RESUMO
Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand the changes in the tumor microenvironment (TME) accompanying transition to IBC, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to interrogate 79 clinically annotated surgical resections using machine learning tools for cell segmentation, pixel-based clustering, and object morphometrics. Comparison of normal breast with patient-matched DCIS and IBC revealed coordinated transitions between four TME states that were delineated based on the location and function of myoepithelium, fibroblasts, and immune cells. Surprisingly, myoepithelial disruption was more advanced in DCIS patients that did not develop IBC, suggesting this process could be protective against recurrence. Taken together, this HTAN Breast PreCancer Atlas study offers insight into drivers of IBC relapse and emphasizes the importance of the TME in regulating these processes.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Diferenciação Celular , Estudos de Coortes , Progressão da Doença , Células Epiteliais/patologia , Epitélio/patologia , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/patologia , Fenótipo , Análise de Célula Única , Células Estromais/patologia , Microambiente TumoralRESUMO
Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-ß, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.
Assuntos
Granuloma/imunologia , Tuberculose/imunologia , Antígeno B7-H1/imunologia , Células Cultivadas , Citocinas/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Pulmão/imunologia , Mycobacterium tuberculosis/imunologia , Células Mieloides/imunologiaRESUMO
The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.
Assuntos
Linfócitos/imunologia , Espectrometria de Massas , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/imunologia , Antígenos CD/metabolismo , Antígeno B7-H1/metabolismo , Análise por Conglomerados , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Estimativa de Kaplan-Meier , Linfócitos/citologia , Linfócitos/metabolismo , Aprendizado de Máquina , Análise de Componente Principal , Receptor de Morte Celular Programada 1/metabolismo , Análise Espacial , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/mortalidade , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.
Assuntos
Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Subpopulações de Linfócitos T/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Medula Óssea/imunologia , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Tecido Linfoide/imunologia , Masculino , Melanoma/imunologia , Melanoma/terapia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Microambiente TumoralRESUMO
Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.
Assuntos
Biologia Computacional/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatologia , Análise de Célula Única/métodos , Medula Óssea/patologia , Criança , Estudos de Coortes , Heterogeneidade Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , TranscriptomaRESUMO
B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.
Assuntos
Subpopulações de Linfócitos B/classificação , Subpopulações de Linfócitos B/imunologia , Isotipos de Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , 5'-Nucleotidase/metabolismo , Apirase/metabolismo , Antígeno CD11c/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Memória Imunológica/imunologia , Antígenos Comuns de Leucócito/metabolismo , Pessoa de Meia-Idade , Transdução de Sinais/imunologia , Receptor fas/metabolismoRESUMO
Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and "corrupted" developmental processes including cancer.
Assuntos
Algoritmos , Linfócitos B/citologia , Linfopoese , Humanos , Interleucina-7/metabolismo , Células Precursoras de Linfócitos B/citologia , Fator de Transcrição STAT5/metabolismo , Recombinação V(D)JRESUMO
High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.
Assuntos
Dendrímeros , Polímeros , Polímeros/química , Antracenos , Anticorpos/química , Fatores de Transcrição ForkheadRESUMO
Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institute's (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2 = 0.94 ± 0.04) and frequency (R2 = 0.95 ± 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2 = 0.85 ± 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between serial sections yielded an average correlation of R2 = 0.94 ± 0.05. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.
Assuntos
Diagnóstico por Imagem , Neoplasias , Anticorpos , Diagnóstico por Imagem/métodos , Humanos , Imuno-Histoquímica , Íons , Espectrometria de Massas/métodosRESUMO
Improvements in single-cell protein analysis are required to study the cell-to-cell variation inherent to diseases, including cancer. Single-cell immunoblotting (scIB) offers proteoform detection specificity, but often relies on fluorescence-based readout and is therefore limited in multiplexing capability. Among rising multiplexed imaging methods is multiplexed ion beam imaging by time-of-flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectral overlap to the same degree as fluorophore-tagged antibodies. We report for the first-time MIBI-TOF of single-cell immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein immunoblotting on a microscale device consisting of a 50- to 75-µm thick hydrated polyacrylamide (PA) gel matrix for protein immobilization prior to in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and confirm that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging â¼42% of PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB.
Assuntos
Proteínas , Análise de Célula Única , Immunoblotting , Íons , Espectrometria de MassasRESUMO
Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.
Assuntos
Anticorpos , Sinaptossomos , Citometria de Fluxo , HumanosRESUMO
Transforming growth factor ß (TGF-ß) is well known for its important function in hematopoietic stem cell (HSC) quiescence. However, the molecular mechanism underlining this function remains obscure. Endoglin (Eng), a type III receptor for the TGF-ß superfamily, has been shown to selectively mark long-term HSCs; however, its necessity in adult HSCs is unknown due to embryonic lethality. Using conditional deletion of Eng combined with serial transplantation, we show that this TGF-ß receptor is critical to maintain the HSC pool. Transplantation of Eng-deleted whole bone marrow or purified HSCs into lethally irradiated mice results in a profound engraftment defect in tertiary and quaternary recipients. Cell cycle analysis of primary grafts revealed decreased frequency of HSCs in G0, suggesting that lack of Eng impairs reentry of HSCs to quiescence. Using cytometry by time of flight (CyTOF) to evaluate the activity of signaling pathways in individual HSCs, we find that Eng is required within the Lin-Sca+Kit+-CD48- CD150+ fraction for canonical and noncanonical TGF-ß signaling, as indicated by decreased phosphorylation of SMAD2/3 and the p38 MAPK-activated protein kinase 2, respectively. These findings support an essential role for Eng in positively modulating TGF-ß signaling to ensure maintenance of HSC quiescence.
Assuntos
Endoglina/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Ciclo Celular , Endoglina/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de SinaisRESUMO
Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in â¼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.
Assuntos
Antígenos CD34/metabolismo , Linhagem da Célula , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteoma/análise , Análise de Célula Única/métodos , Diferenciação Celular , Células Cultivadas , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteoma/metabolismoRESUMO
Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Citocinas/metabolismo , Antígenos Virais de Tumores/imunologia , Citometria de Fluxo , Humanos , Espectrometria de Massas , Fenótipo , Análise de Componente PrincipalRESUMO
An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple ([Formula: see text]40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Biomarcadores Tumorais/metabolismo , Simulação por Computador , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células HeLa , HumanosRESUMO
Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging. Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology. Availability and implementation: The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Biomarcadores/análise , Citometria de Fluxo , SoftwareRESUMO
Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (â¼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and ß-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and ß-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Fatores de TranscriçãoRESUMO
Alzheimer's disease (AD) differentially and specifically affects brain regions and neuronal cell types in a predictable pattern. Damage to the brain appears to spread and worsens with time, taking over more regions and activating multiple stressors that can converge to promote vulnerability of certain cell types. At the same time, other cell types and brain regions remain intact in the face of this onslaught of neuropathology. Although neuropathologic descriptions of AD have been extensively expanded and mapped over the last several decades, our understanding of the mechanisms underlying how certain regions and cell populations are specifically vulnerable or resistant has lagged behind. In this review, we detail what is known about the selectivity of local initiation of AD pathology in the hippocampus, its proposed spread via synaptic connections, and the diversity of clinical phenotypes and brain atrophy patterns that may arise from different fibrillar strains of pathologic proteins or genetic predispositions. We summarize accumulated and emerging knowledge of the cellular and molecular basis for neuroanatomic selectivity, consider potential disease-relevant differences between vulnerable and resistant neuronal cell types and isolate molecular markers to identify them.