Prostate cancer is a common cancer among men and typically progresses slowly for several decades before becoming aggressive and spreading to other organs, leaving few treatment options. While large ...animals have been studied, the dog’s prostate is anatomically similar to humans and has been used to study spontaneous prostate cancer. However, most research currently focuses on the mouse as a model organism due to the ability to genetically modify their prostatic tissues for molecular analysis. One milestone in this research was the identification of the prostate-specific promoter Probasin, which allowed for the prostate-specific expression of transgenes. This has led to the generation of mice with aggressive prostatic tumors through overexpression of the SV40 oncogene. The Probasin promoter is also used to drive Cre expression and has allowed researchers to generate prostate-specific loss-of-function studies. Another landmark moment in the process of modeling prostate cancer in mice was the orthoptic delivery of viral particles. This technology allows the selective overexpression of oncogenes from lentivirus or the use of CRISPR to generate complex loss-of-function studies. These genetically modified models are complemented by classical xenografts of human prostate tumor cells in immune-deficient mice. Overall, pre-clinical models have provided a portfolio of model systems to study and address complex mechanisms in prostate cancer for improved treatment options. This review will focus on the advances in each technique.
A switch from oxidative phosphorylation to glycolysis is frequently observed in cancer cells and is linked to tumor growth and invasion, but the underpinning molecular mechanisms controlling the ...switch are poorly understood. In this report we show that Notch signaling is a key regulator of cellular metabolism. Both hyper- and hypoactivated Notch induce a glycolytic phenotype in breast tumor cells, although by distinct mechanisms: hyperactivated Notch signaling leads to increased glycolysis through activation of the phosphatidylinositol 3-kinase/AKT serine/threonine kinase pathway, whereas hypoactivated Notch signaling attenuates mitochondrial activity and induces glycolysis in a p53-dependent manner. Despite the fact that cells with both hyper- and hypoactivated Notch signaling showed enhanced glycolysis, only cells with hyperactivated Notch promoted aggressive tumor growth in a xenograft mouse model. This phenomenon may be explained by that only Notch-hyperactivated, but not -hypoactivated, cells retained the capacity to switch back to oxidative phosphorylation. In conclusion, our data reveal a role for Notch in cellular energy homeostasis, and show that Notch signaling is required for metabolic flexibility.
Background
In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various ...metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including
18
F-fluoro-2-deoxyglucose (
18
FFDG) and hypoxia-selective 2-nitroimidazoles (e.g.,
18
F-fluoroazomycin-arabinoside,
18
FFAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention.
Methods
Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2 h to 21%, 0.5% or 0% O
2
and
18
F FDG or
18
F FAZA was added, followed by cell harvest and analysis of radioactivity 1 h (
18
FFDG) or 3 h (
18
FFAZA) after. Experiments were conducted with/without orbital shaking.
Results
The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven
18
F FAZA, and to some extent
18
F FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy.
Conclusions
Tracer retention was similar under stagnant and forced convection conditions suggesting that the former approach may be appropriate even when accurate control of oxygen and tracer availability is required. In contrast, conventional plasticware should be used with caution when studying tracers and drugs that are metabolized and retained or activated at low O
2
levels. Downscaling of cell number, by reducing the effective growth area, was feasible, without compromising accuracy.
The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and ...lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis.
Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.
Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined.
We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.
Background
Correct classification of estrogen receptor (ER) status is essential for prognosis and treatment planning in patients with breast cancer (BC). Therefore, it is recommended to sample tumor ...tissue from an accessible metastasis. However, ER expression can show intra- and intertumoral heterogeneity. 16α-
18
Ffluoroestradiol (
18
FFES) Positron Emission Tomography/Computed Tomography (PET/CT) allows noninvasive whole-body (WB) identification of ER distribution and is usually performed as a single static image 60 min after radiotracer injection. Using dynamic whole-body (D-WB) PET imaging, we examine
18
FFES kinetics and explore whether Patlak parametric images (
K
i
) are quantitative and improve lesion visibility.
Results
This prospective study included eight patients with metastatic ER-positive BC scanned using a D-WB PET acquisition protocol. The kinetics of
18
FFES were best characterized by the irreversible two-tissue compartment model in tumor lesions and in the majority of organ tissues.
K
i
values from Patlak parametric images correlated with
K
i
values from the full kinetic analysis, r
2
= 0.77, and with the semiquantitative mean standardized uptake value (SUV
mean
), r
2
= 0.91. Furthermore, parametric
K
i
images had the highest target-to-background ratio (TBR) in 162/164 metastatic lesions and the highest contrast-to-noise ratio (CNR) in 99/164 lesions compared to conventional SUV images. TBR was 2.45 (95% confidence interval (CI): 2.25–2.68) and CNR 1.17 (95% CI: 1.08–1.26) times higher in
K
i
images compared to SUV images. These quantitative differences were seen as reduced background activity in the
K
i
images.
Conclusion
18
FFES uptake is best described by an irreversible two-tissue compartment model. D-WB
18
FFES PET/CT scans can be used for direct reconstruction of parametric
K
i
images, with superior lesion visibility and
K
i
values comparable to
K
i
values found from full kinetic analyses. This may aid correct ER classification and treatment decisions.
Trial registration
ClinicalTrials.gov: NCT04150731,
https://clinicaltrials.gov/study/NCT04150731
Background: This pre-clinical study was designed to refine a dissection method for validating the use of a 15-gene hypoxia classifier, which was previously established for head and neck squamous cell ...carcinoma (HNSCC) patients, to identify hypoxia in prostate cancer. Methods: PC3 and DU-145 adenocarcinoma cells, in vitro, were gassed with various oxygen concentrations (0–21%) for 24 h, followed by real-time PCR. Xenografts were established in vivo, and the mice were injected with the hypoxic markers 18F-FAZA and pimonidazole. Subsequently, tumors were excised, frozen, cryo-sectioned, and analyzed using autoradiography (18F-FAZA) and immunohistochemistry (pimonidazole); the autoradiograms used as templates for laser capture microdissection of hypoxic and non-hypoxic areas, which were lysed, and real-time PCR was performed. Results: In vitro, all 15 genes were increasingly up-regulated as oxygen concentrations decreased. With the xenografts, all 15 genes were up-regulated in the hypoxic compared to non-hypoxic areas for both cell lines, although this effect was greater in the DU-145. Conclusions: We have developed a combined autoradiographic/laser-guided microdissection method with broad applicability. Using this approach on fresh frozen tumor material, thereby minimizing the degree of RNA degradation, we showed that the 15-gene hypoxia gene classifier developed in HNSCC may be applicable for adenocarcinomas such as prostate cancer.
Abstract Purpose Tumor hypoxia is a known cause of resistance to radiotherapy. The aim of this study was to investigate the prognostic value of hypoxia measured by18 F-fluoroazomycin arabinoside (18 ...F-FAZA) PET or the Eppendorf oxygen electrode in a pre-clinical tumor model. Material/methods Pretreatment18 F-FAZA PET scans and blood sampling was conducted in 92 Female CDF1 mice with subcutaneous C3H mammary carcinomas grown in the right foot. Similarly, oxygenation status of 80 equivalent tumors was assessed using an invasive oxygen sensitive electrode. Tumors were then irradiated with a single dose of 55 Gy and local tumor control up to 90 days after the treatment was determined. Results A significant difference in local tumor control between “more hypoxic” or “less hypoxic” groups separated either by a median18 F-FAZA PET determined tumor-to-blood ratio ( P = 0.007; hazard ratio, HR = 0.21 95% CI: 0.06–0.74), or the fraction of oxygen partial pressure (pO2 ) values ⩽2.5 mmHg ( P = 0.018; HR = 0.31 95% CI: 0.11–0.87), was found. Both assays showed that the more hypoxic tumors had significantly lower tumor control. Conclusion18 F-FAZA PET analysis showed that pre treatment tumor hypoxia was prognostic of radiation response. Similar results were obtained when oxygenation status was assessed by the Eppendorf pO2 Histograph. The results of this study support the role of18 F-FAZA as a non-invasive prognostic marker for tumor hypoxia.
Organic cation transporters (OCTs) in the kidney proximal tubule (PT) participate in renal excretion of drugs and endogenous compounds. PT function is commonly impaired in kidney diseases, and ...consequently quantitative measurement of OCT function may provide an important estimate of kidney function. Metformin is a widely used drug and targets OCT type 2 located in the PT. Thus, we hypothesized that (11)C-labeled metformin would be a suitable PET tracer for quantification of renal function.
(11)C-metformin was prepared by (11)C-methylation of 1-methylbiguanide. In vitro cell uptake of (11)C-metformin was studied in LLC-PK1 cells in the presence of increasing doses of unlabeled metformin. In vivo small-animal PET studies in Sprague-Dawley rats were performed at baseline and after treatment with OCT inhibitors to evaluate renal uptake of (11)C-metformin. Kidney and liver pharmacokinetics of (11)C-metformin was investigated in vivo by dynamic (11)C-metformin PET/CT in 6 anesthetized pigs, and renal clearance of (11)C-metformin was compared with renal clearance of (51)Cr-ethylenediaminetetraacetic acid (EDTA). Formation of (11)C metabolites was investigated by analysis of blood and urine samples.
The radiochemical yield of (11)C-metformin was 15% ± 3% (n= 40, decay-corrected), and up to 1.5 GBq of tracer were produced with a radiochemical purity greater than 95% in less than 30 min. Dose-dependent uptake of (11)C-metformin in LLC-PK1 cells was rapid. Rat small-animal PET images showed (11)C-metformin uptake in the kidney and liver, the kinetics of which were changed after challenging animals with OCT inhibitors. In pigs, 80% of the injected metformin dose was rapidly present in the kidney, and a high dose of metformin caused a delayed renal uptake and clearance compared with baseline consistent with transporter-mediated competition. Renal clearance of (11)C-metformin was approximately 3 times the renal clearance of (51)Cr-EDTA.
We successfully synthesized an (11)C-metformin tracer, and PET studies in rats and pigs showed a rapid kidney uptake from the blood and excretion into the bladder similar to other radiopharmaceuticals developed for γ-camera renography.
Abstract Background and purpose Tumor cells are recognized as being highly glycolytic. However, recently it was suggested that lactate produced in hypoxic tumor areas may be taken up by the ...monocarboxylate transporter MCT1 and oxidized in well-oxygenated tumor parts. Furthermore, it was shown that inhibition of lactate oxidation using the MCT1 inhibitor α-cyano-hydroxycinnamate (CHC) can radio-sensitize tumors possibly by forcing a switch from lactate oxidization to glycolysis in oxygenated cells, which in turn improves tumor oxygenation and indirectly kills radio-resistant hypoxic tumor cells from glucose starvation. Material and methods To provide direct evidence for the existence of a targetable energetic symbiosis, mice bearing SiHa or FaDudd tumors were treated with CHC for different time periods. One hour prior to sacrifice, mice were administered with the glucose analog fluorodeoxyglucose (FDG) and the hypoxia-marker pimonidazole. Tumor cryosections were analyzed for regional glucose retention (FDG autoradiograms), hypoxia (pimonidazole retention) and glucose and lactate levels (bioluminescence imaging). Results Treatment did not influence metabolite concentrations, necrosis or extent of hypoxia, but pixel-by-pixel analysis comparing FDG retention and hypoxia (a measure of the apparent in vivo Pasteur effect) showed that CHC treatment caused a transient reduction in the Pasteur effect in FaDudd 1.5 h following CHC administration whereas a reduction was only observed in SiHa following repeated treatments. Conclusions In summary, our data show that CHC is able to influence the intratumoral distribution of glucose use between hypoxic and non-hypoxic tumor areas. That is in accordance with a functional tumor lactate-shuttle, but the absence of any detectable changes in hypoxic extent and tissue metabolites was unexpected and warrants further investigation.
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![Dynamic whole-body [18F]FES...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/2191-219X/small "Dynamic whole-body [18F]FES PET/CT increases lesion visibility in patients with metastatic breast cancer")
