1. IntroductionHuman papillomavirus (HPV) infection-related malignancy remains a severe public health problem worldwide, although HPV prophylactic vaccine has been introduced for more than a decade [1,2]. Worldwide, infection is responsible for approximately 15% of all new cancer cases annually, while HPV infection is the second most important cancer-causing infection, which accounted for 28.8% of all infection-related cancers in 2012 [3,4]. This proportion increased to 31.4% in 2018 [5]. Infections with HPV16 and 18 are responsible for 70% of cervical cancers (CC), which are the fourth most common cancer affecting women worldwide [6]. Moreover, head and neck epithelial carcinoma is increasingly attributed to infection with high-risk HPV, especially HPV16, in both developed and undeveloped countries [7]. Besides, virtually all HPV-attributable cancer in men is caused by infections with HPV16 and 18 [5]. HPV infection-related cancers are most severe in developing countries, where HPV prophylactic vaccination rates are low [8,9].Clinically, the International Federation of Gynecology and Obstetrics (FIGO) Cervical cancer staging system is an effective prognostic factor in patients with cervical cancer and useful guidance for treatment [10]. Stage I CC cells have grown from the surface of the cervix into deeper tissues. Stage II CC (4 cm or larger) grows beyond the cervix and uterus but has not spread to the walls of the pelvis or the lower part of the vagina [11]. Surgery is often the treatment option followed by chemotherapy for these stages; however, the immunopathological profiles between different stages have been less studied.Immunotherapy is becoming a routine modality for the management of cancers, following the introduction of immune checkpoint inhibitors and the transfer of CAR-T cells for cancer management [12]. However, the efficacy of immune checkpoint inhibitor monotherapy, such as the blockade of PD-1 [13] and CTLA-4 [14], is relatively low in the management of advanced CC [15]. Therapeutic vaccines aiming at eliciting tumour-specific T cells have been shown partially effective in CIN3 and VIN3 pre-cancer, but are ineffective against advanced cervical cancers [16,17].In both prophylactical and therapeutic settings, therapeutic vaccination with the simultaneous blocking of interleukin 10 (IL-10) increased antigen-specific CD8+ T cell responses, which consequently improved tumour growth inhibition [18]. The efficacy of this therapy strategy is superior to that of the same vaccination without IL-10 signalling blockade [19]. PD-1 blockade combined with therapeutic vaccines are synergistic in animal models and clinical trials [20,21,22]. The efficacy of PD-1 blockade and therapeutic vaccination becomes more significant after intra-tumoral injection of host-defence caerin 1.1/1.9 peptides [23].Caerin 1.1/1.9 peptides are able to inhibit the proliferation of TC-1 [24,25], HeLa [26] and A549 [27] cells in vitro, and induce cell apoptosis, probably through the stimulation of the TNF-α signalling pathway [26,28]. Caerin 1.1/1.9 inhibited TC-1 tumour growth and modified the functions of tumour infiltrating immune cells, such as T cells, NK cells, and dendritic cells in vivo [23,29], and more activated CD8+ T cells and NK cells were recruited to the tumour sites [23,28,29]. Caerin 1.1/1.9 also modulated macrophage heterogeneity within the tumour, drastically reducing the M2 type while increasing the M1 type macrophages [23,29].Macrophages are one of the essential components of the tumour microenvironment (TME) [30]. The high density of certain tumour-associated macrophage (TAM) phenotypes in the TME is often associated with key processes in tumour progression, such as angiogenesis, immunosuppression, metastasis, and/or a poor prognosis [31,32,33]. TAMs can display opposing phenotypes and functions that are either tumoricidal, M1-like, or immune-suppressive/tumour-supportive, M2-like [34]. M1-like MΦs are activated by toll-like receptor (TLR) agonists and Th1 cytokines, such as interferon-gamma (IFN-γ), which empowers them with the ability to kill and phagocytose pathogens. These MΦs normally show upregulated proinflammatory cytokines such as interleukin (IL)-1β and IL-12, as well as the elevation of tumour necrosis factor-α (TNF-α) and reactive molecular species. M1-like MΦs present antigens via major histocompatibility complex (MHC) class II molecules. Th2 cytokines, like IL-4 and 13, stimulate monocytes/macrophages to express an M2 activation state. M2-like MΦs produce high levels of anti-inflammatory cytokines, such as IL-10, which amplifies metabolic pathways that can suppress adaptive immune responses [35]. Single-cell RNA sequencing (scRNA-seq) found that TAM is composed of heterogenous populations of MΦs with different biological functions. Targeting macrophages achieves better or no antitumour responses [30,36,37,38,39,40], indicating the functional status of the tumour macrophage is of great importance for the outcome of immunotherapy.In this work, we revisited two scRNA-seq studies on the intratumoural injection [23] and topical application [29] of caerin 1.1/1.9 in treating TC-1 tumour in mice, to reveal the signalling pathway(s) regulated by caerin 1.1/1.9 in different macrophage phenotypes. We demonstrated that caerin 1.1/1.9 significantly activated the IFN-α response pathway of multiple macrophage types present in the TME. The reprogramming of M2 to M1-like phenotypes was induced by caerin 1.1/1.9, which became the dominant population in the TME and significantly increased the efficacy of the specific immunotherapy developed in this work. In addition, we compared the heterogeneity and functions of macrophages identified from the TME of cervical cancer (CC) stage I (AIII, CIN3) and stage II (IIB) patients using scRNA-seq analysis. The macrophage phenotypes were characterised, with those derived from the stage I patients showing significant activation of the IFN-α response pathway. These results pinpointed comparative immunological footprints of macrophages between the treatments containing caerin 1.1/1.9 in murine models and human CC stage I patients, strongly suggesting that caerin 1.1/1.9 are promising agents to be included in cancer immunotherapy that could alter the TME to be more immune active. 2. Materials and Methods 2.1. Cervical Cancer Patient Information

Seven patients with pathological confirmed cervical cancer who underwent surgical removal of cervical cancer at the Department of Obstetrics and Gynecology at Foshan First People’s Hospital, between 2019 and 2021 were included in the current study, with three at AIII, one at CIN3 (referred to as CCI) and three patients at IIB (referred as CCII). The ethical approval code was L2016 (13).

2.2. Mice

Experiments were approved by Animal Experimentation Ethics Committee (Ethics Approval Number: FAHGPU20160316). Specific pathogen-free (SPF) six to eight weeks old adult female C57BL/6 (H-2b) mice were ordered from the Animal Resource Centre of Guangdong Province China. Mice were kept under SPF conditions at the Animal Facility of the first affiliated hospital of Guangdong Pharmaceutical University. Five mice were kept in each cage at 22 °C and 75% humidity and provided with sterilised standard mouse food and water and a 12-h light/12-h dark cycle.

2.3. Cell Line, Peptide Synthesis and AntibodiesTransformed with HPV16 E6/E7, a murine TC-1 cell line was purchased from Shanghai Institute for Cell Resources Centre of Chinese Academy of Sciences. Cell culture was following the procedure described previously [28].

Caerin 1.1 (GLLSVLGSVAKHVLPHVVPVIAEHL-NH2), caerin 1.9 (GLFGVLGSIAKHVLPHVVPVIAEKL-NH2), and a control peptide P3 (GTELPSPPSVWFEAEFK-OH) were synthesised by Mimotopes Proprietary Limited (Wuxi, China), with a purity above 99% and the concentrations of lipopolysaccharide below 0.44 EU mL−1.

HPV16 E7 CTL epitope RAHYNIVTF-OH, and four overlapping peptides representing the entire HPV 16 E7 protein, EX (MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE-OH, LNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKC-OH, DRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-OH, CVQSTHVDIRTLEDLLMGTLGIVCPICSQKP-OH were synthesised (purity > 99%) by Mimotopes Proprietary Limited (Wuxi, China).

Rat anti-mouse anti-IL10 receptor (1B1.3), Anti-PD1 (J43) Hamster anti-mouse monoclonal antibody and IgG Isotype control antibody (LTF-2) were purchased from BioXcell, USA. Rat Anti-Mouse fluorescent conjugated antibodies were purchased from BD, Biolegend and eBioscienc, including FITC-CD45.2 (clone: 104), PE-F4/80 (clone:BM8), APC-Ly6C (clone: HK1.4), PerCP-Cyanine5.5-CD11b (clone: M1/70), BV421-MHCII (clone: M5/114.15.2), PE-Cy7-CD11c (clone: HL3), PE-Cy7-CD86 (clone: GL1), APC-Cy7-CD3 (clone: 145-2C11), PE-Cy7-PD-L1 (clone: 10F.9G2), and Fixable Viability Stain 510 (FVS510). They were stored at −80 °C until further use.

2.4. Tumour Challenge

TC-1 cells, at approximately 70% confluency, were harvested with 0.25% trypsin and washed repeatedly with PBS. About 5 × 105 cells/mouse in 0.2 mL of PBS were injected subcutaneously into the left flank. The average diameter of tumour size was assessed every 3 days using callipers, calculated as width × width × length. Mice were given 1% sodium pentobarbital by i.p. injection when treatment was performed. At the end of each experiment, mice were sacrificed when the tumour diameter reached 15 mm (for survival curve), by CO2 inhalation.

2.5. Immunisation of Mice

Three days post-TC-1 challenge, when tumours were palpable, mice were immunised intramuscularly (i.m.) with the vaccine on day 3, day 9 and 18, respectively. The vaccine contains 40 µg of four overlapping HPV16E7 peptides (EX) (10 µg of each peptide), 10 µg of monophosphoryl lipid A (MPLA) (Sigma-Aldrich Corp., St. Louis, MI, USA), with or without 300 µg of anti-IL10R antibodies, dissolved in 100 µL of PBS; 300 µg of anti-PD-1 was administered intraperitoneally (i.p.) on days 9 and 21 after the tumour challenge.

2.6. Tumour Local Administration of Caerin Peptide

Three days post-TC-1 challenge, the tumour diameters reached 3 to 5 mm and the mice were intratumourally injected with 30 µg of caerin peptides (caerin 1.1/1.9) in PBS daily for six consecutive days.

2.7. Depletion of Macrophage

Mice were injected intraperitoneally (i.p.) with 200 μL of Clodronate Liposomes (5 mg/mL, Clodronate Liposomes Organization, Amsterdam, The Netherlands) or Control Liposomes, and 24 h later injected subcutaneously (s.c.) with 5 × 105 TC-1 cells/mouse in 0.2 mL of PBS and into the right flank. Systemic and local macrophage depletion was achieved by injection of Clodronate Liposomes concomitantly intraperitoneally (200 μL) and subcutaneously near the tumour cell injection site (100 μL) per 4 days until the end of the experiment.

2.8. Flow Cytometry

To obtain single cells, TC-1 tumour tissues were isolated and homogenised with a tumour dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Single cells were stained with FITC-CD45.2 (clone: 104), APC-Cy7-CD3 (clone: 145-2C11), PE-Cy7-CD8a (clone: 53–6.7), BV421- INFg (clone: XMG1.2), PE-B220 (clone: RA3-6B2) and FVS510. Viable cells were separated and analysed on a flow cytometer (FACS Aria II; BD Biosciences, San Jose, CA, USA). Data were analysed with Flow Jo v10.0 software (Tree Star Inc., Ashland, OR, USA).

2.9. Cytokine ELISA for IL-12, IL-10, IL-6 and TNF-α

Murine cytokine ELISA kits were purchased from BioLegend (San Diego, CA, USA), and were performed following the instructions of the manufacturer.

2.10. Single Cell RNA Sequencing Data AnalysisPreviously, we performed single-cell RNA sequencing of CD45+ TC-1 tumour infiltrating cells and quantitative analysis of TC-1 tumours of application of caerin 1.1/1.9 and combination therapy of caerin 1.1/1.9 with ICB and therapeutic vaccination [23,29]. The gene expression data were downloaded from https://singlecell.broadinstitute.org/single_cell (accessed on the 13 May 2022), the accession numbers were SCP1371 and SCP1093.For the scRNA-seq analysis of CC patients, the tumour tissues were collected with consent from the Human Ethnic Committee of the Foshan First People’s Hospital (No: L2016). Cells were washed once with ice-cold PBS containing 10% foetal bovine serum post-sorting and counted using a haemocytometer. The cells were then loaded onto a 10× chromium machine (10× Genomics, San Francisco, CA, USA) and run through the library preparation procedures following guidance from the Chromium Single Cell 3′ Reagent Kits v2 (10× Genomics, Pleasanton, CA, USA). The data were analysed using the pipeline described elsewhere [23,29]. 2.11. Protein-Protein Interaction (PPI) AnalysisInteractions among the proteins encoded by significantly regulated genes were predicted using STRING [41]. A required interaction score of 0.700 was used. Neither the first nor second shell of the PPI was included. Topological and statistical analyses were performed to explore the potential functions in our constructed network using the NetworkAnalyzer plugin in Cytoscape 3.7.1 [42]. Proteins without any interaction were excluded from the final network, which was visualised using Cytoscape. 2.12. GSEA AnalysisThe genes differentially expressed between different treatments were analysed by Gene Set Enrichment Analysis (GSEA) with p-value 43], to predict the Hallmark pathways enriched and the ranking of the genes associated with each pathway. 2.13. SCENIC Analysis of Transcription FactorThe transcription factor network inference was analysed using the SCENIC R package [44]. Briefly, a log-normalised expression matrix generated using Seurat was used as input. The gene co-expression network was identified by GENIE3 [45]. Each module was pruned based on a regulatory motif near a transcription start site via RcisTarget. The networks were retained if the TF-binding motif was enriched among its targets. The target genes without direct TF-binding motifs were removed. The retained networks were considered regulons. The activity of each regulon for every single cell was scored via the AUC scores using the AUCell R package. 2.14. Real Time PCRThe total RNA of tumour tissues was isolated with TRIzol reagent (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesised from 1 μg total RNA using a HiScript® II Q RT SuperMix for qPCR Kit (Vazyme, Nanjing, China). The ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for StepOnePlus™ Real-Time PCR System with Tower (Applied Biosystems Inc, Foster City, CA, USA). The cycling conditions were: 90 s at 95 °C, 40 cycles at 95 °C for 5 s, 60 °C for 30 s and 72 °C for 20 s. β-actin (Actb) was used as an internal control. The relative level was calculated by the relative quantification 2-ΔΔCT method. The primer sequences were listed in Table 1. 4. Discussion

In this study, we demonstrated that the DT of ICB and therapeutic vaccination plus caerin 1.1/1.9 increased the proinflammatory cytokine IL12, while reducing IL10, IL6 and TNF-α secretion in tumour macrophages; the depletion of macrophages greatly attenuated the anti-tumour responses by DT boosted by caerin 1.1/1.9. By mining our previously published scRNA-seq data, we showed that tumour local administration (both topical application and intratumoural injection) of caerin 1.1/1.9 significantly activated the IFN-α response pathway in different phenotypes of tumour macrophages, in general, similar to that exhibited by the tumour macrophages of early CC patients. Caerin 1.1/1.9 also promoted the expression of several TFs playing key roles in the regulation of genes that result in a proinflammatory response.

Immunotherapy has become a routine modality for the treatment of cancers, especially with the introduction of ICB and CAR-T therapy, therapeutic vaccines in pre-clinical animal models have also demonstrated exciting efficacies. However, a great portion of cancer patients do not respond to ICB treatment. One of the most important caveats is that the immunosuppressive TME stunts the therapeutic effects of immunotherapy. Disturbing the TME is, therefore, a key issue to consider for immunotherapy. Given that caerin 1.1/1.9 can directly kill tumour cells and make the tumour cells release inflammatory cytokines we, therefore, set out to investigate whether caerin 1.1/1.9 can alter the TME and the efficacy of immunotherapy.

The enhanced efficacy of immunotherapy mediated by tumour local treatment with caerin peptides was mediated by macrophage, most likely through the IFNα signalling pathway as demonstrated by the scRNA-seq analysis of the tumour infiltrating macrophages. scRNA-seq analysis of macrophages between early and later-stage cervical cancer tumour samples also indicate that the IFNα signalling pathway was more activated in the early-stage cervical cancer.

Type I IFNs (IFN-α and IFN-β) constitute the first line of defence against both viruses and cancer cells, although it plays dual roles that can either prevent or promote tumour development [49]. Viruses have been found to use different mechanisms to disturb type I IFN signalling. HPV18E6 directly binds to Tyk2 and impairs Jak-STAT activation by IFN-α [50]. Respiratory syncytial virus impairs IFN-β mediated signal transducer and activator of transcription Stat1 phosphorylation through a mechanism that involves the inhibition of tyrosine kinase 2 phosphorylation [51,52,53]. The INF-α and INF-γ response pathways were highly activated in nearly all macrophage phenotypes with the treatment containing the caerin peptides; the expressions of many associated genes were largely upregulated in TAMs (Figure 3 and previously reported [29]). These two pathways were also remarkably activated in stage I cervical cancer patients compared to those in stage II (Figure 6). The most activated signalling pathway of stage II tumour macrophages is the TNF-α pathway, this accords with a recent study showing the serum level of TNF-α expression in patients with cervical cancer was noticeably elevated, which gradually became normal after the surgical treatment [54].IFN-α has been used to treat cancers; however, the systemic administration of type I IFNs leads to significant side effects for patients [55]. Recently, the ER-associated molecule STING has been found to stimulate the production of type I IFN in the tumour [56,57] and results in anti-tumour responses through the activation of IFN-α signalling pathways in macrophages [58,59]. Moreover, targeting the IFN-α signalling pathway augments immune checkpoint inhibitors in ICB therapy-resistant tumours [60,61]. Interestingly and surprisingly, caerin 1.1/1.9 enhanced the anti-tumour responses of ICB and therapeutic vaccination therapy [23], also through activating the IFN-Stat1 signalling pathway of macrophages in TC-1 model (Figure 3 and Figure S3), and in the B16 tumour model (). A recent study has shown that IFN-α induces the differentiation and exhaustion of chronic myeloid leukemia and myeloproliferative neoplasm stem cells via the processes mediated by Cebpb, and IFN-α upregulates C/EBPβ by recruiting Stat1 and Stat5 to the novel 3′ distal enhancers of Cebpb [62]. The upregulated expression of Cebpb was identified with significance in most MΦ phenotypes of the topical treatment containing the caerin peptides, which was also present as one of the differential TFs in the MΦs of the caerin + DT. The significant upregulation of Stat1 was confirmed by qPCR in this study and was previously detected in both the topical application and intratumoural injection treatments containing the caerin peptides [23,29]. It was demonstrated that C/EBPβ promotes immunity to mucosal candidiasis during cortisone immunosuppression in a manner associated with the expression of β-defensin 3 [63]. Additionally, C/EBPβ regulates numerous genes involved in inflammation [64]. Cebpb and Gm8797 (ubiquitin B pseudogene) were detected as the marker genes of the MΦs of Vsir knockout mice, which showed an exacerbated inflammatory phenotype [65]. The expression of Gm8797 was remarkably upregulated in the caerin + DT. This further suggested Cebpb may play an important role in the proinflammatory state induced by caerin 1.1/1.9.The depletion of macrophages or avoidance of macrophage tumour infiltration in the TC-1 tumour model results in either a beneficial or detrimental anti-tumour response. The depletion of myeloid cells by injecting clodronate liposome increased the efficacy of the vaccination, in terms of reducing tumour size and prolonging the survival of tumour-bearing mice [39]. A recent study showed that the vaccination with a long peptide plus incomplete Freund’s adjuvant-induced tumour regression, which was abrogated rather than enhanced by macrophage depletion [66]. These results indicated that whether macrophages promote tumour rejection or growth depends on their biological characteristics during cancer therapy. Compared with a control peptide, caerin 1.1/1.9 significantly activated tumour infiltrating macrophages, both F4/80+Ly6C+ or F/40+Ly6C− macrophages secreted significantly more IL12, less IL10 and IL6 (Figure 1), coinciding with our previous results that caerin 1.1/1.9 repolarise the tumour macrophage to the M1-like type while reducing the M2-like macrophages. Therefore, the caerin treatment resulted in macrophages becoming more proinflammatory, it was thus not surprising that the depletion of macrophages in our model attenuated the antitumor effect of therapeutic vaccination and ICB treatment. The depletion of tumour macrophages greatly reduced the enhanced anti-tumour responses of immunotherapy mediated by caerin 1.1/1.9 (Figure 2 and Figure S2). Our results and others point to the importance of activated macrophage type I IFN signalling pathways for the outcome of better immunotherapy. The induction of pro-inflammatory cytokines might result in the clonal selection of tumor cells and possible disease relapse in the context of caerin treatment; however, at least in the TC-1 model, the proinflammatory macrophages contribute to the tumor regression. To the best of our knowledge, this is the first time to demonstrate that natural host-defence peptides derived from amphibian skin secretion stimulate type I IFN signalling pathways in tumour MΦs. However, the molecular mechanism underlying this phenomenon is yet to be clear, it is likely that macrophages were activated indirectly, and that caerin peptides result in TC-1 tumour cell death, which release biomolecules that subsequently activate macrophages. We are currently investigating the underlying mechanism by using two murine tumour models and seeking to identify the target molecules of caerin 1.1/1.9.Macrophage infiltration of some solid cancers commonly correlates with poor prognosis [67]. Targeting macrophages has long been used in different tumour models for cancer therapy with varied outcomes [37,39,68,69]. The regulation of macrophage polarisation via stimulating macrophage proinflammatory gene expression has been used to tackle cancer immunosuppression, and consequently activate cytotoxic T cell antitumor responses [70]. It has been reported that the expression of Fos/Jun enhances inflammatory responses in macrophages [71,72]. Jund was previously shown to be positively associated with inflammation, the knockdown of which resulted in significantly reduced macrophage activity and cytokine secretion in rat and human primary macrophages [73]; whereas Junb was found to play the key role in the full expression of Il1b and the genes involved in classical inflammation in macrophages treated with LPS and other immunostimulatory molecules [74]. A very recent study has discovered that the overexpression of Fosl2 induced a systemic inflammatory phenotype with immune infiltrates in multiple organs in mice, by repressing the development of regulatory T cells [75]. These TFs were significantly elevated in the DT + caerin therapy compared to the untreated and control + DT groups, indicating more inflammatory phenotypes of MΦs formed in the TME. It is not surprising that several TFs associated with anti-inflammatory function were also elevated in the DT + caerin group, such as Usf1, Nfil3, and Mafb (Figure 4 and Figure S4).On the other hand, several TFs negatively associated with inflammation were downregulated in the caerin + DT group compared to the DT treatments, such as Ets2, Hif1a, Mitf, Runx1, and Elk3. The negative regulatory role of Ets2 in LPS- and VSV-induced inflammation through the suppression of MAPK/NF-κB signalling was revealed, via the inhibition of transcription of IL-6 with Ets2 direct binding to the promoter [76]. The Mitf− cells were shown to produce larger amounts of IL-1α and IL-1β, compared to Mitf+ cells; in addition, the supernatant of Mitf- melanoma cells reduced Mitf expression in positive cells via the signalling of IL-1R, which we previously found was significantly upregulated in the caerin group [23]. The suppression of Mitf in melanoma cells triggers an inflammatory secretome comprising the proinflammatory cytokines [77,78]. It has been reported that the expression of Runx1 is negatively associated with the production of inflammatory cytokine production by neutrophils in response to toll-like receptor signalling [79]. Besides, the downregulation of Elk3 and the downstream induction of Ho1 appeared crucial for the inflammatory response of macrophage function to infection [80]. Thus, the suppression of these TFs by the caerin + DT further supports the formation of a remarkably more proinflammatory TME.Future experimentation with a higher number of CC patients at each stage is recommended to confirm the findings of this study and elucidate the underlying mechanism of the formation of distinct phenotypes. This may be achieved by a large-scale comparative transcriptomic analysis, in conjunction with the silencing of marker genes in murine models to verify the pathway(s) involved. At the protein level, antibody-mediated signalling blockade or activation by agonists could be performed, with a particular focus on macrophage phenotypes. With respect to how caerin 1.1/1.9 activate IFN signalling of macrophages, the knockout of key regulators on the pathway, e.g., Stat1, Stat2, Jak1, and Ifnar, as well as the use of condition knockout mice, could be employed, followed by in-depth multi-omic analysis. Besides, it should also be noted that angiogenesis plays a significant role in tumor progression, and its constant crosstalk with immune cells, including macrophages [81,82]. It has been shown that anti-angiogenic treatments could induce the normalisation of the tumor vasculature [83], which enhances anti-tumor immunity by increasing antigen presentation in dendritic cells, the populations of M1-like macrophages and active CD8+ T-cells [84]. These were also observed in the treatments containing caerin 1.1/1.9. Thus, caerin 1.1/1.9 in combination with anti-angiogenesis and immune checkpoint inhibitors in the treatment of different cancers warrant further investigation.