Hervé Tiriac

Organoid Models of Human and Mouse Ductal Pancreatic Cancer

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy.

The poor clinical outcome in pancreatic ductal adenocarcinoma (PDA) is attributed to intrinsic chemoresistance and a growth-permissive tumor microenvironment. Conversion of quiescent to activated pancreatic stellate cells (PSCs) drives the severe stromal reaction that characterizes PDA. Here, we reveal that the vitamin D receptor (VDR) is expressed in stroma from human pancreatic tumors and that treatment with the VDR ligand calcipotriol markedly reduced markers of inflammation and fibrosis in pancreatitis and human tumor stroma. We show that VDR acts as a master transcriptional regulator of PSCs to reprise the quiescent state, resulting in induced stromal remodeling, increased intratumoral gemcitabine, reduced tumor volume, and a 57% increase in survival compared to chemotherapy alone. This work describes a molecular strategy through which transcriptional reprogramming of tumor stroma enables chemotherapeutic response and suggests vitamin D priming as an adjunct in PDA therapy. PAPERFLICK:

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of &agr;-smooth muscle actin (&agr;SMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated &agr;SMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

Organoid profiling identifies common responders to chemotherapy in pancreatic cancer

Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection.Significance: New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. Cancer Discov; 8(9); 1112-29. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Conjugation to the sigma-2 ligand SV119 overcomes uptake blockade and converts dm-Erastin into a potent pancreatic cancer therapeutic.

Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the capacity to deliver small molecules as drug cargoes. We utilized the sigma-2-based drug delivery concept to convert Erastin, a clinically underperforming drug, into a potent pancreatic cancer therapeutic. The Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to sigma-2 ligand SV119 to create SW V-49. Conjugation increased the killing capacity of dm-Erastin by nearly 35-fold in vitro and reduced the size of established tumors and doubled the median survival in syngeneic and patient-derived xenograft models when compared to non-targeted dm-Erastin. Mechanistic analyses demonstrated that cell death was associated with robust reactive oxygen species production and could be efficiently antagonized with antioxidants. Mass spectrometry was employed to demonstrate selective uptake into pancreatic cancer cells. Thus, targeted delivery of dm-Erastin via conjugation to the sigma-2 ligand SV119 produced efficient tumor control and prolonged animal survival with minimal off-target toxicities, and SW V-49 represents a promising new therapeutic with the potential to advance the fight against pancreatic cancer.

Patient‐derived organoid models help define personalized management of gastrointestinal cancer

The prognosis of patients with different gastrointestinal cancers varies widely. Despite advances in treatment strategies, such as extensive resections and the addition of new drugs to chemotherapy regimens, conventional treatment strategies have failed to improve survival for many tumours. Although promising, the clinical application of molecularly guided personalized treatment has proven to be challenging. This narrative review focuses on the personalization of cancer therapy using patient‐derived three‐dimensional ‘organoid’ models.

Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening:

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.

Dynamic changes during the treatment of pancreatic cancer

This manuscript follows a single patient with pancreatic adenocarcinoma for a five year period, detailing the clinical record, pathology, the dynamic evolution of molecular and cellular alterations as well as the responses to treatments with chemotherapies, targeted therapies and immunotherapies. DNA and RNA samples from biopsies and blood identified a dynamic set of changes in allelic imbalances and copy number variations in response to therapies. Organoid cultures established from biopsies over time were employed for extensive drug testing to determine if this approach was feasible for treatments. When an unusual drug response was detected, an extensive RNA sequencing analysis was employed to establish novel mechanisms of action of this drug. Organoid cell cultures were employed to identify possible antigens associated with the tumor and the patient’s T-cells were expanded against one of these antigens. Similar and identical T-cell receptor sequences were observed in the initial biopsy and the expanded T-cell population. Immunotherapy treatment failed to shrink the tumor, which had undergone an epithelial to mesenchymal transition prior to therapy. A warm autopsy of the metastatic lung tumor permitted an extensive analysis of tumor heterogeneity over five years of treatment and surgery. This detailed analysis of the clinical descriptions, imaging, pathology, molecular and cellular evolution of the tumors, treatments, and responses to chemotherapy, targeted therapies, and immunotherapies, as well as attempts at the development of personalized medical treatments for a single patient should provide a valuable guide to future directions in cancer treatment.

Testing Susceptibility of Patient-Derived Organoid Cultures to Therapies: Pharmacotyping

Increasingly, patient models of disease are being utilized to facilitate precision medicine approaches through molecular characterization or direct chemotherapeutic testing. Organoids, 3-dimensional (3D) cultures of neoplastic cells derived from primary tumor specimens, represent an ideal platform for these types of studies because benchtop protocols previously developed for 2-dimensional cell lines can be adapted for use. These protocols include directly testing the survival of these organoid cultures when exposed to clinically relevant chemotherapeutic agents, a process we have called pharmacotyping. In this protocol, established tumor-derived organoid cultures are dissociated into single cells, plated in a 3D gel matrix, and exposed to pharmacologic agents. While our protocol has been developed for use with patient-derived pancreatic ductal adenocarcinoma organoids, with minor modifications to the dissociation and medium conditions, this protocol could be adapted for use with a wide range of organoid cultures. We further describe our standard ATP-based assay to determine cellular survival. This protocol can be scaled for use in high-throughput assays.

Abstract A27: Exploring the role of glycosylation in pancreatic disease

Pancreatic ductal adenocarcinoma (PDA) is almost uniformly lethal and surgical intervention is the only cure. Unfortunately, most patients are ineligible for resection because of the advanced stage of disease by the time of diagnosis. This is due in part to the lack of diagnostic tools, especially for families with elevated risk. The PDA biomarker, CA19-9, is measured in the blood to follow tumor burden longitudinally, but is neither sensitive nor specific enough to be used for diagnosis. The use of CA19-9 in PDA diagnosis is problematic given the elevation of CA19-9 in benign pancreatic disease, such as pancreatitis. While CA19-9 has been traditionally used as a diagnostic, retrospective studies reported that PDA patients who maintain a CA19-9 negative/low status have a significantly longer survival relative to those with higher CA19-9 levels in multivariate analyses. The functional significance of CA19-9 to PDA initiation, maintenance, and progression remains unclear due in part to the absence of this carbohydrate modification in mice. We found that expression of CA19-9 in the mouse pancreas is sufficient to induce pancreatitis, a benign proliferative condition that often confounds the diagnosis of PDA. Specifically, CA19-9 elevation resulted in rapid elevation of pancreatic enzymes in the blood, pancreatic infiltration of immune cells, acinar-to-ductal metaplasia and atrophy, as well as increased proliferation. Furthermore, we explored the utility of CA19-9 as a therapeutic target for both acute and chronic pancreatitis. This avenue of treatment strategy exhibits potential given that a pilot study demonstrated that turning off CA19-9 expression results in the normalization of pancreatic enzyme levels within four days following an acute episode of pancreatitis. Future work will focus on how elevation of this glycosylation modification mediates the development of pancreatitis by identifying the signaling pathways that are altered upon CA19-9 expression. In addition, we will explore the efficacy of therapeutically targeting CA19-9 in both pancreatitis and PDA with a larger goal of delineating the role of CA19-9 in pancreatic disease