Spatiotemporal modulation of growth factors directs the generation of multilineage mouse embryonic stem cell-derived mammary organoids.

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment.

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

Nesprin-1 LINC complexes recruit microtubule cytoskeleton proteins and drive pathology in Lmna-mutant striated muscle.

Mutations in LMNA, the gene encoding A-type lamins, cause laminopathies-diseases of striated muscle and other tissues. The aetiology of laminopathies has been attributed to perturbation of chromatin organization or structural weakening of the nuclear envelope (NE) such that the nucleus becomes more prone to mechanical damage. The latter model requires a conduit for force transmission to the nucleus. NE-associated Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes are one such pathway. Using clustered regularly interspaced short palindromic repeats to disrupt the Nesprin-1 KASH (Klarsicht, ANC-1, Syne Homology) domain, we identified this LINC complex protein as the predominant NE anchor for microtubule cytoskeleton components, including nucleation activities and motor complexes, in mouse cardiomyocytes. Loss of Nesprin-1 LINC complexes resulted in loss of microtubule cytoskeleton proteins at the nucleus and changes in nuclear morphology and positioning in striated muscle cells, but with no overt physiological defects. Disrupting the KASH domain of Nesprin-1 suppresses Lmna-linked cardiac pathology, likely by reducing microtubule cytoskeleton activities at the nucleus. Nesprin-1 LINC complexes thus represent a potential therapeutic target for striated muscle laminopathies.

ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model.

ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.

T Cell-Mediated Antitumor Immunity Cooperatively Induced By TGFßR1 Antagonism and Gemcitabine Counteracts Reformation of the Stromal Barrier in Pancreatic Cancer.

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Novel Humanized Mesothelin-Expressing Genetically Engineered Mouse Models Underscore Challenges in Delivery of Complex Therapeutics to Pancreatic Cancers.

Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.

LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies.

Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 × 100 mm, 3.5 µm XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng to 1000 ng in mouse plasma with the lower limit of detection for RP-182 at 1 ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Generation of a Transgenic BALB/c Mouse Line With Selective Expression of Human Mesothelin in Thyroid Gland: Application in Mesothelin-targeted Immunotherapy.

Despite encouraging clinical results with immune checkpoint inhibitors and other types of immunotherapies, the rate of failure is still very high. The development of proper animal models which could be applied to the screening of effective preclinical antitumor drugs targeting human tumor antigens, such as mesothelin (MSLN), is a great need. MSLN is a 40 kDa cell-surface glycoprotein which is highly expressed in a variety of human cancers, and has great value as a target for antibody-based therapies. The present study reports the establishment of an immunocompetent transgenic mouse expressing human MSLN (hMSLN) only in thyroid gland by utilizing an expression vector containing a thyroid peroxidase (TPO) promoter. These mice do not reject genetically modified tumor cells expressing hMSLN on the cell membrane, and tolerate high doses of hMSLN-targeted immunotoxin. Employing this TPO-MSLN mouse model, we find that the combination treatment of LMB-100 and anti-CTLA-4 induces complete tumor regression in 91% of the mice burdened with 66C14-M tumor cells. The combination therapy provides a significant survival benefit compared with both LMB-100 and anti-CTLA-4 monotherapy. In addition, the cured mice reject tumor cells when rechallenged, indicating the development of long-term antitumor immunity. This novel TPO-MSLN mouse model can serve as an important animal tool to better predict tumor responses to any immunomodulatory therapies that target MSLN.

Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones.

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.

Protein Synthesis Inhibition Activity of Mesothelin Targeting Immunotoxin LMB-100 Decreases Concentrations of Oncogenic Signaling Molecules and Secreted Growth Factors.

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24⁻48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endothelial Growth Factor (VEGF) following treatment with cytostatic LMB-100 doses both in culture and in mouse tumors. Decreased VEGF concentration did not change tumor vasculature density, however, LMB-100 caused tissue-specific changes in concentrations of secreted factors made by non-cancer cells. In summary, our data indicate that PSI caused by cytostatic LMB-100 doses preferentially depletes short-lived proteins such as oncogenic signaling molecules and CCSGFs.

Pharmacokinetic evaluation of the PNC disassembler metarrestin in wild-type and Pdx1-Cre;LSL-KrasG12D/+;Tp53R172H/+ (KPC) mice, a genetically engineered model of pancreatic cancer.

PURPOSE: Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers. METHODS: PK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC-MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice. RESULTS: Metarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC0-∞ of 14400 ng h/mL, Cmax of 810 ng/mL and half-life (t1/2) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC50 of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC0-24h ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC0-24h and C24h. AUC0-24h MD to AUC0-24h SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors. CONCLUSIONS: Metarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.

Animal models for analysis of immunological responses to nanomaterials: Challenges and considerations.

Nanotechnology provides many solutions to improve conventional drug delivery and has a unique niche in the areas related to the specific targeting of the immune system, such as immunotherapies and vaccines. Preclinical studies in this field rely heavily on the combination of in vitro and in vivo methods to assess the safety and efficacy of nanotechnology platforms, nanoparticle-formulated drugs, and vaccines. While certain types of toxicities can be evaluated in vitro and good in vitro-in vivo correlation has been demonstrated for such tests, animal studies are still needed to address complex biological questions and, therefore, provide a unique contribution to establishing nanoparticle safety and efficacy profiles. The genetic, metabolic, mechanistic, and phenotypic diversity of currently available animal models often complicates both the animal choice and the interpretation of the results. This review summarizes current knowledge about differences in the immune system function and immunological responses of animals commonly used in preclinical studies of nanomaterials. We discuss challenges, highlight current gaps, and propose recommendations for animal model selection to streamline preclinical analysis of nanotechnology formulations.

Id1 Ablation Protects Hematopoietic Stem Cells from Stress-Induced Exhaustion and Aging.

Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1-/- HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1-/- HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.