Collaborating single-cell and bulk RNA sequencing for comprehensive characterization of the intratumor heterogeneity and prognostic model development for bladder cancer.

INTRODUCTION: Gaining a deeper insight into the single-cell RNA sequencing (scRNA-seq) results of bladder cancer (BLCA) provides a transcriptomic profiling of individual cancer cells, which may disclose the molecular mechanisms involved in BLCA carcinogenesis. METHODS: scRNA data were obtained from GSE169379 dataset. We used the InferCNV software to determine the copy number variant (CNV) with normal epithelial cells serving as the reference, and performed the pseudo-timing analysis on subsets of epithelial cell using Monocle3 software. Transcription factor analysis was conducted using the Dorothea software. Intercellular communication analysis was performed using the Liana software. Cox analysis and LASSO regression were applied to establish a prognostic model. RESULTS: We investigated the heterogeneity of tumors in four distinct cell types of BLCA cancer, namely immune cells, endothelial cells, epithelial cells, and fibroblasts. We evaluated the transcription factor activity of different immune cells in BLCA and identified significant enrichment of TCF7 and TBX21 in CD8+ T cells. Additionally, we identified two distinct subtypes of cancer-associated fibroblasts (CAFs), namely iCAFs and myoCAFs, which exhibited distinct communication patterns. Using sub-cluster and cell trajectory analyses, we identified different states of normal-to-malignant cell transformation in epithelial cells. TF analysis further revealed high activation of MYC and SOX2 in tumor cells. Finally, we identified five model genes (SLCO3A1, ANXA1, TENM3, EHBP1, LSAMP) for the development of a prognostic model, which demonstrated high effectiveness in stratifying patients across seven different cohorts. CONCLUSIONS: We have developed a prognostic model that has demonstrated significant efficacy in stratifying patients with BLCA.

Single-cell transcriptome analysis revealing the intratumoral heterogeneity of ccRCC and validation of MT2A in pathogenesis.

Clear-cell renal cell carcinoma (ccRCC) appears as the most common type of kidney cancer, the carcinogenesis of which has not been fully elucidated. Tumor heterogeneity plays a crucial role in cancer progression, which could be largely deciphered by the implement of scRNA-seq. The bulk and single-cell RNA expression profile is obtained from TCGA and study conducted by Young et al. We utilized UMAP, TSNE, and clustering algorithm Louvain for dimensionality reduction and FindAllMarkers function for determining the DEGs. Monocle2 was utilized to perform pseudo-time series analysis. SCENIC was implemented for transcription factor analysis of each cell subgroup. A series of WB, CFA, CCK-8, and EDU analysis was utilized for the validation of the role of MT2A in ccRCC carcinogenesis. We observed higher infiltration of T/NK and B cells in tumorous tissues, indicating the role of immune cells in ccRCC carcinogenesis. Transcription factor analysis revealed the activation of EOMES and ETS1 in CD8 + T cells, while CAFs were divided into myo-CAFs and i-CAFs, with i-CAFs showing distinct enrichment of ATF3, JUND, JUNB, EGR1, and XBP1. Through cell trajectory analysis, we discerned three distinct stages of cellular evolution, where State2 symbolizes normal renal tubular cells that underwent transitions into State1 and State3 as the CNV score ascended. Functional enrichment examination revealed an amplification of interferon gamma and inflammatory response pathways within tumor cells. The consensus clustering algorithm yielded two molecular subtypes, with cluster 2 being associated with advanced tumor stages and an abundance of infiltrated immune cells. We identified 17 prognostic genes through Cox and LASSO regression models and used them to construct a prognostic model, the efficacy of which was verified in multiple cohorts. Furthermore, we investigated the role of MT2A, one of our hub genes, in ccRCC carcinogenesis, and found it to regulate proliferation and migration of malignant cells. We depicted a detailed single-cell landscape of ccRCC, with special focus on CAFs, endothelial cells, and renal tubular cells. A prognostic model of high stability and accuracy was constructed based on the DEGs. MT2A was found to be actively implicated in ccRCC carcinogenesis, regulating proliferation and migration of the malignant cells.

Towards human-like speed control in autonomous vehicles: A mountainous freeway case.

A driving strategy for autonomous vehicles (AVs) that is consistent with human behavior while demonstrating superior performance seems to have a good chance to be accepted by early AV users and be successful in the long run. Most of the past research focused on motion strategies affected by the presence of other vehicles. On the other hand, AV not constrained by other vehicles must select a safe and comfortable speed that is perceived as such by its occupants. This line of research is not well covered by the published work. The baseline speed, which is the speed AVs will follow without interaction with other vehicles, implemented via cruise control (CC) in modern vehicles is a constant speed consistent with speed limits and design speeds. A more advanced strategy of road-limiting speed control (RC) responds to influencing geometric features ahead of the AV's current position. Neither of the two strategies considers AV occupants' preferences. The current void in research is particularly obvious for free-flow conditions where baseline speeds must be implemented for extended periods of travel. Although the existing strategies have not been yet evaluated on roadways with demanding alignments and operating in free-flow conditions, the principles on which they are based provide a basis for skepticism if they can be acceptable to AV occupants. This study used the Tongji University driving simulator to evaluate the CC and RC strategies and their potential limitations in free-flow conditions on a mountainous freeway with complex alignments. Human speed-selection behavior was observed among a group of participating drivers. The clustering analysis of the data revealed three distinct driving styles: slow, fast, and consistent. The resulted analytical models provided human-focused road-dependent baseline speed profiles- a key element of the proposed human-like speed control (HC) strategy. The comparison of the existing speed-control strategies CC and RC with the proposed HC confirmed the limitations of the two existing ones if applied to roads with complex alignments. Considerable discrepancies were revealed between the baseline speeds produced with the existing and the proposed ones.

Transferability analysis of the freeway continuous speed model.

Operating speed is often used to evaluate consistency in road geometric design. In the China, the Specifications for Highway Safety Audit includes a spot-based speed model that predicts operating speed by dividing the road into homogeneous segments and observing the speeds at sparsely spaced spots. This paper presents a continuous speed model as a more representative alternative for roads with complex alignments, and can be applied to tunnel sections as one general model. The model considers the road geometric characteristics not only at the vehicle's current position, but also in its neighborhood by including the effects of adjacent segments. Before such a model can be confidently used, however, its transferability must be confirmed for roads other than those used for the model's development. This study therefore used data collected at two freeways to demonstrate transferability, as well as the advantages of the continuous speed model over the spot-based model. Results of the spot-based model showed large prediction errors, and changes in the predicted speeds along the road were abrupt and discontinuous. On the other hand, the continuous model's prediction errors were smaller and the predicted speed profile was, as expected, continuous. The continuous model performed well at estimating operating speed on the studied freeway and, most importantly, it can predict operating speeds for out-of-sample roads of the same type as the studied roads. That is, it passed the transferability test. This finding opens an opportunity for evaluating roads in the design stage while minimizing the number of costly driving simulation experiments. Transferring a continuous speed model is a recommended alternative, particularly when high-priced construction is required for roads with challenging conditions such as mountainous terrain.