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Additional file 1. Supplementary figures and supplementary table captions

ObjectiveTo investigate the effect of the 131I-labeled high-affinity peptides Caerin 1.1 and Caerin 1.9 for the treatment of A549 human NSCLC cells.Methods① 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and plate clone formation assays were performed to confirm the in vitro anti-tumor activity of Caerin 1.1 and Caerin 1.9. ② Chloramine-T was used to label Caerin 1.1 and Caerin 1.9 with 131I, and the Cell Counting Kit 8 assay was performed to analyze the inhibitory effect of unlabeled Caerin 1.1, unlabeled Caerin 1.9, 131I-labeled Caerin 1.1, and 131I-labeled Caerin 1.9 on the proliferation of NSCLC cells. An A549 NSCLC nude mouse model was established to investigate the in vivo anti-tumor activity of unlabeled Caerin 1.1, unlabeled Caerin 1.9, 131I-labeled Caerin 1.1, and 131I-labeled Caerin 1.9.Results① Caerin 1.1 and Caerin 1.9 inhibited the proliferation of NSCLC cells in vitro in a concentration-dependent manner. The half-maximal inhibitory concentration was 16.26 µg/ml and 17.46 µg/ml, respectively, with no significant intergroup difference (P>0.05). ② 131I-labeled Caerin 1.1 and 131I-labeled Caerin 1.9 were equally effective and were superior to their unlabeled versions in their ability to inhibit the proliferation and growth of NSCLC cells (P>0.05).Conclusions131I-labeled Caerin 1.1 and 131I-labeled Caerin 1.9 inhibit the proliferation and growth of NSCLC cells and may become potential treatments for NSCLC.

Additional file 1: Table S1. Enrichment analysis of KEGG pathways by using GSVA.

LL-37, also called cathelicidin, is an important part of the human immune system, which can resist various pathogens. A plethora of experiments have demonstrated that it has the multifunctional effects of immune regulation, in addition to antimicrobial activity. Recently, there have been increasing interest in its immune function. It was found that LL-37 can have two distinct functions in different tissues and different microenvironments. Thus, it is necessary to investigate LL-37 immune functions from the two sides of the same coin. On the one side, LL-37 promotes inflammation and immune response and exerts its anti-infective and antitumor effects; on the other side, it has the ability to inhibit inflammation and promote carcinogenesis. This review presents a brief summary of its expression, structure, and immunomodulatory effects as well as brief discussions on the role of this small peptide as a key factor in the development and treatment of various inflammation-related diseases and cancers.

Effective cancer therapy is one of the biggest global challenges. Conventional cancer therapies have been at the forefront of combating cancers, but more evidence showed considerable side effects, limiting their use. There are various new therapies in development, but combined approaches for treating cancer are much expected. Natural herbs had been traditionally in use for cancer therapy in most parts of the world. In this review, we have examined ten commonly used Chinese herbs that have, for centuries, shown effectiveness in treating cancers. They demonstrated the abilities to promote the apoptosis of cancer cells, inhibit their metastasis, activate the patient’s anticancer immunity, and synergistically increase the efficacy of conventional chemotherapy and radiation therapy when used in combination. Clinical experiences had proved that these herbs and their bioactive compounds were effective against a plethora of cancers through a variety of mechanisms, effectively improving patients’ quality of life without significant side effects. These advantages indicate that there are huge potentials in the development of Chinese herbs into cancer medicine as part of a promising, holistic cancer treatment modality.

Background: Cancer is a class of diseases characterised by uncontrolled proliferation of cells. Cancer represents as a major public health problem accounting for approximately 8.8 million deaths each year worldwide. Current treatments include surgical removal of tumour mass, chemotherapy, radiotherapy, or a combination of these therapies. Often these options result in side effects that can be quite detrimental to the patients. Therefore, new treatments are urgently needed. Cancer therapeutic vaccines are aiming at killing tumour cells without affecting normal cells. Vaccine induced cytotoxic T lymphocyte (CTL) responses are pivotal for the killing tumour cells. Interleukin 10 (IL-10) is a cytokine with multiple biological functions. Blocking IL-10 signalling with an anti-IL-10 receptor antibody at the time of immunisation drastically increases vaccine induced immune responses and prevents tumour growth in animal models. However, it is not clear whether there are unwanted side effects with such a blockade, and no clinical grade IL-10 inhibitor for human is available. Advanced cancers show a significant increase in angiogenesis resulting in the formation of abnormal blood vessels, which restrict blood supply and result in reduced oxygen levels within the cancer. Therefore, some areas of the late stage tumour are hypoxic and necrotic. Also, the solid tumour microenvironment (TME) is immunosuppressive. Our laboratory has developed an anaerobic Clostridium ghonii strain. When these clostridial spores are administered and regerminated in hypoxic and necrotic cancer central region, they cause significant oncolysis, leading to tumour regression in animal models. The oncolytic activity may also destroy the TME. Hypothesis and Aims: The hypothesis of the project is (1) Blocking IL-10 at the time of immunisation should increase vaccine induced T cell responses without eliciting unwanted side effects; (2) Humanised and active IL-10 inhibitors could be designed by using computerised programs; (3) Clostridial spores induce oncolysis of cancer should also improve immune cell penetrations to cancer in situ, thus achieving a robust anti-cancer immune response. To test these hypothesis, the thesis is specifically aimed: 1) To investigate whether an antibody-mediated blocking of IL-10 signalling at the time of an immunisation will generate any unwanted side effects; 2) To establish whether new anti-IL-10 peptides designed through computer modelling are bioactive in both vitro and in vivo; 3) To determine whether oncolytic events in anaerobic Clostridium ghonii spore treatment of modelled cancers will improve the immune suppressive TME. Results and discussion: (1) In a mouse TC-1 tumour model, blocking of IL-10 with an antibody at the time of immunisation increases the numbers of IL-10 producing CD4+ T cells in the spleen and draining lymph nodes, and does not result in blood cell infiltration to the intestines and does not cause intestinal pathological changes. These results indicate that immunisation with an IL-10 inhibitor may facilitate a generation of safe and effective therapeutic vaccines against chronic viral infection and cancer. (2) Computer designed Peptides P1, FFKKF FKKFF KKFFK K-OH and P2, FFRRF FRRFF RRFFR R-OH are based on the hydrophobic and hydrophilic pattern of the IL10R-binding helix with IL-10. It could be bound with either IL- 10R1 or IL-10, and inhibit inflammatory signals for a long duration and result in negligible cytotoxicity in vitro. Furthermore, P2 can enhance the vaccine induced antigen specific CD8+ T cell’s responses in mice. As evidenced by both experimental and molecular dynamic simulation, the N-terminal hydrophobic peptide constructed with repeating hydrophobic and hydrophilic pattern of residues is more likely to inhibit IL-10. In addition, the sequence length and the ability of protonation are also important for inhibition activity. (3) Intravenous administration of a derivative of Clostridium ghonii (DCG) spore leads to pro-inflammatory responses characterised by increased levels of IFN-γ or/and Interleukin-9 (IL-9), depending on the dosage of DCG administration. Antigen specific CD8+ T cell responses are elicited after DCG spore administration, and IFN-γ secreting T cells were attracted to the tumour site. Conclusion: Taken together, blocking IL-10 signalling at the time of immunisation does not increase unwanted side effects in mice; a peptide P2 designed through computer modelling is able to inhibit IL-10-mediated signalling both in vitro and in vivo; the length of the peptide and the ability of protonation are critical for the peptide’s function. Finally, systematic administration of DCG spore attracts more IFN-γ secreting T cells to the tumour site. Thus, it is believed that if administration of DCG spore is combined with a therapeutic vaccine, more antigen specific T cells may be attracted to the tumour site, therefore a better outcome for cancer treatment could be achieved. In this project, it is the first time to demonstrate that blocking IL-10 at the time of immunisation increases immune response without significant side effects; Also, the newly developed peptide based IL-10 signalling inhibitors are active, paving the way for the testing of a humanised IL-10 inhibitor; the project also showed a Clostridium ghonii spore-mediated oncolysis could disrupt the TME, may improve vaccine mediated anti-cancer immune responses. If such approaches are combined, an exciting new way of cancer therapy may be developed for advanced cancers.

Interleukin 10 is an anti-inflammatory cytokine which limits immune responses to both self and foreign antigens. Blocking IL10 at the time of immunization increases cytotoxic T cell responses in antigen experienced host, a situation similar to therapeutic vaccination for cancer and chronic viral infection, where patients usually develop ineffective immune responses to tumour or viral antigens before immunotherapy starts. Graphene oxide (GO) is a nano material often used for drug delivery and tissue engineering. In the current paper, we demonstrated that GO is able to absorb anti-IL10 receptor antibodies. The anti-IL10R antibodies absorbed in GO are slowly released and the release of absorbed antibodies is pH dependent. GO absorbed anti-IL10R antibodies are bioactive both in vitro and in vivo. GO absorbed anti-IL10R antibodies are more efficient than free anti-IL10R antibodies at eliciting LPS stimulated CD8 T cell responses. Our results suggest that GO is able to absorb anti-IL10R antibodies and absorbed anti-IL10R antibody may be useful as an adjuvant for vaccination and ideal for delivering to tumour site, and breaking of suppressive tumour environment.