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Transcriptomics is an important OMICs method that is often unavailable in biobank research. Frozen blood samples are routinely collected and stored in medical biobanks, but transcriptional studies have been limited due to technical difficulties of extracting high-quality RNA from blood frozen in standard tubes (without RNA preservatives). We aimed to determine whether biobanked buffy coat samples stored at -80°C for up to 23 years could be successfully used for mRNA sequencing. We used a CryoXtract CXT 350 to remove frozen sample cores, which were immersed in RNA preservative during thawing prior to RNA extraction. RNA sequencing was then performed on extractions from pooled samples as well as from 23 buffy coat samples from prospective colorectal cancer cases and 23 matched controls included in the population-based, prospective Northern Sweden Health and Disease Study (NSHDS). For all samples, two library preparation methods were used (Illumina TruSeq Stranded mRNA poly-A selection and Illumina Stranded Total RNA with Ribo-Zero Globin). RNA yields of over 1 µg were obtained from the majority of NSHDS samples (mean = 2.57 µg), and over 92% of samples had RIN values of ≥ 6, indicating suitability for downstream analyses. In conclusion, we developed a method for successfully extracting and sequencing high-quality mRNA from frozen buffy coat samples stored long term in tubes with no RNA preservative.

African Swine Fever (ASF) is caused by a DNA virus (AFSV) maintained and transmitted by the Argasid ticks. The re-emergence of the disease in Africa coupled with its rapid spread globally is a threat to the pig industry, food security and livelihoods. The ecology and epidemiology of the ASFV sylvatic cycle, especially in the face of changing land use and land cover, further compounds the menace and impacts of this disease in Kenya. The study aimed to determine the occurrence and distribution of ASFV seroprevalence in warthog populations, the tick vectors and extent of tick infestation of warthog burrows, and the genotypes of ASFV in soft ticks in Kenya. Warthogs from different parts of Kenya were captured and venous blood was centrifuged to harvest sera. Warthog burrows were examined for their conditions and to extract ticks. Sera were analyzed for antibodies against ASFV using a commercial ELISA kit coated with p32 ASFV recombinant protein. Ticks were pooled, DNA extracted and the p72 gene of the ASFV was amplified by qPCR and conventional PCR. The overall seroprevalence of ASFV in warthogs was 87.5 %. A total of 228 warthog burrows were examined and 2154 argasid ticks were extracted from the burrows. Tick pools from Kigio Farm and Lewa Wildlife Conservancies were ASFV-positive by qPCR and conventional PCR. ASFV was further confirmed by the Twist Comprehensive Viral Research Panel (TCVRP), which also identified the argasid ticks as Ornithodoros porcinus. The ticks were infected with virus genotype IX, and their occurrence overlaps with regions of previous ASF outbreaks in domestic pigs. Further, Viruses that could be tick endosymbionts/commensals or due to bloodmeal were detected in ticks by TCVRP; Porcine type-C oncovirus; Pandoravirus neocaledonia; Choristoneura fumiferana granulovirus; Enterobacteria phage p7; Leporid herpesvirus 4 isolate; 5; Human Lymphotropic virus; Human herpesvirus 5. In conclusion, our results suggest that infected Ornithodoros spp. seems to have a rich virome, which has not been explored but could be exploited to inform ASF control in Kenya. Further, the ecology of Ornithodoros spp. and burrow-use dynamics are complex and more studies are needed to understand these dynamics, specifically in the spread of ASFV at the interface of wild and domestic pigs. Further, our results provide evidence of genotype IX ASFV sylvatic cycle which through O. porcinus tick transmission has resulted in high exposure of adult common warthogs. Finally, the co-circulation of ASFV genotype IX in the same location with past ASF outbreaks in domestic pigs and presently in ticks brings to focus the role of the interface and ticks on virus transmission to pigs and warthogs.

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5–40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced.

We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein.

Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes.

MicroRNAs (miRNAs) are aberrantly expressed in prostate cancer (PC), but comprehensive knowledge about their levels and function in metastatic PC is lacking. Here, we explored the differential expression of miRNA profiles during PC progression to bone metastasis, and further focused on the downregulation of miRNA-23c and -4328 and their impact on PC growth in experimental models. Using microarray screening, the levels of 1510 miRNAs were compared between bone metastases (n = 14), localized PC (n = 7) and benign prostate tissue (n = 7). Differentially expressed miRNAs (n = 4 increased and n = 75 decreased, p < 0.05) were identified, of which miRNA-1, -23c, -143-3p, -143-5p, -145-3p, -205-5p, -221-3p, -222-3p and -4328 showed consistent downregulation during disease progression (benign > localized PC > bone metastases). The downregulation of miRNA-23c and -4328 was confirmed by reverse transcription and quantitative polymerase chain reaction analysis of 67 metastasis, 12 localized PC and 12 benign prostate tissue samples. The stable overexpression of miRNA-23c and -4328 in the 22Rv1 and PC-3 cell lines resulted in reduced PC cell growth in vitro, and in the secretion of high levels of miRNA-23c (but not -4328) in extracellular vesicles. However, no tumor suppressive effects were observed from miRNA-23c overexpression in PC-3 cells subcutaneously grown in mice. In conclusion, bone metastases display a profound reduction of miRNA levels compared to localized PC and benign disease. The downregulation of those miRNAs, including miRNA-23c and -4328, may lead to a loss of tumor suppressive effects and provide biomarker and therapeutic possibilities that deserve to be further explored.

Background: Patients with metastatic prostate cancer (PC) are treated with androgen deprivation therapy (ADT) that initially reduces metastasis growth, but after some time lethal castration-resistant PC (CRPC) develops. A better understanding of the tumor biology in bone metastases is needed to guide further treatment developments. Subgroups of PC bone metastases based on transcriptome profiling have been previously identified by our research team, and specifically, heterogeneities related to androgen receptor (AR) activity have been described. Epigenetic alterations during PC progression remain elusive and this study aims to explore promoter gene methylation signatures in relation to gene expression and tumor AR activity.

Materials and methods: Genome-wide promoter-associated CpG methylation signatures of a total of 94 tumor samples, including paired non-malignant and malignant primary tumor areas originating from radical prostatectomy samples (n = 12), and bone metastasis samples of separate patients with hormone-naive (n = 14), short-term castrated (n = 4) or CRPC (n = 52) disease were analyzed using the Infinium Methylation EPIC arrays, along with gene expression analysis by Illumina Bead Chip arrays (n = 90). AR activity was defined from expression levels of genes associated with canonical AR activity.

Results: Integrated epigenome and transcriptome analysis identified pronounced hypermethylation in malignant compared to non-malignant areas of localized prostate tumors. Metastases showed an overall hypomethylation in relation to primary PC, including CpGs in the AR promoter accompanied with induction of AR mRNA levels. We identified a Methylation Classifier for Androgen receptor activity (MCA) signature, which separated metastases into two clusters (MCA positive/negative) related to tumor characteristics and patient prognosis. The MCA positive metastases showed low methylation levels of genes associated with canonical AR signaling and patients had a more favorable prognosis after ADT. In contrast, MCA negative patients had low AR activity associated with hypermethylation of AR-associated genes, and a worse prognosis after ADT.

Conclusions: A promoter methylation signature classifies PC bone metastases into two groups and predicts tumor AR activity and patient prognosis after ADT. The explanation for the methylation diversities observed during PC progression and their biological and clinical relevance need further exploration.

The human kallikrein-related peptidase 4 (KLK4) and the transcribed pseudogene KLKP1 are reported to be highly expressed in the prostate. When trying to clone transcripts of KLKP1, we partly failed. Instead, we identified an androgen-regulated transcript, KLK4T2, which appeared to be a splice variant of KLK4 that also contained exons of KLKP1. Expression analysis of KLK4, KLK4T2, and KLKP1 transcripts in prostate cancer cell lines showed high levels of KLKP1 transcripts in the nucleus and in unfractionated cell extract, whereas it was almost completely absent in the cytoplasmatic fraction. This was in contrast to KLK4 and KLK4T2, which displayed high to mod-erate levels in the cytoplasm. In patient cohorts we found significantly higher expression of both KLK4T2 and KLK4 in benign prostatic hyperplasia compared to both primary prostate cancer and bone metastasis. Analysis of tissue panels demonstrated the highest expression of KLK4T2 in the prostate, but in contrast to the classical KLK4, relatively high levels were also found in placenta. So far, the function of KLK4T2 is still to be explored, but the structure of the translation product indicated that it generates a 17.4 kDa intracellular protein with possible regulatory function.

Background: Taxane treatment may be a suitable therapeutic option for patients with castration‐resistant prostate cancer and high expression of constitutively active androgen receptor variants (AR‐Vs). The aim of the study was to compare the effects of cabazitaxel and androgen deprivation treatments in a prostate tumor xenograft model expressing high levels of constitutively active AR‐V7. Furthermore, mechanisms behind acquired cabazitaxel resistance were explored.

Methods: Mice were subcutaneously inoculated with 22Rv1 cells and treated with surgical castration (n = 7), abiraterone (n = 9), cabazitaxel (n = 6), castration plus abiraterone (n = 8), castration plus cabazitaxel (n = 11), or vehicle and/or sham operation (n = 23). Tumor growth was followed for about 2 months or to a volume of approximately 1000 mm3. Two cabazitaxel resistant cell lines; 22Rv1‐CabR1 and 22Rv1‐CabR2, were established from xenografts relapsing during cabazitaxel treatment. Differential gene expression between the cabazitaxel resistant and control 22Rv1 cells was examined by whole‐genome expression array analysis followed by immunoblotting, immunohistochemistry, and functional pathway analysis.

Results: Abiraterone treatment alone or in combination with surgical castration had no major effect on 22Rv1 tumor growth, while cabazitaxel significantly delayed and in some cases totally abolished 22Rv1 tumor growth on its own and in combination with surgical castration. The cabazitaxel resistant cell lines; 22Rv1‐CabR1 and 22Rv1‐CabR2, both showed upregulation of the ATP‐binding cassette sub‐family B member 1 (ABCB1) efflux pump. Treatment with ABCB1 inhibitor elacridar completely restored susceptibility to cabazitaxel, while treatment with AR‐antagonists bicalutamide and enzalutamide partly restored susceptibility to cabazitaxel in both cell lines. The cholesterol biosynthesis pathway was induced in the 22Rv1‐CabR2 cell line, which was confirmed by reduced sensitivity to simvastatin treatment.

Conclusions: Cabazitaxel efficiently inhibits prostate cancer growth despite the high expression of constitutively active AR‐V7. Acquired cabazitaxel resistance involving overexpression of efflux transporter ABCB1 can be reverted by bicalutamide or enzalutamide treatment, indicating the great clinical potential for combined treatment with cabazitaxel and anti‐androgens.

Bone metastasis is the lethal end-stage of prostate cancer (PC), but the biology of bone metastases is poorly understood. The overall aim of this study was therefore to explore molecular variability in PC bone metastases of potential importance for therapy. Specifically, genome-wide expression profiles of bone metastases from untreated patients (n = 12) and patients treated with androgen-deprivation therapy (ADT, n = 60) were analyzed in relation to patient outcome and to morphological characteristics in metastases and paired primary tumors. Principal component analysis and unsupervised classification were used to identify sample clusters based on mRNA profiles. Clusters were characterized by gene set enrichment analysis and related to histological and clinical parameters using univariate and multivariate statistics. Selected proteins were analyzed by immunohistochemistry in metastases and matched primary tumors (n = 52) and in transurethral resected prostate (TUR-P) tissue of a separate cohort (n = 59). Three molecular subtypes of bone metastases (MetA-C) characterized by differences in gene expression pattern, morphology, and clinical behavior were identified. MetA (71% of the cases) showed increased expression of androgen receptor-regulated genes, including prostate-specific antigen (PSA), and glandular structures indicating a luminal cell phenotype. MetB (17%) showed expression profiles related to cell cycle activity and DNA damage, and a pronounced cellular atypia. MetC (12%) exhibited enriched stroma-epithelial cell interactions. MetB patients had the lowest serum PSA levels and the poorest prognosis after ADT. Combined analysis of PSA and Ki67 immunoreactivity (proliferation) in bone metastases, paired primary tumors, and TUR-P samples was able to differentiate MetA-like (high PSA, low Ki67) from MetB-like (low PSA, high Ki67) tumors and demonstrate their different prognosis. In conclusion, bone metastases from PC patients are separated based on gene expression profiles into molecular subtypes with different morphology, biology, and clinical outcome. These findings deserve further exploration with the purpose of improving treatment of metastatic PC.

Advanced prostate cancer frequently metastasizes to bone and induces a mixed osteoblastic/osteolytic bone response. Standard treatment for metastatic prostate cancer is androgen-deprivation therapy (ADT) that also affects bone biology. Treatment options for patients relapsing after ADT are limited, particularly in cases where castration-resistance does not depend on androgen receptor (AR) activity. Patients with non-AR driven metastases may, however, benefit from therapies targeting the tumor microenvironment. Therefore, the current study specifically investigated bone cell activity in clinical bone metastases in relation to tumor cell AR activity, in order to gain novel insight into biological heterogeneities of possible importance for patient stratification into bone-targeting therapies. Metastasis tissue obtained from treatment-naïve (n = 11) and castration-resistant (n = 28) patients was characterized using whole-genome expression analysis followed by multivariate modeling, functional enrichment analysis, and histological evaluation. Bone cell activity was analyzed by measuring expression levels of predefined marker genes representing osteoclasts (ACP5, CTSK, MMP9), osteoblasts (ALPL, BGLAP, RUNX2) and osteocytes (SOST). Principal component analysis indicated a positive correlation between osteoblast and osteoclast activity and a high variability in bone cell activity between different metastases. Immunohistochemistry verified a positive correlation between runt-related transcription factor 2 (RUNX2) positive osteoblasts and tartrate-resistant acid phosphatase (TRAP, encoded by ACP5) positive osteoclasts lining the metastatic bone surface. No difference in bone cell activity was seen between treatment-naïve and castration-resistant patients. Importantly, bone cell activity was inversely correlated to tumor cell AR activity (measured as AR, FOXA1, HOXB13, KLK2, KLK3, NKX3-1, STEAP2, and TMPRSS2 expression) and to patient serum prostate-specific antigen (PSA) levels. Functional enrichment analysis indicated high bone morphogenetic protein (BMP) signaling in metastases with high bone cell activity and low tumor cell AR activity. This was confirmed by BMP4 immunoreactivity in tumor cells of metastases with ongoing bone formation, as determined by histological evaluation of van Gieson-stained sections. In conclusion, the inverse relation observed between bone cell activity and tumor cell AR activity in prostate cancer bone metastasis may be of importance for patient response to AR and/or bone targeting therapies, but needs to be evaluated in clinical settings in relation to serum markers for bone remodeling, radiography and patient response to therapy. The importance of BMP signaling in the development of sclerotic metastasis lesions deserves further exploration.