The Combination of Anti-CD47 Antibody with CTLA4 Blockade Enhances Anti-Tumor Immunity in Non-Small Cell Lung Cancer via Normalization of Tumor Vasculature and Reprogramming of the Immune Microenvironment.

In solid tumors, the formidable anti-tumor impact resulting from blocking the "don't eat me" signal, arising from CD47-SIRPα interaction, is constrained, especially compared to its efficacy in hematopoietic malignancies. Activating macrophage anti-tumor activity not only necessitates the inhibition of the "don't eat me" signal, but also the activation of the "eat me" (pre-phagocyte) signal. Intriguingly, the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody (Ab) has been identified to stimulate Fc receptor-mediated active phagocytes in the tumor microenvironment, thereby generating "eat me" signals. This study postulates that concurrently targeting CD47 and CTLA4 could intensify the anti-tumor effects by simultaneously blocking the "don't eat me" signal while triggering the "eat me" signal. The experimental data from this investigation confirm that the combined targeting of CD47 and CTLA4 enhances immunity against solid tumors in LLC cell-transplanted tumor-bearing mice. This effect is achieved by reducing myeloid-derived suppressor cell infiltration while increasing the presence of effector memory CD8+ T cells, NK1.1+ CD8+ T cells, and activated natural killer T cells. Meanwhile, combination therapy also alleviated anemia. Mechanistically, the anti-CD47 Ab is shown to upregulate CTLA4 levels in NSCLC cells by regulating Foxp1. Furthermore, targeting CD47 is demonstrated to promote tumor vascular normalization through the heightened infiltration of CD4+ T cells. These findings suggest that the dual targeting of CD47 and CTLA4 exerts anti-tumor effects by orchestrating the "eat me" and "don't eat me" signals, reshaping the immune microenvironment, and fostering tumor vascular normalization. This combined therapeutic approach emerges as a potent strategy for effectively treating solid tumors.

scRNA-seq and proteomics reveal the distinction of M2-like macrophages between primary and recurrent malignant glioma and its critical role in the recurrence.

AIMS: Tumor-associated macrophages (TAMs) in the immune microenvironment play an important role in the increased drug resistance and recurrence of malignant glioma, but the mechanism remains incompletely inventoried. The focus of this study was to investigate the distinctions of M2-like TAMs in the immune microenvironment between primary and recurrent malignant glioma and its influence in the recurrence. METHODS: We employed single-cell RNA sequencing to construct a single-cell atlas for a total of 23,010 individual cells from 6 patients with primary or recurrent malignant glioma and identified 5 cell types, including TAMs and malignant cells. Immunohistochemical techniques and proteomics analysis were performed to investigate the role of intercellular interaction between malignant cells and TAMs in the recurrence of malignant glioma. RESULTS: Six subgroups of TAMs were annotated and M2-like TAMs were found to increase in recurrent malignant glioma significantly. A pseudotime trajectory and a dynamic gene expression profiling during the recurrence of malignant glioma were reconstructed. Up-regulation of several cancer pathways and intercellular interaction-related genes are associated with the recurrence of malignant glioma. Moreover, the M2-like TAMs can activate the PI3K/Akt/HIF-1α/CA9 pathway in the malignant glioma cells via SPP1-CD44-mediated intercellular interaction. Interestingly, high expression of CA9 can trigger the immunosuppressive response in the malignant glioma, thus promoting the degree of malignancy and drug resistance. CONCLUSION: Our study uncovers the distinction of M2-like TAMs between primary and recurrent glioma, which offers unparalleled insights into the immune microenvironment of primary and recurrent malignant glioma.

Effect of DanQi Pill on PPARα, lipid disorders and arachidonic acid pathway in rat model of coronary heart disease.

BACKGROUND: Danqi pill (DQP) is one of the most widely prescribed formulas and has been shown to have remarkable protective effect on coronary heart disease (CHD). However, its regulatory effects on lipid metabolism disorders haven't been comprehensively studied so far. We aimed to explore the effects of DQP on Peroxisome Proliferator activated receptors α (PPARα), lipid uptake-transportation-metabolism pathway and arachidonic acid (AA)-mediated inflammation pathway in rats with CHD. METHODS: 80 Sprague-Dawley (SD) Rats were randomly divided into sham group, model group, positive control group and DQP group. Rat model of CHD was induced by ligation of left ventricle anterior descending artery and fed with high fat diet in all but the sham group. Rats in sham group only underwent thoracotomy. After surgery, rats in the positive control and DQP group received daily treatments of pravastatin and DQP respectively. At 28 days after surgery, rats were sacrificed and plasma lipids were evaluated by plasma biochemical detection. Western blot and PCR were applied to evaluate the expressions of PPARα, proteins involved in lipid metabolism and AA pathways. RESULTS: Twenty eight days after surgery, dyslipidemia developed in CHD model rats, as illustrated by elevated plasma lipid levels. Expressions of apolipoprotein A-I (ApoA-I), cluster of differentiation 36 (CD36) and fatty acid binding protein (FABP) in the heart tissues of model group were down-regulated compared with those in sham group. Expressions of carnitine palmitoyl transferase I (CPT-1A) and lipoproteinlipase (LPL) were also reduced significantly. In addition, levels of phospholipase A2 (PLA2) and cyclooxygenase 2 (COX-2) were up-regulated. Expressions of Nuclear factor-κB (NF- κB) and signal transducer and activator of transcription 3 (STAT3) also increased. Furthermore, Expression of PPARα decreased in the model group. DQP significantly up-regulated expressions of ApoA-I and FABP, as well as the expressions of CPT-1A and CD36. In addition, DQP down-regulated expressions of PLA2, COX-2 and NF-κB in inflammation pathway. Levels of STAT3 and LPL were not affected by DQP treatment. In particular, DQP up-regulated PPARα level significantly. CONCLUSIONS: DQP could effectively regulate lipid uptake-transportation-metabolism process in CHD model rats, and the effect is achieved mainly by activating ApoA-I-CD36-CPT-1A molecules. Interestingly, DQP can up-regulate expression of PPARα significantly. The anti-inflammatory effect of DQP is partly exerted by inhibiting expressions of PLA2-COX2 -NF-κB pathway.

[Study on role of Chengqi Shengxue prescription in anti-tumor and immunoregulation].

OBJECTIVE: To investigate the role of Chengqi Shengxue prescription in anti-tumor and immunoregulation and to evaluate its effect on apoptosis and T lymphocyte subsets of tumor-bearing mice. METHOD: S180 ascites tumor and Lewis lung carcinoma tumor-bearing mice were used in the screening. Then 55 mice were treated randomly with the model, cyclophosphamide (30 mg x kg(-1)), or three different dosages of Chengqi Shengxue prescription (2. 4, 1.2, 0.6 g x kg(-1). After the treatment apoptosis of tumor cell and peripheral T lymphocyte subsets of tumor-bearing mice was analyzed by flow cytometry. RESULT: Lewis lung carcinoma was a nsitive tumor cell line to Chengqi Shengxue prescription. Compared with the model group, significantly increased apoptosis was observed after administration of high and medium dose of Chengqi Shengxue prescription (P < 0. 05) by PI staining. Increased early apoptosis in cancer cells was observed in all experimental doses of Chengqi Shengxue prescription by Annexin V and PI double staining (P < 0.01) . The analysis of T lymphocyte subsets showed that the percentage of CD3, CD4 and CD4/CD8 ratio decreased significantly in model group when compared with the normal ones (P < 0.01), while no change was observed in CD8. In administration groups, CD3, CD4 and CD8 were significantly lower than normal ones (P < 0.01) , but CD4/CD8 ratio did not change significantly. CONCLUSION: Chengqi Shengxue prescription has selectively inhibitive effect on the growth of mouse Lewis lung carcinoma and takes an antitransfer role. Its anti-tumor effect may be owing to inducing tumor cell apoptosis. Chengqi Shengxue prescription improves cellular immune function through enhancing CD4/CD8 ratio.

[Rat alveolar type II injured by bleomycin].

OBJECTIVE: To explore dysfunction mechanism of rat alveolar type II (AT-II) injured by bleomycin (BLM). METHODS: SD rats were injected with a single intratracheal dose of bleomycin or control saline. On day 7, 14, and 28 following intratracheal bleomycin or saline instillation, animals were killed under overdose of 1.5% sodium pentobarbital (0.25 ml/100 g, i.p.) and bronchoalveolar lavage fluid (BALF) from the lung was tested for the activity of pulmonary surfactant (PS) by the Whihelmy Film Balance. Several concentrations of bleomycin stimulated the culture of rat AT-II cells, and surfactant protein (SP) A, B, and aquaporin-1 (AQP) mRNA were analyzed by fluorescent quantitative polymerase chain reaction (FQ-PCR). RESULTS: The activity of PS and hypoxemia significantly decreased on day 7 and improved on day 14 and completely recovered to normal status on day 28. SP-A, B, and AQP-1 mRNA expression in BLM-stimulated group were significantly lower than those in the control group (P<0.001). CONCLUSION: BLM-injured AT-II cells decrease the levels of SP-A, B, and AQP-1 mRNA and cause PS dysfunction, resulting in hypoxemia and pneumonedema.