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Abstract Enchondromas are a common tumor in bone that can occur as multiple lesions in enchondromatosis, which is associated with deformity of the affected bone. These lesions harbor somatic mutations in IDH and driving expression of a mutant Idh1 in Col2 expressing cells in mice causes an enchondromatosis phenotype. Here we compared growth plates from E18.5 mice expressing a mutant Idh1 with control littermates using single cell RNA sequencing. Data from Col2 expressing cells were analysed using UMAP and RNA pseudo-time analyses. A unique cluster of cells was identified in the mutant growth plates that expressed genes known to be upregulated in enchondromas. There was also a cluster of cells that was underrepresented in the mutant growth plates that expressed genes known to be important in longitudinal bone growth. Immunofluorescence showed that the genes from the unique cluster identified in the mutant growth plates were expressed in multiple growth plate anatomic zones, and pseudo-time analysis also suggested these cells could arise from multiple growth plate chondrocyte subpopulations. This data supports the notion that a subpopulation of chondrocytes become enchondromas at the expense of contributing to longitudinal growth.

Summary: Intratumoral heterogeneity is common in cancer, particularly in sarcomas like undifferentiated pleomorphic sarcoma (UPS), where individual cells demonstrate a high degree of cytogenic diversity. Previous studies showed that a small subset of cells within UPS, known as the metastatic clone (MC), as responsible for metastasis. Using a CRISPR-based genomic screen in-vivo, we identified the COMPASS complex member Setd1a as a key regulator maintaining the metastatic phenotype of the MC in murine UPS. Depletion of Setd1a inhibited metastasis development in the MC. Transcriptome and chromatin sequencing revealed COMPASS complex target genes in UPS, such as Cxcl10, downregulated in the MC. Deleting Cxcl10 in non-MC cells increased their metastatic potential. Treating mice with human UPS xenografts with a COMPASS complex inhibitor suppressed metastasis without affecting tumor growth in the primary tumor. Our data identified an epigenetic program in a subpopulation of sarcoma cells that maintains metastatic potential.

Sarcomas contain a subpopulation of tumor-propagating cells (TPCs) with enhanced tumor-initiating and self-renewal properties. However, it is unclear whether the TPC phenotype in sarcomas is stable or a dynamic cell state that can derive from non-TPCs. In this study, we utilized a mouse model of undifferentiated pleomorphic sarcoma (UPS) to trace the lineage relationship between sarcoma side population (SP) cells that are enriched for TPCs and non-SP cells. By cotransplanting SP and non-SP cells expressing different endogenous fluorescent reporters, we show that non-SP cells can give rise to SP cells with enhanced tumor-propagating potential in vivo. Lineage trajectory analysis using single-cell RNA sequencing from SP and non-SP cells supports the notion that non-SP cells can assume the SP cell phenotype de novo. To test the effect of eradicating SP cells on tumor growth and self-renewal, we generated mouse sarcomas in which the diphtheria toxin receptor is expressed in the SP cells and their progeny. Ablation of the SP population using diphtheria toxin did not impede tumor growth or self-renewal. Altogether, we show that the sarcoma SP represent a dynamic cell state and targeting TPCs alone is insufficient to eliminate tumor progression.

The rate of repair declines with age; however, exposure to young circulations can rejuvenate fracture repair, but how this is accomplished is unknown. Here, the authors identify proteins, including low-density lipoprotein receptor-related protein 1 (Lrp1), as being secreted from young macrophages and rejuvenating fracture repair in mice.

Summary: Cellular heterogeneity is frequently observed in cancer, but the biological significance of heterogeneous tumor clones is not well defined. Using multicolor reporters and CRISPR-Cas9 barcoding, we trace clonal dynamics in a mouse model of sarcoma. We show that primary tumor growth is associated with a reduction in clonal heterogeneity. Local recurrence of tumors following surgery or radiation therapy is driven by multiple clones. In contrast, advanced metastasis to the lungs is driven by clonal selection of a single metastatic clone (MC). Using RNA sequencing (RNA-seq) and in vivo assays, we identify candidate suppressors of metastasis, namely, Rasd1, Reck, and Aldh1a2. These genes are downregulated in MCs of the primary tumors prior to the formation of metastases. Overexpression of these suppressors of metastasis impair the ability of sarcoma cells to colonize the lungs. Overall, this study reveals clonal dynamics during each step of tumor progression, from initiation to growth, recurrence, and distant metastasis. : Tang et al. demonstrate that distinct clonal architecture underlies different stages of tumor progression, from tumor initiation, local recurrence after therapy, to advanced metastasis. Keywords: clonal evolution, cancer metastasis, suppressors of metastasis, sarcoma, tumor heterogeneity, lineage tracing

Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

Graded Hedgehog (Hh) signaling governs the balance of Gli transcriptional activators and repressors to specify diverse ventral cell fates in the spinal cord. It remains unclear how distinct intracellular Gli activity is generated. Here, we demonstrate that Sufu acts universally as a negative regulator of Hh signaling, whereas Kif7 inhibits Gli activity in cooperation with, and independent of, Sufu. Together, they deter naïve precursors from acquiring increasingly ventral identity. We show that Kif7 is also required to establish high intracellular Gli activity by antagonizing the Sufu-inhibition of Gli2. Strikingly, by abolishing the negative regulatory action of Sufu, diverse ventral cell fates can be specified in the absence of extracellular Hh signaling. These data suggest that Sufu is the primary regulator of graded Hh signaling and establish that the antagonistic and cooperative actions of Kif7 and Sufu are responsible for setting up distinct Gli activity in ventral cell fate specification.