Journal: bioRxiv
Article Title: Co-evolution of Oncogenic KRAS Signaling and LILRB high Macrophages Drives Pancreatic Cancer Recurrence
doi: 10.64898/2026.03.05.709991
Figure Lengend Snippet: (A) Volcano plot of differential expressed genes in macrophages between primary tumors (PT) and matched recurrent tumors (RT), highlighting LILRB4 as a recurrence-associated marker. (B) Dot plot of LILRB family genes’ expression across macrophage subtypes. Dot size indicates P value significance and color denotes expression change between RT and PT (RT–PT), identifying LILRB4 as selectively increased within LILRB⁺ macrophages. (C) Representative multiplex immunofluorescence (mIF) images showing LILRB4 + CD68 + macrophage distribution in matched PT and RT tissues; stains include LILRB4 (orange), CD68 (magenta), PanCK (light blue), and DAPI (blue). Scale bars, 100 µm. (D) Quantification of LILRB4 + CD68 + macrophage density in matched PT and RT samples (left) and paired change (RT–PT) (right); lines connect matched samples; P value from paired t-test. (E) Kaplan–Meier analyses of progression-free/disease-free survival (PFS/DFS; top) and overall survival (OS; bottom) stratified by intratumoral LILRB4 + macrophage density (High vs Low, separated by top quarter) in an independent 190 patient’s cohort. P values, log-rank test. (F) LILRB4 + macrophage density across clinical recurrence categories (non-recurrent, regional recurrence, and distant metastasis); boxes show median and interquartile range with whiskers indicating 1.5× IQR; significance is indicated ( P value, Wilcoxon test; ns, not significant). (G) Representative mIF images showing spatial co-localization of LILRB4 + macrophages (LILRB4 + CD68 + ) with Basal_KRAS high (panCK + CK5 + p-Erk + GATA6 - ) tumor cells in matched PT and RT tissues; scale bars, 100 µm. (H) Representative flow cytometry plots (left) showing LILRB4 expression in tumor-infiltrating macrophages from matched non-tumor adjacent tissue (NAT) and PDAC tumor tissue. LILRB4⁺ macrophages were rare in NAT and enriched in PDAC. Right, paired quantification of LILRB4 + macrophage frequency (%) of all macrophages in NAT versus PDAC across samples (****, P < 10 -4 ; paired test). (I) Immunoblot analysis of PANC-1 and BxPC-3 cells under monoculture or co-culture with TAMs, including control TAMs and LILRB4-knockdown TAMs (LILRB4 kd#1 and LILRB4 kd#2 ). Blots show epithelial/plasticity and lineage markers (E-cadherin, N-cadherin, Vimentin, KRT5, GATA6, TP63) and ERK–MEK pathway activation (ERK, p-ERK, MEK, p-MEK); β-actin, loading control. (J) Representative bright-field time-course images (Days 1–4) of KPC PDAC organoids (Pdx1-Cre; LSL-Kras G12D/+; LSL-Trp53 R172H/+ ) co-cultured with LILRB4⁻ versus LILRB4⁺ TAMs, showing enhanced organoid growth, morphologic plasticity, and invasive outgrowth in the LILRB4⁺ TAM condition. (K) Representative transwell invasion assay images and quantification of invaded PDAC cells after treatment with TAM-derived conditioned medium (CM) from LILRB4⁺ or LILRB4⁻ TAMs. LILRB4⁺ TAM-derived CM increased tumor-cell invasion (*** P < 10 -4 ). PT, primary tumor; RT, recurrent tumor; TAM, tumor-associated macrophage; mIF, multiplex immunofluorescence; PanCK, pan-cytokeratin; PFS/DFS, progression-free/disease-free survival; OS, overall survival. TAM, tumor-associated macrophage; NAT, non-tumor adjacent tissue; CM, conditioned medium. See also Fig. S6 .
Article Snippet: The treatment arms included: Isotype control, LILRB4 mAb (250 μg/time/mouse), the KRAS G12D inhibitor (HRS-4642, 1.5mg/kg/mouse, Jiangsu Hengrui Pharmaceuticals Co., Ltd.), and a combination of LILRB4 mAb and KRAS G12D inhibitor.
Techniques: Marker, Expressing, Multiplex Assay, Immunofluorescence, Flow Cytometry, Western Blot, Co-Culture Assay, Control, Knockdown, Activation Assay, Cell Culture, Transwell Invasion Assay, Derivative Assay
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![a - c , RNA-seq analysis was performed on KS (AsPC1) and KR (PANC-1) PDAC cells treated -/+ MRTX for 24 h. ( a ) Venn diagram showing the overlap of differentially expressed genes between MRTX- and Veh-treated cells and between MRTX-treated AsPC1 and MRTX-treated PANC-1 cells. ( b ) KEGG enrichment analysis of selected overlapping genes (n = 1,339 genes) from ( a ), representing genes whose expression is specifically altered in intrinsic resistant cells upon MRTX treatment. ( c ) Heatmap showing mRNA expression of lipid metabolism-related genes. Blue, replicates with low expression; red, replicates with high expression. d , e , qPCR analysis of indicated mRNAs in the indicated cell lines or organoids treated -/+ MRTX for 24 h. f , LC-MS quantification of intracellular acyl-coenzyme A levels in PANC-1 cells treated -/+ MRTX for 24 h. g , Fractional labelling of TCA cycle intermediates in PANC-1 cells cultured with [U- 13 C]-PA, -/+ MRTX for 12 h. h , OCR of KS and AR AsPC-1 cells treated -/+ MRTX for 24 h before and after treatment with Omy, FCCP, and rotenone/antimycin A (ROT/AA). i , Total cellular ATP in indicated KS and KR/AR cells treated -/+ MRTX for 48 h. Data are presented relative to untreated cells. KP-4 is intrinsically KRASi resistant (KR). j , OCR of KR PANC-1 cells expressing DOX ACSBG1Δ treated -/+ DOX for 48h, followed with -/+ MRTX for 24 h before and after treatment with Omy, FCCP, and rotenone/antimycin A. k , Total cellular ATP in the indicated cells treated -/+ DOX for 48 h, followed with -/+ MRTX or HRS for 48 h. Data are presented relative to untreated cells. l , IB analysis of the indicated proteins in the indicated cells treated -/+ DOX for 48 h. Data in ( f , h - k ) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using two-sided unpaired t -test ( f , i KP-4) or one-way ANOVA with Tukey post-hoc tests ( i , k ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. Exact P values are shown in Source Data.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__04__01__715565/10__64898_slash_2026__04__01__715565___F7.large.jpg)
![a , LC-MS analysis of intracellular acyl-coenzyme A species in sensitive (KS) and acquired (AR) AsPC-1 cells -/+ 100 nM MRTX1133 (MRTX) for 24 h. Veh, Vehicle. b , Right: Fractional labelling of TCA cycle intermediates in KS/AR AsPC-1 cells incubated with [U- 13 C]-PA and -/+ MRTX for 12 h. α-KG, α-ketoglutarate. Left: A schematic illustration of PA-derived acetyl-CoA fuels the TCA cycle. Blue, replicates with low expression; red, replicates with high expression. c , Oxygen consumption rate (OCR) of KS and AR AsPC-1 cells incubated -/+ PA and 100 nM HRS-4642 (HRS) for 24 h before and after addition of 40 μM Etomoxir (Eto), followed by oligomycin (Omy), FCCP, and rotenone/antimycin A (ROT/AA) treatments. d , KS/AR HPAC cells expressing doxycycline (DOX)-inducible CPT1 deletion (DOX CPT1Δ ) were treated -/+ 0.2 μg/mL DOX for 48 h, and then -/+ HRS for 48 h. Total cellular ATP is relative to untreated KS cells. e , Representative images of mitochondria (TIM23) and BODIPY-labelled lipid droplets (LD) in parental KS No.6 and AR No.6 human PDAC organoids treated -/+ MRTX for 24 h. f , Quantification of mitochondria, LD, and their co-localization in ( e ). g , Pancreas morphology 3 weeks after orthotopic transplantation of KS and AR KC6141 (KC) cells expressing DOX CPT1Δ and host treatment at 72 h post-transplantation -/+ 30 mg/kg MRTX (i.p.), 0.2 mg/mL DOX (drinking water), or DOX + MRTX. h , Pancreas weight relative to body weight (P/B weight) of mice in ( g ). i , Representative H&E and IHC staining for CK19 in pancreata from ( g ). Boxed areas are further magnified. Tumor areas are at the bottom. j , Representative AFADESI MSI images and quantification of endogenous C16:0 L-carnitine (m/z 400.3436) and C18:1 L-carnitine (m/z 426.3559) distribution in pancreata two weeks after orthotopic transplantation of KS/AR KC cells expressing DOX CPT1Δ . Host were treated starting 10 days post-transplantation with MRTX, DOX, or DOX + MRTX. AR tumors exhibit elevated FAO and increased sensitivity to CPT1 ablation compared to KS tumors. Boxed regions indicate tumor areas. k , Pancreata from mice in ( j ) were sectioned and cultured with 100 μM [U- 13 C]-PA for 4 h. Representative MALDI MSI images and quantification of unlabeled citrate (M + 0, m/z 191.019) and 13 C-citrate (M + 2, m/z 193.026) distribution in pancreata, showing a similar trend to ( j ). Data in ( a , c , d , k ) (n=3 independent experiments), ( f ) (n=25 fields), ( h ) (n = 5 mice), ( i ) (n=7 fields), and ( j ) (n=6 fields) are mean ± s.e.m. Statistical significance was determined using one-way analysis of variance (ANOVA) with Tukey post-hoc tests ( a , d , f , h - k ) based on data normality distribution. Exact P values are shown in the Source Data. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Scale bars ( e ) 40 μm, ( g ) 1 cm, ( i ) 100 μm, ( j , k ) 500 μm.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__04__01__715565/10__64898_slash_2026__04__01__715565___F1.large.jpg)
![a , b , Genes differentially expressed between KS (AsPC-1, SW1990) and KR/AR (PANC-1, SW1990) cells treated -/+ MRTX for 24 h. Blue, replicates with low expression; red, replicates with high expression. c , d , IB analysis of ADGRB1 in indicated organoids or cell lines treated -/+ HRS for 24 h ( c ) or -/+ DOX for 48 h ( d ). e , KS and AR cells expressing DOX ADGRB1Δ , SRF-responsive luciferase reporter (SRF-RE), and a Renilla-luciferase control were treated -/+ DOX for 48 h, followed by incubation with D-Luciferin. Normalized Firefly/Renilla luciferase ratios, reflecting ADGRB1 (Gα12/13) activity, are shown relative to untreated KS cells. f , Fractional labelling of TCA cycle intermediates in KS and AR HPAC cells expressing DOX ADGRB1Δ treated -/+ DOX for 48 h, and then incubated with [U- 13 C]-PA and MRTX for 12 h. Blue, replicates with low expression; red, replicates with high expression. g , Representative IHC of resected ADGRB1 hi (#556) and ADGRB1 lo (# 835) human PDAC tissues. Boxed areas are further magnified. h , Numbers of human PDAC specimens (n = 42) positive for the indicated proteins, indicated as low and high expression. i , Correlation between expression levels of the indicated proteins in human specimens examined by a two-tailed Chi-square test. j , Comparisons of overall survival between patients with PDAC stratified according to ADGRB1 and pS144-PAK1. Significance was determined by log-rank test. Data in ( e ) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__04__01__715565/10__64898_slash_2026__04__01__715565___F10.large.jpg)
![a . Mice with mutant G6pd , that mimics human G6PD-deficiency, were bred into the KC ( LSL-Kras G12D ; Ptf1a Cre ) line. Cre was always maintained in female breeders. Experiments used both male and female G6pd mutant mice, where females were homozygous for mutant G6pd ( G6pd mut/mut ) and males were hemizygous for mutant G6pd ( G6pd mut/y ), as G6pd is an X-linked gene. G6pd wildtype mice used in experiments were obtained in the same colony and age matched littermates of G6pd mutant mice when possible. Female G6pd wildtype mice have two wildtype copies of G6pd ( G6pd wt/wt ) and males have one wildtype copy (their only copy) of G6pd ( G6pd wt/y ). In the schematics and labelling, “y” in male mice refers to the y chromosome, which does not contain a copy of G6pd . b . Schematic of 14 C-labeling experiment. [1- 14 C]glucose (blue) can be used in both the oxidative pentose phosphate pathway (ox PPP) and the TCA cycle. [6- 14 C]glucose (red) can be used in the TCA cycle. Released CO 2 represented as clouds; G6PD mutation represented as star. c . Graph depicting the relative amount of [ 14 C]-labeled CO 2 derived from glucose tracing in G6pd mut mice relative to G6pd wt mice (n = 2 biological replicates). Blue bar shows the relative [ 14 C]-labeled CO 2 derived from [1- 14 C]glucose, which is generated from either the oxidative pentose phosphate pathway (oxPPP) or the TCA cycle. Red bar shows the relative amount of [ 14 C]-labeled CO 2 derived from [6- 14 C]glucose, which is only generated from the TCA cycle. Grey bar shows the ratio of [ 14 C]-labeled CO 2 derived from [1- 14 C]O 2 to [6- 14 C]O 2 representing flux from the oxPPP. Bars represent the biological replicates’ mean. d . Hematoxylin & eosin (H&E) staining and immunostaining for amylase (AMY; acinar cells), cytokeratin 19 (CK19; ductal, metaplastic, neoplastic cells), and KI67 (proliferation) in pancreata from 8-week-old KC;G6pd wt and KC;G6pd mut mice. Scale bar = 50μm. e . Percent of Amylase+ area in pancreas as quantified from male (closed circle) and female (open circle) mice in KC;G6pd wt (grey bar) and KC;G6pd mut (blue bar) mice at 8 weeks. n = 4 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). f . Percent of CK19+ cells and g . Ki67+ cells in the pancreas as quantified from male (closed circle) and female (open circle) KC;G6pd wt (grey bar) and KC;G6pd mut (blue bar) mice at 8 weeks. n = 5 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). h . Immunostaining for Amylase (AMY), in 8-week and 16-week-old KC;G6pd wt and KC;G6pd mut pancreas. Alcian blue (PanIN-produced mucin) & nuclear fast red counterstain in pancreas of 26-week-old KC;G6pd wt and KC;G6pd mut mice. Scale bar = 500μm. i . Pancreas weight (PW) to body weight (BW) ratios in 8-, 16-, and 26-week-old KC;G6pd wt and KC;G6pd mut mice. Bars represent the mean with standard deviation. Ratios are not significantly different, as calculated using a Student’s t-test (unpaired, two-tailed) for each age. Sample sizes for each age are as follows: 8 week, n = 11 KC;G6pd wt and n = 8 KC;G6pd mut ; 16 week, n = 9 KC;G6pd wt and n = 8 KC;G6pd mut ; 26 week, n = 8 KC;G6pd wt and n = 8 KC;G6pd mut . j . Pathological grading of 26-week-old KC;G6pd wt and KC;G6pd mut pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 5 KC;G6pd wt mice; n = 7 KC;G6pd mut mice. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test. k . Pathological grading of 1-year-old KC;G6pd wt and KC;G6pd mut pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 5 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1030/pmc13121030/pmc13121030__42255_2026_1496_Fig11_ESM.jpg)
