Gray, G.K., et al. A human breast atlas integrating single-cell proteomics and transcriptomics. Developmental Cell 57, 1-21 (2022). Publisher's VersionAbstract
The breast is a dynamic organ whose response to physiological and pathophysiological conditions alters its disease susceptibility, yet the specific effects of these clinical variables on cell state remain poorly annotated. We present a unified, high-resolution breast atlas by integrating single-cell RNA-seq, mass cytometry, and cyclic immunofluorescence, encompassing a myriad of states. We define cell subtypes within the alveolar, hormone-sensing, and basal epithelial lineages, delineating associations of several subtypes with cancer risk factors, including age, parity, and BRCA2 germline mutation. Of particular interest is a subset of alveolar cells termed basal-luminal (BL) cells, which exhibit poor transcriptional lineage fidelity, accumulate with age, and carry a gene signature associated with basal-like breast cancer. We further utilize a medium-depletion approach to identify molecular factors regulating cell-subtype proportion in organoids. Together, these data are a rich resource to elucidate diverse mammary cell states.
Kuiken, H.J., et al. Clonal populations of a human TNBC model display significant functional heterogeneity and divergent growth dynamics in distinct contexts. Oncogene 41, 1, 112-124 (2022).Abstract
Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.
Badrinath, S., et al. A vaccine targeting resistant tumours by dual T cell plus NK cell attack. Nature (2022).Abstract
Most cancer vaccines target peptide antigens, necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore, tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells, but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding, enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably, this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary, highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations.
Plana, D., Fell, G., Alexander, B.M., Palmer, A.C. & Sorger, P.K. Cancer patient survival can be parametrized to improve trial precision and reveal time-dependent therapeutic effects. Nat Commun 13, 1, 873 (2022).Abstract
Individual participant data (IPD) from oncology clinical trials is invaluable for identifying factors that influence trial success and failure, improving trial design and interpretation, and comparing pre-clinical studies to clinical outcomes. However, the IPD used to generate published survival curves are not generally publicly available. We impute survival IPD from ~500 arms of Phase 3 oncology trials (representing ~220,000 events) and find that they are well fit by a two-parameter Weibull distribution. Use of Weibull functions with overall survival significantly increases the precision of small arms typical of early phase trials: analysis of a 50-patient trial arm using parametric forms is as precise as traditional, non-parametric analysis of a 90-patient arm. We also show that frequent deviations from the Cox proportional hazards assumption, particularly in trials of immune checkpoint inhibitors, arise from time-dependent therapeutic effects. Trial duration therefore has an underappreciated impact on the likelihood of success.
Tsvetkov, P., et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science 375, 6586, 1254-1261 (2022).Abstract
Copper is an essential cofactor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copper-dependent, regulated cell death is distinct from known death mechanisms and is dependent on mitochondrial respiration. We show that copper-dependent death occurs by means of direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss, which leads to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms.
Oreskovic, E., et al. Genetic analysis of cancer drivers reveals cohesin and CTCF as suppressors of PD-L1. Proc Natl Acad Sci U S A 119, 7, (2022).Abstract
Immune evasion is a significant contributor to tumor evolution, and the immunoinhibitory axis PD-1/PD-L1 is a frequent mechanism employed to escape tumor immune surveillance. To identify cancer drivers involved in immune evasion, we performed a CRISPR-Cas9 screen of tumor suppressor genes regulating the basal and interferon (IFN)-inducible cell surface levels of PD-L1. Multiple regulators of PD-L1 were identified, including IRF2, ARID2, KMT2D, and AAMP. We also identified CTCF and the cohesin complex proteins, known regulators of chromatin architecture and transcription, among the most potent negative regulators of PD-L1 cell surface expression. Additionally, loss of the cohesin subunit RAD21 was shown to up-regulate PD-L2 and MHC-I surface expression. PD-L1 and MHC-I suppression by cohesin were shown to be conserved in mammary epithelial and myeloid cells. Comprehensive examination of the transcriptional effect of STAG2 deficiency in epithelial and myeloid cells revealed an activation of strong IFN and NF-κB expression signatures. Inhibition of JAK-STAT or NF-κB pathways did not result in rescue of PD-L1 up-regulation in RAD21-deficient cells, suggesting more complex or combinatorial mechanisms at play. Discovery of the PD-L1 and IFN up-regulation in cohesin-mutant cells expands our understanding of the biology of cohesin-deficient cells as well as molecular regulation of the PD-L1 molecule.
Lu, D., Jambhekar, A. & Lahav, G. Louder for longer: Myc amplifies gene expression by extended transcriptional bursting. Cell Rep 38, 9, 110470 (2022).Abstract
Transcription is a complex, dynamic process. Using live single-cell measurements, Patange et al. show, in a recent issue of Cell Reports, that elevated levels of the transcription factor MYC enhance target gene RNA production by increasing the duration but not frequency of transcriptional bursts.
Schapiro, D., et al. MITI minimum information guidelines for highly multiplexed tissue images. Nat Methods 19, 3, 262-267 (2022).
Miller, T.E., et al. Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations. Nat Biotechnol (2022).Abstract
The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.
Hemming, M.L., et al. Preclinical modeling of leiomyosarcoma identifies susceptibility to transcriptional CDK inhibitors through antagonism of E2F-driven oncogenic gene expression. Clin Cancer Res (2022).Abstract
PURPOSE: Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize LMS patient derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies. EXPERIMENTAL DESIGN: We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA-seq and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling. RESULTS: We show that LMS PDX maintain the histologic appearance, copy-number alterations and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX co-cluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional CDK inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth. CONCLUSIONS: Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for LMS patients.
DePasquale, E.A.K., et al. Single-Cell Multiomics Reveals Clonal T-Cell Expansions and Exhaustion in Blastic Plasmacytoid Dendritic Cell Neoplasm. Front Immunol 13, 809414 (2022).Abstract
The immune system represents a major barrier to cancer progression, driving the evolution of immunoregulatory interactions between malignant cells and T-cells in the tumor environment. Blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare acute leukemia with plasmacytoid dendritic cell (pDC) differentiation, provides a unique opportunity to study these interactions. pDCs are key producers of interferon alpha (IFNA) that play an important role in T-cell activation at the interface between the innate and adaptive immune system. To assess how uncontrolled proliferation of malignant BPDCN cells affects the tumor environment, we catalog immune cell heterogeneity in the bone marrow (BM) of five healthy controls and five BPDCN patients by analyzing 52,803 single-cell transcriptomes, including 18,779 T-cells. We test computational techniques for robust cell type classification and find that T-cells in BPDCN patients consistently upregulate interferon alpha (IFNA) response and downregulate tumor necrosis factor alpha (TNFA) pathways. Integrating transcriptional data with T-cell receptor sequencing via shared barcodes reveals significant T-cell exhaustion in BPDCN that is positively correlated with T-cell clonotype expansion. By highlighting new mechanisms of T-cell exhaustion and immune evasion in BPDCN, our results demonstrate the value of single-cell multiomics to understand immune cell interactions in the tumor environment.
Launonen, I.-M., et al. Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer. Nat Commun 13, 1, 835 (2022).Abstract
The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generate spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes. We identify a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we report a prognostic role of a proliferative tumor-cell subpopulation, which associates with enhanced spatial tumor-immune interactions by CD8+ and CD4 + T-cells in the BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the potential to improve immunotherapeutic strategies and patient stratification in HGSC.
Gaglia, G., et al. Temporal and spatial topography of cell proliferation in cancer. Nat Cell Biol 24, 3, 316-326 (2022).Abstract
Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.
Baker, A.R., et al. Transcriptome profiling of ADAR1 targets in triple-negative breast cancer cells reveals mechanisms for regulating growth and invasion. Mol Cancer Res (2022).Abstract
ADARs catalyze Adenosine-to-Inosine (A-to-I) editing of double-stranded RNA and regulate global gene expression output through interactions with RNA and other proteins. ADARs play important roles in development and disease, and previous work has shown that ADAR1 is oncogenic in a growing list of cancer types. Here we show that ADAR1 is a critical gene for triple-negative breast cancer cells, as ADAR1 loss results in reduced growth (viability and cell cycle progression), invasion, and mammosphere formation. Whole transcriptome sequencing analyses demonstrate that ADAR1 regulates both coding and non-coding targets by altering gene expression level, A-to-I editing, and splicing. We determine that a recoding edit in filamin B (FLNB chr3:58156064) reduces the tumor suppressive activities of the protein to promote growth and invasion. We also show that several tumor suppressor microRNAs are upregulated upon ADAR1 loss and suppress cell cycle progression and invasion. Implications: This work describes several novel mechanisms of ADAR1-mediated oncogenesis in triple-negative breast cancer, providing support to strategies targeting ADAR1 in this aggressive cancer type that has few treatment options.
Pan, R., Ryan, J., Pan, D., Wucherpfennig, K.W. & Letai, A. Augmenting NK cell-based immunotherapy by targeting mitochondrial apoptosis. Cell 185, 9, 1521-1538.e18 (2022).Abstract
Interest in harnessing natural killer (NK) cells for cancer immunotherapy is rapidly growing. However, efficacy of NK cell-based immunotherapy remains limited in most trials. Strategies to augment the killing efficacy of NK cells are thus much needed. In the current study, we found that mitochondrial apoptosis (mtApoptosis) pathway is essential for efficient NK killing, especially at physiologically relevant effector-to-target ratios. Furthermore, NK cells can prime cancer cells for mtApoptosis and mitochondrial priming status affects cancer-cell susceptibility to NK-mediated killing. Interestingly, pre-activating NK cells confers on them resistance to BH3 mimetics. Combining BH3 mimetics with NK cells synergistically kills cancer cells in vitro and suppresses tumor growth in vivo. The ideal BH3 mimetic to use in such an approach can be predicted by BH3 profiling. We herein report a rational and precision strategy to augment NK-based immunotherapy, which may be adaptable to T cell-based immunotherapies as well.
Tang, S., Stokasimov, E., Cui, Y. & Pellman, D. Breakage of cytoplasmic chromosomes by pathological DNA base excision repair. Nature (2022).Abstract
Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridge5-8. These structures have fragile nuclear envelopes (NEs) that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here, we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by ADAR enzymes (adenine deaminases acting on RNA) to generate deoxyinosine (dI). dI is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, MPG (N-methyl-purine DNA glycosylase)11,12. These abasic sites are cleaved by the BER endonuclease, APE1 (apurinic/apyrimidinic endonuclease)12, creating single-strand DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the dI base from the DNA strand of RNA-DNA hybrids, which we demonstrate using pure proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with preexisting defects such as the DNA base abnormality described here.
Gil Del Alcazar, C.R., et al. Insights into immune escape during tumor evolution and response to immunotherapy using a rat model of breast cancer. Cancer Immunol Res (2022).Abstract
Animal models are critical for the preclinical validation of cancer immunotherapies. Unfortunately, mouse breast cancer models do not faithfully reproduce the molecular subtypes and immune environment of the human disease. In particular, there are no good murine models of estrogen receptor-positive (ER+) breast cancer, the predominant subtype in patients. Here, we show that Nitroso-N-methylurea-induced mammary tumors in outbred Sprague-Dawley rats recapitulate the heterogeneity for mutational profiles, ER expression, and immune evasive mechanisms observed in human breast cancer. We demonstrate the utility of this model for preclinical studies by dissecting mechanisms of response to immunotherapy using combination TGFBR inhibition and PD-L1 blockade. Short-term treatment of early-stage tumors induced durable responses. Gene expression profiling and spatial mapping classified tumors as inflammatory and non-inflammatory, and identified IFN, TCR and BCR signaling, CD74/MHC II, and epithelium-interacting CD8+ T cells as markers of response, whereas the complement system, M2 macrophage phenotype, and translation in mitochondria were associated with resistance. We found that the expression of CD74 correlated with leukocyte fraction and TCR diversity in human breast cancer. We identified a subset of rat ER+ tumors marked by expression of antigen-processing genes that had an active immune environment and responded to treatment. A gene signature characteristic of these tumors predicted disease-free survival in ER+ Luminal A breast cancer patients and overall survival in metastatic breast cancer patients receiving anti-PD-L1 therapy. We demonstrate the usefulness of this preclinical model for immunotherapy and suggest examination to expand immunotherapy to a subset of patients with ER+ disease.
Mysore, V., et al. Monocytes transition to macrophages within the inflamed vasculature via monocyte CCR2 and endothelial TNFR2. J Exp Med 219, 5, (2022).Abstract
Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.
Baldominos, P., et al. Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche. Cell (2022).Abstract
Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC.
Nirmal, A.J., et al. The spatial landscape of progression and immunoediting in primary melanoma at single cell resolution. Cancer Discov (2022).Abstract
Cutaneous melanoma is a highly immunogenic malignancy, surgically curable at early stages, but life-threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis involving precursor states, melanoma in situ, and invasive tumor. Hallmarks of immunosuppression are already detectable in precursor regions. When tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary. This environment is established by cytokine gradients that promote expression of MHC-II and IDO1, and by PD1-PDL1 mediated cell contacts involving macrophages, dendritic cells, and T cells. A few millimeters away, cytotoxic T cells synapse with melanoma cells in fields of tumor regression. Thus, invasion and immunoediting can co-exist within a few millimeters of each other in a single specimen.