DNA methylation and gene expression as determinants of genome-wide cell-free DNA fragmentation.

Circulating cell-free DNA (cfDNA) is emerging as an avenue for cancer detection, but the characteristics of cfDNA fragmentation in the blood are poorly understood. We evaluate the effect of DNA methylation and gene expression on genome-wide cfDNA fragmentation through analysis of 969 individuals. cfDNA fragment ends more frequently contained CCs or CGs, and fragments ending with CGs or CCGs are enriched or depleted, respectively, at methylated CpG positions. Higher levels and larger sizes of cfDNA fragments are associated with CpG methylation and reduced gene expression. These effects are validated in mice with isogenic tumors with or without the mutant IDH1, and are associated with genome-wide changes in cfDNA fragmentation in patients with cancer. Tumor-related hypomethylation and increased gene expression are associated with decrease in cfDNA fragment size that may explain smaller cfDNA fragments in human cancers. These results provide a connection between epigenetic changes and cfDNA fragmentation with implications for disease detection.

Transfer Learning Reveals Cancer-Associated Fibroblasts Are Associated with Epithelial-Mesenchymal Transition and Inflammation in Cancer Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.

Genome-wide repeat landscapes in cancer and cell-free DNA.

Genetic changes in repetitive sequences are a hallmark of cancer and other diseases, but characterizing these has been challenging using standard sequencing approaches. We developed a de novo kmer finding approach, called ARTEMIS (Analysis of RepeaT EleMents in dISease), to identify repeat elements from whole-genome sequencing. Using this method, we analyzed 1.2 billion kmers in 2837 tissue and plasma samples from 1975 patients, including those with lung, breast, colorectal, ovarian, liver, gastric, head and neck, bladder, cervical, thyroid, or prostate cancer. We identified tumor-specific changes in these patients in 1280 repeat element types from the LINE, SINE, LTR, transposable element, and human satellite families. These included changes to known repeats and 820 elements that were not previously known to be altered in human cancer. Repeat elements were enriched in regions of driver genes, and their representation was altered by structural changes and epigenetic states. Machine learning analyses of genome-wide repeat landscapes and fragmentation profiles in cfDNA detected patients with early-stage lung or liver cancer in cross-validated and externally validated cohorts. In addition, these repeat landscapes could be used to noninvasively identify the tissue of origin of tumors. These analyses reveal widespread changes in repeat landscapes of human cancers and provide an approach for their detection and characterization that could benefit early detection and disease monitoring of patients with cancer.

Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment.

Three-dimensional (3D) in vitro models are essential in cancer research, but they often neglect physical forces. In our study, we combined patient-derived tumor organoids with a microfluidic organ-on-chip system to investigate colorectal cancer (CRC) invasion in the tumor microenvironment (TME). This allowed us to create patient-specific tumor models and assess the impact of physical forces on cancer biology. Our findings showed that the organoid-on-chip models more closely resembled patient tumors at the transcriptional level, surpassing organoids alone. Using 'omics' methods and live-cell imaging, we observed heightened responsiveness of KRAS mutant tumors to TME mechanical forces. These tumors also utilized the γ-aminobutyric acid (GABA) neurotransmitter as an energy source, increasing their invasiveness. This bioengineered model holds promise for advancing our understanding of cancer progression and improving CRC treatments.

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response.

Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg-expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses.

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells' (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo-derived senescence signature (SenSig) using a foreign body response-driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and "cartilage-like" fibroblasts as senescent and defined cell type-specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34-CSF1R-TGFßR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

Distinct Myeloid Derived Suppressor Cell Populations Promote Tumor Aggression in Glioblastoma.

The diversity of genetic programs and cellular plasticity of glioma-associated myeloid cells, and thus their contribution to tumor growth and immune evasion, is poorly understood. We performed single cell RNA-sequencing of immune and tumor cells from 33 glioma patients of varying tumor grades. We identified two populations characteristic of myeloid derived suppressor cells (MDSC), unique to glioblastoma (GBM) and absent in grades II and III tumors: i) an early progenitor population (E-MDSC) characterized by strong upregulation of multiple catabolic, anabolic, oxidative stress, and hypoxia pathways typically observed within tumor cells themselves, and ii) a monocytic MDSC (M-MDSC) population. The E-MDSCs geographically co-localize with a subset of highly metabolic glioma stem-like tumor cells with a mesenchymal program in the pseudopalisading region, a pathognomonic feature of GBMs associated with poor prognosis. Ligand-receptor interaction analysis revealed symbiotic cross-talk between the stemlike tumor cells and E-MDSCs in GBM, whereby glioma stem cells produce chemokines attracting E-MDSCs, which in turn produce growth and survival factors for the tumor cells. Our large-scale single-cell analysis elucidated unique MDSC populations as key facilitators of GBM progression and mediators of tumor immunosuppression, suggesting that targeting these specific myeloid compartments, including their metabolic programs, may be a promising therapeutic intervention in this deadly cancer. One-Sentence Summary: Aggressive glioblastoma harbors two unique myeloid populations capable of promoting stem-like properties of tumor cells and suppressing T cell function in the tumor microenvironment.

FOXM1 acts sexually dimorphically to regulate functional ß-cell mass.

The transcription factor FOXM1 regulates ß-cell proliferation and insulin secretion. Our previous work demonstrates that expressing an activated form of FOXM1 (FOXM1*) in ß cells increases ß-cell proliferation and mass in aged male mice. Additionally, FOXM1* enhances ß-cell function even in young mice, in which no ß-cell mass elevation occurs. Here, we demonstrate that FOXM1 acts in a sexually dimorphic manner in the ß cell. Expression of FOXM1* in female mouse ß cells does not affect ß-cell proliferation or glucose tolerance. Transduction of male but not female human islets with FOXM1* enhances insulin secretion in response to elevated glucose. Estrogen contributes to diabetes susceptibility differences between males and females, and the estrogen receptor (ER)α is the primary mediator of ß-cell estrogen signaling. We show that FOXM1* can rescue impaired glucose tolerance in female mice with a pancreas-wide ERα deletion. Further, FOXM1 and ERα binding sites overlap with each other and with other ß-cell-enriched transcription factors, including ISL1, PAX6, MAF, and GATA. These data indicate that FOMX1 and ERα cooperate to regulate ß-cell function and suggest a general mechanism contributing to the lower incidence of diabetes observed in women.

Persistent mutation burden drives sustained anti-tumor immune responses.

Tumor mutation burden is an imperfect proxy of tumor foreignness and has therefore failed to consistently demonstrate clinical utility in predicting responses in the context of immunotherapy. We evaluated mutations in regions of the genome that are unlikely to undergo loss in a pan-cancer analysis across 31 tumor types (n = 9,242) and eight immunotherapy-treated cohorts of patients with non-small-cell lung cancer, melanoma, mesothelioma, and head and neck cancer (n = 524). We discovered that mutations in single-copy regions and those present in multiple copies per cell constitute a persistent tumor mutation burden (pTMB) which is linked with therapeutic response to immune checkpoint blockade. Persistent mutations were retained in the context of tumor evolution under selective pressure of immunotherapy and tumors with a high pTMB content were characterized by a more inflamed tumor microenvironment. pTMB imposes an evolutionary bottleneck that cancer cells cannot overcome and may thus drive sustained immunologic tumor control in the context of immunotherapy.

Genomic Landscapes and Hallmarks of Mutant RAS in Human Cancers.

The RAS family of small GTPases represents the most commonly activated oncogenes in human cancers. To better understand the prevalence of somatic RAS mutations and the compendium of genes that are coaltered in RAS-mutant tumors, we analyzed targeted next-generation sequencing data of 607,863 mutations from 66,372 tumors in 51 cancer types in the AACR Project GENIE Registry. Bayesian hierarchical models were implemented to estimate the cancer-specific prevalence of RAS and non-RAS somatic mutations, to evaluate co-occurrence and mutual exclusivity, and to model the effects of tumor mutation burden and mutational signatures on comutation patterns. These analyses revealed differential RAS prevalence and comutations with non-RAS genes in a cancer lineage-dependent and context-dependent manner, with differences across age, sex, and ethnic groups. Allele-specific RAS co-mutational patterns included an enrichment in NTRK3 and chromatin-regulating gene mutations in KRAS G12C-mutant non-small cell lung cancer. Integrated multiomic analyses of 10,217 tumors from The Cancer Genome Atlas (TCGA) revealed distinct genotype-driven gene expression programs pointing to differential recruitment of cancer hallmarks as well as phenotypic differences and immune surveillance states in the tumor microenvironment of RAS-mutant tumors. The distinct genomic tracks discovered in RAS-mutant tumors reflected differential clinical outcomes in TCGA cohort and in an independent cohort of patients with KRAS G12C-mutant non-small cell lung cancer that received immunotherapy-containing regimens. The RAS genetic architecture points to cancer lineage-specific therapeutic vulnerabilities that can be leveraged for rationally combining RAS-mutant allele-directed therapies with targeted therapies and immunotherapy. SIGNIFICANCE: The complex genomic landscape of RAS-mutant tumors is reflective of selection processes in a cancer lineage-specific and context-dependent manner, highlighting differential therapeutic vulnerabilities that can be clinically translated.

Peripheral blood immune cell dynamics reflect antitumor immune responses and predict clinical response to immunotherapy.

BACKGROUND: Despite treatment advancements with immunotherapy, our understanding of response relies on tissue-based, static tumor features such as tumor mutation burden (TMB) and programmed death-ligand 1 (PD-L1) expression. These approaches are limited in capturing the plasticity of tumor-immune system interactions under selective pressure of immune checkpoint blockade and predicting therapeutic response and long-term outcomes. Here, we investigate the relationship between serial assessment of peripheral blood cell counts and tumor burden dynamics in the context of an evolving tumor ecosystem during immune checkpoint blockade. METHODS: Using machine learning, we integrated dynamics in peripheral blood immune cell subsets, including neutrophil-lymphocyte ratio (NLR), from 239 patients with metastatic non-small cell lung cancer (NSCLC) and predicted clinical outcome with immune checkpoint blockade. We then sought to interpret NLR dynamics in the context of transcriptomic and T cell repertoire trajectories for 26 patients with early stage NSCLC who received neoadjuvant immune checkpoint blockade. We further determined the relationship between NLR dynamics, pathologic response and circulating tumor DNA (ctDNA) clearance. RESULTS: Integrated dynamics of peripheral blood cell counts, predominantly NLR dynamics and changes in eosinophil levels, predicted clinical outcome, outperforming both TMB and PD-L1 expression. As early changes in NLR were a key predictor of response, we linked NLR dynamics with serial RNA sequencing deconvolution and T cell receptor sequencing to investigate differential tumor microenvironment reshaping during therapy for patients with reduction in peripheral NLR. Reductions in NLR were associated with induction of interferon-γ responses driving the expression of antigen presentation and proinflammatory gene sets coupled with reshaping of the intratumoral T cell repertoire. In addition, NLR dynamics reflected tumor regression assessed by pathological responses and complemented ctDNA kinetics in predicting long-term outcome. Elevated peripheral eosinophil levels during immune checkpoint blockade were correlated with therapeutic response in both metastatic and early stage cohorts. CONCLUSIONS: Our findings suggest that early dynamics in peripheral blood immune cell subsets reflect changes in the tumor microenvironment and capture antitumor immune responses, ultimately reflecting clinical outcomes with immune checkpoint blockade.

Durvalumab with platinum-pemetrexed for unresectable pleural mesothelioma: survival, genomic and immunologic analyses from the phase 2 PrE0505 trial.

Mesothelioma is a rare and fatal cancer with limited therapeutic options until the recent approval of combination immune checkpoint blockade. Here we report the results of the phase 2 PrE0505 trial ( NCT02899195 ) of the anti-PD-L1 antibody durvalumab plus platinum-pemetrexed chemotherapy for 55 patients with previously untreated, unresectable pleural mesothelioma. The primary endpoint was overall survival compared to historical control with cisplatin and pemetrexed chemotherapy; secondary and exploratory endpoints included safety, progression-free survival and biomarkers of response. The combination of durvalumab with chemotherapy met the pre-specified primary endpoint, reaching a median survival of 20.4 months versus 12.1 months with historical control. Treatment-emergent adverse events were consistent with known side effects of chemotherapy, and all adverse events due to immunotherapy were grade 2 or lower. Integrated genomic and immune cell repertoire analyses revealed that a higher immunogenic mutation burden coupled with a more diverse T cell repertoire was linked to favorable clinical outcome. Structural genome-wide analyses showed a higher degree of genomic instability in responding tumors of epithelioid histology. Patients with germline alterations in cancer predisposing genes, especially those involved in DNA repair, were more likely to achieve long-term survival. Our findings indicate that concurrent durvalumab with platinum-based chemotherapy has promising clinical activity and that responses are driven by the complex genomic background of malignant pleural mesothelioma.

Interleukin-36γ-producing macrophages drive IL-17-mediated fibrosis.

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)-4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17-dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.

A biologic scaffold-associated type 2 immune microenvironment inhibits tumor formation and synergizes with checkpoint immunotherapy.

Biomaterials in regenerative medicine are designed to mimic and modulate tissue environments to promote repair. Biologic scaffolds (derived from decellularized tissue extracellular matrix) promote a wound-healing (proregenerative) immune phenotype and are used clinically to treat tissue loss, including in the context of tumor resection. It is unknown whether a biomaterial microenvironment that encourages tissue formation may also promote tumor development. We implanted a urinary bladder matrix (UBM) scaffold, which is used clinically for wound management, with syngeneic cancer cell lines in mice to study how wound-healing immune responses affect tumor formation and sensitivity to immune checkpoint blockade. The UBM scaffold created an immune microenvironment that inhibited B16-F10 melanoma tumor formation in a CD4+ T cell-dependent and macrophage-dependent manner. In-depth immune characterization revealed an activated type 2-like immune response that was distinct from the classical tumor microenvironment, including activated type 2 T helper T cells, a unique macrophage phenotype, eosinophil infiltration, angiogenic factors, and complement. Tumor growth inhibition by PD-1 and PD-L1 checkpoint blockade was potentiated in the UBM scaffold immune microenvironment. Engineering the local tumor microenvironment to promote a type 2 wound-healing immune signature may serve as a therapeutic target to improve immunotherapy efficacy.

Steroid signaling promotes stem cell maintenance in the Drosophila testis.

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.

Epigenetic regulation of stem cell maintenance in the Drosophila testis via the nucleosome-remodeling factor NURF.

Regulation of stem cells depends on both tissue-specific transcriptional regulators and changes in chromatin organization, yet the coordination of these events in endogenous niches is poorly understood. In the Drosophila testis, local JAK-STAT signaling maintains germline and somatic stem cells (GSCs and cyst progenitor cells, or CPCs) in a single niche. Here we show that epigenetic regulation via the nucleosome-remodeling factor (NURF) complex ensures GSC and CPC maintenance by positively regulating JAK-STAT signaling, thereby preventing premature differentiation. Conversely, NURF is not required in early differentiating daughter cells of either lineage. Because three additional ATP-dependent chromatin remodelers (ACF, CHRAC, and dMi-2/NuRD) are dispensable for stem cell maintenance in the testis, epigenetic regulation of stem cells within this niche may rely primarily on NURF. Thus, local signals cooperate with specific chromatin-remodeling complexes in intact niches to coordinately regulate a common set of target genes to prevent premature stem cell differentiation.

Design, development, and implementation of the radiological physics center's pelvis and thorax anthropomorphic quality assurance phantoms.

The Radiological Physics Center (RPC) developed two heterogeneous anthropomorphic quality assurance phantoms for use in verifying the accuracy of radiation delivery: one for intensity-modulated radiation therapy (IMRT) to the pelvis and the other for stereotactic body radiation therapy (SBRT) to the thorax. The purpose of this study was to describe the design and development of these two phantoms and to demonstrate the reproducibility of measurements generated with them. The phantoms were built to simulate actual patient anatomy. They are lightweight and water-fillable, and they contain imageable targets and organs at risk of radiation exposure that are of similar densities to their human counterparts. Dosimetry inserts accommodate radiochromic film for relative dosimetry and thermoluminesent dosimetry capsules for absolute dosimetry. As a part of the commissioning process, each phantom was imaged, treatment plans were developed, and radiation was delivered at least three times. Under these controlled irradiation conditions, the reproducibility of dose delivery to the target TLD in the pelvis and thorax phantoms was 3% and 0.5%, respectively. The reproducibility of radiation-field localization was less than 2.5 mm for both phantoms. Using these anthropomorphic phantoms, pelvic IMRT and thoracic SBRT radiation treatments can be verified with a high level of precision. These phantoms can be used to effectively credential institutions for participation in specific NCI-sponsored clinical trials.