Introduction
Breast cancer is the leading cause of cancer-related death among women worldwide [
1]. Triple-negative breast cancer (TNBC) is the most aggressive subtype, accounting for 10–20% of all breast cancer types. There are very only few therapeutic options [
2,
3] and a very poor prognosis still prevails, Thus, the improvement of the treatment of TNBC is an urgent need [
4].
Transplantable mouse mammary carcinoma 4T1 cells induce a deadly metastatic tumor that closely resembles human TNBC [
5,
6]. This malignancy is accompanied by splenomegaly with splenic granulocytopoiesis and leukemoid reaction and granulocytosis. Indeed, 4T1 tumor cells secrete growth factors that can stimulate extramedullary myelopoiesis, and increased levels of Gr-1 (Ly6G) and CD11b
high myeloid-derived suppressor cells (MDSCs) [
7]. These cells can inhibit the anti-tumor immunity, promoting tumor progression and metastatic niches [
8,
9].
Breast cancer cells have developed various mechanisms for immune evasion, characterized by a reduction of its immunogenicity by different means, such as an increased expression of inhibitory receptors and an enrichment of the tumor microenvironment with T regulatory cells (Tregs), MDSCs, tumor-associated M2 macrophages, and T cell exhausted (Tex), among other cell types that promote tumor expansion and metastasis, worsening the patient condition [
10]. Furthermore, tumor cells can induce their own blood supply from the preexisting vasculature in a process that mimics normal angiogenesis [
11]. Currently, tumor angiogenesis is considered a critical target to treat breast cancer, as it is highly correlated with metastasis [
12,
13]. Indeed, antiangiogenic therapy with bevacizumab, antibodies against vascular endothelial growth factor A (VEGF-A), or tyrosine kinase inhibitors such as sunitinib and pazopanib have been shown to effectively control metastatic breast cancer [
14].
T cell exhaustion is associated with a prolonged exposure to antigen and/or inflammatory signals. Tex overexpress PD-1, CTLA-4, LAG-3, and TIM-3 among other inhibition receptors, and they are characterized by a progressive loss of effector functions, including an impaired production of effector cytokines and reduced proliferation and cytotoxic activity [
10,
15]. Tex (PD-1
high) can be used as a predictor of the responsiveness to immunotherapy with anti-PD-1/PDL-1 antibodies [
16,
17]. However, the benefit of these immunotherapies is still controversial. While some studies show positive results, others are less encouraging [
18‐
20]. Other approaches, such as use of recombinant IL-2 and IFN-γ [
21,
22] or immunomodulatory molecules to activate the adaptive and innate immune responses have also been proposed to treat cancer [
23,
24].
On the other hand, angiogenesis plays a central role in both local tumor growth and metastasis in breast cancer. Thus, anti-angiogenesis therapies for cancer have raised interest. However, only a low to moderate response in the outcome of TNBC patients has been observed. [
25]
GK-1, an 18-aa immunomodulatory peptide originally identified in
Taenia crassiceps and shared by other phylogenetically close cestodes, induces protection against cysticercosis [
26]. It is possible that the adaptive immune response against cysticerci is mediated by MHC, since different motifs were theoretically predicted in the GK-1 sequence [
27]. On the other hand, GK-1 promotes the activation of NFκB in macrophages and dendritic cells through MyD88 [
28], which in turn promote an inflammatory environment that result in an anti-tumoral response.
Its anti-tumor properties were evaluated on murine experimental melanoma [
29] and breast cancer [
30]. In the latter, GK-1- immunotherapy reduced tumor growth, increased immune surveillance and dramatically reduced the number of macro-metastases. This study was designed to deepen on the anti-tumor and anti-metastatic properties of GK-1 in the murine model of mammary carcinoma 4T1.
Material and methods
Mice
Female BALB/c mice, aged 4–6 weeks, were obtained from the animal facilities at the Instituto de Investigaciones Biomédicas (IIB), Universidad Nacional Autónoma de México (UNAM). The animals were acclimated in the animal house and kept under controlled light (12 light /12 dark hours) and temperature (22–24 °C) conditions throughout the experiment. Food and water were allowed ad libitum.
GK-1
GK-1 peptide (GYYYPSDPNTFYAPPYSA) was purchased from USV LTD, Mumbai Maharashtra, India (Lot No. RD0001). Mice were administered with 100 μL of a 1-mg/mL solution of the peptide in isotonic saline solution (ISS).
4T1 cell line
The 4T1 cell linepurchased from the American Type Culture Collection (ATCC, Manassas, VA), were grown in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% serum fetal bovine (SFB, Gibco), and 1% penicillin and streptomycin (Gibco). Cells were maintained at 37 °C and 5% CO2 were detached with 0.05% trypsin/0.5 mM EDTA (Gibco).
4T1 implantation
4T1 cell viability was assessed by the trypan blue exclusion method [
31], and 1000 cells/50 μL of sterile ISS were implanted subcutaneously into the lower mammary fat pad of each mouse. Mice were checked three times a week, monitoring tumor growth and the general condition of each animal. Tumors sized were measured as previously reported [
30].
GK-1 treatment
GK-1 was first i.v. administered once the tumor was 1 mm × 1 mm of size (Day 0), and then administrated then every 7 days for 28 days. (Supplementary Fig. 1).
Lymphoid tissue and primary tumor processing
Spleen, primary tumors, and tumor-draining lymph nodes were collected on 7, 14, 21, and 28 days after first treatment (daft). The spleen and all primary tumors were weighed. For functional tests, the cells were extracted by macerating the tissues between two 70-µm membranes and, the suspension was passed through a 50-µm filter. Mononucleated cells were obtained from this suspension by density gradient with Ficoll-Plaque Plus®, following the manufacturer’s instructions and incubated with red blood lysis buffer for 5 min and washed. Cells were counted and used with > 90% of viability.
Histopathology
After mice were euthanized with sevoflurane, lungs and regional lymph nodes were fixed by intratracheal perfusion with 700 µL of 10% buffered Zamboni solution at day 21 and 28. The tissues were dissected and kept in the same fixative at room temperature (RT) for 24 h. Histological Sects. (4-μm) were obtained and stained with hematoxylin & eosin (H&E). The slides were analyzed and photographed under a Nikon® DS-Ri1 and Zeiss® Axiocam 506 color light microscope. Metastatic foci were counted blindly in lymph nodes and in the five pulmonary lobes (four right, one left) at coronal and horizontal section planes, respectively. In the lungs, metastatic foci were classified as pleural/subpleural or parenchymal foci.
Spleens from both mice groups were used to demonstrate the erythrocyte pseudo-peroxidase activity, to define the limits between splenic cords (red pulp) and the periarteriolar lymphoid sheath (the white pulp, T- and B-cell compartment).
Hematological evaluation
Blood (100 µL) was collected in EDTA-coated tubes on 7, 21, and 28 daft. Leukocyte numbers were obtained with an EXIGO automated veterinary hematology analyzer (Boule Medical A.B., Sweden). Differential leukocyte counts were blindly determined in methanol-fixed, Wright-stained peripheral blood smears under a light microscope (40X).
Angiogenesis
Prior to euthanasia, mice were injected in the lateral tail vein with 25 μL of 2% (w/v) Evans blue dye (EBD) solution per gram of body weight to stain the vasculature, and then processed as described elsewhere [
32]. Cryostat cutting was performed at − 15 °C to obtain 4-μm sections of primary tumors. Slide mounting and nucleus blue counterstain were performed with DAPI.
Image analysis
Images were analyzed as described elsewhere, with slight modifications [
33]. All images were adjusted by deblurring and deconvolution. Then, the software was trained to detect vessels using the Instellesis Trainable Segmentation program. Briefly, six random images per condition were chosen, and three classes were distinguished in those images: background, vascular region, and avascular region. A deep segmentation of 120 features was selected, and a suitable classification and identification of vascular regions were performed. Then, the images were uploaded, image analysis was performed, identifying each class. Vascular regions are expressed in pixels
2 and µm
2. Statistical analysis was performed with the software ZEN v.3.5 (Carl Zeiss) [
33].
Flow cytometry
The immunophenotype of spleen, tumor-draining lymph node, and primary tumor cells was determined by flow cytometry. T lymphocytes were counted and phenotypes were described every 7 days until 28 daft. All assays were performed using samples with > 95% of viability, as measured by trypan blue exclusion. Effector (CD3+ CD44+ CD62L−) and regulatory (CD3+ CD4+ CD25+ FoxP3+) T lymphocytes, monocytes (CD11b+, Ly6Chigh), and granulocytic MDSCs (CD11b+, Ly6G+) were identified (Supplementary Table 1).
The cells were fixed with 4% paraformaldehyde for 20 min at 4 °C and permeabilized with the BD CytoFix/CytoPerm kit for intracellular staining and with eBioscience™ Foxp3/Transcription Factor Fixation/Permeabilization for nuclear staining. The cells were read in a NxT Attune cytometer with two lasers (red and blue) and seven reading channels. Data were analyzed with the software FlowJo vX 10.0v.
Cytotoxicity assay
Spleen and tumor CD8+ T lymphocytes were purified by density gradient, and sorted with a FACS® ARIA cytometer, using a positive selection for CD3+ CD8+ (> 95% purity); cells were co-cultured with 4T1 cells stained with 1 µM calcein AM (Cat. No. C1430, Life Technologies, Carlsbad, CA) for 30 min. Tumor purified lymphocytes were cultured in a 1:1 ratio with tumor cells, while spleen lymphocytes were cultured in a 5:1 ratio. Cells were incubated for 4 h in RPMI-1640 plus 10% SFB at 37 °C and 5% CO2. Then, the cells were centrifuged, and the supernatant were analyzed by fluorimetry (excitation, 470 nm; emission, 535 nm). Fluorescence intensity was measured in 100 µL of supernatant for 5 min in a Synergy H1 Biotek® fluorimeter, and the mean fluorescence intensity (MFI) was recorded. Cytotoxicity rates were based on readings of maximum release controls (tumor cells stained and treated with 0.1% Triton X-100 for 45 min) and free release control (tumor cells only stained).
Intracellular cytokines
The functional activity of T lymphocytes was evaluated with a non-specific stimulus, using the Leukocyte Activation Cocktail with BD GolgiPlug™ (PMA/Ionomycin + Brefeldin) using 1 μL of activator for every 106 cells in 96-well plates. Cells were incubated at 37 °C under 5% CO2 for 6 h, then washed, fixed with BD Cytofix/Cytoperm™ for 45 min at 4 °C and read.
Primary tumors were immersed in liquid nitrogen and stored at − 80 °C. Proteins were extracted from 5 mg of tissue with 300 ml of lysis buffer containing protease inhibitors homogenized with an electric homogenizer (IKA, model T 10 Basic S1, fitted with dispersion tools S10N-5G, Cincinnati, OH) under constant agitation for 2 h at 4 °C. The homogenate was centrifuged for 20 min at 11,000 × g at 4 °C and protein concentration was determined by Lowry in the supernatant. Extracts were stored at –80 °C until used.
Angiogenic factors
Angiogenic factors were quantified in tissue protein extract samples (n = 5 per group), randomly selected from three independent experiments. A commercial multiplex kit (Milliplex MAP Mouse Angiogenesis Growth Factor Magnetic Bead Panel, Cat. No. MAGPMAG-24 K, Merck Millipore, Burlington, MA) was used, and the samples were read in the Luminex Magpix (Xponent Software) system.
Statistical analysis
Data were analyzed either by parametric or non-parametric tests, depending on the results of a Shapiro–Wilk normality test. Tumor progression, PD-1 expression, and cytotoxicity were compared by a two-tailed Mann Whitney U test. Area under the curve (AUC) values were compared with a Kruskal–Wallis’s test. Cytokine production was analyzed by MANOVA with Hotelling’s T-squared comparison. Differences were considered as statistically significant when P < 0.05* or < 0.01** or < 0.001***. All analyses were performed either in GraphPad Prism v.7.0 or R-Studio v.1.3.
Discussion
Breast cancer is the most prevalent and deadly malignancy among women worldwide; thus, finding novel therapeutic options and means of early detection is crucial to reduce the morbidity and mortality of the disease. The 4T1 cell line-induced breast cancer model is a highly metastatic and poorly immunogenic triple-negative tumors, useful for testing novel therapeutic approaches for TNBC [
38,
39], especially for patients with both primary tumors and metastases [
19,
20,
40]. We previously reported that GK-1 slowed 4T1 tumor growth and reduced the number of macrometastases [
30].
In this study, we demonstrate that GK-1 immunotherapy dramatically decreased lung and lymph node micrometastases. Both effects are particularly relevant, since metastasis is responsible for about 90% of human deaths from breast cancer, [
41] and lymph node infiltrating cells are markers of poor prognosis [
42]. Moreover, GK-1 increased the number of intratumoral CD8
+ T cells and its anti-4T1 cytotoxic and effector activity, evinced by the increased expression of IFNγ and granzyme in GK1-treated mice, all indicators of good prognosis in solid tumors [
43]. GK-1 also increased the production of IFNγ and IL-2 in CD4
+ T cells. These effects in the functional activity of T lymphocytes are accompanied by decreased levels of the inhibitory receptor PD-1 [
44], an effect also observed. Altogether, these findings indicate that GK-1 could effectively enhance antitumor T cell immunity by promoting a robust, functionally active T cell infiltration into the breast tumor mass. The changes in tumor microenvironment induced by GK-1 could trigger an immunological conversion from a cold to a hot tumor, by remodeling the immunosuppressive microenvironment of 4T1tumors [
45]. Cold tumors are characterized by a null or very low T cell infiltration, while hot tumors, with a better prognosis, have a high density of CD3
+ and CD8
+ T cells [
46]. Thus, the conversion to hot tumors favored by GK-1 may be associated with a higher sensibility to immune checkpoint inhibitor therapies (ICITs) [
47]. Furthermore, the effectiveness of ICITs such as anti-CTLA-4 and anti-PD-1/PD-L1 could represent a major improvement in life expectancy for patients with a variety of advanced cancer types [
48]. However, as single agents, ICITs are only effective in a small subset of breast cancer patients [
49]. Considering the results shown herein, it could be of interest to evaluate the therapeutic potential of GK-1 when co-administered with ICITs to improve the anti-tumor response. In this respect, promising results have been reported in a mouse model of melanoma [
29].
Another result that merit comments is the capacity of GK-1 to decrease granulocytosis, leukemoid reaction, splenic myelopoiesis and megakaryocytes, factors related to splenomegaly [
50,
51] and tumor progression. The decrease MDSCs—which induce pre-metastatic niches [
52]— and red pulp hyperplasia could explain the reduction in spleen size and weight, both indicators of a reduction in extramedullary hematopoiesis, which in turn could be associated with the reduction in pulmonary metastasis. This reduction could be further favored by the decreased formation of new blood vessels in GK-1-treated mice, in clear contrast with the highly branched blood vessels in control mice, which promote a premature, abnormal angiogenesis [
53].
Proangiogenic molecules, which could regulate endothelial cell proliferation and migration, were searched among 13 candidates in a multiplex assay. A significant decrease in the levels of angiopoietin 2, endothelin 1, SDF-1, and VEGF-C, all of them angiogenic factors involved in angiogenesis and vasculogenesis [
11], was observed in GK-1-treated mice. VEGF-C, a growth factor of the vascular endothelial growth factor family receptors, endothelin 1, and SDF-1 exhibit a high pro-angiogenic activity by promoting mitosis and inhibiting apoptosis on endothelial cells, resulting in an increased vascular permeability and a promotion of cell migration, favoring tumor angiogenesis. In addition, VEGF promotes the recruitment and proliferation of immunosuppressive cells like Treg cells and MDSCs [
54]. Thus, the reduction in the counts of these two suppressor cells in GK-1-treated mice could be partly mediated by the decrease in VEGF levels.
It is important to mention that angiopoietin 2 is one of the best characterized factors of an important family of proteins that promote a weakening of newly formed blood vessels branches. High concentrations of these factors have been reported to correlate with a poor patient prognosis [
55,
56]. Interestingly, IFN-γ secreted by CD8
+ CTLs has been linked to a suppression of tumor angiogenesis by reprogramming tumor-associated macrophages from an M2- to an M1-like type [
57]. Thus, the presence of IFN-γ
+ CD8
+ T lymphocytes in GK1-treated mice may be contributing to control tumor-promoted vascular remodeling.
Overall, these evidences indicate that GK-1 could decrease tumor growth by activating intra-tumoral T cell immunity, whilst inhibiting angiogenesis and extra-medullar myelopoiesis, exerting both immune and vascular functions, which currently are two major targets to improve the prognosis in TNBC cases. These properties make GK-1 a potential next-generation therapeutic agent against the highly mortal TNBC.
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