Immunotherapy in the form of anticancer vaccination relies on the mobilization of the patient's immune system against specific cancer antigens. Instead of focusing on an autologous cell lysate, which is not always available in clinical practice, the present study investigates vaccines utilizing xenogeneic foetal tissue that are rich in oncofoetal antigens. Lewis lung carcinoma (LLC)-challenged C57BL/6 mice were treated with either a xenogeneic vaccine made from chicken whole embryo, or a xenogeneic vaccine made from rat embryonic brain tissue, supplemented with a
Through the immunosurveillance process, the immune system is the main line of defence against cancer (
The inherent tolerogenicity/low immunogenicity of TAAs is an obstacle to effective spontaneous and vaccination-induced antitumour immunity. Since all TAAs (apart from oncoviral TAAs) are derived from self-proteins, TAAs possess a certain degree of tolerance, depending on their type (
Of the various TAAs, oncofoetal (
The present study investigated the immunological and therapeutic (micrometastases-suppressing) efficacy of postoperative xenovaccination in a murine LLC model. In spite of the reports raising the question whether LLC and 3LL cell lines are actually the same cell line, all sources cited in the present study were using the LLC-labelled cell line for the LLC model, therefore this variant was used to maintain consistency across the studies. Two xenogeneic vaccines were investigated, a patented rat embryonic nervous tissue-based xenovaccine adjuvanated with a protein-containing metabolite of
A total of 30 C57BL/6 mice (8–12 weeks old; female) were obtained from the State Research Institute Centre for Innovative Medicine (Vilnius, Lithuania). The mice were housed in plastic cages (≤15 mice per cage) under normal daylight conditions with
The murine metastatic Lewis lung carcinoma LLC1 cell line was a gift from the RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology (Kiev, Ukraine). The cells were cultivated in Dulbecco's modified Eagle's medium (Lonza Group, Ltd., Basel, Switzerland) containing 2 mM L-glutamine, 10% foetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere containing 5% CO2 at 37°C.
The rat embryo nervous tissue vaccine (‘xeno rat’ vaccine) used in the present study has been patented by the Ukrainian Intellectual Property Institute (
On day 0, 30 C57BL/6 mice received a subcutaneous injection of 3×105 LLC cells in the left hind foot. The foot was chosen as the site of tumour inoculation owing to the ease of resection, which contributed to the 100% surgical survival rate in the present study. The hind foot was specifically chosen as mice recover better with their front legs intact. Although the tumours were located in the foot, no bleeding or self-harm was noted at the tumour site. On day 14 following injection, primary tumours were surgically removed by amputating the foot under general anaesthesia, using intraperitoneal ketamine (100 mg/kg)-xylazine (10 mg/kg) injections. The mice were allowed to recover under a heat lamp and were monitored for 2 h or until they were able to uphold an upright position, at which point they were returned to standard cages. The mice with the primary tumour removed were subsequently treated with either the xeno rat vaccine (n=10) or the xeno chicken vaccine (n=10). The mice that underwent surgical LLC removal and were receiving saline injections (n=10) served as the control group. The mice in each group were vaccinated as follows: Each vaccine was injected subcutaneously into the nape of the neck on days 17, 20 and 23. Internal organs were analysed for detection and characterization of LLC metastases in animals that has succumbed to disease. Blood samples were collected at various time points for analysis of cluster of differentiation 8+ (CD8+) T-lymphocyte population. The experimental design is depicted in
To assess the anticancer effects of the vaccines, mice lung and blood samples were obtained (
The survival of mice was observed daily throughout the experiment. Owing to the primary tumour resection, tumour growth could not be used to establish a humane endpoint of the study. Therefore, a time limit of 70 days was introduced. The humane endpoint for individual mice was set subjectively by the technical staff, which were blinded to the purpose of the experiment, and euthanized the animal when they appeared to be in a critical condition. This procedure could only be applied within working hours (07:00-17:00) and at two time points during the weekends (mornings and evenings). Owing to these time constraints, one control mouse was not euthanized, but found to have succumbed at the beginning of the working day, which was fixed as the date of mortality. This event contributed to 8% of the total of mice that succumbed to disease by the end of the experiment.
For histological analysis, lung tissue was fixed in 10% neutral buffered formalin for 24 h at room temperature, dehydrated in alcohol baths at room temperature (70% for 12 h; 90% for 12 h; 100% for 24 h) and embedded in paraffin. The paraffinized samples were serially cut into sections (thickness, 3 µm) using a microtome. The sections were deparaffinized, rehydrated and stained with haematoxylin (15 min) and eosin (5 sec) at room temperature. Each section was examined under a light microscope to identify the tumour-infiltrated areas. The images were captured using an automated Leica DM50000 B microscope equipped with a Leica DFC420 C digital camera (Leica Microsystems, GmbH, Wetzlar, Germany). Images were processed and analysed using the ImageJ image analysis program (version 1.48 k; National Institutes of Health, Bethesda, MD, USA), as described previously (
For analysis of CD8a+ T cell population, 100 µl blood were collected from the hip vein from each mouse. Whole blood was stained with anti-CD8-phycoerythrin (PE) (cat no. 552877; BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD3-allophycocyanin (APC) (cat no. 553066, BD Biosciences) antibodies (1 µl/100 µl blood, 25 min incubation at room temperature). Erythrocytes were lysed using ACK Lysing buffer (Gibco; Thermo Fisher Scientific, Inc.). For FACS, 20,000 events were collected using BD FACSDiva™ 7.0 flow cytometer (BD Biosciences) and analyzed with FlowJo™ (version 10.2; FlowJo LLC, USA) software.
P≤0.05 was considered to indicate a statistically significant difference. For statistical analysis Statistica 12 (Tibco Software, Inc., La Jolla, CA, USA) software was used. Non-parametric Kruskal-Wallis was used for statistical survival data analysis of the animals surviving at the end of the experiment. Once this test was performed, Kaplan-Meier analysis followed by long-rank analysis was used for group analysis as it is the most widely used test for survival analysis (
A total of 30 C57BL/6 female mice were subcutaneously injected with 3×105 LLC cells in the left hind-foot, and all of the mice developed visually detectable tumours at the injection site. On day 14, the foot with the primary tumour was surgically resected. No surgery-associated causalities were recorded. The mice were subsequently treated with either the xeno rat vaccine (n=10) or the xeno chicken vaccine (n=10) or injected with saline solution (n=10).
Mice treated with the xeno chicken vaccine maintained a 100% survival rate over the observation period of 70 days. The xeno chicken vaccine-treated mice survived significantly longer compared with the mice treated with the xeno rat-vaccine and control group animals [Kruskal-Wallis analysis, H (2, n=31)=9.644; P=0.008;
In each experimental group, blood samples were collected from the hip vein at three time points: i) On day 16 (i.e., two days after tumour removal and one day prior to the start of therapeutic xenovaccination); ii) on day 26 (two days after completion of therapeutic xenovaccination), and iii) on day 70 (at the end of the experiment for the surviving mice).
As shown in
As indicated in
Effect of xenogeneic vaccines on metastatic spread into the lungs. All mice that succumbed to disease in any experimental arm during the observation period had metastatic infiltrates in the lungs. Additionally, all control mice had metastatic foci in the liver. On day 70 (at the end of the observation/experiment), metastatic foci in the lungs were detected in 3 out of 10 of the surviving mice that were treated with the xeno chicken vaccine and in 1 out of 4 of the surviving mice that were treated with the xeno rat vaccine. All surviving mice in the control group (n=4) had metastatic infiltrates in lung tissue (see
The metastatic LLC model was introduced in the present study since the handling of oncological patients following primary tumour removal remains a challenge in modern oncology (
Survival analysis (
The long-surviving xeno chicken vaccination group was characterized by the highest proportion of CD8a+ in circulating blood from all investigated groups (18.8% of T lymphocytes vs. 12,4% in Xeno Rat and 11,2% in control groups). This was a larger percentage compared with the value reported in a previous study (
It is already recognized that individual xenogeneic vaccines do not work equally well against all types of cancer, as previously demonstrated for xeno chicken vaccine (
Moreover, in a previously investigated metastatic LLC model, where
Therapeutic cancer vaccinations are the source of a great deal of interest and following various clinical successes and failures continues to be promising for oncological patients (
Various preventive and therapeutic vaccines are generally used with vaccine adjuvants that shape and potentiate the induced immune responses, thereby increasing the efficacy of vaccination (
The results of the present study provide a further insight into the therapeutic potential of xenogeneic therapeutic cancer vaccinations, and may aid the direction of preclinical research and clinical trials in this field.
Experimental anticancer vaccination and sampling scheme for C57BL/6 mice challenged with LLC cells. Experiment design depicted from the time of tumor challenge (day 0) to the end of the survival observation (day 70). Figure presented previously (
Kaplan-Meier survival curves for mice receiving adjuvant treatment with different xenogeneic vaccines following the removal of primary LLC-derived tumors. The mice that were treated with the xeno chicken vaccine survived significantly longer (mOS, 70 days) compared with the mice that were treated with the xeno rat vaccine (mOS, 60 days; P=0.003) and control group animals (mOS, 55 days; P=0.003). There was no significant difference in survival between the xeno rat and control groups. For each treatment arm, n=10. LLC, Lewis lung carcinoma; mOS, mean overall survival.
Percentage of CD8a+ T cells in circulating peripheral blood monocytes prior to therapeutic xenovaccination (day 16, white column), 3 days after the completion of xenovaccination (day 26, black columns) and at the end of the study (day 70, grey columns). There were no significant changes in the levels of circulating CD8a+ T cells 3 days after the completion of xenovaccination, compared with the levels prior to the start of vaccination (within and between the experimental arms). However, on day 70 (at the end of the observation period), the levels of circulating CD8a+ T cells were significantly higher in the surviving mice vaccinated with the xeno chicken vaccine (n=10) compared with surviving mice (n=4) in the control group (P=0.002) or in the Xeno Rat group (P=0.049). In xeno chicken vaccine-treated mice, the level of CD8a+ T cells on day 70 was significantly higher compared with the level on day 16 (following tumor removal, prior to xenovaccination) (P=0.036) and on day 26 (3 days after the completion of xenovaccination) (P=0.003). There were no significant differences in the levels of circulating CD8a+ T cells in the mice that were treated with xeno rat vaccines evaluated at different time points or comparing T-cell levels with those in control group animals. Additionally, there was a significantly lower CD8a+ T-cell population in the control mice at the end of the study (day 70) compared with the level following the completion of vaccination (day 16) (P=0.028). CD8a, cluster of differentiation 8a. *P≤0.05.
CD8a+ flow cytometric analysis. Histograms for the (A) control, (B) xeno rat and (C) xeno chicken treatment groups. CD8a, cluster of differentiation 8a.
Histological analysis of lung samples from mice surviving the experiment (day 70). Histological slides from (A) control mice, (B) mice in the xeno rat experimental arm and (C) mice in the xeno chicken experimental arm. Metastatic foci (A and B) fill the whole field of view or are (C) indicated by an arrow. Magnification, ×100.