Power-law growth models explain incidences and sizes of pancreatic cancer precursor lesions and confirm spatial genomic findings.

Pancreatic ductal adenocarcinoma is a rare but lethal cancer. Recent evidence suggests that pancreatic intraepithelial neoplasia (PanIN), a microscopic precursor lesion that gives rise to pancreatic cancer, is larger and more prevalent than previously believed. Better understanding of the growth-law dynamics of PanINs may improve our ability to understand how a miniscule fraction makes the transition to invasive cancer. Here, using three-dimensional tissue mapping, we analyzed >1000 PanINs and found that lesion size is distributed according to a power law. Our data suggest that in bulk, PanIN size can be predicted by general growth behavior without consideration for the heterogeneity of the pancreatic microenvironment or an individual's age, history, or lifestyle. Our models suggest that intraductal spread and fusing of lesions drive our observed size distribution. This analysis lays the groundwork for future mathematical modeling efforts integrating PanIN incidence, morphology, and molecular features to understand tumorigenesis and demonstrates the utility of combining experimental measurement with dynamic modeling in understanding tumorigenesis.

High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment.

Enrichment of tumor-associated macrophages (TAMΦs) in the tumor microenvironment correlates with worse clinical outcomes in triple-negative breast cancer (TNBC) patients, prompting the development of therapies to inhibit TAMΦ infiltration. However, the lackluster efficacy of CCL2-based chemotaxis blockade in clinical trials suggests that a new understanding of monocyte/macrophage infiltration may be necessary. Here we demonstrate that random migration, and not only chemotaxis, drives macrophage tumor infiltration. We identified tumor- associated monocytes (TAMos) that display a dramatically enhanced migration capability, induced rapidly by the tumor microenvironment, that drives effective tumor infiltration, in contrast to low-motility differentiated macrophages. TAMo, not TAMΦ, promotes cancer cell proliferation through activation of the MAPK pathway. IL-6 secreted both by cancer cells and TAMo themselves enhances TAMo migration by increasing dendritic protrusion dynamics and myosin- based contractility via the JAK2/STAT3 signaling pathway. Independent from CCL2 mediated chemotaxis, IL-6 driven enhanced migration and pro-proliferative effect of TAMo were validated in a syngeneic TNBC mouse model. Depletion of IL-6 in cancer cells significantly attenuated monocyte infiltration and reversed TAMo-induced cancer cell proliferation. This work reveals the critical role random migration plays in monocyte driven TAMΦ enrichment in a tumor and pinpoints IL-6 as a potential therapeutic target in combination with CCL2 to ameliorate current strategies against TAMΦ infiltration.

qMAP enabled microanatomical mapping of human skin aging.

Aging is a major driver of diseases in humans. Identifying features associated with aging is essential for designing robust intervention strategies and discovering novel biomarkers of aging. Extensive studies at both the molecular and organ/whole-body physiological scales have helped determined features associated with aging. However, the lack of meso-scale studies, particularly at the tissue level, limits the ability to translate findings made at molecular scale to impaired tissue functions associated with aging. In this work, we established a tissue image analysis workflow - quantitative micro-anatomical phenotyping (qMAP) - that leverages deep learning and machine vision to fully label tissue and cellular compartments in tissue sections. The fully mapped tissue images address the challenges of finding an interpretable feature set to quantitatively profile age-related microanatomic changes. We optimized qMAP for skin tissues and applied it to a cohort of 99 donors aged 14 to 92. We extracted 914 microanatomic features and found that a broad spectrum of these features, represented by 10 cores processes, are strongly associated with aging. Our analysis shows that microanatomical features of the skin can predict aging with a mean absolute error (MAE) of 7.7 years, comparable to state-of-the-art epigenetic clocks. Our study demonstrates that tissue-level architectural changes are strongly associated with aging and represent a novel category of aging biomarkers that complement molecular markers. Our results highlight the complex and underexplored multi-scale relationship between molecular and tissue microanatomic scales.

AI and immunology.

AI is rapidly becoming part of many aspects of daily life, with an impact that reaches all fields of research. We asked investigators to share their thoughts on how AI is changing immunology research, what is necessary to move forward, the potential and the pitfalls, and what will remain unchanged as the field journeys into a new era.

PanIN or IPMN? Redefining Lesion Size in 3 Dimensions.

Pancreatic ductal adenocarcinoma (PDAC) develops from 2 known precursor lesions: a majority (∼85%) develops from pancreatic intraepithelial neoplasia (PanIN), and a minority develops from intraductal papillary mucinous neoplasms (IPMNs). Clinical classification of PanIN and IPMN relies on a combination of low-resolution, 3-dimensional (D) imaging (computed tomography, CT), and high-resolution, 2D imaging (histology). The definitions of PanIN and IPMN currently rely heavily on size. IPMNs are defined as macroscopic: generally >1.0 cm and visible in CT, and PanINs are defined as microscopic: generally <0.5 cm and not identifiable in CT. As 2D evaluation fails to take into account 3D structures, we hypothesized that this classification would fail in evaluation of high-resolution, 3D images. To characterize the size and prevalence of PanINs in 3D, 47 thick slabs of pancreas were harvested from grossly normal areas of pancreatic resections, excluding samples from individuals with a diagnosis of an IPMN. All patients but one underwent preoperative CT scans. Through construction of cellular resolution 3D maps, we identified >1400 ductal precursor lesions that met the 2D histologic size criteria of PanINs. We show that, when 3D space is considered, 25 of these lesions can be digitally sectioned to meet the 2D histologic size criterion of IPMN. Re-evaluation of the preoperative CT images of individuals found to possess these large precursor lesions showed that nearly half are visible on imaging. These findings demonstrate that the clinical classification of PanIN and IPMN fails in evaluation of high-resolution, 3D images, emphasizing the need for re-evaluation of classification guidelines that place significant weight on 2D assessment of 3D structures.

3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Generative interpolation and restoration of images using deep learning for improved 3D tissue mapping.

The development of novel imaging platforms has improved our ability to collect and analyze large three-dimensional (3D) biological imaging datasets. Advances in computing have led to an ability to extract complex spatial information from these data, such as the composition, morphology, and interactions of multi-cellular structures, rare events, and integration of multi-modal features combining anatomical, molecular, and transcriptomic (among other) information. Yet, the accuracy of these quantitative results is intrinsically limited by the quality of the input images, which can contain missing or damaged regions, or can be of poor resolution due to mechanical, temporal, or financial constraints. In applications ranging from intact imaging (e.g. light-sheet microscopy and magnetic resonance imaging) to sectioning based platforms (e.g. serial histology and serial section transmission electron microscopy), the quality and resolution of imaging data has become paramount. Here, we address these challenges by leveraging frame interpolation for large image motion (FILM), a generative AI model originally developed for temporal interpolation, for spatial interpolation of a range of 3D image types. Comparative analysis demonstrates the superiority of FILM over traditional linear interpolation to produce functional synthetic images, due to its ability to better preserve biological information including microanatomical features and cell counts, as well as image quality, such as contrast, variance, and luminance. FILM repairs tissue damages in images and reduces stitching artifacts. We show that FILM can decrease imaging time by synthesizing skipped images. We demonstrate the versatility of our method with a wide range of imaging modalities (histology, tissue-clearing/light-sheet microscopy, magnetic resonance imaging, serial section transmission electron microscopy), species (human, mouse), healthy and diseased tissues (pancreas, lung, brain), staining techniques (IHC, H&E), and pixel resolutions (8 nm, 2 µm, 1mm). Overall, we demonstrate the potential of generative AI in improving the resolution, throughput, and quality of biological image datasets, enabling improved 3D imaging.

A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

Magnetic Resonance Imaging-Based Assessment of Pancreatic Fat Strongly Correlates With Histology-Based Assessment of Pancreas Composition.

OBJECTIVE: The aim of the study is to assess the relationship between magnetic resonance imaging (MRI)-based estimation of pancreatic fat and histology-based measurement of pancreatic composition. MATERIALS AND METHODS: In this retrospective study, MRI was used to noninvasively estimate pancreatic fat content in preoperative images from high-risk individuals and disease controls having normal pancreata. A deep learning algorithm was used to label 11 tissue components at micron resolution in subsequent pancreatectomy histology. A linear model was used to determine correlation between histologic tissue composition and MRI fat estimation. RESULTS: Twenty-seven patients (mean age 64.0 ± 12.0 years [standard deviation], 15 women) were evaluated. The fat content measured by MRI ranged from 0% to 36.9%. Intrapancreatic histologic tissue fat content ranged from 0.8% to 38.3%. MRI pancreatic fat estimation positively correlated with microanatomical composition of fat (r = 0.90, 0.83 to 0.95], P < 0.001); as well as with pancreatic cancer precursor ( r = 0.65, P < 0.001); and collagen ( r = 0.46, P < 0.001) content, and negatively correlated with pancreatic acinar ( r = -0.85, P < 0.001) content. CONCLUSIONS: Pancreatic fat content, measurable by MRI, correlates to acinar content, stromal content (fibrosis), and presence of neoplastic precursors of cancer.

Engineering self-propelled tumor-infiltrating CAR T cells using synthetic velocity receptors.

Chimeric antigen receptor (CAR) T cells express antigen-specific synthetic receptors, which upon binding to cancer cells, elicit T cell anti-tumor responses. CAR T cell therapy has enjoyed success in the clinic for hematological cancer indications, giving rise to decade-long remissions in some cases. However, CAR T therapy for patients with solid tumors has not seen similar success. Solid tumors constitute 90% of adult human cancers, representing an enormous unmet clinical need. Current approaches do not solve the central problem of limited ability of therapeutic cells to migrate through the stromal matrix. We discover that T cells at low and high density display low- and high-migration phenotypes, respectively. The highly migratory phenotype is mediated by a paracrine pathway from a group of self-produced cytokines that include IL5, TNFα, IFNγ, and IL8. We exploit this finding to "lock-in" a highly migratory phenotype by developing and expressing receptors, which we call velocity receptors (VRs). VRs target these cytokines and signal through these cytokines' cognate receptors to increase T cell motility and infiltrate lung, ovarian, and pancreatic tumors in large numbers and at doses for which control CAR T cells remain confined to the tumor periphery. In contrast to CAR therapy alone, VR-CAR T cells significantly attenuate tumor growth and extend overall survival. This work suggests that approaches to the design of immune cell receptors that focus on migration signaling will help current and future CAR cellular therapies to infiltrate deep into solid tumors.

Three-dimensional assessments are necessary to determine the true, spatially-resolved composition of tissues.

Methods for spatially resolved cellular profiling using thinly cut sections have enabled in-depth quantitative tissue mapping to study inter-sample and intra-sample differences in normal human anatomy and disease onset and progression. These methods often profile extremely limited regions, which may impact the evaluation of heterogeneity due to tissue sub-sampling. Here, we applied CODA, a deep learning-based tissue mapping platform, to reconstruct the three-dimensional (3D) microanatomy of grossly normal and cancer-containing human pancreas biospecimens obtained from individuals who underwent pancreatic resection. To compare inter- and intra-sample heterogeneity, we assessed bulk and spatially resolved tissue composition in a cohort of two-dimensional (2D) whole slide images (WSIs) and a cohort of thick slabs of pancreas tissue that were digitally reconstructed in 3D from serial sections. To demonstrate the marked under sampling of 2D assessments, we simulated the number of WSIs and tissue microarrays (TMAs) necessary to represent the compositional heterogeneity of 3D data within 10% error to reveal that tens of WSIs and hundreds of TMA cores are sometimes needed. We show that spatial correlation of different pancreatic structures decay significantly within a span of microns, demonstrating that 2D histological sections may not be representative of their neighboring tissues. In sum, we demonstrate that 3D assessments are necessary to accurately assess tissue composition in normal and abnormal specimens and in order to accurately determine neoplastic content. These results emphasize the importance of intra-sample heterogeneity in tissue mapping efforts.

Power-law growth models explain incidences and sizes of pancreatic cancer precursor lesions and confirm spatial genomic findings.

Pancreatic ductal adenocarcinoma is a rare but lethal cancer. Recent evidence reveals that pancreatic intraepithelial neoplasms (PanINs), the microscopic precursor lesions in the pancreatic ducts that can give rise to invasive pancreatic cancer, are significantly larger and more prevalent than previously believed. Better understanding of the growth law dynamics of PanINs may improve our ability to understand how a miniscule fraction of these lesions makes the transition to invasive cancer. Here, using artificial intelligence (AI)-based three-dimensional (3D) tissue mapping method, we measured the volumes of >1,000 PanIN and found that lesion size is distributed according to a power law with a fitted exponent of -1.7 over > 3 orders of magnitude. Our data also suggest that PanIN growth is not very sensitive to the pancreatic microenvironment or an individual's age, family history, and lifestyle, and is rather shaped by general growth behavior. We analyze several models of PanIN growth and fit the predicted size distributions to the observed data. The best fitting models suggest that both intraductal spread of PanIN lesions and fusing of multiple lesions into large, highly branched structures drive PanIN growth patterns. This work lays the groundwork for future mathematical modeling efforts integrating PanIN incidence, morphology, genomic, and transcriptomic features to understand pancreas tumorigenesis, and demonstrates the utility of combining experimental measurement of human tissues with dynamic modeling for understanding cancer tumorigenesis.

Digitize your Biology! Modeling multicellular systems through interpretable cell behavior.

Cells are fundamental units of life, constantly interacting and evolving as dynamical systems. While recent spatial multi-omics can quantitate individual cells' characteristics and regulatory programs, forecasting their evolution ultimately requires mathematical modeling. We develop a conceptual framework-a cell behavior hypothesis grammar-that uses natural language statements (cell rules) to create mathematical models. This allows us to systematically integrate biological knowledge and multi-omics data to make them computable. We can then perform virtual "thought experiments" that challenge and extend our understanding of multicellular systems, and ultimately generate new testable hypotheses. In this paper, we motivate and describe the grammar, provide a reference implementation, and demonstrate its potential through a series of examples in tumor biology and immunotherapy. Altogether, this approach provides a bridge between biological, clinical, and systems biology researchers for mathematical modeling of biological systems at scale, allowing the community to extrapolate from single-cell characterization to emergent multicellular behavior.

Mechano-induced homotypic patterned domain formation by monocytes.

Matrix stiffness and corresponding mechano-signaling play indispensable roles in cellular phenotypes and functions. How tissue stiffness influences the behavior of monocytes, a major circulating leukocyte of the innate system, and how it may promote the emergence of collective cell behavior is less understood. Here, using tunable collagen-coated hydrogels of physiological stiffness, we show that human primary monocytes undergo a dynamic local phase separation to form highly patterned multicellular multi-layered domains on soft matrix. Local activation of the ß2 integrin initiates inter-cellular adhesion, while global soluble inhibitory factors maintain the steady-state domain pattern over days. Patterned domain formation generated by monocytes is unique among other key immune cells, including macrophages, B cells, T cells, and NK cells. While inhibiting their phagocytic capability, domain formation promotes monocytes' survival. We develop a computational model based on the Cahn-Hilliard equation, which includes combined local activation and global inhibition mechanisms of intercellular adhesion suggested by our experiments, and provides experimentally validated predictions of the role of seeding density and both chemotactic and random cell migration on pattern formation.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Tissue clearing and 3D reconstruction of digitized, serially sectioned slides provide novel insights into pancreatic cancer.

Pancreatic cancer is currently the third leading cause of cancer death in the United States. The clinical hallmarks of this disease include abdominal pain that radiates to the back, the presence of a hypoenhancing intrapancreatic lesion on imaging, and widespread liver metastases. Technologies such as tissue clearing and three-dimensional (3D) reconstruction of digitized serially sectioned hematoxylin and eosin-stained slides can be used to visualize large (up to 2- to 3-centimeter cube) tissues at cellular resolution. When applied to human pancreatic cancers, these 3D visualization techniques have provided novel insights into the basis of a number of the clinical characteristics of this disease. Here, we describe the clinical features of pancreatic cancer, review techniques for clearing and the 3D reconstruction of digitized microscope slides, and provide examples that illustrate how 3D visualization of human pancreatic cancer at the microscopic level has revealed features not apparent in 2D microscopy and, in so doing, has closed the gap between bench and bedside. Compared with animal models and 2D microscopy, studies of human tissues in 3D can reveal the difference between what can happen and what does happen in human cancers.

Intraparenchymal metastases as a cause for local recurrence of pancreatic cancer.

Using CODA, a technique for three-dimensional reconstruction of large tissues, Kiemen et al. report observation of a microscopic focus of pancreatic cancer found in the vasculature of grossly normal human pancreas tissue resected adjacent to a large tumour. They use TP53 and SMAD4 staining to relate the small focus to the primary tumour. This report describes a represents a probable case of intraparenchymal metastasis of pancreatic cancer, revealing a probable cause of local recurrence.

CODA: quantitative 3D reconstruction of large tissues at cellular resolution.

A central challenge in biology is obtaining high-content, high-resolution information while analyzing tissue samples at volumes relevant to disease progression. We address this here with CODA, a method to reconstruct exceptionally large (up to multicentimeter cubed) tissues at subcellular resolution using serially sectioned hematoxylin and eosin-stained tissue sections. Here we demonstrate CODA's ability to reconstruct three-dimensional (3D) distinct microanatomical structures in pancreas, skin, lung and liver tissues. CODA allows creation of readily quantifiable tissue volumes amenable to biological research. As a testbed, we assess the microanatomy of the human pancreas during tumorigenesis within the branching pancreatic ductal system, labeling ten distinct structures to examine heterogeneity and structural transformation during neoplastic progression. We show that pancreatic precancerous lesions develop into distinct 3D morphological phenotypes and that pancreatic cancer tends to spread far from the bulk tumor along collagen fibers that are highly aligned to the 3D curves of ductal, lobular, vascular and neural structures. Thus, CODA establishes a means to transform broadly the structural study of human diseases through exploration of exhaustively labeled 3D microarchitecture.

Characterization of tumor-associated macrophages in prostate cancer transgenic mouse models.

BACKGROUND: Tumor-associated macrophages (TAMs) are critical components of the tumor microenvironment (TME) in prostate cancer. Commonly used orthotopic models do not accurately reflect the complete TME of a human patient or the natural initiation and progression of a tumor. Therefore, genetically engineered mouse models are essential for studying the TME as well as advancing TAM-targeted therapies. Two common transgenic (TG) models of prostate cancer are Hi-Myc and transgenic adenocarcinoma of the mouse prostate (TRAMP), but the TME and TAM characteristics of these models have not been well characterized. METHODS: To advance the Hi-Myc and TRAMP models as tools for TAM studies, macrophage infiltration and characteristics were assessed using histopathologic, flow cytometric, and expression analyses in these models at various timepoints during tumor development and progression. RESULTS: In both Hi-Myc and TRAMP models, macrophages adopt a more pro-tumor phenotype in higher histological grade tumors and in older prostate tissue. However, the Hi-Myc and TRAMP prostates differ in their macrophage density, with Hi-Myc tumors exhibiting increased macrophage density and TRAMP tumors exhibiting decreased macrophage density compared to age-matched wild type mice. CONCLUSIONS: The macrophage density and the adenocarcinoma cancer subtype of Hi-Myc appear to better mirror patient tumors, suggesting that the Hi-Myc model is the more appropriate in vivo TG model for studying TAMs and TME-targeted therapies.