PrefacePhenotypic and functional heterogeneity arise among cancer cells within the same tumor as a consequence of genetic change, environmental differences, and reversible changes in cellular properties. Some cancers also contain a hierarchy in which tumorigenic cancer stem cells differentiate into non-tumorigenic progeny. However, it remains unclear what fraction of cancers follow the stem cell model and what clinical behaviors the model explains. In this review we will evaluate the implications of recent lineage tracing and deep-sequencing studies for the cancer stem cell model and the extent to which it accounts for therapy resistance and disease progression. IntroductionThe cancer stem cell model provides one explanation for the phenotypic and functional heterogeneity among cancer cells in some tumors [1][2][3][4][5] . The model posits that some cancers are hierarchically organized into subpopulations of tumorigenic cancer stem cells and their nontumorigenic progeny. In these cases, cancer stem cells are thought to drive tumor growth and disease progression, perhaps including therapy resistance 6-8 and metastasis 9,10 . However, difficulty replicating solid cancer stem cell markers, variability from patient to patient, and variation in results from different xenograft models have meant that it remains unclear what fraction of cancers follow this model -most, or only a minority 11 ?Even in cancers that clearly contain a hierarchy of tumorigenic and non-tumorigenic cells, this hierarchy must co-exist with other sources of heterogeneity including clonal evolution 12 , heterogeneity in the microenvironment 13,14 , and reversible changes in cancer cell properties that can occur independently of hierarchical organization [15][16][17][18] . Under these circumstances it is not necessarily clear which phenotypic and functional differences among cells arise from which sources of heterogeneity. To what extent do metastasis, therapy resistance, and disease progression reflect intrinsic properties of cancer stem cells as opposed to genetic evolution or other sources of heterogeneity? Integration of results from multiple experimental approaches will be necessary to distinguish the relative contributions of these sources of heterogeneity to disease progression.New experimental approaches have provided perspective and insight into these questions. Genetic approaches to fate-map the contributions of cancer cells to tumor growth in mice HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscripthave provided evidence in support of the cancer stem cell model in some contexts and evidence against the model in other contexts [19][20][21][22][23] . Since transplantation assays evaluate the potential of cancer cells to form tumors, rather than their actual fate in the native tumor, fate-mapping complements what we have learned from transplantation assays (Figure 1). High-coverage sequencing of human tumors has also provided new insights into genetic heterogeneity within tumors and the cells responsible for relapse after ...

Stem cell fate can be influenced by metabolite levels in culture but it is unknown whether physiological variations in metabolite levels in normal tissues regulate stem cell function in vivo. We developed a metabolomics method for analysis of rare cell populations isolated directly from tissues and used it to compare haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which declined with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, partly by reducing Tet2 function, a dioxygenase tumor suppressor. Ascorbate depletion cooperated with Flt3ITD leukaemic mutations to accelerate leukaemogenesis, though cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate thus accumulates within HSCs to promote Tet function in vivo, limiting HSC frequency and suppressing leukaemogenesis.

Mouse models have dramatically improved our understanding of cancer development and tumor biology. However, these models have shown limited efficacy as tractable systems for unbiased genetic experimentation. Here, we report the adaptation of loss of function screening to mouse models of cancer. Specifically, we have been able to introduce a library of shRNAs into individual mice using transplantable Eμ-myc lymphoma cells. This approach has allowed us to screen nearly 1000 genetic alterations in the context of a single tumor-bearing mouse. Results from these experiments have identified a central role for regulators of actin dynamics and cell motility in lymphoma cell homeostasis in vivo, and validation experiments confirmed that these proteins represent bona fide lymphoma drug targets. Additionally, suppression of two of these targets, Rac2 and Twinfilin, potentiated the action of the front-line chemotherapeutic vincristine, suggesting a critical relationship between cell motility and tumor relapse in hematopoietic malignancies.

We performed a genome-scale shRNA screen for modulators of B-cell leukemia progression in vivo. Results from this work revealed dramatic distinctions between the relative effects of shRNAs on the growth of tumor cells in culture versus in their native microenvironment. Specifically, we identified many “context-specific” regulators of leukemia development. These included the gene encoding the zinc finger protein Phf6. While inactivating mutations in PHF6 are commonly observed in human myeloid and T-cell malignancies, we found that Phf6 suppression in B-cell malignancies impairs tumor progression. Thus, Phf6 is a “lineage-specific” cancer gene that plays opposing roles in developmentally distinct hematopoietic malignancies.