Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.

Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition.

Dendrimer-Inspired Nanomaterials for the in Vivo Delivery of siRNA to Lung Vasculature.

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from in vivo screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer-lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics.

A conditional system to specifically link disruption of protein-coding function with reporter expression in mice.

Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

Strategies to achieve conditional gene mutation in mice.

The laboratory mouse is an ideal model organism for studying disease because it is physiologically similar to human and also because its genome is readily manipulated. Genetic engineering allows researchers to introduce specific loss-of-function or gain-of-function mutations into genes and then to study the resulting phenotypes in an in vivo context. One drawback of using traditional transgenic and knockout mice to study human diseases is that many mutations passed through the germline can profoundly affect development, thus impeding the study of disease phenotypes in adults. New technology has made it possible to generate conditional mutations that can be introduced in a spatially and/or temporally restricted manner. Mouse strains carrying conditional mutations represent valuable experimental models for the study of human diseases and they can be used to develop strategies for prevention and treatment of these diseases. In this article, we will describe the most widely used DNA recombinase systems used to achieve conditional gene mutation in mouse models and discuss how these systems can be employed in vivo.

Whole-mount X-Gal staining of mouse tissues.

Although the development of improved mouse models, including conditional deletions, marks an exciting time in mouse genetics, it is important to characterize and validate these models. Cre reporter strains allow researchers to assess the recombinase expression profile and function in individual Cre mouse lines. These strains are engineered to express a reporter gene (usually LacZ) following the removal of a floxed STOP cassette, thus marking cell lineages that can be targeted with a given Cre line. This protocol provides a detailed method for the histochemical detection of ß-galactosidase activity in Cre mouse strains.

Producing and concentrating lenti-Cre for mouse infections.

Lentiviral vectors offer versatility as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome, which results in long-term expression of the transgene (Cre) both in vitro and in vivo. This protocol describes how lentiviral particles are produced, purified, and concentrated.

In vivo delivery of lenti-Cre or adeno-Cre into mice using intranasal instillation.

Lung cancer remains the leading cause of cancer deaths among both men and women, with a lower rate of survival than both breast and prostate cancer. Development of the Cre/lox system and improved mouse models have allowed researchers to gain a better understanding of human disease, including lung cancer. Through the viral delivery of Cre, gene function in adult mice can be precisely studied at a specific developmental stage or in a specific cell/tissue type of choice. This protocol describes how to produce adenovirus-Cre precipitate. Using this adeno-Cre (or lentivirus-Cre), Cre can be expressed in mouse lungs. The virus is delivered by intranasal instillation.

A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis.

Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3ß1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways.

When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

Somatic activation of a conditional KrasG12D allele causes ineffective erythropoiesis in vivo.

Somatic activation of a conditional targeted Kras(G12D) allele induces a fatal myeloproliferative disease in mice that closely models juvenile and chronic myelomonocytic leukemia. These mice consistently develop severe and progressive anemia despite adequate numbers of clonogenic erythroid progenitors in the bone marrow and expanded splenic hematopoiesis. Ineffective erythropoiesis is characterized by impaired differentiation. These results demonstrate that endogenous levels of oncogenic Ras have cell lineage-specific effects and support efforts to modulate Ras signaling for therapy of anemia in patients with myelodysplastic syndromes and myeloproliferative disorders.

Therapy-induced malignant neoplasms in Nf1 mutant mice.

Therapy-induced cancers are a severe complication of genotoxic therapies. We used heterozygous Nf1 mutant mice as a sensitized genetic background to investigate tumor induction by radiation (RAD) and cyclophosphamide (CY). Mutagen-exposed Nf1(+/-) mice developed secondary cancers that are common in humans, including myeloid malignancies, sarcomas, and breast cancers. RAD cooperated strongly with heterozygous Nf1 inactivation in tumorigenesis. Most of the solid tumors showed loss of the wild-type Nf1 allele but retained two Trp53 alleles. Comparative genomic hybridization demonstrated distinct patterns of copy number aberrations in sarcomas and breast cancers from Nf1 mutant mice, and tumor cell lines showed deregulated Ras signaling. Nf1(+/-) mice provide a tractable model for investigating the pathogenesis of common mutagen-induced cancers and for testing preventive strategies.

Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder.

RAS mutations are common in myeloid malignancies; however, it is not known whether oncogenic RAS can initiate leukemia. We show that expressing mutant K-Ras(G12D) protein from the endogenous murine locus rapidly induces a fatal myeloproliferative disorder with 100% penetrance characterized by tissue infiltration, hypersensitivity to growth factors, and hyperproliferation. Hematopoietic cells from diseased mice demonstrated increased levels of Ras-GTP, but effector kinases were not constitutively phosphorylated and responded normally to growth factors. Oncogenic RAS is sufficient to initiate myeloid leukemogenesis in mice, and this provides an in vivo system for biologic and preclinical studies.