Probes for INS

The epithelial lining of the Fallopian tube is vital for fertility, providing nutrition to gametes, and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions are primarily consisting of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing Fallopian tube epithelial homoeostasis are currently unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and different Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple transgenic mouse models, we showed that Wnt/β-catenin signaling is essential for self-renewal as well as differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.

Abstract

Objectives

The aim of the present study was to investigate the molecular basis for the use of immune checkpoint inhibitors to treat salivary gland carcinomas (SGC).

Materials and methods

We examined the clinical and prognostic significance of programed death ligands 1 and 2 (PD-L1 and -L2) expression using immunohistochemistry and in situ hybridization, as well as microsatellite instability (MSI) status using polymerase chain reaction, along with tumor-infiltrating lymphocytes (TILs) in 30 cases of SGC.

Results

The SGC cases studied included adenoid cystic carcinoma (AdCC, 36.7%), salivary duct carcinoma (SDC, 26.7%), mucoepidermoid carcinoma (MEC, 23.3%), and carcinoma ex pleomorphic adenoma (CxPA, 13.3%). Either PD-L1 or PD-L2 overexpression was observed in 36.7% patients. PD-L2 expression was associated with reduced disease-specific survival (DSS) and disease-free survival (DFS) (P = 0.0266 and P = 0.0209, respectively). Simultaneous PD-L1 and PD-L2 overexpression was detected in 13.3% of cases, and was correlated with reduced DSS (P = 0.0113). Among non-AdCCs, all cases that developed distant metastasis were positive for PD-L2 (P = 0.001). Cases showing low-TILs that were positive for either PD-L1 or L2 were associated with poor DFS. No MSI was detected in the SGC cases studied.

Conclusion

To our knowledge, this is the first comprehensive study examining PD-L1 and PD-L2 status, MSI status, and TILs in SGC. Our results indicate that the PD-1/PD-L1 or PD-L2 pathway, which is associated with poor clinical outcomes, may provide promising therapeutic targets against SGC in selected patients. Further experimental and clinical studies are encouraged.

Spermatogonial stem cells (SSCs) fuel the production of male germ cells but the mechanisms behind SSC self-renewal, proliferation, and differentiation are still poorly understood. Using the Wnt target gene Axin2 and genetic lineage-tracing experiments, we found that undifferentiated spermatogonia, comprising SSCs and transit amplifying progenitor cells, respond to Wnt/β-catenin signals. Genetic elimination of β-catenin indicates that Wnt/β-catenin signaling promotes the proliferation of these cells. Signaling is likely initiated by Wnt6, which is uniquely expressed by neighboring Sertoli cells, the only somatic cells in the seminiferous tubule that support germ cells and act as a niche for SSCs. Therefore, unlike other stem cell systems where Wnt/β-catenin signaling is implicated in self-renewal, the Wnt pathway in the testis specifically contributes to the proliferation of SSCs and progenitor cells.

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.