A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 () and SPARC-related modular calcium binding 2 (). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

IntroductionChemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells.MethodsWe performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations.ResultsHigh expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial–mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy.ConclusionsIn this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0652-6) contains supplementary material, which is available to authorized users.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, while less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5.LGR5-positive cells were isolated from 4 adenoma organoid lines and were subjected to RNA-sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA-seq data from adenoma and normal organoids, we found correlations between LGR5 and CRCspecific genes, including DKK4 (dickkopf WNT signaling pathway inhibitor 4) and SMOC2 (SPARC related modular calcium binding 2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Fibroblasts are thought to be key players in the tumor microenvironment. Means to identify and isolate fibroblasts as well as an understanding of their cancer-specific features are essential to dissect their role in tumor biology. To date, the identification of cancer-associated fibroblasts is widely based on generic markers for activated fibroblasts in combination with their origin in tumor tissue. This study was focused on a deep characterization of the cell surface marker profile of cancer-associated fibroblasts in widely used mouse tumor models and defining aberrant expression profiles by comparing them to their healthy counterparts. We established a generic workflow to isolate healthy and cancer-associated fibroblasts from solid tissues, thereby reducing bias, and background noise introduced by non-target cells. We identified CD87, CD44, CD49b, CD95, and Ly-6C as cancer-associated fibroblast cell surface markers, while CD39 was identified to mark normal fibroblasts from healthy tissues. In addition, we found a functional association of most cancer-related fibroblast markers to proliferation and a systemic upregulation of CD87, and CD49b in tumor-bearing mice, even in non-affected tissues. These novel markers will facilitate the characterization of fibroblasts and shed further light in their functions and implication in cancer progression.