MicroRNA 21 (miR-21) has been demonstrated to be significantly elevated in many types of cancers, including the hepatocellular carcinoma (HCC). In the present study, we investigated the role of miR-21 in HCC by identifying its novel targets, as well as its underlying molecular mechanism.

The adoption of three-dimensional (3D) cell culture systems represents a critical advancement in biomedical research, better mimicking complex 3D tissue environments than traditional two-dimensional (2D) models. However, variability in experimental outcomes has limited their reproducibility and clinical translation. Here, we systematically analyzed 32,000 spheroid images to identify key parameters influencing 3D model reliability. Our large-scale analysis revealed that oxygen levels significantly affect spheroid size and necrosis, while media composition (e.g., glucose and calcium concentrations) and serum levels (0–20%) critically regulate cell viability and structural integrity. For instance, spheroids cultured in 3% oxygen exhibited reduced dimensions and increased necrosis, whereas serum concentrations above 10% promoted dense spheroid formation with distinct necrotic and proliferative zones. By integrating single-cell RNA sequencing and automated image analysis, we uncovered dynamic gene expression patterns linked to spheroid maturation and hypoxia. These findings provide actionable guidelines for standardizing 3D culture protocols, addressing critical reproducibility challenges. Our work establishes a robust framework to enhance the reliability of 3D models in drug testing, personalized medicine, and tumor biology, facilitating their broader adoption in translational research.

Background: Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells.Methods: CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function.Results: The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%.Conclusions: Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Background: The role of T cell Ig and ITIM domain (TIGIT) and programmed cell death-1 (PD-1) in colorectal cancer (CRC) with mismatch repair deficiency is unknown. Methods: This was a study of 60 CRC patients with mismatch repair deficiency and 30 healthy controls between June 2015 and October 2015. Results: The expression of Foxp3, PD-1, and TIGIT was higher in cancer tissues compared with adjacent mucosa (all P < .05). Patients with advanced TNM stage had a significantly higher expression of TIGIT (P = .025) and PD-1 (P = .020) than patients with early-stage CRC. The disease-free survival (DFS) of patients with high TIGIT (HR = 3.96, 95%CI: 1.34–11.69, P = .013) or PD-1 (HR = 214.8, 95%CI: 49.88–925.2, P < .001) expression were better. The overall survival (OS) of the patients with CRC and high expression of PD-1 was worse than those with low expression (HR = 4.01, 95%CI:1.26–12.69, P = .019). Conclusion: TIGIT and PD-1 are upregulated in CRC with mismatch repair deficiency and associated with TNM stage and DFS.

Chemokine C‐C motif chemokine ligand 3 (CCL3) plays an important role in the invasion and metastasis of malignant tumors. For developing new therapeutic targets and antitumor drugs, the effect of chemokine CCL3 and the related cytokine network on colorectal cancer should be investigated. This study used cell, tissue, and animal experiments to prove that CCL3 is highly expressed in colorectal cancer and confirmed that CCL3 can promote the proliferation of cancer cells, and its expression is closely related to TRAF6/NF‐κB molecular pathway. In addition, protein chip technology was used to examine colorectal cancer tissue samples and identify the key factors of chemokine CCL3 and the toll‐like receptors/nuclear factor‐κB (TLR/NF‐κB) pathway in cancer and metastatic lymph nodes. Furthermore, the lentiviral vector technology was employed for transfection to construct interference and overexpression cell lines. The experimental results reveal the mechanism of CCL3 and TNF receptor‐associated factor 6 (TRAF6)/NF‐κB pathway‐related factors and their effects on the proliferation of colon cancer cells. Finally, the expression and significance of CCL3 in colorectal cancer tissues and its correlation with clinical pathology were studied by immunohistochemistry. Also, the results confirmed that CCL3 and C‐C motif chemokine receptor 5 (CCR5) were expressed in adjacent tissues, colorectal cancer tissues, and metastatic cancer. The expression level was correlated with the clinical stage and nerve invasion. The expression of chemokine CCL3 and receptor CCR5 was positively correlated with the expression of TRAF6 and NF‐κB and could promote the proliferation, invasion, and migration of colorectal cancer cells through TRAF6 and NF‐κB.

Tumor-infiltrating lymphocytes (TILs) are vital elements of the tumor microenvironment (TME), and the anti-tumor activity of TILs on colorectal cancer (CRC) has been a topic of concern. However, the characteristics and prognosis of the various types of lymphocyte infiltration in CRC have not been fully explained. Our study aimed to identify distinct features and prognosis of TILs. We integrated multiple-cohort databases to illustrate the features, proportions, and prognosis of TILs on CRC. We found that macrophages were significantly enriched in CRC. When we used the scRNA-seq database to further evaluate the proportion of TILs, we noticed markedly higher numbers of CD4 + T cell, B cell, and CD8 + T cell in four Gene Expression Omnibus Series (GSE) CRC cohorts. Interestingly, we found that the infiltrating level of TIL subgroups from highest to lowest is always dendritic cells, CD8 + T cells, CD4 + T cells, neutrophils, B cells, and macrophages; the proportion of infiltration is largely constant regardless of mutations in specific genes or somatic copy number variation (sCNV). In addition, the data corroborated that CD4+ TILs and CD8+ TILs have certain application values in the prognosis of CRCs, and age negatively related to CD8+ TILs and B plasma infiltration. Finally, patients with CRC who are older than 70 years have a better response to immune-checkpoint blockade. Graphic Abstract