Immunotherapeutic efficacy of a Lactobacillus casei lysate as an adjuvant combined with a heated-4T1 mammary carcinoma cell lysate in a murine model of breast cancer
Article Category: Original article
Publié en ligne: 31 janv. 2017
Pages: 327 - 334
DOI: https://doi.org/10.5372/1905-7415.1004.494
Mots clés
© 2016 Ruhollah Dorostkar, Mohammad Sadegh Hashemzadeh, Sajjad Jafari, Mahdi Tat, Majdedin Ghalavand, Mohammad Hossein Asghari, Milad Moloudizargari
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Malignancies are among the major health problems for humans and not only cause widespread mortality, but also impose a heavy financial burden on health care systems of all societies [1]. Breast cancer is one of the most common malignancies, accounting for approximately one-third of all cancers occurring in women. It is expected that 1 in every 8 women in the USA will develop breast cancer in her lifetime [2]. Increasing prevalence of breast cancer and its immense impact on the quality of life of affected individuals [3] has led to numerous efforts in its treatment, control, and prevention.
A relatively new understanding of the interactions between the immune system and tumors, has given rise to the development of a modern therapeutic approach to malignancies known as immunotherapy [4]. Cancer immunotherapy includes a range of manipulations during which a potent antitumor immune response is activated [5]. This can target both the innate and adaptive immunities, and can be achieved either by augmenting the antitumor response or the disrupting the regulatory mechanisms used by the immune system [6]. This can be elicited to various extents by distinct methods such as the delivery of adjuvants to the tumor milieu and immunization with tumor associated antigens (TAA) in isolated form or as whole cell preparations. For this purpose, various types of vaccines contain tumor cell lysates [7] and heated tumor cell lines have been used as rich sources of TAAs. For instance, dendritic cell-based immunotherapy of tumors has been shown to be a promising method to provoke the antitumor immune response, especially when incorporated with whole tumor antigens [8]. Because of a variety of identified and unidentified epitopes that could be recognized by CD4+ and CD8+ T cells, whole tumor antigens seem to be more effective compared with individual antigens, such as tumor-derived peptides or recombinant tumor proteins [9].
The 4T1 mammary carcinoma cell line is widely used in animal model breast cancer studies because of its strong tumorigenic and highly metastatic properties, and can be readily transplanted into animals where it can develop palpable tumors [3, 5, 10, 11].
According to a definition by the World Health Organization, probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [12]. Lactobacilli are one of the most frequently used probiotics for controlling intestinal microflora with the potential to suppress harmful bacteria with high safety [12]. Among several strains,
The present study aimed to investigate the immunotherapeutic efficiency of anew cancer vaccine using a combination of sublethally-heated-4T1-cell lysate as a source of TAAs and
Phytohemagglutinin (PHA), dioxan, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FCS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco/Life Technologies (Gaithersburg, MD, USA), and the enzyme-linked immunoassay (ELISA) kits were from Qiagen (Hilden, Germany).
The 4T1 cell line was provided by the Pasteur Institute of Iran and cultures were grown in a humidified atmosphere at 37°C under an atmosphere containing 5% CO2. The cells were maintained as monolayers in DMEM supplemented with 10% FBS. To prepare the vaccine, the cells were treated as follows. To induce the production of heat shock proteins (HSPs) in the tumor cells, the cells were treated with sublethal heat at 43°C for 30 min in a bain-marie, as described previously by Huang et al. [17]. The cells were then incubated for 12–48 h and a suspension of 1 x 106 cells/mL was prepared [18]. Subsequently, the cells underwent 4 freeze-thaw cycles by placing them in liquid nitrogen and alternately incubating at 37°C to produce the required cell lysate. As the final step, the lysate was centrifuged at 4000 rpm and the supernatant was passed through a 0.2 μm filter to be used as the vaccine [19].
Forty BALB/c mice, 6-8 weeks of age, were purchased from the Pasteur Institute of Iran and were acclimatized to their housing for one week. The animals were kept under standard housing conditions at a temperature of 22-24°C under a 12 h light-dark cycle and were allowed water and standard laboratory rodent food ad libitum during both the adaptation and experimental periods. All animal studies were performed in compliance with the regulations of the Ministry of Health, Iran and were approved by the Medical Ethics Committee of Urmia University for Animal Studies (approval No. 1432, October 16, 2015).
Subcutaneous challenge of the animals in all groups with 50 μL PBS suspension containing 1 X 104 viable tumor cells was considered as the onset of the experiment. The animals were observed for the development of palpable tumors and were equally distributed in 4 groups of 10 following confirmation of tumor development, including a control group, and 3 treatment groups each receiving immunization with a different vaccine.
Vaccination was started when all mice had developed a palpable tumor (day 12). The left flank of each mouse was chosen as the injection site and all vaccines were delivered subcutaneously. Each mouse in the control group was immunized with 2 injections 100 L of PBS at a one-week interval. Mice in the first treatment group received heated-4T1-cell lysate alone; the mice were immunized with 2 injections of the freeze-thaw lysate of 104 heated 4T1 cells in 100 μLPBS at a one-week interval. Mice in second treatment group were immunized with 2 injections of
At the end of the experimental period, 62 days from the onset challenge by viable tumor cells, the mice were humanely killed and following the aseptic isolation of their spleens for cell culture, the tumors were measured for size and weighed as 2 indicators of tumor growth. The size was measured using the formula of an ellipsoid (length x width x height x 0.5236) and then expressed as mm3. Each tumor was resected and weighed separately and the weights were recorded in grams.
In brief, splenocytes were aseptically isolated from mice and single-cell suspensions of splenocytes were prepared in DMEM supplemented with 10% FCS. The red blood cells were removed using a lysis buffer [21, 22]. Next, cell suspensions (2 x 106 cells/mL) were incubated in 24-well culture plates and pulsed with antigens derived from tumor cells by the freeze-thaw method (100 g/mL) and 25 μLPHA solution (1 mg/mL). Tumor antigen was prepared as described previously. The culture supernatants were collected after 72 h. Tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12, IL-17, and IL-13 levels were determined by ELISA according to the assay manufacturer’s instructions.
Statistical analyses were performed using a one-way analysis of variance (ANOVA) following by a Tukey test to determine differences between the different groups. Results are shown as mean ± standard deviation (SD).
Tumor size and weight studies revealed that immunization of the animals with a combination of
Comparison of lung weight (upper left), tumor necrosis factor (TNF)-α levels (upper right), tumor size (lower left), and tumor weight (lower right), and after immunization with a heated-4T1-cell lysate alone (hatched bar), the Figure 1
Studies of metastasis showed that treatment with the combination of heated-4T1-cell lysate and
The results of all ex vivo cytokine assays included in this study were in parallel with the results for tumor growth and metastasis, indicating that the combined vaccine had the strongest antitumor properties. The splenocytes of the mice from the combined treatment group significantly upregulated production of TNF-α. IL-2, IL-12, and IL-17, while they significantly downregulated production of IL-13, compared with splenocytes from mice in the control group. Immunization with the heated-4T1-cell lysate alone resulted in significant upregulation of IL-2, IL-17, and TNF-α, while it downregulated the production of IL-13. Its effect on IL-2 was comparable to that of the combination vaccine. With the exception of IL-2. immunization with the
Levels of IL-2 (upper left), IL-12 (upper right), IL-13 (lower left), and IL-17 (lower right) after immunization with a heated-4T1-cell lysate alone (hatched bar), the Figure 2
It has been previously shown that exposure of 4T1 cells to sublethal heat not only impairs their ability to proliferate and metastasize, but also results in increased expression of HSP72, a member of the HSP70 family [23]. Moderate hyperthermia (39°C to 43°C) induces a 500-1000-fold increase in the in vitro expression of a heterologous gene in the promoter of HSP70 [17]. This increase might be responsible, at least in part, for the increased HSP production following exposure to sublethal heat. The HSP70 family facilitates the recognition and uptake of TAAs by antigen presenting cells, thus enhancing the immunogenicity of tumors [24]. Therefore, the 4T1 cell line used in this study was subjected to sublethal heat to achieve the desired stimulus of the immune response by the tumor cell lysate, and control of the growth of the tumors. Changes in tumor weight and size were deemed as indices of tumor growth. As shown by
Stimulus of the innate immune system was not found, no specific immune response is seen initiated by such treatment [25]. Pattern recognition receptors such as toll-like receptors are functional compartments of the immune system that recognize specific microbial patterns and in response to this recognition, induce the expression of certain costimulatory molecules and cytokines that are involved in initiating potent antitumor Th1 and cytotoxic T cell responses [26]. Regrettably, tumor cells lack the ability to induce the expression of such costimulatory molecules, which necessitates the application of adjuvants with an antigen source to elicit potent antitumor responses. Adjuvants modulate the immune reaction to specific antigens [27]. A desirable adjuvant in cancer immunotherapy is one that can polarize the immune response towards Th1-mediated immunity [28]. Another concern regarding the application of adjuvants is the level of safety for use in humans [27]. Although various compounds such as Th1-promoting cytokines [29] and CpG DNA [30] have been shown to possess such adjuvant-like properties; finding adjuvants with low toxicity remains a challenge for researchers.
The antitumor efficacy of this strain of Lactobacilli has also been shown against gastric cancer with a similar mechanism proposed to induce apoptosis. The efficacy was attributed to inhibiting nuclear factor (NF)-kB and mTOR-mediated signaling (15). The direct treatment of a cervical cancer cell line with a cell-free extract of
In a rat model of mammary cancer induced by 2-amino- l-methyl-6-phenylimidazo[4,5-b]pyridine, the coadministration of
The present study shows that