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Disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk invoked at perivascular sites is still rudimentary. Here, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in the lung. We show that specific secreted factors, induced in metastasis-associated endothelial cells (ECs), promote metastasis in mice by enhancing stem cell properties and the viability of cancer cells. Perivascular macrophages, activated via tenascin C (TNC) stimulation of Toll-like receptor 4 (TLR4), were shown to be crucial in niche activation by secreting nitric oxide (NO) and tumor necrosis factor (TNF) to induce EC-mediated production of niche components. Notably, this mechanism was independent of vascular endothelial growth factor (VEGF), a key regulator of EC behavior and angiogenesis. However, targeting both macrophage-mediated vascular niche activation and VEGF-regulated angiogenesis resulted in added potency to curb lung metastasis in mice. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis.

Within the last decade CAR-T cells have changed the landscape of treatment regimen for leukemia and myeloma, which is reflected by the first FDA approval of this class of living drugs in 2017. As of today, the clinical trial situation aims on CAR-T applicability in solid tumors, which is more arduous due to antigen heterogeneity and limited CAR-T persistence. More personalized and multi-target oriented cellular products may offer a solution to overcome these problems but then CAR identification and selection display the major bottleneck in the drug development process. Usually, matching scFvs are selected from targeting screens of a phage-display library and hits are subsequently cloned in CAR backbones and tested for functionality and possible limiting factors as the occurrence of tonic signaling. This makes the whole process very time consuming and laborious. Within this project, we propose a novel CAR-T selection method that rapidly shortens the discovery procedure. We have developed a full length CAR library in nS/MARt DNA vectors that is electroporated in a Jurkat reporter cell line reflecting entirely its full diversity. By this, we can quickly identify the amount of tonic signaling CARs and exclude them from further selection. For on-target selection we first perform a bulk pre-selection followed by a single cell functionality screening using the Berkeley Lights Lightning™ device. This allows us to export hits as clonal viable cells that undergo long length Nanopore CAR-RNA sequencing. Our findings show the feasibility of our approach and that it can shorten the timeframe needed for the full selection process from weeks to days. Citation Format: Eren Boga, Luisa Berger, Alice De Roia, Inka Zörnig, Robert Embacher, Stefan B. Eichmüller, Dirk Jäger, Richard Harbottle, Patrick Schmidt. A combination approach of a cellular library and single cell microfluidics analysis for the rapid selection of CAR-T cells [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 1769.

Autosomally replicating episomal DNA nanovectors allow clinical-scale CAR-T cell manufacturing., The compelling need to provide adoptive cell therapy (ACT) to an increasing number of oncology patients within a meaningful therapeutic window makes the development of an efficient, fast, versatile, and safe genetic tool for creating recombinant T cells indispensable. In this study, we used nonintegrating minimally sized DNA vectors with an enhanced capability of generating genetically modified cells, and we demonstrate that they can be efficiently used to engineer human T lymphocytes. This vector platform contains no viral components and is capable of replicating extrachromosomally in the nucleus of dividing cells, providing persistent transgene expression in human T cells without affecting their behavior and molecular integrity. We use this technology to provide a manufacturing protocol to quickly generate chimeric antigen receptor (CAR)–T cells at clinical scale in a closed system and demonstrate their enhanced anti-tumor activity in vitro and in vivo in comparison to previously described integrating vectors.

We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity., Graphical Abstract, Bozza and et al. describe minimally sized DNA vectors that can be used to generate genetically modified isogenic human cells that produce persistent transgene expression while avoiding insertional mutagenesis and vector-mediated toxicity. They show the restoration of the tumor suppressor gene SMAD4 into patient-derived pancreatic cells reversing the cellular and molecular phenotype.

The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by isocitrate dehydrogenase (IDH) mutations promotes gliomagenesis via DNA and histone methylation. Here, we identify an additional activity of R-2-HG: tumor cell-derived R-2-HG is taken up by T cells where it induces a perturbation of nuclear factor of activated T cells transcriptional activity and polyamine biosynthesis, resulting in suppression of T cell activity. IDH1-mutant gliomas display reduced T cell abundance and altered calcium signaling. Antitumor immunity to experimental syngeneic IDH1-mutant tumors induced by IDH1-specific vaccine or checkpoint inhibition is improved by inhibition of the neomorphic enzymatic function of mutant IDH1. These data attribute a novel, non-tumor cell-autonomous role to an oncometabolite in shaping the tumor immune microenvironment.

Pancreatic adenocarcinoma is a highly aggressive malignancy with dismal prognosis and limited curative options. We investigated the influence of organ environments on gene expression in RNU rats by orthotopic and intraportal infusion of Suit2-007luc cells into the pancreas, liver and lung respectively. Tumor tissues from these sites were analyzed by chip array and histopathology. Generated data was analyzed by Chipster and Ingenuity Pathway Analysis (±1.5 expression fold change and p

Gliomas are tumors with low mutational burden with the majority of them being resistant to checkpoint inhibition due to few immunogenic antigens. Multicenter vaccine trials targeting personalized neoantigens in gliomas demonstrated feasibility and illustrated the challenges of retrieving neoepitope-specific T cells based on the prediction of immunogenic neoepitopes. Here we took an entirely different T cell-centric approach and established a single cell sequencing-based high-throughput T cell receptor (TCR) retrieval platform, exploiting the therapeutic potential of spontaneous intratumoral T cell clonotypes for the development of adoptive cell therapy. We conducted direct ex vivo TCR single cell sequencing from freshly sorted human glioma-infiltrating T cell samples. High fidelity PCR was established to clone TCRs from single cell libraries directly into episomal expression vectors further optimized for T cell therapy. In parallel to standard therapy, patient-derived xenografts were developed and characterized. Tumor-reactivity of retrieved TCRs was demonstrated against patient-derived cell lines. Collectively, we provide a novel sequencing-based platform for high-throughput identification and validation of endogenous glioma-targeting TCRs and demonstrate their therapeutic applicability.

Ly6/neurotoxin1 (Lynx1) functions as a brake for nicotinic receptors and was defined as a tumor suppressor in lung cancer. As pancreatic cancer development may be slowed down by cholinergic signaling, we investigated the role of Lynx1 in pancreatic ductal adenocarcinoma (PDAC) cell lines, both in vitro and in vivo. Lynx1 knockout cell clones were generated by transfecting the CRISPRCas9 plasmid - (pSpCas9 (BB)-2A-Puro) into Miapacaluci and BXPC3luci PDAC cells using jet Pei and jet Prime as transfecting agents, respectively. The annealed gRNA was directed towards exon 3 of the Lynx1 gene. Control clones were transfected with the plasmid alone. Clones from both cell lines were tested by qRT-PCR and Western blot for knockout efficiency as well as by proliferation assay. In addition, the expression levels of p-MEK, p-MAPK, p-mTOR and p-Rictor were verified in two BXPC3 cell clones in relation to Lynx1 expression. Furthermore, 4x106 cells of four BXPC3 cell clones (one control, 3 knockout clones) were injected, respectively, into the portal vein of nude rats to control for a possibly reduced tumor growth in the liver. The knockdown of Lynx1 was incomplete at mRNA level and ranged from 20 to 60% in BXPC3 clones and from 0 to 30% in Miapaca clones. At protein level, the respective values ranged from 0 to 90% in BXPC3 and from 22 to 50% in Miapaca clones. However, Lynx1 protein levels increased at later time points. All BXPC3 clones proliferated less quickly than the respective control when tested by MTT assay. Miapaca clones, however, didn't show a significant difference from the respective control, although their growth was clearly disturbed shortly after transfection. Concomitantly with reduced Lynx1 protein levels, there was reduction of p-mTOR (90%), p-Rictor (30%) and p-MEK (40%) in BXPC3 cells. In vivo, the BXPC3 clones showed a lag period of 1 to 2 weeks till the appearance of a first bioluminescence signal indicating tumor growth. Rats injected with cells from the control clone showed a steady increase in the bioluminescence signal (n=7 of 10) as compared to the most sensitive knockdown clone, which didn't show any signal in any of 4 injected rats (p=0.05). Cells of 2 other BXPC3 clones showed a reduced growth rate at best in 2 of 2 rats used for each clone, respectively. In conclusion, knockout of Lynx1 was incomplete at both mRNA and protein levels. Nevertheless, the respective BXPC3 clones exhibited reduced proliferation in vitro, which was associated with diminished p-mTOR, p-Rictor and p-MEK levels. In addition, they failed to establish a tumor in vivo or showed a reduced tumor growth rate. These findings suggest that Lynx1 is a vital gene and may play an important role in the growth and establishment of PDAC cells. Citation Format: Doaa Ali, Michael Zepp, Matthias Bozza, Maria Nikolova, Richard Harbottle, Martin R. Berger. Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4717.

The capability to introduce Chimeric Antigen Receptors (CARs) into naïve Human T-Cells represents one of the most promising therapeutic strategies for the treatment of cancer. However, virus mediated adoptive cell therapy (ACT) remain severely limited by two factors: the long lead time and high cost of GMP virus manufacture, and the virus safety profiles. What if the entire ACT process could be sped up, made safer and more cost-effective by at least an order of magnitude? We have invented a novel DNA Vector platform based on scaffold/matrix attachment region (S/MAR) component that provides the opportunity to efficiently generate genetically engineered T-cells. This system is based on a nanovector technology. It contains no immunogenic and comprises only clinically approved sequences. It is easy, simple and cost-efficient to produce. Critically, it does not integrate and replicates autonomously and extrachromosomally in the nuclei of dividing primary human cells, thus avoiding the inherent risk of integrative mutagenesis. Through a process of iterative CpG depletion, selection marker minimalisation, empirical promoter design and elimination of cryptic eukaryotic signals our nano-S/MARt DNA Vector (nS/MARt) can be efficiently transfected into primary human T Cells. nS/MARt vectors are designed to remain stably expressed, and in addition to having the best in class safety profile, they also demonstrate enhanced performance as a biopharmaceutical. Human T-cells engineered to express the CAR receptor against the carcinoembryonic antigen (CEA) using a nS/MARt vector provide more effective killing of human cancer cells in vitro than those engineered with integrative lentivirus. These results hold in vivo, where nS/MARt transfected CAR Tcells outperform the lentivirally transduced cells, attenuating tumour growth and extending mouse survival. Moreover, in pre-clinical studies, the comparison with the FDA approved drug Kymriah®,T cells modified with nS/MARt vectors harbouring the expression of a CD19 CAR are comparable to those engineered with the viral vector. Notably, we have also taken steps to evaluate nS/MARt's scalability and have succeeded in manufacturing a clinically relevant number of CAR-T Cells (2 × 107CAR+ T-cells per kilo, we estimate the production for an individual of 80 Kg). The extension of the results from mice to patients-scale required a 1000x scale up for the processing of T-cell transfection while halving the time for production to hit a meaningful therapeutic window. We have developed a novel manufacturing protocol where nS/MARt vectors can be used "off the shelf" for CAR-T therapy to generate a clinically relevant number of modified cells in just seven days. The delivery of our DNA to CD3+ cells, reaches ~60-70% with cell viability of 60%, that increases in the days that follow the cell electroporation. Thus, the most significant benefit will be for the patients that will be able to access the nS/MARt mediated therapy in 1 week. To translate this technology into a clinical reality a fermentation process that allows the preparation of 2.6 g/L of pure, supercoiled DNA was optimised. There is a pressing need to offer ACT to more oncology patients, and we believe that this novel DNA Vector system provides a unique and innovative approach to this therapeutic strategy for cancer therapy. Citation Format: Matthias Bozza, Alice De Roia, Aileen Berger, Alexandra Tuch, Patrick Schmidt, Richard Harbottle. A non-integrating, non-viral DNA Nanovector platform for the safe, persistent, and rapid manufacture of recombinant T-cells for Adoptive cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4066.

Endothelial to hematopoietic transition (EHT) is a crucial step in the formation of definitive hematopoietic stem cells during embryonic development. Holding such a critical developmental role not many details regarding molecular changes and cell surface marker (CSM) expression on these transitional cells are known. We think that sub-segregating the process of EHT holds great therapeutic potential by giving new indications to understand the hematopoietic stem cell maturation. We generated embryonic stem (ES) cell lines from Hoxb4-YFP reporter mice (Hills, 2011), in which YFP expression marks functional HSCs in adult and embryonic mice. When these cell lines were subject to embryoid body differentiation assays, we observed a transient Hoxb4/YFP+ cell population, which corresponded to the emergence of hemogenic endothelial cells in culture. We then analyzed the gene expression profile of the Hoxb4+ cells and compared it to immediate precursor (Hoxb4-Flk1+) and daughter (Hoxb4-CD41+) cell populations. As well as documenting the expression of numerous molecular markers previously associated with EHT, we also observed an elevated inflammatory gene expression signature that has previously been characterized as a mediator of hematopoietic specification in vivo. We were additionally able to identify 45 novel cell surface markers that could potentially be used to prospectively isolate and sub segregate cells undergoing EHT. 26 of these targets were subsequently verified using MRM mass spectrometry. To establish if any of these markers were functionally relevant, we generated knockout (KO) cell lines using CRISPR/Cas9. Upon EB differentiation, we observed a profound block in hematopoietic differentiation for ES cells that were KO for the membrane proteins Evi2a and Lyve1. This block was manifest at the EHT stage, as verified by time-lapse video imaging, and was equivalent in magnitude to Runx1 KO, suggesting a crucial role of Evi2a and Lyve1 during the EHT stage of hematopoietic development. Notably, this defect could be rescued by genetic replacement of the deleted gene. When comparing our data to human fetal liver data sets Evi2a is highly expressed in fetal liver, as well as in HSCs isolated from murine AGM and fetal liver.

Adoptive immunotherapy is one of the most encouraging therapeutic strategies for the treatment of a range of cancers. One particularly promising avenue of research is the functional introduction of Chimeric Antigen Receptors (CARs) into naive Human T-Cells for autologous-immunotherapy. Currently, the genetic engineering of these cells is achieved through the use of proprietary integrating vector systems such as lentiviruses or the sleeping beauty transposon system which present a risk of genotoxicity associated with their random genomic integration. We have invented a novel DNA Vector platform for the safe and efficient generation of genetically engineered T-Cells for Human Immunotherapy. This DNA vector system contains no viral components and comprises only clinically approved sequences, it does not integrate into the target cell's genome but it can replicate autonomously and extrachromosomally in the nucleus of dividing human primary cells. These DNA Vectors offer several advantages over currently used vector systems; they are not subject to commercial licences, they are cheaper and easier to produce, and they can more quickly genetically modify human cells without the inherent risk of integrative mutagenesis. In preclinical experiments we have successfully generated genetically engineered human T-Cells expressing the CAR receptor against several epitopes and have demonstrated their viability and capability in targeting and killing human cancer cells which express these epitopes. The long term anti-tumor activity of our DNA-CAR-T cells has been confirmed in vivo using xenotransplanted cell lines in immunodeficient mice. We believe that this novel DNA Vector system provides a unique and innovative approach to this exciting therapeutic strategy for cancer therapy. We estimate that this novel methodology will provide a simpler method of CAR T-cell manufacturing, resulting in a 10-fold reduction in the cost of the CART-product. Citation Format: Patrick Schmidt, Matthias Bozza, Aileen Berger, Claudia Luckner-Minden, Alexandra Tuch, Inka Zörnig, Dirk Jäger, Richard Harbottle. Novel DNA vectors encoding a chimeric antigen receptor mediate long term expression without genomic integration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3573.

Bioluminescent-labeling imaging (BLI) allows sensitive non-invasive sequential imaging of tumor development and early metastasis. Current methods for the genetic modification of cells typically use integrating genotoxic viruses that can disrupt the molecular behavior of cancer cell lines due to their random nature of integration. A primary aim of the study was to utilize a non-integrating DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably genetically modify pancreatic cancer cells to persistently express the reporter gene luciferase without altering the molecular behavior of the cell or altering its sensitivity to therapeutic drug treatments. Once a novel isogenic cell line is generated the cells can subsequently be used in xenograft studies. A second aim was to validate these established models with gemcitabine and test the efficacy of VAL401, formulation of risperidone in rumenic acid. Human BxPC3, Capan-1, MiaPaCa-2 and Panc-1 pancreatic cancer cells were stably transfected with a pSMARt-UBC-Luc DNA vector and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and the molecular integrity of the DNA vector. Efficacy of gemcitabine was tested in these new luciferase expressing cell lines and VAL401 was tested in Capan-1-luc cells. For in vivo studies, BxPC3-luc cells were inoculated orthotopically into the pancreas of athymic nude mice and stratified into groups: control, gemcitabine, VAL401 (1mg/kg, p.o. daily) and VAL401 (2mg/kg, p.o. daily). In vitro validation results indicated that the luciferase transfected cells maintained their original properties with stable expression. Gemcitabine inhibited cell proliferation in all established cell lines. VAL401 inhibited cell proliferation of Capan-1-luc cells at 50 μM concentration. BxPC3-luc cells inoculated orthotopically into the pancreas were followed for 5 weeks with BLI by IVIS, and the results demonstrated high-quality follow-up of tumor growth. BxPC3-luc cells induced growth of pancreatic tumors with high take rate in all groups. Gemcitabine and both studied doses of VAL401 decreased tumor volume, and the same trend was seen in tumor weight and the BLI during the study. In conclusion, both gemcitabine and VAL401 decreased tumor volume and the same trend was observed using BLI. Our results demonstrated that S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and high levels of sustained transgene expression. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essentially non-invasive imaging platform that substantially improves the efficacy of testing anticancer drug candidates. Citation Format: Jenni Bernoulli, Matthias Bozza, Katja M. Fagerlund, Johanna Tuomela, Mari I. Suominen, Suzanne Dilly, George Morris, Jussi M. Halleen, Richard Harbottle. Utilizing a novel luciferase labeling technique to establish and validate preclinical models of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 644.

Bioluminescent-labelling allows sensitive non-invasive sequential imaging of tumor development and early metastasis. Current methods for the genetic modification of cells typically use integrating genotoxic viruses that can potentially disrupt the molecular behavior of cancer cell lines due to their random nature of integration. VAL401 is the reformulation of a clinical drug to enable use in the treatment of cancer. Preclinical data indicate potential use of the reformulated drug in lung cancer, where many subsets of patients have currently a high unmet medical need. We have utilized a non-viral DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably modify cells to be further used in xenograft studies to allow long term expression without affecting cell behavior or silencing over cell divisions. Human BxPC3 pancreatic cancer cells were stably transfected with a pSMARt-UBC-Luc and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and molecular integrity of the DNA vector. For in vivo studies, BxPC3 cells were inoculated orthotopically into the pancreas of athymic nude mice. Four experimental groups were included in the study: 1) Control group receiving vehicle; 2) Reference compound gemcitabine (60 mg/kg, q3dx4 i.p, one week pause, q3dx4 i.p.); 3) Test compound VAL401 (1 mg/kg, p.o. daily); 4) Test compound VAL401 (2 mg/kg, p.o. daily). The luciferase transfected cells maintained their original properties with stable expression. Luciferase-labelled BxPC3 cells inoculated orthotopically into the pancreas were successfully followed for 5 weeks with non-invasive bioluminescence imaging by IVIS, and the results demonstrated high-quality follow-up of tumor growth compared with tumor models using non-labelled cells. BxPC3-luc cells induced growth of pancreatic tumors with high take rate in all groups. Gemcitabine and both studied doses of VAL401 decreased tumor volume, and the same trend was seen in tumor weight and the BLI parameters (total flux, area and average radiance) during the study. In conclusion, both gemcitabine and VAL401 decreased tumor volume and same trend was seen in BLI. Our results demonstrated that S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and sustained transgene expression at high level. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essential non-invasive imaging platform that improves substantially efficacy testing of anticancer drug candidates. Citation Format: Mari I. Suominen, Jenni Bernoulli, Suzanne Dilly, Johanna Tuomela, Matthias Bozza, Katja M. Fagerlund, George Morris, Jussi M. Halleen, Richard Harbottle. VAL401 decreases tumor volume in a xenograft model of pancreatic cancer utilizing a novel improved luciferase labelling technique. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B69.

Bioluminescent-labelling allows sensitive non-invasive sequential imaging of tumor development and early metastasis. However, current methods for the genetic modification of cells typically use integrating genotoxic viruses that can potentially disrupt the molecular behavior of cancer cell lines due to their random nature of integration. Here, we utilized a non-viral DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably modify cells to be further used in xenograft studies to allow long term expression without affecting cell behavior or silencing over cell divisions. Human PC-3 prostate cancer cells and BxPC-3 pancreatic cancer cells were stably transfected with a pCAG-LUC-S/MAR and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and molecular integrity of the DNA vector. For in vivo studies, PC-3 cells were inoculated orthotopically into the prostate, and BxPC-3 cells into the pancreas using athymic and BALB/c nude mice. For comparison, similar experiments with the corresponding study designs were performed with the non-labelled parental cell lines. The luciferase transfected cells maintained their original properties with stable expression. Luciferase-labelled PC-3 and BxPC-3 cells inoculated orthotopically into the prostate and pancreas, respectively, were successfully followed for 5 weeks with non-invasive bioluminescence imaging by IVIS. The results demonstrated high-quality follow-up of tumor growth compared with tumor models using non-labelled cells. In conclusion, S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and sustained transgene expression at high level. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essential non-invasive imaging platform that improves substantially efficacy testing of anticancer drug candidates. Citation Format: Jenni Bernoulli, Johanna Tuomela, Matthias Bozza, Katja M. Fagerlund, Mari I. Suominen, George Morris, Jussi M. Halleen, Richard Harbottle. Novel luciferase labelling technique to improve imaging of orthotopic prostate and pancreatic cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2015-3246