1 Killing of Kras mutant colon cancer cells via Rac-independent actin remodeling by the βGBP cyto... more 1 Killing of Kras mutant colon cancer cells via Rac-independent actin remodeling by the βGBP cytokine a physiological PI3K inhibitor therapeutically effective in vivo
Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldw... more Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-func...
Abstract Background LRIG1 is a negative regulator of epidermal growth factor receptor (EGFR) sign... more Abstract Background LRIG1 is a negative regulator of epidermal growth factor receptor (EGFR) signalling, and regulates stem-cell compartments in skin and gut epithelial cells. Interestingly, LRIG1 is lost in samples from patients with preinvasive squamous cell lung cancer. Basal cells are the upper airway stem cells and the putative origin of this cancer. We aimed to delineate LRIG1 expression within the airway, its effect on the stem-cell compartment, and whether its loss causes preinvasive disease. Methods Lrig1 (EGFP-IRES-CreERT2) mice were used to determine LRIG1 expression in the airways with immunofluorescence and flow cytometry. Basal stem-cell expression of LRIG1 and correlation with cell proliferation were examined. LRIG1+ cells were isolated with flow cytometry, and colony-formation and spheroid-formation assays were used to compare LRIG1+ and LRIG1– basal cells. To lineage trace LRIG+ cells, Lrig1 mice were crossed with reporters and were transgene activated. In-vivo and ex-vivo studies were performed. Data are given as mean (SE). Findings LRIG1 expression was detected throughout the airway epithelium and within 66·3% (3·01) of basal cells. This LRIG1+ subpopulation of basal cells showed increased proliferation compared with LRIG1– cells (12·52% [1·82] vs 6·27 [2·13], p=0·0156). When plated in colony-formation assays, flow-sorted LRIG1+ expressing basal cells showed increased colony-forming efficacy and also had a greater spheroid-forming capacity (spheroid-forming capacity 3·3% [0·7] vs 0·56 [0·26], p=0·031). Ex-vivo lineage tracing assessed with spheroid assays indicated the development of clonal patches derived from LRIG+ basal cells. Formation of in-vivo clonal patches also occurred. Interpretation Our initial data indicate that LRIG1 has a role in airway stem-cell progenitor regulation and that it marks a more proliferative basal cell population. These data highlight the need to further investigate how the loss of LRIG1 leads to epithelial dysregulation and development of malignancy in patients. Funding The Wellcome Trust, Cancer Research UK.
The pandemic caused by SARS-CoV-2 and its associated illness COVID-19 has had an almost unprecede... more The pandemic caused by SARS-CoV-2 and its associated illness COVID-19 has had an almost unprecedented impact on humanity. First detected in late 2019 in China, this novel coronavirus has spread worldwide, causing (at time of writing) over 50 million infections and just over 1.3 million deaths (1). Pandemics such as this occur infrequently, although in the last 100 years, Spanish ‘flu, Human Immunodeficiency Virus, Severe Acute Respiratory Syndrome 1, Middle East Respiratory Syndrome, and Bird ‘flu have been classified as such, and pandemics inevitably lead to a rush to characterize the infectious agents and describe curative or preventative measures. Multiple groups worldwide are currently developing candidate vaccines, and several drugs have been repurposed (2). While we await an effective protective vaccine or novel therapeutic regimes, we have seen restrictive public health measures in most countries in the world. These have been of varying stringency and varying length but, in all cases, have caused severe disruption, not just to normal society but also to research in all workplace settings—basic research, clinical, pharma, manufacturing, and so on. At the same time, it has been important that research into this novel virus, and other areas of critical research, was not halted. This led to the adoption of new, or modified, workplace practices, which would enable at the least the most critical and/or time-constrained research endeavors to continue. This Special Issue collates some of the creative approaches taken globally within flow cytometry shared resource facilities. Shared Resource Laboratories (SRLs) are responsible for continuity and access to research technologies as much as reasonably possible. However, the current times have presented unique and substantial challenges. Many of us who work in SRLs will have had Business Continuity Plans (or Disaster Plans) in place prior to 2020, and a global pandemic, in many cases, was included as a possible eventuality. Despite these plans, the level of emergency preparedness may not have been adequate. Certainly, many workplaces instigated a denial of service within the context of work being able to be carried on elsewhere or outsourced. In addition, planning and implementing are quite different, and as a result, there is much we can learn from the way SRLs have managed multiple facets of operation during this present pandemic, which we hope will serve us well in the future. With a starting point of the 2016 Best Practices publication (3), we thought it would be beneficial to revisit this from the perspective of meeting these Best Practices under very different circumstances, such as the current global pandemic. Lessons learned from the approaches taken to answer these new challenges will, we hope, stand us in good stead in the event of a resurgence of SARS-CoV-2 or even the emergence of a new, different threat. SRL managers and staff have always been an integral part of International Society for the Advancement of Cytometry (ISAC), and as such, we have this unique opportunity to share their expertise and experiences to lay some groundwork for future pandemics or other disasters when normal working conditions are not possible. Within ISAC, there are several SRL committees and task forces (Table 1) that are populated by experienced leaders in the SRL community, with the aim to support ISAC's SRL membership. We asked the ISAC SRL community to reflect on some of these changes and to share those approaches with the wider global community. It is a testament to the dedication and engagement of this community that these manuscripts, gathered and presented in this special issue, were so rapidly completed (Figure 1). Cytometry is not the only technology that operates as a shared resource, and many of the problems and proposed solutions found in this issue would be equally applicable to other types of SRL, such as light microscopy, electron microscopy, or any other facility that has a hybrid client interface that needs to consider training and interactions, equipment maintenance, and biosafety. Indeed, Auger et al. have taken a perspective from the administration of these facilities that considers all aspects across shared resource types, including finance, procurement, and capital equipment budgets (4). As ISAC is an international society, and the pandemic is a global issue, efforts were made to ensure that manuscripts comprised geographical as well as workplace setting diversity. This issue seeks to ensure that the policies and procedures now put in place will allow SRLs to be more prepared for any subsequent periods of forced inaction regardless of location or workplace setting. Out of necessity, these manuscripts are, for the most part, not original articles but rather commentaries on how SRLs have had to adapt to a new situation to be able to continue their function of providing research tools, training, and advice to end users under extenuating circumstances.…
Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteris... more Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteristics including ATP-dependent drug efflux and elevated tumorigenic potential. To determine whether aerodigestive squamous cell carcinomas (SCCs) contain a subpopulation of cancer stem cell-like cells, we performed Hoechst dye efflux assays using four independent cell lines. Results revealed the presence of a rare, drug effluxing stem cell-like side population (SP) of cells within all cell lines tested (SCC-SP cells). These cells resembled previously characterised epithelial stem cells, and SCC-SP cell abundance was positively correlated with overall cellular density and individual cell quiescence. Serial SCC-SP fractionation and passaging increased their relative abundance within the total cell population. Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo. Despite this, SCC-SP cells remained chemotherapeutically s...
Activin is a member of the transforming growth factor beta superfamily, which is known to have ac... more Activin is a member of the transforming growth factor beta superfamily, which is known to have activities involved in regulating differentiation and development. By using reverse transcription-PCR analysis on immunoaffinity-purified human breast cells, we have found that activin beta a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we have found only four (HBL-100, MCF10-A, PMC-42, and BT 20) to be positive for activin beta a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF- 7 cells (at 2 ng/ml), where it causes an arrest in G(1). Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we demonstrate that activin is an inhibitor of tubule formation by human mammary organoids in vitro. These are the first observations of activi...
1 Killing of Kras mutant colon cancer cells via Rac-independent actin remodeling by the βGBP cyto... more 1 Killing of Kras mutant colon cancer cells via Rac-independent actin remodeling by the βGBP cytokine a physiological PI3K inhibitor therapeutically effective in vivo
Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldw... more Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-func...
Abstract Background LRIG1 is a negative regulator of epidermal growth factor receptor (EGFR) sign... more Abstract Background LRIG1 is a negative regulator of epidermal growth factor receptor (EGFR) signalling, and regulates stem-cell compartments in skin and gut epithelial cells. Interestingly, LRIG1 is lost in samples from patients with preinvasive squamous cell lung cancer. Basal cells are the upper airway stem cells and the putative origin of this cancer. We aimed to delineate LRIG1 expression within the airway, its effect on the stem-cell compartment, and whether its loss causes preinvasive disease. Methods Lrig1 (EGFP-IRES-CreERT2) mice were used to determine LRIG1 expression in the airways with immunofluorescence and flow cytometry. Basal stem-cell expression of LRIG1 and correlation with cell proliferation were examined. LRIG1+ cells were isolated with flow cytometry, and colony-formation and spheroid-formation assays were used to compare LRIG1+ and LRIG1– basal cells. To lineage trace LRIG+ cells, Lrig1 mice were crossed with reporters and were transgene activated. In-vivo and ex-vivo studies were performed. Data are given as mean (SE). Findings LRIG1 expression was detected throughout the airway epithelium and within 66·3% (3·01) of basal cells. This LRIG1+ subpopulation of basal cells showed increased proliferation compared with LRIG1– cells (12·52% [1·82] vs 6·27 [2·13], p=0·0156). When plated in colony-formation assays, flow-sorted LRIG1+ expressing basal cells showed increased colony-forming efficacy and also had a greater spheroid-forming capacity (spheroid-forming capacity 3·3% [0·7] vs 0·56 [0·26], p=0·031). Ex-vivo lineage tracing assessed with spheroid assays indicated the development of clonal patches derived from LRIG+ basal cells. Formation of in-vivo clonal patches also occurred. Interpretation Our initial data indicate that LRIG1 has a role in airway stem-cell progenitor regulation and that it marks a more proliferative basal cell population. These data highlight the need to further investigate how the loss of LRIG1 leads to epithelial dysregulation and development of malignancy in patients. Funding The Wellcome Trust, Cancer Research UK.
The pandemic caused by SARS-CoV-2 and its associated illness COVID-19 has had an almost unprecede... more The pandemic caused by SARS-CoV-2 and its associated illness COVID-19 has had an almost unprecedented impact on humanity. First detected in late 2019 in China, this novel coronavirus has spread worldwide, causing (at time of writing) over 50 million infections and just over 1.3 million deaths (1). Pandemics such as this occur infrequently, although in the last 100 years, Spanish ‘flu, Human Immunodeficiency Virus, Severe Acute Respiratory Syndrome 1, Middle East Respiratory Syndrome, and Bird ‘flu have been classified as such, and pandemics inevitably lead to a rush to characterize the infectious agents and describe curative or preventative measures. Multiple groups worldwide are currently developing candidate vaccines, and several drugs have been repurposed (2). While we await an effective protective vaccine or novel therapeutic regimes, we have seen restrictive public health measures in most countries in the world. These have been of varying stringency and varying length but, in all cases, have caused severe disruption, not just to normal society but also to research in all workplace settings—basic research, clinical, pharma, manufacturing, and so on. At the same time, it has been important that research into this novel virus, and other areas of critical research, was not halted. This led to the adoption of new, or modified, workplace practices, which would enable at the least the most critical and/or time-constrained research endeavors to continue. This Special Issue collates some of the creative approaches taken globally within flow cytometry shared resource facilities. Shared Resource Laboratories (SRLs) are responsible for continuity and access to research technologies as much as reasonably possible. However, the current times have presented unique and substantial challenges. Many of us who work in SRLs will have had Business Continuity Plans (or Disaster Plans) in place prior to 2020, and a global pandemic, in many cases, was included as a possible eventuality. Despite these plans, the level of emergency preparedness may not have been adequate. Certainly, many workplaces instigated a denial of service within the context of work being able to be carried on elsewhere or outsourced. In addition, planning and implementing are quite different, and as a result, there is much we can learn from the way SRLs have managed multiple facets of operation during this present pandemic, which we hope will serve us well in the future. With a starting point of the 2016 Best Practices publication (3), we thought it would be beneficial to revisit this from the perspective of meeting these Best Practices under very different circumstances, such as the current global pandemic. Lessons learned from the approaches taken to answer these new challenges will, we hope, stand us in good stead in the event of a resurgence of SARS-CoV-2 or even the emergence of a new, different threat. SRL managers and staff have always been an integral part of International Society for the Advancement of Cytometry (ISAC), and as such, we have this unique opportunity to share their expertise and experiences to lay some groundwork for future pandemics or other disasters when normal working conditions are not possible. Within ISAC, there are several SRL committees and task forces (Table 1) that are populated by experienced leaders in the SRL community, with the aim to support ISAC's SRL membership. We asked the ISAC SRL community to reflect on some of these changes and to share those approaches with the wider global community. It is a testament to the dedication and engagement of this community that these manuscripts, gathered and presented in this special issue, were so rapidly completed (Figure 1). Cytometry is not the only technology that operates as a shared resource, and many of the problems and proposed solutions found in this issue would be equally applicable to other types of SRL, such as light microscopy, electron microscopy, or any other facility that has a hybrid client interface that needs to consider training and interactions, equipment maintenance, and biosafety. Indeed, Auger et al. have taken a perspective from the administration of these facilities that considers all aspects across shared resource types, including finance, procurement, and capital equipment budgets (4). As ISAC is an international society, and the pandemic is a global issue, efforts were made to ensure that manuscripts comprised geographical as well as workplace setting diversity. This issue seeks to ensure that the policies and procedures now put in place will allow SRLs to be more prepared for any subsequent periods of forced inaction regardless of location or workplace setting. Out of necessity, these manuscripts are, for the most part, not original articles but rather commentaries on how SRLs have had to adapt to a new situation to be able to continue their function of providing research tools, training, and advice to end users under extenuating circumstances.…
Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteris... more Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteristics including ATP-dependent drug efflux and elevated tumorigenic potential. To determine whether aerodigestive squamous cell carcinomas (SCCs) contain a subpopulation of cancer stem cell-like cells, we performed Hoechst dye efflux assays using four independent cell lines. Results revealed the presence of a rare, drug effluxing stem cell-like side population (SP) of cells within all cell lines tested (SCC-SP cells). These cells resembled previously characterised epithelial stem cells, and SCC-SP cell abundance was positively correlated with overall cellular density and individual cell quiescence. Serial SCC-SP fractionation and passaging increased their relative abundance within the total cell population. Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo. Despite this, SCC-SP cells remained chemotherapeutically s...
Activin is a member of the transforming growth factor beta superfamily, which is known to have ac... more Activin is a member of the transforming growth factor beta superfamily, which is known to have activities involved in regulating differentiation and development. By using reverse transcription-PCR analysis on immunoaffinity-purified human breast cells, we have found that activin beta a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we have found only four (HBL-100, MCF10-A, PMC-42, and BT 20) to be positive for activin beta a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF- 7 cells (at 2 ng/ml), where it causes an arrest in G(1). Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we demonstrate that activin is an inhibitor of tubule formation by human mammary organoids in vitro. These are the first observations of activi...
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Papers by Derek Davies