Spatial insights into immunotherapy response in non-small cell lung cancer (NSCLC) by multiplexed tissue imaging.

The spatial localisation of immune cells within tumours are key to understand the intercellular communications that can dictate clinical outcomes. Here, we demonstrate an analysis pipeline for highly multiplexed CODEX data to phenotype and profile spatial features and interactions in NSCLC patients that subsequently received PD1 axis immunotherapy. We found that regulatory T cells (Tregs) are enriched in non-responding patients and this was consistent with their localization within stromal and peripheral tumour-margins. Proximity-based interactions between Tregs and both monocytes (p = 0.009) and CD8+ T cells (p = 0.009) were more frequently found in non-responding patients, while macrophages were more frequently located in proximity to HLADR+ tumour cells (p = 0.01) within responding patients. Cellular neighbourhoods analysis indicated that both macrophages (p = 0.003) and effector CD4+ T cells (p = 0.01) in mixed tumour neighbourhoods, as well as CD8+ T cells (p = 0.03) in HLADR+ tumour neighbourhoods were associated with favorable clinical response. Evaluation of the inferred regulatory functions between immune cells relative to the tumour suggested that macrophages exhibit an immunosuppressive phenotype against both CD4+ and CD8+ T cells, and that this association scores more highly in ICI refractory patients. These spatial patterns are associated with overall survival in addition to ICI response and may thus indicate features for the functional understanding of the tumour microenvironment.

In situ single-cell profiling sheds light on IFI27 localisation during SARS-CoV-2 infection.

The utilization of single-cell resolved spatial transcriptomics to delineate immune responses during SARS-CoV-2 infection was able to identify M1 macrophages to have elevated expression of IFI27 in areas of infection.

Whole transcriptome profiling of placental pathobiology in SARS-CoV-2 pregnancies identifies placental dysfunction signatures.

Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a 'preeclampsia-like syndrome'. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9). Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls. Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection.

Inhibition of Aurora B kinase (AURKB) enhances the effectiveness of 5-fluorouracil chemotherapy against colorectal cancer cells.

BACKGROUND: 5-Fluorouracil (5-FU) remains a core component of systemic therapy for colorectal cancer (CRC). However, response rates remain low, and development of therapy resistance is a primary issue. Combinatorial strategies employing a second agent to augment the therapeutic effect of chemotherapy is predicted to reduce the incidence of treatment resistance and increase the durability of response to therapy. METHODS: Here, we employed quantitative proteomics approaches to identify novel druggable proteins and molecular pathways that are deregulated in response to 5-FU, which might serve as targets to improve sensitivity to chemotherapy. Drug combinations were evaluated using 2D and 3D CRC cell line models and an ex vivo culture model of a patient-derived tumour. RESULTS: Quantitative proteomics identified upregulation of the mitosis-associated protein Aurora B (AURKB), within a network of upregulated proteins, in response to a 24 h 5-FU treatment. In CRC cell lines, AURKB inhibition with the dihydrogen phosphate prodrug AZD1152, markedly improved the potency of 5-FU in 2D and 3D in vitro CRC models. Sequential treatment with 5-FU then AZD1152 also enhanced the response of a patient-derived CRC cells to 5-FU in ex vivo cultures. CONCLUSIONS: AURKB inhibition may be a rational approach to augment the effectiveness of 5-FU chemotherapy in CRC.

The in situ transcriptomic landscape of breast tumour-associated and normal adjacent endothelial cells.

BACKGROUND AND AIMS: Triple Negative Breast Cancer (TNBC) is associated with increased angiogenesis, which is known to aid tumour growth and metastasis. Anti-angiogenic therapies that have been developed to target this feature have mostly generated disappointing clinical results. Further research into targeted approaches is limited by a lack of understanding of the in situ molecular profile of tumour-associated vasculature. In this study, we aimed to understand the differences in the molecular profiles of tumour endothelial cells vs normal-adjacent endothelial cells in TNBC tissues. METHOD: We have applied unbiased whole transcriptome spatial profiling of in situ gene expressions of endothelial cells localized in full-face patient TNBC tissues (n = 4) and normal-adjacent regions of the same patient breast tissues. RESULTS: Our comparative analysis revealed that 2412 genes were differentially expressed (padj < 0.05) between the tumour endothelial cells and normal-adjacent endothelial cells. Pathway enrichment showed the enrichment of gene sets related to cell-cell, cell-ECM adhesion, chromatin organization and remodeling, and protein-DNA complex subunit organization. CONCLUSION: Overall, the results revealed unique molecular profiles and signalling pathways of tumour-associated vasculature, which is a critical step towards larger cohort studies investigating potential targets for TNBC prognosis and anti-angiogenic treatments.

A robust platform for integrative spatial multi-omics analysis to map immune responses to SARS-CoV-2 infection in lung tissues.

The SARS-CoV-2 (COVID-19) virus has caused a devastating global pandemic of respiratory illness. To understand viral pathogenesis, methods are available for studying dissociated cells in blood, nasal samples, bronchoalveolar lavage fluid and similar, but a robust platform for deep tissue characterization of molecular and cellular responses to virus infection in the lungs is still lacking. We developed an innovative spatial multi-omics platform to investigate COVID-19-infected lung tissues. Five tissue-profiling technologies were combined by a novel computational mapping methodology to comprehensively characterize and compare the transcriptome and targeted proteome of virus infected and uninfected tissues. By integrating spatial transcriptomics data (Visium, GeoMx and RNAScope) and proteomics data (CODEX and PhenoImager HT) at different cellular resolutions across lung tissues, we found strong evidence for macrophage infiltration and defined the broader microenvironment surrounding these cells. By comparing infected and uninfected samples, we found an increase in cytokine signalling and interferon responses at different sites in the lung and showed spatial heterogeneity in the expression level of these pathways. These data demonstrate that integrative spatial multi-omics platforms can be broadly applied to gain a deeper understanding of viral effects on cellular environments at the site of infection and to increase our understanding of the impact of SARS-CoV-2 on the lungs.

Compartmentalized spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma identifies immune checkpoint molecules and tumor necrosis factor receptor superfamily members as biomarkers of response to immunotherapy.

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures.

Multi-omic and spatial dissection of immunotherapy response groups in non-small cell lung cancer.

The composition and activation status of the cellular milieu contained within the tumour microenvironment (TME) is becoming increasingly recognized as a driving factor for immunotherapy response. Here, we employed multiplex immunohistochemistry (mIHC), and digital spatial profiling (DSP) to capture the targeted immune proteome and transcriptome of tumour and TME compartments from an immune checkpoint inhibitor (ICI)-treated (n = 41) non-small cell lung cancer (NSCLC) patient cohort. We demonstrate by mIHC that the interaction of CD68+ macrophages with PD1+ , FoxP3+ cells is enriched in ICI refractory tumours (p = 0.012). Patients responsive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within their tumour compartments, which corresponded with increased IL2 mRNA (p = 0.001) within their stroma. In addition, stromal IL2 mRNA levels positively correlated with the expression of pro-apoptotic markers cleaved caspase 9 (p = 2e-5 ) and BAD (p = 5.5e-4 ) and negatively with levels of memory marker, CD45RO (p = 7e-4 ). Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were suppressed in ICI-responsive patients. Tumour expression of CD44 was depleted in the responsive patients (p = 0.02), while higher stromal expression of one of its ligands, SPP1 (p = 0.008), was observed. Cox survival analysis also indicated tumour CD44 expression was associated with poorer prognosis (hazard ratio [HR] = 1.61, p = 0.01), consistent with its depletion in ICI-responsive patients. Through multi-modal approaches, we have dissected the characteristics of NSCLC immunotherapy treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.

Transcriptomic profiling of cardiac tissues from SARS-CoV-2 patients identifies DNA damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to present with pulmonary and extra-pulmonary organ complications. In comparison with the 2009 pandemic (pH1N1), SARS-CoV-2 infection is likely to lead to more severe disease, with multi-organ effects, including cardiovascular disease. SARS-CoV-2 has been associated with acute and long-term cardiovascular disease, but the molecular changes that govern this remain unknown. In this study, we investigated the host transcriptome landscape of cardiac tissues collected at rapid autopsy from seven SARS-CoV-2, two pH1N1, and six control patients using targeted spatial transcriptomics approaches. Although SARS-CoV-2 was not detected in cardiac tissue, host transcriptomics showed upregulation of genes associated with DNA damage and repair, heat shock, and M1-like macrophage infiltration in the cardiac tissues of COVID-19 patients. The DNA damage present in the SARS-CoV-2 patient samples, were further confirmed by γ-H2Ax immunohistochemistry. In comparison, pH1N1 showed upregulation of interferon-stimulated genes, in particular interferon and complement pathways, when compared with COVID-19 patients. These data demonstrate the emergence of distinct transcriptomic profiles in cardiac tissues of SARS-CoV-2 and pH1N1 influenza infection supporting the need for a greater understanding of the effects on extra-pulmonary organs, including the cardiovascular system of COVID-19 patients, to delineate the immunopathobiology of SARS-CoV-2 infection, and long term impact on health.

Dissecting Tissue Compartment-Specific Protein Signatures in Primary and Metastatic Oropharyngeal Squamous Cell Carcinomas.

Head and neck squamous cell carcinoma (HNSCC) often presents with locoregional or distant disease, despite multimodal therapeutic approaches, which include surgical resection, chemoradiotherapy, and more recently, immunotherapy for metastatic or recurrent HNSCC. Therapies often target the primary and nodal regional HNSCC sites, and their efficacy at controlling occult distant sites remains poor. While our understanding of the tumor microenvironment conducive to effective therapies is increasing, the biology underpinning locoregional sites remains unclear. Here, we applied targeted spatial proteomic approaches to primary and lymph node metastasis from an oropharyngeal SCC (OPSCC) cohort to understand the expression of proteins within tumors, and stromal compartments of the respective sites in samples of both matched and unmatched patients. In unmatched analyses of n = 43 primary and 11 nodal metastases, our data indicated that tumor cells in nodal metastases had higher levels of Ki-67, PARP, BAD, and cleaved caspase 9, suggesting a role for increased proliferation, DNA repair, and apoptosis within these metastatic cells. Conversely, in matched analyses (n = 7), pro-apoptotic markers BIM and BAD were enriched in the stroma of primary tumors. Univariate, overall survival (OS) analysis indicated CD25 in tumor regions of primary tumors to be associated with reduced survival (HR = 3.3, p = 0.003), while progesterone receptor (PR) was associated with an improved OS (HR = 0.33, p = 0.015). This study highlights the utility of spatial proteomics for delineating the tumor and stromal compartment composition, and utility toward understanding these properties in locoregional metastasis. These findings indicate unique biological properties of lymph node metastases that may elucidate further understanding of distant metastatic in OPSCC.

Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in late 2019 has spread globally, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). A better definition of the pulmonary host response to SARS-CoV-2 infection is required to understand viral pathogenesis and to validate putative COVID-19 biomarkers that have been proposed in clinical studies. METHODS: Here, we use targeted transcriptomics of formalin-fixed paraffin-embedded tissue using the NanoString GeoMX platform to generate an in-depth picture of the pulmonary transcriptional landscape of COVID-19, pandemic H1N1 influenza and uninfected control patients. RESULTS: Host transcriptomics showed a significant upregulation of genes associated with inflammation, type I interferon production, coagulation and angiogenesis in the lungs of COVID-19 patients compared to non-infected controls. SARS-CoV-2 was non-uniformly distributed in lungs (emphasising the advantages of spatial transcriptomics) with the areas of high viral load associated with an increased type I interferon response. Once the dominant cell type present in the sample, within patient correlations and patient-patient variation, had been controlled for, only a very limited number of genes were differentially expressed between the lungs of fatal influenza and COVID-19 patients. Strikingly, the interferon-associated gene IFI27, previously identified as a useful blood biomarker to differentiate bacterial and viral lung infections, was significantly upregulated in the lungs of COVID-19 patients compared to patients with influenza. CONCLUSION: Collectively, these data demonstrate that spatial transcriptomics is a powerful tool to identify novel gene signatures within tissues, offering new insights into the pathogenesis of SARS-COV-2 to aid in patient triage and treatment.

IFI27 transcription is an early predictor for COVID-19 outcomes, a multi-cohort observational study.

Purpose: Robust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness. Methods: We conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients. Results: We show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients. Conclusion: These data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.

Endothelial cells are not productively infected by SARS-CoV-2.

OBJECTIVES: Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious. METHODS: Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier. RESULTS: We show that primary human endothelial cells express very low levels of the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS-CoV-2. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. CONCLUSIONS: Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

The identification of circulating tumour DNA using MassARRAY technology in non-small-cell lung cancer (NSCLC).

OBJECTIVES: Attaining tumour material from lung cancer patients can be challenging with limited sample availability. Therefore, non-invasive means of assessing tumour material is becoming increasingly more important. Circulating tumour DNA (ctDNA), extracted from a blood sample is appealing for the patient, and can be performed serially over the course of treatment. MATERIALS AND METHODS: Here, we describe an approach for profiling the blood samples of 103 NSCLC patients for 73 variants in ctDNA across a panel of actionable lung cancer mutations using the UltraSEEK lung Panel (Agena Biosciences). RESULTS: Our cross-sectional study showed tumour and blood concordance in the detection of KRAS mutations (G12C, G12D, G12A/V, G12R, G12RC, Q61H) in 17/27 (63%), EGFR mutations (e746_a750del, e747_A750, T790M, L861Q) in 16/20 (80%) with additional PIK3CA_p545K mutations across both cohorts. In patients without reported tumour mutations, 11/56 (19.6%) presented with plasma mutations across EGFR, KRAS and PIK3CA. Where ctDNA mutations were measured longitudinally (n = 4 patients), the individual mutations mirrored the response to therapy/progression of disease. CONCLUSION: Whilst preliminary, this study demonstrates the utility of detecting clinically actionable mutations in the blood samples of NSCLC patients at the time of presentation, and over the course of therapy.

Spatial Profiling Identifies Prognostic Features of Response to Adjuvant Therapy in Triple Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has few effective treatment options due to its lack of targetable hormone receptors. Whilst the degree of tumour infiltrating lymphocytes (TILs) has been shown to associate with therapy response and prognosis, deeper characterization of the molecular diversity that may mediate chemotherapeutic response is lacking. Here we applied targeted proteomic analysis of both chemotherapy sensitive and resistant TNBC tissue samples by the Nanostring GeoMx Digital Spatial Platform (DSP). By quantifying 68 targets in the tumour and tumour microenvironment (TME) compartments and performing differential expression analysis between responsive and non-responsive tumours, we show that increased ER-alpha expression and decreased 4-1BB and MART1 within the stromal compartments is associated with adjuvant chemotherapy response. Similarly, higher expression of GZMA, STING and fibronectin and lower levels of CD80 were associated with response within tumour compartments. Univariate overall-survival (OS) analysis of stromal proteins supported these findings, with ER-alpha expression (HR=0.19, p=0.0012) associated with better OS while MART1 expression (HR=2.3, p=0.035) was indicative of poorer OS. Proteins within tumour compartments consistent with longer OS included PD-L1 (HR=0.53, p=0.023), FOXP3 (HR=0.5, p=0.026), GITR (HR=0.51, p=0.036), SMA (HR=0.59, p=0.043), while EPCAM (HR=1.7, p=0.045), and CD95 (HR=4.9, p=0.046) expression were associated with shorter OS. Our data provides early insights into the levels of these markers in the TNBC tumour microenvironment, and their association with chemotherapeutic response and patient survival.

Understanding the tumor microenvironment for effective immunotherapy.

Advances in immunotherapy have led to durable and long-term benefits in a subset of patients across a number of solid tumor types. Understanding of the subsets of patients that respond to immune checkpoint inhibitors at the cellular level, and in the context of their tumor microenvironment (TME) is becoming increasingly important. The TME is composed of a heterogeneous milieu of tumor and immune cells. The immune landscape of the TME can inhibit or promote tumor initiation and progression; thus, a deeper understanding of tumor immunity is necessary to develop immunotherapeutic strategies. Recent developments have focused on characterizing the TME immune contexture (type, density, and function) to discover mechanisms and biomarkers that may predict treatment outcomes. This has, in part, been powered by advancements in spatial characterization technologies. In this review article, we address the role of specific immune cells within the TME at various stages of tumor progression and how the immune contexture determinants affecting tumor growth are used therapeutically.

High-Plex and High-Throughput Digital Spatial Profiling of Non-Small-Cell Lung Cancer (NSCLC).

Profiling the tumour microenvironment (TME) has been informative in understanding the underlying tumour-immune interactions. Multiplex immunohistochemistry (mIHC) coupled with molecular barcoding technologies have revealed greater insights into the TME. In this study, we utilised the Nanostring GeoMX Digital Spatial Profiler (DSP) platform to profile a non-small-cell lung cancer (NSCLC) tissue microarray for protein markers across immune cell profiling, immuno-oncology (IO) drug targets, immune activation status, immune cell typing, and pan-tumour protein modules. Regions of interest (ROIs) were selected that described tumour, TME, and normal adjacent tissue (NAT) compartments. Our data revealed that paired analysis (n = 18) of matched patient compartments indicate that the TME was significantly enriched in CD27, CD3, CD4, CD44, CD45, CD45RO, CD68, CD163, and VISTA relative to the tumour. Unmatched analysis indicated that the NAT (n = 19) was significantly enriched in CD34, fibronectin, IDO1, LAG3, ARG1, and PTEN when compared to the TME (n = 32). Univariate Cox proportional hazards indicated that the presence of cells expressing CD3 (hazard ratio (HR): 0.5, p = 0.018), CD34 (HR: 0.53, p = 0.004), and ICOS (HR: 0.6, p = 0.047) in tumour compartments were significantly associated with improved overall survival (OS). We implemented both high-plex and high-throughput methodologies to the discovery of protein biomarkers and molecular phenotypes within biopsy samples, and demonstrate the power of such tools for a new generation of pathology research.

The evolving landscape of predictive biomarkers in immuno-oncology with a focus on spatial technologies.

Immunotherapies have shown long-lasting and unparalleled responses for cancer patients compared to conventional therapy. However, they seem to only be effective in a subset of patients. Therefore, it has become evident that a greater understanding of the tumor microenvironment (TME) is required to understand the nuances which may be at play for a favorable outcome to therapy. The immune contexture of the TME is an important factor in dictating how well a tumor may respond to immune checkpoint inhibitors. While traditional immunohistochemistry techniques allow for the profiling of cells in the tumor, this is often lost when tumors are analysed using bulk tissue genomic approaches. Moreover, the actual cellular proportions, cellular heterogeneity and deeper spatial distribution are lacking in characterisation. Advances in tissue interrogation technologies have given rise to spatially resolved characterisation of the TME. This review aims to provide an overview of the current methodologies that are used to profile the TME, which may provide insights into the immunopathology associated with a favorable outcome to immunotherapy.

Circulating tumor cell clusters: Insights into tumour dissemination and metastasis.

Introduction: Metastasis results in more than 90% of cancer-related deaths globally. The process is thought to be facilitated by metastatic precursor cells, commonly termed circulating tumor cells (CTCs). CTCs can exist as single cells or cell clusters and travel through the lymphovasculature to distant organs where they can form overt metastasis. Areas covered: Studies have highlighted that CTC clusters, which may be homotypic or heterotypic in composition, have a higher metastatic potential compared to single CTCs. The characterization of CTC clusters is becoming important as heterotypic clusters can provide a mechanism for immune evasion. This review summarizes the latest advances in CTC cluster-mediated metastasis and clinical significance. Expert opinion: Comprehensive characterization of CTC clusters is needed to understand the cell types and interactions within clusters, in order to identify ways in which to reduce CTC cluster-mediated metastasis. The role of CTC clusters in prognosticating disease progression needs to be determined by documenting CTC clusters from the time of diagnosis over the course of therapy.

Identifying Therapies to Combat Epithelial Mesenchymal Plasticity-Associated Chemoresistance to Conventional Breast Cancer Therapies Using An shRNA Library Screen.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. METHODS: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute's Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. RESULTS: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. CONCLUSIONS: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.