Preclinical Drug Efficacy Research Articles

Closed‐perfusion transretinal erg (tERG) on mouse and human retinas

Aims: The isolated mouse and postmortem human retina can serve as a sensitive biosensor for preclinical drug testing. To facilitate testing with minimal amounts of potential drug molecules or nanostructures, we developed a closed‐perfusion tERG setup. The major challenge with small perfusate volumes was the healthiness and stability of the retina in prolonged experiments.Methods: We conducted ex vivo tERG experiments using WT C57BL/6J and Gnat1‐/‐ mouse, and human donor retinas (Lion's Eye Bank, Utah). The isolated mouse or punches of human retinas were embedded in a specimen holder and the retina was perfused with a constant flow (5‐6 ml/min) from photoreceptor side, circulated with a peristaltic pump. Added Penicillin‐Streptomycin to prevent bacterial growth. 50 μM BaCl2 inhibited the K+ currents in Müller glial cells, and 20 μΜ D, L‐2‐amino‐4‐phosphonobutyric acid was added to isolate the rod component. Stimulated the dark‐adapted retina with 530 nm full‐field flashes. The electrical connection through the perfusate from the peristaltic pump to the specimen holder was cut off with burettes that also attenuated the pulsation from the pump. The stimulus and recording devices were remotely controlled by an automatic setup implemented with LabVIEW software.Results: The minimum perfusate needed in our closed perfusate circulation was 50 ml. Pen‐Strep was indispensable for long recordings. With mice, rod responses were stable for more than 24 hours and rod and cone bipolar cell responses for up to 12 hours. Rod responses from human donor tissue were stable up to 12 hours. We used our setup to test the zwitterionic polymer poly (sulfobetaine methacrylate), serving as a promising material for thermoresponsive nanostructures, for possible harmful effects on mouse rod and bipolar cell functioning. No effects on rod and bipolar cell responses were observed.Conclusions: Our method enables long, stable tERG recordings with closed perfusion on mouse and human retinas, allowing fast preclinical drug efficacy and biocompatibility testing with tiny amounts of drug molecules, materials, or nanostructures. Sulfobetaine as monomer or linear polymer did not harm rod, ON‐bipolar, or synapse functioning, suggesting the material can safely be used for constructing thermoresponsive nanostructures for drug delivery.

Closed‐perfusion transretinal erg (tERG) on mouse and human retinas

Aims: The isolated mouse and postmortem human retina can serve as a sensitive biosensor for preclinical drug testing. To facilitate testing with minimal amounts of potential drug molecules or nanostructures, we developed a closed‐perfusion tERG setup. The major challenge with small perfusate volumes was the healthiness and stability of the retina in prolonged experiments.Methods: We conducted ex vivo tERG experiments using WT C57BL/6J and Gnat1‐/‐ mouse, and human donor retinas (Lion's Eye Bank, Utah). The isolated mouse or punches of human retinas were embedded in a specimen holder and the retina was perfused with a constant flow (5–6 ml/min) from photoreceptor side, circulated with a peristaltic pump. Added Penicillin‐Streptomycin to prevent bacterial growth. 50 μM BaCl2 inhibited the K+ currents in Müller glial cells, and 20 μΜ D, L‐2‐amino‐4‐phosphonobutyric acid was added to isolate the rod component. Stimulated the dark‐adapted retina with 530 nm full‐field flashes. The electrical connection through the perfusate from the peristaltic pump to the specimen holder was cut off with burettes that also attenuated the pulsation from the pump. The stimulus and recording devices were remotely controlled by an automatic setup implemented with LabVIEW software.Results: The minimum perfusate needed in our closed perfusate circulation was 50 ml. Pen‐Strep was indispensable for long recordings. With mice, rod responses were stable for more than 24 hours and rod and cone bipolar cell responses for up to 12 hours. Rod responses from human donor tissue were stable up to 12 hours. We used our setup to test the zwitterionic polymer poly (sulfobetaine methacrylate), serving as a promising material for thermoresponsive nanostructures, for possible harmful effects on mouse rod and bipolar cell functioning. No effects on rod and bipolar cell responses were observed.Conclusions: Our method enables long, stable tERG recordings with closed perfusion on mouse and human retinas, allowing fast preclinical drug efficacy and biocompatibility testing with tiny amounts of drug molecules, materials, or nanostructures. Sulfobetaine as monomer or linear polymer did not harm rod, ON‐bipolar, or synapse functioning, suggesting the material can safely be used for constructing thermoresponsive nanostructures for drug delivery.

Cisplatin-Induced Metabolic Responses Measured with Raman Spectroscopy in Cancer Cells, Spheroids, and Canine-Derived Organoids.

Ex vivo assessment of drug response with conventional cell viability assays remains the standard practice for guiding initial therapeutic choices. However, such ensemble approaches fail to capture heterogeneities in treatment response and cannot identify early markers of response. Here, we leverage Raman spectroscopy (RS) as an accurate, low-cost, extraction-free, and label-free approach to track metabolic changes in cancer cells, spheroids, and organoids in response to cisplatin treatment. We identified 12 statistically significant metabolites in cells and 19 metabolites in spheroids and organoids as a function of depth. We show that the cisplatin treatment of 4T1 cells and spheroids results in a shift in metabolite levels; metabolites including nucleic acids such as DNA, 783 cm-1 with p = 0.00021 for cells; p = 0.02173 for spheroids, major amino acids such as threonine, 1338 cm-1 with p = 0.00045 for cells; p = 0.01022 for spheroids, proteins such as amide III, 1248 cm-1 with p = 0.00606 for cells; p = 0.00511 for spheroids serve as early predictors of response. Our RS findings were also applicable to canine-derived organoids, showing spatial variations in metabolic changes as a function of organoid depth in response to cisplatin. Further, the metabolic pathways such as tricarboxylic acid (TCA)/citric acid cycle and glyoxylate and dicarboxylate metabolism that drive drug response showed significant differences based on organoid depth, replicating the heterogeneous treatment response seen in solid tumors where there is a difference from the periphery to the tumor core. Our study showcases the versatility of RS as a predictive tool for treatment response applicable from cells to organotypic cultures, that has the potential to decrease animal burden and readout time for preclinical drug efficacy.

Dynamic changes in immune cells in humanized liver metastasis and subcutaneous xenograft mouse models

Preclinical drug efficacy and tumor microenvironment (TME) investigations often utilize humanized xenograft mouse models, yet these models typically fall short in replicating the intricate TME. We developed a humanized liver metastasis (LM) model by transplanting human peripheral blood mononuclear cells (PBMCs) and assessed it against the conventional subcutaneous (SC) xenograft model, focusing on immune cell dynamics post-transplantation and immunotherapy response. NOD-scid IL2Rgammanull(NSG) were inoculated with PBMCs to create humanized models. We induced SC and LM models using HCT116 cells, to investigate and compare the distributions and transformations of immune cell subsets, respectively. Both models were subjected to anti-PD-L1 therapy, followed by an analysis the TME analysis. The LM model demonstrated enhanced central tumor infiltration by tumor-infiltrating lymphocytes (TILs) compared to the peripheral pattern of SC model. TIL subpopulations in the LM model showed a progressive increase, contrasting with an initial rise and subsequent decline in the SC model. Post-anti-PD-L1 therapy, the LM model exhibited a significant rise in central and effector memory T cells, a response absents in the SC model. Our study highlights differential TME responses between SC and LM models and introduces a robust humanized LM model that swiftly indicates the potential efficacy of immunotherapies. These insights could streamline the preclinical evaluation of TME-targeting immunotherapeutic agents.

Abstract 2101: Patient-derived cells (PDCs) and organoids (PDOs) as critical platforms for developing next therapeutic strategies for NSCLC

Abstract Purpose: Patient-derived cells (PDCs) and organoids (PDOs) are constructed from pleural effusions to mimic the biological features of patients. In cancer research, PDC/PDOs could be a critical tool because they can recapitulate tumor mutations. Since commercial cell lines have difficulty mimicking the heterogeneity of tumors, PDC/PDOs are widely used for predicting the preclinical drug efficacy. The aim of this study is to show that the PDC/PDOs can be used as effective tools for developing new therapeutic strategy on non-small cell lung cancer (NSCLC). Experimental design: PDC/PDO models from malignant effusions were established as following. We succeeded the establishment with samples which are positive for malignancy in cytology tests and tumor colony formation. Mutations that cell harbors are confirmed by Sanger sequencing, Whole exome sequencing, or RNA-sequencing. Following the patient treatment history, we confirmed how does established patient derived model response to drugs that patient was treated. Results: A total of 54 PDCs and 171 PDOs were established from NSCLC patients, including models harboring EGFR mutations, KRAS mutations, uncommon EGFR mutations. 8 models of PDC and 7 models of PDO harbor various mutations of resistance mechanisms to EGFR-TKIs (T790M, C797S/C797G, MET amplification). Also, 4 models of PDC and PDO harbor uncommon EGFR mutations (EGFR G719S, L861Q, G719C/S768I, G719S/S768I) and 12 models of PDO harbors KRAS mutation. YUO-139 and YU-1092 are patient derived models that harbors uncommon EGFR mutation G719S and L861Q respectively. The models response to afatinib sensitively. IC50 of YUO-139 and YU-1092 to afatinib were 2.1 nM and 23.8 nM respectively. YUO-143 is a PDO model that harbors EGFR E19del/T790M/C797S which was derived from gefitinib and mavelertinib resistant patient revealed sensitivity to BLU-945 (IC50, 43 nM), a novel fourth-generation EGFR-TKI. Conclusions: Patient derived models could be a critical tool for developing therapeutic strategies for NSCLC. Citation Format: Sewon Park, Yunjoo Joo, Ju-hyeon Lee, Mi Ran Yun, Mi Ra Yu, Seung Yeon Oh, Eun Ji Lee, Dong Kwon Kim, Seul Lee, Kyumin Lim, Min Hee Hong, Sun Min Lim, Jii Bum Lee, Byoung Chul Cho. Patient-derived cells (PDCs) and organoids (PDOs) as critical platforms for developing next therapeutic strategies for NSCLC [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 2101.

CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

An antibody-drug conjugate (ADC) targeting CD46 conjugated to monomethyl auristatin has a potent anti-myeloma effect in cell lines in vitro and in vivo, and patient samples treated ex vivo. Here, we tested if CD46-ADC may have the potential to target MM-initiating cells (MM-ICs). CD46 expression was measured on primary MM cells with a stem-like phenotype. A patient-derived xenograft (PDX) model was implemented utilizing implanted fetal bone fragments to provide a humanized microenvironment. Engraftment was monitored via serum human light chain ELISA, and at sacrifice via bone marrow and bone fragment flow cytometry. We then tested MM regeneration in PDX by treating mice with CD46-ADC or the nonbinding control-ADC. MM progenitor cells from patients that exhibit high aldehyde dehydrogenase activity also have a high expression of CD46. In PDX, newly diagnosed MM patient samples engrafted significantly more compared to relapsed/refractory samples. In mice transplanted with newly diagnosed samples, CD46-ADC treatment showed significantly decreased engraftment compared to control-ADC treatment. Our data further support the targeting of CD46 in MM. To our knowledge, this is the first study to show preclinical drug efficacy in a PDX model of MM. This is an important area for future study, as patient samples but not cell lines accurately represent intratumoral heterogeneity.

On-chip modeling of physiological and pathological blood-brain barrier microenvironment for studying glial responses to neuroinflammation

The blood-brain barrier (BBB), a unique multicellular physical structure, maintains the central nervous system (CNS) homeostasis, and its disruption along with neurotoxic blood-derived molecules deposition is involved in various neurodegenerative diseases. Studying how the biological reactions of glial cells exacerbate the brain injury in the leaky BBB upon the insult of neuroinflammation is in urgent need for an in vitro physiologically relevant BBB-mimetic model. Here, we developed a microfluidic BBB chip model comprised of endothelial cells, astrocytes and microglia co-culture, which could innovatively recapitulate the physiological and pathological states when combined with particular hydrogels. Glia cells in the functional BBB with Matrigel as the matrix composition underwent significant activation by lipopolysaccharide (LPS) and specific aberrant protein aggregates stimulation, manifesting as astrocyte reactivity, higher microglial migration speed and the upregulated expression of activation marker, while the fibrin-mediated pathological extracellular microenvironment primed the glial cells to exhibit blunted responses to inflammatory irritation. We further verified the neuroprotective effect of the clinically sedative agent dexmedetomidine against neuroinflammation utilizing our BBB chip model. The BBB-on-a-chip may facilitate the elucidation of sophisticated cell-cell interactions in the progression of brain neurodegenerative diseases and the evaluation of preclinical drug efficacy.

A proof-of-concept assay for quantitative and optical assessment of drug-induced toxicity in renal organoids

Kidneys are complex organs, and reproducing their function and physiology in a laboratory setting remains difficult. During drug development, potential compounds may exhibit unexpected nephrotoxic effects, which imposes a significant financial burden on pharmaceutical companies. As a result, there is an ongoing need for more accurate model systems. The use of renal organoids to simulate responses to nephrotoxic insults has the potential to bridge the gap between preclinical drug efficacy studies in cell cultures and animal models, and the stages of clinical trials in humans. Here we established an accessible fluorescent whole-mount approach for nuclear and membrane staining to first provide an overview of the organoid histology. Furthermore, we investigated the potential of renal organoids to model responses to drug toxicity. For this purpose, organoids were treated with the chemotherapeutic agent doxorubicin for 48 h. When cell viability was assessed biochemically, the organoids demonstrated a significant, dose-dependent decline in response to the treatment. Confocal microscopy revealed visible tubular disintegration and a loss of cellular boundaries at high drug concentrations. This observation was further reinforced by a dose-dependent decrease of the nuclear area in the analyzed images. In contrast to other approaches, in this study, we provide a straightforward experimental framework for drug toxicity assessment in renal organoids that may be used in early research stages to assist screen for potential adverse effects of compounds.

Abstract 2822: Maximizing radiotherapy impact: a novel pre-clinical platform for screening of radiosensitizing agents in patient-derived tumor organoids

Abstract Introduction: Radiotherapy (RT) is widely used for various cancer types, however, radiotoxicity in healthy neighbouring tissues remains challenging. To optimize effectiveness of current therapeutic regimens, there is a clinical need to enhance radiotherapy-mediated killing by radiosensitizing agents. Radiosensitizers show great potential by accelerating DNA damage, production of free radicals and disturbing cell cycle processes by induction of cytostatic and -toxic effects, but pre-clinical drug efficacy testing methods are limited. Patient-derived organoids (PDOs) have emerged as clinically relevant in vitro screening tools to enable drug discovery for specific mutational and molecular subtypes of many cancer indications. Although some radiosensitizing agents have been identified, potential radiosensitizing effects of standard of care as well as novel anti-cancer drugs remain largely unknown. Here, we present a unique screening platform that enables radiosensitizer discovery by showing in-depth insight in cytostatic-and toxic changes and RT-drug synergy. Methods: Twelve PDO models from colorectal, pancreas, breast, lung, melanoma, cervical and ovarian tumors were screened for responsiveness to potential radiosensitizing agents using our organoid screening platforms. PDOs were seeded in 384-wells format in suspension or 3D gel (for cell-titer-glo (CTG) or imaging purposes) and compound exposure (three concentrations) was performed prior to irradiation (0, 2, 4, 6, 10 Gy). Cell viability data was obtained using both CTG-based assays and high content imaging (HCI)-based assay. Assay robustness and reproducibility were ensured (Z-scores <0.6, reproducible IC50 values). To assess radiosensitizing effects, data was normalized to non-irradiated control conditions. Morphological changes were assessed using HCI. Results: Clear differences in radiosensitivity were observed, with six out of twelve PDOs showing sensitivity to 10 Gy RT (<30% viability), one breast, two melanoma and two lung PDOs showing limited sensitivity (47-86% viability) and one ovarian PDO showing no response to RT (98% viability). A wide range of responses to the chemo-irradiation treatments were observed, with oxaliplatin exhibiting radiosensitizing effects in cervical and colorectal PDOs, gemcitabine in cervical PDOs and docetaxel showing radioprotective properties in melanoma PDO. Conclusion and Discussion: Our novel platform for testing potential radiosensitizing anti-cancer agents holds great promise for advanced drug discovery in a clinically relevant and readily available PDO panel, harbouring a wide range of RT-sensitive to insensitive models. CTG and HCI read-outs offer both fast and in-depth assessment of cytotoxic and cytostatic effects and organoid morphology, creating omnipotent potential for tailored compound screening of radiosensitizers. Citation Format: Liza Wijler, Jara Garcia, Muntaser Abdulrahman, Linda van Seters, My Nguyen, Annelot Staes, Bram Herpers, Leo Price, Marrit Putker. Maximizing radiotherapy impact: a novel pre-clinical platform for screening of radiosensitizing agents in patient-derived tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2822.

Abstract 2857: Patient-derived cells (PDCs) and organoids (PDOs) as platforms for screening novel therapeutics for NSCLC

Abstract Purpose: Patient-derived cells (PDCs) and organoids (PDOs) are constructed from patient tissue to mimic the biological features of patients. In cancer research, PDC/PDOs have been crucial as they can recapitulate tumor mutations. As heterogeneity between traditional cell lines and the human body leads to clinical trial failure, PDC/PDOs are widely used for predicting the preclinical drug efficacy. The aim of this study is to show that the PDC/PDOs can be used as effective tools for screening novel therapies on non-small cell lung cancer (NSCLC). Experimental design: PDC/PDO models from malignant effusions were established as following. We succeeded the establishment with samples which are positive for malignancy in cytology tests and tumor colony formation. Genotypes are analyzed by Sanger sequencing, Whole exome sequencing, or RNA-sequencing. Cell viability assay was performed using currently approved drugs or drugs in clinical development or their combinations. Results: A total of 46 PDCs and 150 PDOs was established from NSCLC patients, including models harboring sensitizing EGFR mutations, ALK fusions, ROS1 fusions, EGFR exon20 insertion, BRAF V600E, and those harboring various resistance mechanisms to EGFR-TKIs (T790M, C797S/C797G, MET amplification), to ALK-TKIs (G1202R), and to ROS1 TKI (G2032R). Osimertinib-resistant YU-1097 harboring EGFR resistance mutation (E19del/T790M/C797S) revealed sensitivity to BLU-945 (IC50, 108nM), a novel fourth-generation EGFR-TKI. A similar inhibition of cell viability was observed with repotrectinib (IC50, 21nM), a next-generation ROS1-TKI and lorlatinib (IC50, 9nM) in YU-1078 harboring CD74-ROS1, whereas more robust tumor regression was seen with repotrectinib in YU1078-derived xenograft model. Amivantamab, a EGFR-MET bispecific antibody, showed a robust activity in YU-1163 and YUO-036 in vitro and in vivo. YU-1077 harboring ALK G1202R solvent-front mutation showed sensitivity to NVL-655, a next-generation ALK-TKI, with a potency > 10-fold than that of lorlatinib. YUO-010 with MET amplification following osimertinib was sensitive to RGEN 5093-M114, a METxMET bispecific antibody-drug conjugate. Conclusions: PDC/PDO models can be utilized for evaluating activity of novel agents and will accelerate novel drug development in NSCLC. Citation Format: Yunjoo Joo, Sewon Park, Ju-hyeon Lee, Mi Ran Yun, Mi Ra Yu, Chun-Bong Synn, Seung Yeon Oh, Eun Ji Lee, Dong Kwon Kim, Seul Lee, Kyumin Lim, Min Hee Hong, Sun Min Lim, Chang Gon Kim, Ji Yun Lee, Jii Bum Lee, Byoung Chul Cho. Patient-derived cells (PDCs) and organoids (PDOs) as platforms for screening novel therapeutics for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2857.

A high-throughput, 28-day, microfluidic model of gingival tissue inflammation and recovery

Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

Motor deficit and lack of overt dystonia in Dlx conditional Dyt1 knockout mice

DYT1 or DYT-TOR1A dystonia is early-onset generalized dystonia caused by a trinucleotide deletion of GAG in the TOR1A or DYT1 gene leads to the loss of a glutamic acid residue in the resulting torsinA protein. A mouse model with overt dystonia is of unique importance to better understand the DYT1 pathophysiology and evaluate preclinical drug efficacy. DYT1 dystonia is likely a network disorder involving multiple brain regions, particularly the basal ganglia. Tor1a conditional knockout in the striatum or cerebral cortex leads to motor deficits, suggesting the importance of corticostriatal connection in the pathogenesis of dystonia. Indeed, corticostriatal long-term depression impairment has been demonstrated in multiple targeted DYT1 mouse models. Pappas and colleagues developed a conditional knockout line (Dlx-CKO) that inactivated Tor1a in the forebrain and surprisingly displayed overt dystonia. We set out to validate whether conditional knockout affecting both cortex and striatum would lead to overt dystonia and whether machine learning-based video behavioral analysis could be used to facilitate high throughput preclinical drug screening. We generated Dlx-CKO mice and found no overt dystonia or motor deficits at 4 months. At 8 months, retesting revealed motor deficits in rotarod, beam walking, grip strength, and hyperactivity in the open field; however, no overt dystonia was visually discernible or through the machine learning-based video analysis. Consistent with other targeted DYT1 mouse models, we observed age-dependent deficits in the beam walking test, which is likely a better motor behavioral test for preclinical drug testing but more labor-intensive when overt dystonia is absent.

Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development.

Background: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of M. tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. Objectives: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. Methods: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. Results: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. Conclusions: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development.

Development of topical chitosan/ β-glycerophosphate-based hydrogel loaded with levofloxacin in the treatment of keratitis: An ex-vivo study

Staphylococcus species are responsible for most cases of post-operative endophthalmitis. Topical ocular drug was applied for post-operative infection prevention, but the way of delivery encounters many challenges in terms of patient's compliance, drug efficacy, and drug penetration. We used the levofloxacin-loaded chitosan/gelatin/β-glycerophosphate hydrogel sustained releasing system with good in vitro anti-bacterial efficacy and biocompatibility, which we had previously designed, for ex vivo keratitis model to test the preclinical drug efficacy and to determine drug level in the anterior chamber of the eye. The result showed that the ex-vivo corneal keratitis model with S. aureus infection revealed mild opacity over the central cornea with stromal infiltrate, but without obvious stromal infiltration post levofloxacin-loaded hydrogel treatment after 24 h of infection. Quantification of viable bacteria showed a significant anti-bacterial activity. The histological evidence also showed no visible S. aureus after levofloxacin-loaded hydrogel treatment, with a significant anti-inflammatory effect. We also examined the drug concentration in the aqueous humor 24 h after instilling one drop of the levofloxacin-loaded hydrogel. The concentration achieved to a desired drug level. These results suggested that by the ex-vivo model, levofloxacin-loaded hydrogel can be applied for treatment in post-operative endophthalmitis or keratitis after the ophthalmic surgery.

TB-9 An attempt to establish a patient-derived brain tumor culture model by organoid culture method

Background: Molecular heterogeneity among and within tumors are one of the reasons for the poor survival rate of brain tumors even with the current standard therapy. However, monolayer culture and neuro-sphere culture (NS) use exogenous growth factors, so may not show the true nature of the tumor. And the culture establishment rate is low, especially low-grade tumors. Therefore, we used the glioblastoma organoid (GBO) culture method showed by Fadi to create culture models of various brain tumors and investigated their characteristics. Methods: We examined the establishment rate in pathological and genotypic types of 56 patients who underwent brain tumor resection at our hospital between January 2020 and June 2021 and were cultured with GBO or NS. If tumor cells are increased visually at 1 month after culture, we defined establishment. Results: There were 15 cases of glioblastoma, 7 cases of anaplastic astrocytoma, 7 cases of diffuse astrocytoma, 3 cases of diffuse midline glioma, 2 cases of anaplastic oligodendroglioma, 5 cases of oligodendroglioma, and 16 cases of others. The establishment rate was 76.5% by the GBO method and 40% by the N S method. By histological type, GBO: 80% in glioblastoma, NS: 58.3% in glioblastoma, GBO: 83.3% in AA, NS: 40% in AA, and GBO: 100% in DA. The IDH mutation and pTERT mutation were investigated in GBO: IDHwt/TERT+ 87.5%, IDHwt/TERT- 64.3%, IDHmt/TERT- 100%, and in NS: IDHwt/TERT+ 75%, IDHwt/TERT- 33.3%, IDHmt/ TERT- 20% in NS. In addition, establishment was observed in GBO 2 case in medulloblastoma, 1 case in ependymoma. Discussion and Conclusion: This suggest that GBO can be used to establish culture models for low-grade tumors. In addition, GBO can establish culture earlier, so it is expected to be applicable to personalized therapies such as preclinical drug efficacy studies tailored to individual patients.

Dendrimeric nanosystem consistently circumvents heterogeneous drug response and resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with no efficacious treatment. The application of nanomedicine is expected to bring new hope to PDAC treatment. In this study, we report a novel supramolecular dendrimeric nanosystem carrying the anticancer drug doxorubicin, which demonstrated potent anticancer activity, markedly overcoming the heterogeneity of drug response and resistance of primary cultured tumor cells derived from PDAC patients. This dendrimer nanodrug was constructed with a fluorinated amphiphilic dendrimer, which self-assembled into micelles nanostructure and encapsulated doxorubicin with high loading. Because of the fine nanosize, stable formulation and acid-promoted drug release, this dendrimeric nanosystem effectively accumulated in tumor, with deep penetration in tumor tissue and rapid drug uptake/release profile in cells, ultimately resulting in potent anticancer activity and complete suppression of tumor growth in patient-derived xenografts. Most importantly, this dendrimer nanodrug generated uniform and effective response when treating 35 primary pancreatic cancer cell lines issued from patient samples as a robust platform for preclinical drug efficacy testing. In addition, this dendrimer nanodrug formulation was devoid of adverse effects and showed excellent tolerability. Given all these uniquely advantageous features, this simple and convenient dendrimer nanodrug holds great promise as a potential candidate to treat the deadly PDAC.

Operationalizing the Use of Biofabricated Tissue Models as Preclinical Screening Platforms for Drug Discovery and Development

A wide range of complex in vitro models (CIVMs) are being developed for scientific research and preclinical drug efficacy and safety testing. The hope is that these CIVMs will mimic human physiology and pathology and predict clinical responses more accurately than the current cellular models. The integration of these CIVMs into the drug discovery and development pipeline requires rigorous scientific validation, including cellular, morphological, and functional characterization; benchmarking of clinical biomarkers; and operationalization as robust and reproducible screening platforms. It will be critical to establish the degree of physiological complexity that is needed in each CIVM to accurately reproduce native-like homeostasis and disease phenotypes, as well as clinical pharmacological responses. Choosing which CIVM to use at each stage of the drug discovery and development pipeline will be driven by a fit-for-purpose approach, based on the specific disease pathomechanism to model and screening throughput needed. Among the different CIVMs, biofabricated tissue equivalents are emerging as robust and versatile cellular assay platforms. Biofabrication technologies, including bioprinting approaches with hydrogels and biomaterials, have enabled the production of tissues with a range of physiological complexity and controlled spatial arrangements in multiwell plate platforms, which make them amenable for medium-throughput screening. However, operationalization of such 3D biofabricated models using existing automation screening platforms comes with a unique set of challenges. These challenges will be discussed in this perspective, including examples and thoughts coming from a laboratory dedicated to designing and developing assays for automated screening.

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor’s contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Automated Detection of Mouse Pain Behavioral Readouts by Alternating Bottom-Up Pose and Paw Contact Measurements

SUMMARYThe lack of robust quantifiable behavioral assessments of pain in pre-clinical models, especially of ongoing pain, has been a major limitation for both pain neurobiology research and development of novel analgesics. Current methods typically require extensive animal-human observer interaction and are often followed by manual, thus subjective, scorings, which are susceptible to bias and variability, and are labor-intensive and time-consuming. Here we demonstrate that, by leveraging modern machine vision and machine learning tools, quantifiable features of the spontaneous behavior, or the “body language” of an animal recorded in a dark and observer-free environment from a bottom-up view, can be directly used to automatically detect and infer both the internal state of pain and analgesia in an animal, and from surface contact measurements the presence of mechanical hypersensitivity, in an objective manner. This approach minimizes animal handling, reduces numbers of animals needed and the potential for biased assessment, making it a useful tool for exploring the biology of pain and other neurological disorders, one readily scalable for pre-clinical drug efficacy assessment.

Hydroxychloroquine prophylaxis and treatment is ineffective in macaque and hamster SARS-CoV-2 disease models.

We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in 2 animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg given daily) did not significantly benefit clinical outcome, nor did it reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment, neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these 2 preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.