Chemo-Immunotherapy, a Combination Approach for the Treatment of HER2-Positive Breast Cancer in a Mouse Model

Chemo-Immunotherapy, a Combination Approach for the Treatment of HER2-Positive Breast Cancer in a Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chemo-Immunotherapy, a Combination Approach for the Treatment of HER2-Positive Breast Cancer in a Mouse Model Cenk Serhan, KILIÇ Kubilay Doğan, UYANIKGİL Yiğit, KARABOZ İsmail, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3943744/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The discovery of tumor-associated antigens has been a major milestone for cancer therapy as the current conventional therapies (radiotherapy, chemotherapy) were found to be insufficient in terms of cancer treatment. Human epidermal growth factor receptor (HER2) is a tumor-associated antigen, aberrantly found in various cancers, including breast cancer. Today, most of the treatment strategies are skewed towards combination therapies rather than monotherapies, they were found to be ineffective mostly because of patients' resistance mechanisms. A combination of more than one therapeutic approach (passive/active immunotherapy, conventional therapies) was found to be more effective in the clinic. Here, in this study, various vaccine combinations were investigated against the 4T1-HER2 xenograft model of Balb/c mice. Eight different groups of vaccine formulations were tested to find the best synergistic combination including combinations of BM-DC-based vaccine, Peptide-based vaccine, anti-PD-L1, Doxorubicin, and QS-21 adjuvant. Triple combination groups of immunotherapies exerted better activities in terms of tumor dimensions as Doxorubicin+BM-DC-HER2/Neu+QS-21+anti-PD-L1 and Doxorubicin+ HER2/Neu+QS-21+anti-PD-L1 groups significantly decreased tumor dimensions. Similar groups also demonstrated a better stimulation of lymphocytes and IFNγ cytokine in the flow cytometry study, inducing a HER2/Neu specific antibody response in ELISA studies. Doxorubicin+BM-DC-HER2/Neu+QS-21+anti-PD-L1 combination demonstrated significantly higher specific cytotoxicity in lactate dehydrogenase data, as well as the CD4+ and CD8+ responses in the immunohistochemical study. In conclusion, these data indicated that the Doxotubicin+BM-DC+HER2/Neu+QS-21+anti-PD-L1 vaccine combination synergistically generated a promising activity in terms of tumor dimension and immune response stimulation against HER2 overexpressing breast cancer model in mice. HER2+ breast cancer BM-DC immunotherapy anti-PD-L1 chemotherapy combination therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Immune responses to cancers are initiated upon the presence of aberrantly expressed proteins on cancer cells. This immune response is tightly regulated by a balance between immunostimulatory and immunosuppressive mechanisms, preventing unwanted inflammation and autoimmune disease progression [ 1 ]. The majority of cancer immunotherapies in development for breast cancer aim to modulate immune regulation, directing immune responses toward tumor-associated antigens (TAAs), especially to human epidermal growth factor receptor 2 (HER2), or to combine them with immune checkpoint blockade therapeutics together [ 1 – 2 ]. There are also some recent publications combining conventional radiotherapy, or chemotherapeutic drugs along with these immunotherapies to overcome patients' resistance mechanisms against drugs. The immune system plays an important role in the body's defense against various diseases, including cancer. In this study, we aim to investigate the impact of several key parameters including QS-21 adjuvant, Doxorubicin, and PD-L1 on the immune system and its responses. Understanding the complex interactions between these components is crucial for elucidating the mechanisms of immune response modulation and developing effective therapeutic strategies. QS-21 is a potent immunostimulant widely used in vaccine development and immunotherapies. It boosts the body's immune response by stimulating the activation of antigen-presenting cells such as dendritic cells. Furthermore, QS-21 has been shown to promote the production of proinflammatory cytokines and facilitate the formation of antigen-specific T cells. Its precise role in shaping the immune response will be the focus of our research. Doxorubicin is a widely used chemotherapy agent with known immunomodulatory properties. This anthracycline-based drug not only targets cancer cells but can also affect the immune system. By understanding how doxorubicin affects immune cells, we can gain valuable insights into the balance between its cytotoxic effects and its potential to stimulate or inhibit immune responses. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint protein that plays a crucial role in regulating immune responses. The interaction between PD-L1 on cancer cells and its receptor PD-1 on T cells may reduce the ability of the immune system to mount an effective anti-tumor response. Investigating the role of PD-L1 in the context of our study will help shed light on the mechanisms by which tumor cells escape immune surveillance. HER2 is well known for its overexpression in various types of cancers including breast cancers promoting uncontrolled proliferation and cell survival [ 3 ]. Overexpression of this TAA confers 25% of total breast cancer incidences and could induce resistance to, chemotherapy, radiotherapy, and hormone therapy [ 4 – 5 ]. These disadvantages in HER2-positive cancer treatment strategies diverted research studies toward targeted and combination immunotherapies [ 1 ]. Trastuzumab (Herceptin, Genentech) was the very first known immunotherapy approved by the US Food and Drug Administration (FDA), suppressing the activity of HER2 receptor via attachment of a monoclonal antibody against it [ 6 ]. Regarding active immunotherapeutic applications, Neu-vax from Galena Biopharma was the most promising cancer vaccine candidate against HER2-positive cancers that was composed of a short antigenic peptide and GM-CSF as an adjuvant. This approach unfortunately failed the phase studies and currently alternative combination approach of E75 peptide is being tried together with Trastuzumab in a phase III study demonstrating the novel strategies on combination immunotherapeutic approaches [ 7 – 8 ]. Dendritic-based vaccine studies, active immunotherapeutic, are regarded as powerful inducers of immunity without drawback problems in autoimmunity and tolerance [ 9 ]. Recent developments in technology further improved the understanding of the evasion mechanisms of cancer cells and the discovery of immune checkpoint inhibitors (PD-1/PD-L1, CTLA-4) creating novel opportunities for treatment [ 1 ]. Conventional therapies including chemotherapeutics, radiotherapies, and hormonal therapies remained insufficient in terms of treatment apart from being harmful to healthy tissues. Chemotherapeutic resistance is another obstacle to these conventional therapies that remains a major problem in treating cancer patients. Monotherapies remained insufficient and current studies are focusing on the development of combinations of therapies for cancer treatment [ 1 ]. In this study, a novel combination of therapies including BM-DC based vaccine supplemented with QS-21 adjuvant, doxorubicin, and anti-PD-L1 monoclonal antibody (mAb) was used as a combination in a HER2/Neu expressing breast cancer model of Balb/c mice. Different combinations of active/passive immunotherapy and chemotherapy were investigated to determine the best combination and synergistic effect of various formulations. 2. Materials and methods 2.1. Materials Here is the list of items organized by their purposes. Cell Culture and Reagents: DMEM/F12 (Gibco, USA), Fetal bovine serum (Gibco, USA), Penicillin-streptomycin (Biological Industries, Israel) Cytotoxicity Assays: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, USA), LDH Cytotoxicity Assay Kit (96 test) (Cayman, USA) Immunological Agents: Concanavalin A (Con A) (Biochrome, Germany), LPS (Sigma, USA), QS-21 (Desert King, San Diego, CA), GM-CSF (Sigma Aldrich, USA) Antibodies and Immunological Assays: Goat anti-mouse IgG, IgG1, and IgG2a peroxidase conjugate (Southern Biotech Assoc., Birmingham, AL, USA), BD Tritest CD4/CD8/CD3 (BD Biosciences, USA), in vivo Mab anti-mouse PD-L1 (B7-H1) antibody (Bioxcell, USA), Anti-mouse CD4 antibody (Santa Cruz, USA), Anti-mouse CD8-a antibody (Santa Cruz, USA), Anti-mouse CD61 antibody (BioLegend, USA), Anti-mouse CD40-FITC antibody (BD Pharmingen, USA), Anti-mouse CD80-PE antibody (BD Pharmingen, USA), Anti-mouse CD11c-FITC monoclonal antibody (EBioscience, USA) Other Materials: Ketamine (100 mg/ml, EgeVet, Turkey), Mouse HER2/ErbB2 (Acro Biosystems, USA), Matrigel Matrix (Corning, USA), Eumaline Mayer (Bio-Optica, Italy), Sensi Tek, HRP (Scy Tek Laboratories, USA), Super Block (Scy Tek Laboratories, USA), Hydrogen peroxide 30% (Merck, Germany) 2.2. Cell Line GFP tagged HER2 expressing mouse breast cancer cell line (4T1-HER2) was kindly provided by Prof. Michael Kershaw (Cancer Immunology Program, Peter MacCallum Cancer Centre, Victoria, Australia) and maintained in DMEM/F12 with penicillin/streptomycin, and 10% FBS [ 10 ]. 2.3. Verification of GFP tagged HER2 Expression on 4T1-HER2 Cells The HER2 expression was verified by using flow cytometry (BD Accuri C5) as the vector expressing HER2 contains a GFP tag and the green fluorescence level would indicate the presence of HER2. 2.4. Experimental Animals Male-female Balb/c mice (6–10 weeks old) weighing 20–26 g were purchased from Ege University Laboratory Animal Research Center (Bornova, İzmir, Türkiye). Mice were maintained in groups of 5 with a total 5 groups under standard conditions of temperature 22 ± 1 ◦C with regular 12 h light and 12 h dark cycles and had free access to standard laboratory food and water. The experimental protocol was approved by the Local Ethics Review Committee for Animal Experimentation of Ege University (Approval number is 2016/006). 2.5. Bone Marrow Derived Dendritic Cell (BM-DC) Generation DC-generation protocol was performed by using the modified protocol of Madaan et al [ 11 ]. 8–10 weeks old male Balb/c mice were euthanized by using Ketamine. Mice were rinsed by using 70% ethanol for disinfection. The Femur and tibia bones of the mice were dissected from the pelvic joint. Bones are then cleared from tissues, muscles, and fur by using sterile dissecting scissors and forceps. Both ends of the femurs and tibias were trimmed by sterile scissors and the marrow was flushed into a Petri by using RPMI 1640 medium (10% FBS) via a sterile insulin injector. This process was repeated several times and the monocytes were centrifuged at 250 g for 5 minutes. This process was repeated 3 times. After the last washing step, cells were counted and dispensed to Petri dishes at a concentration of 2 x 10 5 cells/ml in a BM-DC medium (5 ng/ml GM-CSF, RPMI 1640, 10% FBS). On the day 3 of culture, 10 ml BM-DC medium was added to the cells. Cells were collected and centrifuged for 5 minutes on the day 6. After centrifugation cells were seeded back to the petri dishes in a 10 ml BM-DC medium. On the day 8, 10 ml BM-DC medium was added to the cells. Cells were collected and counted to assess the differentiation efficiency by using a CD11c marker by flow cytometry (BD Accuri C5) on the day 10. 2.6. Verification of DC-Differentiation On the day 10 of the monocyte-DC differentiation protocol, cells were collected and counted. 5 x 10 5 cells/ml were washed by using PBS and anti-CD11c FITC (1:400) monoclonal antibody was added to the cells followed by incubation for 30 minutes at + 4 o C. After incubation cells were analyzed by BD Accuri C5 flow cytometry [ 11 ]. 2.7. Preparation of Samples for Scanning Electron Microscopy BM-DC’s were seeded onto 24 well plates at a density of 1x10 4 cells/ml and incubated for 24 h. Samples were washed 3 x with PBS and incubated with 2.5% gluteraldehyde for 4 h. Samples were dehydrated by being exposed to 25%, 50%, 75% and 100% ethanol concentrations. After incubation at 100% ethanol for 10 mins, samples were placed into desiccator. Samples were then coated with 200A gold particles and images were taken by using scanning electron microscope. 2.8. The Loading of Mouse HER2/Neu Antigen on BM-DCs On the day 10 of the BM-DC protocol, BM-DCs were incubated with 10 µg/ml mouse HER2/Neu overnight at + 37 o C. After incubation, loading success was assigned by measuring the change in the levels of anti-CD40-FITC (1:100) and anti-CD80-PE (1:100) expression. 2.9. Formation of Xenograft Tumor Model by Using 4T1-HER2 Cells 4T1-HER2 tumor cells were cultured in DMEM/F12 (10% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine). A final concentration of 5 x 10 6 cells/ml 4T1-HER2 tumor cells were injected subcutaneously (s.c.) into the flank of Balb/c mice day 0 of the experiment followed by mixing with matrigel in 1:1 dilution total 200 µl. Tumor growth was measured in mm by using a caliper and was recorded as mean diameter values (width x height x length) 2.10. Immunization Studies Mice were immunized with different vaccine formulations including control groups, as sum 7 groups. Group 1: Control Group 2: HER2/Neu (10 µg/ml) + QS-21 (10 µg/ml) in 100 ml saline solution. Group 3: HER2/Neu peptide (10 µg/ml) + QS-21 (10 µg/ml) + anti-PD-L1 mAb in 100 ml saline solution. Group 4: Doxorubicin only (2.5 mg/kg). Group 5: Doxorubicin (2.5 mg/kg) + anti-PD-L1 mAb Group 6: Doxorubicin + HER2/Neu + QS-21 vaccine + anti-PD-L1 mAb Group 7: Combination of HER2/Neu loaded BM-DC vaccine adjuvant with QS-21, anti-PD-L1, and doxorubicin HER2/Neu and BM-DC-based vaccinations were performed through intraperitoneal (i.p.) injection on the day 1 and day 8. Anti-PD-L1 mAb was i.p. injected on the day 4 and day 9. Doxorubicin was i.p. injected on the day 5 and day 11. The study was terminated, and mice were sacrificed by cervical dislocation on the day 14. Blood, tumor, and spleen samples were collected for performing splenocyte proliferation assay, measurement of immune response, co-culture experiment, HER2 specific antibody response, and immunohistochemical (IHC) staining studies. 2.11. Splenocytes Proliferation Assay Splenocyte proliferation assays were performed according to the modified method by Nalbantsoy et al [ 12 ]. Splenocytes were treated with HER2/Neu (1 µg/ml) incubated for 72h at 37°C. The stimulation index (SI) was calculated based on the following formula: SI = the absorbance value for mitogen-stimulated cultures/the absorbance value for non-stimulated cultures. Each experiment was performed in triplicate. 2.12. Determination of HER2/Neu Specific Antibody Response HER2/Neu specific antibodies (IgG, IgG1, IgG2a) were detected by ELISA according to the modified method by Nalbantsoy et al [ 23 ]. ELISA plates were coated with 100 ng/ml HER2/Neu overnight at + 37°C. Following the washing steps, 100 µl of 1:100 diluted serum samples were added to the wells and incubated for 1 hour at a 37°C incubator. 100 µl of horseradish peroxidase-conjugated antibody for IgG, IgG1, or IgG2a (diluted in 1:8000) were added to the wells followed by four times of washing and incubated 1 hour at 37°C incubator. After a washing step, 100 µl substrate solution (TMB-3,3',5,5'-Tetramethylbenzidine) was added and incubated at room temperature for 30 minutes. The reaction was terminated by adding 50 µl/well-stop solution (H2SO4). The optical density (OD) was measured at 492 nm (Versamax Microplate Reader, USA). Data were calculated as the mean OD value of the samples minus the mean OD value of the control. Results were expressed as log 2 titers. 2.13. Determination of Anti-PD-L1 Specific Antibody Response Anti-PD-L1 specific antibodies (IgG) were detected by ELISA according to the modified method by Nalbantsoy et al [ 12 ]. ELISA plates were coated with 10 ng/ml anti-PD-L1 overnight at + 4°C. After the washing steps, 100 µl of 1:100 diluted serum samples were added to the wells and incubated for 1 hour at a 37°C incubator. The remaining protocol is followed in a similar way to the previous study. 2.14. Measurement of CD8, CD3 and IFN γ Levels Splenocytes of sacrificed mice followed by immunizations were isolated. Cells from each mouse belonging to the same group were collected in a pool. 1 x 10 6 cells/ml from each group were treated with HER2/Neu (1 µg/ml) incubated for 72 h at 37°C. Cells were collected and centrifuged at 1200 rpm for 5 minutes and 50 ml (1:1000) PE-labeled anti-mouse CD3 antibody was added. Samples were incubated for 30 minutes at + 4 o C at dark. After incubation, cells were centrifuged and washed with 250 ml PBS containing 3% FBS. Anti-mouse CD8-PerCP antibody was added to cells and further incubated for 30 minutes at + 4 o C in the dark. Cells were then washed, and 100 ml Fixation / Permeabilization solution was added to cells and incubated for 30 minutes at + 4 o C at dark. After incubation cells were washed and anti-mouse IFNγ-FITC antibody was added to cells followed by incubation for 30 minutes at + 4 o C in the dark. The total volume of 250 ml perm wash (1:10) was added to the cells and the cells were washed twice. After the washing steps, cells were analyzed by flow cytometry (BD Accuri C5). 2.15. Lactate dehydrogenase (LDH) Assay Specific cytotoxicity of splenocytes was determined by participating in an LDH assay by following the manufacturer's protocol (Cayman). 4T1-HER2 cells and splenocytes belonging to different immunization groups were incubated at a 1:40 target: effector ratio. For this purpose, 2 x 10 5 cells/ml 4T1-HER2 cells were seeded on 96-well plates and incubated for 24 h at + 37 o C, 5% CO2 incubator. 24 h post incubation 8 x 10 6 cells/ml splenocytes were seeded on 4T1-HER2 cells and incubated for 4 h at 37 o C. After incubation, the 96 well plate was centrifuged at 1200 rpm and 100 ml supernatant was taken from each well followed by placing into a new 96 well plate. 100 ml LDH substrate was added to the wells and incubated further 30 minutes at room temperature. Then, samples were read at 490 nm by using an ELISA plate reader. The cytotoxicity was calculated according to the kit formula mentioned below. 2.16. Immunohistochemistry (IHC) Staining On the day 14 of the study, mice tumor cells were excised and placed into %4 paraffin. Then, tumors were embedded into paraffin and waited for solidification. After that, 0.5 mm tumor sections were taken by using a microtome. Sections were then incubated at 37oC for 1 h. Later, hemotoxylin-eosin staining was performed. Immunohistochemical staining was performed after incubating tumor sections at 37 o C for 1 h. Following incubation samples were placed overnight into Xylol. After this process, samples were placed into serial alcohol dilutions 100% (4 minutes), 95% (2 minutes), 90% (2 minutes), and 80% (2 minutes). Samples were exposed to a microwave set at 90 o C in sodium citrate solution for 30 minutes. Then, samples were incubated for 15 minutes at room temperature and then placed into the ice-filled water bath for a further 15 minutes. Samples were then incubated in distilled water for 5 minutes, in PBS, and 5% H 2 O 2 for 10 minutes respectively. Tumor sections were then washed 3 times with PBS and a blocking solution was applied to them followed by incubation for 30 minutes. After incubation antibodies prepared (1:100 dilution) (anti-mouse CD8, anti-mouse CD4, and anti-mouse CD61) were added to the samples and incubated overnight at + 4oC. After overnight incubation, samples were washed 3 times with PBS, and a biotin-conjugated secondary antibody was added to the samples followed by incubation for 30 minutes. After incubation, samples were washed 3 times with PBS and 1–3 ml DAB was added on top of the sections and incubated for 2 minutes. Samples were then placed into distilled water containing bath, Meyers Hemotoxylin for 50 secs, tap water, and distilled water respectively. Samples were then placed into a series of ascending alcohol concentrations 80%, 90%, 95%, and 100% respectively. Samples were then incubated at room temperature for 15 minutes and placed into Xylol for another 15 minutes. After the Xylol step, samples were dried at room temperature and closed for investigation under microscopy. 2.17. Statistics The data were measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey’s test for multiple comparisons between each group by using GraphPad Prism 5.0 and SPSS for Windows. P values of less than 0.05*, 0.01** and 0.001*** were stated as statistically significant. 3. Results 3.1. Verification of GFP tagged HER2 Expression on 4T1-HER2 Cells The HER2 expression was analyzed by using BD accuri C5 flow cytometry. Investigation of the fluorescence level clearly indicated the presence of GFP-tagged HER2 (Fig. 1). 3.2. BM-DC differentiation Femur and tibia bones of 8–10 week-old Balb/c mice were excised for generating monocyte-derived DC and the process was evaluated by investigating the levels of anti-mouse CD11c via flow cytometry. The obtained results that the differentiation has been successful and the percentage differentiation is around 80–85% (Fig. 2.I.A-B). 3.3. Mouse HER2/Neu Antigen Loading on BM-DCs Mouse HER2/Neu was loaded onto BM-DCs by co-incubation overnight at 37oC and the antigen uptake was verified by observing the changes in the co-stimulatory molecules on the DCs. For this purpose, cells were stained with anti-mouse CD40-FITC and anti-mouse CD80-PE. The percentage of co-stimulatory molecules differentiations, double fold increased upon antigen loading demonstrating the success of antigen loading (Fig. 2.II - III). The maturation status of the dendritic cells was further characterized by taking SEM pictures as shown in Fig. 2.III. Dendritic cells with antigens were shown to mature, enlarge, and underwent a morphological change with induced dendrites around themselves (Fig. 2.III). 3.4. Formation of Xenograft Tumor Model by Using 4T1-HER2 Cells To assess the vaccine efficiency of the different formulations, 5 x 10 6 4T1-HER2 cells/mice together with matrigel in a 1:1 (100 µl cell suspansion:100 µl Matrigel) concentration ratio was injected subcutaneously to develop a xenograft model of breast cancer in Balb/c mice on day 0. Tumors appeared successfully on the day 3 of the study, and all mice successfully developed palpable tumors. 3.5. Immunization Studies On the day 1, the first injection of different vaccine formulations was injected composed of (HER2/Neu + QS-21, HER2/Neu + QS-21 + anti-PD-L1, Doxorubicin, Doxorubicin + anti-PD-L1, Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1, Doxorubicin + DC + HER2/Neu + QS-21 + anti-PD-L1). Later on the day 4, 100 µg/ml anti-PD-L1 was injected into the combination immunotherapy groups to investigate the synergism of various combination groups. The second dose of injection participated on the day 8 and the second dose of anti-PD-L1 was injected on the day 9. Doxorubicin was injected on the day 5 and day 11 for various combination groups. The study was terminated on the day 14 and tumor dimensions were measured by caliper. The obtained data clearly demonstrated that, anti-PD-L1 addition to formulas further supported the vaccine efficacy. (p < 0.001) (Fig. 3). QS-21 adjuvant also enhanced the vaccination formulas in terms of tumor dimensions. Doxorubicin alone group induced a great reduction in tumor dimensions, but its activity further increased synergistically with vaccines, especially BM-DC based vaccine together with anti-PD-L1 formulation reaching the lowest tumor dimension when compared to control and other formulation groups (p < 0.0001) (Fig. 3). 3.6. Tumor Specific Cytotoxicity of Different Vaccine Combinations To assess the tumor-specific cytotoxicity of different vaccine combinations, an LDH assay was performed in a 1:40 target/effector ratio. Data obtained demonstrated that combination groups have a superior effect on tumor-specific cytotoxicity results when compared to monotherapies. The addition of anti-PD-L1 to combinations boosted the specific cytotoxicity demonstrating a synergistic effect with formulations. Doxorubicin combinations also demonstrated a further supportive role in terms of cytotoxicity, thereby synergistic effect on cytotoxicity with other constituents of combinations. The doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 group demonstrated the highest specific cytotoxicity significantly when compared to the control and other groups (p < 0.0001) (Fig. 4). 3.7. HER2 Specific Antibody Response HER2-specific IgG, IgG1, and IgG2a antibody levels in sera were measured by using ELISA. The data suggests that HER2-specific antibody response was developed upon vaccination. All different vaccination groups demonstrated IgG, IgG1, and IgG2a antibody response against HER2 as shown in Fig. 5. IgG1 is an indicator of Th2 whereas IgG2a antibody is a Th1 immune response. All different vaccine combinations demonstrated a superior effect on Th2 type immune response (IgG1) when compared to Th1 type immune response (IgG2a) (Fig. 5B-C). Data demonstrated that combination immunotherapeutic formulations exhibited significant antibody response when compared to control and monotherapies (p < 0.001). The doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 combination group had significantly increased the anti-HER2 titer when compared to control and other vaccination groups (p < 0.0001) (see Fig. 5B). 3.8. Anti-PD-L1 Specific Antibody Response Anti-PD-L1 specific antibody level in sera was measured by using ELISA. Anti-PD-L1 sera were investigated in both vaccinated and control groups, however, no significant differences were detected between the vaccination groups and control group (Fig. 6). 3.9. Splenocytes Proliferation Assay Splenocyte proliferation upon antigen stimulation is an accepted method for cell-mediated immunity. HER2/Neu antigen-stimulated splenocyte proliferation is shown in Fig. 7. SI of a triple combination vaccine group (Doxorubicin + DC + HER2/Neu + QS21 + anti-PD-L1) demonstrated nearly a 1.5-fold increase when compared to control and tumor control groups (p < 0.05). The same group also exhibited greater SIs when compared to peptide-based vaccines and other combination groups, however, it did not reveal any significant differences with Doxorubicin + HER2/Neu + QS21 + anti-PD-L1 combination (Fig. 7). 3.10. Measurement of CD8, CD3 and IFN γ Levels The rate of immune response was measured on splenocytes belonging to different groups of vaccination. Splenocytes were incubated with HER2/Neu antigen for 72 hours and the levels of total T-cells (CD3), CD-8 + T-cells, and IFNγ cytokine were measured by flow cytometry. The data revealed that CD8-positive T-cell populations were significantly increased in combination groups composed of doxorubicin, anti-PD-L1, and peptide (HER2/Neu) based, or BM-DC based vaccines when compared to all other groups tested, demonstrating the synergistic effect of these combinations in terms of immune system stimulation (p < 0.0001) (Fig. 8A). IFNγ secreting CD8 T-cell population seemed to be the highest in the Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 combination followed by Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1 groups (p < 0.0001) (Fig. 8B). The doxorubicin-only group demonstrated an increase in IFN-secreting CD8 + T-cell response however the activity significantly increased upon the addition of anti-PD-L1 mAb to the group (p < 0.0001). The addition of the active immunity stimulating vaccines to the doxorubicin and anti-PD-L1 further increased the response demonstrating the best activity in terms of IFNγ secreting CD8 + T-cells (Fig. 8B). IFNγ secreting total T-cell population seemed to be significantly increased in the Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 and Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1 immunotherapy groups when compared to all other vaccination and control groups (p < 0.0001). Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 combination demonstrated the best activity in terms of increasing CD8 + T-cell population, IFNγ secretion, and the total T-cell population when compared to other groups (Fig. 8). 3.11. IHC Staining The population of CD4 + and CD8 + T-cells were investigated around the tumor niche along with CD61 + cells indicating tumorigenic potential of the 4T1-HER2 tumors. The data suggested that Both CD4 + and CD8 + cells successfully penetrated into the tumor cells, demonstrating the success of the combination vaccine groups ofDoxorubicin + DC + HER2/Neu + QS-21 and Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1. CD8 + cell population did not seem to increase significantly in HER2/Neu + QS-21, Doxorubicin only, HER2/Neu + QS-21 + anti-PD-L1 groups when compared to the control group, however, only HER2/Neu + QS-21 + anti-PD-L1group demonstrated slight up-regulation in the levels of CD4 positive T-cell populations (Fig. 9). CD61 level of the control groups seemed to have the highest level of tumorigenic potential although no significant differences were detected when compared to the other groups. The combination group composed of BM-DC + HER2/Neu + QS-21 + anti-PD-L1 showed lower CD61 + marker expression when compared to the other groups (Fig. 9). 4. Discussion Tumor-associated antigen discoveries initiated new strategies in combating against cancers. These antigens highlighted a new era in terms of personalized therapies, especially immunotherapies [ 1 ]. Cancer immunotherapy has now been regarded as important as radiotherapies, chemotherapies, hormonal therapies, and surgical therapies [ 13 ]. Current immunotherapy strategies were divided into two subgroups which are active and passive immunotherapies [ 14 ]. Active immunotherapies aim to modulate immune regulation, and direct immune responses towards TAA whereas passive immunotherapies mainly focus on immune checkpoint blockade reactions [ 1 , 14 ]. HER2 overexpression is characterized by various cancer types including breast cancer [ 4 , 5 ]. Its overexpression promotes uncontrolled proliferation and cell survival, however, treating these HER2 overexpressing cancers remained a huge problem even though its TAA is well characterized. Patients with this cancer type are prone to gain resistance to chemotherapeutics making treatment procedures ineffective [ 1 ]. Recent studies on cancer therapies focused on combination immunotherapies not only to overcome resistance mechanisms but also because monotherapies become insufficient in terms of cancer treatment [ 15 – 16 ]. Here in this study, various combinations of immunotherapeutic and conventional chemotherapeutic approaches were investigated to determine the synergistic activity of the combinations as well as discover a novel highly efficient therapeutic combination against HER2 overexpressing breast cancer xenograft model of Balb/c mice. The outcome of the study was aimed to boost the immune response via cancer vaccine, block the immune evasion via monoclonal antibody, and further empower the combination with a chemotherapeutic agent. For this purpose, BM-DC were isolated from the Balb/c mice and the differentiation was found to be as high as 80% which was measured by the CD11c level of cells apart from imaging through the SEM, correlating with the literature [ 11 , 17 – 18 ]. Verification of BM-DC differentiation was followed by antigen (mouse HER2/Neu) loading experiments that were participated successfully via measuring the 1.5-2 fold increase in CD40/CD80 co-stimulatory molecule levels [ 19 – 20 ]. On the last day of the study, tumor dimension measurements were performed, and the combination immunotherapy groups demonstrated a 2–5 fold decrease in tumor dimensions, having the most significant reduction in Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 combination group followed by Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1 combination (p < 0.0001). This demonstrated the success of BM-DC-based vaccines over peptide-based vaccines apart from the synergism of anti-PD-L1 mAb and doxorubicin in combinations. The decrease in tumor dimensions was significant when compared to the other studies in the literature although a lower concentration of anti-PD-L1 and doxorubicin was used in the study [ 21 – 24 ]. Specific cell cytotoxicity of combination immunotherapy groups was investigated by performing an LDH assay. The tumor-specific cytotoxicity was an important aspect in terms of CD8 + T-cells against tumor-specific immune response [ 25 ]. The specific cytotoxicity of Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 vaccine combination demonstrated significantly higher cytotoxicity when compared to other vaccine combinations and control groups (p < 0.0001). Although lower effector-target concentration with lower incubation time (4 h) was tested in the study, promising results were obtained in terms of tumor-specific cytotoxicity when compared to the literature [ 20 , 21 , 23 ]. The power of the immune response formed was also assessed by measuring the levels of HER2-specific IgG, IgG1, and IgG2a [ 26 ]. Data demonstrated that the immune response formed was IgG1 subclass indicating a Th2 bias immune response relatively similar to the study of Cruz et al [ 27 ]. Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 combination group demonstrated a significant increase (2.5 fold) in HER2-specific antibody titers compared to other groups including control groups, except Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1 combination. Anti-PD-L1 specific antibody titer was also investigated in the study and no significant differences between groups were indicated. The lymphocyte stimulation of various vaccine groups was also investigated via flow cytometry. The Doxorubicin + BM-xDC + HER2/Neu + QS-21 + anti-PD-L1 and Doxorubicin + HER2/Neu + QS-21 + anti-PD-L1 combination groups demonstrated significantly higher total T-cell, CD8 + T-cell, and IFNγ population when compared to other groups (p < 0.0001). This data clearly demonstrates the success of lymphocyte priming and also a correlation of the tumor dimension, and LDH cytotoxicity studies together. It is well known that DC-based vaccines were better in terms of polarization of CD8 + T-cells and IFNγ secretion when compared to the peptide-based vaccines demonstrating a similar trend with the study [ 20 ]. The data was further underpinned by the immunohistochemical study, and the levels of CD8+, CD4+, and CD61 + cells were investigated around the tumor niche. It was observed that CD4 + and CD8 + T-cells penetrated the tumor cells further supporting the efficacy of the data obtained. The same trend was also observed in the previous studies correlating with the study [ 28 – 30 ]. Doxorubicin + BM-DC + HER2/Neu + QS-21 + anti-PD-L1 was found to be the most efficient in terms of IHC studies with high penetration of CD4 + and CD8 + cells with lower avb3 integrin marker, tumorigenic potential indicator. In conclusion, the study outcomes demonstrated that BM-DC-based vaccinations are more potent than peptide-based vaccines. Combination immunotherapies might be better in terms of cancer therapies especially when monoclonal antibodies are combined with active immunotherapies and chemotherapeutics. The novel immunotherapeutic combination approach used in this study boosted the immune response against target tumor cells specifically and also prevented the immune escape of tumor cells successfully. Declarations Conflict of Interest: The authors declare that they have no conflict of interest. Author Contribution KDK wrote the main manuscript, YU prepared the figures, KDK and YU did the histopathological experiments and evaluation, AN, CSÖ and İK did vaccine preparation experiments. All authors reviewed the manuscript. Acknowledgement The authors would like to thank Ege University Scientific Research Projects Coordination (16-FEN-011) who funded the project. We also thank Dr. Tuğba TÜRK for her support in the SEM evaluation of DC cells. 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Antiviral Res 77:50–55 Müller P, Kreuzaler M, Khan T, Thommen DS, Martin K, Glatz K, Zippelius A (2015) Trastuzumab emtansine (T-DM1) renders HER2 + breast cancer highly susceptible to CTLA-4/PD-1 blockade. Sci Transl Med ; 7(315) Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3943744","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272133789,"identity":"8a9aa0e0-c48f-4025-a1d1-911c252da019","order_by":0,"name":"Cenk Serhan","email":"","orcid":"","institution":"Department of Bioengineering, Faculty of Engineering, Ege University, İzmir, Türkiye","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cenk","middleName":"","lastName":"Serhan","suffix":""},{"id":272133790,"identity":"d2723605-853b-44f4-9624-6e7d18c58607","order_by":1,"name":"KILIÇ Kubilay Doğan","email":"","orcid":"","institution":"Department of Histology and Embryology, Faculty of Medicine, Ege University, İzmir, Türkiye","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"KILIÇ","middleName":"Kubilay","lastName":"Doğan","suffix":""},{"id":272133791,"identity":"4042ea89-ff07-4b2f-9fcd-d593ac46c6e0","order_by":2,"name":"UYANIKGİL Yiğit","email":"","orcid":"","institution":"Department of Histology and Embryology, Faculty of Medicine, Ege University, İzmir, Türkiye","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"UYANIKGİL","middleName":"","lastName":"Yiğit","suffix":""},{"id":272133792,"identity":"eed58d80-259c-45aa-ba4f-190f0476de15","order_by":3,"name":"KARABOZ İsmail","email":"","orcid":"","institution":"Department of Bioengineering, Faculty of Engineering, Ege University, İzmir, Türkiye","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"KARABOZ","middleName":"","lastName":"İsmail","suffix":""},{"id":272133793,"identity":"2ba611a2-e6a6-43ac-bb05-ec76c12625f9","order_by":4,"name":"NALBANTSOY Ayşe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYHACxsMwxgMgwcNHjB6QFgkgZjYAaWEjRQsbiGAgqIV/dvODwwU1d+rk23vMKr/m2MmwMTA/fHQDjxaJO8cMDs849kzC4MwZs9uy25KBDmMzNs7BZ82NBIPDPGyHJQwkcsxuS25jBmrhYZPGp0X+RvqHwzz/DkvIz8gxK5bcVk9Yi8GNHIPDvG2HJRhu5Jgxftx2mLAWwxs5BYdn9h2W3HDmWLE047bjPGzMBPwidyN94+OCb4f55dubN378ua3anp+9+eFjvN5HAA4DZh4QzUycchBgf8D4g3jVo2AUjIJRMIIAAIwMSefWk17BAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Bioengineering, Faculty of Engineering, Ege University, İzmir, Türkiye","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"NALBANTSOY","middleName":"","lastName":"Ayşe","suffix":""}],"badges":[],"createdAt":"2024-02-09 17:45:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3943744/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3943744/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51122701,"identity":"48d08c4c-2bcf-4bc4-b31e-af62a481428e","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24364,"visible":true,"origin":"","legend":"\u003cp\u003eGFP tagged HER2 expression vector was used for verifying HER2 expression on 4T1-HER2 cells via detecting GFP fluorescence on flow cytometry (BD Accuri C5).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/9048a26b87cf93cfac0419b8.png"},{"id":51122702,"identity":"5a0bafc8-2460-4bb7-a29d-5b5a932ebb29","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eCD11c levels of the BM-DC analyzed by flow cytometry (BD Accuri C5) after application of culturing protocol for 10 days. (A) CD11c level of untreated BM-DCs that were used as control. (B) CD11c level of treated BM-DCs.\u003cstrong\u003eII.\u003c/strong\u003e SEM pictures were taken before and after antigen loading experiments in order to further characterize the HER2/Neu antigen uptake of BM-DCs. (A-B) Untreated BM-DC’s, (C-D) 10 mg/ml HER2/Neu loaded BM-DC’s. \u003cstrong\u003eIII.\u003c/strong\u003eThe change in the levels of CD40 and CD80 co-stimulatory receptors upon HER2/Neu loading on BM-DCs analyzed by flow cytometry (BD Accuri C5). (A) Expression of CD40 on BM-DCs that are not treated with 10mg HER2/Neu. (B) Expression of CD40 on BM-DCs that are treated with 10mg HER2/Neu. (C) Expression of CD80 on BM-DCs that are not treated with 10mg HER2/Neu. (D) Expression of CD80 on BM-DCs that are treated with 10mg HER2/Neu.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/dfda685910fd30d269c74d1d.png"},{"id":51122705,"identity":"dbacc269-6ea2-45e1-9955-8d3819ede81c","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95307,"visible":true,"origin":"","legend":"\u003cp\u003eThe final concentration of 1.5 x 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003e4T1-HER2 tumor cells were injected subcutaneously (s.c.) into the flank of Balb/c mice at day 0 of the experiment followed by mixing with matrigel in 1:1 dilution.Tumor dimensions were measured on day 14 by measuring width, height and length of the tumors formed\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe data were measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey’s test for multiple comparisons between each groups\u0026nbsp; by using GraphPad Prism 5.0 and SPSS for Windows (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e), n=5.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/f8d0f9d324d51b2a7fc322bb.png"},{"id":51122703,"identity":"8243f37c-54ac-4c51-a076-ea4fe6da0a9e","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93864,"visible":true,"origin":"","legend":"\u003cp\u003eSplenocytes belonging to different vaccination groups were tested for cytotoxicity by performing a LDH assay. For this purpose, splenocytes were co-cultured with overnight cultured 4T1-HER2 cells in 1:40 effector/target ratio. Cells were then tested for specific cytotoxicity after 4 hours of co-culture. The data were measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey’s test for multiple comparisons between each groups \u0026nbsp;by using GraphPad Prism 5.0 and SPSS for Windows (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e), n=5.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/e2bff5c9b7e66a4fa83d0c32.png"},{"id":51122709,"identity":"95d4a3ab-e02c-4e2a-b01a-ca7cbbf0e9fe","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":277963,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of vaccination of different groups on HER2-specific IgG, IgG1 and IgG2a antibodies. Groups of five Balb/c mice were immunized intraperitoneally with four different formulations\u0026nbsp; on 1\u003csup\u003est\u003c/sup\u003e\u0026nbsp; and 8\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;days. Sera were collected 1 week after the last immunization. HER2-specific IgG, IgG1 and IgG2a antibodies in the sera were measured by an indirect ELISA as described in the text.\u0026nbsp;(A) HER2 specific IgG titers between sera of different vaccination groups. (B) HER2 specific IgG1 titers between sera of different vaccination groups. (C) HER2 specific IgG2a titers between sera of different vaccination groups. The data was measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey’s test for multiple comparisons between each groups\u0026nbsp; by using GraphPad Prism 5.0 and SPSS for Windows (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e), n=5.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/811b5f495b0124c09ec8b4bb.png"},{"id":51122706,"identity":"e1104761-3b72-41c1-9936-c1c4ffb84208","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78891,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of vaccination of different groups on anti-PD-L1 mAb-specific IgG antibodies. Groups of five Balb/c mice were immunized ip with four different formulations\u0026nbsp; on 1\u003csup\u003est\u003c/sup\u003e\u0026nbsp; and 8\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;days. Sera was collected 1 week after the last immunization. Anti-PD-L1 mAb-specific IgG antibodies in the sera were measured by an indirect ELISA as described in the text.\u0026nbsp;The data were measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey’s test for multiple comparisons between each group\u0026nbsp; by using GraphPad Prism 5.0 and SPSS for Windows (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e), n=5.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/5fd78616a5f6eacc47b348de.png"},{"id":51122707,"identity":"08b38e44-2aaa-4fde-a450-9389f7eff542","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":91101,"visible":true,"origin":"","legend":"\u003cp\u003eSplenocytes belong to different vaccination groups were tested for proliferation by stimulating cells via 1 mg/ml HER2/neu. Stimulation indexes of HER2/Neu stimulated splenocyte from mice immunized with various vaccine combinations were calculated by the formula described in the text. The data was measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey’s test for multiple comparisons between each group \u0026nbsp;by using GraphPad Prism 5.0 and SPSS for Windows (*\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e), n=5.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/476c5238d95880ff1bd7034f.png"},{"id":51122711,"identity":"4a38bed8-a114-4796-9db6-d19e76ce9b58","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":223582,"visible":true,"origin":"","legend":"\u003cp\u003eThe rate of CD3, CD8 and IFNg response between different vaccinations groups were analyzed by flow cytometry (BD Accuri C5). Splenocytes belonging to different vaccination groups were stimulated by 1 mg/ml HER2 for 72 hours and the levels of CD3, CD8 and IFNg were analyzed by pursuing flow cytometry. (A) The percentage of CD8 positive T cells within the total T-cell population. \u0026nbsp;(B) The percentage of IFNg secreting CD8 positive T cells. The percentage of IFNg secreting total T cell population. The data was measured with mean standard errors and the statistical significance of differences was\u003cstrong\u003e \u003c/strong\u003eexamined by using\u003cstrong\u003e \u003c/strong\u003eOne-way ANOVA, and Tukey’s test for multiple comparisons between each group by using GraphPad Prism 5.0 and SPSS for Windows (****\u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e), n=5.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/6e6282f414adb55c2df8747e.png"},{"id":51122708,"identity":"c5b5530e-3cad-4e5a-b579-90da5165654c","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":502625,"visible":true,"origin":"","legend":"\u003cp\u003eGroups of five Balb/c mice were immunized intraperitoneally with 8 different formulations. Tumors were collected 1 week after the last immunization. Immunohistochemical profiles of tumors excised from five different vaccination groups, detecting the levels of CD4+, CD8+ and CD61+ cells together with Hemotoxylin-Eosin staining.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/4211782b4c3e8b71627afa78.png"},{"id":51123527,"identity":"ba7b443f-f26c-4756-9724-d83cc335c959","added_by":"auto","created_at":"2024-02-14 14:40:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2005178,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/2ed36579-d421-4c16-9fb0-5fd5d2708897.pdf"},{"id":51122704,"identity":"a47ac1cc-3679-4c3b-8252-6da0c7590e00","added_by":"auto","created_at":"2024-02-14 14:23:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":88550,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3943744/v1/e2c22b83a51b5c5b4e2d7873.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chemo-Immunotherapy, a Combination Approach for the Treatment of HER2-Positive Breast Cancer in a Mouse Model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eImmune responses to cancers are initiated upon the presence of aberrantly expressed proteins on cancer cells. This immune response is tightly regulated by a balance between immunostimulatory and immunosuppressive mechanisms, preventing unwanted inflammation and autoimmune disease progression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The majority of cancer immunotherapies in development for breast cancer aim to modulate immune regulation, directing immune responses toward tumor-associated antigens (TAAs), especially to human epidermal growth factor receptor 2 (HER2), or to combine them with immune checkpoint blockade therapeutics together [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. There are also some recent publications combining conventional radiotherapy, or chemotherapeutic drugs along with these immunotherapies to overcome patients' resistance mechanisms against drugs.\u003c/p\u003e \u003cp\u003eThe immune system plays an important role in the body's defense against various diseases, including cancer. In this study, we aim to investigate the impact of several key parameters including QS-21 adjuvant, Doxorubicin, and PD-L1 on the immune system and its responses. Understanding the complex interactions between these components is crucial for elucidating the mechanisms of immune response modulation and developing effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eQS-21 is a potent immunostimulant widely used in vaccine development and immunotherapies. It boosts the body's immune response by stimulating the activation of antigen-presenting cells such as dendritic cells. Furthermore, QS-21 has been shown to promote the production of proinflammatory cytokines and facilitate the formation of antigen-specific T cells. Its precise role in shaping the immune response will be the focus of our research.\u003c/p\u003e \u003cp\u003eDoxorubicin is a widely used chemotherapy agent with known immunomodulatory properties. This anthracycline-based drug not only targets cancer cells but can also affect the immune system. By understanding how doxorubicin affects immune cells, we can gain valuable insights into the balance between its cytotoxic effects and its potential to stimulate or inhibit immune responses.\u003c/p\u003e \u003cp\u003eProgrammed cell death ligand 1 (PD-L1) is an immune checkpoint protein that plays a crucial role in regulating immune responses. The interaction between PD-L1 on cancer cells and its receptor PD-1 on T cells may reduce the ability of the immune system to mount an effective anti-tumor response. Investigating the role of PD-L1 in the context of our study will help shed light on the mechanisms by which tumor cells escape immune surveillance.\u003c/p\u003e \u003cp\u003eHER2 is well known for its overexpression in various types of cancers including breast cancers promoting uncontrolled proliferation and cell survival [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Overexpression of this TAA confers 25% of total breast cancer incidences and could induce resistance to, chemotherapy, radiotherapy, and hormone therapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These disadvantages in HER2-positive cancer treatment strategies diverted research studies toward targeted and combination immunotherapies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Trastuzumab (Herceptin, Genentech) was the very first known immunotherapy approved by the US Food and Drug Administration (FDA), suppressing the activity of HER2 receptor via attachment of a monoclonal antibody against it [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Regarding active immunotherapeutic applications, Neu-vax from Galena Biopharma was the most promising cancer vaccine candidate against HER2-positive cancers that was composed of a short antigenic peptide and GM-CSF as an adjuvant. This approach unfortunately failed the phase studies and currently alternative combination approach of E75 peptide is being tried together with Trastuzumab in a phase III study demonstrating the novel strategies on combination immunotherapeutic approaches [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDendritic-based vaccine studies, active immunotherapeutic, are regarded as powerful inducers of immunity without drawback problems in autoimmunity and tolerance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recent developments in technology further improved the understanding of the evasion mechanisms of cancer cells and the discovery of immune checkpoint inhibitors (PD-1/PD-L1, CTLA-4) creating novel opportunities for treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Conventional therapies including chemotherapeutics, radiotherapies, and hormonal therapies remained insufficient in terms of treatment apart from being harmful to healthy tissues. Chemotherapeutic resistance is another obstacle to these conventional therapies that remains a major problem in treating cancer patients. Monotherapies remained insufficient and current studies are focusing on the development of combinations of therapies for cancer treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, a novel combination of therapies including BM-DC based vaccine supplemented with QS-21 adjuvant, doxorubicin, and anti-PD-L1 monoclonal antibody (mAb) was used as a combination in a HER2/Neu expressing breast cancer model of Balb/c mice. Different combinations of active/passive immunotherapy and chemotherapy were investigated to determine the best combination and synergistic effect of various formulations.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eHere is the list of items organized by their purposes.\u003c/p\u003e \u003cp\u003eCell Culture and Reagents: DMEM/F12 (Gibco, USA), Fetal bovine serum (Gibco, USA), Penicillin-streptomycin (Biological Industries, Israel)\u003c/p\u003e \u003cp\u003eCytotoxicity Assays: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, USA), LDH Cytotoxicity Assay Kit (96 test) (Cayman, USA)\u003c/p\u003e \u003cp\u003eImmunological Agents: Concanavalin A (Con A) (Biochrome, Germany), LPS (Sigma, USA), QS-21 (Desert King, San Diego, CA), GM-CSF (Sigma Aldrich, USA)\u003c/p\u003e \u003cp\u003eAntibodies and Immunological Assays: Goat anti-mouse IgG, IgG1, and IgG2a peroxidase conjugate (Southern Biotech Assoc., Birmingham, AL, USA), BD Tritest CD4/CD8/CD3 (BD Biosciences, USA), \u003cem\u003ein vivo\u003c/em\u003e Mab anti-mouse PD-L1 (B7-H1) antibody (Bioxcell, USA), Anti-mouse CD4 antibody (Santa Cruz, USA), Anti-mouse CD8-a antibody (Santa Cruz, USA), Anti-mouse CD61 antibody (BioLegend, USA), Anti-mouse CD40-FITC antibody (BD Pharmingen, USA), Anti-mouse CD80-PE antibody (BD Pharmingen, USA), Anti-mouse CD11c-FITC monoclonal antibody (EBioscience, USA)\u003c/p\u003e \u003cp\u003eOther Materials: Ketamine (100 mg/ml, EgeVet, Turkey), Mouse HER2/ErbB2 (Acro Biosystems, USA), Matrigel Matrix (Corning, USA), Eumaline Mayer (Bio-Optica, Italy), Sensi Tek, HRP (Scy Tek Laboratories, USA), Super Block (Scy Tek Laboratories, USA), Hydrogen peroxide 30% (Merck, Germany)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell Line\u003c/h2\u003e \u003cp\u003eGFP tagged HER2 expressing mouse breast cancer cell line (4T1-HER2) was kindly provided by Prof. Michael Kershaw (Cancer Immunology Program, Peter MacCallum Cancer Centre, Victoria, Australia) and maintained in DMEM/F12 with penicillin/streptomycin, and 10% FBS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Verification of GFP tagged HER2 Expression on 4T1-HER2 Cells\u003c/h2\u003e \u003cp\u003eThe HER2 expression was verified by using flow cytometry (BD Accuri C5) as the vector expressing HER2 contains a GFP tag and the green fluorescence level would indicate the presence of HER2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Experimental Animals\u003c/h2\u003e \u003cp\u003eMale-female Balb/c mice (6\u0026ndash;10 weeks old) weighing 20\u0026ndash;26 g were purchased from Ege University Laboratory Animal Research Center (Bornova, İzmir, T\u0026uuml;rkiye). Mice were maintained in groups of 5 with a total 5 groups under standard conditions of temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ◦C with regular 12 h light and 12 h dark cycles and had free access to standard laboratory food and water. The experimental protocol was approved by the Local Ethics Review Committee for Animal Experimentation of Ege University (Approval number is 2016/006).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Bone Marrow Derived Dendritic Cell (BM-DC) Generation\u003c/h2\u003e \u003cp\u003eDC-generation protocol was performed by using the modified protocol of Madaan et al [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 8\u0026ndash;10 weeks old male Balb/c mice were euthanized by using Ketamine. Mice were rinsed by using 70% ethanol for disinfection. The Femur and tibia bones of the mice were dissected from the pelvic joint. Bones are then cleared from tissues, muscles, and fur by using sterile dissecting scissors and forceps. Both ends of the femurs and tibias were trimmed by sterile scissors and the marrow was flushed into a Petri by using RPMI 1640 medium (10% FBS) via a sterile insulin injector. This process was repeated several times and the monocytes were centrifuged at 250 g for 5 minutes. This process was repeated 3 times. After the last washing step, cells were counted and dispensed to Petri dishes at a concentration of 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml in a BM-DC medium (5 ng/ml GM-CSF, RPMI 1640, 10% FBS). On the day 3 of culture, 10 ml BM-DC medium was added to the cells. Cells were collected and centrifuged for 5 minutes on the day 6. After centrifugation cells were seeded back to the petri dishes in a 10 ml BM-DC medium. On the day 8, 10 ml BM-DC medium was added to the cells. Cells were collected and counted to assess the differentiation efficiency by using a CD11c marker by flow cytometry (BD Accuri C5) on the day 10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Verification of DC-Differentiation\u003c/h2\u003e \u003cp\u003eOn the day 10 of the monocyte-DC differentiation protocol, cells were collected and counted. 5 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml were washed by using PBS and anti-CD11c FITC (1:400) monoclonal antibody was added to the cells followed by incubation for 30 minutes at +\u0026thinsp;4\u003csup\u003eo\u003c/sup\u003eC. After incubation cells were analyzed by BD Accuri C5 flow cytometry [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Preparation of Samples for Scanning Electron Microscopy\u003c/h2\u003e \u003cp\u003eBM-DC\u0026rsquo;s were seeded onto 24 well plates at a density of 1x10\u003csup\u003e4\u003c/sup\u003e cells/ml and incubated for 24 h. Samples were washed 3 x with PBS and incubated with 2.5% gluteraldehyde for 4 h. Samples were dehydrated by being exposed to 25%, 50%, 75% and 100% ethanol concentrations. After incubation at 100% ethanol for 10 mins, samples were placed into desiccator. Samples were then coated with 200A gold particles and images were taken by using scanning electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. The Loading of Mouse HER2/Neu Antigen on BM-DCs\u003c/h2\u003e \u003cp\u003eOn the day 10 of the BM-DC protocol, BM-DCs were incubated with 10 \u0026micro;g/ml mouse HER2/Neu overnight at +\u0026thinsp;37\u003csup\u003eo\u003c/sup\u003eC. After incubation, loading success was assigned by measuring the change in the levels of anti-CD40-FITC (1:100) and anti-CD80-PE (1:100) expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Formation of Xenograft Tumor Model by Using 4T1-HER2 Cells\u003c/h2\u003e \u003cp\u003e4T1-HER2 tumor cells were cultured in DMEM/F12 (10% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine). A final concentration of 5 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml 4T1-HER2 tumor cells were injected subcutaneously (s.c.) into the flank of Balb/c mice day 0 of the experiment followed by mixing with matrigel in 1:1 dilution total 200 \u0026micro;l. Tumor growth was measured in mm by using a caliper and was recorded as mean diameter values (width x height x length)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Immunization Studies\u003c/h2\u003e \u003cp\u003eMice were immunized with different vaccine formulations including control groups, as sum 7 groups.\u003c/p\u003e \u003cp\u003eGroup 1: Control\u003c/p\u003e \u003cp\u003eGroup 2: HER2/Neu (10 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;QS-21 (10 \u0026micro;g/ml) in 100 ml saline solution.\u003c/p\u003e \u003cp\u003eGroup 3: HER2/Neu peptide (10 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;QS-21 (10 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;anti-PD-L1 mAb in 100 ml saline solution.\u003c/p\u003e \u003cp\u003eGroup 4: Doxorubicin only (2.5 mg/kg).\u003c/p\u003e \u003cp\u003eGroup 5: Doxorubicin (2.5 mg/kg)\u0026thinsp;+\u0026thinsp;anti-PD-L1 mAb\u003c/p\u003e \u003cp\u003eGroup 6: Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21 vaccine\u0026thinsp;+\u0026thinsp;anti-PD-L1 mAb\u003c/p\u003e \u003cp\u003eGroup 7: Combination of HER2/Neu loaded BM-DC vaccine adjuvant with QS-21, anti-PD-L1, and doxorubicin\u003c/p\u003e \u003cp\u003eHER2/Neu and BM-DC-based vaccinations were performed through intraperitoneal (i.p.) injection on the day 1 and day 8. Anti-PD-L1 mAb was i.p. injected on the day 4 and day 9. Doxorubicin was i.p. injected on the day 5 and day 11. The study was terminated, and mice were sacrificed by cervical dislocation on the day 14. Blood, tumor, and spleen samples were collected for performing splenocyte proliferation assay, measurement of immune response, co-culture experiment, HER2 specific antibody response, and immunohistochemical (IHC) staining studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Splenocytes Proliferation Assay\u003c/h2\u003e \u003cp\u003eSplenocyte proliferation assays were performed according to the modified method by Nalbantsoy et al [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Splenocytes were treated with HER2/Neu (1 \u0026micro;g/ml) incubated for 72h at 37\u0026deg;C. The stimulation index (SI) was calculated based on the following formula: SI\u0026thinsp;=\u0026thinsp;the absorbance value for mitogen-stimulated cultures/the absorbance value for non-stimulated cultures. Each experiment was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Determination of HER2/Neu Specific Antibody Response\u003c/h2\u003e \u003cp\u003eHER2/Neu specific antibodies (IgG, IgG1, IgG2a) were detected by ELISA according to the modified method by Nalbantsoy et al [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. ELISA plates were coated with 100 ng/ml HER2/Neu overnight at +\u0026thinsp;37\u0026deg;C. Following the washing steps, 100 \u0026micro;l of 1:100 diluted serum samples were added to the wells and incubated for 1 hour at a 37\u0026deg;C incubator. 100 \u0026micro;l of horseradish peroxidase-conjugated antibody for IgG, IgG1, or IgG2a (diluted in 1:8000) were added to the wells followed by four times of washing and incubated 1 hour at 37\u0026deg;C incubator. After a washing step, 100 \u0026micro;l substrate solution (TMB-3,3',5,5'-Tetramethylbenzidine) was added and incubated at room temperature for 30 minutes. The reaction was terminated by adding 50 \u0026micro;l/well-stop solution (H2SO4). The optical density (OD) was measured at 492 nm (Versamax Microplate Reader, USA). Data were calculated as the mean OD value of the samples minus the mean OD value of the control. Results were expressed as log 2 titers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Determination of Anti-PD-L1 Specific Antibody Response\u003c/h2\u003e \u003cp\u003eAnti-PD-L1 specific antibodies (IgG) were detected by ELISA according to the modified method by Nalbantsoy et al [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. ELISA plates were coated with 10 ng/ml anti-PD-L1 overnight at +\u0026thinsp;4\u0026deg;C. After the washing steps, 100 \u0026micro;l of 1:100 diluted serum samples were added to the wells and incubated for 1 hour at a 37\u0026deg;C incubator. The remaining protocol is followed in a similar way to the previous study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.14. Measurement of CD8, CD3 and IFN\u003c/b\u003eγ \u003cb\u003eLevels\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eSplenocytes of sacrificed mice followed by immunizations were isolated. Cells from each mouse belonging to the same group were collected in a pool. 1 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml from each group were treated with HER2/Neu (1 \u0026micro;g/ml) incubated for 72 h at 37\u0026deg;C. Cells were collected and centrifuged at 1200 rpm for 5 minutes and 50 ml (1:1000) PE-labeled anti-mouse CD3 antibody was added. Samples were incubated for 30 minutes at +\u0026thinsp;4 \u003csup\u003eo\u003c/sup\u003eC at dark. After incubation, cells were centrifuged and washed with 250 ml PBS containing 3% FBS. Anti-mouse CD8-PerCP antibody was added to cells and further incubated for 30 minutes at +\u0026thinsp;4 \u003csup\u003eo\u003c/sup\u003eC in the dark. Cells were then washed, and 100 ml Fixation / Permeabilization solution was added to cells and incubated for 30 minutes at +\u0026thinsp;4 \u003csup\u003eo\u003c/sup\u003eC at dark. After incubation cells were washed and anti-mouse IFNγ-FITC antibody was added to cells followed by incubation for 30 minutes at +\u0026thinsp;4 \u003csup\u003eo\u003c/sup\u003eC in the dark. The total volume of 250 ml perm wash (1:10) was added to the cells and the cells were washed twice. After the washing steps, cells were analyzed by flow cytometry (BD Accuri C5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Lactate dehydrogenase (LDH) Assay\u003c/h2\u003e \u003cp\u003eSpecific cytotoxicity of splenocytes was determined by participating in an LDH assay by following the manufacturer's protocol (Cayman). 4T1-HER2 cells and splenocytes belonging to different immunization groups were incubated at a 1:40 target: effector ratio. For this purpose, 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml 4T1-HER2 cells were seeded on 96-well plates and incubated for 24 h at +\u0026thinsp;37 \u003csup\u003eo\u003c/sup\u003eC, 5% CO2 incubator. 24 h post incubation 8 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml splenocytes were seeded on 4T1-HER2 cells and incubated for 4 h at 37 \u003csup\u003eo\u003c/sup\u003eC. After incubation, the 96 well plate was centrifuged at 1200 rpm and 100 ml supernatant was taken from each well followed by placing into a new 96 well plate. 100 ml LDH substrate was added to the wells and incubated further 30 minutes at room temperature. Then, samples were read at 490 nm by using an ELISA plate reader. The cytotoxicity was calculated according to the kit formula mentioned below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Immunohistochemistry (IHC) Staining\u003c/h2\u003e \u003cp\u003eOn the day 14 of the study, mice tumor cells were excised and placed into %4 paraffin. Then, tumors were embedded into paraffin and waited for solidification. After that, 0.5 mm tumor sections were taken by using a microtome. Sections were then incubated at 37oC for 1 h. Later, hemotoxylin-eosin staining was performed. Immunohistochemical staining was performed after incubating tumor sections at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 h. Following incubation samples were placed overnight into Xylol. After this process, samples were placed into serial alcohol dilutions 100% (4 minutes), 95% (2 minutes), 90% (2 minutes), and 80% (2 minutes). Samples were exposed to a microwave set at 90 \u003csup\u003eo\u003c/sup\u003eC in sodium citrate solution for 30 minutes. Then, samples were incubated for 15 minutes at room temperature and then placed into the ice-filled water bath for a further 15 minutes. Samples were then incubated in distilled water for 5 minutes, in PBS, and 5% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 minutes respectively. Tumor sections were then washed 3 times with PBS and a blocking solution was applied to them followed by incubation for 30 minutes. After incubation antibodies prepared (1:100 dilution) (anti-mouse CD8, anti-mouse CD4, and anti-mouse CD61) were added to the samples and incubated overnight at +\u0026thinsp;4oC. After overnight incubation, samples were washed 3 times with PBS, and a biotin-conjugated secondary antibody was added to the samples followed by incubation for 30 minutes. After incubation, samples were washed 3 times with PBS and 1\u0026ndash;3 ml DAB was added on top of the sections and incubated for 2 minutes. Samples were then placed into distilled water containing bath, Meyers Hemotoxylin for 50 secs, tap water, and distilled water respectively. Samples were then placed into a series of ascending alcohol concentrations 80%, 90%, 95%, and 100% respectively. Samples were then incubated at room temperature for 15 minutes and placed into Xylol for another 15 minutes. After the Xylol step, samples were dried at room temperature and closed for investigation under microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Statistics\u003c/h2\u003e \u003cp\u003eThe data were measured with mean standard errors and the statistical significance of differences was examined by using One-way ANOVA, and Tukey\u0026rsquo;s test for multiple comparisons between each group by using GraphPad Prism 5.0 and SPSS for Windows. \u003cem\u003eP\u003c/em\u003e values of less than \u003cem\u003e0.05*, 0.01**\u003c/em\u003e and \u003cem\u003e0.001***\u003c/em\u003e were stated as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Verification of GFP tagged HER2 Expression on 4T1-HER2 Cells\u003c/h2\u003e \u003cp\u003eThe HER2 expression was analyzed by using BD accuri C5 flow cytometry. Investigation of the fluorescence level clearly indicated the presence of GFP-tagged HER2 (Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. BM-DC differentiation\u003c/h2\u003e \u003cp\u003eFemur and tibia bones of 8\u0026ndash;10 week-old Balb/c mice were excised for generating monocyte-derived DC and the process was evaluated by investigating the levels of anti-mouse CD11c via flow cytometry. The obtained results that the differentiation has been successful and the percentage differentiation is around 80\u0026ndash;85% (Fig.\u0026nbsp;2.I.A-B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Mouse HER2/Neu Antigen Loading on BM-DCs\u003c/h2\u003e \u003cp\u003eMouse HER2/Neu was loaded onto BM-DCs by co-incubation overnight at 37oC and the antigen uptake was verified by observing the changes in the co-stimulatory molecules on the DCs. For this purpose, cells were stained with anti-mouse CD40-FITC and anti-mouse CD80-PE. The percentage of co-stimulatory molecules differentiations, double fold increased upon antigen loading demonstrating the success of antigen loading (Fig.\u0026nbsp;2.II - III). The maturation status of the dendritic cells was further characterized by taking SEM pictures as shown in Fig.\u0026nbsp;2.III. Dendritic cells with antigens were shown to mature, enlarge, and underwent a morphological change with induced dendrites around themselves (Fig.\u0026nbsp;2.III).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Formation of Xenograft Tumor Model by Using 4T1-HER2 Cells\u003c/h2\u003e \u003cp\u003eTo assess the vaccine efficiency of the different formulations, 5 x 10\u003csup\u003e6\u003c/sup\u003e 4T1-HER2 cells/mice together with matrigel in a 1:1 (100 \u0026micro;l cell suspansion:100 \u0026micro;l Matrigel) concentration ratio was injected subcutaneously to develop a xenograft model of breast cancer in Balb/c mice on day 0. Tumors appeared successfully on the day 3 of the study, and all mice successfully developed palpable tumors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Immunization Studies\u003c/h2\u003e \u003cp\u003eOn the day 1, the first injection of different vaccine formulations was injected composed of (HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21, HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1, Doxorubicin, Doxorubicin\u0026thinsp;+\u0026thinsp;anti-PD-L1, Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1, Doxorubicin\u0026thinsp;+\u0026thinsp;DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1). Later on the day 4, 100 \u0026micro;g/ml anti-PD-L1 was injected into the combination immunotherapy groups to investigate the synergism of various combination groups. The second dose of injection participated on the day 8 and the second dose of anti-PD-L1 was injected on the day 9. Doxorubicin was injected on the day 5 and day 11 for various combination groups. The study was terminated on the day 14 and tumor dimensions were measured by caliper. The obtained data clearly demonstrated that, anti-PD-L1 addition to formulas further supported the vaccine efficacy. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;3). QS-21 adjuvant also enhanced the vaccination formulas in terms of tumor dimensions. Doxorubicin alone group induced a great reduction in tumor dimensions, but its activity further increased synergistically with vaccines, especially BM-DC based vaccine together with anti-PD-L1 formulation reaching the lowest tumor dimension when compared to control and other formulation groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Tumor Specific Cytotoxicity of Different Vaccine Combinations\u003c/h2\u003e \u003cp\u003eTo assess the tumor-specific cytotoxicity of different vaccine combinations, an LDH assay was performed in a 1:40 target/effector ratio. Data obtained demonstrated that combination groups have a superior effect on tumor-specific cytotoxicity results when compared to monotherapies. The addition of anti-PD-L1 to combinations boosted the specific cytotoxicity demonstrating a synergistic effect with formulations. Doxorubicin combinations also demonstrated a further supportive role in terms of cytotoxicity, thereby synergistic effect on cytotoxicity with other constituents of combinations. The doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 group demonstrated the highest specific cytotoxicity significantly when compared to the control and other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. HER2 Specific Antibody Response\u003c/h2\u003e \u003cp\u003eHER2-specific IgG, IgG1, and IgG2a antibody levels in sera were measured by using ELISA. The data suggests that HER2-specific antibody response was developed upon vaccination. All different vaccination groups demonstrated IgG, IgG1, and IgG2a antibody response against HER2 as shown in Fig.\u0026nbsp;5. IgG1 is an indicator of Th2 whereas IgG2a antibody is a Th1 immune response. All different vaccine combinations demonstrated a superior effect on Th2 type immune response (IgG1) when compared to Th1 type immune response (IgG2a) (Fig.\u0026nbsp;5B-C). Data demonstrated that combination immunotherapeutic formulations exhibited significant antibody response when compared to control and monotherapies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination group had significantly increased the anti-HER2 titer when compared to control and other vaccination groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (see Fig.\u0026nbsp;5B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Anti-PD-L1 Specific Antibody Response\u003c/h2\u003e \u003cp\u003eAnti-PD-L1 specific antibody level in sera was measured by using ELISA. Anti-PD-L1 sera were investigated in both vaccinated and control groups, however, no significant differences were detected between the vaccination groups and control group (Fig.\u0026nbsp;6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Splenocytes Proliferation Assay\u003c/h2\u003e \u003cp\u003eSplenocyte proliferation upon antigen stimulation is an accepted method for cell-mediated immunity. HER2/Neu antigen-stimulated splenocyte proliferation is shown in Fig.\u0026nbsp;7. SI of a triple combination vaccine group (Doxorubicin\u0026thinsp;+\u0026thinsp;DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS21\u0026thinsp;+\u0026thinsp;anti-PD-L1) demonstrated nearly a 1.5-fold increase when compared to control and tumor control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The same group also exhibited greater SIs when compared to peptide-based vaccines and other combination groups, however, it did not reveal any significant differences with Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination (Fig.\u0026nbsp;7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.10. Measurement of CD8, CD3 and IFN\u003c/b\u003eγ \u003cb\u003eLevels\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe rate of immune response was measured on splenocytes belonging to different groups of vaccination. Splenocytes were incubated with HER2/Neu antigen for 72 hours and the levels of total T-cells (CD3), CD-8\u0026thinsp;+\u0026thinsp;T-cells, and IFNγ cytokine were measured by flow cytometry. The data revealed that CD8-positive T-cell populations were significantly increased in combination groups composed of doxorubicin, anti-PD-L1, and peptide (HER2/Neu) based, or BM-DC based vaccines when compared to all other groups tested, demonstrating the synergistic effect of these combinations in terms of immune system stimulation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;8A). IFNγ secreting CD8 T-cell population seemed to be the highest in the Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination followed by Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;8B). The doxorubicin-only group demonstrated an increase in IFN-secreting CD8\u0026thinsp;+\u0026thinsp;T-cell response however the activity significantly increased upon the addition of anti-PD-L1 mAb to the group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The addition of the active immunity stimulating vaccines to the doxorubicin and anti-PD-L1 further increased the response demonstrating the best activity in terms of IFNγ secreting CD8\u0026thinsp;+\u0026thinsp;T-cells (Fig.\u0026nbsp;8B). IFNγ secreting total T-cell population seemed to be significantly increased in the Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 and Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 immunotherapy groups when compared to all other vaccination and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination demonstrated the best activity in terms of increasing CD8\u0026thinsp;+\u0026thinsp;T-cell population, IFNγ secretion, and the total T-cell population when compared to other groups (Fig.\u0026nbsp;8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.11. IHC Staining\u003c/h2\u003e \u003cp\u003eThe population of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T-cells were investigated around the tumor niche along with CD61\u0026thinsp;+\u0026thinsp;cells indicating tumorigenic potential of the 4T1-HER2 tumors. The data suggested that Both CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;cells successfully penetrated into the tumor cells, demonstrating the success of the combination vaccine groups ofDoxorubicin\u0026thinsp;+\u0026thinsp;DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21 and Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1. CD8\u0026thinsp;+\u0026thinsp;cell population did not seem to increase significantly in HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21, Doxorubicin only, HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 groups when compared to the control group, however, only HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1group demonstrated slight up-regulation in the levels of CD4 positive T-cell populations (Fig.\u0026nbsp;9). CD61 level of the control groups seemed to have the highest level of tumorigenic potential although no significant differences were detected when compared to the other groups. The combination group composed of BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 showed lower CD61\u0026thinsp;+\u0026thinsp;marker expression when compared to the other groups (Fig.\u0026nbsp;9).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTumor-associated antigen discoveries initiated new strategies in combating against cancers. These antigens highlighted a new era in terms of personalized therapies, especially immunotherapies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cancer immunotherapy has now been regarded as important as radiotherapies, chemotherapies, hormonal therapies, and surgical therapies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Current immunotherapy strategies were divided into two subgroups which are active and passive immunotherapies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Active immunotherapies aim to modulate immune regulation, and direct immune responses towards TAA whereas passive immunotherapies mainly focus on immune checkpoint blockade reactions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. HER2 overexpression is characterized by various cancer types including breast cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Its overexpression promotes uncontrolled proliferation and cell survival, however, treating these HER2 overexpressing cancers remained a huge problem even though its TAA is well characterized. Patients with this cancer type are prone to gain resistance to chemotherapeutics making treatment procedures ineffective [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent studies on cancer therapies focused on combination immunotherapies not only to overcome resistance mechanisms but also because monotherapies become insufficient in terms of cancer treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere in this study, various combinations of immunotherapeutic and conventional chemotherapeutic approaches were investigated to determine the synergistic activity of the combinations as well as discover a novel highly efficient therapeutic combination against HER2 overexpressing breast cancer xenograft model of Balb/c mice. The outcome of the study was aimed to boost the immune response via cancer vaccine, block the immune evasion via monoclonal antibody, and further empower the combination with a chemotherapeutic agent.\u003c/p\u003e \u003cp\u003eFor this purpose, BM-DC were isolated from the Balb/c mice and the differentiation was found to be as high as 80% which was measured by the CD11c level of cells apart from imaging through the SEM, correlating with the literature [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Verification of BM-DC differentiation was followed by antigen (mouse HER2/Neu) loading experiments that were participated successfully via measuring the 1.5-2 fold increase in CD40/CD80 co-stimulatory molecule levels [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the last day of the study, tumor dimension measurements were performed, and the combination immunotherapy groups demonstrated a 2\u0026ndash;5 fold decrease in tumor dimensions, having the most significant reduction in Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination group followed by Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This demonstrated the success of BM-DC-based vaccines over peptide-based vaccines apart from the synergism of anti-PD-L1 mAb and doxorubicin in combinations. The decrease in tumor dimensions was significant when compared to the other studies in the literature although a lower concentration of anti-PD-L1 and doxorubicin was used in the study [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR23\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSpecific cell cytotoxicity of combination immunotherapy groups was investigated by performing an LDH assay. The tumor-specific cytotoxicity was an important aspect in terms of CD8\u0026thinsp;+\u0026thinsp;T-cells against tumor-specific immune response [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The specific cytotoxicity of Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 vaccine combination demonstrated significantly higher cytotoxicity when compared to other vaccine combinations and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Although lower effector-target concentration with lower incubation time (4 h) was tested in the study, promising results were obtained in terms of tumor-specific cytotoxicity when compared to the literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe power of the immune response formed was also assessed by measuring the levels of HER2-specific IgG, IgG1, and IgG2a [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Data demonstrated that the immune response formed was IgG1 subclass indicating a Th2 bias immune response relatively similar to the study of Cruz et al [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination group demonstrated a significant increase (2.5 fold) in HER2-specific antibody titers compared to other groups including control groups, except Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination. Anti-PD-L1 specific antibody titer was also investigated in the study and no significant differences between groups were indicated. The lymphocyte stimulation of various vaccine groups was also investigated via flow cytometry. The Doxorubicin\u0026thinsp;+\u0026thinsp;BM-xDC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 and Doxorubicin\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 combination groups demonstrated significantly higher total T-cell, CD8\u0026thinsp;+\u0026thinsp;T-cell, and IFNγ population when compared to other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This data clearly demonstrates the success of lymphocyte priming and also a correlation of the tumor dimension, and LDH cytotoxicity studies together. It is well known that DC-based vaccines were better in terms of polarization of CD8\u0026thinsp;+\u0026thinsp;T-cells and IFNγ secretion when compared to the peptide-based vaccines demonstrating a similar trend with the study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The data was further underpinned by the immunohistochemical study, and the levels of CD8+, CD4+, and CD61\u0026thinsp;+\u0026thinsp;cells were investigated around the tumor niche. It was observed that CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T-cells penetrated the tumor cells further supporting the efficacy of the data obtained. The same trend was also observed in the previous studies correlating with the study [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR30\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Doxorubicin\u0026thinsp;+\u0026thinsp;BM-DC\u0026thinsp;+\u0026thinsp;HER2/Neu\u0026thinsp;+\u0026thinsp;QS-21\u0026thinsp;+\u0026thinsp;anti-PD-L1 was found to be the most efficient in terms of IHC studies with high penetration of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;cells with lower avb3 integrin marker, tumorigenic potential indicator.\u003c/p\u003e \u003cp\u003eIn conclusion, the study outcomes demonstrated that BM-DC-based vaccinations are more potent than peptide-based vaccines. Combination immunotherapies might be better in terms of cancer therapies especially when monoclonal antibodies are combined with active immunotherapies and chemotherapeutics. The novel immunotherapeutic combination approach used in this study boosted the immune response against target tumor cells specifically and also prevented the immune escape of tumor cells successfully.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKDK wrote the main manuscript, YU prepared the figures, KDK and YU did the histopathological experiments and evaluation, AN, CS\u0026Ouml; and İK did vaccine preparation experiments. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors would like to thank Ege University Scientific Research Projects Coordination (16-FEN-011) who funded the project. We also thank Dr. Tuğba T\u0026Uuml;RK for her support in the SEM evaluation of DC cells.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEsteva FJ, Hubbard-Lucey VM, Tang J, Pusztai L (2019) Immunotherapy and targeted therapy combinations in metastatic breast cancer. Lancet Oncol 20(3)175\u0026thinsp;\u0026ndash;\u0026thinsp;86\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeather L, McArthur MPH, Page DB (2016) Immunotherapy for the Treatment of Breast Cancer: Checkpoint Blockade, Cancer Vaccines, and Future Directions in Combination Immunotherapy. Clin Adv Hematol Oncol ; 14(11)922\u0026thinsp;\u0026ndash;\u0026thinsp;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrari D, Yarden Y (2000) Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. 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Sci Transl Med ; 7(315)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HER2+, breast cancer, BM-DC, immunotherapy, anti-PD-L1, chemotherapy, combination therapy","lastPublishedDoi":"10.21203/rs.3.rs-3943744/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3943744/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe discovery of tumor-associated antigens has been a major milestone for cancer therapy as the current conventional therapies (radiotherapy, chemotherapy) were found to be insufficient in terms of cancer treatment. Human epidermal growth factor receptor (HER2) is a tumor-associated antigen, aberrantly found in various cancers, including breast cancer. Today, most of the treatment strategies are skewed towards combination therapies rather than monotherapies, they were found to be ineffective mostly because of patients' resistance mechanisms. A combination of more than one therapeutic approach (passive/active immunotherapy, conventional therapies) was found to be more effective in the clinic.\u003c/p\u003e\n\u003cp\u003eHere, in this study, various vaccine combinations were investigated against the 4T1-HER2 xenograft model of Balb/c mice. Eight different groups of vaccine formulations were tested to find the best synergistic combination including combinations of BM-DC-based vaccine, Peptide-based vaccine, anti-PD-L1, Doxorubicin, and QS-21 adjuvant. Triple combination groups of immunotherapies exerted better activities in terms of tumor dimensions as Doxorubicin+BM-DC-HER2/Neu+QS-21+anti-PD-L1 and Doxorubicin+ HER2/Neu+QS-21+anti-PD-L1 groups significantly decreased tumor dimensions. Similar groups also demonstrated a better stimulation of lymphocytes and IFNγ cytokine in the flow cytometry study, inducing a HER2/Neu specific antibody response in ELISA studies. Doxorubicin+BM-DC-HER2/Neu+QS-21+anti-PD-L1 combination demonstrated significantly higher specific cytotoxicity in lactate dehydrogenase data, as well as the CD4+ and CD8+ responses in the immunohistochemical study.\u003c/p\u003e\n\u003cp\u003eIn conclusion, these data indicated that the Doxotubicin+BM-DC+HER2/Neu+QS-21+anti-PD-L1 vaccine combination synergistically generated a promising activity in terms of tumor dimension and immune response stimulation against HER2 overexpressing breast cancer model in mice.\u003c/p\u003e","manuscriptTitle":"Chemo-Immunotherapy, a Combination Approach for the Treatment of HER2-Positive Breast Cancer in a Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-14 14:23:52","doi":"10.21203/rs.3.rs-3943744/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ea1c17c-c0d3-4d93-a904-f7615e214160","owner":[],"postedDate":"February 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-14T14:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-14 14:23:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3943744","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3943744","identity":"rs-3943744","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}