Characterising neutrophil subtypes in cancer using scRNA sequencing demonstrates the importance of IL-1β/CXCR2 axis in generation of metastasis specific neutrophils

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand–receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1β/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1β/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states

Characterising neutrophil subtypes in cancer using human and murine single-cell RNA sequencing datasets

Single cell RNA sequencing data generated by 10xGenomics for Neutrophils derived from colorectal cancer (CRC) KPN tumours (CRC_KPN_counts.csv) and normalised counts (CRC_KPN_NormalisedCounts.csv) as well as from other mouse models of CRC carrying AKPT, BPN, BP and KP mutations (CRC_other_counts.csv and CRC_other_NormalisedCounts.csv), together with the relevant metadata (CRC_KPN_metadata.csv and CRC_other_metadata.csv)

Investigating the effects of 16p11.2 Microdeletion on progenitors and interneuron development using region-specific brain organoids

Copy number variations (CNVs) of chromosomal region 16p11.2 are genetically linked to 1% of Autism Spectrum Disorder (ASD) cases. This 600kbp region contains 29 genes, but the functions of many genes in this CNV remain poorly defined. Similarly, the underlying molecular mechanisms linking the deletion to ASD pathophysiology remain largely unknown. To better understand the nature and presentation of the syndrome from birth and throughout development, we first conducted a case study, presenting three different, unrelated clinical cases of children with 16p11.2 deletion. Patients presented a wide range of clinical symptoms which generally include developmental and language delay, cognitive impairment, seizures, ASD and sometimes, macrocephaly, further supporting previous accounts on the clinical presentation of this syndrome. In parallel, we systematically reviewed the evidence from post-mortem studies of ASD, and of related disorders that present with autistic features. Sufficient replicated evidence was available to implicate GABAergic and glutamatergic dysfunction in ASD pathogenesis. The findings also propose several putative pathophysiological mechanisms that could play a role in ASD pathophysiology, such as excitatory-inhibitory imbalance and disrupted neurogenesis. Based on this, we undertook an exploratory pilot study to investigate whether aberrant progenitor proliferation and differentiation into interneurons are potential mechanisms that contribute to some of the clinical phenotypes of 16p11.2 deletion, namely: macrocephaly and ASD. We generated ventral organoids from 16p11.2deletion-CRISPR/Cas9-iPSC lines and isogenic controls to mimic early-to-mid-foetal ventral telencephalic development. Both deletion and control organoids expressed forebrain, ventral telencephalic and GABAergic markers. Deletion organoids were significantly larger in size. Preliminary cell cycle analysis revealed that a higher proportion of proliferating cells were in S-phase in deletion organoids at 35 days, while the number of Ki67+ proliferating cells declined by 65 days. Gene expression analysis showed a significant increase in SOX2 (expressed in progenitors) and NEUN (expressed in neurons) mRNA expression at earlier stages in deletion organoids, around day 46. Moreover, altered expression levels of genes encoding transcription factors essential for interneuron development together with perturbations in the mRNA expression of several interneuron subtype markers were also found. Our preliminary results suggested that larger organoid size, which mimicked the macrocephalic phenotype of patients, could initially be associated with increased numbers of proliferating cells. The subsequent decline in numbers of proliferating cells could imply a premature depletion of the progenitor pool and a shift towards asymmetric neurogenic divisions. From the results of our pilot study and power calculations, we then performed a main experiment using different deletion lines and control lines. Organoids were analysed by flow cytometry and immunohistochemistry. Expression of forebrain, ventral and GABAergic markers was still evident in both deletion and controls. Larger organoid size in the deletion was dependent on the iPSC-line used, where one line was remarkably larger than all controls and the other was smaller. Significantly larger neural rosettes (circular, radial arrangements of neural progenitor cells) were found in deletion organoids. In addition, deletion organoids revealed a significant increase in COUPTFII expression, a marker of the caudal ganglionic eminences and a transcription factor that is essential for regulating embryonic stem cell differentiation and neural induction. Moreover, flow cytometric analysis revealed no differences in progenitor and immature neuronal populations at 35 days. Using flowcytometry and double IdU/BrdU labelling, we performed a detailed cell cycle analysis to investigate the proportion of cells in the different cell cycle phases, the duration of the phases and total cell cycle length. Unlike the findings in our pilot study, no significant difference in the proportion of cells in G1, S and G2M phases was found, although a trend towards reduced S-phase fractions was noticed in the deletion organoids. Finally, deletion organoids demonstrated significant aberrations in their cell cycle kinetics, revealing a significant increase in cell cycle duration, as well as increased durations of G1 and G2M phases. In line with our findings, analysis of published RNA-seq data in a 16p11.2 mouse model revealed the differential expression of a number of genes associated with S phase and G2M phases. Collectively, our findings suggest that 16p11.2 deletion impairs the proliferation of ventral progenitors. Prolonged cell cycle and G1 lengths suggest that a shift towards asymmetric neurogenic divisions in the progenitor populations is more evident. Whether the interneuron progeny is immature, dysfunctional, or favours a certain interneuron subtype over another is yet to be investigated in detail

Supplementary Figure S7 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Figure S7. Exogenous application of IL-1β and GM-CSF alter transcription of Cxcr2 and Txnip in wild type neutrophils ex vivo.</p