Single-cell and spatial transcriptomics reveal metastasis mechanism and microenvironment remodeling of lymph node in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and is highly prone to metastasis. OS can metastasize to the lymph node (LN) through the lymphatics, and the metastasis of tumor cells reestablishes the immune landscape of the LN, which is conducive to the growth of tumor cells. However, the mechanism of LN metastasis of osteosarcoma and remodeling of the metastatic lymph node (MLN) microenvironment is not clear. METHODS: Single-cell RNA sequencing of 18 samples from paracancerous, primary tumor, and lymph nodes was performed. Then, new signaling axes closely related to metastasis were identified using bioinformatics, in vitro experiments, and immunohistochemistry. The mechanism of remodeling of the LN microenvironment in tumor cells was investigated by integrating single-cell and spatial transcriptomics. RESULTS: From 18 single-cell sequencing samples, we obtained 117,964 cells. The pseudotime analysis revealed that osteoblast(OB) cells may follow a differentiation path from paracancerous tissue (PC) → primary tumor (PT) → MLN or from PC → PT, during the process of LN metastasis. Next, in combination of bioinformatics, in vitro and in vivo experiments, and immunohistochemistry, we determined that ETS2/IBSP, a new signal axis, might promote LN metastasis. Finally, single-cell and spatial dissection uncovered that OS cells could reshape the microenvironment of LN by interacting with various cell components, such as myeloid, cancer-associated fibroblasts (CAFs), and NK/T cells. CONCLUSIONS: Collectively, our research revealed a new molecular mechanism of LN metastasis and clarified how OS cells influenced the LN microenvironment, which might provide new insight for blocking LN metastasis.

Interaction gene set between osteoclasts and regulatory CD4+ T cells can accurately predict the prognosis of patients with osteosarcoma.

Osteoclasts (OCs) and regulatory CD4+ T cells (CD4+ Tregs) are important components in the tumor microenvironment (TME) of osteosarcoma. In this study, we collected six osteosarcoma samples from our previous study (GSE162454). We also integrated a public database (GSE152048), which included single cell sequencing data of 11 osteosarcoma patients. We obtained 138,192 cells and then successfully identified OCs and CD4+ Tregs. Based on the interaction gene set between OCs and CD4+ Tregs, patients from GSE21257 were distinguished into two clusters by consensus clustering analysis. Both the tumor immune microenvironment and survival prognosis between the two clusters were significantly different. Subsequently, five model genes were identified by protein-protein interaction network based on differentially upregulated genes of cluster 2. Quantitative RT-PCR was used to detect their expression in human osteoblast and osteosarcoma cells. A prognostic model was successfully established using these five genes. Kaplan-Meier survival analysis found that patients in the high-risk group had worse survival (p = 0.029). Therefore, our study first found that cell-cell communication between OCs and CD4+ Tregs significantly alters TME and is connected to poor prognosis of OS. The model we constructed can accurately predict prognosis for osteosarcoma patients.

A novel molecular signature for predicting prognosis and immunotherapy response in osteosarcoma based on tumor-infiltrating cell marker genes.

Background: Tumor infiltrating lymphocytes (TILs), the main component in the tumor microenvironment, play a critical role in the antitumor immune response. Few studies have developed a prognostic model based on TILs in osteosarcoma. Methods: ScRNA-seq data was obtained from our previous research and bulk RNA transcriptome data was from TARGET database. WGCNA was used to obtain the immune-related gene modules. Subsequently, we applied LASSO regression analysis and SVM algorithm to construct a prognostic model based on TILs marker genes. What's more, the prognostic model was verified by external datasets and experiment in vitro. Results: Eleven cell clusters and 2044 TILs marker genes were identified. WGCNA results showed that 545 TILs marker genes were the most strongly related with immune. Subsequently, a risk model including 5 genes was developed. We found that the survival rate was higher in the low-risk group and the risk model could be used as an independent prognostic factor. Meanwhile, high-risk patients had a lower abundance of immune cell infiltration and many immune checkpoint genes were highly expressed in the low-risk group. The prognostic model was also demonstrated to be a good predictive capacity in external datasets. The result of RT-qPCR indicated that these 5 genes have differential expression which accorded with the predicting outcomes. Conclusions: This study developed a new molecular signature based on TILs marker genes, which is very effective in predicting OS prognosis and immunotherapy response.

Regioselectivity for condensation reactions of quinonoid models of tryptophan tryptophylquinone: a density functional theory study.

The model compounds of tryptophan tryptophylquinone (TTQ), o-benzoquinone (OBQ), 3-methyl-6,7-dihydro-1H-6,7-indoledione (MIQ), and 3-methyl-4-(3-methyl-1H-2-indolyl)-6,7-dihydro-1H-6,7-indoledione (IIQ), all of which are characteristic of o-quinone groups, have been studied with density functional theory. The dihedral angle of the two indole rings (chi) of IIQ is calculated to be 49.6 degrees for the global minimum. Another local minimum, 0.74 kcal/mol higher in energy, with a chi value of 123.5 degrees is also fully optimized. The transition state connecting the two minima, with a chi value of 97.9 degrees, has been located and the rotation barrier is 1.71 kcal/mol. A scan of the potential energy surface along this dihedral angle showed that the difference of the total energy was within 1.0 kcal/mol at a range of the dihedral angle from 30 degrees to 75 degrees. Hence, IIQ is flexible for the rotation of inter-indole rings. The origin of regioselectivity for the condensation reactions of the models MIQ and IIQ with NH(3) has been elucidated. It is shown that the energy difference between the two different types of carbinolamine intermediates (Delta E) and their corresponding transition structures (Delta E(++)) should be responsible for the regioselectivity. To assess the effect of the fused ring on regioselectivity of the condensation reaction, a series of models were designed. A good linear correlation has been found between the energy difference of the two different carbinolamine intermediates (Delta E) and that of the corresponding transition states (Delta E(++)), suggesting that the factors that stabilize the carbinolamine intermediate also favor the stability of the corresponding transition structure. The pair, 6-amino-6-hydroxy-8-methyl-6H-quinolin-5-one and 5-amino-5-hydroxy-8-methyl-5H-quinolin-6-one (7/8), deviates from the correlation and represents some anomalous behavior, which may be due to their structural particularity. It also has been shown that the tricyclic models, which consist of OBQ and two fused heterocyclic rings, represent more regioselectivity in contrast to the bicyclic systems. Moreover, the fused electron-donating pyrrole and the fused electron-withdrawing pyridine or pyrimidine show a somewhat synergistic effect on each other via the medial OBQ molecule. The barrier of the condensation reaction for pyrrolo[2,3-f]quinoline-4,5-dione is calculated to be ca. 22 kcal/mol. This is lower than that for MIQ (ca. 33 kcal/mol) and IIQ (ca. 32 kcal/mol) by as much as 10.0 kcal/mol, explaining reasonably the larger catalytic effect of pyrroloquinolinequinone (PQQ) relative to TTQ.