Evaluation of [18F]F-DPA as a target for TSPO in head and neck cancer under normal conditions and after radiotherapy.

BACKGROUND: Many malignant tumours have increased TSPO expression, which has been related to a poor prognosis. TSPO-PET tracers have not comprehensively been evaluated in peripherally located tumours. This study aimed to evaluate whether N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([18F]F-DPA) can reflect radiotherapy (RT)-induced changes in TSPO activity in head and neck squamous cell carcinoma (HNSCC). METHODS: RT was used to induce inflammatory responses in HNSCC xenografts and cells. [18F]F-DPA uptake was measured in vivo in non-irradiated and irradiated tumours, followed by ex vivo biodistribution, autoradiography, and radiometabolite analysis. In vitro studies were performed in parental and TSPO-silenced (TSPO siRNA) cells. TSPO protein and mRNA expression, as well as tumour-associated macrophages (TAMs), were also assessed. RESULTS: In vivo imaging and ex vivo measurement revealed significantly higher [18F]F-DPA uptake in irradiated, compared to non-irradiated tumours. In vitro labelling studies with cells confirmed this finding, whereas no effect of RT on [18F]F-DPA uptake was detected in TSPO siRNA cells. Radiometabolite analysis showed that the amount of unchanged [18F]F-DPA in tumours was 95%, also after irradiation. PK11195 pre-treatment reduced the tumour-to-blood ratio of [18F]F-DPA by 73% in xenografts and by 88% in cells. TSPO protein and mRNA levels increased after RT, but were highly variable. The proportion of M1/M2 TAMs decreased after RT, whereas the proportion of monocytes and migratory monocytes/macrophages increased. CONCLUSIONS: [18F]F-DPA can detect changes in TSPO expression levels after RT in HNSCC, which does not seem to reflect inflammation. Further studies are however needed to clarify the physiological mechanisms regulated by TSPO after RT.

Head and neck squamous cell carcinoma cell lines have an immunomodulatory effect on macrophages independent of hypoxia and toll-like receptor 9.

BACKGROUND: A low tissue oxygen level, < 1% O2, is a typical characteristic inside of solid tumors in head and neck cancer (HNSCC) affecting a wide array of cell populations, such as macrophages. However, the mechanisms of how hypoxia influences macrophages are not yet fully elucidated. Our research aimed to study the effect of soluble mediators produced by hypoxic cancer cells on macrophage polarization. Furthermore, we studied the effect of a hypoxic microenvironment on the expression of tumorigenic toll-like receptor 9 (TLR9) and the consecutive macrophage polarization. METHODS: Conditioned media (CMNOX or CMHOX) from cell lines UT-SCC-8, UT-SCC-74A, FaDu, MDA-MB-231 and HaCat cultured under normoxic (21% O2) and hypoxic (1% O2) conditions were used to polarize human monocyte-derived macrophages. Macrophage polarization was measured by flow cytometry and the production of cytokine mRNA using Taqman qPCR. To study the role of TLR9 in macrophage polarization, the lentiviral CRISPR/Cas9 method was used to establish a stable FaDuTLR9def clone. RESULTS: Our results demonstrate that the soluble mediators produced by the cancer cells under normoxia polarize macrophages towards a hybridized M1/M2a/M2c phenotype. Furthermore, the results suggest that hypoxia has a limited role in altering the array of cancer-produced soluble factors affecting macrophage polarization and cytokine production. Our data also indicates that increased expression of TLR9 due to hypoxia in malignant cells does not markedly influence the polarization of macrophages. TLR9 transcriptional response to hypoxia is dissimilar to a HIF1-α-regulated LDH-A. This may indicate a context-dependent expression of TLR9 under hypoxia. CONCLUSIONS: HNSCC cell lines affect both macrophage activity (polarization) and functionality (cytokines), but with exception to iNOS expression, the effects appear independent of hypoxia and TLR9.

γ-(S)-Guanidinylmethyl-Modified Triplex-Forming Peptide Nucleic Acids Increase Hoogsteen-Face Affinity for a MicroRNA and Enhance Cellular Uptake.

γ-Modified (i.e., (S)-aminomethyl, (S)-acetamidomethyl, (R)-4-(hydroxymethyl)triazol-1-ylmethyl, and (S)-guanidinylmethyl) triplex-forming peptide nucleic acids (TFPNAs) were synthesized and the effect of the backbone modifications on the binding to a miR-215 model was studied. Among the modifications, an appropriate pattern of three γ-(S)-guanidinylmethyl modifications increased the affinity and Hoogsteen-face selectivity for the miR-215 model without ternary (PNA)2 /RNA complex formation. Moreover, the γ-(S)-guanidinylmethyl groups were observed to facilitate internalization of the TFPNAs into living PC-3 prostate cancer cells.

Chronic nonbacterial prostate inflammation in a rat model is associated with changes of gut microbiota that can be modified with a galactoglucomannan-rich hemicellulose extract in the diet.

OBJECTIVES: To investigate dietary effects on the gut microbiota composition in a rat model of nonbacterial chronic prostate inflammation (CPI). MATERIALS AND METHODS: Nonbacterial CPI was induced in the Wistar rat strain with subcutaneous testosterone and 17ß-oestradiol (E2 ) hormone pellets for 18 weeks. Rats with placebo pellets served as healthy controls. Rats with CPI were stratified into two groups, which drank either plain tap water (control group) or tap water supplemented with 2% galactoglucomannan-rich hemicellulose extract (GGM group) from Norway spruce (Picea abies) for 5 weeks. Faecal samples were collected at the end of the study, total DNA was extracted, and the bacterial composition was analysed by 16S rRNA gene sequencing. In addition, faecal samples were assayed for short-chain fatty acid (SCFA) concentrations using gas chromatography. Lipopolysaccharide-binding protein (LBP) was measured in serum samples, as an indirect indicator for bacterial lipopolysaccharide (LPS) load in blood. RESULTS: The microbial biodiversity was significantly different between the treatment groups. In the rats with CPI, there was a significant increase in gut microbial populations Rikenellaceae, Odoribacter, Clostridiaceae, Allobaculum and Peptococcaceae compared with healthy rats. Conversely, levels of Bacteroides uniformis, Lactobacillus and Lachnospiraceae were decreased in rats with CPI. SCFA butyric-, valeric- and caproic-acid concentrations were also decreased in the faecal samples of the rats with CPI. In contrast, acetic acid concentrations and serum LBP were significantly elevated in CPI rats compared with healthy ones. Amongst rats with CPI, treatment with the GGM extract significantly reduced the abundance of Odoribacter and Clostridiaceae levels, and increased the B. uniformis levels compared with CPI rats drinking tap water only. In addition, GGM significantly increased the levels of butyric acid and caproic acid, and reduced the levels of LBP in serum. CONCLUSIONS: Hormone-induced nonbacterial CPI in rats is associated with specific changes in gut microbiota and secondary changes in SCFAs and LPS due to gut microbiota alteration. Our results further suggest that fermentable compounds may have a beneficial effect on CPI.

Evaluation of 68Ga-labeled peptide tracer for detection of gelatinase expression after myocardial infarction in rat.

BACKGROUND: Matrix metalloproteinases 2 and 9 (MMP-2/9) play a role in extracellular matrix remodeling after an ischemic myocardial injury. We evaluated 68Ga-DOTA-peptide targeting MMP-2/9 for the detection of gelatinase expression after myocardial infarction (MI) in rat. METHODS: Rats were injected with 43 ± 7.7 MBq of 68Ga-DOTA-peptide targeting MMP-2/9 at 7 days (n = 7) or 4 weeks (n = 8) after permanent coronary ligation or sham operation (n = 5 at both time points) followed by positron emission tomography (PET). The left ventricle was cut in frozen sections for autoradiography and immunohistochemistry 30 minutes after tracer injection. RESULTS: Immunohistochemical staining showed MMP-2 and MMP-9 expressing cells, CD31-positive endothelial cells, and CD68-positive macrophages in the infarcted myocardium. Autoradiography showed increased tracer uptake in the infarcted area both at 7 days and 4 weeks after MI (MI-to-remote area ratio 2.5 ± 0.46 and 3.1 ± 1.0, respectively). Tracer uptake in damaged tissue correlated with the amount of CD68-positive macrophages at 7 days after MI, and CD31-positive endothelial cells at 7 days and 4 weeks after MI. The tracer was rapidly metabolized, radioactivity in the blood exceeded that of the myocardium, and tracer accumulation in the heart was not detectable by in vivo PET. CONCLUSIONS: 68Ga-DOTA-peptide targeting MMP-2/9 accumulates in the damaged rat myocardium after an ischemic injury, but tracer instability and slow clearance in vivo make it unsuitable for further evaluation.

Evaluation of [68Ga]Ga-DOTA-TCTP-1 for the Detection of Metalloproteinase 2/9 Expression in Mouse Atherosclerotic Plaques.

Background: The expression of matrix metalloproteinases 2/9 (MMP-2/9) has been implicated in arterial remodeling and inflammation in atherosclerosis. We evaluated a gallium-68 labeled peptide for the detection of MMP-2/9 in atherosclerotic mouse aorta. Methods: We studied sixteen low-density lipoprotein receptor deficient mice (LDLR-/-ApoB100/100) kept on a Western-type diet. Distribution of intravenously-injected MMP-2/9-targeting peptide, [68Ga]Ga-DOTA-TCTP-1, was studied by combined positron emission tomography (PET) and contrast-enhanced computed tomography (CT). At 60 min post-injection, aortas were cut into cryosections for autoradiography analysis of tracer uptake, histology, and immunohistochemistry. Zymography was used to assess MMP-2/9 activation and pre-treatment with MMP-2/9 inhibitor to assess the specificity of tracer uptake. Results: Tracer uptake was not visible by in vivo PET/CT in the atherosclerotic aorta, but ex vivo autoradiography revealed 1.8 ± 0.34 times higher tracer uptake in atherosclerotic plaques than in normal vessel wall (p = 0.0029). Tracer uptake in plaques correlated strongly with the quantity of Mac-3-positive macrophages (R = 0.91, p < 0.001), but weakly with MMP-9 staining (R = 0.40, p = 0.099). Zymography showed MMP-2 activation in the aorta, and pre-treatment with MMP-2/9 inhibitor decreased tracer uptake by 55% (p = 0.0020). Conclusions: The MMP-2/9-targeting [68Ga]Ga-DOTA-TCTP-1 shows specific uptake in inflamed atherosclerotic lesions; however, a low target-to-background ratio precluded in vivo vascular imaging. Our results suggest, that the affinity of gelatinase imaging probes should be steered towards activated MMP-2, to reduce the interference of circulating enzymes on the target visualization in vivo.

Oncolytic alphavirus SFV-VA7 efficiently eradicates subcutaneous and orthotopic human prostate tumours in mice.

BACKGROUND: Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies. METHODS: We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1. Therapeutic potency of SFV-VA7 was evaluated in subcutaneous and orthotopic mouse LNCaP xenograft models. RESULTS: SFV-VA7 infected and killed the tested human prostate cancer cell lines irrespective of their hormone response status, while the nonmalignant prostate epithelial cell line RWPE-1 proved highly virus resistant. Notably, a single peritoneal dose of SFV-VA7 was sufficient to eradicate all subcutaneous and orthotopic LNCaP tumours. CONCLUSIONS: Our results indicate that SFV-VA7 is a novel, promising therapeutic virus against prostate cancer warranting further testing in early clinical trials.

Lipid Bilayer-Gated Mesoporous Silica Nanocarriers for Tumor-Targeted Delivery of Zoledronic Acid in Vivo.

Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate used for the treatment of bone diseases and calcium metabolism. Anticancer activity of ZOL has been established, but its extraskeletal effects are limited due to its rapid uptake and accumulation to bone hydroxyapatite. In this work, we report on the development of tethered lipid bilayer-gated mesoporous silica nanocarriers (MSNs) for the incorporation, retention, and intracellular delivery of ZOL. The in vitro anticancer activity of ZOL-loaded nanocarriers was evaluated by cell viability assay and live-cell imaging. For in vivo delivery, the nanocarriers were tagged with folic acid to boost the affinity for breast cancer cells. Histological examination of the liver revealed no adverse off-target effects stemming from the nanocarriers. Importantly, nonspecific accumulation of ZOL within bone was not observed, which indicated in vivo stability of the tethered lipid bilayers. Further, the intravenously administered ZOL-loaded nanocarriers showed tumor growth suppression in breast cancer xenograft-bearing mice.

Increased expression of fibroblast growth factor 13 in prostate cancer is associated with shortened time to biochemical recurrence after radical prostatectomy.

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high-risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co-labeled cells suggested a possible interaction of FGF13 with α-tubulin and the voltage-gated sodium channel proteins (Na(V)s/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR-free survival, in particular if combined with other clinical parameters.

Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.

DNA from dead cancer cells induces TLR9-mediated invasion and inflammation in living cancer cells.

TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple-negative, human MDA-MB-231 breast cancer cells stably expressing control, or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy.

Estrogen receptor-α and sex steroid hormones regulate Toll-like receptor-9 expression and invasive function in human breast cancer cells.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor, which is widely expressed in cancer. Synthetic TLR9-ligands induce cancer cell invasion in vitro, but the role of TLR9 in cancer pathophysiology remains unclear. Increased TLR9 expression has been, however, detected in estrogen receptor negative (ER-) breast cancers. In this study, we investigated the effects of ERα expression and sex steroid hormones on TLR9 expression in human ER+ (MCF-7, T47-D) and ER- (MDA-MB-231) breast cancer cell lines in vitro. We also studied TLR9 mRNA expression in archival breast cancer specimens (n = 12) with qRT-PCR, using primer sets that detect only the TLR9A isoform or the isoforms A and B (TLR9A/B). The TLR9 mRNA expression was detected in 10/12 specimens with both primer sets, and in 1/12 with only the TLR9A or the TLR9A/B primer sets. The basal TLR9 mRNA expression levels were significantly lower in the ER+ cell lines as compared with the ER- MDA-MB-231 cells. The transfection of ERα cDNA into MDA-MB-231 cells also resulted in down-regulation of TLR9 expression. While sex steroids had no effect on TLR9 expression in MCF-7 cells, testosterone (10(-8) M) induced TLR9 expression in MDA-MB-231 and T47-D cells. Although bicalutamide blocked this testosterone effect in MDA-MB-231 cells, in T47-D cells bicalutamide increased TLR9 expression and only partially blocked the testosterone effects. Estradiol (10(-8) M) induced TLR9 expression in T47-D cells. The invasive effects of synthetic TLR9-ligands were augmented by testosterone in vitro. This effect was lost in TLR9 siRNA MDA-MB-231 cells and also decreased by over-expression of ERα, which also inhibited NF-κB activation by TLR9-ligands. In conclusion, expression of TLR9 isoforms A and B can be detected in clinical breast cancer specimens. The ERα and sex steroid hormones regulate TLR9 expression and invasive effects in the breast cancer cells. Also, the commonly used hormonal cancer therapy bicalutamide affects TLR9 expression.

Low TLR9 expression defines an aggressive subtype of triple-negative breast cancer.

Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.

Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression.

Fibroblast growth factor receptors (FGFRs) 1-4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor-stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer.

CD73 is a cell surface ecto-5'-nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5'-(α, ß-methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial-mesenchymal transition.

Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.

Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.

Fast growth associated with aberrant vasculature and hypoxia in fibroblast growth factor 8b (FGF8b) over-expressing PC-3 prostate tumour xenografts.

BACKGROUND: Prostate tumours are commonly poorly oxygenated which is associated with tumour progression and development of resistance to chemotherapeutic drugs and radiotherapy. Fibroblast growth factor 8b (FGF8b) is a mitogenic and angiogenic factor, which is expressed at an increased level in human prostate tumours and is associated with a poor prognosis. We studied the effect of FGF8b on tumour oxygenation and growth parameters in xenografts in comparison with vascular endothelial growth factor (VEGF)-expressing xenografts, representing another fast growing and angiogenic tumour model. METHODS: Subcutaneous tumours of PC-3 cells transfected with FGF8b, VEGF or empty (mock) vectors were produced and studied for vascularity, cell proliferation, glucose metabolism and oxygenation. Tumours were evaluated by immunohistochemistry (IHC), flow cytometry, use of radiolabelled markers of energy metabolism ([18F]FDG) and hypoxia ([18F]EF5), and intratumoral polarographic measurements of pO2. RESULTS: Both FGF8b and VEGF tumours grew rapidly in nude mice and showed highly vascularised morphology. Perfusion studies, pO2 measurements, [18F]EF5 and [18F]FDG uptake as well as IHC staining for glucose transport protein (GLUT1) and hypoxia inducible factor (HIF) 1 showed that VEGF xenografts were well-perfused and oxygenised, as expected, whereas FGF8b tumours were as hypoxic as mock tumours. These results suggest that FGF8b-induced tumour capillaries are defective. Nevertheless, the growth rate of hypoxic FGF8b tumours was highly increased, as that of well-oxygenised VEGF tumours, when compared with hypoxic mock tumour controls. CONCLUSION: FGF8b is able to induce fast growth in strongly hypoxic tumour microenvironment whereas VEGF-stimulated growth advantage is associated with improved perfusion and oxygenation of prostate tumour xenografts.

Critical evaluation of the subcutaneous engraftments of hormone naïve primary prostate cancer.

BACKGROUND: Patient-derived xenografts (PDXs) are considered to better recapitulate the histopathological and molecular heterogeneity of human cancer than other preclinical models. Despite technological advances, PDX models from hormone naïve primary prostate cancer are scarce. We performed a detailed analysis of PDX methodology using a robust subcutaneous model and fresh tissues from patients with primary hormone naïve prostate cancer. METHODS: Clinical prostate tumor specimens (n=26, Gleason score 6-10) were collected from robotic-assisted laparoscopic radical prostatectomies at Turku University Hospital (Turku, Finland), cut into pieces, and implanted subcutaneously into 84 immunodeficient mice. Engraftments and the adjacent material from prostatic surgical specimens were compared using histology, immunohistochemistry and DNA sequencing. RESULTS: The probability of a successful engraftment correlated with the presence of carcinoma in the implanted tissue. Tumor take rate was 41%. Surprisingly, mouse hormone supplementation inhibited tumor take rate, whereas the degree of mouse immunodeficiency did not have an effect. Histologically, the engrafted tumors closely mimicked their parental tumors, and the Gleason grades and copy number variants of the engraftments were similar to those of their primary tumors. Expression levels of androgen receptor, prostate-specific antigen, and keratins were retained in engraftments, and a detailed genomic analysis revealed high fidelity of the engraftments with their corresponding primary tumors. However, in the second or third passage of tumors, the carcinoma areas were almost completely replaced by benign tissue with frequent degenerative or metaplastic changes. CONCLUSIONS: Subcutaneous primary prostate engraftments preserve the phenotypic and genotypic landscape. Thus, they serve a potential model for personalized medicine and preclinical research but their use may be limited to the first passage.

FGF-8b induces growth and rich vascularization in an orthotopic PC-3 model of prostate cancer.

Fibroblast growth factor 8 (FGF-8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF-8b on proliferation of PC-3 prostate cancer cells and growth of PC-3 tumors, and to identify FGF-8b-associated molecular targets. Expression of ectopic FGF-8b in PC-3 cells caused a 1.5-fold increase in cell proliferation in vitro and a four- to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF-8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT-qPCR of FGF-8b mRNA. Microarray analyses revealed significantly altered, up- and downregulated, genes in PC-3 cell cultures (169 genes) and in orthotopic PC-3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell-to-cell signaling and energy production, and the downregulated genes associated with differentiation (DKK-1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT-qPCR. In conclusion, our results demonstrate that FGF-8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF-8b actions on prostate cancer progression and metastasis.

Overexpression of vascular endothelial growth factor C increases growth and alters the metastatic pattern of orthotopic PC-3 prostate tumors.

BACKGROUND: Prostate cancer metastasizes to regional lymph nodes and distant sites but the roles of lymphatic and hematogenous pathways in metastasis are not fully understood. METHODS: We studied the roles of VEGF-C and VEGFR3 in prostate cancer metastasis by blocking VEGFR3 using intravenous adenovirus-delivered VEGFR3-Ig fusion protein (VEGFR3-Ig) and by ectopic expression of VEGF-C in PC-3 prostate tumors in nude mice. RESULTS: VEGFR3-Ig decreased the density of lymphatic capillaries in orthotopic PC-3 tumors (p < 0.05) and inhibited metastasis to iliac and sacral lymph nodes. In addition, tumor volumes were smaller in the VEGFR3-Ig-treated group compared with the control group (p < 0.05). Transfection of PC-3 cells with the VEGF-C gene led to a high level of 29/31 kD VEGF-C expression in PC-3 cells. The size of orthotopic and subcutaneous PC-3/VEGF-C tumors was significantly greater than that of PC-3/mock tumors (both p < 0.001). Interestingly, while most orthotopic PC-3 and PC-3/mock tumors grown for 4 weeks metastasized to prostate-draining lymph nodes, orthotopic PC-3/VEGF-C tumors primarily metastasized to the lungs. PC-3/VEGF-C tumors showed highly angiogenic morphology with an increased density of blood capillaries compared with PC-3/mock tumors (p < 0.001). CONCLUSION: The data suggest that even though VEGF-C/VEGFR3 pathway is primarily required for lymphangiogenesis and lymphatic metastasis, an increased level of VEGF-C can also stimulate angiogenesis, which is associated with growth of orthotopic prostate tumors and a switch from a primary pattern of lymph node metastasis to an increased proportion of metastases at distant sites.