Journal: Nature Cancer

Article Title: Cryoablation plus sintilimab and lenvatinib in advanced or metastatic intrahepatic cholangiocarcinoma: a phase 2 trial

doi: 10.1038/s43018-025-01058-2

Figure Lengend Snippet: a , Heatmap showing expression of cluster-defining gene modules by CD8 + T cells at single cell level in the scRNA-seq data. b , Sub-clustering of CD8 + T cells, colored and labeled by subclusters (left) and corresponding Tumor-reactive T cell signature score of each subcluster (right). c , RNA velocities overlaid on the UMAP of CD8 + PD-1 hi T cells in the scRNA-seq data. The arrow represents a spatial trajectory. Dots are colored by CD8 + PD-1 hi T cell subclusters. d , Pseudotime-ordered analysis of T1 and T4 clusters inferred by Monocle2 in the scRNA-seq data. Dots are colored by Pseudotime (left) and celltype (right). e , Scatterplots showing exhaustion score (left) and progenitor score (right) of T1 and T4 along Pseudotime. f , Box plot of scRNA-seq data showing the dynamic proportional changes of CD8 + PD-1 low T-cell subtypes in CD8 + T cells of paired samples stratified by three time points; each time point, n =12. g , Heatmap showing CD8 + T celltype annotation based on average expression of cluster-defining gene expression at the population level in IMC data obtained from 5 patients. h , Box plot of IMC data showing the dynamic proportional changes of CD8 + PD-1 hi Teff in CD8 + T cells of paired samples stratified by three time points; each time point, n =5. i , Box plot illustrating frequencies of clonal (left) and singlet (right) clones of CD8 + PD1 hi Teff stratified by three time points (each timepoint n =9) in scTCR data. j , Heatmap showing expression of cluster-defining gene modules by CD4 + T cells at single cell level (upper panel) and standardized average expression of each subcluster at the population level (lower panel) in the scRNA-seq data. k , Sub-clustering of CD4 + T cells, colored and labeled by subclusters (left) and corresponding Tumor-reactive T cell signature score of each subcluster (right). l , Box plot showing the dynamic proportional changes of CD4 + T cell subtypes of paired samples at three time points; each time point n =13 in the scRNA-seq data. m , Scatterplots showing significant correlations in the scRNA-seq data between CD4 + CXCL13 + Tfh proportion in CD4 + T cells with percentage of CD8 + PD-1 hi Teff in CD8 + T cells(left); percentage of B cells in all cells(middle); and percentage of Plasma in all cells(right). Analyzed by two-sided Spearman correlation test, the shaded area indicates the 95% CI of the fit. n , Representative H&E-stained images depicting intratumoral and stromal lymphocytes from P02 (left), who achieved partial response (PR); and P03 (right), who achieved complete response (CR), at three distinct time points. Scale bar, 100μm. o , p , Boxplots showing the dynamic changes in intratumoral ( o ) and stromal ( p ) lymphocyte densities identified in H&E-stained images at three time points; pre n =21, cryo n =16, lenva+sint n =11. q , Representative image of a tertiary lymphoid structure (TLS)-like formation observed in P02 following lenva+sint. Scale bar, 100μm. r , Box plot displaying the Cabrita’s signature score in bulk RNA-seq samples among three time points; pre n =22, cryo n =13, lenva+sint n =11. For a-e, j, k, m , data was obtained from 45 scRNA-seq samples at three time points (each time point, n =15). For b, f, h, i, k, l, o, p, r , center line indicates the median value, bottom and top hinges represent the 25th and 75th percentiles, respectively and whiskers denote 1.5 × interquartile range. For f, h, i, l , paired two-sided Wilcoxon test was applied. For o, p, r , unpaired two-sided Wilcoxon test was applied. For n, q , representative images from 48 H&E-stained images of 20 patients.

Article Snippet: These mice received intraperitoneal injection of anti-mouse PD1 antibody (BioXCell, BE0146; 10 mg kg −1 ) every 3 days, oral administration of lenva (Selleck, S1164, 3 mg kg −1 ) every day, a combination of both or their respective isotype controls (BioXCell, BP0089; Selleck, S6703).

Techniques: Expressing, Labeling, Gene Expression, Clone Assay, Clinical Proteomics, Staining, RNA Sequencing

Journal: Nature Cancer

Article Title: Cryoablation plus sintilimab and lenvatinib in advanced or metastatic intrahepatic cholangiocarcinoma: a phase 2 trial

doi: 10.1038/s43018-025-01058-2

Figure Lengend Snippet: a , Heat map showing standardized average expression values of cluster-defining genes in CD8 + T cell clusters on population level. b , Spearman correlation map showing associations of the proportions of CD8 + T cell subtypes among 15 pretreatment samples in the scRNA-seq data. c , Box plot of scRNA-seq data showing the intraparticipant dynamic changes in the frequency of CD8 + PD1 hi T cell subtypes in all CD8 + T cells of paired samples stratified by three time points: pre, cryo and lenva + sint (each time point, n = 12). d , Representative IMC images showing the distribution of CD8 + PD1 hi GZMK + Teffs in participant P02 at the three time points. Scale bars, 100 µm and 50 µm. e , Box plot of IMC data showing the dynamic changes in proportion of CD8 + PD1 hi Teffs in all CD8 + T cells at the inter-ROI level stratified by the three time points (pre, n = 32; cryo, n = 27; lenva + sint, n = 29; obtained from five participants each time point). f , Heat map displaying standardized average expression values in typical genes of T cell-related modules of CD8 + PD1 hi T cells at the three time points on population level. g , Violin plots showing scores for exhaustion, progenitor, proliferation and effector or memory signatures of CD8 + PD1 hi T cells at the three time points. h , Intraparticipant clonal expansion levels (left) and clonal transition levels (right) of CD8 + PD1 hi Teffs in paired samples at the three time points, quantified using the STARTRAC index. i , Alluvial diagram displaying CD8 + PD1 hi clonotype-mediated cluster associations. A flow or association among the three time points represents all pairs of cells sharing the same TCR clonotype but may transit to different phenotypes during treatment. j , k , Violin plot ( j ) revealing the change in clone size of persistent CD8 + PD1 hi Teff clones at the three time points at a TCR level (each time point, n = 40 clones) and UMAP plot ( k ) displaying the distribution of these clones over time (representing as blue dots). l , UMAP distribution of novel clones of CD8 + PD1 hi Teffs at the three time points (representing as blue dots). m , Pie charts showing the percentage of novel clones of CD8 + PD1 hi Teffs among the total preexisting cells (left) and TCR clones (right) at the lenva + sint time point. n , Clone size of novel and preexisting clones of CD8 + PD1 hi Teffs at the lenva + sint time point at a TCR level. o , Dot plots showing the expression levels of selected genes in novel and preexisting clones at the lenva + sint time point. Dot size indicates the percentage of cells in which gene expression was detected; color indicates the mean expression level. p , q , Frequencies of the TCR nucleotide sequences of novel clones identified in peripheral blood bulk TCR samples at the three time points illustrated by a heat map ( p ) and a box plot ( q ) (each time point, n = 24 clones). For a , f , g , data were obtained from 45 scRNA-seq samples at three time points (each time point, n = 15). For h – l , data were obtained from 27 scTCR-seq samples at three time points (each time point, n = 9). For m – o , data were analyzed in nine scTCR-seq samples after lenva + sint treatment. For c , e , g , h , j , q , the center line indicates the median value, bottom and top hinges represent the 25th and 75th percentiles, respectively, and whiskers denote 1.5× the interquartile range. For c , h , j , q , a paired two-sided Wilcoxon test was applied. For e , g , n , an unpaired two-sided Wilcoxon test was applied.

Article Snippet: These mice received intraperitoneal injection of anti-mouse PD1 antibody (BioXCell, BE0146; 10 mg kg −1 ) every 3 days, oral administration of lenva (Selleck, S1164, 3 mg kg −1 ) every day, a combination of both or their respective isotype controls (BioXCell, BP0089; Selleck, S6703).

Techniques: Expressing, Clone Assay, Gene Expression

Journal: Nature Cancer

Article Title: Cryoablation plus sintilimab and lenvatinib in advanced or metastatic intrahepatic cholangiocarcinoma: a phase 2 trial

doi: 10.1038/s43018-025-01058-2

Figure Lengend Snippet: a , Violin plots showing the signature scores of Kyoto Encyclopedia of Genes and Genomes (KEGG) antigen processing and presenting, Hallmark IFNγ response, IFNα response and TGFβ signaling in EPs at three time points. b , Representative IMC images showing the distribution of HLAI + EPs of participant P03 at the three time points. Scale bars, 100 µm and 50 µm. c , Box plot of IMC data showing the dynamic changes in proportion of HLA + EPs in all EPs at the inter-ROI level stratified by the three time points from five participants. d , e , UMAP plots of EP clusters colored by gene signatures identified in bulk RNA data related to poor PFS ( d ) and poor OS ( e ). f , Comparative gene set enrichment analysis of pretreatment gene sets in the EP1 cluster and other malignant EP clusters in 15 pretreatment scRNA-seq samples. NES, normalized enrichment score. P values were determined using a one-tailed permutation test. g , Heat map displaying standardized average expression values in typical genes of immune-related modules in EP1 cells at the three time points on population level. h , Heat map showing abundance of pathways and TME components per sample in pretreatment bulk RNA-seq samples stratified by median EP1 signature scores into high-EP1 and low-EP1 signature groups. Significantly differentially expressed pathways are indicated (unpaired two-sided t -test, * P < 0.05). i , Kaplan–Meier survival curves for PFS (left) and OS (right), grouped by EP1 signature scores of pretreatment bulk RNA-seq samples, analyzed by the log-rank test. j , Volcano plots showing the genes with differential expression between the time points before treatment and after cryoablation in all DCs in the scRNA-seq data (each time point n = 15). The x axis and y axis values were calculated by the Seurat method. Blue and green dots indicate genes with significant upregulation at the time points after cryoablation and before treatment, respectively. Significant genes of interest are labeled. k , Dot plots showing the expression levels of selected genes in all DCs at three time points. Dot size indicates the percentage of cells in which the gene was detected. Color indicates the mean expression. l , Numbers of enriched genes (count) of enriched pathways in all DCs at the time points before treatment and after cryoablation (each time point, n = 15) in the scRNA-seq data, analyzed by the hypergeometric test. m , Representative IMC images showing the distribution of CD11c + HLA-DR + IDO1 + APCs of participant P21 at the three time points. Scale bars, 100 µm and 50 µm. n , Quantification of the percentages of APCs in myeloid cells in IMC data at the inter-ROI level stratified by the three time points. o , Box plot of IMC data showing the dynamic changes in IDO1 expression in APCs at the inter-ROI level, stratified by the three time points (pre, n = 17; cryo, n = 20; lenva + sint, n = 23; obtained from five participants each time point); ROIs without IDO1 + APCs were excluded from analysis. p , Interaction score between CD8 + PD1 + Teffs (middle) and APCs (neighbor) in IMC at the inter-ROI level, stratified by the three time points. Black bars indicate the mean and 95% CI. q , Quantification of the distance of CD8 + PD1 + Teffs to the nearest APC in IMC, stratified by the three time points. For a , d , e , g , k , data were obtained from 45 scRNA-seq samples at three time points (each time point, n = 15). For a , c , j , n – q , an unpaired two-sided Wilcoxon test was applied. For c , n , p , q , ROIs obtained from five participants each time point (pre, n = 32; cryo, n = 27; lenva + sint, n = 29) were analyzed. For a , c , n , o , q , the center line indicates the median value, bottom and top hinges represent the 25th and 75th percentiles, respectively, and whiskers denote 1.5× the interquartile range.

Article Snippet: These mice received intraperitoneal injection of anti-mouse PD1 antibody (BioXCell, BE0146; 10 mg kg −1 ) every 3 days, oral administration of lenva (Selleck, S1164, 3 mg kg −1 ) every day, a combination of both or their respective isotype controls (BioXCell, BP0089; Selleck, S6703).

Techniques: One-tailed Test, Expressing, RNA Sequencing, Quantitative Proteomics, Labeling

Journal: Nature Cancer

Article Title: Cryoablation plus sintilimab and lenvatinib in advanced or metastatic intrahepatic cholangiocarcinoma: a phase 2 trial

doi: 10.1038/s43018-025-01058-2

Figure Lengend Snippet: a , Heat map showing standardized average expression values of cluster-defining genes in eight EC clusters on population level. b , Box plot showing dynamic proportional changes in all EC subtypes in paired samples at three time points of scRNA-seq data (each time point, n = 11). Data were analyzed using a paired two-sided Wilcoxon test. c , Pseudotime-ordered analysis of EC clusters EC1–EC4 in scRNA-seq data. Dot colors indicate pseudotime inferred by Monocle2 (top), cell type (middle) and time point (bottom). d , Heat map of the dynamic changes in gene expression along the pseudotime of the EC1–EC4 transition (divided into three phases), showing the distribution of the four EC subtypes over pseudotime. Top: subclusters are labeled by color. Genes showing differential expression during pseudotime (left) and enriched pathways (right) are labeled. e , Heat map showing scaled mean activities of selected modules in the eight EC clusters on population level. GO, Gene Ontology. f – i , Dynamic changes in transcriptional characteristics in EC subclusters at the three time points. Heat maps showing scaled average activities of selected modules in stalk and tip cells ( f ) and PCV cells ( h ) on population level. Dot plots showing the expression levels of selected genes in stalk and tip cells ( g ) and PCV cells ( i ). Dot size indicates the percentage of cells in which the gene was detected. Color indicates the mean expression. j , Scatter plots showing significant correlations between the proportion of CD8 + PD1 hi Teffs in CD8 + T cells and the percentages of stalk cells (top), tip cells (middle) and PCV cells (bottom) in all ECs. Data analyzed by the two-sided Pearson correlation test (top) or the two-sided Spearman correlation test (middle and bottom). k , Scatter plots showing significant correlations between the proportion of CD8 + PD1 hi Teffs in CD8 + T cells of scRNA-seq data and the TVNS in bulk RNA-seq samples (pre, n = 12; cryo, n = 9; lenva + sint, n = 9). Data were analyzed using a two-sided Pearson correlation test. l , Kaplan–Meier survival curves for PFS and OS, grouped by high and low TVNSs of 11 bulk RNA-seq samples from the CASTLE-01 discovery cohort after lenva + sint treatment, analyzed using the log-rank test. m , Box plot of IMC data showing the dynamic proportional changes in PCV ECs and stalk and tip cells of all ECs at the inter-ROI level, stratified by the three time points. n , Representative IMC images showing the distributions of CD31 + SELP + HLA-DR + PCV ECs and CD31 + VEGFR2 + stalk and tip cells of participant P03 at the three time points. Red arrows indicate the distribution of CD8 + PD1 hi Teff. Scale bars, 100 µm and 50 µm. o , Box plot of IMC data showing the dynamic changes in HLA-DR expression in PCV ECs and stalk and tip cells at the inter-ROI level stratified by the three time points (PCV HLA-DR expression calculation: pre, n = 26; cryo, n = 19; lenva + sint, n = 28; stalk and tip HLA-DR expression calculation: pre, n = 31; cryo, n = 24; lenva + sint, n = 28). ROIs without PCV ECs or stalk and tip cells were excluded for analysis. p , Interaction scores of CD8 + PD1 hi Teffs with PCV cells (neighbor; left) and stalk and tip cells (neighbor; right) in IMC data at the inter-ROI level, stratified by the three time points. Black bars indicate the mean values and 95% CI. For a , c – j , data were obtained from 45 scRNA-seq samples at three time points (each time point, n = 15). For j , k , the shaded area indicates the 95% CI of the fit. For m , p , ROIs obtained from five participants each time point (pre, n = 32; cryo, n = 27; lenva + sint, n = 29) were analyzed. For b , m , o , the center line indicates the median value, bottom and top hinges represent the 25th and 75th percentiles, respectively, and whiskers denote 1.5× the interquartile range. For m , o , p , an unpaired two-sided Wilcoxon test was applied.

Article Snippet: These mice received intraperitoneal injection of anti-mouse PD1 antibody (BioXCell, BE0146; 10 mg kg −1 ) every 3 days, oral administration of lenva (Selleck, S1164, 3 mg kg −1 ) every day, a combination of both or their respective isotype controls (BioXCell, BP0089; Selleck, S6703).

Techniques: Expressing, Gene Expression, Labeling, Quantitative Proteomics, RNA Sequencing

Journal: Nature Cancer

Article Title: Cryoablation plus sintilimab and lenvatinib in advanced or metastatic intrahepatic cholangiocarcinoma: a phase 2 trial

doi: 10.1038/s43018-025-01058-2

Figure Lengend Snippet: a , Heat map showing standardized average expression values of cluster-defining genes in mesenchymal cell clusters on population level. b , Heat map showing scaled mean activities of selected module in CAF subgroups on population level. c , Box plot of scRNA-seq data showing dynamic proportional changes in mesenchymal cell subtypes of paired samples stratified by three time points, analyzed by the two-sided paired Wilcoxon test. d , e , Dynamic changes in transcriptional characteristics of CAF1 cells at three time points, as illustrated by a heat map showing scaled average activities of selected modules on population level ( d ) and a dot plot showing the expression levels of selected genes ( e ). Dot size indicates the percentage of cells in which the gene was detected. Color indicates the mean expression. f , Scatter plots showing significant correlations of the percentage of CAF1 among mesenchymal cells with the proportion of CD8 + PD1 hi Tex cells among CD8 + T cells (left) and the exhaustion score in CD8 + PD1 hi T cells (right). Data were analyzed using a two-sided Spearman correlation test (left) or two-sided Pearson correlation test (right); the shaded area indicates the 95% CI of the fit. g , Heat maps showing NicheNet analysis in the scRNA-seq data of the regulatory potential of inferred prioritized ligands in CAF1 to the predicted target genes in CD8 + PD1 hi T cells and the inferred prior interaction potential of these ligands to receptor genes in CD8 + PD1 hi T cells (middle), scaled mean expression of ligands in CAF1 (left) and scaled mean expression of selected genes in CD8 + PD1 hi T cells (bottom) at the three time points. h , i , Dynamic changes in transcriptional characteristics of CAF3 cells at the three time points on a population level, as illustrated by a heat map showing scaled average activities of selected modules ( h ) and a dot plot showing the expression levels of selected genes ( i ). Dot size represents the percentage of cells in which the gene was detected. Color indicates the mean expression. j , Receptor–ligand pairs with significant differences inferred using scRNA-seq data between the three time points, based on CAF3 with CD8 + PD1 hi Tex cells and CD8 + PD1 hi Teffs. The larger dot size indicates a significant P value. Colors illustrate the communication probability. k , Representative IMC images showing the distribution of FAP + SMA + collagen + myCAFs, FAP + FBLN5 + iCAFs and FAP + FBLN5 + HLA + apCAFs of participant P02 at the three time points. Red arrows indicate the distribution of CD8 + PD1 hi Teff. Scale bars, 100 µm and 50 µm. l , Box plot of IMC data showing the dynamic proportional changes in myCAFs, iCAFs and apCAFs in mesenchymal cells at the inter-ROI level, stratified by the three time points. m , Violin plots of the distance between CD8 + PD1 hi Teffs and the nearest myCAF (left) and EPs and the nearest CD8 + PD1 hi Teff (right) at the three time points. n , Interaction scores of CD8 + PD1 hi Teffs with myCAFs (neighbor; left) and EPs with CD8 + PD1 hi Teffs (neighbor; right) in IMC at the inter-ROI level, stratified by the three time points. Black bars indicate the mean values and 95% CI. For a – j , data were obtained from 45 scRNA-seq samples at three time points (each time point, n = 15). For c , l , m , the center line indicates the median value, bottom and top hinges represent the 25th and 75th percentiles, respectively, and whiskers denote 1.5× the interquartile range. For l – n , ROIs obtained from five participants each time point (pre, n = 32; cryo, n = 27; lenva + sint, n = 29) were analyzed and an unpaired two-sided Wilcoxon test was applied.

Article Snippet: These mice received intraperitoneal injection of anti-mouse PD1 antibody (BioXCell, BE0146; 10 mg kg −1 ) every 3 days, oral administration of lenva (Selleck, S1164, 3 mg kg −1 ) every day, a combination of both or their respective isotype controls (BioXCell, BP0089; Selleck, S6703).

Techniques: Expressing

Journal: Nature Cancer

Article Title: Cryoablation plus sintilimab and lenvatinib in advanced or metastatic intrahepatic cholangiocarcinoma: a phase 2 trial

doi: 10.1038/s43018-025-01058-2

Figure Lengend Snippet: a , Schematic illustrating the evaluation of cryoablation plus lenva and anti-PD1 treatment effectiveness in an ectopic mouse ICC model. b , Differences in tumor growth between various treatment groups ( n = 6 mice per group). Data are shown as the mean ± s.e.m. and were analyzed using a two-way analysis of variance (ANOVA). c , Tumor images on day 28 ( n = 6 mice per group). d , OS in mice with ectopic ICC tumors ( n = 6 mice per group). Data were analyzed using a log-rank test. e – k , Flow cytometry quantification of CD11c + DCs ( e ), IFNγ + CD8 + T cells ( f ) and Ki67 + CD8 + T cells ( g ) from TdLNs and MHC I + EPs ( h ), CD8 + T cells ( i ), IFNγ + CD8 + T cells ( j ) and SELP + ECs ( k ) from tumors of mice with different treatment groups. Mice were killed on day 21 ( n = 5 mice per group). Data are shown as the mean ± s.d. and were analyzed using a one-way ANOVA. l , Schematic illustration of the cellular composition dynamics and potential therapeutic mechanism of cryoablation followed by lenva + sint.

Article Snippet: These mice received intraperitoneal injection of anti-mouse PD1 antibody (BioXCell, BE0146; 10 mg kg −1 ) every 3 days, oral administration of lenva (Selleck, S1164, 3 mg kg −1 ) every day, a combination of both or their respective isotype controls (BioXCell, BP0089; Selleck, S6703).

Techniques: Flow Cytometry