Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer.

PURPOSE: Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. EXPERIMENTAL DESIGN: We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. RESULTS: In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. CONCLUSIONS: Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

Establishment of Pancreatobiliary Cancer Zebrafish Avatars for Chemotherapy Screening.

BACKGROUND: Cancers of the pancreas and biliary tree remain one of the most aggressive oncological malignancies, with most patients relying on systemic chemotherapy. However, effective biomarkers to predict the best therapy option for each patient are still lacking. In this context, an assay able to evaluate individual responses prior to treatment would be of great value for clinical decisions. Here we aimed to develop such a model using zebrafish xenografts to directly challenge pancreatic cancer cells to the available chemotherapies. METHODS: Zebrafish xenografts were generated from a Panc-1 cell line to optimize the pancreatic setting. Pancreatic surgical resected samples, without in vitro expansion, were used to establish zebrafish patient-derived xenografts (zAvatars). Upon chemotherapy exposure, zAvatars were analyzed by single-cell confocal microscopy. RESULTS: We show that Panc-1 zebrafish xenografts are able to reveal tumor responses to both FOLFIRINOX and gemcitabine plus nanoparticle albumin-bound (nab)-paclitaxel in just 4 days. Moreover, we established pancreatic and ampullary zAvatars with patient-derived tumors representative of different histological types. CONCLUSION: Altogether, we provide a short report showing the feasibility of generating and analyzing with single-cell resolution zAvatars from pancreatic and ampullary cancers, with potential use for future preclinical studies and personalized treatment.

Innate immune evasion revealed in a colorectal zebrafish xenograft model.

Cancer immunoediting is a dynamic process of crosstalk between tumor cells and the immune system. Herein, we explore the fast zebrafish xenograft model to investigate the innate immune contribution to this process. Using multiple breast and colorectal cancer cell lines and zAvatars, we find that some are cleared (regressors) while others engraft (progressors) in zebrafish xenografts. We focus on two human colorectal cancer cells derived from the same patient that show contrasting engraftment/clearance profiles. Using polyclonal xenografts to mimic intra-tumor heterogeneity, we demonstrate that SW620_progressors can block clearance of SW480_regressors. SW480_regressors recruit macrophages and neutrophils more efficiently than SW620_progressors; SW620_progressors however, modulate macrophages towards a pro-tumoral phenotype. Genetic and chemical suppression of myeloid cells indicates that macrophages and neutrophils play a crucial role in clearance. Single-cell-transcriptome analysis shows a fast subclonal selection, with clearance of regressor subclones associated with IFN/Notch signaling and escaper-expanded subclones with enrichment of IL10 pathway. Overall, our work opens the possibility of using zebrafish xenografts as living biomarkers of the tumor microenvironment.

Zebrafish xenografts as a fast screening platform for bevacizumab cancer therapy.

Despite promising preclinical results, average response rates to anti-VEGF therapies, such as bevacizumab, are reduced for most cancers, while incurring in remarkable costs and side effects. Currently, there are no biomarkers available to select patients that can benefit from this therapy. Depending on the individual tumor, anti-VEGF therapies can either block or promote metastasis. In this context, an assay able to predict individual responses prior to treatment, including the impact on metastasis would prove of great value to guide treatment options. Here we show that zebrafish xenografts are able to reveal different responses to bevacizumab in just 4 days, evaluating not only individual tumor responses but also the impact on angiogenesis and micrometastasis. Importantly, we perform proof-of-concept experiments where clinical responses in patients were compared with their matching zebrafish Patient-Derived Xenografts - zAvatars, opening the possibility of using the zebrafish model to screen bevacizumab therapy in a personalized manner.