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Open AccessArticlePost Publication Peer ReviewVersion 2, Revised

Differentiation of Heterogeneous Mouse Liver from HCC by Hyperpolarized 13C Magnetic Resonance (Version 2, Revised)

Department of Radiology, Hadassah Medical Center, The Faculty of Medicine, Hebrew University of Jerusalem, Kalman Mann 1, Jerusalem 9112001, Israel
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Received: 13 December 2019 / Revised: 10 January 2020 / Accepted: 10 January 2020 / Published: 10 June 2020
Peer review status: 2nd round review Read review reports

Reviewer 1 Rodolfo Sacco Cisanello Hospital, Pisa, Italy Reviewer 2 Melissa Edwards Colorado State University
Version 1
Approved with revisions
Authors' response
Approved with revisions
Authors' response
Version 2
Reviewer invited Approved
Version 2, Revised
Published: 10 June 2020
DOI: 10.3390/sci2020043
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Version 1, Original
Published: 13 January 2020
DOI: 10.3390/sci2010003
Download Full-text PDF
The clinical characterization of small hepatocellular carcinoma (HCC) lesions in the liver and differentiation from heterogeneous inflammatory or fibrotic background is important for early detection and treatment. Metabolic monitoring of hyperpolarized 13C-labeled substrates has been suggested as a new avenue for diagnostic magnetic resonance. The metabolism of hyperpolarized [1-13C]pyruvate was monitored in mouse precision-cut liver slices (PCLS) of aged MDR2-KO mice, which served as a model for heterogeneous liver and HCC that develops similarly to the human disease. The relative in-cell activities of lactate dehydrogenase (LDH) to alanine transaminase (ALT) were found to be 0.40 ± 0.06 (n = 3) in healthy livers (from healthy mice), 0.90 ± 0.27 (n = 3) in heterogeneously inflamed liver, and 1.84 ± 0.46 (n = 3) in HCC. Thus, the in-cell LDH/ALT activities ratio was found to correlate with the progression of the disease. The results suggest that the LDH/ALT activities ratio may be useful in the assessment of liver disease. Because the technology used here is translational to both small liver samples that may be obtained from image-guided biopsy (i.e., ex vivo investigation) and to the intact liver (i.e., in a non-invasive MRI scan), these results may provide a path for differentiating heterogeneous liver from HCC in human subjects. View Full-Text
Keywords: lactate dehydrogenase; alanine transaminase; MDR2 knockout; dissolution dynamic nuclear polarization; perfused precision cut liver slices lactate dehydrogenase; alanine transaminase; MDR2 knockout; dissolution dynamic nuclear polarization; perfused precision cut liver slices
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Figure 1

MDPI and ACS Style

Lev-Cohain, N.; Sapir, G.; Uppala, S.; Nardi-Schreiber, A.; Goldberg, S.N.; Adler-Levy, Y.; Sosna, J.; Gomori, J.M.; Katz-Brull, R. Differentiation of Heterogeneous Mouse Liver from HCC by Hyperpolarized 13C Magnetic Resonance. Sci 2020, 2, 43.

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Reviewer 1

Sent on 17 Apr 2020 by Rodolfo Sacco | Approved with revisions
Cisanello Hospital, Pisa, Italy

The paper is interesting and worth of mentioning.  I would like to  ask Authors:

-if they have data on larger nodules from the same mouse.  

-if Authors think the ratio LDH/ALT could be useful (using 13C MRI) to monitor efficacy of targeted therapy for HCC (see the role of monitoring LDH levels during Sorafenib therapy).   Please, The authors should speculate on this matter.

Response to Reviewer 1

Sent on 10 Jul 2020 by Naama Lev-Cohain, Gal Sapir, Sivaranjan Uppala, Shraga Nahum Goldberg, Atara Nardi-Schreiber, Yael Adler-Levy, Jacob Sosna, J. Moshe Gomori, Rachel Katz-Brull

During surgery, when the nodules appeared large in gross examination, we included only those nodules (with minimal surrounding tissue) and the tissue was classified as part of the HCC group. In fact, we observed that in this cohort of MDR2-KO mice, the livers either presented small nodules (included here in the “heterogeneously inflamed liver” group) or large tumors (HCC group). Therefore, the “larger nodules” are already included in this study as they comprise the HCC group.

This is a very interesting aspect. High serum LDH levels are considered to be correlated with hypoxia within the tumor microenvironment. It has been shown that patients with low LDH levels pre-treatment with sorafenib have better progression-free survival (PFS) and overall survival (OS), both in HCC [1] and in pancreatic adenocarcinoma [2]. In HCC, It was shown that a decrease in the serum LDH levels during the treatment predicts better patient survival (both PFS and OS) [1]. It was later confirmed on a much larger cohort of patients (more than 5,000) that a decrease in serum LDH during sorafenib treatment in HCC predicts improved clinical outcome, while baseline LDH levels do not [3], suggesting that LDH dynamics during treatment may be a better predictor of clinical outcome than one pre-treatment measurement. Current guidelines by the NCCN support the systemic therapy (with sorafenib as a first-line agent) for inoperable local HCC, and for advanced HCC (although TACE is considered superior, if possible)[4]. Clinical (dermatologic adverse effects [5]) and biochemical (baseline plasma angiotensin 2 and VEGF levels [6]) markers were suggested as predictors for response to sorafenib, but so far none has made it into clinical practice guidelines. It is indeed possible that the LDH/PDH ratio could outperform previously suggested markers and provide information on early response to sorafenib to predict clinical outcome, once acquisition methods to determine these enzymatic rates can be applied on patients. References [1] L Faloppi, et al., BMC Cancer 2014. [2] L Faloppi, et al., Oncotarget 2015. [3] R Sacco, et al., The International Journal of Biological Markers 2015. [4] NCCN, (accessed 7/6/2020) 2020. [5] M Reig, et al., Journal of Hepatology 2014. [6] JM Llovet, et al., Clinical Cancer Research 2012.

Reviewer 2

Sent on 10 May 2020 by Melissa Edwards | Approved with revisions
Colorado State University

ONLY requested revision: Figure 1 would benefit from the inclusion of an image of the healthy liver tissue as all other figures compare the healthy, heterogeneously inflamed, and HCC tumor livers.


Are there other sources of these liver enzymatic rates (from either healthy or diseased animals) that can be re-calculated to expand your study and exhibit the consistent ratios? In your M&M you mentioned that the info for your healthy mice in Table 1. was obtained from your previous work, but are there other resources for similar information?


Understanding that the primary method is particularly to focus on in vivo and ex vivo models, do you know of any in vitro based methods that further support your results in being able to so clearly differentiate between the inflamed and HCC tissue types?


I understand how and why it was not possible for this study and I would be interested to know if the ratios would be comparable between male and female mice as all male mice were used in this study. I would also like to say that I appreciate your use of available mice and the minimization of sacrificed animals.


Overall, a fascinating study and rather sound.

Response to Reviewer 2

Sent on 10 Jul 2020 by Naama Lev-Cohain, Gal Sapir, Sivaranjan Uppala, Shraga Nahum Goldberg, Atara Nardi-Schreiber, Yael Adler-Levy, Jacob Sosna, J. Moshe Gomori, Rachel Katz-Brull


In our previous work on PCLS from healthy mice (DOI: 10.1002/nbm.4043), we compared LDH and PDH activities measured in biochemical methods to our results. The following is an excerpt from the discussion section relating to this aspect: “[1‐ 13C]lactate production in liver slices was found here to range between 1.1 and 4.1 nmole/s/g wet weight. Previously, in whole perfused rat liver, the LDH activity was determined to be 13 nmole/s/g wet weight (during 15 min of aerobic perfusion, converting from 0.78 μmol/min/g, published in Woods HF and Krebs HA. Lactate production in the perfused rat liver. Biochem J. 1971;125(1):129‐139). This determination was made using a standard biochemical assay, which relies on the quantification of NADH concentration by UV absorption (at 340 nm). This comparison suggests that the LDH activity determined in the current study is lower but within the same order of magnitude as determined previously using a standard biochemical assay. We note that the LDH activity determined in the current study reflects the actual metabolic rate as influenced by the intracellular microenvironment of the hepatocytes in real time, and that the measurement is direct—i.e., the actual level of lactate produced is determined. However, the standard biochemical assay for LDH activity is performed on lysed cells and therefore may be less correlated to the activity in the intact cell. In addition, the latter method is indirect. The [1‐ 13C]alanine production rate determined here was 2.6 to 9.7 nmole/s/g wet weight. Previously, using a commercially available kit, ALT activity was determined in flash‐frozen and then processed liver tissues by a coupled enzyme assay, which results in a colorimetric (570 nm) product proportional to the pyruvate generated. The resulting ALT activity in these homogenized liver tissues was 2.7 μmol/min/g (Josan S, et al. Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized 1‐C‐13 pyruvate. NMR Biomed. 2015;28(12):1671‐1677), which can be converted to 45 nmol/s/g tissue. Our results appear to be lower but of the same order of magnitude as the results determined with this classical biochemical assay. Nevertheless, the previous reports using classical biochemical assays do support the conclusion of the current study: that in the healthy rodent liver, under aerobic conditions, the ALT activity is higher than the LDH activity, or, in other words, that an ALT/LDH activity ratio larger than one can be regarded as a characteristic of the healthy liver.” Most importantly, in short, the LDH/ALT ratio that can be calculated based on these prior publications is ~0.29. Even though this is only a rough estimation, this ratio seems consistent with the results in livers from healthy mice presented in the current manuscript (0.40 ± 0.06).

Below please find an example for another study that showed a difference in activation of proteins in the presence of active inflammation in the liver and their relation to HCC tumor growth. Cohen S, Stemmer SM, Zozulya G, et al. CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver. J Cell Physiol. 2011;226(9):2438‐2447. doi:10.1002/jcp.22593.

We agree with the reviewer that further studies should include both male and female mice. We would expect the results to be similar between male and female mice because we are not aware of any enzymatic HCC characteristic that differs between genders in humans. In clinical practice there is no different cutoff used for abnormal levels of these enzymes in males or females. However, there is some indication for differences in serum ALT levels that is mostly attributed to Body Mass Index which differs between males and females. Please see: Bussler S, et al. Hepatology (2018) 68(4):1319-1330. doi: 10.1002/hep.29542. New pediatric percentiles of liver enzyme serum levels (alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase): Effects of age, sex, body mass index, and pubertal stage. However, we could not find any indication for sex differences in the in-cell enzyme activity within hepatocytes.

Thank you.

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