Assessment Of Drug Efficacy Research Articles

Abstract 2548: Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples

Abstract Background: Assessing drug efficacy in primary acute myeloid leukemia (AML) patient cells is crucial for pinpointing drug targets and rationally selecting therapies for patients. We have previously shown that our flow cytometry-based predictive precision medicine platform (PPMP) predicts patient outcomes for standard-of-care (SOC) AML treatments using fresh blood or bone marrow samples with high precision. Here, we wanted to test the feasibility and efficacy of the PPMP on cryopreserved patient-derived leukemic samples. However, cryopreserved leukemic samples are known for exhibiting low viability and cell recovery, rendering their practical utilization often challenging. In this study, we adapt an existing methodology for cryopreservation for patient-derived leukemic cells to investigate the potential of cryopreserved samples as an alternative to fresh samples for ex vivo prediction of treatment outcomes on our PPMP. Methods: We screened freshly isolated white blood cells from AML patients on the PPMP to assess their response to multiple SOC single agents and combinations. The assay was also run without drug treatments immediately after receiving the fresh sample (≤ 2 days from draw) to establish a phenotype baseline. The cryopreserved samples were processed using a similar protocol, with post-thaw culture times of either 24 or 48 hours in two types of cytokine-containing media. A comparative analysis between untreated fresh and cryopreserved samples determined the optimal post-thaw culture time for cryopreserved samples. Once culture time was optimized, we screened cryopreserved samples on the PPMP with identical SOC drug conditions as the fresh samples and compared the response of leukemic blasts to drug treatment as well as impacts on other phenotypic markers. Results: Based on our preliminary data, we found that culturing cryopreserved cells for 24 hours post-thaw was sufficient to maintain a minimum cell viability of 80% while closely maintaining a phenotype that recapitulated that of the corresponding fresh samples. In response to drug treatments within the ex vivo assays, preliminary data support a correlation (Decitabine single agent, R2=0.81) between the response profiles of cryopreserved and fresh patient samples as gauged from AUC calculations. Conclusion: While the preferred method for screening is to use fresh patient samples, many practical constraints make it challenging or unfeasible. The ability of Notable Lab’s high-throughput platform to use cryopreserved samples opens the doors to enhanced retrospective studies leveraging clinically annotated data from biobanks or clinical trials. These retrospective studies provide a substantial reduction in the time needed to conclude such investigations. Citation Format: Lokesh Patil, Christine J. Gu, Wade Anderson, Nicola Long, Kara Johnson, Cristina Tognon, Markus D. Lacher. Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2548.

Abstract 2115: Assessing BCL2 protein complex level in peripheral blood mononuclear cells as a surrogate pharmacodynamic biomarker of BCL2 inhibitors in hematological cancers

Abstract In the last decade, cancer therapeutics have adhered to the practice of administering drugs at the maximum tolerated dose (MTD) selected from limited patient cohorts. The emergence of targeted therapies has disrupted this dose selection paradigm, as optimal efficacy may be achievable at lower dose than MTD. Recently, a regulatory agency initiated "OPTIMUS" to redefine the dose selection process, recommending quantitative assessments of drug efficacy in relation to the target protein throughout development and clinical trials. ABT-199 is a protein interaction inhibitor that specifically disrupts the interaction of BCL2 protein. Consequently, the assessment of changes in the level of BCL2 protein complex is necessary to evaluate the ABT-199 efficacy for optimal dose selection. In this study, we adapted the Single-molecule Protein Interaction Detection platform for evaluating pharmacodynamics of BCL2 inhibitors in clinical samples. We assessed the dissociation of two BCL2-related biomarkers, BCL2-BAX and BCL2-BIM, from a model system under various ABT-199 doses. Following one hour treatment with 300 nM of ABT-199, the BCL2-BAX complex dissociated almost by 40%, while 80% of the BCL2-BIM complex remained. The reduction of the BCL2-BIM complex was not changed when extending the treatment for 3 hours, indicating that the BCL2-BAX complex may serve as a more sensitive biomarker for assessing the target engagement of ABT-199. To determine the assay sensitivity, we evaluated the ABT-199 response across different cell numbers and found that 100,000 cells are sufficient for performing the BCL2-BAX assay. Subsequently, we validated the utility of our method in assessing clinical samples, specifically BMMCs and PBMCs. We collected 4 PBMCs and the corresponding BMMCs from hematological cancer patients and treated them with various doses of ABT-199 ex vivo. With 100,000 BMMCs and 1,000,000 PBMCs, we can generate dose-response curves for the BCL2-BAX complex at doses of ABT-199 ranging from 1 to 1,000 nM. To explore the feasibility of employing the BCL2-BAX complex in PBMCs as a surrogate biomarker for assessing ABT-199 engagement with BCL2 in BMMCs, we compared the averaged dose-response curves from 4 PBMCs with that of BMMCs. Remarkably, the two curves showed a coherent dissociation trend, indicating that the biochemical effect of ABT-199 on the BCL2-BAX complex may be conserved across different sample types. Collectively, we have developed a target engagement assay for BCL2 inhibitors by measuring the BCL2-BAX complex. Notably, we observed a consistent dissociation pattern of the BCL2-BAX complex by ABT-199 treatment in both PBMCs and BMMCs. This molecular information offers an alternative way to assess the pharmacodynamics of BCL2 inhibitors, particularly in the case where collecting a series of BMMCs with sufficient amounts may not be feasible. Citation Format: Hongwon Lee, Byungsan Choi, Saem Hong, Yunseo Lee, Tae-Young Yoon, Janghee Woo. Assessing BCL2 protein complex level in peripheral blood mononuclear cells as a surrogate pharmacodynamic biomarker of BCL2 inhibitors in hematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2115.

Zebrafish live imaging: a strong weapon in anticancer drug discovery and development.

Developing anticancer drugs is a complex and time-consuming process. The inability of current laboratory models to reflect important aspects of the tumor in vivo limits anticancer medication research. Zebrafish is a rapid, semi-automated in vivo screening platform that enables the use of non-invasive imaging methods to monitor morphology, survival, developmental status, response to drugs, locomotion, or other behaviors. Zebrafish models are widely used in drug discovery and development for anticancer drugs, especially in conjunction with live imaging techniques. Herein, we concentrated on the use of zebrafish live imaging in anticancer therapeutic research, including drug screening, efficacy assessment, toxicity assessment, and mechanism studies. Zebrafish live imaging techniques have been used in numerous studies, but this is the first time that these techniques have been comprehensively summarized and compared side by side. Finally, we discuss the hypothesis of Zebrafish Composite Model, which may provide future directions for zebrafish imaging in the field of cancer research.

Dynamics of Plasmodium species and genotype diversity in adults with asymptomatic infection in Gabon

ObjectivesWe investigated the diversity and dynamics of Plasmodium infection in serially collected samples from asymptomatic participants of a clinical trial assessing the efficacy and safety of ivermectin in Gabon. We checked whether the baseline sample reflected the P. falciparum genotype and Plasmodium species diversity seen over 7 days of follow-up. MethodsBlood samples were collected at inclusion, every 8 hours until hour 72, daily until day 7, and on day 14. Plasmodium species was determined by qPCR and pfmsp1 length polymorphism was assessed for P. falciparum genotyping. ResultsIn 17/48 (35%) individuals, all pfmsp1 genotypes identified during the assessed period were detected at baseline; in 31/48 (65%), new genotypes were found during follow-up. Additional sampling at hour 24 allowed the identification of all genotypes seen over 7 days in 50% of the individuals. Ivermectin did not impact the genotype dynamics. Mixed Plasmodium spp. infections were detected in 28/49 (57%) individuals at baseline, and detection of non-falciparum infections during follow-up varied. ConclusionsOur results reveal complex intra-host dynamics of P. falciparum genotypes and Plasmodium species and underscore the importance of serial sampling in clinical trials for antimalarial drugs with asymptomatically P. falciparum-infected individuals. This might allow a more accurate identification of genotypes in multiple infections, impacting the assessment of drug efficacy.

Combinatorial Inhibition of Complement Factor D and BCL2 for Early-Onset Colorectal Cancer.

The tumor immune microenvironment is distinct between early-onset and late-onset colorectal cancer, which facilitates tumor progression. We previously identified several genes, including complement factor D, as having increased expression in patients with early-onset colorectal cancer. This study aimed to assess and validate the differential expression of immune genes in early-onset and late-onset colorectal cancer. We also aimed to test known drugs targeting genes increased in early-onset colorectal cancer in preclinical mouse models. A retrospective cohort study with analysis was performed using tumor RNA from formalin-fixed paraffin-embedded cell culture and immunohistochemistry to validate gene expression and function and in vivo preclinical tumor study to assess drug efficacy. The Oregon Colorectal Cancer Registry was queried to identify patients with colorectal cancer. The study included 67 patients with early-onset colorectal cancer and 54 patients with late-onset colorectal cancer. Preclinical animal models using the HCT-116 colon cancer cell line were treated with the complement factor D inhibitor danicopan and the BCL2 inhibitor venetoclax, or with vehicle controls. Elevated RNA signatures using NanoString data were evaluated by the retrospective cohort. When inhibiting these markers in the mouse preclinical model, tumor volume and weight were the main outcome measures. After updating our sample size from our previously published data, we found that complement factor D and BCL2, genes with known function and small molecule inhibitors, are elevated in patients with early-onset colorectal cancer. When inhibiting these markers with the drugs danicopan and venetoclax in a mouse model, we found that the combination of these drugs decreased tumor burden but also resulted in toxicity. This study is limited by a small sample size and a subcutaneous tumor model. Combinatorial inhibition of early-onset associated genes complement factor D and BCL2 slows the growth of early-onset colorectal cancer in a mouse preclinical model. See Video Abstract . ANTECEDENTES:El microambiente inmunológico del tumor es distinto entre el cáncer colorrectal de aparición temprana y el de aparición tardía, lo que facilita la progresión de dicho tumor. Anteriormente identificamos varios genes, incluidos el factor D-Complementario, con una mayor expresión en pacientes con cáncer colorrectal de aparición temprana.OBJETIVO:El presente estudio tuvo como objetivo el evaluar y validar la expresión diferenciada de genes inmunes en casos de cáncer colorrectal de aparición temprana y tardía. También nos propusimos evaluar los fármacos conocidos dirigidos sobre los genes aumentados en el cáncer colorrectal de aparición temprana en modelos pre-clínicos en ratones.DISEÑO:Estudio de cohortes con análisis retrospectivo utilizando el ARN tumoral procedente de cultivos celulares fijados con formalina e incluidos en parafina, y el analisis por inmunohistoquímica para validar la expresión y la función genética. Se realizó el estudio pre-clínico de los tumores in vivo para evaluar la eficacia de los fármacos.AJUSTES:Se consultó el Registro de Oregon de casos de Cáncer Colorrectal para encontrar los pacientes afectados.SUJETOS:67 pacientes con cáncer colorrectal de aparición temprana y 54 pacientes con cáncer colorrectal de aparición tardía.INTERVENCIONES (SI LAS HUBIESE):Los modelos animales pre-clínicos que utilizaron la línea celular de cáncer de colon HCT-116 se trataron con el inhibidor del factor D-Complementario o Danicopan y con el inhibidor de BCL-2 o Venetoclax, ambos con control del transportador.PRINCIPALES MEDIDAS DE RESULTADO:Se evaluaron las firmas de ARN elevadas utilizando los datos del NanoString a partir de la cohorte retrospectiva. Al inhibir estos marcadores del modelo pre-clínico en los ratones, el volumen y el peso del tumor fueron las principales medidas de resultado.RESULTADOS:Después de actualizar el tamaño de nuestra muestra a partir de datos publicados con anterioridad, encontramos que el factor D-Complementario y BCL-2, genes con función conocida e inhibidores de moléculas pequeñas, se encuentran elevados en aquellos pacientes con cáncer colorrectal de aparición temprana. Al inhibir estos marcadores con los medicamentos Danicopan y Venetoclax en el modelo de ratones vivos, encontramos que la combinación de estos dos farmacos disminuyó la carga tumoral pero también produjo toxicidad.LIMITACIONES:Estudio limitado por un tamaño de muestra pequeño y el modelo de tumor subcutáneo.CONCLUSIONES:La inhibición combinada de genes asociados de aparición temprana, el factor D-Complementario y el BCL-2, enlentecen el crecimiento del cáncer colorrectal de aparición temprana del modelo preclínico en ratones. (Traducción-Dr. Xavier Delgadillo ).

Abstract B059: A patient-derived xenograft (PDX) orthotopic mouse model of rare gynecologic mesonephric-like adenocarcinoma as a platform of personalized medicine

Abstract Background: Mesonephric adenocarcinoma (MA) is an uncommon, HPV-independent, malignant neoplasm with mesonephric (Wolffian) differentiation that mainly occurs in the uterine cervix. Recently, mesonephric-like adenocarcinoma (MLA) is characterized as a rare tumor showing similar characteristics to MA in the uterine body and the ovary (WHO Classification of tumors Female Genital Tumors 5th Edition). Although no large-size epidemiological data are available for MA and MLA, a multi-institutional study showed that MA and MLA were aggressive subtypes, typically diagnosed at advanced stages, with a high rate of pulmonary recurrence (Pors J, et al. Am J Surg Pathol 2021;45:498–506). Because of the rarity, there are no cell lines, no in vitro/in vivo preclinical models, no clinical trials conducted as well as no standard treatment established for MA and MLA. To find a more effective treatment than standard chemotherapy (paclitaxel plus carboplatin), here we aim to develop a personalized medicine platform for MLA using the patient-derived xenograft (PDX) model. Methods: We collected a patient's MLA tumor arising from the left broad ligament during the surgery and orthotopically implanted it into nude mice. When the PDX tumor developed over 1000 mm3, we euthanize the mouse, harvested the tumor, minced it, and transplanted the PDX tumor chunks into other mice to expand the PDX tumors. To investigate genomic characterization, whole exome sequencing (WES) of the primary MLA tumor was performed. For drug testing, we performed a patient-derived explant (PDE) model using sliced PDX tumors on medium-soaked gelatin sponges exposed to the drug (paclitaxel, carboplatin, cisplatin, doxorubicin, trametinib (MEK inhibitor), and omipalisib (PI3K/mTOR inhibitor)) for 72 hours. As another assessment of drug efficacy, we sorted MLA cells from PDX tumors using magnet-based mouse cell depletion and incubated them in a 3D-organoid culture method with each treatment (paclitaxel, carboplatin, trametinib, omipalisib, and trametinib plus omipalisib) for 72 hours. In vivo anti-tumor effect was tested using the PDX mouse model (control, carboplatin, trametinib plus omipalisib). Results: We established a rare gynecologic MLA PDX model and the PDX tumors have been collected stably up to the eighth passage. PDX tumors represented similar histology to the original patient tumor. WES results showed KRAS and PIK3CA pathogenic variants from the patient's tumor. In PDE and 3D-organoid models, MLA showed a significant response to carboplatin, paclitaxel, omipalisib, and trametinib compared with the control. In vivo PDX model demonstrated the efficacy of a combination of trametinib plus omipalisib. Conclusion: We made a PDX model of MLA and utilized the PDX for personalized medicine. Based on the driver genes, KRAS and PIK3CA, we found a combination of trametinib plus omipalisib is an effective treatment for MLA. This PDX-based method helps us to find effective therapeutics for many types of rare cancer. Citation Format: Yasuto Kinose, Kanako Kasuya, Yan Wang, Mai Koizumi, Aska Toda, Koji Nakamura, Mahiru Kawano, Michiko Kodama, Kae Hashimoto, Kenjiro Sawada, Tadashi Kimura. A patient-derived xenograft (PDX) orthotopic mouse model of rare gynecologic mesonephric-like adenocarcinoma as a platform of personalized medicine [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B059.

Development of Drug Efficacy Testing Platform for Glomerulonephritis.

We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and endothelial cells with a bidirectional flow in the bottom channel. Using puromycin-induced GN, we observed decreased cell viability, increased albumin permeability, and reduced WT1 and nephrin compared to the normal GFB. Tacrolimus restored cell viability, reduced albumin permeability, and increased WT1 expression. Using serum from five membranous nephropathy (MN) patients, we created MN models using a GFB-mimicking chip. A notable decline in cell viability was observed in the serum-induced MN1 and MN2 models. However, tacrolimus restored it. Albumin permeability was reduced in the MN1, MN2, and MN5 models by tacrolimus treatment. MN1 displayed the best clinical response to tacrolimus, exhibiting increased expression of WT1 in chip-based evaluations after tacrolimus treatment. We successfully evaluated the efficacy of tacrolimus using puromycin-induced and serum-induced GN models on a chip that mimicked the structure and function of the GFB. The GFB-mimicking chip holds promise as a personalized platform for assessing drug efficacy using patient serum samples.

The new era of cardiovascular research: revolutionizing cardiovascular research with 3D models in a dish.

Cardiovascular research has heavily relied on studies using patient samples and animal models. However, patient studies often miss the data from the crucial early stage of cardiovascular diseases, as obtaining primary tissues at this stage is impracticable. Transgenic animal models can offer some insights into disease mechanisms, although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression. In recent years, a promising breakthrough has emerged in the form of invitro three-dimensional (3D) cardiovascular models utilizing human pluripotent stem cells. These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment. This advancement is pivotal as it addresses the existing gaps in cardiovascular research, allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models. In this review, we first outline various approaches employed to generate these models. We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions. Moreover, we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety. Despite their immense potential, challenges persist, particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs. However, overcoming these challenges could revolutionize cardiovascular research. It has the potential to offer comprehensive mechanistic insights into human-specific disease processes, ultimately expediting the development of personalized therapies.

Microfluidics in High-Throughput Drug Screening: Organ-on-a-Chip and C. elegans-Based Innovations.

The development of therapeutic interventions for diseases necessitates a crucial step known as drug screening, wherein potential substances with medicinal properties are rigorously evaluated. This process has undergone a transformative evolution, driven by the imperative need for more efficient, rapid, and high-throughput screening platforms. Among these, microfluidic systems have emerged as the epitome of efficiency, enabling the screening of drug candidates with unprecedented speed and minimal sample consumption. This review paper explores the cutting-edge landscape of microfluidic-based drug screening platforms, with a specific emphasis on two pioneering approaches: organ-on-a-chip and C. elegans-based chips. Organ-on-a-chip technology harnesses human-derived cells to recreate the physiological functions of human organs, offering an invaluable tool for assessing drug efficacy and toxicity. In parallel, C. elegans-based chips, boasting up to 60% genetic homology with humans and a remarkable affinity for microfluidic systems, have proven to be robust models for drug screening. Our comprehensive review endeavors to provide readers with a profound understanding of the fundamental principles, advantages, and challenges associated with these innovative drug screening platforms. We delve into the latest breakthroughs and practical applications in this burgeoning field, illuminating the pivotal role these platforms play in expediting drug discovery and development. Furthermore, we engage in a forward-looking discussion to delineate the future directions and untapped potential inherent in these transformative technologies. Through this review, we aim to contribute to the collective knowledge base in the realm of drug screening, providing valuable insights to researchers, clinicians, and stakeholders alike. We invite readers to embark on a journey into the realm of microfluidic-based drug screening platforms, fostering a deeper appreciation for their significance and promising avenues yet to be explored.

Polydopamine-Based Resveratrol-Hyaluronidase Nanomedicine Inhibited Pancreatic Cancer Cell Invasive Phenotype in Hyaluronic Acid Enrichment Tumor Sphere Model.

The dense storm microenvironment formed by an excessively cross-linked extracellular matrix, such as hyaluronic acid and collagens, serves as a major barrier that prevents drugs from reaching the deeper tumor. Current traditional two-dimensional (2D) cultures are not capable of modeling this drug delivery barrier in vitro. Thus, tumor spheroids have become increasingly important in cancer research due to their three-dimensional structure. Currently, various methods have been developed to construct tumor spheroids. However, there are still challenges, such as lengthy construction time, complex composition of added growth factors, and high cultivation costs. To address this technical bottleneck, our study combined the GelMA hydrogel system to develop a rapid and high-yield method for tumor spheroids generation. Additionally, we proposed an evaluation scheme to assess the effects of drugs on tumor spheroids. Building on the hyaluronic acid-rich pathological tumor microenvironment, we constructed a resveratrol-loaded nano-drug delivery system with tumor stroma modulation capability and used a three-dimensional (3D) tumor sphere model to simulate in vivo tumor conditions. This process was utilized to completely evaluate the ability of the nano-drug delivery system to enhance the deep penetration of resveratrol in the tumor microenvironment, providing new insights into future oncology drug screening, efficacy assessment, and drug delivery methods.

Ex vivo model of pathological calcification of human aortic valve.

The development of drug therapy for the pathological calcification of the aortic valve is still an open issue due to the lack of effective treatment strategies. Currently, the only option for treating this condition is surgical correction and symptom management. The search for models to study the safety and efficacy of anti-calcifying drugs requires them to not only be as close as possible to in vivo conditions, but also to be flexible with regard to the molecular studies that can be applied to them. The ex vivo model has several advantages, including the ability to study the effect of a drug on human cells while preserving the original structure of the valve. This allows for a better understanding of how different cell types interact within the valve, including non-dividing cells. The aim of this study was to develop a reproducible ex vivo calcification model based on valves from patients with calcific aortic stenosis. We aimed to induce spontaneous calcification in valve tissue fragments under osteogenic conditions, and to demonstrate the possibility of significantly suppressing it using a calcification inhibitor. To validate the model, we tested a Notch inhibitor Crenigacestat (LY3039478), which has been previously shown to have an anti-calcifying effect on interstitial cell of the aortic valve. We demonstrate here an approach to testing calcification inhibitors using an ex vivo model of cultured human aortic valve tissue fragments. Thus, we propose that ex vivo models may warrant further investigation for their utility in studying aortic valve disease and performing pre-clinical assessment of drug efficacy.

Three-Dimensional In Vitro Cell Cultures as a Feasible and Promising Alternative to Two-Dimensional and Animal Models in Cancer Research.

Cancer represents one of the diseases with the highest mortality rate worldwide. The burden of cancer continues to increase, not only affecting the health-related quality of life of patients but also causing an elevated global financial impact. The complexity and heterogeneity of cancer pose significant challenges in research and clinical practice, contributing to increase the failure rate of clinical trials for antitumoral drugs. This is partially due to the fact that preclinical models still present important limitations in faithfully recapitulating human tumors to serve as reliable indicators of drug effectiveness. Up to now, research and development strategies employ expensive animal models (including the so-called "humanized mice") that not only raise ethical concerns, but also frequently fail to accurately predict responses to anticancer drugs because they do not faithfully replicate human physiology as well as the patient's tumor microenvironment. On the other side, traditional two-dimensional (2D) cell cultures fail to adequately reproduce the structural organization of tumor and the cellular heterogeneity found in vivo. The growing necessity to develop more accurate cancer models has increasingly emphasized the importance of three-dimensional (3D) in vitro cell cultures, such as cancer-derived spheroids and organoids, as promising alternatives to bridge the gap between 2D and animal models. In this review, we provide a brief overview focusing on 3D in vitro cell cultures as preclinical models capable of properly reproducing the tissue organization, biological composition, and complexity of in vivo tumors in a fine-tuned microenvironment. Despite their limitations, these models collectively enhance our understanding of the mechanisms underlying cancer and may offer the potential for a more reliable assessment of drug efficacy before clinical testing and, consequently, improve therapeutic outcomes for cancer patients.

High Specificity but Low Sensitivity of Lab-on-a-Disk Technique in Detecting Soil-Transmitted Helminth Eggs among Pre- and School-Aged Children in North-Western Tanzania.

An estimated 1.5 billion people are infected with soil-transmitted helminths (hookworms, Ascaris lumbricoides and Trichuris trichiura). These infections are targeted for elimination by the World Health Organization (WHO) by 2030, with the main interventions being mass drug administration using albendazole or mebendazole. Tanzania is one of the endemic countries; it has been implementing MDA to school-aged children for more than a decade and the infection prevalence and intensity of infection have declined. Thus, at this point, the monitoring and evaluation of infection prevalence and intensity of infections, and assessing drug efficacy is crucial and requires accurate diagnostic tests. The currently used standard diagnostic test, the Kato-Katz (KK) technique, has several limitations and the WHO is calling for the development and evaluation of new diagnostic tests. The Lab-on-a-disk (LOD) was developed and tested in the endemic areas of north-western Tanzania to evaluate its sensitivity and specificity using KK and the formol-ether concentration technique. The results showed that when using a duplicate KK slide, the LOD had a sensitivity and specificity of 37.2% (95% CI: 30.7-43.9) and 67.3% (95% CI: 63.1-71.3%). Using four KK slides as a standard technique, the overall sensitivity and specificity were 37.7% (95% CI: 33.1-42.6) and 70.7% (95% CI: 65.5-75.6). The LOD attained high specificity but low sensitivity especially in detecting eggs of Trichuris trichiura. The LOD technique has potential as a promising diagnostic test, but its sensitivity still requires improvement.

Development of a Highly Differentiated Human Primary Proximal Tubule MPS Model (aProximate MPS Flow).

The kidney proximal tubule (PT) mediates renal drug elimination in vivo and is a major site of drug-induced toxicity. To reliably assess drug efficacy, it is crucial to construct a model in which PT functions are replicated. Current animal studies have proven poorly predictive of human outcome. To address this, we developed a physiologically relevant micro-physiological system (MPS) model of the human PT, the aProximate MPS Flow platform (Patent No: G001336.GB). In this model, primary human PT cells (hPTCs) are subjected to fluidic media flow and a shear stress of 0.01-0.2 Pa. We observe that these cells replicate the polarity of hPTCs and exhibit a higher expression of all the key transporters of SLC22A6 (OAT1), SLC22A8 (OAT3), SLC22A2 (OCT2), SLC47A1 (MATE1), SLC22A12 (URAT1), SLC2A9 (GLUT9), ABCB1 (MDR1), ABCC2 (MRP2), LRP2 (megalin), CUBN (cubilin), compared with cells grown under static conditions. Immunofluorescence microscopy confirmed an increase in OAT1, OAT3, and cilia protein expression. Increased sensitivity to nephrotoxic protein cisplatin was observed; creatinine and FITC-albumin uptake was significantly increased under fluidic shear stress conditions. Taken together, these data suggest that growing human PT cells under media flow significantly improves the phenotype and function of hPTC monolayers and has benefits to the utility and near-physiology of the model.

Label‐free Raman spectroscopy for the assessment of liver fibrosis

AbstractThe enormous incidence and dire consequences of liver fibrosis highlight the need for biologically plausible markers as readouts of pathology and drug efficacy. The classic reference standard for diagnosis of liver fibrosis is histopathological examinations. However, it is burdened by time‐consuming steps, subjectivity, and the static nature of information. Raman spectroscopy is a rapid, objective, and label‐free analytical tool that provides numerous molecular information. Thus, we utilized Raman spectroscopy to characterize the pathological features of liver fibrosis and to evaluate the efficacy of drugs (obeticholic acid [OCA] and gigantol [YS30]). Principal component analysis with subsequent linear discriminant analysis could basically discriminate between fibrotic mice and healthy controls and showed that animals with OCA or YS30 treatment resembled those with vehicle treatment. Moreover, we identified several potential biomarkers, including cytochrome C, for liver fibrosis development based on their Raman “fingerprints.” Raman imaging provided quantitative and spatial distribution of collagen and cytochrome C in situ. In addition, we demonstrated the role of OCA or YS30 in revitalizing mitochondrial function to alleviate liver fibrosis using Raman spectroscopy. Collectively, Raman spectroscopy succeeded in tracking liver fibrosis progression, assessing drug efficacy, and yielding significant molecular information useful for quantitatively linking pathological assessment and mechanism of drug action.

Recent Contributions of Mass Spectrometry-Based "Omics" in the Studies of Breast Cancer.

Breast cancer (BC) is one of the most heterogeneous groups of cancer. As every biotype of BC is unique and presents a particular "omic" signature, they are increasingly characterized nowadays with novel mass spectrometry (MS) strategies. BC therapeutic approaches are primarily based on the two features of human epidermal growth factor receptor 2 (HER2) and estrogen receptor (ER) positivity. Various strategic MS implementations are reported in studies of BC also involving data independent acquisitions (DIAs) of MS which report novel differential proteomic, lipidomic, proteogenomic, phosphoproteomic, and metabolomic characterizations associated with the disease and its therapeutics. Recently many "omic" studies have aimed to identify distinct subsidiary biotypes for diagnosis, prognosis, and targets of treatment. Along with these, drug-induced-resistance phenotypes are characterized by "omic" changes. These identifying aspects of the disease may influence treatment outcomes in the near future. Drug quantifications and characterizations are also done regularly and have implications in therapeutic monitoring and in drug efficacy assessments. We report these studies, mentioning their implications toward the understanding of BC. We briefly provide the MS instrumentation principles that are adopted in such studies as an overview with a brief outlook on DIA-MS strategies. In all of these, we have chosen a model cancer for its revelations through MS-based "omics".

Clinical pharmacology facing the real-world setting: Pharmacovigilance, pharmacoepidemiology and the economic evaluation of drugs

Traditionally, clinical pharmacology has focused its activities on drug-organism interaction, from an individual or collective perspective. Drug efficacy assessment by performing randomized clinical trials and analysis of drug use in clinical practice by carrying out drug utilization studies have also been other areas of interest. From now on, Clinical pharmacology should move from the analysis of the drug-individual interaction to the analysis of the drug-individual-society interaction. It should also analyze the clinical and economic consequences of the use of drugs in the conditions of normal clinical practice, beyond clinical trials. The current exponential technological development that facilitates the analysis of real-life data offers us a golden opportunity to move to all these other areas of interest. This review describes the role that clinical pharmacology has played at the beginning and during the evolution of pharmacovigilance, pharmacoepidemiology and economic drug evaluations in Spain. In addition, the challenges that clinical pharmacology is going to face in the following years in these three areas are going to be outlined too.

Single-cell metabolic profiling reveals subgroups of primary human hepatocytes with heterogeneous responses to drug challenge

BackgroundXenobiotics are primarily metabolized by hepatocytes in the liver, and primary human hepatocytes are the gold standard model for the assessment of drug efficacy, safety, and toxicity in the early phases of drug development. Recent advances in single-cell genomics demonstrate liver zonation and ploidy as main drivers of cellular heterogeneity. However, little is known about the impact of hepatocyte specialization on liver function upon metabolic challenge, including hepatic metabolism, detoxification, and protein synthesis.ResultsHere, we investigate the metabolic capacity of individual human hepatocytes in vitro. We assess how chronic accumulation of lipids enhances cellular heterogeneity and impairs the metabolisms of drugs. Using a phenotyping five-probe cocktail, we identify four functional subgroups of hepatocytes responding differently to drug challenge and fatty acid accumulation. These four subgroups display differential gene expression profiles upon cocktail treatment and xenobiotic metabolism-related specialization. Notably, intracellular fat accumulation leads to increased transcriptional variability and diminishes the drug-related metabolic capacity of hepatocytes.ConclusionsOur results demonstrate that, upon a metabolic challenge such as exposure to drugs or intracellular fat accumulation, hepatocyte subgroups display different and heterogeneous transcriptional responses.

Automation, live-cell imaging, and endpoint cell viability for prostate cancer drug screens.

Androgen deprivation therapy (ADT) is the standard of care for high risk and advanced prostate cancer; however, disease progression from androgen-dependent prostate cancer (ADPC) to lethal and incurable castration-resistant prostate cancer (CRPC) and (in a substantial minority of cases) neuroendocrine prostate cancer (NEPC) is common. Identifying effective targeted therapies is challenging because of acquired resistance to established treatments and the vast heterogeneity of advanced prostate cancer (PC). To streamline the identification of potentially active prostate cancer therapeutics, we have developed an adaptable semi-automated protocol which optimizes cell growth and leverages automation to enhance robustness, reproducibility, and throughput while integrating live-cell imaging and endpoint viability assays to assess drug efficacy in vitro. In this study, culture conditions for 72-hr drug screens in 96-well plates were established for a large, representative panel of human prostate cell lines including: BPH-1 and RWPE-1 (non-tumorigenic), LNCaP and VCaP (ADPC), C4-2B and 22Rv1 (CRPC), DU 145 and PC3 (androgen receptor-null CRPC), and NCI-H660 (NEPC). The cell growth and 72-hr confluence for each cell line was optimized for real-time imaging and endpoint viability assays prior to screening for novel or repurposed drugs as proof of protocol validity. We demonstrated effectiveness and reliability of this pipeline through validation of the established finding that the first-in-class BET and CBP/p300 dual inhibitor EP-31670 is an effective compound in reducing ADPC and CRPC cell growth. In addition, we found that insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib is a potential pharmacological agent against highly lethal and drug-resistant NEPC NCI-H660 cells. This protocol can be employed across other cancer types and represents an adaptable strategy to optimize assay-specific cell growth conditions and simultaneously assess drug efficacy across multiple cell lines.

A Markov model of fibrosis development in nonalcoholic fatty liver disease predicts fibrosis progression in clinical cohorts.

Disease progression in nonalcoholic steatohepatitis (NASH) is highly heterogenous and remains poorly understood. Fibrosis stage is currently the best predictor for development of end-stage liver disease and mortality. Better understanding and quantifying the impact of factors affecting NASH and fibrosis is essential to inform a clinical study design. We developed a population Markov model to describe the transition probability between fibrosis stages and mortality using a unique clinical nonalcoholic fatty liver disease cohort with serial biopsies over 3 decades. We evaluated covariate effects on all model parameters and performed clinical trial simulations to predict the fibrosis progression rate for external clinical cohorts. All parameters were estimated with good precision. Age and diagnosis of type 2 diabetes (T2D) were found to be significant predictors in the model. Increase in hepatic steatosis between visits was the most important predictor for progression of fibrosis. Fibrosis progression rate (FPR) was twofold higher for fibrosis stages 0 and 1 (F0-1) compared to fibrosis stage 2 and 3 (F2-3). A twofold increase in FPR was observed for T2D. A two-point steatosis worsening increased the FPR 11-fold. Predicted fibrosis progression was in good agreement with data from external clinical cohorts. Our fibrosis progression model shows that patient selection, particularly initial fibrosis stage distribution, can significantly impact fibrosis progression and as such the window for assessing drug efficacy in clinical trials. Our work highlights the increase in hepatic steatosis as the most important factor in increasing FPR, emphasizing the importance of well-defined lifestyle advise for reducing variability in NASH progression during clinical trials.