Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening.

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine

Lead Identification using 3D Models of Pancreatic Cancer: Development of 3D Tumor Models for High-throughput Screening.

Recent technological advances have enabled 3D tissue culture models for fast and affordable HTS. We are no longer bound to 2D models for anti-cancer agent discovery, and it is clear that 3D tumor models provide more predictive data for translation of preclinical studies. In a previous study, we validated a microplate 3D spheroid-based technology for its compatibility with HTS automation. Small-scale screens using approved drugs have demonstrated that drug responses tend to differ between 2D and 3D cancer cell proliferation models. Here, we applied this 3D technology to the first ever large-scale screening effort completing HTS on over 150K molecules against primary pancreatic cancer cells. It is the first demonstration that a screening campaign of this magnitude using clinically relevant, ex-vivo 3D pancreatic tumor models established directly from biopsy, can be readily achieved in a fashion like traditional drug screen using 2D cell models. We identified four unique series of compounds with sub micromolar and even low nanomolar potency against a panel of patient derived pancreatic organoids. We also applied the 3D technology to test lead efficacy in autologous cancer associated fibroblasts and found a favorable profile for better efficacy in the cancer over wild type primary cells, an important milestone towards better leads. Importantly, the initial leads have been further validated in across multiple institutes with concordant outcomes. The work presented here represents the genesis of new small molecule leads found using 3D models of primary pancreas tumor cells

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of alpha-smooth muscle actin (alphaSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated alphaSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca(2+)- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling

Characterization of a novel role for the Bur cyclin dependent kinase complex in pre-mRNA splicing

Pre-messenger RNA (pre-mRNA) splicing is carried out by a dynamic ribonucleoprotein machine called the spliceosome. From budding yeast to mammalian cells, the majority of splicing occurs cotranscriptionally. Such spatial and temporal coupling suggest coordinated regulation. To begin to understand this coupled regulation we must first identify factors that play functional roles both in transcription and in splicing, and elucidate their mechanism of action. This motivated a directed genetic screen to identify canonical transcription factors that affect splicing in vitro. Here we describe the discovery that the essential Saccharomyces cerevisiae cyclin dependent kinase Bur1/2, which has previously been shown to regulate transcription elongation, is necessary for efficient pre-mRNA splicing in vivo. Remarkably, the complex also plays a transcription-independent role in splicing that can be observed in vitro. Using in vitro spliceosome assembly assays we demonstrate that integrity of the Bur complex is essential for early prespliceosome formation as well as catalytic spliceosome formation and activation. Our data indicates that the Bur complex associates with prespliceosomes and interacts with the pre -mRNA substrate as well as the U1 snRNA. We further show that the Bur1 kinase can phosphorylate known splicing factors both in vitro as well as in extracts. Furthermore we show that the kinase likely mediates numerous phosphorylation events leading to phosphorylated protein in prespliceosomes and spliceosomes. Finally with the use of a modular analog sensitive Bur1 kinase we uncover a role for Bur1 activation through phosphorylation in spliceosome formation. These results provide the first example in budding yeast of a cyclin dependent kinase affecting pre-mRNA splicing and spliceosomal formation through physical interactions with the spliceosome. This highlights the importance of post translational modifications, and the proteins that mediate them, in regulating critical spliceosome rearrangements. Furthermore, our data paint a picture of a model whereby a transcription factor plays a previously uncharacterized and important regulatory role in pre-mRNA splicing, and hint at an intricate coupling mechanism between multiple RNA processe

Modeling Pancreatic Cancer with Organoids

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy for which new treatment and diagnostic approaches are urgently needed. For such breakthroughs to be discovered, researchers require systems that accurately model the development and biology of PDA. While cell lines, genetically engineered murine models, and xenografts have all led to valuable clinical insights, organotypic culture models have emerged as tractable systems to recapitulate the complex 3D organization of PDA. Recently, multiple methods for modeling PDA using organoids have been reported. This review aims to summarize these organoid methods in the context of other PDA models. While each model system has unique benefits and drawbacks, ultimately, organoids hold special promise for the development of personalized medicine approaches

Dual inhibition of KRASG12D and pan-ERBB is synergistic in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a low survival rate. Recently, new drugs that target KRASG12D, a common mutation in PDAC, have been developed. We studied one of these compounds, MRTX1133, and found it was specific and effective at low nanomolar concentrations in patient-derived organoid models and cell lines harboring KRASG12D mutations. Treatment with MRTX1133 upregulated the expression and phosphorylation of EGFR and HER2, indicating that inhibition of ERBB signaling may potentiate MRTX1133 antitumor activity. Indeed, the irreversible pan-ERBB inhibitor, afatinib, potently synergized with MRTX1133 in vitro, and cancer cells with acquired resistance to MRTX1133 in vitro remained sensitive to this combination therapy. Finally, the combination of MRTX1133 and afatinib led to tumor regression and longer survival in orthotopic PDAC mouse models. These results suggest that dual inhibition of ERBB and KRAS signaling may be synergistic and circumvent the rapid development of acquired resistance in patients with KRAS mutant pancreatic cancer.SignificanceKRAS-mutant pancreatic cancer models, including KRAS inhibitor-resistant models, show exquisite sensitivity to combined pan-ERBB and KRAS targeting, which provides the rationale for testing this drug combination in clinical trials

Table S3 from Dual Inhibition of KRAS^G12D and Pan-ERBB Is Synergistic in Pancreatic Ductal Adenocarcinoma

Table S3. Results of RPPA and transcriptomic analyses.</p

ABA- and Ca²⁺-Induced Stomatal Closure Is Partially Impaired in <i>cpk3cpk6</i> Mutants

<div><p>(A) ABA-induced stomatal closing (white bars: wild-type; shaded bars: <i>cpk3-1cpk6–1</i> double mutant; black bars: <i>cpk3-2cpk6–2</i> double mutant). Average data from representative experiments are shown (wild-type: WT, <i>n</i> = 13 experiments, 260 total stomata; <i>cpk3-1cpk6–1</i> double mutant: <i>n</i> = 7 experiments, 140 stomata; <i>cpk3-2cpk6–2 n</i> = 4 experiments, <i>n</i> = 120 stomata).</p> <p>(B) External Ca²⁺-induced stomatal closing (white bars: wild-type, shaded bars: <i>cpk3-1cpk6–1</i> double mutant, black bars: <i>cpk3-2cpk6–2</i> double mutant). Average data from representative experiments are shown (wild-type: WT, <i>n</i> = 9 experiments including 4 blind experiments, 180 total stomata; <i>cpk3-1cpk6–1</i> double mutant, <i>n</i> = 9 experiments including 4 blind experiments, 180 total stomata; <i>cpk3-2cpk6–2</i>, <i>n</i> = 4 experiments, <i>n</i> = 80 stomata). Stomatal aperture widths are illustrated. Error bars represent SEM.</p></div

Guard Cell Expression of <i>CPK3</i> and <i>CPK6</i> CDPKs

<div><p>(A) Expression of <i>CPK3</i> and <i>CPK6</i> in guard cell (GC) and mesophyll cell (MC) protoplasts was examined by RT-PCR. Control amplifications of the guard cell-expressed <i>KAT1</i> gene and the mesophyll-expressed <i>CBP</i> marker genes [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040327#pbio-0040327-b054" target="_blank">54</a>] (Leonhardt et al., 2004) were used to test the purity of cell preparations (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040327#s2" target="_blank">Results</a>). <i>ACTIN2</i> was used for an internal loading control. To amplify each <i>CDPK</i>-specific band, RT-PCR was performed with primer sets as indicated by arrowheads in (B) for 36 cycles. Plants were sprayed with water (−ABA) or 100 μM ABA (+ABA) 4 h before isolation of protoplasts and RNA extraction.</p> <p>(B) Cartoon showing the T-DNA insertion positions in <i>cpk3</i> and <i>cpk6</i> T-DNA insertion alleles. PCR was performed with a left boarder primer of the T-DNA and a gene-specific primer, and the PCR products were sequenced to determine the T-DNA insertion positions. Arrowheads indicate primer locations for RT-PCR in (A) and (C). ATG and TGA indicate start and stop codons. White boxes indicate exons.</p> <p>(C) RT-PCR confirmed that <i>cpk3–1</i> and <i>cpk6–1</i> alleles were disruption mutants. PCRs (32 cycles) were performed with primer sets as indicated in (B) (black arrowheads) in the left three panels. Transcripts of wild-type (WT) and <i>cpk6–2</i> were examined with two sets of primers [white and black arrowheads in (B)] showing that <i>cpk6–2</i> lacks exon 1 and that the <i>cpk6–2</i> has 8% or less the mRNA level of wild-type based on densitometry analyses (<i>n</i> = 2). RNA was extracted from leaves of WT, homozygous <i>cpk3–1</i>, <i>cpk6–1,</i> and <i>cpk6–2</i> single mutants, and the <i>cpk3-1cpk6–1</i> double mutant.</p></div