Colorectal Cancer Stem Cell Subtypes Orchestrate Distinct Tumor Microenvironments.

Several classification systems have been developed to define tumor subtypes in colorectal cancer (CRC). One system proposes that tumor heterogeneity derives in part from distinct cancer stem cell populations that co-exist as admixtures of varying proportions. However, the lack of single cell resolution has prohibited a definitive identification of these types of stem cells and therefore any understanding of how each influence tumor phenotypes. Here were report the isolation and characterization of two cancer stem cell subtypes from the SW480 CRC cell line. We find these cancer stem cells are oncogenic versions of the normal Crypt Base Columnar (CBC) and Regenerative Stem Cell (RSC) populations from intestinal crypts and that their gene signatures are consistent with the "Admixture" and other CRC classification systems. Using publicly available single cell RNA sequencing (scRNAseq) data from CRC patients, we determine that RSC and CBC cancer stem cells are commonly co-present in human CRC. To characterize influences on the tumor microenvironment, we develop subtype-specific xenograft models and we define their tumor microenvironments at high resolution via scRNAseq. RSCs create differentiated, inflammatory, slow growing tumors. CBCs create proliferative, undifferentiated, invasive tumors. With this enhanced resolution, we unify current CRC patient classification schema with TME phenotypes and organization.

EED is required for mouse primordial germ cell differentiation in the embryonic gonad.

Development of primordial germ cells (PGCs) is required for reproduction. During PGC development in mammals, major epigenetic remodeling occurs, which is hypothesized to establish an epigenetic landscape for sex-specific germ cell differentiation and gametogenesis. In order to address the role of embryonic ectoderm development (EED) and histone 3 lysine 27 trimethylation (H3K27me3) in this process, we created an EED conditional knockout mouse and show that EED is essential for regulating the timing of sex-specific PGC differentiation in both ovaries and testes, as well as X chromosome dosage decompensation in testes. Integrating chromatin and whole genome bisulfite sequencing of epiblast and PGCs, we identified a poised repressive signature of H3K27me3/DNA methylation that we propose is established in the epiblast where EED and DNMT1 interact. Thus, EED joins DNMT1 in regulating the timing of sex-specific PGC differentiation during the critical window when the gonadal niche cells specialize into an ovary or testis.

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

Disruption of ß-Catenin-Dependent Wnt Signaling in Colon Cancer Cells Remodels the Microenvironment to Promote Tumor Invasion.

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin-dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer. IMPLICATIONS: Decreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation.

Human primordial germ cells (hPGCs) are the first embryonic progenitors in the germ cell lineage, yet the molecular mechanisms required for hPGC formation are not well characterized. To identify regulatory regions in hPGC development, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to systematically characterize regions of open chromatin in hPGCs and hPGC-like cells (hPGCLCs) differentiated from human embryonic stem cells (hESCs). We discovered regions of open chromatin unique to hPGCs and hPGCLCs that significantly overlap with TFAP2C-bound enhancers identified in the naive ground state of pluripotency. Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity.

TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency.

Transition from primed to naive pluripotency is associated with dynamic changes in transposable element (TE) expression and demethylation of imprinting control regions (ICRs). In mouse, ICR methylation and TE expression are each regulated by TRIM28; however, the role of TRIM28 in humans is less clear. Here, we show that a null mutation in TRIM28 causes significant alterations in TE expression in both the naive and primed states of human pluripotency, and phenotypically this has limited effects on self-renewal, instead causing a loss of germline competency. Furthermore, we discovered that TRIM28 regulates paternal ICR methylation and chromatin accessibility in the primed state, with no effects on maternal ICRs. Taken together, our study shows that abnormal TE expression is tolerated by self-renewing human pluripotent cells, whereas germline competency is not.

An integration-free, virus-free rhesus macaque induced pluripotent stem cell line (riPSC90) from embryonic fibroblasts.

The rhesus macaque induced pluripotent stem cell (riPSC) line, UCLAi090-A (riPSC90), was generated from rhesus embryonic fibroblast (REF) cells called REF90. REF90 cells and the riPSC90 line were authenticated by short tandem repeat analysis and had a normal male (42, XY) karyotype. The riPSC90 line expressed markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA4, and generated teratomas after transplantation into immunocompromised mice. riPSC90 could be used in parallel with riPSC89, which was derived from REFs cultured from a different rhesus macaque embryo (Sosa et al. 2016).

An integration-free, virus-free rhesus macaque induced pluripotent stem cell line (riPSC89) from embryonic fibroblasts.

We generated a rhesus macaque induced pluripotent stem cell (riPSC) line, riPSC89, from rhesus embryonic fibroblasts (REFs). Fibroblasts were expanded from the skin of a rhesus macaque embryo at embryonic day 47. REFs and riPSCs had a normal male (42, XY) karyotype. The riPSC89 line was positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA4. Pluripotency was demonstrated through the generation of teratomas using transplantation into immunocompromised mice. The riPSC89 line may be a useful non-human primate resource to uncover developmental origins of disease, or used as a basic model to understand lineage specification in the primate embryo.

PGC Reversion to Pluripotency Involves Erasure of DNA Methylation from Imprinting Control Centers followed by Locus-Specific Re-methylation.

Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However, when removed from their embryonic niche, PGCs undergo reversion to pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) is variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. Here we show that reverting PGCs to EGCs involved stable ICC methylation erasure at Snrpn, Igf2r, and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes erasure followed by de novo re-methylation. PGCs differentiated in vitro from ESCs completed Snrpn ICC erasure. However, the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC was abnormally hypermethylated in ESCs, this is not erased in PGCs differentiated from ESCs. Therefore, launching PGC differentiation from ESC lines with appropriately methylated ICCs is critical to the generation of germline cells that recapitulate endogenous ICC erasure.

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice.

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1-Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.