Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1ß/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 IL1ß/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 IL1ß/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. SIGNIFICANCE: We identify two recurring neutrophil populations and demonstrate their staged evolution from health to malignancy through the IL1ß/CXCL8/CXCR2 axis, allowing for immunotherapeutic neutrophil-targeting approaches to counteract immunosuppressive subtypes that emerge in metastasis.

Pathway level subtyping identifies a slow-cycling biological phenotype associated with poor clinical outcomes in colorectal cancer.

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5+ stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1+ stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers.

KRAS allelic imbalance drives tumour initiation yet suppresses metastasis in colorectal cancer in vivo.

Oncogenic KRAS mutations are well-described functionally and are known to drive tumorigenesis. Recent reports describe a significant prevalence of KRAS allelic imbalances or gene dosage changes in human cancers, including loss of the wild-type allele in KRAS mutant cancers. However, the role of wild-type KRAS in tumorigenesis and therapeutic response remains elusive. We report an in vivo murine model of colorectal cancer featuring deletion of wild-type Kras in the context of oncogenic Kras. Deletion of wild-type Kras exacerbates oncogenic KRAS signalling through MAPK and thus drives tumour initiation. Absence of wild-type Kras potentiates the oncogenic effect of KRASG12D, while incidentally inducing sensitivity to inhibition of MEK1/2. Importantly, loss of the wild-type allele in aggressive models of KRASG12D-driven CRC significantly alters tumour progression, and suppresses metastasis through modulation of the immune microenvironment. This study highlights the critical role for wild-type Kras upon tumour initiation, progression and therapeutic response in Kras mutant CRC.

Driver gene combinations dictate cutaneous squamous cell carcinoma disease continuum progression.

The molecular basis of disease progression from UV-induced precancerous actinic keratosis (AK) to malignant invasive cutaneous squamous cell carcinoma (cSCC) and potentially lethal metastatic disease remains unclear. DNA sequencing studies have revealed a massive mutational burden but have yet to illuminate mechanisms of disease progression. Here we perform RNAseq transcriptomic profiling of 110 patient samples representing normal sun-exposed skin, AK, primary and metastatic cSCC and reveal a disease continuum from a differentiated to a progenitor-like state. This is accompanied by the orchestrated suppression of master regulators of epidermal differentiation, dynamic modulation of the epidermal differentiation complex, remodelling of the immune landscape and an increase in the preponderance of tumour specific keratinocytes. Comparative systems analysis of human cSCC coupled with the generation of genetically engineered murine models reveal that combinatorial sequential inactivation of the tumour suppressor genes Tgfbr2, Trp53, and Notch1 coupled with activation of Ras signalling progressively drives cSCC progression along a differentiated to progenitor axis. Taken together we provide a comprehensive map of the cSCC disease continuum and reveal potentially actionable events that promote and accompany disease progression.

Return visit rates after an emergency department discharge for children with sickle cell pain episodes.

BACKGROUND: High return visit rates after hospitalization for people with sickle cell disease (SCD) have been previously established. Due to a lack of multicenter emergency department (ED) return visit rate data, the return visit rate following ED discharge for pediatric SCD pain treatment is currently unknown. PROCEDURE: A seven-site retrospective cohort study of discharged ED visits for pain by children with SCD was conducted using the Pediatric Emergency Care Applied Research Network Registry. Visits between January 2017 and November 2021 were identified using previously validated criteria. The primary outcome was the 14-day return visit rate, with 3- and 7-day rates also calculated. Modified Poisson regression was used to analyze associations for age, sex, initial hospitalization rate, and a visit during the COVID-19 pandemic with return visit rates. RESULTS: Of 2548 eligible ED visits, approximately 52% were patients less than 12 years old, 50% were female, and over 95% were non-Hispanic Black. The overall 14-day return visit rate was 29.1% (95% confidence interval [CI]: 27.4%-30.9%; site range 22.7%-31.7%); the 7- and 3-day return visit rates were 23.0% (95% CI: 21.3%-24.6%) and 16.7% (95% CI: 15.3%-18.2%), respectively. Younger children had slightly lower 14-day return visit rates (27.3% vs. 31.1%); there were no associations for site hospitalization rate, sex, and a visit occurring during the pandemic with 14-day returns. CONCLUSION: Nearly 30% of ED discharged visits after SCD pain treatment had a return visit within 14 days. Increased efforts are needed to identify causes for high ED return visit rates and ensure optimal ED and post-ED care.

Therapeutic targeting of tumour myeloid cells.

Myeloid cells are pivotal within the immunosuppressive tumour microenvironment. The accumulation of tumour-modified myeloid cells derived from monocytes or neutrophils - termed 'myeloid-derived suppressor cells' - and tumour-associated macrophages is associated with poor outcome and resistance to treatments such as chemotherapy and immune checkpoint inhibitors. Unfortunately, there has been little success in large-scale clinical trials of myeloid cell modulators, and only a few distinct strategies have been used to target suppressive myeloid cells clinically so far. Preclinical and translational studies have now elucidated specific functions for different myeloid cell subpopulations within the tumour microenvironment, revealing context-specific roles of different myeloid cell populations in disease progression and influencing response to therapy. To improve the success of myeloid cell-targeted therapies, it will be important to target tumour types and patient subsets in which myeloid cells represent the dominant driver of therapy resistance, as well as to determine the most efficacious treatment regimens and combination partners. This Review discusses what we can learn from work with the first generation of myeloid modulators and highlights recent developments in modelling context-specific roles for different myeloid cell subtypes, which can ultimately inform how to drive more successful clinical trials.

MmCMS: mouse models' consensus molecular subtypes of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

Consensus definition of essential, optimal, and suggested components of a pediatric sickle cell disease center.

Sickle cell disease (SCD) requires coordinated, specialized medical care for optimal outcomes. There are no United States (US) guidelines that define a pediatric comprehensive SCD program. We report a modified Delphi consensus-seeking process to determine essential, optimal, and suggested elements of a comprehensive pediatric SCD center. Nineteen pediatric SCD specialists participated from the US. Consensus was predefined as 2/3 agreement on each element's categorization. Twenty-six elements were considered essential (required for guideline-based SCD care), 10 were optimal (recommended but not required), and five were suggested. This work lays the foundation for a formal recognition process of pediatric comprehensive SCD centers.

Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.

Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia.

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer.

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

Oncogenic BRAF, unrestrained by TGFß-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis.

RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma Determine Response to SLC7A11 Inhibition.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.

The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

Implications of Peak Selection in the Interpretation of Unsupervised Mass Spectrometry Imaging Data Analyses.

Mass spectrometry imaging can produce large amounts of complex spectral and spatial data. Such data sets are often analyzed with unsupervised machine learning approaches, which aim at reducing their complexity and facilitating their interpretation. However, choices made during data processing can impact the overall interpretation of these analyses. This work investigates the impact of the choices made at the peak selection step, which often occurs early in the data processing pipeline. The discussion is done in terms of visualization and interpretation of the results of two commonly used unsupervised approaches: t-distributed stochastic neighbor embedding and k-means clustering, which differ in nature and complexity. Criteria considered for peak selection include those based on hypotheses (exemplified herein in the analysis of metabolic alterations in genetically engineered mouse models of human colorectal cancer), particular molecular classes, and ion intensity. The results suggest that the choices made at the peak selection step have a significant impact in the visual interpretation of the results of either dimensionality reduction or clustering techniques and consequently in any downstream analysis that relies on these. Of particular significance, the results of this work show that while using the most abundant ions can result in interesting structure-related segmentation patterns that correlate well with histological features, using a smaller number of ions specifically selected based on prior knowledge about the biochemistry of the tissues under investigation can result in an easier-to-interpret, potentially more valuable, hypothesis-confirming result. Findings presented will help researchers understand and better utilize unsupervised machine learning approaches to mine high-dimensionality data.