uPAR CAR T细胞靶向实体瘤及其支持性基质

Figure 1. uPAR marks a distinct cell state enriched in TP53 mutations and EMT programs (A) Multiplex immunofluorescence (multi-IF) across human tumor samples. Left: representative cores stained for uPAR, tumor cells (PanCK, p53, or MSLN), fibroblasts (αSMA), immune cells (CD45), and nuclei (DAPI). Right: donut plots showing the fraction of samples per tumor type stratified by the percentage of uPAR+ tumor cells: high (>50%), intermediate (30%–50%), low (5%–30%), and negative (<5%), and n indicates the number of patients analyzed per tumor type or number of tumors for OV and BLCA (patient information in Table S1A). (B–D) The Cancer Genome Atlas (TCGA) analysis (n = 10,071) showing (B) PLAUR expression stratified by mutation status across 456 cancer-relevant genes (MSK-IMPACT)31; (C) pathway enrichment in PLAUR-high (>66th percentile) versus PLAUR-low (<33rd percentile) tumors; and (D) correlation of uPAR activity with the indicated transcriptional signatures across cancer types (each dot represents one cancer type). (E) scRNA-seq analysis of epithelial tumor cells from liver (n = 79), lung (n = 9), ovarian (n = 41), and pancreatic (n = 16) cancers comparing PLAUR-high (>66th percentile) and PLAUR-low (<33rd percentile) for the indicated signatures.BLCA, bladder urothelial carcinoma; BRAIN, brain tumor (multiple subtypes); BRCA, breast invasive carcinoma; CCA, cholangiocarcinoma; CRC, colorectal cancer; MESO, mesothelioma; NSCLC, non-small-cell lung cancer; OV, ovarian cancer; PDAC, pancreatic ductal adenocarcinoma; PRAD, prostate adenocarcinoma; SARC, sarcoma. Statistics: Wilcoxon test (B and E), permutation test (C), and Pearson correlation test (D). Scale bars, 100 or 250 μm. See also Figure S1.

Figure 2. uPAR+ tumor, CAF, and myeloid cells define a fibrotic, immunosuppressive niche (A and B) Spatial transcriptomics (10× xenium) of a treatment-naive human PDAC specimen showing the cell-type map (A) and H&E staining from an adjacent section with xenium feature plots of PLAUR expression, PLAUR+ cells, and senescence signature scores (B). Full sections are shown in Data S1.3E. (C) Multi-IF-based spatial map from an adjacent section showing enrichment of uPAR+ PDAC cells near uPAR+/p16+ stromal cells. Cell types: tumor cells (PanCK+), fibroblasts (α SMA+), monocytes/macrophages (Mo/Mac and CD68+), and basal PDAC cells (KRT17+PanCK+). Full sections are shown in Data S1.3H. (D) Senescence scores in PLAUR+ versus PLAUR− Mo/Mac and CAF populations identified in (A). (E) Spatial co-localization heatmap for selected cell populations (complete heatmap in Figure S2E). (F) Frequency of neighboring cell types around basal versus classical PDAC neighborhoods. (G and H) Hyperplex 24-plex IF images from PDAC tumors (n = 101 cases and 649,581 cells analyzed) (G) and Spearman correlation analysis among cell populations (quantification in Data S1.4A) (H). Statistics: Wilcoxon test (D) and Spearman correlation (H) (p values shown in Figure S2H). Scale bar, 100 μm. See also Figures S2 and S3.

Figure 3. uPAR CAR T cells exhibit potent antitumor activity across multiple cancer models (A) Schematic of uPAR proteolytic cleavage and release of soluble uPAR (suPAR). Created with BioRender.com. (B) Quantification of uPAR surface density across 71 human cancer cell lines by flow cytometry. Non-cancer cells are indicated in green, uPAR-KO (uPARKO) in blue, and cell lines exhibiting durable in vivo tumor regression in red (see D and E). (C) Multi-IF staining for uPAR in subcutaneous xenografts of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), high-grade serous ovarian carcinoma (HGSOC), and pancreatic ductal adenocarcinoma (PDAC). (D–F) Antitumor efficacy of uPAR CAR T cells expressing the uPAR-binding scFv (T1-h.28z) versus a CD19 control CAR (h.19-h.28z), administered intravenously (2 × 106 cells per mouse): (D) tumor volume, (E) overall survival, and (F) CAR T cell infiltration assessed by anti-CD3 multi-IF staining 7 days after treatment. Statistics: multiple t test (D) and log-rank test (E). Error bars indicate SEM. Scale bar, 100 μm. See also Figures S4 and S5.

Figure 4. Efficacy of uPAR CAR T cells in ovarian cancer xenografts (A) Representative multi-IF images from the MSK SPECTRUM HGSOC cohort. (B) Orthotopic ovarian cancer model (Tyk-nuluc) treated with control CD19 CAR T cells (h.19-h.28z; 2 × 106) or uPAR CAR T cells (T1-h.28z or A1-h.28z; 2 × 106). Tumor burden was monitored by bioluminescence (images, left; quantification, middle) and overall survival (Kaplan-Meier, right). (C) Multi-IF of a human HGSOC specimen showing uPAR-expressing tumor cells (left) and higher magnification views highlighting uPAR+ tumor cells within detached tumor-cell clusters (right). (D) Spontaneous metastasis model using orthotopic Tyk-nuluc xenografts. Left: in vivo imaging following oophorectomy and ex vivo bioluminescence of met astatic dissemination (see schematic in Figure S7E). Right: metastatic tumor burden (middle) and Kaplan-Meier survival analysis (right) after treatment with control or uPAR CAR T cells. (E) Immunohistochemistry for GFP and quantification of tumor burden in the adnexa (left) and metastatic lesions in the lung (middle) and liver (right). (F) Longitudinal 89Zr-labeled uPAR PET imaging of primary (orthotopic; left) and metastatic (intraperitoneal; right) Tyk-nu tumors. Schematic created with BioRender.com. (G) Longitudinal 64Cu-labeled uPAR PET imaging of Tyk-nu tumors following treatment with control or uPAR CAR T cells at the indicated time points. Schematic created with BioRender.com. Statistics: multiple t test (B, middle; D, middle), Student’s t test (E), and log-rank test (B, right; D, right). Error bars indicate SEM (B and D) or SD (E). See also Figures S6 and S7.

Figure 5. uPAR CAR T cells target both tumor cells and their fibrotic stroma (A) Multi-IF images of orthotopic Tyk-nu (top) and COV644 (bottom) xenograft tumors showing human uPAR+ tumor cells and αSMA+ fibrotic murine stroma (left panel; scale bar, 50 μm) and murine uPAR+ stromal cells, including fibroblasts (αSMA+, right panel; scale bar, 10 μm). (B) In vivo bioluminescence imaging of Gaussia-luciferase (Gluc)-labeled CAR T cells expressing a murine uPAR-targeting CAR (m.uPAR-h.28z) or control CD19 CAR (h.19-h.28z) in mice bearing orthotopic Tyk-nuluc tumors expressing firefly luciferase (Fluc). Mice were imaged using a Gluc substrate (stromal targeting) or a Fluc substrate (tumor burden) 24 h after intravenous CAR T cell infusion and 4 weeks after tumor implantation, and radiance quantification is shown (n = 5 mice). (C) Fluc tumor burden in mice bearing orthotopic Tyk-nuluc tumors treated with h.19-h.28z, h.uPAR-h.28z, m.uPAR-h.28z, or combinations, and CAR T cell infusion timing is indicated (n = 5 mice) (D) Sirius red staining of lung metastasis sections from Tyk-nuluc-bearing mice treated with the indicated CAR T cells, with quantification of fibrosis area per field of view (FoV) using Fiji (see STAR Methods) (n = 5 mice). Scale bars, 1 mm (top) and 50 μm (bottom). (E) Multi-IF of Tyk-nu lung metastases showing m.uPAR+ stromal cells (red), GFP+ tumor cells (green), and CD3+ CAR T cells (blue) after treatment. Pseudocolor images and quantification of CAR T cell infiltration per FoV (n = 5 mice). Scale bars, 10 μm. (F–I) Flow cytometry of dissociated human HGSOC tumors after 16 h co-culture with the indicated CAR T cells (representative gating and quantification; n = 4 patients): (F) EpCAM+ tumor cells and the fraction of uPAR+EpCAM+ cells within EpCAM+ cells; (G) PDPN+ CAFs and the fraction of uPAR+PDPN+ cells within PDPN+ cells. Parental gate: live+CD45-CD31-EpCAM−; (H) MDSCs (CD45+Lin−CD11b+CD14−HLA−DR−CD15+; Lin− excludes CD3/CD19/CD56) and the fraction of uPAR+MDSCs; (I) GPNMB+CD11b+ myeloid cells (CD45+Lin−CD11b+GPNMB+; Lin− excludes CD3/CD19/CD56) and the fraction of uP AR+GPNMB+ cells within GPNMB+CD11b+ myeloid cells. (J and K) Flow cytometry analysis of CAR T cells following co-culture with primary HGSOC CAFs (n = 4). (J) Representative flow cytometry plots showing the percentages of CD4+ and CD8+ CAR T cells, as well as CD69+/CD62L+ cells within the CD4+ or CD8+ CAR T populations. (K) Quantification of the data shown in (J). CAR expression was assessed by LNGFR staining. In all flow cytometry plots, positive gates were defined using fluorescence minus one (FMO) controls. Statistics: Student’s t test (B and F–I), multiple t tests (C), and one-way ANOVA (D, E, and K). Error bars indicate SEM (C) or SD (F–K). See also Figures S8 and S9.

Figure 6. uPAR CAR T cells exhibit a favorable safety profile (A) Schematic of the electroporation-based genetically engineered mouse model (EPO-GEMM) enabling transposon-mediated oncogene integration (MYC) and CRISPR-Cas9-mediated tumor suppressor KO (Trp53 and Brca1) in the ovary/fallopian tube to generate autochthonous ovarian tumors. Created with BioRender.com. (B) Representative images of established MYCOE; Trp53KO EPO-GEMM tumors, and spontaneous metastases. (C) Representative H&E and immunohistochemistry staining for uPAR and WT1 in MYCOE; Trp53KO (MP) tumors. (D–F) Mice bearing autochthonous MP tumors were treated with 2 × 106 control untransduced T cells (UTD) or murine uPAR CAR T cells (m.uPAR-m.28z): (D) tumor weight at 2 weeks post-treatment (left) and overall survival (right), (E) body weight over time post treatment, and (F) complete blood counts (at day 40 post treatment). (G) Schematic for testing CAR T cells in mice with a humanized immune system (see STAR Methods). (H) Flow cytometry of bone marrow from humanized mice 6 weeks after HSPC transplantation and prior to tumor implantation (see STAR Methods). (I) Effects of the indicated TRAC-edited CAR T cells in humanized mice bearing Tyk-nuCD19 xenografts: bioluminescence images (left), radiance quantification of tumor (middle), and overall survival (right). (J) Flow cytometry showing relative abundance of human immune cell populations in bone marrow at tumor remission (day 7 post CAR T treatment) normalized to mouse CD45+ cells. (K) uPAR expression in human granulocytes (CD16+SSAhigh) and monocytes (CD14+) was assessed by flow cytometry using a human-specific uPAR antibody (gated as hCD45+CD19−CD3−). TRAC denotes TRAC-locus-edited CAR T cells. 1XX denotes CARs with inactivation of immunoreceptor tyrosine-based activation motifs 2 and 3 (ITAM2/3).50 UTD, untransduced T cells. Statistics: Student’s t test (D and F), multiple t test (I, middle), log-rank test (D, right; I, right), and one-way ANOVA (J and K). Er ror bars indicate SD (D) or SEM (E and I–K). See also Figures S10 and S11.

Figure 7. Therapy-induced senescence enhances CAR T cell efficacy (A) Representative multi-IF images of HGSOC tumors from treatment-naive (n = 23) or post-chemotherapy (n = 13) patients. (B) Quantification of uPAR+ cells among PanCK+ (tumor) or PanCK− (stromal) populations from (A) (each dot represents one tumor). (C and D) Bulk RNA-seq analysis of matched pre- and post-cisplatin-treated ovarian cancer samples61 showing PLAUR expression (C) and cell-cycle signature scores (D). (E) qPCR analysis of the indicated genes in MPB1, 136p, and OVCAR8 cells after 72 h exposure to the indicated senescence-inducing treatments in vitro. (F) Proliferation curves and immunoblotting showing uPAR upregulation in MPB1 cells 72 h after cisplatin (1 μM). (G) Flow cytometry analysis of surface uPAR expression in MPB1 cells treated with vehicle or cisplatin (1 μM) for 72 h. (H) Incucyte-based viability analysis of proliferating (vehicle) or senescent (cisplatin; 1 μM) MPB1 cells co-cultured with control (h.19-h.28z) or murine uPAR CAR T cells (m.uPAR-h.28z). (I) Multi-IF images (left) and quantification (right) of GFP+ MPB1 tumor cells (green) and m.uPAR-m.28z CAR T cells (white) 1 week after treatment of mice bearing metastatic MPB1 tumors (2 × 106 CAR T cells). (J–L) Response of mice bearing MPB1luc tumors treated with cisplatin (3 mg/kg), UTD T cells, m.uPAR-m.28z CAR T cells (2 × 106), or the combination. Tumor burden was monitored by bioluminescence (J), overall survival was shown by Kaplan-Meier plot (K), and body weight (L, left) and temperature (L, right) were assessed as an indicator of systemic toxicities. Statistics: two-way ANOVA (B and E), Student’s t test (C, D, F, and I), multiple t test (J), and log-rank test (K). Error bars indicate SD (B, E, F, and I) or SEM (H and J– L). See also Figure S12.

Figure S5. In vitro and in vivo characterization of uPAR CAR T cells, related to Figure 3 (A) Tumor growth curves of human cancer xenograft models treated with control h.19-h.28z CAR T cells or h.uPAR-h.28z CAR T cells. Shown are LoVo (CRC), MDA-MB-231 (triple-negative breast cancer [TNBC]), PC3 (double-negative prostate cancer [DNPC]), MDA-MB-436 (TNBC), DU145 (DNPC), and U87-MG (GBM) xenografts. Tumor volumes were measured over the indicated time courses. (B) Bright-field images of patient-derived organoids (PDOs) from mucinous ovarian cancer (MOC, left) and colorectal cancer (CRC, right) after co-culture with h.19-h.28z or h.uPAR-h.28z CAR T cells. Boxes highlight representative regions of organoid disruption, shown at higher magnification below. Scale bars, 500 μm. (C) Quantification of PDO cell lysis from MOC and CRC organoids after co-culture with h.19-h.28z or h.uPAR-h.28z CAR T cells at the indicated effector-to-target ratios. (D) Representative IF staining of CRC tissue sections showing expression of uPAR (red), αSMA (cyan), and p53 (green). Scale bar, 100 μm. (E) Tumor growth curves (left) and Kaplan-Meier survival analysis (right) for CRC PDO xenografts treated with h.19-h.28z or h.uPAR-h.28z CAR T cells. (F) Immunofluorescence staining of HGSOC PDX tumors showing merged channels (left), WT1 (green, middle), and uPAR (red, right). Scale bars, 100 μm. The graphs show tumor growth curves and Kaplan-Meier survival analysis for the HGSOC PDX xenografts treated with h.19-h.28z or h.uPAR-h.28z CAR T cells. (G) Immunofluorescence staining of MOC PDX tumors showing merged channels (left), p53 (green, middle), and uPAR (red, right). Scale bars, 100 μm. The graphs show tumor growth curves and Kaplan-Meier survival analysis for the MOC PDX xenografts treated with h.19-h.28z, h.uPAR-h.28z, or h.uPAR-h.28z 1XX CAR T cells. (H) Representative IF staining of relapsed tumor samples from NCI-H1703 (relapse at day 284) and PDX-MOC (relapse at day 200), showing uPAR (red), αSMA (cyan), and p53 (green). Scale bars, 100 μm. (I) Flow cytometry histograms showing uPAR surface expression in parental and relapsed tumor cells from MPB1 (MycOE;Trp53−/−;Brca1−/−) and MP (MycOE;Trp53−/−) models. Overlays compare parental isotype, parental uPAR, relapsed isotype, and relapsed uPAR signals. (J) Tumor growth curves (left) of MP (MycOE;Trp53KO) mouse ovarian tumors, either WT or uPARKO. Bar plot (right) shows the distribution of metastatic and non- metastatic disease for mice with WT versus uPARKO tumors. (K) Kaplan-Meier analysis shows survival of mice bearing MP or MPB1 tumors with and without uPAR KO. p values compare uPARKO to uPAR WT. (L) Tumor growth curve of Tyk-nu human HGSOC xenografts comparing WT versus uPARKO tumors. Statistical significance was determined using multiple t test (A; E, left; F, middle; G, middle; and J) and log-rank (Mantel-Cox) test (E, right; F, right; G, right; and K). Error bars indicate SEM.

Figure S11. Safety evaluation using mice with humanized immune system, related to Figure 6 (A) Flow cytometry analysis of human CD45+ cells showing uPAR expression in immune cell subpopulations from bone marrow in humanized immune system mice at baseline. (B) Flow cytometry analysis of Tyk-nu cells with ectopic CD19 expression, confirming uPAR and CD19 expression. (C) Schematic showing human uPAR CAR T cell designs: 28ζ (CD28-CD3ζ) and 28ζ-1XX (CD28-CD3ζ with ITAM2/3 inactivated).50 (D) Flow cytometry plot showing expression of CD3 and LNGFR (marker for CAR) in TRAC-edited human T cells. (E) Pie charts showing the proportions of T cells with surface expression of CD3 and CAR after TRAC editing: CD3-CAR+ (TRAC-edited CAR T), CD3+CAR+ (unedited CAR T cells), CD3+CAR− (WT T cells), and CD3-CAR− (TRAC-edited WT T cells). (F) Cytotoxic activity of CAR T cells derived from TRAC-edited human T cells against luciferase-expressing Tyk-nu cells, assessed using an 18 h bioluminescence assay. (G) Radiance measurement reflecting tumor burden in mice with a humanized immune system and rechallenged with Tyk-nu or Tyk-nu-uPARKO cells. (H) Representative flow cytometry histograms showing uPAR expression on the indicated cell types at 1 week post-treatment with 1 × 106 of control (TRAC-UTD), TRAC-edited CD19-CAR T (TRAC-h.19-h.28z.1XX), or TRAC-edited uPAR-CAR T (TRAC-h.uPAR-h.28z.1XX CAR T cells [T1 or A1]). (I) Bar plot showing uPAR median fluorescence intensity (MFI) in human immune cells 1 week post-treatment with TRAC-edited untransduced or CAR T cells. (J) Bar plot showing uPAR expression (MFI) in uPAR+ or uPAR- granulocytes and monocytes 1 week post-treatment with TRAC-edited UTD or CAR T cells. (K and L) Representative flow cytometry plots showing effects of co-culturing freshly isolated human primary CD14+ monocytes from peripheral blood mono nuclear cells with (K) h.19-h.28z or (L) h.uPAR-h.28z CAR T cells. (M) Flow cytometry plots showing expression of uPAR in CD14neg, CD14low, and CD14high monocyte populations from bone marrow of humanized mice. Error bars indicate SD (F) or SEM (I and J).