Biology subjects

Allocca, G.

Publications and source records attributed to Allocca, G..

6 recordsLinked to original sources

bioRxiv2026

ORTHOTOPIC TUMOUR INDUCTION PROTOCOL SHAPES TUMOUR MICROENVIRONMENT

Robust preclinical cancer models are essential for studying tumour progression and evaluating therapeutic strategies. Orthotopic implantation of tumour cells into the mammary gland is widely used to model triple-negative breast cancer (TNBC), yet substantial variation exists in the number of cells used to initiate tumours, often without explicit consideration of how this parameter shapes tumour biology. Here, we systematically assess how tumour cell number at implantation influences the tumour microenvironment using the 4T1 orthotopic TNBC model. Mammary tumours initiated with different cell numbers exhibited comparable early growth kinetics and gross size, yet diverged markedly in cellular density, hypoxia, necrosis, proliferation, vascular integrity, and immune cell infiltration. High-density tumours displayed increased HIF-1 staining and necrosis, alongside differences in vascular-associated markers and increased macrophage recruitment. Conversely, low-density tumours remained highly proliferative, less necrotic and exhibited higher PDGFR{beta} staining. Importantly, tumour cells disseminated systemically within days of implantation across all conditions, although differences in colony recovery and ex vivo expansion were not statistically significant. Together, these findings demonstrate that tumour initiating protocols are powerful determinants of tumour state, with potential implications for dissemination frequency and tumour cell fate at distant sites. Rather than representing a source of experimental variability alone, this understanding provides an opportunity to intentionally tailor orthotopic models to investigate specific stages of disease progression and therapeutic response, thereby enhancing cross-study reproducibility and translational relevance.

cancer biology↗

bioRxiv2025

Sleep-Wake Transitions Are Impaired in the AppNL-G-FMouse Model of Early Onset Alzheimers Disease

Poor sleep quality and reduced sleep duration are associated with Alzheimers disease (AD)-related {beta}-amyloid (A{beta}) pathologies. We conducted two studies of sleep/wake, activity and body temperature in AppNL-G-F mice, a strain that exhibits three mutations in the human App gene associated with elevated risk for early onset AD. First, AppNL-G-F mice were compared to wildtype (WT) littermates at 14-18 and 18-22 months of age and, at both ages, were found to exhibit more Wake and less NREM and REM sleep than WT littermates. This long wake/short sleep phenotype was evident during the dark phase at 14-18 months but occurred in both the light and dark phases at 18-22 months. AppNL-G-F mice had fewer short (<60 sec) and more long (>260 sec) Wake bouts and were hyperactive at 18-22 months, which undoubtedly contributed to the increased Wake/reduced sleep. Despite this reduced sleep phenotype, AppNL-G-F mice were no sleepier than WT mice and the sleep homeostat was functional in both strains. In the second study, sex differences in these parameters were assessed at 18-24 months. Reduced sleep was evident in both sexes of AppNL-G-F mice but was clearly more evident in females. Wake and REM sleep bout durations were longer in both sexes of AppNL-G-F mice than in WT littermates. EEG spectral power during NREM sleep was reduced in female AppNL-G-F mice between 4.88-10.50 Hz compared to WT mice whereas, during REM sleep, both male and female AppNL-G-F mice exhibited reduced spectral power in the theta range. These results suggest that A{beta} deposition may impair state transition mechanism(s) in AppNL-G-F mice and demonstrate that, as in human AD patients, the long wake/short sleep phenotype was more evident in female AppNL-G-F mice, thus supporting the use of this strain as a model to investigate interventions that mitigate AD burden during early disease stages.

neuroscience↗

bioRxiv2025

Automated sleep scoring in hibernating and non-hibernating American black bears

Hibernating bears show remarkable metabolic suppression. Their decline in core body temperature (Tb) is moderate (from 38{degrees}C to 30-35{degrees}C), but their metabolism declines as much as 75%. To understand the role of sleep in this hypometabolic state, we recorded biotelemetrically EEG, EOG and EMG data over 3500 days from 16 captive American black bears in and out of hibernation under semi-natural conditions. This data set is too large to score manually for Wake, REM- and NREM sleep, so we tested two machine learning classifiers: (1) Somnotate trained on multiple one-day recordings, and (2) Somnivore, trained on a small subset from each recording. As automated scoring methods have not been applied to hibernating species before, a major concern is the effect changing brain temperature has on the EEG and on the machine learning based detection. Therefore, we selected reference data using consensus by 3 manual sleep scorers from each of 6 bears, two one-day recordings at the highest and lowest body temperatures during hibernation when Tb was oscillating in multiday cycles, and a non-hibernating one-day recording in summer. Somnotate results were excellent when trained separately for hibernating and non-hibernating data. Training Somnotate separately for high and low Tb within hibernation did not improve results further. Sleep times in hibernation were about 2x that in summer for both automated scores and manual scores (p<0.0001). There were no significant differences in occupancy of vigilance states between automated and manual scores in hibernation (p>0.05), but a small overestimate of sleep time in summer (p<0.05). Both applications yielded F-measures against manual scores in the 0.90-0.98 range. Outliers in the 0.67-0.88 range were correlated between the two applications, indicating that specific files are more challenging to annotate. We conclude that both applications have accuracies approaching that of manual scorers when trained on high quality data.

neuroscience↗

bioRxiv2024

An EEG Signature of MCH Neuron Activities Predicts Cocaine Seeking

BackgroundIdentifying biomarkers that predict substance use disorder (SUD) propensity may better strategize anti-addiction treatment. The melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH) critically mediates interactions between sleep and substance use; however, their activities are largely obscured in surface electroencephalogram (EEG) measures, hindering the development of biomarkers. MethodsSurface EEG signals and real-time Ca2+ activities of LH MCH neurons (Ca2+MCH) were simultaneously recorded in male and female adult rats. Mathematical modeling and machine learning were then applied to predict Ca2+MCH using EEG derivatives. The robustness of the predictions was tested across sex and treatment conditions. Finally, features extracted from the EEG-predicted Ca2+MCH either before or after cocaine experience were used to predict future drug-seeking behaviors. ResultsAn EEG waveform derivative - a modified theta-to-delta ratio (EEG Ratio) - accurately tracks real-time Ca2+MCH in rats. The prediction was robust during rapid eye movement sleep (REMS), persisted through REMS manipulations, wakefulness, circadian phases, and was consistent across sex. Moreover, cocaine self-administration and long-term withdrawal altered EEG Ratio suggesting shortening and circadian redistribution of synchronous MCH neuron activities. In addition, features of EEG Ratio indicative of prolonged synchronous MCH neuron activities predicted lower subsequent cocaine seeking. EEG Ratio also exhibited advantages over conventional REMS measures for the predictions. ConclusionsThe identified EEG Ratio may serve as a non-invasive measure for assessing MCH neuron activities in vivo and evaluating REMS; it may also serve as a potential biomarker predicting drug use propensity.

neuroscience↗

bioRxiv2021

The Development of Sleep/Wake Disruption and Cataplexy as Hypocretin/Orexin Neurons Degenerate in Male vs. Female Orexin/tTA; TetO-DTA Mice

Narcolepsy Type 1 (NT1), a sleep disorder with similar prevalence in both sexes, is thought to be due to loss of the hypocretin/orexin (Hcrt) neurons. Several transgenic strains have been created to model this disorder and are increasingly being used for preclinical drug development and basic science studies, yet most studies have solely used male mice. We compared the development of narcoleptic symptomatology in male vs. female orexin-tTA; TetO-DTA mice, a model in which Hcrt neuron degeneration can be initiated by removal of doxycycline (DOX) from the diet. EEG, EMG, body temperature, gross motor activity and video recordings were conducted for 24-h at baseline and 1, 2, 4 and 6 weeks after DOX removal. Female DTA mice exhibited cataplexy, the pathognomonic symptom of NT1, by Week 1 in the DOX(-) condition but cataplexy was not consistently present in males until Week 2. By Week 2, both sexes showed an impaired ability to sustain long wake bouts during the active period, the murine equivalent of excessive daytime sleepiness in NT1. Body temperature appeared to be regulated at lower levels in both sexes as the Hcrt neurons degenerated. During degeneration, both sexes also exhibited the "Delta State", characterized by sudden cessation of activity, high delta activity in the EEG, maintenance of muscle tone and posture, and the absence of phasic EMG activity. Since the phenotypes of the two sexes were indistinguishable by Week 6, we conclude that both sexes can be safely combined in future studies to reduce cost and animal use. Statement of SignificanceAlthough narcolepsy is a disorder that affects both men and women with similar frequency, most basic research and preclinical development studies of sleep have utilized male experimental subjects. The identification of the hypocretin/orexin (Hcrt) neuron loss as the likely cause of human narcolepsy has led to the development of transgenic mouse strains that model this disorder. Here, we compare the emergence of narcoleptic symptoms in male vs. female bigenic orexin-tTA; TetO DTA mice, a state-of-the-art narcolepsy model in which degeneration of the Hcrt neurons can be triggered by dietary manipulation. We find that female mice develop the narcoleptic phenotype more rapidly than males but that both sexes are equally symptomatic by the end of the degeneration period.

neuroscience↗

bioRxiv2020

Circulating levels of epirubicin cause endothelial senescence while compromising metabolic activity and vascular function

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.

cell biology↗