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Abstract Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3’ and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

Abstract Tumour-Infiltrating Lymphocytes (TILs) are pivotal in the immune response against cancer cells. Existing deep learning methods for TIL analysis in whole-slide images (WSIs) demand extensive patch-level annotations, often requiring labour-intensive specialist input. To address this, we propose a framework named annotation-efficient segmentation and attention-based classifier (ANSAC). ANSAC requires only slide-level labels to classify WSIs as having high vs. low TIL scores, with the binary classes divided by an expert-defined threshold. ANSAC automatically segments tumour and stroma regions relevant to TIL assessment, eliminating extensive manual annotations. Furthermore, it uses an attention model to generate a map that highlights the most pertinent regions for classification. Evaluating ANSAC on four breast cancer datasets, we demonstrate substantial improvements over three baseline methods in identifying TIL-relevant regions, with up to 8% classification improvement on a held-out test dataset. Additionally, we propose a pre-processing modification to a well-known method, enhancing its performance up to 6%.

Abstract Background Distant relapse of breast cancer complicates management of the disease and accounts for 90% of breast cancer‐related deaths. Monocyte chemoattractant protein‐1 (MCP‐1) has critical roles in breast cancer progression and is widely accepted as a pro‐metastatic chemokine. Methods This study explored MCP‐1 expression in the primary tumour of 251 breast cancer patients. A simplified ‘histoscore’ was used to determine if each tumour had high or low expression of MCP‐1. Patient breast cancers were retrospectively staged based on available patient data. p

The distribution and organisation of matrix molecules in the tumour stroma help shape solid tumour progression. Here they perform temporal proteomic profiling of the matrisome during breast cancer progression and show that collagen XII secreted from CAFs provides a pro-invasive microenvironment.

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

Abstract Background Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.

Background: Despite advances in immunotherapy and targeted therapy, platinum-based chemotherapy remains crucial for many patients with advanced non-small cell lung cancer (NSCLC). Resistance to platinum chemotherapy is common, and predictive biomarkers are needed to tailor treatment to patients likely to respond. In vitro evidence implicates the transforming growth factor-β (TGF-β) superfamily ligands activin-A and growth differentiation factor 11 (GDF-11) in innate platinum resistance. We performed a validation study to assess their utility as predictive biomarkers of platinum chemotherapy response in advanced NSCLC. Patients and Methods: Our study included 123 adult patients with advanced NSCLC without a driver mutation treated with platinum chemotherapy. 98 patients were from a retrospective cohort and 25 from a prospective cohort. We performed immunohistochemistry staining for Activin-A, GDF-11 and TGF-β on tumour samples for each patient and analysed IHC expression with objective radiological response and overall survival. Results: The overall median survival was 14.8 months. We performed statistical analysis around a cytoplasmic score of 8/18 for Activin-A and GDF-11 based on previously published work, and 110/30 for TGF-β based on a calculated cutpoint for significance. No survival difference was detected between these groups for Activin-A (p=0.35), GDF-11 (p=0.57) or TGF-β (p=0.34). There was no association between rates of progressive disease and high Activin-A expression (p=0.43), high GDF-11 expression (p=1.0) or high TGF-β expression p=0.89). Conclusion: Within the confines of our study, Activin-A, GDF-11 and TGF-β expression was not a predictor of objective radiological response to chemotherapy or overall survival.

Stromal cell recruitment, activation and crosstalk with cancer cells is poorly understood. Here, the authors demonstrate that cancer cell-derived Hedgehog ligand triggers stromal remodeling that in turn induces a cancer-stem-cell like, drug-resistant phenotype of nearby cancer cells while treatment with smoothened inhibitors reverses these phenotypes.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation.

Purpose Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA) mutations are frequently observed in primary breast cancer. We evaluated their prognostic relevance by performing a pooled analysis of individual patient data. Patients and Methods Associations between PIK3CA status and clinicopathologic characteristics were tested by applying Cox regression models adjusted for age, tumor size, nodes, grade, estrogen receptor (ER) status, human epidermal growth factor receptor 2 (HER2) status, treatment, and study. Invasive disease-free survival (IDFS) was the primary end point; distant disease-free survival (DDFS) and overall survival (OS) were also assessed, overall and by breast cancer subtypes. Results Data from 10,319 patients from 19 studies were included (median OS follow-up, 6.9 years); 1,787 patients (17%) received chemotherapy, 4,036 (39%) received endocrine monotherapy, 3,583 (35%) received both, and 913 (9%) received none or their treatment was unknown. PIK3CA mutations occurred in 32% of patients, with significant associations with ER positivity, increasing age, lower grade, and smaller size (all P < .001). Prevalence of PIK3CA mutations was 18%, 22%, and 37% in the ER-negative/HER2-negative, HER2-positive, and ER-positive/HER2-negative subtypes, respectively. In univariable analysis, PIK3CA mutations were associated with better IDFS (HR, 0.77; 95% CI, 0.71 to 0.84; P < .001), with evidence for a stronger effect in the first years of follow-up (0 to 5 years: HR, 0.73; 95% CI, 0.66 to 0.81; P < .001; 5 to 10 years: HR, 0.82; 95% CI, 0.68 to 0.99; P = .037); > 10 years: (HR, 1.15; 95% CI, 0.84 to 1.58; P = .38; P heterogeneity = .02). In multivariable analysis, PIK3CA genotype remained significant for improved IDFS ( P = .043), but not for the DDFS and OS end points. Conclusion In this large pooled analysis, PIK3CA mutations were significantly associated with a better IDFS, DDFS, and OS, but had a lesser prognostic effect after adjustment for other prognostic factors.