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Advanced solid cancers are complex assemblies of tumor, immune, and stromal cells characterized by high intratumoral variation. We use highly multiplexed tissue imaging, 3D reconstruction, spatial statistics, and machine learning to identify cell types and states underlying morphological features of known diagnostic and prognostic significance in colorectal cancer. Quantitation of these features in high-plex marker space reveals recurrent transitions from one tumor morphology to the next, some of which are coincident with long-range gradients in the expression of oncogenes and epigenetic regulators. At the tumor invasive margin, where tumor, normal, and immune cells compete, T cell suppression involves multiple cell types and 3D imaging shows that seemingly localized 2D features such as tertiary lymphoid structures are commonly interconnected and have graded molecular properties. Thus, while cancer genetics emphasizes the importance of discrete changes in tumor state, whole-specimen imaging reveals large-scale morphological and molecular gradients analogous to those in developing tissues. [Display omitted] • Multiplexed analysis shows intermixed tumor morphologies and molecular gradients • Various cancer characteristic cellular features are large, interconnected structures • 3D TLS networks show intra-TLS patterning variation • PD1-PDL1 interactions are primarily between T and myeloid cells in this CRC cohort Multiplexed whole-slide imaging analysis characterizes intermixed and graded morphological and molecular features in human colorectal cancer samples, highlighting large-scale cancer characteristic structural features and variations in intra-tertiary lymphoid cellular compositions and structural patterning. [ABSTRACT FROM AUTHOR]

Summary: Mammalian cells have a remarkable capacity to compensate for heterozygous gene loss or extra gene copies. One exception is Down syndrome (DS), where a third copy of chromosome 21 mediates neurogenesis defects and lowers the frequency of solid tumors. Here we combine live-cell imaging and single-cell analysis to show that increased dosage of chromosome 21-localized Dyrk1a steeply increases G1 cell cycle duration through direct phosphorylation and degradation of cyclin D1 (CycD1). DS-derived fibroblasts showed analogous cell cycle changes that were reversed by Dyrk1a inhibition. Furthermore, reducing Dyrk1a activity increased CycD1 expression to force a bifurcation, with one subpopulation of cells accelerating proliferation and the other arresting proliferation by costabilizing CycD1 and the CDK inhibitor p21. Thus, dosage of Dyrk1a repositions cells within a p21-CycD1 signaling map, directing each cell to either proliferate or to follow two distinct cell cycle exit pathways characterized by high or low CycD1 and p21 levels. [Copyright &y& Elsevier]

Summary: Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders. [ABSTRACT FROM AUTHOR]

Summary: Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation. [ABSTRACT FROM AUTHOR]

Summary: Postreplication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis. [Copyright &y& Elsevier]

Ovarian cancer is resistant to immunotherapy, and this is influenced by the immunosuppressed tumor microenvironment (TME) dominated by macrophages. Resistance is also affected by intratumoral heterogeneity, whose development is poorly understood. To identify regulators of ovarian cancer immunity, we employed a spatial functional genomics screen (Perturb-map), focused on receptor/ligands hypothesized to be involved in tumor-macrophage communication. Perturb-map recapitulated tumor heterogeneity and revealed that interleukin-4 (IL-4) promotes resistance to anti-PD-1. We find ovarian cancer cells are the key source of IL-4, which directs the formation of an immunosuppressive TME via macrophage control. IL-4 loss was not compensated by nearby IL-4-expressing clones, revealing short-range regulation of TME composition dictating tumor evolution. Our studies show heterogeneous TMEs can emerge from localized altered expression of cancer-derived cytokines/chemokines that establish immune-rich and immune-excluded neighborhoods, which drive clone selection and immunotherapy resistance. They also demonstrate the potential of targeting IL-4 signaling to enhance ovarian cancer response to immunotherapy. [Display omitted] • Perturb-map enables CRISPR screens modeling inter- and intratumor heterogeneity • Regional differences in cytokine expression create immunosuppressed tumor neighborhoods • Cancer-derived CCL7 acts locally to organize immune composition around tumor clones • Cancer cell-produced IL-4 mediates resistance to immunotherapy via macrophage control Cancer-derived paracrine factors create distinct "neighborhoods" in the tumor microenvironment, promoting clonal selection and tumor heterogeneity. In ovarian cancer, IL-4 originating from a minority of cells within the tumor can promote immune suppression and tumor resistance to immune checkpoint blockade therapy. [ABSTRACT FROM AUTHOR]

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease characterized by keratinized epithelial tunnels that grow deeply into the dermis. Here, we examined the immune microenvironment within human HS lesions. Multi-omics profiling and multiplexed imaging identified tertiary lymphoid structures (TLSs) near HS tunnels. These TLSs were enriched with proliferative T cells, including follicular helper (Tfh), regulatory (Treg), and pathogenic T cells (IL17A + and IFNG +), alongside extensive clonal expansion of plasma cells producing antibodies reactive to keratinocytes. HS fibroblasts express CXCL13 or CCL19 in response to immune cytokines. Using a microfluidic system to mimic TLS on a chip, we found that HS fibroblasts critically orchestrated lymphocyte aggregation via tumor necrosis factor alpha (TNF-α)-CXCL13 and TNF-α-CCL19 feedback loops with B and T cells, respectively; early TNF-α blockade suppressed aggregate initiation. Our findings provide insights into TLS formation in the skin, suggest therapeutic avenues for HS, and reveal mechanisms that may apply to other autoimmune settings, including Crohn's disease. [Display omitted] • TLSs near tunnel epithelium of HS lesional skin support local immune cell proliferation • Extensive clonal expansion of plasma cells producing antibodies against keratinocytes • HS fibroblasts promote lymphocyte aggregation through TNF-α-CXCL13 and TNF-α-CCL19 loops • TNF-α blockade is more effective at the initiation of lymphocyte aggregation Hidradenitis suppurativa (HS) is a severe chronic inflammatory disease without effective treatment options. Yu et al. identify and characterize tertiary lymphoid structures (TLSs) in HS, highlighting fibroblast-driven TLS formation via cytokine feedback loops with immune cells and lymphocyte clonal expansion and antibody production against keratinocytes. Their findings have implications for autoimmune pathogenesis and treatment options. [ABSTRACT FROM AUTHOR]

Lymphocytes are key for immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. We used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of lung tumors from a Kras/Trp53-mutant mouse model and human resections. Networks of interacting lymphocytes ("lymphonets") emerged as a distinctive feature of the anti-cancer immune response. Lymphonets nucleated from small T cell clusters and incorporated B cells with increasing size. CXCR3-mediated trafficking modulated lymphonet size and number, but T cell antigen expression directed intratumoral localization. Lymphonets preferentially harbored TCF1 + PD-1 + progenitor CD8 + T cells involved in responses to immune checkpoint blockade (ICB) therapy. Upon treatment of mice with ICB or an antigen-targeted vaccine, lymphonets retained progenitor and gained cytotoxic CD8 + T cell populations, likely via progenitor differentiation. These data show that lymphonets create a spatial environment supportive of CD8 + T cell anti-tumor responses., Competing Interests: Declaration of interests P.K.S. is a BOD member of Applied Biomath and Glencoe Software (co-founder), SAB member for RareCyte, NanoString, and Montai Health, and consultant for Merck. T.J. is a BOD member of Amgen and ThermoFisher, co-founder of Dragonfly Therapeutics and T2-Biosystems, SAB member of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics, and President of Break-Through-Cancer. Jacks lab receives funding from J&J Lung Cancer Initiative and Lustgarten Foundation (not supporting research in this manuscript). These affiliations do not represent a conflict of interest in this manuscript with respect to design/execution/interpretation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests J.S.B. is a scientific advisory board (SAB) member of Frontier Medicines and eFFECTOR Therapeutics and was an Agios Pharmaceuticals SAB member until January 2022. D.A.D. is on the SAB for Oncology Analytics, Inc., has consulted for Novartis, and receives research support from Canon, Inc. J.E.G. is a paid consultant for Helix and an uncompensated consultant for Konica Minolta and Earli. K.L.N. is a Nest Genomics SAB member. R.C.J.S. and W.L.G. are current employees of Genmab B.V. and Carcell Therapeutics, respectively. There are no conflicts of interest with this work. P.K.S. is an SAB or Board of Directors member of Applied Biomath, Nanostring, RareCyte Inc., and Glencoe Software, which distributes a commercial version of OMERO. In the last five years, P.K.S. has received research funding from Novartis and Merck. S.S. is a consultant for RareCyte Inc. A.R. is a co-founder and equity holder of Celsius Therapeutics and an equity holder in Immunitas. She was an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics, and Asimov until July 31, 2020. She has been an employee of Genentech since August 1, 2020 and has equity in Roche. P.K.S., S.S., and A.R. declare that these relationships have not influenced this manuscript’s content. G.K.G. is of no relation to the Gray Foundation leadership. J.S.B. is on the Advisory Board of Developmental Cell., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sirtuin 3 (SIRT3) is a NAD + -dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability., (Copyright © 2017 Elsevier Inc. All rights reserved.)