Micro Regional Heterogeneity of ⁶⁴Cu-ATSM and ¹⁸F-FDG Uptake in Canine Soft Tissue Sarcomas: Relation to Cell Proliferation, Hypoxia and Glycolysis

Tumour microenvironment heterogeneity is believed to play a key role in cancer progression and therapy resistance. However, little is known about micro regional distribution of hypoxia, glycolysis and proliferation in spontaneous solid tumours. The overall aim was simultaneous investigation of micro regional heterogeneity of 64Cu-ATSM (hypoxia) and 18F-FDG (glycolysis) uptake and correlation to endogenous markers of hypoxia, glycolysis, proliferation and angiogenesis to better therapeutically target aggressive tumour regions and prognosticate outcome.Exploiting the different half-lives of 64Cu-ATSM (13 h) and 18F-FDG (2 h) enabled simultaneous investigation of micro regional distribution of hypoxia and glycolysis in 145 tumour pieces from four spontaneous canine soft tissue sarcomas. Pairwise measurements of radioactivity and gene expression of endogenous markers of hypoxia (HIF-1α, CAIX), glycolysis (HK2, GLUT1 and GLUT3), proliferation (Ki-67) and angiogenesis (VEGFA and TF) were performed. Dual tracer autoradiography was compared with Ki-67 immunohistochemistry.Micro regional heterogeneity in hypoxia and glycolysis within and between tumour sections of each tumour piece was observed. The spatial distribution of 64Cu-ATSM and 18F-FDG was rather similar within each tumour section as reflected in moderate positive significant correlations between the two tracers (ρ = 0.3920-0.7807; p = 0.0180 -<0.0001) based on pixel-to-pixel comparisons of autoradiographies and gamma counting of tumour pieces. 64Cu-ATSM and 18F-FDG correlated positively with gene expression of GLUT1 and GLUT3, but negatively with HIF-1α and CAIX. Significant positive correlations were seen between Ki-67 gene expression and 64Cu-ATSM (ρ = 0.5578, p = 0.0004) and 18F-FDG (ρ = 0.4629-0.7001, p = 0.0001-0.0151). Ki-67 gene expression more consistently correlated with 18F-FDG than with 64Cu-ATSM.Micro regional heterogeneity of hypoxia and glycolysis was documented in spontaneous canine soft tissue sarcomas. 64Cu-ATSM and 18F-FDG uptakes and distributions showed significant moderate correlations at the micro regional level indicating overlapping, yet different information from the tracers.18F-FDG better reflected cell proliferation as measured by Ki-67 gene expression than 64Cu-ATSM

Simultaneous hyperpolarized 13C-pyruvate MRI and 18F-FDG-PET in cancer (hyperPET):feasibility of a new imaging concept using a clinical PET/MRI scanner

In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and (18)F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We have named this concept hyper PET. Intravenous injection of the hyperpolarized (13)C-pyruvate results in an increase of (13)C-lactate, (13)C-alanine and (13)C-CO(2) ((13)C-HCO(3)) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization (DNP) and use of (13)C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of (13)C-pyruvate to (13)C-lactate. In this study, we combined it with (18)F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified local recurrence of a liposarcoma on the right forepaw was imaged using a combined PET/MR clinical scanner. PET was performed as a single-bed, 10 min acquisition, 107 min post injection of 310 MBq (18)F-FDG. (13)C-chemical shift imaging (CSI) was performed just after FDG-PET and 30 s post injection of 23 mL hyperpolarized (13)C-pyruvate. Peak heights of (13)C-pyruvate and (13)C-lactate were quantified using a general linear model. Anatomic (1)H-MRI included axial and coronal T1 vibe, coronal T2-tse and axial T1-tse with fat saturation following gadolinium injection. In the tumor we found clearly increased (13)C-lactate production, which also corresponded to high (18)F-FDG uptake on PET. This is in agreement with the fact that glycolysis and production of lactate are increased in tumor cells compared to normal cells. Yet, most interestingly, also in the muscle of the forepaw of the dog high (18)F-FDG uptake was observed. This was due to activity in these muscles prior to anesthesia, which was not accompanied by a similarly high (13)C-lactate production. Accordingly, this clearly demonstrates how the Warburg Effect directly can be demonstrated by hyperpolarized (13)C-pyruvate MRSI. This was not possible with (18)F-FDG-PET imaging due to inability to discriminate between causes of increased glucose uptake. We propose that this new concept of simultaneous hyperpolarized (13)C-pyruvate MRSI and PET may be highly valuable for image-based non-invasive phenotyping of tumors. This methods may be useful for treatment planning and therapy monitoring

Ectomycorrhizal necromass turnover is one-third of biomass turnover in hemiboreal Pinus sylvestris forests

Societal Impact Statement Efficient mitigation of climate change requires predictive models of forest ecosystems as sinks for atmospheric carbon. Mycorrhizal fungi are drivers of soil carbon storage in boreal forests, yet they are typically excluded from ecosystem models, because of a lack of information about their growth and turnover. Closing this knowledge gap could help us better predict future responses to climate change and guide policy decisions for sustainable management of forest ecosystems. This study provides new estimates of the production and turnover of mycorrhizal mycelial biomass and necromass. This information can facilitate the integration of mycorrhizal fungi into new predictive models of boreal forest soils. Summary center dot In boreal forests, turnover of biomass and necromass of ectomycorrhizal extraradical mycelia (ERM) are important for mediating long-term carbon storage. However, ectomycorrhizal fungi are usually not considered in ecosystem models, because data for parameterization of ERM dynamics is lacking. center dot Here, we estimated the production and turnover of ERM biomass and necromass across a hemiboreal Pinus sylvestris chronosequence aged 12 to 100 years. Biomass and necromass were quantified in sequentially harvested in-growth bags, and incubated in the soil for 1-24 month, and Bayesian calibration of mathematical models was applied to arrive at parametric estimates of ERM production and turnover rates of biomass and necromass. center dot Steady states were predicted to be nearly reached after 160 and 390 growing season days, respectively, for biomass and necromass. The related turnover rates varied with 95% credible intervals of 1.7-6.5 and 0.3-2.5 times yr-1, with mode values of 2.9 and 0.9 times yr-1, corresponding to mean residence times of 62 and 205 growing season days. center dot Our results highlight that turnover of necromass is one-third of biomass. This together with the variability in the estimates can be used to parameterize ecosystem models, to explicitly include ERM dynamics and its impact on mycorrhizal-derived soil carbon accumulation in boreal forests

Data from: Carbon use efficiency of mycorrhizal fungal mycelium increases during the growing season but decreases with forest age across a Pinus sylvestris chronosequence

1. In boreal forest soils, mycelium of mycorrhizal fungi is pivotal for regulating soil carbon (C) cycling and storage. The carbon use efficiency (CUE), a key parameter in C cycling models, can inform on the partitioning of C between microbial biomass, and potential soil storage, and respiration. Here we test the dependency of mycorrhizal mycelial CUE on stand age and seasonality in managed boreal forest stands. 2. Based on mycelial production and respiration estimates, derived from sequentially incubated ingrowth mesh bags, we estimated CUE on an ecosystem-scale during a seasonal cycle and across a chronosequence of eight, 12- to 158-years-old, managed Pinus sylvestris forest stands characterised by decreasing pH and nitrogen (N) availability with increasing age. Mycelial respiration was related to total soil respiration, and by using eddy covariance flux measurements, primary production (GPP) was estimated in the 12- and 100-years-old forests, and related to mycelial respiration and CUE. 3. As hypothesized, mycelial CUE decreased significantly with increasing forest age by c. 65%, supposedly related to a shift in mycorrhizal community composition and a metabolic adjustment to reduce their own biomass N demand with declining soil N availability. Furthermore, mycelial CUE increased by a factor of five over the growing season; from 0.03 in May to 0.15 in November, and we propose that the seasonal change in CUE is regulated by a decrease in photosynthate production and temperature. The respiratory contribution of mycorrhizal mycelium ranged from 14 to 26% of total soil respiration, and was on average 17% across all sites and occasions. 4. Synthesis: Carbon is retained more efficiently in mycorrhizal mycelium late in the growing season, when fungi have access to a more balanced C and nutrient supplies. Earlier in the growing season, at maximum host plant photosynthesis, when belowground C availability is high in relation to N, the fungi respire excess C resulting in lower mycelial CUE. Additionally, C is retained less efficiently in mycorrhizal fungal biomass in older forest stands characterized by more nutrient depleted soils than younger forest stands

Mycorrhizal fungal mycelium - carbon use efficiency

Data on respiration, carbon use efficiency, mycelial biomass, mycelial production, gross primary production and sporocarp production. Biomass estimates are based on amounts of ergosterol (a fungal-specific biomass marker), quantified using liquid chromatography. Growth estimates are derived from biomass estimates and mathematical models. Estimates of carbon use efficiency are based on growth and respiration estimates. Gross primary production estimates are based on eddy covariance flux measurements

Ki-67 IHC versus ⁶⁴Cu-ATSM and ¹⁸F-FDG autoradiography.

<p>Visual example of the comparison between a Ki-67 IHC image and ⁶⁴Cu-ATSM and ¹⁸F-FDG autoradiography images for tumour 2. First column: Ki-67 IHC image rescaled to the same pixel size (42 μm) as the autoradiographies (AR). A selection is chosen for illustration of correlations between Ki-67 IHC and ¹⁸F-FDG and ⁶⁴Cu-ATSM AR respectively (columns 2–4).</p

Correlation between gene expression of Ki-67 and ⁶⁴Cu-ATSM and ¹⁸F-FDG uptake.

<p>Spearman’s Rank correlations (ρ) and p-values for comparison of gene expression for Ki-67 and tumour uptake of ¹⁸F-FDG (right column) and ⁶⁴Cu-ATSM (left column) calculated as standardized uptake value (SUV) from gamma counts of tumour pieces. Row <b>A</b>, <b>B</b> and <b>C</b> show data for tumour pieces from canine cancer patient 2, 3 and 4 respectively. n is the number of tumour pieces included in the final analysis. <i>n</i>.<i>s</i>. not significant. Significant correlations are written in bold.</p