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Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.
Prostate cancer (PCa) is the second most commonly diagnosed cancer type and the fifth leading cause of cancer death in men worldwide1. Androgen deprivation therapy has been used to hamper tumor growth due to the hormone sensitivity of the prostate. However, a subset of tumors will acquire resistance and reoccur as castration-resistant prostate cancer (CRPC). Novel classes of androgen inhibitors, such as enzalutamide2 and abiraterone3, are used for CRPC cases, however acquisition of resistance and intratumor heterogeneity limits their efficiency, thus compelling the use of drugs with different mechanisms of action. The lack of available experimental models of early stage, treatment-naive PCa is a major restriction in preclinical PCa research.
Patient-derived xenografts (PDX) are used to address intra-tumor characteristics and drug response since they model the original tumor in a more representative manner than other models such as two-dimensional cell culture4. Various PDX study programs have evaluated the tumor take of various PDX models of primary and metastatic PCa5,6,7, with the use of different immunocompromised mouse strains, sites of implantations (subrenal8, subcutaneous9, orthotopic5, intrafemoral10) and grafted material (biopsies, cells, circulatory tumor cells11 and patient-derived organoids (PDOs)9,12). Cohorts of available PCa PDXs have expanded in recent years13,14,15. Overall, tumor take proved to be consistently higher for PDXs from advanced and therapy-resistant metastatic cases than for primary PCa, thus underrepresenting early stage cases that can reflect disease progression events5,16,17.
Tumor-derived organoids recapitulate features of naturally occurring tumors such as cellular phenotype, heterogeneity, drug response, and overall complexity more efficiently than 2D cell lines, while providing an alternative to animal models18,19. Several studies have demonstrated that drug response in organoids correlates with concomitant genomic profile and may predict clinical outcome9,20,21. Large scale drug screening on primary PCa organoids have not been yet performed, in contrast to CRPC neuroendocrine PCa12, mainly due to the low proliferation rate of PCa organoids and limited availability of material (e.g. needle biopsies). The extent to which PCa PDX and organoids can model key features of therapy resistance and drug response remains unclear.
In this study, we describe the development of a PDX model derived from a treatment-naïve soft tissue metastasis (PNPCa), with androgen-sensitive characteristics. Molecular characterization revealed distinctive genomic features including CHD1 and BRCA2 mutations as well as high microsatellite instability (MSI-H). To assess whether therapy resistance preexists in this treatment-naïve PCa case, we developed a method for organoid derivation that facilitates in vitro immunological assays and drug screening. Using PDX organoids from three different models, we established a pipeline for medium-throughput organoid drug screen and implemented the use of a clinically relevant, near-patient in vitro tool.
Establishment of clinically relevant models for human PCa
We have generated a PDX model from soft tissue metastasis maintained in immunodeficient mice (PNmet) (Fig. 1a). Tissue morphology (Fig. 1b, Supplementary Fig. 1a), presence of luminal markers PSA, NKX3.1, AR, CK8 expression, and absence of CK5+ basal cells (Fig. 1b, Supplementary Fig. 1b–d) indicated stable luminal epithelial morphology among the primary TUR-P tumor (T1), the PNmet, and PDX1–6 passages. Presence of prostatic ducts was observed in the primary T1, PNmet, and PDXs, with a more distinctive ductal morphology in the PDXs (Fig. 1b, Supplementary Fig. 1a). The PDX was established in two different immunocompromised strains, with and without testosterone supplementation (Supplementary Fig. 1e). Flow cytometric analysis showed that PNPCa PDX cells are positive for prostate-specific membrane antigen (PSMA), E-cadherin (E-Cad), and Integrin α-6 (CD49f), supporting the epithelial and prostatic origin of the tissue. In addition, 37% of cells were CD44+, while a minor fraction stained positively for CD36 (6%) and CD146 (1%) (Fig. 1c).
Tumor growth properties in response to androgen levels, were addressed in vivo. The PDX (passage 6) was implanted subcutaneously and received weekly testosterone injections, reaching ~1000 mm3 volume by day 67 (Fig. 1d, Intact + Testost, black line). Surgical castration resulted in progressive tumor regression, reaching non-palpable tumors by day 151 (Fig. 1d, Castration, red line). Spontaneous, androgen-independent tumor regrowth was not detected in castrated mice (up to 206 days post-castration, Fig. 1d, day 273), including a setting of prolonged androgen deprivation (up to 289 days post-castration; Supplementary Fig. 2a, Prolonged Castrated + testost, blue line). Testosterone supplementation consistently induced tumor regrowth in all castrated groups, with statistically significant tumor burden after 42 days (Fig. 1d, Castrated + Testosterone, green line; day 231 p = 0.0005 (***), day 238 onwards p ˂ 0.0001 (****); Supplementary Table 1) and after 49 days (Supplementary Fig. 2a, Prolonged Castrated + Testost, day 392 and 399 p = 0.0161, day 403 p = 0.0018) of androgen replacement, in the castrated and prolonged castration groups, respectively.
At endpoint, tumors were subjected to histological and transcriptomic analysis. Castration-induced reduction of epithelial NKX3.1+, CK8+, AR+ glands (Fig. 1e; Castrated), which was reversible upon testosterone administration (Fig. 1e; Castrated + Testost). Castrated and androgen-replaced tumors were genomically homogeneous as they shared the majority of mutations with the intact tumors (Fig. 1f, Supplementary Data 1). Whereas, after prolonged castration, testosterone-replaced tumors acquired additional mutations in JAK1 (p.Ala723Ser), AR (p.Thr878Ala), and RET (p.Ser891Leu) genes (Supplementary Fig. 2b, Supplementary Data 1). Analysis of different organs (liver, lung, prostate, lymph node, femur, and tibia) at end point indicated macroscopic foci on all lung tissues (Supplementary Fig. 2c), loss of normal epithelial architecture in anterior prostate (Supplementary Fig. 2d), human panCK cell infiltration of a lymph node in one case (Supplementary Fig. 2d) and potential micrometastases areas of scattered panCK+ (human specific) cells residing in the bone (Supplementary Fig. 2e), although no apparent lesion was detectable by X-ray (Supplementary Fig. 2f).
Principal component analysis (PCA) and unsupervised hierarchical cluster analysis of RNASeq data (Fig. 1g and Supplementary Fig. 3a) indicated high transcriptomic correlation among tumors from intact and testosterone-replaced hosts, while tumors from castrated hosts further diverged from the aforementioned groups (Fig. 1g). Differential expression analysis revealed lower expression of genes in metabolic pathways, mTOR, MYC, and AR pathways in the Castrated group compared to the Intact tumor groups (Fig. 1h). In particular, cluster analysis of AR pathway showed the differential expression of multiple genes in the castrated group, compared to both the intact and the androgen-replaced groups (Supplementary Fig. 3b). The latter groups shared similar transcriptomic profile and androgen sensitivity, however, pathway analysis indicated that tumors from androgen-replaced hosts had significant downregulation of cell cycle, apoptosis and hypoxia-related pathways (NES ≤ −1), and upregulation of interferon response, protein secretion and androgen response pathways (NES ≥ 1) (Supplementary Fig. 3c).
Androgen ablation by castration caused tumor regression, whereas testosterone re-administration induced tumor regrowth, rendering the PNPCa an androgen-dependent, treatment-naïve model.
Molecular analysis revealed genomic and transcriptomic stability among the PCa xenograft and organoid models
To further explore the in vitro characteristics of the PDX tumor cells, we developed an organoid culture method from bulk tumor tissue that allows organoids to grow in suspension conditions, with no requirement for extracellular matrix support (e.g. Matrigel). Two previously established bone-metastatic PCa PDX models, BM1822 and LAPC923 were used for direct comparison with the PNPCa, representing respectively, androgen-sensitive and androgen-independent, advanced PCa models (Supplementary Fig. 4a, b). Organoids derived from all investigated PDXs displayed budding acinar and adenocarcinoma-like morphology, with the expression of the luminal markers CK8, PSA and AR (Fig. 2a). Gene expression of basal markers (CD49f/ITGA6, KRT5, KRT6) was detected but less abundant than luminal ones (NKX3.1, AR, CK18) in all organoid and PDX models (Supplementary Fig. 5a). Tumorigenic potential of BM18 and LAPC9 organoids was confirmed by subrenal grafting (Supplementary Fig. 4c–h) or intraprostatic inoculation (Supplementary Fig. 4i–n). Organoid viability was enhanced when cultured in dihydrotestosterone (DHT)-supplemented organoid media (Fig. 2b).
Next, we analyzed the transcriptomic and genomic profiles of PDX organoids and matched tissues, assessing its stability across multiple samples (Supplementary Fig. 5b). PCA indicated that the three PDX models were clearly distinct, while the matched PDX tissues and organoids were highly similar (Fig. 2c). This was further demonstrated by the high gene expression correlation scores in PDX tissues and organoids, generated using the developed culture system (Fig. 2d).
We next performed gene set enrichment analysis (GSEA) using Hallmark and KEGG (C2 subset) gene sets (Supplementary Fig. 6, log2FC (NES) ≥ | 1 |, FDR > 0.05). In all samples compared, we found a significant upregulation of pathways involved in cell growth (E2F targets, G2M checkpoint, DNA repair mechanisms, MYC targets), metabolic activity (amino acid and fatty acid metabolism, oxidative phosphorylation, protein secretion) and androgen-regulated pathways (androgen response, spermatogenesis). Downregulated pathways included epithelial plasticity (epithelial–mesenchymal transition, adhesion molecules regulation), angiogenesis (angiogenesis, coagulation) and cellular interactions with immune system (antigen presentation, interferons, and cytokines pathways).
We then assessed the genomic landscape of PNPCa PDX model by whole exome sequencing (WES) (Fig. 2e, Supplementary Figs. 5b, 7a, b, Supplementary Data 2, Supplementary Table 2). Copy-number analysis showed that PDX-derived organoids retained the overall patterns of copy number alterations (CNAs) of tissues (Supplementary Fig. 8a). Of the total somatic non-synonymous mutations found, 51% were overlapping between all samples (Supplementary Fig. 8b, Supplementary Data 2). WES was additionally performed on the LAPC9 and BM18 PDXs and organoids, which recapitulated the majority of the somatic mutations detected in the matched PDX tissues and organoids (Fig. 2e, Supplementary Fig. 7c, d, Supplementary Data 2, Supplementary Table 2). Frameshift mutations of high impact detected in the BM18 model were in ATM, ZHFX3, and in the chromatin modifiers KDM6A and ARID1A genes (Fig. 2e). LAPC9 PDX returned an overall higher mutational burden, which included a NKX3.1 deletion, along with frameshift mutations in TP53, TMPRSS2, and PI3K and in genes of the Wnt pathway (APC, CTNNB1) (Fig. 2e). Nearly all non-synonymous somatic mutations in bona fide cancer genes were preserved in the PNPCa models, including truncating mutations in CHD1, ACVR2A, RNF43, APC, and BRCA2 (Fig. 2e, Suppementary Fig. 7a). Three frameshift mutations in cancer genes were lost in the PDX tumors: HGF, SPEN, and PIK3CG (Fig. 2e, Supplementary Fig. 7a). No mutations in the AR gene were identified, however mutations in the AR pathway-associated gene UBE3A were detected, while mutations in PMEPA1 and KDM3A were observed sporadically (Supplementary Fig. 7b). We determined the cancer cell fraction (CCF) of the reported mutations to determine their stability over PDX passaging and organoid generation. Mutations with homogeneously high prevalence in all samples (≥80% CCF) were found in CHD1, APC, RNF43, and KMT2D (Supplementary Fig. 9). BRCA2 and ACVR2A mutations were found in 60–90% CCF in P2, P3, P4, and Org2 and in ≤20% CCF of T1 (Supplementary Fig. 9). We next analysed the mutational variation of the sequenced PNPCa samples by performing clonal analysis. Different clone dynamics were detected for the most abundant clones and are reported in Fig. 2f. Of relevance, CHD1 mutation was shared among all subclones while two main subclones captured discrete subsets of truncal mutations from the original primary tumor (T1) and stably transmitted across PDX passages and organoid samples.
We compared gene expression of PNPCa samples to that of genetically defined subgroups within the Cancer Genome Atlas (TCGA). The PCA plot reports the variance of PCa cases with CHD1 homozygous deletion (Supplementary Fig. 10a) or with mutant FOXA1, SPOP, CHD1, ETS rearrangements (ERG, ETV1, ETV4) (Supplementary Fig. 10b). We further cross-compared those samples for their expression of genesets specific of different signatures of PCa subtypes using single-sample gene-set enrichment analysis (ssGSEA). While the signatures of ETS and SPOP/FOXA1 subgroups were divergent from each other, all PNPCa samples, clustered between the two categories (Supplementary Fig. 10c) and closely to CHD1 homozygous-deletion group (Supplementary Fig. 10a), in agreement with presence of a truncating CHD1 mutation (Fig. 2d).
Additionally, we used transcriptomic analysis to compare the PNPCa PDX to a subcohort of the LuCaP PDXs series13. Unsupervised hierarchical cluster analysis placed PNPCa closely with LuCaP-78 which, despite being classified as androgen-sensitive adenocarcinoma-like PCa, was derived from a CRPC patient already exposed to different lines of treatment, including taxanes (Supplementary Fig. 10d). From a genomic standpoint, LuCaP-23.1 and 145.2 contain heterozygous loss of BRCA2, a trait associated with progression to CRPC24, while only LuCaP-147 is reported to have a hypermutation profile associated to MSI-H25. Thus, PNPCa PDX represents a model of treatment-naïve, early-stage advanced disease, recapitulating features commonly found in more progressed stages of PCa.
Functional testing of targeted treatments according to the genomic profile of the PNPCa PDX and organoids
We next characterized the functional implications of mutations in genes linked to DNA stability, detected in the PNPCa models. In vitro viability assays on irradiated PNPCa organoids showed a significant reduction of viability already after 48 h compared to control PNPCa organoids (Fig. 3a). Conversely, no difference in viability was evident in irradiated organoids derived from BM18 or LAPC9, confirming the sensitivity of PNPCa to irradiation in this experimental setup (Fig. 3a–c).
The mutational landscape of PNPCa primary tumor, PDX and organoids was largely driven by mutational processes associated with signatures of MSI (Fig. 3d), consistent with the observations that this tumor had a higher mutation rate compared to other PCa26, especially to those with an overall flat copy number profile (Supplementary Fig. 8a) and an elevated proportion of small indels27 (Supplementary Fig. 11a). We did not observe genomic markers indicative of homologous recombination deficiency, neither in terms of mutational signature (Fig. 3e) nor of large-scale transitions28. The MSI status was further evaluated using the MSIsensor algorithm29 and the Bethesda MSI test. MSIsensor classified all the samples except the T1 tumor as MSI-H (Fig. 3e, gray dotted line). Four out of the six loci of the Bethesda panel were altered in the T1 tumor, confirming the tumor itself as MSI-H (Supplementary Fig. 11b).
Considering that MSI score/defective mismatch repair (dMMR) mechanism are used as biomarkers for PD-L1/PD-1 immunotherapy response30,31, and correlate with PD-L1 overexpression32, we assessed the expression level of immune-related markers and the overall immunomodulatory properties of PNPCa organoids. Compared to the normal tissue N1, all the PDXs, Org2, and the primary tumor showed a reduction of transcript abundance of the major histocompatibility complex (HLA-A and HLA-B) and of galectin-9, the main ligand of the inhibitory receptor Tim-3. While the primary tumor, the soft tissue metastasis, and the PDX-derived organoids showed a moderate, epithelial-specific staining for PD-L1, PDX1, and PDX2 tissues revealed a loss of expression of this marker in the epithelial compartment (Fig. 3f). The increased level of expression of PD-L1 in the primary tumor compared to healthy tissue N1 was not preserved in the PDX and in the organoid samples (Supplementary Fig. 11c, d).
We further assessed the expression of immune checkpoint inhibitors after 48 h stimulation of PNPCa organoids with 50 ng/ml IFN-γ. Treatment with IFN-γ was sufficient to significantly evoke the upregulation of PD-L1 (p < 0.001) and galectin-9 (p = 0.0011), linked to immune evasion (Fig. 3g, Supplementary Fig. 11e)33,34. However, as the molecular and histological data could not provide a unitary picture for these markers, we further investigated their functional role by co-culturing PNPCa organoids with allogeneic CD3+ lymphocytes and mature dendritic cells (mDC). Despite the upregulation of immune checkpoint inhibitors at the molecular level, PNPCa organoids did not modulate the proliferation, Treg polarization or PD-1 expression of CD3+ lymphocytes, even after 48 h pre-treatment with IFN-γ (Fig. 3h–j). Overall, the current results do not support the presence of an active immune evasion machinery in PNPCa organoids.
Organoid drug response to standard-of-care and repurposing of FDA-approved compounds on a medium-throughput automated screen
We next implemented the PDX-derived PCa organoid in a medium-throughput automated drug screening pipeline. A total of 74 compounds, including routinely used PCa standard-of-care drugs as well as different FDA-approved drugs with indications for other cancer types were assayed, at multiple concentrations (Supplementary Data 3). Overall, the tested compounds targeted several distinct cellular processes and pathways, with a specific focus on signaling mediators including growth factor receptors and androgen response.
Pre-screens were run for every PDX-derived organoid model to optimize cell density, positive controls and time of drug exposure, according to a pipeline established using PNPCa organoids (Supplementary Fig. 12a, b). Screens were run in at least three independent replicates for each PDX model, validating each replicate before data integration (Supplementary Fig. 12c, d). Once automatically seeded, organoids were allowed to form for 48 h before adding the drugs; cell viability was assessed on-plate after 48–72 h from initial drugs exposure (Fig. 4a).
PNPCa organoids showed resistance to the majority of the tested drugs, however 14 drug compounds significantly reduced their viability (Fig. 4b, Table 1, FDR < 0.05). The most effective compounds targeted the AR pathway (enzalutamide), EGFR/HER2 (afatinib, erlotinib, gefitinib), mTOR (rapamycin, temsirolimus), DNA replication (doxorubicin, daunorubicin, and epirubicin), multiple tyrosine kinase pathways (sorafenib, ponatinib, sunitinib), c-Met (crizotinib), and Src (bosutinib). Performing the drug screen on multiple PCa PDX-derived organoid models identified drugs that were both exclusively effective in only one of the models as well as drugs broadly effective across all tested PDX-derived organoids (Fig. 4b, Supplementary Fig. 13). As expected, the amount of significant hits inversely correlated with tumor aggressiveness. In particular, the androgen-sensitive BM18 showed a specific sensitivity to taxanes, while the castration-resistant LAPC9 to mTOR inhibition by everolimus. All models were sensitive to the tested anthracyclines (doxorubicin, daunorubicin, and epirubicin) targeting DNA replication and to the tyrosine kinase inhibitor ponatinib.
We next performed targeted pathway analysis to elucidate if the pathways targeted by the screened drugs (e.g. mTOR, cell cycle, AR, TKI targets; EGFR, FGFR, Jak/STAT, Src, PDGFRB) were enriched in the PDX tissues as well as in their derived organoids. A curated list of Hallmark and KEGG C2 pathways significantly enriched in the PDX tissues and organoids is reported in Fig. 4c. Differential expression and fGSEA analysis indicated a significant upregulation of pathways linked to AR signaling, DNA repair, mTOR signaling, and cell cycle progression across all PDX models and organoids, compared to normal tissue (Fig. 4c, pathways with NES ≥ 1). Overall, pathway analysis supported the results of drug screening on organoids and highlighted the molecular correlation of tissue/organoids on pharmacologically relevant pathways.
In order to validate the drug candidates, we performed drug screens on ex vivo tissue slices from PNPCa, BM18, and LAPC9 PDX. We tested 13 compounds on PNPCa tissue slices including 12 of the effective compounds and docetaxel, which did not significantly affect organoid viability in the PNPCa drug screen. Of these, we were able to validate 11 out of the 13 tested compounds (Supplementay Fig. 14a, b). We then tested a selection of drugs, both effective and ineffective in the organoid screening, on ex vivo tissue slices of LAPC9 and BM18 PDX. We validated the effectiveness of 7 out of 11 tested compounds on LAPC9 PDX and of 4 out of 5 tested compounds on BM18 PDX (Supplementary Fig. 14c). Overall, the effective compounds identified were part of three drug classes: anthracyclines, mTOR inhibitors, and TKIs.
Among the multiple TKIs effective on the different organoid models tested (Fig. 4b), ponatinib showed a clear dose-dependent effect, both in the organoid and in ex vivo tissue slice assay, while it has not been previously studied in the context of PCa. We validated the in vivo efficacy of ponatinib on the most aggressive PDX, LAPC9. The vehicle formulation was optimized for intraperitoneal (IP) injection and matched the solubility profile of vehicle DMSO (Supplementary Fig. 15a, b). Mice treated with ponatinib had a significantly lower tumor burden compared to controls already after 18 days from PDX implantation, resulting in significantly lower tumor weight and volume in the treated group at the end of the experiment (Fig. 4d, Supplementary Fig. 15c, d). Moreover, treatment with ponatinib reduced the average mouse weight loss seen in the control group and likely ascribable to the effects of PDX growth (Supplementary Fig. 15e). Histological morphology and quantification of Ki67 indicated no significant treatment-dependent differences in proliferation rates of the LAPC9 xenografts (Supplementary Fig. 15f, g). The concordance of the in vivo data with the ex vivo and in vitro drug screens is supporting the feasibility of an organoid-based drug assay as a surrogate tool for in vivo response, therefore we further investigated the efficacy of the identified compounds on patient-derived material.
Defining a drug panel for therapy resistant PCa (PDXs and patient-derived material) for routine organoid screens and treatment decision
In order to develop a precision medicine approach for PCa patients, we established PDOs from needle biopsies of radical prostatectomy and metastatic specimens. As a control for PDO formation efficiency, we sampled areas macroscopically unaffected by cancer in radical prostatectomy specimens, based on the evaluation of board-certified pathologists. On average, the matched control tissues formed fewer organoids compared to samples from malignant PCa (Fig. 5a, “benign” and “tumor”). PCa PDOs showed two main morphological phenotypes in vitro: organoids with a more acinar or cystic morphology and organoids with an adenocarcinoma-like phenotype (Fig. 5a, “acinar” and “adenocarcinoma”). Although PDO cultures showed both inter- and intra-patient morphological heterogeneity, each PDO culture showed consistent morphology across passages. Tumorigenic potential of PCa organoids was assessed by in vivo intraprostatic injections (Supplementary Fig. 16, representative cases).
Matched patients’ blood, bioptic tissue, and PDOs were subjected to targeted RNA and DNA sequencing using a panel of clinically relevant cancer-related genes and according to specimen abundance (Fig. 5b–d, Supplementary Table 3). The demographic characteristics of this cohort are included in Supplementary Table 4. In 54.5% of cases, the somatic mutations identified in PDOs were matching those identified in the matched tissue (Fig. 5b, purple), in 27.3% of cases no genomic concordance/mutations in the organoids were found (Fig. 5b, dark gray), and in 18.2% of the cases no mutation was identified in both the organoids and the original tissue (Fig. 5b, light gray), possibly due to sampling a no-tumor or low-tumor area of the prostate. A binary heatmap plot details the different mutations detected in the analyzed samples (Fig. 5c). Comparison of transcriptomic profile of PCa bioptic tissues with matched PDOs resulted in high correlation in the cases evaluated (Fig. 5d, cases P61 and P62). Moreover, the analysis confirmed the prevalence of luminal over basal markers in the analyzed PDOs (Supplementary Fig. 17).
Of the effective drugs short-listed from the PDX-derived organoids drug screening, we selected a panel of 13 most effective compounds (based on statistical significance), together with 4 PCa standard-of-care compounds, to develop an in vitro, multi-drug, PDO-based precision medicine assay. PDOs from three advanced PCa cases as well as from two primary PCa cases were screened with the assay (Fig. 5e). Confirmation of tumor content was done either by genomic sequencing (Fig. 5c) or after histopathological evaluation by certified pathologist. Normalized viability scores for each assay are reported on the heatmap in Fig. 5e and unsupervised hierarchical cluster analysis identified compound classes with differential effectiveness. AR-interfering drugs (abiraterone and enzalutamide) and docetaxel clustered together with the controls, indicating overall a low efficacy on PDOs. Drugs targeting mTOR (rapamycin, temsirolimus, everolimus) and EGFR-targeting erlotinib significantly reduced viability of PDOs only in some cases, showing differential efficacy. A third group, consisting of anthracyclines, the mTKIs ponatinib, bosutinib, and sunitinib, together with crizotinib, were broadly effective, reducing PDO viability in most cases. From a clinical standpoint, PDOs from patients receiving in their treatment lines taxanes (case P89) or ADT (cases P80, P82, P89, P133) confirmed resistance to these compounds in vitro with the exception of cases P82 and P134, which were sensitive and resistant, respectively, to enzalutamide despite undergoing (case P89) or a lack of ADT treatment (case P134). Although larger sample size would increase statistical robustness, this proof-of-principle data endorses the applicability of a near-patient PDO-based approach for personalized drug screening for PCa patients.
In this study, we describe the establishment of a PCa PDX model, from an early onset and treatment-naïve patient, representing a critical stage of PCa between initial relapse and CRPC development. We established organoid cultures from this and from more advanced PCa PDXs (BM18, LAPC9), to develop an organoid-based drug screening pipeline. We then further adapted organoid cultures to patient-derived bioptic material, implementing a clinically relevant, patient-tailored organoids drug screening assay.
PCa is generally a slow proliferating tumor with few key mutations and genetic alterations commonly found in patients such as TMPRSS-ERG, SPOP, FOXA1, PTEN35,36. SPOP is associated with DNA repair errors and a higher number of genomic rearrangements37. Although no SPOP mutation was identified in the PNPCa, there was a higher transcriptomic correlation with SPOP-mutated cases compared to other subtypes and specifically to the ERG-mutated ones: as characterized in previous studies, SPOP mutations and ERG fusion are fundamentally mutually exclusive38,39. Nevertheless, PNPCa PDX carries an inactivating mutation in CHD1, an event frequently co-occuring with SPOP mutations40 (15% of PCa cases show loss of heterozygosity for CHD1) and driving PCa-specific growth in transgenic mice41.
The PNPCa model displays an heterozygous frameshift mutation in the BRCA2 gene and high MSI, combining in one model two of the four genomic subtypes of metastatic CRPC26. Patients with germline BRCA2 defects have earlier disease onset, a higher rate of 5-year metastatic progression and poor survival compared to non-carriers42,43,44. PNPCa organoids exhibited sensitivity to irradiation, a clinical treatment for patients harboring BRCA2 mutations42. Tumors with BRCA1/2 defects and defective DNA repair mechanism are particularly sensitive to DNA damaging agents such as radiotherapy45 and to PARP inhibitors such as olaparib, in use for breast and ovarian cancer46 and currently in a phase III clinical trial for CRPC patients harboring BRCA1/2 or ATM mutations (PROfound, NCT02987543).
To our knowledge, PNPCa is the only reported MSI-H case of an early metastasis, retaining androgen sensitivity that has been modeled in vivo. Hypermutation and MSI are rare and sporadic features of PCa47 that are associated with hereditary cancer predisposition. Despite the high tumor mutational burden (18–20 mut/MB), no deleterious alterations were detected in key MMR genes MSH2, MSH6, MLH1, or PMS2 in PNPCa samples. Albeit rare, MSI-H features lacking a dMMR signature have been clinically observed30,48. Other molecular mechanisms, such as silencing of MLH1 gene promoter or other epigenetic regulations may be responsible for the MSI-H status49,50.
In PCa, high MSI is associated with poorly differentiated stage51,52 ranging from 1% of primary tumor cases to up to 12% metastatic cases53. Among the patients with defined MSI-H/dMMR molecular phenotype, ~50% respond to anti-PD-1/PD-L1 immunotherapy54. Given the lack of biomarkers for CRPC and the higher MSI prevalence in metastatic cases compared to primary cases, screening of patients for MSI status during initial diagnosis could determine whether anti-PD-1 treatment is the optimum treatment option. However, the lack of pressure of an immune system, common in both in vitro cultures and PDX passaging, could mask the pathophysiological role of this axis.
Organoid medium composition and overall cultural techniques were adapted from well-established and previously characterized studies55,56, coherently with other recent research studies in the field12,57. However, in order to develop a translational assay, we eliminated extracellular matrix components to; increase drug availability, facilitate drug screening throughput and elimination of stromal cells. A drawback of this approach is that in our established methodology, organoids were generally used within a few passages. The preservation of high molecular correlation between PDX tissue and PDX-derived organoids was preferred over the opportunity to perform prolonged serial organoids propagation. Moreover, the predominant luminal phenotype observed in these PDX tumors was maintained in the organoid culture models.
We cross-validated drug responses in a complementary ex vivo assay on tissue slices, derived from the established PDXs based on our previously developed methodology58. The findings from both assays were similar, however organoid drug screenings are more amenable to standardization and can be performed routinely, in a shorter time frame and using limited amount of tissue from patient-derived material.
The implementation of PCa organoids in translational assays could facilitate the identification of effective therapies in non-responders to anti-androgens or chemotherapy, especially if repurposing drugs approved for other malignancies59. Differential responses to AR-blockers in LAPC9 and BM18/PNPCa organoids suggests that drug response correlates with individual tumor phenotypes. PNPCa was the only model showing sensitivity to EGFR-inhibitors: deregulation of EGFR signaling is found in a subset of PCa cases, however EGFR inhibitors have showed limited effectiveness60,61. Among the TKI inhibitors identified in our screen, ponatinib emerged as broadly effective in metastatic PCa PDX as well as in PDOs. While sorafenib and sunitinib have been tested in phase II/III clinical trials for CRPC62,63, ponatinib has not been yet investigated in PCa. Interestingly, both sorafenib and ponatinib were identified as CRPC candidate compounds based on a computational gene expression tool for drug identification64. and while its use in solid tumors is being currently investigated65, it is approved mainly for patients with acute or chronic leukemia (PACE Trial66) and with acquired resistance to other TKIs67. When ultimately validated in vivo, ponatinib resulted in significant tumor growth inhibition and a very good tolerability for the duration of the experiment. These results corroborate the potential of organoids-based assays on drug screening applications in PCa.
We further developed a patient-derived organoid (PDOs)-based drug screening on a limited cohort of PCa patients, resulting in a descriptive, proof-of-principle study. PDOs recapitulated in most cases the genomic alteration of the matching tissues, both in metastatic and in primary lesions. In some cases, however we could not detect mutations in the PDOs or in both PDOs and matching tissues. This could be due to the low (or no) tumor content of the initial biopsy as well as to oncogenic mutations in regions outside those covered by our targeted DNA analysis. The developed PDO-based drug assay has an average duration of two weeks from initial organoid formation until readout, a time frame compatible with clinical decision-making. Individual inter-patient drug-sensitivity profiles, indicating the most effective compounds, even within the same drug subclass, are suggested by the results of the personalized drug screenings. Addressing intra-patient reproducibility was hampered due to the limited amount of material and of matching tumor-adjacent tissue available. In line with the high heterogeneity of PCa, the drug profiles correlated with disease stage, however they did not always match with the clinical history of the patient, highlighting the need to implement a near-patient assay. Our results highlight the applicability of patient-derived organoid drug screenings to predict clinical outcome21 and their correlation with genomic and transcriptomic features of the primary tumor, as shown in recent studies for lung68, gastrointestinal cancer21, hepatocellular carcinoma69, and ovarian cancer70.
In summary, we presented a translational pipeline designed by cross-platform analysis of an early onset and treatment-naïve PCa xenograft model. The specific biologic and genetic landscape of this model may provide insights into tumor growth, metastasis, and drug resistance profile at an earlier stage of the disease. Comparison of its drug response profile with those of more advanced PCa PDXs allowed the generation of a highly translational tool for the evaluation of drug response in PDO, thus supporting a precision medicine approach to clinical decision-making.
The established PDX was originated from a patient who presented with primary PCa (Gleason 9) and underwent transurethral resection of the prostate (TUR-P) procedure. After 6 months, biopsy sampling was performed and the patient was diagnosed with a soft tissue metastasis; histopathology showed an infiltration of an adenocarcinoma from the prostate, PSA 91 ng/ml. Orchiectomy was performed directly after biopsy sampling, thus the tumor was androgen-dependent at the time of collection. No biochemical relapse was observed up to 18 months since the diagnosis (PSA < 1 ng/ml). All patients included in this study provided written informed consent (Cantonal Ethical approval KEK 06/03 and 2017-02295).
Tumor sample preparation and xenograft surgery procedure
Needle biopsy from soft tissue metastasis was collected in Dulbecco’s MEM (Gibco, 61965-026) media containing primocin (InVivoGen). For xenograft implantation, needle biopsies were implanted subcutaneously in male, NOD-scid IL2Rgammanull (NSG) mouse, under aneasthesia (Domitor® 0.5 mg/kg, Dormicum 5 mg/kg, Fentanyl 0.05 mg/kg). Animal license BE 55/16 and BE 68/20. Weekly subcutaneous injections of testosterone propionate dissolved in castor oil (Sigma, 86541-5 G) were performed (2 mg per dosage, 25 G needle) starting 1 week after the surgery. For PDX passaging, serial subcutaneous implantations of tumor pieces into new recipients (NSG or CB17 SCID mice) was performed. Abiraterone acetate (Selleckchem, S2246) treatment was administered once daily (i.p.) for 5 days per week over a duration of 4 weeks at 0.5 mmol/kg/d (5% benzyl alcohol and followed by 95% safflower oil solution).
DNA isolation from organoids and tissue samples
For DNA extraction from organoids and tissue samples the DNeasy Blood and tissue kit (Qiagen, 69504) was used. DNA from FFPE material was extracted Maxwell® 16 LEV RNA FFPE Purification Kit (Promega, AS1260).
RNA isolation and RT-qPCR
RNA isolation from organoids was performed using the PicoPure Arcturus (Thermo Scientific, KIT0204) kit method. Tissue RNA was extracted using standard protocol of Qiazol (Qiagen) tissue lysis by TissueLyser (2 min, 20 Hz). Quality of RNA was assessed by Bioanalyzer (Agilent). RNA from FFPE material was extracted using the Maxwell® 16 LEV RNA FFPE Purification Kit (Promega, AS1260). Total RNA was used for cDNA synthesis using random primers and RNAse H-MML-V reverse transcriptase first-strand cDNA synthesis system (Promega AG, Dübendorf, Switzerland). For qPCR, cDNA (10 ng per reaction) was amplified in a CFX Real Time Detection system (Bio-Rad, Cressier, Switzerland) using SYBR Green Supermix reagent (Bio-Rad). Expression levels were normalized to the transcripts of HPRT and ACTB. Primer sequences are indicated in Supplementary Table 5.
Tissue dissociation and organoid culture
Tumor tissue was collected in Basis medium (Advanced DMEM F12 Serum Free medium (Thermo Fisher Scientific, 12634010) containing 10 mM Hepes (Thermo Fisher Scientific, 15630080), 2 mM GlutaMAX supplement (Thermo Fisher Scientific, 35050061), and 100 μg/ml Primocin (InVivoGen, ant-pm-1). After mechanical disruption the tissue was washed in Basis medium (220rcf, 5 min) and incubated in enzyme mix for tissue dissociation (collagenase type II enzyme mix (Gibco, 17101-015), 5 mg/ml dissolved in Basis medium, DNase: 15 μg/ml (Roche, 10104159001) and 10 μM Y-27632-HCl Rock inhibitor (Selleckchem, S1049). Enzyme mix volume was adjusted so that the tissue volume does not exceed 1/10 of the total volume and tissue was incubated at 37 °C for 1–2 h with mixing every 20 min. After digestion of large pieces was complete, the suspension was passed through 100 μm cell strainer (Falcon®, VWR 734-0004) attached to a 50 ml Falcon tube. Using a syringe rubber the tissue was minced against the strainer and washed in 5 ml basic medium (220rcf, 5 min). Cell pellet was incubated in 5 ml precooled EC lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubated for 10 min, washed in equal volume of basis medium followed by centrifugation (220rcf, 5 min). Pellet was resuspended in 2–5 ml accutase™ (StemCell Technologies, 07920), depending on the sample amount; biopsies or tissue, and incubated for 10 min at room temperature. The cell suspension was passed through 40 μm pore size strainer (Falcon®, VWR 734-0004), and the strainer was washed by adding 2 ml of accutase on the strainer. Single cell suspension was counted to determine seeding density, washed in 5 ml of basis medium and spun down 220rcf, 5 min. Cell pellet was reconstituted in organoid medium and seeded in ultra low attachment (ULA) plates; e.g. 30,000 cells per well in 96-well plates with 100 μl media, 100,000 cells per well of 24-well plate with 750 μl media, 300,000–500,000 cells per well of 6-well plate (2 ml) (Corning, Costar #3471, 3473, 3474). Organoid culture media contains the following reagents: Basis medium containing 10 μM Y-27632-HCl (Selleckchem, S1049), 5% fetal calf serum (Gibco #10270-106, LOT 42G7277K), 1× B-27 supplement (Thermo Fisher Scientific, 17504044), 10 mM Nicotinamide (Sigma, N0636 100G), 500 ng/ml Rspondin (Peprotech, 120-38), 1.25 mM N-acetyl-cysteine (Sigma, A9165), 10 μM SB202190 (Selleckchem, S1077), 100 ng/ml Noggin (Peprotech, 250-38), 500 nM A83-01 (Tocris, 2939), 10 nM DHT (Fluka Chemica, 10300), 10 ng/ml Wnt3a (Peprotech, 315-20), 50 ng/ml HGF (Peprotech, 100-39), 50 ng/ml EGF (Peprotech, AF-100-15), 10 ng/ml FGF10 (Peprotech, 100-26), 1 ng/ml FGF2 (Peprotech, 100-18B), 1 μM PGE2 (Tocris, 2296). Media is prepared and kept at 4 °C for no longer than 7 days.
Medium-throughput organoid drug screen at NEXUS personalized health technologies automation platform
A drug library was compiled based on predicted activity against PCa (Selleck Chemicals, Houston, TX, USA) as 96-well format sample storage tubes with drugs in 10 mM concentration. Using a Tecan EVO 100 (Tecan AG, Männedorf, Switzerland), this drug library was aliquoted over 96-well plates (#651261, Greiner Bio-One, Kremsmünster, Austria) and further diluted to yield stock plates with a concentration of 10 mM, 1 mM, and 0.1 mM in DMSO (Sigma Aldrich, cat. D8418). After aliquotting, plates were sealed under argon gas using an Agilent PlateLoc (Agilent Technologies, Santa Clara CA, USA) with peelable aluminium heat-sealing foil (Agilent, cat. 24210-001) for 1 s at 170 °C. An overview of the purchased drugs and their known targets can be found in Supplementary Data 3. Control molecules enzalutamide, docetaxel, and doxorubicin were purchased at Sellechekchem (Lubio Science, Zürich, Switzerland, #S1250, #S1148, #S1208). Staurosporine was purchased at Toronto Research Chemicals (Toronto, Canada, #S685000).
Automated drug screening with PCa organoids
Automated screening procedures were performed at NEXUS Personalized Health Technologies (ETH Zürich, Zürich, Switzerland) using an automated screening platform (HighRes Biosolutions, Beverley, MA, USA). PCa organoids of BM18, LAPC9, or PNPCa origin were prepared and expanded from murine tumor tissue as for 5–7 days to allow organoid formation using Costar ultra-low attachment plates (#3471, Corning, New York, NY, USA). For the drug screens, organoids were dissociated into single cell suspension by both enzymatic (TrypLE incubation) and mechanical separation (22 G needle), counted and seeded in ULA 384 well plates at appropriate cell density for each tumor model; 3500 c/well (LAPC9, BM18) or 5000 c/well (PNPCa). Cells were seeded 25 µL per well in 384-well flat-bottom ultra-low attachment plates (#3827, Corning) using a BioTek EL406 with wide-bore 5 µL peristaltic pump tubing (BioTek Instruments Inc., Winooski, VT, USA). After cell seeding, plates were shaken for 2 min, incubated for 1 h at room temperature and subsequently transferred to a 37 °C incubator with 95% humidity and 5% CO2. 48 h after cell seeding, 96-well plates (#651261, Greiner Bio-One) containing 1000-times concentrated compound stock solutions at differing concentrations (10, 1, and 0.1 mM) in DMSO were centrifuged at 250rcf for 10 s (HiG 5000, BioNex Solutions Inc., San Jose, CA, USA) and de-sealed using a Brooks Xpeel (Brooks Life Sciences, Chelmsford, MA, USA) and subsequently diluted 1:125 in culture medium and added to quadrant 1, 2, and 3 (respectively for 10, 1, and 0.1 mM stock plates) of deepwell 384-well plates (#781271, Greiner Bio-One). DMSO-stock solutions of control molecules (100% DMSO as negative control and as positive controls we included 1000-times concentrated docetaxel [30 µM], enzalutamide [6 mM], and doxorubicin [10 mM] for LAPC9 and BM18 organoids, or staurosporine [2.5 mM] instead of docetaxel for PNPCa organoids) were added to plate quadrant 4. An additional 1:1 dilution step was done prior to adding 20 µL of diluted drugs to the cell culture plates (containing 20 µL cell suspension after correction for evaporation). Compound dilutions were performed using automated liquid handling equipment (Tecan AG, Männedorf, Switzerland) in technical triplicate (LAPC9 and BM18) or technical quadruplicate (PNPCa). A Schematic representation of the compound dilution and addition procedure is shown in Supplementary Fig. 18a. After compound exposure, compound DMSO stock plates were sealed under argon gas using an Agilent PlateLoc as described above, and organoid culture plates were transferred back to the 37 °C incubator with a 95% humidity and 5% CO2 atmosphere. 48 h after compound exposure, a CellTiter-Glo 3D assay (#G9682, Promega, Madison, WI, USA) was used to measure ATP levels as a proxy for cell viability. This assay is lytic and thus maximized readout from all cells composing large organoid structures. The cell viability readout was performed according to manufacturer’s instructions using the automation equipment. Briefly, 40 µL room-temperature CellTiter-Glo 3D reagent was added per well to the assay plates using an automated liquid handler (Tecan AG). Plates were subsequently shaken for 5 min on a BioTek EL406 and incubated in a temperature-controlled incubator at 22 °C for 25 min. After incubation, luminescence was measured using a Tecan M1000 Pro plate reader (Tecan AG) with 1000 ms integration time. Results were collected as spreadsheets and coupled to plate layouts.
Data have been normalized using the median of the negative control conditions (DMSO 0.1%) and values have been log2 transformed (each plate with its own internal negative control). For the statistical analysis the different plates have been considered as replicates. p-value and FDR were calculated for all drugs after removing DMSO and no-treated control conditions).
All unique materials are readily available upon request to the corresponding author.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
All relevant data are available from the corresponding author. Sequencing data have been deposited at the European Genome-phenome Archive (EGA), under accession number EGAS00001004673 and EGAS00001004675. RNASeq related to Supplementary Fig. 10d were deposited at the ENA (European Nucleotide Archive/Arrayexpress) under accession number E-MTAB-9656.
Ferlay, J. et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries and 25 major cancers in 2018. Eur. J. Cancer 103, 356–387 (2018).
Beer, T. M. et al. Enzalutamide in metastatic prostate cancer before chemotherapy. N. Engl. J. Med. 371, 424–433 (2014).
de Bono, J. S. et al. Abiraterone and increased survival in metastatic prostate cancer. N. Engl. J. Med. 364, 1995–2005 (2011).
Byrne, A. T. et al. Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nat. Rev. Cancer 17, 254–268 (2017).
Wang, Y. et al. Development and characterization of efficient xenograft models for benign and malignant human prostate tissue. Prostate 64, 149–159 (2005).
Wetterauer, C. et al. Early development of human lymphomas in a prostate cancer xenograft program using triple knock-out Immunocompromised mice. Prostate 75, 585–592 (2015).
Navone, N. M. et al. Movember GAP1 PDX project: an international collection of serially transplantable prostate cancer patient-derived xenograft (PDX) models. Prostate 78, 1262–1282 (2018).
Lin, D. et al. High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development. Cancer Res. 74, 1272–1283 (2014).
Pauli, C. et al. Personalized In vitro and in vivo cancer models to guide precision medicine. Cancer Discov. 7, 462–477 (2017).
Raheem, O. et al. A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions. J. Transl. Med. 9, 185 (2011).
Williams, E. S. et al. Generation of prostate cancer patient derived xenograft models from circulating tumor cells. J. Vis. Exp. 105, 53182 (2015).
Puca, L. et al. Patient derived organoids to model rare prostate cancer phenotypes. Nat. Commun. 9, 2404 (2018).
Nguyen, H. M. et al. LuCaP prostate cancer patient-derived xenografts reflect the molecular heterogeneity of advanced disease and serve as models for evaluating cancer therapeutics. Prostate 77, 654–671 (2017).
Navone, N. M. et al. Movember GAP1 PDX project: an international collection of serially transplantable prostate cancer patient-derived xenograft (PDX) models. Prostate 78, 1262–1282 (2018).
Palanisamy, N. et al. The MD Anderson Prostate Cancer Patient-derived xenograft series (MDA PCa PDX) captures the molecular landscape of prostate cancer and facilitates marker-driven therapy development. Clin. Cancer Res. 26, 4933–4946 (2020).
Priolo, C. et al. Establishment and genomic characterization of mouse xenografts of human primary prostate tumors. Am. J. Pathol. 176, 1901–1913 (2010).
Klein, K. A. et al. Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice. Nat. Med. 3, 402–408 (1997).
Kondo, J. & Inoue, M. Application of cancer organoid model for drug screening and personalized therapy. Cells 8, 470 (2019).
Bleijs, M. et al. Xenograft and organoid model systems in cancer research. EMBO J. 38, e101654 (2019).
Granat, L. M. et al. The promises and challenges of patient-derived tumor organoids in drug development and precision oncology. Anim. Models Exp. Med. 2, 150–161 (2019).
Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920–926 (2018).
McCulloch, D. R. et al. BM18: a novel androgen-dependent human prostate cancer xenograft model derived from a bone metastasis. Prostate 65, 35–43 (2005).
Craft, N. et al. Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process. Cancer Res. 59, 5030–5036 (1999).
Chakraborty, G. Significance of BRCA2 and RB1 co-loss in aggressive prostate cancer progression. Clin. Cancer Res. 26, 2047 (2020).
Pritchard, C. C. et al. Complex MSH2 and MSH6 mutations in hypermutated microsatellite unstable advanced prostate cancer. Nat. Commun. 5, 4988 (2014).
van Dessel, L. F. et al. The genomic landscape of metastatic castration-resistant prostate cancers reveals multiple distinct genotypes with potential clinical impact. Nat Commun. 10, 5251 (2019).
Alexandrov, L. B. et al. Signatures of mutational processes in human cancer. Nature 500, 415–421 (2013).
Popova, T. et al. Ploidy and large-scale genomic instability consistently identify basal-like breast carcinomas with BRCA1/2 inactivation. Cancer Res. 72, 5454–5462 (2012).
Niu, B. et al. MSIsensor: microsatellite instability detection using paired tumor-normal sequence data. Bioinformatics 30, 1015–1016 (2014).
Abida, W., et al. Analysis of the prevalence of microsatellite instability in prostate cancer and response to immune checkpoint blockade. 2018.
Barata, P. et al. Clinical activity of pembrolizumab in metastatic prostate cancer with microsatellite instability high (MSI-H) detected by circulating tumor DNA. J. Immunother. Cancer 8, e001065 (2020).
Nava Rodrigues, D. et al. Immunogenomic analyses associate immunological alterations with mismatch repair defects in prostate cancer. J. Clin. Investig. 128, 4441–4453 (2018).
Rabinovich, G. A. & Conejo-García, J. R. Shaping the immune landscape in cancer by galectin-driven regulatory pathways. J. Mol. Biol. 428, 3266–3281 (2016).
O’Donnell, J. S., Teng, M. W. L. & Smyth, M. J. Cancer immunoediting and resistance to T cell-based immunotherapy. Nat. Rev. Clin. Oncol. 16, 151–167 (2019).
Robinson, D. et al. Integrative clinical genomics of advanced prostate cancer. Cell 161, 1215–1228 (2015).
Grasso, C. S. et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239 (2012).
Boysen, G. et al. SPOP mutation leads to genomic instability in prostate cancer. Elife 4, e09207 (2015).
Cancer Genome Atlas Research, N. The molecular taxonomy of primary prostate cancer. Cell 163, 1011–1025 (2015).
Barbieri, C. E. et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat. Genet. 44, 685–689 (2012).
Rescigno, P. et al. Molecular and clinical implications of CHD1 loss and SPOP mutations in advanced prostate cancer. J. Clin. Oncol. 36, 5064–5064 (2018).
Augello, M. A. et al. CHD1 loss alters AR binding at lineage-specific enhancers and modulates distinct transcriptional programs to drive prostate tumorigenesis. Cancer Cell 35, 817–819 (2019).
Castro, E. et al. Germline BRCA mutations are associated with higher risk of nodal involvement, distant metastasis, and poor survival outcomes in prostate cancer. J. Clin. Oncol. 31, 1748–1757 (2013).
Risbridger, G. P. et al. Patient-derived xenografts reveal that intraductal carcinoma of the prostate is a prominent pathology in BRCA2 mutation carriers with prostate cancer and correlates with poor prognosis. Eur. Urol. 67, 496–503 (2015).
Taylor, R. A. et al. Germline BRCA2 mutations drive prostate cancers with distinct evolutionary trajectories. Nat. Commun. 8, 13671 (2017).
Vesprini, D. et al. The therapeutic ratio is preserved for radiotherapy or cisplatin treatment in BRCA2-mutated prostate cancers. Can. Urol. Assoc. J. = J. Assoc. Urol. Can. 5, E31–E35 (2011).
Ma, Y. et al. Response to olaparib in metastatic castration-resistant prostate cancer with germline BRCA2 mutation: a case report. BMC Med. Genet. 19, 185–185 (2018).
Prtilo, A. et al. Tissue microarray analysis of hMSH2 expression predicts outcome in men with prostate cancer. J. Urol. 174, 1814–1818 (2005). discussion 1818.
Graham, L. S. et al. Mismatch repair deficiency in metastatic prostate cancer: response to PD-1 blockade and standard therapies. PLoS ONE 15, e0233260–e0233260 (2020).
Deng, G. et al. Methylation of hMLH1 promoter correlates with the gene silencing with a region-specific manner in colorectal cancer. Br. J. Cancer 86, 574–579 (2002).
Kane, M. F. et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 57, 808–811 (1997).
Uchida, T. et al. Microsatellite instability in prostate cancer. Oncogene 10, 1019–1022 (1995).
Egawa, S. et al. Genomic instability of microsatellite repeats in prostate cancer: relationship to clinicopathological variables. Cancer Res. 55, 2418–2421 (1995).
Hempelmann, J. A. et al. Microsatellite instability in prostate cancer by PCR or next-generation sequencing. J. Immunother. Cancer 6, 29–29 (2018).
Abida, W. et al. Analysis of the prevalence of microsatellite instability in prostate cancer and response to immune checkpoint blockade. JAMA Oncol. 5, 471–478 (2019).
Gao, D. et al. Organoid cultures derived from patients with advanced prostate cancer. Cell 159, 176–187 (2014).
Drost, J. et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat. Protoc. 11, 347–358 (2016).
Beshiri, M. L. et al. A PDX/Organoid Biobank of advanced prostate cancers captures genomic and phenotypic heterogeneity for disease modeling and therapeutic screening. Clin. Cancer Res. 24, 4332–4345 (2018).
Karkampouna, S. et al. CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma. J. Pathol. 245, 297–310 (2018).
Bertolini, F., Sukhatme, V. P. & Bouche, G. Drug repurposing in oncology—patient and health systems opportunities. Nat. Rev. Clin. Oncol. 12, 732 (2015).
Sridhar, S. S. et al. A multicenter phase II clinical trial of lapatinib (GW572016) in hormonally untreated advanced prostate cancer. Am. J. Clin. Oncol. 33, 609–613 (2010).
Guerin, O. et al. EGFR targeting in hormone-refractory prostate cancer: current appraisal and prospects for treatment. Pharmacology 3, 2238–2247 (2010).
Beardsley, E. K. et al. A phase II study of sorafenib in combination with bicalutamide in patients with chemotherapy-naive castration resistant prostate cancer. Investig. New Drugs 30, 1652–1659 (2012).
Zurita, A. J. et al. Sunitinib in combination with docetaxel and prednisone in chemotherapy-naive patients with metastatic, castration-resistant prostate cancer: a phase 1/2 clinical trial. Ann. Oncol. 23, 688–694 (2012).
Kim, I. W., Kim, J. H. & Oh, J. M. Screening of drug repositioning candidates for castration resistant prostate cancer. Front. Oncol. 9, 661 (2019).
Tan, F. H. et al. Ponatinib: a novel multi-tyrosine kinase inhibitor against human malignancies. OncoTargets Ther. 12, 635–645 (2019).
Cortes, J. E. et al. Ponatinib efficacy and safety in Philadelphia chromosome-positive leukemia: final 5-year results of the phase 2 PACE trial. Blood 132, 393–404 (2018).
Sanford, D. et al. Phase II trial of ponatinib in patients with chronic myeloid leukemia resistant to one previous tyrosine kinase inhibitor. Haematologica 100, e494–e495 (2015).
Kim, M. et al. Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening. Nat. Commun. 10, 3991 (2019).
Nuciforo, S. et al. Organoid models of human liver cancers derived from tumor needle biopsies. Cell Rep. 24, 1363–1376 (2018).
Phan, N. et al. A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids. Commun. Biol. 2, 78 (2019).
We thank all patients who participated in our study, all involved clinical personnel and study nurses, in particular we would like to thank Anna-Katharina Herrmann and Sofia Bonné. We would like to acknowledge the Microscopy Facility of University of Bern, Viola Paradiso and Sina Maletti for the ion torrent sequencing. Swiss National Science Foundation 31003A_169352; 310030_189149; UniBe Initiator Grant 2016; KWF 2015_7599; Novartis 17B076; SOCIBP SPHN Driver Project (SAM).
The authors declare no competing interests.
Peer review information: Nature Communications thanks Adam Sowalsky and the other, anonymous, reviewer for their contribution to the peer review of this work. Peer reviewer reports are available.
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Karkampouna, S., La Manna, F., Benjak, A. et al. Patient-derived xenografts and organoids model therapy response in prostate cancer. Nat Commun 12, 1117 (2021). https://doi.org/10.1038/s41467-021-21300-6
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text:
Established in 2005, this festival is dedicated to various styles and techniques that make guitar one of the most versatile and influent instruments in the world. From bluegrass to blues, jazz, rock folk and acoustic fingerstyle, everything you can have from a guitar.
Location: Clonakilty Co. Cork, Ireland
The Cork Jazz Festival is one of Ireland’s most successful Arts Cultural Events and one of the top three Jazz music festivals in the world. It attracts over 40,000 visitors to Cork City each October Bank Holiday Weekend to hear over 1,000 musicians perform in concerts, clubs and pubs around the city.
Annual festival including performances, workshops, events and theatre in the Galway City Museum and in the street. Their objective is to bring the music and dances of the 12th -17th centuries alive through concerts performed by performers and bring awareness and interest to the general public of Galway’s medieval heritage.

domain: artistic


text:
The ACLU of Louisiana is calling on federal prosecutors to launch an investigation into the Jefferson Parish Sheriff’s Office following a report by WWNO/WRKF and ProPublica that revealed stark racial disparities in shootings by deputies and systemic transparency problems.
The news organizations’ ongoing investigation into the Sheriff’s Office found that more than 70% of people who deputies shot at during the past eight years were Black; that figure is more than double the 27% of the parish's population that is Black. In addition, 12 of the 16 people who died after being shot or restrained by deputies during that time were Black men.
In response to public records requests, the office could not account for how often its deputies use force. It denied a request for copies of all complaints against Sheriff’s Office employees during the past two years and couldn’t even say how many had been filed, stating such a number “does not exist.”
Alanah Odoms, the ACLU of Louisiana’s executive director, asked the U.S. Attorney’s Office for the Eastern District of Louisiana to “scrutinize the data and evidence collected” by the news organizations and to review three pending lawsuits filed by the ACLU against the Sheriff’s Office.
“It is no secret that the Jefferson Parish Sheriff’s Office has a deep-rooted history of racial discrimination and cruelty toward residents of color,” Odoms said. “The harsh political reality is the sheriff of Jefferson Parish is wholly unaccountable to the people.”
This article was co-published with ProPublica and produced for ProPublica’s Local Reporting Network in partnership with WRKF and WWNO.
The Sheriff’s Office did not immediately respond to a reporter’s voicemail or email about the ACLU statement. Sheriff Joe Lopinto previously declined to be interviewed about the news organization’s findings, saying only that when his deputies commit serious misconduct, they are arrested, noting that at least nine had been booked since he became sheriff in 2017.
The U.S. Attorney’s Office declined to comment on Odoms’ request for an investigation.
The U.S. Department of Justice, the parent agency of the U.S. Attorney’s Office, launched an investigation into the neighboring New Orleans Police Department over a decade ago and found problems similar to those identified by the news organizations’ investigation into Jefferson Parish. The NOPD had failed to properly track and review when its officers used force, its internal investigation system was deeply flawed and its officers were disproportionately shooting and killing Black people, the DOJ reported.
But, despite years of complaints by Black community leaders in Jefferson Parish, the DOJ has not intervened.
U.S. Rep. Troy Carter, D-New Orleans, called the news organizations’ findings “disturbing.”
“I do not, and our society should not, condone any violence, misconduct or excessive use of force by those sworn to protect and serve,” said Carter, who this month called for a DOJ investigation into the Louisiana State Police, which has come under heavy scrutiny recently over a pattern of violence directed at Black arrestees.
“I am hopeful that the Jefferson Parish Sheriff’s Office will use all resources available to thoroughly and transparently seek out the truth, wherever it may lead,” Carter said.

domain: news


text:
New Report: Best and Worst States for Doctors
Earlier this month, personal finance site WalletHub published a report ranking the best and worst states for physicians. They based the rankings on a variety of factors, including the cost of medical malpractice insurance, the amount of malpractice award payouts per capita in the previous year and average physician wages, as well as public health measures, like the number of state residents with health insurance and the number of hospitals in the state. Click here to view their full report.
Coming out on top was Mississippi, which had the highest wages for physicians when adjusted for cost-of-living. Mississippi is also undergoing a physician shortage, especially in rural areas, so there is plenty of demand for doctors. Other top-ranking states included Iowa (2), Minnesota (3) and North Dakota (4). Both Iowa and Minnesota were among the least expensive states for medical liability insurance, while North Dakota was number one on the list of states with the lowest malpractice award payouts per capita.
The bottom of the rankings included many states in the northeast and mid-Atlantic regions, with Washington, D.C. (51) ranking last, just ahead of New York (50), Rhode Island (49), Maryland (48) and Connecticut (47). Maryland, New York and Rhode Island were among the states with the highest malpractice award payouts per capita, while Washington, D.C. and New York had some of the most expensive medical liability insurance. Connecticut, New York and Washington, D.C. also had some of the lowest wages for physicians, when adjusted for cost-of-living.
While WalletHub’s report provides some interesting insights for physicians, to get a clearer picture of the cost of medical malpractice insurance in the 50 states and Washington, D.C., see our Historic Medical Malpractice Rate Data. Cunningham Group has partnered with the Medical Liability Monitor to publish historic rate data for all 50 states. Click here to see insurance rates for your state!

domain: geography


Going on the list of new car for 2011 by Mitsubishi
Mitsubishi Eclipse Not much changes in Mitsubishi Eclipse for this year. The ride height has been reduced by half an inch and all coupes now feature a blacked-out top in place of a body-coloured roof. Alloy wheels are now standard on the GS base model, while the GS Sport gets standard heated leather front seats and a power driver seat.
Mitsubishi Eclipse Spyder Modifications to the Eclipse Spyder represent those of the coupe model, with the exception of the blacked-out roof. The Spyder's power soft top with rear glass window and defroster continues to bury itself under the hard tonneau cover in less than 20 seconds.
Mitsubishi Endeavor This year Endeavor lists the previously optional Navigation and Sunroof package as standard equipment on SE models. Moreover it adds a DVD-based touch screen navigation system, rear-view back-up camera, power sunroof and a cargo cover.
Mitsubishi Galant Company reduced the Galant's ride height by a half inch. Extra standard features are added to the SE trim too, with a power sunroof, auto-dimming rear-view mirror, illuminated vanity mirrors and a programmable garage door opener. Leather seating is added as an SE option.
Mitsubishi Lancer This year model Lancer adds electric-assist power steering and regenerative braking to its base engine models, proposing a small improve in fuel economy, and the base model replaces the former rear disc brakes with drum brakes. Finally, the performance-oriented Lancer GTS and Ralliart editions now offer Mitsubishi's Fuse voice-activation system and a USB port.
Mitsubishi Lancer Evolution The Lancer Evolution's exhaust system has been revised for an enhanced, sportier-sounding exhaust note. Evos equipped with the available 40GB touch screen HDD-based navigation system now benefit from real-time traffic. A USB auxiliary input port has been added, as well as the Fuse system that coordinates hands-free calls on your Bluetooth-enabled cell phone, controls your iPod and allows drivers to access a USB drive by simple voice commands.
Mitsubishi Lancer Sportback Automaker lowered the Sportback ES's price by replacing the 2.4-liter four-cylinder engine found in the Lancer Sportback GTS model with a smaller 2.0-liter four-cylinder engine that produces 148 horsepower (down 20 hp from the Sportback GTS model's engine). The 2011 Lancer Sportback ES also receives Mitsubishi's electric power steering and a five-speed manual or a continuously variable transmission (CVT). Mitsubishi's Fuse voice-activation system becomes standard on the top-of-the-line GTS.
Mitsubishi Outlander The Outlander is accessible in five trim levels — the 2WD ES, SE and XLS models as well as 4WD SE and GT editions — and upgrades to the five- or seven-passenger 2011 Outlander's engine management system have led to improved EPA ratings across the line-up.
Mitsubishi Outlander Sport The new for this year Outlander Sport becomes the third crossover in the Mitsubishi line-up. It's powered by a revamped version of the Lancer's 2.0-liter inline four-cylinder engine, rated at 148 hp and 145 pound-feet of torque. (State emissions regulations lower those ratings to 143 hp and 143 lb-ft in California.)
The Outlander Sport is available in a two-wheel-drive configuration (ES model) or in either two-wheel or with a driver-selected electronically controlled four-wheel-drive system (SE model) that is identical to that found in the slightly larger Mitsubishi Outlander. And while the two-wheel-drive ES model is available with either a five-speed manual or continuously variable transmission (CVT) with magnesium-alloy steering-column-mounted paddle shifters, the two-wheel- and four-wheel-drive SE versions come equipped exclusively with the CVT. While the ES includes a long list of standard features and equipment, the more upscale SE edition adds 18-inch aluminium-alloy wheels, super-wide-beam HID (S-HID) headlamps, rain-sensing windshield wipers, auto on/off headlamps, upgraded interior fabric, one-touch Start/Stop (OSS) engine switch, automatic climate control with pollen filter and the FAST-key passive entry system.


Sam Houston State University Opera and Kobe College presents “Dido and Aeneas,” a tragedy by Henry Purcell, in the dance theatre of the James and Nancy Gaertner Performing Arts Center March 23-25.
“Dido and Aeneas” is presented in collaboration with SHSU Department of Dance, SHSU Center for Early Music and Research and Performance and Kobe College Dance and Music from Japan. This is one of the first English operas ever written, composed in approximately 1688 by Henry Purcell with a libretto by Nahum Tate.
“The opera is a tragedy in three acts and is based on Book IV of Virgil’s Aeneid,” Associate Professor and Director of Opera Workshop Rebecca Grimes said. “The story outlines the love affair between Dido, Queen of Carthage and the Trojan hero Aeneas, and her despair when she is abandoned by him in his quest for the founding of Rome.”
Although this opera is over 350 years old, according to Grimes, “Dido and Aeneas” has themes of love and loss portrayed by complex characters that the audience will still find relevant.
“This is a great first opera to see,” Grimes said. “It is only about an hour long, and is in English, and the music is beautiful! It also features dance as well as early musical instruments that folks may not be familiar with.”
To make this possible, Grimes is collaborating with SHSU Director of the School of Music Scott Plugge, Professor of Dance Dana Nicolay Director of CEMRAP Mario Aschauer, Professor of Cello and acting coordinator of this event Daniel Saenz, and Professor of Saxophone and project translator Masahito Sugihara.
“In March, students and faculty from Kobe College [are traveling] from Japan to participate in the opera production at SHSU,” Grimes said.
The first performance was held March 13 at The Centrum/John Wesley Methodist Church in Houston. March 23-25 will be performed in the SHSU dance theatre of the James and Nancy Gaertner Performing Arts Center at 7:30 p.m.
“SHSU students and faculty will travel to Kobe, Japan in May for a repeat performance on the Kobe campus,” Grimes said. “During each exchange, students and faculty will participate in joint recitals and master classes. In a time of so much uncertainty and social fracture, this project represents several groups of people coming together to create something beautiful that is greater than ourselves. It also provides the students an opportunity to learn about people who have a different world experience, which is invaluable. The creative arts are a vehicle that allows these things to happen.”
The opera will be performed at Kobe College on June 1 and 2.
Tickets for the SHSU performances are $15 general, $12 seniors and $5 for SHSU students.
To purchase tickets visit www.shsu.edu/boxoffice or call 936-294-2339.


At Jackson & Phillips we’ve always tried to ensure we’ve got the skills and equipment needed to deal with any vehicle that comes through our gates.
Hybrid and electric vehicle repairs and servicing were once considered a rarity but that’s fast changing.
We now have two technicians in our workshop capable of working on them, many of which are sent our way by leasing companies.
Electric vehicles aren’t always obvious though.
For years we have carried out MOTs and repairs on a Ferrari 308 GTS, which until recently was powered by a V8 engine.
It’s now thought to be the world’s first Tesla-powered Ferrari.
Our customer has owned it for 28 years but, with the V8 in poor shape, he was faced with an expensive engine strip-down and rebuild.
The owner, Peter, who just so happens to be an electrical engineer who already owned a Tesla, contacted Electric Classic Cars and made the decision to have his 1982 Ferrari 308 GTS converted to house a large Tesla drive unit, batteries and chargers.
The conversion was carried-out carefully and almost all the original interior features have been retained.
The iconic ‘H’ gate transmission selector trim, for example, now houses micro switches which are activated via the selector for reverse and drive.
Expert engineering means the Tesla drivetrain fits perfectly into the rear of the car where the V8 was once housed.
The new motor sits much lower than the combustion engine did, which together with suspension and brake upgrades and a wider rear track, means the handling has been improved greatly.
What’s more, it’s now practical too.
A full charge provides a range of around 180 miles – that’s just 20 miles less than a full tank of petrol.
That said, it’s clearly not as quick and easy to fill/charge as it previously was.
But a near perfect 50:50 weight distribution makes it a pleasure to drive and Peter now has room for a set of golf clubs in a beautifully crafted luggage compartment behind the drive unit in the rear.
When it comes to performance, 450bhp and 600nm of torque propels it from 0-60mph in a blistering 3.8 seconds.
Top speed? That’s limited to 165mph.
Peter used the car on track both before and after the conversion, so he’ll know more than anyone about the gains that have been had and he’s very satisfied with the results.
I have to admit though, when it turned up for its first visit after the conversion, it was a little odd with the absence of that beautiful V8 soundtrack.
Peter assures me that the driving experience isn’t hampered one bit by the lack of engine roar.
He told me he missed the noise for all of five minutes.
The cost of the conversion? It was an eye watering £40,000-£50,000, which is about twice the cost of an engine rebuild.
That said, Peter now has the reliability of a new car and greatly reduced running and maintenance costs.
Love it or hate it, this certainly seems to be the way forward and you can’t deny the amazing level of engineering that has gone into this build.
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- PhD–1997 (University of Minnesota)
VSCI V 768 Special Topics in Vision Science: Quantitative Data Analysis Methods in the Biological Sciences (v768/index.htm)
Dr. Nicholas Port completed his undergraduate training at NYU's Center for Neural Science (1990) and completed his pre-graduate training at the Centre for Cognitive Neuroscience at Oxford University (1991). He received his Ph.D. in Neuroscience from the University of Minnesota in 1997. Dr. Port did his postdoctoral training at the Laboratory for Sensorimotor Research at the National Eye Institute. Dr. Port joined the faculty of the IU School of Optometry in August 2005.
- Port NL and Wurtz RH (2003) Sequential activity of simultaneously recorded neurons in the superior colliculus during during curved saccades. J Neurophys 90: 1887-1903.
- Kruse W, Port NL, Lee D, Georgopoulos, AP (2001) Neural mechanisms of catching: translating moving target information into hand interception movement. In: Cognitive neuroscience perspectives on the problem of intentional action (Johnson SH, ed). Cambridge, MA: MIT Press.
- Lee DN, Georgopoulos AP, Clark MJO, Craig CM, Port NL (2001) Guiding contact by coupling the taus of gaps. Exp Brain Res 139:151-159.
- Port NL, Kruse W, Lee, D, Georgopoulos, AP (2001) Motor cortical activity during interception of moving targets. J Cog Neurosci 13: 306-318.
- Lee D, Port NL, Kruse W, Georgoploulos AP (2001) Neuronal clusters in the primate motor cortex during interception of moving targets. J Cogn Neurosci 13: 319-331.
- Port NL, Sommer MA, Wurtz RH (2000) Multielectrode evidence for spreading activity across the superior colliculus movement map. J Neurophys 84: 344-57.
- Lee D, Port NL, Kruse W, Georgopoulos AP (1998) Neuronal population coding: Multielectrode recordings in primate cerebral cortex. In: Neuronal ensembles: strategies for recording and decoding (Eichenbuam H, Davis J, ed), pp 117-136 New York: Wiley.
- Lee D, Port NL, Kruse W, Georgopoulos AP (1998) Variability and correlated noise in the discharge of neurons in motor and parietal areas of the primate cortex. J Neurosci 18: 1161-70.
- Port NL, Lee D, Dassonville P, Georgopoulos AP (1997) Manual Interception of Moving Targets. I. Performance and movement initiation. Exp Brain Res 116:406-420.
- Lee D, Port NL, Georgopoulos AP (1997) Manual interception of moving targets. II. Online control of overlapping submovements. Exp Brain Res 116:421-43.
- Port NL, Pellizer G, Georgopoulos AP (1996) Intercepting real and path-guided apparent motion targets. Exp Brain Res 110: 298-307.
- Kettner R, Marcario JK, Port NL (1996) Control of remembered reaching sequences in monkey. II. Storage and preparation before movement in motor and pre-motor cortex. Exp Brain Res 112: 347-358.
- Kettner R, Marcario JK, Port N (1993) A neural network model of motor cortex activity during reaching. J Cognitive Neurosci 5: 14-33.

domain: medical


Sliders UK has recently added the Veka Imagine to its bi-folding door range, part of a new and rapidly expanding PVC-U bi-fold offer.
“At an industry-wide level, we have seen the launch of two or three very accomplished PVC-U bi-fold door systems. These have redefined the potential of PVC-U as a material type in retail but also commercial markets”, says Steve Mines, Sales Director Sliders UK.
He continues: “As a specialist door fabricator we’re working with leading PVC-U systems companies to bring those products to market alongside established aluminium systems. The point is that the gap between aluminium and PVC-U bifold systems is closing.”
Sliders UK’s growth – it reported that it was 30 per cent up year on year Q1 2016 – has gone hand-in-hand with product development. The niche manufacturer became the first company to fabricate a Secured by Design accredited inline sliding door in 2008, adding PVC-U and aluminium bi-fold doors to its staple product range in 2009.
It followed this up in 2011 with its manufacture of the ALUK aluminium bi-folding door offer – the first aluminium bifold to be accredited with SBD status. “It’s one of the reasons why we opted to bring the Veka Imagine on board”, continues Mines.
“It can be manufactured to an SBD and PAS24:2012 standard and so there’s a natural fit with our Ultimate Range of SBD accredited aluminium and PVC-U inline sliding, bi-folding and composite door ranges.
“But it’s also a product which delivers some very strong sightlines and technical innovations and in doing it at the price-point of PVC-U, rather than aluminium, we believe that it generates distinct opportunities for installers.”
According to research agency D&G Consulting, the window industry may have so far underestimated the market potential of PVC-U as a material for bi-fold manufacture.
The authors of the UK Annual PVC Window Door & Conservatory Industry Market Report, predict that bi-fold door sales will top 30,000 per year by the end of 2016 and 40,000 per year by 2018.
‘We forecast that volumes of bi-fold doors will continue to increase in volume and by 2018 PVC bifold doors will represent 39% of the total volume of PVC sliding patio and bifold doors – this despite considerable competition from timber and aluminium products. . . [The question is] . . . can and will PVC bifold doors take volume from the other materials?’ . . . they continue.
The launch of the Veka Imagine, gives Sliders UK a second PVC-U bi-fold system in addition to its established Eurocell Ultimate Aspect PVC-U offer. With lead times of just two weeks, Mines argues, that the combined offer puts Sliders UK and as a consequence its customers, in a unique position.
He says: “We’ll leave detailed market forecasts to the experts but what’s clear is that predictions for PVC-U products are promising. What’s yet to be seen is, if they will and if so how much, market share will they take from other material types.
“From our perspective because we offer PVC-U and aluminium bi-folds, regardless of where demand comes from, we can support our customers in meeting it. From a retail sales perspective you’re keeping the door open. If aluminium is too much, you’re giving the home owner an alternative.”
The fully suited Veka Imagine system is available in 19 different panel configurations to a maximum of seven sashes, closing apertures of up to 6000mm wide and up to 2400mm height. The maximum sash width is 1000mm, while doors can be set to open inward or outward.
Featuring custom designed hardware from ERA, the PAS24:2012 accredited Veka Imagine from Sliders UK also delivers top U-values of up to 0.94W/m2k and an indicative DSER rating of A+13 .
Available in 18 foiled finishes the 70mm system is also available in a choice of beveled or sculptured sashes and beads. “This for me is another key differential in new generation bi-fold doors” continues Mines.
“The massively improved aesthetics from sculptured and beveled systems like the Ultimate Imagine, to the contemporary and flush finish of the Ultimate Aspect bi-fold combined with foils – it’s really delivered a step-change in end user appeal of PVC-U products.”
The Ultimate Imagine also features other technical benefits. Hinges are fully adjustable and again come in a choice of white or black, while adjustable locks shootbolts and bi-directional hook locks, also provide additional security and compression.
“What we’re seeing is new generation PVC-U products become more capable, deliver better aesthetics and become easier to fit”, says Mines. It’s not just the Imagine but is also evident in the Ultimate Aspect bifold.
“90 per cent of the glazed screen can be opened, the system can be manufactured in a two to seven panel configuration, multi-point locking, anti-jemmy pins, SBD and PAS24, low threshold options – it’s all there in a PVC-U door.
“It’s this innovation which is narrowing the gap between the performance of PVC-U and aluminium. The Ultimate Aspect and Ultimate Imagine doors are examples of how far PVC-U products have evolved and the aesthetic that they can deliver.
“As a specialist manufacturer I think it’s where the real value of our offer kicks in, because it gives our customers access to multiple aluminium and PVC-U systems from a single point of supply and on a single delivery.
“Predicting exactly where the market will go is difficult but the point is you always need to maximize return on each and every opportunity, which is what we’re geared up to supporting our customers in doing.”
For more information contact firstname.lastname@example.org


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Cannonville / Bryce Valley KOA is located in Cannonville, Utah and offers great camping sites!
2: 13, the work of the Holy Spirit in our life is to save us " through the sanctifying work of the Spirit and through belief in the truth. Bryce Harper will be one of the biggest free agents in MLB history this offseason, and he has the potential to change the fate of the lucky franchise that lands him.
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The name of Fabiola Letelier will remain inscribed on the indelible wall of Chilean memory. Deceased on Thursday at the age of 92, accompanied by her family, the lawyer is part of the faces of brave, prepared women, full of humanity and consequence who took the lead in the fight against human rights violations of the dictatorship of Augusto Pinochet (1973-1990). From the political and moral stock of other great Chilean women, such as Ángela Jeria or Ana González, Fabiola made her life a cause. “History forced me to live as a social fighter and today I can say that I am happy with my life, even if it has been hard,” she said three years ago, when the National Institute of Human Rights (INDH) awarded her for her tireless work on behalf of truth and justice.
He lived the horror firsthand. His younger brother, Orlando Letelier, was assassinated by the dictatorship in Washington DC in 1976, in one of the most ferocious crimes of the regime. She had been chancellor in the government of the socialist Salvador Allende and from the United States she was one of the main opponents of Pinochet from abroad. It was the reason why the dictator’s secret police, the DINA, dared to attack Letelier with a bomb attached to the car in the heart of the diplomatic quarter of the American capital. Letelier and Ronni Moffit, his American collaborator, died. Since then, her sister Fabiola has devoted herself to the case. “In the face of one of the most heinous crimes committed in the history of this country, Fabiola Letelier had a courage and loyalty to her brother that are exemplary. She was also – and what is an honor and a source of pride for us – a most excellent lawyer from our university. His name will remain inscribed in our history ”, reflected in these days the rector of the University of Chile, Ennio Vivaldi.
Fresh out of university, Fabiola Letelier joined humanitarian work. In the 1960s he joined the Inter-American Commission on Human Rights and, after the coup, he returned to Chile after living eight years in the United States. Born on July 17, 1929 in Temuco, in the south of the country, the lawyer was part of the Pro Paz Committee, a body founded only a few days after the democratic breakdown by Cardinal Raúl Silva Henríquez, who from the first moment resisted dictatorial brutality and played a central role in helping the persecuted and their families. It was the Committee for Peace that quickly began to organize the defense of the opposition with visits to the detention camps, assistance to relatives to try to locate relatives, defense of those who were being subjected to Councils of War and tours of the prisons .
With its dissolution due to tensions with the dictatorship, in October 1975 the cardinal managed to found the Vicariate of Solidarity, through a decree of Pope Paul VI. Attorney Letelier, again, was there. The organization not only faced the military and the paralyzed courts of justice, but was also a counterintelligence apparatus before the regime. “Liberation Theology, a name forged by the Peruvian theologian Gustavo Gutiérrez, who defended the preferential option for the poor, profoundly marks the Chilean Church of the sixties and seventies,” explained the lawyer of the Vicaría de la Solidaridad, Carmen Hertz , to understand the antecedents of the position of Chilean Catholicism.
The human rights project that the Vicariate represented has been perhaps the most powerful globally developed during the dictatorship and was explained in his memoirs by Cardinal Silva Henríquez himself, who died in 1999. “It was an original creation, yes. It did not exist elsewhere, it was unprecedented. An extraordinary situation caused her, no doubt! But it was not by chance, nor was it a sudden inspiration: the Vicariate was born from a long and long-suffering reflection on what we lived through day after day, ”he indicated in the book by journalist Ascanio Cavallo. The group was joined by lawyers and social workers, among others, most of them young. This building, located in front of the Plaza de Armas, became a symbol of the anti-dictatorial resistance since the late 1970s and the main place of reception for the victims and their families. “Along with the monthly report, which recounted all the abuses that had been committed in the last month, once a year we presented the state of the human rights situation to the Judiciary. We hoped that the president of the Supreme Court would make some reference, but it never happened ”, recalled María Luis Sepúlveda, who became executive secretary of the Vicariate.
Lawyer Letelier was there that morning of September 21, 1976, when the news of the attack on her brother Orlando reached the Vicarage. The lawyer Rosemarie Bornand remembers it: “No one who was in the Vicarage that morning of September 21 will forget those moments,” she points to the phone. “We all knew the history of repression against Orlando Letelier, who was in exile in the United States, after passing through Venezuela. He had an immense political activity of solidarity with Chile and denouncing crimes. That September morning, Fabiola received a call, while she was working at her desk. And she only said: ‘They murdered Orlando’, immensely moved. It was shocking. We had heard of the assassination of General Carlos Prats in Argentina the previous year, but a crime in the heart of Washington was surreal. That and Fabiola’s pain, that we experienced it as in our own flesh, because she was one of us. It was tremendous, frightening, “says the lawyer.
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Bornand recalls that Letelier began in the Vicariate, but that in his biography the role he played in the Committee for the Defense of the Rights of the People (CODEPU), a secular body that he founded in the early eighties and focused on at work with the social organization, but in political terms. “Fabiola was very warm, loving, yet formal. Her work in court, where she was always highly presented, was admirable because it showed her perseverance. It is unforgettable how he worked on his brother’s case. He got the prosecution of Manuel Contreras, the number one of the DINA, in a historic ruling, in which she has a leading role ”, recalls her partner about the decision of the Supreme Court of May 1995, where Brigadier Pedro Espinoza, another of the police chiefs, was also convicted. secret. “Most of the people gathered in the streets near the Courts, many of them relatives of the disappeared, hugged each other and sang the national anthem when they heard the ruling,” reported EL PAIS.
He never walked away from human rights work. She was president and legal director of CODEPU until 1998, in democracy. In the courts of Justice they remember having seen her alleging at least until 2010, in her 80s. He had a special interest in cases where the victims were women.
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Rest in Power Fabiola Letelier


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The off white walls impede perplexing, pressing thoughts, time and space. Like a dusty digit worthy of a baked suffix, I'm freezing in this room. but then conversely I yearn to be boxed in close the door but smash the mirror. Fear what is outside then hide the oyster shell... read more about room.
Listing posts for July 2013
As the conversation reverberates off the punctured walls, the drinks flow easy and free. You'll find me in the corner notebook hand in, penning this poem happily. As the guests the line the walls, seated in chairs, sharing tales, both tall and true I recline and relax, and satiated in... read more about engagement party.
Not settling into the rat race Salt and light in the daily pepper grind Humility, honesty and humour Meeting then surpassing expectations Creativity, courage and courtesy Christ dependency and fortitude Early nights and early mornings Prayer, scripture and gratitude Adventure, focus and proactivity Intentional and grace-filled life Flank living read more about Flank living.
Return to the land of milk and honey Open fires, kitchen table, full fridge Rolling hills, green winter pastures Dirt road leads to a dear friend Small town supermarkets boast monopolising paraphernalia Cold pews, stained glass windows Liturgy found on page six Seemingly inattentive father Allegiance lies with arduous toil... read more about Familiar place.
The pack is stacked Clothes laundered Snap lock bags stowed Photos shared, stories told Bruises fading, grazes healing I return to my notebook and pen e xchange rock hopping for full stopping. In these words in the motions of pen on paper fingers on keyboard just as out on the... read more about After the trail.
As the ocean laps solemnly onto the shore the tide of my hearts drifts away. I'll be here waiting, biding my time Hoping I'll meet you one day. As the sunshine brings shimmer to the morning dew the clouds open the sky to expanse. A new day is dawning as... read more about place & hope.
I broke the camera on the first day so photos of my trip may be hard to find so all I have to recall of my trek are memories, objects and images in my mind. The rope on the beach at sunset as day one drew to a close. Fear,... read more about Hinchinbrook.
Humble yourselves in the sight of the Lord, and He will lift you up. read more about James 4:10.


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