Rationale: Although immunotherapy through programmed death 1 / programmed death ligand 1 (PD-1/PD-L1) checkpoint blockade has shown impressive clinical outcomes, not all patients respond to it. Recent studies have demonstrated that the expression level of PD-L1 in tumors is one of the factors that correlate with the PD-1/PD-L1 checkpoint blockade therapy. Herein, a novel ⁶⁸Ga-labeled nanobody tracer, ⁶⁸Ga-NOTA-Nb109, was designed and developed for specific and noninvasive imaging of PD-L1 expression in the melanoma-bearing mice model. Methods: The nanobody Nb109 was labeled with the radionuclide ⁶⁸Ga through a NOTA chelator. In vitro binding assay was performed to assess the affinity and binding epitope of Nb109 to PD-L1. The clinical application value of ⁶⁸Ga-NOTA-Nb109 was evaluated by the stability assay, biodistribution and pharmacokinetics studies as well as positron emission tomography (PET) imaging, autoradiography and immunohistochemical staining studies on the tumor-bearing models with different PD-L1 expression. Results: The tracer ⁶⁸Ga-NOTA-Nb109 was obtained with the radiochemical yield > 95% and the radiochemical purity > 98% in 10 min. It showed a high specific affinity for PD-L1 with the equilibrium dissociation constant (KD) of 2.9 × 10-9 M. A competitive binding assay indicated a different binding epitope for Nb109 compared with PD-1 and PD-L1 antibody. All the biodistribution, PET imaging, autoradiography and immunohistochemical staining studies revealed that the tracer ⁶⁸Ga-NOTA-Nb109 specifically accumulated in A375-hPD-L1 tumor with a maximum uptake of 5.0 ± 0.35 %ID/g at 1 h. Conclusion: The tracer ⁶⁸Ga-NOTA-Nb109 holds great potential for noninvasive PET imaging of the PD-L1 status in tumors and timely evaluating the therapeutic effect of immune checkpoint targeting treatment.

Anlotinib is a new multitarget tyrosine kinase inhibitor for tumor angiogenesis, and its monotherapy exhibits a decent clinical efficacy. However, the process of combining Anlotinib and immune checkpoint therapy to achieve optimal antitumor effects while limiting side effects remains unclear. In this study, we found that effective low-dose Anlotinib was sufficient to inhibit tumor growth while reducing side effects compared with high doses. Effective low-dose Anlotinib treatments induced durable tumor vascular normalization and improved anti-PD-1 therapy in both short- and long-term treatment regimens. Mechanistically, the combination therapy increased the proportions of intratumoral CD4⁺ T, CD8⁺ T, and NK cells. Anlotinib-associated antitumor effects were independent of interferon γ; however, the combination therapy required CD8⁺ T cells to suppress tumor growth. Together, these results suggest that the combination of effective low-dose Anlotinib and PD-1 blockade induces durable antitumor effects with fewer side effects. Our findings indicate that antiangiogenic treatments combined with immune checkpoint therapy at an effective low-dose, rather than a tolerable high dose, would be more efficacious and safer.

Claudin 18.2 (CLDN18.2)-targeting chimeric antigen receptor (CAR)-modified T cells are one of the few cell therapies currently producing an impressive therapeutic effect in treating solid tumors; however, their long-term therapeutic efficacy is not satisfactory with a short duration of response. Transgenic expression of interleukin (IL)-15 has been reported to promote T-cell expansion, survival, and function and enhance the antitumor activity of engineered T cells in vitro and in vivo. Therefore, this study aimed to explore whether IL-15 modification would increase the antitumor activity of CLDN18.2-targeting CAR-modified T (CAR-T) cells in immunocompetent murine tumor models. CLDN18.2-specific CAR-T cells with (H9 CAR-IL15) or without transgenic IL-15 expression (H9 CAR) were generated by retroviral transduction of mouse splenic T cells. In vitro, compared with H9 CAR T cells, H9 CAR-IL15 T cells exhibited better expansion and viability in the absence of antigen stimulation, with a less differentiated and T-cell exhausted phenotype; although IL-15 modification did not affect the production of effector cytokines and cytotoxic activity in the short-term killing assay, it moderately improved the in vitro recursive killing activity of CAR-T cells against CLDN18.2-expressing tumor cells. In vivo, H9 CAR T cells showed no antitumor activity against CLDN18.2-expressing pancreatic tumors in immunocompetent mice without lymphodepleting pretreatment; however, H9 CAR-IL15 T cells produced significant tumor-suppressive effects. Furthermore, H9 CAR-IL15 T cells exhibited greater in vivo expansion and tumor infiltration when combined with lymphodepleting preconditioning, resulting in superior antitumor activity in two murine tumor models and a survival advantage in one tumor model. We further demonstrated that recurrent tumors following H9 CAR-IL15 T-cell therapy downregulated CLDN18.2 expression, suggesting immune escape through the selection of antigen-negative cells under persistent CAR-T-cell immune pressure. In conclusion, our findings provide preclinical evidence supporting the clinical evaluation of IL-15-expressing CLDN18.2 CAR-T cells in patients with CLDN18.2-positive tumors.

Endometrial cancer (EC)) is one of the most common malignant tumors of the female genital system, with an increasing incidence and mortality, worldwide. Although the therapeutic strategy of EC is still complicated and challenging, further understanding of carcinogenesis from a gene perspective would allow an effort to improve therapeutic precision in this complex malignancy. DNA methylation is the most widely studied epigenetic alteration in human tumors. Aberrant DNA methylation events, resulting in altered gene expression, are features of many tumor types. In this review, we provide an update on evidence about the roles of aberrant DNA methylation within some classical tumor suppressor genes and oncogenes in endometrial carcinogenesis, and report on recent advances in the understanding of the contribution of aberrant DNA methylation to EC, as well as opportunities and challenges of DNA methylation in EC management and prevention.

Non‐small‐cell lung cancer (NSCLC), with an aggressive biological behavior, is one of the most diagnosed cancers. Tumor‐associated inflammatory cells play important roles in the interaction between chronic inflammation and lung cancer. However, the mechanisms involved are far from defined. In the present study, by developing an orthotopic NSCLC mouse model on the basis of chronic inflammation, we proved that inflammatory microenvironment accelerated growth of orthotopic xenografts in vivo. Tumor associated macrophages (TAMs) the most abundant population of inflammatory cells were identified. Treatment of macrophage conditional medium (MCM) promoted growth and migration of NSCLC cells. By using bioinformatics analysis, we identified down‐regulated PP2Ac expression in NSCLC cells upon treatment of MCM. We further confirmed that this down‐regulation was executed through NF‐κB pathway dependent manner. Since IKK has been proved to be a substrate of PP2Ac, inhibition on PP2Ac could result in an amplification of NF‐κB pathway signaling. Overexpression of PP2Ac, or the dominant negative forms of IKK or IκB, attenuated the acceleration on growth and metastasis by MCM. By using bioinformatics analysis, we further identified CXCL1 and COL6A1 could be downstreams of NF‐κB/PP2Ac pathway. Luciferase assay and ChIP assay further confirmed location of response elements on the promoter regions of CXCL1 and COL6A1. Elevated CXCL1 facilitated angiogenesis, while up‐regulated COL6A1 promoted proliferation and migration.

Background: Immunotherapy has become the standard of treatment for recurrent metastatic esophageal cancer (EC), and the value of efficacy predictive markers represented by programmed death-ligand 1 (PD-L1) is limited. The purpose of this study is to analyze the prognostic value of peripheral blood absolute lymphocyte count (ALC) at baseline in patients with recurrent metastatic EC treated with immunotherapy, and to further investigate the relationship between the minimal ALC value (Min ALC) and radiotherapy (RT) parameters. Methods: The main inclusion criteria were: histologically or imaging confirmed recurrent or metastatic EC; complete routine blood test data. A total of 105 patients were included in a single-center institution, 65 of whom had previously received RT. The optimal cut-off value for baseline lymphopenia was determined by the receiver operating characteristic (ROC) curve. The prognostic value of baseline phase lymphopenia for immunotherapy were determined by cox regression analysis and the associated factors affecting lymphopenia were explored by logistic regression analysis. Results: The cut-off value for baseline ALC predicting 1-year overall survival (OS) was 625 cells/µL. The OS was significantly lower in the lymphopenia group (ALC ≤625 cells/µL) than in the non-lymphopenia group (ALC >625 cells/µL) (median OS: 6 vs. 12 months, P=0.002). Multivariate analysis showed that pre-immunotherapy lymphopenia was an important factor influencing patient prognosis [hazard ratio (HR): 1.771, 95% confidence interval (CI): 1.051–2.985; P=0.032)] (adjusted for clinical factors including sex, age, tumor location, histology, degree of differentiation, distant metastasis, use of RT). Patients with a previous grade 4 (G4) Min ALC during RT were more likely to develop pre-immunotherapy lymphopenia following diagnosis of recurrent metastasis [odds ratio (OR): 10.809, 95% CI: 2.185–53.471; P=0.004]. Planning target volume (PTV) volume greater than 521.2 cm³ (OR: 19.981, 95% CI: 1.372–290.985; P=0.028) was an independent risk factor affecting the G4 Min ALC during RT. Conclusions: Lymphopenia is associated with a poorer immunotherapy prognosis in patients with recurrent metastatic EC and those with previous G4 Min ALC after RT. RT-related parameters, especially irradiation volume, can significantly affect lymphocyte counts.