Identification of Functional Heterogeneity of Carcinoma-Associated Fibroblasts with Distinct IL6-Mediated Therapy Resistance in Pancreatic Cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of fibroblasts as part of the host response to cancer. Using single-cell RNA sequencing, multiplex immunostaining, and several genetic mouse models, we identify carcinoma-associated fibroblasts (CAF) with opposing functions in PDAC progression. Depletion of fibroblast activation protein (FAP)+ CAFs results in increased survival, in contrast to depletion of alpha smooth muscle actin (αSMA)+ CAFs, which leads to decreased survival. Tumor-promoting FAP+ CAFs (TP-CAF) and tumor-restraining αSMA+ CAFs (TR-CAF) differentially regulate cancer-associated pathways and accumulation of regulatory T cells. Improved efficacy of gemcitabine is observed when IL6 is deleted from αSMA+ CAFs but not from FAP+ CAFs using dual-recombinase genetic PDAC models. Improved gemcitabine efficacy due to lack of IL6 synergizes with anti-PD-1 immunotherapy to significantly improve survival of PDAC mice. Our study identifies functional heterogeneity of CAFs in PDAC progression and their different roles in therapy response. SIGNIFICANCE: PDAC is associated with accumulation of dense stroma consisting of fibroblasts and extracellular matrix that regulate tumor progression. Here, we identify two distinct populations of fibroblasts with opposing roles in the progression and immune landscape of PDAC. Our findings demonstrate that fibroblasts are functionally diverse with therapeutic implications. This article is highlighted in the In This Issue feature, p. 1397.

Stabilized epithelial phenotype of cancer cells in primary tumors leads to increased colonization of liver metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is therapeutically recalcitrant and metastatic. Partial epithelial to mesenchymal transition (EMT) is associated with metastasis; however, a causal connection needs further unraveling. Here, we use single-cell RNA sequencing and genetic mouse models to identify the functional roles of partial EMT and epithelial stabilization in PDAC growth and metastasis. A global EMT expression signature identifies ∼50 cancer cell clusters spanning the epithelial-mesenchymal continuum in both human and murine PDACs. The combined genetic suppression of Snail and Twist results in PDAC epithelial stabilization and increased liver metastasis. Genetic deletion of Zeb1 in PDAC cells also leads to liver metastasis associated with cancer cell epithelial stabilization. We demonstrate that epithelial stabilization leads to the enhanced collective migration of cancer cells and modulation of the immune microenvironment, which likely contribute to efficient liver colonization. Our study provides insights into the diverse mechanisms of metastasis in pancreatic cancer and potential therapeutic targets.

Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.

The exact nature and dynamics of pancreatic ductal adenocarcinoma (PDAC) immune composition remains largely unknown. Desmoplasia is suggested to polarize PDAC immunity. Therefore, a comprehensive evaluation of the composition and distribution of desmoplastic elements and T-cell infiltration is necessary to delineate their roles. Here we develop a novel computational imaging technology for the simultaneous evaluation of eight distinct markers, allowing for spatial analysis of distinct populations within the same section. We report a heterogeneous population of infiltrating T lymphocytes. Spatial distribution of cytotoxic T cells in proximity to cancer cells correlates with increased overall patient survival. Collagen-I and αSMA+ fibroblasts do not correlate with paucity in T-cell accumulation, suggesting that PDAC desmoplasia may not be a simple physical barrier. Further exploration of this technology may improve our understanding of how specific stromal composition could impact T-cell activity, with potential impact on the optimization of immune-modulatory therapies.

Morphologically constrained spectral unmixing by dictionary learning for multiplex fluorescence microscopy.

MOTIVATION: Current spectral unmixing methods for multiplex fluorescence microscopy have a limited ability to cope with high spectral overlap as they only analyze spectral information over individual pixels. Here, we present adaptive Morphologically Constrained Spectral Unmixing (MCSU) algorithms that overcome this limitation by exploiting morphological differences between sub-cellular structures, and their local spatial context. RESULTS: The proposed method was effective at improving spectral unmixing performance by exploiting: (i) a set of dictionary-based models for object morphologies learned from the image data; and (ii) models of spatial context learned from the image data using a total variation algorithm. The method was evaluated on multi-spectral images of multiplex-labeled pancreatic ductal adenocarcinoma (PDAC) tissue samples. The former constraint ensures that neighbouring pixels correspond to morphologically similar structures, and the latter constraint ensures that neighbouring pixels have similar spectral signatures. The average Mean Squared Error (MSE) and Signal Reconstruction Error (SRE) ratio of the proposed method was 39.6% less and 8% more, respectively, compared to the best of all other algorithms that do not exploit these spatial constraints. AVAILABILITY AND IMPLEMENTATION: Open source software (MATLAB). CONTACT: broysam@central.uh.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer.

Pancreatic cancer presents with a dismal mortality rate and is in urgent need of methods for early detection with potential for timely intervention. All living cells, including cancer cells, generate exosomes. We previously discovered double stranded genomic DNA in exosomes derived from the circulation of pancreatic cancer patients, which enabled the detection of prevalent mutations associated with the disease. Here, we report a proof-of-concept study that demonstrates the potential clinical utility of circulating exosomal DNA for identification of KRASG12D and TP53R273H mutations in patients with pancreas-associated pathologies, including pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP) and intraductal papillary mucinous neoplasm (IPMN), and in healthy human subjects. In 48 clinically annotated serum samples from PDAC patients, digital PCR analyses of exosomal DNA identified KRASG12D mutation in 39.6% of cases, and TP53R273H mutation in 4.2% of cases. KRASG12D and TP53R273H mutations were also detected in exosomal DNA from IPMN patients (2 out of 7 with KRASG12D, one of which also co-presented with TP53R273H mutation). Circulating exosomal DNA in 5 out of 9 CP patients enabled the detection of KRASG12D mutation. In 114 healthy subject-derived circulating exosomal DNA, 2.6% presented with KRASG12D mutation and none with TP53R273H mutation. This study highlights the value of circulating exosomal DNA for a rapid, low-cost identification of cancer driving mutations. The identification of mutations in IPMN patients and healthy subjects suggests that liquid biopsies may allow potential assessment of cancer risk but with a cautionary note that detection of clinical cancer cannot be assumed.

Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis.

OBJECTIVE: Angiogenesis is crucial in RA disease progression. Lymphotoxin ß receptor (LTßR)-induced activation of the non-canonical nuclear factor-κB (NF-κB) pathway via NF-κB-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-κB in this process. METHODS: Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LTßR ligands LTß and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LTßR-Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated. RESULTS: RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LTß and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LTßR-induced vessel formation (P < 0.05). LTßR-Ig not only blocked LTß- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LTß, LIGHT, growth factors (P < 0.05) and RASF (P < 0.01). CONCLUSION: We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-κB signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.

A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis.

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.