Single cell RNA-seq identified six different macrophage populations in TC-1 tumour
In this study, untreated TC-1 tumour bearing mice and tumour bearing mice immunised with a HPV16 E7 peptide-based vaccine containing anti-IL-10 receptor antibody and PD-1 blockade were locally injected with a mixture of caerin 1.1 and 1.9 peptides (molar ratio 1:1) (“caerin”) or a control peptide. The two treatment groups showed significantly reduced tumour mass when compared with the untreated group, with a reduction of 69% (caerin, P=0.0011) and 42% (control peptide, P=0.0352). An additional injection of caerin further reduced the tumour weights, though the reduction was insignificant (P=0.11). Elispot results from spleen and draining lymph nodes (LN) of individual mice (3 mice) indicate that two immunotherapy and peptide treated groups demonstrated similar E7 specific CD8+ T cells in the spleen and draining lymph nodes (Fig. 1A).
Thirty days after TC-1 tumour challenge, total viable CD45+ leukocytes were isolated from tumours using a tumour infiltrating cell isolation kit (Fig. 1B and Supplementary Data 1). Gene expression data from cells extracted from the control tumours and the two treated tumours were aligned and projected in a 2-dimensional space through t-stochastic neighbour embedding (t-SNE) to allow identification of tumour associated immune cell populations and the overlapping patterns associated with the control tumours and the two treated tumour groups (Fig. 1C and Supplementary Fig. 1). There were 19 distinct cell clusters (Cluster 0 to 18) detected by using unsupervised graph-based clustering method (see Methods) (Fig. 1C through 1E; Supplementary Fig. 2A to 2D). The presence of lymphocyte lineages was supported by the established canonical markers, such as Nkg7, Cd19, Fcmr, Cd8b1 and Cd79a, as well as myeloid cells that were determined by the identifications of Cd11c, Cd14, Cd68, Cd209a, Adgre1, Itgax, Csf1r, Lgals3, Ccr2 and Ly6c223, 24. The populations and expression patterns of a few marker genes were shown in Fig. 1D and 1E (see Supplementary Data 2 for the full list of all marker genes detected).
Nonmacrophage cell populations were identified as monocytes (cluster 0; marker genes: Ly6a, Ly6c2, Fcgr1 and Dpep2), neutrophils (cluster 7; Retnlg, S100a8, Cxcl2, and Hdc), B cells (cluster 11; Cd79a, Fcmr, Ly6d, and Mzb1), conventional DC type 2 (cDC2) (cluster 12; Cd209a, Flt3, Ctnnd2, and Epcam)25, natural killer (NK) cells (cluster 14; Gzma, Xcl1, Ncr1, and Klrb1c), migratory DCs (migDC) (cluster 16; Ccl22, Bcl2l14, Fscn1, and Cacnb3)26, plasmacytoid dendritic cells (pDCs) (cluster 18; D13Ertd608e, Siglech, Ccr9, and Pacsin1)25 and four clusters with gene signatures suggesting various phenotypes of T cells (clusters 5, 10, 13 and15). Fibroblast (cluster 4; 2810417H13Rik, Tk1, Birc5, and Cdca3), adipogenic stem and precursor cells (ASPCs) (cluster 6; Gas1, Igfbp3, Col3a1, Mgp and Cyr61)27 were also identified as contaminants (also see Supplementary Data 2 and Supplementary File 1).
The macrophage populations were examined in more detail, and expression of the top 20 marker genes of each macrophage population was compared to their expression across all macrophage cell populations, and to the expression of other genes in the same macrophage population (Fig. 1F). Specific gene expression patterns differentiating these MΦ populations and certain overlaps could be determined. Many of the top 20 marker genes of cluster 1, including Pf4, Arg1, Fabp5, and Mmp12, were associated with M2 MΦ (Fig. 1F; Supplementary Data 2), and similar numbers of these cells were detected in tumours treated with caerin peptides or control peptide. (Supplementary Fig. 2C and 2D). The top three highly expressed genes with significance in cluster 2 were H2-Eb1, Cd74 and C1qc (Supplementary Data 2 and Supplementary File 1), confirming a MΦ signature. These genes were found to mark MHCIIhi border associated MΦ in mouse brain26. The high expressions of several H-2 (MHCII) members, including H2-Eb1, H2-Ab1, H2-Aa, H2-DMb1 and H2-DMa, were also confirmed in cluster 2, thus we considered these as MHCIIhi MΦ hereafter. Cluster 3 had a mixed cell phenotype, including proinflammatory MΦ (Cxcl10, Gbp2, and Thbs1), Ly6chi infiltrating MΦ (Chil3 and Plac8), and dendritic cells (Rsad2, Ifit1, Ifit2 and Ifi205). Thus, this cluster was labelled as MΦ/DCs. Increase in the size of this cluster in caerin peptide and control peptide treated tumours suggested that analysis of its subpopulations might help to further clarify its function. It was mainly composed of macrophages (Nop16, Gatm and Pf4) and NKT cells (Ntpcr, Mrpl28 and Commd10), and was identified as MΦ/NKT. Due to the exclusive high expression of Ear2 in cluster 9, it was assigned as Ear2hi MΦ (Chil3, Adgre5, Ace, and Ifitm6)28, 29. Cluster 17 exhibited the signatures of MΦ (Adgre1, Csf1r, Fcgr1, and Cd68) and there was upregulated chemokine gene expression (Ccl8, Ccl24, Ccl6, and Ccl9) (Supplementary Data 2 and Supplementary File 1), suggesting that these might be tumour associated MΦ (TAM) as Ccl8 and Siglec1 are markers for TAMs in breast cancer30, and both were highly expressed in this cluster, with Ccl8 expression exclusive to cluster 17 (Supplementary Data 2). The presence of other significantly upregulated genes, e.g., Cd209f, Clec10a and Cd163, also indicated the existence of activated M2 MΦ. Thus, we consider cluster 17 as TAMs. In comparison to other clusters, the top 20 marker genes of cluster 8 appeared less significant as indicated by their relatively higher P-values (P>1.0E-34) (Supplementary Data 2).
We found that, in comparison to the untreated tumours, the tumours from the two immunised and peptide treated groups had significantly increased populations of monocytes, MHCIIhi MΦ, MΦ/DCs, Ear2hi MΦ, CD8+ T cells, and pDCs and substantially reduced M2 MΦ and B cell populations (Supplementary Data 1; Supplementary Fig. 2C and Fig. 2D). CD8+ T cells appeared nearly exclusive to tumours from immunised animals, and the M2 MΦ population was reduced in these tumours, possibly due to the use of anti-IL10 antibody in the immunisation, since it directly associates with the secretion of IL1031. Numbers of MHCIIhi MΦ and NK cells were greatest in caerin peptide treated tumours. Notably, B cells were almost depleted from tumours treated with caerin peptide (15/5,685 cells), when compared to tumours from untreated animals (194/4,011 cells) and control peptide and immunisation treated animals (87/5,415 cells) (Supplementary Data 1).
Intratumor injection of caerin peptides significantly increased Arg1- tumour infiltrating macrophages
Six macrophage populations were detected, including M2 MΦ, MHCIIhi MΦ, MΦ/DCs, Ear2hi MΦ, MΦ/NKT and TAMs. Altogether, these macrophages represented the largest cell populations (Supplementary Fig. 3), constituting 49.58% of total cells in the untreated TC-1 tumour, and a similar fraction of total cells in immunised animals treated with caerin peptide (47.47%) or control peptide (42.00%) (Supplementary Data 2). The correlation between each group of MΦs were derived based on the expression of significantly upregulated genes (Fig. 2A). Notably, M2 MΦ and MHCIIhi MΦ were highly correlated with MΦ/NKT, suggesting close development of these MΦ lineages. The normalised numbers of different MΦs in the tumours were compared between the control and treated animals, and M2 MΦ were reduced in tumours in the immunised and peptide treated animals by about 80% (Fig. 2B). Injection of caerin largely induced MHCIIhi and Ear2hi MΦs in tumours, while reducing TAMs, when compared to tumours from untreated animals or receiving control peptide treatment. M2 MΦ was reduced from 73.2% of macrophages in untreated tumour to 16.2% in caerin and 18.9% in control peptide treated tumours (Fig. 2C). In contrast, the proportions of MHCIIhi MΦ were elevated by approximately 5-fold (caerin peptide) and 4-fold (control peptide) treated tumours, and MΦ/DCs and Ear2hi MΦ were similarly increased in the caerin and control peptide treated tumours.
We next sought to unravel the phenotype and functions behind the specific gene expression patterns of each macrophage. The expressions of key lineage-associated genes of M2 MΦ were compared in parallel with their expressions in other macrophages (Fig. 2D). Several marker genes appeared exclusive to M2 MΦ, such as Mmp12, Arg1, Mmp13 and Slc6a8, for which expression is associated with tumour angiogenesis and invasiveness32, 33, 34. The role of Arg1 in immunosuppression has been described elsewhere35. Some marker genes of M2 MΦ were also highly expressed in TAMs, suggesting potential similarity between these two macrophages in terms of cellular function.
The distribution of M2 MΦ in untreated tumours and tumours injected with caerin peptides was compared in Fig. 2E. The expression of some marker genes (Rcgg, Ndrg1 and Egln3), aligned well with those of M2 MΦ after peptide treatment. The relative expression of the top 40 marker genes of M2 MΦ were hierarchically clustered and compared amongst untreated and peptide treated tumours (Fig. 2F). All genes were significantly downregulated in caerin and control peptide treated tumours, except Mmp13 in the control peptide treated tumours and Ndrg1 in the caerin treated tumours. To evaluate the significance of this observation, the expression values of selected genes were further compared, where significant downregulation of Arg1, Mmp13, Pf4 and Hmox1 were present in caerin treated tumours when compared to control peptide treated tumours (Fig. 2G). Most of the biological processes enriched in M2 MΦ represented by the marker genes unique to caerin treated tumours related to immune responses including apoptosis, responses to stimulus with organic substance, cytokine production and secretion, and T cell differentiation (Fig. 2H). Different gene expression patterns relating to metabolism and transport of macromolecules (Supplementary Fig. 4B), and responses to heat and wounding (Supplementary Fig. 4C) were found in the untreated tumours and those treated with control peptide.
MHCIIhi MΦ were significantly increased in control and caerin peptide treated tumours, more significantly with caerin treatment. The expression of selected marker genes across six macrophage populations were displayed in Fig. 3A, which suggests Lira5, Cxcl9, Dnase1l3 and Cd300e as the signatures exclusive to MHCIIhi MΦ. Cadm1, Cxcl9 and Cd300e expression was increased in macrophages, and Dnase1l3 has been reported as a signature of CD141+CLEC9A+ DCs36. In addition, Clec12a was recently found to highly expressed in myeloid cells including macrophages and DC subsets37. Thus, the MHCIIhi MΦ cluster displayed a characteristic of mixing phenotypes and its subpopulations were further investigated.
A total of five subpopulations of MHCIIhi MΦ were identified and projected in a 2-dimensional tSNE space, with the subpopulation 0, 1 and 2 possessing the highest cell numbers (Fig. 3B). Subpopulation 3 was present in control and caerin peptide treated tumours in similar numbers, while subpopulation 4 was negligible in untreated tumours (Supplementary Data 3). The normalised cell numbers of five subpopulations with different treatments were compared in Fig. 3C, and caerin peptide treatment stimulated much higher number of subpopulations 0, 1 and 2 when compared to control peptide treatment (group C), with fold changes of 4.22, 8.14 and 3.89 relative to the untreated group. The expressions (Log2 FC) of the top 10 marker genes of each subpopulation were compared across all five subpopulations (Fig. 3D). The first subpopulation, C0_MHCIIhi-CXCL2, characterised by Cxcl2, Nfil3 and Osm, had the phenotype of activated macrophages38, 39, 40. The second subpopulation showed significant expressions of Ifit3b, Ifit3 and Ifit2, typical of polarised M1 macrophages41, suggesting pro-inflammatory function. In addition, this subpopulation also showed significant upregulation of several other interferon-induced protein relevant genes, thus we labelled it as C1_MHCIIhi-IFIT.
The third subpopulation had the lowest number of significantly upregulated genes compared to other four subpopulations, and was characterised by expression of Fcrls, Ptgs1, Mrc1 and Igfbp4, the signature of resident-like macrophages26, 42, 43. Thus, this subpopulation is referred as C2_MHCIIhi-ResMΦ. The fourth subpopulation, named C3_MHCIIhi-DCs, possessed the highest number of marker genes and the top marker genes, including Ankrd33b, Xrc1, and Asb2 were typical of DCs. Also, the signature gene of B cells, Fcrl5, was significantly expressed in C3_MHCIIhi-DCs, suggesting antigen presentation capacity. The fifth subpopulation was characterised by several marker genes of progenitor cells, such as Birc544, Cdkn345, Ccnb246 and Kif20a47, which was referred as C4_MHCIIhi-PROG. The H-2 (MHCII) members were mainly elevated in C1_MHCIIhi-IFIT and C4_MHCIIhi-PROG.
The correlations amongst these subpopulations were evaluated based on the upregulated genes (Fig. 3E). C1_MHCIIhi-IFIT and C3_MHCIIhi-DCs were correlated to a much higher degree, compared to the connections amongst other three clusters. Genes that were shared between C1_MHCIIhi-IFIT and C3_MHCIIhi-DCs revealed biological processes mutually exerted by these two subpopulations, including metabolism of lipids and lipoproteins, G-protein signalling and FCGR activation. Reactome pathways based on marker genes that were unique to each subpopulation were analysed (Supplementary Fig. 5). C0_MHCIIhi-CXCL2 showed an enrichment in caspase-mediated cleavage of cytoskeletal proteins, immune system, apoptotic cleavage of cellular proteins and apoptotic execution phase. The signalling of interferon, interferon gamma and cytokine in immune system were detected in C1_MHCIIhi-IFIT with significance. The pathways found in C2_MHCIIhi-ResMΦ were less significant (high P-values) than those in other subpopulations, such as GPCR ligand binding, chemokine receptors bind chemokines and collagen formation, which were less relevant to activating immune response. C3_MHCIIhi-DCs showed enrichment in haemostasis, GPVI-mediated activation cascade and adaptive immune system. Many cell cycle related pathways were found enriched in C4_MHCIIhi-PROG (Supplementary Data 3).
The populations of Ear2hi MΦ were remarkably elevated in control peptide and particularly in caerin peptide treated tumours (Supplementary Fig. 6A). The distribution of cells expressing selected pro-inflammatory marker genes appeared aligned well with Ear2hi MΦ in caerin peptide compared to untreated and control peptide treated tumours. Significant upregulation of Ear2, Ace, Adgre4, Serpinb2 and Prtn3 in Ear2hi MΦ was identified in the caerin treated tumours compared to untreated tumours (Supplementary Fig. 6B). A High degree of gene expression concordance c was present amongst Ear2hi MΦs, yet distinct biological processes were found in subgroups Ear2hi MΦ (Supplementary Fig. 6C). Ear2hi MΦ showed the suppression of many biological processes, such as transferase activity, phosphorylation, and cellular protein metabolism in untreated tumours, when compared to the activation of metabolic processes in control peptide treated tumours. Caerin treated tumours had activated cellular structure remodelling and immune response genes including those suggesting myeloid cell differentiation.
More CD8+ T cells infiltrate to TC-1 tumour following vaccination and PD-1 blockade, and CD8+ T cells are more activated in caerin 1.1/1.9 treatment group B
In control peptide and caerin peptide treated tumours, the population of CD8+ T cells infiltrating to TC-1 tumour was was 3.73% (caerin) and 4.58% (control peptide) of the total CD45+ cells compared to only 0.27% in untreated tumours (Supplementary Data 1). With peptide treatment, CD8+ T cells were relatively separated from other three T cell populations on the tSNE graph (Fig. 4A), indicating a possible variation in function. Also, all T cell types showed a more than 10-fold increase between untreated tumours to 40% (caerin) and 37% (control peptide) (Fig. 4B). Analysis of the gene expression pattern in the four T cell populations (Fig. 4C.) showed that expression of most of these genes was higher in CD8 T cells than that in any other T cell type (see Supplementary Fig. 5 for the expression of marker genes in other T cells), including genes that enhance the activation of CD8+ T cells, such as Ucp2, Fth1, Apoe, Fcer1g and Calm3.
Since pDCs present antigens (Ag) and induce immunogenic T cell responses through differentiation of cytotoxic CD8+ T cells and effector CD4+ T cells48, 49, we compared the gene expression of signature genes of CD8+ T cells across four types of T cells and pDCs (Fig. 4D). It shows that Cd8a, Klrc1 and Lag3 were almost exclusive to CD8+ T cells, while comparable expression of Cxcr6 was observed in CD4-CD8- T cells, and lower expression of Cd8a, Cd8b1 and Lag3 was observed in pDCs. Ribosome was determined to be the most enriched KEGG pathway, followed by T cell receptor signalling and natural killer cell mediated cytotoxicity (Fig. 4E). The top 25 enriched biological processes in CD4+CD8+, CD8+, CD4+CD25+ and CD4-CD8- T cells were compared in Fig. 4F. Translation was found to be the most enriched process in the T cell subsets except CD4+CD25+, and was subsequently excluded to highlight the difference amongst other enriched processes (Supplementary Data 5). Since these cells share similar T cell lineage development, overlaps of certain biological processes were observed, such as T cell differentiation, T cell receptor signalling, and innate immune response was observed as expected. However, T cell relevant processes were more enriched in CD8+ T cells suggested by lower P-values compared to other three subtypes. Furthermore, there were a set of processes only enriched in CD8+ T cells, such as positive regulation of histone deacetylation, regulation of translational initiation, chromosome organisation, activation of cysteine-type endopeptidase activity involved in apoptotic process and regulation of cytokine production (Fig. 4F and Supplementary Data 5).
The expression of marker genes, including Cd8a, Cd8b1, Tox, Lag3, Ifng, Nkg7, Nrgn, Gldc, Prf1, Abcb9, Nrn1 and Rgs16, were compared amongst untreated, caerin, and control peptide treated tumours (Fig. 5A), where significant upregulations of these genes induced by peptide treatment were observed, except Nrn1 for the control peptide treatment. In addition, Cd8a, Cd8b1, Tox, Ifng, Prf1 and Rgs16 were significantly elevated by caerin when compared to control peptide, suggesting that CD8+ T cells were more activated by the caerin peptide. The subpopulations of CD8+ T cells were further investigated to reveal the changes of heterogeneity due to peptide treatment, and five subpopulations were identified (Fig. 5B; Supplementary Fig. 8). We found that the signatures representing naïve T cells, including Sell, Lef1 and Tcf7, had higher expression in untreated tumour (Fig. 5C). Peptide treatment caused elevation of signatures for exhausted T cells, such as Tigit, Lag3, Tox and Pdcd1, while the effector T cells were stimulated by two treatments, suggested by the upregulation of Gzmb and Prf1. The expression of these genes in the five CD8 subpopulations was also compared to reveal their possible functions. The average expression of the top 20 markers genes of five subpopulations were compared in Supplementary Fig. 8E, where the marker genes of subpopulation 2 and 3 were more exclusive.
The first cluster C0_CD8-CCL5 cells characterised by marker genes Ccl550, Cd3e51, Cxcr652 and Gzmk53, were considered as memory T cells. Most of the top 20 highly expressed genes in the second cluster were various ribosomal proteins, such as Rpl32, Rpl26, Rpl23 and Rpl28. It has been reported that translation is upregulated during effector CD8+ T cell expansion54. In addition, Tnfrsf955 and Prf156 appeared to highly express in this subpopulation. Thus, these were likely effector CD8+ T cells and were named C1_CD8-TNFRSF9. The third cluster, C2_CD8-CDCA5, was characterised by significant upregulation of Cdca5, Cdc6 and Ccna2 (Supplementary Data 5), commonly associated with dividing T cells57. Additionally, several histones and regulators, including Tmsb10 and Ptma, were among those genes with highest expression in C2_CD8-CDCA5. The fourth cluster possessed more than 1,500 genes with significant upregulation (Supplementary Fig. 8) and was characterised by Fth1, Cd74, and Ifitm3. The relevance of Fth1 to CD8+ effector T cell response was reported, which revealed that it played an immunomodulatory role in cytokine signalling, adaptive immunity, and cell death58. The high expression of Cd74 and Ifitm3 was detected in memory T cell53, 59. Thus, this cluster was comprised of effector-memory T cells, referred as C3_CD8-FTH1 hereafter. The remaining cell cluster, C4_CD8-MS4A4B, was characterised by MS4a4B, Ly6a, Cd8b1, Ly6e, and were naïve T cells. Notably, the CD8+ T cells of untreated tumours only contained two of five subpopulations, i.e., C0_CD8-CCL5 and C2_CD8-CDCA5, while the control peptide and caerin peptides induced all five subpopulations (Fig. 5B; Supplementary Fig. 8A; Supplementary Data 5). Caerin treated tumours possessed higher number of C0_CD8-CCL5 and C3_CD8-FTH1 compared to the control peptide treated tumours.
We then projected CD8+ T cells onto the two-dimensional state-space defined by Monocle3 for sample similarity and pseudotime analysis, to obtain the information inferring
lineage trajectories from expression data (Fig. 5C). Most cells from each subpopulation aggregated based on expression similarities, and different clusters formed into a relative process in pseudotime that began with C2_CD8-CDCA5 (dividing CD8+ T cells), then developed in separate directions, with one direction developing to C3_CD8-FTH1 cells (effector-memory CD8+ T cells). It appeared that C0_CD8-CCL5 (memory CD8+ T cells), C1_CD8-TNFRSF9 (effector CD8+ T cells) and C4_CD8-MS4A4B (naïve CD8+ T cells) started to emerge at approximately similar pseudotime on the other direction, gradually overlapping on three branches along the pseudotime trajectory, two of which also included certain amount of C2_CD8-CDCA5 cells, indicating functional divergence of this subpopulation. On these two branches, C2_CD8-CDCA5 aggregated with C0_CD8-CCL5 and C1_CD8-TNFRSF9, which suggested close correlation between regulatory, effector and memory CD8+ T cells, and different functions might be executed. C4_CD8- MS4A4B was diversely present together with C0_CD8-CCL5 and C1_CD8-TNFRSF9 along the pseudotime, especially at the middle area (Fig. 5C), implying their close association.
Seven states were thus identified based on pseudotime analysis (Fig. 5D), where cells in transitional state 2 and state 5 exclusively corresponded to C0_CD8-CCL5 and C2_CD8-CDCA5, respectively. Most cells of state 1, 3 and 7 were C0_CD8-CCL5, C1_CD8-TNFRSF9 and C2_CD8-CDCA5. A transitional state 4 was identified, which consisted of all clusters except C3_CD8-FTH1. The predicted developmental trajectory was also confirmed by the marker genes with similar expression pattern, which hierarchically clustered these markers along the pseudotime in each state (Supplementary Fig. 8). States 1, 6 and 7 included genes with expression gradually increasing with the time. The potential divergence of cell functions in different state cells were investigated (Supplementary Data 5). Notably, state 1 showing significantly elevation of genes enriched in the signalling pathways of IL-2 and IL-3, G protein, and G13 at a later stage, and caerin treated tumours had a higher population in this state compared to untreated and control peptide treated tumours (Fig. 5D and Supplementary Data 5). Apoptosis was the only pathway enriched in state 3, and signalling by EGFR1, chemokine and TGF-β was present in state 7. The transition state 2 between state 3 and 7 had very different functions, such as the enrichments of macrophage markers, ApoE and miR-146 in inflammation and atherosclerosis, and antigen processing and presentation. The presence of more state 6 cells at late pseudotime potentially associated with caerin treatment correlated with the marker genes playing roles in TNFα, NF-kB signalling and inflammatory response.
The genes significantly differentiating the branches were also analysed, with expression variation of top 10 genes along the pseudotime trajectory compared amongst different groups (Fig. 5F to 5H). Most of these genes were expressed around pseudotime zero in the untreated tumours but had a significantly prolonged expression with control or caerin peptide treatment. During the first transition, the genes highly associated with immune system, and their expression, declined at an early stage in states 5 and 6 possibly due to low cell numbers, then increased sharply onwards pseudotime 25, where more cells expressing these genes were observed in caerin treated tumours. There was also a slow increase of expression of Apoe, C1qb, Cd74, H2-Aa, H2-Ab1and H2-Eb1 along the pseudotime on the branch involving state 1, 2, 3, 4, 5 and 7, which also correlated with higher cell number stimulated by caerin (Fig. 5F). Most of genes such as Rps11, 15a, 36, 24 and 26 during the branching displayed in Fig. 5G appeared downregulated along the pseudotime in state 1, 2, 4 and 5, indicating the deactivation of translation, which was also the case in state 1, 2, 4, 5 and 7 for the third trajectory separation (Fig. 5H). In addition, the expression trend of these genes in state 3, 4 and 5 aligned well with the cell distribution in caerin treated tumours.
TMT10plex labelling quantitative proteomics revealed higher immune response induced by the injection of caerin 1.1/1.9
To validate our scRNA-seq data and capture treatment-dependent alterations in protein content for the TC-1 tumour, we performed quantitative proteomic analysis of tumours using the TMT labelling method (details of protein quantitation and annotations are in Table S6). The pairwise comparison showed that significantly more proteins were regulated with caerin treatment when compared to control peptide (Supplementary Fig. 9A and 9B). The hierarchical clustering of quantified proteins implied consistency between biological triplicates of each group. A total of 238 proteins were uniquely upregulated in with caerin treatment, while the upregulation of 51 proteins were observed with both caerin and control peptide treatments (Supplementary Fig. 9C).
The gene ontology (biological process, molecular function, and cellular component) enrichment of upregulated proteins with each treatment was carried out (Supplementary Data 6; Supplementary Fig. 10; Supplementary Fig. 11). With caerin, the enrichment of many biological processes was more significant than with control peptide, including immune system process (FDR=8.10E-28 with caerin versus 1.29E-12 with control peptide), and innate immune response (caerin, FDR=9.85E-22; control, FDR=3.60E-05). The top 60 proteins significantly regulated by caerin were shown in Fig. 6A. Caerin induced significant upregulation of proteins involved in immune response and regulation processes, such as Gzma, Gzmc, Irf5, Tgtp1, Prg2 and Ighg1. The quantities of selected proteins uniquely regulated by caerin is shown in Fig. 6B.
The protein-protein interaction (PPI) analysis of upregulated proteins identified intensive interactions in both treatments (Fig. 6C and 6D; the complete predicted PPIs were recorded in Supplementary Data 6; the statistical analysis of PPIs was presented in Supplementary File 2). Similar nodes can be found on two networks with different interaction degrees, with Stat1 being the node with highest degrees with caerin and B2m with control peptide treatment. The fold changes of top 10 most interacting protein nodes on two PPIs were compared with respect to internal references (Fig. 6E). Most of these proteins showed higher contents with caerin than with control peptide. Stat1 was upregulated by 2.2-fold compared to untreated tumour (Supplementary Data 6) and detected as a marker gene only in monocytes, MΦ/DCs and CD4+CD25+ T cells with caerin treatment (Supplementary Data 6) with no significant change in protein or mRNA with control peptide treatment.
The top 40 enriched biological processes were compared in Fig. 6F. Caerin treatment caused significant upregulation of proteins involved in immune response and regulation processes in the tumour, while many proteins upregulated appeared to play important roles in the processes related to antigen processing and presentation following control peptide treatment. This correlated with the finding that the injection of caerin largely reduced the population of B cells as suggested by the scRNA-seq (Supplementary File 2). A correlation was observed between proteins significantly upregulated only with the injection of caerin and cell populations identified in scRNA-seq (Fig. 6G). Of those proteins showing a fold change greater than 2, many were closely correlated with normalised expressions of genes in the populations of monocytes, MΦ and DCs, such as Iigp1, Gbp2, Irf5 and Parvg. There were a few proteins more closely associated with their gene expression in T cell and NK populations, including Satb1, Spn, Dok2 and Hip1r. Marker gene Gzmc appeared exclusive to NK cells, and protein upregulation was only considered significant with injection of caerin. Stat1 was detected as an upregulated gene in nearly all cell populations except B cells and was elevated with caerin treatment in the proteomic analysis, suggesting that Stat1 was largely regulated by caerin.
The KEGG pathways enriched (P-value<0.05) in upregulated proteins were compared for the different treatment in Fig. 6H, and more pathways were significantly identified with the caerin treatment, including apoptosis, natural killer cell mediated cytotoxicity, necroptosis, the signalling of nod-like receptor (NLR), TNF, chemokine, NF-Kappa B, RIG-I like receptor and toll-like receptor and several disease-related pathways. Amongst these KEGG pathways, NLR signalling was determined as the most enriched pathway (P-value=1.55E-10), supported by the significantly increased concentrations of Gbp2, Gbp5, Nlrc4, Ccl2, Tlr4 and so forth by caerin treatment; the genes of many of these proteins were detected as signatures for MΦ/DCs by scRNA-seq. Notably, the antigen processing and presenting KEGG pathway was less significantly changed with caerin treatment (P-value=6.0E-4) compared to control peptide treatment (P-value=1.0E-11), in accordance with the observation that the population of B cells was remarkably reduced by the injection of caerin peptides.