Vascular-targeted TNFα and IFNγ inhibits orthotopic colorectal tumor growth
© The Author(s) 2016
Received: 17 March 2016
Accepted: 15 June 2016
Published: 24 June 2016
Tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) were originally identified to show potent anti-tumor activity and immunomodulatory capability. Unfortunately, several clinical studies of relevant cancer therapy did not observe significant response in maximum tolerated dose whether given alone or in combination. We have identified a tumor vasculature homing peptide (TCP-1 peptide) which targets only the vasculature of colorectal tumors but not normal blood vessels in animals and humans. In the current study, the antitumor effect of TCP-1/TNFα and TCP-1/IFNγ alone or in combination was studied in orthotopic colorectal tumor model.
TCP-1/TNFα and TCP-1/IFNγ recombinant proteins were prepared and i.v. injected to study the in vivo anticancer effect in orthotopic colorectal tumor model. Tumor apoptosis was determined by TUNEL staining and cleaved caspase-3 immunofluorescent staining. Tumor infiltrating lymphocytes were analyzed by immunofluorescent staining and flow cytometry. Western-blot was performed to examine the expression of proteins. Cell apoptosis was measured by Annexin V/PI flow cytometry.
Targeted delivery of TNFα or IFNγ by TCP-1 peptide exhibited better antitumor activity than unconjugated format by inducing more tumor apoptosis and also enhancing antitumor immunity shown by increased infiltration of T lymphocytes inside the tumor. More importantly, combination therapy of TCP-1/TNFα and TCP-1/IFNγ synergistically suppressed tumor growth and alleviated systematic toxicity associated with untargeted therapy. This combination therapy induced massive apoptosis/secondary necrosis in the tumor.
Taken together, our data demonstrate TCP-1 is an efficient drug carrier for targeted therapy of colorectal cancer (CRC). TCP-1/TNFα combined with TCP-1/IFNγ is a promising combination therapy for CRC.
KeywordsVascular targeting TNFα IFNγ Colorectal cancer Drug delivery
Despite a slight decline in the mortality rate over the past decade, colorectal cancer (CRC) remains the third most common cancer and a leading cause of cancer deaths worldwide. Tumor necrosis factor alpha (TNFα) consists of three noncovalently linked TNFα monomers, ~17.5 kDa each, which forms a compact bell-shaped homotrimer [1, 2]. TNFα is a powerful anti-tumor cytokine as well as a potent inflammatory cytokine which can induce complex immune responses . It exerts its anti-tumor activity through complex mechanisms including induction of inflammatory and immune responses, tumor cell apoptosis/necrosis and extensive thrombosis and destruction of tumor vasculature [4, 5]. Although TNFα shows potent anti-tumor activity and produces impressive results in various animal cancer models, clinical use of TNFα as an anticancer drug is hampered by severe systemic toxicity [6, 7]. To date, the clinical use of TNFα has been limited to cancer treatment in the isolated limb perfusion (ILP) setting for soft tissue sarcoma and melanoma intrinsic metastases confined to the limb [8, 9].
It has been shown that tumor vascular-targeted delivery of TNFα is capable of increasing tumor concentration of TNFα and directing TNFα specifically to the tumor site [6, 10–12]. This strategy has resulted in several tumor vascular ligands fused to TNFα for cancer therapy research, and even clinical trials are underway [13–16]. We have previously established an orthotopic colorectal tumor model and identified a cyclic peptide known as TCP-1. This peptide can specifically target the vasculature of orthotopic colorectal tumors . Targeted delivery of TNFα by TCP-1 peptide displayed more potent antitumor activity than unconjugated TNFα by inducing more apoptosis and destructing neovasculature in orthotopic colorectal tumors at 24 h with the dose 5 μg/mouse. Furthermore, low-dose TCP-1/TNFα (1 ng/mouse) potentiated the antitumor effect of 5-fluorouracil (5-FU) by normalizing the tumor vasculature, facilitating the infiltration of immune cells to the tumor as well as improving 5-FU penetration into the tumor mass. More importantly, TCP-1/TNFα attenuated the immunosuppressing effects of TNFα in bone marrow and spleen with marked reduction in systemic toxicity . These findings provide a solid proof that TCP-1/TNFα could be used to treat CRC through synergistic effects with standard chemotherapeutic agents as targeted therapy.
Interferon gamma (IFNγ) is a pleiotropic cytokine produced by immune cells and plays physiologically important roles in promoting innate and adaptive immune responses [18, 19]. It is also indicated that IFNγ could induce the antiproliferative and proapoptotic effects on various tumor cells, repress tumor angiogenesis and produce antitumor cellular responses through activation of natural killer cells, macrophages and CD8+ cytotoxic T cells . But similar to TNFα, IFNγ lacks a unique selectivity as an antitumor agent and can bind to most of cells due to ubiquitous expression of its receptor . Therefore, IFNγ failed in several clinical trials as a sole antitumor agent due to low maximal tolerated dose in patients [19, 21]. Tumor vasculature targeting approach also has been used to deliver IFNγ to tumor tissues for targeted therapy. Several studies of targeted IFNγ have obtained promising results [21–23]. Low dose of targeted IFNγ can produce significant tumor growth inhibition in different tumor types .
TNFα and IFNγ have been shown to have synergistic antitumor effect in a few cell line studies [24–27]. Relevant study also demonstrated that co-administration of targeted TNFα and nontargeted IFNγ resulted in significant synergistic tumoricidal activity in renal cell carcinoma. However, early clinical studies failed in patients with advanced gastrointestinal cancers through co-administration of non-targeted TNFα and IFNγ [28–31]. Targeted delivery of TNFα or IFNγ not only reduces the effective dose of individual cytokines and enhances their local concentration in tumors, but also enlarges their therapeutic window. More importantly it is likely that the combination of targeted TNFα and IFNγ might produce more effective antitumor activity than either agent given alone. This could further reduce the dosage of each agent, which will markedly reduce the potential side effects produced by these agents and make the treatment more viable in cancer patients. Here, we explored the antitumor effect of targeted TNFα or IFNγ in CRC through using fusion protein of TNFα or IFNγ with TCP-1 peptide, a novel ligand which can specifically bind to the vasculature of orthotopic colorectal tumors [24–27]. Furthermore, the antitumor activity and mechanism of action for each fusion protein alone or in combination were also assessed.
Cells and animals
The murine CRC cell Colon 26 was obtained from the Health Science Research Resources Bank (Osaka, Japan). Mouse fibroblast cell line L929 for IFNγ activity determination was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in RPMI 1640 supplemented with 100 U/mL penicillin G, 100 μg/mL streptomycin, and 10 % fetal bovine serum (FBS) and maintained at 37 °C in a humidified atmosphere containing 5 % CO2. Male BALB/c mice aged 6 weeks were maintained at the Chinese University of Hong Kong Animal Facility. Animal experiments in this project had been approved by the Laboratory Animals Ethics Committee of the Chinese University of Hong Kong.
Reagents and antibodies
Anti-mouse CD31 (553274), anti-mouse CD4 (553043) and anti-mouse CD8 (550281) monoclonal antibodies were purchased from BD Pharmingen. Anti-caspase-3 (9662), anti-PARP (9542), anti-β-actin (4967) antibodies were obtained from Cell Signaling Technology. PE-Cy™ 7 anti-mouse CD3 (100320), PE anti-mouse CD4 (103405) and FITC anti-mouse CD8a (100706), APC anti-mouse CD34 (128612), PE-Cy™ 7 anti-mouse CD45 (103222) antibodies, and isotype IgG were purchased from Biolegend. In Situ Cell Death Detection Kit (Fluorescein) was brought from Roche. Dead Cell Apoptosis Kit with Annexin V FITC and PI was purchased from ThermoFisher.
Orthotopic CRC model
Animals were anesthetized with a mixture of ketamine and xylazine. A 29-gauge syringe was used to inject 2.5 × 104 colon 26 cells, suspended in RPMI 1640 with 10 % FBS, submucosally into the distal, posterior rectum. The injection was performed approximately 1–2 mm beyond the anal canal and into the rectal mucosa, which minimized the chance of establishing anal tumors. Tumors were developed at 1.5–2 weeks. Successful models were used for various in vivo anti-cancer experiments after tumors were formed.
The IFNγ and TCP-1/IFNγ plasmids were constructed using method similar to that previously described for TNFα and TCP-1/TNFα . The mouse IFNγ fragment was amplified by PCR from mouse spleen cDNA with primer pair 5′-CAT GGT ACC CAC GGC ACA GTC ATT GAA AGC CTA-3′and 5′-CAT GGA TCC TCA GCA GCG ACT CCT TTT CCG CTT C-3′ flanked by KpnI and BamHI restriction enzyme sites. The PCR product was then cloned into a modified pET-14b vector. Subsequently, the TCP-1 gene was introduced into constructed pET-14b/IFNγ plasmid by PCR-mediated site-directed mutagenesis with the primer pair 5′-TTT TCG CAT TGC GGA GGT ACC CAC GGC ACA GTC ATT GAA AGC CTA-3′ and 5′-AGG ACT AGG CGT ACA AGC GGG CCC CAT ATG GCT GCC GCG CGG-3′. All the constructs were finally confirmed by DNA sequencing. The flow chart of plasmid construction is shown in Additional file 1: Fig. S1.
Protein expression, purification and verification
The protein expression and purification procedure was performed as previously described . Proteins with >90 % purity based on SDS-PAGE image were used for various examinations. The quantitative chromogenic Limulus amebocyte lysate (LAL) test was used to quantitate Gram-negative bacterial endotoxin. The endotoxin concentration in the purified proteins used in the study is approximately 0.1 EU/μg.
IFNγ activity assay
The activities of IFNγ and TCP-1/IFNγ were determined in L929 and Colon 26 cells by MTT assay. Briefly, 2 × 103 cells/well were seeded in a 96-well plate with 100 μL growth medium. After 24 h, different concentrations of IFNγ and TCP-1/IFNγ were added for an additional 72 h. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to detect the cell viability.
To investigate whether TCP-1 peptide is able to deliver IFNγ to tumor blood vessels, 50 nmol TCP-1/IFNγ fusion protein or equal molar IFNγ was i.v. injected through tail vein into mice bearing orthotopic CRC to examine the distribution of IFNγ. The proteins were allowed to circulate for 1 h. Tumor and control tissues were collected and prepared for frozen section. Blood vessels were stained with anti-CD31 antibody. IFNγ signal was amplified by biotin-labeled anti-His tag antibody. To study the short-term effect of IFNγ or TCP-1/IFNγ, 5 μg of IFNγ or equal dose of IFNγ in TCP-1/IFNγ, was i.v. injected into mice bearing orthotopic CRC. Animals were sacrificed 24 h after injection. For antitumor experiment, mice bearing orthotopic CRC were randomized into different group (n ≥ 4): PBS, TNFα (1 μg/mouse), TCP-1/TNFα (1 μg TNFα/mouse), IFNγ (5 μg/mouse), TCP-1/IFNγ (5 μg IFNγ/mouse), TNFα (0.5 μg/mouse) plus IFNγ (2.5 μg/mouse) and TCP-1/TNFα (0.5 TNFα μg/mouse) plus TCP-1/IFNγ (2.5 μg IFNγ/mouse). Treatment was given by i.v. injection again through tail vein once. Mice were euthanized at 7 days after drug administration. Tumors and control organs were dissected and prepared for frozen sections. Tumor microvessel density and apoptosis were assessed.
At the end of experiment, mice were heart-perfused with 4 % neutral-buffered paraformaldehyde and tumors were obtained for frozen section. Frozen sections were processed and immunofluorescent and immunohistochemical staining were performed as previously described .
Western blots and flow cytometry
Western blots and flow cytometry were performed following the standard laboratory protocol as previously reported .
Hochest and PI double staining
Frozen tissue sections were washed in PBS and double-stained with propidium iodide (PI, 2.5 mg/mL) and Hoechst 33342 (2.5 mg/mL) for 10 min. Intact blue nuclei, condensed/fragmented blue nuclei, condensed/fragmented pink nuclei, and intact pink nuclei were considered viable, early apoptotic, late apoptotic and necrotic cells, respectively.
The results are expressed as mean ± SEM. GraphPad Prism 5 (GraphPad Software) was used for statistical analysis. Two tailed Student’s t test was applied for paired data analysis. For the in vivo treatment experiment with TCP-1 fusion proteins, comparisons among all groups were analyzed by one-way ANOVA followed by the Tukey’s test. P value below 0.05 was considered statistically significant.
Targeted delivery of IFNγ by TCP-1 in the orthotopic CRC model
TCP-1/TNFα or TCP-1/IFNγ given alone inhibited orthotopic colorectal tumor growth
TCP-1/TNFα combined with TCP-1/IFNγ dramatically inhibited orthotopic colorectal tumor growth
TCP-1/TNFα and TCP-1/IFNγ enhanced antitumor immunity
Mechanism of action of TCP-1/TNFα combined with TCP-1/IFNγ
Tumor vasculature undergoing angiogenesis expresses specific endothelial surface markers which are absent or barely detectable in mature vessels . Peptide has many advantages over antibody as drug carrier . Therefore, tumor-homing peptides (THPs) that target tumor vasculature are important and promising imaging agent and drug delivery vectors . Using phage display biopanning, we previously identified a novel cyclic peptide TCP-1 which can specifically bind to the vasculature of colorectal tumor in both animals and humans but not normal blood vessels. We have also shown that TCP-1 is useful for targeted delivery of imaging agent and pro-apoptotic peptide . This peptide is advantageous over other THPs because it exhibits a unique homing ability to the vasculature only in the CRC, indicating its specificity and accuracy as a carrier in CRC diagnosis and therapy. The TNFα and IFNγ synergism has been reported under many biological conditions including their tumor inhibitory effect [25, 26, 36–38]. Early clinical trials conducted in 1990s tried to use the combination of these two cytokines to treat advanced gastrointestinal cancer patients [27–30]. Unfortunately only modest beneficial effects but severe side effects were seen. In vivo study showed that combination of nontargeted TNFα and IFNγ often show significant toxicity . Targeted TNFα and nontargeted IFNγ exerted additive tumoricial activity in renal cell carcinoma . However, direct proof of the antitumor effect of targeted TNFα combined with targeted IFNγ in vivo has not been reported before.
In this study, we extended to study the effect of TCP-1/TNFα and TCP-1/IFNγ either alone or in combination in orthotopic colorectal tumor model in immune-competent mice. We found that TNFα (1 μg/mouse) or IFNγ (5 μg/mouse) slightly increased tumor apoptosis while conjugation with TCP-1 peptide significantly inhibited tumor growth and increased tumor cell apoptosis. TNFα exerts different actions in cancer therapy depending on the dosage used . High dose of TNFα mainly inhibits tumor angiogenesis, leading to vessel destruction which decreases the blood flow and oxygen required for the progression of tumor growth. Low dose of TNFα could induce vessel remodeling and increase vessel perfusion, thereby enhancing drug accumulation in the tumor. Our previous study has demonstrated that low dose TCP-1/TNFα could normalize tumor blood vessel, enhance the intratumoral accumulation of anticancer drug 5-FU, thus potentiating its antitumor activity . In the present study, there was no significantly vessel destruction observed for TNFα or TCP-1/TNFα. Our previous result demonstrated that TNFα or TCP-1/TNFα at 5 μg/mouse could lead to acute vessel destruction. We here chose 1 μg/mouse for TNFα which could partly inhibit tumor growth and avoid masking the action of combined treatment of TCP-1/TNFα and TCP-1/IFNγ. This dosage may not be sufficient to induce vessel destruction. For IFNγ, consistent with previous findings that a dose–response curve of IFNγ-NGR is bell-shaped , our result also showed that TCP-1/IFNγ achieved optimal antitumor effect at the dose of 5 μg/mouse. Elevating the dose did not ensure better response. In addition to inducing tumor apoptosis, TCP-1/TNFα or TCP-1/IFNγ also increased the infiltration of CD8+ and CD4+ T cell in the tumor leading to enhanced antitumor immunity. Previous study has shown that the antitumor effect of targeted IFNγ in animal study mainly involves inhibition of angiogenesis and induction of apoptosis but not infiltration of immune cells [22, 40], which is different from our result. This difference may be due to the different dose, treatment time and animal model.
Most interestingly, combination treatment by TCP-1/TNFα and TCP-1/IFNγ dramatically inhibited tumor growth. At the beginning we used TCP-1/TNFα (1 μg/mouse) combined with TCP-1/IFNγ (5 μg/mouse). The antitumor effect was very drastic, however a little myelosuppression was observed (data not shown). After that, we used half the dose: TCP-1/TNFα (0.5 μg/mouse) combined with TCP-1/IFNγ (2.5 μg/mouse). The inhibitory effect of combination treatment on tumor growth was dose-dependent (data not shown) and no myelosuppression or toxicity to other organs was observed for the latter combination. TCP-1/TNFα and TCP-1/IFNγ combined treatment also significantly improved maximum tolerance of two cytokines related to side effects compared with untargeted TNFα combined with IFNγ indicating alleviation of systematic toxicity. Morphologically, condensed and fragmented nucleus (pyknosis and karyorrhexis) were found in high percentage (>90 %) inside the tumor treated by combined treatment which might be due to late apoptosis or necrosis. Mechanism study using mouse colon cancer cells, colon 26 cells reveals that the cells treated with TNFα and IFNγ pass through early apoptosis to late apoptosis/secondary necrosis but not through primary necrosis.
Although we have shown that combined treatment of TCP-1/TNFα and TCP-1/IFNγ induced late apoptosis/secondary necrosis, the molecular mechanism remains elusive. According to previous studies, TNFα induced NF-κB signaling could counteract TNFα-induced apoptosis [41, 42] while IFNγ could inhibit NF-κB activation and the expression of downstream apoptosis inhibitors, finally sensitizing the cancer cells to TNFα treatment [24, 26]. However, our results showed that the expression of apoptosis inhibitor downstream of NF-κB including XIAP and c-IAP-1 were not changed by either TNFα or combined treatment (Additional file 6: Fig. S6). It has also been proposed that TNFα and IFNγ synergistically inhibit cancer cell growth because IFNγ increases the expression of TNFα receptors [43, 44]. However, it is also argued that increase of TNFα receptors is not the major mechanism underlying the synergism between TNFα and IFNγ. STAT1/IRF-1 pathways initiated by IFNγ had been shown to be important in TNFα and IFNγ synergism in inducing cervical cancer cell apoptosis . However, neither STAT1 nor IRF-1 could totally explain the priming effect of IFNγ in TNFα-indiced apoptosis. It seems that the TNFα and IFNγ synergism requires multiple factors, and research evidence so far has not pointed to a conclusion. Further studies are warranted to delineate the potential molecular mechanism.
Our results demonstrate for the first time that TCP-1/TNFα and TCP-1/IFNγ combination is very promising as potential CRC therapy. The synergistic antitumor activity of TNFα combined with IFNγ has been exploited in early clinical trials, but the result was disappointing due to systematic toxicity and limited beneficial effect. Our study could overcome this drawback by enhancing the anticancer action of TNFα combining with IFNγ so that lower doses of both cytokines can be given to patients to achieve promising outcome with less systemic side effects. Since both cytokines are being used in human to treat different diseases, the approach suggested from the current study would provide the likelihood of using drug combination of TCP-1/TNFα and TCP-1/IFNγ for future clinical trials to treat CRC.
tumor necrosis factor alpha
isolated limb perfusion
nuclear factor kappa-light-chain-enhancer of activated B cells
X-linked inhibitor of apoptosis protein
cellular inhibitor of apoptosis protein-1
signal transducer and activator of transcription 1
interferon regulatory factor 1
JS carried out most of the experiment and drafted the manuscript. ZJL and WKKW and CHC contributed to the idea and manuscript revision. LFL contributed to the flow cytometry experiment. LL prepared some of the recombinant protein. LL, ZGX, WH and KMC helped in animal experiment. LZ and MXL helped in data analysis. All authors read and approved the final manuscript.
We thank Ms. Chun Yin for her technical support in flow cytometry.
The authors declare that they have no competing interests.
Availability of data
The datasets supporting the conclusions of this article are included within the article (and its additional files).
The study was supported by the Innovation and Technology Support Programme and the General Research Fund from the Innovation and Technology Commission, the Research Grant Council from Hong Kong and the start-up fund from Southwest Medical University. We thank Chun Yin for the technical assistance in this study.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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