Targeting CXCL12 Research Articles

CXCL12+ Tumor-associated Endothelial Cells Promote Immune Resistance in Hepatocellular Carcinoma.

BackgroundThe tumor microenvironment (TME) plays a crucial role in the limited efficacy of existing treatments for hepatocellular carcinoma (HCC), with tumor-associated endothelial cells (TECs) serving as fundamental TME components that substantially influence tumor progression and treatment efficacy. However, the precise roles and mechanisms of TECs in HCC remain inadequately understood. MethodsWe employed a multi-omics profiling strategy to investigate the single-cell and spatiotemporal evolution of TECs within the microenvironment of HCC tumors showcasing varied responses to immunotherapy. Through an analysis of a clinical cohort of HCC patients, we explored the correlation between TEC subpopulations and immunotherapy outcomes. The influence of TEC subsets on the immune microenvironment was confirmed through comprehensive in vitro and in vivo studies. To further explore the mechanisms of distinct TEC subpopulations in microenvironmental modulation and their impact on immunotherapy, we utilized TEC subset-specific knockout mouse models as well as humanized mouse models. ResultsIn this research, we identified a new subset of CXCL12+ TECs that exert a crucial role in immune suppression within the HCC TME. Functionally, CXCL12+ TECs impede the differentiation of CD8+ naïve T cells into CD8+ cytotoxic T cells by secreting CXCL12. Furthermore, they attract myeloid-derived suppressor cells (MDSCs). A bispecific antibody was developed to target both CXCL12 and PD1 specifically, showing significant promise in bolstering anti-tumor immune responses and advancing HCC therapy. ConclusionsCXCL12+ TECs are pivotal in mediating immunosuppression within HCC microenvironment and targeting CXCL12+ TECs presents a promising approach to augment the efficacy of immunotherapies in HCC patients. Impact and implicationThis investigation reveals a pivotal mechanism in the HCC TME, where CXCL12+ TECs emerge as crucial modulators of immune suppression. The discovery of CXCL12+ TECs as inhibitors of CD8+ naïve T cell activation and recruiters of MDSCs significantly advances our grasp of the dynamic between HCC and immune regulation. Moreover, the development and application of a bispecific antibody precisely targeting CXCL12 and PD1 has proven to enhance immune responses in a humanized mouse HCC model. This finding underscores a promising therapeutic direction for HCC, offering the potential to amplify the impact of current immunotherapies.

CXCL12 and CD3E as Indicators for Tumor Microenvironment Modulation in Bladder Cancer and Their Correlations With Immune Infiltration and Molecular Subtypes.

Bladder cancer (BLCA) represents the ninth most common malignant tumor in the world and is characterized by high recurrence risk. Tumor microenvironment (TME) plays an important role in regulating the progression of BLCA. Immunotherapy, including Bacillus Calmette-Guerin (BCG) and programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1), is closely associated with TME and is widely used for treating BLCA. But parts of BLCA patients have no response to these treatment ways, thus a better understanding of the complex TME of BLCA is still needed. We downloaded the gene expression profile and corresponding clinical information of 414 BLCA patients from the TCGA database. Via the ESTIMATE and CIBERSORT algorithm, we identified the two hub genes (CXCL12 and CD3E) and explored their correlations with immune infiltration. We found that BLCA patients with higher expression of CXCL12 and lower expression of CD3E had prolonged survival. Gene set enrichment analysis (GSEA) revealed that both CXCL12 and CD3E were enriched in immune-related pathways. We also discovered that stromal score and the level of CXCL12 were higher in luminal subtype, and immune score and the level of CD3E were higher in the basal subtype. Furtherly, we found that CXCL12 was associated with naive B cells, resting mast cell, M2 macrophages, follicular helper T cells, and dendritic cells. CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), and macrophages were correlated with CD3E. In conclusions, we found that CXCL12 and CD3E might serve as indicators of TME modulation in BLCA. Therapy targeting CXCL12 and CD3E had the potential as novel therapeutic strategy.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as important mediators of intercellular communication in response to cartilage damage. In this study, we sought to characterize the inhibitory role of microRNA (miR)-31 encapsulated in synovial MSC (SMSC)-derived EVs in knee osteoarthritis (OA). The expression of miR-31, lysine demethylase 2A (KDM2A), E2F transcription factor 1 (E2F1), and pituitary tumor transforming gene 1 (PTTG1) was validated in cartilage tissues of knee OA patients. Following SMSC-EV extraction and identification, chondrocytes with the miR-31 inhibitor were added with SMSC-EVs, whereupon the effects of miR-31 on proliferation and migration of chondrocytes were assessed. The interaction among miR-31, KDM2A, E2F1, and PTTG1 in chondrocyte activities was probed in vitro, along with an in vivo mouse knee OA model. We identified downregulated miR-31, E2F1, and PTTG1 and upregulated KDM2A in cartilage tissues of knee OA patients. SMSC-EV-packaged miR-31 potentiated chondrocyte proliferation and migration as well as cartilage formation by targeting KDM2A. Mechanistically, KDM2A bound to the transcription factor E2F1 and inhibited its transcriptional activity. Enrichment of E2F1 in the PTTG1 promoter region activated PTTG1 transcription, accelerating chondrocyte proliferation and migration. SMSC-EVs and EVs from miR-31-overexpressed SMSCs alleviated cartilage damage and inflammation in knee joints in vivo. SMSC-EV-encapsulated miR-31 ameliorates knee OA via the KDM2A/E2F1/PTTG1 axis.

CXCR4‐Enriched Nano‐Trap Targeting CXCL12 in Lung for Early Prevention and Enhanced Photodynamic Therapy of Breast Cancer Metastasis

AbstractBreast cancer metastasis is strongly correlated with CXCR4‐CXCL12 axis, in which the CXCR4 positive cancer cells are recruited to target organs rich in CXCL12. Although various agents have been developed to inhibit CXCR4, few strategies are reported for targeting and perturbation of CXCL12 to control breast cancer metastasis. Inspired by the increasing popularity of cell membrane (CM)‐derived therapeutics, herein, CXCR4‐enriched 4T1 CMs loaded with copper‐indium‐sulfide quantum dots (QDs) nanoparticles are employed as Nano‐trap to occupy CXCL12 and block breast cancer lung metastasis. CMs fused onto QDs cores faithfully inherit CXCR4 expression of the source cells. CXCR4‐upregulated Nano‐trap binds CXCL12 protein more efficiently than the CXCR4‐silenced counterparts, which effectively abrogate CXCL12‐mediated cancer cell invasion in vitro. In vivo fluorescent imaging reveals preferential distribution of Nano‐trap in lungs with abundant CXCL12 expression. Further interrogation of the in vivo efficacy finds lung metastasis is successfully delayed in breast cancer models pre‐injected with Nano‐trap, which reduces CXCL12 exposure in lung. For the already formed lung metastasis, Nano‐trap can alleviate hypoxia by regulating alpha‐smooth muscle actin, thus improving photodynamic therapy in the metastatic tumor. This proof‐of‐concept study sheds light on exploiting more functionalities of CM proteins for metastasis management.

Hypoxic preconditioning improves the survival and neural effects of transplanted mesenchymal stem cells via CXCL12/CXCR4 signalling in a rat model of cerebral infarction.

The treatment of neural deficiency after cerebral infarction is challenging, with limited therapeutic options. The transplantation of mesenchymal stem cells (MSCs) to the ischemic penumbra is a potential therapeutic approach. In the present study, a cerebral infarction model was generated by performing middle cerebral artery occlusion (MCAO) in SD rats. The expression of CXCR4 increased, and the number of MSCs migrating to the peri-infarct area was higher in rats transplanted with preconditioned MSCs than in rats transplanted with untreated MSCs. The rate of apoptosis, as evaluated by TUNEL staining and immunoblotting assays, was reduced in rats receiving preconditioned MSCs. A significant amelioration of neural regeneration and improved neurological function were observed in rats injected with preconditioned MSCs compared with those injected with untreated MSCs. However, the application of an siRNA targeting CXCL12 significantly inhibited the protective role of preconditioned MSCs against apoptosis and promoted the migration of MSCs to the ischemic area, leading to impaired neuronal regeneration and limited recovery of neuronal function. Hypoxic preconditioning of MSCs prior to transplantation suppressed apoptosis and increased their migration abilities, leading to the promotion of neuronal regeneration and improvement in neural function after transplantation. This preconditioning strategy may be considered as a potential approach for the modification of MSCs prior to cell transplantation therapy in patients with cerebral infarction. SIGNIFICANCE OF THE STUDY: We found that hypoxic preconditioning of MSCs improved their ability to promote neuronal regeneration and the recovery of neuronal function. Moreover, we showed that CXCR4 inhibited apoptosis, improved cell homing, and promoted neuronal differentiation, without influencing angiogenesis. Our study provides a relatively safe preconditioning method for potential use for cell transplantation therapy in ischemic cerebral infarction. The results presented here will facilitate the development of novel strategies and techniques to improve the tolerance and migration ability of transplanted cells for the treatment of cerebral infarction sequelae.

Abstract A156: PDA tumor cell death as following combination anti-PD-1 blockade and CXCR4 blockade is a direct effect of CD8+ T-cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States. Despite the fact that the human PDA microenvironment contains a mixture of infiltrating immune cells, including activated effector CD8+ T-cells, attempts to apply immune checkpoint inhibitor therapy have not yet been successful in achieving a clinical response (1). Combining blockade of the immune checkpoint receptor PD-1 with blockade of the chemokine receptor CXCR4 in a mouse model of PDA produced T-cell-mediated tumor cell killing (2). We therefore hypothesized that, in a human PDA slice culture model system that maintains the intact tumor microenvironment (3), combination of CXCR4 blockade with PD-1 blockade would lead to anti-tumor T-cell activation. Methods: Cores (6 mm) were taken from freshly resected, sterile human PDA specimens and cut into 250 µm thick slices using a vibratome. The slices were treated with either IgG antibody isotype control, AMD3100 (a CXCR4 blocking small molecule drug) plus antibody isotype control, anti-PD-1 antibody, or a combination of anti-PD-1 antibody and AMD3100 for two days. The slices were then stained with fluorescently labelled antibodies for CD8 and EpCAM, as well as with SR-FLICA, a reagent that produces a fluorescent signal when bound to activated Caspase 3 and 7 enzymes. Live slices were maintained in a humidified, temperature-controlled CO2 chamber for time-lapse confocal imaging for 1 hour. Similarly, untreated slices were imaged for 90 minutes prior to and following combined anti-PD-1 and AMD3100 drug treatment. Each slice was imaged at 3 different positions to observe CD8+ T-cells, EpCAM+ tumor cells, and SR-FLICA+ apoptotic cells throughout the slice. Each cell type was counted at each position over the entire time course imaged. Results: Compared to antibody isotype control or monotherapy, slices treated with combined PD-1 and CXCR4 blockade contained a greater fraction of EpCAM+ tumor cells that were undergoing apoptosis, indicated by SR-FLICA positivity. This treatment also increased the proportion of EpCAM+ tumor cells with a CD8+ T-cell within 20 µm, and EpCAM+ cells had increased evidence of apoptosis when a CD8+ cell was nearby. When individual slices were imaged before and after combined PD-1 and CXCR4 blockade, a greater proportion of EpCAM+ cells were found to have a CD8+ cell nearby after treatment compared to before treatment, and the tumor cells with a CD8+ cell nearby were more like to be undergoing apoptosis compared to before treatment. Conclusion: Direct visualization of live pancreatic cancer slice cultures demonstrates that combined PD-1 and CXCR4 blockade enhances both the migration of CD8+ T-cells towards epithelial tumor cells and their anti-tumor cytotoxic activity.

Epigenetic Regulation of CXCL12 Plays a Critical Role in Mediating Tumor Progression and the Immune Response In Osteosarcoma.

The mechanism by which osteosarcomas metastasize is elusive, and challenges remain regarding its treatment with modalities including immunotherapy. CXCL12 is deeply involved in the process of tumor metastasis and T-cell homing, which is driven by a chemokine gradient, but healthy bones are supposed to preferentially express CXCL12. Here, we show for the first time that osteosarcomas epigenetically downregulate CXCL12 expression via DNA methyltransferase 1 (DNMT1) and consequently acquire the ability to metastasize and to impair cytotoxic T-cell homing to the tumor site. Analysis of human osteosarcoma cases further revealed that CXCL12 expression strongly correlated with overall survival. Evaluations on fresh human chemotherapy-free osteosarcoma samples also showed a positive correlation between CXCL12 concentration and the number of intratumoral lymphocytes. Critically, treatment targeting DNMT1 in immunocompetent mouse models significantly elevated expression of CXCL12 in tumors, resulting in a robust immune response and consequently eradicating early lung metastases in addition to suppressing subcutaneous tumor growth. These antitumor effects were abrogated by CXCL12-CXCR4 blockade or CD8+ T-cell depletion. Collectively, our data show that CXCL12 regulation plays a significant role in both tumor progression and immune response, and targeting CXCL12 is promising for therapeutics against osteosarcoma.Significance: Epigenetic regulation of CXCL12 controls metastasis and immune response in osteosarcoma, suggesting epigenetic therapies or therapies targeting CXCL12 have potential for therapeutic intervention in osteosarcoma. Cancer Res; 78(14); 3938-53. ©2018 AACR.

Abstract 4502: Cxcl-12-cxcr4 inhibition enhances immune response in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma, one of the most aggressive malignancies and is refractory to Immunotherapy. This, in part, is mediated by the fibrotic stroma, creating an immune-suppressive microenvironment. CXCL12/SDF-1 has been shown to act as a chemo-repellant in several cancers. Therefore, we evaluated the role of stromal CXCL-12 in mediating an immunosuppressive phenotype within the pancreatic tumor. Methods: Pancreatic cancer cells harvested from KPC, a genetically engineered mouse model for PDAC, were either injected or alone or co-injected with pancreatic stellate cells (PSCs) extracted from WT (C57/BL6) mice to induce tumors. SDF-1 expression levels were detected by IF and q-RT PCR in tumor sections. An SDF-1 inhibitor, AMD3100 (2mg/kg), or vehicle was administered i.p for 21 days in tumor bearing mice and tumor growth and immune infiltration were assessed as an end point by flow cytometry. To evaluate the T cell migration in vitro, PSCs were pre-treated with SDF-1 neutralizing antibody, educated with KPC cancer cells and further co-cultured with splenic CD3+ T cells extracted from KPC mice. Results: In vitro results showed markedly increased secretion of SDF-1 by PSCs and neutralization of SDF-1 led to increased T-cell migration when cultured with cancer cells alone. We also evaluated the effect of inhibition of SDF-CXCR4 axis on tumor growth. Co-injection of KPC with WT PSC formed larger tumors with increased section of CXCL-12 as compared to when KPC cells were injected alone. Interestingly, Inhibition of SDF-1-CXCR4 interaction with AMD3100 led to reduced tumor growth with increased infiltration of activated CD8+ cytotoxic T cells as compared to the saline-treated mice. Conclusion: Our results indicate that stromal cells contribute to tumor progression by blocking the infiltration of cytotoxic T cells via secretion of CXCL-12. Targeting CXCL-12 could pave the way for designing better therapeutic interventions against pancreatic cancer. Citation Format: Bharti Garg, Anthony Ferrantella, Bhuwan Giri, Shrey Modi, Vrisketan Sethi, Saba Kurtom, Sulagna Banerjee, Ramakrishnan Sundaram, Eli Gilboa, Ashok Saluja, Vikas Dudeja. Cxcl-12-cxcr4 inhibition enhances immune response in pancreatic ductal adenocarcinoma [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 4502.

Cardiomyocyte-derived CXCL12 is not involved in cardiogenesis but plays a crucial role in myocardial infarction.

The chemokine CXCL12/SDF-1 is crucial for heart development and affects cardiac repair processes due to its ability to attract leukocytes and stem cells to injured myocardium. However, there is a great controversy whether CXCL12 is beneficial or detrimental after myocardial infarction (MI). The divergence in the reported CXCL12 actions may be due to the cellular source and time of release of the chemokine after MI. This study was designed to evaluate the role of cardiomyocyte-derived CXCL12 for cardiogenesis and heart repair after MI. We generated two rodent models each targeting CXCL12 in only one cardiac cell type: cardiomyocyte-specific CXCL12-overexpressing transgenic (Tg) rats and CXCL12 conditional knockout (cKO) mice. Animals of both models did not show any signs of cardiac abnormalities under baseline conditions. After induction of MI, cKO mice displayed preserved cardiac function and remodeling. Moreover, fibrosis was less pronounced in the hearts of cKO mice after MI. Accordingly, CXCL12 Tg rats revealed impaired cardiac function post-MI accompanied by enhanced fibrosis. Furthermore, we observed decreased numbers of infiltrating Th1 cells in the hearts of cKO mice. Collectively, our findings demonstrate that cardiomyocyte-derived CXCL12 is not involved in cardiac development but has adverse effects on the heart after injury via promotion of inflammation and fibrosis. • CXCL12 in cardiomyocytes is not involved in cardiac development. • CXCL12 deficiency in cardiomyocytes improves outcome of myocardial infarction. • CXCL12 overexpression in cardiomyocytes worsens outcome of myocardial infarction. • CXCL12 increases fibrosis and invasion of Th1 cells in the heart after infarction.

Pancreatic cancer microenvironment, to target or not to target?

We have collectively been spoiled by the astounding clinical benefit of antimicrobials. Much like the discovery and use of penicillin to eradicate once deadly infections, we continue to desperately search for the next “magic bullet” to kill cancer while sparing the non‐transformed cells. Greater appreciation for the molecular intricacies of malignancy has resulted in dedicated pursuit of cancer genomics and large‐scale informatics to identify “drugable” targets within the cancer cell itself. However, studies at the bench elucidating a dynamic relationship between tumor and microenvironment have become more common and demonstrate promise for novel therapeutic intervention.

Tumor-Promoting Effects of Myeloid-Derived Suppressor Cells Are Potentiated by Hypoxia-Induced Expression of miR-210.

Myeloid-derived suppressor cells (MDSC) contribute significantly to the malignant characters conferred by hypoxic tumor microenvironments. However, selective biomarkers of MDSC function in this critical setting have not been defined. Here, we report that miR-210 expression is elevated by hypoxia-inducible factor-1α (HIF1α) in MDSC localized to tumors, compared with splenic MDSC from tumor-bearing mice. In tumor MDSC, we determined that HIF1α was bound directly to a transcriptionally active hypoxia-response element in the miR-210 proximal promoter. miR-210 overexpression was sufficient to enhance MDSC-mediated T-cell suppression under normoxic conditions, while targeting hypoxia-induced miR-210 was sufficient to decrease MDSC function against T cells. Mechanistic investigations revealed that miR-210 modulated MDSC function by increasing arginase activity and nitric oxide production, without affecting reactive oxygen species, IL6, or IL10 production or expression of PD-L1. In splenic MDSC, miR-210 regulated Arg1, Cxcl12, and IL16 at the levels of both mRNA and protein, the reversal of which under normoxic conditions decreased T-cell-suppressive effects and IFNγ production. Interestingly, miR-210 overexpression or targeting IL16 or CXCL12 enhanced the immunosuppressive activity of MDSC in vivo, resulting in increased tumor growth. Taken together, these results provide a preclinical rationale to explore miR-210 inhibitory oligonucleotides as adjuvants to boost immunotherapeutic responses in cancer patients.

MiR-101 represses lung cancer by inhibiting interaction of fibroblasts and cancer cells by down-regulating CXCL12.

Cancer-associated fibroblasts (CAFs) are the main component of tumor stroma which support tumor progression. Here, we set out to determine the factors that may be involved in dramatic alteration of microRNAs (miRNAs) expression pattern in CAFs. miRNAs analyses identified differential expression of 15 microRNAs, with miR-101 being the most downregulated miRNA in CAFs which were different from the normal fibroblasts. We examined several putative miR-101 target genes identified by microarray analysis and demonstrated that miR-101 directly targets CXCL12, which play important roles in CAFs. Overexpression of miR-101 significantly impaired the ability of CAFs to stimulate tumor cell proliferation, sphere formation migration and invasion, and enhanced apoptosis. Further research showed that the cellular biological behavior was regulated by miR-101 targeting CXCL12. These findings provide new insights miR-101 down-regulation in CAFs could inhibit lung cancer proliferation and metastasis via targeting CXCL12.

Abstract 3187: Depleting cells expressing fibroblast activation protein disrupts tumor-promoting desmoplasia

Abstract Solid tumors recruit and activate a heterogeneous population of non-transformed stromal cells (CASCs) in the tumor microenvironment (TME). There has been growing interest in the field to develop strategies to treat cancer and circumvent resistance to therapy by targeting CASCs. Defining the phenotypic subsets and the function of distinct subpopulations including myofibroblasts (defined by the expression of SMA) and the overlapping but distinct population of CASCs expressing Fibroblast Activation Protein (FAP) will be critical to informing the rational design of stromal-targeted therapies. FAP is a membrane serine protease that is selectively up-regulated in CASCs including cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and a subset of M2 macrophages, of virtually all common human epithelial cancers. Based on its selective expression on CASCs across a wide range of tumor types, we engineered FAP-specific chimeric antigen receptor expressing T cells (FAP-CAR T cells) and adoptively transferred them into tumor bearing mice to: 1) define the requirement for FAP+ cells for the accumulation and maintenance of a desmoplastic matrix; 2) define the relationship between the degree of desmoplasia, the capacity of FAP-CAR T cells to disrupt the tumor stroma and to inhibit tumor growth; and 3) determine whether FAP-CAR T cells can inhibit tumor growth through immune independent mechanisms. We found that FAP+ CACSs are responsible for maintaining the desmoplastic response and that conditional depletion following treatment with FAP-CAR T cells inhibits growth of human lung cancer xenografts and mouse pancreatic tumors in an immune-independent fashion. Taken together with recently published studies (1-3), our data provide important distinctions between the function of FAP+ CASCs and SMA+ myofibroblasts and provide support for further development of FAP+ stromal cell-targeted therapies for a wide range of solid tumors. Reference: 1. Feig, C., Jones, J. O., Kraman, M., Wells, R. J., Deonarine, A., Chan, D. S., et al. (2013). Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA 110, 20212-20217. 2. Ozdemir, B. C., Pentcheva-Hoang, T., Carstens, J. L., Zheng, X., Wu, C. C., Simpson, T. R., et al. (2014). Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25, 719-734. 3. Wang, L. C., Lo, A., Scholler, J., Sun, J., Majumdar, R. S., Kapoor, V., et al. (2014). Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity. Cancer Immunol Res 2, 154-166. Citation Format: Albert Lo, Liang-Chuan S Wang, John Scholler, James Monslow, Diana Avery, Rebecca A. Evans, David J. Bajor, Amy C. Durham, Elizabeth L. Buza, Robert H. Vonderheide, Carl H. June, Steven M. Albelda, Ellen Puré. Depleting cells expressing fibroblast activation protein disrupts tumor-promoting desmoplasia. [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 3187. doi:10.1158/1538-7445.AM2015-3187

Abstract A61: Exploring the PDAC-subtype-associated microenvironment in PDX models and patients

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis (1, 2). Despite extensive research and the discovery of several promising drug candidates, little progress in PDAC treatment has been reported in the last years (3, 4). Two facts can be behind these disappointing results. In one hand, although PDAC is still clinically treated as a single disease, three distinct molecular subtypes (classical, quasi-mesenchymal and exocrine) were recently described based on expression profiling of microdissected epithelial tumor cells of PDAC samples (5). Interestingly, in vitro studies revealed differences in drug response of two of the proposed subtypes for which cell lines were available. These results highlight the importance of considering PDAC as a heterogeneous disease and point to the stratification of patients as a possible way to improve PDAC treatment response. On another hand, an additional reason behind the limited efficacy of PDAC treatment might be the tumor microenvironment. PDAC is the solid tumor with the highest stromal content, which can account for up to 90% of the total tumor mass. The PDAC microenvironment is known to actively affect tumorigenesis (6) and to impair drug delivery (7). Thus, rendering the PDAC microenvironment as an appealing therapeutic target to improve PDAC care (8). We have developed a novel workflow to efficiently generate patient-derived orthotopic xenografts (PDX) and serum-free cell cultures from primary resected PDAC tumors. The established primary cell lines comprise for first time all three described PDAC subtypes. Additionally, when re-injected into immunodeficient mice, these cells generate xenografts with high pathological similarity to the original patient tumor, including a prominent stromal presence. To explore the differences in the microenvironment associated to the individual PDAC subtypes we have now generated gene expression profiles for the stroma of a number of xenografts from our PDX model representing all three subtypes. Besides, RNA sequencing from different sub-populations isolated from fresh primary human PDAC tumors (as separated by fluorescent activated cell sorting according to surface markers) may reveal interesting interactions between the different tumor compartments. We have developed a set of immunohistochemical markers to identify the PDAC-subtypes that can be used in patient paraffin sections. Hence, the RNAseq data of the different tumor populations can be also easily studied in the context of the different subtypes. We believe that these approaches will shed some light on how different stromal expression patterns are interconnected with different epithelial expression profiles and vice versa, and how this information can be ultimately exploited for patient stratification and therapy. 1. Hidalgo M. Pancreatic cancer. N Engl J Med 2010 2. Vincent A et al. Pancreatic cancer. Lancet, 2011 3. Werner J et al. Advanced-stage pancreatic cancer: therapy options. Nature Reviews Clinical Oncology, 2013 4. Hidalgo M et al. Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas. Clin Cancer Res, 2012 5. Collisson EA et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med, 2011 6. Feig C et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblastssynergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA, 2013 7. Provenzano PP et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell, 2012 8. Neesse A et al. Emerging concepts in pancreatic cancer medicine: targeting the tumor stroma. Onco Targets Ther, 2013 Citation Format: Elisa Espinet Espinet, Christian Eisen, Elisa M. Noll, Vanessa Vogel, Corinna Klein, Zuguang Gu, Matthias Schlesner, Tobias Bauer, Nathalia Giese, Roland Eils, Jens Werner, Wilko Weichert, Martin R. Sprick, Andreas Trumpp. Exploring the PDAC-subtype-associated microenvironment in PDX models and patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A61.

Therapeutic aptamers: developmental potential as anticancer drugs.

Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a second targeting CXCL12 - are currently undergoing clinical trials for treating cancer patients, and many more are under study. In this mini-review, we present the current clinical status of aptamers and aptamer-based cancer therapeutics. We also discuss advantages, limitations, and prospects for aptamers as cancer therapeutics. [BMB Reports 2015; 48(4): 234-237]

Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12

Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12β, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.

Mode of Action of the SDF-1/CXCL12 Inhibiting Spiegelmer® Nox-A12 and Its Impact On Chronic Lymphocytic Leukemia (CLL) Cell Motility and Chemosensitization

Abstract 318NOX-A12 is a novel Spiegelmer®-based antagonist of SDF-1/CXCL12, a chemokine involved in the regulation of chronic lymphocytic leukemia (CLL) cell trafficking. Spiegelmers® are mirror-image oligonucleotides that are identified to specifically bind to proteins in a manner conceptually similar to antibodies. Unlike aptamers, however, Spiegelmers® are built from the non-natural L-isomer form of nucleotides which confers resistance to the action of nucleases and avoids potential immunogenicity.CXCL12 is constitutively secreted and presented by bone marrow stromal cells (BMSC) via glycosaminoglycans (GAG) and acts as a homing factor for normal and malignant hematopoietic cells to the bone marrow (BM) and secondary lymphoid tissues via CXCR4 receptors that are expressed at high levels on circulating CLL cells. The microenvironment in the BM and secondary lymphoid tissues, in particular the CXCL12-CXCR4 axis, favors survival and chemotherapy-resistance of leukemic cells. We therefore investigated the effects of NOX-A12 in an in vitro co-culture system to model the interaction of CLL cells with their microenvironment.Surprisingly we observed that NOX-A12 increased pseudoemperipolesis in vitro, i.e. spontaneous leukemia cell migration beneath BMSC. Interestingly, this NOX-A12 induced trans-migration of CLL cells was completely inhibited by the CXCR4 antagonist AMD3100, suggesting a CXCL12/CXCR4 dependent mechanism. We postulated that this observation might result from a direct effect of NOX-A12 on CXCL12 release by the stromal cells. Therefore, we investigated this hypothesis in different BMSC lines (MS-5, R15C, and TSt-4) and we found that NOX-A12 induced a significant CXCL12 release in all three tested cell lines.We asked whether this NOX-A12 dependent increase of CXCL12 of BMSCs is due to release from either intracellular or extracellular storages. Intracellular staining of CXCL12 using flow cytometry did not reveal significant changes when BMSCs were incubated with NOX-A12. Furthermore, the transcription of CXCL12 was not found to be altered after NOX-A12 incubation over a period of three days as shown by quantitative RT-PCR. Rather, CXCL12 is released from extracellular storages of BMSCs. First hints were obtained through a rapid CXCL12 release within five minutes of incubation with NOX-A12. To confirm that CXCL12 is bound to the extracellular surface (by GAGs like heparin) and is being detached by NOX-A12 we first incubated BMSCs with NOX-A12, followed by a wash step and the addition of recombinant CXCL12. Recombinant CXCL12 was bound by BMSCs that were pre-incubated with NOX-A12 but not with a non-functional control (revNOX-A12), indicating that NOX-A12 strips off CXCL12. To corroborate the findings we incubated the BMSCs with heparin which also led to the release of CXCL12 in a dose dependent manner. Of note, the EC50 of heparin regarding CXCL12 release was much higher compared to the EC50 of NOX-A12 (≈ 12 μM vs. 5 nM) revealing the high affinity of NOX-A12 to CXCL12. The competition of NOX-A12 with heparin regarding CXCL12 binding was confirmed by Biacore experiments.Based on these findings, we developed a novel adapted co-culture approach to examine the ability of NOX-A12 to chemosensitize CLL cells. In this setting, we first strip off CXCL12 from BMSCs by NOX-A12 and subsequently add CLL cells which will be either non-treated or treated with chemotherapy (fludarabine combined with bendamustine). We found that NOX-A12 slightly decreased CLL cell viability. As expected, a strong viability decrease was observed with chemotherapy, which could be even further decreased by the combination with NOX-A12, suggesting synergistic effects.In conclusion, we propose that NOX-A12's mode of action is the release of extracellular bound CXCL12 and its subsequent inhibition. Since CXCL12 induces leukemia cell trafficking and homing to tissue microenvironment and also favors leukemia cell survival, we believe that targeting CXCL12 is an attractive approach to remove the protective effects of CXCL12-secreting BMSCs in order to sensitize CLL cells for subsequent chemotherapy. Thus, NOX-A12 represents a very promising agent to significantly improve the treatment of CLL. The compound is currently being tested in a Phase IIa study in relapsed CLL patients. Disclosures:Zboralski:NOXXON Pharma AG, Berlin, Germany: Employment. Maasch:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Burger:NOXXON Pharma AG: Consultancy, Research Funding.

Gap Junction–Mediated Import of MicroRNA from Bone Marrow Stromal Cells Can Elicit Cell Cycle Quiescence in Breast Cancer Cells

Bone marrow (BM) metastasis of breast cancer (BC) can recur even decades after initial diagnosis and treatment, implying the long-term survival of disseminated cancer cells in a dormant state. Here we investigated the role of microRNAs (miRNA) transmitted from BM stroma to BC cells via gap junctions and exosomes in tumor cell quiescence. MDA-MB-231 and T47D BC cells arrest in G(0) phase of the cell cycle when cocultured with BM stroma. Analyses of miRNA expression profiles identified numerous miRNAs implicated in cell proliferation including miR-127, -197, -222, and -223 targeting CXCL12. Subsequently, we showed that these CXCL12-specific miRNAs are transported from BM stroma to BC cells via gap junctions, leading to reduced CXCL12 levels and decreased proliferation. Stroma-derived exosomes containing miRNAs also contributed to BC cell quiescence, although to a lesser degree than miRNAs transmitted via gap junctions. This study shows that the transfer of miRNAs from BM stroma to BC cells might play a role in the dormancy of BM metastases.