PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.

This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

3D histology reveals that immune response to pancreatic precancers is heterogeneous and depends on global pancreas structure.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer for which few effective therapies exist. Immunotherapies specifically are ineffective in pancreatic cancer, in part due to its unique stromal and immune microenvironment. Pancreatic intraepithelial neoplasia, or PanIN, is the main precursor lesion to PDAC. Recently it was discovered that PanINs are remarkably abundant in the grossly normal pancreas, suggesting that the vast majority will never progress to cancer. Here, through construction of 48 samples of cm 3 -sized human pancreas tissue, we profiled the immune microenvironment of 1,476 PanINs in 3D and at single-cell resolution to better understand the early evolution of the pancreatic tumor microenvironment and to determine how inflammation may play a role in cancer progression. We found that bulk pancreatic inflammation strongly correlates to PanIN cell fraction. We found that the immune response around PanINs is highly heterogeneous, with distinct immune hotspots and cold spots that appear and disappear in a span of tens of microns. Immune hotspots generally mark locations of higher grade of dysplasia or locations near acinar atrophy. The immune composition at these hotspots is dominated by naïve, cytotoxic, and regulatory T cells, cancer associated fibroblasts, and tumor associated macrophages, with little similarity to the immune composition around less-inflamed PanINs. By mapping FOXP3+ cells in 3D, we found that regulatory T cells are present at higher density in larger PanIN lesions compared to smaller PanINs, suggesting that the early initiation of PanINs may not exhibit an immunosuppressive response. This analysis demonstrates that while PanINs are common in the pancreases of most individuals, inflammation may play a pivotal role, both at the bulk and the microscopic scale, in demarcating regions of significance in cancer progression.

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.

E-cadherin Induces Serine Synthesis to Support Progression and Metastasis of Breast Cancer.

The loss of E-cadherin, an epithelial cell adhesion molecule, has been implicated in metastasis by mediating the epithelial-mesenchymal transition (EMT), which promotes invasion and migration of cancer cells. However, recent studies have demonstrated that E-cadherin supports the survival and proliferation of metastatic cancer cells. Here, we identified a metabolic role for E-cadherin in breast cancer by upregulating the de novo serine synthesis pathway (SSP). The upregulated SSP provided metabolic precursors for biosynthesis and resistance to oxidative stress, enabling E-cadherin+ breast cancer cells to achieve faster tumor growth and enhanced metastases. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, significantly and specifically hampered proliferation of E-cadherin+ breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. These findings reveal that E-cadherin reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers.

Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis.

Aged patients with melanoma (>65 years old) have more aggressive disease relative to young patients (<55 years old) for reasons that are not completely understood. Analysis of the young and aged secretome from human dermal fibroblasts identified >5-fold levels of IGF-binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to those co-cultured with young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts or overexpressing IGFBP2 in melanoma cells promoted lipid synthesis and accumulation in melanoma cells. Treatment of young mice with rIGFBP2 increases tumor growth. Neutralizing IGFBP2 in vitro reduces migration and invasion in melanoma cells, and in vivo studies demonstrate that neutralizing IGFBP2 in syngeneic aged mice reduces tumor growth and metastasis. Our results suggest that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment. SIGNIFICANCE: The aged microenvironment drives metastasis in melanoma cells. This study reports that IGFBP2 secretion by aged fibroblasts induces lipid accumulation in melanoma cells, driving an increase in tumor invasiveness. Neutralizing IGFBP2 decreases melanoma tumor growth and metastasis.

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.

A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity.

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment.

Small Extracellular Vesicles Promote Stiffness-mediated Metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiologic matrix stiffness affects the quantity and protein cargo of small extracellular vesicles (EV) produced by cancer cells, which in turn aid cancer cell dissemination. Primary patient breast tissue released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα2ß1, ITGα6ß4, ITGα6ß1, CD44) compared with EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix proteins including collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer-associated fibroblast phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. SIGNIFICANCE: Here we show that the quantity, cargo, and function of breast cancer-derived EVs vary with mechanical properties of the extracellular microenvironment.

MLL1 regulates cytokine-driven cell migration and metastasis.

Cell migration is a critical contributor to metastasis. Cytokine production and its role in cancer cell migration have been traditionally associated with immune cells. We find that the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) controls 3D cell migration via cytokines, IL-6, IL-8, and TGF-ß1, secreted by the cancer cells themselves. MLL1, with its scaffold protein Menin, controls actin filament assembly via the IL-6/8/pSTAT3/Arp3 axis and myosin contractility via the TGF-ß1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1 also regulates cell proliferation via mitosis-based and cell cycle-related pathways. Mice bearing orthotopic MLL1-depleted tumors exhibit decreased lung metastatic burden and longer survival. MLL1 depletion leads to lower metastatic burden even when controlling for the difference in primary tumor growth rates. Combining MLL1-Menin inhibitor with paclitaxel abrogates tumor growth and metastasis, including preexistent metastasis. These results establish MLL1 as a potent regulator of cell migration and highlight the potential of targeting MLL1 in patients with metastatic disease.

E-cadherin interacts with EGFR resulting in hyper-activation of ERK in multiple models of breast cancer.

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success.

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.

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.

Small extracellular vesicles promote stiffness-mediated metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα 2 ß 1 , ITGα 6 ß 4 , ITGα 6 ß 1 , CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment.

Serine synthesis pathway upregulated by E-cadherin is essential for the proliferation and metastasis of breast cancers.

The loss of E-cadherin (E-cad), an epithelial cell adhesion molecule, has been implicated in the epithelial-mesenchymal transition (EMT), promoting invasion and migration of cancer cells and, consequently, metastasis. However, recent studies have demonstrated that E-cad supports the survival and proliferation of metastatic cancer cells, suggesting that our understanding of E-cad in metastasis is far from comprehensive. Here, we report that E-cad upregulates the de novo serine synthesis pathway (SSP) in breast cancer cells. The SSP provides metabolic precursors for biosynthesis and resistance to oxidative stress, critically beneficial for E-cad-positive breast cancer cells to achieve faster tumor growth and more metastases. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, significantly and specifically hampered the proliferation of E-cad-positive breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. Our findings reveal that E-cad adhesion molecule significantly reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers.

Tumor proliferation and invasion are intrinsically coupled and unraveled through tunable spheroid and physics-based models.

Proliferation and invasion are two key drivers of tumor growth that are traditionally considered independent multicellular processes. However, these processes are intrinsically coupled through a maximum carrying capacity, i.e., the maximum spatial cell concentration supported by the tumor volume, total cell count, nutrient access, and mechanical properties of the tissue stroma. We explored this coupling of proliferation and invasion through in vitro and in silico methods where we modulated the mechanical properties of the tumor and the surrounding extracellular matrix. E-cadherin expression and stromal collagen concentration were manipulated in a tunable breast cancer spheroid to determine the overall impacts of these tumor variables on net tumor proliferation and continuum invasion. We integrated these results into a mixed-constitutive formulation to computationally delineate the influences of cellular and extracellular adhesion, stiffness, and mechanical properties of the extracellular matrix on net proliferation and continuum invasion. This framework integrates biological in vitro data into concise computational models of invasion and proliferation to provide more detailed physical insights into the coupling of these key tumor processes and tumor growth. STATEMENT OF SIGNIFICANCE: Tumor growth involves expansion into the collagen-rich stroma through intrinsic coupling of proliferation and invasion within the tumor continuum. These processes are regulated by a maximum carrying capacity that is determined by the total cell count, tumor volume, nutrient access, and mechanical properties of the surrounding stroma. The influences of biomechanical parameters (i.e., stiffness, cell elongation, net proliferation rate and cell-ECM friction) on tumor proliferation or invasion cannot be unraveled using experimental methods alone. By pairing a tunable spheroid system with computational modeling, we delineated the interdependencies of each system parameter on tumor proliferation and continuum invasion, and established a concise computational framework for studying tumor mechanobiology.

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.