Dalia Martinez-Marin

AFM nano-mechanics and calcium dynamics of prostate cancer cells with distinct metastatic potential

BACKGROUND Despite recent advances, it is not clear to correlate the mechanical compliances and the metastatic potential of cancer cells. In this study, we investigated combined signatures of mechanical compliances, adhesions, and calcium dynamics correlated with the metastatic potential of cancer cells. SCOPE OF REVIEW We used the lowly (LNCaP) and highly (CL-1, CL-2) metastatic human prostate cancer cells. The AFM-based nanomechanics was performed to determine the elastic moduli and the cell-to-substrate adhesion. The intracellular calcium dynamics was evaluated by fluorescence spectroscopy. Cell migration and the distribution of cytoskeleton were evaluated using the wounded monolayer model and immunofluorescence, respectively. The elastic moduli, the calcium dynamics, and the migratory ability are greater in CL-1 and CL-2 than LNCaP. CL-1 and CL-2 also display a significantly larger area of cell-to-substrate adhesions while the LNCaP displays a limited adhesion. These properties were slightly reduced in CL-2 compared with CL-1 cells. The enhanced elastic moduli and calcium dynamics found in CL-1 and CL-2 can be consistently explained by the intensified tensile stress generated by actin cytoskeletons anchored at more focal adhesion sites. MAJOR CONCLUSIONS Although the suppressed mechanical compliance of highly metastatic cells may not support the enhanced cancer metastasis, the enhanced adhesion and calcium dynamics are favorable for invasion and extra-vasation required for malignant progression. GENERAL SIGNIFICANCE Our results suggest that the mechanical compliance alone may fail to indicate the metastatic progression, but the combined biomechanical signatures of mechanical compliance, adhesion, and calcium dynamics can provide critical clues to determine the metastatic potential of cells.

Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These “non-metastatic” exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.Exosomes are extracellular vesicles that can favor tumor development and metastasis. Here, the authors show that cancer exosomes may also exert a suppressive function; in fact, exosomes from non-metastatic melanoma cells can lead to the recruitment of patrolling monocytes, which clear cancer cells at the pre-metastatic niche.

Positive correlation between PEDF expression levels and macrophage density in the human prostate.

In this study, we investigated the capacity of pigment epithelium‐derived factor (PEDF) to modulate the recruitment and the differentiation of monocytes/macrophages both in vitro and in human prostate.

Pigment epithelium-derived factor expression prolongs survival and enhances the cytotoxicity of low-dose chemotherapy in castration-refractory prostate cancer

There is currently no cure for advanced castration-refractory prostate cancer (CRPC) despite the recent approval of several new therapeutic agents. We report here the anti-tumor effect of the angio-inhibitory pigment epithelium-derived factor (PEDF) in the metastatic LNCaP-derivative CRPC CL1 model and explore PEDF anti-neoplasic efficacy in combination with low-dose chemotherapy. Androgen-sensitive LNCaP and CRPC PC3 cell lines were examined as comparison. Using a retroviral expression system, we showed that PEDF limited the proliferation of all prostatic cell lines tested; an effect attributed to interleukin 8 (IL8)-CXCR1/IL8RA inhibition. PEDF also reduced the number and size of 3D tumor spheroids in vitro, but only induced cell differentiation in CRPC spheroids. Similarly, PEDF inhibited the migration of CRPC cells suggesting both anti-proliferative and anti-migratory functions. In vivo, PEDF decreased by 85% and 65% the growth of subcutaneous (s.c.) PC3 and CL1 tumors, respectively. In the CL1 orthotopic model, tumor intake with lethal metastases was found in all animals; nevertheless, PEDF prolonged the median survival of tumor-bearing mice (95% confidence interval: 53±0.001 to 57±1 days). Accordingly, PEDF delayed the emergence of skeletal-related event in intra-tibial xenografts. Next, we evaluated low-dose docetaxel (DTX; 5, 1, 0.5 mg/kg) or cyclophosphamide (CTX; 10–20 mg/kg) on established s.c. PC3 tumors that conditionally express PEDF anti-tumoral epitope/NT3. Although NT3–DTX-5 mg/kg combination was inefficient, NT3–DTX-1 mg/kg and -0.5 mg/kg inhibited by 95% and 87.8%, respectively, tumor growth compared with control and induced tumor stasis. Both NT3–CTX combinations were advantageous. Inversely, PEDF–DTX-5 mg/kg and PEDF–CTX-10 mg/kg delayed the most CL1 tumor growth (15, 11 and 5 days for PEDF–DTX-5 mg/kg, PEDF–CTX-10 mg/kg and single treatments, respectively) with elevated apoptosis and serum thrombospondin-1 as possible mechanism and marker, respectively. As well, both PEDF–CTX-10 mg/kg and PEDF–DTX-5 mg/kg prolonged significantly the survival of tumor-bearing mice compared with single treatments. Metastases were reduced in PEDF–DTX-5 mg/kg compared with other treatments, suggesting that PEDF–DTX delayed metastases formation. Our results advocate that PEDF/low-dose chemotherapy may represent a new therapeutic alternative for CRPC.

PEDF increases the tumoricidal activity of macrophages towards prostate cancer cells in vitro

Background Although inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment in vitro, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis. Methods RAW 264.7 and THP-1 cells, and BMDMs were grown in vitro as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry. Results We found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects were observed with P18 PEDF-derived peptide. Conclusions These data established that modulating the molecular interactions between macrophages and PCa cells using PEDF may be a promising strategy for PCa treatment.

Cabazitaxel regimens inhibit the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with various efficacy and toxicity

Taxanes chemotherapies represent the major therapeutic alternative for symptomatic mCRPC. While docetaxel is the most commonly prescribed Taxane for mCRPC; cabazitaxel has been approved for patients unresponsive to docetaxel. Still mCRPC remains incurable and patients often experience severe side effects. Recently, the FIRSTANA trial first demonstrated the absence of superiority in overall survival between cabazitaxel and docetaxel in mCRPC patients. Inversely, different toxicity were reported suggesting that cabazitaxel may provide a first line treatment option for some patients urging for a deeper characterization of cabazitaxel mechanisms of action as well as a re‐evaluation of cabazitaxel conventional dose and schedule. In this study, our goal was therefore to evaluate the anti‐tumor efficacy of various cabazitaxel regimens delivered as monotherapy or in combination with PEDF, a known anti‐angiogenic and anti‐neoplastic agent.

Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

Abstract 288: Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel

Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Still mCRPC continues to be incurable. Furthermore, patients often experience severe side effects, prompting a re-evaluation of conventional regimen. Past studies have demonstrated promise for Low-Dose Metronomic (LDM) chemotherapy; schedule defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Using CRPC cell lines (PC3, Du145 and the LNCaP-derivative CL1), we have shown that cabazitaxel-treated cells have a significantly lower EC50 compared to docetaxel, with Du145 cells presenting the greatest differences. In cell cycle analysis, both low-dose taxanes increased the sub-G1 cells population. However, the sub-G1 increase was significantly greater in cabazitaxel- than in docetaxel-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cabazitaxel than docetaxel validating a higher efficacy due to increased apoptosis. As other possible cell death mechanisms, autophagy and necrosis were investigated. Beclin1 expression levels remain unchanged in all three cell lines. In contrast, necrosis was stimulated in all the taxanes-treated cell lines in a dose dependent manner. In vivo, LDM cabazitaxel was significantly more efficient in delaying tumor growth than docetaxel. This effect was markedly increased when cabazitaxel was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF). Other results showed that PEDF and LDM chemotherapy combination induces more phagocytosis of CRPC cells when compared to single treatments. In conclusion, our data demonstrate a higher efficacy of cabazitaxel on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC. Citation Format: Courtney L. Jarvis, Thomas Nelius, Dalia Martinez-Marin, Srirupa Cheerla, Stephanie Filleur. Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel. [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 288.

MP66-04 CABAZITAXEL INHIBITS THE PROLIFERATION OF HUMAN CASTRATION-REFRACTORY PROSTATE CANCER CELLS IN VITRO AND ENHANCES THE ANTI-TUMOR PROPERTIES OF THE ANGIO-INHIBITORY PIGMENT EPITHELIUM-DERIVED FACTOR IN VIVO WITH A GREATER EFFICACY THAN DOCETAXEL

therapeutic response. The goal of this study was to evaluate and genetically characterize exosome derived RNA (exoRNA) isolated from blood of metastatic CRPC patients. METHODS: Whole blood samples from 18 consented clinically annotated mCRPC patients and 1 normal control were collected. Exosomes were isolated with ultracentrifugation and exoRNA extracted. Following library prep, paired-end sequencing was performed using Illumina Hi-Seq 2000. A bioinformatics pipeline was used for data prepossessing including alignment, duplicate removal, normalization and variant calling. Visualization and differential analyses were performed with SNP & Variation Suite v8.x. RESULTS: In exoRNA there is evidence of extensive chromosomal rearrangement resulting in a myriad of gene fusions and isoforms. Through preliminary analyses we identified 39 genes commonly expressed in the exosomes of these mCRPC patients. These include PDPK1, USP9X, MAGI2, HMGA2 and PTGFR all of which have been previously expressed in prostate cancer tissue. A diverse variety of lcnRNA, ncRNA and miRNA were also identified in circulating exosomes. Validation of these alterations and additional analyses evaluating translocations and splice variants, PCR validation, and/or direct tumor based nucleic acid assays is ongoing. CONCLUSIONS: These preliminary analyses of circulating exoRNA have identified gene expression signatures of several prostate cancer associated transcripts. Ultimately, the identification of PCa associated transcripts in plasma derived exosomes provides evidence that exosomes and exosomal cargo may serve as biomarker in CRPC patients. Exosomes and exoRNA may provide otherwise unattainable insight into tumor evolution and disease progression. Additional studies evaluating the clinical relevance and prognostic value of exosomal RNA will be critical for biomarker development.

Effect of cabazitaxel on the growth of human castration-refractory prostate cancer cells in vitro compared to docetaxel and on the anti-tumor properties of the angio-inhibitor PEDF in vivo.

205 Background: Docetaxel/DTX and cabazitaxel/CBZ have shown promise in the treatment of metastatic Castration-Refractory Prostate Cancer/mCPRC however, comparative studies are missing. Toxicities of these drugs are significant, urging the need to modify taxane regimens. Recently, low-dose metronomic/LDM treatments using conventional chemotherapeutic drugs have shown benefits in CPRC in improving the effect of anti-angiogenic agents. Previously, we have demonstrated that LDM-DTX in combination with PEDF curbs significantly CRPC growth, limits metastases formation and prolongs survival in vivo. In this study, we intended to compare the cytotoxic effect of CBZ and DTX on CRPC cells in vitro and CL1 tumors in vivo. Methods: PC3, DU145 cell lines were from ATCC.CL1 cells were obtained from androgen-deprived LNCaP cells. Cell proliferation was assessed by crystal violet staining and cell cycle analyses. In vitro cytotoxicity assays were performed on CL1 cells/RAW264.7 macrophages co-cultures treated with PEDF ...