Biology subjects

Bullock, A.

Publications and source records attributed to Bullock, A..

5 recordsLinked to original sources

bioRxiv2024

Long-term maintenance of patient-specific characteristics in tumoroids from six cancer indications in a common base culture media system

Tumoroids, also known as cancer organoids, are patient-derived cancer cells grown as 3D, self-organized multicellular structures that maintain key characteristics (e.g., genotype, gene expression levels) of the tumor from which they originated. These models have emerged as valuable tools for studying tumor biology, cytotoxicity, and response of patient-derived cells to cancer therapies. However, the establishment and maintenance of tumoroids has historically been challenging, labor intensive, and highly variable from lab to lab, hindering their widespread use. Here, we characterize the establishment and/or expansion of colorectal, lung, head and neck, breast, pancreas, and endometrial tumoroids using the standardized, serum-free Gibco OncoPro Tumoroid Culture Medium. Newly derived tumoroid lines (n=20) were analyzed by targeted genomic profiling and RNA sequencing and were representative of tumor tissue samples. Tumoroid lines were stable for over 250 days in culture and freeze-thaw competent. Previously established tumoroid lines were also transitioned to OncoPro medium and exhibited, on average, similar growth rates and conserved donor-specific characteristics when compared to original media systems. Additionally, OncoPro medium was compatible with both embedded culture in extracellular matrix and growth in a suspension format for facile culture and scale up. An example application of these models for assessing the cytotoxicity of a natural killer cell line and primary natural killer cells over time and at various doses demonstrated the compatibility of these models with assays used in compound and cell therapy development. We anticipate that the standardization and versatility of this approach will have important benefits for basic cancer research, drug discovery, and personalized medicine and help make tumoroid models more accessible to the cancer research community.

cancer biology↗

bioRxiv2022

Differential remodelling in small and large murine airways revealed by novel whole lung airway analysis.

Airway remodelling occurs in chronic asthma leading to increased airway smooth muscle (ASM) mass and extra-cellular matrix (ECM) deposition. Whilst extensively studied in murine airways; studies report only selected larger airways at one time point meaning the spatial distribution and resolution of remodelling are poorly understood. Here we use a new method allowing comprehensive assessment of the spatial and temporal changes in ASM, ECM and epithelium in large numbers of murine airways after allergen challenge. Using image processing to analyse 20-50 airways from a whole lung section revealed increases in ASM and ECM after allergen challenge were greater in small and large rather than intermediate airways. ASM predominantly accumulated adjacent to the basement membrane whereas ECM was distributed across the airway wall. Epithelial hyperplasia was most marked in small and intermediate airways. Post challenge, ASM changes resolved over seven days whereas ECM and epithelial changes persisted. The new method suggests large and small airways remodel differently and the long-term consequences of airway inflammation may depend more on ECM and epithelial changes than ASM. The method reduces the number of animals needed, reveals important spatial differences in remodelling and could set new analysis standards for murine asthma models.

physiology↗

bioRxiv2021

Parkinson' disease medication alters rat small intestinal motility and microbiota composition

Parkinsons disease (PD) is known to be associated with altered gastrointestinal function and microbiota composition. Altered gastrointestinal function is key in the development of small intestinal bacterial overgrowth, which is a comorbidity often observed in PD patients. Although PD medication could be an important confounder in the reported alterations, its effect per se on the microbiota composition or the gastrointestinal function at the site of drug absorption has not been studied. To this end, healthy (i.e., not PD model) wild-type Groningen rats were employed and treated with dopamine, pramipexole (in combination with levodopa/carbidopa), or ropinirole (in combination with levodopa/carbidopa) for 14 sequential days. Rats treated with dopamine agonists showed a significant reduction in the small intestinal motility and an increase in bacterial overgrowth in the distal small intestine. Importantly, significant alterations in microbial taxa were observed between the treated and vehicle groups, analogous to the changes previously reported in human PD vs HC microbiota studies. These microbial changes included an increase in Lactobacillus and Bifidobacterium, and decrease in Lachnospiraceae and Prevotellaceae. Importantly, certain Lactobacillus species correlated negatively with the plasma levels of levodopa. Overall, the results highlight the significant effect of PD medication per se on the gut microbiota, and the disease-associated comorbidities, including gastrointestinal dysfunction and small intestinal bacterial overgrowth. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSIn the last years many studies focused on the faecal microbiota profiles of Parkinsons disease (PD) patients and compared them to healthy control (HC) subjects and reported a difference between the microbiota profiles. Although some bacterial taxa have been reported to be differential abundant across studies there is a low consensus between the overall changes observed. It remains to be elucidated whether and how the disease status itself, the PD medication or the gastrointestinal dysfunction, an often reported comorbidity, plays a role in the altered microbiota profiles of Parkinsons disease patients. Added value of this studyTwo major confounding factors potentially affecting the microbiota profiles differentiating PD patients from HC subjects are the PD medication and gastrointestinal dysfunction. We showed that PD medication elicits an decreasing effect on the small intestinal gut motility in a healthy (i.e. non Parkinson) rat model which was a contributing factor to the altered microbiota profile observed. We found that taxa reported previously to be differentially abundant were mirrored in our healthy rat model showing the impact of PD medication on microbiota profiles. Implications of all the available evidenceThis study showed that PD medication and gastrointestinal motility are important factors in the microbiota profiles and could explain some of the differential abundant taxa reported in the cross-sectional PD microbiota studies. Future studies should take the potential side effects of medication that could elicit alterations of the microbiota composition into account when seeking for microbial biomarkers of disease status.

microbiology↗

bioRxiv2019

Microsatellite instability as a tool to diagnose genome instability in CHO cell culture

As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10s of milligrams to over 10s of grams per liter. Most of these gains in productivity have been due to increasing cell numbers in the bioreactors, and with those increases in cell numbers, strategies have been developed to minimize metabolite waste accumulation, such as lactate and ammonia. Unfortunately, cell growth cannot occur without some waste metabolite accumulation, as central metabolism is required to produce the biopharmaceutical. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a less than optimal culture environment, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the effects on Chinese hamster ovary (CHO) cells genome sequences and physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo mutations, in addition to variants in functional regions of certain genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and eventual genome instability. Additionally, we characterized the presence of microsatellites against the most recent Chinese Hamster genome assembly and discovered certain loci are not replicated faithfully in the presence of elevated ammonia, which represents microsatellite instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cultures.

genomics↗

bioRxiv2019

β-Caryophyllene Enhances the Transcriptional Upregulation of Cholesterol Biosynthesis in Breast Cancer Cells

{beta}-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. Here, we examine the hypothesis that BCP induces membrane remodeling. Our data show that high concentrations of BCP increase membrane permeability of human breast cells (hBrC) causing detachment and cell death. At a sub-lethal concentration of BCP, we show that BCP induces a striking upregulation of genes involved in cholesterol biosynthesis, including the gene that encodes for HMGCoA reductase (HMGCR), the rate-determining step in cholesterol biosynthesis. In addition, stearoyl-CoA desaturase (SCD) is also upregulated which would lead to the enhanced formation of monounsaturated fatty acids, specifically oleate and palmitoleate from stearoyl CoA and palmitoyl CoA, respectively. These fatty acids are major components of membrane phospholipids and cholesterol esters. Together, these data suggest that cells respond to BCP by increasing the synthesis of components found in membranes. These responses could be viewed as a repair mechanism and/or as a mechanism to mount resistance to the cytotoxic effect of BCP. Blocking HMGCR activity enhances the cytotoxicity of BCP, suggesting that BCP may provide an additional therapeutic tool in controlling breast cancer cell growth.

cancer biology↗