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Background: Gallbladder cancer (GC) is a malignant disease characterized with highly cellular heterogeneity and poor prognosis. Determining the intratumoral heterogeneity and microenvironment (TME) can provide novel therapeutic strategies for GC. Methods: We performed the single‐cell RNA sequencing on the primary and lymph node metastatic gallbladder tumors and the adjacent normal tissues of five patients. The transcriptomic atlas and ligand–receptor‐based intercellular communication networks of the single cells were characterized. Results: The transcriptomic landscape of 24,887 single cells was obtained and characterized as 10 cellular clusters, including epithelial, neuroendocrine tumor cells, T&NK cells, B cells, RGS5+ fibroblasts, POSTN+ fibroblasts, PDGFRA+ fibroblasts, endothelial, myeloid cells, and mast cells. Different types of GC harbored distinct epithelial tumor subpopulations, and squamous cell carcinoma could be differentiated from adenocarcinoma cells. Abundant immune cells infiltrated into adenocarcinoma and squamous cell carcinoma, rather than neuroendocrine neoplasms, which showed significant enrichment of stromal cells. CD4+/FOXP3+ T‐reg and CD4+/CXCL13+ T helper cells with higher exhausting biomarkers, as well as a dynamic lineage transition of tumor‐associated macrophages from CCL20hi/CD163lo, CCL20lo/CD163hi to APOE+, were identified in GC tissues, suggesting the immunosuppressive and tumor‐promoting status of immune cells in TME. Two distinct endothelial cells (KDR+ and ACKR1+), which were involved in angiogenesis and lymphangiogenesis, showed remarkable ligand–receptor interactions with primary GC cells and macrophages in gallbladder tumors. Conclusions: This study reveals a widespread reprogramming across multiple cell populations in GC progression, dissects the cellular heterogeneity and interactions in gallbladder TME, and provides potential therapeutic targets for GC. [ABSTRACT FROM AUTHOR]

Use of immune index is a new potential approach for cancer classification and prediction. To investigate the status and clinical effect of immune index in gallbladder cancer (GBC), 238 GBC patients from Zhongshan Hospital affiliated to Fudan University were involved in the present study, including 113 patients in a training set and 125 patients in a validation set. Five immune cells (macrophages, neutrophils, regulatory T cells, cytotoxic T cells and mast cells) were selected based on a literature review and the immune index for each patient was calculated using the LASSO regression. A low immune index (<1) was defined as immunotype A and a high immune index (≥1) was defined as immunotype B. The 5‐year overall survival rate for immunotype A was higher than that for immunotype B in the training set and the validation set (70.0% vs 37.0%, P < 0.001; 68.9% vs 47.5%, P = 0.002; respectively). Moreover, the immune index showed higher prediction efficiency compared with all the single immune cells which we selected. When combined with the immune index, the areas under the curve (AUC) of the TNM staging system in both sets were elevated from 0.677 to 0.787 and from 0.631 to 0.694, respectively. Interestingly, gemcitabine‐based chemotherapy only benefits stage II patients of immunotype B and stage III patients of both immunotype A and immunotype B (P = 0.015, P = 0.030, P = 0.011, respectively) but does not work in stage II patients of immunotype A (P =.307). Taken together, the immune index could effectively predict prognosis and the benefits of gemcitabine‐based chemotherapy and might improve on the TNM staging system. [ABSTRACT FROM AUTHOR]

Gallbladder carcinoma (GBC) is a considerable challenge because of its high metastatic potential. The tumor microenvironment is characterized by nutrient starvation, which promotes tumor metastasis. Stathmin1, an important microtubuleregulating protein, is overexpressed and promotes metastasis in GBC. It remains unclear how the harsh tumor microenvironment regulates stathmin1 expression to affect GBC metastasis. We employed glucose deficiency to mimic nutrient starvation and found that glucose deficiency upregulated stathmin1 transcription. However, glucose deficiency also promoted p27 degradation. There was a significant negative correlation between stathmin1 and p27 protein levels under glucose deficiency. Further study revealed that, under glucose deficiency, human kinase interacting with stathmin (hKIS) induced phosphorylation at Ser10 of p27 and its translocation to the cytoplasm for degradation, which upregulated the transcription factor E2F1 to promote stathmin1 transcription. hKIS knockdown significantly inhibited p27 cytoplasmic translocation and its consequent degradation. Stathmin1 knockdown significantly inhibited GBC cell migration and invasion in vitro. Our study revealed the role of the hKIS/p27/E2F1 axis in upregulating stathmin1 transcription to promote GBC cell migration and invasion under glucose deficiency conditions • We model the nutrition starvation conditions with glucose deficiency; • We found increased levels of stathmin1 of GBC in nutrition starvation condition; • Glucose deficiency induced phosphorylation of p27Kip1 at Ser10 and consequently degradation; • Phosphorylation of p27 at Ser10 promoted GBC migration and invasion; • hKIS/p27/E2F1 axis regulated stathmin1 expression and GBC cell migration and invasion under glucose deficiency conditions. [ABSTRACT FROM AUTHOR]