Small RNAs (sRNAs) are short (∼50-200 nucleotides) noncoding RNAs that regulate cellular activities across bacteria. Salmonella enterica starved of a carbon-energy (C) source experience a host of genetic and physiological changes broadly referred to as the starvation-stress response (SSR). In an attempt to identify novel sRNAs contributing to SSR control, we grew log-phase, 5-h C-starved and 24-h C-starved cultures of the virulent Salmonella enterica subspecies enterica serovar Typhimurium strain SL1344 and comprehensively sequenced their small RNA transcriptomes. Strikingly, after employing a novel strategy for sRNA discovery based on identifying dynamic transcripts arising from "gene-empty" regions, we identify 58 wholly undescribed Salmonella sRNA genes potentially regulating SSR averaging an ∼1,000-fold change in expression between log-phase and C-starved cells. Importantly, the expressions of individual sRNA loci were confirmed by both comprehensive transcriptome analyses and northern blotting of select candidates. Of note, we find 43 candidate sRNAs share significant sequence identity to characterized sRNAs in other bacteria, and ∼70% of our sRNAs likely assume characteristic sRNA structural conformations. In addition, we find 53 of our 58 candidate sRNAs either overlap neighboring mRNA loci or share significant sequence complementarity to mRNAs transcribed elsewhere in the SL1344 genome strongly suggesting they regulate the expression of transcripts via antisense base-pairing. Finally, in addition to this work resulting in the identification of 58 entirely novel Salmonella enterica genes likely participating in the SSR, we also find evidence suggesting that sRNAs are significantly more prevalent than currently appreciated and that Salmonella sRNAs may actually number in the thousands.

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Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bi-transgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in β-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. The utility of this model is enhanced by the ability to form tumor-derived allografts. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

No abstract

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community’s ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

No abstract

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bitransgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in beta-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. The utility of this model is enhanced by ability to form tumor-derived allografts. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer related death with a 5-year survival rate at 11%. New systemic therapies for patients with PDAC are desperately needed. Upregulation or exposure of a new protein on the tumor cell surface can serve as a therapeutic or diagnostic target. Here, we highlight our recently developed strategy to induce localization of the reticular protein calreticulin (CALR) to the cell surface in PDAC cells and its subsequent detection using a novel radiolabeled peptide. Methods: Surface translocation of CALR was detected by flow cytometry, western blot, and total internal reflection fluorescence (TIRF) microscopy in PDAC cells treated with either doxorubicin or gemcitabine. The radionuclide-binding chelator ‘DOTA’ was covalently linked to a CALR-specific peptide ‘KLGFFKR’ and then labeled with 68Ga. Samples were analyzed on HPLC with an average radiolabeling efficiency of 93%. Mice bearing Panc02 allografts were treated with doxorubicin for 24h, injected with ~3 MBq (5 μg) of radiopeptide, and sacrificed after 1 hour to determine biodistribution. Results: Using flow cytometry, we found that treating PDAC cells with doxorubicin or gemcitabine increased both the total number and the median fluorescence intensity of surface staining for live cells expressing CALR. When membrane proteins were isolated from PDAC cells treated with doxorubicin at various time points, a peak in surface CALR protein was detected at 30 minutes with persistent expression lasting for 24 hours. TIRF microscopy showed that Panc02 cells treated with doxorubicin had approximately a 2-fold higher surface CALR expression as detected by enhanced membrane fluorescence compared to controls. In vivo, our novel [68Ga]-CALR peptide showed rapid clearance through the kidneys with no significant uptake in vital organs (n=4). In Panc02 allograft-bearing mice treated intratumorally with either vehicle or doxorubicin, biodistribution analysis after radiopeptide injection showed a significant increase in radiopeptide uptake in the treated tumors (n=6, p < 0.05). Conclusions: CALR is translocated to the cell surface in PDAC cells, where it can subsequently be targeted by a novel radiopeptide agent. Future studies are needed to determine if induced CALR can be targeted for therapeutic effect. Citation Format: Rachael Guenter, Maxwell Ducharme, Brendon Herring, Tejeshwar C. Rao, Odalyz Montes, Tyler McCaw, Herbert Chen, Suzanne E. Lapi, Benjamin Larimer, J. Bart Rose. Using a novel [68Ga]-radiolabeled peptide to detect cell surface expression of calreticulin in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3578.