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1. Introduction

Cancer-related fatigue (CRF) is a form of physical, emotional and/or cognitive fatigue associated with cancer itself or its treatment. It is a remarkably distressing subjective sensation that persists for an extended duration, disproportionate to recent activity, and significantly interferes with normal functioning, thereby impacting patients' quality of life (1,2). CRF is prevalent among patients with cancer, with nearly all patients reporting symptoms of fatigue (3,4). Currently, the treatment of CRF primarily involves non-specific medications (such as antidepressants, analgesics and stimulants), psychological interventions (such as cognitive behavioral therapy) and physical activity interventions. However, due to the unclear pathophysiology of CRF, these symptomatic treatments are of limited effectiveness or are accompanied by side effects.

Mice are commonly used in experimental studies of CRF, and the establishment of mouse models that accurately simulate the fatigue symptoms experienced by patients with cancer is crucial for investigating the mechanisms of CRF. Currently, several researchers have developed various mouse models of CRF using different approaches. However, there is inconsistency across these models in terms of strain selection, modeling methods and fatigue assessment criteria, and standardized protocols have yet to be established. Furthermore, although technologies such as metabolomics and proteomics have made significant progress in exploring the mechanisms of CRF, specific therapeutic targets for CRF remain unclear. A substantial gap still exists between clinical research and experimental studies. The present review summarizes and discusses the modeling methods for CRF in mice and the underlying mechanisms of fatigue, with the aim of providing insights for further research on CRF.

2. Developing a CRF model using tumor-bearing mice

Colon cancer-induced CRF mouse models

The Colon Tumor 26 (CT26) cell line is a commonly used mouse colon cancer cell line that is characterized by rapid proliferation and the ability to form both solid and metastatic tumors. Pin et al (5) subcutaneously implanted 1x106 CT26 cells into the dorsal area of male CD2F1 mice, while Peters et al (6) performed subcutaneous implantation of the same number of CT26 cells in the inguinal region. Shen et al (7) conducted a similar experiment but selected BALB/c mice instead. These researchers successfully established cancer-induced fatigue models, with experimental results showing that CT26 tumor-bearing mice exhibited a decrease in both body weight and tumor-free body weight, reduced skeletal muscle mass and strength, and significant fatigue symptoms. Other researchers used higher doses of CT26 cells for subcutaneous implantation, with several researchers subcutaneously implanting 5x106 CT26 cells into different mouse strains to establish tumor-bearing mouse fatigue models (8-13). For example, Roberts et al (12) and Murphy et al (10) used male CD2F1 mice, while Lee et al (9) and Song et al (13) used male BALB/c mice, and Norden et al (11) and Clark et al (8) conducted experiments with female CD2F1 mice. Norden et al (11,14) suggested that female mice bearing tumors could better maintain food intake, exhibit less weight loss and reduce the likelihood of local inflammation caused by mice gnawing at the tumor site, making them more suitable for studying cancer-induced fatigue.

In addition to subcutaneous implantation, intravenous injection of CT26 cells can also be used to establish a tumor-bearing mouse model. Ferdaos et al (15) injected 5x106 CT26 cells intravenously into one group of BALB/c mice, while another group received 2x106 CT26 cells subcutaneously in the back. Both groups exhibited fatigue symptoms and lung metastases, with a comparable tumor count. Compared with mice subjected to subcutaneous or intravenous injection of tumor cells, colon orthotopic tumor-bearing mice may simulate more closely the real conditions of patients with colon cancer. Zhang et al (16) reported a method for colon cancer orthotopic tumor modeling: Tumors formed by subcutaneously implanted CT26 cells were cut into 2x2x1 mm pieces and surgically implanted in the cecum of mice, with a success rate of 62.5%. Orthotopic tumors may better mimic the clinical scenario of colon cancer, but the procedure is more complex and has a lower success rate. Additionally, ApcMin/+(MIN) mice carrying a mutant Apc gene can also be used for colon cancer research. VanderVeen et al (17) found that male C57BL/6J MIN mice exhibited a 6% weight loss at 18 weeks of age, with an 8% reduction in forelimb grip strength and tibialis anterior muscle mass, and fatigue symptoms appeared before significant weight loss. Hetzler et al (18) observed that these mice showed sex differences, with female MIN mice experiencing less weight loss and lower blood IL-6 levels. This mouse model does not require tumor cell implantation, thus offering improved experimental stability; however, it is more expensive than CRF mouse models requiring tumor cell implantation.

Regardless of whether subcutaneous implantation, intravenous injection of CT26 cells, or orthotopic tumor transplantation is used, researchers have reported that tumor-bearing mice exhibit important fatigue behaviors, such as reduced grip strength and motor ability, decreased body weight, muscle mass and fat content, and elevated levels of inflammatory factors. Murphy et al (10) also found that injecting the same number of CT26 cells from different laboratories induced varying degrees of cachexia in mice, suggesting that researchers should ensure the use of cells from the same source within a single experiment to avoid excessive experimental error. Furthermore, to meet experimental objectives, it is recommended to use the minimum number of cancer cells necessary for implantation, reduce the tumor burden in mice, and perform euthanasia when tumors become excessively large, in compliance with animal ethics guidelines and to protect the welfare of laboratory animals.

Lung cancer-reduced CRF mouse models

Lewis Lung Carcinoma (LLC) is a commonly used mouse lung adenocarcinoma cell line, known for its high invasiveness and ability to simulate the progression of lung adenocarcinoma in mice. Scott et al (19) and Zhu et al (20) established lung cancer mouse models by subcutaneously implanting 5x105 LLC cells in the backs of male C57BL/6J and BALB/c mice, respectively. Zhou et al (21) subcutaneously implanted 3x105 LLC cells in the right axilla of male C57BL/6 mice, while Vichaya et al (22) implanted 1x106 LLC cells in the axilla, successfully creating lung CRF models in mice. Although the location and number of subcutaneously implanted LLC cells varied among the aforementioned studies, all reported that tumor-bearing mice exhibited significant fatigue symptoms, such as reduced wheel running, decreased forelimb grip strength and swimming time, and elevated levels of inflammatory factors.

Apart from subcutaneous implantation of LLC cells, intraperitoneal injection has also been utilized to establish a CRF model of lung cancer. Wu et al (23) injected 1x107 LLC cells into female ICR mice to create a fatigue model. Similarly, Kim et al (24) administered 0.5x106 LLC cells via intraperitoneal injection in both male and female C57BL/6J mice to establish a fatigue model. Additionally, another group of mice received an injection of 0.1x106 TC-1 cells (tumorigenic mouse lung epithelial cells), which also exhibited symptoms of fatigue, such as lethargy and reduced grooming behavior, although the level of fatigue was milder compared with that of the LLC group. While there are studies reporting the use of LLC cells via intrathoracic injection, these have not been applied to CRF research. Previous studies have employed varying cell numbers and implantation methods to develop mouse models of CRF, all reporting different fatigue symptoms. However, the differences in cell quantities, implantation sites and fatigue assessment methods make it challenging to compare the effects of tumor burden on the onset and severity of fatigue. Therefore, standardization of cell dosage and monitoring parameters is necessary to improve evaluation of fatigue levels in mice, and to facilitate comparison and reference across studies.

Head and neck cancer-reduced CRF mouse models

HPV16 E6/E7 and H-ras (mEER) cells are commonly used cancer cell lines closely associated with the development of head and neck cancer. Vichaya et al (25) established an ectopic syngeneic mouse model of HPV-related head and neck cancer using male oropharyngeal epithelial cells derived from stable expression of mEER cells. The method involved subcutaneously injecting 1x106 mEER cells into the right hind leg of adult male C57BL/6J mice and Ido1-/- mice, resulting in reduced wheel running and fatigue symptoms in both groups. In another study, Vichaya et al (22) used the same method to establish a fatigue model in Tlr4-/- mice on a C57BL/6J background. Grossberg et al (26,27) reported the successful establishment of an HPV-positive head and neck cancer model by subcutaneously implanting 1x106 mEER cells into male C57BL/6J mice, while another group of mice was injected with the same number of shPTPBL/hRas cells to establish an HPV-negative head and neck cancer model, with both groups showing fatigue symptoms. Grossberg et al (26,27) also found that metabolic competition between skeletal muscle and tumors may represent a non-inflammatory mechanism of cancer-induced fatigue, offering new insights for its treatment. However, it is important to note that mEER cells are derived from the tonsillar epithelium of male mice, and injecting these cells into female mice may result in unsuccessful tumor implantation.

Other cancer-reduced CRF mouse models

The KPC cell line (a pancreatic cancer cell line derived from mouse pancreatic tissue with Kras deletion and p53 mutation, KrasG12D/p53R172H) is commonly used for pancreatic cancer research. Zhu et al (28) divided C57BL/6J mice into three groups: Two groups received either 3x106 KPC cells for pancreatic orthotopic implantation or intraperitoneal injection, and the third group was subcutaneously injected with 10x106 KPC cells into the shoulder joint space. Fatigue behaviors were observed as early as 6 days post-injection. Zhu et al (28) concluded that the orthotopic pancreatic implantation model is the most stable and reliable, while the subcutaneous model yielded inconsistent results and is not recommended. Delitto et al (29) also reported that, compared with intraperitoneal injection, orthotopic implantation of KPC cells led to more severe muscle atrophy and systemic inflammation.

The 4T1 cell line is a commonly used breast cancer cell line. Yang et al (30) successfully established a cancer-induced fatigue model by injecting 5x105 4T1 cells into the right fourth mammary fat pad of female BALB/c mice. Consistent with the results of Norden et al (14), Yang et al (30) also found that food intake and appetite were more stable in female mice compared with male mice after cancer cell inoculation, making them more suitable for cancer-induced fatigue studies.

The B16-F10 cell line is a melanoma cell line, and the ID8 cell line is a commonly used mouse ovarian cancer cell line, both of which are highly invasive. Kim et al (24) intraperitoneally injected 0.3x106 B16-F10 and 1x106 ID8 cells into two groups of C57BL/6J mice, respectively, and both groups displayed fatigue symptoms such as lethargy, reduced grooming behavior and lower hemoglobin levels, which indicate successful establishment of cancer-induced fatigue models.

Despite various cancer cell lines can be used to establish cancer-induced fatigue mouse models, most are ectopically implanted and may not fully capture the fatigue symptoms caused by organ-specific tumors. Future research should focus on cancer-induced fatigue models established by orthotopic cancer cell implantation. Detailed information on mouse models of CRF induced by tumor bearing is shown in Table I.

Table I Tumor-bearing mouse models of cancer-related fatigue.

Table I

Tumor-bearing mouse models of cancer-related fatigue.

First author, year	Mice sex and strain	Mice age	Cell line	Cell number	Site and method of cell injection	(Refs.)
Pin et al, 2022	Male CD2F1 mice	11 weeks	C26	1x106	Subcutaneous injection into the flank	(5)
Peters et al, 2011	Male CD2F1 mice	6-7 weeks	C26	1x106	Subcutaneous injection into the right inguinal flank	(6)
Shen et al, 2019	Male BALB/c mice	6-8 weeks	C26	1x106	Subcutaneous injection into the flank	(7)
Clark et al, 2015	Female CD2F1 mice	Adult	C26	5x105	Subcutaneous injection between the scapulae	(8)
Lee et al, 2021	BALB/c mice	6 weeks	C26	5x105	Subcutaneous injection into the right flank	(9)
Murphy et al, 2012	Male CD2F1 mice	12 weeks	C26	5x105	Subcutaneous injection into the dorsal side	(10)
Roberts et al, 2013	Male CD2F1 mice	Adult	C26	5x106	Subcutaneous injection into each flank	(12)
Song et al, 2024	Male BALB/c mice	6 weeks	C26	5x105	Subcutaneous injection in the dorsal region	(13)
Norden et al, 2015	Female CD2F1 mice	10 weeks	C26	5x105	Subcutaneous injection between the scapulae	(11,14)
Ferdaos et al, 2023	Male BALB/c mice	8 weeks	C26	5x105, 2x106	Intravenous injection, subcutaneous injection into the right flank	(15)
Zhang et al, 2019	Male BALB/c mice	6-7 weeks	C26 subcutaneous tumor tissue	2x2x1 mm	Intraperitoneal orthotopic implantation	(16)
Scott et al, 2022	Male C57BL/6J mice	10 weeks	LLC	5x105	Subcutaneous injection into the right flank	(19)
Zhu et al, 2019	CD2F1 mice	4-6 weeks	LLC	5x105	Subcutaneous injection in the scapula	(20)
Zhou et al, 2020	Male C57BL/6J mice	6 weeks	LLC	3x106	Subcutaneous injection into the right underarm	(21)
Vichaya et al, 2020	C57BL6/J mice	Adult	LLC	1x106	Subcutaneous injection into the right flank	(22)
Wu et al, 2022	C57BL/6J mice	5 weeks	LLC	1x106	Subcutaneous injection in the right flank	(23)
Kim et al, 2014	C57BL/6J mice	6 weeks	LLC, TC-1, B16-F10, ID8	0.5x106, 0.1x106, 0.3x106, 1x106	Intraperitoneal injection	(24)
Vichaya et al, 2019	Male C57BL/6J mice, Ido1-/- mice	Adult	mEER, luciferase- tagged mEER	1x105, 1x106	Intramuscular injection into the right hind leg, subcutaneous injection into the right flank	(25)
Grossberg et al, 2020	Male C57BL/6J mice	10-13 weeks	mEER	1x106	Subcutaneous injection into the right flank	(26)
Grossberg et al, 2018	Male C57BL/6J	10-13 weeks	mEER, LLC, ID8, IG10, shPTPBL/hRas	1.1x106, 5x105, 1x106, 1x106, 1x106	Subcutaneous injection into the right flank	(27)
Zhu et al, 2019	C57BL/6J mice	Adult	KPC	3x106, 3x106, 10x106	Orthotopic implantation, intraperitoneal injection, subcutaneous injection into interscapular space	(28)
Delitto et al, 2017	Female NSG mice	8 weeks	PANC-1, L3.6pl	1x106	Orthotopic injections into the pancreas, subcutaneous injection	(29)
Yang et al, 2021	Female BALB/c mice	10 weeks	4T1	5x105	Subcutaneous injection into the right fourth mammary fat pad	(30)

[i] LLC, Lewis lung carcinoma; mEER, HPV16 E6/E7 and H-ras; sh, small hairpin.

3. Chemotherapy-induced CRF mouse models

5-Fluorouracil (5-FU)-induced CRF mouse model

5-FU is a commonly used antineoplastic drug that can cause severe gastrointestinal reactions and bone marrow suppression, leading to significant fatigue symptoms in patients. C57BL/6 mice are frequently used in fatigue experiments induced by intraperitoneal injection of 5-FU. Wolff et al (31) induced fatigue-like behavior in mice by administering 50 mg/kg 5-FU intraperitoneally for 3 consecutive days. Fatigue symptoms, including reduced body weight and spontaneous wheel running activity, were observed 1 week later. Dougherty et al (32) injected 60 mg/kg 5-FU intraperitoneally for 5 consecutive days, resulting in decreased body weight day 6, with voluntary wheel running activity and treadmill fatigue test confirming fatigue in the mice. Mahoney et al (33,34) reported in two studies that intraperitoneal injections of 5-FU at doses of 20, 40 or 60 mg/kg for 5 consecutive days led to milder fatigue and faster recovery in mice treated with lower doses, suggesting that fatigue may be dose-dependent. Hsu et al (35) reported that intraperitoneal injections of 60 mg/kg 5-FU for 5 consecutive days resulted in significant reductions in skeletal muscle and epididymal fat weight, as well as a marked decrease in exercise distance. Researchers have successfully established CRF mouse models using intraperitoneal injection of 5-FU. This method is simple, has a high success rate, and produces stable and reliable models. However, the appropriate 5-FU dose should be selected based on the experimental objectives to avoid excessive symptom burden and toxic reactions in the mice due to high doses of 5-FU.

Platinum-based chemotherapy-induced CRF mouse models

Cisplatin and oxaliplatin are commonly used platinum-based drugs in clinical practice, both of which can cause severe gastrointestinal reactions, bone marrow suppression and neurotoxicity, leading to significant fatigue symptoms in patients. Feather et al (36) established a fatigue model by administering oxaliplatin to C57BL/6J mice using both short- and long-term protocols. The short-term protocol involved administering 5 mg/kg oxaliplatin every other day for a total of four injections, with a cumulative dose of 20 mg/kg. The long-term protocol consisted of administering 2.5 mg/kg oxaliplatin 12 times over 17 days, with a cumulative dose of 30 mg/kg. The results showed that fatigue symptoms, including weight loss and reduced activity, appeared as early as the fourth day after oxaliplatin injection, with the severity of fatigue significantly correlated with the drug dose. Sakai et al (37) administered 3 mg/kg cisplatin intraperitoneally to C57BL/6J mice once daily for 4 consecutive days, resulting in muscle atrophy and pronounced fatigue, suggesting that fatigue may be associated with chemotherapy-induced muscle atrophy. Scott et al (38) and Chelette et al (39) administered 2.83 mg/kg cisplatin intraperitoneally to mice once daily for a total of five doses, leading to fatigue symptoms characterized by reduced wheel running activity and weight loss, but no decrease in food intake. This finding suggests that cisplatin-induced fatigue may be due to muscle fatigue rather than mental fatigue. Researchers generally use similar cumulative doses of platinum-based drugs and prefer intraperitoneal injection due to its simplicity. However, injecting these drugs may not be suitable for studying mental fatigue in mice. It is recommended that the method of fatigue induction is selected appropriately according to the research objectives.

Doxorubicin chemotherapy-induced CRF mouse models

Doxorubicin is a broad-spectrum antitumor drug with potent cytotoxicity, leading to severe bone marrow suppression and cardiotoxicity. Gilliam et al (40) administered doxorubicin to C57BL/6 mice through both intravenous and intraperitoneal routes at a dose of 20 mg/kg (equivalent to the clinical dose for patients) for 3 consecutive days. The study found that doxorubicin caused hind limb muscle impairment in mice, with both administration routes leading to respiratory muscle weakness, although intraperitoneal injection resulted in more severe diaphragm weakness. Mackay et al (41) conducted an experiment with a single intraperitoneal injection of 15 mg/kg doxorubicin and observed no significant muscle strength decline after 3 days, possibly due to the short observation period, during which skeletal muscle dysfunction had not yet fully developed. Zombeck et al (42) administered doxorubicin to female Hsd-ICR (CD1) mice at a dose of 2.5 mg/kg per day for 6 days. By the second week of treatment, the mice exhibited weight loss and reduced afternoon wheel running activity, similar to the afternoon fatigue exacerbation observed in patients with cancer. Wang et al (43) created a circadian rhythm disorder environment by altering light exposure and administered doxorubicin intraperitoneally at 3.75 mg/kg on days 1, 2, 9 and 10. The mice showed weight loss, decreased wheel running activity and extended rest periods, with fatigue symptoms associated with immunosuppression and circadian rhythm disturbances. Wang et al (44) further reported that, when comparing doxorubicin administered at 3.75 mg/kg intraperitoneally with 3.75 mg/kg daunorubicin and 11.25 mg/kg etoposide using the same method, the doxorubicin group exhibited more severe fatigue symptoms. Although numerous researchers have used doxorubicin to establish CRF mouse models, due to the severe cytotoxicity of doxorubicin and the more pronounced toxic reactions observed with intraperitoneal injection, caution must be exercised regarding the dosage and duration of experiments following doxorubicin administration. Some researchers have reported that a 20 mg/kg intraperitoneal injection of doxorubicin can result in 60-70% mortality in wild-type mice within 1 week (45,46).

Taxane drugs or cyclophosphamide-induced CRF mouse models

Taxane drugs are commonly used in clinical chemotherapy and can cause bone marrow suppression, neurotoxicity and allergic reactions. Ray et al (47) divided female BALB/c mice into two groups, and administered paclitaxel and albumin-bound paclitaxel (Abraxane) via the retro-orbital sinus vein at a dose of 10 mg/kg once daily for 5 consecutive days. The results showed that, while both groups of mice exhibited reduced activity indicative of fatigue, the paclitaxel group showed muscle damage and more severe fatigue. Loman et al (48) administered paclitaxel at 30 mg/kg (80% of the intravenous dose used in patients) to female BALB/c mice via tail vein injection every other day for a total of 6 doses. The study found that the mice's physical activity significantly decreased. Grant et al (49) used the same method and dosage to administer paclitaxel to female C57BL/6 mice, and observed that paclitaxel-induced fatigue in mice gradually recovered over time. Chaillou et al (50) administered a single dose of 20 mg/kg docetaxel and weekly injections of 20 mg/kg for 3 weeks via tail vein injection to female FVB/NRj mice. The results showed no weight difference between the two groups, but muscle mass significantly decreased in mice that underwent multiple chemotherapy sessions compared with that in mice that received a single dose. All the aforementioned researchers used intravenous injection of taxane drugs to establish fatigue models in mice. Compared with other taxane drugs, paclitaxel-induced fatigue is more severe. Furthermore, taxane-induced fatigue is generally milder and more easily recoverable compared with doxorubicin-induced fatigue, suggesting that researchers should select appropriate taxane drugs to establish CRF mouse models based on their research objectives.

Cyclophosphamide is also a commonly used clinical chemotherapy drug, which can cause bone marrow suppression and acute skeletal muscle effects. Crouch et al (51) administered a single tail vein injection of 300 mg/kg cyclophosphamide to female C57BL/6 mice, resulting in reduced activity indicative of fatigue. A total of 6 weeks after administration, treadmill running time was still significantly reduced. Our systematic literature review revealed that studies on inducing fatigue in mice using cyclophosphamide alone are relatively scarce.

Combination chemotherapy-induced CRF mouse models

Combination chemotherapy regimens are the most commonly used treatments for patients with cancer, and inducing fatigue in mice with combination chemotherapy may improve simulation of clinical scenarios compared with single-agent chemotherapy. Smith et al (52) used the CAF regimen (cyclophosphamide 167 mg/kg, doxorubicin 4 mg/kg and 5-fluorouracil 167 mg/kg) administered via intraperitoneal injection. The method involved intraperitoneal injection of cyclophosphamide and doxorubicin, followed by intraperitoneal injection of 5-FU 1 h later, for a total of four treatments, with each chemotherapy session spaced 20 days apart. The mice showed significant weight loss and a marked reduction in voluntary wheel running activity, with pronounced fatigue symptoms. Weymann et al (53) also used the CAF regimen to establish a mouse model of combination chemotherapy-induced fatigue, finding that this regimen induced fatigue/somnolence and decreased voluntary motor activity in mice. Wong et al (54) administered the CA regimen (cyclophosphamide 167 mg/kg and doxorubicin 4 mg/kg) via intraperitoneal injection to C57BL/6 mice every 2 weeks for a total of four treatments. The mice exhibited weight loss and reduced voluntary wheel running time, but their weight returned to baseline levels after the treatment ended.

FOLFOX (5-fluorouracil, leucovorin and oxaliplatin) is also a commonly used chemotherapy regimen, known to cause fatigue, weakness and skeletal muscle dysfunction. Halle et al (55,56) reported in two studies that intraperitoneal injection of 30 mg/kg 5-FU, 6 mg/kg oxaliplatin and 90 mg/kg leucovorin was administered to male C57BL/6 mice every 2 weeks for a total of four treatments. The results showed a decrease in running time and forelimb grip strength in mice, but no weight loss was observed. Barreto et al (57) also reported a study where male CD2F1 mice were divided into FOLFOX and FOLFIRI (5-FU, leucovorin, irinotecan) groups, and underwent five cycles of chemotherapy. Both groups showed quadriceps atrophy and muscle weakness, but only the FOLFIRI group exhibited progressive weight loss. The FOLFOX regimen was shown to induce fatigue symptoms in mice, with minimal impact on body weight, suggesting that studies focusing primarily on weight changes should carefully consider the FOLFOX chemotherapy regimen as an intervention factor.

Although both single-agent and combination chemotherapy can induce fatigue symptoms in mice and successfully establish chemotherapy-related CRF models, these experiments were conducted without tumor burden and therefore only reflect the effects of chemotherapy drugs on fatigue. Additionally, the drug dosages and injection routes used by different researchers vary. It is recommended that researchers, based on their study objectives and considering animal welfare, select appropriate methods and routes to establish CRF mouse models for research purposes. Datailed data on mouse models of CRF induced by chemotherapy are shown in Table II.

Table II Chemotherapy-induced cancer-related fatigue mouse models.

Table II

Chemotherapy-induced cancer-related fatigue mouse models.

First author, year	Mice sex and strain	Mice age	Chemotherapy drugs and dosage	Chemotherapy duration	Administration method	(Refs.)
Wolff et al, 2022	BNDF mice	7-18 weeks	5-FU 50 mg/kg	3 consecutive days	Intraperitoneal injection	(31)
Mahoney et al, 2013	C57BL/6 mice, MCP-1-/- mice	6 weeks	5-FU 20 mg/kg, 5-FU 40 mg/kg, 5-FU 60 mg/kg	5 consecutive days	Intraperitoneal injection	(33)
Hsu et al, 2020	Male C57BL/6 mice	8-9 weeks	5-FU 60 mg/kg	5 consecutive days	Intraperitoneal injection	(35)
Feather et al, 2018	Male C57BL/6 mice	8 weeks	Oxaliplatin 5 mg/kg, oxaliplatin 2.5 mg/kg	Once every other day, total of four doses, 3 consecutive days followed by a 3-day break for a total of nine doses	Intraperitoneal injection	(36)
Sakai et al, 2014	Male C57BL/6J mice	8-9 weeks	Cisplatin 1 mg/kg, cisplatin 3 mg/kg	4 consecutive days	Intraperitoneal injection	(37)
Scott et al, 2023	C57BL/6J mice	8 weeks	Cisplatin 2.83 mg/kg	5 consecutive days	Intraperitoneal injection	(38)
Chelette et al, 2023	C57BL/6J mice	10-12 weeks	Cisplatin 2.83 mg/kg	5 consecutive days	Intraperitoneal injection	(39)
Gilliam et al, 2011	Male C57BL/6 mice	8 weeks	Doxorubicin 20 mg/kg	One single dose	Intravenous administration, intraperitoneal injection	(40)
Mackay et al, 2021	C57BL/6 mice	5 weeks	Doxorubicin 15 mg/kg	One single dose	Intraperitoneal injection	(41)
Zombeck et al, 2013	Female Hsd-ICR mice	7 weeks	Doxorubicin 2.5 mg/kg	3 consecutive days, repeated after 1 week for a total of six doses	Intraperitoneal injection	(42)
Wang et al, 2024	Male C57BL/6J mice	10 weeks	Doxorubicin 3.75 mg/kg, aclarubicin 3.75 mg/kg, etoposide 11.25 mg/kg	Days 1, 2, 9 and 10	Intraperitoneal injection	(43)
Ray et al, 2011	Female BALB/c mice	Adult	Paclitaxel 10 mg/kg, Abraxane 10 mg/kg	5 consecutive days	Retro-orbital sinus vein injection	(47)
Loman et al, 2019	Female BALB/c mice	7-8 weeks	Paclitaxel 30 mg/kg	Once every other day for a total of six doses	Caudal vein injection	(48)
Grant et al, 2023	Female C57BL/6 mice	12 weeks	Paclitaxel 30 mg/kg	Once every other day for a total of six doses	Tail vein injection	(49)
Chaillou et al, 2017	Female mice on FVB/NRj strain background	8-11 weeks	Docetaxel 20 mg/kg	One single dose, days 1 and 21	Tail vein injection	(50)
Crouch et al, 2017	Female C57BL/6J mice	16 weeks	Cyclophosphamide 300 mg/kg	One single dose	Tail vein injection	(51)
Smith et al, 2014	Female mice lacking TNFR1-/- and/or IL-1R1-/-	Adult	CAF regimen (cyclophos- phamide 167 mg/kg, doxorubi- cin 4 mg/kg, 5-FU 167 mg/kg)	Four doses of CAF at 10-20 days intervals	Intraperitoneal injection	(52)
Weymann et al, 2014	Female C57BL/6J mice	8-12 weeks	CAF regimen (cyclophos- phamide 167 mg/kg, doxoru- bicin 4 mg/kg, 5-FU 167 mg/kg)	Four cycles at 3-week intervals	Intraperitoneal injection	(53)
Wong et al, 2018	Female C57BL/6 mice	10-12 weeks	CA regimen (cyclophosphamide 167 mg/kg, doxorubicin 4 mg/kg)	One single dose	Intraperitoneal injection	(54)
Halle et al, 2023	Male C57BL6/J mice	10 weeks	FOLFOX regimen (5-FU 30 mg/kg oxaliplatin, 6 mg/kg, leucovorin 90 mg/kg)	Four cycles at 2-week intervals	Intraperitoneal injection	(56)

[i] 5-FU, 5-fluorouracil.

4. Tumor-bearing mice with chemoradiotherapy-induced CRF mouse models

Colon cancer cell-bearing mice with chemotherapy-induced CRF models

Compared with chemotherapy-induced fatigue in healthy mice, chemotherapy in tumor-bearing mice may improve simulation of the CRF symptoms observed in patients with cancer. Abulizi et al (58) implanted 2.5x105 CT26 cells subcutaneously into female BALB/c mice to establish tumor-bearing mice. A total of 8 days later, 30 mg/kg of 5-FU was administered intraperitoneally every other day for a total of eight injections. Compared with the tumor-bearing mice without chemotherapy, the 5-FU chemotherapy group exhibited significantly increased immobility time in the forced swim test, indicating more severe fatigue. Furthermore, muscle histology showed disordered and ruptured skeletal muscle fibers with shortened sarcomere length. Park et al (59) subcutaneously inoculated 5x106 HT-29 cells (human-derived colon cancer cells) into immunodeficient female BALB/c-nu/nu mice. A total of 14 days later, 30 mg/kg of 5-FU was administered intraperitoneally 3 times a week for a total of 12 injections. The results similarly demonstrated that the 5-FU chemotherapy group exhibited significantly lower performance in both the running wheel activity and forced swim test compared with the tumor-bearing group without chemotherapy. Additionally, the 5-FU chemotherapy group had lower white and red blood cell counts. Compared with tumor-bearing mice without chemotherapy, 5-FU chemotherapy exacerbated fatigue in CT26 tumor-bearing mice. This model of CRF induced by 5-FU in colon cancer-bearing mice better replicates the clinical situation of chemotherapy in patients with colon cancer, and is more suitable for exploring the mechanisms of CRF and for developing new drugs.

In a study on alleviating fatigue induced by weighted swimming in rats, Wang et al (60) employed gas chromatography-mass spectrometry (MS)-based metabolomics and found significant metabolic disturbances in fatigued rats. Specifically, lipid peroxidation products, such as malondialdehyde, were markedly elevated, while levels of glutathione and superoxide dismutase were decreased, indicating an imbalance in skeletal muscle energy metabolism. In a study on postoperative fatigue syndrome, Lu et al (61) used ultra-high performance liquid chromatography-quadrupole time-of-flight MS-based metabolomics and identified hippocampal metabolic fluctuations in postoperative fatigued rats. The authors identified 15 hippocampal metabolites that could serve as potential drug targets, highlighting metabolomics as an important tool in the study of fatigue mechanisms. Future research could further explore the application of metabolomics in understanding the mechanisms of fatigue, providing a more diverse range of tools for fatigue mechanism research.

Lung cancer cell-bearing mice with chemotherapy-induced CRF models

Lung cancer-bearing mice treated with chemotherapy are also commonly used to induce CRF. Wu et al (23) subcutaneously implanted 1x106 LLC cells into C57BL/6 mice. When the tumor size reached 80-100 mm³, cisplatin was administered intraperitoneally at a dose of 5 mg/kg once a week for 3 weeks. Compared with tumor-bearing mice without chemotherapy, the cisplatin-treated group showed significantly reduced swimming time and distance, indicating more pronounced fatigue. Yoshizawa et al (62) established a similar tumor-bearing model by subcutaneously implanting cancer cells and, 8 days later, administering cisplatin intraperitoneally at 10 mg/kg for 4 consecutive days. The cisplatin-treated mice exhibited significantly reduced running time on the treadmill test and experienced weight loss compared with the tumor-bearing group without chemotherapy; however, their weight returned to baseline by day 16. Ouyang et al (63) subcutaneously implanted 5x106 A549 human lung adenocarcinoma cells into male BALB/c-nu nude mice. When the tumor size reached 80-100 mm³, cisplatin was administered intraperitoneally at 10 mg/kg every other day for a total of 10 injections. Compared with the tumor-bearing group without chemotherapy, the cisplatin-treated group experienced earlier onset of fatigue and higher levels of inflammatory cytokines. Although the cisplatin dosage and treatment duration varied across studies, the results consistently showed that cisplatin exacerbated and accelerated the onset of fatigue in tumor-bearing mice, with its effects on body weight gradually reversing over time.

Wood et al (64) subcutaneously implanted 5x105 LLC cells into female C57BL/6 mice to establish a tumor-bearing mouse model. A total of 8 days after cancer cell implantation, the mice were administered etoposide intraperitoneally at a dose of 50 mg/kg every 3 days for a total of five doses. Compared with the tumor-bearing group without chemotherapy, the etoposide-treated group showed no significant differences in voluntary wheel-running activity or body weight. However, the chemotherapy group had lower hemoglobin levels and higher serum IL-6 levels, suggesting that etoposide has a limited impact on fatigue in tumor-bearing mice. Therefore, caution is advised when selecting this drug for modeling CRF.

Although several researchers have studied fatigue models in lung cancer-bearing mice treated with chemotherapy, there are considerable variations in the number of cancer cells implanted, as well as in the dosage and duration of the chemotherapy regimens used. These differences make it difficult to compare the extent of fatigue across studies or to determine the specific impact of chemotherapy dosage on fatigue. Future research should focus on exploring the effects of varying chemotherapy doses on fatigue in lung cancer-bearing mice. Currently, gene editing technologies have been applied in fatigue mouse models, but their use has largely been limited to the establishment of CRF models by implanting human-derived tumor cells into nude mice. Future research should further explore the application of gene editing techniques to knock out or overexpress genes associated with CRF and observe their effects on fatigue symptoms. This approach could provide potential therapeutic targets for subsequent targeted treatments of CRF.

Breast cancer cell tumor-bearing and chemotherapy-induced CRF mouse models

Lv et al (65) subcutaneously injected 2x105 4T1 cells into female BALB/c mice. When the tumor volume reached ~0.5 cm³, cyclophosphamide was administered intraperitoneally at a dose of 100 mg/kg once daily for 3 consecutive days. Mice that were both tumor bearing and received chemotherapy exhibited symptoms of fatigue, including weight loss, reduced appetite, lethargy, slowed movement, prolonged immobility in the forced swim test and decreased scores in both horizontal and vertical movement in the open field test. Additionally, there was a significant increase in inflammatory cytokines in both the intestine and hippocampus.

Ouyang et al (63) used a similar approach, injecting 1x105 4T1 cells into mice. When the tumor volume reached 50-100 mm³, paclitaxel was administered intraperitoneally at a dose of 10 mg/kg every other day for a total of 10 doses. Compared with the group with only tumors, the combined tumor-bearing and chemotherapy group showed a more pronounced reduction in weight-loaded swimming time, indicating more severe fatigue, along with higher serum levels of inflammatory cytokines. Although the number of 4T1 cells implanted subcutaneously differed by a factor of two between these studies, both showed a high tumor formation rate. Further research is needed to determine the optimal number of tumor cells that can meet research requirements without causing excessive tumor growth to ensure animal welfare.

Currently, mouse fatigue in CRF studies is assessed using subjective indicators such as anorexia, drowsiness and sluggish movement, which may lead to inconsistencies in judgment standards among different researchers. Rafaih and Ari (66) applied artificial intelligence (AI)-driven technology through machine learning algorithms, wearable devices and real-time feedback systems to analyze the physiological, behavioral and environmental data of surgeons for comprehensive fatigue monitoring. However, there is limited research on using AI technology to monitor mouse fatigue behavior. Future studies could explore the use of AI technology to more accurately and comprehensively assess and monitor mouse fatigue.

Head and neck tumor cell-bearing with chemoradiotherapy-induced CRF mouse models

Chemoradiotherapy is a common treatment strategy for head and neck cancer, and patients may develop fatigue within the first few days of combined treatment (67). Vichaya et al (68,69) injected 1x105 mEER cells into the right hind limb of male C57BL/6 mice. On the seventh day post-cell implantation, cisplatin was administered intraperitoneally at a dose of 5.28 mg/kg, combined with 8 Gy of localized leg radiation therapy once a week for 4 weeks. The mice exhibited burrowing deficits, weight loss and increased inflammatory cytokines in liver and tumor tissues. Vichaya et al (70) also reported implanting mEERL95 cells into C57BL/6J mice, noting that this cell line is applicable to both male and female mice. The study found that female mice had smaller tumors and exhibited fatigue behaviors later and less frequently than males, suggesting the importance of considering sex differences in the fatigue levels of head and neck tumor-bearing mice.

Phan et al (71) divided mice into two groups: One was injected with mEER cells and the other one with LLC cells. Post-chemoradiotherapy, both groups experienced weight loss and reduced running wheel activity, with fatigue symptoms worsening as the number of chemoradiotherapy sessions increased. These results indicate that chemoradiotherapy exacerbates fatigue in a dose-dependent manner, highlighting the need to tailor the dose and regimen of chemotherapeutic agents according to the research objectives.

Other tumor cell-bearing with chemotherapy-induced CRF mouse models

Wang et al (72) injected 1x107 H22 hepatocellular carcinoma cells subcutaneously into Swiss mice. When the subcutaneous tumor reached 80-100 mm³, cisplatin was administered intraperitoneally at a dose of 10 mg/kg every other day for a total of seven doses. Compared with tumor-bearing mice without chemotherapy, the chemotherapy group exhibited more significant reductions in body weight, load-bearing swim time and activity levels, along with higher serum BUN and IL-1β levels.

Zhang et al (73) divided male C57BL/6 mice into two groups. One group was subcutaneously implanted with 5x105 YUMMER1.7 malignant melanoma cells, and on day 13 post-implantation, the mice received intraperitoneal injections of anti-mouse PD-1 antibody (BioXCell, InVivoMab series, clone RMP1-14) at 10 mg/kg three times a week. The other group was subcutaneously implanted with 1x106 MC38 colon cancer cells, and on day 12 post-implantation, the mice received intraperitoneal injections of 5-FU at 100 mg/kg for chemotherapy three times a week. The study showed that anti-PD-1 immunotherapy impaired the motivation for activity, leading to reduced forced swim time, while chemotherapy impaired muscle function, resulting in decreased forelimb grip strength and a reduction in maximum treadmill speed. These results suggest that, although both immunotherapy and chemotherapy can induce fatigue symptoms in mice, the underlying mechanisms differ, leading to distinct fatigue behaviors. Given the limited research on immunotherapy-related fatigue, further studies are needed to elucidate the mechanisms and behavioral manifestations of fatigue induced by immunotherapy. Detailed data on mouse models of CRF induced by chemotherapy and/or radiotherapy with tumor burden are shown in Table III.

Table III Chemotherapy and/or radiotherapy-induced cancer-related fatigue mouse models with tumor burden.

Table III

Chemotherapy and/or radiotherapy-induced cancer-related fatigue mouse models with tumor burden.

First author/s, year	Mice sex and strain	Mice age	Cell line and number	Cell injection site and method	Chemotherapy drugs and/or dosage	Chemotherapy and/or radiotherapy duration	(Refs.)
Wu et al, 2022	C57BL/6J mice	5 weeks	LLC 1x106	Subcutaneous injection in the right flank	Cisplatin 5 mg, intraperitoneal injection	Once per week over a period of 3 weeks	(23)
Wang et al, 2024	Swiss mice	Adult	H22 1x107	Subcutaneous injection into the right flank	Cisplatin 10 mg/kg, intraperitoneal injection	Once every other day for a total of seven doses	(43)
Abulizi et al, 2021	Female BALB/c mice	6-7 weeks	C26 2.5x105	Subcutaneous injection over the scapula	5-FU 30 mg/kg, intraperitoneal injection	Every other day for a total of eight doses	(58)
Park et al, 2015	Female BALB/c- nu/nu mice	5 weeks	HT-29 5x106	Subcutaneous injection in the right flank	5-FU 30 mg/kg, intraperitoneal injection	3 consecutive days per week over a period of 4 weeks	(59)
Yoshizawa et al, 2021	Male C57BL/ 6N mice	7 weeks	LLC 1x106	Subcutaneous injection in the right flank	Cisplatin 10 mg/kg, intraperitoneal injection	One single dose	(62)
Ouyang et al, 2016	Male BALB/c- nu mice	5-6 weeks	A549 5x106	Subcutaneous injection in the right flank	Cisplatin 10 mg/kg, intraperitoneal injection	Once every 2 days for a total of 10 doses	(63)
Ouyang et al, 2016	Female BALB/c mice	7 weeks	4T1 1x105	Subcutaneous injection into the fourth breast pad area	Paclitaxel 10 mg/kg, intraperitoneal injection	Once every other day for a total of 10 doses	(63)
Wood, et al, 2006	Female C57BL/6J mice	8 weeks	LLC 5x105	Subcutaneous injection in the scapular region	VP-16 50 mg/kg, intraperitoneal injection	Once every 3 days for a total of five doses	(64)
Lv et al, 2022	Female BALB/c mice	6-8 weeks	4T1 2x105	Subcutaneous injection into the fourth breast pad area	Cyclophosphamide 100 mg/kg, intraperitoneal injection	Once daily for 3 conse- cutive days	(65)
Vichaya et al, 2021	Male C57BL/ 6J mice	Adult	mEER 1x106	Intramuscular injection into the right hind leg	Cisplatin 5.28 mg/kg, intraperitoneal injection and local radiation 8 Gy	Once per week over a period of 4 weeks	(70)
Phan et al, 2024	C57BL/6J mice	8 weeks	mEER 1x106, LLC 5x105	Intramuscular injection into the right hind leg, subcutaneous injection in the right flank	Cisplatin 5.28 mg/kg, intraperitoneal injection and local radiation 8 Gy	Once per week over a period of 4 weeks	(71)

[i] LLC, Lewis lung carcinoma; mEER, HPV16 E6/E7 and H-ras; 5-FU, 5-fluorouracil.

Chen et al (74) administered anti-PD-1 antibody (12.5 µg/g every 5 days for 6 injections) and anti-PD-L1 antibody (10 µg/g once a week for 6 weeks) to 6-8-week-old C57BL/6 mice, resulting in significant cardiac toxicity symptoms, including myocarditis. Cao et al (75) found that anti-PD-1 treatment led to DNA and mitochondrial damage in cardiac macrophages, and could induce macrophage polarization towards a pro-inflammatory phenotype. Other researchers also observed that anti-PD-1 treatment caused heart failure, left ventricular dilation and enhanced thymic inflammation signaling in mice. Inflammation in the myocardium and thymus may further exacerbate fatigue symptoms in mice. Currently, animal models related to immune checkpoint inhibitor therapy mainly focus on myocarditis models, with fewer studies investigating the induction of fatigue by immune checkpoint inhibitors in mice. Further attention is needed to explore the mechanisms underlying fatigue induced by immunotherapy.

5. Peripheral irradiation-induced CRF mouse models

Peripheral irradiation may exacerbate fatigue in patients with cancer. Wolff et al (76) performed localized irradiation on the lower abdominal/pelvic region of anesthetized male C57BL/6 mice with a dose of 8 Gy for 3 consecutive days. The results indicated reduced activity and the appearance of fatigue symptoms in the mice, accompanied by weight loss. Wolff et al (31,77) also reported that voluntary running wheel distance was minimized on day 3 post-radiation therapy, with a concomitant reduction in brain-derived neurotrophic factor (BDNF) levels in pelvic-irradiated mice. The decrease in BDNF may be causally linked to fatigue. Renner et al (78) used a similar peripheral irradiation method to establish a murine model of fatigue. Their findings showed that the fatigue trajectory in mice closely mirrored that observed in clinical patients, with reduced running distance and speed on the voluntary running wheel in the afternoon or evening, indicating exacerbated fatigue during this period. This animal model realistically simulates clinical conditions and offers an suitable model for studying fatigue induced by peripheral irradiation.

McDonald et al (79) conducted external beam radiation therapy (EBRT) on male C57BL/6J mice, delivering a dose of 2.84 Gy five times a week for a total of 24 sessions. This treatment regimen mimicked the radiotherapy protocols used in clinical patients with prostate cancer. The results indicated that the mice subjected to EBRT exhibited symptoms of fatigue, including reduced voluntary wheel running time and distance, weight loss, decreased food intake, and elevated levels of inflammatory markers in blood and tissues. Similarly, Wood et al (80) employed an analogous pelvic irradiation method, supporting these findings. Huang et al (81) anesthetized male BALB/c mice and surgically exposed the left adrenal gland, administering a radiation dose of 2 Gy. The results demonstrated that irradiated mice experienced decreased swimming endurance and significant reductions in nighttime cortisol levels. The models of fatigue induced by localized irradiation in healthy mice established by the aforementioned researchers may not adequately reflect clinical scenarios. Additionally, the radiation doses, treatment frequencies and targeted sites varied among studies. Future research should focus on fatigue induced by localized irradiation in tumor-bearing mice, as this situation aligns more closely with clinical conditions. Detailed information on mouse models of CRF induced by peripheral irradiation is shown in Table IV.

Table IV Peripheral irradiation-induced cancer-related fatigue mouse models.

Table IV

Peripheral irradiation-induced cancer-related fatigue mouse models.

First author/s, year	Mice sex and strain	Mice age	Peripheral irradiation dosage and area	Duration of peripheral irradiation	(Refs.)
Renner et al, 2016	Male C57BL/6 mice	5 weeks	8 Gy external lower abdominal irradiation	Once daily for a total of 3 days	(78)
McDonald et al, 2016	Male C57BL/6 mice	10-12 weeks	2.84 Gy external pelvic irradiation	5 consecutive days per week for a total of 24 times	(79)
Wood et al, 2008	Male C57BL/6 mice	Adult	2.84 Gy external pelvic irradiation	5 consecutive days per week for a total of 24 times	(80)
Huang et al, 2022	Male BALB/c mice	4 weeks	2 Gy internal left adrenal gland irradiation	One single time	(81)

6. Conclusions

Despite the development of fatigue mouse models through various methods such as tumor cell implantation, chemotherapy and radiation therapy, and subsequent behavioral and biological studies, the molecular mechanisms underlying CRF remain insufficiently understood. Future research could explore the integration of genomic, transcriptomic and metabolomic data through multi-omics analysis. By performing data correlation analyses at a holistic level, researchers may identify fatigue-related biomarkers and potential molecular targets associated with CRF. These findings could be validated using gene editing techniques and applied to the early prediction and monitoring of CRF, thereby providing a theoretical foundation for drug development.

Despite the development of CRF mouse models through various methods such as tumor cell implantation, chemotherapy and radiation therapy, these models often fail to accurately replicate the full spectrum of fatigue symptoms experienced by patients with cancer. Numerous models rely on ectopic tumor implantation, which does not mimic the systemic effects of orthotopic tumors. Additionally, current CRF mouse models assess fatigue symptoms in the absence of comorbidities and without considering the psychological stress responses associated with cancer and its treatments, such as anxiety, depression and pain. This limitation hinders the simulation of the psychological factors that significantly contribute to fatigue in patients with cancer. Furthermore, there is a lack of consensus regarding the assessment methods for fatigue in mice. Existing evaluations predominantly focus on physical fatigue, with minimal attention given to emotional and cognitive dimensions. This narrow focus may impede a comprehensive understanding of CRF mechanisms. Future research should aim to develop more clinically relevant mouse models by integrating cancer-induced effects with psychological stressors, such as isolation-induced anxiety or chronic social defeat-induced depression. Employing multidimensional assessment tools, including biomarkers such as inflammatory cytokines and metabolites, physiological evaluations, behavioral assays and cognitive tests (Morris water maze) can facilitate a holistic evaluation of fatigue across physical, emotional and cognitive domains. This approach will enhance the current understanding of CRF mechanisms and inform the development of targeted therapeutic strategies.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by Shandong Traditional Chinese Medicine Research Program of Science and Techology (grant no. 2021Z037).

Availability of data and materials

Not applicable.

Authors' contributions

CX designed the study and reviewed the manuscript. HG was responsible for the literature review and drafted the initial manuscript. LC conducted the literature search and acquired, analyzed and interpreted the data. All authors read and approved the final version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References