Sadanandam, Anguraj

ABSTRACT The normal adult male breast has not been characterized at single-cell resolution, leaving the cellular basis of male breast cancer (MBC) biology undefined. Here we present an integrated single-cell RNA sequencing atlas of the adult human breast comprising 174,471 cells from 17 donors (3 male, 14 female), including 18,117 male-derived cells. This revealed that the male breast retains all three epithelial populations, basal (BC), luminal progenitor (LP), and luminal committed cells (LC), but with an increase in LC at the expense of BC and LP across all three male donors. Male LC were distinguished from female by elevated ESR1 and PGR mRNA, enrichment of RNA processing and ribosome biogenesis programs, reduced inflammatory cytokine and growth factor signaling, elevated estradiol gene set enrichment scores, and higher inferred activity of developmental patterning transcription factors. This pattern was observed across differential expression, gene ontology, ligand profiling, and regulon-based analyses, and was not restricted to sex chromosome-linked gene expression. This is consistent with the near-universal estrogen receptor (ER) positivity that characterizes MBC clinically. This atlas provides the first cellular and transcriptional reference for the normal male breast and a resource for investigating sex differences in mammary biology, germline susceptibility variant interpretation, and modeling breast malignancies.

Abstract The role of maternal microbiome transmitted at birth in cancer control is poorly understood. We have developed the first germfree B6 mouse model of breast cancer to investigate the role of the maternal microbiome in controlling oncogenic/metastatic frequencies of pro-oncogenic mammary cells. In this model, a DNA barcoded, primitive normal mouse mammary epithelial cell encoding MMTV-PyMT oncogene, was transplanted in large numbers into conventional or germfree B6 mice. Next-gen sequencing analysis of the DNA barcodes in tissues enabled us to clonally track millions of cells and measure the frequency and growth dynamics of clones at the primary site and their systemic distribution in circulation and all vital organs, generating an unprecedented high-definition map of cancer progression. Our results show that in conventional B6 mice with maternal microbial transmission at-birth, a small fraction (~0.01%) of transplanted cells transform de novo and produce slow growing, late-onset benign tumors (mouse median survival of > 1 year). In contrast, in germfree B6 mice, a >10-fold higher frequency of cells transform de novo and generate early-onset, highly aggressive metastatic clones, and are frequently associated with features leading to early euthanasia or endpoint (i.e. sudden death, intracardiac metastasis, paralysis, swollen abdomen, early multiorgan aggressive metastasis) (median survival of ~4 months; p<0.005 vs conventional mice). However, postnatal intervention with healthy fecal implants reconstituted gut microbiota in germfree B6 mice and significantly restored protective effects against cancer progression and sudden death, as well as improved quality/duration of life (median survival of >1 year; p<0.005 vs germfree mice; p=ns vs conventional mice). We found maternal microbial transmission fully protected against systemic metastasis. GC-MS analysis identified significantly distinct primary tumor metabolome associated with conventional (non-metastatic) mice vs germfree (metastatic) mice. Metagenomic analysis of fecal samples from a subset of tumor bearing conventional (non-metastatic) mice vs fecal implanted (late-onset, less aggressive metastatic) germfree mice identified significant association between metastasis and altered microbial communities. These findings using novel germfree mouse model of metastasis and cellular DNA barcoding technology provide first direct evidence that diverse microbial communities transmitted at birth play a critical role in protecting the host from clonal de novo mammary cell transformation and progression to systemic disease and thereby improving quality and duration of life. Citation Format: Nagarajan Kannan, Syed Mohammed Musheer Aalam, Xiaojia Tang, Krishna Kalari, Purna Kashyap, Jun Chen, Stephen Johnson, Anguraj Sadanandam, Mark Sherman. Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB226.

e23014 Background: The number of annual global cancer deaths is rising and the majority of this burden, for a multitude of reasons, falls in low- and middle- income countries (LMICs). With the United Nations’ 3rd and 17th Sustainable Development Goals in mind (which include by 2030 “reduce by one third premature mortality from non-communicable diseases” and “partnership for the goals”) a survey was undertaken at the UK’s two largest comprehensive cancer centres to scope individual and team endeavours to work with colleagues in less well-resourced countries. Methods: Employees at the Royal Marsden Foundation Trust (RM) and Institute of Cancer Research (ICR) in London and Surrey, UK and The Christie NHS Foundation Trust (Christie), Manchester were invited to complete a survey to capture collaborative clinical care, research, education and training. Results: Responses were received from 520 multidisciplinary individuals across the 2 centres to two similar questionnaires. A large number had experience of working in some capacity in, or in collaboration with an LMIC. At the RM 14.62% of respondees were currently working with colleagues in LMICs. At The Christie 13.22% of staff had experience of working in LMICs in a supportive capacity. Those currently collaborating with colleagues in LMICs were working in a wide range of countries across Asia, Africa and South America in a range of initiatives spanning clinical care, research, education and training. Of those who answered the survey 64% at The Christie said they’d like to hear more about opportunities to be involved in supporting global health care, and 89% at RM/ICR said they’d be interested in joining a collaborative group working on global oncology initiatives at the institutions. Conclusions: This survey highlights the body of willing, interested individuals keen to work with colleagues in LMICs to improve cancer outcomes. The launch of the UK Global Cancer Network in 2020 will build upon these two surveys with a planned national survey of global health and cancer work undertaken by individuals in 2021.

e18055 Background: Entrectinib, a TRK kinase inhibitor, has been approved for the treatment of tissue agnostic rare tumors positive for TRK fusions. A very low frequency molecular subset of TRK fusion tumors dubbed as secretory carcinoma (SC) are characterized by organ-agnostic epithelial origin, ETV6-NTRK3 (EN) fusion and distinguishable secretory-type tumor cells. These tumors have been frequently miscategorized as acinic cell carcinoma or adenocarcinoma. A previous mouse study identified EGF dependent epithelial progenitors as putative cell-of-origin for SC. Methods: To test the role of EGFR signaling in EN mediated transformation and therapy resistance, we expressed EN, kinase-dead EN-K380M, and drug-resistant EN-G623R in human epithelial MCF10A cells with EGF-dependent primitive function and investigated their ability to grow in the presence and absence of EGF and/or entrectinib. To understand the significance of findings based on our model, we analyzed a total of 22 ‘rare’ patients from Mayo Clinic Tissue Registry and analyzed TCGA PanCan datasets. Results: We report herein that EGF signaling is essential for normal growth but dispensable during EN driven transformation. Our findings suggest that levels equivalent to circulating EGF (0.5-1ng/ml) is sufficient to drive 100% resistance to entrectinib in vitro. Three different strategies to blockade EGF/EGFR axis including depletion of EGF in culture system, genetic depletion of EGFR using shRNA as well as cetuximab antibody-based EGFR neutralization potentiated oncogene-addiction and hypersensitivity to entrectinib in our models. As predicted, models with G623R mutation in EN was refractory to entrectinib under all experimental conditions. Further omics analysis of TCGA PanCan suggested that EN and EGFR mutations are mutually exclusive and entrectinib-resistant G623R mutation were uncommon. Interestingly, nearly all EN tumors from Mayo Clinic Tissue Registry immunostained weakly or strongly for EGFR and showed perfect concordance with pEGFR suggesting pathway activation. Conclusions: Together, these findings raise an important question whether blockade of ‘wildtype’ EGFR signaling could improve medical intervention in SC patients presenting with wildtype EGFR and no drug-resistant mutation in entrectinib, by improving oncogene-addiction and attendant hypersensitization of transformed cells to entrectinib.

Abstract COVID-19 patients show significant clinical heterogeneity in presentation and outcomes that makes pandemic control and strategy difficult; optimising management requires a systems biology approach of understanding the disease. Here we sought to understand and infer complex system-wide changes in patients infected with coronaviruses (SARS-CoV and SARS-CoV-2; n=38 and 57 samples) at two different disease stages compared with healthy individuals (n=16) and patients with other infections (n=144). We applied inferential statistics/machine-learning approaches (the COVID-engine platform) to RNA profiles derived from peripheral blood mononuclear cells (PBMCs). Compared to healthy individuals, an integrated blood-based gene signatures distinguished acute-like (mimicking coronavirus-infected patients with prolonged hospitalisation) from recovering-like patients. These signatures also hierarchically represented systems-level parameters associated with PBMC including dysregulated cytokines, genes, pathways, networks of pathways/concepts, immune status, and cell types. Proof-of-principle confirmatory observations included PBMC-associated increases in ACE2, cytokine storm-associated IL6, enhanced innate immunity (macrophages and neutrophils), and lower adaptive T and B cell immunity in patients with acute-like disease compared to those with recovery-like disease. Patients in the recovery-like stage had significantly enhanced TNF, IFN-g, anti-viral, HLA-DQA1, and HLA-F gene expression and cytolytic activity, and reduced pro-viral gene expression compared to those in the acute-like stage in PBMC. Besides, PBMC-derived surrogate-based approach revealed overlapping genes associated with comorbidities (associated diabetes), and disease-like symptoms (associated with thromboembolism, pneumonia, lung disease and septicaemia). Overall, our study involving PBMC-based RNA profiling may further help understand complex and variable systems-wide responses displayed by coronavirus-infected patients.