Relevant Preclinical Models Research Articles

Abstract 27: Characterizing 3q26 copy number gain in lung squamous cell carcinoma through a novel genetically engineered mouse model

Abstract Lung squamous cell carcinoma (LUSC) is a major histological subtype of lung cancer accounting for ~30% of all lung cancer diagnoses. LUSC patients suffer from poor therapeutic response, high relapse rate, and poor prognosis. A major reason for this bleak outcome is the lack of well characterized oncogenic drivers of LUSC. This has stalled the generation of relevant genetic pre-clinical mouse models amenable to studying LUSC tumorigenesis in vivo and developing targeted therapeutics to treat LUSC patients. Our group has identified concomitant 3q26 copy number gain (CNG) and TP53 loss as defining genetic alterations present in ~90% of LUSC tumors. Furthermore, we have identified three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a potential cell of origin for LUSC) is both necessary and sufficient to transform these cells into orthotopic LUSC tumors in syngeneic mice. These findings indicate that 3q26 amplification and TP53 loss play a critical role in the initiation and progression of LUSC. Based on these data, we have developed the first autochthonous genetically engineered mouse model (GEMM) to study the role of 3q26 CNG in LUSC tumorigenesis in vivo. Our GEMM allows inducible overexpression of Prkci, Ect2, and Sox2 and knockout of Trp53 (PES/Trp53−/−) in lung tissue. Tumorigenesis will be initiated in PES/Trp53−/− mice using an established protocol to target transgene recombination to LBSCs in vivo. These mice will be assessed for tumor latency, survival, tumor burden, and metastatic potential. In related studies, we used bioinformatics analyses on primary LUSC tumors from the Cancer Genome Atlas’ database to identify 13 genes in addition to PRKCI, ECT2 and SOX2, whose expression is driven by 3q26 amplification. These genes were functionally characterized by assessing the effect of shRNA-mediated gene silencing on soft-agar growth of human H520 LUSC cells, a cell line that harbors 3q26 amplification. Our results revealed that knockdown of each of these genes independently resulted in a significant decrease in soft agar colony formation. We further assessed these genes through RT-qPCR mRNA expression profiling comparing PES/Trp53−/- transformed LBSCs to non-transformed LBSCs. These genes were significantly upregulated in PES/Trp53−/- transformed LBSCs, suggesting that they play important roles in LBSC transformation. Future work will further assess the molecular mechanisms by which these additional 3q26 genes contribute to LUSC cell transformation and assess the role of alternate cells of origin (Club and AT2) in 3q26-driven LUSC tumor formation using our PES/Trp53−/− GEMM. Citation Format: Kayleah M. Meneses, Capella Weems, Yi Liu, Verline Justilien, Nicole R. Murray, Alan P. Fields. Characterizing 3q26 copy number gain in lung squamous cell carcinoma through a novel genetically engineered mouse model [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 27.

Abstract 2969: Development of a canine PD-L1 antibody and caninized PD-L1 mouse model: an essential translational research tool to raise the success rate of immunotherapy in humans

Abstract Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, the response rates in cancer patients are limited (~20-40%). To improve immune checkpoint blockade therapy, relevant preclinical animal models are an essential component in the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs are convertible into knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies that target canine immune checkpoint molecules, such as canine PD-L1, have not been commercially available. Here, we developed a new canine PD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties by multiple assays. Furthermore, the therapeutic efficacy of cPD-L1 antibodies was evaluated in our unique caninized PD-L1 mice. Altogether, these in vitro and in vivo data including an initial safety profile in laboratory dogs, support the development of our canine PD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. In conclusion, our new therapeutic antibody and caninized PD-L1 mouse model will be an essential translational research tool in raising the success rate of immunotherapy in both dogs and humans. Citation Format: Wonkyung Oh, Alyssa Kim, Deepika Dhawan, Perry M. Kirkham, Deborah W. Knapp, Seung-Oe Lim. Development of a canine PD-L1 antibody and caninized PD-L1 mouse model: an essential translational research tool to raise the success rate of immunotherapy in humans [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 2969.

Abstract 1197: B7-H3-CAR T cells for the treatment of pediatric adrenocortical carcinoma

Abstract Outcomes for pediatric patients with unresectable, metastatic, or relapsed adrenocortical carcinoma (ACC) are dismal despite aggressive management with multimodality therapies. CAR T cell therapy (CAR T) is a promising approach to improve outcomes for patients with this disease. However, progress is hindered by a lack of targetable surface antigens and limited availability of relevant preclinical models. B7-H3 is a promising immunotherapeutic target but little is known about its expression in pediatric ACC. Thus, we sought to i) evaluate B7-H3 expression in primary pediatric ACC samples and ii) assess B7-H3-CAR T activity in two novel pediatric patient-derived xenograft (PDX) models. First, we assessed B7-H3 expression in 44 pediatric adrenocortical tumors (ACTs) using a CLIA-approved immunohistochemistry (IHC) assay. ACTs had a range of B7-H3 positivity, including 24 (55%) with expression above the threshold for eligibility on our active B7-H3-CAR T clinical trial (H-score ≥ 100; NCT04897321). Univariate analyses demonstrated B7-H3 expression was correlated with the presence of germline TP53 mutations and poor prognostic factors including the inability to achieve complete resection, Cushing phenotype, and the absence of TP53 by IHC. Given a dearth of in vivo models, we next established two clinically relevant pediatric ACC PDX models. SJ-ACC4 was derived from a metastatic tumor and had somatic mutations in TP53 (p.C135Lfs*14) and CTNNB1 (p.S45F). SJACT030812_X1 (www.stjude.org/cstn) was derived from a primary tumor and had a germline TP53 mutation (p.R110Pfs*39). Both had partial ATRX deletions (exons 11-30; 3-28), uniparental disomy at chromosome 11p15, and were B7-H3-positive. Notably, SJ-ACC4 was resistant to a battery of chemotherapeutic agents. To evaluate B7-H3-CAR T anti-ACC activity in vivo, SJ-ACC4 tumor fragments were implanted subcutaneously in NOD SCID gamma (NSG) mice and treated with B7-H3-CAR or control T cells. Strikingly, B7-H3-CAR T eradicated 100% of SJ-ACC4 tumors and mice survived long term compared to controls that met endpoints within 35 days (n=8 per group, p< 0.0001). Finally, we asked if B7-H3-CAR T had antitumor activity in an orthotopic pediatric ACC PDX model. SJACT030812_X1 cells were injected into the para-adrenal region of NSG mice under ultrasound guidance and monitored by serial volumetric ultrasonography. B7-H3-CAR T again had significant antitumor activity, and mice survived long-term compared to controls that met endpoints within 42 days (n=5 per group, p< 0.01). In conclusion, B7-H3 is expressed in a majority of pediatric ACTs and B7-H3-CAR T have potent anti-ACC activity. These findings provide a strong rationale for clinically evaluating B7-H3-CAR T for pediatric patients with chemotherapy-resistant ACC. Additionally, the newly established pediatric ACC PDX models provide a valuable resource for investigating different aspects of this rare and aggressive disease. Citation Format: Rebecca Epperly, Emilia M. Pinto, Teresa Santiago, Heather Sheppard, Michael R. Clay, Christopher Morton, Phuong Nguyen, Yinmei Zhou, Mary Woolard, Carla O'Reilly, Julie Justice, Walter Akers, Melissa Johnson, Asa Karlstrom, Cheng Cheng, Gerard Zambetti, Andrew Davidoff, Raul Ribeiro, Christopher DeRenzo. B7-H3-CAR T cells for the treatment of pediatric adrenocortical carcinoma [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 1197.

Abstract 2424: Development of radiation sensitivity evaluation platform in esophageal cancer patient derived organoids

Abstract Radiation therapy (Radiotherapy) is a highly effective treatment with wide range of cancer curing. Radiotherapy allows the cancer cells to be killed or reduced in number while protecting the majority of other organs and tissues in the body. Radiotherapy is commonly used with chemotherapy or immunotherapy or radiosensitizer drugs for enhancing the treatment efficacy. Predicting individual radiation sensitivity is very important in determining the direction of future treatment after cancer diagnosis. However, it is lack of accurate and relevant preclinical model for evaluation of radiation sensitivity.In this study, we developed the platform for radiosensitivity evaluation using the Esophageal cancer patient derived organoids. Biopsy tissues from Esophageal cancer were digested to single cells and then dispensed with ECM on pillar plate (MBD co.) to forming organoids. After organoid formation, the organoids were exposed to radiation with increasing dosage. Cell viability and radiation sensitivity were calculated by 3D Celltiter glo method and Calcein AM staining. Radiation sensitivity index was determined based on radiation response and organoid growth.We included 22 patients with ESCC treat with radiation therapy from Samsung Medical Center. After surgery, we compared radiation sensitivity index with pathological grade for sensitivity and specificity of radio sensitivity evaluation platform. The sensitivity was 75.00% (95% CI 34.91% - 86.81%) and the specificity was 71.43% (95% CI 41.90% - 91.61%). This resulted in an accuracy in this population of 72.73% (95% CI 49.78% - 89.27%).This study has a very high correlation to patient matched clinical outcomes of radiation sensitive group or non-sensitive group. This radiation sensitivity evaluation platform could contribute to precision medicine in Esophageal cancer patients and it can be helpful to make decision how to cure of cancer. Citation Format: Jung Eun Kim, Mi-Hyeon Jeong, Thunyaporn Ravangnam, Sunghun Ju, Dong Woo Lee, Bosung Ku, Dongryul Oh. Development of radiation sensitivity evaluation platform in esophageal cancer patient derived organoids [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 2424.

Abstract 3648: MSU-42011, alone and in combination with selumetinib, reduces pERK levels in NF1 cancer cells and decreases CCL2 expression in THP-1 macrophages

Abstract Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes approximately 50% of affected individuals to develop plexiform neurofibromas (PNFs), which can progress to highly aggressive malignant peripheral nerve sheath tumors (MPNSTs) in approximately 10% of patients. NF1 is caused by mutations in the tumor suppressor gene NF1, which encodes for neurofibromin, a negative regulator of RAS activity. Selumetinib, a specific inhibitor of MEK1/2, is the only FDA-approved drug for NF1-associated PNFs. However, the anti-tumor effects of selumetinib are limited in MPNSTs and have dose-limiting side effects. Deficiency of the NF1 gene not only promotes tumorigenesis but also has broad effects on the immune cells and cytokine signaling driven by hyperactive RAS signaling. Because macrophages account for almost half of cells in NF1 lesions and their infiltration highly correlates with disease progression, we hypothesized that targeting tumor-promoting immune cells could be an alternative approach for NF1 treatment. The novel retinoid X receptor (RXR) agonist MSU-42011 reduces tumor growth in experimental Kras-driven cancers by decreasing pERK expression, reducing tumor-promoting immune cells like CD206+ macrophages and regulatory T cells, and increasing activated cytotoxic T cells. Here, we tested MSU-42011 and selumetinib, either alone or in combination, to evaluate their efficacy against NF1 cancer cells and macrophages using monoculture and conditioned media (CM) treatments. Treatment with 200 nM MSU-40211, 50 nM selumetinib, and the combination for 3 hours reduced pERK protein levels by approximately 40%, 70% and 90%, respectively vs. untreated controls. Treatment for 72 hours with selumetinib and the combination reduced the viability of PNF cells in a dose-dependent manner. Moreover, CM from human PNF cells increased monocyte chemoattractant CCL2 (C-C motif chemokine ligand 2) mRNA expression in human THP1 monocytes and THP1 macrophages differentiated by PMA. Notably, MSU-42011 and selumetinib alone similarly inhibited CCL2 mRNA expression by 25% in THP1 macrophages stimulated with CM from PNF cells, and the inhibition of CCL2 mRNA expression was enhanced to 50% with combination treatment. Taken together, our data suggest that MSU-42011 should be tested in relevant preclinical models of NF1. Citation Format: Pei-Yu Hung, Lizbeth Lockwood, Karen T. Liby. MSU-42011, alone and in combination with selumetinib, reduces pERK levels in NF1 cancer cells and decreases CCL2 expression in THP-1 macrophages. [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 3648.

Abstract 23: Development of a biallelicDicer1mutant mouse strain for studying the pathogenesis of DICER1 syndrome-associated cancer

Abstract DICER1 encodes a RNase III enzyme that post-transcriptionally controls gene expression through governing microRNA (miRNA) biogenesis. We and others have demonstrated that somatic mutations of DICER1 at its RNase IIIb domain metal binding sites, impairing its capability to produce mature miRNAs from the 5P strand of miRNA precursors, occur recurrently in a broad spectrum of cancers associated with the DICER1 syndrome that affects children and youths predominantly. The biallelic DICER1 mutations in these cancers challenge the two-hit hypothesis theory of traditional tumour suppressors, but it remains unproven whether these mutations are sufficient to drive tumorigenesis. The lack of biology relevant preclinical models has also hindered the understanding the pathogenesis. Here, we developed a genetically engineered mouse strain that expresses an inducible knockin Dicer1 missense mutation (Dicer1+/fl-D1693N), equivalent to the DICER1 RNase IIIb hotspot mutation D1709N in human cancers. This conditional strain behaves as a null allele due to an aberrant splicing event without Cre-mediated recombination. When crossed with a well-characterized conditional null Dicer1 strain (Dicer1fl/fl), the resulting compound heterozygous strain (Dicer1fl/fl-D1693N), acts as a hemizygous wildtype allele that is converted to a hemizygous missense mutation allele upon Cre-mediated recombination, reflecting the genetic status of DICER1 in DICER1 syndrome-associated cancer. When crossing with the anti-Mullerian hormone receptor 2 (Amhr2) driven cre strain (Amhr2+/cre), the biallelic Dicer1 mutation resulted in infertility in females by impairing the development of oviduct and endometrium and ultimately drove the development of multicystic tubal and intra-uterine tumours. Histologically, these murine tumors resemble human Müllerian adenosarcoma and other DICER1 syndrome-associated sarcomas. Molecular analysis of these murine tumours validated the miRNA biogenesis defects in 5P-miRNA production, uncovered the activation of the myc signaling and identified that loss of let-7 family miRNAs may drive the transcriptomic rewiring and malignant transformation. Thus, this first DICER1 syndrome-associated murine cancer model faithfully recapitulates the biology of human cancer and provides a unique tool for future investigation and therapeutic development. Citation Format: Yemin Wang, Shary Chen, Janine Senz, Maxwell Douglas, Shelby Thornton, Yana Moscovitz, C. Blake Gilks, David Huntsman, Gregg Morin. Development of a biallelicDicer1mutant mouse strain for studying the pathogenesis of DICER1 syndrome-associated cancer [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 23.

Abstract 2387: Characterization of orthotopic bioluminescent patient-derived xenograft model of glioma for drug discovery

Abstract Introduction: Gliomas account for majority of primary brain tumors and are composed of different grades, which present distinct molecular alterations, histological characteristics, and response to treatments. Patient-derived xenografts (PDXs) are the most “close-to-patient” models for oncology studies which largely maintain the histo- and molecular- pathology of the original tumors and are widely applied in preclinical evaluations. Subcutaneously inoculated glioma PDX models provide convenient evaluation of treatments’ efficacy but are difficult to mimic the in-situ microenvironment and diffuse invasive growth to peripheral tissue in the brain. Orthotopic implantation of bioluminescent labelled glioma models provides a more relevant preclinical model to evaluate and quantify treatment response. Methods: A glioma PDX model was established by subcutaneously engrafting tumor directly from patient following surgery into immunodeficient mice. Once established and expanded through passaging, glioma cells were isolated from fresh PDX tumor and transduced with pLVX-IRES-ZsGreen1 lentiviral vector, which expressed both luciferase and GFP, rapidly. Cells were then subcutaneously inoculated into immunodeficient mice directly without in vitro culture and selection. Cell sorting of the following tumors based on GFP expression was performed to exclude the luciferase negative cells and enrich for stable luciferase signal in subsequent passages. The in vivo expansion and sorting of GFP+ tumor cells were repeated for two rounds. Cells from third passages of subcutaneous tumor were orthotopically inoculated in the brain of NOD.scid mice. The tumor progression in the brain was further confirmed by the IVIS® Spectrum In Vivo Imaging System. Response of orthotopically inoculated BN2289-Luc to temozolomide was also evaluated according to the bioluminescent signal in brain. Results: We successfully established a luciferase-GFP dual-labeled glioma PDX model BN2289-Luc which was stably passaged in vivo. The successful transduction of luciferase-GFP cascade was detected by in vitro luciferase assay and fluorescence imaging. Stable tumor progression and bioluminescent signal growth of BN2289-Luc were validated orthotopically and subcutaneously in immunodeficient mice, and a histological study revealed the consistency between orthotopic and subcutaneous tumors. Efficacy studies showed that orthotopically inoculated BN2289-Luc responded to temozolomide (25mg/kg, p.o., Day1-5/wkx4wks). Conclusion: An orthotropic glioma PDX model, BN2289-Luc displayed luciferase signal of in situ tumor progression and response to temozolomide treatment, providing an ideal approach for orthotopic investigation of gliomas and preclinical testing of new drugs. Citation Format: Jinxi Wang, Ling Chen, Jun Zhou, Likun Zhang, Jinyu Huang, Xueluan Zhu, Ludovic Bourre, Jingjing Wang. Characterization of orthotopic bioluminescent patient-derived xenograft model of glioma for drug discovery [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 2387.

Zooming in and out of ferroptosis in human disease.

Ferroptosis is defined as an iron-dependent regulated form of cell death driven by lipid peroxidation. In the past decade, it has been implicated in the pathogenesis of various diseases that together involve almost every organ of the body, including various cancers, neurodegenerative diseases, cardiovascular diseases, lung diseases, liver diseases, kidney diseases, endocrine metabolic diseases, iron-overload-related diseases, orthopedic diseases and autoimmune diseases. Understanding the underlying molecular mechanisms of ferroptosis and its regulatory pathways could provide additional strategies for the management of these disease conditions. Indeed, there are an expanding number of studies suggesting that ferroptosis serves as a bona-fide target for the prevention and treatment of these diseases in relevant pre-clinical models. In this review, we summarize the progress in the research into ferroptosis and its regulatory mechanisms in human disease, while providing evidence in support of ferroptosis as a target for the treatment of these diseases. We also discuss our perspectives on the future directions in the targeting of ferroptosis in human disease.

Bioprinting of hydrogel beads to engineer pancreatic tumor-stroma microtissues for drug screening

Pancreatic ductal adenocarcinoma (PDAC) having features of dense fibrotic stromal and extracellular matrix (ECM) components has poor clinical outcome. In vitro construction of relevant preclinical PDAC models recapitulating the tumor-stroma characteristics is therefore in great need for the development of pancreatic cancer therapy. In this work, a three-dimensional (3D) heterogeneous PDAC microtissue based on a dot extrusion printing (DEP) system is reported. Gelatin methacryloyl (GelMA) hydrogel beads encapsulating human pancreatic cancer cells and stromal fibroblasts were printed, which demonstrated the capacity of providing ECM-mimetic microenvironments and thus mimicked the native cell-cell junctions and cell-ECM interactions. Besides, the spherical structure of the generated hydrogel beads, which took the advantage of encapsulating cells in a reduced volume, enabled efficient diffusion of oxygen, nutrients and cell waste, thus allowing the embedded cells to proliferate and eventually form a dense pancreatic tumor-stroma microtissue around hundred microns. Furthermore, a tunable stromal microenvironment was easily achieved by adjusting the density of stromal cells in the hydrogel beads. Based on our results, the produced heterogeneous pancreatic microtissue recapitulated the features of cellular interactions and stromal-like microenvironments, and displayed better anti-cancer drug resistance than mono-cultured pancreatic cancer spheroids. Together, the DEP system possesses the ability to simply and flexibly produce GelMA hydrogel beads, providing a robust manufacturing tool for the pancreatic cancer drug screening platform fabrication. In addition, the engineered pancreatic tumor-stroma microtissue based on bioprinted GelMA hydrogel beads, other than being ECM-biomimetic and stroma-tunable, can be used for observation in situ and may serve as a new drug screening platform.

Open questions in human lung organoid research.

Organoids have become a prominent model system in pulmonary research. The ability to establish organoid cultures directly from patient tissue has expanded the repertoire of physiologically relevant preclinical model systems. In addition to their derivation from adult lung stem/progenitor cells, lung organoids can be derived from fetal tissue or induced pluripotent stem cells to fill a critical gap in modelling pulmonary development in vitro. Recent years have seen important progress in the characterisation and refinement of organoid culture systems. Here, we address several open questions in the field, including how closely organoids recapitulate the tissue of origin, how well organoids recapitulate patient cohorts, and how well organoids capture diversity within a patient. We advocate deeper characterisation of models using single cell technologies, generation of more diverse organoid biobanks and further standardisation of culture media.

Bioprinting of hydrogel beads to engineer pancreatic tumor-stroma microtissues for drug screening.

Pancreatic ductal adenocarcinoma (PDAC) having features of dense fibrotic stromal and extracellular matrix (ECM) components has poor clinical outcome. In vitro construction of relevant preclinical PDAC models recapitulating the tumor-stroma characteristics is therefore in great need for the development of pancreatic cancer therapy. In this work, a three-dimensional (3D) heterogeneous PDAC microtissue based on a dot extrusion printing (DEP) system is reported. Gelatin methacryloyl (GelMA) hydrogel beads encapsulating human pancreatic cancer cells and stromal fibroblasts were printed, which demonstrated the capacity of providing ECM-mimetic microenvironments and thus mimicked the native cell-cell junctions and cell-ECM interactions. Besides, the spherical structure of the generated hydrogel beads, which took the advantage of encapsulating cells in a reduced volume, enabled efficient diffusion of oxygen, nutrients and cell waste, thus allowing the embedded cells to proliferate and eventually form a dense pancreatic tumor-stroma microtissue around hundred microns. Furthermore, a tunable stromal microenvironment was easily achieved by adjusting the density of stromal cells in the hydrogel beads. Based on our results, the produced heterogeneous pancreatic microtissue recapitulated the features of cellular interactions and stromal-like microenvironments, and displayed better anti-cancer drug resistance than mono-cultured pancreatic cancer spheroids. Together, the DEP system possesses the ability to simply and flexibly produce GelMA hydrogel beads, providing a robust manufacturing tool for the pancreatic cancer drug screening platform fabrication. In addition, the engineered pancreatic tumor-stroma microtissue based on bioprinted GelMA hydrogel beads, other than being ECM-biomimetic and stroma-tunable, can be used for observation in situ and may serve as a new drug screening platform.

Fibroblasts Impair Migration and Antitumor Activity of NK-92 Lymphocytes in a Melanoma-on-Chip Model.

Adoptive cell therapy in solid tumors, such as melanoma, is impaired, but little is known about the role that the fibroblasts present in the tumor microenvironment could exert. However, the mechanism at play is not well understood, partly due to the lack of relevant pre-clinical models. Three-dimensional culture and microfluidic chips are used to recapitulate the dynamic interactions among different types of cells in the tumor microenvironment in controlled and physiological settings. In this brief report, we propose a reductionist melanoma-on-a-chip model for evaluating the essential role of fibroblasts in the antitumor activity of lymphocytes. To this end, 3D melanoma spheroids were monocultured and co-cultured with human dermal fibroblasts and the NK-92 cell migration towards the tumor compartment was tested in a commercially available microfluidic device. Utilizing confocal microscopy, we observed the different recruitment of NK-92 cells in the presence and absence of fibroblasts. Our results show that fibroblasts' presence inhibits immune effector recruiting by exploiting a 3D pre-clinical tumor model.

Celiac Disease: Disease Models in Understanding Pathogenesis and Search for Therapy

Celiac disease is a complex polygenic systemic disorder caused by dietary gluten exposure that selectively occurs in genetically susceptible people. The potential celiac disease is defined by the presence of celiac disease-specific antibodies and compatible human leukocyte antigen but without histological abnormalities in duodenal biopsies. At present, the only treatment is lifelong adherence to a gluten-free diet. Despite its effectiveness, the diet is difficult to maintain due to its cost, availability of gluten-free foods, and hidden gluten. The need to develop non-dietary treatment methods is widely recognized, but this is prevented by the absence of a pathophysiologically relevant preclinical model. Nonetheless, in vitro and in vivo models have made it possible to investigate the mechanisms of the disease and develop new treatment approaches: The use of foods with neutralized gluten, microbiota correction, cocktails of specific endoproteinase, polymer gluten binders, specific inhibitors of transglutaminases and inflammatory cytokines, and a vaccine based on allergen-specific therapy.

Charting co-mutation patterns associated with actionable drivers in intrahepatic cholangiocarcinoma

In recent years, intrahepatic cholangiocarcinoma (iCCA) has evolved as a "role model" for precision oncology in gastrointestinal cancers. However, its rarity, paired with its genomic heterogeneity, challenges the development and evolution of targeted therapies. Interrogating large datasets drives better understanding of the characteristics of molecular subgroups of rare cancers and enables the identification of genomic patterns that remain unrecognized in smaller cohorts. We performed a retrospective analysis of 6,130 patients diagnosed with iCCA from the FoundationCORE database who received diagnostic panel sequencing on the FoundationOne platform. Short variants/fusion-rearrangements and copy number alterations in >300 tumor-associated genes were evaluated, and the tumor mutational burden (TMB) as well as the microsatellite instability (MSI) status were available for the majority of the cohort. We provide a highly representative cartography of the genomic landscape of iCCA and outline the co-mutational spectra of seven therapeutically relevant oncogenic driver genes: IDH1/2, FGFR2, ERBB2, BRAF, MDM2, BRCA1/2, MET and KRASG12C. We observed a negative selection of RTK/RAS/ERK pathway co-alterations, and an enrichment of epigenetic modifiers such as ARID1A and BAP1 in patients with IDH1/2 and FGFR2 alterations. RNF43 as well as KMT2D occurred with high frequency in MSIhigh and TMBhigh tumors. Detailed knowledge of the most prevalent genomic constellations is key to the development of effective treatment strategies for iCCA. Our study provides a valuable resource that could be used to assess the feasibility of clinical trials and subgroup analyses, spurs the development of translationally relevant preclinical models, and serves as a knowledge base to predict potential mechanisms of resistance to targeted therapies in genomically defined subgroups. Due to the high frequency of targetable alterations, molecular diagnostics is recommended in patients with biliary tract cancers, and especially in those with iCCA. The identification of an actionable lesion, however, does not guarantee therapeutic success, and the co-mutational spectrum may act as a critical modifier of drug response. Using a large dataset of comprehensive panel sequencing results from 6,130 patients with iCCA, we provide a detailed analysis of the co-mutational spectrum of the most frequent druggable genetic alterations, which is meant to serve as a reference to establish genetically relevant preclinical models, develop hypothesis-driven combination therapies and identify recurrent genetic profiles.

Infantile epileptic spasms syndrome in children with cardiofaciocutanous syndrome: Clinical presentation and associations with genotype.

Gene variants that dysregulate signaling through the RAS-MAPK pathway cause cardiofaciocutaneous syndrome (CFCS), a rare multi-system disorder. Infantile epileptic spasms syndrome (IESS) and other forms of epilepsy are among the most serious complications. To investigate clinical presentation, treatment outcomes, and genotype-phenotype associations in CFCS patients with IESS, molecular genetics and clinical neurological history were reviewed across two large clinical research cohorts (n=180). IESS presented in 18/180 (10%) cases, including 16 patients with BRAF variants and 2 with MAP2K1 variants. Among IESS patients with BRAF variants, 16/16 (100%) had sequence changes affecting the protein kinase domain (exons 11-16), although only 57% of total BRAF variants occurred in this domain. Clinical onset of spasms occurred at a median age of 5.4months (range: 1-24 months). Among 13/18 patients whose IESS resolved with anti-seizure medications, 10 were treated with ACTH and/or vigabatrin. A substantial majority of CFCS patients with IESS subsequently developed other epilepsy types (16/18; 89%). In terms of neurodevelopmental outcomes, gross motor function and verbal communication were more limited in patients with a history of IESS compared to those without IESS. These findings can inform clinical neurological care guidelines for CFCS and development of relevant pre-clinical models for severe epilepsy phenotypes.

Utility of Human Relevant Preclinical Animal Models in Navigating NAFLD to MAFLD Paradigm.

Fatty liver disease is an emerging contributor to disease burden worldwide. The past decades of work established the heterogeneous nature of non-alcoholic fatty liver disease (NAFLD) etiology and systemic contributions to the pathogenesis of the disease. This called for the proposal of a redefinition in 2020 to that of metabolic dysfunction-associated fatty liver disease (MAFLD) to better reflect the current understanding of the disease. To date, several clinical cohort studies comparing NAFLD and MAFLD hint at the relevancy of the new nomenclature in enriching for patients with more severe hepatic injury and extrahepatic comorbidities. However, the underlying systemic pathogenesis is still not fully understood. Preclinical animal models have been imperative in elucidating key biological mechanisms in various contexts, including intrahepatic disease progression, interorgan crosstalk and systemic dysregulation. Furthermore, they are integral in developing novel therapeutics against MAFLD. However, substantial contextual variabilities exist across different models due to the lack of standardization in several aspects. As such, it is crucial to understand the strengths and weaknesses of existing models to better align them to the human condition. In this review, we consolidate the implications arising from the change in nomenclature and summarize MAFLD pathogenesis. Subsequently, we provide an updated evaluation of existing MAFLD preclinical models in alignment with the new definitions and perspectives to improve their translational relevance.

Improving Preclinical Development of Novel Interventions to Treat Pain: Insanity Is Doing the Same Thing Over and Over and Expecting Different Results.

Preclinical pain research has applied state-of-the-art methods over the past 40 years to describe, characterize, and image molecules, cells, and circuits in rodents to understand the pathophysiology of chronic pain. Despite generating a plethora of novel analgesic targets, pharmaceuticals for chronic pain treatment remain largely limited to the same 6 drug classes as present 40 years ago. It is possible that 40 years of effort has brought us to the verge of a paradigm shift and an explosion of novel analgesic drug classes with remarkable safety, efficacy, and tolerability. We think it more likely that advances will not occur until we follow the description of exciting discoveries with hypothesis testing using clinically relevant preclinical animal models and ethologically relevant outcome measures, which better reflect the clinical characteristics of chronic pain syndromes. Furthermore, to be valuable, experiments using such models must be conducted to the highest levels of internal validity, rigor, and reproducibility. Efforts by funders, most recently the Helping End Addiction Long-Term by the National Institutes of Health, aim to address some of these challenges and enhance communication and collaboration between preclinical and clinical investigators. However, the greater problem is a culture that emphasizes novelty and number of publications over scientific rigor and robust replication leading to a high likelihood of false-positive results. A path forward is provided by the evolution of clinical research beginning 50 years ago that resulted in methods to reduce bias and enhance transparency and ethics of reporting, moving from case reports to randomized controlled trials to innovative study designs with a focus on rigor, generalizability, and reproducibility. We argue that culture changed in clinical science in part because powerful forces outside the peer review system, especially from federal regulators that approve new drugs and human studies committees that addressed ethical failures of earlier research, mandated change in studies within their purview. Whether an external force will affect change in peclinical pain research is unclear.

Intravital Microscopy of the Native Thymus

Hematopoietic Cell Transplantation (HCT) is a common treatment for patients suffering from a variety of malignant or benign diseases, reconstituting the hematopoietic system after preconditioning treatment. In patients undergoing HCT, the capacity for the thymus to produce functional T cells is inhibited due to damage from the cytotoxic preconditioning. Endogenous thymus regeneration depends on the complex relationship between thymus stromal cells (including vascular endothelial cells (EC)) and the recruitment of de novo "seeding" thymic progenitor cells (TPCs) from the regenerated bone marrow (BM). It has been demonstrated that ECs are radio-resistant, however in the context of HCT, it is not clear which type of EC (arterial, venous, or capillary) can survive following injury. Moreover, ECs are critical cells in endogenous thymic regeneration and are involved in recruiting TPCs from the BM, but functional damage to the vascular network may alter the hemodynamics and negatively impact TPC homing and thymus regeneration. Flow cytometry, immunohistochemistry, and ex vivo imaging techniques have been applied to study the thymus in preclinical mouse models since direct visualization of the native thymus in live mice was deemed impossible. Teleost fish are an alternative model organism for direct thymus observation in vivo but redundant cytokine pathways are present which do not exist in humans and mice. Unlike fish models, in mammals T cell development critically depends on the IL-7 cytokine signaling pathway. Therefore, tools to directly visualize the mouse thymus are needed in order to study functional changes to the vascular system after cytotoxic preconditioning in an immunologically relevant preclinical animal model. We hypothesize that cytotoxic preconditioning causes functional and anatomical changes in blood vessel architecture, especially cortical vasculature, that negatively impacts TPC homing and leads to long-term changes in the thymus microenvironment. In this project, we developed intravital imaging techniques to visualize the native thymus in live mice. Using our methodology, we were able to quantify the changes to the blood vessel network after sublethal irradiation (4.5 Gy). Our results showed an increase in blood vessel diameter in mice one day after receiving irradiation. We validated this result using tissue clearing and ex vivo imaging. In addition, most cortical blood flow velocity is <500 µm/s in both control and one day after sublethal irradiation, although no significant changes were observed in blood flow velocity and shear rate between the groups at this timepoint. Taken together, our study suggests that native intravital thymus imaging is a powerful technique enabling functional and anatomical characterization of the thymus vascular network. We believe, further work will help clarify the changes to the vascular system at later timepoints and in the context of higher irradiation doses. This method enables a whole new paradigm for studying thymus biology not previously possible.

Combination of Menin and IDH Mutant Inhibition in Acute Myeloid Leukemia

The interaction between menin and mixed lineage leukemia 1 (MLL1) protein contributes to abnormal proliferation in the MLL1-(KMT2A) rearranged and nucleophosmin 1 (NPM1)-mutated acute myeloid leukemia (AML). Indeed, small molecule inhibition of the menin/MLL1 interaction was shown to inhibit leukemia in both AML subtypes. Approximately 25% of NPM1 mutated AML also carry an IDH 1 or 2 mutation. Carrying both NPM1- and IDH mutations is associated with worse overall survival and relapse free survival, compared to having only the NPM1 mutation. MI-3454, a small molecule inhibitor of MLL1/menin interaction, has been shown to be effective in AML with KMT2A rearrangements and NPM1 mutations. A close analogue of this compound, KO-539 (Ziftomenib) is now being tested in clinical trials in refractory and relapsed AML patients (NCT 04067336). The aim of this study is to determine the efficacy of the combination of MI-3454 and IDH mutant inhibitors in AML carrying concurrent NPM1 and IDH1/2 mutations. Blood or bone marrow aspirate samples from patients with AML harboring both NPM1 and IDH1 and/or IDH2 mutations were obtained and used to assess the effect of combinatorial treatment of menin and IDH mutant inhibitors. Colony formation assays with these AML patient samples were performed in methylcellulose media with cells treated with a concentration range of MI-3454, ivosidenib (IDH1 inhibitor) or enasidenib (IDH2 inhibitor) as single agents and in combinations. At the end point of the experiments, colonies were counted. Cells were collected for 1) cytohistology to assess cell morphology 2) flow cytometry to evaluate markers of differentiation and apoptosis and 3) gene expression studies to assess the effect of the combinations on the expression level of genes implicated in leukemogenesis and differentiation. In the samples with concurrent NPM1 and IDH1 mutations, the combination of MI-3454 and ivosidenib caused synergistic reduction in colony number compared to the single agents, with combination indices (CI) ranging from 0.44 to 0.94. Furthermore, cells treated with this combination demonstrated more differentiating phenotypes than single agent treated cells. In the samples with concurrent NPM1 and IDH2 mutations, the combination of MI-3454 and enasidenib caused synergistic reduction in colony formation versus either single agent MI-3454 or enasidenib, with CI values 0.12-0.64. The combination of these agents also yielded stronger differentiation than single agents alone based on both morphologic assessment and enhanced expression of CD11B differentiation marker. Mechanistically, the combination of MI-3454 and IDH1/2 mutant inhibitors also more significantly decreased the expression of HOXA9, MEIS1, and HOXB2, which are oncogenes implicated in leukemogenesis, over single agents. The expression of MNDA was also significantly enhanced by these combinations, supporting differentiation. Overall, our study demonstrates that combination of MI-3454 and IDH mutant inhibitors outperforms the effect of single agents with respect to their anti-leukemic effects in clinically relevant pre-clinical models. These results support that combination of menin and IDH mutant inhibitors could be utilized in clinical trials to improve the ability of patients with AML to achieve remission.

Automated whole slide image analysis for a translational quantification of liver fibrosis

Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to their discontinuous values and reader-dependent variability. Here we used an automated image analysis software to measure fibrosis deposition in two relevant preclinical models of liver fibrosis, and established correlation with other quantitative fibrosis descriptors. Longitudinal quantification of liver fibrosis was carried out during progression of post-necrotic (CCl4-induced) and metabolic (HF-CDAA feeding) models of chronic liver disease in mice. Whole slide images of picrosirius red-stained liver sections were analyzed using a fully automated, unsupervised software. Fibrosis was characterized by a significant increase of collagen proportionate area (CPA) at weeks 3 (CCl4) and 8 (HF-CDAA) with a progressive increase up to week 18 and 24, respectively. CPA was compared to collagen content assessed biochemically by hydroxyproline assay (HYP) and by standard histological staging systems. CPA showed a high correlation with HYP content for CCl4 (r = 0.8268) and HF-CDAA (r = 0.6799) models. High correlations were also found with Ishak score or its modified version (r = 0.9705) for CCl4 and HF-CDAA (r = 0.9062) as well as with NASH CRN for HF-CDAA (r = 0.7937). Such correlations support the use of automated digital analysis as a reliable tool to evaluate the dynamics of liver fibrosis and efficacy of antifibrotic drug candidates in preclinical models.