Mapping the long road to cancer.

Every cell in our body accumulates mutations throughout life, and sometimes an unfortunate combination of mutations drives the initiation of cancer. A new study infers extraordinarily detailed timelines of pre-cancerous evolution by sequencing single-cell genomes in patients with blood malignancies-finding that key mutations can arrive decades before diagnosis.

Increased stem cell proliferation in atherosclerosis accelerates clonal hematopoiesis.

Clonal hematopoiesis, a condition in which individual hematopoietic stem cell clones generate a disproportionate fraction of blood leukocytes, correlates with higher risk for cardiovascular disease. The mechanisms behind this association are incompletely understood. Here, we show that hematopoietic stem cell division rates are increased in mice and humans with atherosclerosis. Mathematical analysis demonstrates that increased stem cell proliferation expedites somatic evolution and expansion of clones with driver mutations. The experimentally determined division rate elevation in atherosclerosis patients is sufficient to produce a 3.5-fold increased risk of clonal hematopoiesis by age 70. We confirm the accuracy of our theoretical framework in mouse models of atherosclerosis and sleep fragmentation by showing that expansion of competitively transplanted Tet2-/- cells is accelerated under conditions of chronically elevated hematopoietic activity. Hence, increased hematopoietic stem cell proliferation is an important factor contributing to the association between cardiovascular disease and clonal hematopoiesis.

B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis.

Autonomic nerves control organ function through the sympathetic and parasympathetic branches, which have opposite effects. In the bone marrow, sympathetic (adrenergic) nerves promote hematopoiesis; however, how parasympathetic (cholinergic) signals modulate hematopoiesis is unclear. Here, we show that B lymphocytes are an important source of acetylcholine, a neurotransmitter of the parasympathetic nervous system, which reduced hematopoiesis. Single-cell RNA sequencing identified nine clusters of cells that expressed the cholinergic α7 nicotinic receptor (Chrna7) in the bone marrow stem cell niche, including endothelial and mesenchymal stromal cells (MSCs). Deletion of B cell-derived acetylcholine resulted in the differential expression of various genes, including Cxcl12 in leptin receptor+ (LepR+) stromal cells. Pharmacologic inhibition of acetylcholine signaling increased the systemic supply of inflammatory myeloid cells in mice and humans with cardiovascular disease.

Profound Tissue Specificity in Proliferation Control Underlies Cancer Drivers and Aneuploidy Patterns.

Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma mouse models.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer.

BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.

A bilateral tumor model identifies transcriptional programs associated with patient response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) is efficacious in many diverse cancer types, but not all patients respond. It is important to understand the mechanisms driving resistance to these treatments and to identify predictive biomarkers of response to provide best treatment options for all patients. Here we introduce a resection and response-assessment approach for studying the tumor microenvironment before or shortly after treatment initiation to identify predictive biomarkers differentiating responders from nonresponders. Our approach builds on a bilateral tumor implantation technique in a murine metastatic breast cancer model (E0771) coupled with anti-PD-1 therapy. Using our model, we show that tumors from mice responding to ICB therapy had significantly higher CD8+ T cells and fewer Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) at early time points following therapy initiation. RNA sequencing on the intratumoral CD8+ T cells identified the presence of T cell exhaustion pathways in nonresponding tumors and T cell activation in responding tumors. Strikingly, we showed that our derived response and resistance signatures significantly segregate patients by survival and associate with patient response to ICB. Furthermore, we identified decreased expression of CXCR3 in nonresponding mice and showed that tumors grown in Cxcr3-/- mice had an elevated resistance rate to anti-PD-1 treatment. Our findings suggest that the resection and response tumor model can be used to identify response and resistance biomarkers to ICB therapy and guide the use of combination therapy to further boost the antitumor efficacy of ICB.

Consecutive seeding and transfer of genetic diversity in metastasis.

During metastasis, only a fraction of genetic diversity in a primary tumor is passed on to metastases. We calculate this fraction of transferred diversity as a function of the seeding rate between tumors. At one extreme, if a metastasis is seeded by a single cell, then it inherits only the somatic mutations present in the founding cell, so that none of the diversity in the primary tumor is transmitted to the metastasis. In contrast, if a metastasis is seeded by multiple cells, then some genetic diversity in the primary tumor can be transmitted. We study a multitype branching process of metastasis growth that originates from a single cell but over time receives additional cells. We derive a surprisingly simple formula that relates the expected diversity of a metastasis to the diversity in the pool of seeding cells. We calculate the probability that a metastasis is polyclonal. We apply our framework to published datasets for which polyclonality has been previously reported, analyzing 68 ovarian cancer samples, 31 breast cancer samples, and 8 colorectal cancer samples from 15 patients. For these clonally diverse metastases, under typical metastasis growth conditions, we find that 10 to 150 cells seeded each metastasis and left surviving lineages between initial formation and clinical detection.

Reprogramming the microenvironment with tumor-selective angiotensin blockers enhances cancer immunotherapy.

Cancer-associated fibroblasts (CAFs) can either suppress or support T lymphocyte activity, suggesting that CAFs may be reprogrammable to an immunosupportive state. Angiotensin receptor blockers (ARBs) convert myofibroblast CAFs to a quiescent state, but whether ARBs can reprogram CAFs to promote T lymphocyte activity and enhance immunotherapy is unknown. Moreover, ARB doses are limited by systemic adverse effects such as hypotension due to the importance of angiotensin signaling outside tumors. To enhance the efficacy and specificity of ARBs in cancer with the goal of revealing their effects on antitumor immunity, we developed ARB nanoconjugates that preferentially accumulate and act in tumors. We created a diverse library of hundreds of acid-degradable polymers and chemically linked ARBs to the polymer most sensitive to tumor pH. These tumor microenvironment-activated ARBs (TMA-ARBs) remain intact and inactive in circulation while achieving high concentrations in tumors, wherein they break down to active ARBs. This tumor-preferential activity enhances the CAF-reprogramming effects of ARBs while eliminating blood pressure-lowering effects. Notably, TMA-ARBs alleviate immunosuppression and improve T lymphocyte activity, enabling dramatically improved responses to immune-checkpoint blockers in mice with primary as well as metastatic breast cancer.

Bone Marrow Endothelial Cells Regulate Myelopoiesis in Diabetes Mellitus.

BACKGROUND: Diabetes mellitus is a prevalent public health problem that affects about one-third of the US population and leads to serious vascular complications with increased risk for coronary artery disease. How bone marrow hematopoiesis contributes to diabetes mellitus complications is incompletely understood. We investigated the role of bone marrow endothelial cells in diabetic regulation of inflammatory myeloid cell production. METHODS: In 3 types of mouse diabetes mellitus, including streptozotocin, high-fat diet, and genetic induction using leptin-receptor-deficient db/db mice, we assayed leukocytes, hematopoietic stem and progenitor cells (HSPC). In addition, we investigated bone marrow endothelial cells with flow cytometry and expression profiling. RESULTS: In diabetes mellitus, we observed enhanced proliferation of HSPC leading to augmented circulating myeloid cell numbers. Analysis of bone marrow niche cells revealed that endothelial cells in diabetic mice expressed less Cxcl12, a retention factor promoting HSPC quiescence. Transcriptome-wide analysis of bone marrow endothelial cells demonstrated enrichment of genes involved in epithelial growth factor receptor (Egfr) signaling in mice with diet-induced diabetes mellitus. To explore whether endothelial Egfr plays a functional role in myelopoiesis, we generated mice with endothelial-specific deletion of Egfr (Cdh5CreEgfrfl/fl). We found enhanced HSPC proliferation and increased myeloid cell production in Cdh5CreEgfrfl/fl mice compared with wild-type mice with diabetes mellitus. Disrupted Egfr signaling in endothelial cells decreased their expression of the HSPC retention factor angiopoietin-1. We tested the functional relevance of these findings for wound healing and atherosclerosis, both implicated in complications of diabetes mellitus. Inflammatory myeloid cells accumulated more in skin wounds of diabetic Cdh5CreEgfrfl/fl mice, significantly delaying wound closure. Atherosclerosis was accelerated in Cdh5CreEgfrfl/fl mice, leading to larger and more inflamed atherosclerotic lesions in the aorta. CONCLUSIONS: In diabetes mellitus, bone marrow endothelial cells participate in the dysregulation of bone marrow hematopoiesis. Diabetes mellitus reduces endothelial production of Cxcl12, a quiescence-promoting niche factor that reduces stem cell proliferation. We describe a previously unknown counterregulatory pathway, in which protective endothelial Egfr signaling curbs HSPC proliferation and myeloid cell production.

Stress granule-associated protein G3BP2 regulates breast tumor initiation.

Breast tumors contain tumorigenic cancer cells, termed "tumor-initiating cells" (TICs), which are capable of both replenishing themselves and giving rise to populations of nontumorigenic breast cancer cells (non-TICs). However, the molecular mechanisms responsible for breast tumor initiation remain poorly understood. Here we describe a chemical screening strategy to identify small molecules that enhance the effect of chemotherapeutic agents on TIC-enriched breast cancer cells. We identified proteins that interact with the lead compound C108, including the stress granule-associated protein, GTPase-activating protein (SH3 domain)-binding protein 2, G3BP2. G3BP2 regulates breast tumor initiation through the stabilization of Squamous cell carcinoma antigen recognized by T cells 3 (SART3) mRNA, which leads to increased expression of the pluripotency transcription factors Octamer-binding protein 4 (Oct-4) and Nanog Homeobox (Nanog). Our findings suggest that G3BP2 is important for the process of breast cancer initiation. Furthermore, these data suggest a possible connection between stress granule formation and tumor initiation in breast cancer cells.

Hypermutable DNA chronicles the evolution of human colon cancer.

Intratumor genetic heterogeneity reflects the evolutionary history of a cancer and is thought to influence treatment outcomes. Here we report that a simple PCR-based assay interrogating somatic variation in hypermutable polyguanine (poly-G) repeats can provide a rapid and reliable assessment of mitotic history and clonal architecture in human cancer. We use poly-G repeat genotyping to study the evolution of colon carcinoma. In a cohort of 22 patients, we detect poly-G variants in 91% of tumors. Patient age is positively correlated with somatic mutation frequency, suggesting that some poly-G variants accumulate before the onset of carcinogenesis during normal division in colonic stem cells. Poorly differentiated tumors have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We generate poly-G mutation profiles of spatially separated samples from primary carcinomas and matched metastases to build well-supported phylogenetic trees that illuminate individual patients' path of metastatic progression. Our results show varying degrees of intratumor heterogeneity among patients. Finally, we show that poly-G mutations can be found in other cancers than colon carcinoma. Our approach can generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

TGF-ß blockade improves the distribution and efficacy of therapeutics in breast carcinoma by normalizing the tumor stroma.

Although the role of TGF-ß in tumor progression has been studied extensively, its impact on drug delivery in tumors remains far from understood. In this study, we examined the effect of TGF-ß blockade on the delivery and efficacy of conventional therapeutics and nanotherapeutics in orthotopic mammary carcinoma mouse models. We used both genetic (overexpression of sTßRII, a soluble TGF-ß type II receptor) and pharmacologic (1D11, a TGF-ß neutralizing antibody) approaches to block TGF-ß signaling. In two orthotopic mammary carcinoma models (human MDA-MB-231 and murine 4T1 cell lines), TGF-ß blockade significantly decreased tumor growth and metastasis. TGF-ß blockade also increased the recruitment and incorporation of perivascular cells into tumor blood vessels and increased the fraction of perfused vessels. Moreover, TGF-ß blockade normalized the tumor interstitial matrix by decreasing collagen I content. As a result of this vessel and interstitial matrix normalization, TGF-ß blockade improved the intratumoral penetration of both a low-molecular-weight conventional chemotherapeutic drug and a nanotherapeutic agent, leading to better control of tumor growth.