Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

Chemokine Pathways in Cutaneous Melanoma: Their Modulation by Cancer and Exploitation by the Clinician

The incidence of cutaneous malignant melanoma is rising globally and is projected to continue to rise. Advances in immunotherapy over the last decade have demonstrated that manipulation of the immune cell compartment of tumours is a valuable weapon in the arsenal against cancer; however, limitations to treatment still exist. Cutaneous melanoma lesions feature a dense cell infiltrate, coordinated by chemokines, which control the positioning of all immune cells. Melanomas are able to use chemokine pathways to preferentially recruit cells, which aid their growth, survival, invasion and metastasis, and which enhance their ability to evade anticancer immune responses. Aside from this, chemokine signalling can directly influence angiogenesis, invasion, lymph node, and distal metastases, including epithelial to mesenchymal transition-like processes and transendothelial migration. Understanding the interplay of chemokines, cancer cells, and immune cells may uncover future avenues for melanoma therapy, namely: identifying biomarkers for patient stratification, augmenting the effect of current and emerging therapies, and designing specific treatments to target chemokine pathways, with the aim to reduce melanoma pathogenicity, metastatic potential, and enhance immune cell-mediated cancer killing. The chemokine network may provide selective and specific targets that, if included in current therapeutic regimens, harbour potential to improve outcomes for patients.

Surviving Stress with the Tumour Microenvironment: Understanding Complex Cell - Cell Interactions

<p><b>Each year more than 18 million people are diagnosed with cancer. Stress survival plays a key role in tumour resistance which is a leading cause of cancer deaths. Understanding how cells respond to stress is key to understanding and treating cancer. Within a tumour, cells interact with many other cell types in the surrounding tumour microenvironment. The tumour microenvironment acts to both assist and resist tumorigenesis and metastasis depending on these cellular interactions.</b></p> <p>Rho Zero cells are cells that contain no mitochondrial DNA and therefore lack a functional electron transport chain and rely on glycolysis for energy metabolism, these cells require supplementation to survive in cell culture. When deprived of essential supplementation rho zero cells exhibit severe metabolic stress. Yet when implanted into mice they form tumours after a 2-3 week delay. After this they contain new mitochondrial DNA acquired from the tumour microenvironment.</p> <p>This thesis explored how cells under stress interact with a model tumour microenvironment and exploit normal cellular processes for continued survival. Using confocal microscopy, IncuCyte microscopic videography, and MinION sequencing techniques we have established that rho zero cells under metabolic stress in a co-culture environment uptake mitochondrial DNA from the surrounding microenvironment and that this cell survival is contact associated. Nars1 was identified as a potential signal from the rho zero cells to the tumour microenvironment when placed under metabolic stress. This identified Nars1 as potential part of a novel stress response mechanism for severe metabolic stress. I then investigated if these findings could be replicated in cisplatin and doxorubicin treated wild type cells. While contact dependant survival was implied in the doxorubicin treated cells no Nars1 signal was found. However, an up-regulation of Cxcl5 and Cxcl1 was identified as a commonly up-regulated transcript between the rho zero and both chemotherapy treatments in 4T1 breast cancer implicating a conserved chemokine signalling response to stress.</p> <p>Together these results illustrate the importance of chemokine signalling in complex cellular interactions as part of a cellular survival stress response.</p>

Surviving Stress with the Tumour Microenvironment: Understanding Complex Cell - Cell Interactions

<p><b>Each year more than 18 million people are diagnosed with cancer. Stress survival plays a key role in tumour resistance which is a leading cause of cancer deaths. Understanding how cells respond to stress is key to understanding and treating cancer. Within a tumour, cells interact with many other cell types in the surrounding tumour microenvironment. The tumour microenvironment acts to both assist and resist tumorigenesis and metastasis depending on these cellular interactions.</b></p> <p>Rho Zero cells are cells that contain no mitochondrial DNA and therefore lack a functional electron transport chain and rely on glycolysis for energy metabolism, these cells require supplementation to survive in cell culture. When deprived of essential supplementation rho zero cells exhibit severe metabolic stress. Yet when implanted into mice they form tumours after a 2-3 week delay. After this they contain new mitochondrial DNA acquired from the tumour microenvironment.</p> <p>This thesis explored how cells under stress interact with a model tumour microenvironment and exploit normal cellular processes for continued survival. Using confocal microscopy, IncuCyte microscopic videography, and MinION sequencing techniques we have established that rho zero cells under metabolic stress in a co-culture environment uptake mitochondrial DNA from the surrounding microenvironment and that this cell survival is contact associated. Nars1 was identified as a potential signal from the rho zero cells to the tumour microenvironment when placed under metabolic stress. This identified Nars1 as potential part of a novel stress response mechanism for severe metabolic stress. I then investigated if these findings could be replicated in cisplatin and doxorubicin treated wild type cells. While contact dependant survival was implied in the doxorubicin treated cells no Nars1 signal was found. However, an up-regulation of Cxcl5 and Cxcl1 was identified as a commonly up-regulated transcript between the rho zero and both chemotherapy treatments in 4T1 breast cancer implicating a conserved chemokine signalling response to stress.</p> <p>Together these results illustrate the importance of chemokine signalling in complex cellular interactions as part of a cellular survival stress response.</p>

Cell-Specific Transcriptome of the COPD Alveolar Niche

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of death worldwide. To identify cell-specific mechanisms underlying COPD pathobiology, we analysed single-cell RNA sequencing (scRNAseq) profiles of explanted lung tissue from subjects with advanced COPD or control lungs. Findings were validated with scRNAseq of lungs from mice exposed to 10 months of cigarette smoke (CS), isolated human alveolar epithelial cells, and immunostaining of human lung tissue samples. We identified a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance, exemplified by decreased expression of the stress-response gene NUPR1. Network analyses identified an important role for inflamed capillary endothelial cells in COPD, particularly through CXCL-motif chemokine signalling. Finally, we detected a metallothionein expressing macrophage subpopulation unique to COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

ENDOTHELIAL CELL-DERIVED EXTRACELLULAR VESICLES ELICIT NEUTROPHIL DEPLOYMENT FROM THE SPLEEN FOLLOWING ACUTE MYOCARDIAL INFARCTION

Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release of neutrophils from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 minutes) release of extracellular vesicles (EVs), which are enriched in vascular cell adhesion molecule-1 (VCAM-1) and miRNA-126, and are thus a potential mechanism for communicating with remote tissues. Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to their arrival at injured tissue. Ischemic myocardium leads to the generation and release of EC-derived-EVs bearing VCAM-1. EC-EV delivery to the spleen alters inflammatory gene and chemokine protein expression, and mobilises neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EV and showed that its deletion removed the ability of EC-EV to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Our findings show a novel mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve, independent of classical chemokine signalling, and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Cytotoxic T cells swarm by homotypic chemokine signalling

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Clinical and functional characterization of CXCR1/CXCR2 biology in the relapse and radiotherapy resistance of primary PTEN-deficient prostate carcinoma

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTENLOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.