Animal Research Research Articles

INSaFLU-TELEVIR: an open web-based bioinformatics suite for viral metagenomic detection and routine genomic surveillance

BackgroundImplementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification.ResultsThe routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a “generic” build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis.ConclusionsThe accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code).

Characteristic of a group of genes with low level of expression in the pancreas of rats under conditions of multi-day intermittent hypoxia influence

In modern medical science great attention is paid to the clarification of the molecular mechanisms, which are the basis of adaptation to environmental factors of unusual origin and/or extraordinary strength. The aim of the study is to determine the features of a group of genes with low expression level, associated with hypoxia in the pancreas of Wistar rats under conditions of intermittent hypoxia. Materials and methods. The study was conducted on 10 white, sexually mature Wistar rats, which were divided into 2 groups (5 animals in each). Animals of group 1 were part of the control (intact) group. The animals of the 2nd group were subjected to hypoxic training according to the following scheme: for 15 days, 6 hours daily, namely on days 1–5 they simulated an ascent to a height of one to five kilometers above sea level under the conditions of a barometer, and the last 10 days 6 km above the sea level. To analyze gene expression, we used the polymerase chain reaction method with real-time reverse transcription (PCR) CFX-96 Touch™ (Bio-Rad, USA) and the RT2 Profiler™ PCR Array Rat Hypoxia Signaling Pathway kit (QIAGEN, Germany), where 84 genes were the subject of research in experimental animals. Results. According to the results of the PCR study of genes in the pancreas samples of intact animals and animals exposed to hypoxic training, it was established that out of 84 genes associated with hypoxia, a group of 5 genes with a low expression level (∆∆Ct < 30) was found. This pattern includes Bhlhe40 genes, Ctsa, Hif1a, Lox, and Slc16a3, the expression of which is statistically reduced. Thus, compared to the level of their expression in intact animals, the expression of Bhlhe40 decreased by 2.59 times, Ctsa by 6.02 times, Hif1a by 3.85 times, Lox by 3.01 times, and Slc16a3 by 2.40 times. Conclusions. Intermittent hypoxia reduces the expression of the Bhlhe40 gene by 2.59 times, which can be considered as an element of adaptation of cells to a low level of oxygen and modulation of genetic programs. The decrease in Ctsa gene expression by 6.02, Hif1a by 3.85, and Lox by 3.01 times during intermittent hypoxia demonstrates, that these effects can be used as sanogenic factors in insulin resistance and type 2 diabetes. The 2.40-fold decreased expression level of Slc16a3 is probably an element of metabolic adaptation and adaptation of the metabolic pathway of cells to hypoxia conditions.

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics.

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel insecure and lack knowledge because of inadequate training and teaching methods. Therefore, the objective of this article is to outline the steps involved in generating an ex vivo porcine lung model to be used in the future, to study and teach lung mechanics. To generate the model, five porcine lungs were carefully removed from the thorax following the guidelines of the Animal Research Ethics Committee with adequate care and were connected to the mechanical ventilator through a tracheal cannula. These lungs were then subjected to the alveolar recruitment maneuver. Respiratory mechanics parameters were recorded, and video cameras were used to obtain videos of the lungs during this process. This process was repeated for five consecutive days. When not used, the lungs were kept refrigerated. The model showed different lung mechanics after the alveolar recruitment maneuver every day; not being influenced by the days, only by the maneuver. Therefore, we conclude that the ex vivo lung model can provide a better understanding of lung mechanics and its effects, and even of the alveolar recruitment maneuver through visual feedback during all stages of the process.

Human Brain Organoid Transplantation: Testing the Foundations of Animal Research Ethics

Alongside in vitro studies, researchers are increasingly exploring the transplantation of human brain organoids (HBOs) into non-human animals to study brain development, disease, and repair. This paper focuses on ethical issues raised by such transplantation studies. In particular, it investigates the possibility that they might yield enhanced brain function in recipient animals (especially non-human primates), thereby fundamentally altering their moral status. I assess the critique, raised by major voices in the bioethics and science communities, according to which such concerns are premature and misleading. I identify the assumptions underlying this skeptical critique, and mention some objections against them, followed by some possible replies. I proceed to argue that the skeptical position is ultimately implausible, because it presupposes an unreasonably high standard of full moral status. My argument appeals to David DeGrazia’s idea of a “borderline person”, and to the need for consistency with existing animal research regulations. I outline the practical implications of my view for the conduct of studies that might result in the development of full moral status in a transplanted animal. I also discuss some of the ethical implications of animal enhancement (particularly of rodents) below the threshold associated with full moral status. I conclude that far from being premature, further debate on these issues is urgently needed to help clarify the prospects that a neural chimera might attain full moral status in the foreseeable future, and the level of quality of life required to make it acceptable to knowingly create such a being via HBO transplantation.

Professional quality of life in animal research personnel is linked to retention & job satisfaction: A mixed-methods cross-sectional survey on compassion fatigue in the USA.

Working with research animals can be both rewarding and challenging. The rewarding part of the work is associated with understanding the necessity for animal research to improve the health of humans and animals and the knowledge that one can provide care and compassion for the animals. Challenges with animal research include witnessing stress/pain in animals necessitated by scientific requirements, end of study euthanasia, and societal stigmatization about animal research. These challenges could be compounded with more general workplace stresses, in turn, impacting job retention and satisfaction. However, these factors have yet to be formally evaluated. Therefore, the purpose of this survey was to comprehensively evaluate professional quality of life's correlation with key workplace metrics. Six institutions were recruited to participate in a longitudinal intervention trial on compassion fatigue resiliency. This manuscript reports key baseline metrics from this survey. A cross-sectional mixed methods survey was developed to evaluate professional quality of life, job satisfaction, retention, and factors influencing compassion fatigue resiliency. Quantitative data were analyzed via general linear models and qualitative data were analyzed by theme. Baseline data was collected from 198 participants. Personnel who reported higher compassion satisfaction also reported higher retention and job satisfaction. Conversely, personnel who reported higher burnout also reported lower job satisfaction. In response to open-ended questions, participants said their compassion fatigue was impacted by institutional culture (70% of participants), animal research (58%), general mental health (41%), and specific compassion fatigue support (24%). In conclusion, these results show that professional quality of life is related to important operational metrics of job satisfaction and retention. Furthermore, compassion fatigue is impacted by factors beyond working with research animals, including institutional culture and general mental health support. Overall, this project provides rationale and insight for institutional support of compassion fatigue resiliency.

Organoid models: the future companions of personalized drug development.

High failure rates of the current drug development process are driving exemplary changes toward methodologies centered on human disease in-vitro modeling. Organoids are self-organized tissue sub-units resembling their organ of origin and are widely acknowledged for their unique potential in recapitulating human physio-pathological mechanisms. They are transformative for human health by becoming the platform of choice to probe disease mechanisms and advance new therapies. Furthermore, the compounds' validation as therapeutics represents another point of the drug development pipeline where organoids may provide key understandings and help pharma organizations replace or reduce animal research. In this review, we focus on gastrointestinal organoid models, which are currently the most advanced organoid models in drug development. We focus on experimental validations of their value, and we propose avenues to enhance their use in drug discovery and
development, as well as precision medicine and diagnostics.

Phytoestrogens: Chemistry, potential health benefits, and their medicinal importance.

Phytoestrogens, also known as xenoestrogens, are secondary metabolites derived from plants that have similar structures and biological effects as human estrogens. These compounds do not directly affect biological functions but can act as agonists or antagonists depending on the level of endogenous estrogen in the body. Phytoestrogens may have an epigenetic mechanism of action independent of estrogen receptors. These compounds are found in more than 300 plant species and are synthesized through the phenylpropanoid pathway, with specific enzymes leading to various chemical structures. Phytoestrogens, primarily phenolic compounds, include isoflavonoids, flavonoids, stilbenes, and lignans. Extensive research in animals and humans has demonstrated the protective effects of phytoestrogens on estrogen-dependent diseases. Clinical trials have also shown their potential benefits in conditions such as osteoporosis, Parkinson's disease, and certain types of cancer. This review provides a concise overview of phytoestrogen classification, chemical diversity, and biosynthesis and discusses the potential therapeutic effects of phytoestrogens, as well as their preclinical and clinical development.

The (misleading) role of animal models in drug development

Animals like mice and rats have long been used in medical research to help understand disease and test potential new treatments before human trials. However, while animal studies have contributed to important advances, too much reliance on animal models can also mislead drug development. This article explains for a general audience how animal research is used to develop new medicines, its benefits and limitations, and how more accurate and humane techniques—alternatives to animal testing—could improve this process.

Pharmacological Screening of Cholinergic Drugs Using <i>Daphnia magna Straus</i> Hydrobionts as a Biological Test Object

This work presents the results of primary pharmacological screening of anticholinergic drugs and comparative evaluation of their specific activity in equitoxic concentrations using Daphnia magna Straus hydrobionts as a biological test object. We determine the protective index and the minimum effective concentration in terms of preventing atypical motor hyperactivity of pharmacological substances when poisoning with a reversible acetylcholinesterase inhibitor. On the basis of the results obtained, the list of promising candidates for further research in warm-blooded animals is extended by all of the anticholinergic drugs previously selected in experiments on mammals.

Current regulations in the Animal Welfare Act and the significance for animal research

Due to the legal implantation of the 3Rprinciple, the number of laboratory animals decreased significantly over the past 10years. In this article, the historical development of animal experiments over the last decade will be presented in the context of the current regulations of the Animal Welfare Act. It points out bureaucratic obstacles to the approval of animal experiments, which jeopardize Germany as aresearch location for both academia and industry. The article presents constructive proposals for solutions. This should be done in accordance with the DFG recommendation to ensure efficient biomedical research while maintaining the highest animal welfare standards.

Variation in the mu-opioid receptor gene (OPRM1) moderates the influence of maternal sensitivity on child attachment

The endogenous opioid system is thought to play an important role in mother-infant attachment. In infant rhesus macaques, variation in the μ-opioid receptor gene (OPRM1) is related to differences in attachment behavior that emerges following repeated separation from the mother; specifically, infants carrying at least one copy of the minor G allele of the OPRM1 C77G polymorphism show heightened and more persistent separation distress, as well as a pattern of increased contact-seeking behavior directed towards the mother during reunions (at the expense of affiliation with other group members). Research in adult humans has also linked the minor G allele of the analogous OPRM1 A118G polymorphism with greater interpersonal sensitivity. Adopting an interactionist approach, we examined whether OPRM1 A118G genotype and maternal (in)sensitivity are associated with child attachment style, predicting that children carrying the G allele may be more likely to develop an ambivalent attachment pattern in response to less sensitive maternal care. The sample consisted of 191 mothers participating with their children (n = 223) in the Maternal Adversity, Vulnerability and Neurodevelopment (MAVAN) project, a community-based, birth cohort study of Canadian mothers and their children assessed longitudinally across the child’s development. Maternal sensitivity was coded from at-home mother-child interactions videotaped when the child was 18 months of age. Child attachment was assessed at 36 months using the Strange Situation paradigm. As predicted, G allele carriers, but not AA homozygotes, showed increasing odds of being classified as ambivalently attached with decreasing levels of maternal sensitivity. Paralleling earlier non-human animal research, this work provides support for the theory that endogenous opioids contribute to the expression of attachment behaviors in humans.

Functioning human lung organoids model pulmonary tissue response from carbon nanomaterial exposures

Human lung organoids (HLOs) are increasingly used to model development and infectious diseases, however their ability to recapitulate functional pulmonary tissue response to nanomaterial (NM) exposures has yet to be demonstrated. Here, we established a lung organoid exposure model that utilises microinjection to present NMs into the lumen of organoids. Our model assures efficient, reproducible and controllable exposure of the apical pulmonary epithelium, emulating real-life human exposure scenario. By comparing the impact of two well studied carbon-based NMs, graphene oxide sheets (GO) and multi-walled carbon nanotubes (MWCNT), we validated lung organoids as tools for predicting pulmonary NM-driven responses. In agreement with established in vivo data, we demonstrate that MWCNT, but not GO, elicit adverse effects on lung organoids, leading to a pro-fibrotic phenotype. Our findings reveal the capacity and suitability of HLOs for hazard assessment of NMs, aligned with the much sought-out 3Rs (animal research replacement, reduction, refinement) framework.

Potential antidepressant effects of Traditional Chinese botanical drug formula Chaihu-Shugan-San and its active ingredients.

Background: Depression is a severe mental disorder that poses a significant threat to both the physical and mental wellbeing of individuals. Currently, there are various methods for treating depression, including traditional Chinese herbal formulations like Chaihu-Shugan-San (CSS), which have shown effective antidepressant effects in both clinical and animal research. Objective: This review aims to provide a comprehensive synthesis of evidence related to CSS, considering both preclinical and clinical studies, to uncover its potential multi-level, multi-pathway, and multi-target mechanisms for treating depression and identify its active ingredients. Methods: A thorough search was conducted in electronic databases, including PubMed, MEDLINE, Web of Science, Google Scholar, CNKI, and Wanfang, using keywords such as "Chaihu Shugan" and "depression" to retrieve relevant literature on CSS and its active ingredients. The review process adhered to the PRISMA guidelines. Results: This review consolidates the mechanisms underlying antidepressant effects of CSS and its active ingredients. It emphasizes its involvement in the regulation of monoaminergic neurotransmitter systems, synaptic plasticity, and the hypothalamic-pituitary-adrenal axis, among other aspects. Conclusion: CSS exerts a pivotal role in treating depression through various pathways, including the monoaminergic neurotransmitter system, the hypothalamic-pituitary-adrenal axis, synaptic plasticity, inflammation, brain-derived neurotrophic factor levels, and the brain-gut axis. This review facilitates a comprehensive understanding of the current state of CSS research, fostering an in-depth exploration of the etiological mechanisms of depression and the potential discovery of novel antidepressant drugs.

Ethical Considerations in Animal Research: A Focus on China

Laboratory animals have played an indispensable role in advancing human knowledge and serve as essential subjects for generating reliable and precise outcomes in life science research. However, the ethical quandaries associated with animal testing, including issues related to their treatment and welfare, demand careful consideration. Similar to numerous developed nations, China has dedicated significant efforts to the protection of laboratory animals. This paper primarily examines China's legislative modifications concerning animal experiments within the cosmetic industry, conducts an ethical analysis of these legislative changes, and applies the 3Rs (Replace, Reduce, Refine) framework to propose practical recommendations for improving China's approach in this context.

Exploring Alternatives for Marine Toxicity Testing: Initial Evaluation of Fish Embryo and Mysid Tests.

Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish-based tests using 3,4-dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;00:1-15. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Experimental Animal Research in Vaccine Studies during the Pandemic Process

The pandemic that emerged in Wuhan, China, in 2019, once again revealed the role of basic sciences and experimental animal research in the world. This situation, also called COVID-19, has spread rapidly all over the world in a short period of 3 months. It has been revealed that vaccination is more important than treatment in reducing mass deaths during the fight against the pandemic. The fact that RNA viruses constantly change their genetic properties was also a major challenge in finding an effective vaccine. However, increasing progress in basic sciences (especially mRNA technology) and the use of humanized mice have contributed greatly to reducing mass deaths. The fact that the Sars-Cov-2 virus does not cause infection in conventional mice and that it can create an infection model in mutant mice to which human lung epithelial genes are transferred has given great impetus to vaccine and new drug treatment studies. The first vaccine model was developed using these mice in vaccine studies. Nearly 176 vaccine types have been developed in the Covid-19 pandemic. These vaccines; viron vaccines are nucleic acid-based (mRNA), viral vector and protein-based vaccines. The mutant mice used in the first step of these vaccines created the disease model almost perfectly and ensured that the results in the target species (Human) progressed with fewer errors. Since the disease occurs in mice carrying human lung epithelium very close to the target species, they have played a major role in testing the protective effects of vaccines. The vaccines prepared towards the end of 2020 were made ready for use. Vaccine production has been carried out at an almost record speed in pandemics in world history. Although vaccines may have different effects and protection levels, the role of vaccines in the pandemic is undeniable. More than 2.5 billion doses of COVID-19 vaccines have been administered worldwide, and approximately 35 million doses continue to be administered every day. Experimental animal research, genetic research and gene transfer that started in the 1970s were used to obtain vaccines that were prepared in a short time and made available to humanity. technologies and mutant mouse technology play a huge role. Although the pandemic process is still not over, it is expected to ease and continue to have effects similar to seasonal flu. In this process, humanity has gained important experience in acting together and rapid vaccine development. The use of experimental animals is of great importance in these processes.

Assembling of a cost-effective and adaptable motorised rodent exercise wheel

Objectives: Exercise physiology is one of the leading branches of applied physiology. It is concerned with studies related to the effect of acute and chronic exercise on mental and physical health research in human subjects and animals. There are various methods of physical exercise which have been used in animal studies including rodents. However, the machines available for research purposes are sophisticated and expensive, which also requires an additional annual maintenance cost. The objective is to assemble an efficient, reliable, cost-effective, and humane motorized exercise wheel setup for the study of acute and chronic physical exercise in rodents. Materials and Methods: The motorized rodent exercise wheel was assembled using affordable locally available materials. Results: A cost-effective, efficient model for rodent exercise was built and the total cost of this setup was 32 USD or 2860 INR only. Conclusion: This cost-effective rodent exercise wheel works efficiently for the conduction of exercise-related studies in rodents.

Decoding the secrets of longevity: unraveling nutraceutical and miRNA-Mediated aging pathways and therapeutic strategies.

MicroRNAs (miRNAs) are short RNA molecules that are not involved in coding for proteins. They have a significant function in regulating gene expression after the process of transcription. Their participation in several biological processes has rendered them appealing subjects for investigating age-related disorders. Increasing data indicates that miRNAs can be influenced by dietary variables, such as macronutrients, micronutrients, trace minerals, and nutraceuticals. This review examines the influence of dietary factors and nutraceuticals on the regulation of miRNA in relation to the process of aging. We examine the present comprehension of miRNA disruption in age-related illnesses and emphasize the possibility of dietary manipulation as a means of prevention or treatment. Consolidating animal and human research is essential to validate the significance of dietary miRNA control in living organisms, despite the abundance of information already provided by several studies. This review elucidates the complex interaction among miRNAs, nutrition, and aging, offering valuable insights into promising areas for further research and potential therapies for age-related disorders.

Active forgetting and neuropsychiatric diseases.

Recent and pioneering animal research has revealed the brain utilizes a variety of molecular, cellular, and network-level mechanisms used to forget memories in a process referred to as "active forgetting". Active forgetting increases behavioral flexibility and removes irrelevant information. Individuals with impaired active forgetting mechanisms can experience intrusive memories, distressing thoughts, and unwanted impulses that occur in neuropsychiatric diseases. The current evidence indicates that active forgetting mechanisms degrade, or mask, molecular and cellular memory traces created in synaptic connections of "engram cells" that are specific for a given memory. Combined molecular genetic/behavioral studies using Drosophila have uncovered a complex system of cellular active-forgetting pathways within engram cells that is regulated by dopamine neurons and involves dopamine-nitric oxide co-transmission and reception, endoplasmic reticulum Ca2+ signaling, and cytoskeletal remodeling machinery regulated by small GTPases. Some of these molecular cellular mechanisms have already been found to be conserved in mammals. Interestingly, some pathways independently regulate forgetting of distinct memory types and temporal phases, suggesting a multi-layering organization of forgetting systems. In mammals, active forgetting also involves modulation of memory trace synaptic strength by altering AMPA receptor trafficking. Furthermore, active-forgetting employs network level mechanisms wherein non-engram neurons, newly born-engram neurons, and glial cells regulate engram synapses in a state and experience dependent manner. Remarkably, there is evidence for potential coordination between the network and cellular level forgetting mechanisms. Finally, subjects with several neuropsychiatric diseases have been tested and shown to be impaired in active forgetting. Insights obtained from research on active forgetting in animal models will continue to enrich our understanding of the brain dysfunctions that occur in neuropsychiatric diseases.

Abstract 4305: Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice

Abstract Cancer is a complex disease that involves both malignant cells and the surrounding microenvironment. The tumor microenvironment (TME) is comprised of various components, including cancer-associated fibroblasts, immune cells, blood vessels, and extracellular matrix elements, all of which interact with cancer cells. It is increasingly recognized that the components of the TME are a critical determinant of therapeutic response and resistance, and as such targeting the TME has emerged as a promising strategy in cancer therapy. Syngeneic models are necessary to study the TME, and more specifically the tumor infiltrating immune cell populations, as these models allow us to monitor various aspects of the immune response. Another important aspect to consider is tumor heterogeneity, which can cause issues during analysis due to immune cell populations not being evenly distributed throughout the tumor. The decision of whether to analyze whole tumors or halved tumors when monitoring changes in immune cell populations is an important consideration. When considering multiple endpoints from a single tumor sample, halved tumors are oftentimes considered as we remain mindful of the 3Rs (replacement, reduction and refinement) in animal research. As such, it is important to understand the effects of splitting samples and how it can affect the data that is generated. To determine the effects of analyzing whole and halved tumor samples on immune cell subsets, studies were designed to subcutaneously implant either CT26.WT or 4T1-Luc2-1A4 cells into female BALB/cAnNHsd mice and harvest tumor samples after the completion of treatment. Mice were treated with isotype control (Clone MPC-11) or anti-mCTLA-4 (Clone 9D9). At the time of sample collection, tumors were kept whole intact or halved and analyzed via flow cytometry to quantify lymphoid and myeloid immune cell subsets within tumor samples. The data revealed differences in immune cell subset populations for both isotype vs. anti-CTLA-4 treated tumors and whole vs. halved tumors. Changes in the lymphoid cell compartment were observed for whole vs. halved tumors of ± ~25-100% and ± ~0-40% for isotype treated and of ± ~0-30% and ± ~25-2600% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively. Changes in the myeloid cell compartment of ± ~5-45% and ± ~10-65% for isotype treated and of ± ~15-100% and ± ~0-70% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively, were also observed. Additionally, differences were observed between the two tumor halves, not just between whole and halved tumors. Taken together, the data support that splitting tumor samples can negatively impact the analysis of the immune cell populations. Tumors can be split if required to perform multiple analyses, however, this should be done with caution, as it can result in trend differences and increases variability within the dataset. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Sheri Barnes, David Draper, Scott Wise. Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice [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 4305.