Analysis of Osmotic Pump-Administered Xylitol in a Syngeneic Mouse Melanoma Model

1. Introduction

Xylitol is a prebiotic polyol that has been used for preventive dental care for many decades. It is considered natural and is generally regarded as a safe food additive or supplement [1]. It has long been used as a substitute for ordinary table sugar due to its sweetness and taste profile, similar to sucrose [2]. In 1975, Mäkinen et al., first reported that xylitol significantly reduced dental caries by inhibiting the growth of Streptococcus mutans [3]. Since then, clinical studies have shown that xylitol products reduce the oral microbiome levels of S. mutans, decrease plaque accumulation, and decrease the incidence of dental caries in children [4,5,6]. Total or partial substitution of sucrose with xylitol in the human diet reportedly results in more than an 85% reduction in the incidence of dental caries [7]. According to Mäkinen et al., most S. mutans strains transport xylitol into the cell via the phosphotransferase system, which is then phosphorylated to xylitol-5-phosphate and expelled from the cell [8,9]. This energy-consuming pathway is thought to inhibit S. mutans and other energy-dependent pathogens [10]. Numerous studies have demonstrated the efficacy of xylitol in preventing caries and inhibiting the growth of periodontal pathogens [11,12]. Additionally, xylitol provides microbial balance by maintaining a healthy gut microbiome, starting with the oral gateway microbiome, which supports innate immunity and disease resistance [13,14,15,16].

The microbiome’s influence on cancer development and treatment has recently been recognized [17]. As a result, research on complex microbial communities and the mechanisms through which microbiota influence cancer prevention, carcinogenesis, and anticancer therapy has significantly increased. Therefore, researchers are considering the urgent development of next-generation prebiotics and probiotics designed to target specific diseases [18]. Healthcare professionals can utilize prebiotics, probiotics, and postbiotics to prevent and treat diseases such as cancer [19]. The hallmarks of cancer are immune elimination and escape, both of which can be partly bacteria-dependent, shaping immunity by mediating host immunomodulation [20]. Additionally, host immunity regulates the microbiome by modulating bacterial-associated signaling to influence tumor surveillance [21]. Cancer immunotherapy, including immune checkpoint blockade, appears to have heterogeneous therapeutic effects in different individuals, partially attributed to the microbiota [22]. Personalized medicine will require a better understanding of the microbiota and their interaction with cancer cells. The manipulation of the gut microbiota with prebiotics to improve cancer therapeutic responses may prove indispensable for future cancer treatment [23].

When administered orally and systemically, xylitol inhibits the growth of cancer cells [24,25,26]. Because xylitol is utilized by healthy human cells and exhibits almost no side effects, it may be a safe supplement for inhibiting the proliferation of cancer cells [27,28]. Xylitol also decreased tumor vascularization by inhibiting angiogenesis. Increased vascularization supports tumor growth and possibly cancer metastasis [29]. Xylitol is also a natural substance, and humans are reported to produce approximately 15 g of xylitol per day in the liver [30]. Xylitol is converted by mitochondrial xylitol dehydrogenase into a precursor of the tricarboxylic acid cycle. Xylitol dehydrogenase on the cristae metabolizes xylitol to xylulose, which converts NADP to NADPH [31]. Many plants naturally contain measurable amounts of xylitol, including blueberries, strawberries, plums, cauliflower, and oatmeal [32,33]. This is in addition to the production of xylitol by the human liver from glucose via the pentose phosphate pathway [32]. Interestingly, there is some overlap between the list of xylitol-containing foods and the American Heart Association’s list of “heart-healthy” foods [33]. Also, xylitol has been suggested to help prevent diabetes and act as an anti-inflammatory agent [24,35].

Previously published research on animal models has reported positive results in inhibiting cancer cell lines and cancer xenografts with xylitol supplementation [36]. Combination treatments with olive oil phenolic compounds and xylitol have also been reported to be potentially beneficial [37]. The first phase of cancer research, which involves identifying potential therapeutic agents, typically utilizes animal models [38,39]. Therefore, the present study used a syngeneic mouse model for human malignant melanoma to evaluate the inhibitory efficacy of xylitol (a commercial product from Xlear, American Fork, USA) delivered via Alzet osmotic mini pumps. However, this approach later proved to be suboptimal. An additional study is currently underway using a 10% xylitol solution administered in drinking water, as the initial study also revealed that the Alzet osmotic mini pumps may clog due to the crystallization of the xylitol solution.

2. Materials and Methods

The B16F10 Syngeneic Model consisted of 6–8-week-old C57BL/6 male mice (Charles River Laboratories) subcutaneously injected with 5 × 10⁵ B16F10 cells (ATCC CRL-6475) suspended in 100 μl PBS into the right flank. Mice were monitored for tumor development and randomized according to tumor volume and body weight for implantation surgery. The mice were randomly assigned to two groups: Group 1 was the treatment group, receiving 10% xylitol-loaded minipumps (n = 10), while Group 2 was the control group, receiving saline-loaded minipumps (n = 10). Alzet mini pumps (Model 2004, Cupertino, USA) were implanted in B16F10 injected mice once more than 50% of the mice developed palpable tumors. Alzet osmotic pumps were loaded with a 10% xylitol solution in saline or saline vehicle controls. The pumps were primed at 37°C for 48 hours before the implantation surgery. All mini pump surgeries had to be completed in one day, and the mini pumps were loaded more than 40 hours before surgery. The minipumps should have released xylitol for 28 days until pump depletion. The dose was 0.5 (mg/g/day) for a total of 10 mg/day average with a delivery rate (mg/hr) of 0.417, the pump rate of solution was 0.25 (ul/hr), at a concentration of 1.67 (mg/ul) and the total pump volume (ul) was 200. All animal procedures were conducted in accordance with the ethical standards and approved protocols of Northwestern University. Mice were housed under a 12:12-hour light:dark cycle with access to standard chow (Envigo 7912) and water ad libitum.

Twenty mice underwent surgery to implant Alzet minipumps and were group-housed before and after surgery. Each mouse was placed under isoflurane anesthesia (~3%) and then given preoperative analgesia (SQ: 20 mg/kg meloxicam, line block of 2 mg/kg 0.1% bupivacaine). Under sterile conditions, an incision of approximately 1 cm was made in the lower left flank. Pumps were placed subcutaneously in the lower left flank, and for most mice, the surgical incision was located caudal to the mini pump. The incisions were closed using 2 to 3 wound clips. The mice were monitored 2x per day for the first 48 hours post-operation. After minipump implantation surgery, the mice were monitored and weighed twice to thrice weekly. Tumor sizes were measured with calipers twice to three times per week, and all mice were euthanized when their tumors reached a diameter of 20 mm on any axis or a volume of 2,000 mm³. The excised tumors (Figure 1) were measured and then carefully cut in half, with one half sent for histological analysis and the other for metabolomic analysis. In addition to tumor tissue, draining lymph nodes were collected and sent for histological analysis. The osmotic minipumps were collected and weighed for data analysis. After euthanasia, tumor tissue was collected for two studies: metabolomics (tissue flash-frozen in liquid nitrogen) and histopathology, where the tissue was fixed in formalin for H&E staining to assess general morphology and IHC staining to evaluate microvascular density.

Rodent CO₂ Euthanasia was performed according to IACUC protocol. Animals were not combined from different cages, and when euthanizing an entire cage, the animals remained in their original housing. The maximum number of mice per cage was five, and the CO2 flow rate per mouse cage was 3 L/min until one minute after breathing stopped. Euthanasia was confirmed by cervical dislocation. Immediately following euthanasia, tumors were harvested and fixed in 10% neutral-buffered formalin for 48 hours. Tumors were embedded in paraffin, sectioned into 4-5 µm-thick sections, placed on microscopic slides, and stained with hematoxylin and eosin (H&E) by the Mouse Histology and Phenotyping Laboratory of Northwestern University. Microscopic slides were imaged using an Olympus BX45 microscope with an Olympus DP28 digital camera. Digital images were visualized using Olympus cellSens imaging software (version 4.2).

Metabolomic samples were analyzed using high-performance liquid chromatography, high-resolution mass spectrometry, and Tandem mass spectrometry (HPLC-MS/MS). Specifically, the system consisted of a Thermo Q-Exactive, in line with an electrospray source, and an Ultimate3000 (Thermo) series HPLC, comprising a binary pump, degasser, and autosampler outfitted with an Xbridge Amide column (Waters; dimensions: 3.0 mm × 100 mm, particle size: 3.5 µm). The mobile phase A contained 95% (vol/vol) water, 5% (vol/vol) acetonitrile, 10 mM ammonium hydroxide, 10 mM ammonium acetate, pH = 9.0; B was 100% Acetonitrile. The gradient was as follows: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A with a flow rate of 150 μL/min. The capillary of the ESI source was set to 275 °C, with sheath gas at 35 arbitrary units, auxiliary gas at five arbitrary units, and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen, and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106, and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented in a data-dependent manner, using the higher-energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution of 17,500. Besides matching m/z, metabolites are identified by matching either retention time with analytical standards and/or MS2 fragmentation pattern. Xcalibur 4.1 software and Tracefinder 4.1 software from Thermo Fisher Scientific were used for data acquisition and analysis. MetaboAnalyst software 6.0 can be used for statistical and functional data analysis and Mendelian randomization for causal inference.

3. Results

The survival of the xylitol-treated group appeared 40% longer than that of the control group, but not by a statistically significant amount (Chi-square 2.610, df 1, P =0.1062) – see Figure 1. The difference in the survival rate was not statistically significant, perhaps due to the sample size. The Kaplan-Meier Survival curve analysis demonstrated an insignificant difference between the groups. The tumor volumes were significantly different on days 8 and 15 (P < 0.05) but not on day 10, suggesting a possible adaptation of the cancer cell line to xylitol or inconsistent osmotic pump operations. The growth of the tumor was reduced by approximately 35% by volume (See Figure 2) in the xylitol group, with a statistically significant difference, but only on certain days of the tumor growth. The weight gain in the xylitol groups was also significantly less than in the vehicle group (see Figure 3), likely due to the decreased tumor size. The average weight of the mice at the start of the study was 23.8g, and there was no significant difference between the xylitol and the control groups. The mice had been randomized into the control and xylitol groups according to the size or volume of the tumor present. Upon initiation of the intervention with osmotic pumps, the average weight of the mice was 25.9g, the xylitol group being slightly less in weight but not significantly so. All mice had the same diet and water supplied.

The metabolomic analysis heatmap (Figure 4) demonstrated that xylitol reduced the tumor production of histidine, acetylcholine, GTP, ATP/dGTP, and UDP, possibly improving the host’s immune response to tumor cells by reactive oxygen species production [40]. Significant differences existed between the experimental (xylitol) and vehicle-treated (control) groups. Note that proline, NMN, and XMP increase with the vehicle group. Additionally, the xylitol group exhibited decreases in dopamine, epinephrine, and putrescine levels compared to the control group. The results of the statistical analysis (t-test) comparing the vehicle to the xylitol groups are shown in Table 1. Xylitol significantly increases dATP, ATP/dGTP, CDP, SAICAR, acetoacetic acid, N1-acetylspermine, F-1, 6/2, 6-DP, and spermidine metabolites in the tumor compared to the saline control.

The histological samples (see Figure 5a,b) demonstrated areas of extensive intratumoral necrosis (arrows) with viable tumor cells surrounding blood vessels (asterisks). Scattered mitotic figures were present throughout the viable fraction of tumors (arrowheads). There were no discernible histopathological differences between the vehicle and the xylitol groups; however, metabolomic changes that result in increased cancer cell sensitivity to innate immunity may not be apparent in histological sections. However, the tumor size in the vehicle group was measurably larger (Figure 3). Tumors in both treatment groups exhibited considerable variability in intratumoral necrosis (viable tumor fraction) and mitotic indices. None of the xenograft tumors showed accompanying inflammation, increased apoptotic tumor cells, or intratumoral thrombosis. Histological sections that included adjacent tissue suggested reduced infiltration by tumor cancer cells and angiogenesis, consistent with previous studies [24,25,26]. The nature of the necrosis in the tumors was characteristic of aggressive tumor cells that outgrow their blood supply, resulting in necrosis of the cells farthest from the blood vessels. Lymph node tissue demonstrated metastasis in one lymph node in each group, with no significant differences.

4. Discussion

The present study was part of a series of syngeneic models aimed at evaluating the effects of xylitol on tumor cell proliferation and determining any improvement in the survival of the subject mice. The use of Alzet pumps was suggested due to the inadequacy of the intratumoral injection protocol. Intratumor injections resulted in stromal disintegration and subsequent xylitol leakage. Additionally, the 20% solution may have been too concentrated, resulting in local areas of crystallization and hindering proper dissemination. Alzet pumps exhibited crystal deposition above a 10% solution, which occasionally led to poor wound healing, potentially reducing the potential for increased survivability. However, metabolomics revealed a significant change in metabolites within the tumor when xylitol was administered instead of a saline control. This finding is consistent with our previous research and other published scientific articles [19,26,27]. The metabolomic analysis revealed that xylitol induced a metabolic response in the tumor, potentially enhancing the host immune response to reactive oxygen species generated by innate immune cells.

N1-acetylspermine is a polyamine derivative that plays a crucial role in cellular metabolism and function. Formed through the acetylation of spermine, it is involved in various physiological processes, including regulating gene expression, stabilizing nucleic acids, and modulating enzyme activities [41]. This metabolite is associated with cell growth, differentiation, and apoptosis. The clinical significance of N1-acetylspermine is being increasingly recognized. Abnormal levels of this metabolite have been linked to cancer, neurodegenerative diseases, and other metabolic disorders. Its measurement in biological fluids such as urine and blood can provide valuable insights into disease states and therapeutic responses [42]. N1-acetylspermine has been found to play a significant role in cancer development by promoting tumor growth and metastasis through its influence on cell proliferation and apoptosis [43]. Additionally, it can modulate the host immune response, potentially impacting the tumor microenvironment. Elevated levels of N1-acetylspermine can suppress immune cell function, aiding in tumor immune evasion [44].

The balance between F-1,6-BP and F-2,6-BP is crucial for maintaining metabolic homeostasis. These metabolites facilitate ATP production and influence various cellular processes such as apoptosis, cell proliferation, and response to hypoxia. Dysregulation of their levels can lead to metabolic disorders, including diabetes and cancer [45]. F-1,6-BP and F-2,6-BP play critical roles in cancer metabolism by supporting the high glycolytic rates observed in tumor cells, known as the Warburg effect [46]. This enhanced glycolysis provides energy and biosynthetic precursors necessary for rapid cell proliferation. Furthermore, these metabolites can influence the host's immune response by altering the metabolism and function of immune cells [47].

Spermidine has been implicated in cancer development due to its role in cell proliferation and the inhibition of apoptosis. It can also modulate the host immune response, enhancing the antitumor activity of immune cells. Increased spermidine levels have been shown to support tumor growth, whereas reducing its levels can impair tumor progression. Furthermore, spermidine can influence autophagy, which can promote or inhibit cancer, depending on the context [48].

Histidine, acetylcholine, GTP, ATP, and UDP also enhance the host's immune response to tumor cells by producing reactive oxygen species (ROS) [49]. The mice that received xylitol via osmotic pumps exhibited higher levels of these metabolites, as mentioned earlier, in their tumors. Histidine, an amino acid, serves as a precursor for synthesizing histamine, which can activate immune cells and trigger ROS production, thereby contributing to the immune-mediated killing of cancer cells [50,51,52]. Acetylcholine, a neurotransmitter, has been shown to modulate immune cell activity and promote ROS generation, enhancing immune cells' cytotoxicity against tumor cells [53]. GTP and its derivatives, such as dGTP, are essential for activating G-proteins, which are involved in various signaling pathways, including those that regulate ROS production. Elevated levels of GTP and dGTP can enhance the immune response by promoting ROS-mediated tumor cell death [54,55,56,57]. ATP, a critical energy molecule, can also act as a signaling molecule that induces ROS production in immune cells, thereby increasing their antitumor activity [58,59,60]. UDP, a nucleotide sugar, is involved in glycosylation processes that can modulate immune cell function and ROS production, enhancing the immune response to tumor cells [61].

Reactive oxidative species (ROS) play a pivotal role in the death of cancer cells. ROS are highly reactive molecules that can cause significant damage to cellular components, including DNA, proteins, and lipids. This oxidative damage can lead to apoptosis, necrosis, and autophagy in cancer cells, inhibiting their proliferation and survival [62]. Various factors, including immune cell activation, chemotherapy, and radiation therapy, can stimulate the production of ROS. Immune cells, such as macrophages and T cells, generate ROS to eliminate cancer cells as part of the body's natural defense mechanism [63]. These ROS-induced cytotoxic effects can be further enhanced by therapeutic interventions that increase ROS levels in the tumor microenvironment [64]. Immune cells, such as macrophages and T cells, generate ROS to eliminate cancer cells as part of the body's natural defense mechanism [65]. These ROS-induced cytotoxic effects can be further enhanced by therapeutic interventions that increase ROS levels in the tumor microenvironment [66]. By influencing these pathways, ROS can promote the apoptosis of cancer cells and inhibit their resistance to therapy [67,68,69].

AGP/ATP are listed in this specific format in metabolomic studies due to their distinct yet complementary roles in cellular energy metabolism [70]. AGP (Adenine Guanine Phosphate) and ATP (Adenosine Triphosphate) are involved in energy transfer and storage within cells. AGP serves as a precursor to ATP, and their combined study provides a comprehensive understanding of energy dynamics and metabolic processes. In metabolic studies, the ATP/dGTP ratio is a significant parameter that reflects the energy status and nucleotide balance within cells [71]. Miriam et al. provided insights into how the interplay between these two nucleotides influences energy metabolism and cellular processes. They highlighted how variations in this ratio can affect cellular functions and potentially alter the metabolic pathways involved in cancer progression and immune regulation [72].

Osmotic minipumps could, theoretically, be inserted into large tumors to deliver a constant supply of xylitol, which inhibits the growth of specific cancer cell lines without any side effects, if a beneficial effect on tumor growth is discovered. Alternatively, xylitol could be systemically administered during the treatment phase along with standard-of-care therapy, followed by using dietary xylitol supplements to prevent recurrence. Xylitol is absorbed in the small intestine through passive diffusion, which does not require energy. The efficiency of xylitol absorption is influenced by several factors, including other nutrients and the overall health of the intestinal mucosa [73]. This absorption mechanism is consistent with the behavior of other sugar alcohols, which are also absorbed through passive diffusion [74]. Xylitol has been shown to influence the gut microbiota composition in mice. Uebanso et al. reported that xylitol consumption changes the populations of beneficial and harmful bacteria in the gut [75]. These changes can influence digestive health and immune function, as gut microbiota plays a crucial role in these processes [75].

The digestion of xylitol primarily occurs in the liver, where it is converted into D-xylulose through the action of xylitol dehydrogenase. The half-life of xylitol in plasma is relatively short, typically ranging from 30 minutes to 1 hour [76]. This rapid clearance is attributed to its efficient utilization in metabolic pathways and excretion through the kidneys. This metabolic pathway was detailed by Amo et al. (2011), who highlighted the role of xylitol dehydrogenase in facilitating the conversion process [77]. The conversion of xylitol into D-xylulose allows it to enter the pentose phosphate pathway, which is essential for cellular metabolism and the production of nucleotides [78]. In the liver, xylitol is phosphorylated and metabolized to xylulose 5-phosphate (Xu5P), an intermediate of the nonoxidative branch of the pentose phosphate pathway [79]. The xylitol metabolite Xu5P specifically activates both nuclear transport and the DNA-binding activities of carbohydrate response element binding protein. Studies have shown that xylitol can influence metabolic pathways, particularly those involved in energy production and immune regulation [76,77]. It was reported that variations in the ATP/dGTP ratio can affect the efficiency of xylitol metabolism, thereby altering the overall energy status of cells [76]. The interplay between xylitol and cellular metabolism is significant for understanding its potential health implications [80].

These changes can influence digestive health and immune function as the gut microbiota plays a crucial role in these processes. Delivering xylitol directly via Alzet mini osmotic pumps eliminated dietary variables and reduced potential confounders, especially those originating from the gut microbiome. The absorption and digestion of xylitol in mice involve complex processes influenced by various factors, including the presence of other nutrients, the health of the intestinal mucosa, and the animal's overall metabolic state [81]. However, xylitol may be a safe substitute for sucrose for patients desiring sweet-tasting foods. Additionally, the consumption of high-sucrose foods should be discouraged, and xylitol-containing products, as well as fruits and vegetables, should be encouraged among patients with cancer. Previous studies have demonstrated that xylitol inhibits the growth of certain cancers [24,25,26] and is readily incorporated into the diet, replacing sucrose and fructose. In the present study, the survival times of xylitol-treated mice were up to 35% higher than those of control mice, although the difference was not statistically significant. A larger sample size may yield statistically significant results in this type of biological study, which often involves numerous potential confounders.

Extensive clinical trials should be considered to evaluate the benefits of xylitol, following confirmation by further research using cancer cell lines and syngeneic mouse models. No noticeable side effects of the xylitol treatment were found in the experimental group.

5. Conclusions

The results of this preliminary study suggest that xylitol has the potential to be an adjunct to oncological treatment and is being further investigated. An osmotic minipump (Alzet) provided systemic exposure to 10% xylitol solution, which temporarily reduced the growth of malignant melanoma tumors and may have increased the survivability of xylitol-treated mice; however, the results were not statistically significant. The sample size or blockage of the mini pumps by crystallization may have affected the statistical results. Future large-scale studies with syngeneic models are warranted to evaluate the safety and efficacy of supplemental xylitol as an adjunct in cancer treatment.