Profiling the heterogeneity of colorectal cancer consensus molecular subtypes using spatial transcriptomics.

The consensus molecular subtypes (CMS) of colorectal cancer (CRC) is the most widely-used gene expression-based classification and has contributed to a better understanding of disease heterogeneity and prognosis. Nevertheless, CMS intratumoral heterogeneity restricts its clinical application, stressing the necessity of further characterizing the composition and architecture of CRC. Here, we used Spatial Transcriptomics (ST) in combination with single-cell RNA sequencing (scRNA-seq) to decipher the spatially resolved cellular and molecular composition of CRC. In addition to mapping the intratumoral heterogeneity of CMS and their microenvironment, we identified cell communication events in the tumor-stroma interface of CMS2 carcinomas. This includes tumor growth-inhibiting as well as -activating signals, such as the potential regulation of the ETV4 transcriptional activity by DCN or the PLAU-PLAUR ligand-receptor interaction. Our study illustrates the potential of ST to resolve CRC molecular heterogeneity and thereby help advance personalized therapy.

Reproductive findings in male animals exposed to selective survival of motor neuron 2 (SMN2) gene splicing-modifying agents.

Risdiplam is a daily, orally dosed, survival of motor neuron 2 (SMN2) mRNA splicing-modifying agent approved for the treatment of spinal muscular atrophy (SMA). RG7800 is a closely related SMN2 mRNA-splicing compound. Effects on secondary mRNA splice targets such as Forkhead Box M1 (FOXM1) and MAP kinase-activating death domain protein (MADD), which have been implicated in cell-cycle regulation, were observed in non-clinical studies with both risdiplam and RG7800. Potential effects of risdiplam on male fertility via FOXM1 and MADD are important as these secondary splice targets exist in humans. This publication reports the findings from 14 in vivo studies that investigated the reproductive tissues of male animals in various stages of development. Exposure to risdiplam or RG7800 induced changes within the germ cells in the testes of male cynomolgus monkeys and rats. Germ-cell changes included both cell-cycle gene changes (alteration of mRNA-splicing variants) and seminiferous tubule degeneration. In monkeys treated with RG7800, there was no evidence of damage to spermatogonia. Observed testicular changes were stage-specific with spermatocytes in the pachytene stage of meiosis and were fully reversible in monkeys following a sufficient recovery period of eight weeks following cessation of RG7800. In rats, seminiferous tubule degeneration was present, and full reversibility of germ-cell degeneration in the testes was observed among half of the rats that were exposed to risdiplam or RG7800 and then allowed to recover. With these results, coupled with histopathological findings, the effects on the male reproductive system are expected to be reversible in humans for these types of SMN2 mRNA-splicing modifiers.

Species selection for nonclinical safety assessment of drug candidates: Examples of current industry practice.

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies.

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-ß-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).

A minimally-invasive serial cerebrospinal fluid sampling model in conscious Göttingen minipigs.

Drug concentrations in cerebrospinal fluid (CSF) are typically used as a as a surrogate measure of their availability in the CNS, and CSF penetration in animal studies are used for assessment of CNS drug delivery in early preclinical drug development. The minipig is a valid alternative to dogs and non-human primates as non-rodent species in preclinical research, but this species presents anatomical peculiarities that make the serial collection of CSF technically challenging. A minimally-invasive serial cerebrospinal fluid collection model via catheterization of the subarachnoid space in conscious minipigs was developed allowing assessment of longitudinal drug pharmacokinetics in the central nervous system in preclinical research. Shortly, the subarachnoid space was accessed in the anesthetized minipig by puncture with a Tuohy needle; when CSF was flowing through the needle a catheter was advanced and thereafter tunneled and fixed on the back. The PK of peptide A administered subcutaneously was performed and CSF could be sampled in the conscious animals for up to 48 h. When compared to the plasma kinetic data, there was a clear difference in the elimination phase of Pept. A from CSF, with an apparent longer average terminal half-life in CSF. The 3Rs are addressed by reducing the number of animals needed for a pharmacokinetic profile in central nervous system and by improving the validity of the model avoiding biases due to anesthesia, blood contamination, and inter-individual variability.

Perspectives From the 12th Annual Minipig Research Forum: Early Inclusion of the Minipig in Safety Assessment Species Selection Should be the Standard Approach.

Over the last decade, the minipig has been established as a species which can be used in biomedical research, including drug development safety assessment. There are no mandatory regulatory guidelines regarding species selection strategy for safety assessment; hence, choice is at the discretion of companies responsible for drug development. A survey of member companies by IQ DruSafe (2016) highlighted inconsistent and low use of the minipig. At the 12th Annual Minipig Research Forum in 2018, presentations and a workshop examined current practices and considered if the minipig could be utilized more from earliest drug development stages. Despite the agreed utility of scientific data and validity of the minipig, especially for small molecules, each company has its own approach in nonrodent species selection, without consistent rationale. The overall objective should be to ensure the most appropriate species is selected and is scientifically based, with the minipig systematically included from early screening stages.

Drug-induced Obstructive and Retrograde Nephropathy Associated with α2u-globulin in Male Rats.

The chemically induced accumulation of α2u-globulin protein in male rats causes specific renal lesions and subsequent nephropathy. Herein, we report additional parallel findings in the kidney of male rats consistent with obstructive and retrograde nephropathy. Kidney and urinary bladder samples were evaluated from Wistar rats treated with RG7129 for 2 week and 8 week and from an 8-week mechanistic study using females, intact and castrated males. Histopathological findings were present in intact males in all studies, including hyaline droplet accumulation and granular casts consistent with α2u-globulin nephropathy. In addition, tubular degeneration and regeneration, tubular changes extending from papilla to cortex, tubular dilation, and interstitial and luminal inflammation were observed consistent with retrograde and obstructive nephropathy. Renal and urinary lesions and their severity increased in a time- and dose-dependent manner. Urinalysis findings, including increases in leukocytes, protein, and in kidney biomarkers, kidney injury molecule 1 and clusterin, were present only in intact males. No treatment-related changes were observed in female rats or in castrated males. These results indicate that RG7129 induces α2u-globulin nephropathy, associated with retrograde and obstructive nephropathy secondary to precipitation in intact male rats only, constituting a species- and sex-specific syndrome that is not expected to occur in humans or other species.

FcRn Expression on Placenta and Fetal Jejunum during Early, Mid-, and Late Gestation in Minipigs.

Developmental toxicity testing of therapeutic antibodies is most often conducted in nonhuman primates owing to lack of cross-reactivity in other species. Minipigs may show cross-reactivity for some humanized antibodies but have not been used for developmental toxicity testing due to an assumed lack of embryo-fetal exposure. Unlike in humans, maternal IgGs do not cross the porcine placenta to reach the fetus. Some humanized IgGs, however, have a higher affinity for the neonatal Fc receptor (FcRn) and are more likely than endogenous antibodies to cross the placenta of animals. The major site of prenatal IgG transfer is the placenta, though FcRn in fetal intestine could also uptake maternal IgGs from swallowed amniotic fluid. Using immunohistochemistry andin situhybridization in this experiment, FcRn was found in minipig placenta and fetal intestine during early, mid-, and late gestation. To date, however, fetal exposure to maternally administered IgGs has never been demonstrated in the minipig.

Pedicled Instep Flap and Tibial Nerve Reconstruction in a Cynomolgus Monkey [Macaca fascicularis].

A male cynomolgus monkey experienced extensive soft tissue trauma to the right caudal calf area. Some weeks after complete healing of the original wounds, the monkey developed a chronic pressure sore on plantar surface of the heel of its right foot. A loss of sensitivity in the sole of the foot was hypothesized. The skin defect was closed by a medial sensate pedicled instep flap followed by counter transplantation of a full thickness graft from the interdigital webspace. The integrity of the tibial nerve was revised and reconstructed by means of the turnover flap technique. Both procedures were successful. This is an uncommon case in an exotic veterinary patient as it demonstrates a reconstructive skin flap procedure for the treatment of a chronic, denervated wound in combination with the successful reconstruction of 2.5 cm gap in the tibial nerve.

Subcutaneous absorption of biotherapeutics: knowns and unknowns.

Subcutaneous administration of biotherapeutics offers several potential advantages compared with intravenous administration. Many biotherapeutics, both marketed or in development, are administered via the subcutaneous route. This minireview provides an overview of the presystemic absorption processes following subcutaneous administration, the resulting pharmacokinetics after subcutaneous administration, and provides recent case examples of the development of subcutaneous administered drugs with a focus on monoclonal antibodies. Subcutaneous absorption of biotherapeutics is relatively slow and mostly incomplete. Knowledge of the subcutaneous tissue is important to understand the absorption kinetics after subcutaneous administration. Transport in the subcutis to the absorbing blood or lymph capillaries appears to be a major contributor to the slow subcutaneous absorption. Larger proteins (>20 kDa) are mostly absorbed via the lymphatic system, although potential species differences are not fully understood yet. Also, the presystemic catabolism leading to incomplete bioavailability is little understood, both the involved enzymes and its translation across species. For IgGs, binding to the neonatal Fc receptor is important to obtain a high bioavailability. Overall, several aspects of subcutaneous absorption are still poorly understood, which hampers, e.g., translation across species. Further research in this area is warranted.

Spontaneously occurring multicentric basal cell carcinoma and keratoacanthomas in a multimammate mouse (Mastomys spp.).

A multicentric basal cell carcinoma was diagnosed in a male multimammate mouse (Mastomys spp.) with widespread cutaneous alterations. Macroscopically, the skin was thickened and extremely wrinkled. Histopathological examination showed multicentric expanding cell-rich tumors composed of basaloid cells interpreted as basal cell carcinoma. Immunohistochemistry detected strong cytokeratin 14 positivity in the epidermal basal layer and in loosely arranged areas of these tumors but only a minimal positive reaction in densely packed areas of tumor cells. Furthermore, samples from the abdomen showed 3 nodular proliferations diagnosed as keratoacanthomas.

Demonstration of Mycoplasma bovis by immunohistochemistry and in situ hybridization in an aborted bovine fetus and neonatal calf.

Mycoplasmas are host-specific commensals on mucous membranes of the genital tract, but infection and clinical disease by Mycoplasma bovis in the genital tract of cattle is not well described. In the current study, 1 aborted bovine fetus and 1 neonatal calf were examined macroscopically and histologically. For the detection of M. bovis, bacterial isolation, immunohistochemistry (IHC), and in situ hybridization (ISH) were performed. For further characterization of the inflammatory infiltrates, IHC was performed using antibodies to cluster of differentiation (CD)3, CD79a, lysozyme, L1, S-100A8, S-100A9, and von Willebrand factor VIII. Gross examination revealed a lobular consolidation of the lung. Histologically, the lungs of both animals showed an interstitial pneumonia associated with suppurative bronchopneumonia, intraalveolar multinucleated giant cells, and lymphocytic aggregates. The expression of L1, S-100A8, and S-100A9 in multinucleated giant cells supports a histiocytic origin. Mycoplasma bovis antigen was detected by IHC in brain, lung, liver, and placenta of the fetus, and M. bovis DNA was detected by ISH in various organs of the fetus, including lung and placenta and within the lung of the calf.

In situ hybridization for the detection of Mycoplasma bovis in paraffin-embedded lung tissue from experimentally infected calves.

Mycoplasma bovis DNA was detected in lung tissue of experimentally infected calves by in situ hybridization (ISH) with a nonradioactive, digoxigenin-labeled DNA probe. The 171-base pair DNA probe targeting part of the gene of the major immunodominant variable surface protein A, which is conserved among all vsp genes, was generated by polymerase chain reaction. Four calves between 57 and 63 days old were inoculated intratracheally with 30 ml of a suspension of M. bovis strain 1067 containing 7 x 10(4) colony forming units per milliliter. Two calves inoculated with 30 ml of sterile medium served as control animals. The calves were euthanized and then examined 21 days after inoculation. The ISH method developed in the current study was suitable for the detection of M. bovis DNA in formalin-fixed, paraffin-embedded lung tissue and may be a valuable tool for diagnostic purposes and for further investigating the pathogenesis of M. bovis infection.

Feasibility and safety of endoscopic full-thickness esophageal wall resection and defect closure: a prospective long-term survival animal study.

BACKGROUND: The first experiences with endoscopic closure of esophageal perforations in animal survival studies encouraged us to extend these procedures to full-thickness resections of pieces of the esophageal wall (FTEW). OBJECTIVE: To learn the feasibility, safety, and long-term effects of FTEW removal and defect closure. DESIGN: Feasibility animal study. SETTING: Approved animal facility. INTERVENTIONS: Twelve pigs were used for 3-month survival studies, autopsy, and histologic examination. Resection of a 2-cm piece of wall was performed with needle-knife and forceps/snare. Closure was performed by using prototype endoscopic suturing. MAIN OUTCOME MEASUREMENTS: Feasibility and complication assessment of this new endoscopic method. RESULTS: There were no complications relating to incision, resection, or closure. All pigs recovered quickly. In 2 animals a larger piece of wall causing a larger defect was removed, resulting in much air penetrating into the mediastinum, causing difficult ventilation. This was resolved with thoracic drain. In 3 of 12 animals a toxic substance slipped into the mediastinum, resulting in an abscess in 1 pig and misfire of an anchor as a result of obscured vision. This caused temporary illness of the animal but not death. Autopsy and histologic study confirmed no mediastinitis and well-healed scars in all but one. LIMITATION: Animal study. CONCLUSION: FTEW has proven to be feasible. Long-term survival demonstrated no mediastinitis and only 1 abscess after contamination of the mediastinum. These first experiences encourage further animal studies because the prospect of endoscopic full-thickness removal of esophageal lesions in patients might be very advantageous.

Malignant catarrhal fever-like lesions associated with ovine herpesvirus-2 infection in three goats.

This is the first description of malignant catarrhal fever-like lesions associated with ovine herpesvirus-2 (OvHV-2) infection in goats with central nervous symptoms. The diagnosis was based on typical histological lesions characterized by systemic lymphohistiocytic and fibrinoid vasculitis and confirmed by polymerase chain reaction and subsequent phylogenetic analysis of the detected OvHV-2 sequences.