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Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma, the main cellular constituents of which are cancer-associated fibroblasts (CAFs). Stroma-targeting agents have been proposed to improve the poor outcome of current treatments. However, clinical trials using these agents showed disappointing results. Heterogeneity in the PDAC CAF population was recently delineated demonstrating that both tumor-promoting and tumor-suppressive activities co-exist in the stroma. Here, we aimed to identify biomarkers for the CAF population that contribute to a favorable outcome. RNA-sequencing reads from patient-derived xenografts (PDXs) were mapped to the human and mouse genome to allocate the expression of genes to the tumor or stroma. Survival meta-analysis for stromal genes was performed and applied to human protein atlas data to identify circulating biomarkers. The candidate protein was perturbed in co-cultures and assessed in existing and novel single-cell gene expression analysis from control, pancreatitis, pancreatitis-recovered and PDAC mouse models. Serum levels of the candidate biomarker were measured in two independent cohorts totaling 148 PDAC patients and related them to overall survival. Osteoglycin (OGN) was identified as a candidate serum prognostic marker. Single-cell analysis indicated that Ogn is derived from a subgroup of inflammatory CAFs. Ogn-expressing fibroblasts are distinct from resident healthy pancreatic stellate cells and arise during pancreatitis. Serum OGN levels were prognostic for favorable overall survival in two independent PDAC cohorts (HR = 0.47, P = .042 and HR = 0.53, P = .006). Altogether, we conclude that high circulating OGN levels inform on a previously unrecognized subgroup of CAFs and predict favorable outcomes in resectable PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Effective treatment options are currently lacking, and targeted agents have so far demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs are characterized by expression of genes associated with immature cells, and demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, the unique relevance of CSCs in established PDAC tissue has not been determined. Here, we used a marker-independent stochastic clonal labeling system, in combination with a quantitative model of tumor expansion, to uncover the growth dynamics of PDAC tissue. We found that in situ all PDAC cells display clonogenic potential, which is in stark contrast to a strict hierarchical CSC model. Furthermore, we uncover that the proximity to activated cancer-associated fibroblasts determines which tumor cells actively drive tumor expansion. This means that the microenvironment is a dominant actor in defining the clonogenic activity of PDAC cells. Indeed, manipulating the PDAC associated stroma by Hedgehog pathway inhibition, altered the mode of tumor growth. These results reveal how tumor-stroma crosstalk shapes the growth dynamics and clonal architecture of these malignancies, and how perturbance of the stroma impacts on important aspects of PDAC biology.

Significance We found that cancer-associated fibroblasts, the most abundant noncancer cell type in esophageal cancer tissue, contribute to the resistance of tumors against currently applicable treatments. These cancer-associated fibroblasts do so by producing and secreting IL-6. IL-6 induces a mesenchymal, resistant tumor cell state. Inhibition of IL-6 reverted the mesenchymal cell state and resensitized tumor cells to therapy. Serum-borne proxies for the presence of IL-6–producing fibroblasts were identified and found to be predictive for the response to neoadjuvant therapy., Esophageal adenocarcinoma (EAC) has a dismal prognosis, and survival benefits of recent multimodality treatments remain small. Cancer-associated fibroblasts (CAFs) are known to contribute to poor outcome by conferring therapy resistance to various cancer types, but this has not been explored in EAC. Importantly, a targeted strategy to circumvent CAF-induced resistance has yet to be identified. By using EAC patient-derived CAFs, organoid cultures, and xenograft models we identified IL-6 as the stromal driver of therapy resistance in EAC. IL-6 activated epithelial-to-mesenchymal transition in cancer cells, which was accompanied by enhanced treatment resistance, migratory capacity, and clonogenicity. Inhibition of IL-6 restored drug sensitivity in patient-derived organoid cultures and cell lines. Analysis of patient gene expression profiles identified ADAM12 as a noninflammation-related serum-borne marker for IL-6–producing CAFs, and serum levels of this marker predicted unfavorable responses to neoadjuvant chemoradiation in EAC patients. These results demonstrate a stromal contribution to therapy resistance in EAC. This signaling can be targeted to resensitize EAC to therapy, and its activity can be measured using serum-borne markers.

Background & Aims: Esophageal cancer is the fifth most common solid cancer globally, and esophageal adenocarcinoma (EAC) is the predominant histological subtype in the western world. Patients often present at an advanced stage and overall-5-year survival rates are less than 15%. In many patients, the response to neoadjuvant therapy is encouraging at first, but most will develop metastatic disease several years later. Recently, we observed plasticity along the epithelial-to-mesenchymal (EMT) axis in EAC tumor cells upon therapeutic pressure. The last decade has seen the discovery of static molecular subtypes in cancers and a poor-prognosis mesenchymal subtype has been reported in multiple gastrointestinal cancers, but not in EAC. Here, we set out to identify transcriptomic subtypes and the frequency of their occurrence during EAC progression, and exposure to therapy in a clinical setting. Methods: A cohort comprising of 174 esophageal cancer cases (n=100 pre-treatment biopsies, n=46 neoadjuvantly-treated resection specimens, and n=28 metastatic biopsies) with 12 esophageal cancer cell lines (8 primary and 4 ATCC cell lines) was RNA-sequenced. To circumvent non-tumor signals confounding the analysis, we leveraged non-negative matrix factorization (NMF) as a virtual microdissection tool. Patients were subtyped by consensus clustering the top 50 exemplar genes per tumor-cell intrinsic signature. Next, the subtypes were validated in two independent cohorts and clinical correlates were analyzed. In addition, we perturbed subtype-specific regulators to functionally assess the existence of the subtypes. Results: Here, we identified 7 biological signatures involved in primary and metastatic EAC. Two of these were unambiguously tumor-intrinsic signals. All seven signatures associated with tumor cellularity scores estimated by an experienced pathologist confirming the reliability of NMF as a virtual microdissection tool. Upon clustering two EAC subtypes emerged: Intestinal-like (IL) and Mesenchymal-like (ML). ML-subtyped patients were observed with increased frequency after neoadjuvant treatment, and following metastatic dissemination. This association was validated in an independent cohort. Lastly, we identified HMGA2 as a key transcription factor for the ML-subtype. Genetic perturbation of HMGA2 induced an IL-associated phenotype. Conclusion: Our study demonstrate for the first time from clinical transcriptomics data that EAC cells can exist in distinct cell states. In addition, the abundance of either cell states, that constitute to tumor subtype, alter when exposed to chemoradiation or following metastatic spread. These subtypes offer a valuable method to design more informed personalized treatment approaches for EAC patients; for existing and future subtype-directed treatments. Citation Format: Mark P.G. Dings, Amber P. Zalm, Marjolein F. Lansbergen, César Oyarce, Sybren L. Meijer, Cynthia Waasdorp, Jan Paul Medema, Hanneke van Laarhoven, Maarten F. Bijlsma. Transcriptomic signatures in esophageal adenocarcinoma define distinct subtypes with therapeutic relevance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4008.

Background and Aims: The incidence of esophageal adenocarcinoma (EAC) has increased six-fold in Western countries over the last decades, and 5-year survival rates remain low at 5-20%. While multimodality treatment strategies for curative treatment of esophageal cancer, including the CROSS regimen (chemoradiotherapy followed by surgery) have increased median overall survival, the majority of patients develop recurrences after several months. Epithelial to mesenchymal transition (EMT) has been recently shown by our group to be one of the major underlying mechanisms of resistance to therapy. Paradoxically, therapeutic pressure of effective therapies such as the CROSS regimen are found to instruct a mesenchymal, resistant phenotype in models for EAC. In this study, the aim was to delineate the heterogeneity for the propensity to undergo EMT after chemoradiation and which mechanisms underpin this propensity. Methods: A panel of 8 EAC cell lines (5 primary and 3 ATCC cell lines) were treated with chemoradiotherapy and ranked by their propensity to undergo EMT, based on morphology when EMT occurred and protein marker expression. Next, the cell line panel as well as 44 pre-treated esophageal biopsies were RNA-sequenced. Expression data of the cell line panel were linked to their ranked in vitro EMT response. By means of Leave-one-out cross validation with Ridge Regression, EMT score prediction in pre-treated biopsies was validated. Gene expression profiles were related to clinical outcome data to identify markers that associated with propensity for EMT in patients. Results: In the panel of in vitro EAC models, a strong heterogeneity was observed for the propensity to EMT after chemoradiation. For each marker, Ridge regression analysis identified the top 50 highly correlating genes. Combining all positively correlating genes of days to EMT, NCAD and ZEB1, known key transcription factors of pluripotency including NANOG and OCT4 emerged. Expression of NANOG and OCT4 in pre-treatment biopsies was highly predictive for response to neoadjuvant chemoradiation, occurrence of recurrences, and survival in patients. Genetic perturbation by knockout and inhibition of NANOG and OCT4 reduced the onset of EMT and sensitized cells for chemoradiation. Conclusions: In conclusion, we were able to identify patients who are disproportionally prone to develop EMT in response to chemoradiation. Moreover, stemness factors NANOG and OCT4 are crucial regulators in plasticity of EAC and are promising predictive markers in pre-treatment biopsies of patients. By targeting NANOG and OCT4 in vitro, cells were sensitized to chemoradiation, holding promise for stemness inhibition to prevent therapy resistance in EAC. Citation Format: Amber Perenna van der Zalm, Mark P. Dings, Reimer Janssen, Peter Bailey, Jan Koster, Danny Zwijnenburg, Richard Volckmann, Cynthia Waasdorp, Jeroen Blokhuis, César Oyarce, Gerrit Hooijer, Sybren L. Meijer, Jan Paul Medema, Hanneke W. van Laarhoven, Maarten F. Bijlsma. Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2616.

Background: Gemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma.Methods: We stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS.Findings: Gemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4(+) T-helper, CD8(+) Tand NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS.Interpretation: Gemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies. (C) 2020 The Authors. Published by Elsevier B.V.

Purpose Targeting tumor-infiltrating macrophages limits progression and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma (PDAC). Protease-activated receptor (PAR)1 drives monocyte/macrophage recruitment, and stromal ablation of PAR1 limits cancer growth and enhances gemcitabine sensitivity in experimental PDAC. However, the functional interplay between PAR1, macrophages and tumor cells remains unexplored. Here we address the PAR1-macrophage-tumor cell crosstalk and assess its contributions to tumor progression. Methods PAR1 expression and macrophage infiltration were correlated in primary PDAC biopsies using gene expression datasets and tissue microarrays. Medium transfer experiments were used to evaluate the functional consequences of macrophage-tumor cell crosstalk and to assess the contribution of PAR1 to the observed responses. PAR1 cleavage assays were used to identify a macrophage-secreted PAR1 agonist, and the effects of candidate proteases were assessed in medium transfer experiments with specific inhibitors and/or recombinant agonist. Results PAR1 expression correlates with macrophage infiltration in primary PDACs, and macrophages induce mesenchymal transition of PDAC cells through PAR1 activation. Protease profiling identified macrophage-secreted matrix metalloprotease 9 (MMP9) as the relevant PAR1 agonist in PDAC. PAR1 and/or MMP9 inhibition limited macrophage-driven mesenchymal transition. Likewise, preventing mesenchymal transition by silencing ZEB1 or by pharmacological inhibition of the MMP9/PAR1 axis significantly reduced the ability of tumor cells to survive the anti-tumor activities of macrophages. Conclusion Macrophages secrete MMP9, which acts upon PDAC cell PAR1 to induce mesenchymal transition. This macrophage-induced mesenchymal transition supports the tumor-promoting role of macrophage influx, explaining the dichotomous contributions of these immune cells to tumor growth.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundance of stroma. Multiple molecular classification efforts have identified a mesenchymal tumor subtype that is consistently characterized by high‐grade growth and poor clinical outcome. The relation between PDAC stroma and tumor subtypes is still unclear. Here, we aimed to identify how PDAC cells instruct the main cellular component of stroma, the pancreatic stellate cells (PSCs). We found in primary tissue that high‐grade PDAC had reduced collagen deposition compared to low‐grade PDAC. Xenografts and organotypic co‐cultures established from mesenchymal‐like PDAC cells featured reduced collagen and activated PSC content. Medium transfer experiments using a large set of PDAC cell lines revealed that mesenchymal‐like PDAC cells consistently downregulated ACTA2 and COL1A1 expression in PSCs and reduced proliferation. We identified colony‐stimulating factor 1 as the mesenchymal PDAC‐derived ligand that deactivates PSCs, and inhibition of its receptor CSF1R was able to counteract this effect. In conclusion, high‐grade PDAC features stroma that is low in collagen and activated PSC content, and targeting CSF1R offers direct options to maintain a tumor‐restricting microenvironment., This study reveals that PDAC cells counteract mechanical restrictions of tumor stroma by secreting CSF‐1, which could be a potential therapeutic target.

Purpose Basal cell carcinoma (BCC) is one of the most common skin cancers, and is typically driven by an aberrantly activated Hedgehog (Hh) pathway. The Hh pathway is regulated by interactions between the Patched-1 (Ptch1) and Smoothened (Smo) receptors. Smo is an activating receptor and is subject to inhibition by Ptch1. Following ligand binding to Ptch1, its inhibitory action is relieved and pathway activation occurs. This receptor interaction is pivotal to restraining uncontrolled cellular growth. Both receptors have been found to be frequently mutated in BCCs. Ptch2 is a Ptch1 paralog that exhibits overlapping functions in both normal development and tissue homeostasis. As yet, its contribution to cancer growth is poorly defined. Here we set out to assess how Ptch2 inhibits BCC growth. Methods We used several in vitro readouts for transcriptional and chemotactic Hh signaling in BCC-derived ASZ001 cells, and a novel xenograft model to assess in vivo BCC tumor growth. Gene editing by TALEN was used to untangle the different Ptch2-dependent responses to its ligand sonic hedgehog (Shh). Results We first defined the signaling competence of Ptch2 in Ptch1-deficient ASZ001 cells in vitro, and found that Ptch2 ligand binding drives their migration rather than eliciting a transcriptional response. We found that subsequent targeting of Ptch2 abrogated the chemotaxic effect. Next, we tested the contribution of Ptch2 to in vivo tumor growth using a xenograft model and found that reduced Ptch function results in increased tumor growth, but that selective pressure appatently acts against complete Ptch2 ablation. Conclusions We conclude that like Ptch1, Ptch2 exerts a tumor-suppressive function in BCC cells, and that after targeting of both paralogs, ligand-independent activation of the Hh pathway contributes to tumor growth. Electronic supplementary material The online version of this article (10.1007/s13402-018-0381-9) contains supplementary material, which is available to authorized users.

Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate–pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate–pulsed DC immunotherapy is effective in mice and safe in humans. Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte–derived DCs pulsed with tumor lysate from five mesothelioma cell lines. Results: In mice, allogeneic lysate–pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1–20.3] and median OS not reached (median follow-up = 22.8 months). Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766–76. ©2017 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is marked by an abundant stromal deposition. This stroma is suspected to harbor both tumor‐promoting and tumor‐suppressing properties. This is underscored by the disappointing results of stroma targeting in clinical studies. Given the complexity of tumor–stroma interaction in PDAC, there is a need to identify the stromal proteins that are predominantly tumor‐promoting. One possible candidate is SPOCK1 that we previously identified in a screening effort in PDAC. We extensively mined PDAC gene expression datasets, and used species‐specific transcript analysis in mixed‐species models for PDAC to study the patterns and driver mechanisms of SPOCK1 expression in PDAC. Advanced organotypic coculture models with primary patient‐derived tumor cells were used to further characterize the function of this protein. We found SPOCK1 expression to be predominantly stromal. Expression of SPOCK1 was associated with poor disease outcome. Coculture and ligand stimulation experiments revealed that SPOCK1 is expressed in response to tumor cell‐derived transforming growth factor‐beta. Functional assessment in cocultures demonstrated that SPOCK1 strongly affects the composition of the extracellular collagen matrix and by doing so, enables invasive tumor cell growth in PDAC. By defining the expression pattern and functional properties of SPOCK1 in pancreatic cancer, we have identified a stromal mediator of extracellular matrix remodeling that indirectly affects the aggressive behavior of PDAC cells. The recognition that stromal proteins actively contribute to the protumorigenic remodeling of the tumor microenvironment should aid the design of future clinical studies to target specific stromal targets.

Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow‐up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell‐free DNA from plasma samples of 58 treatment‐naive mPDAC patients was isolated and sequenced using a custom‐made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross‐check with droplet digital PCR showed good agreement in Bland–Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p, What's new? Circulating tumor DNA (ctDNA) attracts much interest as a possible prognostic tool for cancer. Here, the authors showed that the quantity of ctDNA correlated strongly with tumor volume in metastatic pancreatic ductal adenocarcinoma (mPDAC). They conducted a retrospective analysis using samples collected from 58 untreated mPDAC patients. For this study, the authors designed a pancreatobiliary NGS panel, which they used to test the patients’ cell‐free DNA, along with droplet digital PCR. Both ctDNA variant allele frequency and tumor volume predicted overall survival, they found.

Esophageal adenocarcinoma (EAC) has a dismal prognosis, and survival benefits of recent multimodality treatments remain small. Cancer-associated fibroblasts (CAFs) are known to contribute to poor outcome by conferring therapy resistance to various cancer types, but this has not been explored in EAC. Importantly, a targeted strategy to circumvent CAF-induced resistance has yet to be identified. By using EAC patient-derived CAFs, organoid cultures, and xenograft models we identified IL-6 as the stromal driver of therapy resistance in EAC. IL-6 activated epithelial-to-mesenchymal transition in cancer cells, which was accompanied by enhanced treatment resistance, migratory capacity, and clonogenicity. Inhibition of IL-6 restored drug sensitivity in patient-derived organoid cultures and cell lines. Analysis of patient gene expression profiles identified ADAM12 as a noninflammation-related serum-borne marker for IL-6-producing CAFs, and serum levels of this marker predicted unfavorable responses to neoadjuvant chemoradiation in EAC patients. These results demonstrate a stromal contribution to therapy resistance in EAC. This signaling can be targeted to resensitize EAC to therapy, and its activity can be measured using serum-borne markers.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma that harbors tumor-promoting properties. No good biomarkers exist to monitor the effect of stromal targeting therapies or to predict response. We set out to identify such non-invasive markers for PDAC stroma and predict response to therapy. Gene expression datasets, co-culture experiments, xenografts, and patient samples were analyzed. Serum samples were measured from a cohort of 58 resected patients, and 87 metastatic or locally advanced PDAC patients. Baseline and follow-up levels were assessed in 372 additional metastatic PDAC patients who received nab-paclitaxel with gemcitabine (n = 184) or gemcitabine monotherapy (n = 188) in the phase III MPACT trial. Increased levels of ADAM12 were found in PDAC patients compared to healthy controls (p n = 157 and n = 38). High levels of ADAM12 significantly associated with poor outcome in resected PDAC (HR 2.07, p = 0.04). In the MPACT trial survival was significantly longer for patients who received nab-paclitaxel and had undetectable ADAM12 levels before treatment (OS 12.3 m vs 7.9 m p = 0.0046). Consistently undetectable or decreased ADAM12 levels during treatment significantly associated with longer survival as well (OS 14.4 m and 11.2 m, respectively vs 8.3, p = 0.0054). We conclude that ADAM12 is a blood-borne proxy for stromal activation, the levels of which have prognostic significance and correlate with treatment benefit.