RNA-seq based transcriptomic map reveals new insights into mouse salivary gland development and maturation

Heatmap depicting the hierarchical clustering of the 45 genes that are conserved between the mouse adult salivary gland gene signature and the RNA-seq data obtained from the Human Protein Atlas. The values reported represent Z-scores of the conserved genes in their respective datasets. (PDF 737 kb

The Reign of Follistatin in Tumors and Their Microenvironment: Implications for Drug Resistance

Follistatin (FST) is a potent neutralizer of the transforming growth factor-β superfamily and is associated with normal cellular programs and various hallmarks of cancer, such as proliferation, migration, angiogenesis, and immune evasion. The aberrant expression of FST by solid tumors is a well-documented observation, yet how FST influences tumor progression and therapy response remains unclear. The recent surge in omics data has revealed new insights into the molecular foundation underpinning tumor heterogeneity and its microenvironment, offering novel precision medicine-based opportunities to combat cancer. In this review, we discuss these recent FST-centric studies, thereby offering an updated perspective on the protean role of FST isoforms in shaping the complex cellular ecosystem of tumors and in mediating drug resistance

A Systemic and Integrated Analysis of p63-Driven Regulatory Networks in Mouse Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity and is linked to tobacco exposure, alcohol consumption, and human papillomavirus infection. Despite therapeutic advances, a lack of molecular understanding of disease etiology, and delayed diagnoses continue to negatively affect survival. The identification of oncogenic drivers and prognostic biomarkers by leveraging bulk and single-cell RNA-sequencing datasets of OSCC can lead to more targeted therapies and improved patient outcomes. However, the generation, analysis, and continued utilization of additional genetic and genomic tools are warranted. Tobacco-induced OSCC can be modeled in mice via 4-nitroquinoline 1-oxide (4NQO), which generates a spectrum of neoplastic lesions mimicking human OSCC and upregulates the oncogenic master transcription factor p63. Here, we molecularly characterized established mouse 4NQO treatment-derived OSCC cell lines and utilized RNA and chromatin immunoprecipitation-sequencing to uncover the global p63 gene regulatory and signaling network. We integrated our p63 datasets with published bulk and single-cell RNA-sequencing of mouse 4NQO-treated tongue and esophageal tumors, respectively, to generate a p63-driven gene signature that sheds new light on the role of p63 in murine OSCC. Our analyses reveal known and novel players, such as COTL1, that are regulated by p63 and influence various oncogenic processes, including metastasis. The identification of new sets of potential biomarkers and pathways, some of which are functionally conserved in human OSCC and can prognosticate patient survival, offers new avenues for future mechanistic studies

Functional characterization and genomic studies of a novel murine submandibular gland epithelial cell line

<div><p>A better understanding of the normal and diseased biology of salivary glands (SG) has been hampered, in part, due to difficulties in cultivating and maintaining salivary epithelial cells. Towards this end, we have generated a mouse salivary gland epithelial cell (mSGc) culture system that is well-suited for the molecular characterization of SG cells and their differentiation program. We demonstrate that mSGc can be maintained for multiple passages without a loss of proliferation potential, readily form 3D-spheroids and importantly express a panel of well-established salivary gland epithelial cell markers. Moreover, mSGc 3D-spheroids also exhibit functional maturation as evident by robust agonist-induced intracellular calcium signaling. Finally, transcriptomic characterization of mSGc by RNA-seq and hierarchical clustering analysis with adult organ RNA-seq datasets reveal that mSGc retain most of the molecular attributes of adult mouse salivary gland. This well-characterized mouse salivary gland cell line will fill a critical void in the field by offering a valuable resource to examine various mechanistic aspects of mouse salivary gland biology.</p></div

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance

Abstract Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance

Additional file 7: Table S1. of RNA-seq based transcriptomic map reveals new insights into mouse salivary gland development and maturation

Developmental gene signature. Table S2. Common genes expressed between salivary gland, pancreas, skin, bladder and placenta. Table S3. Common genes expressed between salivary gland and pancreas. Table S4. Adult gene signature. Table S5. HPA data. Table S6. Human CAGE data. (XLSX 80 kb

Growth analysis and 3D-spheroid formation of a spontaneously immortalized mouse salivary gland epithelial cell line (mSGc).

<p>(A-B) Early (~P20) and late passage (~P70) mSGc were seeded in serum free media (SFM) or serum containing media (SCM) as indicated, and a single-cell colony forming assay was performed. Within 5 days, large colonies were clearly visible in all cell and media conditions tested. (C) Quantification of colony forming assay performed in panel A and B. Data are represented as mean ± Standard Deviation (S.D.), <i>n = 5</i>. (D) mSGc stained with Rhodamine B revealed no differences in clonogenic potential. (E) Quantification of clonogenic results obtained in panel D above. Data are represented as mean ± Standard Deviation (S.D.), <i>n = 4</i>. (F) Late passage mSGc grown on matrigel organize into 3D-spheroid structures. Scale bar, 100μm.</p

3D-spheroids express salivary gland epithelial markers.

<p>(A) Immunofluorescence staining of a sub-set of salivary gland cell markers in mSGc grown as monolayers (upper panel) and 10 day 3D-spheroids (lower panel). Scale bar 18.75 μm. (B) Quantitative RT-PCR analysis demonstrating the mRNA expression levels of a panel of salivary gland cell markers in 3D-spheroids grown for 10 days as compared to mSGc grown as monolayers. C) Immunofluorescence staining of acinar cell markers in 3D-spheroids grown for 10 days. Values were normalized to the housekeeping gene Hprt. Data are represented as the mean ±S.E. of three independent experiments. *P<0.005, Student’s <i>t</i>-test.</p

Hierarchical clustering of mSGc and mouse tissues.

<p>Transcripts per million (TPM) values from the top 1500 genes with the highest mean absolute deviation were used to cluster mSGc and adult mouse tissues (Pearson Correlation, Average Linkage). The resulting heat map demonstrates that mSGc cluster closely with the mouse salivary gland (green box).</p