the appropriate dose. We demonstrated pre-clinically that ruxolitinib interferes with both CAR-T cells and the other immune cells that play an important role in triggering sCRS reactions. This work provides useful and important scientific data for clinicians on the question of whether ruxolitinib has an effect on CAR-T cell function loss causing CAR-T treatment failure when applied in the treatment of sCRS, the answer to which is of great clinical significance.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 acts as a protein kinase to regulate glioblastoma progression by activating the AKT/forkhead box O1 pathway.

Abnormal expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) is closely related to the occurrence and development of tumors, and PFKFB4 has been shown to function as a protein kinase. However, the molecular mechanisms through which PFKFB4 functions in glioblastoma (GBM) remain poorly understood. Accordingly, in this study, we assessed the roles of PFKFB4 in GBM. Compared to in adjacent tissues, PFKFB4 was highly expressed in GBM, and its expression level was negatively correlated with the overall survival time. In addition, knockdown of PFKFB4 inhibited the proliferation and invasion of GBM cells and promoted apoptosis. In a xenograft tumor model, tumor growth was inhibited by knockdown of PFKFB4 using short hairpin RNA. Further studies demonstrated that PFKFB4 is involved in regulating the AKT signaling pathway. Thus, PFKFB4 acts as a protein kinase to regulate GBM progression by activating the AKT/forkhead box O1 pathway, which may be a potential therapeutic target in GBM.

ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation.

Rho family mechano-signaling through the actin cytoskeleton positively regulates physiological TEAD/YAP transcription, while the evolutionarily conserved Hippo tumor suppressor pathway antagonizes this transcription through YAP cytoplasmic localization/degradation. The mechanisms responsible for oncogenic dysregulation of these pathways, their prevalence in tumors, as well as how such dysregulation can be therapeutically targeted are not resolved. We demonstrate that p53 DNA contact mutants in human tumors, indirectly hyperactivate RhoA/ROCK1/actomyosin signaling, which is both necessary and sufficient to drive oncogenic TEAD/YAP transcription. Moreover, we demonstrate that recurrent lesions in the Hippo pathway depend on physiological levels of ROCK1/actomyosin signaling for oncogenic TEAD/YAP transcription. Finally, we show that ROCK inhibitors selectively antagonize proliferation and motility of human tumors with either mechanism. Thus, we identify a cancer driver paradigm and a precision medicine approach for selective targeting of human malignancies driven by TEAD/YAP transcription through mechanisms that either upregulate or depend on homeostatic RhoA mechano-signaling.

Protocol for chronic hepatitis B virus infection mouse model development by patient-derived orthotopic xenografts.

BACKGROUND: According to the World Health Organization, more than 250 million people worldwide are chronically infected with the hepatitis B virus, and almost 800.000 patients die annually of mediated liver disorders. Therefore, adequate biological test systems are needed that could fully simulate the course of chronic hepatitis B virus infection, including in patients with hepatocellular carcinoma. METHODS: In this study, we will assess the effectiveness of existing protocols for isolation and cultivation of primary cells derived from patients with hepatocellular carcinoma in terms of the yield of viable cells and their ability to replicate the hepatitis B virus using isolation and cultivation methods for adhesive primary cells, flow cytometry and quantitative polymerase chain reaction. Another part of our study will be devoted to evaluating the effectiveness of hepatocellular carcinoma grafting methods to obtain patient-derived heterotopic and orthotopic xenograft mouse avatars using animal X-ray irradiation and surgery procedures and in vivo fluorescent signals visualization and measurements. Our study will be completed by histological methods. DISCUSSION: This will be the first extensive comparative study of the main modern methods and protocols for isolation and cultivation primary hepatocellular carcinoma cells and tumor engraftment to the mice. All protocols will be optimized and characterized using the: (1) efficiency of the method for isolation cells from removed hepatocellular carcinoma in terms of their quantity and viability; (2) efficiency of the primary cell cultivation protocol in terms of the rate of monolayer formation and hepatitis B virus replication; (3) efficiency of the grafting method in terms of the growth rate and the possibility of hepatitis B virus persistence and replication in mice. The most effective methods will be recommended for use in translational biomedical research.

Gαi2-induced conductin/axin2 condensates inhibit Wnt/ß-catenin signaling and suppress cancer growth.

Conductin/axin2 is a scaffold protein negatively regulating the pro-proliferative Wnt/ß-catenin signaling pathway. Accumulation of scaffold proteins in condensates frequently increases their activity, but whether condensation contributes to Wnt pathway inhibition by conductin remains unclear. Here, we show that the Gαi2 subunit of trimeric G-proteins induces conductin condensation by targeting a polymerization-inhibiting aggregon in its RGS domain, thereby promoting conductin-mediated ß-catenin degradation. Consistently, transient Gαi2 expression inhibited, whereas knockdown activated Wnt signaling via conductin. Colorectal cancers appear to evade Gαi2-induced Wnt pathway suppression by decreased Gαi2 expression and inactivating mutations, associated with shorter patient survival. Notably, the Gαi2-activating drug guanabenz inhibited Wnt signaling via conductin, consequently reducing colorectal cancer growth in vitro and in mouse models. In summary, we demonstrate Wnt pathway inhibition via Gαi2-triggered conductin condensation, suggesting a tumor suppressor function for Gαi2 in colorectal cancer, and pointing to the FDA-approved drug guanabenz for targeted cancer therapy.

Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

Antibody-Drug Conjugate Targeting c-Kit for the Treatment of Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15-25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 × 10-9 M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment.

KHDRBS3 promotes paclitaxel resistance and induces glycolysis through modulated MIR17HG/CLDN6 signaling in epithelial ovarian cancer.

Paclitaxel (PTX) resistance contributes to mortality in epithelial ovarian cancer (EOC). Aerobic glycolysis is elevated in the tumor environment and may influence resistance to PTX in EOC. KH domain-containing, RNA-binding signal transduction-associated protein 3 (KHDRBS3) is an RNA binding protein that is up-regulated in EOC, but its underlying mechanism in EOC is unclear. Here, we investigate the role of KHDRBS3 in glycolysis and increased resistance to PTX. Expression of KHDRBS3 and Claudin (CLDN6) were measured in EOC tissue and cells by quantitative real-time PCR, western blotting and immunohistochemistry. The biological functions of KHDRBS3, MIR17HG and CLDN6 were examined using MTT, colony formation, apoptosis and seahorse assays in vitro. For in vivo experiments, a xenograft model was used to investigate the effects of KHDRBS3 and MIR17HG in EOC. Here, we investigate the role of KHDRBS3 in glycolysis and increased resistance to PTX. The expression of KHDRBS3 was up-regulated in PTX-resistant cells. KHDRBS3 knockdown restrained the IC50 of PTX, cell proliferation, colony formation and glycolysis in SKOV3-R and A2780-R cells in vitro and enhanced PTX sensitivity in a xenograft mouse model in vivo. KHDRBS3 interacts with lncRNA MIR17HG, which is down-regulated in EOC tissue and cells. The effect of KHDRBS3 overexpression on PTX resistance and glycolysis was rescued by MIR17HG overexpression. Additionally, MIR17HG interacts with the 3'UTR of CLDN6 and negatively regulates CLDN6 expression. MIR17HG overexpression suppressed the IC50 of PTX and glycolysis by targeting CLDN6. Our results reveal a KHDRBS3-MIR17HG-CLDN6 regulatory axis that contributes to enhanced glycolysis in EOC and represents a potential target for therapy.