Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC ...cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5− tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
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•rRNA and proteins in CRCs are produced in dedicated tumor domains•Differentiated tumor cells experience an irreversible loss of biosynthetic capacities•POLR1A-high CRC cells reside at the top of the tumor cell hierarchy•Both LGR5+ and LGR5− tumor cells within biosynthetic niches fuel tumor growth
Morral and colleagues discovered that most rRNA and proteins synthesized in colorectal cancers (CRCs) are contributed by a limited subset of tumor cells that reside adjacent to the stroma. This architecture defines a common stem cell hierarchy. In some CRCs, the biosynthetic tumor cell population renders LGR5+ tumor cells dispensable.
The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, ...we devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus. Analysis of LGR5‐EGFP+ cells isolated from organoid‐derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage‐tracing experiments showed that LGR5+ CRC cells self‐renew and generate progeny over long time periods that undergo differentiation toward mucosecreting‐ and absorptive‐like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
Synopsis
To overcome technical and conceptual limitations in the analysis of tumor cell heterogeneity, the genomes of human cancer organoids were edited to integrate reporter and lineage tracing cassettes that enable tracking selected tumor cell populations in vivo.
CRISPR/Cas9‐mediated genome editing facilitated the integration of cassettes at marker genes in colorectal cancer (CRC) patient‐derived organoids (PDOs).
A GFP reporter integrated at the LGR5 locus labeled a population of ISC‐like tumor cells in human CRCs.
The LGR5‐GFP+ cell population was enriched in tumor‐initiating cells.
Lineage tracing analysis using CreERT2 knock‐in PDOs demonstrated that in intact tumors LGR5+ CRC cells are multipotent, long‐lived and produce cell clones that scale proportional to tumor growth.
Double edited LGR5‐GFP/Ki67‐RFP PDOs revealed the existence of proliferating and quiescent LGR5+ CRC cells.
To overcome technical and conceptual limitations in the analysis of tumor cell heterogeneity, the genomes of human cancer organoids were edited to integrate reporter and lineage tracing cassettes that enable tracking selected tumor cell populations in vivo.
Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, ...particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (T
1) activity and reduced immune cytotoxicity or increased TGFβ levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFβ-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the T
1-effector phenotype. Immunotherapies directed against TGFβ signalling may therefore have broad applications in treating patients with advanced colorectal cancer.
Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years
. Therapies to prevent disease relapse ...remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5
stem-like tumour cells
, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1
cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.
Against a backdrop of a looming labor shortage and changing demographics, this study explored what is important to older workers. Job attitudes, preferences, and concerns were investigated through a ...survey of employees from a dozen U.S. based organizations. Findings from 415 older workers (older than 45) and 663 younger workers (age 30 and younger) suggested that both groups of employees care about work they do, the relationship they have with their supervisor, and the organization's policies and practices around rewards, fairness, and flexibility. Compared to younger workers, however, older workers care more about schedule and task control, as well as task variety and challenge. They are less concerned with the social aspects of work.
Business statistics courses are not typically popular, and students often find the examples and textbook problems tedious and irrelevant. In this article, the author describes a pedagogy project that ...keeps students interested and working effectively in a large class. Through a student survey, the author gathered data on students' university experience and demographics. The class used the Statistical Package for the Social Sciences (SPSS) data set for demonstration of a range of statistical techniques. The student survey instrument and data set provided examples and problems for each of the major topics of the course. Student comments and performance demonstrated the project's positive results.
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