Prostate Cancer Spheroids Research Articles

Abstract 2877: Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation

Abstract Introduction: docetaxel (DTX) represents the standard of care first line treatment of castration-resistant prostate cancer (PCa). However, the onset of systemic side effects hampers patient's compliance and DTX resistance invariably emerges, leading to disease relapse, suggesting the need of novel combination strategies. Cancer stem cells (CSC) drive PCa survival and metastasis and are thought to be responsible for the development of resistance to DTX. The mevalonate pathway (MVP) plays a critical role in PCa progression and is implicated in cell stemness, proliferation, and organ size regulation through the YAP-TAZ signaling axis. Here, we evaluate the combination treatment of valproic acid (VPA), an histone deacetylase (HDAC) inhibitor and the cholesterol-lowering drug simvastatin (SIM), an inhibitor of HMGCoA reductase (HMGCR), the rate limiting enzyme in MVP, alone and plus DTX in PCa models. Method: synergistic antiproliferative effects were assessed on LNCAP, 22Rv1, DU145, PC3 and DU14580 SIM-resistant cell lines and normal epithelial prostate EPN cells, by calculating combination index (CI) according to Chou and Talalay method. Apoptosis was measured by FACS analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. Proteins and genes expression was assessed by western blot and real time PCR analysis. In vivo experiments were performed on xenograft models in athymic mice. Cholesterol content was evaluated by nuclear magnetic resonance (1H-NMR). Results: synergistic antiproliferative and proapoptotic effect of VPA-SIM was observed in all PCa cell lines tested, except in EPN cells and was confirmed in PCa spheroids. SIM-dependent cholesterol content downmodulation was potentiated by VPA and mevalonic acid, the product of HMGCR activity, reverts all the antitumor combined effect, suggesting the involvement of MVP in the synergistic interaction. Mechanistically, the combination is able to induce a reduction of HMGCR mRNA expression and the inhibitory phosphorylation of HMGCR and YAP, followed by a reduction of CTGF, BRCA5 and Cyr61 YAP-target genes, as well as a reduction of a CSC-marker gene such as NANOg. Notably, the VPA-SIM combination, sensitizes PCa cells to DTX treatment in sensitive and DTX-resistant cells, as shown by CI calculation, apoptosis, and CSC enriched-spheroid experiments. The synergistic interaction of the triple combination was also confirmed in vivo in both DU145R80 and 22Rv1 xenograft models. Conclusions: Overall, this study suggests that the combination of two safe generic drugs such as VPA and SIM, may improve the therapeutic index of DTX and revert DTX-resistance, representing an innovative and feasible antitumor strategy for the treatment of prostate cancer that warrants further clinical evaluation. Citation Format: Federica Iannelli, Maria Serena Roca, Chiara Ciardiello, Simona De Rienzo, Rita Lombardi, Angela Sorice, Susan Costantini, Tania Moccia, Maria Rita Milone, Biagio Pucci, Alfredo Budillon, Francesca Bruzzese. Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation [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 2877.

Abstract 1165: A tissue-engineered bone mimetic in vitro model for monitoring metastatic PCa growth and therapy response

Abstract Despite recent advances in prostate cancer (PCa) treatment, the outcome of metastatic disease remains frequently fatal and the underlying biology poorly understood. Thus, the development of clinically relevant in vitro models to monitor PCa biology in organotypic bone-like environment is critical to uncover mechanisms of therapy resistance and identify more effective treatments. To establish a bone-mimetic culture, we combined the following components: (i) bioactive osteoblasts depositing bone-like calcified extracellular matrix, (ii) complex 3D surface geometries, (iii) multicellular tumor application as spheroids/organoids, and (iv) applicability for live-cell microscopy to monitor the development of lesions over time. Calcified polycaprolactone (PCL) scaffolds were functionalized with bone-derived human mesenchymal stem cells (hMSCs) differentiated to osteoblasts, to generate a 3D niche-like calcified scaffold. PCa spheroids (PC3, C4-2B, patient-derived xenografts) were on-planted and their growth and invasion longitudinally monitored by advanced microscopy. PCa spheroids seeded on the organotypic bone model could be maintained and expanded over weeks, sufficient for monitoring therapy response to docetaxel, a first-line therapy for advanced PCa. The bone mimetic culture further revealed resistance to docetaxel mainly at the invasive edges, through a mechanism depending on the presence of osteoblasts. Thus, this 3D in vitro organotypic model will be suitable for dissecting the physical and molecular PCa cell-osteoblast interaction involved in PCa growth and therapy resistance. Citation Format: Claudia Paindelli, Dietmar Hutmacher, Peter Friedl, Eleonora Dondossola. A tissue-engineered bone mimetic in vitro model for monitoring metastatic PCa growth and therapy response [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 1165.

Targeted AKT Inhibition in Prostate Cancer Cells and Spheroids Reduces Aerobic Glycolysis and Generation of Hyperpolarized [1-13C] Lactate.

The PI3K/AKT/mTOR (PAM) signaling pathway is frequently mutated in prostate cancer. Specific AKT inhibitors are now in advanced clinical trials, and this study investigates the effect of MK2206, a non-ATP-competitive inhibitor, on the cellular metabolism of prostate cancer cells. We observed a reduction in cell motility and aerobic glycolysis in prostate cancer cells with treatment. These changes were not accompanied by a reduction in the ratio of high-energy phosphates or a change in total protein levels of enzymes and transporters involved in glycolysis. However, a decreased ratio of NAD+/NADH was observed, motivating the use of hyperpolarized magnetic resonance spectroscopy (HP-MRS) to detect treatment response. Spectroscopic experiments were performed on tumor spheroids, 3D structures that self-organize in the presence of an extracellular matrix. Treated spheroids showed decreased lactate production with on-target inhibition confirmed using IHC, demonstrating that HP-MRS can be used to probe treatment response in prostate cancer spheroids and can provide a biomarker for treatment response. Mol Cancer Res; 16(3); 453-60. ©2018 AACR.

Patient-derived multicellular prostate cancer spheroids for in vitro cell culture and drug testing

Patient-derived multicellular prostate cancer spheroids for in vitro cell culture and drug testing

The Microwell-mesh: A high-throughput 3D prostate cancer spheroid and drug-testing platform

Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.

Abstract B51: Targeted nanoparticle approach to treating metastatic prostate cancer

Abstract Introduction: Treatment outcomes of bone metastatic prostate cancer remain poor despite an increasing number of therapeutic options approved in recent years. Limitations exist in the drugs that are approved, such as no overall survival benefits or serious dose-limiting toxicities. We have engineered a targeted nanoparticle (NP) system to deliver cabazitaxel to bone lesions. The amino-bisphosphonate, alendronate, was used as the bone-targeting moiety on the surface of the NP. The objective of this approach is to increase therapeutic payload at the site of bone metastasis to achieve an improved therapeutic index. Methods: Poly(lactic-co-glycolic acid) NPs were synthesized using a modified water-in-oil-in-water double-emulsion solvent evaporation technique followed by binding of alendronate to the surface of the NP. We characterized NPs for size and zeta potential using dynamic light scattering. Cabazitaxel encapsulation efficiency, drug loading, and release kinetics were optimized by quantification with HPLC analysis. In vitro cell viability studies were performed on C4-2B and PC3 prostate cancer cell lines. 3D prostate cancer spheroids were utilized as well to test cell viability and NP penetration. Ex vivo bone binding studies were used to test nanoparticle affinity for hydroxyapatite structure. In vivo tumor efficacy studies were carried out with male athymic nude mice implanted with intraosseous tumors consisting of PC3-luciferase cells. After bone tumors were established for 1 week, mice were treated via tail vein injection with either saline, free cabazitaxel, non-targeted NPs, or targeted NPs for one month (starting n=6 per group). Response was measured with in vivo bioluminescence imaging, tumor limb weight at conclusion of experiment, and mouse x-ray monitoring. In addition, animal behavior experiments were performed on all treatment groups to assess functional status through gait analysis and pain through von Frey filament assay. Results: NPs were successfully synthesized to mean size of 236 nm with a PDI of .120. Highest encapsulation of cabazitaxel into nanoparticle was 55.8% with a drug loading of 3.7%. Release kinetics showed a strong burst phase release over the first 8 hours and subsequent sustained release up to 72 hours. In vitro cell viability and 3D spheroid experiments showed targeted and nontargeted NP to be equivalent to free drug. Ex vivo bone binding experiment showed targeted NP had a 4-fold increase in binding to bone at 6 hours and an 8-fold increase in bone binding at 72 hours compared to nontargeted NP. Tumor efficacy experiment showed targeted NP and nontargeted NP had a statistically significant overall reduction in tumor measured by bioluminescence (P value < 0.005). Interestingly, mice treated with targeted NP had no bone lesions on x-ray, whereas 100% of mice in saline group, 100% of mice in cabazitaxel group, and 33% of mice in nontargeted NP group had bone lesions. Gait analysis did not show a statistically significant difference between groups. However, von Frey assay showed a significant reduction in relative response in the targeted NP group (P value < 0.005). Conclusion: We have successfully synthesized a bone-targeted NP system. In this project, we have shown that targeted NPs help maintain bone structure in tumor-burdened limbs as well as decrease tumor size. We have also shown that these targeted NPs reduce the relative response to von Frey filament pain stimulation in the tumor limb. This bone-targeted NP system is a promising potential therapeutic in developing improved treatments for bone-metastatic prostate cancer patients. Note: This abstract was not presented at the conference. Citation Format: Andrew Gdowski, Amalendu Ranjan, Marjana Sarker, Jamboor Vishwanatha. Targeted nanoparticle approach to treating metastatic prostate cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B51.

Abstract 4465: Glypican-1: A tumor suppressor or an oncogene in human bone metastatic prostate cancer cells

Abstract Glypican-1 (GPC-1) is a cell surface heparan sulfate proteoglycan that mediates cell proliferation, differentiation and migration. It also possess oncogenic functions in breast, pancreatic and lung cancer cells, and is a proposed biomarker for prostate cancer. Despite these data the function of GPC-1 in prostate cancer is not fully known. We addressed this gap-in-knowledge using in vitro and in vivo mouse models. We first showed that GPC-1 was overexpressed in bone metastatic prostate cancer cells (PC-3) but was not detected in lymph node metastatic cancer cells (LNCaP) and non-cancerous prostate cells (RWPE-1). Next, we inhibited GPC-1 expression using shRNA in bone metastatic cancer cells (PC-3), and verified inhibition using western blot analysis and qRT-PCR. Crystal violet staining, trans-well assays, matrigel colony cultures and adhesion assays were used to determine the effect of GPC-1 inhibition on cancer cell growth, migration, spheroid formation and cell adhesion. Inhibition of GPC-1 reduced prostate cancer cell growth, migration and spheroid formation; however, there were no changes in cell adhesion. These in vitro data suggested that GPC-1 played a role in prostate cancer progression by acting as an oncogene. We further investigated this possibility in vivo using athymic (NCr) mice xenografts. Interestingly, inhibition of GPC-1 in PC-3 cells appeared to increase tumor size as compared to scrambled controls. Further analysis showed that that inhibition of GPC-1 increased the tumor expression of MMP-9 and N-cadherin, but not E-cadherin. We hypothesized that the discrepancy between the in vitro and in vivo results may be mediated by interaction of PC-3 cells with the tumor microenvironment. We addressed this hypothesis by testing the effect of tumor conditioned media (TCM) from control and GPC-1 knockdown cells on human bone marrow derived mesenchymal stem cells (hMSC) and fibroblast (HS-27). Treatment of both hMSC and HS-27 cells with TCM from GPC-1 knockdown PC-3 cells increased MMP-9 and interestingly MMP-2 expression and activity as compared to control cells. We also detected increases in the expression of N-Cadherin in a co-culture model. Collectively, these data suggest that GPC-1 acts as an oncogene to enhance prostate cancer cell growth in vitro; however, these data also suggest the GPC-1 may act as a tumor suppressor in vivo. This discrepancy may be due to the interaction off GPC-1 with the tumor microenvironment, which would not be detected in single-cell culture models. Citation Format: Nhat D. Quach, Matthew Eggert, Deepraj Ghosh, Michelle R. Dawson, Robert Arnold, Brian S. Cummings. Glypican-1: A tumor suppressor or an oncogene in human bone metastatic prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2017-4465

Dormant cancer cells accumulate high protoporphyrin IX levels and are sensitive to 5-aminolevulinic acid-based photodynamic therapy.

Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to drive the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. However, the tendency to accumulate PpIX is not well understood. Patients with cancer can develop recurrent metastatic disease with latency periods. This pause can be explained by cancer dormancy. Here we created uniformly sized PC-3 prostate cancer spheroids using a 3D culture plate (EZSPHERE). We demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture. Dormant cancer cells accumulated high PpIX levels and were sensitive to ALA-PDT. In dormant cancer cells, transporter expressions of PEPT1, ALA importer, and ABCB6, an intermediate porphyrin transporter, were upregulated and that of ABCG2, a PpIX exporter, was downregulated. PpIX accumulation and ALA-PDT cytotoxicity were enhanced by G0/G1-phase arrestors in non-dormant cancer cells. Our results demonstrate that ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor.

Noninvasive Ablation of Prostate Cancer Spheroids Using Acoustically-Activated Nanodroplets.

We have developed acoustically activated nanodroplets (NDs) using an amphiphilic triblock copolymer, which self-assembles and encapsulates different perfluorocarbons including perfluoropentane (PFP) and perfluorohexane (PFH). Applying histotripsy pulses (i.e., short, high pressure, ultrasound pulses) to solutions of PFP- and PFH-NDs generated bubble clouds at a significantly reduced acoustic pressure compared to the cavitation pressure observed for histotripsy treatment alone. In this report, we summarize the results of combining histotripsy at low frequency (345 and 500 kHz) with PFP-NDs and PFH-NDs on the ablation of PC-3 and C4-2B prostate cancer cells. Using custom built histotripsy transducers coupled to a microscope and a high speed recording camera, we imaged the generation of a cavitation bubble cloud in response to different ultrasound regimes in solution and in tissue-mimicking gel phantoms. We quantified the associated ablation of individual cancer cells and 3D spheroids suspended in solution and embedded in tissue phantoms to compare the ablative capacity of PFP-NDs and PFH-NDs. Results show that histotripsy pulses at high acoustic pressure (26.2 MPa) ablated 80% of prostate cancer spheroids embedded in tissue-mimicking gel phantoms. In comparison, combining histotripsy pulses at a dramatically lower acoustic pressure (12.8 MPa) with PFP-NDs and PFH-NDs caused an ablation of 40% and 80% of the tumor spheroid volumes, respectively. These results show the potential of acoustically activated NDs as an image-guided ablative therapy for solid tumors and highlight the higher ablative capacity of PFH-NDs, which correlates with the boiling point of the encapsulated PFH and the stability of the formed bubble cloud.

Abstract 4745: Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance

Abstract Although docetaxel (DTX) remains a standard of care for advanced prostate cancer (PCa), limited long-term responses, side effects and resistant disease suggested the need of novel combination strategies. Increased expression of histone deacetylases (HDAC) and alteration of the mevalonate pathway (MVP) are common aberrations in PCa. In this study, we analyzed the antitumor effect of DTX in combination with valproic acid (VPA), an anticonvulsant with HDAC inhibitory activity, and simvastatin (SIM), a cholesterol-lowering drug inhibiting the rate-limiting enzyme of MVP HMG-CoA reductase, on androgen-dependent 22RV1 and LNCAP and androgen-independent PC3 and DU145 PCa cells, as well as on the highly aggressive SIM-resistant DU145R80 subline developed in our laboratory from DU145 cells (Milone MR et al. Cell Death Dis. 2013; Milone MR et al. Oncotarget 2014). We first showed a potent synergistic anti-proliferative effect of VPA/SIM combination, assessed by calculating combination index (CI) according to the method of Chou and Talalay, on all cell lines, including SIM-resistant cells, whatever schedule of administration (simultaneous vs sequential) we used, confirming our previous data on the combination between the HDAC inhibitor (HDACi) panobinostat and zoledronic acid, the latter also targeting the MVP pathway (Bruzzese F. et al, Cell Death Dis. 2013). Notably, exposure to triple combinations (VPA/SIM/DTX) resulted in a further strong synergistic anti-proliferative effect, with sequential exposure with 24h delay between VPA/SIM and DTX as the best schedule. The synergistic interaction of VPA/SIM and DTX combination involved apoptotic effect, measured by FACS analysis of sub-diploid DNA content and caspase 3/7 cleavage, and DNA damage induction, assessed by γH2AX expression. Synergistic effect of VPA/SIM and DTX combination was confirmed by soft agar clonogenic assay and by 3D culture on self-assembled PCa spheroids. Significantly, VPA/SIM combination was also able to revert DTX-resistance in DTX-resistant PC3 and DU145 sublines developed in our laboratory from the parental cells. All together these findings suggested that the combination of two safe generic drugs such as VPA and SIM can improve DTX efficacy, representing a novel therapeutic approach that warrant clinical investigation in advanced PCa patients. Our study also suggests a new strategy to overcome resistance to standard taxane-based therapy in PCa patients. Citation Format: Federica Iannelli, Rita Lombardi, Biagio Pucci, Maria Rita Milone, Chiara Ciardiello, Alessandra Leone, Elena Di Gennaro, Alfredo Budillon, Francesca Bruzzese. Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance. [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 4745.

Arum Palaestinum with isovanillin, linolenic acid and β-sitosterol inhibits prostate cancer spheroids and reduces the growth rate of prostate tumors in mice.

BackgroundArum palaestinum is a plant commonly found in the Middle East that is ingested as an herbal remedy to fight cancer. However, no studies have examined the direct effect of the plant/plant extract on tumor growth in an animal model.MethodsVerified prostate cancer cells were plated as 3D spheroids to determine the effect of extract from boiled Arum Palaestinum Boiss roots. In addition, male NU/NU mice (8 weeks old) with xenograft tumors derived from the prostate cancer cell line were treated daily with 1000 mg/kg body weight gavage of the suspension GZ17. The tumor growth was measured repeatedly with calipers and the excised tumors were weighed at the termination of the 3 week study. Control mice (10 mice in each group) received vehicle in the same manner and volume.ResultsThe number of live prostate cancer cells declined in a dose/dependent manner with a 24 h exposure to the extract at doses of 0.015 to 6.25 mg/mL. A fortified version of the extract (referred to as GZ17) that contained higher levels of isovanillin, linolenic acid and β-sitosterol had a stronger effect on the cell death rate, shifting the percentage of dead cells from 30 % to 55 % at the highest dose while the vehicle control had no effect on cell numbers. When GZ17 was applied to non-cancer tissue, in this case, human islets, there was no cell death at doses that were toxic to treated cancer cells. Preliminary toxicity studies were conducted on rats using an up-down design, with no signs of toxic effect at the highest dose. NU/NU mice with xenograft prostate tumors treated with GZ17 had a dramatic inhibition of tumor progression, while tumors in the control group grew steadily through the 3 weeks. The rate of tumor volume increase was 73 mm3/day for the vehicle group and 24 mm3/day for the GZ17 treated mice. While there was a trend towards lower excised tumor weight at study termination in the GZ17 treatment group, there was no statistical difference.ConclusionsFortified Arum palaestinum Boiss caused a reduction in live cells within prostate cancer spheroids and blocked tumor growth in xenografted prostate tumors in mice without signs of toxicity.

Effect of PEDF on the migration, M1 polarization, and phagocytic activity in macrophages: A new way to inhibit PCa?

e16058 Background: Macrophages have been described as one of the main inflammatory components involved in prostate cancer (PCa) development. PEDF (Pigment Epithelium-Derived Factor) is a secreted angio-inhibitor with differentiation and suggested immune-modulating activities. PEDF expression has been shown to be down-regulated in PCa compared to normal tissues. Previously, we have demonstrated that PEDF re-expression in PCa cells curbs tumor growth in vivo and prolongs the survival of tumor-bearing mice. Others have shown that PEDF increased the recruitment of tumor-cytotoxic macrophages into MatLyLu rat prostate tumors suggesting a link between PEDF and inflammation in PCa. However, PEDF modes of action on macrophages still remain unknown and need further investigation. Methods: THP-1, RAW264.7 and primary macrophages treated with PEDF, its derived active P18 peptide or ctrl were used as models. Migration was assessed using Boyden chamber and 3D PCa spheroid assays. M1/tumor-cytotoxic polarization was te...

Necrosis in DU145 prostate cancer spheroids induces COX‐2/mPGES‐1‐derived PGE2 to promote tumor growth and to inhibit T cell activation

Cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2 ) supports the growth of a spectrum of cancers. The potential benefit of COX-2-inhibiting non-steroidal anti-inflammatory drugs (NSAIDs) for cancer treatment is however limited by their well-known cardiovascular side-effects. Therefore, targeting microsomal PGE synthase 1 (mPGES-1), the downstream enzyme in the COX-2-dependent pathway of PGE2 production might be attractive, although conflicting data regarding a potential tumor-supporting function of mPGES-1 were reported. We determined the impact of mPGES-1 in human DU145 prostate cancer cell growth. Surprisingly, knockdown of mPGES-1 did not alter growth of DU145 monolayer cells, but efficiently inhibited the growth of DU145 multicellular tumor spheroids (MCTS). Opposed to MCTS, monolayer cells did not secrete PGE2 due to a lack of COX-2 expression, which was induced during spheroid formation. Pharmacological inhibition of COX-2 and mPGES-1 supported the crucial role of PGE2 for growth of MCTS. The functionality of spheroid-derived PGE2 was demonstrated by its ability to inhibit cytotoxic T cell activation. When investigating mechanisms of spheroid-induced COX-2 induction, we observed that among microenvironmental factors neither glucose deprivation, hypoxia nor tumor cell apoptosis enhanced COX-2 expression. Interestingly, interfering with apoptosis in spheroids triggered a shift towards necrosis, thus augmenting COX-2 expression. We went on to demonstrate that necrotic cells induced COX-2 mRNA expression and PGE2 secretion from live tumor cells. In conclusion, necrosis-dependent COX-2 upregulation in MCTS promoted PGE2 -dependent tumor growth and inhibited activated cytotoxic T cells. Hence, blocking mPGES-1 as a therapeutic option may be considered for COX-2/mPGES-1-positive solid cancers.

Recreating the tumor microenvironment in a bilayer, hyaluronic acid hydrogel construct for the growth of prostate cancer spheroids

Cancer cells cultured in physiologically relevant, three-dimensional (3D) matrices can recapture many essential features of native tumor tissues. In this study, a hyaluronic acid (HA)-based bilayer hydrogel system that not only supports the tumoroid formation from LNCaP prostate cancer (PCa) cells, but also simulates their reciprocal interactions with the tumor-associated stroma was developed and characterized. HA hydrogels were prepared by mixing solutions of HA precursors functionalized with acrylate groups (HA-AC) and reactive thiols (HA-SH) under physiological conditions. The resultant viscoelastic gels have an average elastic modulus of 234 ± 30 Pa and can be degraded readily by hyaluronidase. The orthogonal and cytocompatible nature of the crosslinking chemistry permits facile incorporation of cytokine-releasing particles and PCa cells. In our bilayer hydrogel construct, the top layer contains heparin (HP)-decorated, HA-based hydrogel particles (HGPs) capable of releasing heparin-binding epidermal growth factor-like growth factor (HB-EGF) in a sustained manner at a rate of 2.5 wt%/day cumulatively. LNCaP cells embedded in the bottom layer receive the growth factor signals from the top, and in response form enlarging tumoroids with an average diameter of 85 μm by day 7. Cells in 3D hydrogels assemble into spherical tumoroids, form close cellular contacts through E-cadherin, and show cortical organization of F-actin, whereas those plated as 2D monolayers adopt a spread-out morphology. Compared to cells cultured on 2D, the engineered tumoroids significantly increased the expression of two pro-angiogenic factors, vascular endothelial growth factor-165 (VEGF165) and interleukin-8 (IL-8), both at mRNA and protein levels. Overall, the HA model system provides a useful platform for the study of tumor cell responses to growth factors and for screening of anticancer drugs targeting these pathways.

Abstract A72: Prostate cancer spheroid formation in a 384 hanging drop array plate.

Abstract Background: Bridging the gap between in vitro and in vivo studies is critical to therapeutic advancement. Achieving more physiological tumor models in vitro will attenuate dependence on preclinical animal testing and facilitate more accurate prediction of a compound's antitumor behavior in vivo. Multicellular tumor spheroids are 3-dimensional, self-assembled clusters of cancer cells formed in an environment where cell-cell interactions dominate in the absence of cell-surface interactions. A spheroid composed of prostate cancer cells, or prostatosphere, not only provides a 3-dimensional tumor model, but also allows the formation of extracellular matrix as would occur in vivo. Thus, drug screening performed on spheroids should yield results of greater physiological significance than screening performed on 2-dimensional tissue cultures. Furthermore, the use of a 384 hanging drop array plate has previously been shown to enable the convenient formation of spheroids from COS7, mES, and A431.H9 cell lines[1]. Here we chronicle the compatibility of the 384 hanging drop array plate with four prostate cancer cell lines for prostatosphere formation. Methods: Prostate cancer cell suspensions were deposited into staggered access holes on the 384 hanging drop array plate, creating 15 to 20 L hanging drops with cell concentrations ranging from 500 to 5000 cells per drop. In order to arrest drop evaporation, the hanging drop array plate was sandwiched between a lid and 96-well plate filled with distilled water and was wrapped in Parafilm. Drops were observed using bright field microscopy and imaged daily (Olympus IX71). The image processing software ImageJ was used to obtain spheroid dimensions, and mean spheroid diameter was calculated using the following equation: d = (a × b)∘(1/2), where a and b are orthogonal diameters of the spheroid. Average size was reported as mean diameter ± standard deviation and was measured on the second day after spheroid formation was observed. Results: Spheroid formation was observed within 24 hours for LNCaP and LAPC4 cells, and within 48 hours for VCaP and DU145 cells. LNCaP, LAPC4, and VCaP spheroids characteristically formed one single spheroid per drop, with larger spheroids obtained at higher cell concentrations. These three cell lines exhibited round and oblong spheroid morphologies at low cell concentration, with spheroid shape becoming more irregular, branched, or partially hollow at higher cell concentrations. Hanging drops containing DU145 cells tended to contain multiple spheroids of smaller diameter. Thus, DU145 spheroids were usually limited to round or oblong morphologies. DU145 spheroids attained an average size of 72.1 ± 43.7 microns. Conclusion: These prostate cancer cell lines are shown to form prostatospheres when cultured in the 384 hanging drop array plate. Preliminary evidence suggests that additional prostate cancer cell lines are also capable of doing so. Our group intends to screen antitumor therapeutics on prostatospheres in comparison to prostate cancer cells under traditional 2-dimensional tissue culture conditions. Co-culture spheroid formation incorporating prostate cancer cells, endothelial cells, and osteoblasts to simulate the tumor microenvironment is expected, as is drug screening on such spheroids.

Abstract 4275: A prostate cancer spheroid related gene and chemotherapy

Abstract Spheroid culture is one of the three-dimensional (3-D) culture models. We have constructed a spheroid culture model for prostate cancer therapy and characterized this model. The change of global gene expression profiles in spheroids and solid tumor tissues were also compared with monolayer cultures, leading to extraction of candidate genes in contexts that more closely mimic tumor growth in vitro under different in vitro growth conditions. It is possible that these genes may be specific to the construction of spheroids and reveal the possibilities of new mechanisms of acquiring anticancer drug resistance or sensitivity. The plk-2 was extracted as a candidate e gene related with spheroid formation. A member of the polo-like kinase (PLK) family, PLK2 is one of highly conserved serine/threonine protein kinases. In this study, we analyzed effects of the PLK2 on the spheroid characterization and anticancer drug resistance in DU-145 spheroids by suppressing plk2 with RNAi. When docetaxel (DTX) was exposed for siRNA-transfected spheroids, the viability changed, the volume decreased at each concentration of DTX, and spheroids were collapsed at last. The result indicates that the plk2 may be involved in acquiring anticancer drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4275. doi:10.1158/1538-7445.AM2011-4275

Abstract 3331: New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in colon and prostate CSCs

Abstract In vast majority of human cancers, malignant stem cells (CSCs) are responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that colon and prostate CSCs are not only functionally and morphologically different from their bulk counterparts, but themselves may represent heterogeneous phenotypic populations. In contrast to the major (bulk) colon and prostate cancer cells expressing low levels of CD133 and CD44, cells with a high combined expression of these markers, as well as colon cancer cells lacking CD133, possessed several characteristic stem cell features, including high tumor initiating potential, profound self-renewal capacity, and an ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating multicellular spheroids induced by candidate phenotypic cell populations; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214 in 3D culture. Experimental: Selected CSC phenotypes were isolated from highly invasive colon cancer HCT116 cells and prostate cancer PC3MM2 cells. A stem cell-specific PCR array (SABiosciences) revealed that 3D spheroids induced by either colon CD133high/CD44high or CD133neg cells, as well as by CD133high/CD44high prostate cancer cells expressed profound up-regulation of stem cell-related genes compared to their differentiated counterparts (68%, 56% and 61%, correspondently). The FACS analysis has shown that both colon and prostate spheroids contained minority cell populations with high levels of expression of Oct4, Sox2, c-Myc and Nanog, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 0.01-0.1 μg/ml for 48 hr not only induced growth inhibition and apoptotic cell death in these two types of cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities. Summay: We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon and prostate cancer spheroids induced by and enriched with drug resistant tumorigenic cell populations and efficiently inhibited expression of the majority of stem cell-related genes. Conclusion: Our data indicates that the long-term efficacy of the SB-T-1214 against several drug resistant tumors in vivo may be explained by down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3331.

The Biological Behavior of Prostate Cancer Cells in 3D Culture Systems

Prostate cancer is the most common non-cutaneous malignant neoplasm in men in Western countries. In Japan, the number of afflicted men has been increasing although it is still low compared with Western countries. One of the most important problems in prostate cancer patients is treatment for hormone-refractory prostate cancer (HRPC). Although docetaxel is considered as a first-line chemotherapeutic option in patients with HRPC in the USA, it is still necessary to search and develop new drugs. Spheroid culture models have an invaluable role in tumor biology or drug screening. Characteristics of cancer cells in three-dimensional (3D) culture, especially spheroid culture, differ dramatically from those in two-dimensional (2D) culture. Spheroid culture models appear to be an ideal tool, however, their models have not been incorporated in drug screening. In this article, we demonstrate characterization of prostate cancer spheroids including chemo-resistance compared with 2D culture and xenograft models. Prostate cancer cells except PC-3 formed E-cadherin-mediated spheroids. An immunocytochemical analysis of the spheroids revealed that cells showing Ki-67 were localized in the peripheral layer and the intermediate zone cells showed p27 and poly (ADP-ribose) polymerase-1 (PARP-1), suggesting quiescent cell character. Prostate cancer cells acquired resistance to most agents when grown as spheroids, but not to all of the anticancer agents tested. This article also attempts to provide up-to-date information about spheroids, especially quiescent cells as therapeutic targets and the involvement of genetics and epigenetics in forming spheroids.

Spatial Composition of Prostate Cancer Spheroids in Mixed and Static Cultures

Spatial Composition of Prostate Cancer Spheroids in Mixed and Static Cultures

Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids.

Neoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing, from 0.21 +/- 0.03 to 0.13 +/- 0.03 in LNCaP multicellular spheroids, and from 0.13 +/- 0.04 to 0.03 +/- 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 micro m within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. Thus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.