Multiplex imaging of localized prostate tumors reveals altered spatial organization of AR-positive cells in the microenvironment.

Mapping the spatial interactions of cancer, immune, and stromal cell states presents novel opportunities for patient stratification and for advancing immunotherapy. While single-cell studies revealed significant molecular heterogeneity in prostate cancer cells, the impact of spatial stromal cell heterogeneity remains poorly understood. Here, we used cyclic immunofluorescent imaging on whole-tissue sections to uncover novel spatial associations between cancer and stromal cells in low- and high-grade prostate tumors and tumor-adjacent normal tissues. Our results provide a spatial map of single cells and recurrent cellular neighborhoods in the prostate tumor microenvironment of treatment-naive patients. We report unique populations of mast cells that show distinct spatial associations with M2 macrophages and regulatory T cells. Our results show disease-specific neighborhoods that are primarily driven by androgen receptor-positive (AR+) stromal cells and identify inflammatory gene networks active in AR+ prostate stroma.

A community-based approach to image analysis of cells, tissues and tumors.

Emerging multiplexed imaging platforms provide an unprecedented view of an increasing number of molecular markers at subcellular resolution and the dynamic evolution of tumor cellular composition. As such, they are capable of elucidating cell-to-cell interactions within the tumor microenvironment that impact clinical outcome and therapeutic response. However, the rapid development of these platforms has far outpaced the computational methods for processing and analyzing the data they generate. While being technologically disparate, all imaging assays share many computational requirements for post-collection data processing. As such, our Image Analysis Working Group (IAWG), composed of researchers in the Cancer Systems Biology Consortium (CSBC) and the Physical Sciences - Oncology Network (PS-ON), convened a workshop on "Computational Challenges Shared by Diverse Imaging Platforms" to characterize these common issues and a follow-up hackathon to implement solutions for a selected subset of them. Here, we delineate these areas that reflect major axes of research within the field, including image registration, segmentation of cells and subcellular structures, and identification of cell types from their morphology. We further describe the logistical organization of these events, believing our lessons learned can aid others in uniting the imaging community around self-identified topics of mutual interest, in designing and implementing operational procedures to address those topics and in mitigating issues inherent in image analysis (e.g., sharing exemplar images of large datasets and disseminating baseline solutions to hackathon challenges through open-source code repositories).

Toward reproducible, scalable, and robust data analysis across multiplex tissue imaging platforms.

The emergence of megascale single-cell multiplex tissue imaging (MTI) datasets necessitates reproducible, scalable, and robust tools for cell phenotyping and spatial analysis. We developed open-source, graphics processing unit (GPU)-accelerated tools for intensity normalization, phenotyping, and microenvironment characterization. We deploy the toolkit on a human breast cancer (BC) tissue microarray stained by cyclic immunofluorescence and present the first cross-validation of breast cancer cell phenotypes derived by using two different MTI platforms. Finally, we demonstrate an integrative phenotypic and spatial analysis revealing BC subtype-specific features.

Predicting primary site of secondary liver cancer with a neural estimator of metastatic origin.

Purpose: Pathologists rely on relevant clinical information, visual inspection of stained tissue slide morphology, and sophisticated molecular diagnostics to accurately infer the biological origin of secondary metastatic cancer. While highly effective, this process is expensive in terms of time and clinical resources. We seek to develop and evaluate a computer vision system designed to reasonably infer metastatic origin of secondary liver cancer directly from digitized histopathological whole slide images of liver biopsy. Approach: We illustrate a two-stage deep learning approach to accomplish this task. We first train a model to identify spatially localized regions of cancerous tumor within digitized hematoxylin and eosin (H&E)-stained tissue sections of secondary liver cancer based on a pathologist's annotation of several whole slide images. Then, a second model is trained to generate predictions of the cancers' metastatic origin belonging to one of three distinct clinically relevant classes as confirmed by immunohistochemistry. Results: Our approach achieves a classification accuracy of 90.2% in determining metastatic origin of whole slide images from a held-out test set, which compares favorably to an established clinical benchmark by three board-certified pathologists whose accuracies ranged from 90.2% to 94.1% on the same prediction task. Conclusions: We illustrate the potential impact of deep learning systems to leverage morphological and structural features of H&E-stained tissue sections to guide pathological and clinical determination of the metastatic origin of secondary liver cancers.

SHIFT: speedy histological-to-immunofluorescent translation of a tumor signature enabled by deep learning.

Spatially-resolved molecular profiling by immunostaining tissue sections is a key feature in cancer diagnosis, subtyping, and treatment, where it complements routine histopathological evaluation by clarifying tumor phenotypes. In this work, we present a deep learning-based method called speedy histological-to-immunofluorescent translation (SHIFT) which takes histologic images of hematoxylin and eosin (H&E)-stained tissue as input, then in near-real time returns inferred virtual immunofluorescence (IF) images that estimate the underlying distribution of the tumor cell marker pan-cytokeratin (panCK). To build a dataset suitable for learning this task, we developed a serial staining protocol which allows IF and H&E images from the same tissue to be spatially registered. We show that deep learning-extracted morphological feature representations of histological images can guide representative sample selection, which improved SHIFT generalizability in a small but heterogenous set of human pancreatic cancer samples. With validation in larger cohorts, SHIFT could serve as an efficient preliminary, auxiliary, or substitute for panCK IF by delivering virtual panCK IF images for a fraction of the cost and in a fraction of the time required by traditional IF.

SHIFT: speedy histopathological-to-immunofluorescent translation of whole slide images using conditional generative adversarial networks.

Multiplexed imaging such as multicolor immunofluorescence staining, multiplexed immunohistochemistry (mIHC) or cyclic immunofluorescence (cycIF) enables deep assessment of cellular complexity in situ and, in conjunction with standard histology stains like hematoxylin and eosin (H&E), can help to unravel the complex molecular relationships and spatial interdependencies that undergird disease states. However, these multiplexed imaging methods are costly and can degrade both tissue quality and antigenicity with each successive cycle of staining. In addition, computationally intensive image processing such as image registration across multiple channels is required. We have developed a novel method, speedy histopathological-to-immunofluorescent translation (SHIFT) of whole slide images (WSIs) using conditional generative adversarial networks (cGANs). This approach is rooted in the assumption that specific patterns captured in IF images by stains like DAPI, pan-cytokeratin (panCK), or α-smooth muscle actin ( α-SMA) are encoded in H&E images, such that a SHIFT model can learn useful feature representations or architectural patterns in the H&E stain that help generate relevant IF stain patterns. We demonstrate that the proposed method is capable of generating realistic tumor marker IF WSIs conditioned on corresponding H&E-stained WSIs with up to 94.5% accuracy in a matter of seconds. Thus, this method has the potential to not only improve our understanding of the mapping of histological and morphological profiles into protein expression profiles, but also greatly increase the e ciency of diagnostic and prognostic decision-making.