Julienne L. Carstens

Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.

Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer

Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT). Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7, 8, 9, 10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.

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.

Microenvironment-dependent cues trigger miRNA-regulated feedback loop to facilitate the EMT/MET switch.

The metastatic spread of tumor epithelial cells accounts for over 90% of cancer-specific mortality; however, the molecular mechanisms that govern tumor spread and distant recolonization remain unclear. In this issue of JCI, Rokavec and colleagues shine light on this murky aspect of tumor biology by focusing through the lens of microenvironmental contributions, namely inflammation, as driving signals that set off a delicate, intracellular feedback loop among cytokine receptors, transcription factors and miRNAs. This study provides in vivo evidence and identifies molecular players behind the elusive switch that drives the epithelial-to-mesenchymal transition and the mesenchymal-to-epithelial transition.

Erratum to Depletion of Carcinoma-Associated Fibroblasts and Fibrosis Induces Immunosuppression and Accelerates Pancreas Cancer with Reduced Survival [Cancer Cell 25, 719-734; (2014)]

Berna C. Özdemir, Tsvetelina Pentcheva-Hoang, Julienne L. Carstens, Xiaofeng Zheng, Chia-Chin Wu, Tyler R. Simpson, Hanane Laklai, Hikaru Sugimoto, Christoph Kahlert, Sergey V. Novitskiy, Ana De Jesus-Acosta, Padmanee Sharma, Pedram Heidari, Umar Mahmood, Lynda Chin, Harold L. Moses, Valerie M. Weaver, Anirban Maitra, James P. Allison, Valerie S. LeBleu, and Raghu Kalluri* *Correspondence: rkalluri@mdanderson.org http://dx.doi.org/10.1016/j.ccell.2015.11.002

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.

Dual reporter genetic mouse models of pancreatic cancer identify an epithelial‐to‐mesenchymal transition‐independent metastasis program

Epithelial‐to‐mesenchymal transition (EMT) is a recognized eukaryotic cell differentiation program that is also observed in association with invasive tumors. Partial EMT program in carcinomas imparts cancer cells with mesenchymal‐like features and is proposed as essential for metastasis. Precise determination of the frequency of partial EMT program in cancer cells in tumors and its functional role in metastases needs unraveling. Here, we employed mesenchymal cell reporter mice driven by αSMA‐Cre and Fsp1‐Cre with genetically engineered mice that develop spontaneous pancreatic ductal adenocarcinoma (PDAC) to monitor partial EMT program. Both αSMA‐ and Fsp1‐Cre‐mediated partial EMT programs were observed in the primary tumors. The established metastases were primarily composed of cancer cells without evidence for a partial EMT program, as assessed by our fate mapping approach. In contrast, metastatic cancer cells exhibiting a partial EMT program were restricted to isolated single cancer cells or micrometastases (3–5 cancer cells). Collectively, our studies identify large metastatic nodules with preserved epithelial phenotype and potentially unravel a novel metastasis program in PDAC.

Abstract IA15: Functional diversity of the pancreas tumor stroma

The desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC) is composed of functionally diverse cellular components embedded in a complex and dynamic fibrotic matrix. In this context, the marked accumulation of stromal myofibroblasts has long been associated with pro-tumorigenic functions, by enhancing tumor cell growth, and limiting immune infiltration and chemotherapeutic drug delivery. Testing of some of these concepts in the clinical care of PDAC patients was however met with paradoxical results. Precise functional auditing of the stromal fibroblasts using genetic targeting in genetically engineered mouse models of PDAC depicted a more complex picture, wherein myofibroblast emerged in this context as tumor-limiting stromal cells. Depletion of the stromal fibroblasts in the early and late stages of PDAC in mice led to more invasive tumors with a negative impact on overall survival, and failed to improve response to Gemcitabine therapy. While our result indicated that suppression of the desmoplastic reaction was associated with decreased tumor angiogenesis, increased intratumoral hypoxia, and epithelial-to-mesenchymal transition of cancer cells with heightened tumorigenicity, our study also informed on the immunomodulatory functions of myofibroblasts in murine PDAC. Depletion of myofibroblasts supported an enhanced response to anti-CTLA4 checkpoint blockade therapy with prolonged survival of mice. Citation Format: Valerie S. LeBleu, Berna C. Ozdemir, Tsvetelina Pentcheva-Hoang, Julienne L. Carstens, Xiaofeng Zheng, Chin-Chia Wu, Tyler Simpson, Hanane Laklai, Hikaru Sugimoto, Christoph Kahlert, Sergey V. Novitskiy, Ana DeJesus Acosta, Padmanee Sharma, Pedram Heidari, Umar Mahmood, Lynda Chin, Harold Moses, Valerie Weaver, Anirban Maitra, James P. Allison and Raghu Kalluri. Functional diversity of the pancreas tumor stroma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA15.