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Table S2. List of primers used for RT-qPCR analysis.

Figure S2. Late TGF-β blockade does not affect tumor outgrowth.

The absence of T cells in the tumor microenvironment of solid tumors is a major barrier to cancer immunotherapy efficacy. Oncolytic viruses, including reovirus type 3 Dearing (Reo), can recruit CD8+ T cells to the tumor and thereby enhance the efficacy of immunotherapeutic strategies that depend on high T-cell density, such as CD3-bispecific antibody (bsAb) therapy. TGF-β signaling might represent another barrier to effective Reo&CD3-bsAb therapy due to its immunoinhibitory characteristics. Here, we investigated the effect of TGF-β blockade on the antitumor efficacy of Reo&CD3-bsAb therapy in the preclinical pancreatic KPC3 and colon MC38 tumor models, where TGF-β signaling is active. TGF-β blockade impaired tumor growth in both KPC3 and MC38 tumors. Furthermore, TGF-β blockade did not affect reovirus replication in both models and significantly enhanced the Reo-induced T-cell influx in MC38 colon tumors. Reo administration decreased TGF-β signaling in MC38 tumors but instead increased TGF-β activity in KPC3 tumors, resulting in the accumulation of α-smooth muscle actin (αSMA+) fibroblasts. In KPC3 tumors, TGF-β blockade antagonized the antitumor effect of Reo&CD3-bsAb therapy, even though T-cell influx and activity were not impaired. Moreover, genetic loss of TGF-β signaling in CD8+ T cells had no effect on therapeutic responses. In contrast, TGF-β blockade significantly improved therapeutic efficacy of Reo&CD3-bsAb in mice bearing MC38 colon tumors, resulting in a 100% complete response. Further understanding of the factors that determine this intertumor dichotomy is required before TGF-β inhibition can be exploited as part of viroimmunotherapeutic combination strategies to improve their clinical benefit.Significance:Blockade of the pleiotropic molecule TGF-β can both improve and impair the efficacy of viro-immunotherapy, depending on the tumor model. While TGF-β blockade antagonized Reo&CD3-bsAb combination therapy in the KPC3 model for pancreatic cancer, it resulted in 100% complete responses in the MC38 colon model. Understanding factors underlying this contrast is required to guide therapeutic application.

Supplementary Methods 1. Methods for immunohistochemistry, Western Blotting, TGF-β1 ELISA and in vitro assays.

FcγRIII receptor blockade impairs reoNAb loading of monocytes

Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus–antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcγRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 6(10); 1161–73. ©2018 AACR.

Flow chart of patients with BRCA1/2 alterations submitted to the study team between September 2016 and December 2019, and reasons for drop-out, rejection or screen failure. Abbreviations: VUS = Variant of Unknown Significance, LOH = Loss of Heterozygosity.

This file contains supplementary information on dendrimer synthesis and characterization and on the histopathological analysis of tumor perfusion.

Supplementary Figure from Patients with Rare Cancers in the Drug Rediscovery Protocol (DRUP) Benefit from Genomics-Guided Treatment

Purpose:Patients with rare cancers (incidence less than 6 cases per 100,000 persons per year) commonly have less treatment opportunities and are understudied at the level of genomic targets. We hypothesized that patients with rare cancer benefit from approved anticancer drugs outside their label similar to common cancers.Experimental Design:In the Drug Rediscovery Protocol (DRUP), patients with therapy-refractory metastatic cancers harboring an actionable molecular profile are matched to FDA/European Medicines Agency–approved targeted therapy or immunotherapy. Patients are enrolled in parallel cohorts based on the histologic tumor type, molecular profile and study drug. Primary endpoint is clinical benefit (complete response, partial response, stable disease ≥ 16 weeks).Results:Of 1,145 submitted cases, 500 patients, including 164 patients with rare cancers, started one of the 25 available drugs and were evaluable for treatment outcome. The overall clinical benefit rate was 33% in both the rare cancer and nonrare cancer subgroup. Inactivating alterations of CDKN2A and activating BRAF aberrations were overrepresented in patients with rare cancer compared with nonrare cancers, resulting in more matches to CDK4/6 inhibitors (14% vs. 4%; P ≤ 0.001) or BRAF inhibitors (9% vs. 1%; P ≤ 0.001). Patients with rare cancer treated with small-molecule inhibitors targeting BRAF experienced higher rates of clinical benefit (75%) than the nonrare cancer subgroup.Conclusions:Comprehensive molecular testing in patients with rare cancers may identify treatment opportunities and clinical benefit similar to patients with common cancers. Our findings highlight the importance of access to broad molecular diagnostics to ensure equal treatment opportunities for all patients with cancer.

Supplementary Table from Patients with Rare Cancers in the Drug Rediscovery Protocol (DRUP) Benefit from Genomics-Guided Treatment

Supplementary Table 1

Dynamic contrast–enhanced MRI (DCE-MRI) is a promising technique for assessing the response of tumor vasculature to antivascular therapies. Multiagent DCE-MRI employs a combination of low and high molecular weight contrast agents, which potentially improves the accuracy of estimation of tumor hemodynamic and vascular permeability parameters. In this study, we used multiagent DCE-MRI to assess changes in tumor hemodynamics and vascular permeability after vascular-disrupting therapy. Multiagent DCE-MRI (sequential injection of G5 dendrimer, G2 dendrimer, and Gd-DOTA) was performed in tumor-bearing mice before, 2 and 24 hours after treatment with vascular disrupting agent DMXAA or placebo. Constrained DCE-MRI gamma capillary transit time modeling was used to estimate flow F, blood volume fraction vb, mean capillary transit time tc, bolus arrival time td, extracellular extravascular fraction ve, vascular heterogeneity index α−1 (all identical between agents) and extraction fraction E (reflective of permeability), and transfer constant Ktrans (both agent-specific) in perfused pixels. F, vb, and α−1 decreased at both time points after DMXAA, whereas tc increased. E (G2 and G5) showed an initial increase, after which, both parameters restored. Ktrans (G2 and Gd-DOTA) decreased at both time points after treatment. In the control, placebo-treated animals, only F, tc, and Ktrans Gd-DOTA showed significant changes. Histologic perfused tumor fraction was significantly lower in DMXAA-treated versus control animals. Our results show how multiagent tracer-kinetic modeling can accurately determine the effects of vascular-disrupting therapy by separating simultaneous changes in tumor hemodynamics and vascular permeability.Significance: These findings describe a new approach to measure separately the effects of antivascular therapy on tumor hemodynamics and vascular permeability, which could help more rapidly and accurately assess the efficacy of experimental therapy of this class. Cancer Res; 78(6); 1561–70. ©2018 AACR.

Supplementary Legend

Clinical studies revealed that single-agent immunotherapies yield limited efficacy in the management of prostate and bladder cancer (PCa, BCa). PCa and BCa often induce immunosuppressive and immune evasion mechanisms that restrict anti-tumor responses. Oncolytic viruses (OVs) offer promising and safe therapeutic options in the treatment of cancer as OVs target and or preferentially replicate in tumor cells, potentially evoking systemic anti-tumor immune responses. Combination of immunotherapy with OVs may, therefore, unleash the full potential of immunotherapy for these cancers. To achieve beneficial anti-tumor responses a more personalized OV therapy approach seems crucial. Strikingly, only few studies describe head-to-head comparison of the effects of different OVs. Together with our partners in the oncolytic viro-immunotherapy (OVIT) consortium we are developing and evaluating multiple OVs for their abilities to induce oncolysis and subsequent immunomodulation. Furthermore, we are developing tools to predict oncolytic virus effectiveness and design strategies to personalize oncolytic virus therapy. The OVs that were generated by our consortium encompass the spontaneous Orthoreovirus mutant (jin-3), a non-human primate Adenovirus (AdV-lumc007dRbD), and a Newcastle Disease Virus variant (NDV-F0).In this study we determined the anti-tumor and immunomodulatory effects of selected OVs in multiple ‘near-patient and patient-derived human PCa and BCa models; 1) tumor cell line cultures, 2) cocultures of tumor cell lines and monocyte-derived dendritic cells (mo-DCs), and 3) ex-vivo cultured tumor tissue slices and explants from patient material or patient-derived xenograft (PDX) models. The selected OVs infected a panel of PCa and BCa cell lines and displayed heterogenous effects on tumor cell viability and expression of immunogenic cell death markers (HSP90, Calreticulin, and HMGB1). For instance, the jin-3 virus showed enhanced tumor tropism in most PCa cells, while AdV-lumc007dRbD was more efficient in BCa cells. Virus infection of tumor cells resulted in maturation and activation of moDCs. Furthermore, OVs were able to infect and replicate in ex-vivo cultured tumor tissue slices and viability of ex-vivo tumor tissue explants was reduced following viral inoculation. Transcriptomic analysis of infected patient tissues revealed heterogenous responses to the tested OVs.Taken together, we established and compared the oncolytic properties of various OVs in multiple preclinical models of PCa and BCa. The data indicate heterogeneity of response for the tested OVs in urological cancers. In order for OV therapy to reach robust clinical efficacy, better appreciation of underlying molecular and cellular mechanisms is crucial for the personalization of oncolytic viro-immunotherapy. Citation Format: Thijs J. Janssen, Geertje van der Horst, Arjanneke F. van der Merbel, Maaike van der Mark, Martine L. Lamfers, Bernadette G. van den Hoogen, Sjoerd S. van der Burg, Dana A. Mustafa, Niven Mehra, Tilly Aalders, Nadine van Montfoort, Jack Schalken, Rob C. Hoeben, Gabri van der Pluijm. Personalization of oncolytic virus therapy: Heterogenous anti-tumor responses in preclinical prostate and bladder cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1802.

Introduction: Both HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) often show activation of the PI3K/AKT/mTOR pathway, due to, amongst others, PI3KCA mutations, PTEN loss, or receptor tyrosine kinase activation. In HPV-negative tumors, CDKN2A (p16) inactivation or CCND1 (cyclin D1) amplification frequently occurs resulting in sustained cyclin dependent kinase (CDK) 4/6 activation. The aim of our study was to investigate the efficacy of PI3K/AKT/mTOR pathway inhibitors (PI3Ki) (alpelisib, buparlisib and gedatolisib) and CDK4/6 inhibitors (CDKi) (palbociclib and ribociclib) in HPV-positive and -negative HNSCC cell lines. Methods: The efficacy of the inhibitors was assessed using MTT assays and changes in PI3K and Cyclin D1/CDK pathway protein expression were determined by Western blotting. Cell cycle analysis was performed with flow cytometry and apoptosis was assessed by Annexin-V staining. Changes in cell metabolism were assessed by Seahorse XF assays. Results: Both HPV-positive and -negative HNSCC cell lines were highly sensitive to the PI3Ki (Gedatolisib, IC50 5-30 nM > Buparlisib, IC50 0.6-3.6 µM > Alpelisib, IC50 3-23 µM). In general, PI3Ki decreased pathway activity, resulted in moderate cell cycle arrest and apoptosis, and decreased oxidative and glycolytic metabolism. CDKi were particularly effective in blocking HPV-negative cell line growth (Palbociclib, IC50 0.5-2 µM > Ribociclib, IC50 4-7 µM). CDK inhibition showed decreased pRb expression and G1 cell cycle arrest, whereas apoptosis was not induced. Conclusion: PI3Ki and CDKi efficiently inhibit their respective pathways and HNSCC cell growth in vitro, the latter only in HPV-negative cell lines. Whereas PI3Ki especially show an effect on oxidative and glycolytic metabolism, CDKi particularly lead to cell cycle arrest. Further research should elucidate whether these inhibitors may be effective therapeutic agents in HNSCC patients. Citation Format: Imke Demers, Femke Verhees, Dion Leegemaate, Robin Jacobs, Ann Hoeben, Frank Hoebers, Bernd Kremer, Ernst-Jan Speel. PI3K/AKT/mTOR pathway and CDK4/6 inhibitors efficiently inhibit cell growth of HPV-positive and HPV-negative head and neck squamous cell carcinoma in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2573.

Background: The brain tumor glioblastoma (GBM) is one of the most aggressive forms of cancer. The dismal prognosis of these patients, with a median survival of less than 15 months despite maximal therapy makes the need for new therapeutic approaches urgent. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, it appears that for each OV only a small group of patients responds to treatment. As inter- and intra-tumoral heterogeneity is a hallmark of GBM, we hypothesized that fresh patient-derived GBM cell cultures will reflect this inter-tumoral variability in response and allow identification of potential biomarkers of susceptibility to specific OVs. Furthermore, we established a co-culture system of primary GBM cultures with autologous peripheral blood mononuclear cells (PBMCs) to capture the degree of OV-induced oncolysis in conjunction with subsequent immune activation. Using these model systems, we attempt to develop tools which may guide future personalized trials of OV treatment for GBM. Methods: We tested the oncolytic potency of four OVs derived from different viral families (DNX2401, rQnestin34.5 V1, wild type Reovirus, lentogenic NDV-f0-GFP) on a panel of 19 molecularly characterized GBM cultures and calculated the half maximal effective concentration (EC50) for each virus on each cell culture. Quantitative PCR was performed to assess cytokine expression in tumor cells after infection with the 4 different OVs. OV-induced changes in the gene and protein expression of immune associated genes were assessed in co-cultures of GBM cells with PBMCs using Nanostring nCounter System and Elisa. Results: Screening of the 4 OVs on the panel of patient-derived GBM cell cultures revealed great inter-tumoral variability in oncolysis and cytokine response to the 4 different OVs with some degree of OV specific cytokine response profiles. Correlation analysis of transcriptome data with susceptibility to the four OVs shows that genes involved in distinct pathways are related to specific OV-sensitivity. In particular, cell cycle and immune related biological processes discriminate responders and non-responders. The co-culture of OV-infected glioma cells with PBMCs suggests that infection with different OVs leads to expression of distinct sets of genes and proteins in PBMCs; indicating that each OV mounts a specific immune response. Conclusion: Heterogeneity in OV sensitivity is demonstrated in primary GBM cultures, in terms of oncolysis, cytokine induction and in virus-specific changes in gene and protein expression in OV-infected tumor cells/PBMCs co-cultures. These results support the hypothesis that improving the response rates in oncolytic virotherapy for GBM may require a personalized approach. Citation Format: Eftychia Stavrakaki, Anne Kleijn, Wouter B. van den Bossche, Rutger K. Balvers, Lisette B. Vogelezang, Jie Ju, Andrew Stubbs, Yunlei Li, Dana Mustafa, Federica Fabro, Bernadette van den Hoogen, Rob Hoeben, William F. Goins, Hiroshi Nakashima, E. Antonio Chiocca, Clemens M. Dirven, Martine L. Lamfers. Towards personalized oncolytic virotherapy: Differential response of four oncolytic viruses in primary glioblastoma cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3560.

Background: Glioblastoma is the most prevalent and severe type of malignant brain tumor in adults. Although the genetic make-up initiating glioblastoma is increasingly better understood, a better understanding in the mechanisms that drive its evolution, heterogeneity and therapy resistance may reveal new directions for therapy development. To get better insights into glioblastoma evolution, we analyzed and de-convoluted transcriptomes of primary and recurrent glioblastoma resections. Material and Methods: Matching primary and secondary resections from n=185 glioblastoma patients were collected as part of EORTC Study 1542. The study was extended with tumor pairs from n=51 patients from the international GLASS study. The datasets were subjected to differential and deconvolution analysis using in-house algorithms. Results: When mapping the tumor samples into a reduced Glioblastoma Intrinsic Transcriptional Subtype space, we visualized subtype traversal, indicating that the CL subtype most often switches. As we found no more transitions from MES to other subtypes than to be expected by chance, we concluded that MES is an end-state. On average, tumor cell percentages decreased from ~67% to ~50% mostly due to an increase in TAM/microglia. Differential expression analysis was performed with correction for tumor cell percentages. While expression of most known oncogenes did not change considerably over time, marker genes of TAM/microglia, neurons and oligodendrocytes were up-regulated whereas endothelial cell markers were down-regulated over time. Furthermore, a cluster of ~30 extracellular matrix-associated genes increase significantly over time. A signature representing the gene-set was significantly associated with poor survival; high signatures were in particular associated to survival in secondary resections (P = 6.613e-06, Kaplan-Meier estimator). This suggests that the increase of extracellular matrix expression fulfils an important role in glioblastoma evolution. Conclusion: Using a large cohort, we interrogated changes in the glioblastoma transcriptome over time and found that in particular the composition of the tumor and its environment changes. The tumor cell percentage drops, suggesting more invasion or recruitment of non-malignant cells or a combination of both. This change is independent of an increase in the prognostic increase in extracellular matrix expression. Citation Format: Youri Hoogstrate, Kaspar Draaisma, Santoesha A. Ghisai, Iris de Heer, Levi van Hijfte, Wouter Coppieters, Melissa Kerkhof, Astrid Weyerbrock, Marc Sanson, Ann Hoeben, Slávka Lukacova, Giuseppe Lombardi, Sieger Leenstra, Monique Hanse, Ruth Fleischeuer, Colin Watts, Joseph McAbee, Nicos Angelopoulos, Thierry Gorlia, Vassilis Golfinopoulos, Johan M. Kros, Vincent Bours, Martin J. van den Bent, Pierre A. Robe, Pim J. French. Transcriptional evolution of glioblastoma reveals changes in bulk composition, mesenchymal sub-type as end-state, and a prognostic association with increased extracellular matrix gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6140.

Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus–antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcγRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 6(10); 1161–73. ©2018 AACR.

The acquisition of therapy resistance and the formation of distant metastases represent major problems in prostate cancer. To date, immunotherapeutic approaches have been relatively disappointing in the treatment of prostate cancer. Therapies based on oncolytic viruses are being exploited for several tumor entities and have been studied in multiple clinical trials. Mammalian orthoreovirus is a non-enveloped double stranded RNA virus that has not been linked to serious disease in humans. Reovirus is relatively safe to use and several clinical trials are currently ongoing to study the oncolytic effect of reovirus in various tumor types. Type 3 Dearing (T3D) is a promising candidate for the use as an oncolytic agent. Besides wildtype T3D reovirus (R124), spontaneous mutants Jin1, 2 and 3 were generated which harbor mutations in spike protein Sigma-1. In this way, Jin mutants are able to infect cells independently of the Junction Adhesion Molecule-A (JAM-A) entry receptor on the tumor cell surface, which is often downregulated in different types of cancer. In this study, the direct oncolytic effect of wildtype (R124) and Jin mutant reovirus was examined in human prostate cancer cells in vitro and in our ‘near-patient’ model of ex vivo cultured human prostate cancer tissue slices. Both R124 and Jin3 dose-dependently induced prostate cancer cell death in PC-3M-Pro4luc2, DU145 and 22Rv1 cells in vitro. Interestingly, JAM-A negative cancer cells were insensitive for R124, but were effectively killed by Jin1 mutant, indicating the beneficial effects of the Jin mutant in JAM-A low/negative tumors. Moreover, tumor tissue slices were generated from explanted patient prostate cancer tissues and patient-derived xenografts (PDX) and cultured with reovirus. Oncolytic effects were observed in ex vivo cultured tumor slices upon viral replication and subsequent stimulation of apoptosis. Therefore, ex vivo culture of patient-derived tumor tissue allows the assessment of individual anti-tumor response of the oncolytic virotherapy (i.e. personalized therapeutics). Taken together, our results indicate that both wildtype R124 virus and mutant Jin reovirus can infect and replicate in human prostate cancer cells and patient-derived prostate cancer tissues. The Jin mutants are of particular interest in JAM-A negative tumors. The indirect immunomodulatory effects of reovirus in human prostate cancer are currently ongoing. In conclusion, our selected oncolytic reoviruses may represent a promising novel treatment strategy in human prostate cancer alone or combined with immunostimulatory agents. Citation Format: Arjanneke F. van de Merbel, Geertje van der Horst, Maaike H. van der Mark, Diana J. van den Wollenberg, Jack A. Schalken, Anne Collins, Norman J. Maitland, Rob C. Hoeben, Gabri van der Pluijm. Oncolytic reovirus variants induce direct oncolysis in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 349.

Colorectal tumors frequently contain activating mutations in KRAS. ReovirusT3D is an oncolytic virus that preferentially kills tumor cells with an activated Ras pathway. Here we have assessed the contribution of endogenous mutant KRAS in human colorectal cancer cell lines to ReovirusT3D replication and to tumor cell oncolysis. In addition, treatment combinations involving ReovirusT3D, oxaliplatin, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) were tested for their efficacy in tumor cell killing. The mutation status of KRAS did not predict the sensitivity of a panel of human colorectal cancer cell lines to ReovirusT3D. Virus replication was observed in all cell lines tested regardless of KRAS status and was not affected by deletion of endogenous mutant KRASD13. However, deletion of KRASD13 or p53 did reduce apoptosis induction by ReovirusT3D whereas deletion of β-cateninΔS45 had no effect. Likewise, KRASD13- or p53-deficient cells display reduced sensitivity to oxaliplatin but not to death receptor activation by TRAIL. Finally, the treatment of colorectal cancer cells with ReovirusT3D combined with either oxaliplatin or TRAIL resulted in a nonsynergistic increase in tumor cell killing. We conclude that oncolysis of human tumor cells by ReovirusT3D is not determined by the extent of virus replication but by their sensitivity to apoptosis induction. Oncogenic KRASD13 increases tumor cell sensitivity to activation of the cell-intrinsic apoptosis pathway without affecting ReovirusT3D replication. (Cancer Res 2006; 66(10): 5403-8)

Objective: Prostate cancer (PCa) preferentially spreads to bone with additional metastasis to regional lymph nodes, lung and liver in a subset of patients. Understanding the determining factors of this metastatic pattern and to elucidate the involved pathways and interplay between epithelial and the stromal niche, we established a patient-derived xenograft (PDX) model showing spontaneous metastatic spread to all relevant organs and metastatic lesions in liver. Methods: PDX of PCa were obtained from NMRI tumor-bearing mice and 3D cell cultures were established by collagenase treatment o/n in prostate growth medium (PGM) containing 2% fetal calf serum. 3D cultures of PDX PC339 were established (PC339C) that could be passaged and stored in liquid nitrogen. PC339C was transfected with a lentiviral construct containing luc2-GFP fused protein (M21) and upon FACS-sorting the PC339C-M21 was retrieved. PC339C-M21 cells were inoculated subcutaneously in NSG mice and when tumors were established mice underwent tumorectomy (700-1000 mm3) to extend the life span of the animal and allow metastastic outgrowth. Mice were monitored weekly for metastatic outgrowth using IVIS, microCT and MRI. At sacrifice, M21-positive organs, including liver, lymph nodes, lung and bone, were sampled. Part of the tissue was used for culturing, part was fresh frozen for RNA and protein isolation to allow qPCR and western blot analyses, and part was formalin-fixed and paraffin-embedded for histology and immunohistochemistry. Results: PC339C-M21 tumors were established in NSG mice and after tumorectomy a spontaneous liver metastasis was established. Culturing of this tumor resulted in the PC339C-M21-L cell line. This cell line was inoculated subcutaneously in NSG mice to result in tumor formation and spontaneous liver metastasis in all mice. Tumorectomy was performed at 25-30 days after inoculation. Luciferase-positive signals in liver were detected by IVIS as early as 7 days after tumorectomy with MRI showing first visible lesions at 21 days and microCT at 28 days. 3D reconstructed microCT images and MRI-detected lesions confirmed the signals and location as seen by IVIS. When inoculated orthotopically, liver metastatic lesions became visible by IVIS at 40 days, which was not different from the subcutaneous tumors. However, these mice could not be followed longer due to the orthotopic tumor burden. On top of the PC339C-M21-L cell line, culturing of luciferase-positive organs resulted in cell lines from lymph node (PC339C-M21-L-LN), lung (PC339C-M21-L-LU), and bone (PC339C-M21-L-B) together constituting a cell line panel of metastatic PCa. This cell line panel is being further characterized in vitro and in vivo for their unique properties and metastatic potential. Conclusion: A new spontaneous PCa liver metastasis xenograft model, PCL339-M21-L, was generated. This model was extended to yield a panel of metastatic sublines from different organ origin. Together, this platform may serve as a tool to study the biology of spontaneous metastatic outgrowth in liver and other organs. Furthermore, we demonstrated the feasibility of non-invasive imaging techniques to follow metastatic spread and outgrowth allowing to monitor responses to anti-cancer therapies. Citation Format: Hanneke J.A.A. van Zoggel, Sigrun E. Erkens-Schulze, Corrina M.A. De Ridder, Wilma Teubel, Hamza Saleem, Stefan J. Roobol, Sander A.H. Hoeben, Yanto R. Ridwan, Joost C. Haeck, Wytske M. van Weerden{Authors}. Establishment of a novel spontaneous prostate cancer liver metastasis model. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B21.