Probes for INS

Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.

Abstract

BACKGROUND:

Craniopharyngiomas are neoplasms of the sellar/parasellar region that are classified into adamantinomatous (ACP) and papillary (PCP) subtypes. Surgical resection of craniopharyngiomas is challenging, and recurrence is common, frequently leading to profound morbidity. BRAF V600E mutations render PCP susceptible to BRAF/MEK inhibitors, but effective targeted therapies are needed for ACP. We explored the feasibility of targeting the PD-1/PD-L1 immune checkpoint pathway in ACP and PCP.

METHODS:

We mapped and quantified PD-L1 and PD-1 expression in ACP and PCP resections using immunohistochemistry, immunofluorescence, and RNA in situ hybridization. We used tissue-based cyclic immunofluorescence (t-CyCIF) to map the spatial distribution of immune cells and characterize cell cycle and signaling pathways in ACP tumor cells which intrinsically express PD-1.

RESULTS:

All ACP (15±14% of cells, n=23, average±S.D.) and PCP (35±22% of cells, n=18) resections expressed PD-L1. In ACP, PD-L1 was predominantly expressed by tumor cells comprising the cyst-lining. In PCP, PD-L1 was highly-expressed by tumor cells surrounding the stromal fibrovascular cores. ACP also exhibited tumor cell-intrinsic PD-1 expression in whorled epithelial cells with nuclear-localized beta-catenin. These cells exhibited evidence of elevated mTOR and MAPK signaling. Profiling of immune populations in ACP and PCP showed a modest density of CD8+ T-cells.

CONCLUSIONS:

ACP exhibit PD-L1 expression in the tumor cyst-lining and intrinsic PD-1 expression in cells proposed to comprise an oncogenic stem-like population. In PCP, proliferative tumor cells express PD-L1 in a continuous band at the stromal-epithelial interface. Targeting PD-L1 and/or PD-1 in both subtypes of craniopharyngioma might therefore be an effective therapeutic strategy.

PD-L1 expression may be a predictive marker for anti-PD-1 therapeutic efficacy. No standard detection method of PD-L1 expression was available for advanced gastric cancer (AGC), which would be investigated in this study using RNA in situ hybridization and immunohistochemistry. Patients (N = 165) with AGC treated at Peking University Cancer Hospital from October 2008 to February 2013 were retrospectively studied. Tissue samples prior to chemotherapy were assessed for PD-L1 expression using RNA in situ hybridization (an RNAscope assay) and immunohistochemistry (IHC). The correlations of PD-L1 expression to patient characteristics and clinical outcomes were statistically analyzed. PD-L1 mRNA signals were located in tumor compartments or the mesenchyme in a brown dotted or clustered pattern, and PD-L1 mRNA expression in gastric cancer was heterogeneous. PD-L1-positive expressions were observed in 33.9% (56/165) and 35.1% (46/131) patients in mRNA level and protein level, respectively. A positive relationship was found between PD-L1 mRNA and PD-L1 protein, and compared to IHC, RNAscope assay could provide an intuitional and quantitative data with potential clinical application. No statistically significant differences occurred between PD-L1 expression and clinical response to chemotherapy, or survival. However, we found that PD-L1 expression was higher in intestinal type than in diffuse type. These findings suggested that the RNAscope assay may be a promising method for patient assessment in gastric cancer clinical trials, which would be illustrated in further study.

ABSTRACT

Triple-negative breast cancer (TNBC) represents a more aggressive and difficult subtype of breast cancer where responses to chemotherapy occur, but toxicity is significant and resistance often follows. Immunotherapy has shown promising results in various types of cancer, including breast cancer. Here, we investigated a new combination strategy where histone deacetylase inhibitors (HDACi) are applied with immune checkpoint inhibitors to improve immunotherapy responses in TNBC.

Testing different epigenetic modifiers, we focused on the mechanisms underlying HDACi as priming modulators of immunotherapy. Tumor cells were co-cultured with human peripheral blood mononuclear cells (PBMCs) and flow cytometric immunophenotyping was performed to define the role of epigenetic priming in promoting tumor antigen presentation and immune cell activation. We found that HDACi up-regulate PD-L1 mRNA and protein expression in a time-dependent manner in TNBC cells, but not in hormone responsive cells. Focusing on TNBC, HDACi up-regulated PD-L1 and HLA-DR on tumor cells when co-cultured with PBMCs and down-regulated CD4+ Foxp3+ Treg in vitro. HDACi significantly enhanced the in vivo response to PD-1/CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model, the only currently available experimental system with functional resemblance to human TNBC. This resulted in a significant decrease in tumor growth and increased survival, associated with increased T cell tumor infiltration and a reduction in CD4+Foxp3+ T cells in the tumor microenvironment. Overall, our results suggest a novel role for HDAC inhibition in combination with immune checkpoint inhibitors and identify a promising therapeutic strategy, supporting its further clinical evaluation for TNBC treatment.

Chemokine (C-C motif) ligand 5 (CCL5) belongs to a group of chemokines that play a role in the peripheral immune system, mostly as chemoattractant molecules, and mediate tactile allodynia. In the central nervous system (CNS), CCL5 and its receptors have multiple functions, including promoting neuroinflammation, insulin signaling, neuromodulator of synaptic activity and neuroprotection against a variety of neurotoxins. Evidence has also suggested that this chemokine may regulate opioid response. The multifunctional profile of CCL5 might correlate with its ability to bind different chemokine receptors, as well as with its unique cellular expression. In this work, we have used fluorescence in situ hybridization combined with immunohistochemistry to examine the expression profile of CCL5 mRNA in the adult rat brain and provide evidence of its cellular localization. We have observed that the highest expression of CCL5 mRNA occurs in all major fiber tracts, including the corpus callosum, anterior commissure, and cerebral peduncle. In these tracts, CCL5 mRNA was localized in oligodendrocytes, astrocytes and microglia. Astrocytic and microglial expression was also evident in several brain areas including the cerebral cortex, caudate/putamen, hippocampus, and thalamus. Furthermore, using a specific neuronal marker, we observed CCL5 mRNA expression in discrete layers of the cortex and hippocampus. Interestingly, in the midbrain, CCL5 mRNA co-localized with tyrosine hydroxylase (TH) positive cells of the ventral tegmental area, suggesting that CCL5 might be expressed by a subset of dopaminergic neurons of the mesolimbic system. The expression of CCL5 mRNA and protein, together with its receptors, in selected brain cell populations proposes that this chemokine could be involved in neuronal/glial communication.

Lung adenosquamous cell carcinomas (ASCs) is a rare variant of NSCLC with a poorer prognosis and fewer treatment option than the more common variants. PD-L1 expression is reported to be the predictor of clinical response in trials of NSCLC. In our study, PD-L1 expression was evaluated via immunohistochemistry using a specific monoclonal antibody (SP263), and PD-L1 mRNA expression was evaluated via in situ hybridization. This study included 51 ASCs, 133 lung adenocarcinomas, and 83 lung squamous cell carcinomas (SCC). Similar results were obtained for PD-L1 expression measured at the mRNA and protein level (k coefficient, 0.851, P = 1.000). PD-L1 expression was significantly higher in the squamous versus glandular component of the 36 ASCs in which the components were analyzed separately. The PD-L1 expression rate was similar in the squamous cell component of ASCs and lung SCC (38.89% vs. 28.92%, P = 0.293), so does the adenocarcinoma component of ASCs and lung adenocarcinomas (11.11% vs 13.53%, P = 1.000). PD-L1 expression correlated significantly with lymphovascular invasion (P = 0.016), but not with EGFR, KRAS, and ALK mutations in lung ASCs. Anit-PD-L1 is a promising treatment option in lung ASC cases in which PD-L1 upregulated and EGFR mutations are present.