Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular ...state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f + cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell—based therapeutics.
In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation ...potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.
Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through ...large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover “subTMEs,” histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. “Reactive” subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich “deserted” subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units.
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•PDAC regional heterogeneity stems from sub-tumor microenvironments (subTMEs)•SubTMEs exhibit distinct immune phenotypes and CAF differentiation states•SubTMEs execute distinct tumor-promoting and chemoprotective functions•Intratumoral subTME co-occurrence links stromal heterogeneity to patient outcome
Intratumoral heterogeneity in the human pancreatic tumor microenvironment is not random but originates in well-definable regional tissue states. The underlying sub-tumor microenvironments shape regional epithelial and immune phenotypes and influence key clinical metrics of disease progression.
To determine the impact of basal-like and classical subtypes in advanced pancreatic ductal adenocarcinoma (PDAC) and to explore GATA6 expression as a surrogate biomarker.
Within the COMPASS trial, ...patients proceeding to chemotherapy for advanced PDAC undergo tumor biopsy for RNA-sequencing (RNA-seq). Overall response rate (ORR) and overall survival (OS) were stratified by subtypes and according to chemotherapy received. Correlation of
with the subtypes using gene expression profiling,
hybridization (ISH) was explored.
Between December 2015 and May 2019, 195 patients (95%) had enough tissue for RNA-seq; 39 (20%) were classified as basal-like and 156 (80%) as classical. RECIST response data were available for 157 patients; 29 basal-like and 128 classical where the ORR was 10% versus 33%, respectively (
= 0.02). In patients with basal-like tumors treated with modified FOLFIRINOX (
= 22), the progression rate was 60% compared with 15% in classical PDAC (
= 0.0002). Median OS in the intention-to-treat population (
= 195) was 9.3 months for classical versus 5.9 months for basal-like PDAC (HR, 0.47; 95% confidence interval, 0.32-0.69;
= 0.0001).
expression by RNA-seq highly correlated with the classifier (
< 0.001) and ISH predicted the subtypes with sensitivity of 89% and specificity of 83%. In a multivariate analysis, GATA6 expression was prognostic (
= 0.02). In exploratory analyses, basal-like tumors, could be identified by keratin 5, were more hypoxic and enriched for a T-cell-inflamed gene expression signature.
The basal-like subtype is chemoresistant and can be distinguished from classical PDAC by GATA6 expression.
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The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. The validity of this model in the mouse has been questioned; however, ...little is known about the lineage potential of human progenitors. Here we provide a comprehensive analysis of the human hematopoietic hierarchy by clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow. Human multilymphoid progenitors, identified as a distinct population of Thy-1(neg-lo)CD45RA(+) cells in the CD34(+)CD38(-) stem cell compartment, gave rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells, which indicated that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation.
Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are ...functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.
Although p53 inactivation promotes genomic instability
and presents a route to malignancy for more than half of all human cancers
, the patterns through which heterogenous TP53 (encoding human p53) ...mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.
Combination chemotherapy, either modified FOLFIRINOX (mFFX) or gemcitabine-nabpaclitaxel, are used in the treatment of most patients with advanced pancreatic ductal adenocarcinoma (PDAC), yet robust ...biomarkers of outcome are currently lacking to guide regimen selection. Here, we tested GATA6 immunohistochemistry (IHC) as a putative biomarker in advanced PDAC. GATA6 is a transcription factor in normal pancreas development. Two pathologists, blinded to clinical and molecular data, independently assessed GATA6 IHC in biopsy specimens of 130 patients with advanced PDAC, in 2 distinct phases (without and with computer assistance using the open source software QuPath). Low GATA6 IHC expression was associated with shorter overall survival median OS 6.2 months for patients with GATA6 low tumors vs. 11.5 months for patients with GATA6 high tumors, HR 1.66 (95% CI 1.15-2.40), P = 0.007. Progression appears to be higher in GATA6-low tumors compared to GATA6-high tumors in patients treated with mFFX (P = 0.024) but not in patients treated with gemcitabine regimens. GATA6 IHC expression was significantly associated with molecular subtypes (P = 0.0003). Digital assistance markedly improved interrater concordance (Cohen's kappa scores of 0.32 vs. 0.95). Our results provide strong evidence that GATA6 IHC can be used as a single biomarker in the clinic to predict clinical outcome in advanced PDAC, warranting further investigation in prospective clinical trials. These results provide the basis for an improved classification of PDAC and future biomarker design using digital pathology workflow.
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