Senescent cancer-associated fibroblasts in pancreatic adenocarcinoma restrict CD8+ T cell activation and limit responsiveness to immunotherapy in mice

Senescent cells within tumors and their stroma exert complex pro- and anti-tumorigenic functions. However, the identities and traits of these cells, and the potential for improving cancer therapy through their targeting, remain poorly characterized. Here, we identify a senescent subset within previously-defined cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinomas (PDAC) and in premalignant lesions in mice and humans. Senescent CAFs isolated from mouse and humans expressed elevated levels of immune-regulatory genes. Depletion of senescent CAFs, either genetically or using the Bcl-2 inhibitor ABT-199 (venetoclax), increased the proportion of activated CD

Deconstructing tumor heterogeneity: The stromal perspective

Significant advances have been made towards understanding the role of immune cell-tumor interplay in either suppressing or promoting tumor growth, progression, and recurrence, however, the roles of additional stromal elements, cell types and/or cell states remain ill-defined. The overarching goal of this NCI-sponsored workshop was to highlight and integrate the critical functions of non-immune stromal components in regulating tumor heterogeneity and its impact on tumor initiation, progression, and resistance to therapy. The workshop explored the opposing roles of tumor supportiv

DNA2 and EXO1 in replication-coupled, homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway

Axonal growth arrests after an increased accumulation of Schwann cells expressing senescence markers and stromal cells in acellular nerve allografts

Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated β-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100β(+)) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin(+)/S100β(−)/CD68(−) cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest (“stressed” ANAs). These stressed ANAs contained mainly S100β(+)/p16(+) cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no deficits in axonal extension. Overall, this evidence demonstrates that long ANAs are repopulated with increased p16(+) Schwann cells and stromal cells compared to short ANAs, suggesting a role for these cells in poor axonal regeneration across nerve constructs

Gene expression predicts dormant metastatic breast cancer cell phenotype

BACKGROUND: Breast cancer can recur months to decades after an initial diagnosis and treatment. The mechanisms that control tumor cell dormancy remain poorly understood, making it difficult to predict which patients will recur and thus benefit from more rigorous screening and treatments. Unfortunately, the extreme rarity of dormant DTCs has been a major obstacle to their study. METHODS: To overcome this challenge, we developed an efficient system to isolate and study rare dormant breast cancer cells from metastatic organs including bones, which represent a major site of metastasis. After isolation of cells from the long bones, we used single cell RNA-sequencing (scRNA-seq) to profile proliferative and dormant PyMT-Bo1 breast cancer cells. We also compared this signature to dormant versus proliferative tumor cells isolated from the lungs. Finally, we compared our dormant signature to human datasets. RESULTS: We identified a group of genes including Cfh, Gas6, Mme and Ogn that were highly expressed in dormant breast cancer cells present in the bone and lung. Expression of these genes had no impact on dormancy in murine models, but their expression correlated with disease-free survival in primary human breast cancer tumors, suggesting that these genes have predictive value in determining which patients are likely to recur. CONCLUSIONS: Dormant breast cancer cells exhibit a distinct gene expression signature regardless of metastatic site. Genes enriched in dormant breast cancer cells correlate with recurrence-free survival in breast cancer patients

Human Dna2 is a nuclear and mitochondrial DNA maintenance protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance

A Cdh3-β-catenin-laminin signaling axis in a subset of breast tumor leader cells control leader cell polarization and directional collective migration

Carcinoma dissemination can occur when heterogeneous tumor and tumor-stromal cell clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system mimicking native breast tumor microenvironment, we developed 3D computational models, which hypothesize that leader cells need to generate high protrusive forces and overcome extracellular matrix (ECM) resistance at the leading edge. From single-cell sequencing analyses, we find that leader cells are heterogeneous and identify and isolate a keratin 14- and cadherin-3-positive subpopulation sufficient to lead collective migration. Cdh3 controls leader cell protrusion dynamics through local production of laminin, which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration