Preclinical antitumor evaluation of a tetrahydrocannabinol and cannabidiol (1:6) cannabis extract in an MCF-7 xenograft model of estrogen receptor-positive breast cancer

Source / Quelle: https://veterinaryworld.org/Vol.19/June-2026/17.php

ABSTRACT

Background and Aim: Breast cancer remains one of the leading causes of cancer-related mortality worldwide, despite advances in surgery, chemotherapy, endocrine therapy, and targeted treatments. Cannabinoids derived from Cannabis sativa, particularly tetrahydrocannabinol (THC) and cannabidiol (CBD), have demonstrated anticancer properties in several experimental models; however, in vivo evidence in estrogen receptor (ER)-positive breast cancer remains limited. This study aimed to evaluate the antitumor effects of a THC:CBD (1:6) cannabis extract in a Michigan Cancer Foundation-7 breast cancer cell line (MCF-7) xenograft mouse model of ER-positive breast cancer.

Materials and Methods: Female BALB/c nude mice bearing MCF-7 xenograft tumors were randomly assigned into five groups (n = 5/group): negative control (sesame oil), positive control treated with 5-fluorouracil (5-FU; 20 mg/kg), and three treatment groups receiving oral THC:CBD (1:6) extract at doses of 2, 10, or 20 mg/kg body weight for 30 consecutive days. Tumor growth was monitored throughout the experiment. Histopathological examination and immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) expression were performed to evaluate apoptosis-related morphology and tumor cell proliferation. Hematological and biochemical parameters were assessed to determine systemic safety.

Results: Cannabinoid-treated groups exhibited significant suppression of tumor growth compared with the negative control group. Tumor volume reduction was observed in all treatment groups, with the greatest reduction detected in the high-dose THC:CBD group. Histopathological evaluation revealed increased numbers of tumor cells exhibiting morphological features consistent with apoptosis in cannabinoid-treated mice. Immunohistochemical analysis demonstrated significantly lower PCNA expression scores in all THC:CBD-treated groups compared with both negative and positive controls, indicating reduced tumor cell proliferation. Hematological parameters remained within normal physiological ranges in cannabinoid-treated animals. However, elevated alanine aminotransferase and aspartate aminotransferase levels were observed in the high-dose group, suggesting potential dose-related hepatic stress.

Conclusion: The THC:CBD (1:6) cannabis extract demonstrated significant antitumor activity in an MCF-7 xenograft model by suppressing tumor progression primarily through inhibition of tumor cell proliferation, with supportive apoptosis-related histological features. These findings provide novel in vivo evidence supporting the potential of cannabinoid-based formulations as adjunctive therapeutic approaches for ER-positive breast cancer.

Keywords: apoptosis, breast cancer, cannabidiol, cannabinoids, estrogen receptor-positive, MCF-7, tetrahydrocannabinol, xenograft model.

INTRODUCTION

Breast cancer is one of the most common cancers in women worldwide and remains a leading cause of cancer-related mortality. According to hospital-based cancer registries, it continues to be the most prevalent cancer among women in Thailand, with incidence rates rising in parallel with global trends [1, 2]. Despite advances in screening and treatment, mortality rates remain high. The prognosis strongly depends on the stage at diagnosis, with early-stage cancers generally associated with more favorable outcomes than advanced-stage disease [3]. Management of breast cancer typically requires a multidisciplinary approach, incorporating surgery, radiotherapy, systemic chemotherapy, endocrine therapy, and targeted biological therapies, often used in combination depending on tumor subtype and stage. Treatment decisions are largely guided by the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). However, receptor status can change over time, which may significantly affect therapy selection. Triple-negative breast cancers (TNBC), lacking all three receptors, generally have a poorer prognosis and rely primarily on chemotherapy, with few targeted therapeutic options [4, 5].

While these treatments have improved survival, they are frequently associated with adverse effects such as alopecia, nausea, vomiting, and immunosuppression, which can negatively impact quality of life [3, 6]. Consequently, there is a pressing need to identify novel, safer, and more effective therapeutic strategies. In recent years, herbal-based medicinal compounds, such as cannabis, have attracted attention as potential alternative or adjunct therapies for cancer patients. Cannabis contains several bioactive compounds, the main ones being cannabinoids, including delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), which are responsible for most of its biological effects [7]. Cannabinoids have been reported not only to reduce symptoms associated with cancer or its treatments, such as nausea, vomiting, pain, and loss of appetite, but also to enhance the efficacy of conventional chemotherapeutic agents while mitigating their side effects [8, 9]. Beyond these palliative effects, preclinical studies indicate that cannabinoids can directly inhibit tumor progression by targeting multiple stages, including proliferation, angiogenesis, invasion, and metastasis, as well as inducing apoptosis and autophagy [10, 11]. As mentioned above, breast cancer treatment is largely guided by receptor status, which can change over time and potentially limit the effectiveness of receptor-dependent therapies. In contrast, cannabinoids appear to exert antitumor effects independently of receptor expression. This is supported by recent in vitro meta-analytic evidence showing that CBD potently inhibits breast cancer cell growth regardless of receptor status, suggesting a receptor-independent mechanism of action [12]. These findings are further supported by in vivo studies in mouse xenograft models using MDA-MB-231 TNBC cells, in which both CBD and THC individually reduced primary tumor growth, suppressed lung metastasis, and prolonged survival [13].

Although the anticancer properties of cannabinoids have been increasingly reported in both in vitro and in vivo studies, several important gaps remain in the current literature. Most previous breast cancer studies have focused on either THC or CBD as single agents, while limited evidence is available regarding the combined use of these cannabinoids in defined ratios [14]. In addition, most in vivo breast cancer studies have been conducted using TNBC models, particularly MDA-MB-231 xenografts, whereas studies investigating ER-positive breast cancer models remain scarce. This is clinically important because ER-positive breast cancer represents one of the most common subtypes in women and exhibits distinct biological behavior and therapeutic responses compared with TNBC. Furthermore, the antitumor effects of whole-cannabis extracts containing multiple phytocannabinoids have not been adequately evaluated in Michigan Cancer Foundation-7 breast cancer cell line (MCF-7) breast cancer xenograft models. Existing studies have also provided limited comparisons between cannabinoid-based treatments and conventional chemotherapeutic agents such as 5-fluorouracil (5-FU), restricting the translational relevance of the findings [15]. Moreover, the influence of a pharmacokinetically optimized THC:CBD ratio on tumor proliferation and apoptosis-related responses in receptor-positive breast cancer remains poorly understood. Therefore, further in vivo investigation is required to clarify the therapeutic potential, biological effects, and safety profile of combined cannabinoid formulations in ER-positive breast cancer models.

This study aimed to evaluate the antitumor effects of a defined-ratio THC:CBD (1:6) whole-cannabis extract in an MCF-7 xenograft mouse model of ER-positive breast cancer. Specifically, the study investigated the effects of the cannabinoid extract on tumor growth, histopathological alterations, apoptosis-related morphology, and tumor cell proliferation by analyzing proliferating cell nuclear antigen (PCNA) expression. In addition, the study assessed hematological and biochemical parameters to determine the treatment’s systemic safety profile. The therapeutic efficacy of the cannabinoid extract was also compared with the conventional chemotherapeutic agent 5-FU to provide clinically relevant preclinical evidence regarding the potential use of cannabinoid-based formulations as adjunctive therapeutic approaches for ER-positive breast cancer.

MATERIALS AND METHODS

Ethical approval

All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the National Cancer Institute, Thailand (Protocol No. 272_2019RB_IN602), and Lerdsin Hospital, Department of Medical Services (Protocol No. AEC-F-v03-02). All animal experiments were conducted in accordance with relevant institutional guidelines and regulations for the care and use of laboratory animals. The study also complied with the ARRIVE 2.0 guidelines for reporting animal research. Humane endpoints were established before study initiation and included severe weight loss, impaired mobility, ulceration or infection at the tumor site, or signs of severe clinical deterioration [16]. Animals were monitored regularly throughout the experimental period to minimize pain and distress. No animals reached the predefined humane endpoints during the study.

Study period and location

The study was conducted at the accredited animal research facility of Lerdsin Hospital, Department of Medical Services, Bangkok, Thailand. Cell culture experiments and xenograft preparation were performed in collaboration with the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand. Cannabinoid extracts were prepared and supplied by the Government Pharmaceutical Organization (GPO), Thailand.

Study design

Female BALB/cAJcl-nu nude mice, four weeks of age, were obtained from Nomura Siam International (Bangkok, Thailand). Animals were housed under controlled environmental conditions (22 ± 2°C, 50%–70% humidity, 12 h light/dark cycle) with free access to food and water. A one-week acclimatization period was provided before experimentation.

To support estrogen-dependent tumor growth, mice received weekly subcutaneous injections of 17β-estradiol prepared from β-estradiol powder (Sigma E2758, Sigma-Aldrich, St. Louis, MO, USA). Estradiol was initially dissolved in sterile sesame oil to a stock concentration of 2 mg/mL and subsequently diluted to deliver 5 µg per 0.1 mL per mouse. The solution was freshly prepared weekly and administered subcutaneously under aseptic conditions. Injections began 1 week before tumor cell inoculation and continued weekly throughout the study period [17]. However, serum estradiol concentrations were not measured.

MCF-7 breast cancer cells at a concentration of 1 × 107 viable cells were mixed with Matrigel (Corning, NY, USA) at a 1:1 ratio (v/v) and subcutaneously injected into the right flank of each mouse under aseptic conditions. Body weight (BW) and tumor dimensions were recorded every 3 days. Tumor length and width were measured using a caliper, and tumor volume was calculated using the following formula: Tumor volume (mm³) = 1/2 (length × width²) [18].

Mice were randomly assigned to treatment groups once tumors became established, defined as the presence of a palpable tumor mass at the injection site approximately 19 days post-inoculation, using simple randomization. Tumor sizes were comparable among groups at baseline (day 0). Blinding was not performed because treatment groups received different doses.

The sample size was determined using a priori power analysis performed in Minitab (Minitab LLC, State College, PA, USA). Based on an effect size (f = 0.827) derived from a previous study [19], with a significance level of 0.05, statistical power of 0.80, and five experimental groups, the minimum total sample size required to detect significant differences was calculated to be 25 animals. Accordingly, 25 mice (n = 5 per group) were included in the study. This sample size is consistent with previous preclinical studies using cannabinoid-based treatments, in which 5–8 animals per group were sufficient to detect significant differences in tumor growth, proliferation, and apoptosis [20].

The animals were randomly allocated into five groups: (1) negative control group receiving sesame oil by oral gavage, (2) positive control group treated intraperitoneally with 5-FU (20 mg/kg) three times weekly, and (3–5) experimental groups receiving cannabinoid extract (THC:CBD, 1:6) supplied by the GPO at doses of 2, 10, or 20 mg/kg BW by oral gavage for 30 consecutive days. All animals with successfully established tumors were included in the study. Three animals died during the experimental period, including one mouse each from groups 1–3. One animal displayed severe weight loss, one was found dead without an apparent cause, and one developed rectal prolapse. These events were considered incidental and unrelated to tumor burden or treatment. No additional animals or data points were excluded from the final analysis.

Cannabinoid formulation

The GPO, Thailand, prepared and supplied a THC and CBD solution at a 1:6 ratio using Cannabis sativa L. cultivated in the GPO medical cannabis greenhouse. The extraction process followed modified methods from previous studies [21]. Briefly, cannabis flowers (cola) were harvested, and cannabinoids were isolated using cold ethanol extraction. After quantification of THC and CBD concentrations, the extract was adjusted to achieve the desired THC:CBD ratio of 1:6.

The selection of this ratio was based on pharmacokinetic considerations to achieve approximately balanced systemic exposure. In rodent pharmacokinetic studies, oral administration of THC and CBD at equivalent doses showed that THC remained detectable in plasma at later time points, whereas CBD concentrations declined more rapidly and became undetectable at the same time point, indicating faster systemic clearance of CBD than THC in vivo [22]. In addition, previous studies in mice demonstrated that CBD inhibits cytochrome P450-mediated metabolism of THC, resulting in a prolonged half-life and increased persistence of THC and its metabolites, particularly in brain tissue, compared with THC administered alone [23]. These findings indicate that co-administration of THC and CBD alters the pharmacokinetic profiles of both compounds. Consistent with these observations, co-administration studies in experimental animals have reported reduced maximum serum concentrations of CBD and increased THC exposure compared with single-compound administration.

Oral administration of the whole-cannabis extract was selected to better reflect potential clinical application compared with intraperitoneal administration of single compounds. Therefore, a THC:CBD ratio of 1:6 was selected to compensate for the faster clearance of CBD and the altered metabolism of THC, with the aim of achieving an approximately 1:1 effective systemic exposure of THC and CBD in vivo, comparable to the ratio used in the in vitro cell culture experiments (unpublished data). Following extraction, the solvent was removed by rotary evaporation, and the concentrated cannabinoid extract was dissolved in pharmaceutical-grade oil for in vivo administration. The stock solution was prepared at a concentration of 5 mg/mL and further diluted in sesame oil to achieve the desired dosing concentrations based on BW (mg/kg). All experiments were conducted using the same production batch of the extract to minimize variability.

Exact concentrations of individual cannabinoids beyond THC and CBD, as well as full compositional profiling, including terpene content, residual solvents, and stability data, were not available. Therefore, the presence of additional constituents in the extract cannot be excluded. However, the extract was produced under standardized conditions by the GPO in Thailand to ensure batch consistency and quality control.

In vitro screening for animal dose selection

A cell viability assay was performed to assess the antiproliferative effects of cannabis extracts, building on previous studies conducted in multiple cancer cell lines. A cannabis extract with a 1:1 THC:CBD ratio was used for the in vitro experiments. In contrast to in vivo conditions, in vitro experiments do not involve pharmacokinetic processes such as absorption, distribution, metabolism, and elimination; therefore, a 1:1 ratio was directly applied to reflect the intended effective exposure at the cellular level.

The half-maximal inhibitory concentration (IC50), defined as the concentration required to reduce cell proliferation by 50%, was determined to guide dose selection for subsequent in vivo studies. In MCF-7 breast cancer cells, the IC50 of this extract was approximately 1 µg/mL (unpublished data). Dose selection for the xenograft study was based on a combination of in vitro potency (IC50) and in vivo pharmacokinetic considerations, with dose calculations performed in consultation with pharmacists from the GPO. Based on in vivo pharmacokinetic data (unpublished), oral administration of THC at 10 mg/kg was estimated to achieve a maximum blood concentration of approximately 0.4 µg/mL in rodents. To achieve a target systemic concentration closer to the in vitro IC50 value (~1 µg/mL), a higher dose was therefore required. Accordingly, 20 mg/kg was selected as the high-dose level, and 2 and 10 mg/kg were selected as the low- and intermediate-dose levels, respectively.

Cell line expansion and preparation for transplantation

The human breast cancer cell line MCF-7 (CRL-3435™, American Type Culture Collection [ATCC], Manassas, VA, USA) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (ATCC™ 30-2002, ATCC), supplemented with 10% fetal bovine serum (cell-culture tested, ATCC™ 30-2020, ATCC), in accordance with the guidelines provided by ATCC. Cells were maintained in 25 cm³ sterile culture flasks (NUNC EasyFlask, Thermo Scientific, Shanghai, China) under standard conditions at 37°C in a humidified incubator with 5% CO2. Cells were used at low passage numbers (<20 passages) to maintain biological consistency. Short tandem repeat profiling and mycoplasma testing were not performed; however, culture conditions were maintained in accordance with ATCC guidelines to minimize variability.

The medium was renewed every 3 days to maintain nutrient balance and cell health. Upon reaching 70%–80% confluence, adherent cells were detached using 0.25% Trypsin-EDTA solution (Gibco, Thermo Scientific, Waltham, MA, USA), pelleted by centrifugation, and resuspended in fresh medium. Expanded cultures were subsequently transferred into 75 cm³ flasks for large-scale propagation until adequate cell numbers were obtained for transplantation.

For xenograft preparation, cells were harvested, washed twice with sterile phosphate-buffered saline (PBS, pH 7.2, Gibco), and resuspended in complete DMEM. Cell concentration and viability were assessed using the 0.4% Trypan blue staining method and counted using a hemocytometer. The suspension was then adjusted to 1 × 107 viable cells in 0.1 mL for subcutaneous injection into the right flank of nude mice.

Histopathological evaluation

At the end of the 30-day treatment period, mice were euthanized by carbon dioxide inhalation in a dedicated chamber in accordance with institutional animal care guidelines. Animals were exposed to carbon dioxide until respiration ceased and death was confirmed. Blood samples were collected for hematological and biochemical evaluation. Tumors and major internal organs were excised, fixed in 10% neutral-buffered formalin for 24 h, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) to evaluate histopathological features, detect potential metastases, and quantify necrotic and apoptotic areas following procedures described previously [24].

Briefly, the percentage of necrotic area was calculated by dividing the necrotic region by the total tumor area using an image analysis system (NIS-Elements Analysis D, Nikon, Tokyo, Japan). Apoptotic cells were manually counted in five randomly selected high-power fields (HPFs) at 40× magnification, and the results were reported as the average number of apoptotic cells per field.

Immunohistochemistry

Tumor cell proliferation was assessed using immunohistochemistry. Proliferation was evaluated using a monoclonal mouse anti-PCNA antibody (clone PC10, Dako, Hamburg, Germany) at a dilution of 1:400. For each mouse, five HPF at 40× magnification were randomly selected for analysis. PCNA immunoreactivity was scored according to staining intensity and the proportion of positive cells as follows: 0 = no staining, 1 = weak staining or 0%–25% positive cells, 2 = 26%–50% positive cells, 3 = 51%–75% positive cells, and 4 = >75% positive cells [25].

Statistical analysis

Statistical analysis was conducted using SPSS software version 22.0 (IBM, Chicago, IL, USA). Before statistical comparisons, data were assessed for normality using the Shapiro-Wilk test, and normally distributed values were presented as mean ± standard deviation. Differences among groups were analyzed using one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons. A p < 0.05 was considered statistically significant.

RESULTS

BW and tumor growth

The mean BW of mice across all five groups before the experiment showed no significant differences (p = 0.832). By day 30, at the conclusion of the study, BWs remained comparable among groups (p = 0.848). All groups exhibited an overall increase in BW during the experimental period, with no significant differences observed among groups (p = 0.693) (Table 1).

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