Community assessment of methods to deconvolve cellular composition from bulk gene expression.

We evaluate deconvolution methods, which infer levels of immune infiltration from bulk expression of tumor samples, through a community-wide DREAM Challenge. We assess six published and 22 community-contributed methods using in vitro and in silico transcriptional profiles of admixed cancer and healthy immune cells. Several published methods predict most cell types well, though they either were not trained to evaluate all functional CD8+ T cell states or do so with low accuracy. Several community-contributed methods address this gap, including a deep learning-based approach, whose strong performance establishes the applicability of this paradigm to deconvolution. Despite being developed largely using immune cells from healthy tissues, deconvolution methods predict levels of tumor-derived immune cells well. Our admixed and purified transcriptional profiles will be a valuable resource for developing deconvolution methods, including in response to common challenges we observe across methods, such as sensitive identification of functional CD4+ T cell states.

Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity.

The tumor microenvironment (TME) has a significant impact on tumor growth and immunotherapy efficacies. However, the precise cellular interactions and spatial organizations within the TME that drive these effects remain elusive. Using advanced multiplex imaging techniques, we have discovered that regulatory T cells (Tregs) accumulate around lymphatic vessels in the peripheral tumor stroma. This localized accumulation is facilitated by mature dendritic cells enriched in immunoregulatory molecules (mregDCs), which promote chemotaxis of Tregs, establishing a peri-lymphatic Treg-mregDC niche. Within this niche, mregDCs facilitate Treg activation, which in turn restrains the trafficking of tumor antigens to the draining mesenteric lymph nodes, thereby impeding the initiation of anti-tumor adaptive immune responses. Disrupting Treg recruitment to mregDCs inhibits tumor progression. Our study provides valuable insights into the organization of TME and how local crosstalk between lymphoid and myeloid cells suppresses anti-tumor immune responses.

Tracing unknown tumor origins with a biological-pathway-based transformer model.

Cancer of unknown primary (CUP) represents metastatic cancer where the primary site remains unidentified despite standard diagnostic procedures. To determine the tumor origin in such cases, we developed BPformer, a deep learning method integrating the transformer model with prior knowledge of biological pathways. Trained on transcriptomes from 10,410 primary tumors across 32 cancer types, BPformer achieved remarkable accuracy rates of 94%, 92%, and 89% in primary tumors and primary and metastatic sites of metastatic tumors, respectively, surpassing existing methods. Additionally, BPformer was validated in a retrospective study, demonstrating consistency with tumor sites diagnosed through immunohistochemistry and histopathology. Furthermore, BPformer was able to rank pathways based on their contribution to tumor origin identification, which helped to classify oncogenic signaling pathways into those that are highly conservative among different cancers versus those that are highly variable depending on their origins.

ConTIG: Continuous representation learning on temporal interaction graphs.

Representation learning on temporal interaction graphs (TIG) aims to model complex networks with the dynamic evolution of interactions on a wide range of web and social graph applications. However, most existing works on TIG either (a) rely on discretely updated node embeddings merely when an interaction occurs that fail to capture the continuous evolution of embedding trajectories of nodes, or (b) overlook the rich temporal patterns hidden in the ever-changing graph data that presumably lead to sub-optimal models. In this paper, we propose a two-module framework named ConTIG, a novel representation learning method on TIG that captures the continuous dynamic evolution of node embedding trajectories. With two essential modules, our model exploits three-fold factors in dynamic networks including latest interaction, neighbor features, and inherent characteristics. In the first update module, we employ a continuous inference block to learn the nodes' state trajectories from time-adjacent interaction patterns using ordinary differential equations. In the second transform module, we introduce a self-attention mechanism to predict future node embeddings by aggregating historical temporal interaction information. Experiment results demonstrate the superiority of ConTIG on temporal link prediction, temporal node recommendation, and dynamic node classification tasks of four datasets compared with a range of state-of-the-art baselines, especially for long-interval interaction prediction.

cgMSI: pathogen detection within species from nanopore metagenomic sequencing data.

BACKGROUND: Metagenomic sequencing is an unbiased approach that can potentially detect all the known and unidentified strains in pathogen detection. Recently, nanopore sequencing has been emerging as a highly potential tool for rapid pathogen detection due to its fast turnaround time. However, identifying pathogen within species is nontrivial for nanopore sequencing data due to the high sequencing error rate. RESULTS: We developed the core gene alleles metagenome strain identification (cgMSI) tool, which uses a two-stage maximum a posteriori probability estimation method to detect pathogens at strain level from nanopore metagenomic sequencing data at low computational cost. The cgMSI tool can accurately identify strains and estimate relative abundance at 1× coverage. CONCLUSIONS: We developed cgMSI for nanopore metagenomic pathogen detection within species. cgMSI is available at https://github.com/ZHU-XU-xmu/cgMSI .

Dear-DIAXMBD: Deep Autoencoder Enables Deconvolution of Data-Independent Acquisition Proteomics.

Data-independent acquisition (DIA) technology for protein identification from mass spectrometry and related algorithms is developing rapidly. The spectrum-centric analysis of DIA data without the use of spectra library from data-dependent acquisition data represents a promising direction. In this paper, we proposed an untargeted analysis method, Dear-DIAXMBD, for direct analysis of DIA data. Dear-DIAXMBD first integrates the deep variational autoencoder and triplet loss to learn the representations of the extracted fragment ion chromatograms, then uses the k-means clustering algorithm to aggregate fragments with similar representations into the same classes, and finally establishes the inverted index tables to determine the precursors of fragment clusters between precursors and peptides and between fragments and peptides. We show that Dear-DIAXMBD performs superiorly with the highly complicated DIA data of different species obtained by different instrument platforms. Dear-DIAXMBD is publicly available at https://github.com/jianweishuai/Dear-DIA-XMBD.

SC-AIR-BERT: a pre-trained single-cell model for predicting the antigen-binding specificity of the adaptive immune receptor.

Accurately predicting the antigen-binding specificity of adaptive immune receptors (AIRs), such as T-cell receptors (TCRs) and B-cell receptors (BCRs), is essential for discovering new immune therapies. However, the diversity of AIR chain sequences limits the accuracy of current prediction methods. This study introduces SC-AIR-BERT, a pre-trained model that learns comprehensive sequence representations of paired AIR chains to improve binding specificity prediction. SC-AIR-BERT first learns the 'language' of AIR sequences through self-supervised pre-training on a large cohort of paired AIR chains from multiple single-cell resources. The model is then fine-tuned with a multilayer perceptron head for binding specificity prediction, employing the K-mer strategy to enhance sequence representation learning. Extensive experiments demonstrate the superior AUC performance of SC-AIR-BERT compared with current methods for TCR- and BCR-binding specificity prediction.

Hybrid Graph Convolutional Network With Online Masked Autoencoder for Robust Multimodal Cancer Survival Prediction.

Cancer survival prediction requires exploiting related multimodal information (e.g., pathological, clinical and genomic features, etc.) and it is even more challenging in clinical practices due to the incompleteness of patient's multimodal data. Furthermore, existing methods lack sufficient intra- and inter-modal interactions, and suffer from significant performance degradation caused by missing modalities. This manuscript proposes a novel hybrid graph convolutional network, entitled HGCN, which is equipped with an online masked autoencoder paradigm for robust multimodal cancer survival prediction. Particularly, we pioneer modeling the patient's multimodal data into flexible and interpretable multimodal graphs with modality-specific preprocessing. HGCN integrates the advantages of graph convolutional networks (GCNs) and a hypergraph convolutional network (HCN) through node message passing and a hyperedge mixing mechanism to facilitate intra-modal and inter-modal interactions between multimodal graphs. With HGCN, the potential for multimodal data to create more reliable predictions of patient's survival risk is dramatically increased compared to prior methods. Most importantly, to compensate for missing patient modalities in clinical scenarios, we incorporated an online masked autoencoder paradigm into HGCN, which can effectively capture intrinsic dependence between modalities and seamlessly generate missing hyperedges for model inference. Extensive experiments and analysis on six cancer cohorts from TCGA show that our method significantly outperforms the state-of-the-arts in both complete and missing modal settings. Our codes are made available at https://github.com/lin-lcx/HGCN.

MuRCL: Multi-Instance Reinforcement Contrastive Learning for Whole Slide Image Classification.

Multi-instance learning (MIL) is widely adop- ted for automatic whole slide image (WSI) analysis and it usually consists of two stages, i.e., instance feature extraction and feature aggregation. However, due to the "weak supervision" of slide-level labels, the feature aggregation stage would suffer from severe over-fitting in training an effective MIL model. In this case, mining more information from limited slide-level data is pivotal to WSI analysis. Different from previous works on improving instance feature extraction, this paper investigates how to exploit the latent relationship of different instances (patches) to combat overfitting in MIL for more generalizable WSI classification. In particular, we propose a novel Multi-instance Rein- forcement Contrastive Learning framework (MuRCL) to deeply mine the inherent semantic relationships of different patches to advance WSI classification. Specifically, the proposed framework is first trained in a self-supervised manner and then finetuned with WSI slide-level labels. We formulate the first stage as a contrastive learning (CL) process, where positive/negative discriminative feature sets are constructed from the same patch-level feature bags of WSIs. To facilitate the CL training, we design a novel reinforcement learning-based agent to progressively update the selection of discriminative feature sets according to an online reward for slide-level feature aggregation. Then, we further update the model with labeled WSI data to regularize the learned features for the final WSI classification. Experimental results on three public WSI classification datasets (Camelyon16, TCGA-Lung and TCGA-Kidney) demonstrate that the proposed MuRCL outperforms state-of-the-art MIL models. In addition, MuRCL can achieve comparable performance to other state-of-the-art MIL models on TCGA-Esca dataset.

Prioritizing prognostic-associated subpopulations and individualized recurrence risk signatures from single-cell transcriptomes of colorectal cancer.

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies. There are few recurrence risk signatures for CRC patients. Single-cell RNA-sequencing (scRNA-seq) provides a high-resolution platform for prognostic signature detection. However, scRNA-seq is not practical in large cohorts due to its high cost and most single-cell experiments lack clinical phenotype information. Few studies have been reported to use external bulk transcriptome with survival time to guide the detection of key cell subtypes in scRNA-seq data. We proposed scRankXMBD, a computational framework to prioritize prognostic-associated cell subpopulations based on within-cell relative expression orderings of gene pairs from single-cell transcriptomes. scRankXMBD achieves higher precision and concordance compared with five existing methods. Moreover, we developed single-cell gene pair signatures to predict recurrence risk for patients individually. Our work facilitates the application of the rank-based method in scRNA-seq data for prognostic biomarker discovery and precision oncology. scRankXMBD is available at https://github.com/xmuyulab/scRank-XMBD. (XMBD:Xiamen Big Data, a biomedical open software initiative in the National Institute for Data Science in Health and Medicine, Xiamen University, China.).

Opioid-induced fragile-like regulatory T cells contribute to withdrawal.

Dysregulation of the immune system is a cardinal feature of opioid addiction. Here, we characterize the landscape of peripheral immune cells from patients with opioid use disorder and from healthy controls. Opioid-associated blood exhibited an abnormal distribution of immune cells characterized by a significant expansion of fragile-like regulatory T cells (Tregs), which was positively correlated with the withdrawal score. Analogously, opioid-treated mice also showed enhanced Treg-derived interferon-γ (IFN-γ) expression. IFN-γ signaling reshaped synaptic morphology in nucleus accumbens (NAc) neurons, modulating subsequent withdrawal symptoms. We demonstrate that opioids increase the expression of neuron-derived C-C motif chemokine ligand 2 (Ccl2) and disrupted blood-brain barrier (BBB) integrity through the downregulation of astrocyte-derived fatty-acid-binding protein 7 (Fabp7), which both triggered peripheral Treg infiltration into NAc. Our study demonstrates that opioids drive the expansion of fragile-like Tregs and favor peripheral Treg diapedesis across the BBB, which leads to IFN-γ-mediated synaptic instability and subsequent withdrawal symptoms.

Multiplexed imaging mass cytometry reveals distinct tumor-immune microenvironments linked to immunotherapy responses in melanoma.

Background: Single-cell technologies have enabled extensive analysis of complex immune composition, phenotype and interactions within tumor, which is crucial in understanding the mechanisms behind cancer progression and treatment resistance. Unfortunately, knowledge on cell phenotypes and their spatial interactions has only had limited impact on the pathological stratification of patients in the clinic so far. We explore the relationship between different tumor environments (TMEs) and response to immunotherapy by deciphering the composition and spatial relationships of different cell types. Methods: Here we used imaging mass cytometry to simultaneously quantify 35 proteins in a spatially resolved manner on tumor tissues from 26 melanoma patients receiving anti-programmed cell death-1 (anti-PD-1) therapy. Using unsupervised clustering, we profiled 662,266 single cells to identify lymphocytes, myeloid derived monocytes, stromal and tumor cells, and characterized TME of different melanomas. Results: Combined single-cell and spatial analysis reveals highly dynamic TMEs that are characterized with variable tumor and immune cell phenotypes and their spatial organizations in melanomas, and many of these multicellular features are associated with response to anti-PD-1 therapy. We further identify six distinct TME archetypes based on their multicellular compositions, and find that patients with different TME archetypes responded differently to anti-PD-1 therapy. Finally, we find that classifying patients based on the gene expression signature derived from TME archetypes predicts anti-PD-1 therapy response across multiple validation cohorts. Conclusions: Our results demonstrate the utility of multiplex proteomic imaging technologies in studying complex molecular events in a spatially resolved manner for the development of new strategies for patient stratification and treatment outcome prediction.

Protocol to estimate cell type proportions from bulk RNA-seq using DAISM-DNNXMBD.

Computational protocols for cell type deconvolution from bulk RNA-seq data have been used to understand cellular heterogeneity in disease-related samples, but their performance can be impacted by batch effect among datasets. Here, we present a DAISM-DNN protocol to achieve robust cell type proportion estimation on the target dataset. We describe the preparation of calibrated samples from human blood samples. We then detail steps to train a dataset-specific deep neural network (DNN) model and cell type proportion estimation using the trained model. For complete details on the use and execution of this protocol, please refer to Lin et al. (2022).

Deepfakes: A new threat to image fabrication in scientific publications?

There is an increasing risk of people using advanced artificial intelligence, particularly the generative adversarial network (GAN), for scientific image manipulation for the purpose of publications. We demonstrated this possibility by using GAN to fabricate several different types of biomedical images and discuss possible ways for the detection and prevention of such scientific misconducts in research communities.

DAISM-DNNXMBD: Highly accurate cell type proportion estimation with in silico data augmentation and deep neural networks.

Understanding the immune cell abundance of cancer and other disease-related tissues has an important role in guiding disease treatments. Computational cell type proportion estimation methods have been previously developed to derive such information from bulk RNA sequencing data. Unfortunately, our results show that the performance of these methods can be seriously plagued by the mismatch between training data and real-world data. To tackle this issue, we propose the DAISM-DNNXMBD (XMBD: Xiamen Big Data, a biomedical open software initiative in the National Institute for Data Science in Health and Medicine, Xiamen University, China.) (denoted as DAISM-DNN) pipeline that trains a deep neural network (DNN) with dataset-specific training data populated from a certain amount of calibrated samples using DAISM, a novel data augmentation method with an in silico mixing strategy. The evaluation results demonstrate that the DAISM-DNN pipeline outperforms other existing methods consistently and substantially for all the cell types under evaluation in real-world datasets.

A downsampling method enables robust clustering and integration of single-cell transcriptome data.

The random noises, sampling biases, and batch effects often confound true biological variations in single-cell RNA-sequencing (scRNA-seq) data. Adjusting such biases is key to the robust discoveries in downstream analyses, such as cell clustering, gene selection and data integration. Here we propose a model-based downsampling algorithm based on minimal unbiased representative points (MURPXMBD). MURPXMBD is designed to retrieve a set of representative points by reducing gene-wise random independent errors, while retaining the covariance structure of biological origin hence provide an unbiased representation of the cell population. Subsequent validation using benchmark datasets shows that MURPXMBD can improve the quality and accuracy of clustering algorithms, and thus facilitate the discovery of new cell types. Besides, MURPXMBD also improves the performance of dataset integration algorithms. In summary, MURPXMBD serves as a useful noise-reduction method for single-cell sequencing analysis in biomedical studies.

Application of individualized differential expression analysis in human cancer proteome.

Liquid chromatography-mass spectrometry-based quantitative proteomics can measure the expression of thousands of proteins from biological samples and has been increasingly applied in cancer research. Identifying differentially expressed proteins (DEPs) between tumors and normal controls is commonly used to investigate carcinogenesis mechanisms. While differential expression analysis (DEA) at an individual level is desired to identify patient-specific molecular defects for better patient stratification, most statistical DEP analysis methods only identify deregulated proteins at the population level. To date, robust individualized DEA algorithms have been proposed for ribonucleic acid data, but their performance on proteomics data is underexplored. Herein, we performed a systematic evaluation on five individualized DEA algorithms for proteins on cancer proteomic datasets from seven cancer types. Results show that the within-sample relative expression orderings (REOs) of protein pairs in normal tissues were highly stable, providing the basis for individualized DEA for proteins using REOs. Moreover, individualized DEA algorithms achieve higher precision in detecting sample-specific deregulated proteins than population-level methods. To facilitate the utilization of individualized DEA algorithms in proteomics for prognostic biomarker discovery and personalized medicine, we provide Individualized DEP Analysis IDEPAXMBD (XMBD: Xiamen Big Data, a biomedical open software initiative in the National Institute for Data Science in Health and Medicine, Xiamen University, China.) (https://github.com/xmuyulab/IDEPA-XMBD), which is a user-friendly and open-source Python toolkit that integrates individualized DEA algorithms for DEP-associated deregulation pattern recognition.

Deep representation features from DreamDIAXMBD improve the analysis of data-independent acquisition proteomics.

We developed DreamDIAXMBD (denoted as DreamDIA), a software suite based on a deep representation model for data-independent acquisition (DIA) data analysis. DreamDIA adopts a data-driven strategy to capture comprehensive information from elution patterns of peptides in DIA data and achieves considerable improvements on both identification and quantification performance compared with other state-of-the-art methods such as OpenSWATH, Skyline and DIA-NN. Specifically, in contrast to existing methods which use only 6 to 10 selected fragment ions from spectral libraries, DreamDIA extracts additional features from hundreds of theoretical elution profiles originated from different ions of each precursor using a deep representation network. To achieve higher coverage of target peptides without sacrificing specificity, the extracted features are further processed by nonlinear discriminative models under the framework of positive-unlabeled learning with decoy peptides as affirmative negative controls. DreamDIA is publicly available at https://github.com/xmuyulab/DreamDIA-XMBD for high coverage and accuracy DIA data analysis.

Dice-XMBD: Deep Learning-Based Cell Segmentation for Imaging Mass Cytometry.

Highly multiplexed imaging technology is a powerful tool to facilitate understanding the composition and interactions of cells in tumor microenvironments at subcellular resolution, which is crucial for both basic research and clinical applications. Imaging mass cytometry (IMC), a multiplex imaging method recently introduced, can measure up to 100 markers simultaneously in one tissue section by using a high-resolution laser with a mass cytometer. However, due to its high resolution and large number of channels, how to process and interpret the image data from IMC remains a key challenge to its further applications. Accurate and reliable single cell segmentation is the first and a critical step to process IMC image data. Unfortunately, existing segmentation pipelines either produce inaccurate cell segmentation results or require manual annotation, which is very time consuming. Here, we developed Dice-XMBD, a Deep learnIng-based Cell sEgmentation algorithm for tissue multiplexed imaging data. In comparison with other state-of-the-art cell segmentation methods currently used for IMC images, Dice-XMBD generates more accurate single cell masks efficiently on IMC images produced with different nuclear, membrane, and cytoplasm markers. All codes and datasets are available at https://github.com/xmuyulab/Dice-XMBD.