Predictive biomarkers for response to TGF- ß inhibition in resensitizing chemo(radiated) esophageal adenocarcinoma.

Epithelial-mesenchymal transition (EMT) has been identified as a driver of therapy resistance, particularly in esophageal adenocarcinoma (EAC), where transforming growth factor beta (TGF-ß) can induce this process. Inhibitors of TGF-ß may counteract the occurrence of mesenchymal, resistant tumor cell populations following chemo(radio)therapy and improve treatment outcomes in EAC. Here, we aimed to identify predictive biomarkers for the response to TGF-ß targeting. In vitro approximations of neoadjuvant treatment were applied to publicly available primary EAC cell lines. TGF-ß inhibitors fresolimumab and A83-01 were employed to inhibit EMT, and mesenchymal markers were quantified via flow cytometry to assess efficacy. Our results demonstrated a robust induction of mesenchymal cell states following chemoradiation, with TGF-ß inhibition leading to variable reductions in mesenchymal markers. The cell lines were clustered into responders and non-responders. Genomic expression profiles were obtained through RNA-seq analysis. Differentially expressed gene (DEG) analysis identified 10 positively- and 23 negatively-associated hub genes, which were bioinformatically identified. Furthermore, the correlation of DEGs with response to TGF-ß inhibition was examined using public pharmacogenomic databases, revealing 9 positively associated and 11 negatively associated DEGs. Among these, ERBB2, EFNB1, and TNS4 were the most promising candidates. Our findings reveal a distinct gene expression pattern associated with the response to TGF-ß inhibition in chemo(radiated) EAC. The identified DEGs and predictive markers may assist patient selection in clinical studies investigating TGF-ß targeting.

High-grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1.

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.

Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma.

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.

Chemoradiation induces epithelial-to-mesenchymal transition in esophageal adenocarcinoma.

Multimodality treatment has advanced the outcome of esophageal adenocarcinoma (EAC), but overall survival remains poor. Therapeutic pressure activates effective resistance mechanisms and we characterized these mechanisms in response to the currently used neoadjuvant treatment against EAC: carboplatin, paclitaxel and radiotherapy. We developed an in vitro approximation of this regimen and applied it to primary patient-derived cultures. We observed a heterogeneous epithelial-to-mesenchymal (EMT) response to the high therapeutic pressure exerted by chemoradiation. We found EMT to be initiated by the autocrine production and response to transforming growth factor beta (TGF-ß) of EAC cells. Inhibition of TGF-ß ligands effectively abolished chemoradiation-induced EMT. Assessment of TGF-ß serum levels in EAC patients revealed that high levels after neoadjuvant treatment predicted the presence of fluorodeoxyglucose uptake in lymph nodes on the post-chemoradiation positron emission tomography-scan. Our study shows that chemoradiation contributes to resistant metastatic disease in EAC patients by inducing EMT via autocrine TGF-ß production. Monitoring TGF-ß serum levels during treatment could identify those patients at risk of developing metastatic disease, and who would likely benefit from TGF-ß targeting therapy.

The pluripotency factor NANOG contributes to mesenchymal plasticity and is predictive for outcome in esophageal adenocarcinoma.

BACKGROUND: Despite the advent of neoadjuvant chemoradiotherapy (CRT), overall survival rates of esophageal adenocarcinoma (EAC) remain low. A readily induced mesenchymal transition of EAC cells contributes to resistance to CRT. METHODS: In this study, we aimed to chart the heterogeneity in cell state transition after CRT and to identify its underpinnings. A panel of 12 esophageal cultures were treated with CRT and ranked by their relative epithelial-mesenchymal plasticity. RNA-sequencing was performed on 100 pre-treatment biopsies. After RNA-sequencing, Ridge regression analysis was applied to correlate gene expression to ranked plasticity, and models were developed to predict mesenchymal transitions in patients. Plasticity score predictions of the three highest significant predictive models were projected on the pre-treatment biopsies and related to clinical outcome data. Motif enrichment analysis of the genes associated with all three models was performed. RESULTS: This study reveals NANOG as the key associated transcription factor predicting mesenchymal plasticity in EAC. Expression of NANOG in pre-treatment biopsies is highly associated with poor response to neoadjuvant chemoradiation, the occurrence of recurrences, and median overall survival difference in EAC patients (>48 months). Perturbation of NANOG reduces plasticity and resensitizes cell lines, organoid cultures, and patient-derived in vivo grafts. CONCLUSIONS: In conclusion, NANOG is a key transcription factor in mesenchymal plasticity in EAC and a promising predictive marker for outcome.

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Although chemotherapy combined with radiotherapy (chemoradiotherapy) followed by surgery has improved survival, tumor recurrence and metastatic disease (that has spread to other parts of the body) are often observed after several months. In this study, we assessed the effect of chemoradiotherapy on esophageal cells in the lab to predict the effect in patients with esophageal cancer. To investigate this, genes were assessed from 12 different cell lines and 100 patient tissues. We revealed that levels of one of the genes, NANOG, associates with poor response in patients. NANOG could be a promising marker to predict outcome in patients with esophageal cancer. This knowledge might help clinicians to treat patients with esophageal cancer appropriately, or may lead to new or optimized treatments.

Local irradiation of patient-derived tumors in immunodeficient mice.

Severe combined immunodeficient mice are typically used for xenografting experiments and show reliable tumor engraftment; however, their Prkdscid mutation renders them highly sensitive to irradiation. Here, we describe a protocol that allows safe local irradiation of tumor xenografts in immunodeficient mice. We detail the steps for the establishment and handling of patient-derived cancer cultures, subcutaneous injection of cancer cells on the mouse hind limb, localized irradiation in mice, tumor monitoring, and tumor characterization via histological and immunohistochemical assessment. For complete details on the use and execution of this protocol, please refer to Dings et al. (2022).1.

Estrogen-related receptor alpha drives mitochondrial biogenesis and resistance to neoadjuvant chemoradiation in esophageal cancer.

Neoadjuvant chemoradiotherapy (nCRT) improves outcomes in resectable esophageal adenocarcinoma (EAC), but acquired resistance precludes long-term efficacy. Here, we delineate these resistance mechanisms. RNA sequencing on matched patient samples obtained pre-and post-neoadjuvant treatment reveal that oxidative phosphorylation was the most upregulated of all biological programs following nCRT. Analysis of patient-derived models confirms that mitochondrial content and oxygen consumption strongly increase in response to nCRT and that ionizing radiation is the causative agent. Bioinformatics identifies estrogen-related receptor alpha (ESRRA) as the transcription factor responsible for reprogramming, and overexpression and silencing of ESRRA functionally confirm that its downstream metabolic rewiring contributes to resistance. Pharmacological inhibition of ESRRA successfully sensitizes EAC organoids and patient-derived xenografts to radiation. In conclusion, we report a profound metabolic rewiring following chemoradiation and demonstrate that its inhibition resensitizes EAC cells to radiation. These findings hold broader relevance for other cancer types treated with radiation as well.

Soluble Compounds Released by Hypoxic Stroma Confer Invasive Properties to Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma and a hypoxic microenvironment. Pancreatic stellate cells (PSC) are activated by hypoxia and promote excessive desmoplasia, further contributing to the development of hypoxia. We aimed to explore how hypoxia and stroma interact to contribute to invasive growth in PDAC. [18F]HX4 PET/CT was found to be a feasible non-invasive method to assess tumor hypoxia in 42 patients and correlated with HIF1α immunohistochemistry in matched surgical specimens. [18F]HX4 uptake and HIF1α were strong prognostic markers for overall survival. Co-culture and medium transfer experiments demonstrated that hypoxic PSCs and their supernatant induce upregulation of mesenchymal markers in tumor cells, and that hypoxia-induced stromal factors drive invasive growth in hypoxic PDACs. Through stepwise selection, stromal MMP10 was identified as the most likely candidate responsible for this. In conclusion, hypoxia-activated PSCs promote the invasiveness of PDAC through paracrine signaling. The identification of PSC-derived MMP10 may provide a lead to develop novel stroma-targeting therapies.