ObjectiveThe prognosis of advanced gastrointestinal cancer is poor. There are studies indicating that gut microbes might have the predictive ability to evaluate the outcome of cancer therapy, especially immunotherapy. There is limited evidence to date on the influence of microbes on chemotherapeutic response.DesignIn total, 130 patients with advanced or metastatic esophageal (n=40), gastric (n=46), and colorectal cancer (n=44) were enrolled. We included 147 healthy people as controls and used 16S rRNA sequencing to analyze the fecal microbiota.ResultsSignificant differences in the abundance of fecal microbiota between patients with gastrointestinal cancer and controls were identified. The abundance of Bacteroides fragilis, Escherichia coli, Akkermansia muciniphila, Clostridium hathewayi, and Alistipes finegoldii were significantly increased in the patient group. Faecalibacterium prausnitzii, Roseburia faecis, Clostridium clostridioforme, Blautia producta, Bifidobacterium adolescent, and Butyricicoccus pullicaecorum taxa were significantly more abundant in the controls. The amount of R. faecis in non-responders (NR) was more likely to decrease significantly after chemotherapy, while the amount mostly increased in responders (R) (P=0.040). The optimal abundance variation of R. faecis may be a predictor for distinguishing patients with PD from those with non-PD in all patients with gastrointestinal cancer, with a sensitivity of 75.0% and a specificity of 93.9%.ConclusionThe gut microbiome of patients with esophageal cancer, gastric cancer, and colorectal cancer differs from those of healthy people. The abundance alteration of R. faecis in patients with GI cancer might be a predictor of chemotherapy efficacy.

Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse embryonic stem cells (ESCs) can directly differentiate into advanced male germ cell lineage in the same conditions. Here, we showed rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by the activation of spermatogenesis-associated genes such as Mvh, Dazl, Prdm14, Stella, Scp1, Scp3, Stra8 and Rec8. In contrast, for cells cultured in control medium, the activation of the above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin-expressing cells and the positive ratio reached a peak at day 6. Our work thus establishes a simple and cost-efficient approach for male germ like cell differentiation from mouse PSCs and may propose a useful strategy for studying spermatogenesis in vitro.

The incidence and mortality of colorectal cancer (CRC) have been rising rapidly in China. A number of miRNAs have been confirmed to be involved in diverse biological processes of CRC. However, whether miR‑197 plays a role in migration and invasion of CRC has never been explored. In the present study Transwell chambers were used in in vitro migration and invasion assays. Dual-luciferase reporter assay was employed to confirm the target of miR‑197. RT‑PCR and IHC staining were performed to quantify miR‑197 and IGFBP3 expression, respectively. Clinicopathological features were collected for statistical analysis. We observed that the overexpression of miR‑197 significantly promoted migration and invasion in 3 CRC cell lines including HCT8, HCT116 and SW480 (P<0.05), while the inhibition of miR‑197 weakened both biological processes (P<0.05). In bioinformatics and dual-luciferase reporter assay, luciferase activities of IGFBP3‑WT‑transfected cells significantly decreased upon miR‑197 overexpression and this inhibitory effect was abolished when miR‑197 binding region in IGFBP3 3'‑UTR was mutated, which indicated that miR‑197 directly suppressed the expression of IGFBP3 in CRC cells by targeting its 3'UTR. Downregulation of the expression of IGFBP3 by using targeted siRNA led to significant enhancement of cell migration and invasion in two CRC cell lines including HCT8 and HCT116 (P<0.05). Finally, in cancerous tissues of CRC patients, the miR‑197 level was inversely correlated with the expression of IGFBP3 (P=0.026), which indicated that miR‑197 may modulate cell migration and invasion by targeting IGFBP3 in CRC patients. In conclusion, we revealed that miR‑197 modulates IGFBP3 and therefore plays a critical role in regulating CRC migration and invasion.

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

BackgroundProgrammed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) inhibitors works by reactivating immune cells. Considering the accessibility of noninvasive liquid biopsies, it is advisable to employ peripheral blood lymphocyte subsets to predict immunotherapy outcomes.MethodsWe retrospectively enrolled 87 patients with available baseline circulating lymphocyte subset data who received first-line PD-1/PD-L1 inhibitors at Peking Union Medical College Hospital between May 2018 and April 2022. Immune cell counts were determined by flow cytometry.ResultsPatients who responded to PD-1/PD-L1 inhibitors had significantly higher circulating CD8+CD28+ T-cell counts (median [range] count: 236 [30-536] versus 138 [36-460]/μL, p &lt; 0.001). Using 190/μL as the cutoff value, the sensitivity and specificity of CD8+CD28+ T cells for predicting immunotherapy response were 0.689 and 0.714, respectively. Furthermore, the median progression-free survival (PFS, not reached versus 8.7 months, p &lt; 0.001) and overall survival (OS, not reached versus 16.2 months, p &lt; 0.001) were significantly longer in the patients with higher CD8+CD28+ T-cell counts. However, the CD8+CD28+ T-cell level was also associated with the incidence of grade 3-4 immune-related adverse events (irAEs). The sensitivity and specificity of CD8+CD28+ T cells for predicting irAEs of grade 3-4 were 0.846 and 0.667, respectively, at the threshold of CD8+CD28+ T cells ≥ 309/μL.ConclusionsHigh circulating CD8+CD28+ T-cell levels is a potential biomarker for immunotherapy response and better prognosis, while excessive CD8+CD28+ T cells (≥ 309/μL) may also indicate the emergence of severe irAEs.

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Background Treatment for non-small cell lung cancer (NSCLC) has greatly improved in recent years. However, noninvasive early screening for carcinogenesis and progression unclear. The aim of this study was to explore the predictive value of peripheral blood immune cells in untreated NSCLC patients. Methods We retrospectively enrolled 305 untreated NSCLC patients and 132 healthy participants from February 2016 to August 2019 in Peking Union Medical College Hospital. Immune cell levels were determined by flow cytometry and routine blood tests. Results NSCLC patients had lower levels of T lymphocytes, NK cells, CD8+ T cells, naïve CD4+/CD4+, naïve CD4+ T cells and higher levels of CD4+ T cells, memory CD4+/CD4+ T cells, memory CD4+ T cells, CD4+CD28+/CD4+ T cells, CD4+CD28+ T cells, CD8+CD28+/CD8+ T cells, CD8+HLA-DR+/CD8+ T cells, CD8+HLA-DR+ T cells T cells, CD8+CD38+/CD8+ T cells, CD8+CD38+ T cells and CD4+/CD8+ T cells than those in controls. The percentages of specific lymphocyte subtypes were significantly different in cancer patients versus healthy individuals. For instance, cancer patients had lower levels of B cells, CD4+ T cells, naïve CD4+/CD4+ T cells, naïve CD4+ T cells, CD4+CD28+ T cells, CD8+CD28+ T cells and higher levels of NK cells, white blood cells (WBC), monocytes, neutrophils, eosinophils, basophils, monocytes to lymphocyte ratio (MLR), neutrophils to lymphocyte ratio (NLR), eosinophil to lymphocyte ratio (ELR), basophil to lymphocyte ratio (BLR), and blood platelet to lymphocyte ratio (PLR). Conclusions Abnormal T cell levels can be used as an independent predictive biomarker for noninvasive early screening in NSCLC occurrence and progression.

BRAF wild-type recurrent indeterminate dendritic cell tumour presenting with leonine facies Editor Indeterminate dendritic cell tumour (IDCT) was a rare histiocytosis originating from stromal dendritic accessory cells. 1 Only two studies have described leonine facies as the dominant feature of IDCTs. 2 Here, we report another IDCT presenting with leonine facies. A 37-year-old man presented with 4-year history of diffuse papules on his cheeks and then gradually spreading to his face, back and extremities. Some facial papules fused into bumps and nodules, accompanied by slight pruritus. Skin examinations showed multiple pinkish red nodular lesions coalesced into irregularshaped or rod-like protuberances on his forehead, both cheeks, eyelids, temples and pinnas of ears (Fig. 1a,b). Diffuse red papules scattered throughout his legs, posterior surface of forearms and lower back, with old greyish and wrinkled lesions among them (Fig. 1c). Dermatoscopy revealed patchy, branchy and spiral vessel dilations with keratosis and scales scattered on red background (Fig. 1d). Biopsy showed dermis diffusely infiltrated by histiocytoid cells with nuclear grooves and eosinophilic cytoplasm but atrophic and uninvolved epidermis (Fig. 1e). The histiocytoid cells were CD1a and CD68 positive (Fig. 1f,g), S-100 focally positive and Langerin negative (Fig. 1h). Ki-67 index was 25%. The diagnosis was revised from previous Langerhans cell histiocytosis (LCH) to IDCT based on the absence of Langerin. Since BRAF V600E mutation and BRAF fusions have been reported in malignant histiocytosis, 3,4 a Human BRAF V600E ARMS-PCR Kit and a next-generation sequencing (NGS) array were applied but neither BRAF V600E mutation nor BRAF fusions were found. The details and outcomes of