Cell Type of Pancreatic Ductal Adenocarcinoma Origin: Implications for Prognosis and Clinical Outcomes

<b><i>Background:</i></b> Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease that has no effective early detection method or treatment to date. <b><i>Summary:</i></b> The normal cell type that initiates PDAC, or its cellular origin, is still unknown. To investigate the contribution of distinct normal epithelial cell types to PDAC tumorigenesis, genetically engineered mouse models were used to show that both acinar and ductal cells are capable of giving rise to PDAC. These studies indicated that genetic mutations and pancreatic injury interact differently with each cellular origin to affect their predilection and process for forming PDAC. In this review, we summarize recent findings using various genetically engineered mouse models in the identification and characterization of the PDAC cell of origin. We also discuss potential implications for cellular origin on tumor development, PDAC transcriptional subtype, and disease prognosis of patients. <b><i>Key Message:</i></b> Although it is clear that both ductal and acinar cells have the potential to form PDAC, whether cellular origin can indeed influence patient prognosis and whether knowledge of cellular origin will aid in the diagnosis or treatment of patients in the future will need further study.

Genetic Mutations of Pancreatic Cancer and Genetically Engineered Mouse Models

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, and the seventh leading cause of cancer-related deaths worldwide. An improved understanding of tumor biology and novel therapeutic discoveries are needed to improve overall survival. Recent multi-gene analysis approaches such as next-generation sequencing have provided useful information on the molecular characterization of pancreatic tumors. Different types of pancreatic cancer and precursor lesions are characterized by specific molecular alterations. Genetically engineered mouse models (GEMMs) of PDAC are useful to understand the roles of altered genes. Most GEMMs are driven by oncogenic Kras, and can recapitulate the histological and molecular hallmarks of human PDAC and comparable precursor lesions. Advanced GEMMs permit the temporally and spatially controlled manipulation of multiple target genes using a dual-recombinase system or CRISPR/Cas9 gene editing. GEMMs that express fluorescent proteins allow cell lineage tracing to follow tumor growth and metastasis to understand the contribution of different cell types in cancer progression. GEMMs are widely used for therapeutic optimization. In this review, we summarize the main molecular alterations found in pancreatic neoplasms, developed GEMMs, and the contribution of GEMMs to the current understanding of PDAC pathobiology. Furthermore, we attempted to modify the categorization of altered driver genes according to the most updated findings.

Programmable in vivo CRISPR activation elucidates the oncogenic and immunosuppressive role of MYC in lung adenocarcinoma

Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. Here, we describe the development of a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Unlike conventional GEMMs, our model system allows for flexible, sustained and timed activation of one or more target genes using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we demonstrate gene activation in primary pancreatic organoid cultures in vitro and enhanced tumorigenic potential in Myc-activated organoids when transplanted orthotopically. By implementing our model as an autochthonous lung cancer model, we show that transduction-mediated Myc activation leads to accelerated tumor progression and significantly reduced overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation led to the acquisition of an immune suppressive cold tumor microenvironment. Through cross-species validation of our results using publicly available RNA/DNA-seq data sets, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Overall, our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening.

Pre-clinical Models of Metastasis in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a hostile solid malignancy coupled with an extremely high mortality rate. Metastatic disease is already found in most patients at the time of diagnosis, resulting in a 5-year survival rate below 5%. Improved comprehension of the mechanisms leading to metastasis is pivotal for the development of new targeted therapies. A key field to be improved are modeling strategies applied in assessing cancer progression, since traditional platforms fail in recapitulating the complexity of PDAC. Consequently, there is a compelling demand for new preclinical models that mirror tumor progression incorporating the pressure of the immune system, tumor microenvironment, as well as molecular aspects of PDAC. We suggest the incorporation of 3D organoids derived from genetically engineered mouse models or patients as promising new tools capable to transform PDAC pre-clinical modeling and access new frontiers in personalized medicine.

Atlas of Lobular Breast Cancer Models: Challenges and Strategic Directions

Invasive lobular carcinoma (ILC) accounts for up to 15% of all breast cancer (BC) cases and responds well to endocrine treatment when estrogen receptor α-positive (ER+) yet differs in many biological aspects from other ER+ BC subtypes. Up to 30% of patients with ILC will develop late-onset metastatic disease up to ten years after initial tumor diagnosis and may experience failure of systemic therapy. Unfortunately, preclinical models to study ILC progression and predict the efficacy of novel therapeutics are scarce. Here, we review the current advances in ILC modeling, including cell lines and organotypic models, genetically engineered mouse models, and patient-derived xenografts. We also underscore four critical challenges that can be addressed using ILC models: drug resistance, lobular tumor microenvironment, tumor dormancy, and metastasis. Finally, we highlight the advantages of shared experimental ILC resources and provide essential considerations from the perspective of the European Lobular Breast Cancer Consortium (ELBCC), which is devoted to better understanding and translating the molecular cues that underpin ILC to clinical diagnosis and intervention. This review will guide investigators who are considering the implementation of ILC models in their research programs.

Multiplexed identification of RAS paralog imbalance as a driver of lung cancer growth

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we used multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung tumors, we identified HRAS and NRAS as key suppressors of KRASG12D-driven tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogs interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signaling. HRAS mutations identified in KRAS-driven human tumors partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRAS- and BRAF-driven lung cancer models confirmed that RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven lung cancer.

Integrating Cancer Vaccines in the Standard-of-Care of Ovarian Cancer: Translating Preclinical Models to Human

As the majority of ovarian cancer (OC) patients are diagnosed with metastatic disease, less than 40% will survive past 5 years after diagnosis. OC is characterized by a succession of remissions and recurrences. The most promising time point for immunotherapeutic interventions in OC is following debulking surgery. Accumulating evidence shows that T cells are important in OC; thus, cancer vaccines capable of eliciting antitumor T cells will be effective in OC treatment. In this review, we discuss different cancer vaccines and propose strategies for their incorporation into the OC standard-of-care regimens. Using the murine ID8 ovarian tumor model, we provide evidence that a cancer vaccine can be effectively combined with OC standard-of-care to achieve greater overall efficacy. We demonstrate several important similarities between the ID8 model and OC patients, in terms of response to immunotherapies, and the ID8 model can be an important tool for evaluating combinatorial regimens and clinical trial designs in OC. Other emerging models, including patient-derived xenograft and genetically engineered mouse models, are continuing to improve and can be useful for evaluating cancer vaccination therapies in the near future. Here, we provide a comprehensive review of the completed and current clinical trials evaluating cancer vaccines in OC.

A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm v2

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. This protocol is compatible with frozen sperm from all major repositories and the IVF can easily be adapted to accommodate freshly harvested sperm.

Two Types of Mouse Models for Sarcopenia Research: Senescence Acceleration and Genetic Modification Models

Sarcopenia leads to loss of skeletal muscle mass, quality, and strength due to aging; it was recently given a disease code (International Classification of Diseases, Tenth Revision, Clinical Modification, M62.84). As a result, in recent years, sarcopenia-related research has increased. In addition, various studies seeking to prevent and treat sarcopenia by identifying the various mechanisms related to the reduction of skeletal muscle properties have been conducted. Previous studies have identified muscle synthesis and breakdown; investigating them has generated evidence for preventing and treating sarcopenia. Mouse models are still the most useful ones for determining mechanisms underlying sarcopenia through correlations and interventions involving specific genes and their phenotypes. Mouse models used to study sarcopenia often induce muscle atrophy by hindlimb unloading, denervation, or immobilization. Though it is less frequently used, the senescence-accelerated mouse can also be useful for sarcopenia research. Herein, we discuss cases where senescence-accelerated and genetically engineered mouse models were used in sarcopenia research and different perspectives to use them.