Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the ...global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1βhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.
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•Stereotyped keratinocyte subpopulations modularly comprise human epidermis•Scalp keratinocytes exhibit an inherent inflammatory transcriptional program•Myeloid dendritic cells predominate APCs in psoriatic epidermis•Macrophages represent major APCs in foreskin epidermis
Cheng et al. report single-cell RNA sequencing of normal and inflamed human epidermis, revealing a discrete set of specialized keratinocytes that exhibit a distinct composition at different anatomic sites. Myeloid dendritic cells and macrophages also vary sharply with epidermal anatomic site and inflammation, indicating dynamic programming of antigen-presenting cells.
The success of immunotherapy with immune checkpoint inhibitors (ICIs) in a subset of individuals has been very exciting. However, in many cancers, responses to current ICIs are modest and are seen ...only in a small subsets of patients. Herein, a widely applicable approach that increases the benefit of ICIs is reported. Intratumoral administration of augmenting immune response and inhibiting suppressive environment of tumors—AIRISE‐02 nanotherapeutic that co‐delivers CpG and STAT3 siRNA—results in not only regression of the injected tumor, but also tumors at distant sites in multiple tumor model systems. In particular, three doses of AIRISE‐02 in combination with systemic ICIs completely cure both treated and untreated aggressive melanoma tumors in 63% of mice, while ICIs alone do not cure any mice. A long‐term memory immune effect is also reported. AIRISE‐02 is effective in breast and colon tumor models as well. Lastly, AIRISE‐02 is well tolerated in mice and nonhuman primates. This approach combines multiple therapeutic agents into a single nanoconstruct to create whole‐body immune responses across multiple cancer types. Being a local therapeutic, AIRISE‐02 circumvents regulatory challenges of systemic nanoparticle delivery, facilitating rapid translation to the clinic. AIRISE‐02 is under investigational new drug (IND)‐enabling studies, and clinical trials will soon follow.
Augmenting immune response and inhibiting suppressive environment of tumors (AIRISE‐02) is a nano‐immunotherapeutic candidate that co‐delivers CpG and STAT3 siRNA to a local tumor, generating anti‐tumor immune response against cancer everywhere in the body (both treated and untreated tumors). Combination of AIRISE‐02 and standard immune checkpoint inhibitors cures 63% of mice with melanoma tumors, while the inhibitors alone cure none.
We consider the problem of reconstructing a gene regulatory network structure from limited time series gene expression data, without any a priori knowledge of connectivity. We assume that the network ...is sparse, meaning the connectivity among genes is much less than full connectivity. We develop a method for network reconstruction based on compressive sensing, which takes advantage of the network's sparseness.
For the case in which all genes are accessible for measurement, and there is no measurement noise, we show that our method can be used to exactly reconstruct the network. For the more general problem, in which hidden genes exist and all measurements are contaminated by noise, we show that our method leads to reliable reconstruction. In both cases, coherence of the model is used to assess the ability to reconstruct the network and to design new experiments. We demonstrate that it is possible to use the coherence distribution to guide biological experiment design effectively. By collecting a more informative dataset, the proposed method helps reduce the cost of experiments. For each problem, a set of numerical examples is presented.
The method provides a guarantee on how well the inferred graph structure represents the underlying system, reveals deficiencies in the data and model, and suggests experimental directions to remedy the deficiencies.
This study explores the roles of genome copy number abnormalities (CNAs) in breast cancer pathophysiology by identifying associations between recurrent CNAs, gene expression, and clinical outcome in ...a set of aggressively treated early-stage breast tumors. It shows that the recurrent CNAs differ between tumor subtypes defined by expression pattern and that stratification of patients according to outcome can be improved by measuring both expression and copy number, especially high-level amplification. Sixty-six genes deregulated by the high-level amplifications are potential therapeutic targets. Nine of these (
FGFR1,
IKBKB,
ERBB2,
PROCC,
ADAM9,
FNTA,
ACACA,
PNMT, and
NR1D1) are considered druggable. Low-level CNAs appear to contribute to cancer progression by altering RNA and cellular metabolism.
To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array ...analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.
Systematically identifying synergistic combinations of targeted agents and immunotherapies for cancer treatments remains difficult. In this study, we integrated high-throughput and high-content ...techniques-an implantable microdevice to administer multiple drugs into different sites in tumors at nanodoses and multiplexed imaging of tumor microenvironmental states-to investigate the tumor cell and immunological response signatures to different treatment regimens. Using a mouse model of breast cancer, we identified effective combinations from among numerous agents within days. In vivo studies in three immunocompetent mammary carcinoma models demonstrated that the predicted combinations synergistically increased therapeutic efficacy. We identified at least five promising treatment strategies, of which the panobinostat, venetoclax and anti-CD40 triple therapy was the most effective in inducing complete tumor remission across models. Successful drug combinations increased spatial association of cancer stem cells with dendritic cells during immunogenic cell death, suggesting this as an important mechanism of action in long-term breast cancer control.
We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer.
We measured messenger RNA (mRNA) levels of Dicer and Drosha in specimens of invasive epithelial ovarian ...cancer from 111 patients, using a quantitative reverse-transcriptase-polymerase-chain-reaction assay, and compared the results with clinical outcomes. Validation was performed with the use of published microarray data from cohorts of patients with ovarian, breast, and lung cancer. Mutational analyses of genomic DNA from the Dicer and Drosha genes were performed in a subgroup of ovarian-cancer specimens. Dicer-dependent functional assays were performed by means of in vitro transfection with small interfering RNA (siRNA) and short hairpin RNA (shRNA).
Levels of Dicer and Drosha mRNA correlated with the levels of expression of the corresponding protein and were decreased in 60% and 51% of ovarian-cancer specimens, respectively. Low Dicer expression was significantly associated with advanced tumor stage (P=0.007), and low Drosha expression with suboptimal surgical cytoreduction (P=0.02). Cancer specimens with both high Dicer expression and high Drosha expression were associated with increased median survival (>11 years, vs. 2.66 years for other subgroups; P<0.001). We found three independent predictors of reduced disease-specific survival in multivariate analyses: low Dicer expression (hazard ratio, 2.10; P=0.02), high-grade histologic features (hazard ratio, 2.46; P=0.03), and poor response to chemotherapy (hazard ratio, 3.95; P<0.001). Poor clinical outcomes among patients with low Dicer expression were validated in additional cohorts of patients. Rare missense mutations were found in the Dicer and Drosha genes, but their presence or absence did not correlate with the level of expression. Functional assays indicated that gene silencing with shRNA, but not siRNA, may be impaired in cells with low Dicer expression.
Our findings indicate that levels of Dicer and Drosha mRNA in ovarian-cancer cells have associations with outcomes in patients with ovarian cancer.
Recent studies suggest that thousands of genes may contribute to breast cancer pathophysiologies when deregulated by genomic or epigenomic events. Here, we describe a model “system” to appraise the ...functional contributions of these genes to breast cancer subsets. In general, the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors, although some significant differences are documented. The cell lines that comprise the system also exhibit the substantial genomic, transcriptional, and biological heterogeneity found in primary tumors. We show, using Trastuzumab (Herceptin) monotherapy as an example, that the system can be used to identify molecular features that predict or indicate response to targeted therapies or other physiological perturbations.
The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes ...is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments.
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•Bioprinted tumor tissue is a scaffold-free model of tumor-stromal interactions•Cells within bioprinted tissues mature, self-organize, and deposit matrix proteins•Heterogeneity in therapeutic response, migration, and signaling can be assessed•Primary patient tissue can be bioprinted into tissues for translational studies
Langer et al. use three-dimensional bioprinting to incorporate multiple cell types, including patient-derived cells, into scaffold-free in vitro tumor tissues. They show that cells within these tissues self-organize, secrete extracellular matrix factors, and respond to extrinsic signals and that multiple tumorigenic phenotypes can be assessed simultaneously.
Oncogenic RAS mutations drive cancers at many sites. Recent reports suggest that RAS dimerization, multimerization, and clustering correlate strongly with activation of RAS signaling. We have found ...that re-expression of DIRAS3, a RAS-related small GTPase tumor suppressor that is downregulated in multiple cancers, inhibits RAS/mitogen-activated protein kinase (MAPK) signaling by interacting directly with RAS-forming heteromers, disrupting RAS clustering, inhibiting Raf kinase activation, and inhibiting transformation and growth of cancer cells and xenografts. Disruption of K-RAS cluster formation requires the N terminus of DIRAS3 and interaction of both DIRAS3 and K-RAS with the plasma membrane. Interaction of DIRAS3 with both K-RAS and H-RAS suggests a strategy for inhibiting oncogenic RAS function.
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•Re-expression of DIRAS3 (ARHI) inhibits ovarian, lung, and pancreatic cancer growth•DIRAS3 (ARHI) interacts directly with RAS at the plasma membrane•Re-expression of DIRAS3 inhibits RAS clustering and downstream MAPK signaling•The N-terminal extension of DIRAS3 is required for its tumor-suppressive function
Sutton et. al. show that re-expression of DIRAS3 can inhibit the growth of multiple cancer types driven by K-RAS mutations by a direct interaction and disruption of K-RAS higher ordered clusters. This phenotype is driven by an N-terminal extension, which distinguishes DIRAS3 from other RAS-related small GTPases.
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