Animal ethics misconduct: mice with very large tumors

In the past few years I have found some examples of papers showing photos of mice or rats with very large tumors. Some of these tumors appear to go far beyond what animal ethics guidelines consider to be acceptable.

This post contains images that might be disturbing to some viewers. So, please proceed with caution.

Before I continue this post, I want to clearly state that I am not against animal experiments per se. Cancer research, drug development and other areas of research are often dependent on using animals to test drugs on, before they can continue to test their therapies on humans. Microbiome research – the field I have work in – has benefitted from the development of germ-free animals to test the effect of lack of microbes on organ development and behavior.

I also realize that different institute have different guidelines for what they think is acceptable animal suffering. What is allowed in one institute would not be acceptable in another institute. This post is just based looking at photos and thinking about how (un)comfortable the animal was at that time.

Animal Ethics Committees oversee animal work at research institutes

To make sure that animals are not unnecessarily used in experiments, research institutes have an Animal Ethics Committee (AEC, names might vary per institute) that provides guidelines, reviews and approves protocols, and oversees that animal handlers have the appropriate certifications and are following the guidelines.

Keywords often used by these committees are Replacement, Refinement, and Reduction, which reflect the ultimate goal of trying to find alternative methods that use fewer animals.

A researcher cannot just order a bunch of mice and start experimenting on them. Researches need to show their institutional AEC that they will use enough animals to show significant results, but not more animals than needed, that they will treat the animals well (enough water and food, clean facilities, pain management, etc), and that they will put down animals humanely at any point in time where they feel an animal suffers.

Guidelines for tumor sizes

Animal ethics guidelines usually include rules about the maximum size of tumors with respect to an animal’s body weight or size. You can imagine that large tumors might be painful, limit an animal’s mobility, or ability to eat and clean itself. At some point, a tumor is too big, and the animal should be put down before it suffers anymore. Such guidelines are sometimes called “human endpoint criteria”.

Although institutes might differ in specific guidelines, and although specific rules are not always easily accessible for people at other institutes, there are some general guidelines that are available. Here are some published guidelines.

• Guidelines for the welfare and use of animals in cancer research – P. Workman et al. – British Journal of Cancer (2010).
• Guidelines for the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35892‐2018 – MacArthur Clark JA and Sun D – Animal Models and Experimental Medicine (2020)
• Laboratory Guidelines for Animal Care – Couto M and Cates C – Vertebrate Embryogenesis (2019)
• Washington State University, Institutional Animal Care and Use Committee – Tumor Burden Guide (2017)

Animal ethics in scientific publishing

Just like research institutes need to make sure that animal experiments are done in an ethical way, scientific publishers need to make sure that the papers they publish include experiments that were performed following those guidelines.

Most journals have guidelines for how to publish animal research. Most of these focus on providing enough experimental details (e.g. breed, age, sex of animals used). Here are some general publications that focus on how to properly report animal experiments in scientific papers:

• Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research – Kilkenny C et al. – PLOS Biology (2010)
• A gold standard publication checklist to improve the quality of animal studies, to fully integrate the Three Rs, and to make systematic reviews more feasible – Hooijmans CR et al. – Altern Lab Anim (2010)
• Guidance for the Description of Animal Research in Scientific Publications – Institute for Laboratory Animal Research – National Academies of Sciences, Engineering Medicine (2011)

In addition, most publishers and their journals offer their own guidelines to ensure that animal experiments are performed according to the rules.

The Committee for Publication Ethics, COPE, is an organization that supports and educates most major scientific journals and publishers. COPE has many guidelines and flowcharts for how to handle e.g. allegations of misconduct and how to retract papers. Most PubMed-indexed journals (but not all) are COPE members. Unfortunately, although COPE features some specific cases on their website, it does not appear to have specific guidelines on how to handle concerns about animal ethics in papers.

From the examples I will give below, it appears as if some journals or publishers do not check for animal welfare during the editorial process, which is unfortunate.

A mouse with a huge uterus tumor

Here is a paper I discovered yesterday.

Establishment and characterization of novel human primary endometrial cancer cell line (ZJB-ENC1) and its genomic characteristic – Xiaozhen Liu et al. – Journal of Cancer (2019) – PubPeer comment

In this paper, a new human cell line was created from a 58-year old woman with endometrioid adenocarcinoma (cancer of the uterus lining). To show the tumorigenicity of this new cell line, the uterus tumor cells were injected into 6 “nude” mice.

Nude mice have greatly reduced T-cells and therefore a lack of immune response to tumors. They are often used as a test model for tumor growth. They also are nearly hairless, hence the name.

Figure 4A shows a photo of a mouse with a huge tumor. The length of the tumor is about half the length and width of the animal. Figure 4B shows the average size of the tumor diameter in 6 mice, reaching 29 mm after 35 days.

It appears that the size of the tumor in this mouse was far beyond animal ethics guidelines. Looking at the photo, it is almost as if they carry a baby mouse on their back. How would a human feel if they had a tumor the size of a baby?

The Methods states that “All animal studies were performed according to protocols approved by the Institutional Animal Study Committee of Zhejiang University of Traditional Chinese Medicine Animal Testing Center”. According to the guidelines of Zhejiang University, a mouse cannot carry a tumor more than 10% of its body weight.

Looking at Figure 4A it appears that the tumor is about half the length and half the width of the body of the animal, which would put the tumor at 20-25% of the mouse’s weight. That is much more than the suggested 10%.

Workman et al. (Br J Cancer 2010) “Guidelines for the Welfare and Use of Animals in Cancer Research” states “For an animal carrying a single tumour, the mean diameter should not normally exceed 1.2 cm in mice.”

In this Liu et al. paper the diameter was more than double of that, i.e. 2.9 cm.

Of note, this work was supported by grants from the National Natural Science Foundation of China (comparable to the US National Institute of Health or National Science Foundation), the Natural Science Foundation of Zhejiang Province, and the Zhejiang Provincial Health Department Foundation. It is sad to see that there was no apparent oversight in the animal ethics aspect of this research.

The black and white tumors

SWAP-70 contributes to spontaneous transformation of mouse embryo fibroblasts – Yu-Tzu Chang et al. – Experimental Cell Research (2016), DOI: 10.1016/j.yexcr.2015.06.011 – PubPeer comment

This 2016 paper describes an embryonic cell line that showed unlimited growth and that formed tumors in nude mice. As stated in the Methods, “This study was carried out in strict accordance with the recommendations in the guide for the care and use of Laboratory Animal center of the National Health Research Institutes (NHRI). (…) The protocol was approved by the committee on the ethics of Animal Experiments of the NHRI (Permit Number: NHRI-IACUC-101028-A). (…) Maximum weight of the cancer was 0.87 g.“

Assuming that a tumor of 1 cm³ weighs about 1 gram, that suggests that no tumors were permitted to grow larger than 1 cm³ , which is roughly the same as in the previous paper that stated that tumors in mice should not exceed 1.2 cm diameter.

Some individual tumors in Figures 6 and 7 of this paper, however, appear to be much bigger. In addition, animals appear to be carrying multiple tumors on both sides of the body. The size of the tumors thus appears to exceed animal guidelines.

I posted my concerns on PubPeer in January of this year, but none of the authors or journal editors have answered yet.

The repetitive mice

Targeted Hsp70 expression combined with CIK-activated immune reconstruction synergistically exerts antitumor efficacy in patient-derived hepatocellular carcinoma xenograft mouse models – Huanzhang Hu et al. – Oncotarget (2015) – DOI: 10.18632/oncotarget.2835 – PubPeer comments.

Figure 3C shows lots of mice corpses, and many of these bear large tumors that were likely interfering with mobility and feeding of the animals. In addition, some of these mice appeared to be duplicated between the top and the bottom panels.

An anonymous PubPeer user already raised the alarm about this figure in 2015, writing “A grotesque example of the wild west in biomedical research. The tumor sizes are much larger than allowed.”

An other user argued that several guidelines suggest a maximum tumor size of a diameter of 20 mm (= 10 mm radius), which would measure 4,200 cubic mm.

A third user (presumably one of the authors) commented that experiments had to be stopped if a tumor was 3,000 mm3, and that the paper appear to adhere to that. After I reported this paper to the journal in August 2019, the authors showed that most of the tumors were between 2,000-3,200 cubic mm.

Still, 3,000 or even 4,200 cubic mm is much larger than the guidelines as posted by Workman et al. above, which had an upper limit of a diameter of 1.2 cm (12 mm, 1,700 cubic mm). Clearly, different institutes have very different limits of tumor sizes that can vary 3-4 fold.

Meanwhile, after I reported this paper in August 2019, the journal published a statement around December 2019 that the paper is “currently undergoing investigation”.

The Ova66 mice

OVA66, a Tumor Associated Protein, Induces Oncogenic Transformation of NIH3T3 Cells – Wei Rao et al. – PLOS ONE (2014), DOI: 10.1371/journal.pone.0085705 – PubPeer comments

This 2014 PLOS ONE paper shows two mice in Figure 5 that have very large tumors. Looking at the panel below, some of the tumor sizes exceeded 3 or even 3.5 cubic cm.

Again, it is not clear what the institute’s maximum tumor size rule is, but looking at both animals it appears clear that they could hardly move (look at the vein/skin attached between the left mouse’s tumor and its hind legs).

The proteomics mice

Establishment of a new OSCC cell line derived from OLK and identification of malignant transformation-related proteins by differential proteomics approach – Yang Dong – Scientific Reports (2015) – DOI: 10.1038/srep12668 – PubPeer comments

Figure 5 of this paper shows two mice, one of which carries a large tumor on it side, created by injecting an oral cancer cell line. The text states that the tumors were about 1 cm in size – but they appear to be much larger on the photo.

The Methods state that “The animal experiments were carried out in accordance with the approved guidelines and all experimental protocols were approved by the Animal Care and Use Committee of Cancer Hospital and Cancer Institute in Chinese Academy of Medical Sciences and Peking Union Medical College.”

A call for more unity and scrutiny

Based on posting these images on Twitter, the comments appear to range from “This is acceptable, why are you being so problematic?” to “Tumor size are clearly beyond the size allowed by our institute”.

Clearly, institutes differ widely in the acceptable maximum tumor size. One of the problems in interpreting the rules is that they are not consistent in units, both in the guidelines as well as in the publications. Tumor sizes are listed as percentage of body weight, weight, diameter, or cubic cm/mm. There are also different rules for different tumor types, and how to calculate the maximum allowable size if a mouse has multiple tumors.

Looking at these photos and hearing that some authors state that these were within range of their institutional AEC, one could wonder if publishers should create some uniform and strict animal ethics rules to govern the maximum size of tumors and other inhumane experiments. This might also prevent some clarity about the use of a (in my opinion) particular cruel rat model on depression (see an earlier blog post).

The welfare of laboratory animals would benefit greatly if publishers would start paying more attention to these animal ethics guidelines.