Consensus subtypes of hepatocellular carcinoma associated with clinical outcomes and genomic phenotypes.

BACKGROUND AND AIMS: Although many studies revealed transcriptomic subtypes of HCC, concordance of the subtypes are not fully examined. We aim to examine a consensus of transcriptomic subtypes and correlate them with clinical outcomes. APPROACH AND RESULTS: By integrating 16 previously established genomic signatures for HCC subtypes, we identified five clinically and molecularly distinct consensus subtypes. STM (STeM) is characterized by high stem cell features, vascular invasion, and poor prognosis. CIN (Chromosomal INstability) has moderate stem cell features, but high genomic instability and low immune activity. IMH (IMmune High) is characterized by high immune activity. BCM (Beta-Catenin with high Male predominance) is characterized by prominent ß-catenin activation, low miRNA expression, hypomethylation, and high sensitivity to sorafenib. DLP (Differentiated and Low Proliferation) is differentiated with high hepatocyte nuclear factor 4A activity. We also developed and validated a robust predictor of consensus subtype with 100 genes and demonstrated that five subtypes were well conserved in patient-derived xenograft models and cell lines. By analyzing serum proteomic data from the same patients, we further identified potential serum biomarkers that can stratify patients into subtypes. CONCLUSIONS: Five HCC subtypes are correlated with genomic phenotypes and clinical outcomes and highly conserved in preclinical models, providing a framework for selecting the most appropriate models for preclinical studies.

The design, analysis and application of mouse clinical trials in oncology drug development.

BACKGROUND: Mouse clinical trials (MCTs) are becoming wildly used in pre-clinical oncology drug development, but a statistical framework is yet to be developed. In this study, we establish such as framework and provide general guidelines on the design, analysis and application of MCTs. METHODS: We systematically analyzed tumor growth data from a large collection of PDX, CDX and syngeneic mouse tumor models to evaluate multiple efficacy end points, and to introduce statistical methods for modeling MCTs. RESULTS: We established empirical quantitative relationships between mouse number and measurement accuracy for categorical and continuous efficacy endpoints, and showed that more mice are needed to achieve given accuracy for syngeneic models than for PDXs and CDXs. There is considerable disagreement between methods on calling drug responses as objective response. We then introduced linear mixed models (LMMs) to describe MCTs as clustered longitudinal studies, which explicitly model growth and drug response heterogeneities across mouse models and among mice within a mouse model. Case studies were used to demonstrate the advantages of LMMs in discovering biomarkers and exploring drug's mechanisms of action. We introduced additive frailty models to perform survival analysis on MCTs, which more accurately estimate hazard ratios by modeling the clustered mouse population. We performed computational simulations for LMMs and frailty models to generate statistical power curves, and showed that power is close for designs with similar total number of mice. Finally, we showed that MCTs can explain discrepant results in clinical trials. CONCLUSIONS: Methods proposed in this study can make the design and analysis of MCTs more rational, flexible and powerful, make MCTs a better tool in oncology research and drug development.

[Effect of oridonin on apoptosis and intracellular reactive oxygen species level in triple-negative breast cancer MDA-MB-231 cells].

Oridonin, which is an ent-kaurene diterpenoid isolated from traditional Chinese medicine Rabdosia rubescens, displays various bioactivities, including anti-inflammation, anti-bacteria and anti-tumor. This study aimed to investigate the effect of oridonin on apoptosis of triple-negative breast cancer MDA-MB-231 cells and its underlying mechanisms. The inhibitory effect of oridonin on proliferation of MDA-MB-231 cells was measured by MTT assay; Apoptosis was analyzed by flow cytometry with PI staining and Annexin V-FITC/PI staining; Intracellular reactive oxygen species (ROS) level was determined by ROS detection kit, and expressions of PARP, Bcl-2, caspase-3 were analyzed by Western blot. The results showed that oridonin exhibited a significant effect in inducing apoptosis of MDA-MB-231 cells, enhancing intracellular ROS level, down-regulating expression of Bcl-2 protein, and promoting cleavage of caspase-3 and its substrate PARP. These results indicated that the apoptosis-inducing effect of oridonin on MDA-MB-231 cells might be correlated with increase of intracellular ROS level, down-regulation of Bcl-2 protein and activation of caspase-3.

NSCLC harboring EGFR exon-20 insertions after the regulatory C-helix of kinase domain responds poorly to known EGFR inhibitors.

Anecdote clinical observations hint that non-small cell lung cancer (NSCLC) with exon-20 insertions might respond poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), contrasting to those with classic mutations. Lack of patient-derived experimental models has been a major hurdle for the discovery of new treatment for the diseases. We established two NSCLC-PDXs harboring two different exon-20 insertions, LU0387-adenocarcinoma (ADC) with a nine-base insertion at 2319 (H773-V774insNPH) and LU3075-squamous cell carcinoma (SCC) with a nine-base insertion at 2316 (P772-H773insDNP). Both insertions immediately follow the regulatory C-helix of the kinase domain. Contrary to the generally good responses to EGFR inhibitors observed in PDXs with classic mutations, both exon-20 insertions are largely resistant to cetuximab and TKIs in vivo, suggesting fundamental difference from the classic EGFR mutations, consistent with the poor response rate to TKI seen in anecdotal clinic reports. It is worth noting that although responses are generally poor, they differ between the two exon-20 mutants depending on the type of TKI. In vitro drug sensitivity assays using established primary cell lines from our two PDXs largely confirmed the in vivo data. Our data from patient-derived experimental models confirmed that exon-20 insertions in domain immediately following the C-helix confer poor response to all known EGFR inhibitors, and suggested that these models can be utilized to facilitate the discovery of new therapies targeting NSCLC harboring exon-20 insertions.

An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

Narazaciclib, a novel multi-kinase inhibitor with potent activity against CSF1R, FLT3 and CDK6, shows strong anti-AML activity in defined preclinical models.

CSF1R is a receptor tyrosine kinase responsible for the growth/survival/polarization of macrophages and overexpressed in some AML patients. We hypothesized that a novel multi-kinase inhibitor (TKi), narazaciclib (HX301/ON123300), with high potency against CSF1R (IC50 ~ 0.285 nM), would have anti-AML effects. We tested this by confirming HX301's high potency against CSF1R (IC50 ~ 0.285 nM), as well as other kinases, e.g. FLT3 (IC50 of ~ 19.77 nM) and CDK6 (0.53 nM). An in vitro proliferation assay showed that narazaciclib has a high growth inhibitory effect in cell cultures where CSF1R or mutant FLT3-ITD variants that may be proliferation drivers, including primary macrophages (IC50 of 72.5 nM) and a subset of AML lines (IC50 < 1.5 µM). In vivo pharmacology modeling of narazaciclib using five AML xenografts resulted in: inhibition of MV4-11 (FLT3-ITD) subcutaneous tumor growth and complete suppression of AM7577-PDX (FLT3-ITD/CSF1Rmed) systemic growth, likely due to the suppression of FLT3-ITD activity; complete suppression of AM8096-PDX (CSF1Rhi/wild-type FLT3) growth, likely due to the inhibition of CSF1R ("a putative driver"); and nonresponse of both AM5512-PDX and AM7407-PDX (wild-type FLT3/CSF1Rlo). Significant leukemia load reductions in bone marrow, where disease originated, were also achieved in both responders (AM7577/AM8096), implicating that HX301 might be a potentially more effective therapy than those only affecting peripheral leukemic cells. Altogether, narazaciclib can potentially be a candidate treatment for a subset of AML with CSF1Rhi and/or mutant FLT3-ITD variants, particularly second generation FLT3 inhibitor resistant variants.

HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models.

Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009's further clinical development for treating NHLs.

A living biobank of matched pairs of patient-derived xenografts and organoids for cancer pharmacology.

Patient-derived tumor xenograft (PDX)/organoid (PDO), driven by cancer stem cells (CSC), are considered the most predictive models for translational oncology. Large PDX collections reflective of patient populations have been created and used extensively to test various investigational therapies, including population-trials as surrogate subjects in vivo. PDOs are recognized as in vitro surrogates for patients amenable for high-throughput screening (HTS). We have built a biobank of carcinoma PDX-derived organoids (PDXOs) by converting an existing PDX library and confirmed high degree of similarities between PDXOs and parental PDXs in genomics, histopathology and pharmacology, suggesting "biological equivalence or interchangeability" between the two. Here we demonstrate the applications of PDXO biobank for HTS "matrix" screening for both lead compounds and indications, immune cell co-cultures for immune-therapies and engineering enables in vitro/in vivo imaging. This large biobank of >550 matched pairs of PDXs/PDXOs across different cancers could become powerful tools for the future cancer drug discovery.

Pharmacological characterization of chemically synthesized monomeric phi29 pRNA nanoparticles for systemic delivery.

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

Tumor Purity in Preclinical Mouse Tumor Models.

Tumor biology is determined not only by immortal cancer cells but also by the tumor microenvironment consisting of noncancerous cells and extracellular matrix, together they dictate the pathogenesis and response to treatments. Tumor purity is the proportion of cancer cells in a tumor. It is a fundamental property of cancer and is associated with many clinical features and outcomes. Here we report the first systematic study of tumor purity in patient-derived xenograft (PDX) and syngeneic tumor models using next-generation sequencing data from >9,000 tumors. We found that tumor purity in PDX models is cancer specific and mimics patient tumors, with variation in stromal content and immune infiltration influenced by immune systems of host mice. After the initial engraftment, human stroma in a PDX tumor is quickly replaced by mouse stroma, and tumor purity then stays stable in subsequent transplantations and increases only slightly by passage. Similarly, in syngeneic mouse cancer cell line models, tumor purity also turns out to be an intrinsic property with model and cancer specificities. Computational and pathology analysis confirmed the impact on tumor purity by the diverse stromal and immune profiles. Our study deepens the understanding of mouse tumor models, which will enable their better and novel uses in developing cancer therapeutics, especially ones targeting tumor microenvironment. Significance: PDX models are an ideal experimental system to study tumor purity because of its distinct separation of human tumor cells and mouse stromal and immune cells. This study provides a comprehensive view of tumor purity in 27 cancers in PDX models. It also investigates tumor purity in 19 syngeneic models based on unambiguously identified somatic mutations. It will facilitate tumor microenvironment research and drug development in mouse tumor models.

Targeting brain lesions of non-small cell lung cancer by enhancing CCL2-mediated CAR-T cell migration.

Metastatic non-small cell lung cancer (NSCLC) remains largely incurable and the prognosis is extremely poor once it spreads to the brain. In particular, in patients with brain metastases, the blood brain barrier (BBB) remains a significant obstacle for the biodistribution of antitumor drugs and immune cells. Here we report that chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR) exhibit antitumor activity in vitro against tumor cell lines and lung cancer organoids, and in vivo in xenotransplant models of orthotopic and metastatic NSCLC. The co-expression of the CCL2 receptor CCR2b in B7-H3.CAR-T cells, significantly improves their capability of passing the BBB, providing enhanced antitumor activity against brain tumor lesions. These findings indicate that leveraging T-cell chemotaxis through CCR2b co-expression represents a strategy to improve the efficacy of adoptive T-cell therapies in patients with solid tumors presenting with brain metastases.

Different syngeneic tumors show distinctive intrinsic tumor-immunity and mechanisms of actions (MOA) of anti-PD-1 treatment.

Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various tumor-infiltrate leukocytes (TILs). We modeled loss of function (LOF) in four common anti-PD-1 antibody-responsive syngeneic tumors, MC38, Hepa1-6, CT-26 and EMT-6, by systematical depleting a series of TIL lineages to explore the mechanisms of tumor immunity and treatment. CD8+-T-cells, CD4+-T-cells, Treg, NK cells and macrophages were individually depleted through either direct administration of anti-marker antibodies/reagents or using DTR (diphtheria toxin receptor) knock-in mice, for some syngeneic tumors, where specific subsets were depleted following diphtheria toxin (DT) administration. These LOF experiments revealed distinctive intrinsic tumor immunity and thus different MOAs in their responses to anti-PD-1 antibody among different syngeneic tumors. Specifically, the intrinsic tumor immunity and the associated anti-PD-1 MOA were predominately driven by CD8+ cytotoxic TILs (CTL) in all syngeneic tumors, excluding Hepa1-6 where CD4+ Teff TILs played a key role. TIL-Treg also played a critical role in supporting tumor growth in all four syngeneic models as well as M2-macrophages. Pathway analysis using pharmacodynamic readouts of immuno-genomics and proteomics on MC38 and Hepa1-6 also revealed defined, but distinctive, immune pathways of activation and suppression between the two, closely associated with the efficacy and consistent with TIL-pharmacodynamic readouts. Understanding tumor immune-pathogenesis and treatment MOAs in the different syngeneic animal models, not only assists the selection of the right model for evaluating new immunotherapy of a given MOA, but also can potentially help to understand the potential disease mechanisms and strategize optimal immune-therapies in patients.

A recombinant sialidase fusion protein effectively inhibits human parainfluenza viral infection in vitro and in vivo.

BACKGROUND: The first step in infection by human parainfluenza viruses (HPIVs) is binding to the surface of respiratory epithelial cells via interaction between viral receptor-binding molecules and sialic acid-containing receptors. DAS181, a recombinant sialidase protein containing the catalytic domain of Actinomyces viscosus sialidase, removes cell surface sialic acid, and we proposed that it would inhibit HPIV infection. METHODS: Depletion of sialic acid receptors by DAS181 was evaluated by lectin-binding assays. Anti-HPIV activity in cultured cell lines and in human airway epithelium was assessed by the reduction in viral genomes and/or plaque forming units on treatment. In vivo efficacy of intranasally administered DAS181 was assessed using a cotton rat model. RESULTS: DAS181-mediated desialylation led to anti-HPIV activity in cell lines and human airway epithelium. Intranasal DAS181 in cotton rats, a model for human disease, significantly curtailed infection. CONCLUSIONS: Enzymatic removal of the sialic acid moiety of HPIV receptors inhibits infection with all tested HPIV strains, both in vitro and in cotton rats. Enzyme-mediated removal of sialic acid receptors represents a novel antiviral strategy for HPIV. The results of this study raise the possibility of a broad spectrum antiviral agent for influenza virus and HPIVs.

Preclinical pharmacology modeling of chimeric antigen receptor T therapies.

Chimeric antigen receptor (CAR) T cells have largely been successful in treating hematological malignancies in the clinic but have not been as effective in treating solid tumors, in part, owing to poor access and the immunosuppressive tumor microenvironment. In addition, CAR-T therapy can cause potentially life-threatening side effects, including cytokine release syndrome and neurotoxicity. Current preclinical testing of CAR-T therapy efficacy is typically performed in mouse tumor models, which often fails to predict toxicity. Recent developments in humanized models and transgenic mice as well as in vitro three-dimensional organoids in early development and nonhuman primate models are being adopted for CAR-T cell efficacy and toxicity assessment. However, because no single model perfectly recapitulates the human immune system and tumor microenvironment, careful model selection based on their respective pros and cons is crucial for adequate evaluation of different CAR-T treatments, so that their clinical development can be better supported.

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials ("Phase II-like mouse clinical trials"); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 µm cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 µM Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis.

OBJECTIVES: The influenza virus (IFV) infection models commonly used to evaluate antiviral agents (e.g. MDCK cell line and mice) are limited by physiological differences from the human respiratory tract in vivo. Here we report the pharmacodynamics of DAS181, a sialidase fusion protein that inhibits influenza infection, in the model systems of well-defined human airway epithelium (HAE) culture and ex vivo culture of fresh human bronchial tissue, both of which are close mimics of the human respiratory tract in vivo. METHODS: HAE culture and ex vivo human bronchi were used to evaluate the sialic acid removal and regeneration efficiency and IFV inhibition after various DAS181 treatment levels and regimens. RESULTS: DAS181 effectively desialylates HAE cultures and ex vivo bronchi tissues and therefore potently inhibits replication of different IFV strains. The treatment effect of DAS181 occurs immediately upon application to the epithelial surface and is unaffected by the respiratory mucus. In both HAE and human bronchial tissue, the inhibitory effect of DAS181 treatment lasts for at least 2 days. Approximately 80% epithelial surface desialylation and significant anti-IFV efficacy can be achieved at topical concentrations of DAS181 in the range of 5-10 microg/cm(2) when applied once daily. An additional treatment or a higher loading dose of DAS181 on the first day provides significant additional treatment benefit. Comparing the effect of DAS181 versus its two analogues, DAS180 and DAS185, has confirmed that sialidase function is critical for DAS181, and the cell-binding domain (amphiregulin tag) prolongs DAS181 retention and potentiates its function. CONCLUSIONS: These results provide valuable insights into DAS181 treatment dose and potential regimens in the clinical setting.

Authentication, characterization and contamination detection of cell lines, xenografts and organoids by barcode deep NGS sequencing.

Misidentification and contamination of biobank samples (e.g. cell lines) have plagued biomedical research. Short tandem repeat (STR) and single-nucleotide polymorphism assays are widely used to authenticate biosamples and detect contamination, but with insufficient sensitivity at 5-10% and 3-5%, respectively. Here, we describe a deep NGS-based method with significantly higher sensitivity (≤1%). It can be used to authenticate human and mouse cell lines, xenografts and organoids. It can also reliably identify and quantify contamination of human cell line samples, contaminated with only small amount of other cell samples; detect and quantify species-specific components in human-mouse mixed samples (e.g. xenografts) with 0.1% sensitivity; detect mycoplasma contamination; and infer population structure and gender of human samples. By adopting DNA barcoding technology, we are able to profile 100-200 samples in a single run at per-sample cost comparable to conventional STR assays, providing a truly high-throughput and low-cost assay for building and maintaining high-quality biobanks.

In Vivo Pharmacology Models for Cancer Target Research.

Experimental animal tumor models have been broadly used to evaluate anticancer drugs in the preclinical setting. They have also been widely applied for drug target discovery and validation, which usually follows four experimental strategies: first, assess the roles of putative drug targets using in vivo tumorigenicity and tumor growth kinetics assays of transplanted tumors, engineered through gain-of-function (GOF) by overexpressing transgene or knock-in (KI) or loss-of-function by gene silencing using knockdown (KD) or knockout (KO) or mutation via mutagenesis procedures; second, similarly genetically engineered mouse models (GEMM), through either germline or somatic cell procedures, are used to test the roles of potential targets in spontaneous tumorigenicity assays; third, patient-derived xenografts (PDXs), which most closely resemble patient genetics and histopathology, are used in tumor inhibition assays for evaluating target-/pathway-specific inhibitors, including large and small molecules, thus assessing the drug target; and fourth, the targets can be assessed in population-based trials, mouse clinical trials (MCT), so that the validation can be generally meaningful as performed in human clinical trials. This chapter outlines the commonly used protocols in cancer drug target research: the first four sections describe four sets of different, specific pharmacology protocols used in the respective cancer modeling stages, with the last section summarizing the common protocols applicable to all four pharmacology modeling steps.

Ribozyme technology for cancer gene target identification and validation.

Ribozymes are naturally occurring RNAs with catalytic activities including cis- or trans- cleavage of RNA at predefined sequence sites. This activity has been exploited for specific gene inactivation in cells during the last two decades, and ribozymes have been important functional genomics tools, especially in the pre-RNAi era. It has also been broadly applied in drug target identification and validation in pharmaceutical R&D. This chapter covers many application principles and case studies of ribozyme technology in the areas of cancer research. We also described RNAi applications in some of the same studies for comparison. Although RNAi may be more effective than ribozymes in many respects, they are nonetheless built on many of the same principles.