miR-802 Suppresses Acinar-to-Ductal Reprogramming During Early Pancreatitis and Pancreatic Carcinogenesis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor that is almost uniformly lethal in humans. Activating mutations of KRAS are found in >90% of human PDACs and are sufficient to promote acinar-to-ductal metaplasia (ADM) during tumor initiation. The roles of miRNAs in oncogenic Kras-induced ADM are incompletely understood. METHODS: The Ptf1aCre/+LSL-KrasG12D/+ and Ptf1aCre/+LSL-KrasG12D/+LSL-p53R172H/+ and caerulein-induced acute pancreatitis mice models were used. mir-802 was conditionally ablated in acinar cells to study the function of miR-802 in ADM. RESULTS: We show that miR-802 is a highly abundant and acinar-enriched pancreatic miRNA that is silenced during early stages of injury or oncogenic KrasG12D-induced transformation. Genetic ablation of mir-802 cooperates with KrasG12D by promoting ADM formation. miR-802 deficiency results in de-repression of the miR-802 targets Arhgef12, RhoA, and Sdc4, activation of RhoA, and induction of the downstream RhoA effectors ROCK1, LIMK1, COFILIN1, and EZRIN, thereby increasing F-actin rearrangement. mir-802 ablation also activates SOX9, resulting in augmented levels of ductal and attenuated expression of acinar identity genes. Consistently with these findings, we show that this miR-802-RhoA-F-actin network is activated in biopsies of pancreatic cancer patients and correlates with poor survival. CONCLUSIONS: We show miR-802 suppresses pancreatic cancer initiation by repressing oncogenic Kras-induced ADM. The role of miR-802 in ADM fills the gap in our understanding of oncogenic Kras-induced F-actin reorganization, acinar reprogramming, and PDAC initiation. Modulation of the miR-802-RhoA-F-actin network may be a new strategy to interfere with pancreatic carcinogenesis.

Identification of HIF-dependent alternative splicing in gastrointestinal cancers and characterization of a long, coding isoform of SLC35A3.

Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify many HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We correlate the discovered hypoxia-driven events with available sequencing data from pan-cancer TCGA patient cohorts to select a narrow set of putative biologically relevant splice events for experimental validation. We validate a small set of candidate HIF-dependent alternative splicing events in multiple human gastrointestinal cancer cell lines as well as patient-derived human pancreatic cancer organoids. Lastly, we report the discovery of a HIF-dependent mechanism to produce a hypoxia-dependent, long and coding isoform of the UDP-N-acetylglucosamine transporter SLC35A3.

rBPI21 (Opebacan) Promotes Rapid Trilineage Hematopoietic Recovery in a Murine Model of High-Dose Total Body Irradiation.

The complexity of providing adequate care after radiation exposure has drawn increasing attention. While most therapeutic development has focused on improving survival at lethal radiation doses, acute hematopoietic syndrome (AHS) occurs at substantially lower exposures. Thus, it is likely that a large proportion of such a radiation-exposed population will manifest AHS of variable degree and that the medical and socioeconomic costs of AHS will accrue. Here, we examined the potential of rBPI21 (opebacan), used without supportive care, to accelerate hematopoietic recovery after radiation where expected survival was substantial (42-75%) at 30 days). rBPI21 administration was associated with accelerated recovery of hematopoietic precursors and normal marrow cellularity, with increases in megakaryocyte numbers particularly marked. This translated into attaining normal trilineage peripheral blood counts 2-3 weeks earlier than controls. Elevations of hematopoietic growth factors observed in plasma and the marrow microenvironment suggest the mechanism is likely multifactorial and not confined to known endotoxin-neutralizing and cytokine down-modulating activities of rBPI21 . These observations deserve further exploration in radiation models and other settings where inadequate hematopoiesis is a prominent feature. These experiments also model the potential of therapeutics to limit the allocation of scarce resources after catastrophic exposures as an endpoint independent of lethality mitigation. This article is protected by copyright. All rights reserved.

Myeloid sarcoma with NPM1 mutation may be clinically and genetically distinct from AML with NPM1 mutation: a study from the Bone Marrow Pathology Group.

Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.

First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1^Mut MS may be genetically distinct from NPM1^Mut AML.NPM1^Mut MS may have inferior overall survival compared to NPM1^Mut AML.

Tumor Organoids as a Research Tool: How to Exploit Them.

Organoid models allow for the study of key pathophysiological processes such as cancer biology in vitro. They offer insights into all aspects covering tumor development, progression and response to the treatment of tissue obtained from individual patients. Tumor organoids are therefore not only a better tumor model than classical monolayer cell cultures but can be used as personalized avatars for translational studies. In this review, we discuss recent developments in using organoid models for cancer research and what kinds of advanced models, testing procedures and readouts can be considered.

Molecular Diagnostic Review of Diffuse Large B-Cell Lymphoma and Its Tumor Microenvironment.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. It is a clinically and morphologically heterogeneous entity that has continued to resist complete subtyping. Molecular subtyping efforts emerged in earnest with the advent of gene expression profiling (GEP). This molecular subtyping approach has continued to evolve simultaneously with others including immunohistochemistry and more modern genomic approaches. Recently, the veritable explosion of genomic data availability and evolving computational methodologies have provided additional avenues, by which further understanding and subclassification of DBLCLs is possible. The goal of this review is to provide a historical overview of the major classification timepoints in the molecular subtyping of DLBCL, from gene expression profiling to present day understanding.

SF3B1 facilitates HIF1-signaling and promotes malignancy in pancreatic cancer.

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

Magnetic Curvatures of a Uniformly Magnetized Tesseroid Using the Cartesian Kernels.

In recent years, the gravitational curvatures, the third-order derivatives of the gravitational potential (GP), of a tesseroid have been introduced in the context of gravity field modeling. Analogous to the gravity field, magnetic field modeling can be expanded by magnetic curvatures (MC), the third-order derivatives of the magnetic potential (MP), which are the change rates of the magnetic gradient tensor (MGT). Exploiting Poisson's relations between ( n + 1 ) th-order derivatives of the GP and nth-order derivatives of the MP, this paper derives expressions for the MC of a uniformly magnetized tesseroid using the fourth-order derivatives of the GP of a uniform tesseroid expressed in terms of the Cartesian kernel functions. Based on the magnetic effects of a uniform spherical shell, all expressions for the MP, magnetic vector (MV), MGT and MC of tesseroids have been examined for numerical problems due to singularity of the respective integral kernels (i.e., near zone and polar singularity problems). For this, the closed analytical expressions for the MP, MV, MGT and MC of the uniform spherical shell have been provided and used to generate singularity-free reference values. Varying both height and latitude of the computation point, it is found numerically that the near zone problem also exists for all magnetic quantities (i.e., MP, MV, MGT and MC). The numerical tests also reveal that the polar singularity problems do not occur for the magnetic quantity as a result of the use of Cartesian as opposed to spherical integral kernels. This demonstrates that the magnetic quantity including the newly derived MC 'inherit' the same numerical properties as the corresponding gravitational functional. Possible future applications (e.g., geophysical information) of the MC formulas of a uniformly magnetized tesseroid could be improved modeling of the Earth's magnetic field by dedicated satellite missions.

Maintenance of Primary Human Colorectal Cancer Microenvironment Using a Perfusion Bioreactor-Based 3D Culture System.

Colorectal cancer (CRC) is a leading cause of cancer-related death. Conventional chemotherapeutic regimens have limited success rates, and a major challenge for the development of novel therapies is the lack of adequate in vitro models. Nonmalignant mesenchymal and immune cells of the tumor microenvironment (TME) are known to critically affect CRC progression and drug responsiveness. However, tumor drug sensitivity is still evaluated on systems, such as cell monolayers, spheroids, or tumor xenografts, which typically neglect the original TME. Here, it is investigated whether a bioreactor-based 3D culture system can preserve the main TME cellular components in primary CRC samples. Freshly excised CRC fragments are inserted between two collagen scaffolds in a "sandwich-like" format and cultured under static or perfused conditions up to 3 d. Perfused cultures maintain tumor tissue architecture and densities of proliferating tumor cells to significantly higher extents than static cultures. Stromal and immune cells are also preserved and fully viable, as indicated by their responsiveness to microenvironmental stimuli. Importantly, perfusion-based cultures prove suitable for testing the sensitivity of primary tumor cells to chemotherapies currently in use for CRC. Perfusion-based culture of primary CRC specimens recapitulates TME key features and may allow assessment of tumor drug response in a patient-specific context.

Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers.

Infiltration of human cancers by T cells is generally interpreted as a sign of immune recognition, and there is a growing effort to reactivate dysfunctional T cells at such tumor sites1. However, these efforts only have value if the intratumoral T cell receptor (TCR) repertoire of such cells is intrinsically tumor reactive, and this has not been established in an unbiased manner for most human cancers. To address this issue, we analyzed the intrinsic tumor reactivity of the intratumoral TCR repertoire of CD8+ T cells in ovarian and colorectal cancer-two tumor types for which T cell infiltrates form a positive prognostic marker2,3. Data obtained demonstrate that a capacity to recognize autologous tumor is limited to approximately 10% of intratumoral CD8+ T cells. Furthermore, in two of four patient samples tested, no tumor-reactive TCRs were identified, despite infiltration of their tumors by T cells. These data indicate that the intrinsic capacity of intratumoral T cells to recognize adjacent tumor tissue can be rare and variable, and suggest that clinical efforts to reactivate intratumoral T cells will benefit from approaches that simultaneously increase the quality of the intratumoral TCR repertoire.

Growth of Epithelial Organoids in a Defined Hydrogel.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.

Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed "in vivo".

Anticancer compound screening on 2D cell cultures poorly predicts "in vivo" performance, while conventional 3D culture systems are usually characterized by limited cell proliferation, failing to produce tissue-like-structures (TLS) suitable for drug testing. We addressed engineering of TLS by culturing cancer cells in porous scaffolds under perfusion flow. Colorectal cancer (CRC) HT-29 cells were cultured in 2D, on collagen sponges in static conditions or in perfused bioreactors, or injected subcutaneously in immunodeficient mice. Perfused 3D (p3D) cultures resulted in significantly higher (p < 0.0001) cell proliferation than static 3D (s3D) cultures and yielded more homogeneous TLS, with morphology and phenotypes similar to xenografts. Transcriptome analysis revealed a high correlation between xenografts and p3D cultures, particularly for gene clusters regulating apoptotic processes and response to hypoxia. Treatment with 5-Fluorouracil (5-FU), a frequently used but often clinically ineffective chemotherapy drug, induced apoptosis, down-regulation of anti-apoptotic genes (BCL-2, TRAF1, and c-FLIP) and decreased cell numbers in 2D, but only "nucleolar stress" in p3D and xenografts. Conversely, BCL-2 inhibitor ABT-199 induced cytotoxic effects in p3D but not in 2D cultures. Our findings advocate the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed "in vivo" and to test anticancer compounds.

Absence of myeloperoxidase and CD8 positive cells in colorectal cancer infiltrates identifies patients with severe prognosis.

Colorectal cancer (CRC) infiltration by cells expressing myeloperoxidase (MPO) or CD8 positive T lymphocytes has been shown to be independently associated with favorable prognosis. We explored the relationship occurring between CD8+ and MPO+ cell CRC infiltration, its impact on clinical-pathological features and its prognostic significance in a tissue microarray (TMA) including 1,162 CRC. We observed that CRC showing high MPO+ cell infiltration are characterized by a prognosis as favorable as that of cancers with high CD8+ T cell infiltration. However, MPO+ and CD8+ CRC infiltrating cells did not synergize in determining a more favorable outcome, as compared with cancers showing MPOhigh/CD8low or MPOlow/CD8high infiltrates. Most importantly, we identified a subgroup of CRC with MPOlow/CD8low tumor infiltration characterized by a particularly severe prognosis. Intriguingly, although MPO+ and CD8+ cells did not co-localize in CRC infiltrates, an increased expression of TIA-1 and granzyme-B was detectable in T cells infiltrating CRC with high MPO+ cell density.

"In vitro" 3D models of tumor-immune system interaction.

Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Experimental animal models and conventional "in vitro" studies have provided a wealth of information on this interaction, currently used to develop immune-mediated therapies. Studies utilizing three-dimensional culture technologies have emphasized that tumor architecture dramatically influences cancer cell-immune system interaction by steering cytokine production and regulating differentiation patterns of myeloid cells, and decreasing the sensitivity of tumor cells to lymphocyte effector functions. Hypoxia and increased production of lactic acid by tumor cells cultured in 3D architectures appear to be mechanistically involved. 3D culture systems could be further developed to (i) include additional cell partners potentially influencing cancer cell-immune system interaction, (ii) enable improved control of hypoxia, and (iii) allow the use of freshly derived clinical cancer specimens. Such advanced models will represent new tools for cancer immunobiology studies and for pre-clinical assessment of innovative treatments.

High frequency of CD8 positive lymphocyte infiltration correlates with lack of lymph node involvement in early rectal cancer.

AIMS: A trend towards local excision of early rectal cancers has prompted us to investigate if immunoprofiling might help in predicting lymph node involvement in this subgroup. METHODS: A tissue microarray of 126 biopsies of early rectal cancer (T1 and T2) was stained for several immunomarkers of the innate and the adaptive immune response. Patients' survival and nodal status were analyzed and correlated with infiltration of the different immune cells. RESULTS: Of all tested markers, only CD8 (P = 0.005) and TIA-1 (P = 0.05) were significantly more frequently detectable in early rectal cancer biopsies of node negative as compared to node positive patients. Although these two immunomarkers did not display prognostic effect "per se," CD8+ and, marginally, TIA-1 T cell infiltration could predict nodal involvement in univariate logistic regression analysis (OR 0.994; 95% CI 0.992-0.996; P = 0.009 and OR 0.988; 95% CI 0.984-0.994; P = 0.05, resp.). An algorithm significantly predicting the nodal status in early rectal cancer based on CD8 together with vascular invasion and tumor border configuration could be calculated (P < 0.00001). CONCLUSION: Our data indicate that in early rectal cancers absence of CD8+ T-cell infiltration helps in predicting patients' nodal involvement.

GM-CSF Production by Tumor Cells Is Associated with Improved Survival in Colorectal Cancer.

PURPOSE: Colorectal cancer infiltration by CD16(+) myeloid cells correlates with improved prognosis. We addressed mechanistic clues and gene and protein expression of cytokines potentially associated with macrophage polarization. EXPERIMENTAL DESIGN: GM-CSF or M-CSF-stimulated peripheral blood CD14(+) cells from healthy donors were cocultured with colorectal cancer cells. Tumor cell proliferation was assessed by (3)H-thymidine incorporation. Expression of cytokine genes in colorectal cancer and autologous healthy mucosa was tested by quantitative, real-time PCR. A tumor microarray (TMA) including >1,200 colorectal cancer specimens was stained with GM-CSF- and M-CSF-specific antibodies. Clinicopathological features and overall survival were analyzed. RESULTS: GM-CSF induced CD16 expression in 66% ± 8% of monocytes, as compared with 28% ± 1% in cells stimulated by M-CSF (P = 0.011). GM-CSF but not M-CSF-stimulated macrophages significantly (P < 0.02) inhibited colorectal cancer cell proliferation. GM-CSF gene was expressed to significantly (n = 45, P < 0.0001) higher extents in colorectal cancer than in healthy mucosa, whereas M-CSF gene expression was similar in healthy mucosa and colorectal cancer. Accordingly, IL1ß and IL23 genes, typically expressed by M1 macrophages, were expressed to significantly (P < 0.001) higher extents in colorectal cancer than in healthy mucosa. TMA staining revealed that GM-CSF production by tumor cells is associated with lower T stage (P = 0.02), "pushing" growth pattern (P = 0.004) and significantly (P = 0.0002) longer survival in mismatch-repair proficient colorectal cancer. Favorable prognostic effect of GM-CSF production by colorectal cancer cells was confirmed by multivariate analysis and was independent from CD16(+) and CD8(+) cell colorectal cancer infiltration. M-CSF expression had no significant prognostic relevance. CONCLUSIONS: GM-CSF production by tumor cells is an independent favorable prognostic factor in colorectal cancer.

Colorectal carcinoma infiltration by myeloperoxidase-expressing neutrophil granulocytes is associated with favorable prognosis.

The prognostic relevance of innate immune cells infiltrating colorectal carcinoma lesions is highly debated. By evaluating the expression of myeloperoxidase (MPO) as a marker of neutrophil granulocytes in a large cohort of colorectal carcinoma specimens, we have observed that robust tumor-infiltration by MPO+ cells correlates with improved patient survival independently of other histopathological parameters, including disease stage.

High IL-17-positive tumor immune cell infiltration is indicative for chemosensitivity of ovarian carcinoma.

PURPOSE: Ovarian carcinoma in most instances is diagnosed in an advanced stage which cannot be cured by surgical tumor debulking alone. Standard adjuvant chemotherapy usually follows surgical procedures. Yet, few reliable predictive tissue markers exist for the response of ovarian carcinoma to chemotherapy. In this study, we evaluated the predictive value of IL-17- and FOXP3-positive tumor immune cell infiltration (TICI) for response to chemotherapy in ovarian carcinoma. METHODS: Formalin fixed and paraffin embedded biopsies of mostly high-grade primary serous ovarian carcinomas and their matched recurrences were immunostained with IL-17 and FOXP3 on a tissue microarray. Chemoresistance was defined as tumor recurrence within 6 months of the completion of platinum-based chemotherapy. In 94 and 90 biopsies, conclusive data for IL-17 and FOXP3 were available, respectively. RESULTS: IL-17, but not FOXP3-positive TICI, displayed a significantly higher density in biopsies of chemosensitive tumors (p = 0.01). No significant difference in the expression of IL-17 and FOXP3 TICI was observed in all paired primary and recurrent biopsies without respect to chemoresponse (p = 0.77 and p = 0.87, respectively). However, significantly more IL-17-positive recurrences were encountered in the group of patients with chemosensitive tumors (p = 0.008). CONCLUSIONS: High IL-17-positive TICI is indicative for response to chemotherapy in ovarian carcinoma. Higher frequency of IL-17-positive TICI might persist in recurrent tumor tissues of chemosensitive biopsies, suggesting repetitive platinum-based chemotherapy as an appropriate therapy for patients with IL-17-positive recurrences.