Tumour neovascularisation, a pivotal process for tumour growth and metastasis, involves angiogenesis and vasculogenesis (17). By definition, angiogenesis corresponds to new vascularisation generated from mature endothelial cells in existing vessels, whereas in vasculogenesis blood vessels arise from endothelial progenitor cells (EPCs) of the bone marrow (18–20). In fact, tumours may employ both mechanisms simultaneously (21). To foster endothelial proliferation and new vessel formation, tumours trigger the release of proangiogenic substances and suppress endogenous antiangiogenic factors like endostatin and thrombospondin-1 (TSP-1) (2, 19, 22, 23). This phenomenon, called “angiogenic switch,” enables tumours to exit their dormant state, occurring at diverse tumour progression stages (17, 19, 24). Hypoxia is one of its main triggers, since low concentrations of oxygen in the TME typically promote the production of proangiogenic factors (22). Several of these factors have already been identified, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), hypoxia-inducible factor-1α (HIF-1α) and angiopoietin-1 (25–29).

These antiangiogenic effects are evidenced by studies suggesting that tumour angiogenesis and vasculogenesis can be inhibited by MC through multiple pathways, including the reduction of endothelial and EPC proliferation and circulation, hindering immature endothelial cell differentiation, and modulating proangiogenic and antiangiogenic factors (6, 15, 30–33).

The administration of immunostimulating cytokines, such as interleukin (IL)-12 can potentially enhance the antiangiogenic properties of MC, according to experimental studies in mice (34, 35). Evidence of this benefit in companion animals is still scarce, but a preliminary study showed promising therapeutic results in a small group of dogs (23).

Finally, considering that some VEGF isoforms have been associated with the formation of new intra- and peritumoral lymphatic vessels, it could be hypothesised that MC may also have a crucial effect on inhibiting lymphangiogenesis, preventing neoplastic spread through the lymphatic route (36, 37).

Neoplasms have several strategies to escape the immune surveillance, such as the activation of myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs) that induce an immunosuppressive state in the TME (19, 22). In turn, these cells contribute to immune evasion and tumour progression by promoting macrophage and neutrophil polarisation, compromising the activation of dendritic cells, suppressing effector cells (e.g., cytotoxic and helper T-cells and natural killer cells) and stimulating the secretion of immunosuppressive cytokines such as interleukin (IL)-10 and transforming growth factor-β (TGF-β) (22, 27, 32, 38).

According to several studies, MC has as an immunomodulatory effect, counteracting the aforementioned immune evasion strategies, namely suppressing MDSCs and Tregs function, increasing lymphocyte, memory T-cell and natural killer cell proliferation and upregulating dendritic cells (23, 39–44).

Another mechanism of action that has been attributed to MC is the direct cytotoxic effect on tumour cells, particularly cancer stem cells (CSCs) (14, 15, 45, 46). These CSCs, known for their role in therapeutic resistance through their capacity for self-renewal and differentiation into diverse cancer cell types, are fundamental in tumour proliferation, invasion, and metastasis (47). Unlike traditional high-dose chemotherapy, MC has demonstrated efficacy in diminishing CSC populations, potentially by limiting angiogenesis and directly influencing VEGF expression (48, 49).

Additionally, MC’s impact extends to the three compartments of the tumour microenvironment (immune system, tumour cells and vasculature). The intricate interplay within these compartments may induce and maintain a state of tumour dormancy, a dynamic stability between cell proliferation and cellular apoptosis, potentially ensuring long-term asymptomatic control of the disease (15, 32, 50, 51).

The drug-driven dependence/deprivation effect (also known as the 4D effect) can be achieved through long-term exposure of the tumour to cytotoxic agents followed by abrupt withdrawal (52). This effect, as per in vitro studies, leaves drug-dependent cells more hypersensitive and thus more vulnerable to therapeutic strategies, a phenomenon observed in breast cancer cells resistant to anti-hormonal treatments (53, 54). Hence, it is suggested that a temporary interruption introduced after a prolonged course of chemotherapy, a strategy frequently employed in MC protocols, could break tumour resistance and trigger cancer cells death (52–54). This approach suggests a strategic manipulation of drug administration to enhance treatment efficacy, optimising cancer therapy. Nonetheless, the 4D effect has only been investigated in human medicine, so further research is required to validate the advantages of this effect in in vivo animal models undergoing MC protocols.

Several cytotoxic drugs have been used in MC protocols in veterinary oncology patients over the last few years. The most commonly used is oral cyclophosphamide, whose dose ranges between 6 and 27 mg/m² once daily to once every other day, according to several published clinical trials (23, 42–44, 55–57, 59–67, 69–72, 74–82). Although less frequently, oral chlorambucil has been described as the main drug at the dose of 4 mg/m² daily in dogs (58, 62, 83–85) and 0.4 to 0.6 mg/kg or 4 mg/m² every other day in cats (79, 82) for the treatment of some neoplasms in these two species. It has also been used as a substitute for cyclophosphamide when sterile haemorrhagic cystitis occurs (57, 70, 73, 74, 76, 80). In turn, metronomic prescription of lomustine (68, 86), temozolomide (42), and etoposide (55, 57, 87) has also been described in the oncological treatment of some canine patients at daily doses of 2.84 mg/m², 6.6 mg/m² and 50 mg/m², respectively.

Despite the drug doses and schedules mentioned above, there are currently no standard recommendations for drug doses, as published data are still scarce and quite heterogenous for most canine and feline tumour types. Even so, several authors have shown that the prescribed dose has a significant impact on the patient’s therapeutic response. For example, Burton et al. (43) reported significantly greater immunomodulatory and antiangiogenic effects when using a higher dose of oral cyclophosphamide (15 mg/m² versus 12.5 mg/m²) in dogs with soft tissue sarcoma. However, it should be noted that higher doses are often associated with earlier and more frequent manifestation of adverse effects, as has already been described in canine patients treated with higher doses of chlorambucil (6 to 8 mg/m² versus 4 mg/m²) (88). Further research is still required to establish the minimum effective drug dose for treating each specific tumour type.

Several clinical trials based on MC have been published in the last two decades, presenting different doses and schedules, as represented in Table 1.

In order to potentiate its antiangiogenic and immunomodulatory effects, MC is often combined with the administration of cytotoxic and non-cytotoxic agents (4, 19, 89).

In the first scenario, MC can be used in combination with MTDC to reduce the risk of neoplastic regrowth between administrations. In fact, several authors have suggested the possibility of an additive and synergistic effect of this combined use, which could potentially improve the prognosis of these patients (57, 72). The two intravenous drugs that have been most commonly used in a MTDC setting, in combination with MC, are doxorubicin (57, 71, 72, 74) and carboplatin (64, 71). Additionally, tyrosine kinase inhibitors have also been prescribed in association with metronomic cyclophosphamide, particularly oral toceranib at a dose of 2.4 to 2.75 mg/kg every other day or three times a week (44, 56, 65, 80, 90).

Regarding non-cytotoxic agents, non-steroidal anti-inflammatory drugs (NSAIDs) are the most prescribed in association with MC, due to their ability to inhibit cyclooxygenase isoform-2 (COX-2), whose expression is considered a negative prognostic factor in various types of canine and feline tumours (91). This inhibitory effect compromises endothelial cell tube formation and VEGF expression, preventing tumour progression (73, 78, 80) Thus, several COX-2 inhibitors have been included in MC protocols, such as piroxicam (55, 56, 61, 62, 67, 69–71, 73–75, 77, 79–81, 86, 90), meloxicam (56, 61–63, 70, 73–76, 82, 85, 86, 90), firocoxib (56, 59, 62, 65, 73, 76, 86, 90), carprofen (68, 70, 86), deracoxib (74, 75, 86), celecoxib (78), and cimicoxib (65). Amongst these, piroxicam, an oxicam derivate, is the NSAID whose efficacy as anticancer drug has been most recognised, at a recommended dose of 0.3 mg/kg per day or every other day (69, 79, 92). There are also a few reported cases of the combined use of MC with corticosteroids, such as prednisone (58, 86).

Other non-cytotoxic drugs that have also been described in patients undergoing MC are thalidomide (56, 61, 62, 67, 69, 73, 81) and doxycycline (60, 74, 75, 90). Thalidomide has been associated with anti-inflammatory and antiangiogenic effects by inhibiting the expression of VEGF, FGF and TNF- α, although the mechanism of action is not yet fully understood (59, 69, 93). This drug was considered well-tolerated in canine patients at a daily dose of 10 mg/kg (94), although, it has typically been used at 2 to 4 mg/kg per day, in combination with cyclophosphamide and piroxicam (61, 69, 73, 81). Care must be taken with the timing of administration, as food intake seems to affect its bioavailability, delaying but increasing its absorption (95). Despite this, the accessibility of thalidomide on the global market has been limited due to the severe teratogenic effects reported in humans (96), which may compromise its routine use in veterinary metronomic protocols in some geographical territories. In turn, doxycycline is a tetracycline antibiotic with reported antiangiogenic and cytotoxic activity on tumour cells (97, 98). However, evidence on its specific therapeutic effect in canine and feline neoplastic conditions is still lacking, with recommended doses for anticancer purposes not yet being established.

Currently, the toxicity of antineoplastic therapies in dogs and cats is estimated based on adverse events (AEs), according to the criteria published by the Veterinary Cooperative Oncology Group (VCOG-CTCAE) (99). Each AE can be classified with a grade, according to its severity: grade 1 (mild), grade 2 (moderate), grade 3 (severe), grade 4 (life-threatening) and grade 5 (death). This classification system allows the clinician to define the recommended intervention according to the severity of each AE, as well as understand its impact on activities of daily living (ADL) and the consequences on the patient’s health status. Although this toxicity is typically low grade, several AEs have been reported in veterinary patients, with gastrointestinal signs, sterile haemorrhagic cystitis (SHC) and haematological toxicity being the most frequent (44, 62, 64, 68, 75, 86).

Regarding gastrointestinal toxicity, vomiting, diarrhoea, anorexia and nausea have been the main signs recorded in animals treated with metronomic cyclophosphamide (44, 56, 61–63, 65, 69–71, 73–77, 80–82), chlorambucil (83, 84), and lomustine (86). These signs tend to appear in the short term, typically within the first month of treatment, and are generally low grade (1 or 2) and self-limiting, requiring only supportive treatment (44, 62, 71, 73, 77, 79, 80, 83, 86). Moreover, NSAIDs have also been associated with gastrointestinal disturbances, especially piroxicam, which could potentially limit their long-term use in some cancer-bearing dogs and cats (92, 100). Still, it appears to be generally well-tolerated in feline patients even after one month and particularly if used as sole therapy (101).

In turn, sterile haemorrhagic cystitis has been described in dogs treated with oral metronomic cyclophosphamide, due to the formation of acrolein through liver metabolism, which accumulates and causes irritation in the bladder mucosa (55, 76, 90). This toxicity can affect up to 58% of canine patients (55, 59, 61, 62, 70, 73, 75–77, 81, 90) and must be prevented by administering it in the morning and encouraging water intake and frequent urination, in order to reduce urinary stasis. In line with this, the concomitant use of diuretics, such as furosemide, has also been advised (90, 102). Furthermore, if this urinary AE occurs, cyclophosphamide is generally replaced by chlorambucil (57, 70, 73, 74, 76, 80). The time required for its development differs depending on the dose of cyclophosphamide. Lower doses, such as 10 mg/m², have been associated with a later onset of this AE, particularly when compared to doses of 15 to 25 mg/m² (74, 75).

Haematological toxicity has been also associated with MC, as result of bone marrow suppression caused by the continued use of these drugs, and can be expressed as anaemia, thrombocytopenia and neutropenia of different grades (44, 56, 58, 62, 63, 65, 68, 86). These cytopenias are generally mild to moderate and transient, and may develop within the first few weeks or only after several months (44, 58, 86).

Finally, mild to severe renal toxicity has been reported in both dogs (65, 69, 81) and cats (56, 63, 82) treated with metronomic cyclophosphamide. This potential nephrotoxic effect may be worsened by the concomitant use of NSAIDs, such as piroxicam, requiring close monitoring, especially in older patients (100, 101).

Other undesired harmful effects may be described as the application of MC continues to increase in veterinary medicine, mainly with drugs whose toxicological profiles in companion animals have been less studied. For example, in human patients, continued administration of etoposide and thalidomide have been, respectively, associated with an increased risk of secondary leukaemia (103) and thromboembolic events (104), although a similar association has not yet been described in dogs and cats.

Despite all the potential AEs discussed above, it should be noted that MC has been associated with significantly fewer AEs than MTDC, as described by Marconato et al. (61) in a multi-institutional retrospective study (15.8% versus 43.5%, respectively).

The AEs reported in the various clinical trials published to date are described in detail in Table 2, along with the respective management.

Unlike MTDC, which must be administered by a qualified veterinary professional in appropriate facilities with the necessary protective equipment, MC relies on oral administration of cytotoxic drugs to the animal in a home setting by the owner. Therefore, the person responsible for administering the drug is at greater risk of toxic exposure, which is particularly relevant with this type of chemotherapeutic protocols, as this procedure typically has to be performed daily for an extended period of time or even chronically (105). According to the International Agency for Research on Cancer (IARC), most cytostatics applied metronomically to canine and feline patients are considered carcinogenic to humans (group 1), such as cyclophosphamide, chlorambucil and etoposide, or at least probably carcinogenic (group 2A), such as lomustine (106). Therefore, to prevent health hazards, the owner must be adequately educated on the safety measures that must be followed when administering these medications, such as preserving the integrity of pills and capsules until adequate ingestion, using chemotherapy-rated gloves and washing hands afterwards (105). Although the potential risk of exposure through excretions, such as urine, faeces and vomit, has not been addressed in the literature in animals undergoing MC, care must be taken, and immediate cleaning using gloves is recommended (105, 107).

Total splenectomy is recommended in patients with splenic hemangiosarcoma, which has been associated with adjuvant chemotherapy protocols, particularly doxorubicin-based, in order to improve the prognosis of these patients (57, 118). More recently, several authors have suggested the addition of metronomic cyclophosphamide to the adjuvant treatment of these animals, but its potential therapeutic benefit is not yet completely clear, according to the literature. In fact, some authors have described a significant positive effect on prognosis (55, 73), whilst others have not (60–62, 74).

Two previous studies concluded that dogs with hemangiosarcoma treated with metronomic cyclophosphamide, either following conventional chemotherapy with doxorubicin (73) or as an alternative to it (55), lived significantly longer compared to those treated with adjuvant MTDC only. On the contrary, five others reported no significant improvement in outcome when MC was added to surgery (57), administered following surgery plus MTDC (60, 62, 74), or used as an alternative to adjuvant conventional chemotherapy (61, 62). Still, one of them suggested a therapeutic benefit at least in the short term (first 4 months) (57). There are also reports of the use of metronomic lomustine (86) and chlorambucil (62), but there is still no evidence to support its beneficial effect in these patients.

Therefore, as doxorubicin continues to be considered the only effective cytotoxic drug in the adjuvant treatment of this malignant neoplasm, MC should currently be reserved for cases in which the previous one is contraindicated (e.g., dogs with heart disease), or when a better quality of life with less therapy-related toxicity is prioritised at the potential expense of survival time (119).

Amputation of the affected limb and subsequent carboplatin chemotherapy is the treatment of choice for appendicular osteosarcomas, but in order to improve its effectiveness, the adjuvant use of MC in these animals was investigated (68, 71, 76, 80). However, according to three studies, the addition of metronomic cyclophosphamide and an NSAID to conventional treatment did not offer any significant benefit in prolonging disease-free interval or survival time (71, 76, 80), not even when toceranib was also added (80). Metronomic lomustine has also been used in dogs with appendicular osteosarcoma, but despite being well tolerated (68, 86), it did not appear to significantly improve the survival of these patients, compared to other treatments such as radiotherapy (68). Thus, the data obtained so far do not support the use of MC in dogs diagnosed with appendicular osteosarcomas.

Mastectomy remains the treatment of choice for malignant mammary tumours in dogs, however adjuvant MTDC with single or multiple cytotoxic drugs, such as cyclophosphamide, 5-fluorouracil, mitoxantrone, carboplatin and gemcitabine, has been described for lesions at risk of local recurrence or metastasis (120). Given the complexity of these tumours, novel therapeutic approaches have been investigated, namely multi-targeted therapies, such as MC combined with NSAIDs.

In line with that, De Campos et al. and Machado et al. showed that metronomic cyclophosphamide following surgery and four (59) to six (64) cycles of intravenous carboplatin resulted in a better outcome than surgery alone (59) or surgery plus conventional chemotherapy only (59, 64). Interestingly, the only adjuvant drug that allowed a better prognosis in these patients when added instead of cyclophosphamide was thalidomide, according to one of these studies (59). Thus, according to these two studies, MC appears to be associated with a clinical benefit in the therapeutic management of canine mammary carcinomas.

In turn, inflammatory mammary carcinoma, the most aggressive form of mammary cancer in dogs, has been associated with a very poor prognosis despite the therapeutic approaches carried out, with no consensus regarding the benefit of chemotherapy (121). Even so, as these tumours were associated with a higher expression of COX-2, the use of its inhibitors, such as piroxicam, has been described (122, 123). In line with this, Alonso-Miguel et al. (65) recently evaluated the potential benefit of adding metronomic cyclophosphamide and toceranib phosphate to COX-2 inhibitor therapy alone, showing a significant increase in survival. However, the small number of dogs assessed and the retrospective nature of the study prevent further clinical conclusion. In fact, based on the current scientific evidence, an effective medical therapy has yet to be found and will probably be based on new therapeutic targets (121, 124).

Soft tissue sarcomas (STS) must be excised with wide margins in order to avoid local recurrence and potentially achieve therapeutic cure. In addition, adjunctive chemotherapy and/or radiotherapy protocols have been applied for incompletely resected and high-grade tumours, even though the level of scientific evidence is still considered low (125).

Nevertheless, metronomic cyclophosphamide has been described as effective in preventing the recurrence of these incompletely resected tumours, allowing longer disease-free times compared to surgery alone (77). A survival benefit was also reported by Cancedda et al. (67) in their retrospective study on dogs with macroscopic STS treated with hypofractionated radiotherapy followed or not by MC with oral cyclophosphamide, piroxicam and thalidomide. In this study, dogs that underwent adjuvant MC lived significantly longer (p = 0.023), although no significant difference in the progression-free interval was obtained. Metronomic use of chlorambucil was also evaluated in these patients. Leach et al. (83) reported that one dog with a STS of the flank was still in complete remission 35 weeks after starting this drug. According to these studies, MC seems to be a valid option in the adjuvant treatment of these patients.

The administration of chemotherapeutic agents, such as mitoxantrone, carboplatin or vinblastine, in combination with NSAIDs, has been considered the treatment of choice for urinary tract tumours in dogs due to the typically challenging location that often prevents surgery from being feasible (126).

Two studies described the metronomic use of chlorambucil in dogs diagnosed with malignant neoplasms affecting the bladder (84, 85) and/or urethra (85). According to Schrempp et al. (84), a chlorambucil-based metronomic protocol appears to be a well-tolerated and effective option for dogs with transitional cell carcinoma of the urinary bladder, particularly when other therapies have failed. More recently, Gedon et al. (85) showed that oral administration of chlorambucil combined with meloxicam in patients with urothelial carcinoma appears to be a good therapeutic option compared to NSAID treatment alone.

More studies are needed, but given these results, this protocol should be considered at least in three clinical scenarios: (1) when conventional chemotherapy has failed; (2) when an alternative chemotherapeutic approach with lower toxicity is intended; and (3) when NSAID therapy is elected, since the addition of metronomic chlorambucil appears to enhance its therapeutic effect.

Although surgery and/or radiotherapy are the preferred approaches to treat malignant oral tumours in dogs, oral metronomic cyclophosphamide could be beneficial as a palliative option when owners refuse them, as suggested by Milevoj et al. (70). In that study, half of the animals achieved partial response or stable disease after 1 month. However, it has several limitations, such as the small number of animals enrolled, the variability of histological types and the lack of a control group, which lower the level of evidence In contrast, Garcia et al. (66) found no clinical benefit in adding metronomic cyclophosphamide to an immunotherapy protocol in dogs with oral melanoma. Thus, randomised and controlled studies are needed to clarify the potential advantage of MC in oral malignant tumours in dogs.

Surgical removal of primary pulmonary tumours is the therapeutic approach that allows a better prognosis, however given their location and size it may not be possible, requiring systemic treatment as alternative, such as MTDC with carboplatin, vinorelbine or gemcitabine (69, 127, 128). A MC protocol based on cyclophosphamide, piroxicam and thalidomide was also described by Polton et al. (69), which was associated with a significant therapeutic benefit in dogs diagnosed with advanced primary lung carcinoma. According to the multivariable survival analysis performed, patients who did not receive MC and underwent surgery, MTDC or no oncological treatment, had a 1.7 and 1.5 increased risk of tumour disease progression and death, respectively. Considering this outcome, the low toxicity reported and the improvement in quality of life described in most patients (91.3%), MC appears to be a good therapeutic alternative in unresectable and/or metastatic primary pulmonary tumours, although more studies are needed to strengthen this evidence.

A single study was published on the therapeutic use of MC in hepatocellular carcinomas in dogs (81). The authors’ aim was to investigate an effective and well-tolerated chemotherapy alternative for this tumour type, as the prognosis is typically poor when complete surgical resection is not possible. However, animals treated with metronomic cyclophosphamide, piroxicam and thalidomide had a poor outcome. Therefore, a potentially effective systemic treatment (such as MC) for the management of canine hepatocellular carcinoma remains to be found.

For the treatment of canine intracranial tumours, surgery and/or radiotherapy are the recommended options. By contrast, chemotherapy has a very limited therapeutic value given the heterogeneity of these tumours and the fact that the blood–brain barrier often compromises exposure to cytotoxic drugs in adequate doses (129). Even so, Bentley et al. (58) described the metronomic use of daily chlorambucil, associated with prednisone and monthly lomustine, after microsurgical resection of canine cerebral gliomas. This adjuvant approach was well tolerated and these patients had a better outcome compared to others previously treated with symptomatic approach and lomustine alone (130, 131). However, these promising results must be interpreted carefully given the small number of dogs enrolled in the study. Therefore, further studies are needed, not only to evaluate this protocol, but also to investigate the potential benefit of combining MC with other therapies typically recommended in these patients, such as adjuvant radiotherapy.

Feline mammary carcinomas are highly malignant, requiring an aggressive approach that typically involves radical mastectomy followed by doxorubicin (132, 133), carboplatin (134) or mitoxantrone (135). In order to find adjuvant alternatives associated with fewer AEs and that could avoid potentially stressful treatment sessions for cats, metronomic protocols have recently been suggested. However, according to these studies, female cats treated with metronomic cyclophosphamide (63, 82) or chlorambucil (82) did not live significantly longer than cats that underwent surgery alone (63), surgery plus doxorubicin-based MTDC (63, 82) or even surgery plus toceranib phosphate administration (82). Thus, to date, there is no scientific evidence to support the use of MC in this group of patients.

MC has also been applied in the treatment of canine metastatic tumours, whether with cyclophosphamide (78, 79), chlorambucil (83) or lomustine (86). Considering the typically guarded prognosis of these patients, promising results have been described with MC as some animals have achieved stable disease (78, 83, 86) or even partial (79, 86) and complete responses (78, 79). Feline advanced or metastatic tumours have also been treated with metronomic cyclophosphamide (56, 82) or chlorambucil (79), with some cats achieving stable disease as well (56). Therefore, according to these studies, MC should be considered in patients with metastatic neoplastic disease, not only as palliative therapy, but also as an adjuvant or even first-line approach, depending on the specific case.