Single Cell Analysis of Cultivated Fibroblasts from Chronic Pancreatitis and Pancreatic Cancer Patients.

Cancer-associated fibroblasts (CAFs) play a major role in the progression and drug resistance of pancreatic cancer. Recent studies suggest that CAFs exhibit functional heterogeneity and distinct transcriptomic signatures in pancreatic cancer. Pancreatic fibroblasts also form an integral component in pancreatic diseases such as chronic pancreatitis named disease-associated fibroblasts (DAFs). However, intra-tumoral heterogeneity of CAFs in pancreatic cancer patients and their pivotal role in cancer-related mechanisms have not been fully elucidated. Further, it has not been elucidated whether CAF subtypes identified in pancreatic cancer also exist in chronic pancreatitis. In this study, we used primary isolated fibroblasts from pancreatic cancer and chronic pancreatitis patients using the outgrowth method. Single-cell RNA sequencing (scRNA-seq) was performed, and bioinformatics analysis identified highly variable genes, including factors associated with overall survival of pancreatic cancer patients. The majority of highly variable genes are involved in the cell cycle. Instead of previously classified myofibroblastic (myCAFs), inflammatory (iCAFs), and antigen-presenting (ap) CAFs, we identified a myCAFs-like subtype in all cases. Most interestingly, after cell cycle regression, we observed 135 highly variable genes commonly identified in chronic pancreatitis and pancreatic cancer patients. This study is the first to conduct scRNAseq and bioinformatics analyses to compare CAFs/DAFs from both chronic pancreatitis and pancreatic cancer patients. Further studies are required to select and identify stromal factors in DAFs from chronic pancreatitis cases, which are commonly expressed also in CAFs potentially contributing to pancreatic cancer development.

1. Introduction

Pancreatic cancer is currently the fourth leading cause of cancer deaths and is projected to become the second most common cancer death in the United States by 2030 [1,2]. The dense stromal tumor microenvironment, which accounts for up to ~90% of the pancreatic tumor mass, is a major cause of resistance to chemotherapy and radiotherapy [3,4,5]. The major stromal compartment consists of CAFs, which are a source of extracellular matrix proteins and potential therapeutic targets [6]. Fibrotic stroma and generally termed disease-associated fibroblasts (DAFs) form an integral component in pancreatic diseases including pancreatic cancer (CAFs) and chronic pancreatitis [7]. CAF depletion can also have tumor-promoting effects, suggesting that CAFs exhibit distinct functional heterogeneity [5]. CAFs, which are derived from several cell types such as pancreatic stellate cells or mesenchymal stem cells, play a major role in the progression and drug resistance of pancreatic cancer [8,9]. Recent studies suggest that CAF subtypes exhibit distinct transcriptomic signatures in pancreatic cancer. Previous human and murine studies described several CAF subtypes such as myofibroblastic CAFs (myCAFs) expressing Acta2, (coding α-smooth muscle actin), Vim (vimentin), and Ctgf (connective tissue growth factor), Col1a1, Col5a1, and Col6a1, inflammatory CAFs (iCAFs) expressing Il1, Il6, Il11, and Lif (Leukemia inhibitory factor) [10], as well as antigen-presenting CAFs (apCAFs) expressing Slpi, Saa3, Cd74, H2-Ab1, Nkain4 (Sodium/Potassium Transporting ATPase Interacting 4), and Irf5 [11]. MyCAFs are associated with an extracellular matrix signature, while iCAFs are suggested to establish an immunosuppressive microenvironment [9]. ApCAFs may play an immunomodulatory role in pancreatic cancer [9]. In the current study, we used CAFs/DAFs isolated from pancreatic cancer and chronic pancreatitis patients via the outgrowth method to evaluate the presence of myCAF, iCAF, or apCAF subtypes in these specimens, and for further stratification of fibroblast subtypes associated with pancreatic disease development and progression.

2. Materials and Methods

2.1. Human Subjects

Human pancreas samples from patients with pancreatic cancer or chronic pancreatitis were obtained from the Department of Surgery, University Medical Center Halle, Martin-Luther-University Halle-Wittenberg, Germany. The study on human material was approved by the institutional review board of the Medical Faculty of the Martin-Luther-University Halle-Wittenberg and designated in accordance with the Declaration of Helsinki (Approval number: 2019-037). Written informed consent was obtained from all patients.

2.2. Isolation and Cultivation of Fibroblasts

CAFs/DAFs from patient specimens were obtained by the outgrowth method, which has been described previously with modifications [12]. Briefly, pancreatic tissue was cut into ca. 20 mm3 pieces, seeded in a plate in the presence of Dulbecco’s Modified Eagle’s Medium (DMEM) low glucose and F12, supplemented with 16% fetal bovine serum, 1% penicillin-streptomycin, and 1% amphotericin, and maintained at 37 °C, 5% CO2 until fibroblasts grew out. Single-cell RNA sequencing (10X Genomics, Pleasanton, CA, USA) was performed with cells in passage-3.

2.3. Single-Cell RNA Sequencing and Bioinformatics Analysis

Isolated CAFs/DAFs were processed with the 10X Genomics Single Cell 3’ v3 Reagent Kit [13]. Samples were processed according to the manufacturer’s user guide (10X Genomics). Final libraries were sequenced with the NextSeq 500 system (Illumina, San Diego, CA, USA) in high-output mode (paired end, 75 base pairs). Single-cell RNA sequencing and bioinformatics analyses were performed with cellranger 6.0.0. Gene expression was quantified using the default 10X Cell Ranger v3 pipeline using the GRCh37/hg19 Reference provided by 10X Genomics. UMI and genes were filtered for each sample and, in general, cells with a minimum number of 2000 genes were kept. Cells containing more than 5% of mitochondrial reads were excluded from downstream analyses. Downstream analyses were performed using the R package Seurat version 4.0. Each scRNA-seq dataset was scaled by library size and log-transformed and for each sample the top 2000 most variable genes were identified. Data were scaled to unit variance and zero mean and the dimensionality of the data was reduced by principal component analysis (PCA) (15 components) and visualized with t-distributed stochastic neighbor embedding (t-SNE) or Uniform Minifold Approximation and Projection (UMAP). Clustering was performed using the Louvain algorithm on the 15 principal components (resolution = 0.5). Cluster-specific markers were identified with the “FindAllMarkers” function.

2.4. Immunocytochemistry

Approximately 2.5 × 104 of CAFs/DAFs were seeded on the slides and incubated overnight. PANC-1 cells were used in addition as control for epithelial cell adhesion molecule (EpCAM) staining. Cells were then fixed with 4% PFA and sequentially immunostained with antibodies directed against alpha-smooth muscle actin (α-SMA) (Invitrogen, 701457), KRT19 (Invitrogen, MA5-31977, Waltham, MA, USA), C-X-C motif chemokine 12 (CXCL12) (Invitrogen, PA5-114344), or EpCAM (Dako, M0804).

2.5. GEPIA

For gene expression profiling and overall survival analysis, we conducted bioinformatics analysis on the GEPIA platform using the Kaplan–Meier survival plots tool [14], as described previously [15]. Our analysis included 176 to 178 pancreatic cancer samples based on the Cancer Genome Atlas (TCGA) database. Median expression was used as the threshold between high and low expression. p < 0.05 was considered statistically significant.

2.6. Data Access

Sequencing raw data are available at European Genome-Phenome Archive (EGA) box with the accession number EGAS00001005889.

3. Results

3.1. Identification of Highly Variable Genes in Disease-Associated Fibroblasts of Pancreatic Cancer and Chronic Pancreatitis Patients

To study previously described myCAF, iCAF, or apCAF subtypes, and investigate potentially altered CAF states in pancreatic cancer (patient1 and patient2) and chronic pancreatitis patients (patient3) in our cohort (Table 1), we isolated primary disease-associated fibroblasts (DAFs) and performed single-cell RNA sequencing (scRNA-seq) analysis. We selected the outgrowth method, because it is a widely recognized and established method for isolating CAFs. To validate the absence of tumor cells in our samples, we performed immunocytochemistry against EpCAM. We observed positive staining of EpCAM in PANC-1 pancreatic cancer cells, but no positivity was observed in isolated DAFs (Figure 1). After quality control, we obtained 2714, 6764, and 7639 cells, respectively (Table 1). CAFs from patient1 showed several highly variable genes that contribute strongly to cell-to-cell variation, including Insulin Growth Factor Binding Proteins IGFBP3 and IGFBP5, Set- and Mynd Domain Containing 3 (SMYD3), Neurofilament Light Chain (NEFL), Keratin 19 (KRT19), Matrix Metalloprotease 1 (MMP1), MMP3, metastasis suppressor KISS1, Growth Differentiation Factor 15 (GDF15), Pentraxin 3 (PTX3), S100 Calcium Binding Protein A4 (S100A4), and Interferon Alpha Inducible Protein 27 (IFI27) (Figure 2A). In CAFs from patient2, we detected chemokines CXCL1, CXCL3, CXCL8, Interleukin 1B (IL1B), MMP1, MMP3, Heat Shock Protein Family A Member 5 (HSPA5, also known as GRP78 or BiP), Proteoglycan 4 (PRG4), RAS-related Glycolysis Inhibitor And Calcium Channel Regulator (RRAD), and HIST1H4C (Figure 2B). In the fibroblasts from patient3, we detected IGFBP5, SMYD3, MMP1, TIMP1, Serpin Family E Member 2 (SERPINE2), KRT18, G0/G1 Switch 2 (G0S2), ADAM Metallopeptidase with Thrombospondin Type 1 Motif 18 (ADAMTS18), and HIST1H4C (Figure 2C). To examine whether expression of above mentioned 25 factors is associated with pancreatic cancer patient survival, we performed overall survival (OS) analysis on the GEPIA platform. We identified that expression of IFI27, KRT18, KRT19, MMP1, MMP3, and NEFL are associated with a shorter overall survival of pancreatic cancer patients (Figure 3), whereas the expression of the other 16 genes are not associated with the overall survival of pancreatic cancer patients (not shown). It has been previously shown with a lineage-labeling approach that CAFs of non-PSC origin exhibited elevated expression of KRT8, KRT18, and KRT19 [16]. Interestingly, we observed both KRT19-positive and KRT19-negative fibroblasts by immunocytochemistry in all three cases (Figure 4).

Table 1

Information of the patients and analyzed number of single cells.

Title 1	Patient1	Patient2	Patient3
Age at the resection	79	48	25
gender	male	female	male
Disease, Tumor classification	Pancreatic cancer, G2 pT3 pN1 M0, L0 V0 Pn1, UICC-Status IIB	Pancreatic cancer, G2 pT2 pN0 M0, L0 V0 Pn1, UICC-Status IB	Chronic pancreatitis
Number of nuclei	2714	6764	7639

Figure 1

Immunocytochemistry of EpCAM in disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. (A) Panc-1 cells stained as positive control, (B) cells from patient1, (C) from patient2, and (D) from patient3. Bar: 50 μm.

Figure 2

Highly variable features in disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. A subset of features that exhibit high variation between cells in disease-associated fibroblasts (A) from patient1, (B) from patient2, and (C) from patient3.

Figure 3

Analysis of 6 genes and overall survival. Overall survival analyses of 6 genes (IFI27, KRT18, KRT19, MMP1, MMP3, and NEFL) in pancreatic cancer based on the GEPIA database. Median expression was used as the threshold between high and low expression. The 95% confidence interval is plotted by the dotted lines.

Figure 4

Immunocytochemistry of KRT19 in disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. (A) from patient1, (B) from patient2, and (C) from patient3. Bar: 50 μm.

3.2. Cell Cycle-Associated Factors Are Majority of Highly Variable Genes

Next, to identify which factors are co-expressed together in CAFs/DAFs, we performed heat map analyses and show the top differentially expressed genes in 3 panels for each patient (Figure 5). The analyses revealed that the variance was mostly covered by cell-cycle genes including MKI67, CENPF, and CCNB1 (Figure 5). To mitigate the effects of cell cycle heterogeneity in scRNA data, we performed cell cycle regression analysis as previously demonstrated [17]. Prior to cell cycle regression, we observed 9 clusters of cells with similar expression profiles produced by the t-distributed stochastic neighbor embedding (t-SNE) algorithm in CAFs from patient1, 10 clusters in CAFs from patient2, and 11 clusters in fibroblasts from patient3 (Figure 6A–C). After cell cycle regression, we did not observe clearly distinct clusters (Figure 6D–F), suggesting that (1) signals derived from cell cycle-associated factors strongly impact on cluster heterogeneity presentation, and (2) primary isolated and cultured cells are not unique population but exhibit highly similar expression profiling between the clusters. We were still able to divide clusters 0 to 3 in CAFs from patient1, clusters 0 to 3 in CAFs from patient2, as well as clusters 0 to 4 in DAFs from patient3 (Figure 6D–F). We summarize highly variable features after cell cycle regression in Table 2 (patient1), Table 3 (patient2), and Table 4 (patient3). Among 25 highly variable genes identified before cell cycle regression, two factors namely IGFBP3 and IGFBP5 were identified as highly variable genes even after cell cycle regression (Table 2, Cluster 0 in Figure 2D), suggesting that these two genes may contribute to CAF heterogeneity independent of cell cycle stage. Interestingly, a number of factors, cell cycle-associated but also non-cell-cycle-associated factors, are identified as highly variable genes shared between CAFs/DAFs isolated from chronic pancreatitis and pancreatic cancer patients (Table 5).

Figure 5

Heat map analyses of scRNAseq data of disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. Top differentially expressed genes were summarized as 3 selected heat map panels in disease-associated fibroblasts (A) from patient1, (B) from patient2, and (C) from patient3.

Figure 6

Dimensionality reduction of scRNAseq data. The t-SNE plot analysis of scRNAseq data prior to cell cycle regression (A) from patient1, (B) from patient2, and (C) from patient3. The Uniform Minifold Approximation and Projection (UMAP) plot analysis after cell cycle regression (D) from patient1, (E) from patient2, and (F) from patient3.

Table 2

Highly variable genes after cell cycle regression in CAFs isolated from Patient1.

Gene	Cluster	p-Value	Average log2FC	Pct.1	Pct.2	p-Value Adjusted
MXD4	0	5.03 × 10−115	0.52657675335757	0.889	0.508	1.16 × 10−110
PAPPA	0	9.83 × 10−86	1.0109090899262	0.842	0.551	2.27 × 10−81
GDF15	0	2.41 × 10−73	1.16972243694366	0.629	0.281	5.57 × 10−69
CDC42EP5	0	3.35 × 10−69	0.329695153407811	0.651	0.294	7.75 × 10−65
SAT1	0	7.20 × 10−64	0.604752674279049	0.866	0.618	1.66 × 10−59
THBS2	0	3.09 × 10−57	0.420962922589772	0.905	0.656	7.14 × 10−53
CTSK	0	1.11 × 10−56	0.922547366516297	0.713	0.442	2.57 × 10−52
COX7A1	0	1.63 × 10−56	0.481923174187465	0.777	0.502	3.76 × 10−52
SNRPN	0	3.29 × 10−55	0.367555328393367	0.905	0.679	7.62 × 10−51
FADS3	0	2.22 × 10−51	0.372644480541774	0.842	0.593	5.12 × 10−47
PCDH10	0	3.71 × 10−50	0.388362189718403	0.562	0.248	8.58 × 10−46
SCG5	0	1.47 × 10−47	0.897313835683302	0.684	0.414	3.40 × 10−43
IGFBP5	0	6.23 × 10−47	1.89606245760768	0.648	0.388	1.44 × 10−42
AHNAK2	0	1.58 × 10−45	0.342619142825	0.74	0.438	3.66 × 10−41
C5orf46	0	2.85 × 10−44	0.434334936421886	0.602	0.32	6.60 × 10−40
AC097480.1	0	3.92 × 10−44	0.397420295368166	0.376	0.117	9.07 × 10−41
CRYAB	0	4.14 × 10−41	0.719531739217665	0.795	0.554	9.58 × 10−37
IGFBP3	0	2.10 × 10−40	1.30767448451024	0.691	0.435	4.85 × 10−36
ANGPTL4	0	3.34 × 10−40	0.355106223670863	0.543	0.256	7.73 × 10−36
FSTL3	0	9.44 × 10−38	0.332060887611438	0.761	0.491	2.18 × 10−33
COPZ2	0	2.37 × 10−37	0.326611462306643	0.891	0.678	5.48 × 10−33
GPNMB	0	3.63 × 10−35	0.353241339224302	0.761	0.522	8.38 × 10−31
LMCD1	0	4.55 × 10−34	0.505462991451827	0.635	0.395	1.05 × 10−29
LUM	0	7.07 × 10−29	0.407477532269438	0.69	0.454	1.63 × 10−24
COL11A1	0	8.50 × 10−27	0.410716209611568	0.59	0.349	1.96 × 10−23
ANKRD37	0	1.84 × 10−26	0.357421975813258	0.474	0.231	4.26 × 10−22
KIF26B	0	1.09 × 10−24	0.454003954754278	0.697	0.449	2.53 × 10−19
CYP1B1	0	2.93 × 10−21	0.310903502762083	0.473	0.245	6.78 × 10−17
ABI3BP	0	5.40 × 10−21	0.504578102968974	0.402	0.192	1.25 × 10−16
CDK1	1	3.38 × 10−281	1.217773701271	0.923	0.232	7.81 × 10−277
HJURP	1	8.28 × 10−214	0.666094535423477	0.776	0.148	1.91 × 10−210
UBE2C	1	1.45 × 10−211	1.5585090216938	0.937	0.407	3.36 × 10−207
TOP2A	1	1.19 × 10−206	1.54271793537239	0.936	0.435	2.76 × 10−202
AURKB	1	3.29 × 10−205	0.74616142540391	0.847	0.23	7.60 × 10−201
FBXO5	1	1.02 × 10−203	0.67512175613025	0.873	0.249	2.35 × 10−199
HIST1H1B	1	3.44 × 10−195	1.45922164557741	0.777	0.185	7.95 × 10−191
CDCA2	1	1.35 × 10−188	0.692330589773016	0.825	0.222	3.12 × 10−184
KIF18B	1	6.20 × 10−188	0.56145936836675	0.75	0.155	1.43 × 10−183
CDCA8	1	3.90 × 10−179	0.752658739698257	0.837	0.226	9.02 × 10−175
CKAP2L	1	8.64 × 10−179	0.60469745700897	0.821	0.221	2.00 × 10−174
ARHGAP11B	1	7.96 × 10−172	0.550161023821282	0.713	0.152	1.84 × 10−167
CEP128	1	5.52 × 10−170	0.586677178192601	0.82	0.237	1.28 × 10−165
HMGB2	1	7.70 × 10−169	1.24812017681573	0.958	0.53	1.78 × 10−164
LINC01572	1	9.80 × 10−155	0.633946121686711	0.787	0.263	2.27 × 10−151
MELK	1	3.91 × 10−151	0.565960749066164	0.865	0.335	9.05 × 10−147
ESCO2	1	1.19 × 10−147	0.480935013769491	0.754	0.202	2.76 × 10−143
GTSE1	1	6.96 × 10−147	0.727246306246762	0.844	0.317	1.61 × 10−142
NCAPG	1	8.42 × 10−147	0.657784193430886	0.888	0.348	1.95 × 10−142
KIFC1	1	1.29 × 10−145	0.52383057541541	0.803	0.25	2.99 × 10−140
KIF23	1	2.14 × 10−143	0.771038833443001	0.895	0.441	4.95 × 10−139
NEIL3	1	7.34 × 10−143	0.372824444469321	0.626	0.118	1.70 × 10−139
DLEU2	1	4.85 × 10−140	0.628595309288741	0.933	0.5	1.12 × 10−135
KIF11	1	3.37 × 10−137	0.570091739120192	0.833	0.298	7.79 × 10−133
NDC80	1	5.48 × 10−136	0.573873285368215	0.821	0.271	1.27 × 10−131
NSD2	1	6.78 × 10−134	0.683028968844798	0.973	0.604	1.57 × 10−129
UBE2T	1	2.76 × 10−133	0.717575875506757	0.97	0.621	6.38 × 10−129
CDCA5	1	3.45 × 10−129	0.396031325743175	0.706	0.182	7.99 × 10−125
KNL1	1	2.60 × 10−127	0.590067939428105	0.827	0.311	6.02 × 10−124
CENPK	1	1.68 × 10−126	0.595773001526377	0.933	0.489	3.88 × 10−123
BARD1	1	1.05 × 10−124	0.497621617904068	0.847	0.341	2.43 × 10−120
MKI67	1	3.50 × 10−124	0.990647290602104	0.918	0.482	8.09 × 10−120
KIF20B	1	8.54 × 10−122	0.678820111686073	0.915	0.428	1.97 × 10−117
TTK	1	1.72 × 10−118	0.39198516969214	0.633	0.15	3.98 × 10−114
SMC4	1	3.37 × 10−118	0.831072141566686	0.974	0.662	7.79 × 10−114
CDCA4	1	9.86 × 10−118	0.462152550274466	0.818	0.345	2.28 × 10−113
KIF4A	1	5.43 × 10−117	0.506969008189571	0.803	0.299	1.26 × 10−112
FEN1	1	3.23 × 10−116	0.581337824059999	0.91	0.456	7.46 × 10−112
NUF2	1	4.54 × 10−116	0.646107435494053	0.836	0.335	1.05 × 10−110
KIF14	1	5.91 × 10−115	0.463692524219373	0.705	0.212	1.37 × 10−110
DBF4	1	1.95 × 10−114	0.587834146901801	0.858	0.436	4.52 × 10−110
SPC25	1	1.04 × 10−113	0.521305325488545	0.818	0.323	2.40 × 10−109
KIF2C	1	1.08 × 10−113	0.512991064138338	0.727	0.223	2.49 × 10−109
CCNA2	1	1.33 × 10−113	0.58858592921946	0.848	0.337	3.09 × 10−109
FAM83D	1	1.36 × 10−114	0.388259146399628	0.613	0.154	3.14 × 10−109
KIF15	1	4.23 × 10−112	0.308609790169668	0.593	0.134	9.78 × 10−108
HIST1H1E	1	8.84 × 10−112	0.930971071463036	0.719	0.306	2.04 × 10−107
SGO1	1	1.18 × 10−111	0.414278244461817	0.719	0.218	2.73 × 10−107
CLSPN	1	2.91 × 10−111	0.65508914483109	0.956	0.501	6.73 × 10−107
HIST1H2AH	1	7.15 × 10−111	0.375216848297376	0.471	0.077	1.65 × 10−106
TMPO	1	2.97 × 10−110	0.707133223014209	0.973	0.692	6.87 × 10−106
BRIP1	1	3.94 × 10−110	0.610259058781836	0.863	0.392	9.10 × 10−106
HIST1H3B	1	6.36 × 10−110	0.339009976604904	0.492	0.088	1.47 × 10−105
CENPE	1	2.31 × 10−109	0.83868521493331	0.836	0.385	5.35 × 10−105
ATAD2	1	3.36 × 10−110	0.646418548726152	0.944	0.533	7.76 × 10−105
MIR924HG	1	1.87 × 10−108	0.60912425610384	0.734	0.238	4.32 × 10−104
FAM111A	1	4.28 × 10−108	0.480066568863614	0.757	0.321	9.90 × 10−104
POLQ	1	5.56 × 10−107	0.326887174080224	0.619	0.156	1.29 × 10−102
Z94721.1	1	6.30 × 10−106	0.311290952655472	0.568	0.129	1.46 × 10−101
ASPM	1	6.94 × 10−106	0.806476667727835	0.852	0.381	1.60 × 10−101
KIF18A	1	2.04 × 10−105	0.457985114861287	0.764	0.307	4.71 × 10−101
HIST1H3D	1	6.05 × 10−105	0.38895099141706	0.489	0.094	1.40 × 10−100
MIS18BP1	1	8.74 × 10−104	0.543388638803541	0.923	0.59	2.02 × 10−100
NCAPH	1	9.30 × 10−104	0.318441669167983	0.643	0.177	2.15 × 10−99
H2AFX	1	9.41 × 10−104	0.685703332643443	0.94	0.632	2.18 × 10−99
ARHGAP11A	1	4.07 × 10−103	0.432432564600806	0.698	0.243	9.42 × 10−99
TACC3	1	1.13 × 10−102	0.584123376464301	0.865	0.405	2.61 × 10−98
BUB1B	1	1.58 × 10−101	0.450386318125073	0.74	0.257	3.65 × 10−97
CCDC18	1	1.78 × 10−101	0.501883274033725	0.786	0.355	4.11 × 10−97
ANLN	1	8.20 × 10−100	0.686740618692537	0.922	0.505	1.90 × 10−95
RAD51AP1	1	3.03 × 10−99	0.385345488278663	0.758	0.277	7.01 × 10−95
CIT	1	5.55 × 10−99	0.425635672538582	0.678	0.23	1.28 × 10−94
CCNF	1	3.61 × 10−97	0.309720189642715	0.533	0.124	8.35 × 10−93
CENPU	1	3.42 × 10−96	0.517098949441927	0.921	0.482	7.91 × 10−92
USP1	1	4.73 × 10−97	0.529622421208818	0.928	0.59	1.09 × 10−91
ARHGAP11A	1	4.07 × 10−103	0.432432564600806	0.698	0.243	9.42 × 10−99
TACC3	1	1.13 × 10−102	0.584123376464301	0.865	0.405	2.61 × 10−98
BUB1B	1	1.58 × 10−101	0.450386318125073	0.74	0.257	3.65 × 10−97
CCDC18	1	1.78 × 10−101	0.501883274033725	0.786	0.355	4.11 × 10−97
ANLN	1	8.20 × 10−100	0.686740618692537	0.922	0.505	1.90 × 10−95
RAD51AP1	1	3.03 × 10−99	0.385345488278663	0.758	0.277	7.01 × 10−95
CIT	1	5.55 × 10−99	0.425635672538582	0.678	0.23	1.28 × 10−94
CCNF	1	3.61 × 10−97	0.309720189642715	0.533	0.124	8.35 × 10−93
CENPU	1	3.42 × 10−96	0.517098949441927	0.921	0.482	7.91 × 10−92
USP1	1	4.73 × 10−97	0.529622421208818	0.928	0.59	1.09 × 10−91
GAS2L3	1	5.48 × 10−96	0.440448497779858	0.641	0.214	1.27 × 10−91
CENPI	1	1.43 × 10−95	0.313820083241218	0.611	0.176	3.31 × 10−91
RRM2	1	1.66 × 10−95	0.643511970429096	0.945	0.558	3.83 × 10−91
CENPA	1	8.64 × 10−95	0.511135030394721	0.682	0.244	2.00 × 10−90
SKA3	1	5.87 × 10−94	0.324596062484181	0.679	0.221	1.36 × 10−89
ZWINT	1	1.05 × 10−92	0.531799371626574	0.919	0.536	2.43 × 10−88
NUSAP1	1	1.96 × 10−92	0.693560843591611	0.918	0.522	4.53 × 10−88
SGO2	1	1.41 × 10−90	0.580055932148788	0.825	0.394	3.27 × 10−86
SHCBP1	1	3.79 × 10−89	0.448247740254958	0.869	0.404	8.76 × 10−85
DNAJC9	1	8.51 × 10−89	0.519479438857925	0.975	0.77	1.97 × 10−83
ASF1B	1	1.37 × 10−87	0.343159210532476	0.69	0.239	3.16 × 10−83
HIST2H2AC	1	1.63 × 10−87	0.679980454603195	0.723	0.36	3.78 × 10−83
C21orf58	1	1.34 × 10−86	0.308264298086868	0.638	0.203	3.11 × 10−82
BRCA1	1	4.39 × 10−86	0.378210564781065	0.776	0.318	1.02 × 10−81
GMNN	1	3.35 × 10−85	0.483939504250589	0.917	0.56	7.74 × 10−81
ORC6	1	4.22 × 10−85	0.398267082494843	0.801	0.345	9.76 × 10−81
MND1	1	1.25 × 10−84	0.324291743759601	0.679	0.235	2.88 × 10−81
CENPF	1	1.95 × 10−83	0.961460630993616	0.921	0.551	4.50 × 10−79
MXD3	1	1.44 × 10−81	0.36440768070372	0.566	0.177	3.33 × 10−77
RRM1	1	3.17 × 10−81	0.474604869567564	0.94	0.655	7.33 × 10−77
HIST1H1A	1	1.80 × 10−80	0.710605680679022	0.493	0.137	4.16 × 10−76
MAD2L1	1	5.20 × 10−80	0.489488750455522	0.908	0.513	1.20 × 10−75
AURKA	1	1.24 × 10−79	0.63054187607067	0.825	0.42	2.86 × 10−74
ECT2	1	9.18 × 10−78	0.446260241003061	0.837	0.466	2.12 × 10−73
RACGAP1	1	3.16 × 10−77	0.357060353701338	0.732	0.304	7.30 × 10−73
CDC45	1	2.22 × 10−76	0.308840494379532	0.709	0.272	5.14 × 10−72
RFC3	1	3.01 × 10−76	0.343352218848334	0.769	0.321	6.97 × 10−73
PBK	1	3.17 × 10−75	0.567611838947207	0.919	0.499	7.32 × 10−71
PKMYT1	1	3.47 × 10−74	0.306089875792054	0.654	0.235	8.03 × 10−70
BRCA2	1	1.42 × 10−73	0.34965053011471	0.704	0.281	3.28 × 10−69
MYBL1	1	3.34 × 10−72	0.349226656230426	0.682	0.296	7.72 × 10−68
PIF1	1	8.17 × 10−72	0.338413259822857	0.448	0.122	1.89 × 10−67
TPX2	1	9.93 × 10−72	0.664286155940432	0.93	0.605	2.30 × 10−67
FANCI	1	8.02 × 10−71	0.334501015448917	0.798	0.369	1.85 × 10−66
HELLS	1	2.83 × 10−70	0.436294541448265	0.878	0.504	6.55 × 10−66
CENPP	1	3.61 × 10−70	0.429243416895106	0.769	0.386	8.34 × 10−66
PRIM2	1	9.54 × 10−70	0.445663113372904	0.926	0.615	2.21 × 10−65
TCF19	1	2.05 × 10−69	0.306977990083555	0.682	0.265	4.75 × 10−67
LMNB1	1	1.72 × 10−68	0.334939880529808	0.751	0.317	3.99 × 10−64
MIS18A	1	2.62 × 10−68	0.351894600497854	0.77	0.39	6.06 × 10−64
CIP2A	1	3.52 × 10−68	0.35413555558672	0.758	0.351	8.15 × 10−64
NAV2	1	5.25 × 10−68	0.715967019361379	0.907	0.698	1.21 × 10−63
EZH2	1	9.96 × 10−68	0.330376830176061	0.779	0.354	2.30 × 10−63
POLD3	1	1.03 × 10−67	0.346527003387637	0.747	0.358	2.38 × 10−63
SPATA5	1	1.55 × 10−67	0.424438610846798	0.833	0.501	3.58 × 10−63
NCAPG2	1	6.97 × 10−67	0.370094493158939	0.802	0.413	1.61 × 10−62
VRK1	1	1.58 × 10−66	0.350828198823895	0.795	0.411	3.64 × 10−62
SCLT1	1	2.50 × 10−66	0.538567884484186	0.881	0.639	5.78 × 10−62
G2E3	1	1.41 × 10−65	0.434931481714217	0.809	0.518	3.26 × 10−61
HIST1H1C	1	6.02 × 10−65	0.749471025632811	0.792	0.533	1.39 × 10−60
PCLAF	1	7.63 × 10−65	0.548409542540011	0.982	0.726	1.76 × 10−60
CEP55	1	2.28 × 10−64	0.444384877721666	0.883	0.448	5.26 × 10−60
AP001347.1	1	2.65 × 10−64	0.338860490459612	0.697	0.306	6.13 × 10−60
RAD18	1	3.64 × 10−62	0.349511034427767	0.813	0.464	8.42 × 10−58
MYBL2	1	4.80 × 10−62	0.327599270826376	0.701	0.309	1.11 × 10−57
APOLD1	1	1.04 × 10−61	0.482705829625938	0.714	0.356	2.40 × 10−57
PSMC3IP	1	3.62 × 10−60	0.305727970041719	0.775	0.366	8.37 × 10−56
MCM7	1	5.97 × 10−59	0.406425723666096	0.947	0.575	1.38 × 10−54
GPSM2	1	1.11 × 10−57	0.356375237610496	0.653	0.299	2.58 × 10−53
NCAPD3	1	1.47 × 10−55	0.323452379603221	0.81	0.47	3.39 × 10−51
CKAP2	1	1.48 × 10−55	0.549640977439134	0.872	0.602	3.42 × 10−51
CDKN2D	1	5.12 × 10−54	0.441625540592813	0.683	0.387	1.18 × 10−49
SMC2	1	1.35 × 10−53	0.382496233463084	0.93	0.671	3.11 × 10−49
BUB1	1	1.40 × 10−53	0.311722421521002	0.699	0.307	3.24 × 10−49
FOXM1	1	2.19 × 10−53	0.300815092823151	0.75	0.378	5.06 × 10−50
TEX30	1	3.39 × 10−51	0.319135647569659	0.842	0.546	7.84 × 10−47
PRR11	1	6.78 × 10−52	0.383434189793599	0.803	0.444	1.57 × 10−46
GINS2	1	1.38 × 10−50	0.365751194770039	0.885	0.526	3.20 × 10−45
EMP2	1	5.82 × 10−51	0.34924550830157	0.872	0.6	1.34 × 10−44
TRIM59	1	7.84 × 10−48	0.391721961373452	0.772	0.506	1.81 × 10−43
HIST1H1D	1	3.16 × 10−47	0.440065411259682	0.331	0.081	7.30 × 10−43
HMMR	1	5.48 × 10−46	0.406688350508869	0.839	0.441	1.27 × 10−41
SMC1A	1	2.73 × 10−45	0.34372240444355	0.895	0.678	6.31 × 10−41
CCDC14	1	3.02 × 10−46	0.328566007707011	0.801	0.507	6.98 × 10−41
KIF22	1	5.25 × 10−45	0.306036800059592	0.777	0.479	1.21 × 10−40
PLK1	1	3.34 × 10−44	0.417998745939095	0.678	0.336	7.73 × 10−39
LCORL	1	1.35 × 10−42	0.388923389039701	0.776	0.521	3.13 × 10−38
DEPDC1	1	3.55 × 10−42	0.325869872955602	0.742	0.37	8.21 × 10−38
SAC3D1	1	4.47 × 10−41	0.321992720632306	0.895	0.677	1.03 × 10−36
PRC1	1	5.34 × 10−40	0.434659757767406	0.934	0.581	1.23 × 10−35
DHFR	1	3.09 × 10−39	0.315986392775952	0.908	0.686	7.14 × 10−35
CENPM	1	1.11 × 10−38	0.324964822919566	0.906	0.615	2.56 × 10−34
BIRC5	1	7.70 × 10−35	0.40221160065042	0.945	0.655	1.78 × 10−30
RFX8	1	1.72 × 10−34	0.305449206969217	0.654	0.362	3.98 × 10−29
SAP30	1	6.59 × 10−31	0.320896181997167	0.783	0.569	1.52 × 10−26
RAD51B	1	1.46 × 10−29	0.367003320328178	0.813	0.594	3.38 × 10−24
KNSTRN	1	1.59 × 10−22	0.302722797631973	0.764	0.552	3.67 × 10−18
CCNB1	2	1.38 × 10−170	1.28130409189154	0.997	0.65	3.19 × 10−166
CDC20	2	6.96 × 10−149	1.08085310140798	0.975	0.51	1.61 × 10−144
CCNB2	2	1.08 × 10−147	0.79963647553927	0.938	0.428	2.51 × 10−143
DLGAP5	2	2.02 × 10−127	0.776139871899789	0.94	0.427	4.68 × 10−123
PRC1	2	2.18 × 10−117	0.870056869643796	0.987	0.589	5.03 × 10−113
HMMR	2	3.01 × 10−114	0.754271372359569	0.943	0.437	6.95 × 10−110
CDKN3	2	5.32 × 10−111	0.769225621045913	0.993	0.691	1.23 × 10−106
BIRC5	2	3.17 × 10−98	0.735538745956502	0.987	0.662	7.33 × 10−94
CKS1B	2	2.03 × 10−94	0.691059605394196	0.99	0.75	4.70 × 10−88
TPX2	2	4.90 × 10−92	0.80960851990548	0.978	0.612	1.13 × 10−87
CENPF	2	3.71 × 10−91	0.857661764249716	0.992	0.555	8.59 × 10−87
CDCA3	2	6.47 × 10−89	0.545369444372327	0.871	0.419	1.50 × 10−84
PBK	2	3.82 × 10−84	0.6553913393387	0.953	0.517	8.83 × 10−80
DEPDC1	2	9.19 × 10−81	0.562161053568371	0.811	0.374	2.13 × 10−76
CEP55	2	1.67 × 10−77	0.617579552600243	0.891	0.473	3.86 × 10−74
PIMREG	2	1.84 × 10−75	0.469640939018683	0.755	0.352	4.25 × 10−71
PHF19	2	7.36 × 10−73	0.522695499227147	0.946	0.67	1.70 × 10−68
NUSAP1	2	2.76 × 10−59	0.510702502616756	0.943	0.54	6.38 × 10−55
PLK1	2	1.76 × 10−58	0.43795685432261	0.745	0.338	4.06 × 10−54
MAD2L1	2	3.55 × 10−54	0.492908983497593	0.898	0.541	8.21 × 10−50
CKAP2	2	6.02 × 10−49	0.598043842889473	0.864	0.621	1.39 × 10−44
SMC4	2	1.88 × 10−47	0.525458738244071	0.977	0.681	4.34 × 10−43
MKI67	2	7.10 × 10−47	0.460075658603473	0.943	0.503	1.64 × 10−42
ANLN	2	1.47 × 10−46	0.434340895876565	0.921	0.532	3.39 × 10−42
NEK2	2	1.14 × 10−45	0.307580708480862	0.526	0.211	2.63 × 10−41
PRR11	2	4.65 × 10−46	0.368030429515734	0.811	0.465	1.08 × 10−41
BUB1	2	1.91 × 10−36	0.317568378806836	0.657	0.344	4.43 × 10−32
TMPO	2	5.74 × 10−34	0.429872635835613	0.948	0.717	1.33 × 10−29
ASPM	2	1.36 × 10−33	0.396222694173503	0.789	0.429	3.14 × 10−29
TACC3	2	1.11 × 10−32	0.332936107197724	0.801	0.453	2.56 × 10−28
MSH6	2	2.77 × 10−31	0.336669026498678	0.747	0.53	6.40 × 10−27
KNSTRN	2	9.43 × 10−30	0.40640158305418	0.765	0.565	2.18 × 10−25
PCLAF	2	2.54 × 10−28	0.412586221887705	0.987	0.741	5.87 × 10−24
H2AFX	2	5.65 × 10−26	0.345102089485394	0.886	0.667	1.31 × 10−20
CENPE	2	7.96 × 10−24	0.31517477923108	0.757	0.436	1.84 × 10−19
AURKA	2	1.16 × 10−21	0.454990793395169	0.705	0.479	2.68 × 10−17
KIF20B	2	1.19 × 10−23	0.343044424382152	0.781	0.497	2.76 × 10−17
RRM2	2	1.22 × 10−22	0.315412549099177	0.901	0.595	2.81 × 10−17
NUF2	2	1.01 × 10−20	0.31369299247181	0.7	0.405	2.34 × 10−16
MCM7	2	1.11 × 10−18	0.302397358738716	0.844	0.628	2.56 × 10−14
SGO2	2	5.42 × 10−16	0.328146528775139	0.687	0.46	1.25 × 10−11

Table 3

Highly variable genes after cell cycle regression in CAFs isolated from Patient2.

Gene	Cluster	p-Value	Average log2FC	Pct.1	Pct.2	p-Value Adjusted
TOP2A	0	0	1.79605326374694	0.815	0.233	0
UBE2C	0	0	1.63919445211954	0.781	0.211	0
CENPF	0	0	1.34876124117104	0.811	0.292	0
CDK1	0	0	1.33264666202355	0.774	0.138	0
STMN1	0	0	1.32138682451592	0.942	0.547	0
NUSAP1	0	0	1.2286980735027	0.792	0.238	0
DIAPH3	0	0	1.16113907654703	0.851	0.295	0
CKS2	0	0	1.15293101811774	0.922	0.62	0
HMGB2	0	0	1.11538535017322	0.768	0.246	0
TYMS	0	0	1.10000618998348	0.866	0.314	0
MKI67	0	0	1.07580673615101	0.731	0.204	0
SMC4	0	0	0.985921598378538	0.846	0.44	0
PCLAF	0	0	0.968323819792444	0.882	0.296	0
TPX2	0	0	0.924358894514124	0.769	0.266	0
ASPM	0	0	0.897670249420366	0.678	0.182	0
CLSPN	0	0	0.887171912927092	0.757	0.176	0
ATAD2	0	0	0.874751526379846	0.775	0.237	0
PBK	0	0	0.855784944466262	0.735	0.22	0
CENPU	0	0	0.835117757098471	0.782	0.196	0
CKS1B	0	0	0.833435203727637	0.876	0.454	0
UBE2T	0	0	0.825964540809045	0.785	0.275	0
RRM2	0	0	0.82406183500547	0.663	0.161	0
KIF20B	0	0	0.815776478647066	0.736	0.207	0
APOLD1	0	0	0.797140087161004	0.603	0.156	0
CENPK	0	0	0.793137975019939	0.805	0.261	0
CENPE	0	0	0.792614984666222	0.673	0.19	0
TACC3	0	0	0.766915247357903	0.705	0.199	0
TMPO	0	0	0.739947410344141	0.75	0.267	0
ANLN	0	0	0.732349867027408	0.691	0.206	0
SGO2	0	0	0.729181104940837	0.693	0.209	0
TK1	0	0	0.724386338545294	0.783	0.238	0
HMMR	0	0	0.699172842546925	0.645	0.161	0
NCAPG	0	0	0.697319371070576	0.645	0.124	0
FEN1	0	0	0.696892668232285	0.706	0.197	0
SPC25	0	0	0.681763327564178	0.635	0.128	0
HJURP	0	0	0.679793595290009	0.556	0.086	0
FBXO5	0	0	0.671620101318795	0.611	0.113	0
NUF2	0	0	0.640912005859067	0.611	0.148	0
HELLS	0	0	0.63451507811957	0.647	0.17	0
NDC80	0	0	0.621413114006706	0.616	0.141	0
GTSE1	0	0	0.619982966600172	0.6	0.145	0
AURKB	0	0	0.606884764450141	0.558	0.092	0
KIF23	0	0	0.602477769303273	0.605	0.145	0
ZWINT	0	0	0.598416502953059	0.656	0.175	0
KNL1	0	0	0.583367784243798	0.584	0.127	0
KIF11	0	0	0.572709771587086	0.578	0.131	0
CDCA8	0	0	0.538516599881907	0.512	0.089	0
CKAP2L	0	0	0.508486954585748	0.524	0.096	0
CEP128	0	0	0.485831827254704	0.5	0.086	0
MELK	0	0	0.482259210927512	0.538	0.106	0
ESCO2	0	4.69 × 10−295	0.443942217336555	0.469	0.073	1.12 × 10−289
PSMC3IP	0	2.56 × 10−293	0.534713822780204	0.612	0.161	6.12 × 10−289
AURKA	0	5.53 × 10−288	0.69848370289414	0.62	0.164	1.32 × 10−283
NSD2	0	6.07 × 10−287	0.672994436541658	0.719	0.268	1.45 × 10−282
BRIP1	0	1.73 × 10−286	0.500689898963752	0.469	0.078	4.15 × 10−282
MXD3	0	3.37 × 10−286	0.52543794412312	0.488	0.092	8.08 × 10−282
CEP55	0	1.45 × 10−282	0.626250526855352	0.662	0.187	3.46 × 10−278
ARHGAP11B	0	5.82 × 10−282	0.432470373138973	0.426	0.06	1.39 × 10−277
KIF14	0	6.53 × 10−279	0.458459893320424	0.479	0.085	1.56 × 10−275
KPNA2	0	6.13 × 10−277	1.11884776918933	0.869	0.555	1.47 × 10−272
RFC3	0	8.41 × 10−277	0.468635621907317	0.57	0.137	2.01 × 10−272
CCNA2	0	1.27 × 10−276	0.601184117702451	0.581	0.146	3.04 × 10−273
H2AFX	0	4.76 × 10−275	0.772913382345842	0.734	0.314	1.14 × 10−270
BIRC5	0	1.80 × 10−274	0.811277206411767	0.767	0.27	4.31 × 10−269
CENPM	0	3.93 × 10−273	0.542434709391609	0.67	0.202	9.40 × 10−269
CENPW	0	1.75 × 10−272	0.72148469926098	0.816	0.345	4.20 × 10−268
HIST1H1B	0	1.11 × 10−271	0.707208911930734	0.41	0.059	2.66 × 10−268
PRC1	0	2.52 × 10−271	0.745506641367581	0.746	0.261	6.03 × 10−268
PCNA	0	2.34 × 10−270	0.718422723531482	0.809	0.395	5.60 × 10−266
SHCBP1	0	1.48 × 10−268	0.451644802204119	0.559	0.129	3.55 × 10−264
DNAJC9	0	9.37 × 10−267	0.633949672086213	0.781	0.347	2.25 × 10−262
KIFC1	0	3.26 × 10−266	0.41048060928204	0.478	0.089	7.80 × 10−262
DTL	0	9.05 × 10−265	0.404951277172161	0.428	0.068	2.17 × 10−260
RAD51AP1	0	2.77 × 10−264	0.417840591287033	0.478	0.092	6.64 × 10−260
GINS2	0	6.91 × 10−264	0.460387044513258	0.512	0.11	1.65 × 10−259
CDCA5	0	7.00 × 10−262	0.347587617602858	0.409	0.058	1.68 × 10−257
KIF18B	0	3.05 × 10−261	0.382458042816294	0.404	0.057	7.30 × 10−257
BARD1	0	8.79 × 10−260	0.424084384025479	0.462	0.086	2.11 × 10−255
SGO1	0	8.35 × 10−259	0.41290078141843	0.472	0.09	2.00 × 10−254
KIF4A	0	3.33 × 10−255	0.472499859356929	0.5	0.109	7.97 × 10−251
TTK	0	7.37 × 10−255	0.366775451612081	0.419	0.066	1.76 × 10−250
DTYMK	0	1.18 × 10−254	0.7026225521664	0.913	0.593	2.82 × 10−250
ORC6	0	9.10 × 10−254	0.43159577464565	0.53	0.126	2.18 × 10−249
LINC01572	0	4.59 × 10−252	0.424717002777551	0.429	0.074	1.10 × 10−247
MAD2L1	0	6.58 × 10−252	0.577304939125178	0.699	0.247	1.57 × 10−247
MYBL1	0	2.37 × 10−249	0.365784421616595	0.415	0.066	5.67 × 10−245
CKAP2	0	1.65 × 10−248	0.745562712544766	0.747	0.31	3.95 × 10−244
KIF2C	0	6.83 × 10−246	0.436136580958906	0.456	0.09	1.64 × 10−241
NEIL3	0	1.20 × 10−245	0.346115986129678	0.377	0.051	2.88 × 10−241
SMC2	0	8.54 × 10−243	0.608875725169878	0.821	0.411	2.05 × 10−238
MCM7	0	1.14 × 10−241	0.493220488397421	0.652	0.213	2.73 × 10−237
BRCA1	0	7.42 × 10−236	0.422694688818054	0.509	0.124	1.78 × 10−231
DLGAP5	0	2.32 × 10−235	0.584637912644874	0.618	0.185	5.56 × 10−231
DEPDC1	0	2.39 × 10−235	0.497867650448442	0.574	0.156	5.73 × 10−231
RRM1	0	3.05 × 10−235	0.531960719218836	0.724	0.292	7.30 × 10−231
DNMT1	0	7.81 × 10−235	0.610218771091325	0.854	0.474	1.87 × 10−230
FANCI	0	5.13 × 10−233	0.41420243743029	0.521	0.133	1.23 × 10−228
CENPH	0	1.86 × 10−232	0.448572767362875	0.642	0.207	4.46 × 10−228
BUB1B	0	3.53 × 10−231	0.415512381900352	0.481	0.108	8.46 × 10−227
CDC25C	0	5.81 × 10−231	0.380566646887002	0.403	0.07	1.39 × 10−226
NCAPH	0	1.11 × 10−229	0.318274751878298	0.391	0.063	2.66 × 10−226
CENPX	0	1.39 × 10−226	0.639373459416858	0.922	0.651	3.32 × 10−222
PHF19	0	5.15 × 10−226	0.541201400054887	0.696	0.262	1.23 × 10−221
NCAPG2	0	3.70 × 10−224	0.431846455633852	0.539	0.149	8.86 × 10−220
KIF18A	0	1.53 × 10−223	0.421064420210407	0.48	0.114	3.67 × 10−219
CDCA2	0	2.31 × 10−223	0.449341137619628	0.506	0.133	5.54 × 10−219
PRR11	0	1.29 × 10−221	0.487110880247023	0.561	0.161	3.08 × 10−217
E2F1	0	2.84 × 10−221	0.39788386826894	0.473	0.11	6.81 × 10−217
CENPN	0	6.53 × 10−220	0.480054472791842	0.67	0.246	1.56 × 10−215
CDCA3	0	3.89 × 10−218	0.562071082533152	0.59	0.185	9.32 × 10−214
DLEU2	0	7.17 × 10−216	0.496516580212333	0.623	0.218	1.72 × 10−210
GMNN	0	2.80 × 10−214	0.558870724562077	0.729	0.319	6.71 × 10−210
CCDC18	0	3.73 × 10−214	0.468051889873288	0.558	0.173	8.93 × 10−210
CDC45	0	1.24 × 10−213	0.315790294359164	0.383	0.066	2.97 × 10−209
PTTG1	0	6.60 × 10−213	0.777391296315842	0.881	0.517	1.58 × 10−208
RPL39L	0	6.71 × 10−213	0.557396767350297	0.729	0.327	1.61 × 10−208
MND1	0	1.89 × 10−211	0.315714589477428	0.37	0.061	4.52 × 10−207
ITGB3BP	0	3.64 × 10−210	0.516068351304291	0.726	0.318	8.73 × 10−206
BRCA2	0	9.28 × 10−211	0.342272583284152	0.442	0.097	2.22 × 10−206
SPDL1	0	3.71 × 10−209	0.615851855050538	0.826	0.47	8.89 × 10−205
RACGAP1	0	2.33 × 10−204	0.402972574424527	0.5	0.136	5.58 × 10−200
SKA2	0	3.70 × 10−203	0.538645990835765	0.836	0.455	8.87 × 10−199
CENPA	0	1.63 × 10−202	0.492527034567137	0.513	0.149	3.91 × 10−198
BUB1	0	4.01 × 10−202	0.357204472978074	0.459	0.109	9.60 × 10−198
ARHGAP11A	0	1.47 × 10−199	0.353884246564175	0.422	0.093	3.53 × 10−196
NASP	0	7.93 × 10−199	0.579507057462542	0.882	0.54	1.90 × 10−194
TMEM106C	0	2.65 × 10−199	0.555492375381984	0.771	0.397	6.35 × 10−194
CDKN3	0	3.21 × 10−198	0.64734439585374	0.715	0.284	7.69 × 10−194
VRK1	0	4.26 × 10−197	0.380498837312631	0.538	0.167	1.02 × 10−192
USP1	0	3.68 × 10−196	0.487231052109189	0.682	0.284	8.82 × 10−192
PKMYT1	0	2.42 × 10−195	0.322695046925214	0.386	0.076	5.79 × 10−191
CIT	0	2.76 × 10−194	0.36227876018763	0.412	0.091	6.61 × 10−190
TCF19	0	3.14 × 10−194	0.326812235523065	0.392	0.081	7.51 × 10−190
PLK1	0	5.72 × 10−193	0.478566405086608	0.487	0.136	1.37 × 10−188
DBF4	0	7.76 × 10−193	0.476243325691938	0.579	0.205	1.86 × 10−189
MCM5	0	4.10 × 10−188	0.389797623180505	0.508	0.151	9.83 × 10−184
EMP2	0	5.79 × 10−188	0.482573581188507	0.729	0.328	1.39 × 10−183
CCNB2	0	9.87 × 10−187	0.483990292844758	0.604	0.205	2.36 × 10−182
FOXM1	0	1.36 × 10−185	0.347433950574551	0.483	0.132	3.25 × 10−181
CDKN2C	0	5.29 × 10−185	0.390740413257006	0.524	0.165	1.27 × 10−180
CENPP	0	1.73 × 10−184	0.418338769529916	0.521	0.164	4.14 × 10−180
KNSTRN	0	2.07 × 10−183	0.520280464541216	0.606	0.234	4.96 × 10−179
PRIM2	0	4.38 × 10−183	0.499396287232664	0.702	0.329	1.05 × 10−178
CIP2A	0	1.15 × 10−182	0.338827439703043	0.444	0.115	2.76 × 10−179
C21orf58	0	3.86 × 10−183	0.309679968228961	0.397	0.089	9.25 × 10−178
GAS2L3	0	3.93 × 10−182	0.355561254043055	0.383	0.083	9.42 × 10−178
CDC20	0	4.93 × 10−181	0.524729916423815	0.626	0.221	1.18 × 10−175
RPA3	0	3.54 × 10−179	0.508784005186509	0.849	0.518	8.47 × 10−175
PSIP1	0	4.21 × 10−178	0.531844560159837	0.858	0.525	1.01 × 10−173
CBX5	0	3.40 × 10−176	0.526371278230042	0.849	0.522	8.15 × 10−173
MIS18BP1	0	4.87 × 10−175	0.483623163399267	0.682	0.316	1.17 × 10−171
CCNB1	0	8.93 × 10−174	0.628253981701828	0.691	0.295	2.14 × 10−169
DHFR	0	9.04 × 10−174	0.426468651928734	0.635	0.258	2.17 × 10−169
EZH2	0	7.76 × 10−173	0.317640681430593	0.407	0.099	1.86 × 10−168
NCAPD3	0	4.50 × 10−171	0.360000013437414	0.518	0.171	1.08 × 10−167
CDC6	0	5.25 × 10−171	0.30306426739638	0.343	0.067	1.26 × 10−166
FAM111A	0	1.10 × 10−169	0.391990222575551	0.51	0.172	2.63 × 10−165
CDCA4	0	2.26 × 10−167	0.370946302210805	0.484	0.154	5.41 × 10−163
MIR924HG	0	5.76 × 10−166	0.321676526418005	0.286	0.043	1.38 × 10−161
BUB3	0	1.56 × 10−165	0.575824912784537	0.849	0.575	3.74 × 10−161
CENPQ	0	5.28 × 10−165	0.33197679927466	0.456	0.133	1.27 × 10−161
CDKN2D	0	1.41 × 10−156	0.570905800968053	0.664	0.335	3.37 × 10−152
CHAF1A	0	7.21 × 10−156	0.31441445649264	0.412	0.113	1.73 × 10−151
SAC3D1	0	1.94 × 10−155	0.473418133474899	0.702	0.362	4.65 × 10−151
NAV2	0	4.38 × 10−155	0.685637322475529	0.884	0.663	1.05 × 10−150
POLD3	0	2.41 × 10−153	0.403682002890545	0.559	0.218	5.76 × 10−149
HIST1H1A	0	5.94 × 10−153	0.469000700324917	0.311	0.062	1.42 × 10−148
CCDC34	0	5.20 × 10−151	0.440947201576789	0.689	0.342	1.24 × 10−146
LRR1	0	2.48 × 10−150	0.372292987167317	0.621	0.267	5.95 × 10−146
ECT2	0	2.06 × 10−146	0.369035453280601	0.56	0.221	4.92 × 10−142
RAD21	0	8.57 × 10−144	0.495144592786876	0.804	0.508	2.05 × 10−139
PARP1	0	4.44 × 10−139	0.406065423637836	0.649	0.306	1.06 × 10−134
TUBG1	0	6.03 × 10−139	0.433334255101158	0.778	0.459	1.45 × 10−134
FOSL1	0	4.94 × 10−137	0.491250510826917	0.778	0.485	1.18 × 10−133
SRSF2	0	5.02 × 10−138	0.46724198469059	0.912	0.681	1.20 × 10−132
EXOSC8	0	1.27 × 10−136	0.415536195334573	0.727	0.401	3.05 × 10−132
RFC4	0	2.50 × 10−136	0.358148003209805	0.541	0.218	5.98 × 10−134
CMC2	0	1.98 × 10−129	0.453658147952604	0.853	0.606	4.74 × 10−125
TRIM59	0	6.80 × 10−128	0.369853501293669	0.497	0.195	1.63 × 10−123
NCEH1	0	9.76 × 10−126	0.365913285724998	0.518	0.212	2.34 × 10−121
MCM3	0	2.65 × 10−125	0.375861362978072	0.518	0.214	6.34 × 10−121
SMC1A	0	8.46 × 10−125	0.427372190793345	0.75	0.44	2.03 × 10−120
TEX30	0	2.02 × 10−124	0.370736140970149	0.619	0.295	4.84 × 10−120
NUDT1	0	9.64 × 10−123	0.393853592948846	0.741	0.419	2.31 × 10−118
CSE1L	0	1.75 × 10−121	0.334689815978467	0.615	0.288	4.19 × 10−117
SCLT1	0	5.47 × 10−121	0.459845797339858	0.668	0.359	1.31 × 10−116
ALYREF	0	8.72 × 10−121	0.402862238077345	0.779	0.493	2.09 × 10−115
HAT1	0	9.02 × 10−118	0.38773725726055	0.734	0.422	2.16 × 10−113
SMC3	0	6.38 × 10−116	0.436689045824374	0.836	0.579	1.53 × 10−111
KIRREL3	0	1.41 × 10−114	0.452577593279448	0.662	0.357	3.37 × 10−110
BCL2L12	0	3.60 × 10−114	0.332916074952397	0.561	0.254	8.62 × 10−110
GPSM2	0	4.01 × 10−114	0.322807074183221	0.482	0.194	9.59 × 10−109
RAB3B	0	2.04 × 10−112	0.419372269808872	0.807	0.496	4.88 × 10−108
CDC25B	0	4.30 × 10−111	0.335344921243839	0.477	0.193	1.03 × 10−106
WDR34	0	1.19 × 10−110	0.320815379473183	0.587	0.272	2.85 × 10−106
RFX8	0	6.64 × 10−107	0.328403138105245	0.4	0.142	1.59 × 10−102
G2E3	0	5.74 × 10−106	0.373888532232132	0.578	0.284	1.37 × 10−101
CKAP5	0	9.76 × 10−106	0.389144262518308	0.692	0.397	2.34 × 10−101
EXOSC9	0	4.42 × 10−105	0.335587033772145	0.634	0.325	1.06 × 10−99
EZR	0	2.08 × 10−100	0.3749120699691	0.685	0.385	4.99 × 10−97
ANP32E	0	1.16 × 10−99	0.423805784666755	0.77	0.514	2.79 × 10−95
KIF22	0	1.84 × 10−99	0.389035285574824	0.718	0.43	4.41 × 10−95
FUS	0	1.36 × 10−98	0.390642972327249	0.891	0.691	3.26 × 10−93
RBMX	0	3.48 × 10−97	0.380013251798135	0.846	0.611	8.33 × 10−93
HSPB11	0	5.70 × 10−97	0.375582585560417	0.878	0.648	1.37 × 10−93
PLAU	0	2.59 × 10−95	0.484438089791465	0.536	0.261	6.20 × 10−90
CCDC14	0	2.89 × 10−94	0.313033819300279	0.549	0.267	6.92 × 10−90
ACYP1	0	4.27 × 10−94	0.317264238978913	0.618	0.327	1.02 × 10−89
BTG3	0	5.53 × 10−94	0.412283557307453	0.824	0.576	1.33 × 10−89
HMGB3	0	2.40 × 10−92	0.332437957934013	0.588	0.297	5.76 × 10−88
ARHGAP29	0	1.91 × 10−91	0.331087035035293	0.465	0.203	4.57 × 10−87
PGP	0	5.01 × 10−91	0.363745280592072	0.863	0.642	1.20 × 10−86
POP7	0	5.29 × 10−84	0.35041100976186	0.824	0.595	1.27 × 10−79
MOK	0	3.21 × 10−83	0.46737513519966	0.634	0.38	7.69 × 10−79
CBX1	0	6.14 × 10−83	0.352701885583383	0.844	0.629	1.47 × 10−75
FABP5	0	1.85 × 10−78	0.306117925413734	0.582	0.308	4.43 × 10−74
SAP30	0	8.53 × 10−77	0.352635521735067	0.743	0.504	2.04 × 10−72
MAP3K20	0	2.57 × 10−74	0.33640702090946	0.789	0.545	6.15 × 10−70
HIST1H1E	0	3.63 × 10−73	0.339306408869152	0.367	0.157	8.69 × 10−69
LSM2	0	2.41 × 10−70	0.31840562333533	0.83	0.606	5.77 × 10−66
PBX3	0	3.22 × 10−69	0.370031925013218	0.721	0.488	7.72 × 10−65
HYI	0	4.02 × 10−68	0.320373254026447	0.787	0.55	9.63 × 10−64
WNT5B	0	3.78 × 10−66	0.318356399985377	0.6	0.348	9.06 × 10−62
CRIM1	0	9.71 × 10−65	0.366441919630305	0.82	0.601	2.33 × 10−61
FAF1	0	4.79 × 10−63	0.326595080517033	0.757	0.517	1.15 × 10−59
RBBP7	0	4.80 × 10−63	0.312026848537851	0.761	0.535	1.15 × 10−58
CCDC88A	0	2.20 × 10−62	0.326290677753468	0.759	0.536	5.27 × 10−58
HAS2	0	6.61 × 10−54	0.348787908529369	0.765	0.513	1.58 × 10−49
C11orf96	2	6.44 × 10−104	0.740866964874605	0.754	0.515	1.54 × 10−99
PTTG1	3	0	1.68818151464034	0.984	0.579	0
CDKN3	3	0	1.32280170053098	0.928	0.345	0
BIRC5	3	0	1.24448294135578	0.969	0.347	0
CCNB1	3	1.68 × 10−270	1.53989120433647	0.889	0.351	4.02 × 10−266
CDC20	3	5.47 × 10−235	1.19385593452449	0.806	0.281	1.31 × 10−230
PRC1	3	1.56 × 10−225	1.03131500977058	0.89	0.343	3.73 × 10−221
CCNB2	3	8.32 × 10−206	0.89505539193741	0.772	0.266	1.99 × 10−201
CENPW	3	1.90 × 10−196	0.906748625989908	0.918	0.429	4.55 × 10−192
CENPF	3	1.65 × 10−191	1.10946534906002	0.949	0.382	3.95 × 10−187
DLGAP5	3	2.36 × 10−182	0.828327618951242	0.755	0.258	5.66 × 10−178
STMN1	3	1.16 × 10−172	1.04854350049214	0.993	0.623	2.79 × 10−168
KIF20A	3	6.24 × 10−153	0.478111913734825	0.457	0.109	1.49 × 10−148
PBK	3	6.93 × 10−153	0.768087459409072	0.817	0.317	1.66 × 10−148
TROAP	3	3.95 × 10−143	0.535024517798047	0.575	0.183	9.45 × 10−139
CKS1B	3	1.16 × 10−141	0.749209886022839	0.932	0.535	2.79 × 10−137
CDCA3	3	9.83 × 10−139	0.655516371526958	0.696	0.256	2.35 × 10−134
HMGB3	3	2.65 × 10−134	0.686178765495008	0.733	0.338	6.34 × 10−130
CEP55	3	2.18 × 10−129	0.712781591135284	0.702	0.281	5.23 × 10−125
PHF19	3	8.56 × 10−124	0.626704147754039	0.755	0.345	2.05 × 10−119
ANLN	3	1.05 × 10−123	0.620871330821275	0.751	0.3	2.52 × 10−119
SNHG3	3	2.41 × 10−122	0.614603665812925	0.62	0.264	5.77 × 10−118
ARL6IP1	3	3.38 × 10−119	0.952333640124179	0.899	0.651	8.10 × 10−116
DTYMK	3	2.31 × 10−117	0.677527425601535	0.94	0.657	5.54 × 10−113
LSM5	3	5.07 × 10−114	0.624023436144164	0.941	0.724	1.21 × 10−109
PCLAF	3	1.06 × 10−110	0.634544335405642	0.878	0.42	2.53 × 10−107
DEPDC1	3	1.15 × 10−109	0.638099370253651	0.611	0.239	2.76 × 10−104
MAD2L1	3	1.21 × 10−104	0.598964547956272	0.723	0.338	2.91 × 10−100
TPX2	3	1.75 × 10−102	0.719187603010911	0.772	0.371	4.19 × 10−98
PRR11	3	1.43 × 10−96	0.489548530150862	0.598	0.24	3.42 × 10−92
MKI67	3	2.35 × 10−96	0.596910007884026	0.753	0.312	5.62 × 10−92
DDX39A	3	3.47 × 10−96	0.602213577593247	0.808	0.516	8.30 × 10−92
PIMREG	3	7.42 × 10−92	0.399778725345863	0.433	0.139	1.78 × 10−87
SKA2	3	1.39 × 10−89	0.554764456829379	0.84	0.534	3.33 × 10−85
CKAP2	3	9.51 × 10−88	0.844708042203515	0.731	0.404	2.28 × 10−84
CENPN	3	7.43 × 10−87	0.485581470062575	0.679	0.334	1.78 × 10−82
CENPM	3	6.78 × 10−86	0.496753162630272	0.651	0.303	1.62 × 10−81
TK1	3	3.41 × 10−80	0.489549105877891	0.729	0.36	8.16 × 10−76
SMC4	3	4.70 × 10−80	0.625204041290138	0.867	0.522	1.12 × 10−75
NUSAP1	3	1.54 × 10−77	0.423643896935689	0.799	0.353	3.68 × 10−73
HMMR	3	9.63 × 10−77	0.5858548544598	0.606	0.268	2.31 × 10−72
TACC3	3	8.69 × 10−75	0.488847321071564	0.672	0.31	2.08 × 10−70
ASPM	3	3.16 × 10−73	0.587859571128048	0.635	0.292	7.57 × 10−68
ANP32E	3	3.27 × 10−67	0.520532490104568	0.818	0.561	7.83 × 10−63
KIF20B	3	7.51 × 10−66	0.566391401995441	0.658	0.329	1.80 × 10−61
RPL39L	3	1.40 × 10−63	0.449433426665234	0.729	0.412	3.36 × 10−59
HOTAIRM1	3	9.20 × 10−62	0.539538061574298	0.722	0.486	2.20 × 10−56
NASP	3	1.66 × 10−58	0.481350708319828	0.857	0.615	3.98 × 10−54
EMP2	3	7.00 × 10−55	0.437325491780974	0.703	0.416	1.68 × 10−50
SNHG19	3	4.24 × 10−53	0.356176367815828	0.42	0.184	1.02 × 10−48
MZT1	3	2.55 × 10−52	0.45252067702474	0.672	0.425	6.12 × 10−48
PLK1	3	5.14 × 10−52	0.363157432767263	0.48	0.211	1.23 × 10−47
NEK2	3	3.87 × 10−48	0.303008972860681	0.349	0.134	9.28 × 10−44
KPNA2	3	4.96 × 10−45	0.408224476401982	0.855	0.623	1.19 × 10−40
CENPE	3	9.27 × 10−45	0.429963395668457	0.589	0.303	2.22 × 10−40
NOP58	3	1.29 × 10−43	0.398497263591403	0.7	0.482	3.08 × 10−40
CENPH	3	5.09 × 10−43	0.342977126521325	0.562	0.309	1.22 × 10−38
RUVBL2	3	4.62 × 10−43	0.401938142708255	0.643	0.428	1.11 × 10−37
TYMS	3	1.75 × 10−41	0.348245582386649	0.774	0.442	4.20 × 10−37
NUDT1	3	1.27 × 10−40	0.387242464109805	0.712	0.49	3.03 × 10−35
KNSTRN	3	4.48 × 10−39	0.419295075117713	0.557	0.319	1.07 × 10−35
SMC2	3	1.60 × 10−38	0.363784568686639	0.747	0.508	3.82 × 10−33
SNRPA	3	4.23 × 10−36	0.37927864885733	0.584	0.375	1.01 × 10−31
DNMT1	3	2.02 × 10−36	0.394715713862948	0.785	0.564	4.84 × 10−31
NUF2	3	8.63 × 10−30	0.321669355708339	0.493	0.262	2.07 × 10−25
EZR	3	6.54 × 10−28	0.338204074306678	0.665	0.451	1.57 × 10−23
NDC80	3	1.26 × 10−27	0.319825238579957	0.48	0.26	3.01 × 10−23
SGO2	3	5.73 × 10−25	0.392584539338203	0.545	0.331	1.37 × 10−20

Table 4

Highly variable genes after cell cycle regression in CAFs isolated from Patient3.

Gene	Cluster	p-Value	Average log2FC	Pct.1	Pct.2	p-Value Adjusted
GDF15	0	1.00 × 10−124	0.670012236981009	0.517	0.238	2.33 × 10−119
CEMIP	0	4.81 × 10−115	0.790192919194519	0.621	0.347	1.12 × 10−110
DEPTOR	0	5.87 × 10−106	0.354082730670604	0.379	0.139	1.36 × 10−101
MXD4	0	6.26 × 10−90	0.393580712819188	0.642	0.379	1.45 × 10−85
ARMC9	0	9.84 × 10−81	0.453091970457053	0.676	0.438	2.28 × 10−77
PNRC1	0	2.58 × 10−77	0.31718171845308	0.448	0.216	5.98 × 10−74
CACNB4	0	2.37 × 10−74	0.434880339385922	0.543	0.304	5.50 × 10−69
PLXDC2	0	7.28 × 10−68	0.303863251658921	0.427	0.209	1.69 × 10−62
KIF26B	0	1.29 × 10−56	0.466385578865574	0.589	0.376	2.98 × 10−52
ANKRD37	0	2.18 × 10−56	0.502529523475167	0.482	0.279	5.05 × 10−52
FAM43A	0	6.47 × 10−57	0.341495945768616	0.469	0.257	1.50 × 10−51
TOP2A	1	0	1.72704801568497	0.862	0.345	0
UBE2C	1	0	1.33497306832552	0.733	0.217	0
HMGB2	1	0	1.31068678526262	0.911	0.399	0
HIST1H1B	1	0	1.21011344182568	0.614	0.102	0
CDK1	1	0	1.20899649346328	0.737	0.13	0
MKI67	1	0	1.03431981185335	0.81	0.353	0
DIAPH3	1	0	1.00041486814025	0.945	0.6	0
CDCA8	1	0	0.779702240086265	0.595	0.12	0
HJURP	1	0	0.765459493666479	0.573	0.082	0
CDCA2	1	0	0.702274359748242	0.565	0.121	0
GTSE1	1	0	0.693761336263562	0.604	0.16	0
NCAPG	1	0	0.659818505587877	0.627	0.172	0
AURKB	1	0	0.619116521095821	0.539	0.108	0
FBXO5	1	0	0.615389481482515	0.525	0.103	0
CKAP2L	1	0	0.598792006314765	0.541	0.103	0
ARHGAP11B	1	0	0.5812417345836	0.473	0.074	0
KIF18B	1	0	0.580142879752472	0.471	0.071	0
KIF23	1	2.27 × 10−287	0.720638959800598	0.691	0.235	5.27 × 10−283
HIST1H1A	1	6.01 × 10−285	0.913609800701636	0.481	0.106	1.39 × 10−280
SMC4	1	7.58 × 10−283	0.864979422051172	0.9	0.514	1.76 × 10−278
ESCO2	1	2.48 × 10−281	0.502365836533897	0.458	0.084	5.75 × 10−277
APOLD1	1	4.64 × 10−279	0.838368640254338	0.67	0.237	1.07 × 10−274
KIF2C	1	7.56 × 10−278	0.589787736752698	0.49	0.104	1.75 × 10−273
MELK	1	3.52 × 10−274	0.575736739467258	0.583	0.16	8.15 × 10−270
TMPO	1	4.30 × 10−274	0.767947092480434	0.843	0.423	9.96 × 10−270
CLSPN	1	6.95 × 10−274	0.719876368800088	0.743	0.275	1.61 × 10−269
TACC3	1	1.79 × 10−270	0.748458380464173	0.717	0.263	4.16 × 10−266
LINC01572	1	1.38 × 10−268	0.589935230882103	0.527	0.134	3.20 × 10−263
NEIL3	1	4.34 × 10−266	0.437098081258912	0.392	0.061	1.01 × 10−261
BRIP1	1	6.60 × 10−264	0.637712916789726	0.588	0.169	1.53 × 10−259
KIF20B	1	2.62 × 10−259	0.726463701130549	0.699	0.258	6.08 × 10−255
CEP128	1	3.30 × 10−259	0.550822882209233	0.495	0.118	7.64 × 10−255
ASPM	1	5.17 × 10−252	0.820869581321595	0.688	0.251	1.20 × 10−247
KNL1	1	9.01 × 10−249	0.595358414126582	0.607	0.184	2.09 × 10−244
KIF11	1	4.17 × 10−248	0.607181845238362	0.591	0.18	9.66 × 10−244
KIFC1	1	5.13 × 10−246	0.485167534071316	0.476	0.108	1.19 × 10−241
NDC80	1	5.91 × 10−245	0.581545868791417	0.579	0.167	1.37 × 10−240
CENPE	1	1.74 × 10−242	0.830486761428012	0.665	0.245	4.03 × 10−238
NUF2	1	6.57 × 10−242	0.638010395689706	0.612	0.197	1.52 × 10−238
CENPU	1	4.52 × 10−240	0.581598609771616	0.698	0.252	1.05 × 10−235
TTK	1	7.67 × 10−238	0.437592463155119	0.405	0.076	1.78 × 10−233
MIR924HG	1	1.21 × 10−238	0.647701379585797	0.516	0.138	2.81 × 10−233
DBF4	1	7.15 × 10−237	0.632464785381952	0.653	0.242	1.66 × 10−232
CENPK	1	1.91 × 10−233	0.62044231564181	0.718	0.292	4.42 × 10−229
SGO2	1	2.05 × 10−232	0.667120708929706	0.645	0.229	4.75 × 10−228
CCNA2	1	1.22 × 10−231	0.623778974117052	0.631	0.216	2.84 × 10−228
ATAD2	1	3.59 × 10−229	0.658767565339001	0.721	0.297	8.32 × 10−225
ORC6	1	2.23 × 10−227	0.481784925937081	0.52	0.141	5.18 × 10−223
SPC25	1	1.19 × 10−225	0.581833584432011	0.592	0.191	2.75 × 10−221
KIF14	1	2.21 × 10−224	0.490496690709161	0.459	0.109	5.11 × 10−220
UBE2T	1	4.57 × 10−224	0.621313085775268	0.733	0.313	1.06 × 10−219
HIST1H1E	1	3.81 × 10−217	0.722266888541442	0.527	0.175	8.82 × 10−213
KIF4A	1	6.36 × 10−216	0.543875954867782	0.56	0.175	1.47 × 10−212
GMNN	1	1.53 × 10−215	0.583998264523595	0.736	0.317	3.54 × 10−211
FEN1	1	2.71 × 10−213	0.507380219199132	0.583	0.19	6.29 × 10−209
NUSAP1	1	2.89 × 10−212	0.772936396606703	0.763	0.371	6.70 × 10−208
CKS2	1	3.74 × 10−212	0.871013874667081	0.883	0.609	8.66 × 10−208
NSD2	1	1.62 × 10−211	0.630784667555947	0.748	0.36	3.77 × 10−207
AURKA	1	5.76 × 10−210	0.734078391529851	0.582	0.203	1.34 × 10−205
CENPF	1	4.08 × 10−212	0.981493266552953	0.836	0.46	9.46 × 10−205
ANLN	1	5.04 × 10−208	0.665580161160135	0.71	0.305	1.17 × 10−203
CCDC18	1	2.17 × 10−205	0.537992631230034	0.589	0.209	5.03 × 10−201
SGO1	1	2.14 × 10−204	0.42462140086729	0.445	0.109	4.95 × 10−200
BARD1	1	1.55 × 10−204	0.469675415789094	0.51	0.152	3.60 × 10−199
DLEU2	1	9.72 × 10−202	0.600325914395249	0.681	0.297	2.25 × 10−197
HIST1H3B	1	8.08 × 10−198	0.331364514860151	0.281	0.037	1.87 × 10−193
KIF15	1	3.79 × 10−197	0.366987727162554	0.367	0.074	8.79 × 10−193
TPX2	1	3.06 × 10−193	0.732127237168298	0.831	0.458	7.09 × 10−189
KIF18A	1	3.14 × 10−192	0.510434190240487	0.535	0.178	7.28 × 10−188
SPDL1	1	5.37 × 10−191	0.639004145224719	0.805	0.474	1.24 × 10−186
PBK	1	6.00 × 10−188	0.652609738045231	0.774	0.375	1.39 × 10−183
BUB1B	1	6.41 × 10−186	0.447366477932345	0.494	0.146	1.49 × 10−181
RFC3	1	6.72 × 10−185	0.442017047728623	0.501	0.154	1.56 × 10−180
ARHGAP11A	1	2.57 × 10−184	0.44833851287311	0.427	0.114	5.96 × 10−180
CDC25C	1	8.49 × 10−184	0.302666270199418	0.274	0.038	1.97 × 10−179
USP1	1	9.57 × 10−184	0.554036641164941	0.716	0.339	2.22 × 10−179
H2AFX	1	3.44 × 10−183	0.620529441343685	0.712	0.359	7.98 × 10−179
CDCA4	1	1.80 × 10−183	0.421167860141002	0.469	0.141	4.16 × 10−178
RRM2	1	7.34 × 10−182	0.5714047398839	0.659	0.276	1.70 × 10−177
POLQ	1	1.14 × 10−181	0.334881354743659	0.338	0.067	2.65 × 10−176
BRCA1	1	1.27 × 10−179	0.463882543405512	0.533	0.18	2.94 × 10−176
C21orf58	1	3.34 × 10−174	0.349629662415553	0.366	0.084	7.74 × 10−170
CENPA	1	3.60 × 10−173	0.501865111481393	0.454	0.139	8.36 × 10−170
CIT	1	7.99 × 10−173	0.414358233464705	0.423	0.117	1.85 × 10−168
RAD51AP1	1	2.60 × 10−172	0.393885717817882	0.417	0.111	6.02 × 10−168
KPNA2	1	8.94 × 10−171	0.852269471899063	0.858	0.593	2.07 × 10−166
GAS2L3	1	2.47 × 10−169	0.425123516789511	0.376	0.094	5.72 × 10−165
FAM83D	1	1.28 × 10−166	0.355532271296998	0.325	0.068	2.96 × 10−162
DNAJC9	1	9.66 × 10−166	0.539087426376657	0.8	0.458	2.24 × 10−161
MIS18BP1	1	2.18 × 10−163	0.526547560532024	0.672	0.321	5.06 × 10−159
TYMS	1	6.28 × 10−163	0.567080173939906	0.846	0.508	1.46 × 10−158
CKAP2	1	3.26 × 10−162	0.695663722723763	0.709	0.361	7.56 × 10−158
NCAPH	1	8.18 × 10−162	0.310271769913698	0.338	0.074	1.90 × 10−157
BRCA2	1	1.09 × 10−161	0.413729693225856	0.444	0.136	2.53 × 10−157
FAM111A	1	2.02 × 10−161	0.399559533411284	0.439	0.135	4.69 × 10−158
ECT2	1	1.61 × 10−160	0.477856374138588	0.562	0.218	3.74 × 10−156
HMMR	1	1.47 × 10−150	0.551172165466593	0.637	0.275	3.41 × 10−146
MAD2L1	1	1.96 × 10−149	0.451799259093044	0.638	0.272	4.55 × 10−145
SHCBP1	1	3.08 × 10−147	0.423856719181729	0.536	0.201	7.13 × 10−144
HIST1H1C	1	1.37 × 10−145	0.608927235709592	0.559	0.255	3.18 × 10−141
MXD3	1	7.58 × 10−143	0.38986107239568	0.387	0.114	1.76 × 10−138
DNMT1	1	1.44 × 10−142	0.515550569349073	0.9	0.66	3.34 × 10−138
NAV2	1	3.85 × 10−142	0.688091984444401	0.776	0.512	8.92 × 10−138
CDC45	1	6.33 × 10−141	0.310435808801802	0.366	0.098	1.47 × 10−136
PIF1	1	5.35 × 10−139	0.36016834161781	0.292	0.067	1.24 × 10−134
MYBL1	1	1.80 × 10−136	0.318318334241842	0.347	0.093	4.17 × 10−132
VRK1	1	5.73 × 10−136	0.392139040630886	0.502	0.19	1.33 × 10−131
ZWINT	1	6.04 × 10−137	0.451302935397852	0.61	0.277	1.40 × 10−131
DTL	1	3.02 × 10−135	0.367072327396311	0.372	0.108	7.00 × 10−131
SMC2	1	4.91 × 10−133	0.49254004897609	0.802	0.489	1.14 × 10−128
HELLS	1	4.44 × 10−131	0.472491197382194	0.597	0.269	1.03 × 10−126
SCLT1	1	5.28 × 10−131	0.550006155368793	0.677	0.377	1.22 × 10−126
CEP55	1	1.14 × 10−130	0.490879947494692	0.684	0.333	2.64 × 10−126
MND1	1	8.76 × 10−130	0.312301643925559	0.361	0.103	2.03 × 10−125
SKA3	1	4.05 × 10−127	0.310432816536786	0.354	0.102	9.39 × 10−123
MCM7	1	3.48 × 10−125	0.43694530973709	0.662	0.317	8.07 × 10−121
FOSL1	1	1.48 × 10−125	0.579087551857356	0.838	0.614	3.44 × 10−120
RAD18	1	6.07 × 10−124	0.369091089371499	0.493	0.194	1.41 × 10−119
EZH2	1	2.68 × 10−122	0.350087093415413	0.43	0.151	6.21 × 10−118
SPATA5	1	4.54 × 10−121	0.430333880167729	0.539	0.245	1.05 × 10−116
MIS18A	1	5.92 × 10−120	0.338627347295294	0.455	0.167	1.37 × 10−116
NCAPG2	1	8.08 × 10−120	0.388953597980289	0.518	0.212	1.87 × 10−115
HIST2H2AC	1	2.18 × 10−117	0.459827084117736	0.447	0.181	5.04 × 10−113
CIP2A	1	2.48 × 10−118	0.348589258463476	0.443	0.164	5.75 × 10−113
NCAPD3	1	4.68 × 10−118	0.364429029867863	0.498	0.205	1.08 × 10−112
PCLAF	1	3.77 × 10−116	0.458137696175664	0.862	0.584	8.74 × 10−112
CENPP	1	6.27 × 10−116	0.415419981923284	0.506	0.212	1.45 × 10−111
DEPDC1	1	1.61 × 10−113	0.393812693913351	0.516	0.211	3.72 × 10−109
PRIM2	1	4.97 × 10−113	0.422873887917786	0.692	0.372	1.15 × 10−112
RRM1	1	8.12 × 10−113	0.431960647746961	0.665	0.355	1.88 × 10−108
G2E3	1	1.51 × 10−112	0.444785977826341	0.585	0.296	3.50 × 10−108
PCNA	1	3.48 × 10−112	0.476948583142726	0.661	0.369	8.06 × 10−108
AP001347.1	1	1.12 × 10−111	0.315812948836076	0.373	0.125	2.60 × 10−107
CKS1B	1	3.12 × 10−109	0.456582258564046	0.778	0.479	7.24 × 10−105
GPSM2	1	1.17 × 10−108	0.345728943259348	0.417	0.156	2.72 × 10−105
PRC1	1	2.53 × 10−107	0.476953071781039	0.783	0.449	5.87 × 10−103
KNSTRN	1	9.23 × 10−107	0.401461259105815	0.512	0.225	2.14 × 10−102
RACGAP1	1	1.35 × 10−106	0.332736847877824	0.411	0.15	3.13 × 10−103
PLK1	1	2.35 × 10−106	0.412673618186031	0.46	0.184	5.44 × 10−102
FANCI	1	3.69 × 10−107	0.324134569142021	0.433	0.164	8.55 × 10−102
ITGB3BP	1	9.35 × 10−105	0.36277717625636	0.596	0.287	2.17 × 10−100
POLD3	1	1.37 × 10−103	0.348618544428778	0.461	0.189	3.17 × 10−99
DTYMK	1	2.55 × 10−103	0.432705404552704	0.902	0.692	5.91 × 10−99
TRIM59	1	2.03 × 10−98	0.38570544217349	0.509	0.237	4.70 × 10−94
RFC4	1	1.74 × 10−97	0.353796335374352	0.489	0.215	4.03 × 10−93
CHAF1A	1	8.15 × 10−97	0.310489196440483	0.409	0.156	1.89 × 10−92
MCM5	1	1.35 × 10−96	0.34553837929917	0.536	0.241	3.14 × 10−92
GINS2	1	7.46 × 10−95	0.34719563516437	0.518	0.233	1.73 × 10−91
CDKN2D	1	2.50 × 10−94	0.368325034441899	0.391	0.155	5.79 × 10−90
PGP	1	1.02 × 10−93	0.407835180695608	0.798	0.544	2.37 × 10−89
SMC3	1	4.64 × 10−91	0.408844500543907	0.836	0.62	1.08 × 10−86
NASP	1	5.28 × 10−84	0.395402376975593	0.847	0.611	1.22 × 10−80
CKAP5	1	1.46 × 10−83	0.410491352853466	0.67	0.419	3.39 × 10−78
CBX5	1	2.20 × 10−82	0.369518182859071	0.864	0.652	5.10 × 10−78
CCDC14	1	2.74 × 10−81	0.308855146567134	0.455	0.207	6.35 × 10−77
SMC1A	1	1.81 × 10−78	0.376337526793547	0.655	0.389	4.20 × 10−74
CENPM	1	1.94 × 10−77	0.329745149819119	0.597	0.316	4.50 × 10−73
BUB3	1	3.08 × 10−76	0.401293068494684	0.797	0.586	7.15 × 10−73
PRR11	1	9.66 × 10−76	0.356696418545198	0.542	0.28	2.24 × 10−71
SAP30	1	1.49 × 10−75	0.340983727579872	0.555	0.296	3.45 × 10−71
SAC3D1	1	2.96 × 10−74	0.345588725644688	0.588	0.333	6.87 × 10−71
DHFR	1	1.98 × 10−72	0.309844463962329	0.513	0.256	4.60 × 10−68
EMP2	1	5.47 × 10−72	0.308138970460742	0.537	0.275	1.27 × 10−67
CMC2	1	2.34 × 10−70	0.341531617753068	0.725	0.479	5.42 × 10−66
POLE3	1	1.23 × 10−67	0.306632137012119	0.586	0.327	2.85 × 10−64
BIRC5	1	4.30 × 10−66	0.364636043735149	0.827	0.572	9.97 × 10−62
PSIP1	1	4.97 × 10−66	0.335570400412872	0.814	0.591	1.15 × 10−61
CDK5RAP2	1	2.04 × 10−61	0.303897510950978	0.533	0.293	4.74 × 10−57
PARP1	1	4.80 × 10−61	0.337561777573747	0.749	0.529	1.11 × 10−56
LCORL	1	5.83 × 10−61	0.329713081181335	0.489	0.268	1.35 × 10−56
BTG3	1	1.85 × 10−60	0.332337133755457	0.717	0.477	4.28 × 10−56
TK1	1	2.04 × 10−59	0.33510181879942	0.807	0.59	4.72 × 10−55
RAD51B	1	2.52 × 10−58	0.331080583017355	0.566	0.335	5.84 × 10−54
CENPW	1	2.58 × 10−54	0.308998607277912	0.863	0.629	5.97 × 10−50
CDC20	1	5.43 × 10−52	0.318295484247153	0.642	0.382	1.26 × 10−47
EXOSC9	1	6.81 × 10−54	0.304012196032269	0.585	0.364	1.58 × 10−46
KMT5A	1	2.58 × 10−50	0.306185177005478	0.597	0.378	5.98 × 10−46
RAD21	1	3.48 × 10−50	0.319947957948111	0.715	0.503	8.07 × 10−46
NCEH1	1	7.36 × 10−49	0.310793306903988	0.558	0.344	1.71 × 10−44
CCNB1	2	0	1.31153913591001	0.924	0.423	0
CDC20	2	0	1.12129311051783	0.867	0.348	0
CENPF	2	0	1.04026677851946	0.956	0.461	0
PTTG1	2	0	1.03054711379723	0.984	0.7	0
DLGAP5	2	0	0.917754645754222	0.753	0.268	0
CCNB2	2	1.58 × 10−249	0.825103171657037	0.726	0.301	3.67 × 10−246
PRC1	2	3.33 × 10−244	0.871240943557588	0.866	0.455	7.71 × 10−239
TPX2	2	4.54 × 10−235	0.876780210888109	0.888	0.474	1.05 × 10−231
CDKN3	2	5.29 × 10−233	0.865324099602013	0.902	0.553	1.23 × 10−228
BIRC5	2	2.76 × 10−211	0.795052317508702	0.908	0.572	6.40 × 10−206
PBK	2	1.49 × 10−204	0.787661110394609	0.819	0.395	3.45 × 10−200
ARL6IP1	2	6.80 × 10−195	0.816866773874749	0.814	0.494	1.58 × 10−190
HMMR	2	1.85 × 10−183	0.715432914553575	0.685	0.292	4.29 × 10−179
CEP55	2	1.66 × 10−175	0.704510401183117	0.727	0.35	3.86 × 10−171
MKI67	2	2.89 × 10−160	0.644584202642253	0.812	0.388	6.70 × 10−156
NUSAP1	2	2.03 × 10−150	0.648434446726603	0.778	0.398	4.70 × 10−146
CDCA3	2	1.88 × 10−138	0.589370038211954	0.568	0.252	4.36 × 10−134
SMC4	2	2.00 × 10−131	0.608459878088531	0.869	0.551	4.64 × 10−128
CKAP2	2	4.48 × 10−117	0.680097827457623	0.694	0.392	1.04 × 10−112
CKS1B	2	2.31 × 10−108	0.559775788818762	0.77	0.504	5.35 × 10−104
PHF19	2	2.70 × 10−103	0.53926975121795	0.701	0.455	6.27 × 10−98
DEPDC1	2	2.72 × 10−99	0.517613684298651	0.505	0.238	6.30 × 10−95
HMGB3	2	7.45 × 10−90	0.522062416887759	0.722	0.513	1.73 × 10−86
KPNA2	2	1.33 × 10−88	0.442058824158783	0.831	0.62	3.07 × 10−83
SNHG3	2	4.26 × 10−85	0.515930886429753	0.55	0.339	9.87 × 10−81
ANLN	2	1.74 × 10−77	0.455210659452447	0.625	0.357	4.04 × 10−72
PLK1	2	9.38 × 10−72	0.422891321882443	0.444	0.21	2.17 × 10−67
MAD2L1	2	2.06 × 10−71	0.468184987478444	0.55	0.322	4.78 × 10−67
TACC3	2	9.49 × 10−70	0.425832817667313	0.59	0.329	2.20 × 10−64
CENPE	2	8.03 × 10−67	0.433653107799618	0.563	0.302	1.86 × 10−62
ASPM	2	2.51 × 10−62	0.465087211115013	0.558	0.317	5.83 × 10−58
KIF20B	2	7.25 × 10−44	0.359035439133911	0.542	0.33	1.68 × 10−40
PTTG1	3	1.81 × 10−58	0.459413851506037	0.93	0.724	4.19 × 10−54

Table 5

Shared highly variable genes identified from CAFs/DAFs isolated from chronic pancreatitis and pancreatic cancer patients.

ANLN, APOLD1, ARHGAP11A, ARH11B, ASPM, ATAD2, AURKA, AURKB, BARD1, BIRC5, BRCA1, BRCA2, BRIP1, BUB1B, C21orf58, CCDC14, CCDC18, CCNA2, CCNB1, CCNB2, CDC20, CDC45, CDCA2, CDCA3, CDCA4, CDCA8, CDKN2D, CDKN3, CENPA, CENPE, CENPF, CENPK, CENPM, CENPP, CENPU, CEP128, CEP55, CIP2A, CIT, CKAP2, CKAP2L, CLSPN, DBF4, DEPDC1, DHFR, DLEU2, DLGAP5, DNAJC9, ECT2, EMP2, ESCO2, EZH2, FAM111A, FANCI, FBXO5, FEN1, G2E3, GAS2L3, GINS2, GMNN, GPSM2, GTSE1, H2AFX, HELLS, HIST1H1A, HIST1H1B, HIST1H1E, HJURP, HMGB2, HMMR, KIF11, KIF14, KIF18A, KIF18B, KIF20B, KIF23, KIF2C, KIF4A, KIFC1, KNL1, KNSTRN, LINC01572, MAD2L1, MCM7, MELK, MIR924HG, MIS18BP1, MKI67, MND1, MXD3, MYBL1, NAV2, NCAPG, NCAPG2, NCAPH, NDC80, NEIL3, NSD2, NUF2, NUSAP1, ORC6, PBK, PCLAF, PHF19, PLK1, POLD3, PRC1, PRIM2, PRR11, RACGAP1, RAD51AP1, RFC3, RRM1, RRM2, SACD1, SAP30, SCLT1, SGO1, SGO2, SHCBP1, SMC1A, SMC2, SMC4, SPC25, TACC3, TMPO, TOP2A, TPX2, TRIM59, TTK, UBE2C, UBE2T, USP1, VRK1, ZWINT

3.3. Instead of myCAF-, iCAF-, or apCAF-Specific Clusters, Rather a myCAF-Like Subtype with CXCL12 Expression Observed in Primary Isolated Disease-Associated Fibroblasts

We next analyzed potential myCAF, iCAF or apCAF transcriptomes in our primary disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. We used myCAF, iCAF, and apCAF markers as previously described [10,11] (see Table 6). As shown in Figure 7, we did not detect any myCAF-, iCAF-, or apCAF-specific cluster in disease-associated fibroblasts from pancreatic cancer or chronic pancreatitis patients. Yet, all disease-associated fibroblasts showed a gene expression profile similar to the myCAFs described by Öhlund et al. (Figure 7A). We observed positive staining of αSMA in all cases, we also observed positive expression of CXCL12, an iCAF marker (Figure 8).

Table 6

Lists of selected markers for myCAF, iCAF, and apCAF.

CAF Subtype	Selected Markers
myCAF (Öhlund et al.) [10]	Acta2, Vim, Ctgf, Col1a1, Col5a1, Col6a1
myCAF (Elyada et al.) [11]	Tnc, Tgfb1, Thy1, Tagln, Col12a1, Pdgfrb
iCAF (Öhlund et al.) [10]	Il1, Il6, Il11, Lif
iCAF (Elyada et al.) [11]	Clec3b, Col14a1, Gsn, Ly6c1, Cxcl12
apCAF (Elyada et al.) [11]	Slpi, Saa3, Cd74, H2-Ab1, NKain4, Irf5

Figure 7

No myofibroblastic, inflammatory or antigen-presenting cancer-associated fibroblast-specific cluster, but exhibit myCAF-like profiling in disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. Cell with (A,B) myofibroblastic, (C,D) inflammatory, and (E) antigen-presenting cancer-associated fibroblasts are visualized in fibroblasts from pancreatic cancer and chronic pancreatitis patients. Lists of selected markers are listed Table 5, markers were selected from the publications Öhlund et al. [10] for (A,C), Elyada et al. [11] for (B,D,E).

Figure 8

Immunocytochemistry of αSMA and CXCL12 in disease-associated fibroblasts from pancreatic cancer and chronic pancreatitis patients. Cells from (A) patient1, (B) from patient2, and (C) from patient3 for αSMA staining, (D) cells from patient1, (E) from patient2, and (F) from patient3 for CXCL12 staining. Bar: 50 μm.

In summary, our single-cell RNA sequencing and bioinformatics analysis identified several highly variable genes including IFI27, KRT18, KRT19, MMP1, MMP3, and NEFL, whose expression is associated with a shorter overall survival of pancreatic cancer patients. The majority of highly variable genes are however dependent on the cell cycle; therefore, we performed cell cycle regression analyses. We identified IGFBP3 and IGFBP5 after cell cycle regression, which may potentially contribute to CAF heterogeneity. Here, we could not identify myCAF-, iCAF-, or apCAF-specific clusters, but the expression profiling resemble a rather myCAF-like subtype. Most interestingly, after cell cycle regression, we observed large number of overlapping highly variable genes between samples from pancreatic cancer and chronic pancreatitis patients.

4. Discussion

Increasing evidence shows that CAFs represent a highly heterogeneous subpopulations that can be both tumor-promoting and tumor-suppressing, highlighting the importance of the identification and characterization of the diversity of CAF subtypes [4]. In the current study, we isolated primary disease-associated fibroblasts by the outgrowth method from pancreatic cancer and chronic pancreatitis patients. By using single-cell RNA sequencing and bioinformatics analysis, we identified several highly variable genes including IFI27, KRT18, KRT19, MMP1, MMP3, and NEFL. Expression of these genes is associated with shorter overall survival of pancreatic cancer patients (Figure 3). IFI27 has been previously identified as a prognostic marker for pancreatic cancer [18], and involved in pancreatic cancer migration and invasion [19]. The above-mentioned genes are not exclusively expressed in CAFs/DAFs. Yet, our data support that factors expressed in CAFs/DAFs can contribute to pancreatic cancer migration, invasion, and patient outcome.

We selected the outgrowth technique that has been established for isolating pancreatic CAFs and has become a common CAF isolation strategy. In our scRNAseq analysis, we found that the majority of identified highly variable genes are associated with the cell cycle. It was therefore important to conduct cell cycle regression [17], which showed that signals originating from cell cycle-associated factors have a strong impact on cluster heterogeneity (Figure 6). Interestingly, a number of highly variable features after cell cycle regression are commonly found in CAFs/DAFs isolated from chronic pancreatitis and pancreatic cancer patients (Table 5). These 135 factors still include a number of cell cycle-associated factors, but also cell cycle independent factors that are implicated in tumorigenesis. For example, KNSTRN promotes tumorigenesis and gemcitabine resistance in bladder cancer [20]. ZWINT supports pancreatic cancer proliferation [21]. Further studies are required to identify which stromal factors among these 135 genes play a key role in pancreatic cancer. It needs to be further clarified, whether pancreatic cancer-derived CAFs and chronic pancreatitis-derived DAFs have already similar profiling before isolation, or whether they become similar during culture. To that end, it is necessary in the future to establish chronic pancreatitis and pancreatic cancer mouse models with fibroblast lineage-tracing systems, to subsequently isolate CAFs/DAFs, and conduct single-cell RNA sequencing both before and after cell culture. Interestingly, our findings are consistent with the study by Barrera et al. Here, microarray experiments from DAFs isolated by the outgrowth method were performed demonstrating that pancreatic cancer- and chronic pancreatitis-derived fibroblasts share the greatest similarity [7].

One of the goals of the study was to see whether we identify previously described major CAF subtypes, myCAFs-, iCAFs-, and apCAFs. We did not observe any clear myCAFs-, iCAFs-, or apCAFs-specific clusters (Figure 7). Our data are in part consistent with a recent publication, where Grünwald et al. chose the outgrowth method and identified eleven sub-clusters from ten pancreatic cancer patients, but no clear myCAF versus iCAF subpopulation differences [22]. Initially, myCAFs and iCAF subtypes have been described by Öhlund et al. [10]. For the majority of their experiments, quiescent cells were isolated by Collagenase P and DNAse I digestion and co-cultured with pancreatic cancer organoids [10]. Murine late-stage tumors of KPC mice (Pdx1-Cre; lox-stop-lox-Kras) supported the presence of iCAFs and myCAFs, that were isolated by digesting tissue with Pronase, Collagenase P, and DNAse I [23]. Elyada et al. isolated single cells from human pancreatic cancer patients and KPC mice by digestion with Collagenase D, Liberase DL, and DNAse I [11]. Beside myCAFs and iCAFs, the study identified an additional subpopulation of CAFs expressing MHC class II and CD74 (apCAFs) [11]. Notably, in these studies enzymatic digestion methods were applied rather than the outgrowth method for CAF isolation. Whether methodological disparities have an influence on the outcome of these studies, need to be clarified in the future (e.g., via above-mentioned lineage-tracing strategies).

Our study is limited by the small sample size but is the first study to conduct scRNAseq and bioinformatics analyses combined for chronic pancreatitis and pancreatic cancer patients. In summary, our scRNAseq and bioinformatics analysis identified several highly variable genes, some of whose expression is associated with shorter overall survival of pancreatic cancer patients. We could not identify myCAF-, iCAF-, or apCAF-specific clusters, but the expression profiling resemble rather a myCAF-like subtype. Most interestingly, after cell cycle regression, we observed a large number of overlapping highly variable genes between samples from pancreatic cancer and chronic pancreatitis patients, which need to be verified and functionally characterized in both pancreatic cancer and chronic pancreatitis in vivo.