Mutual editing of alternative splicing between breast cancer cells and macrophages

Introduction

Tumor-associated macrophages (TAMs), one main immune cell population found in most solid tumors, contribute to tumor development, progression, drug resistance and suppression of antitumor immune cells (1–4). There are two main TAM subtypes: M1 macrophages, which encourage inflammation, and M2 macrophage, which encourage tissue repair (5). TAMs are mainly of the M2 phenotype (6). TAMs are recruited to the tumor as a response to cancer-associated inflammation (7,8) and acquire M2 properties in response to cytokines such as tumor growth factor (TGF)-β, interleukin (IL)-10 and macrophage colony-stimulating factor (M-CSF). An increased amount of M2 type TAMs is associated with a worse prognosis in several cancer types including breast cancer (9,10). However, recent single-cell sequencing results have shown that both M1 and M2-associated genes are frequently expressed in the same cells and are positively correlated with one another along the same activation trajectory. These results have challenged the model of macrophage polarization wherein M1 and M2 activation states are two discrete states (11). As macrophages affect all therapeutic modalities, further investigation of the interplay between macrophages and tumor cells is urgently needed.

The interplay between tumor cells and macrophage have been revealed. For example, macrophages can release inflammatory compounds such as tumor necrosis factor (TNF)-α which activates nuclear factor (NF)-κB and NF-κB regulates tumor cell apoptosis, proliferation and inflammation. Moreover, macrophages serve as a source for many pro-angiogenic factors including vascular endothelial growth factor (VEGF), TNF-α, M-CSF/CSF1 and IL-1 and IL-6 further contributing to tumor growth and metastasis (12). In turn, tumors produce factors, such as M-CSF/CSF1, monocyte chemoattractant protein-1 (MCP-1)/CCL2 and angiotensin II, triggering the amplification and mobilization of macrophages in tumors (13–15).

Alternative splicing (AS) is a post-transcriptional regulation process during gene expression that results in a single gene coding for multiple proteins. AS has been proved to play important roles in macrophage differentiation and regulation of most tumor hallmarks (16). For example, Liu et al found that splicing factor MBNL1 is the major regulator during the differentiation from monocytes to macrophages (17). Human M-CSF heterogeneity is partially derived from alternative mRNA splicing (18). However, how tumor cells and macrophages interplay at the alternative splicing level and its potential application in cancer therapy remain unknown. Understanding the tumor-macrophage crosstalk at the AS level would reveal the molecular mechanisms underlying tumor progression, drug resistance and suppression of immune cells and further shade light on the identification of new cancer therapeutic strategies.

Generally, TAMs appear to have an unfavorable role in breast cancer (19,20). Moreover, breast cancer is considered to be a highly heterogeneous disease, and many secreted factors may differentially contribute to the macrophage phenotype. Therefore, the aim of the present study was to understand macrophage ‘education’ under the influence of two breast cancer types, estrogen receptor-positive (ER+) breast cancer and triple-negative breast cancer (TNBC) at the AS level in a simplified setting. Through calculation of the number of common and specific altered AS events, the present study aimed to ascertain: i) Whether ER+ breast cancer and TNBC cells exert similar influences on co-cultured macrophage cells; ii) whether ER+ breast cancer and TNBC cells are affected to a similar degree at the AS level in the presence of macrophages; iii) which specific biological pathways are disturbed at the AS level in macrophage cells when cocultured with ER+ breast cancer and TNBC cells; iv) which specific biological pathways are disturbed at the AS level in ER+ breast cancer and TNBC cells at the presence of macrophage cells.

In the present study, we reanalyzed a previously published dataset (GSE75130) and our results showed that differing from transcriptional analysis, DNA repair and DNA damage processes were enriched in both ER+ breast cancer and TNBC after co-culturing with macrophages, which indicated the conserved functional regulation of macrophages on tumor cells in the tumor microenvironment. Meanwhile, macrophages were differentially regulated from the pathway views by co-culturing with ER+ breast cancer and TNBC cells. Sequence features of skipped exons from different conditions were also characterized in our analysis.

Materials and methods

GEO dataset GSE75130 was reanalyzed (21) which was originally used to characterize the differences in macrophage activation under the influence of either ER+ breast cancer or TNBC cells. For detailed treatment procedures please refer to a previous study (21).

Profiling of gene expression

Paired-end reads were mapped to the human genome primary assembly (GRCh37) (22), and the Ensembl human gene annotation for GRCh37 genebuild was used to improve the accuracy of the mapping with STAR software (STAR_2.4.2a) (23). FeatureCounts (version 1.4.6-p5) (24) was used to assign sequence reads to genes. Mitochondrial genes, ribosomal genes, and genes possessing less than five raw reads in half the samples were removed. Normalized gene expression profiles were obtained with the edgeR package 1.6 (25).

Alternative splicing analysis

For AS analysis, MISO software (version 0.5.4) (26) was used to analyze RNA-Seq data and estimate the percentage of splicing isoforms. Firstly, we utilized MISO to achieve the AS profiles for each treated sample and 5 types of AS profiles including alternative 3′/5′ splice site (A3SS, A5SS), skipped exons (SE), mutually exclusive exons (MXE) and retained introns (RI) were obtained. AS profiles were represented by percentage of splice-in (PSI/Ψ) values. Secondly, we identified the delta PSI (ΔPSI) by comparing the cocultured sample with individually cultured sample. Finally, we determine the significantly different AS events by Bayes factor >6 and |ΔPSI|>0.2.

Principal component analysis (PCA)

PCA was performed as previously described (27). A total of 20,169 genes were accounted for in PCA of the whole transcriptome. Expression was normalized with the reads per million mapped reads (RPM) method. The prcomp package from R was used to perform PCA and the default parameters were used (28). The ggplot2 package from R was used to draw the scatter plot (29). A total of 36,377 AS events were utilized in the PCA of the ER+ breast cancer and TNBC transcriptome analysis. A total of 35,476 AS events were utilized in the PCA of the macrophage transcriptome analysis.

Functional enrichment analysis

Gene Ontology (GO) term enrichment analysis was conducted with webserver DAVID 6.8 (30). P<0.05 was considered as statistically significant. Background gene list was set to 16,133 esemble genes which possess alternative splicing isoforms.

Protein-protein interaction analysis

Web server STRING (31) was utilized to explore the protein-protein interactions. Default parameters were applied.

NCBI protein domain analysis

Protein domains of full-length CHEK2 (accession no. CAG30304.1) was analyzed with NCBI webserver (https://www.ncbi.nlm.nih.gov) (32). Two significant protein domains were identified. The first domain is STKc_Chk2 (accession: cd14084) and the second is FHA (accession: cd00060).

Evolutionary conservation

Placental Mammal PhastCons scores were used to represent evolutionary conservation. For average conservation of exons, bigWigAverageOverBed (33) was used to calculate the mean conservation across each exon. The ggplot2 package from R was used to draw the box plot (29).

3′ and 5′splicing strength

To evaluate splice site strength, 3′ of the exon-intron boundary (20 nt into intron and +3 nt into exon) and 5′ of the exon-intron boundary (3 nt into exon and +6 nt into intron), together with the transcript sequences for these regions were obtained by bedtools (34). MaxEntScan (35) was used to calculate the strength of the splice sites for the AS exons.

Repetitive element enrichment

To identify repetitive elements in AS exons, Repeat Masker track was downloaded from UCSC Genome Browser and intersected with AS exons by bedtools intersect (34). Repeats were grouped into families defined by the Pfam database of repetitive DNA elements. Phylostratum scores were used to describe gene age, as previously reported (36).

Association between 222 splicing factors and altered AS events

Overlap of gene-associated events and conditional specific events (macrophages vs. macrophages co-cultured with T47D cells; macrophages vs. macrophages co-cultured with MDA-MB-231 cells; T47D cells vs. T47D cells co-cultured with macrophages; and MDA-MB-231 cells vs. MDA-MB-231 cells co-cultured with macrophages) were used to assess how much of the conditional specific events could be explained by each splicing factor. First, conditional specific ES events were isolated by comparing splicing events from treatment samples against those from the control samples. The significant splicing events were determined based on Bayes factor >6 and |ΔPSI| >0.2. Second, gene-associated ES events were determined by correlation analyses using gene expression levels (RPKM) and PSI values across all samples. Correlation coefficients (R-value) and corresponding P-values were calculated with the Pearson method, and gene-associated events were determined by R>0.2 and P<0.0001. Common events were considered as events that occurred (overlapped) in both gene-associated and conditional specific events. Finally, top 5 splicing factors which explain the most conditional specific events were isolated.

Statistical analyses

Significantly different AS events were determined by Bayes factor >6 and |ΔPSI|>0.2 during comparison of AS events between cocultured state and single cultured state. Fisher exact test was applied to test significance of GO term enrichment and P<0.05 was considered as statistically significant. R>0.2 and P<0.0001 were applied to identify significantly associated AS events to a specific splicing factor across 14 samples in this study. Wilcoxon signed-rank test was applied to test significance in Fig. 6A-D and P<0.05 was set to determine significance. Fisher's exact test was used to test significant enrichment of each elements and P-values were-log10 transformed to plot the heatmap in Fig. 6E.

Figure 6. Sequence features of AS events. (A) Violin plot showing the Phastcon score distribution of constitutive exons, exons from altered ES type events of macrophages co-cultured with MDA-MB-231 cells [Monocyte (M)], exons from altered ES type events of macrophages co-cultured with T47D [Monocyte (T)], exons from altered ES type events of MDA-MB-231 cells co-cultured with macrophages [MDA-MB-231 (M)] and exons from altered ES type AS events of T47D cells cocultured with macrophages [T47D (M)]. (B) Boxplot showing the log2 transformed skip exon length across samples. (C) Boxplot showing the 5′splicing strength across samples. (D) Boxplot showing the 3′ splicing strength across samples. (E) DNA element enrichment in Monocyte (M), Monocyte (T), MDA-MB-231 (M) and T47D (M). Constitutive skip exons were set as background. P-value derived hypergeometric distribution test and -log10(P) value was color scaled. (F) Top 5 splicing factors which were putatively responsible for the altered AS profiles in each condition. AS, alternative splicing.

Results

Confirmation of data reliability

To investigate the interplay between breast cancer cells and macrophages at the alternative splicing level, we downloaded one dataset from a previous study (21). In this previous study, freshly isolated human peripheral monocytes were cultured with two breast cancer cell lines (T47D, ER+ and MDA-MB-231, TNBC) in an in vitro Transwell co-culture assay. Then, at day five, the whole transcriptome of macrophage cells and breast tumor cells were sequenced. Detailed sample information was previously described (22). We firstly evaluated the data reliability by investigating the genetic variance among samples using gene expression profiles based on the assumption that samples from the same cell type and treatment should have smaller genetic distances. PCA was performed with 20,169 genes and the results showed that samples were grouped together according to the types and treatments (Fig. 1A). Then unsupervised clustering based on the same transcriptomes confirmed the dataset reliability (Fig. 1B).

Figure 1. Data quality verification. (A) Principal component analysis (PCA) of whole expression profiles of the individually cultured macrophages [Monocyte (1) and Monocyte (2)] and of T47D [Monocyte (T1), Monocyte (T2)] or MDA-MB-231 [Monocyte (M1), Monocyte (M2), Monocyte (M3)] co-cultured macrophages; individually cultured T47D cells and co-cultured T47D cells [T47D (M1), T47D (M2)]; individually cultured MDA-MB-231 cells and cocultured MDA-MB-231 cells [MDA-MB-231 (M1), (MDA-MB-231 (M2), (MDA-MB-231 (M3)] which showed genetic distances among these samples. (B) Unsupervised clustering analysis of genetic distances of samples from A.

DNA damage and DNA repair pathways are altered in both MDA-MB-231 and T47D cell lines by co-culturing with macrophages

Next, we aimed to ascertain how macrophages educate different breast cancer cells at the AS level. To obtain the AS profile for each sample, we performed the Mixture of Isoforms (MISO) pipeline (26) according to its recommended procedure (https://miso.readthedocs.io/en/fastmiso/). Five types of AS events including skip exon (SE), alternative 3′/5′ splice sites (A3SS, A5SS), mutually exclusive exons (MXE), and retained introns (RI) were analyzed. A total of 36,377 splicing events with values at least in 7 samples were identified. Then, PCA and unsupervised clustering were used to characterize the genetic distances among samples. The PCA result showed that both T47D and MDA-MB-231 individually cultured samples were obviously separated from their macrophage co-cultured states (Fig. 2A). Unsupervised clustering result also supported that co-cultured breast cancer cells were different from the individually cultured states (Fig. 2B). These results indicated that AS profiles of both TNBC and ER+ breast cancer were affected by co-culturing with macrophages.

Figure 2. AS alteration in TNBC (MDA-MB-231) and ER+ (T47D) breast cancer cells following co-culturing with macrophages. (A) Principal component analysis (PCA) of whole AS profiles of individually cultured T47D cells and co-cultured T47D cells; individually cultured MDA-MB-231 cells and cocultured MDA-MB-231 cells. (B) Clustering analysis of genetic distance of samples from (A). (C) Overlap of AS events between events altered in MDA-MB-231 cells cocultured with macrophages and events altered in T47D cells cocultured with macrophages. (D and E) Gene Ontology analysis of genes containing altered AS events in MDA-MB-231 cells co-cultured with macrophages (D) and genes containing altered AS events in T47D cells co-cultured with macrophage (E). AS, alternative splicing; TNBC, triple-negative breast cancer; ER+, estrogen receptor-positive.

Then, it was further determined which AS events were affected in each breast caner type and whether the altered AS events from two breast cancer types were largely similar or different. We isolated those altered splicing events by comparing the co-cultured states with individually cultured states from T47D and MDA-MB-231 cell lines, respectively. Statistically different splicing events with delta percentage of splice-in (ΔPSI) >0.2 and bayes factor >6 were isolated. A total of 101 and 108 different splicing events were identified from T47D and MDA-MB-231 cell lines, respectively (Tables I and II) and there were 10 common splicing events in both T47D and MDA-MB-231 altered splicing events. All of these results indicated that ER+ breast cancer cells and TNBC cells were differently ‘educated’ at the AS level by co-culturing with the macrophages (Fig. 2C).

Table I. Altered AS events in MDA-MB-231 cells co-cultured with macrophages.

Table I.

Altered AS events in MDA-MB-231 cells co-cultured with macrophages.

Gene	Events	ΔPSI (SRR2922607_vs_SRR2922615)	ΔPSI (SRR2922607_vs_SRR2922617)
GPS1	chr17:80009763:80009840:+@chr17:80010135:80010335:+@chr17:80011150:80011242:+	0.68	0.57
NA	chr17:79520100:79520153:-@chr17:79518954:79519078:-@chr17:79517274:79518229:-	0.62	0.61
INO80B, WBP1	chr2:74685527:74685798:+@chr2:74685959:74686046:+@chr2:74686565:74686872:+	0.60	0.48
TSC1	chr9:135786955-135786840:-@chr9:135786500-135786389:-	0.50	0.44
MTRF1	chr13:41837621:41837713:-@chr13:41836547:41836617:-@chr13:41836184:41836467:-@chr13:41834629:41835051:-	0.47	0.49
CTDP1	chr18:77477810:77478016:+@chr18:77488907:77489069:+@chr18:77496355:77496521:+	0.46	0.36
HAUS2	chr15:42851537:42851606:+@chr15:42852980:42853068:+@chr15:42853468:42853600:+	0.45	0.44
VWA9	chr15:65903469:65903134|65903436:-@chr15:65899497:65899763:-	0.44	0.45
PFDN5	chr12:53689235:53689423:+@chr12:53690214|53691634:53691708:+	0.44	0.66
SPAG9	chr17:49054469:49054582:-@chr17:49053224:49053262:-@chr17:49052132:49052308:-	0.43	0.25
PPCDC	chr15:75315927:75315967:+@chr15:75320588:75320794:+@chr15:75335782:75335877: +@chr15:75340894:75341062:+	0.42	0.41
OGG1	chr3:9796388:9796569:+@chr3:9798451:9798500:+@chr3:9807493:9808353:+	0.42	0.43
ATG12	chr5:115177087:115177548:-@chr5:115176515:115176631:-@chr5:115176194:115176309:-@chr5:115173325:115173461:-	0.41	0.34
RHOC, PPM1J	chr1:113249700:113250025:-@chr1:113247790|113248874:113247722:-	0.41	0.41
RBM25	chr14:73566375-73566458:+@chr14:73569900-73570186:+	0.40	0.39
KIAA1551	chr12:32123123:32123290:+@chr12:32133812:32138891:+@chr12:32140173:32140256:+	0.39	0.37
WDR26	chr1:224619283:224619179|224619227:-@chr1:224612220:224612356:-	0.37	0.33
PRPF4B	chr6:4060647-4060920:+@chr6:4061249-4061381:+	0.37	0.34
PRDX5	chr11:64085560:64085858:+@chr11:64087206:64087340:+@chr11:64088337:64088375:+	0.37	0.37
PFKM	chr12:48528726:48528821|48529166:+@chr12:48531504:48531629:+	0.36	0.35
MAP2K7	chr19:7968765:7968953:+@chr19:7970693:7970740:+@chr19:7974640:7974781:+	0.35	0.31
NFATC4	chr14:24845500:24845760|24846084:+@chr14:24846844:24848810:+	0.34	0.33
NAV3	chr12:78515720:78516208:+@chr12:78520947:78520988:+@chr12:78522486:78522646:+	0.34	0.34
APEX1	chr14:20923737-20923932:+@chr14:20924073-20924260:+	0.33	0.21
VWA9	chr15:65903486:65903024|65903134:-@chr15:65899497:65899780:-	0.33	0.32
TAGLN	chr11:117070040:117070148|117070545:+@chr11:117073718:117073909:+	0.33	0.33
SOD2	chr6:160103670-160103506:-@chr6:160100330-160100096:-	0.33	0.40
UBXN1	chr11:62444990:62445106:-@chr11:62444111|62444477:62443972:-	0.32	0.28
SDR39U1	chr14:24911552:24911658:-@chr14:24911384:24911466:-@chr14:24910880:24911001:-	0.32	0.29
ITGA6	chr2:173362703:173362828:+@chr2:173366500:173366629:+@chr2:173368819:173371181:+	0.32	0.29
TSPAN4	chr11:842824:842915:+@chr11:847201:847300:+@chr11:850288:850367:+@chr11:862550:862741:+	0.32	0.37
C16orf13	chr16:686094:686347:-@chr16:685612:685774:-@chr16:685281:685340:-	0.31	0.32
VWA9	chr15:65903469:65903134|65903436:-@chr15:65899497:65899780:-	0.3	0.33
RPAIN	chr17:5329291-5329619:+@chr17:5331391-5331531:+	0.29	0.27
SUV420H1	chr11:67980944:67981068:-@chr11:67957384:67957619:-@chr11:67953248:67953395:-	0.29	0.38
COX7A2	chr6:75950892:75950981:-@chr6:75949993:75950101:-@chr6:75947391:75947704:-	0.28	0.32
ZNRD1	chr6:30029036-30029175:+@chr6:30029292-30029446:+	0.28	0.38
GNB2L1	chr5:180669170:180669345:-@chr5:180668563|180668639:180668492:-	0.27	0.29
ERRFI1	chr1:8075555:8075752:-@chr1:8075368:8075460:-@chr1:8071779:8074456:-	0.27	0.30
SLC25A35	chr17:8192961-8192892:-@chr17:8191750-8191082:-	0.27	0.33
GOLGA2	chr9:131036129:131036251:-@chr9:131035064:131035144:-@chr9:131030699:131030803:-	0.27	0.39
NA	chr14:105180540:105181193:+@chr14:105181621:105181677:+@chr14:105185132:105185947:+	0.26	0.20
RFWD3	chr16:74700684:74700779:-@chr16:74698522:74698643:-@chr16:74694830:74695349:-@chr16: 74685818:74686020:-	0.26	0.20
SEC23A	chr14:39572797:39573033:-@chr14:39572236:39572432:-@chr14:39565102:39565343:-@chr14:39562391:39562448:-	0.26	0.26
RPS21	chr20:60962899-60962970:+@chr20:60963365-60963576:+	0.26	0.30
TXNRD1	chr12:104680460:104680887:+@chr12:104682709:104682818:+@chr12:104705068:104705190: +@chr12:104707023:104707095:+	0.26	0.39
ALG13	chrX:110928193:110928331:+@chrX:110929365:110929402:+@chrX:110931115:110933623:+	0.25	0.20
MTRF1	chr13:41837621:41837713:-@chr13:41836184:41836467:-@chr13:41834629:41835051:-	0.25	0.21
C16orf13	chr16:686094:686347:-@chr16:685518:685774:-@chr16:685281:685340:-	0.24	0.24
PHC2	chr1:33799893:33799691|33799786:-@chr1:33797876:33798005:-	0.24	0.26
C16orf13	chr16:686094:686347:-@chr16:685518:685774:-@chr16:685281:685340:-@chr16:684719:684797:-	0.24	0.30
PTPN3	chr9:112189230:112189402:-@chr9:112184998:112185132:-@chr9:112182704:112182880:-	0.23	0.22
BAX	chr19:49464067-49464171:+@chr19:49464789-49465055:+	0.23	0.22
PFKM	chr12:48528726:48528821:+@chr12:48529074:48529166:+@chr12:48531504:48531629:+	0.23	0.23
TMEM41B	chr11:9305140-9304918:-@chr11:9302588-9302201:-	0.23	0.23
NCAPG2	chr7:158444985:158445172:-@chr7:158443524:158443664:-@chr7:158439152:158439255:-	0.23	0.25
MTRF1L	chr6:153316454:153316271|153316379:-@chr6:153315648:153315811:-	0.23	0.30
ORMDL1	chr2:190648995:190649097:-@chr2:190647740:190647849:-@chr2:190647148:190647328:-	0.22	0.21
RHOC, PPM1J	chr1:113249700:113250025:-@chr1:113247722:113248874:-@chr1:113246266:113246428:-	0.22	0.21
BCAS3	chr17:59093112:59093262:+@chr17:59104227:59104271:+@chr17:59112027:59112151:+	0.22	0.23
TSPAN4	chr11:844081:844186:+@chr11:847201:847300:+@chr11:850288:850367:+@chr11:862550:862741:+	0.22	0.27
NFATC4	chr14:24845500-24845760:+@chr14:24846844-24848810:+	0.21	0.25
DRAM2	chr1:111663315:111663138|111663154:-@chr1:111662499:111662581:-	0.20	0.23
ANKRD49	chr11:94229770-94230117:+@chr11:94231237-94232744:+	0.20	0.28
MEF2A	chr15:100185766:100185969:+@chr15:100211528:100211659:+@chr15:100211725:100211862: +@chr15:100214598:100214817:+	−0.2	−0.33
NCOR2	chr12:124812179:124811955|124812093:-@chr12:124810737:124810916:-	−0.20	−0.24
TSPAN4	chr11:847201:847300:+@chr11:850288:850367:+@chr11:862550:862741:+	−0.20	−0.22
CYB5B	chr16:69458498:69458760:+@chr16:69481053:69481181:+@chr16:69492996:69493024:+@chr16: 69496333:69500167:+	−0.20	−0.21
MIR3654, EEF1G	chr11:62342380-62342149:-@chr11:62340214-62340056:-	−0.21	−0.22
MKNK2	chr19:2040133:2040176:-@chr19:2037828|2039855:2037470:-	−0.22	−0.29
NA	chr19:3638882:3639014:-@chr19:3633435:3633518:-@chr19:3630179:3633167:-	−0.22	−0.24
YKT6	chr7:44250622:44250723:+@chr7:44251153:44251203:+@chr7:44251846:44253893:+	−0.22	−0.23
FN1	chr2:216246935:216247048:-@chr2:216245534:216245803:-@chr2:216243853:216244040:-	−0.22	−0.22
MBNL2	chr13:98009736:98009889:+@chr13:98017390:98017425:+@chr13:98018713:98018807: +@chr13:98043576:98046374:+	−0.22	−0.22
ARL6IP4, RP11-197N18.2	chr12:123465676:123465813|123465846:+@chr12:123466118:123466426:+	−0.23	−0.21
UBC	chr12:125398665:125398551|125398587:-@chr12:125398042:125398320:-	−0.24	−0.27
C9orf9	chr9:135763677-135763824:+@chr9:135765344-135765418:+	−0.24	−0.26
MRRF	chr9:125026882:125027217:+@chr9:125027741:125027824:+@chr9:125033143:125033354:+	−0.25	−0.32
SPNS1	chr16:28986096:28986713:+@chr16:28986813:28986878:+@chr16:28989229:28989365: +@chr16:28990476:28990627:+	−0.25	−0.26
AC124789.1	chr17:36607870:36608071:-@chr17:36607550:36607769:-@chr17:36606638:36606967:-	−0.25	−0.23
TBRG1	chr11:124495567:124495799:+@chr11:124496369:124496505:+@chr11:124496800:124496946:+	−0.25	−0.21
H2AFY	chr5:134696187:134696297:-@chr5:134688636:134688735:-@chr5:134686513:134686603:- @chr5:134681658:134681747:-	−0.26	−0.38
NAV3	chr12:78513017:78513725:+@chr12:78515720:78516208:+@chr12:78522486:78522646:+	−0.26	−0.27
AC124789.1	chr17:36607870:36608071:-@chr17:36607550:36607637:-@chr17:36606638:36606967:-	−0.26	−0.25
LRRC28	chr15:99796103:99796330:+@chr15:99816781:99816821:+@chr15:99827462:99827499:+	−0.30	−0.42
OSBPL3	chr7:24903115:24903218:-@chr7:24902819:24902911:-@chr7:24901232:24901388:-	−0.30	−0.33
TBRG1	chr11:124495567:124495799:+@chr11:124496369:124496505:+@chr11:124496589: 124496647:+@chr11:124496800:124496946:+	−0.30	−0.32
NA	chr17:57232800:57232317|57232492:-@chr17:57187308:57189706:-	−0.30	−0.29
EEF1D	chr8:144679518:144679845:-@chr8:144674817:144675063:-@chr8:144671161:144672251:-	−0.31	−0.32
NBPF12	chr1:146395307:146395516:+@chr1:146397359:146397461:+@chr1:146398293:146398507:+	−0.32	−0.32
NA	chr6:30524486:30525227:+@chr6:30525927:30525989:+@chr6:30529105:30529285:+@chr6: 30529611:30529901:+	−0.32	−0.31
UBAP2L	chr1:154241233:154241430:+@chr1:154241838:154241888:+@chr1:154242676:154243329:+	−0.33	−0.20
ASB8	chr12:48551242:48551377:-@chr12:48547308|48547312:48547151:-	−0.35	−0.33
PARP3	chr3:51976361:51976724:+@chr3:51977365|51977370:51977554:+	−0.35	−0.33
EEF1D	chr8:144679518:144679845:-@chr8:144674817:144675063:-@chr8:144672815:144672908:- @chr8:144671161:144672251:-	−0.36	−0.40
MBNL2	chr13:98009736:98009889:+@chr13:98018713:98018807:+@chr13:98043576:98046374:+	−0.36	−0.37
ATG12	chr5:115176515:115176631:-@chr5:115176194:115176309:-@chr5:115173325:115173461:-	−0.37	−0.46
GCOM1, POLR2M	chr15:57967166:57967266:+@chr15:57976600:57976872:+@chr15:57998132:57999153: +@chr15:58001383:58001556:+	−0.37	−0.41
COPS7B	chr2:232646417:232646593:+@chr2:232653265:232653442:+@chr2:232658973:232659061: +@chr2:232660816:232661018:+	−0.37	−0.34
ZNF507	chr19:32836514:32836689:+@chr19:32838151:32838244:+@chr19:32843735:32845863:+	−0.40	−0.41
EEF1D	chr8:144679518:144679845:-@chr8:144674817:144675063:-@chr8:144672778:144672908:-	−0.41	−0.43
C17orf70	chr17:79519078:79518758|79518954:-@chr17:79517274:79518229:-	−0.43	−0.48
SAFB	chr19:5667057:5667175:+@chr19:5667358|5667364:5667461:+	−0.47	−0.54
FGFR1	chr8:38314874:38315052:-@chr8:38287200:38287466:-@chr8:38285864:38285953:-	−0.47	−0.32
PIK3CD	chr1:9711790:9711860:+@chr1:9751525:9751629:+@chr1:9770163:9770338:+@chr1:9770482:9770654:+	−0.53	−0.47
ACADVL	chr17:7127132-7127194:+@chr17:7127287-7127388:+	−0.57	−0.62
STAG2	chrX:123095164:123095285:+@chrX:123095636:123095706:+@chrX:123155217:123155281: +@chrX:123156381:123156521:+	−0.57	−0.61
NME3	chr16:1821411-1821270:-@chr16:1821172-1821071:-	−0.67	−0.49

[i] AS, alternative splicing; PSI, percentage of splice-in. NA: there is no HUGO gene symbol corresponding for the gene.

Table II. Altered AS events in T47D cells co-cultured with macrophages.

Table II.

Altered AS events in T47D cells co-cultured with macrophages.

Gene	Events	ΔPSI (SRR2922618_vs_SRR2922619)	ΔPSI (SRR2922618_vs_SRR2922620)
UBQLN1	chr9:86284100:86284242:-@chr9:86281265:86281309:-@chr9:86279945:86280060:-	−0.49	−0.57
PFDN5	chr12:53689235:53689423:+@chr12:53690214|53691634:53691708:+	−0.64	−0.54
ARMC8	chr3:137906148:137906441:+@chr3:137907297:137907372:+@chr3:137928659:137928735:+	−0.61	−0.53
SENP2	chr3:185316200:185316333:+@chr3:185316780:185316812:+@chr3:185318553:185318643:+	−0.59	−0.52
RRN3	chr16:15185172:15185228:-@chr16:15180222:15180311:-@chr16:15179986:15180115:-	−0.32	−0.51
SULF1	chr8:70378859:70379185:+@chr8:70408000:70408161:+@chr8:70414109:70414203:+	−0.44	−0.50
NA	chr5:10236590:10236727:-@chr5:10235323:10235373:-@chr5:10225620:10227759:-	−0.45	−0.49
NA	chr6:30524486:30525227:+@chr6:30525927:30525989:+@chr6:30529611:30529901:+	−0.47	−0.47
VAMP1	chr12:6574107-6574056:-@chr12:6572008-6571404:-	−0.43	−0.45
ZFAND6	chr15:80364903:80364989:+@chr15:80390758:80390920:+@chr15:80412670:80412840:+	−0.38	−0.45
CNOT1	chr16:58663632:58663790:-@chr16:58658557:58658698:-@chr16:58657186:58657313:- @chr16:58633140:58633415:-	−0.38	−0.45
C16orf13	chr16:686094:686347:-@chr16:685612:685774:-@chr16:685281:685340:-	−0.31	−0.45
43349	chrX:118759298:118759342:-@chrX:118752749|118754014:118750909:-	−0.28	−0.45
SP140L	chr2:231253274:231253348:+@chr2:231254634:231254738:+@chr2:231256802:231256944:+	−0.39	−0.44
TMEM91, BCKDHA, CTC-435M10.3	chr19:41888677:41888826:+@chr19:41889466:41889537:+@chr19:41889768:41889987:+	−0.29	−0.44
VAMP1	chr12:6574107-6574056:-@chr12:6572008-6571403:-	−0.40	−0.43
IKBKB	chr8:42128820:42128987:+@chr8:42129601:42129723:+@chr8:42146152:42146246: +@chr8:42147674:42147791:+	−0.75	−0.41
FKBP14	chr7:30062281:30062432:-@chr7:30059829:30059920:-@chr7:30058612:30058739:-	−0.40	−0.41
NCOR2	chr12:124862783:124862930:-@chr12:124858959:124859009:-@chr12:124856568:124857156:-	−0.33	−0.41
ANAPC11	chr17:79849599:79849709|79849717:+@chr17:79852343:79852462:+	−0.36	−0.40
MYO5A	chr15:52638558:52638658:-@chr15:52635314:52635394:-@chr15:52632393:52632591:-	−0.37	−0.39
C16orf13	chr16:686094:686347:-@chr16:685518:685774:-@chr16:685281:685340:-@chr16:684719:684797:-	−0.27	−0.38
PTPN3	chr9:112189230:112189402:-@chr9:112184998:112185132:-@chr9:112182704:112182880:-	−0.29	−0.37
43349	chrX:118763281:118763471:-@chrX:118752749|118754014:118750909:-	−0.29	−0.37
ATP6V0D1	chr16:67514860:67515089:-@chr16:67478431:67478609:-@chr16:67477002:67477081:-	−0.31	−0.36
PILRB, CTB-161A2.4	chr7:99950187-99950537:+@chr7:99950620-99950746:+	−0.37	−0.35
NA	chr3:37396592:37396678:+@chr3:37402734:37402796:+@chr3:37407571:37408370:+	−0.26	−0.35
MUC1	chr1:155160639:155160707:-@chr1:155160198:155160334:-@chr1:155159701:155159850:-	−0.37	−0.34
DALRD3	chr3:49058279:49058467:-@chr3:49055833:49055986:-@chr3:49055456:49055751:-	−0.33	−0.34
RP11-66N24.4	chr14:24025198:24025552:+@chr14:24025952:24026243:+@chr14:24027904:24028790:+	−0.31	−0.34
ZNRD1	chr6:30029036-30029175:+@chr6:30029292-30029446:+	−0.31	−0.33
NA	chr7:75633076:75633173:-@chr7:75630208:75630272:-@chr7:75625655:75625917:-	−0.36	−0.32
DLG1	chr3:196921296:196921460:-@chr3:196888511:196888609:-@chr3:196876614:196876667:-	−0.3	−0.32
NEIL2	chr8:11627172:11627300:+@chr8:11627683:11627844:+@chr8:11628955:11629094:+	−0.29	−0.32
C16orf13	chr16:686094:686347:-@chr16:685518:685774:-@chr16:685281:685340:-	−0.21	−0.32
TPD52L1	chr6:125574863:125574901:+@chr6:125578244:125578304:+@chr6:125583980:125584644:+	−0.37	−0.31
ORMDL1	chr2:190648995:190649097:-@chr2:190647740:190647849:-@chr2:190647148:190647328:-	−0.36	−0.31
ORMDL1	chr2:190648995:190649097:-@chr2:190647328|190647849:190647148:-	−0.40	−0.29
SMPD4	chr2:130921947:130922018:-@chr2:130918759:130918845:-@chr2:130914824:130914969:-	−0.34	−0.29
AP001055.7, C21orf33	chr21:45553494:45553720:+@chr21:45553984:45554036:+@chr21:45555942:45556055:+	−0.26	−0.28
NA	chr22:50964675:50964905:-@chr22:50964430:50964570:-@chr22:50961997:50962853:-	−0.25	−0.28
NA	chr17:5326089:5326149:+@chr17:5331391:5331531:+@chr17:5335862:5336340:+	−0.34	−0.27
NA	chr10:14920782:14920918:+@chr10:14923499:14923644:+@chr10:14938845:14939516:+	−0.29	−0.27
C1orf52	chr1:85725041:85725355:-@chr1:85724618:85724744:-@chr1:85724207:85724405:-	−0.28	−0.27
RP11-66N24.4	chr14:24025198:24025552:+@chr14:24025952:24026248:+@chr14:24027904:24028790:+	−0.27	−0.27
ISYNA1	chr19:18548670:18548798:-@chr19:18547289|18547915:18547140:-	−0.26	−0.27
ENTPD6	chr20:25176339:25176503:+@chr20:25187158:25187226:+@chr20:25187712:25188033:+	−0.23	−0.26
NA	chr11:72983352:72983511:+@chr11:73006775:73006860:+@chr11:73007530:73009664:+	−0.31	−0.25
MBD1	chr18:47801347:47801415:-@chr18:47800556:47800723:-@chr18:47799934:47800233:-	−0.26	−0.25
CRYZ	chr1:75175782:75175931:-@chr1:75172787:75172888:-@chr1:75172583:75172678:-	−0.25	−0.25
SLMAP	chr3:57908616:57908750:+@chr3:57911572:57911661:+@chr3:57913023:57914894:+	−0.24	−0.25
ZMYM2	chr13:20567203-20567704:+@chr13:20567966-20568059:+	−0.32	−0.24
CHEK2	chr22:29095826:29095925:-@chr22:29092889:29092975:-@chr22:29091698:29091861:-	−0.28	−0.24
MBD1	chr18:47801347:47801415:-@chr18:47800556:47800720:-@chr18:47799934:47800233:-	−0.25	−0.24
ATP5J	chr21:27107965:27107164|27107626:-@chr21:27101942:27102112:-	−0.24	−0.24
UBE2D3	chr4:103749101:103749307:-@chr4:103748749:103748898:-@chr4:103747642:103747793:-	−0.22	−0.24
ATP13A3	chr3:194134488:194134568:-@chr3:194132928:194133017:-@chr3:194123403:194126845:-	−0.27	−0.23
MBD1	chr18:47801499:47801615:-@chr18:47801347:47801415:-@chr18:47800556:47800723:- @chr18:47799934:47800233:-	−0.25	−0.23
SDR39U1	chr14:24911552:24911658:-@chr14:24911384:24911466:-@chr14:24910880:24911001:-	−0.23	−0.23
CTAGE5, MIA2	chr14:39788413:39788495:+@chr14:39790132:39790260:+@chr14:39796068:39796226:+	−0.27	−0.22
PAK4	chr19:39660172:39660397:+@chr19:39663558:39663746:+@chr19:39664216:39664650:+	−0.22	−0.22
ZFAND5	chr9:74979612:74980163:-@chr9:74978386:74978522:-@chr9:74975544:74975703:-	−0.22	−0.22
CLDND1, CPOX	chr3:98241693:98241910:-@chr3:98240497:98240547:-@chr3:98239977:98240286:-	−0.36	−0.21
NA	chr7:75039605:75039743:+@chr7:75039889:75040009:+@chr7:75044163:75044301:+	−0.33	−0.21
HSPA14	chr10:14882074:14882156:+@chr10:14884133:14884845:+@chr10:14885370:14886747:+	−0.25	−0.21
TPD52L1	chr6:125569428:125569529:+@chr6:125574863:125574901:+@chr6:125578244:125578304: +@chr6:125583980:125584644:+	−0.26	−0.20
LGMN	chr14:93176017:93176217:-@chr14:93170985:93171052:-@chr14:93170152:93170706:-	−0.24	−0.20
GGCX	chr2:85788509:85788657:-@chr2:85787938:85788108:-@chr2:85786040:85786198:-	−0.22	−0.20
OS9	chr12:58112776:58112965:+@chr12:58113882:58114046:+@chr12:58114189:58114301:+	−0.22	−0.20
UBC	chr12:125398665:125398551|125398587:-@chr12:125398042:125398320:-	0.20	0.20
MRPL9	chr1:151735622:151735431|151735466:-@chr1:151734852:151734976:-	0.20	0.20
EEF1D	chr8:144679518:144679845:-@chr8:144674817:144675063:-@chr8:144672815:144672908:- @chr8:144671161:144672251:-	0.23	0.20
RPL17, RPL17-C18ORF32	chr18:47018628:47018834:-@chr18:47017954|47018203:47017902:-	0.20	0.21
ENOX2	chrX:129790555:129790669:-@chrX:129771378|129771384:129771203:-	0.20	0.22
ARL6IP4, RP11-197N18.2	chr12:123465676:123465813|123465846:+@chr12:123466142:123466426:+	0.21	0.23
MRPL9	chr1:151735622:151735426|151735466:-@chr1:151734852:151734976:-	0.23	0.23
NOP56	chr20:2636580:2636680|2636860:+@chr20:2637047:2637195:+	0.20	0.24
CPSF3L	chr1:1249681:1249745:-@chr1:1249301|1249485:1249112:-	0.27	0.24
NA	chr18:674406-674161:-@chr18:673076-670324:-	0.20	0.27
C10orf2	chr10:102747293:102747370|102749210:+@chr10:102749401:102749641:+	0.21	0.28
LIME1, ZGPAT	chr20:62367133-62368064:+@chr20:62368886-62369000:+	0.26	0.28
FLOT1	chr6:30698877-30698696:-@chr6:30698504-30698459:-	0.22	0.29
GYLTL1B	chr11:45944329-45944515:+@chr11:45944609-45944718:+	0.39	0.29
MIIP	chr1:12089280-12089951:+@chr1:12090085-12090181:+	0.27	0.33
SCMH1	chr1:41503034:41503213:-@chr1:41499655:41499774:-@chr1:41494256:41494398:-	0.36	0.33
TAOK2	chr16:29997586-29997825:+@chr16:29999680-29999719:+	0.48	0.34
SRSF5	chr14:70235515-70235613:+@chr14:70235899-70237257:+	0.29	0.35
PQLC3	chr2:11304330:11304387:+@chr2:11312051:11312171:+@chr2:11315094:11315135: +@chr2:11317863:11318998:+	0.34	0.35
APEX1	chr14:20923385-20923487:+@chr14:20923737-20923862:+	0.35	0.35
PGAP2	chr11:3832480:3832654:+@chr11:3838662:3838765:+@chr11:3844139:3844223: +@chr11:3845113:3845365:+	0.53	0.39
ARL6IP4, RP11-197N18.2	chr12:123465676:123465813:+@chr12:123466118|123466142:123466426:+	0.33	0.42
TCEA2	chr20:62701118-62701174:+@chr20:62701613-62701767:+	0.51	0.48
PGAP2	chr11:3832480:3832654:+@chr11:3838583:3838765:+@chr11:3844139:3844223: +@chr11:3845113:3845365:+	0.62	0.48
WDR19	chr4:39274600-39274681:+@chr4:39276428-39276578:+	0.41	0.49
ZNF707	chr8:144766622:144766712:+@chr8:144766926:144767237:+@chr8:144771374:144771471: +@chr8:144772227:144772293:+	0.40	0.50
ARSA	chr22:51066601:51066326|51066374:-@chr22:51065984:51066220:-	0.42	0.59
ZBTB8OS	chr1:33116030:33116155:-@chr1:33100369:33100393:-@chr1:33099552:33099673:-	0.59	0.59
RGL2	chr6:33261843-33261811:-@chr6:33261693-33261572:-	0.49	0.60
OSBPL6	chr2:179204399:179204491:+@chr2:179209013:179209087:+@chr2:179213951:179214116:+	0.56	0.60
WDR90	chr16:705773-705889:+@chr16:706302-706537:+	0.28	0.64
TIMM9	chr14:58894110:58894232:-@chr14:58893772:58893960:-@chr14:58878625:58878689:- @chr14:58877561:58877656:-	0.50	0.68

[i] AS, alternative splicing; PSI, percentage of splice-in. NA: there is no HUGO gene symbol corresponding for the gene.

To further investigate the biological processes affected by those altered AS events, Gene Ontology (GO) functional enrichment analysis was performed. GO terms such as DNA repair, nucleotide-excision repair, DNA damage recognition, response to reactive oxygen species, intrinsic apoptotic signaling pathway in response to DNA damage were significantly enriched in the MDA-MB-231-altered AS events (Fig. 2D). Biological processes such as DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest, stress-activated MAPK cascade, signal transduction in response to DNA damage were significantly enriched in T47D-altered alternative splicing events (Fig. 2E). Taken together, although the altered AS events from MDA-MB-231 and T47D cells were different, DNA repair and DNA damage-related biological processes were significantly enriched in both breast cancer cell lines of differing types in the presence of macrophages.

Then, we investigated how the DNA repair and DNA damage-related AS events influence the function of corresponding genes. Fig. 3A and B shows the DNA repair and DNA damage-related AS events identified from MDA-MB-231 and T47D samples, respectively. Sashimi plots showed the AS events ‘chr22:29095826:29095925:-@chr22:29092889:29092975:-@chr22:29091698:29091861:-’ from gene Chek2 in T47D individual cultured sample (SRR2922618) and co-cultured sample (SRR2922619) (Fig. 3C). CHEK2 is a cell cycle checkpoint regulator and putative tumor suppressor (37) which contains a forkhead-associated protein interaction domain essential for activation in response to DNA damage and is rapidly phosphorylated in response to replication blocks and DNA damage. CHEK2 has been shown to stabilize the tumor-suppressor protein p53 (38,39), leading to cell cycle arrest in G1 and it also interacts with and phosphorylates BRCA1, allowing BRCA1 to restore survival after DNA damage (40). According to our domain analysis, we found that CHEK2 contains two crutial functional domains (Fig. 3D): The first one is STKc_Chk2, in which STKs can catalyze the transfer of a γ-phosphoryl group from ATP to serine/threonine residues on protein substrates, and the second one is FHA domain which is a putative nuclear signaling domain. The skipped exon of Chek2 encodes 28 aa which locates in the STKc_Chk2 domain (Fig. 3D). Thus the inclusion or exclusion of the skippd exon changes the STKc_Chk2 domain of Chek2, which may play an important role in the functional regulation of CHEK2. Protein-protein interaction network analysis showed that these DNA repair proteins have extensive interactions and CHEK2 may serve as a hub gene in the network (Fig. 3E).

Figure 3. DNA repair-related AS events altered in MDA-MB-231 and T47D cells by co-culturing with macrophages. (A and B) DNA repair-related AS events altered in MDA-MB-231 cells cocultured with macrophages (A) and T47D cells co-cultured with macrophages (B). (C) Example of AS events from CHEK2 gene in T47D cells individually cultured (SRR2922618) and co-cultured with macrophages (SRR2922619). Numbers indicate read counts supporting the splicing junctions. Ψ indicates PSI (percentage of splice-in). (D) Two functional domains identified in CHEK2: FHA domain and STKc_Chk2 domain. 28 aa was the amino acids encoded by the skip exon which is marked in black. (E) Protein-protein interaction (PPI) among DNA repair-related proteins. Colored edges represent active interaction sources (https://string-db.org/). AS, alternative splicing. AS, alternative splicing.

Altered AS events in macrophages by co-culturing with MDA-MB-231 and T47D cells, respectively

As the differentiation of TAMs is largely regulated by mediators secreted by tumor cells, it was ascertained how different breast cancer cells ‘educate’ macrophages and regulate the differentiation process at the AS level. Similarly, a total of 35,476 splicing events with values in at least 7 samples were identified. Then, PCA and unsupervised clustering were used to characterize the genetic distances among samples. From the PCA result, it was revealed that macrophages individually cultured were separated from their co-cultured states whereas the co-cultured states of macrophage from T47D and MDA-MB-231 cells were not able to be separated (Fig. 4A), which is also supported by the unsupervised clustering result (Fig. 4B). Then, we isolated those altered splicing events by comparing the co-cultured samples with individually cultured samples. A total of 115 and 118 significant splicing events were identified in macrophages co-cultured with MDA-MB-231 and T47D cells, respectively (Tables III and IV). There were 14 common splicing events in the macrophages co-cultured with T47D and MDA-MB-231 cells (P>0.2) which indicated that ER+ breast cancer cells and TNBC cells have different influences on macrophages at the AS level (Fig. 4C).

Figure 4. AS alteration in macrophages co-cultured with TNBC (MDA-MB-231) and ER+ (T47D) breast cancer cells. (A) Principal component analysis (PCA) of whole alternative splicing profiles of individually cultured macrophages and of T47D or MDA-MB-231 cells co-cultured with macrophages. (B) Unsupervised clustering analysis of genetic distance of samples from (A). (C) Overlap of AS events between events altered in macrophages co-cultured with MDA-MB-231 cells and events altered in macrophages co-cultured with T47D cells. (D and E) Gene Ontology analysis of genes containing altered AS events in macrophages cocultured with MDA-MB-231 cells (D) and genes containing altered AS events in macrophages cocultured with T47D cells. (E) AS, alternative splicing; TNBC, triple-negative breast cancer; ER+, estrogen receptor-positive.

Table III. Altered AS events in macrophages co-cultured with MDA-MB-231 cells.

Table III.

Altered AS events in macrophages co-cultured with MDA-MB-231 cells.

Gene	Events	ΔPSI (SRR2922608_vs_SRR2922609)	ΔPSI (SRR2922608_vs_SRR2922611)
WARS	chr14:100841620:100841927:-@chr14:100840473:100840602:-@chr14:100840317:100840401:- @chr14:100835424:100835595:-	−0.44	−0.87
WARS	chr14:100841620:100841927:-@chr14:100840473:100840581:-@chr14:100840317:100840401:- @chr14:100835424:100835595:-	−0.47	−0.85
SLC25A19	chr17:73285170:73285453:-@chr17:73284583:73284672:-@chr17:73284469:73284542:- @chr17:73282714:73282883:-	−0.44	−0.54
AKR1B1	chr7:134132813-134132732:-@chr7:134132133-134132050:-	−0.59	−0.53
NA	chr3:148750159-148749988:-@chr3:148749150-148747904:-	−0.47	−0.53
WDR67	chr8:124153001:124153141:+@chr8:124154502:124154696:+@chr8:124156957:124157095:+	−0.57	−0.50
AKR1B1	chr7:134132813:134132213|134132732:-@chr7:134132050:134132133:-	−0.58	−0.49
PLK3	chr1:45270011-45270173:+@chr1:45270322-45270451:+	−0.35	−0.47
NA	chr13:99852679:99853193:+@chr13:99883688:99883767:+@chr13:99890681:99890808:+	−0.29	−0.45
RABGAP1L	chr1:174780970:174781098:+@chr1:174846530:174846743:+@chr1:174926594:174926686:+	−0.44	−0.44
GBA2	chr9:35737936-35737745:-@chr9:35737601-35736864:-	−0.36	−0.43
AP1G2	chr14:24031497:24031624:-@chr14:24031171:24031275:-@chr14:24030721:24030844:-	−0.31	−0.43
IFFO1	chr12:6658922:6659062:-@chr12:6658642:6658650:-@chr12:6657834:6657991:-	−0.56	−0.42
HYAL2	chr3:50359204:50358797|50358999:-@chr3:50357000:50357966:-	−0.47	−0.42
PQLC3	chr2:11312051:11312171:+@chr2:11315094:11315135:+@chr2:11317863:11318998:+	−0.31	−0.42
APEX1	chr14:20923385-20923487:+@chr14:20923737-20923862:+	−0.46	−0.39
CKB	chr14:103987600:103987771:-@chr14:103986839:103986929:-@chr14:103986459:103986648:-	−0.47	−0.37
RASSF7	chr11:560971:561033:+@chr11:561762:561892:+@chr11:562079:562776:+	−0.42	−0.37
UGP2	chr2:64068098:64068366:+@chr2:64069193:64069338:+@chr2:64069673:64069733: +@chr2:64083440:64083567:+	−0.32	−0.37
UGP2	chr2:64068098:64068366:+@chr2:64069193:64069338:+@chr2:64082018:64082070: +@chr2:64083440:64083567:+	−0.38	−0.36
SPATA20	chr17:48624429:48624646:+@chr17:48625081:48625128:+@chr17:48625644:48625814:+	−0.29	−0.36
UEVLD	chr11:18557953:18558016:-@chr11:18555877:18556000:-@chr11:18552950:18554034:-	−0.41	−0.35
MX1	chr21:42797978:42798190:+@chr21:42799133:42799231:+@chr21:42799682:42799793:+	−0.32	−0.34
SPHK1	chr17:74381532-74381735:+@chr17:74382024-74382218:+	−0.32	−0.34
NIPBL	chr5:37063892-37064121:+@chr5:37064629-37065921:+	−0.38	−0.33
DYSF	chr2:71738937:71739051:+@chr2:71740370:71740462:+@chr2:71740846:71741051:+	−0.38	−0.33
PSTPIP1	chr15:77327849:77327904:+@chr15:77328143|77328152:77328276:+	−0.31	−0.33
PXN	chr12:120703420:120703574:-@chr12:120664642:120664920:-@chr12:120662446:120662530:- @chr12:120661954:120662180:-	−0.30	−0.33
ZSWIM7	chr17:15881477-15881358:-@chr17:15881227-15881047:-	−0.25	−0.33
FAM21C	chr10:46252460:46252587:+@chr10:46254763:46254849:+@chr10:46258836:46258937:+	−0.24	−0.33
STXBP2	chr19:7707869:7707934:+@chr19:7708051:7708131:+@chr19:7709500:7709638:+	−0.25	−0.32
NA	chrX:30742218:30742298:+@chrX:30745583:30745669:+@chrX:30746849:30749577:+	−0.42	−0.31
CEP350	chr1:180080132-180080330:+@chr1:180081572-180082515:+	−0.35	−0.31
PCNP	chr3:101293042:101293123:+@chr3:101298634|101298686:101298848:+	−0.25	−0.31
ZSWIM7	chr17:15881477-15881358:-@chr17:15881227-15879875:-	−0.24	−0.30
DGUOK	chr2:74173846:74174033:+@chr2:74177712:74177859:+@chr2:74185273:74185372:+	−0.33	−0.29
NA	chr12:9845424:9845527:+@chr12:9845630:9845711:+@chr12:9846421:9846544:+@chr12:9847356:9852151:+	−0.50	−0.28
CKB	chr14:103987600:103987771:-@chr14:103986806:103986929:-@chr14:103986459:103986648:-	−0.28	−0.28
C9, DAB2	chr5:39388901:39388954:-@chr5:39388407:39388469:-@chr5:39382720:39383373:-@chr5:39381556:39381718:-	−0.21	−0.28
TBRG1	chr11:124495567:124495799:+@chr11:124496369:124496505:+@chr11:124496800:124496946:+	−0.27	−0.27
NA	chr12:9845424:9845527:+@chr12:9846421:9846544:+@chr12:9847356:9852151:+	−0.45	−0.26
TNIP1	chr5:150460441:150460645:-@chr5:150444521:150444688:-@chr5:150443174:150443308:-	−0.31	−0.26
PARVG	chr22:44568836:44569071:+@chr22:44577622:44577797:+@chr22:44579198:44579288:+	−0.28	−0.26
NIN	chr14:51226575:51227077:-@chr14:51223210:51225348:-@chr14:51221460:51221585:-	−0.22	−0.26
NA	chr6:30181082:30181271:-@chr6:30172433:30172542:-@chr6:30168812:30168915:-	−0.25	−0.25
VPS33B	chr15:91565384:91565833:-@chr15:91561035:91561115:-@chr15:91557614:91557663:-	−0.37	−0.24
HYAL2	chr3:50360084:50360281:-@chr3:50358797:50359204:-@chr3:50357000:50357966:-	−0.35	−0.24
YIPF1	chr1:54325729:54325826:-@chr1:54320674:54320724:-@chr1:54317392:54317943:-	−0.20	−0.24
UBE2D3	chr4:103748749:103749105:-@chr4:103747793|103748001:103747642:-	−0.26	−0.23
PPP1R12A	chr12:80201006:80201110:-@chr12:80199946:80200113:-@chr12:80199372:80199548: -@chr12:80192274:80192364:-	−0.22	−0.22
TANK	chr2:162016855:162016995|162016997:+@chr2:162036125:162036272:+	−0.24	−0.21
NA	chr12:15747871-15747991:+@chr12:15749024-15751265:+	−0.23	−0.21
C17orf76-AS1	chr17:16342641-16342728:+@chr17:16342895-16343567:+	−0.23	−0.21
CPNE1, NFS1	chr20:34252682:34252878:-@chr20:34246852:34246936:-@chr20:34220717:34220845:-	−0.30	−0.20
NA	chr12:9845424:9845527|9846544:+@chr12:9847356:9852151:+	−0.26	−0.20
TNIP1	chr5:150460441:150460645:-@chr5:150444521:150444692:-@chr5:150443174:150443308:-	−0.25	−0.20
CNN2	chr19:1032558:1032695:+@chr19:1036066:1036245:+@chr19:1036415:1036561:+	−0.21	−0.20
TNFRSF12A	chr16:3070313:3070492:+@chr16:3071216:3071320:+@chr16:3071556:3071690:+	−0.20	−0.20
CD44	chr11:35211382:35211612:+@chr11:35219668:35219793:+@chr11:35232793:35232996: +@chr11:35236399:35236461:+	0.20	0.20
GK	chrX:30671476:30671732:+@chrX:30683628:30683701:+@chrX:30695492:30695569:+	0.40	0.20
UNC45A	chr15:91478515:91478560|91478605:+@chr15:91478774:91478935:+	0.20	0.21
43166	chr2:160615737:160615846|160615869:+@chr2:160619391:160619504:+	0.21	0.21
TBC1D15	chr12:72287002:72287104:+@chr12:72288105:72288155:+@chr12:72288466:72288663:+	0.22	0.21
CD44	chr11:35211382:35211612:+@chr11:35226059:35226187:+@chr11:35227659:35227790:+	0.23	0.21
APLP2	chr11:129992200:129992408:+@chr11:129993507:129993674:+@chr11:129996595:129996725:+	0.20	0.22
NUDT22	chr11:63993762-63993899:+@chr11:63994107-63994604:+	0.23	0.22
DCAF6	chr1:167973771:167974031:+@chr1:167992226:167992285:+@chr1:168007609:168007726:+	0.24	0.22
CBFB	chr16:67116116:67116211|67116242:+@chr16:67132613:67134958:+	0.20	0.23
CD44	chr11:35211382:35211612:+@chr11:35229652:35229756:+@chr11:35232793:35232996:+@chr11: 35236399:35236461:+	0.21	0.23
DPP7	chr9:140009170-140009099:-@chr9:140009028-140008915:-	0.28	0.23
ARGLU1	chr13:107212005-107211780:-@chr13:107209479-107209396:-	0.32	0.23
BAG6	chr6:31620477:31620177|31620201:-@chr6:31619433:31619553:-	0.27	0.24
PLD1	chr3:171405161:171405374:-@chr3:171404475:171404588:-@chr3:171395356:171395484:-	0.36	0.24
RFFL, RAD51L3-RFFL	chr17:33353393:33353580:-@chr17:33348390:33348800:-@chr17:33344542:33344625:-	0.37	0.24
NCOR2	chr12:124812179:124811955|124812093:-@chr12:124810737:124810916:-	0.25	0.25
CELF2	chr10:11370889-11371060:+@chr10:11372419-11374591:+	0.32	0.26
CELF2	chr10:11370889-11371060:+@chr10:11372419-11374605:+	0.32	0.26
CD44	chr11:35211382:35211612:+@chr11:35231512:35231601:+@chr11:35232793:35232996: +@chr11:35236399:35236461:+	0.26	0.27
CELF2	chr10:11370889-11371060:+@chr10:11372419-11376357:+	0.34	0.27
RP11-458D21.5	chr1:145281369:145281704:+@chr1:145290429:145290511:+@chr1:145293114:145293272: +@chr1:145293371:145293580:+	0.34	0.27
RNASE1	chr14:21270956:21271036:-@chr14:21270408:21270478:-@chr14:21269515:21270252:-	0.27	0.28
TMX2-CTNND1, RP11-691N7.6,CTNND1	chr11:57529234:57529518|57529591:+@chr11:57556509:57556627:+	0.24	0.29
CD44	chr11:35211382:35211612:+@chr11:35219695:35219793:+@chr11:35232793:35232996: +@chr11:35236399:35236461:+	0.28	0.29
PGAP2	chr11:3832480:3832654:+@chr11:3838662:3838765:+@chr11:3845113:3845365:+	0.31	0.29
SRRM1	chr1:24989151:24989295:+@chr1:24989674:24989715:+@chr1:24993306:24993416:+	0.34	0.29
NSUN4	chr1:46810473:46810816:+@chr1:46812593:46812747:+@chr1:46818540:46818700:+	0.33	0.30
CHFR	chr12:133447310:133447369:-@chr12:133446205:133446384:-@chr12:133438053:133438220:-	0.51	0.30
SNX17	chr2:27593389:27593673:+@chr2:27594136:27594174:+@chr2:27595490:27595607:+	0.23	0.31
ITGA6	chr2:173362703:173362828:+@chr2:173366500:173366629:+@chr2:173368819:173371181:+	0.26	0.32
CHFR	chr12:133447310:133447369:-@chr12:133446205:133446420:-@chr12:133438053:133438220:-	0.46	0.32
NA	chr14:20923290:20923497|20923548:+@chr14:20923737:20923862:+	0.35	0.33
PNPLA8	chr7:108166473:108166762:-@chr7:108161920:108161965:-@chr7:108154880:108156018:-	0.4	0.34
GOLIM4	chr3:167759180:167759262:-@chr3:167758574:167758657:-@chr3:167754624:167754782:-	0.29	0.35
LILRA2, LILRB1	chr19:55087274:55087576:+@chr19:55088932:55089190:+@chr19:55098477:55098527: +@chr19:55098668:55099027:+	0.31	0.35
FLNB	chr3:58124009:58124256:+@chr3:58127585:58127656:+@chr3:58128377:58128479:+	0.41	0.36
CD44	chr11:35211382:35211612:+@chr11:35219695:35219793:+@chr11:35226059:35226187:+ @chr11:35229652:35229753:+	0.47	0.36
STX2	chr12:131283070:131283180:-@chr12:131280540:131280665:-@chr12:131274145:131276522:-	0.29	0.38
POLD1	chr19:50910584:50910672:+@chr19:50911964|50912042:50912158:+	0.37	0.38
NA	chr13:99852679:99853193:+@chr13:99853683:99853821:+@chr13:99883688:99883767: +@chr13:99890681:99890808:+	0.46	0.41
AFTPH	chr2:64800080:64800202:+@chr2:64806620:64806680:+@chr2:64812556:64812679:+	0.52	0.41
NA	chr1:120930039:120930293:-@chr1:120928331:120928615:-@chr1:120926128:120927417:-	0.50	0.43
AC069513.3, SDHAP2	chr3:195389442:195389604:+@chr3:195390495:195390703:+@chr3:195390957:195391121:+	0.34	0.44
ELMOD3	chr2:85617261-85617388:+@chr2:85617883-85618875:+	0.43	0.44
ELMOD3	chr2:85617261-85617388:+@chr2:85617883-85618871:+	0.43	0.45
FBXO38	chr5:147805085:147805264:+@chr5:147806776:147807285:+@chr5:147812987:147813087:+	0.31	0.46
FBXO38	chr5:147805085:147805264:+@chr5:147806776:147807510:+@chr5:147812987:147813087:+	0.32	0.48
AC024560.3	chr3:197350091:197350253:-@chr3:197349058:197349201:-@chr3:197348575:197348739:-	0.53	0.50
NBPF14	chr1:148011680:148011788:-@chr1:148010884:148011056:-@chr1:148009344:148009516: -@chr1:148008573:148008624:-	0.20	0.51
NQO2	chr6:3000067:3000319:+@chr6:3002284:3002520:+@chr6:3003903:3004023:+	0.61	0.54
AFTPH	chr2:64800080:64800202:+@chr2:64806620:64808407:+@chr2:64812556:64812679:+	0.57	0.55
IFT88	chr13:21141208:21141395:+@chr13:21141809:21142136:+@chr13:21148519:21148614:+	0.58	0.59
NQO2	chr6:3000067:3000319:+@chr6:3003903:3004023:+@chr6:3004729:3004885:+@chr6:3006702:3006793:+	0.61	0.61
HPCAL1	chr2:10536961:10537046:+@chr2:10546391:10546487:+@chr2:10548688:10548860: +@chr2:10559860:10560261:+	0.44	0.62
SLC25A19	chr17:73284672:73284469|73284583:-@chr17:73282714:73282883:-	0.48	0.62
PDE1B	chr12:54964025-54964141:+@chr12:54966385-54966525:+	0.63	0.63
TRIM39-RPP21	chr6:30312906:30313005:+@chr6:30313075:30313175:+@chr6:30313268:30313350: +@chr6:30314209:30314334:+	0.45	0.67
PTGS1	chr9:125133465:125133551:+@chr9:125133892:125134113:+@chr9:125140178:125140294:+	0.77	0.73
NA	chr17:26646121:26646391:+@chr17:26652529:26652673:+@chr17:26653560:26655711:+	0.61	0.74

[i] AS, alternative splicing; PSI, percentage of splice-in. NA: there is no HUGO gene symbol corresponding for the gene.

Table IV. Altered AS events in macrophages co-cultured with T47D cells.

Table IV.

Altered AS events in macrophages co-cultured with T47D cells.

Gene	Event	ΔPSI (SRR2922612_vs_SRR2922610)	ΔPSI (SRR2922612_vs_SRR2922614)
IL6	chr7:22766766:22766900:+@chr7:22767132:22767253:+@chr7:22768312:22768425:+	−0.68	−0.55
CKB	chr14:103987600:103987771:-@chr14:103986839:103986929:-@chr14:103986459:103986648:-	−0.5	−0.55
SUCO	chr1:172520652:172520766:+@chr1:172522400:172522510:+@chr1:172525009:172525163:+	−0.51	−0.52
SEP3	chr22:42390625-42390718:+@chr22:42392906-42394225:+	−0.47	−0.49
ANKRD49	chr11:94229770:94229994|94230243:+@chr11:94231237:94232744:+	−0.37	−0.47
NA	chr14:70883617:70883807:-@chr14:70855187:70855323:-@chr14:70842393:70842488:-	−0.46	−0.46
IQSEC2	chrX:53310678:53310796:-@chrX:53308746:53308841:-@chrX:53295506:53296246:-	−0.46	−0.46
SP110	chr2:231084589:231084827:-@chr2:231081496:231081536:-@chr2:231079665:231079833:-	−0.51	−0.44
ANXA2	chr15:60688350:60688626:-@chr15:60685237:60685639:-@chr15:60678227:60678285:-	−0.46	−0.44
DCTD	chr4:183837692:183837439|183837572:-@chr4:183836614:183836728:-	−0.35	−0.44
MGA	chr15:42032251:42032401:+@chr15:42034744:42035370:+@chr15:42040835:42041125:+	−0.44	−0.43
C17orf76-AS1	chr17:16342641-16342728:+@chr17:16342895-16343567:+	−0.4	−0.43
KIAA0226L, PPP1R2P4	chr13:46961269:46961635:-@chr13:46952025:46952140:-@chr13:46946076:46946732:-	−0.36	−0.43
HNRNPC	chr14:21737457:21737638:-@chr14:21731495|21731741:21731470:-	−0.36	−0.43
EXOC7	chr17:74087224:74087316:-@chr17:74086410:74086562:-@chr17:74085256:74085401:-	−0.36	−0.42
NA	chr2:3605976:3606588:+@chr2:3606994|3607038:3607319:+	−0.48	−0.41
ARIH2	chr3:48956254:48956431:+@chr3:48960181:48960244:+@chr3:48962151:48962272:+	−0.31	−0.41
NA	chr12:15747871-15747991:+@chr12:15749024-15751265:+	−0.26	−0.41
RPS15A, RP11-1035H13.3	chr16:18801656:18801566|18801632:-@chr16:18800303:18800440:-	−0.47	−0.4
RP11-691N7.6, CTNND1	chr11:57529234:57529518:+@chr11:57556509:57556627:+@chr11:57558857:57559145:+	−0.47	−0.39
UGP2	chr2:64068098:64068366:+@chr2:64069193:64069338:+@chr2:64069673:64069733: +@chr2:64083440:64083567:+	−0.24	−0.39
EXOC7	chr17:74087224:74087316:-@chr17:74086410:74086478:-@chr17:74085256:74085401:-	−0.35	−0.36
SLC41A2	chr12:105352071:105351866|105351968:-@chr12:105321875:105322472:-	−0.46	−0.34
NA	chrX:30742218:30742298:+@chrX:30745583:30745669:+@chrX:30746849:30749577:+	−0.4	−0.34
NHLRC3	chr13:39613701:39613848:+@chr13:39616242:39616442:+@chr13:39618227:39618318:+	−0.35	−0.34
IL6	chr7:22766766:22766900:+@chr7:22767063:22767253:+@chr7:22768312:22768425:+	−0.34	−0.34
ST7L	chr1:113162266:113162405:-@chr1:113161531:113161761:-@chr1:113159435:113159517: -@chr1:113153463:113153625:-	−0.5	−0.33
UGP2	chr2:64068098:64068366:+@chr2:64069193:64069338:+@chr2:64082018:64082070: +@chr2:64083440:64083567:+	−0.22	−0.33
RABGAP1L	chr1:174780970:174781098:+@chr1:174846530:174846743:+@chr1:174926594:174926686:+	−0.46	−0.32
WASH4P	chr16:67427-67291:-@chr16:67051-66916:-	−0.27	−0.32
ZCCHC6	chr9:88916191:88916516:-@chr9:88913682:88913745:-@chr9:88902648:88903659:-	−0.34	−0.31
USP8	chr15:50773678:50774262:+@chr15:50776472:50776558:+@chr15:50781998:50782078:+	−0.31	−0.31
NUBP2	chr16:1836538:1836656|1836956:+@chr16:1837678:1837832:+	−0.27	−0.31
TMEM19	chr12:72094612-72094805:+@chr12:72097757-72097836:+	−0.36	−0.3
HNRNPH1	chr5:179047893:179048036:-@chr5:179046270:179046361:-@chr5:179045146:179045324:-	−0.42	−0.29
SBF1	chr22:50897684:50897821:-@chr22:50895463:50895540:-@chr22:50894921:50895102:-	−0.35	−0.29
BCL2L13	chr22:18171752:18171908:+@chr22:18178907:18178976:+@chr22:18185009:18185152:+	−0.32	−0.29
YWHAZ	chr8:101965078:101965221:-@chr8:101964157:101964353:-@chr8:101960824:101961128:-	−0.3	−0.29
TCOF1	chr5:149769450:149769586:+@chr5:149771107:149771220:+@chr5:149771520:149771739:+	−0.25	−0.29
CHTOP	chr1:153610771:153610924:+@chr1:153614719:153614905:+@chr1:153617540:153618782:+	−0.34	−0.28
GIT2	chr12:110385061:110385309:-@chr12:110383065:110383154:-@chr12:110376970:110377052:-	−0.32	−0.28
PCM1	chr8:17838100:17838264:+@chr8:17840742:17840798:+@chr8:17842956:17843128:+	−0.27	−0.28
SIGLEC11, NUP62	chr19:50432583:50432988:-@chr19:50431072|50431105:50430951:-	−0.21	−0.28
ANKRD12, RP11-21J18.1	chr18:9195549:9195696:+@chr18:9204474:9204542:+@chr18:9208655:9208801:+	−0.28	−0.27
AC092755.4, BNIP2	chr15:59961091:59961189:-@chr15:59960302:59960337:-@chr15:59955062:59956319:-	−0.25	−0.27
CNN2	chr19:1032558:1032695:+@chr19:1036066:1036245:+@chr19:1036415:1036561:+	−0.25	−0.27
NSUN4	chr1:46805849:46806591:+@chr1:46806995:46807169:+@chr1:46810473:46810816:+	−0.25	−0.27
NUBP2	chr16:1836538:1836656:+@chr16:1836739:1836956:+@chr16:1837678:1837832:+	−0.25	−0.27
SRPK2	chr7:104767440:104767509:-@chr7:104766695:104766787:-@chr7:104766229:104766321: -@chr7:104756823:104758469:-	−0.42	−0.26
ITGAM	chr16:31308835:31308975:+@chr16:31309063|31309066:31309275:+	−0.33	−0.26
OSBPL3	chr7:24874355:24873706|24874105:-@chr7:24870387:24870524:-	−0.32	−0.26
YWHAZ	chr8:101965078:101965221:-@chr8:101964157:101964536:-@chr8:101960824:101961128:-	−0.26	−0.26
C17orf76-AS1	chr17:16342641-16342728:+@chr17:16342895-16343017:+	−0.2	−0.26
PXN	chr12:120703420:120703574:-@chr12:120664642:120664920:-@chr12:120662446:120662530: -@chr12:120661954:120662180:-	−0.28	−0.25
NA	chr22:50964675:50964905:-@chr22:50964430:50964585:-@chr22:50961997:50962853:-	−0.53	−0.24
ANXA2	chr15:60690142:60690185:-@chr15:60688350:60688626:-@chr15:60685237:60685639: -@chr15:60678227:60678285:-	−0.27	−0.24
DFFA	chr1:10523266-10523115:-@chr1:10521759-10520603:-	−0.23	−0.24
NA	chr1:171673674-171673527:-@chr1:171671224-171669296:-	−0.29	−0.22
CKB	chr14:103987600:103987771:-@chr14:103986806:103986929:-@chr14:103986459:103986648:-	−0.22	−0.22
MAPK7	chr17:19283921-19284999:+@chr17:19285094-19285779:+	−0.22	−0.22
SLC11A2	chr12:51375586-51375422:-@chr12:51373841-51373566:-	−0.39	−0.21
MPZL1	chr1:167742473:167742605:+@chr1:167745301:167745403:+@chr1:167757057:167761156:+	−0.25	−0.21
ABI1	chr10:27054147:27054247:-@chr10:27047991:27048164:-@chr10:27040527:27040712:-	−0.33	−0.2
ABI1	chr10:27054147:27054247:-@chr10:27047991:27048167:-@chr10:27040527:27040712:-	−0.32	−0.2
NT5C2	chr10:104899163:104899236:-@chr10:104871502:104871562:-@chr10:104866346:104866463:-	0.22	0.2
ILK	chr11:6624964:6625052:+@chr11:6625410|6625456:6625590:+	0.29	0.2
SRRT	chr7:100479674:100479862:+@chr7:100480386:100480711:+@chr7:100481691:100481860:+	0.24	0.21
MFSD10	chr4:2933880-2933772:-@chr4:2933663-2933552:-	0.2	0.22
ACTR6	chr12:100594555:100594697|100594838:+@chr12:100598718:100598835:+	0.24	0.22
NCOR1	chr17:16118676:16118874:-@chr17:16101915:16102033:-@chr17:16097776:16097953:-	0.33	0.22
TXNRD1	chr12:104680460:104680887:+@chr12:104682711:104682818:+@chr12:104705068:104705190: +@chr12:104707023:104707095:+	0.39	0.22
LILRA2, LILRB1	chr19:55087274:55087576:+@chr19:55088932:55089190:+@chr19:55098477:55098527: +@chr19:55098668:55099027:+	0.21	0.23
RPL17, RPL17-C18orf32	chr18:47018628:47018834:-@chr18:47017954|47018203:47017902:-	0.29	0.23
TXNRD1	chr12:104680727:104680887|104681124:+@chr12:104682709:104682818:+	0.32	0.23
NA	chr1:44679125-44679199:+@chr1:44679381-44679530:+	0.22	0.24
MSRB1	chr16:1991406:1990779|1991258:-@chr16:1988234:1989144:-	0.31	0.24
WARS	chr14:100842597:100842680:-@chr14:100841620:100841883:-@chr14:100828045:100828258:-	0.32	0.24
NA	chr10:105156166:105156270:-@chr10:105155503:105155789:-@chr10:105153956:105154151: -@chr10:105152128:105152223:-	−0.37	0.25
IRF7	chr11:615647-615345:-@chr11:615259-615097:-	0.33	0.25
RREB1	chr6:7229230:7232140:+@chr6:7240671:7240835:+@chr6:7246657:7247454:+	0.43	0.25
DCAF6	chr1:167973771:167974031:+@chr1:167992226:167992285:+@chr1:168007609:168007726:+	0.23	0.26
PXN	chr12:120663712:120663872:-@chr12:120662446:120662530:-@chr12:120661954:120662180:-	0.39	0.26
SRSF7	chr2:38976671:38976847:-@chr2:38976040:38976488:-@chr2:38975721:38975795:-	0.37	0.27
MLL3	chr7:151855948:151856157:-@chr7:151854846:151855010:-@chr7:151853290:151853431:-	0.45	0.27
WARS	chr14:100842597:100842680:-@chr14:100841620:100841883:-@chr14:100835424:100835595:-	0.24	0.28
FBXO38	chr5:147805085:147805264:+@chr5:147806776:147807510:+@chr5:147812987:147813087:+	0.26	0.28
NT5C3	chr7:33102180:33102409:-@chr7:33075546:33075600:-@chr7:33066429:33066527:-	0.31	0.28
NCOR2	chr12:124812179:124811955|124812093:-@chr12:124810737:124810916:-	0.37	0.28
NA	chr6:30181082:30181271:-@chr6:30168812:30168915:-@chr6:30166443:30166930:-	0.3	0.31
SRSF6	chr20:42087001:42087149:+@chr20:42087793:42088060:+@chr20:42088411:42088535:+	0.33	0.31
C10orf2	chr10:102750626:102750767|102750811:+@chr10:102752947:102754158:+	0.27	0.32
DYRK1B	chr19:40317925:40318065:-@chr19:40317507|40317627:40317312:-	0.38	0.32
HNRNPH1	chr5:179050670:179050596|179050637:-@chr5:179050038:179050165:-	0.38	0.33
ANKZF1	chr2:220098855-220099010:+@chr2:220099548-220100034:+	0.36	0.34
ARIH2	chr3:48956254:48956431:+@chr3:48960181:48960244:+@chr3:48962151:48962272: +@chr3:48982569:48982614:+	0.3	0.36
CAMTA2	chr17:4886052:4886187:-@chr17:4885384:4885522:-@chr17:4884973:4885126:-	0.31	0.36
MRRF	chr9:125027741:125027824|125028059:+@chr9:125033143:125033354:+	0.31	0.36
SETMAR	chr3:4344988:4345210:+@chr3:4354582:4354913:+@chr3:4355331:4355445:+	0.42	0.36
RREB1	chr6:7229230:7232140:+@chr6:7240671:7240835:+@chr6:7248744:7252213:+	0.6	0.36
NA	chr1:207095163:207095378:-@chr1:207087104:207087439:-@chr1:207086274:207086387:-	0.43	0.39
RP11-315D16.2	chr15:68497583:68498448:-@chr15:68491879:68492019:-@chr15:68489778:68489966:-	0.48	0.39
NA	chr18:32820994:32821073:+@chr18:32822355:32822848:+@chr18:32823116:32823257:+	0.5	0.4
FOPNL	chr16:15982415:15982447:-@chr16:15977865:15978062:-@chr16:15973661:15973745: -@chr16:15967348:15967484:-	0.38	0.41
AAMDC	chr11:77532208:77532287:+@chr11:77552065:77552106:+@chr11:77553525:77553674:+	0.47	0.41
NA	chrX:1718085:1718325:+@chrX:1719031:1719100:+@chrX:1719552:1721411:+	0.48	0.41
TCTN1	chr12:111078188:111078322:+@chr12:111078829:111078954:+@chr12:111082772:111082934: +@chr12:111085016:111085141:+	0.42	0.44
MON1A	chr3:49950653:49950792:-@chr3:49948959:49949444:-@chr3:49947552:49948317:-	0.47	0.44
TMBIM6	chr12:50135340:50135394:+@chr12:50135740:50135898:+@chr12:50138196:50138325: +@chr12:50146247:50146332:+	0.43	0.45
NA	chr5:80597402:80597489|80597517:+@chr5:80604380:80604530:+	0.48	0.45
TM2D1	chr1:62160369:62160442:-@chr1:62152464:62152593:-@chr1:62149089:62149218:-	0.42	0.46
NA	chr22:18165980:18166087:+@chr22:18185009:18185152:+@chr22:18209443:18213621:+	0.25	0.47
CHD8	chr14:21868572:21868771:-@chr14:21868466|21868490:21868310:-	0.35	0.47
NA	chr22:18171752:18171908:+@chr22:18185009:18185152:+@chr22:18209443:18213621:+	0.23	0.48
SLA	chr8:134114644:134114786:-@chr8:134087098:134087375:-@chr8:134072345:134072445: -@chr8:134063061:134063160:-	0.51	0.62
ARIH2	chr3:48956254:48956431:+@chr3:48962151:48962272:+@chr3:48964895:48965246:+	0.36	0.64

[i] AS, alternative splicing; PSI, percentage of splice-in. NA: there is no HUGO gene symbol corresponding for the gene.

Similarly, GO functional enrichment analysis was applied to investigate the biological processes affected by those altered AS events. GO terms such as signal transduction, cell-matrix adhesion, protein transport, brain development were significantly enriched in the macrophages co-cultured with MDA-MB-231 cells (Fig. 4D). Terms such as RNA export from the nucleus, regulation of angiogenesis, positive regulation of transcription, DNA-templated, mRNA export from the nucleus, negative regulation of apoptotic process were significantly enriched in the macrophages co-cultured with the T47D cells (Fig. 4E). Briefly, protein transport-related processes were altered by co-culturing macrophages with the MDA-MB-231 cells and RNA processing biological processes were affected by co-culturing macrophages with the T47D cells.

The RNA processing and protein transport-related AS events derived from the macrophages co-cultured with the MDA-MB-231 (Fig. 5A) and T47D (Fig. 5B) cells were further analyzed. Vacuolar protein sorting-associated protein 33B (VPS33B) is a Sec1 familiy protein involved in vesicle transport which mediates protein sorting and plays an important role in segregation of intracellular molecules into distinct organelles (41,42). As known, the induced inflammatory responses by microbial ligands is influenced by internalization of Toll-like receptors (TLRs), thus, endosomal maturation in clearing receptors terminates inflammatory responses. Akbar et al found that Vps33B proteins play critical roles in the maturation of phagosomes and endosomes in Drosophila and mammals and therefore affect the process of maturation of macrophages (43). Sashimi plot showed the AS event ‘chr15:91565384:91565833:-@chr15:91561035:91561115:-@chr15:91557614:91557663:-’ of gene VPS33B. The exon was largely excluded from the macrophages co-cultured with MDA-MB-231 cells (Fig. 5C), which may influence the protein function and further the maturation process of macrophages. The protein encoded by SRSF6 belongs to the splicing factor SR and may play a role in the determination of alternative splicing. Boisson et al demonstrated that SRSF6 regulate the AS of the IKBKG gene, which accounts for NF-κB activation and further immune response activation (44). Here, we showed that SRSF6 itself is regulated by AS. Specifically, the event ‘chr20:42087001:42087149:+@chr20:42087793:42088060:+@chr20:42088411:42088535:+’ of SRSF6 was significantly included after coculturing macrophages with T47D cells (Fig. 5C). PPI analysis showed that protein transport-related proteins CPNE1, AP1G2, AFTPH, STX2, VPS33B and STXBP2 were interacted intimately (Fig. 5D). In the interaction network of RNA processing proteins, SRSF6 has extensive interactions with another 6 proteins and may serve as the hub protein in the network (Fig. 5D). These results further indicate that AS events containing genes including Vps33B and SRSF6 may play important roles in the maturation processes of macrophages.

Figure 5. Protein transport and RNA export/splicing/translation-related AS events altered in macrophages co-cultured with MDA-MB-231 and T47D cells, respectively. (A) Protein transport-srelated AS events altered in macrophage co-cultured with MDA-MB-231 cells. (B) RNA export/splicing/translation-related AS in macrophages cocultured with T47D cells. (C) Example of AS event from the VPS33B gene in macrophages individually sampled (SRR2922608) and co-cultured with MDA-MB-231 cells (SRR2922609). AS event from the SRSF6 gene in macrophages individually sampled (SRR2922612) and co-cultured with T47D cells (SRR2922614). Numbers indicate read counts supporting the splicing junctions. Ψ indicates PSI (percentage of splice-in). (D) Protein-protein interaction (PPI) among proteins from A and B. Colored edges represent active interaction sources (https://string-db.org/). AS, alternative splicing.

Sequence feature characterization of the altered skip exons

To investigate whether the altered AS events in different treatments had distinct properties, we first measured the degree of evolutionary conservation of skip exon sequences across placental mammals. Unexpectedly, all the altered skip exons in different treatments showed a higher degree of sequence conservation equivalent compared to that of constitutive exons (Fig. 6A). Altered skip exons in the macrophages co-cultured with T47D cells showed highest evolutionary conservation, whereas macrophages co-cultured with MDA-MB-231 cells showed the lowest. There were no significant differences in skip exon length among the different treatments (Wilcoxon signed-rank test, P>0.05; Fig. 6B). Splicing site strength analysis showed there were no significant differences in splicing strength between constitutive skip and altered skip exons and no significant differences among the different treatments (Wilcoxon signed-rank test, P>0.05; Fig. 6C and D). Repetitive elements such as Alu are known to be stochastically exonized (45), and we found Alu elements were more enriched within skip exons in the macrophages cocultured with the T47D cells, were fewer in skip exons of macrophages cocultured with the MDA-MB-231 cells, and were almost absent from skip exons of MDA-MB-231 co-cultured with macrophages and T47D co-cultured with macrophages (Fisher's exact test; Fig. 6E). We conclude that altered skip exons of macrophages cocultured with T47D cells were mostly enriched with Alu element and had highest evolutionary conservation.

Finally, the splicing factors responsible for the AS events in the different cells and treatments were determined. Here, we calculated the associations between splicing factor expression and the PSI values across the 14 samples. The top 5 splicing factors which possessed the most associated splicing events in each of the 4 cell conditions are shown in Fig. 6F. In the MDA-MB-231 cells co-cultured with macrophages, CPSF7, RBM33, SNRNP48, RBM43 and CSTF2 were the top five. Akman et al confirmed that CSTF2 is a major regulator of 3′UTR shortening in TNBC and contributes to the proliferation phenotype (46). In T47D cells co-cultured with macrophages, CWC25, SNRPE, RBM18, RBM20 and MEX3C were the top five. Meanwhile, in the macrophages co-cultured with MDA-MB-231 cells, SF3B4, NAA38, SNRPG, ELAVL3 and RBM34 were the top five and in the macrophages co-cultured with T47D cells, PRPF3, SF3B2, SNRNP48, CELF1 and RBM46 were the top five. Lin et al found that knockdown of CELF1 in primary human macrophages led to increased inflammatory response to M1 stimulation (47). Subsequently, the expression pattern of candidate splicing factors among normal and 4 breast cancer subtypes was explored. Generally, splicing factors responsible for each condition showed similar expression pattern among the samples (Fig. 7A). In addition, we characterized the expression pattern of these splicing factors among samples from the GSE75130 dataset. Splicing factors such as CPSF7, RBM33, SNRNP48 responsible for the altered AS events in MDA-MB-231 cells cocultured with macrophages showed a similar expression pattern among the different treatments (Fig. 7B).

Figure 7. Expression pattern of candidate splicing factors. (A) Expression pattern of candidate splicing factors across normal breast samples and 4 breast cancer subtypes. Expression profiles were derived from BRCA cohort of the TCGA dataset (https://portal.gdc.cancer.gov/). (B) Expression pattern of candidate splicing factors across different treatments from the GSE75130 dataset.

Discussion

In the present study, mutual editing of alternative splicing (AS) between tumor cells and macrophage was investigated by re-exploring a previous dataset. Importantly, it was found that ER+ and triple-negative breast cancer (TNBC) were differentially regulated from the AS events view when co-cultured with macrophages. However, DNA repair and DNA damage processes were enriched in both ER+ and TNBC after co-culturing with macrophages. Meanwhile, macrophages were also differentially regulated from the biological processes view by co-culturing with ER+ and TNBC. These results revealed a new view of the mutual regulation between tumor cells and macrophages, in which biological pathways were dramatically different identified at the expression level.

At the expression level, macrophages co-cultured with MDA-MB-231 cells were enriched in the superpathway of citrulline metabolism, cellular effects of sildenafil (Viagra), GNRH signaling, citrulline biosynthesis, factors promoting cardiogenesis in vertebrates and bladder cancer signaling. Whereas at the AS level, RNA processing and translation-related processes were enriched. In macrophages co-cultured with T47D cells, biological pathways such as granulocyte adhesion and diapedesis, inhibition of matrix metalloproteases, agranulocyte adhesion and diapedesis, VDR/RXR activation were enriched at the transcription level (21), whereas, biological processes such as protein transport related pathways were enriched at the AS level. In T47D cells co-cultured with macrophages, acute phase response signaling, G-protein coupled receptor signaling, LXR/RXR activation, hepatic fibrosis/hepatic stallate cell activation were enriched at the transcriptional level (21), whereas DNA repair and DNA damage processes were enriched at the AS level. In the MDA-MB-231 cells co-cultured with macrophages, axonal guidance signaling, RhoGDI signaling, relaxin signaling, CCR3 signaling in eosinophil pathways were enriched at the transcriptional level (21), whereas also DNA repair and DNA damage related pathways were enriched at the AS level. Here, we see that the biological pathways affected at the transcription level and alternative splicing level were very different which suggested that AS may provide a new view on the interplay regulation between tumor cells and macrophages.

Another interesting phenomenon was that although the altered AS events from ER+ breast cancer and TNBC cells co-cultured with macrophages were not overlapping, the biological pathways such as DNA repair and DNA damage was enriched in both cell line treatments. It has been established that macrophage cells exert their DNA repair functions to normal cells. For example, double-strand breaks (DSBs) activate the cytosolic DNA sensor stimulator of interferon (IFN) genes, thus inducing the production of IFNα and IFNβ, which promote innate immune responses including macrophages (21). In addition, human monocytes, by releasing macrophage-derived heparin-binding epidermal growth factor (HB-EGF), enhance DNA damage response (DDR) in neighboring cells suffering from DNA damage. Consequently, HB-EGF-treated cells exhibit higher double-strand break (DSB) rejoining and display lower levels of residual DSBs (21). This study also has limitations. For example, we did not experimentally verify the functions of candidate splicing factors in regulation of AS profiles under different treatments. In addition, the roles of key AS events in the cross-talk between breast cancer cells and macrophages warrant further research. Further investigation of the functions of splicing factors and key altered AS events would largely improve our understanding of the molecular mechanisms involved in cancer-macrophage crosstalk.

In short, our results here showed that macrophages play roles in the regulation of DNA damage and DNA repair pathways, which improve our understanding concerning the interplay between macrophages and tumor cells. Thus, the specific roles of macrophages in the regulation of tumor cells require further research, which may shed light on the improvement of cancer immunotherapy.

Acknowledgements

Not applicable.

Funding

The present study was funded by the Yunnan Applied Basic Research Projects (grant no. 2016FB038) and the National Natural Science Foundation of China (grant no. 31801249).

Availability of data and materials

The datasets generated during this study are available from the corresponding author upon reasonable request.

Authors' contributions

LD and HZ designed the experiments, interpreted the results, and wrote the manuscript. WD and DL performed the data analysis. PZ, LS, HD, YL, XB and YW revised this work critically and helped to interpret the data. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

All the authors declare that there was no financial support or relationship that may pose a conflict of interest and all the authors have no conflict of interests.

References