Preclinical Settings Research Articles

Promising Safety and Anti-Lymphoma Efficacy of Autologous Pmb-CT01 (BAFFRCAR T Cell) Therapy in a First-in-Human Phase 1 Study

Introduction: CD19 chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment landscape of B cell lymphoma. However, a significant number of patients with aggressive B cell lymphoma (aBCL), mantle cell lymphoma (MCL) and follicular lymphoma (FL) still relapse or have refractory disease after autologous CD19CAR T cell therapy, highlighting the urgent unmet need to seek alternative approaches to improve the outcomes. BAFF-R signaling is considered a driver of B cell and malignant growth and survival. This feature may limit the capacity of B cell tumors to evade therapy by loss of BAFF-R expression. Recent work (Qin et al., Sci Transl Med. 2019) has shown that BAFF-R expression is independent of CD19 expression on malignant B cells and that CAR T cells targeting BAFF-R were able to effectively eliminate various B cell malignancies in the preclinical setting. Here we report the initial results from the first 3 patients treated on an ongoing phase 1, first-in-human clinical trial (NCT05370430) evaluating the safety and efficacy of autologous BAFFRCAR T cells in patients with B cell lymphoma. Study Design and Methods: Our studyis a phase 1, single center, open label, therapeutic trial. PMB-CT01 is a T N/ SCM-enriched BAFFR.41BB.z.EGFRt-CAR T cell therapy which was previously described (Qin et al. Sci Transl Med. 2019). There are 3 dose levels (DL) in the dose escalation phase including 50x10 6 (DL1), 200x10 6 (DL2), and 600x10 6 (DL3) CAR T cells. The lymphodepletion regimen consists of cyclophosphamide 500 mg/m 2/day and fludarabine 30 mg/m 2/day for 3 days. The primary endpoints are the incidence of adverse events and the determination of the maximum tolerated dose. Secondary endpoints include disease response, incidence of negative minimal residual disease (MRD), progression-free survival, and overall survival. ASTCT consensus criteria are used to grade cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Responses are determined by Lugano 2014 (Cheson et al., JCO 2014). Results: As of July 19, 2023, three patients were enrolled and all received treatment at DL1, 50x10 6 PMB-CT01(BAFFRCAR T cells). The median age was 56 years old (41-75), 100% male. Patients #1 and #2 had MCL and both had received prior treatment with BTK inhibitors, and progressed after CD19CAR T cell therapy. Both patients had bone marrow involvement prior to treatment, and had 4 and 10 prior lines of therapy, respectively. Patient #3 with T cell/histiocyte-rich B cell lymphoma had received 3 prior lines of therapy and his lymphoma was negative for both CD19 and CD20. All 3 patients developed grade 1 CRS only. All CRS events resolved with 2 doses of tocilizumab given to patient #1 and 1 dose to patient #2. Patients #1 and #2 also developed grade 1 ICANS which were self-limited and resolved without pharmacologic intervention. No dose-limiting toxicity (DLT) was observed. Infection was seen in patient #2 with viral pneumonia at month 3. All 3 treated patients responded to treatment. The overall response rate was 100% including MRD-negative complete response (CR) by flow cytometry and next-generation sequencing (NGS) in patient #1 and by flow cytometry in patient #2, and partial response (PR) in patient #3. All responses are ongoing at 8 months (patient #1), 4 months (patient #2) and 1.5 months (patient #3), respectively. Figure 1 shows the clearance of lymphoma cells in the bone marrow of the first patient 28 days post- CAR T cell infusion. Robust CAR T cell expansion was observed in all 3 patients with peak of expansion (between 2.1x10 6 and 8.9x10 6 copies/ml of blood) on day 12 (patient #1) and day 14 (patients #2 and #3) (Figure 1). Conclusions: PMB-CT01(BAFFRCART cell) treatment at DL1 was safe and demonstrated potent anti-lymphoma activity with a 100% ORR (2 CR, 1 PR) in patients with poor prognosis. CRS and ICANS were all grade 1 and reversible.Enrollment is ongoing at DL2, and additional clinical and correlative analyses will be presented at the meeting.

The Reversible BTK Inhibitor Nemtabrutinib Demonstrates Favorable Antitumor Efficacy and Enhances the Function of CAR T Cells in Mantle Cell Lymphoma

Background Covalent Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the therapy of mantle cell lymphoma (MCL). In January 2023, the reversible BTK inhibitor pirtobrutinib was approved by the FDA for the treatment of relapsed or refractory MCL. Nemtabrutinib is another reversible BTK inhibitor which also targets other kinases. The objective of this study was to evaluate the efficacy of nemtabrutinib alone and in combination with anti-CD19 CAR T cells against MCL. Methods Cell viability was measured following a 72-hour treatment with nemtabrutinib in a panel of MCL cell lines using CellTiter-Glo luminescent cell viability assay (Promega). Annexin V/PI staining was utilized to determine whether nemtabrutinib induces cell death through apoptosis. Patient-derived organoids (PDOs) were generated by culturing patient primary cells in 50% Matrigel (Corning) and cultured in cytokine-containing medium. The PDOs were treated for 72 hours, and viability assays were performed. Western blotting was employed to investigate the drug impact on kinase signaling pathways. Nemtabrutinib treated Mino cells were subjected to bulk RNA sequencing to interrogate the transcriptome profiling. The anti-MCL efficacy of nemtabrutinib was tested in an ibrutinib-resistant patient derived xenograft (PDX) mouse model in vivo. Additionally, the potential synergistic effects of nemtabrutinib and anti-CD19 CAR T cells were examined using the luciferase-expressing MCL cells. Results Nemtabrutinib demonstrated comparable growth inhibitory activity to ibrutinib in MCL cell lines with IC 50 values at micromolar concentrations (IC 50 = 0.7-10.1 μM). Remarkably, in the 3D PDO screening system that mimics the tumor microenvironment for MCL, nemtabrutinib displayed superior anti-MCL efficacy compared to ibrutinib (p < 0.01). The apoptotic potential of nemtabrutinib was further investigated using Annexin V/PI staining, revealing a dose-dependent induction of apoptosis, and demonstrated a similar sensitivity profile across cell lines observed with ibrutinib. Western blotting revealed that nemtabrutinib effectively inhibited phosphorylation at both Tyr223 and Tyr551 sites of BTK, and also dramatically suppressed the activation of Src family kinases, Syk and ERK. Bulk RNA-seq was performed to examine the transcriptome profile following nemtabrutinib treatment. 449 genes were identified to be significantly upregulated and 460 genes were downregulated. Gene set enrichment analysis uncovered a significant decrease in TNFα signaling via NF-κB, inflammatory response, and IFNγ response signalings (FDR < 0.05). In an ibrutinib-resistant PDX mouse model, treatment with nemtabrutinib resulted in a significant reduction in tumor burden and effectively attenuated the tumor involvement in spleen, liver and bone marrow. Furthermore, to enhance efficacy, we investigated the combination of nemtabrutinib with anti-CD19 CAR T cells in luciferase-expressing MCL cells. The result demonstrated that nemtabrutinib enhanced the effector function and anti-MCL activity of anti-CD19 CAR T cells. Conclusion Nemtabrutinib demonstrated favorable anti-MCL efficacy in both in vitro and in vivo studies, the promising synergistic effects observed in combination with CAR T cells warrants further investigation in both preclinical and clinical settings.

Emotional Intelligence and Resilience "PROGRAM" Improves Wellbeing and Stress Management Skills in Preclinical Medical Students.

The purpose of this study was to determine if implementation of a new educational curriculum focusing on Emotional Intelligence (EI) and Resilience improved second year medical student scores in these areas. Our EI-Resilience curriculum was offered as an elective for second year medical students to voluntarily enroll in. The elective consisted of six 2-hour sessions taught by a single faculty member over eight months. Sessions focused on development of EI skills and teaching a Resilience "PROGRAM" (Positive thinking, Reframing, Optimism, Gratitude, Reflection, Altruism, Meaning). Participants' EI levels were assessed before and after the elective using the Bar-On Emotional Quotient Inventory 2.0 (EQ-i 2.0). Over a period of 2 years, 70 students participated in the elective. The overall mean EI score significantly improved after the educational elective (100.05 ± 12.94 versus 108.14 ±12.36, p < 0.001). Compared to the baseline scores, there was significant improvement in all EI components, including all five composite scales, all fifteen content subscales, and the well-being score (all p < 0.05). In a post-intervention survey assessing student perception of the elective, most students found the elective to be helpful (95%, 64/67), most students felt the elective should continue to be available for future students (95%, 64/67), and most would recommend the elective to other students (93%, 62/67). An EI-Resilience curriculum offered as an elective to second year medical students was well received by students. Our outcomes showed significant improvement in students' EI scores and all sub-scores, including all components of the stress management composite and well-being score. Teaching EI skills and Resilience strategies in the preclinical setting might be an opportune time for this type of educational intervention.

Research and Patents Status of Selected Phytochemicals Against Cancer: How Close and How Far?

Cancer is a global health issue and economic burden with a continuous increase in incidence and mortality. Over the years, the underlying molecular mechanism of cancers was thoroughly researched, leading to multiple drugs' development. Unfortunately, most drugs have some serious drawbacks, such as therapy resistance and toxicity. Epidemiological studies have shown that a diet rich in fruits and vegetables has cancer prevention properties, which shifted the attention to the potential role of phytochemicals in anti-carcinogenic activity. To review the present status of phytochemicals research and patents in cancer prevention and chemosensitization. We explored the relevant published articles and patents to review the phytochemicals showing cancer preventive role in preclinical settings from 1997 onwards. We summarise the role of phytochemicals on anti-carcinogenic, anti-inflammatory, antiproliferative, anti-metastatic, and pro-apoptotic activities in both in vitro and in vivo. Thus, phytochemicals might be an excellent chemosensitizing agent against chemoresistant cells and possibly one of the safest and most effective options for cancer therapy. However, one of the limitations of phytochemicals is their poor bioavailability and rapid excretion. Several analogs have been introduced to increase bioavailability, better biological efficacy, absorption, and retention. In fact, various phytochemicals and their analogs have been patented for their anti-cancerous properties. This mini-review discusses various phytochemicals and their anti-cancerous and chemosensitizing roles. Due to their clinical relevance, recent trends in phytochemical extraction and exploration have shown that more and more phytochemicals are being patented.

Does statin suppress oxaliplatin-induced peripheral neuropathy in patients with colorectal cancer? A single-center observational study

BackgroundOxaliplatin-induced peripheral neuropathy (OIPN) is a common and dose-limiting toxicity that markedly limits the use of oxaliplatin and affects quality of life. Statins have been shown to exert neuroprotective effects in preclinical settings. The aim of the present study was to clarify whether statins prevented OIPN in patients with colorectal cancer (CRC) receiving adjuvant CAPOX therapy.MethodsWe examined 224 patients who received adjuvant CAPOX therapy for CRC between July 2010 and December 2021 at our hospital. Patients were divided into “Statin” and “Non-statin” groups based on statin use. Details on and the adverse events of adjuvant CAPOX therapy were examined in association with statin use.ResultsThirty-one patients (14%) were treated with statins. There were no intergroup differences in the relative dose intensity or number of CAPOX cycles between the Statin and Non-statin groups. In total, 94% of patients in the Statin group and 95% of those in the Non-statin group developed OIPN (p=0.67). The severity of OIPN was similar between the two groups (p=0.89). The frequency of treatment delays in CAPOX did not significantly differ between the Statin and Non-statin groups (16% vs. 11%, p=0.45).ConclusionsThe efficacy of statins to attenuate OIPN during adjuvant CAPOX therapy was not apparent in the current study. Further studies are needed to confirm the present results.

Assessment of quality improvement of simulation-based learning using an evidence-based framework in dental education.

Simulation-based education is of paramount importance in a dental pre-clinical setting. Hence, continuous quality improvement is crucial to optimize students' knowledge and clinical skills. This study aimed to evaluate the impact of evidence-based simulation learning (EBSL) compared with traditional-based simulation learning (TBSL) using Plan-Do-Study-Act (PDSA) model. This quality improvement project was undertaken at a private university. Guided by the PDSA model, rubber dam application tasks were conducted in the simulation lab in 2 phases. Phase 1 included TBSL and phase 2 included EBSL comprising of 2 PDSA cycles. 'Plan' stage involved obtaining feedback from students and the concerned staff. 'Do' stage included implementation of EBSL in eight steps adopted from Higgins's framework. 'Study' stage evaluated the outcomes and in 'Act' stage amendments were made to the first EBSL cycle. In the second PDSA cycle re-implementation and evaluation of the rubber dam application exercises were carried out. Descriptive data were presented as percentages and mean scores were compared using paired t-test. Thirty-seven year 2 students participated in this study. A significant improvement in the mean scores was observed between TBSL and EBSL (3.02 + 0.16 and 3.91 + 0.27, respectively, p < .001) using PDSA model with the highest mean score for rubber dam application in the second PDSA cycle for EBSL (4.00 + 0.01). Ninety-five per cent of the students provided positive feedback pertaining to EBSL. EBSL was effective in achieving high student satisfaction, desired knowledge, and skills required during simulation-based exercises. The PDSA cycle can be considered to optimize pre-clinical skills learning.

Gamma-secretase modulators: a promising route for the treatment of Alzheimer's disease.

Recent clinical data with three therapeutic anti-Aβ antibodies have demonstrated that removal of Aβ-amyloid plaques in early Alzheimer's disease (AD) can attenuate disease progression. This ground-breaking progress in AD medicine has validated both the amyloid cascade hypothesis and Aβ-amyloid as therapeutic targets. These results also strongly support therapeutic approaches that aim to reduce the production of amyloidogenic Aβ to prevent the formation of Aβ-pathology. One such strategy, so-called gamma-secretase modulators (GSM), has been thoroughly explored in preclinical settings but has yet to be fully tested in clinical trials. Recent scientific progress has shed new light on the role of Aβ in Alzheimer's disease and suggests that GSMs exhibit specific pharmacological features that hold great promise for the prevention and treatment of Alzheimer's disease. In this short review, we discuss the data that support why it is important to continue to progress in this class of compounds.

Advances in mRNA-Based Cancer Vaccines.

Cancer is a leading cause of death worldwide, accounting for millions of deaths every year. Immunotherapy is a groundbreaking approach for treating cancer through harnessing the power of the immune system to target and eliminate cancer cells. Cancer vaccines, one immunotherapy approach, have shown promise in preclinical settings, but researchers have struggled to reproduce these results in clinical settings. However, with the maturity of mRNA technology and its success in tackling the recent coronavirus disease 2019 (COVID-19) pandemic, cancer vaccines are expected to regain attention. In this review, we focused on the recent progress made in mRNA-based cancer vaccines over the past five years. The mechanism of action of mRNA vaccines, advancements in neoantigen discovery, adjuvant identification, and delivery materials are summarized and reviewed. In addition, we also provide a detailed overview of current clinical trials involving mRNA cancer vaccines. Lastly, we offer an insight into future considerations for the application of mRNA vaccines in cancer immunotherapy. This review will help researchers to understand the advances in mRNA-based cancer vaccines and explore new dimensions for potential immunotherapy approaches.

The Molecular Size of Bioengineered Oxygen Carriers Determines Tissue Oxygenation in a Hypercholesterolemia Guinea Pig Model of Hemorrhagic Shock and Resuscitation.

Polymerized human hemoglobin (PolyhHb) has shown promise in preclinical hemorrhagic shock settings. Different synthetic and purification schemes can control the size of PolyhHbs, yet research is lacking on the impact of polymerized hemoglobin size on tissue oxygenation following hemorrhage and resuscitation in specialized animal models that challenge their resuscitative capabilities. Pre-existing conditions that compromise the vasculature and end organs, such as the liver, may limit the effectiveness of resuscitation and exacerbate the toxicity of these molecules, which is an important but minimally explored therapeutic dimension. In this study, we compared the effective oxygen delivery of intermediate molecular weight PolyhHb (PolyhHb-B3; 500-750 kDa) to high molecular weight PolyhHb (PolyhHb-B4; 750 kDa-0.2 μm) for resuscitative effectiveness in guinea pig models subjected to hemorrhagic shock. We evaluated how the size of PolyhHb impacts hemodynamics and tissue oxygenation in normal guinea pigs and guinea pigs on an atherogenic diet. We observed that while PolyhHb-B3 and -B4 equivalently restore hemodynamic parameters of normal-dieted guinea pigs, high-fat-dieted guinea pigs resuscitated with PolyhHb-B4 have lower mean arterial pressures, impaired tissue oxygenation, and higher plasma lactate levels than those receiving PolyhHb-B3. We characterized the plasma of these animals following resuscitation and found that despite similar oxygen delivery kinetics, circulating PolyhHb-B3 and -B4 demonstrated a size-dependent increase in the plasma viscosity, consistent with impaired perfusion in the PolyhHb-B4 transfusion group. We conclude that intermediate-sized PolyhHbs (such as -B3) are ideal for further research given the effective resuscitation of hemorrhagic shock based on tissue oxygenation in hypercholesterolemic guinea pigs.

Autonomous animal heating and cooling system for temperature-regulated magnetic resonance experiments.

Temperature is a hallmark parameter influencing almost all magnetic resonance properties (e.g., T1 , T2 , proton density, and diffusion). In the preclinical setting, temperature has a large influence on animal physiology (e.g., respiration rate, heart rate, metabolism, and oxidative stress) and needs to be carefully regulated, especially when the animal is under anesthesia and thermoregulation is disrupted. We present an open-source heating and cooling system capable of regulating the temperature of the animal. The system was designed using Peltier modules capable of heating or cooling a circulating water bath with active temperature feedback. Feedback was obtained using a commercial thermistor, placed in the animal rectum, and a proportional-integral-derivative controller was used to modulate the temperature. Its operation was demonstrated in a phantom as well as in mouse and rat animal models, where the standard deviation of the temperature of the animal upon convergence was less than a 10th of a degree. An application where brain temperature of a mouse was modulated was demonstrated using an invasive optical probe and noninvasive magnetic resonance spectroscopic thermometry measurements.

Treatment with the dual-incretin agonist DA-CH5 demonstrates potent therapeutic effect in a rat model of Wolfram Syndrome.

Wolfram Syndrome (WS) is a rare condition caused by mutations in Wfs1, with a poor prognosis and no cure. Mono-agonists targeting the incretin glucagon-like-peptide 1 (GLP-1) have demonstrated disease-modifying potential in pre-clinical and clinical settings. Dual agonists that target GLP-1 and glucose-dependent insulinotropic polypeptide (GIP-1) are reportedly more efficacious; hence, we evaluated the therapeutic potential of dual incretin agonism in a loss-of-function rat model of WS. Eight-month-old Wfs1 knock-out (KO) and wild-type control rats were continuously treated with either the dual agonist DA-CH5 or saline for four months. Glycemic profile, visual acuity and hearing sensitivity were longitudinally monitored pre-treatment, and then at 10.5 and 12 months. Pancreata and retina were harvested for immunohistological analysis. DA-CH5 therapy reversed glucose intolerance in KO rats and provided lasting anti-diabetogenic protection. Treatment also reversed intra-islet alterations, including reduced endocrine islet area and β-cell density, indicating its regenerative potential. Although no rescue effect was noted for hearing loss, visual acuity and retinal ganglion cell density were better preserved in DA-CH5-treated rats. We present preclinical evidence for the pleiotropic therapeutic effects of long-term dual incretin agonist treatment; effects were seen despite treatment beginning after symptom-onset, indicating reversal of disease progression. Dual incretins represent a promising therapeutic avenue for WS patients.

Discovery and characterisation of quinazolines and 8-Azaquinazolines as NLRP3 agonists with oral bioavailability in mice

The NLRP3 inflammasome is a multiprotein complex that plays a critical role in activating the immune system in response to danger signals. Small molecule agonists of NLRP3 may offer clinical benefits in cancer immunology either as a monotherapy or in combination with checkpoint blockade, where it is hypothesised that their application can help to initiate an antitumor immune response. In this study, we report the discovery of quinazolines and 8-azaquinazolines as NLRP3 agonists and their chemical optimization to afford compounds with oral bioavailability in mice. We confirm that these compounds engage the NLRP3 inflammasome by verifying their dependence upon lipopolysaccharide (LPS) priming for cytokine release and the activation of Caspase-1. We further demonstrate pathway engagement through loss of activity in an NLRP3-knockout THP1 cell line. Based on their pharmacokinetic profile and biological activity, these compounds represent valuable tools to evaluate the therapeutic potential of NLRP3 activation in a pre-clinical setting.

Calorie restriction partially attenuates sickness behavior induced by viral mimetic poly I:C

Calorie restriction (CR) has been shown to extend the mean and maximum lifespan in both preclinical and clinical settings. We have previously demonstrated that CR attenuates lipopolysaccharide (LPS)-induced fever and sickness behavior. CR also leads to reductions in pro-inflammatory and increases in anti-inflammatory profiles. LPS is a bacterial mimetic; however, few studies have explored this phenomenon utilizing a viral mimetic, such as polyinosinic:polycytidylic acid (poly I:C). Dose-dependently, poly I:C induced an increase in core body temperature (Tb), with the largest dose (5000 µg/kg) resulting in a 1.62 °C ( ± 0.23 °C) Tb increase at 7 h post-injection in ad libitum mice and was associated with reduced home-cage locomotor activity. We then investigated the effect of 50% CR for 28 days to attenuate fever and sickness behavior induced by a poly I:C (5000 µg/kg) viral immune challenge. CR resulted in the partial attenuation of fever and sickness behavior measures post-poly I:C. The freely fed, control mice demonstrated a 2.02 °C ( ± 0.22 °C) increase in Tb at 7 h post-injection compared to the CR poly I:C group which demonstrated an increase in Tb of 0.94 °C ( ± 0.27 °C). Locomotor patterns post-injection were different, CR mice displayed a reduction in activity during the light phase, and the control group displayed a reduction during the dark phase. CR moderately attenuated the neuroinflammatory response with a reduction in microglial density in the ventromedial nucleus of the hypothalamus. The fever and sickness behavior attenuation seen after CR may be driven by similar anti-inflammatory processes as after LPS; however, further investigation is required.

Development and Optimization of 11C-Labeled Radiotracers: A Review of the Modern Quality Control Design Process.

Introduction - Several 11C-tracers have demonstrated high potential in early diagnostic PET imaging applications of neurodegenerative diseases including Alzheimer's and Parkinson's disease. These radiotracers often track critical biomarkers in disease pathogenesis such as tau fibrils ([11C]PBB3) or β-amyloid plaques ([11C]PiB) associated with such diseases. Purpose - The short review aims to serve as a guideline in the future development of radiotracers for students, postdocs and/or new radiochemists who will be synthesizing clinical grade or novel research 11C-tracers, including knowledge of regulatory requirements. We aim to bridge the gap between novel and established 11C-tracer quality control (QC) processes through exploring the design process and regulatory requirements for 11C-pharmaceuticals. Methods - A literature survey was undertaken to identify articles with a detailed description of the QC methodology and characterization for each of the sections of the review. Overview - First a general summary of 11C-tracer production was presented; this was used to establish possible places for contamination or assurances for a sterile final product. The key mandated QC analyses for clinical use were then discussed. Further, we assessed the QC methods used for established 11C-tracers and then reviewed the routine QC tests for preclinical translational and validation studies. Therefore, both mandated QC methods for clinical and preclinical animal studies were reviewed. Last, some examples of optimization and automation were reviewed, and implications of the QC practices associated with such procedures were considered. Conclusion - All of the common QC parameters associated with 11C-tracers under clinical and preclinical settings (along with a few exceptions) were discussed in detail. While it is important to establish standard, peer-reviewed QC testing protocols for a novel 11C-tracer entering the clinical umbrella, equal importance is needed on preclinical applications to address credibility and repeatability for the study.

Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model.

Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.

In-vivo Hepatoprotective Evaluation of Sicyos edulis on Wistar Albino Rats

The liver is known for synthesising enzymes, metabolism, and excretion of drugs and food. However, during biological processes, the abnormality occurs in the liver, which becomes a significant global health burden in humans, characterised by loss of synthetic function, breakdown of blood, irregular vitamin K, and localised, permanent changes to parenchymal cells. The study was designed to research the Phytochemical and biological screening of Sicyos edulis leaf for hepatoprotective activity on laboratory animals using paracetamol and methotrexate model for acute incidence. The study evaluated liver toxicity in healthy Wistar albino rats using two in vivo models. Each study group consists of six animals. In the first model, paracetamol p.o. for seven days. Similarly, in the second model, methotrexate was administered (single dose treatment) to animals with 20mg/kg, b.w., p.o. Both models were challenged with methanolic extract of Sicyos edulis leaf (MESEL) of doses 100mg/kg (low) and 200 mg/kg (high) p.o. for seven days, respectively. On day 8th, the blood samples were collected from the tail vein and analysed for various biochemical parameters. MESEL successfully restored the elevated serum biomarker levels in our study. The decrease in aspartate aminotransferase was observed by removing toxic metabolites, the reduction in alanine aminotransferase was due to an increase in ATP synthesis in mitochondria, thereby modulating the balance of liver energy metabolism, and the decrease in alkaline phosphates is due to tissue regeneration, an increase in total protein denotes the restoration of protein imbalance from acute liver injury. At different concentrations, all these effects strengthen the liver, regulate body metabolism, and ultimately inhibit further liver cell damage in favour of their regeneration. Our study also evidences the protective action of MESEL in rats against the Paracetamol and methotrexate model. The study reveals hepatocyte regeneration followed by hepatic restoration in pre-clinical settings. Keywords: Acute liver disease, Sicyos edulis, Silymarin, Methotrexate, Liver enzymes

Preclinical validation of a novel deep learning-based metal artifact correction algorithm for orthopedic CT imaging.

To validate a novel deep learning-based metal artifact correction (MAC) algorithm for CT, namely, AI-MAC, in preclinical setting with comparison to conventional MAC and virtual monochromatic imaging (VMI) technique. An experimental phantom was designed by consecutively inserting two sets of pedicle screws (size Φ 6.5×30-mm and Φ 7.5×40-mm) into a vertebral specimen to simulate the clinical scenario of metal implantation. The resulting MAC, VMI, and AI-MAC images were compared with respect to the metal-free reference image by subjective scoring, as well as by CT attenuation, image noise, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and correction accuracy via adaptive segmentation of the paraspinal muscle and vertebral body. The AI-MAC and VMI images showed significantly higher subjective scores than the MAC image (all p<0.05). The SNRs and CNRs on the AI-MAC image were comparable to the reference (all p>0.05), whereas those on the VMI were significantly lower (all p<0.05). The paraspinal muscle segmented on the AI-MAC image was 4.6% and 5.1% more complete to the VMI and MAC images for the Φ 6.5×30-mm screws, and 5.0% and 5.1% for the Φ 7.5×40-mm screws, respectively. The vertebral body segmented on the VMI was closest to the reference, with only 3.2% and 7.4% overestimation for Φ 6.5×30-mm and Φ 7.5×40-mm screws, respectively. Using metal-free reference as the ground truth for comparison, the AI-MAC outperforms VMI in characterizing soft tissue, while VMI is useful in skeletal depiction.

Human milk oligosaccharide composition and associations with growth: results from an observational study in the US.

Breast milk is the recommended source of nutrients for newborns and infants. Human milk oligosaccharides (HMO) are the third most abundant solid component in human milk and their composition varies during lactation. Our objective was to investigate longitudinal and cross-sectional changes in HMO composition and whether these changes were associated with infant growth up to 24 months of age. Associations with maternal characteristics were also investigated. 24 HMOs were quantified in samples taken at 2 weeks (n = 107), 6 weeks (n = 97) and 3 months (n = 76), using high performance liquid chromatography. Body length, weight, and head circumference were measured at 8 timepoints, until 24 months. Clusters of breast milk samples, reflecting different HMO profiles, were found through a data-driven approach. Longitudinal associations were investigated using functional principal component analysis (FPCA) and used to characterize patterns in the growth trajectories. Four clusters of samples with similar HMO composition were derived. Two patterns of growth were identified for length, body weight and head circumference via the FPCA approach, explaining more than 90% of the variance. The first pattern measured general growth while the second corresponded to an initial reduced velocity followed by an increased velocity ("higher velocity"). Higher velocity for weight and height was significantly associated with negative Lewis status. Concentrations of 3'GL, 3FL, 6'GL, DSNLT, LNFP-II, LNFP-III, LNT, LSTb were negatively associated with higher velocity for length. We introduced novel statistical approaches to establish longitudinal associations between HMOs evolution and growth. Based on our approach we propose that HMOs may act synergistically on children growth. A possible causal relationship should be further tested in pre-clinical and clinical setting.

Preclinical Intraoperative FLASH Radiotherapy

Intraoperative radiation therapy (IORT) is a method of delivering high radiation doses to an internal target with low dose to adjacent organs. Most organs at risk can often be moved out of the radiation field, so dose is usually limited by in-field vascular and/or nerve structures. It is known that ultra-high (FLASH) dose rate (>40 Gy/s) radiation reduces normal tissue toxicity compared to conventional, while preserving tumor control. We hypothesize that FLASH radiation can increase the dose tolerance of vascular and nerve structures, thus permitting for dose escalation in IORT. Here, we present a novel platform using orthovoltage x-rays to study FLASH effects in preclinical IORT. The experimental setup and dosimetry of the system have been established for FLASH irradiation of the abdominal aorta in rats to investigate dose tolerance. A 150 kVp rotating anode x-ray source with a 75-kW generator was used to deliver FLASH and conventional irradiation. A 3D printed immobilization platform was designed to reproducibly place a surgically exposed rat abdominal aorta at 55mm SSD. A 3-mm thick lead collimator with a 10mm x 10mm aperture was placed into a flange adapter, which docks into the immobilization platform. Phantom and in vivo dosimetry were performed using both calibrated radiographic film and thermoluminescent dosimeters (TLD). Anesthetized 2-month-old Wistar rats underwent laparotomy to expose the abdomen for irradiation. A 10x12x1 mm3 tungsten plate was placed under the aorta to reduce spinal cord dose. Animals were then docked into the irradiator using the immobilization platform. Radiation dose in the range of 20 - 50 Gy were delivered to the abdominal aorta at FLASH and conventional dose-rates. Following treatment, animals undergo MR angiography every two months for 6 months, at which a histological evaluation of aorta wall will be performed. The FLASH dose rate measured at the aorta wall in a rat carcass was 49.7 ± 1.2 Gy/s distal to the collimator. Dose measurements between the films and TLDs showed an agreement within ±3%. Positional uncertainty mainly depends on the visual localization of the aorta on the collimator window, which is verified before irradiation. Vertical uncertainty is less than 1 mm, which is important given the rapid dose fall off of the system. Eight animals went successfully through the procedure, are healthy and currently being follow up with MR angiography. More animals are being treated for meaningful statistical results. MR angiography and IORT are performed on different days to avoid longer times under anesthesia, which initially resulted on mortality. Results of the MR angiography and histological analysis will be presented. The proposed platform has been successfully utilized for precision setup for FLASH IORT in a preclinical laboratory setting. Evaluation of FLASH effects in IORT setup will be an important step in the potential translation of FLASH IORT, where only one high-dose fraction can be delivered.

Brachytherapy-on-Chip: A Microfluidic Setup for In Vitro Interrogation of Hypoxic Spheroids

Despite evidence of its advantages in many cancers, brachytherapy (BT) remains clinically underused and understudied in the pre-clinical setting due to a lack of versatile RT-compatible in vitro tools that can emulate the tumor microenvironment and radiobiology of various cancers. Microfluidic devices use conventional cell culture methods in 3D-tumor models, are radiocompatible and can integrate radiobioassays. However, they are seldom used in pre-clinical BT research. This project engineered the first microfluidic tool for in vitro testing of BT, with applications in translational radiobiology. PDMS microfluidic chips were engineered to grow and culture concentric rows of hypoxic spheroids, and to allow the insertion of a clinical iodine-125 BT seed at the center of the device. FaDu (hypopharyngeal squamous cell carcinoma), SK-LMS-1 (leiomyosarcoma) and HCT116 (colorectal carcinoma) cell lines were selected for their clinical relevance and ability to form spheroids. Presence of hypoxia in spheroids was assessed by immunofluorescence (IF) staining for hypoxic protein CAIX. On-chip dose distribution was calculated using clinical TG-43 parameters and compared to EBT-XD radiographic film. Target dose criteria was fixed at 8 Gy in the center of the first row of spheroids. Treatment response was quantified by DNA damages (γH2AX IF, comet assay) and cell survival (clonogenic assay). Response of hypoxic and normoxic regions of spheroids will be compared in IF. A total of 15 spheroids that are 750µm or larger can be cultured in our device, arranged as 5 rows of 3 spread from 1.5mm to 7.5mm away from a central iodine-125 seed. 48h after cell seeding, hypoxic cores were observed in spheroids derived from FaDu (50 ± 4% of cross-section, N = 3) and SK-LMS-1 (46 ± 4% of cross-section, N = 3) cells, results are pending for HCT116. TG-43 formula predicts that the center of each row receives 8 Gy, 2.6 Gy, 1.2 Gy, 0.7 Gy and 0.4 Gy respectively. Radiographic film analysis confirms on-chip TG-43 calculated doses (N = 5, R2 = 0.999). Similarly, tail moment from comet assays follows predicted dose trends (n>60, N = 3). There was no statistical difference between 8 Gy BT and 8 Gy GammaCell (8 Gy BT vs 8 Gy GammaCell, p = 0.8758), with a statistical difference between 8 Gy BT (8 Gy BT vs 2.6 Gy BT,1.2 Gy BT,0.7 Gy BT,0.4 Gy BT,0 Gy, p < 0.0001), 2.6 Gy BT (2.6 Gy BT vs 1.2 Gy BT,0.7 Gy BT,0.4 Gy BT,0 Gy, p < 0.05) or 8 Gy GammaCell (8 Gy GammaCell vs 2.6 Gy BT,1.2 Gy BT,0.7 Gy BT,0.4 Gy BT,0 Gy, p < 0.0001) and other lower BT doses. For the first time, brachytherapy can be easily integrated on-chip and its effects evaluated on relevant 3D-tumor models. Our system allows simultaneous quantification of BT efficacy on normoxic and hypoxic cells treated at various doses. On-chip combination of BT with antitumor drug will be explored in future work. We hope this device will serve as further proof of the potential of BT/RT-on-chip systems for better drug development, treatment planification and theranostics.