Abstract
Postoperative tumor recurrence and metastasis remain an extreme challenge in breast cancer. Therapies that target cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have provided unprecedented clinical benefits in various types of cancer. The aim of this study was to determine whether the combination of anti-CTLA-4 and anti-PD-1 could prevent postoperative breast tumor recurrence and metastasis in breast tumor–bearing mice. The results indicated that the combination of CTLA-4 and PD-1 inhibitors was more effective compared with single inhibitors for mammary tumor growth and prevention of postsurgical tumor recurrence and pulmonary metastasis (P < 0.05), which resulted in prolonged survival (P < 0.05). Analysis of the underlying mechanism revealed that anti-CTLA-4 and anti-PD-1 in combination synergistically promoted the infiltration of CD8+ and CD4+ T cells into tumors (P < 0.05 vs. single inhibitors), thus boosting the antitumor immune responses. In summary, our results revealed that combination immunotherapy with anti-CTLA-4 and anti-PD-1 may present a new, promising regimen to inhibit postoperative breast cancer relapse and lung metastasis and improve patient outcomes, which warrants further investigation in clinical settings.
Introduction
Breast cancer is one of the most frequent cancer types for women, which has the highest incidence rate among new cancer cases, ranking the second important cause of death in women with malignant tumors (1). Surgical removal is considered as the standard treatment for resectable breast cancer and some of prospective randomized trials have proven that surgery combined with adjuvant therapies such as radiation, endocrine therapy, and chemotherapy can reduce the residual risk of recurrence and prolong overall survival of patients (2–4). However, although surgery plus adjuvant or neoadjuvant therapy have shown promising results in clinical studies, the 5-year recurrence risk indicative of treatment failure remains 7% to 13% (5). Therefore, new therapeutic regimens to prevent postoperative breast cancer relapse are in demand.
It has been shown that breast cancer tumors have higher infiltration of immune cells compared with normal breast tissue (6). As indicated by several clinical trials, in certain subtypes of breast cancer, especially in triple-negative breast cancer (TNBC), high levels of tumor-infiltrating lymphocytes (TIL) are strongly correlated with patients' recurrence-free survival and predict favorable outcomes, thus serving as an effective prognostic biomarker (7–11). These findings suggest that to inhibit postoperative tumor recurrence and metastasis, therapies should aim to activate host antitumor immune responses. Therefore, immunotherapy should provide a solution to a problem of improving patients' outcome in TNBC.
Indeed, immunotherapy enabled to achieve promising clinical responses in patients with several types of solid tumors and has attracted considerable attention as a viable therapeutic approach in cancer (12–14). In recent years, clinical application of immune checkpoint inhibitors in cancer has increased. Among them, immunotherapy inhibitors against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) or programmed cell death protein 1 (PD-1), which promote migration of TILs to tumor sites, demonstrated remarkable efficacy in patients with various cancers such as non–small cell lung cancer, advanced melanoma, and urothelial carcinoma (13, 15, 16). Furthermore, combined treatment with anti-CTLA-4 and anti-PD-1 has demonstrated satisfactory preliminary results, including the application in advanced melanoma, prostate cancer, and renal caner (17–19). Because of limited benefit from monotherapy in breast cancer, current efforts focus on developing promising immunotherapy combinations are now being investigated in the clinically early stage. Recently, two clinical trials are underway, evaluating the efficacy of the combination of anti-CTLA-4 and anti-PD-1 in BC (NCT02536794, NCT03789110), and a follow up study focus on the synergistic effect between anti-CTLA-4 and anti-PD-1 and other agents (e.g., chemotherapy, cytokine, cryoablation) may provide new approaches to further increase responses (NCT02983045, NCT03409198, NCT02833233). Results from combining chemotherapy with both anti-CTLA-4 and anti-PD-1 significantly prolongs survival in BRCA1-mutated breast cancer mice, suggesting a promising therapeutic strategy for breast cancer (20). These completed or ongoing studies mentioned above suggest that combination immunotherapy may elicit synergistic effects and enhance antitumor responses through regulation of the immune system. Tolerance of tumor-specific T cells overexpressing inhibitor receptors, including CTLA-4 and PD-1, is responsible for the immunosuppressive environment, which prevents elimination of cancer cells (21, 22). The antitumor mechanisms elicited by immune checkpoint molecules are different; thus, CTLA-4 is believed to primarily regulate the early stage of T-cell proliferation in lymph nodes, whereas PD-1 mainly suppresses the later stage of T-cell–mediated immune response in the tumor microenvironment (23). Therefore, the combined application of CTLA-4 and PD-1 inhibitors should elicit a synergetic effect, accounting for the effectiveness of combination immunotherapy at the molecular level.
A previous report indicated that anti-CTLA-4 adjuvant therapy was successful in reducing tumor recurrence after resection of primary tumors in prostate cancer (24), suggesting that immunotherapy is a potential solution to prevent cancer relapse through activation of the inhibited immune system and elimination of residual cancer cells. However, this approach to suppress postsurgical tumor recurrence has yet to be evaluated in TNBC. The aim of this study was to determine the effectiveness of combined immunotherapy with anti-CTLA-4 and anti-PD-1 in preventing TNBC recurrence after mastectomy using a mouse model of TNBC (25) and noninvasive fluorescence and bioluminescence imaging (BLI) methods (26). Our results show that the combined immunotherapy with these immune checkpoint inhibitors can inhibit breast tumor growth, prevent postsurgical cancer relapse, and increase survival of breast tumor bearing mice.
Materials and Methods
Cell culture and establishment of a breast tumor mouse model
The mouse TNBC cell line 4T1-fLuc used in this study was derived from the mouse mammary carcinoma cell line 4T1 (ATCC) transfected with the luciferase gene. As shown in our previous study, orthotopic 4T1 cell-derived tumors are prone to pulmonary metastasis and were used here to establish a TNBC mouse model (25). Cells were cultured in RPMI1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator. The 4T1-fLuc cells (1 × 106) were suspended in PBS (Gibco, Thermo Fisher Scientific) and injected into the left mammary gland or subcutaneously into the right hind back of female BALB/c mice (20–25 g, 5–6 weeks; Vital River Laboratory Animal Technology Co. Ltd.). All experimental protocols were approved by Peking Union Medical College Hospital (PUMCH), and all efforts were made to minimize animal sufferings.
Animal surgery
Surgery was performed when the tumor volume reached about 100 to 150 mm3. Mice were anesthetized with 2% isoflurane and spontaneous breathing was maintained. Fur surrounding the incision site was removed with aseptic preparation of surgical site with twice iodophor disinfectant. One of the two doctors performed surgeries with sterilized surgical instruments in the procedure room. Small incisions were sutured quickly and mice were returned to cages after waking up. BLI (IVIS Imaging Spectrum System) was used to examine tumor residues. The postoperative mice were randomly divided into different treatment groups.
Anti-CTLA-4 and anti-PD-1 treatment
All tumor-bearing mice in need of immunotherapy were randomly divided into four treatment groups: (i) PBS (vehicle), (ii) anti-PD-1, (iii) anti-CTLA-4, and (iv) combined anti-PD-1+anti-CTLA-4 therapeutic group. To evaluate the effect of immunotherapy on preventing postoperative recurrence, 40 mice (n = 10 per group) were randomly divided into the above four groups. Postoperative immunotherapy was started immediately after surgery by intraperitoneal injection of 100 μg anti-CTLA-4 (InVivoMab anti-mouse CTLA-4; BioXCell) and/or 200 μg anti-PD-1 (InVivoMab anti-mouse PD-1; BioXCell) twice a week for 3 weeks; the control group was administered an equivalent volume of PBS. Injections were performed by another doctor blinded to the drug administered. For orthotopic tumor treatment without surgery, mice (n = 10 per group) were injected the same doses of anti-CTLA-4 and/or anti-PD-1 as mentioned above 3 days after tumor cell implantation when a palpable tumor formed.
In vivo monitoring of the immunotherapeutic effect and postsurgical pulmonary metastasis by BLI
At the end of the treatment period, a total of 24 mice (n = 6 per group) were administered D-luciferin solution (40 mg/mL; Biotium) by intraperitoneal injection prior to imaging and sacrificed to examine pulmonary metastasis by BLI. Bioluminescence intensity was expressed as photons (p)/cm2/s.
Body weight and survival monitoring
Mouse body weight was measured using an automatic weighing scale twice a week during treatment. Survival time was recorded for postoperative and orthotopic tumor-bearing mice.
Synthesis and characterization of CTLA-4-IRDye800CW and PD-1-IRDye800CW
The CTLA-4-IRDye800CW was prepared by conjugating IRDye800CW succinimidyl ester (LiCor Biosciences) to anti-CTLA-4 mAb. The connection of IRDye800CW and the anti-CTLA-4 mAb was performed according to the IRDye800CW Labeling Kit from LI-COR Corporate according to the manufacturer's instructions. UV-Vis spectrophotometer was used to determine the IRDye800CW-conjugated protein mAb concentrations and the labeling degree. Briefly, the anti-CTLA-4 mAb was first concentrated using centrifugal Ultra-2 filter of 10 kDa to achieve concentration of ∼8.75 mg/mL. The anti-CTLA-4 mAb (10 nmol, 1 mL, 0.01 mmol/L) was diluted with borate buffer (pH 8.5–9.0) and reacted with aqueous IRDye800CW succinimidyl ester (40 nmol, 0.005 mL, 8 mmol/L), and the resulting solution was incubated at room temperature for 1.5 hours. Finally, the purification was performed by centrifugation filter through 10 kDa and washing repeatedly with PBS (pH 7.4) until no free IRDye800CW was detected in the filtrate. The fluorescence probe was then stored at −4 °C before use. PD-1-IRDye800CW and IgG-IRDye800CW were also prepared using similar method. The characterization of the probes was shown in the Supplementary Information.
Biodistribution and FMI with CTLA-4-IRDye800CW and PD-1-IRDye800CW
The dynamic biodistribution of CTLA-4-IRDye800CW and PD-1-IRDye800CW in 4T1-fLuc breast tumor bearing mice was assessed by noninvasive in vivo FMI using the IVIS Imaging Spectrum System and the corresponding software (version 3.0). BLI was performed prior to FMI to confirm tumor location. FMI was carried out from 0 to 48 hours after tail vein injection of CTLA-4-IRDye800CW and PD-1-IRDye800CW probes; the control group was injected IgG-IRDye800CW. A total of 18 mice (n = 6 per group) were randomly divided into three groups to evaluate FMI biodistribution in vivo. Mice were sacrificed by cervical dislocation 24 hours after in vivo imaging, and tissues including the brain, heart, liver, spleen, kidney, and tumors were harvested for ex vivo FMI. The tumor-to-background ratio (TBR) was calculated according to the following formula: TBR = Fluorescence light intensitytumor/Fluorescence light intensitymuscle.
Multispectral imaging analysis
In the multispectral immunofluorescence staining, tumors dissected after the treatment were fixed in 4% formaldehyde solution, sectioned at 4-μm thickness, and stained with the PerkinElmer Opal Kit (Perkin Elmer). Three tumor specimens were collected in each treatment group and three discontinuous slices were stained for each specimen. The technician randomly selected three views to quantify the percentage of CD3+CD8+ and CD3+CD4+ T cells on each slice. Slides were scanned using the PerkinElmer Vectra system (Perkin Elmer) and images were analyzed with the PerkinElmer advanced image analysis software 3.0. Rabbit antimouse CD8, CD4, and CD3 mAbs (ab209775, ab183685, and ab5690, respectively; Abcam) were used for multispectral immunofluorescence staining.
Statistical analysis
GraphPad Prism (version 5.0) was used for statistical analysis. Mouse survival was evaluated by Kaplan–Meier analysis and log-rank test with Bonferroni correction. The tumor volume was analyzed using a two-way ANOVA with multiple comparisons. One-way ANOVA with Tukey test was used for multiple group comparison. P < 0.05 was considered statistically significant.
Results
Targeted FMI of CTLA-4 and PD-1 expression in 4T1 tumor xenografts
Before injecting CTLA-4-IRDye800CW and PD-1-IRDye800CW fluorescence probes, tumor location was identified by BLI. The characterization of CTLA-4-IRDye800CW and PD-1-IRDye800CW probes were shown in supplemental results (Supplementary Fig. S1–S3). Results of FMI performed at different time points revealed that CTLA-4-IRDye800CW and PD-1-IRDye800CW started to appear at the tumor sites 4-hour postinjection, accumulated from 4 to 8 hours, and then steadily decreased from 12 to 48 hours. At 48-hour postinjection, the CTLA-4-IRDye800CW and PD-1-IRDye800CW signals could still be detected at the tumor location (Fig. 1A). Similarly, in the IgG-IRDye800CW control group, the tumor-localized signal was observed at 4 hours after probe injection and gradually increased from 4 to 8 hours, but cannot be detected at 48-hour postinjection (Fig. 1A). To further validate the in vivo results, mice were sacrificed and tumors and major organs dissected at 24-hour postinjection. Ex vivo FMI analysis indicated that fluorescence signals of the targeted probes in tumors were stronger than that of the control probe (Fig. 1B). Quantification of the TBR revealed that the signals produced by the CTLA-4-IRDye800CW and PD-1-IRDye800CW probes were more specific and stable within tumor tissues than that of the control probe, and their intensity at the peak point (8 hours) was about two times higher (P < 0.05; Fig. 1C). Thus, FMI can be used for direct visualization of dynamic tumor CTLA-4 and PD-1 expression in vivo.
Biodistribution of CTLA-4-IRDye800CW, PD-1-IRDye800CW, and IgG-IRDye800CW in 4T1-fLuc breast tumor–bearing mice. Probes were injected into breast heterotopic tumor–bearing mice (n = 6 per group); fluorescence was analyzed by FMI at the indicated time points. A, In vivo sequential time point imaging. B, Ex vivo biodistribution of fluorescent probes in tumors and organs at 24 hours. C, Quantitative analysis of the tumor-to-background fluorescence ratio in vivo. *, P < 0.05.
Suppression of orthotopic 4T1 tumor growth by the combination of anti-CTLA-4 and anti-PD-1
BLI analysis revealed that bioluminescence intensity was significantly decreased in tumors of mice treated with anti-CTLA-4 or anti-PD-1 compared with control (PBS) mice; the decrease was especially pronounced in the combined treatment group (P < 0.01 vs. control and P < 0.05 vs. single inhibitor groups; Fig. 2A and B). The results of tumor volume measurements were consistent with BLI observations (Fig. 2C). Overall, these findings suggest that the use of CTLA-4/PD-1 immune checkpoint inhibitors as a combined treatment regimen provides effective suppression of breast tumor growth.
The effect of combination therapy with anti-CTLA-4 and anti-PD-1 on orthotopic 4T1 tumor growth. Mice were inoculated with tumor cells into the mammary gland and treated with PBS (control), anti-CTLA-4, anti-PD-1, or anti-CTLA-4+anti-PD-1 for 18 days (n = 10 mice per group). A, Continuous observation of the therapeutic effect by BLI. Bioluminescence intensity (B) and tumor volume (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Analysis of TILs in the tumor microenvironment after immunotherapy
To address the immune mechanisms underlying breast tumor inhibition by anti-CTLA-4/anti-PD-1, we analyzed T-cell infiltration into the tumor microenvironment after immunotherapy by multispectral imaging (Fig. 3A and B). The results showed that mice treated with the immune checkpoint inhibitors had significantly increased presence of CD8+ and CD4+ T cells in tumors compared with control; this effect was the highest in the group receiving both anti-CTLA-4 and anti-PD-1 (P < 0.05 vs. single inhibitor groups; Fig. 3C). Furthermore, the accumulation of CD8+ T cells at the tumor margin was also increased in all treatment groups compared with control and was significantly higher in the combination group compared with anti-PD-1 treatment group (P < 0.05; Fig. 4).
Analysis of TILs by multiplexed immunofluorescence. A, Multispectral imaging after treatment: CD3 (red), CD8 (green), CD4 (cyan), and DAPI (blue). Scale bar, 100 μm. B, Representative image of a tumor with high TIL infiltration after combination immunotherapy. The inset presents a magnified view of an area with high numbers of CD3+CD8+ (arrows) and CD3+CD4+ (asterisks) T cells. C, Quantitative analysis of CD3+CD8+ and CD3+CD4+ T-cell infiltration in tumors (n = 3). *, P < 0.05; **, P < 0.01. ns, not significant.
CD8+ T-cell accumulation at the tumor margin with different treatments. A, Representative images of tumors from the indicated groups. B, Quantitative analysis of CD8+ T cells at the tumor margin. *, P < 0.05. ns, not significant.
Combined application of anti-CTLA-4 and anti-PD-1 reduces orthotopic tumor recurrence and prolongs survival after surgery
Our data indicated that the CTLA-4 and PD-1 inhibitors in combination could synergistically suppress orthotopic breast tumor growth, suggesting a new treatment regimen for TNBC therapy. We further examined the effects of the combination immunotherapy on orthotopically postsurgical tumor recurrence using BLI. The results showed that tumor recurrence was observed 6 days after surgery in 60% mice of the PBS and anti-PD-1 groups; however, bioluminescence intensity in the PBS group increased more rapidly than the anti-PD-1 group (Fig. 5A and B). In the anti-CTLA-4 group, tumor recurrence was fist observed 12 days after surgery in 60% mice, whereas in the combination group, only 10% mice had tumor recurrence at the end of the observation period (18 days; Fig. 5A and B), indicating that the combination immunotherapy could significantly inhibit cancer relapse. Survival analysis further confirmed the effectiveness of anti-CTLA-4/anti-PD-1 combination in suppressing TNBC progression. The survival time in the combination group was significantly prolonged compared not only with control but also with the single inhibitor-treated groups (P < 0.05, Fig. 5C).
Combined treatment with anti-CTLA-4 and anti-PD-1 reduced postsurgical tumor recurrence and prolonged survival. Orthotopic mammary tumors were removed and mice were treated with PBS (control), anti-CTLA-4, anti-PD-1, or anti-CTLA- 4+anti-PD-1 for 18 days (n = 10 mice per group). A, Tumor recurrence after surgery monitored by BLI. B, Quantitative analysis of tumor bioluminescence intensity by BLI. C, Kaplan–Meier survival curves showing treatment response. *, P < 0.05; **, P < 0.01.
As incomplete breast tumor resection may result in the invasion of cancer cells into the surrounding tissues, we also analyzed the effects of the immune checkpoint inhibitors in a subcutaneous tumor model. The results revealed that the combination of immune checkpoint inhibitors could slow tumor growth and in some cases even eliminate residual tumors (Supplementary Figs. S4A and S4B). The combination treatment group also had a better survival rate than the control and single inhibitor groups (Supplementary Fig. S4C). These findings indicate that the combined application of CTLA-4 and PD-1 inhibitors is a promising strategy to prevent postsurgical tumor recurrence in TNBC.
Combination of anti-CTLA-4 and anti-PD-1 prevents lung metastasis of 4T1 tumors
As 4T1 cell-derived breast tumors are characterized by frequent lung metastasis (25), we investigated the effect of immunotherapy on the inhibition of pulmonary metastasis after surgery both in vivo and ex vivo. BLI revealed that in the control group, 5 of 6 mice had metastatic lesions in the lung, which progressed rapidly during the treatment period, whereas in the single inhibitor-treated groups, 4 of 6 mice developed lung metastasis and pulmonary lesions grew slower than in the control group (Fig. 6A and B). In contrast, only 1 of 6 mice appeared to have lung lesions in the combination group at the end of treatment (Fig. 6A and B). Quantitative analysis indicated that the anti-CTLA-4 and anti-PD-1 combination treatment group had the lowest luminescence signal intensity in lung metastatic tumors (P < 0.05 vs. single inhibitor groups; Fig. 6C). There was no significant body weight loss during the treatment course (Supplementary Fig. S5), suggesting that the dosing regimen was relatively well tolerated and safe.
Combined treatment with anti-CTLA-4 and anti-PD-1 prevented lung metastasis of 4T1 tumors. Orthotopic mammary tumors were removed and mice were treated with PBS (control), anti-CTLA-4, anti-PD-1, or anti-CTLA-4+anti-PD-1 for 18 days (n = 6 mice per group). A, Postsurgical pulmonary metastasis analyzed by in vivo BLI. B, Pulmonary metastasis analyzed by ex vivo BLI. C, Quantitative analysis of bioluminescence intensity. *, P < 0.05. ns, not significant.
Discussion
Postoperative recurrence in TNBC remains a challenging problem due to cancer resistance to therapies and/or tumor metastasis. Therefore, novel effective treatment, including combination therapy that can elicit good response in patients with TNBC are urgently needed. Recent clinical trials present compelling evidence that immune checkpoint inhibitors such as anti-CTLA-4 and anti-PD-1 can provide efficient and durable tumor control by stimulating antitumor immune response in patients. In this study, we proved that the combination of anti-CTLA-4 and anti-PD-1 caused synergistic antitumor effects superior to those of the single inhibitors and that the underlying mechanism is based on increased infiltration of CD8+ and CD4+ T cells in breast tumors. Moreover, we found that the combined immunotherapy could also inhibit postsurgical tumor recurrence and metastasis.
Rational combination immunotherapy has already been applied to patients with advanced non–small cell lung cancer and melanoma, who showed longer progression-free survival (PFS) and higher objective response rates after receiving anti-CTLA-4 together with anti-PD-1 compared with those treated with the single inhibitors (27, 28). As breast cancer is considered a low-immunogenicity cancer because of moderate mutational load, it does not elicit a sufficient antitumor immune response, which should be stimulated by appropriate therapy. Our present results indicate that anti-CTLA-4 and anti-PD-1 in combination exhibited significantly stronger suppression of orthotopic breast tumor growth compared with the single inhibitors, indicating a more powerful effect of combination therapy. Moreover, using in vivo and ex vivo FMI we revealed that fluorescently labeled CTLA-4 and PD-1 probes were preferentially accumulated in 4T1 cell-derived tumors, suggesting that immunotherapy with these immune checkpoint inhibitors could be effective.
Currently, response evaluation criteria (RECIST) and WHO classification of solid tumor have become general acceptance criteria despite imperfection. Patients treated by immunotherapy may experience tumor pseudo-progression (29), which has led to the development of immune-specific response guidelines such as immunotherapy RECIST (iRECIST). Many patients benefited from immunotherapy; however, there are no proven biomarkers to exactly predict the effect of immune checkpoint inhibitors (9). A study of immunotherapy in patients with metastatic melanoma treated with anti-PD-1 showed that the intratumoral density of CD8+ T cells could serve as a prognostic criterion of favorable treatment response (9, 30). In patients with TNBC, the density of CD8+ T cells was highly related to TILs, which can predict patients' pathologic complete response (pCR; refs. 9, 31). Similar results were also shown in desmoplastic melanomas and synovial sarcoma (32, 33). Furthermore, the increased CD8+ T cell density at the invasive tumor margin was identified as the best biomarker for treatment response to immune checkpoints inhibitors (30). In our study, we observed that tumor infiltration of CD8+ and CD4+ T cells and accumulation of CD8+ T cells at the tumor margin were increased in mice treated with the combination of anti-CTLA-4 and anti-PD-1 compared with monotherapy, suggesting that the effects of the combination immunotherapy in TNBC are due to the activation of TILs.
A previous study showed that a lower recurrence rate and longer pCR after surgery correlated with higher infiltration of TILs (9, 31, 34). Consistent with these data, our results indicated that the combined treatment with CTLA-4 and PD-1 inhibitors prolonged survival compared with monotherapy, which corresponded to increased CD8+ T-cell infiltration. For the patients with metastatic breast cancer, primary tumor resection can reduce tumor burden and improve the survival time (35), followed by chemotherapy or radiotherapy aimed to prevent tumor recurrence and metastasis. In our study, we observed that the dual immunotherapy can inhibit cancer relapse and pulmonary metastasis characteristic for TNBC and improve survival after the removal of primary tumors, indicating that immunotherapy with CTLA-4/PD-1 inhibitors can eliminate residual lesions. The majority of lung metastasis cases are currently treated with radiotherapy, chemotherapy, and video-assisted thoracoscopic surgery (36), and our results on the regression of pulmonary lesions suggest that the combination immunotherapy may provide a new treatment method for patients with pulmonary micro-metastasis to improve PFS.
However, it should be noted that the increasing application of anti-CTLA4 and anti-PD-1 has raised a question of immune-related adverse events (irAE). Previous studies revealed that 75% of patients treated with CTLA-4 inhibitors developed all-grade irAEs, which were dose dependent (37), whereas patients treated with PD-1/PD-L1 inhibitors had a ≤30% risk of developing irAEs (38–40). The irAEs were shown to increase by the dual immunotherapy. Thus, the incidence of diarrhea, which is one of the most common irAEs, was higher in patients receiving the combination of anti-CTLA-4 and anti-PD-1 inhibitors (41), which can also lead to a higher risk of pneumonitis (42), arthritis (43), and neurologic complications (44) compared with single-agent therapy. Therefore, long-term management of irAEs should be considered for patients treated with combinatorial immunotherapy.
Although orthotopic 4T1 mouse model is a powerful representative to research immunotherapy in TNBC, it does not represent every subtypes of breast cancer. Certain types of breast cancer respond differently even to the same immunotherapy inhibitors. In summary, our results revealed that the dual immunotherapy with anti-CTLA-4 and anti-PD-1 can suppress orthotopic breast tumor growth and prevent postsurgical cancer relapse, resulting in extended survival of 4T1-fLuc breast tumor bearing mice. In this study, we used highly sensitive optical imaging methods to monitor the therapeutic effect of combination immunotherapy, including multispectral imaging technology which enabled detection of different types of immune cells infiltrating tumor sites. Our study demonstrates that the combined application of anti-CTLA-4 and anti-PD-1 provides a new treatment strategy to prevent postoperative tumor recurrence by promoting infiltration of CD4+ and CD8+ T cells in tumors. Thus, the combinatorial immunotherapy, in particular with anti-CTLA-4 and anti-PD-1, is a promising treatment strategy in TNBC, which should be further evaluated in clinics.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: Y. Du, J. Tian, H. Xue
Development of methodology: Y. Du, J. Tian
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Sun, W. Zhang, Y. Li
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Sun
Writing, review, and/or revision of the manuscript: T. Sun, Z. Jin, Y. Du, J. Tian, H. Xue
Study supervision: Y. Du, J. Tian, H. Xue
Acknowledgments
This work was supported by the grants from the National Key Research and Development Plan of China (grant number 2017YFA0205200); National Natural Science Foundation of China (grant numbers 81371608, 81871514, 81470083, 81227901); Beijing Natural Science Foundation (grant number 7172170); National Public Welfare Basic Scientific Research Project (grant number 2017PT32004); and Chinese Academy of Medical Sciences Students' Innovation Foundation (grant number 1007-1002-1-12). The authors would like to acknowledge the instrumental and technical support of multimodal biomedical imaging experimental platform, Institute of Automation, Chinese Academy of Sciences.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Mol Cancer Ther 2020;19:802–11
- Received May 18, 2019.
- Revision received September 25, 2019.
- Accepted November 25, 2019.
- Published first December 3, 2019.
- ©2019 American Association for Cancer Research.
References
Volume 19, Issue 3
